CORD-19:22616110b79baaf4c9d23f130a9ee35c64d991f7 / 525944-526011 3 Projects
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R2250 H1N1 virus infection: review of chest radiographic findings
Abstract
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Abstracts accepted for publication only Pathogenesis R2249 Effect of a probiotic dairy product containing Lactobacillus casei Shirota on the gut microbiota in irritable bowel syndrome: a randomised, placebo-controlled, double-blind study
Objectives: It has been hypothesized that disturbances in the intestinal flora could be a factor in the onset and persistence of IBS complaints. The aim of the study was to analyse the effect of Lactobacillus casei Shirota (LcS) on the faecal flora of IBS patients. Methods: In a randomised, placebo controlled, double blind study, IBS patients fulfilling the Rome criteria II were included. Patients took 2 bottles daily for 8 weeks, containing either LcS or placebo. Faecal samples were collected before intervention (week −2/0,S1), at the end of intervention (week 6/8,S2) and during follow-up (week 14/16,S3). Microbial populations (Bacteroides, anaerobes, bifidobacteria, coliforms, clostridia, and lactobacilli) were enumerated using quantitative plating. Total bacterial counts were performed using FISH. Bacterial DNA was analysed by quantitative Real-Time PCR for the detection of Atopobium spp., Bacteroides-Prevotella-Porphyromonas group, Clostridium coccoides-Eubacterium rectale group, Clostridium leptum subgroup, Clostridium difficile, Clostridium perfringens, Desulfovibrio desulfuricans group, and Bifidobacterium spp. Statistical analysis was performed using Mann-Whitney U tests or Paired Wilcoxon tests. P < 0.05 was considered statistically significant. Results: No significant differences in total bacterial counts were seen between the treatment and placebo group. Using quantitative plating, no significant differences were seen for the different bacterial groups between both groups. Significantly lower bacterial counts were seen in both groups for clostridia for S2 compared to S1 and S3. In the placebo group, significantly higher amounts of bifidobacteria and lactobacilli were detected for S2 compared to S1. Using qRT-PCR, significantly lower counts for D. desulfuricans group were seen in the placebo group compared to the treatment group. In the treatment group significant differences were seen only for S2 compared to S3, whereas in the placebo group, no significant differences were detected for the different bacterial groups at the different sampling points. Analysis at the IBS subtype level revealed differences in microbiota between the different subtypes. Conclusions: Although significant changes in the microbiota were seen during intake of LcS in the treatment group, differences in microbial populations between both groups were not significant. Interestingly, specific findings are likely to be associated with a specific IBS subtype rather than with IBS in general.
Objective: To asses the chest radiography findings of patients with H1N1 Influenza virus infection.
Review of radiological findings of 110 patients with confirmed influenza A virus infection admitted to two Hospitals in Cantabria, Spain. The radiological findings were characterized by pattern of opacities, lung distribution and, presence or absence of pleural effusions or hiliar or mediastinal adenopahies.
The initial radiography was abnormal in 39 (34%) patients, but only 28 (25%) were related exclusively to the influenza A virus infection. Characteristic imaging findings in these 28 patients included: parenchymal consolidation 68%, consolidation plus ground glass 25% and ground glass opacities in 7%. Seventeen patients had a diffuse distribution of the opacities, in 61% it was bilateral, and lower and middle zones were those most frequently affected. No pleural effusions or pathological mediastinal lymph nodes were seen in this initial radiography. Patients with abnormal chest X-ray were more frequently men, smokers, had dyspnoea, pleuritic pain and diarrhoea. Conclusions: Although more of patients with influenza virus A infection showed normal chest X-ray, those with abnormal chest radiographies, characteristically showed areas of consolidation with or without ground glass affectation, bilateral and diffuse distribution, and predilection for lower and middle zones. Pleural effusion or mediastinal adenopahty were not findings seen in the initial radiography.
Severe streptococcal infections lead to serious tissue damages. Myositis is an example of these conditions. Systemic antibiotic therapy is the principle course of treatment in necrotizing soft tissue infection. Clinical trials have also shown Hyperbaric Oxygen (HBO) therapy to be effective in streptococcal myositis patients. Ozone (O3), known as a cytotoxic gas, has recently been shown to support immune system by activating oxidative mechanisms and increasing pro-inflammatory cytokines. In this study, we compared the effectiveness of antibiotherapy, HBO and O3 application on bacterial growth and wound recovery in a murine model of streptococcal myositis. Forty-five male Sprague-Dawley rats were divided in five groups. Myositis was caused by inoculation of 0.15 ml 0.5 McFarland streptococcus pyogenes to thigh region in the four groups other than the Sham group. Normal saline was given to the rats in one of the four groups as a control, whereas other three groups were treated by penicillin G (98 mg/kg in 0.25 ml intraperitoneally 2X1), HBO (100% oxygen, 2,5 Atm. 90 min. 2X1) and ozone (1 mg/kg 1X1 intraperitoneally). At the seventh day, histological and bacterial investigations were done on soft tissue specimens taken from thigh region. There was significant difference in response to therapy in all of the three groups compared to control group (p < 0.05). Antibiotic and ozone groups were also more successful than HBO (p < 0.05). Ozone group yielded better results than that of antibiotic group (p < 0.05). Our results conclude that Ozone therapy should be used in addition to antibiotic treatment in serious infections such as myositis.
Introduction: Mutations in the DNA Mismatch Repair (MMR) genes, mutSL (equivalent to hexAB locus of pneumococci), have been associated with hypermutability phenotypes in various bacterial species and may play a significant role in the antibiotic resistance emergence. Objectives: The mutator frequency of 233 consecutive non-redundant Enterococcus faecalis isolates collected in a 2001 extra-hospital survey was investigated. Methods and Results: To detect mutators, a first approach was based on an agar diffusion method using a fosfomycin disk, and the identification of hypermutable strains was achevied by counting the "squatter" colonies growing in the inhibition zone after 24 h of incubation. Four strains (Ef1468, Ef1473, Ef1497 and Ef1591) with more than 50 Colony Forming Units within the fosfomycin inhibition area were considered as mutators. Their mutation frequency was evaluated on agar medium with or without rifampicin. The rates were comprised between 2.2×10 −5 to 7.3×10 −6 (SD, 2.4×10 −5 to 7.3×10 −6 ), i.e. at least 100-fold higher than five other tested strains taken at random in the collection, including the E. faecalis strain ATCC 29212 (frequency, <10 −9 ). The mutators were isolated from urine (Ef1468, Ef1473 and Ef1497) or sperm fluid (Ef1591). The analysis of mutSL was investigated for Ef1497 by PCR amplification and sequencing experiments. Five (A321T, A323S, A397E, K415N, I800S) and three (A437V, A440E, A495T) amino acid (aa) substitutions in MutS (858 aa) and MutL (710 aa) respectively, were identified in comparison to the reference sequence (GenBank accession no. AE016830). In addition, a nonsense mutation of the arginine at position 341 led to the inactivation of MutS; this mutation was associated with the hypermutability of Ef1497. Conclusions: The mutator frequency in a clinical population of E. faecalis was 1.7%. In a previous study, we have shown that linezolid mutants emerged more readily from Ef1497 than from E. faecalis ATCC 29212 (Ba et al., 2010 , Antimicrob. Agent Chemother., 54:1443 . Hypermutation capability of strains should be detected by the simple agar diffusion method using a fosfomycin disk, prior to a prolonged antibiotic monotherapy treatment. Our study gives the first evidence of an association of a MMR gene inactivation with a mutator phenotype in a clinical E. faecalis strain.
S. Najibi, B. Bakhshi*, M. Sattari, M. Alebouyeh, M. Tajbakhsh (Tehran, IR) Objectives: Infectious diarrheal diseases is recognized as the second cause of mortality among infectious diseases in children less than five years. Enteropathogenic Escherichia coli (EPEC) play an important role as a causative agent of children diarrhea. Integrons are DNA elements known to carry genetic cassettes responsible for antibiotic resistance. The objective of this study was to investigate the presence and resistance gene cassette content of class 1 integron among Enteropathogenic E. coli isolated from patients referred to Tehran hospitals. Methods: In this study, 300 stool samples were collected and Enteropathogenic Escherichia coli was detected using biochemical and serological methods and confirmed by PCR amplification of eae, stx1 and stx2 genes. Isolates with eae+/stx1−/stx2− genotype were defined as EPEC and subjected to further analysis. Class 1 integron was investigated with primers specific for the conserved integrase gene (int). The variable resistance gene cassettes were amplified and sequenced. Results: During the study period 28 cases of Enteropathogenic Escherichia coli strains was detected and serotype O86 and O127 were appeared as dominant serotypes. Twenty two strains (79%) appeared to harbor class 1 integron among which the variable region was defined to be dfrA7, aadA1, dfrA1/aadA1, dfrA12/aadA2 which related to amplicons of approximately 750, 1000, 1700 and 2000 bp in sizes, respectively.
The presence of class 1 integron in 79% reveals that, this genetic element plays a very important role in the transfer of antibiotic resistance among the strains studied and may transfer resistance to aminoglycosides and trimetoprim to other Gram-negative bacteria.
D. Plachouras*, I. Galani, Z. Chrysouli, F. Kontopidou, T. Panagea, M. Souli, G. Petrikkos (Haidari, GR) Objectives: Colistin resistance in Salmonella enterica has been associated with mutations in the PmrAB two component regulatory system that controls the composition of the outer membrane lipopolysachharide. The mechanism of recently described colistin resistance in Klebsiella pneumoniae clinical strains has not been elucidated. BasRS, the homologue system to PmrAB in K. pneumoniae, was studied. Methods: Colistin resistant K. pneumoniae strains isolated in clinical and surveillance specimens of 10 patients, who were on colistin treatment, as well as sensitive strains isolated from the same patients before colistin use, were included in the study. Exact MICs for colistin were determined by the E-test strip. Sensitive and resistant isolates were epidemiologically studied by repetitive extragenic palindromic (REP)-PCR methodology. The basR and basS genes from the respective resistant and susceptible isolates were amplified by PCR and sequenced. The primers used were designed by WebPrimer to amplify a 653 bp internal fragment of BasR ( In all other cases no significant differences were observed between the genes from the susceptible and resistant strains.
The mutation observed in the basS gene of one resistant isolate has already been described in the homologue gene pmrA of a colistin resistant laboratory strain of Salmonella enterica. However mutations in the two component regulatory system BasRS in clinical isolates do not explain most of the cases of colistin resistance in Klebsiella pneumoniae strains.
A. Liakopoulos*, K. Chatzigeorgiou, K. Tarpatzi, L. Zerva, E. Petinaki (Larissa, Athens, GR) Objective: To investigate the mechanism of high-level methicillinresistance in a Staphylococcus lugdunensis isolate. Materials and Methods: The microorganism was isolated from blood culture of a patient with endocarditis. Identification to the species level was performed by the Vitek 2 System and was verified by a molecular method based on fbl gene. Susceptibility testing to various antimicrobial agents was assessed by the Vitek 2 system, while the MICs values were determined by Etest. The production of penicillinase was performed by the nitrocephin disk test, while the presence of mecA gene was detected by PCR. In order to detect mutations in PBP1 and PBP4 genes, amplification followed by sequencing analysis was done and the results were compared with those obtained from a clinical S. lugdunensis strain susceptible to oxacillin (MIC: 0.75 mg/L).
The isolate, despite the high-level resistance to oxacillin (MIC: 256 mg/L), did not carry the mecA gene, while no overproduction of penicillinase or mutation of PBP4 were detected. However some mutations were found in PBP1: an alteration in the 482 position (L482P) and an insertion in 570 position of four aminoacids (SAYG). These mutations were not found in the susceptible strain. As b-lactams bind to the penicillin-binding motif KTG in the transpeptidase region, that is located in 582 position, alterations in or around the motif possibly confer resistance due to reduced affinity to b-lactams. Conclusions: This is the first report for mutations in PBP1 resulting in high-level methicillin-resistance in a clinical S. lugdunensis isolate.
Escherichia coli causing septic diarrhoea in calves in Italy: emergence of a multi-resistant O78 clonal group A. Marchese*, E. Coppo, R. Barbieri, S. Zoppi, C. Pruzzo, F. Rossi, S. Bergagna, A. Dondo, E. Debbia (Genoa, Turin, IT) Objectives: The emergence of antibiotic-resistant epidemic clones among animals should be monitored since clonal relatedness among certain O serogroup strains (i.e. E. coli O78) capable of causing disease in different hosts, humans included, has been shown and, although different molecules are employed, cross-resistance exists among fluoroquinolones used in human and veterinary medicine. An increased incidence of enrofloxacin-resistant E. coli associated with septic diarrhoea in calves was recently observed in northern Italy (from 14.3% in 2002 to >30% in 2007) . The aim of this study was to investigate this phenomenon. Methods: A total of 47 consecutive E. coli isolates exhibiting reduced susceptibility to enrofloxacin (intermediately resistant or resistant) causing septic diarrhoea in calves from 45 large-scale farms during 2006-2007, were studied. Phylogenetic group, antibiotic susceptibility and O serogroup were determined with RAPD and PFGE typing providing additional discrimination. Results: The majority (97.8%) of microorganisms (46/47) carried resistance to two or more additional drugs with the pattern: fluoroquinolone -ampicillin -co-trimoxazole -tetracycline -gentamicinthiamphenicol being the most represented (24/47; 51.0%). Plasmidmediated extended-spectrum and AmpC b-lactamases including plasmidmediated fluoroquinolone resistance genetic determinants were not detected. Third generation cephalosporins emerged as the most active antimicrobial agents tested (97.9% of susceptible strains). Conclusion: Overall, 37 different RAPD profiles and 18 different O serogroups could be distinguished among the typable strains indicating a substantial heterogeneity and suggesting the occurence of several independent selection events. However, approximately one fourth (11/47) of the strains belonged to serogroup O78 and PFGE revealed that the majority (7/11) of these were clonally related, indicating the selection of a O78 clonal group.
Since animals have been suggested as a possible source of serogroup O78 E. coli infections in humans, further studies are needed to clearly determine the zoonotic potential of these strains.
A. Barguigua, M. Bouchakour, M. Talmi, K. Zerouali, F. El Otmani, M. Timinouni* (Casablanca, El Jadida, MA) Objectives: The Morocco, like other countries worldwide, has a growing problem with CTX-M b-lactamase producing Enterobacteriaceae. The present study sought to characterize blaCTX-M-15-containing plasmids associated with Klebsiella pneumoniae CTX-M-15 isolates recovered in Moroccan community. Methods: We characterized the multidrug resistance region sequences of three plasmids that encode CTX-M-15 b-lactamases in Klebsiella pneumoniae strains isolated in three Moroccan cities; Casablanca (plasmid A), El Jadida (plasmid B) and Settat (plasmid C). Conjugative mating was attempted on agar. MICs were determined by E-Test method. Antibiotic resistance genes and integrons were identified by PCR and sequenced.
Results: Conjugative transfer of cefotaxime resistance was achieved in all strains. blaCTX-M-15 was carried by a 125 kb plasmid. In addition the plasmid A harboured the following 6 antibiotic resistance genes conferring resistance to seven antibiotic classes: blaOXA-1, blaTEM-1, aac(6 )-Ib-cr, catB4, tet(A), and the qnrB genes; the plasmid B carried blaTEM-1 consistently, also blaOXA-1, catB4, aac(6 )-Ib-cr, and tet(A). By contrast plasmid C carried blaOXA-1, catB4, aac(6 )-Ibcr, and tet(A). The blaCTX-M-15 gene presented the following genetic environment: ISEcp1-blaCTX-M-15-orf477 and blaTEM-1b -TnpA-ISEcp1-blaCTX-M-15-orf477. Conclusions: To our knowledge, this is the first description of genetic environment of CTX-M-15 gene in K. pneumoniae from a Moroccan community. The plasmids encoding CTX-M-15 conferred similar multidrug resistance phenotypes, suggesting that they may share a similar genetic scaffold. Both shared features with plasmids encoding CTX-M-15 b-lactamases in Escherichia coli from Canada.
21st ECCMID/27th ICC, Abstracts accepted for publication only R2303 Macrolide resistance and in vitro selection of antibiotic resistance in different human isolated Lactobacillus strains V. Rodighiero *, E. De Vecchi, R. Mattina, T. Celeste, M. Toscano, L. Drago (Milan, IT) Objectives: Spreading of antibiotic resistance is of concern due to the increasing rate of isolation of multiresistant pathogens. Since commensal bacteria can transfer determinants of resistance to pathogens, studies on resistance should include lactic acid bacteria, especially those intended for use as probiotics. We performed this study aiming to evaluate the capability of some antibiotics to select for resistance in lactobacilli. Methods: Strains of Lactobacillus acidophilus (n = 13), Lactobacillus plantarum (n = 9), Lactobacillus crispatus (n = 6) and Lactobacillus casei (n = 12) isolated from human feces were included. Amoxicillin/clavulanate, erythromycin and tetracycline were tested. Minimum Inhibitory Concentrations (MICs) were measured with the microdilution broth method. Susceptibility was determined according to breakpoints established by EFSA. The frequency of spontaneous mutations was calculated as the number of colonies grown on antibiotic-containing agar plates per inoculum. Selection of resistance was performed at 4× and 8× MIC and peak serum concentration (Cmax). Susceptible isolates were serially subcultured onto agar plates containing a linear gradient of each antibiotic. Bacteria were exposed to ten consecutive passages on antibiotic-gradient plates, then to ten passages on antibiotic-free plates. Acquisition of resistance was defined as >4-fold increase in MIC. Stable mutants with reduced susceptibility to erythromycin were analysed by PCR to detect the presence of erm and mef genes. Results: Resistance to macrolides was observed in 16 strains (8 of them harboured the ermB gene) and to tetracycline in 11 strains; all strains were susceptible to amoxicillin/clavulanate. Frequencies of mutation of susceptible strains (n = 26) were lower at 8× than at 4× MIC. Tetracycline showed the highest frequencies of mutations. After multi-step selection an increase in MICs was generally observed. Such change was stable only in some strains. All tested antibiotics could select stable resistance in all species. Molecular characterization of resistant mutants did not lead to detection of erm nor mef genes. Conclusions: Our results suggest that a decrease in susceptibility following exposure to antibiotics might occur in some lactobacilli. So, evaluation of the ability to acquire resistance to common antibiotics should be performed in parallel with investigations on the presence of resistance determinants in strains intended for human and animal use.
S. Ikeda*, H. Hanaki, Y. Ikeda-Dantsuji, H. Matsui, M. Iwatsuki, K. Shiomi, T. Nakae, K. Sunakawa, S. Omura (Tokyo, Kanagawa, JP) Background: Patients who suffer from MRSA infection are often coinfected with Gram-negative bacteria, and they are likely to be treated with a combination of vancomycin (VAN) and b-lactam antibiotics. However, this combination therapy causes the emergence of VAN-resistant MRSA, designated as b-lactam antibiotic-induced VAN-resistant MRSA (BIVR), which traps a large amount of VAN and lower free drug concentration in the mi-lieu. b-Lactam antibiotics inhibit peptidoglycan synthesis and promote the expression of autolysin resulting in the release of large amounts of peptidoglycan fragments into the extracellular milieu. We hypothesized that peptidoglycan fragments generated by the action of b-lactam antibiotics and autolysins were incorporated into the cytoplasm for recycling and promote synthesis of nascent lipid-II, an analogy to the case in Escherichia coli. Thus, we searched active compound(s), which mimics the role of b-lactam antibiotics in the induction of the BIVR phenomenon. Method: The BIVR strain K744 was used for the indicator cells in the BIVR assay. The compounds tested for the induction of BIVR phenomenon were either prepared from peptidoglycan fragments or the synthetics. The compounds to be tested were impregnated on a paper disc with a diameter of 8 mm, and that was placed on the VAN agar plate streaked with the K744 cells. The plates were incubated overnight. If the test compound was active, the indicator BIVR cells grow around a paper disc forming a hollow, of which diameter was measured. The non-BIVR cell show no growth zone due to the presence of VAN. Results: The purified muropeptide, GlcNAc-MurNAc-L-Ala-D-isoGln-L-Lys-(Gly4)-D-Ala-Gly2 and GlcNAc-1, 6-N, O-diacetyl-MurN-L-Ala-D-isoGln-L-Lys-(Gly4)-D-Ala-Gly2 at 75 mg per disc yielded 17.5 and 11.5 mm, respectively, of the K744 growth zone. The Gly4 peptide was not essential for the activity. The BIVR inducing activity was undetectable by GlcNAc-MurNAc-L-Ala-D-isoGln, MurNAc-L-Ala-D-isoGlu-L-Lys and L-Ala-D-isoGlu-L-Lys.
We concluded that the active compound was composed of the GlcNAc-MurNAc glycan chain and the L-Ala-D-isoGln-L-Lys-D-Ala-Gly2 peptide.
C. Ludden, M. Corcoran, D. Killeen, D. Keady, M. Da Costa, M. Cormican, D. Morris, T.W. Boo* (Galway, IE) Objective: Emergence of carbapenem-resistant Enterobacteriaceae (CRE) has become a significant public health threat. Within this diverse group, VIM-producing Enterobacter cloacae is increasingly reported in countries such as Greece and Spain. In this report, we evaluate the evidence for transmission of carbapenem-resistant E. cloacae in a cardiothoracic intensive care unit (CT-ICU). Methods: Clinical, infection control and microbiological data were collated. Clinical specimens were cultured by rountine methods. Patients (rectal swab) and the environment were screened for CRE. Isolates were identified by Vitek 2 and susceptibility testing was performed by CLSI disc diffusion. Evaluation for carbapenemase was conducted using the modified Hodge test (MHT), metallo-b-lactamase (MBL) Etest and synergy testing. PCR detection of carbapenemase genes and analysis of Xba1-restricted PFGE banding patterns were also performed. Results: Carbapenem-resistant E. cloacae were cultured from sternal wounds of 2 patients within a 6-day period. Both patients had received broad-spectrum antibiotics but neither had a history of recent travel. Their respective stays in the CT-ICU had overlapped for 11 days. Both were treated with appropriate antibiotics while aggressive infection control measures were instituted. CRE was not isolated from other patients, the hospital environment or medical equipment at that time. The isolates were resistant to b-lactams (including carbapenems), gentamicin and tobramycin, but susceptible to ciprofloxacin, amikacin and colistin. Phenotypic carbapenemase screening methods yielded positive but often inconsistent and subtle results. PCR confirmed the presence of blaVIM in both isolates, while PFGE demonstrated indistinguishable banding patterns.
Conclusions: This is the first report of the emergence of VIM-producing E. cloacae in Ireland. Sternal wound infections by such organisms were also not previously reported. Clinical and molecular data indicated that cross-transmission of carbapenem-resistant E. cloacae in CT-ICU was likely. Laboratory detection of VIM-producing Enterobacteriaceae remains challenging, while the need for active surveillance in highrisk patients warrants further consideration. The report highlights the therapeutic and infection control challenges posed by these organisms, and reiterates the importance of prudent antibiotic usage and aggressive infection control measures.
Introduction: Integrons are mobile genetic elements capable of gene capture and expression via site-specific recombination and the action of a promoter. Integrons play a major role in the dissemination of antibiotic resistance genes and are commonly associated with members of the family Enterobacteriaceae.
Objectives: To find out the incidence and the classes of integron associated with ESBL producing Klebsiella pneumoniae isolated from blood stream infections.
This study was carried out on 256 K. pneumoniae isolates over a period of two years. Antimicrobial susceptibility was tested for 14 antibiotics. ESBL detection was done as per CLSI followed by a multiplex PCR. Integrase gene PCR was done to detect class 1, class 2 integrons; similarly for class 3 and class 4 integron using specific primers. Sequencing was done for representative number of strains. Results: Out of the 256 isolates, 167 (65.2%) were ESBL producers. blaSHV (77.2%) and blaCTX-M (85.6%) were the most common. Of the 167 ESBL positive isolates, 121 (72.4%) carried class 1 integron; 51 (42.1%) isolates carried class 2 integron. Both class 1 and class 2 were found in 33 (27.2%) and none had class 3 or class 4 type. Sequencing and blasting results confirmed their identities. The drug resistant rates of integron positive isolates were 23% higher compared to integron negative strains.
Conclusions: A higher percentage of class 2 integrons association with ESBL strains is being noted for the first time from our region, also the co-existence of both class 1 and class 2 types increases the higher risk of multidrug resistant gene transfer rates. These findings strongly suggest that integrons have a major role in the dissemination of ESBL mediated resistance among nosocomial isolates of K. pneumoniae.
T. Alarcon*, E. Aznar, M. Espinola, A. Somodevilla, M. Lopez-Brea (Madrid, ES) Objective: The aim of this study was to detect the presence of prophages by the induction of its lytic cycle after culturing clinical isolates in the presence of low concentrations of mitomycin C and determine its association with the resistance to antimicrobial agents. Methods: 47 H. pylori strains were studied. They were obtained from gastric biopsies following standard methodology. Strains were stored at −80ºC until used. TIGR 26695 was used as negative control as no prophage was detected in its genome. Amoxicillin, clarithromycin, rifampicin, ciprofloxacin, tetracycline, metronidazole resistance was determined by E-test following standard methodology.
For prophage induction, H. pylori strains were subcultured on recently prepared blood agar plates containing 5 ng/ml Mitomycin C, 10 mg/l vancomycin and 5 mg/l amphotericyn B. After 2 to 5 days incubation, plates were examined for the presence of growth inhibition plaques. Results: Porphage induction was observed in 15 out of 47 strains (31.9%) (not in TIGR) by detection of inhibition plaques after culture on mitomycin C containing blood agar plates. The following resistance percentages were detected: 4.2% to amoxicillin, 44.7% to clarithromycin, 10.9% to rifampicin, 4.3% to ciprofloxacin, 2.1% to tetraciclin and 27.6% to metronidazole. Percentage of resistance was analyzed in 2 groups (strains with or without prophages) Resistance was higher in the group with prophages for the following antimicrobial agents: amoxicillin (6,6% vs 3,1%), clarithromycin (53,3% vs 40,6%), ciprofloxacin (7,1% vs 3,1%) and metrronidazol (33,3% vs 25%). Resistance was lower in the group with prophages for the following antimicrobial agents: rifampicin (7,1% vs 12,5%) and tetracycline (0% vs 3,1%). Conclusions: (1) Mitomycin C containing blood agar plates is an easy method to detect prophage carriage among H. pylori strains.
(2) A high percentage of H. pylori strains showed prophage detected by mitomycin C induction and (3) the presence of these prophages seems to be associated with a higher percentage of resistance to the antimicrobial agents most frequently used to treat infection produced by H. pylori.
Objective: To identify the resistance mechanism and determine appropriate antibiotic treatment for a patient whose blood and abdominal fluid isolated an E. coli resistant to ertapenem and the cephalosporins, with borderline imipenem and meropenem resistance. To highlight problems with detection and difficulties with treatment of multi drug resistant Gram negative organisms. Methods: Following hospitalisation for an MI in Egypt, requiring ITU care in The Nile Hospital, the patient was repatriated via air-ambulance to Wythenshawe Hospital for cardiac rehabilitation and subsequently developed an acute abdomen secondary to cholycystitis. Laporotomy and cholecystectomy were carried out and cultures from blood and drain fluid isolated a resistant E. coli. Routine in-house antibiotic susceptibility testing of the isolates was undertaken by VITEK. Resistance was confirmed by the Reference Laboratory who carried out MICs to cephalosporins, carbapenems, aminoglycosides, tigecycline and other agents by agar dilution. Potential carbapenemase activity was examined by a plate assay (Clover Leaf test), followed by enzyme identification by PCR. Results: A pure growth of E. coli with reduced carbapenem susceptibility was isolated. MICs of carbapenems were ertapenem = 8, meropenem = 1 and imipenem = 2. The isolate was clover leaf positive for carbapenemase activity and PCR identified an OXA48. The isolate was pan-b-lactam-resistant due to a CTX-M, seen by the MIC of Cefotaxime potentiated from >256 to 1, whilst ceftazidime fell from 32 to 1. The isolate was sensitive to tigecycline, which the patient received for 10 days resulting in full recovery. A detailed travel history revealed visits to Tunisia, Spain and Portugal but no travel to Greece or Turkey, during the past 5 yrs. Conclusion: OXA48 has not previously been identified in an E. coli in the UK. Detection of this carbapenemase is problematic as resistance to ertapenem may be the only indicator, yet the MIC of ertapenem can be readily raised by impermeability alone, making its use as a single maker uncertain. Dissemination of OXA48 has to date, been geographically and species restricted and not previously associated with E. coli or with travel to Egypt. Tigecycline concentrates well in the gall bladder and should be considered for sensitivity testing in these circumstances; however concerns remain around the achievement of adequate serum levels of the agent in patients with severe sepsis with a single confirmed pathogen.
21st ECCMID/27th ICC, Abstracts accepted for publication only
A. Rafi*, R. Moaddab (Tabriz, IR) Objectives: Study and evaluation of effectiveness of second line drugs against mycobacterial strains has become more important in the past few years, particularly due to the outbreak caused by multi-drug resistant (MDR) strains of Mycobacterium tuberculosis (MT) . The aim of this study was to evaluate the in-vitro susceptibility of M. tuberculosis (MT) and mycobacteria other than tubercle bacilli (MOTT) strains to the two main second line anti-mycobacterial agents (ciprofloxacin and ofloxacin). Methods: In this study, in-vitro activities of ofloxacin and ciprofloxacin against totally 100 mycobacterial strains including 90 Mycobacterium tuberculosis strains (40 strains resistant and 50 strains sensitive to the first line drugs) and 10 mycobacteria other than tubercle bacilli strains (all strains resistant to the first line drugs) were investigated by proportional method on Lowenstein-Jensen (LJ) medium. Results: Out of 90 M. tuberculosis strains, 50 strains that were sensitive to the first line drugs were diagnosed as susceptible to ofloxacin and ciprofloxacin. Of other 40 strains which were resistant to the first line drugs, only one strain was resistant to ofloxacin and 2 strains were found to be resistant to ciprofloxacin. Of 10 mycobacteria other than tubercle bacilli strains, 4 strains were resistant to ofloxacin and 3 strains were found to be resistant to ciprofloxacin. Conclusion: The findings of this study showed that ofloxacin and ciprofloxacin could be effectively used against Mycobacterium tuberculosis strains and also mycobacteria other than tubercle bacilli strains.
R2310 Staphylococcus aureus faecal carriage among healthy humans in Spain. Detection of livestock-associated genetic lineages ST398 and ST133 in methicillin-susceptible isolates D. Benito*, C. Lozano, E. Gómez-sanz, M. Zarazaga, C. Torres (Logroño, ES) Objectives: To determine the rate of S. aureus faecal carriage in healthy humans in Spain and to perform the genetic characterization of recovered isolates. Methods: Fecal samples of 96 healthy humans were recovered in La Rioja (Spain) during September-December 2010. Samples were inoculated into Mannitol-Salt-agar and ORSAB plates for S. aureus and methicillin-resistant S. aureus recovery, respectively. Isolates were identified by biochemical methods and nuc-gene PCR. Antibiotic susceptibility profile was determined by disk-diffusion method for 18 antibiotics. S. aureus isolates were submitted to spa-agr-and MLST typing. The presence of 9 antibiotic resistance genes (msrA, msrB, mphC, ermA, ermB, ermC, ermF, ant-4 , mupA) and 11 virulence factor genes (lukF/lukS-PV, lukE-lukD, lukM, tst-1, eta, etb, hlA, hlB, hlD, hlG, hlgv) were studied by PCR. Results: S. aureus was recovered in 14 of 96 studied samples (14,6%), all of them being methicillin-susceptible (MSSA). A very high diversity of spa-types was detected among our isolates (t084, t002, t209, t012, t021, t216, t136, t3495, t571) , types t084 and t002 being detected in two samples, each one. MLST was performed for isolates with spa-types t571 and t3495, and sequence types ST398 and ST133, respectively, were identified. Isolates were susceptible to most of antibiotic tested with some exceptions: erythromycin-clindamycin (3 isolates, with ermC with/without ermA+mphC), tobramycin and mupirocin (1 isolate with ant(4 ) + mupA genes). All 14 MSSA were negative for lukF/lukS-PV genes encoding Panton-Valentine leucocidin. The lukDE gene was identified in 5 isolates. Interestingly, strain ST133 harboured the lukDE + lukM genes. Five isolates were tst-1 positives (35% of all MSSA).
Others virulence factors detected were (number of isolates): hlA (14), hlB(9), hlD (14), hlG(6), and hlG2(8).
Conclusions: A high prevalence and a high clonal diversity of MSSA isolates were identified. As far as we know, this is the first detection of MSSA ST133 in faecal samples of healthy humans.
R2311 Antimicrobial resistance and vancomycin heteroresistance of MRSA strains from a tertiary hospital in Greece T. Panayea*, M. Souli, I. Galani, I. Karantani, H. Giamarellou, G. Petrikkos (Athens, GR) Methicillin resistant S. aureus strains cause a variety of infections that are sometimes difficult to treat and are spread both in hospital environment and community. Vancomycin heteroresistance (hVISA) is mainly observed among these strains. ESBL-Escherichia coli (EEC) was defined as an E. coli resistant to third generation cephalosporins but susceptible to amoxicillin clavulanate. Thus, most TEM, SHV and CTX-M-type enzymes (molecular class A enzymes or functional group 2be from the Bush-Jacoby-Medeiros classification) were taken into account. ITU were considered different if time between two episodes was greater than 15 days or the isolate presented different susceptibility patterns affecting ESBL classification.
In vitro growth competitions were carried out fivefold by placing the same inoculum of two isolates of E. coli (EEC vs. E. coli with no antimicrobial resistance) into 10ml of thioglicolate (TG). Then, aliquots were sub-cultured every day for both re-growth in 10ml TG and colony identification.
Results: The total number of urinary isolates analyzed was 70,827 belonging to 49,304 different patients. Of these isolates, 5,161 (7.3%) were categorized as EEC. The number of different ITU was 64,472, 4,403 (6.8%) by EEC, and 1701 were discarded because they took place at the end of the study period during the fourth consecutive ITU. The mean time from an EEC ITU to another EEC ITU was 43 days, while the mean time from an EEC ITU to a non-EEC ITU was 75 days. Growth competitions showed a greater than 99% decreased in the EEC population after 5 days. Conclusions: Our data suggests that, given the time and in the absence of antimicrobial pressure, E. coli with fewer antimicrobial resistances will outgrow EEC, probably due to a better fitness of the isolate.
Objective: To examine patterns of antimicrobial susceptibility in Acinetobacter calcoaceticus-baumannii isolates to commonly used drugs at a tertiary care hospital in Riyadh, Saudi Arabia. Methods: A retrospective study was carried out at King Fahad National Guard Hospital (KFNGH) between 2008 and 2010. Organisms were identified and tested by the automated identification and susceptibility system (MicroScan Walk away 96, Siemens ® ) and the Antibiotic susceptibility testing were confirmed by the Etest (AB Biodisk, Sweden). The procedural details interpretations were as recommended by the Clinical laboratory standards Institute (CLSI). Results: Between 2008-2010, a total of 2552 isolates of Acinetobacter baumannii (A. baumannii) were available for analysis. The organism showed high rates of resistance to Pip-Tazo (81% of isolates), Imipenem (66%), Meropenem (83%), Gentamicin (68%), amikacin (65%), ceftazidime (82%), cefepime (70%), ciprofloxacin (80%) and colistin 19%. Multidrug resistance was observed in (65−75%) of Acinetobacter species isolates. Conclusion: Antimicrobial resistance in Acinetobacter baumannii is a major emerging problem particularly in the intensive care unit, Strict infection control measures, judicious prescribing of antibiotics, antibiotic stewardship programs and antibiotic cycling should be adopted to control infections due to these bacteria in patients admitted to intensive care Continuous monitoring of antimicrobial susceptibility and strict adherence to infection prevention guidelines are essential to eliminate major outbreaks in the future.
C. Caneiras*, F. Calisto, R. Moura, M. Augusto, G. Da Silva, J. Melo Cristino, A. Duarte (Lisbon, Coimbra, PT) Objectives: Tigecycline is an antibiotic used in clinical setting where multidrug resistance (MDR) and extended spectrum b-lactamases (ESBL) production is prominent. Due to the constant potential of emergency of resistance, long term survey studies are needed. The aim of this study was to assess the activity of tigecycline against MDR and ESBL Enterobacteriaceae isolates during a period of ten years. Methods: A total of 155 isolates, Escherichia coli (n = 92) and Klebsiella pneumoniae (n = 63), were collected at 5 hospitals in Portugal (1999 Portugal ( -2010 . Susceptibilities to antimicrobial agents were determined by disk diffusion and interpreted according to CLSI guidelines: tigecycline, imipenem, ciprofloxacin, gentamicin, amikacin, ampicillin, cefotaxime, ceftazidime, cefepime and amoxicillin/clavulanic acid. Tigecycline MICs were performed by Etest. The ESBLs were identified by PCR with specific primers for bla-CTX-M, bla-TEM and bla-KPC gene.
Results: According to the breakpoints proposed by CLSI, 25% of K. pneumoniae and 13% of E. coli ESBL-producing were nonsusceptible to tigecycline. Higher prevalence of intermediate susceptibility was found (Klebsiella 63% and E. coli 44%). Longitudinal analysis showed no increase in tigecycline resistance over the 10-years study period and was not influenced by the presence of CTX-M-and TEM-type enzymes. However for the first time in Portugal it was identified a KPC-3 E. coli resistant to tigecycline and nine K. pneumoniae isolates producing KPC-3 showed reduced activity (33%). MDR and ESBLproducing E. coli had tigecycline MIC 90 3 mg/l and K. pneumoniae isolates had tigecycline MIC 90 2 mg/l. The analysis of tigecycline MICs showed a poor correlation with values obtained and disk zone diameters, 38% of the MIC results are concordant. The discordant data occur mostly in the intermediate/susceptible zones. Conclusion: Tigecycline showed good activity against MDR and ESBL isolates. However, the high frequency of isolates in intermediate category found and the discordant correlation between MICs and inhibition zone diameters, suggest that further selective pressure will lead to an overall reduced susceptibility to tigecycline in Enterobacteria. For instances, the tigecycline resistance found in KPC-3 isolates collected in 2010 may indicate an indiscriminate use of tigecycline. These results indicate that the use of tigecycline in hospital setting should be carefully controlled to avoid the emergence of resistance.
R. Mendes *, H. Sader, D. Farrell, R. Jones (North Liberty, US) Objectives: To assess telavancin (TLV) and comparator activities against staphylococci, including strains with decreased susceptibility (S) to vancomycin (VA) and teicoplanin (TE), from Europe (EU). TLV is approved in the United States (US) and Canada for the treatment of adults with complicated skin and skin structure infections (cSSSI). This drug is also under review for the treatment of complicated skin and soft tissue infections in EU and nosocomial pneumonia in the US and EU. Methods: 3 868 S. aureus (SA) and 1 003 coagulase-negative staphylococci (CoNS) were collected from 33 sites in 12 countries, including Turkey and Israel. Isolates were submitted to a central laboratory and identification performed by standard algorithms and Vitek 2. Strains were S tested by CLSI methods (M07-A8, 2009). EUCAST criteria (2010) were applied, when available. Results: SA were from SSSI (41.0%), bacteremia (35.3%), and respiratory tract infections (13.0%), while the majority (68.2%) of CoNS were from bacteremia. TLV (MIC 50/ 90 , 0.12/0.25 mg/L) was 2-fold more potent than daptomycin (DA; MIC 50/ 90 , 0.25/0.5 mg/L) and 4-to 8-fold more active than VA (MIC 50/ 90 , 1/1 mg/L) and linezolid (LZ; MIC 50/ 90 , 1/2 mg/L) against all SA. Similar results were noted for CoNS. 1.6 and 1.0% of SA had higher VA (>1 mg/L; all 2 mg/L) and TE MICs ( activity was observed against CoNS, regardless of country of origin or glycopeptide S. Conclusions: TLV exhibited higher potency (at least 2-fold) than direct comparators (VA, DA and LZ) . SA with decreased S to glycopeptides showed higher TLV MICs, although all strains were inhibited ( 0.5 mg/L) at concentrations below the FDA breakpoint for SA ( 1 mg/L). The TLV activity against SA and CoNS suggests this drug as an option for staphylococcal infections, including those caused by strains with decreased S to glycopeptides.
M. Espínola*, A. García,Á. Somodevilla, M. Martínez, A. Guiu, A. Correa, M. López-Brea (Madrid, ES) Escherichia coli is involved in 85% of community-acquired urinary tract infections (UTIs). For this reason, it is important to know its antimicrobial susceptibility patterns in order to give an appropriate empiric treatment of UTIs. The aim of this study is to evaluate the antibiotic resistance of E. coli strains isolated in community-acquired UTIs, and to see the prevalence of extended-spectrum b-lactamase (ESBL) producing strains in the community. Methods: 2598 E. coli isolates were collected from outpatients with clinical evidence of community-acquired UTIs during the period January to November 2010 in a hospital in Madrid, Spain. Urine samples were cultured in CLED agar and incubated at 37ºC for 48 hours. UTIs were defined as the culture of a single organism from a midstream urine specimen at 105 colony forming units per milliliter. Identification and antibiotic susceptibility tests were made by the routinely used automated system MicroScan (Siemens). Additionally, susceptibility tests were performed by disk diffusion method according to standard procedures. For the study, reduced susceptibility to 8 antibiotics commonly used in UTIs (amoxicillin, amoxicillin/clavulanic acid, cefuroxime, norfloxacin, ciprofloxacin, trimethoprim/sulfamethoxazole, fosfomycin and nitrofurantoin) was evaluated. In addition, extended-spectrum b-lactamase (ESBL) production was examined; a ratio of 8 or greater for the ceftazidime to ceftazidime-clavulanic acid minimal inhibitory concentrations (MICs) and the cefotaxime to cefotaxime-clavulanic acid MICs was considered ESBL-positive.
Results: 2598 E. coli isolates were obtained, 179 of which (6,9%) produced an extended-spectrum b-lactamase. Isolates presenting reduced susceptibility to the different antibiotics evaluated are shown in the table attached.
Conclusion: This study shows the increasing prevalence of ESBL-E. coli in community-acquired infections, being almost 7% of the E. coli strains isolated positive for ESBL. High resistance rates are obtained for quinolones (norfloxacin and ciprofloxacin) in both, ESBL and not ESBL producing E. coli. For this reason, these antibiotics should not be used for the empiric treatment of community-acquired UTIs in our country. However, low resistance rates are shown for nitrofurantoin and fosfomycin, in both ESBL and not ESBL E. coli, which suggests that these antibiotics may be a good option in the empiric treatment of UTIs.
Introduction: Antimicrobial resistance level is high among pathogen bacteria especially hospitals. The aim of this study was to determine antimicrobial susceptibility levels of environmental bacteria from a national park in Turkey.
A total of 72 bacteria were defined by 16S rRNA sequencing (Figure 1 ). The antimicrobial susceptibilities of 72 bacteria isolated were investigated to penicillin, ampicillin, lincomycin, erythromycin, vancomycin, ciprofloxacin, gentamicin, tetracyclin, and chloramphenicol by agar diffusion MIC method. Strains with high MICs for lincomycin, chloramphenicol and penicillin were tested for inactivation by Gots' test. b lactamase positive strains were tested for ESBL production. All strains with high level MICs for gentamicin, tetracyclin, and chloramphenicol were studied for the presence of known gene by PCR. Gram positive bacteria were tested for the presence of erytromycin, and vancomycin resistanve genes. The following genes were tested for their presence: ermA, ermB, ermC, vanA, tetA, aac-aph and cat.
Results: Among isolates high MIC rates were 76.3%, 75%, 70.8%, 68%, 54.1%, and 47.2% for ampicillin, penicillin, gentamicin, chloramphenicol, tetracycline, and ciprofloxacin, respectively among all isolates. MIC values were evaluated for lincomycin, erythromycin and vancomycin among Gram (+) bacteria which showed 88.6%, 45.4%, and 15,9% high level MICs, respectively. Gots' test showed that, 35 isolates inactivates penicillin and only one isolate inactivates chloramphenicol. Among b-lactamase (+) strains one was ESBL producer. Of 20 isolates with high level erythromycin MICs 2 had ermB, 1 ermC, and 0 ermA. Of 51 isolate with high gentamicin MICs only 1 had aac-aph. Of 39 tetracyclin resistant isolates 4 had tetA and 2 had tetM genes but 33 were negative for both. Of 21 vancomycin resistant isolates 1 had vanA. The vanA (+) isolate was E. faecium and ST type of this strain was close to 17 with one allele difference.
Conclusions: Antimicrobial resistance is one of the main public health problem. The use of antimicrobials select resistance strains in hospitals but also the dispersion of antimicrobials to the nature may cause selection of resistance in the nature. Our study showed presence of high level antimicrobial non-susceptibility among environmental isolates in a natural park. The effect of environmental resistant strains on public health should be evaluated. Objectives: Antimicrobial agents are widely used in animal husbandry for treating infectious diseases and ensuring animal welfare and good quality food production. It is well known that antimicrobial usage can select for resistant forms of bacteria and resistant strains can be exchanged between humans, animals and other ecosystems. To the aim of monitoring resistance against selected antimicrobial agents commonly used in sheep producing milk, a survey was carried out on S. aureus strains isolated from milk of animals with mastitis problems in seven years from 87 farms. Methods: 172 strains of Staphylococcus aureus were isolated and collected from milk during 2002-2008 in sicilian livestocks with big problems of ovine mastitis. The isolates in fact were used to prepare autogenous vaccine at the IZS of Sicily. They were tested for antimicrobial susceptibility by disk diffusion test (DDT) against the following agents: ampicillin (AMP 30mcg), amoxicillin+clavulanate (AMC 30mcg), enrofloxacin (ENR 5mcg), eritromycin (ER 15mcg), penicillin (P 10UI), tetracycline (TE 30mcg), vancomycin (VA 30mcg), oxacillin (OX 1mcg), oxytetracycline (OT 30mcg) according to CLSI guidelines. Results: As shown in the table 1, the number of clones resistant to AMP, AMC, P and Te, OT, the antimicrobials commonly used in animal husbandry, increased during 2002 to 2008. The resistance to ER was also found (19−20%). A low level of OX resistance was detected, with a decrease from 2002-2005 to 2006-2008 . A double increase in tetracycline resistence is shown and an substantial increase in OT was also observed. The presence of mecA gene was investigated by PCR and resulted in a 10% of positive clones with similar percentages in the 2 considered periods. None of them showed resistance to VA and ENR. Conclusion: The finding of a general increasing trend in antibiotic resistance against the selected drugs implies that the selective pressure has acted in the ecosystem favouring the espansion of resistant clones; for this reason is important the monitoring of drug resistance in food animals and a prudent usage of antibiotics in the veterinary field, to avoid major risk for increase drug resistance in the human population.
one-year results of a large, multicentre, observational study E. Mantengoli, F. Luzzaro, T. Di Maggio, G. Brigante, B. Pini, A. Goglio, L. Ferrari, P. Pecile, M. Tinelli, E. Tacconelli, R. Cauda, G.M. Rossolini* (Siena, Lecco, Bergamo, Cremona, Florence, Lodi, Rome, IT) Objectives: Italy is among the countries reporting increasing resistance rates to expanded-spectrum cephalosporins (ESCs) in Enterobacteriaceae. Production of extended-spectrum b-lactamases (ESBLs) and
AmpC-type b-lactamases (CBLs) remain the major mechanisms of ESC resistance. We report here on the molecular epidemiology of ESBL-and CBL-producing enterobacterial isolates collected during one-year in a large, multicentric study recently conducted in Italy.
or Proteus mirabilis with reduced susceptibility to ESCs (cefotaxime and/or ceftazidime MICs >1 mg/L) during the period 2007-2008. ESBL production was confirmed using the double-disk synergy test. ESBL and CBL determinants were characterized by DNA hybridization and confirmed by PCR and sequencing. Clonal relatedness was investigated by RAPD.
Results: A total of 444 cases of infection caused by Enterobacteriaceae with reduced susceptibility to ESCs were enrolled (268 by E. coli, 74 by K. pneumoniae and 102 by P. mirabilis). ESBL production was confirmed in 396 (89%) of isolates, CBL production was observed in all ESBLnegative P. mirabilis. Among the ESBL producers, CTX-M-type enzymes were overall the most prevalent (80% overall, 91% in E. coli, 90% in Klebsiella, and 12% in Proteus) and mostly (95.8%) belonged in group 1 (CTX-M-1 and CTX-M-15). However, CTX-Ms of groups 2 (1.1%, CTX-M-2) and 9 (3.1%, CTX-M-14 and CTX-M-27) were also detected. Other ESBLs included TEM-and SHV-type variants (10% and 4.7% overall, respectively, including TEM-92, TEM-72 and SHV-12, SHV-2a). All CBLs belonged in the CMY-2 lineage, being totally represented by CMY-16. Clonal heterogeneity was observed among the ESBL producers of each species, although clonal expansion phenomena caused by CTX-M-producing E. coli ST131 were observed. All CBLproducing P. mirabilis isolates were clonally related with each other.
In comparison with previous studies, a remarkable evolution of the molecular epidemiology of ESC-resistant Enterobacteriaceae was observed in Italy. CTX-M-type enzymes have become the predominant ESBLs, with enzymes of group 2 and 9 also emerging, while CMY-2-like CBLs now significantly contribute to ESC resistance in P. mirabilis.
since the 1970s. It is also recognised that there is a shift to Gram-negative pathogens with increasing age. This study describes the trends in BSI pathogens and antimicrobial susceptibility over 9 years at an Australian adult tertiary referral hospital. Methods: Positive blood cultures from 1st January 2001 till 31st December 2009 were reviewed. Duplicate isolates (within 14 days of the primary culture) and patients <20 years of age were excluded. Patients in haematology (including bone marrow transplantation), respiratory (including lung transplant and cystic fibrosis), oncology and burns units were also excluded as they represented heterogeneous populations. Coagulase negative Staphylococci (CNS) were excluded as it was not possible to retrospectively differentiate between true infections and contamination. Results: We found an overall decline in blood culture positivity rate from 9.48 to 4.80 per 1000 admissions. Similarly, the proportion of positive cultures also decreased from 2.6% to 1.5%. Overall, Grampositive pathogens were isolated in 48.9% of patients (S. aureus 24.0%, Enterococcus spp. 7.1%, S. viridans 3.6%) compared to Gram-negative pathogens in 45.5% of patients (E. coli 20.3%, Klebsiella spp. 6.9%, Pseudomonas spp. 3.8%). There was a trend to an increasing proportion of Gram-negative pathogens during these 9 years. However, age specific proportions of Gram-positives and Gram-negatives remained similar, suggesting this relates to overall aging of the hospital population. The proportion of S. aureus isolates that were methicillin-resistant (MRSA) declined from 54.0% to 27.5%. In Gram-negatives, susceptibility to gentamicin and third generation cephalosporins by Enterobacteriaceae remained stable. Ceftazidime resistance also remained stable, suggesting no increase in extended spectrum b-lactamase (ESBL) producing organisms. Conclusions: Our study demonstrated important trends in epidemiology of BSI pathogens over time and their impact on choices of appropriate antimicrobial therapy, particularly for the elderly hospital inpatient population. b-lactams, gentamicin and quinolones all remain effective antibiotics for our patient population.
Objectives: The primary objective of this study was to determine leading factors influencing the presence of antimicrobial residues in the environment and to rank their predicted environmental concentrations (PEC).
Methods: A mechanistic model is presented for determination of antimicrobial presence in the environment (EXCEL 2003 with @Risk 5.0 ® add-on). The model was tested for six main groups of antimicrobials consumed in Europe: penicillins (PEN), b-lactams (BET), tetracyclines (TET), macrolides (MAC), quinolone/fluoroquinolones (Q/F) and sulfonamides/trimethoprim (S/T). The model simulates the release of antimicrobials into the environment by integrating the effects of antimicrobial use, metabolism, degradation, and dilution. Each input variable was unified and assigned a probability density to represent inherent uncertainty and variability in the parameter. The Monte Carlo simulation model resulted in probability distributions of PECs for each antimicrobial group. Using regression analysis, resulting PECs were ranked in relation to resistance potential, chronic and acute toxicity and hazard quotient (HQ) . Results: The model simulated the mean PEC of PEN, BET, TET, MAC, Q/F and S/T (Table 1) . Degradation was the main input influencing PEN PEC, usage was foremost for BET and S/T, while metabolism was the most critical input for TET, MAC and Q/F PECs. Q/F expressed the highest rate of resistance formation potential (57%). BET expressed a moderate HQ (2.02) with all remaining antimicrobials expressing a low HQ. No antimicrobial group was predicted to express toxicity in the environment.
The observed results infer that current antimicrobial use can lead to levels in the environment which may increase resistance formation. The legislation of new antimicrobials should consider metabolism, as it will greatly influence levels emitted into the environment. The in-depth understanding of antimicrobial presence in the environment is lacking; specifically, with regard to lower limits of minimum inhibitory concentrations acting as selectors for resistance formation. The use of ECOSAR ® for antimicrobial toxicity reference values is limited as the bacteria targeted by antimicrobials are phylogenetically distinct from the cyanobacteria used to calculate the EC/LCsub50 values. The results and limitations presented here accentuate the need for further research into antimicrobials in the environment and the development of antimicrobial resistant strains.
C.C. Lee, K. T. Wong, R.W. Lai, T.K. Ling* (Hong Kong, HK) Objectives: Tigecycline is the first of the glycylcycline antimicrobial active against a wide range of bacterial pathogens. We have participated in the global Tigecycline Evaluation and Surveillance Trial (TEST) to study the in-vitro activity of tigecycline. This report summarizes the antimicrobial susceptibility of bacterial pathogens collected in 2010. Methods: A total of 135 Gram-negative and 65 Gram-positive isolates were collected in the Prince of Wales Hospital. MICs were determined using microdilution trays purchased from TREK Diagnostics Systems, East Grinstead, UK and interpreted using CLSI guidelines. Table 1 and 2 respectively. Shading denotes % resistance value smaller than or equal to 10%. Conclusion: Tigecycline showed excellent activity against all Grampositive and Gram-negative isolates but less active against Acinetobacter spp. and P. aeruginosa.
A. Ergin*,Ö. Köseoglu Eser, G. Hasçelik (Ankara, TR) Objectives: Penicillin resistance concomitant with erythromycin resistance is a risk factor in the mortality of patients with S. mitis and S. oralis bacteremia. The aim of the study was to determine penicillin and macrolide resistance mechanisms in invasive S. mitis and S. oralis bacteremia strains isolated from patients that have haematological malignancies. Methods: S. mitis (n:23) and S. oralis (n:17) isolates from blood cultures of patients (n:40) with haematological malignancies were included in the study. Isolates were identified with BD Phoenix system. Penicillin and erythromycin susceptibilities were performed with E-test. Vancomycin, clindamycin, cefotaxime, levofloxacin and linezolid susceptibilities were determined by broth microdilution. Penicillin and erythromycin resistance genes, pbp1a, pbp2b, pbp2x, ermB and mefA/E were amplified using PCR method. BamHI restriction enzyme was used for discrimination of mefA and mefE. Results: Patient age range was 42−84. Twenty of fourty patients were women. Thirty of the patients have diagnosed as myelogeneous (n:22) and lymphoblastic leukaemia (n:8). Fifteen of the patients have died due to solid cancers. Fourteen (35%) and eighteen (45%) strains were resistant to penicillin (MIC 4 mg/L) and erythromycin (MIC 4 mg/L), respectively. Rate of resistance to clindamycin and cefotaxime were 32.5% and 22.5%. All the isolates were susceptible to vancomycin, levofloxacin and linezolid. Five of penicillin resistant isolates carried pbp2b and three carried pbp2x genes. Among erythromycin resistant and intermediate (n:23) isolates,16 exhibited ermB and 7 mefE genotypes. Conclusion: Erythromycin resistance is highly due to ermB and followed by mefE genes and penicillin susceptibility can partially be explained by the presence of pbp2b and pbp2x genes among our isolates. It is important to be aware of high level resistance of penicillin and erythromycin in S. mitis and S. oralis in patients with haematological malignancies as an emerging threat since susceptible empirical antibiotics may not reduce overall mortality.
R2332 Trend of antimicrobial susceptibility of P. aeruginosa isolates from UTI and RTI of Japanese hospital participating in the levofloxacine surveillance group during 1994-2010
O. Akira*, I. Yoshikazu, T. Kazuhiro, Y. Keizo on behalf of the the Levofloxacin Surveillance group
Objectives: Pseudomonas aeruginosa has become ploblematic because of an outbreak of multidrug-resistant clone producing metallob-lactamases (MBLs). We have already been taken nationwide surveillance for FQ and other antimicrobials resistance against many bacterial clinical isolates since 1994 in Japan. In this study, we report surveillance data for P. aeruginosa isolates from patients with urinary tract infection (UTI) and with respiratory tract infection (RTI) collected between 1994-2010.
Methods: A total of 7,019 clinical isolates (3, 232 isolates from UTI and 3,787 isolates from RTI) were collected from 92 centers participating in the Levofloxacin Surveillance group during 1994-2010 in Japan. Antimicrobial susceptibility testing by broth microdilution methods was based on Clinical and Laboratory Standards Institute (CLSI) guidelines that were updated annually as revised documents were published. Results and Discussions: 1. UTI; The 'Susceptible' rate for levofloxacin has particularly increased over time (from 41.8% in 1994 to 74.2% in 2010) . Although the cause of this increase is obscure, recent request for strict observance of the dosing period is probably implicated because drug use reviews do not show any decrease of the amount of levofloxacin used nationwide in the field of urology. Amikacin and ceftazidime also showed a gradual increase of the 'Susceptible' rate. The susceptibility for imipenem remained unchanged. The rate of 4 drugs resistant isolate to levofloxacin, ceftazidime, amikacin and imipenem was about 1% until 1998, however increased to 4.6% abruptly in 2000 and then shifted at the level of approximately 4%. 2. RTI; The 'Susceptible' rate to levofloxacin, amikacin, ceftazidime and imipenem has been maintained constantly at a level of approximately 80%, 97%, 88% and 70%, respectively. The rate of 4 drugs resistant isolate to levofloxacin, ceftazidime, amikacin and imipenem was in a moderate increase in comparison with that in UTI. 3. Metallo-b-lactamase; The rate of metallo-b-lactamase producing isolates from UTI and RTI was 8.0% in 2002 , 7.2% in 2004 and 5.6% in 2007 , and 1. 5% in 2002 5% in 1.0% in 2004 5% in and 2.2% in 2007 , respectively.
Y. Alikhani*, Z. Karimi tabar, P. Karami, A. Zamani, S. Farajnia (Hamadan, Zanjan, Tabriz, IR) Objectives: Pseudomonas aeruginosa is a leading cause of nosocomial infections worldwide. The infections can be particularly severe in patients with impaired immune systems. The infections of this agent are frequently difficult to treat because of both the natural resistance of the species and its ability to acquire further resistance mechanisms to multiple groups of antimicrobial agents. Resistance of P. aeruginosa strains to the broad-spectrum cephalosporins may be mediated by the extended-spectrum b-lactamases (ESBLs). The aim of this study was to determination of P. aeruginosa antibacterial resistance patterns and the prevalence of ESBLs producing strains as PER-1 and VEB genes. Methods: In our study, a total of 106 clinical isolates of P. aeruginosa were studied. The isolates were collected from two university hospitals in Hamadan, Iran, during 7 month (2009), to assess the current levels of antimicrobial susceptibility. The susceptibility of the investigate P. aeruginosa isolates to 12 antimicrobial agents was determine by the disc diffusion method on Mueller Hinton agar plates and was interpreted according to the Clinical an Laboratory Standards Institute (CLSI) recommendation. ESBLs producing strains have detected by combined disk test and the presence of PER-1 and VEB genes by PCR. Results: The antibiotic resistance rates against the broad-spectrum cephalosporins and monobactames were: cefepime 97%, cefotaxime 92.5% ceftazidime 51%, and aztreonam 27%. Ciprofloxacin (91.5%), imipenem (84.9) and meropenem (82.07) were the most effective antipseudomonal agents. The results revealed that 94 (88.7%) of the isolates were multidrug resistant and 60 (58.25%) of the isolates were ESBL positive. Sixteen (26.6%), 9 (15%) and 3 (5%) strains among 60 ESBLproducing strains amplified blaPER-1, blaVEB and blaPER-1/blaVEB respectively. Conclusion: This study highlights needs to establish antimicrobial resistance surveillance networks for P. aeruginosa to determine the appropriate empirical treatment regimen. Bacterial strains resistant to most classes of antibiotics will continue to emerge unless inappropriate uses of drugs are decreasing a continuous education of infection control practices maintained. The high prevalence of multidrug resistance and production of ESBLS in P. aeruginosa isolates in patient confirm that Protocols considering these issues should be considered in hospitals.
Objectives: To determine the epidemiologic, clinical, and microbiologic data of infections caused by drug-resistant Streptococcus pneumoniae (DRSP) in adult patients.
A retrospective study of all adult patients with pneumococcal infections who were hospitalized at King Chulalongkorn Memorial Hospital, Bangkok, Thailand, was carried out from January 1, 2008 to December 31, 2009. In addition, a trend of prevalence of penicillin-and cephalosporin-non-susceptible S. pneumoniae (PNSP and CNSP) from 1997 to 2009 in our institute was analyzed.
Results: Of 86 pneumococcal isolates, there were 70 (81.40%), 5 (5.81%), and 11 (12.79%) patients with pneumonia, primary bacteremia, and meningitis, respectively. There were 56 (65.11%) males and 30 (34.89%) females with the mean age of 62 + 10 years (range 15-101 years). Of 70 patients with pneumonia, there were 67 (95.70%), 3 (4.30%), 0 (0%), 70 (100%), 0 (0%), and 0 (0%) of penicillin-susceptible S. pneumoniae (PSSP), penicillin-intermediate S. pneumoniae (PISP), penicillin-resistant S. pneumoniae (PRSP), cefotaxime-susceptible S. pneumoniae (CSSP), cefotaxime-intermediate S. pneumoniae (CISP), and cefotaxime-resistant S. pneumoniae (CRSP), respectively. Of 5 patients with primary bacteremia, there were 5 (100%) and 5 (100%) of PSSP and CSSP, respectively. Of 11 patients with meningitis, there were 7 (63.64%), 4 (36.36%), 10 (90.91%), 1 (9.09%), and 0 (0%) of PSSP, PRSP, CSSP, CISP, and CRSP, respectively. All isolates were susceptible to vancomycin. A trend of prevalence of meningitis caused by PNSP and CNSP from 1997 to 2009 in our institute is summarized in Table. Conclusions: There is a relatively high prevalence of infections caused by PNSP and CNSP in adult patients in our institute. This emphasizes an urgent need to strengthen both appropriate use of antimicrobials and strict infection control measures to help reduce the occurrence of DRSP.
Streptococcus pneumoniae isolates before introduction of PCV7 in Ankara, Turkeÿ O. Köseoglu Eser*, H. Uludag Altun, D. Gür, G. Hascelik (Ankara, TR) Objectives: In this study, macrolide resistance mechanisms and the diversity of PBPs 2b, 2x and 1a of invasive Streptococcus pneumoniae (SP) isolates identified from patients admitted to Hacettepe Hospitals before the introduction of heptavalent pneumococcal conjugate vaccine in Turkey between 1996 and 2008 was investigated. Methods: Invasive SP clinical isolates were collected from children and adults admitted to Ihsan Dogramaci Children's Hospital and Adult Hospital of Hacettepe University. Antimicrobial susceptibility testing of all isolates were performed for six antimicrobial agents; penicillin (PEN), ceftriaxone (CRO), levofloxacin (LEV), erythromycin (EM), clindamycin (CD) and vancomycin (VAN) by broth microdilution method according to "Clinical Laboratory Standards Institute, CLSI". Serotypes were determined by Quellung reaction with specific antisera for SP. Isolates with MIC 0.125 mg/ml were examined for the PBP genes pbp2b, pbp2x ve pbp1a by PCR. Resistance genotypes of EM resistant isolates (MIC 0.5mg/ml) were determined by a multiplex erm(B)/mef PCR method for SP. RFLP analysis was done to differentiate mefA/E genes. Results: Of the 182 nonduplicated pneumococcal isolates, 59 were obtained from children and 123 from adults. Of these, 32 were cerebrospinal fluid (CSF) and 150 were blood isolates. In 16 of CSF isolates (50.0%), MICs for PEN were 2 mg/ml. EM resistance (MIC 0.5mg/ml) was found in 23 (12.6%) isolates, of these 11 were resistant to both EM and CD. In EM resistant isolates, 10 (43.5%) had ermB gene, two (8.7%) had mef E gene. In 55 isolates with PEN MIC 0.125 mg/ml, 27 (49.1%) had pbp2x, pbp2x and pbp1a together, four (7.3%) pbp2x and pbp2b, three (5.5%) pbp2b, one pbp2x and pbp2b, one pbp2B and pbp1a and one pbp1a. In all isolates, resistance for LEV was 1% and 0 for CRO and VAN. In the paediatric age group, 17 different serotypes and in the adult group, 23 different serotypes were observed. The most frequent serogroups in both age groups were 6, 3, 23, 9 and 5. Conclusions: Although there was no resistance for PEN among the blood isolates, PEN resistance was high among the CSF isolates. The major serotype associated with PEN resistance in CSF isolates was serotype 23F. Analysis of PBP genes showed predominancy in pbp2B. The predominant mechanism of macrolide resistance is associated with the erm(B) gene with strains showing co-resistance to clindamycin in the majority of cases.
in large conjugative plasmids of enterococci from different ecological niches are scarce. Our goal was to analyze the occurrence of tcrB among enterococci from different ecological niches and to characterize the genetic context of this gene. Methods: We analyzed 214 E. 23 E. gallinarum, 4 E. casseliflavus, 37 E. hirae, 97 Enterococci sp from hospitalized humans (H, n = 103); healthy humans (HV, n = 125), poultry (P, n = 129), piggeries environment/swine (PE, n = 232) and sewage/ river (SR, n = 52) recovered from different Portuguese areas (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) . Genes coding for CuR and ABR were searched by PCR. Mating assays were performed for 45 representative tcrB+ isolates in the presence of tetracycline, erythromycin, vancomycin or gentamicin, using different receptor strains. Clonality was studied by PFGE (SmaI)/MLST. Co-localization of tcrB and ABR genes was assessed by S1 PFGE hybridization.
Results: tcrB was detected in 15% (98/641) of isolates (26% PE, 15% SR, 11% HV, 9% P, 3% H; 59 Efm, 14 Efl, 25 other species). It was co-transferred with ABR genes (ermB-20; vanA-7; tetM-5; tetL-2; aac(6 )-Ie-aph(2 )-Ia-1). A polyclonal population was detected among tcrB+ representative isolates. Among Efm, 32 PFGE types corresponded to CC17 (ST18, ST393, ST431); CC5 (ST5, ST185, ST150) and also ST432, ST434 and ST432. Ten Efl PFGE types were detected. A few common strains were identified within and between niches (PFGE A-PE and H). tcrB was located on plasmids of 90-120kb in Efl and 120-300kb in Efm. The tcrB gene was located alone (5Efm PE, HV; 1Efl P) or with vanA (1Efm; PE), tetL (2Efm PE, HV), tetM (3Efm PE, HV), ermB (2Efm PE, SR; 2Efl PE, P), tetL+ermB (5Efm PE, HV), tetM+ermB (2Efm PE, SR), tetM+tetL (1Efl P), vanA+tetM+ermB (1Efm PE), tetM+tetL+ermB (6Efm PE, HV, SR). In 14 isolates tetM, tetL, ermB or aac(6 )-Ie-aph(2 )-Ia were located in other plasmids (25−90Kb), which co-transferred with those of tcrB (n = 4).
The co-transference of tcrB with other ABR genes located in the same or different plasmids suggest that the intensive use of copper in some niches might favour the selection of ABR enterococci. Both plasmid transfer and clonal expansion play important roles in the spread of tcrB. Objectives: Nosocomial infections remain the main cause of morbidity and mortality in burn patients. Infections caused by A. baumannii have emerged as a significant problem reported in burn units. A. baumannii strains are usually resistant to multiple antimicrobial agents including carbapenems which represent an important option for the treatment of Acinetobacter infections caused by multidrug-resistant isolates. The most common mechanism responsible for carbapenem-resistance are carbapenem-hydrolysing-b-lactamases belonging to molecular class D (OXA enzymes) and also may be associated with the presence of an insertion sequence (ISAba1). The aim of the study was detection of:1) OXA encoding genes; 2) presence of ISAba1. Methods: A total of 32 A. baumannii strains were collected from 2007-2010 in burn unit (BU) of Specialized Hospital in Krakow, Poland.
All strains selected to this study were carbapenem-resistant and were isolated from single patients. Identification and susceptibility testing were performed by VITEK-2 Compact (bioMérieux, Poland) according to CLSI criteria. Multiplex PCR described by Woodford et al. (2006) was applied for detection of OXA carbapenemases encoding genes (blaoxa-51-like, blaoxa-24-like and blaoxa-23-like). All strains were also tested for the presence of 549-bp fragment containing a portion of ISAba1. Results: Antibiotic resistance rate to piperacillin was 100%, piperacillintazobactam 94%, ceftazidime 91%, cefepime 94%, imipenem 100%, meropenem 100%, gentamicin 100%, ciprofloxacin 100% but was only 3% to tobramycin and 25% to amikacin. All carbapenem-resistant isolates contained intrinsic gene encoding b-lactamase belonging to OXA-51-like group. The isolates were also found to encode blaOXA-23-like and blaOXA-24-like genes respectively in 28 (88%) and 2 (6%). ISAba1 insertion sequence gene was detected in all tested strains.
Conclusions: Carbapenem resistance in tested isolates might be associated with: 1) expression of acquired oxacillinases belonging to OXA-23-like and OXA-24-like groups; 2) extended expression of intrinsic oxacillinases belonging to OXA-51-like group supported by the presence of insertion sequence ISAba1.
A. González*, J. Nigri, V. Hernández, R. González, M.A. Ferrús (Valencia, ES) Objectives: Arcobacter butzleri has been associated with human infection. Fluoroquinolones are potential drugs for treatment. However, there is evidence of increasing resistance to these antimicrobial agents. This resistance may occur due to mutations in a quinolone resistancedetermining region (QRDR) of the gyrA gene. Therefore the goal of this study was to look for the mutations associated with quinolone resistance in Arcobacter isolates. Methods: Forty-four A. butzleri isolates and the reference strain A. butzleri DSM 8739 were used in this study. Arcobacter susceptibility to ciprofloxacin and levofloxacin was determined by disc diffusion tests (BD, USA) and E-test ® strips (AB BIODISK, Sweden). As there is not any recommendation about antibiotic resistance of arcobacters, the disc diffusion breakpoints and the minimum inhibitory concentration (MIC) values were determined as recommended by the Clinical and Laboratory Standards Institute (CLSI ® ) for campylobacters. All the isolates were subsequently analysed in order to determine the presence of mutations in the QRDR of gyrA gene. For that purpose, a 344-bp fragment of gyrA gene of Arcobacter spp. was amplified using F-QRDR (5 -TGG ATT AAA GCC AGT TCA TAG AAG-3 ) and R2-QRDR (5 -TCA TMG WAT CAT CAT AAT TTG GWA C-3 ) primers. Finally, PCR products were purified and sequenced on both strands by Sistemas Genómicos S. L. (Valencia, Spain) . Results: Among the 44 isolates tested, 31 were sensitive and the remaining 13 were considered to be resistant to both antibiotics. A disc diffusion zone of 6 mm and a MIC value 4 mg/mL indicates resistance. The MIC values with respect to ciprofloxacin and levofloxacin ranged from 0.064 to 0.38 mg/mL and 0.094 to 0.5 mg/mL, respectively for the sensitive isolates. The 13 resistant isolates presented MICs ranging from 8 to 32 mg/mL for both antibiotics. The sequencing of the 344-bp PCR product revealed a mutation in position 254 of the gyrA gene in the 13 resistant Arcobacter isolates. This C-254 to T mutation could be the cause of quinolones resistance as this change was absent in all the susceptible isolates.
This study shows high rates of quinolone resistance in arcobacters that was always associated to one mutation in the QRDR region of the gyrA gene. The increasing resistance to this class of antibiotics could be a public health concern as they have been reported to be the most commonly used and best performing fluoroquinolones against arcobacters.
A. Ergin *,Ö. Köseoglu Eser, N. Pakasticali, G. Hasçelik (Ankara, TR) Objectives: The aim of this study was to evaluate antimicrobial susceptibility, metallo b-lactamase production (MBL) and dissemination of oxacillinase genes among multidrug resistant (
Salmonella serovars isolated in Iran M.R. Aghasadeghi *, S.D. Siadat, B. Rajaei, F. Badmasti, N. Sepehri Rad, R. Sabohi, S. Javadian, M.R. Razavi, S. Najar Peerayeh, S.M. Zahraei, R.A. Khavari-Nejad, S.M. Sadat, A. Moshiri, A. Rahimi (Tehran, IR) Objective: High levels of multidrug resistance are normally associated with mobile genetic elements that encode specific resistance genes. Among these genetic elements are the integrons, which are structures that can integrate and express resistance genes. Integrons play an important role in the capture and expression of exogenous genetic material. Methods: Eighty five epidemiologically unrelated clinical isolates of Salmonella.spp were collected from different provinces of Iran through 2008-2009. All isolates were serotyped comprising four serovars (A, B, C, D) and tested for the antimicrobial susceptibility for several antibiotic. All isolates were screened for the presence of class 1 integron using primers specific for intI1 gene. The gene cassettes inserted in the variable region of class 1 integrons were amplified using 5 -CS and 3 -CS primer pairs. The PCR products were extracted from agarose gel and purified with the High Pure PCR Product Purification Kit. According to the size of IVR amplified, one representative band of each group were sequenced and compared with the GenBank sequences using online BLAST software ww.ncbi.nlm.nih.gov/BLAST. Following this analysis, sequences were submitted to the EMBL/GenBank database. Results: Forty isolates (47.05%) which were resistant to at least 4 antimicrobial agents considered as MDR Salmonella serovars. Of the 85 isolates, 58.82% (50 isolates) presented class 1 integrons. 68% (34 cases) of these isolates were multidrug resistance. PCR assays and DNA sequencing of internal variable regions (IVRs) of class 1 integron characterized four gene cassette arrays including dhfr7 (0.8 kb), aadA1 (1kp), blaP1 (1.2 kb),dhfr7-aadA1 (1.6 kb) with eight IVR distribution patterns in MDR isolates. The nucleotide sequences of aadA1 gene, the dhfr7 gene, the blaP1 gene, the dhfr1-aadA1 gene cassette and the aadA1 gene in the class 1 integrons have been deposited in the NCBI GenBank sequence databases under the accession numbers HQ132374,HQ132376,HQ132377,HQ132378, HQ132375 respectively. Conclusion: High frequency of MDR Salmonella serovars demonstrates that antimicrobial selection pressure is widespread in our clinical settings. Detection of class 1 integron carrying gene cassettes which confer resistance to different classes of antibiotics such as aminoglycosides,b-lactams and trimethoprim confirms that integronmediated antimicrobial gene cassettes are common in MDR Salmonella serovars isolated in Iran. With regard to biofilm production of isolates studied, all isolates were positive in different degree of production, except of two isolates.
This study shown that the nontypable S. Typhimurium isolates were characterized by multidrug antimicrobial resistance encoded by class 1 integrons in some of them. PFGE analysis suggested presence of the multiple clones of nontypable clinical isolates of this serovar. This work was supported by Ministry of Health of the Slovak Republic under the project "Molecular analysis of antibiotic resistance of nontyphoid salmonella serovars".
I. Gajic *, V. Mijac, I. Lazarevic, M. Stanojevic, N. Opavski (Belgrade, RS) Objective: Group A streptococci (GAS), are the most common cause of bacterial pharyngitis. Penicillin remains the first line therapy for GAS infections, but in the case of allergy, macrolides are used instead. However, resistance to these antibiotics among GAS population is in rise in many European countries. The distribution of resistance pheno/ genotypes as well as the prevalence of different emm types among macrolide resistant strains varies considerably in different regions and with time. Therefore, the aim of this study was to investigate phenotypes, genotypes and emm type distribution among macrolide resistant GAS isolates from pharyngitis patients in Serbia. Methods: Fifty one GAS isolates exhibiting macrolide resistance were collected from various regions from Serbia in the period 2008-2009. Phenotypes of erythromycin resistance (M, iMLS and cMLS phenotype) were evaluated by triple disk diffusion test with erythromycin, clindamycin and spiramycin, as previously described. The corresponding resistance genes: mefA, ermA, and ermB were detected using PCR amplification. The emm genotypes were determined by PCR with "all M" primers following a previously published protocol by Podbielski and co-workers. Results: Out of 51 GAS isolates, the majority (71%) harbored mef A, 25% had ermA, while, ermB was rarely encountered (4%). All strains with M phenotype had mef A gene. Out of 13 iMLS isolates, all were ermA positive, and one harbored additionally mefA gene. ErmB was present in 2 out of 3 strains with cMLS phenotype. Emm genotyping revealed the presence of 5 different emm types: emm 75 (47%), emm 77 (25%), emm 12 (24%), emm 28 (2%) and emm118 (2%). The relation of emm types and particular resistance mechanisms could be observed: all emm 75 type isolates were mefA positive and all emm 77 isolates were ermA positive. Emm 12 was encountered among isolates harboring mefA as well as in one ermA positive strain and one cMLS strain that harbored none of the tested genes. Erm B positive strains were of emm 28 and emm 118 types. Conclusion: Our data show the predominance of efflux mediated resistance (mefA) and homogenous emm type distribution among macrolide resistant GAS in Serbia. Genotypes associated with resistance were mainly emm 75 and emm 77.
H. -Y. Lee*, R.-C. Chang, L.-H. Su, T.-Y. Lin, C.-H. Chiu (Taoyuan, TW) Objectives: A total of 82 A. baumannii clinical isolates from two teaching hospitals in Taiwan in December of 2006 and 2009 were collected and examined in order to elucidate current resistance mechanisms.
The minimum inhibitory concentrations (MIC) of imipenem, ceftazidime, and ceftriaxone were checked by E-test analysis to these isolates. Primers specific for resistance genes (blaIMP-1, blaVIM-1, blaADC) and upstream regions of insertion sequences (ISAba1, ISAba2, ISAba3, ISAba4 or IS1008) were designed for PCR amplification and sequence identification. All 62 A. baumannii isolates in 2009 were genotyped by pulsed-field gel electrophoresis (PFGE). The 30-day mortality data of patients with isolates in 2009 were collected. Results: The upstream ISAba1 was found in 53 isolates with blaOXA-23, including Tn2006 (ISAba1-blaOXA-23-ISAba1, n = 47) and Tn2008 (ISAba1-blaOXA-23, n = 6), and in 9 isolates with blaOXA-51-like. All these isolates expressed full resistance to imipenem (MIC >32) (Table 1) . ISAba3-blaOXA-58-ISAba3 was found in 3 isolates and offered 2 isolates with resistance to imipenem (MIC >12). In the contrast, without upstream ISAba1, or ISAba3, isolates with OXA-type b-lactamases were all susceptible to imipenem. The upstream ISAba1 found in 50 isolates with blaADC-25 and the upstream ISAba1 or ISAba3 found in other 3 isolates with OXA-type b-lactamases offered these isolates with full resistance to both ceftriaxone and ceftazidime.
Isolates with the combination of upstream ISAba1 or ISAba3 and OXAtype b-lactamases or blaADC-25 showed more resistant to imipenem, ceftazidime and ceftriaxone than those without such combination (all P < 0.001 All the strains produced one (77%) or more (23%) b-lactamases of different types. There were 4 types of b-lactamases with isoelectric points (pI) 5.4 (TEM-1), 5.7 (PSE), 7.0 (OXA-1) and 7.6 (SHV-1) detected. Predominant types of enzymes, responsible for the resistance to ampicillin, were OXA-1 (52.2%) and TEM-1 (46.0%). Frequency of other types of b-lactamases was lower: 17.7% for SHV-1 and 8.0% for PSE. Totally ten spectrums of b-lactamases were detected. The prevalence of one enzyme production in the studied strains was revealed − OXA-1 (39.8% strains) or TEM-1 (25.7%). The combination of two enzymes − TEM-1+SHV-1 or TEM-1+OXA-1 produced 9.7% and 8.8% of strains respectively. The other combinations of enzymes were revealed only in 1−2 strains. Few strains produced only one enzyme − PSE or SHV-1 − 6.2% and 5.3% respectively. Only one strain (S. enteritidis) produced three types of enzymes (TEM-1+OXA-1+SHV-1).
1. The resistance to ampicillin in Salmonella spp. and Shigella spp. in St. Petersburg and North-West region of Russia was mostly due to production of one type of b-lactamase. 2. The predominant types of b-lactamases were OXA-1 and TEM-1. 3. The most widespread combinations of these enzymes, detected in 18.5% strains, were TEM-1+SHV-1 and TEM-1+OXA-1. T. Demir*, N. Coplu, H. Bayrak, M. Turan, T. Büyükgüçlü, D. Caliskan, B. Yalcin, U. Cinar, N. Atakan, Z. Karahan, B. Esen (Kirsehir, Ankara, Mersin, Karabuk, TR) Objectives: PVL toxin is commonly associated with community acquired-S. aureus isolates with a wide range of diseases including skin, soft tissue and pulmoner infections, as well. The aim of this study is to assess the prevalence of PVL toxin co-carriage both in wound and nasal samples. Methods: Susceptibility of C. striatum strains to erythromycin and clindamycin was analyzed by Etest (AB BIODISK, Solna, Sweden). For spiramycin the disk diffusion method was used. The type of resistance of the C. striatum strains was elucidated by the double disc diffusion test. The gene(s) associated with resistance were isolated by PCR with primers specific for ermX and mef genes. The region surrounding the resistance gene(s) in two representative C. striatum isolates was cloned by inverse PCR using primers designed from the sequence of their ermX alleles. The transferability of the resistance gene(s) from two C. striatum strains to E. coli was assayed by electroporation and conjugation. Results: We analyzed a collection of 74 C. striatum from clinical specimens responsible for cases of wound infections, skin ulcers and pneumonia. 64 C. striatum were resistant to erythromycin, 64 to clindamycin and 62 to spiramycin; 62 were resistant to both erythromycin and clindamycin; 60 isolates were resistant to the three compounds. The gene ermX was detected in 62 of the 64 C. striatum resistant to erythromycin and clindamycin. This gene was also found in one isolate sensitive to the three compounds. Preliminary results indicate that in our C. striatum isolates the gene ermX is chromosomic and does not take part of transposon Tn5432. Conclusion: Our study revealed a high incidence of resistance to two macrolides and clindamycin in C. striatum clinical strains isolated at hospital Marqués de Valdecilla, Santander, Spain. First results show that in our C. striatum collection the ermX gene is chromosomic and is not associated to transposon Tn5432.
A. Ripoll*, M.C. Turrientes, R. Cantón, M. Tato, L. Martínez-Martínez, F. Baquero, J.C. Galán (Madrid, Santander, ES) Objectives: Unlike TEM and SHV, b-lactam plus b-lactamase inhibitor (BBLIs) resistant variants among CTX-M enzymes have not hitherto been described in the clinical setting. These variants may exist but their phenotypic patterns could be confused with those of IRT or IR-SHV. The objectives of this work were: i) to test if an automatic susceptibility testing system (Wider) and blind expert analysis might identify different CTX-Ms harbouring different laboratory-introduced mutations conferring increased MICs to BBLIs; ii) to compare the efficiency to detect IR-CTX-M enzymes when using CLSI or EUCAST breakpoints. Methods: 14 constructions of hybrid plasmids containing 3 wildtype CTX-M genes and 11 CTX-M laboratory-obtained coding for enzymes resistant to inhibitors were transformed into 4 isogenic E. coli laboratory strains with porin membrane alterations: MKW505 (wild-type), KAEC5 (ompC::Kn), MH621 (ompF::lacZ) and KAECF5 (ompF::lacZ; ompC::Kn). Wild-type CTX-Ms were CTX-M-1, CTX-M-2 and CTX-M-14 and mutated CTX-M belonged to these CTX-Ms but carrying changes (S130G, S237G and K234R) that we previously described conferring increased BBLI MICs. Results: All wild-type CTX-Ms into the four genetic backgrounds were correctly detected by Wider and expert personnel. Those six mutated CTX-Ms carrying S130G were classified by Wider as IRT (with and without overexpression of TEM) or ESBL (2 cases) while expert personnel as IRT (2), overexpression of TEM (1), overexpression of SHV (1) and ESBL (2). Those three mutated CTX-Ms-K234R and two CTX-Ms-S237G mutants were classified as ESBL with Wider and expert personnel. Using current breakpoints, reference CTX-Ms were always identified as ESBL, except KAECF5-CTX-M-1 which was classified as IR phenotype with EUCAST breakpoints. All variants were coincidently identified as IRT in CTX-Ms-S130G and as ESBL in CTX-Ms-K234R and CTX-Ms-S237G when using both CLSI and EUCAST breakpoints. Interestingly, CTX-M-14-K234R and CTX-M-35-S130G were classified as IR-ESBL when using EUCAST but not with CLSI breakpoints. Conclusions: Not a single IR-CTX-M phenotype was detectable either by the automatic system or the blind expert analysis, suggesting that these IR-CTX-M variants could be overlooked in the clinical microbiology setting. When using current breakpoints, EUCAST detected more IR-CTX-M-ESBL phenotypes than CLSI. This result suggests that EUCAST must be considered as reference to detect these IR-CTX-M mutants in the future. -29) . Five of these isolates exhibited a high level resistance to the 3 carbapenems tested (MICs 128 mg/L) whereas 2 strains (producing either IMP-2 or IMP-29) turned out to be moderately resistant to imipenem (MIC of 32 mg/L). No class A or D carbapenemases were found in the collection. Among the 98 remaining strains (93.3%), the decreased susceptibility to imipenem (MIC from 6 to 64 mg/L) was attributed to alteration or loss of porin OprD. In these impermeability mutants, the activity of meropenem and doripenem was generally better than that of imipenem (MICs from 2 to 16 times lower). Conclusion: Seven % of the imipenem non-susceptible P. aeruginosa isolated in French ICUs produce a MBL. This prevalence is lower than in some other European countries. Nevertheless, the epidemic potential of these broad-spectrum b-lactamases should incite ICUs and microbiology laboratories to strengthen surveillance measures to prevent their spread.
O. Köseoglu Eser*, Y.Öcal, B. Sener, G. Hascelik (Ankara, TR) Objectives: Detection of plasmid mediated AmpC b-lactamase producing organisms is important to ensure effective therapeutic intervention and optimal infection control. However, detection of these enzymes is a challenge for laboratories since there is no reliable method for the routine detection of plasmid mediated AmpC enzymes. R2357 Are ready-to-eat salads an important vehicle of pathogenic and commensal bacteria resistant to antibiotics? J. Campos, L. Peixe, J. Mourão, J. Pires, A. Silva, C. Costa, H. Nunes, N. Pestana, C. Novais, P. Antunes* (Porto, PT) Objectives: The increase demand for fresh fruits and vegetables is causing an expansion of the market share for minimally processed vegetables along with recognized food safety problems. We analyzed the microbiological quality of Portuguese ready-to-eat salads (RTS) and their role in the spread of bacteria carrying antibiotic resistance (ABR) genes. Methods: RTS (n = 50; 7 brands; split or mixed leaves, carrot, cornmeal) were collected in 5 of the main supermarkets (2010). The evaluation of microbiological load and quality followed the international standard methods for counting aerobic mesophilic, coliforms, Enterococcus sp and detection of Salmonella sp or Listeria monocytogenes. Samples were also plated in different culture media with/without AB before and after a pre-enrichment step. ABR was studied by agar diffusion method (CLSI) and ESBL expression by double disk synergy test (DDST). Species were identified by PCR (Gram positive), API ID32GN or 16rRNA (Gram negative). ABR genes, integron types and E. coli phylogenetic groups were searched by PCR and clonality by MLST in specific isolates. Results: A high number of RTS presented poor microbiological quality (86% for aerobic mesophilic, 74%-coliforms, 4%-E. coli), but no pathogens. Different ABR phenotypes and genotypes were seen to both Gram positive and Gram negative bacteria. E. coli detected in 13 samples (n = 26; phylogenetic groups A-7, B1−10, B2−1, D-8) presented resistance (%) to tetracycline (73; tetA and/or tetB), streptomycin (50; aadA), sulfametoxazole (46; sul1 and/or sul2), trimethoprim (46; dfrA1 or dfrA12), ampicillin (46; blaTEM), nalidixic acid (27), ciprofloxacin (8) or chloramphenicol (4) . Two integron types (dfrA1 + aadA, dfrA12 + aadA) were detected in 11 isolates. Multidrug resistant E. coli (n = 2; D) belonged to the widespread ST69; the fumC alleles of other E. coli were highly diverse and identified as 8, 48, 65 and 100. DDST gave a positive test for 2 Raoultella sp (2 samples) carrying an ESBL identified as SHV2. Among enterococci (n = 108) ABR (%) was seen for tetracyclines (6; tetM and/or tetL), erythromycin (3; ermB), nitrofurantoin (1) or ciprofloxacin (1). Conclusions: The present study positions RTS within the spectrum of ecological niches that may be reservoirs/vehicles for ABR bacteria/ genes with clinical interest (e.g. E. coli-B2 or ST69; ESBL) being these findings worthy of attention as their spread to humans by ingestion cannot be dismissed. Plasmid characterization included determination of size and content (S1-PFGE), and identification of relaxases (rel), rep initiation proteins (rep) and toxin-antitoxin systems (TA) by PCR, sequencing and hybridization. Results: Efc strains studied were classified in 47 PFGE-types and 38 STs which clustered into CC9 (n = 11, 16%), CC2 (n = 10, 15%), CC16 (n = 9, 13%), CC25 (n = 5, 7%) and CC21 (n = 4, 6%). They were highly resistant to erythromycin (72%), tetracycline (59%), streptomycin (47%), gentamicin (37%), ciprofloxacin (35%) and chloramphenicol (18%). All were susceptible to glycopeptides. Plasmid content of Efc isolates was variable (1−3/cell; 20-100 kb), those ranging from 40 to 60 kb being predominant. They belong to different plasmid families: i) RCR/thetha (15% reppS86/pAMa1; 21% relpS86/pAMa1); ii) repA_N (62% reppAD1, 10% reppAM373, 62% relpAD1, 32% relpCF10, 10% par) iii) Inc18 (18% repInc18, 22% reppRE25, 19% relpRE25, <5% relpEF1). Modules from small theta replicating plasmids (pCIZ2, pEF418 and pEF1071) were rarely found (<5%). Similar rep and rel content was observed among isolates for each CC. Mosaic plasmids containing rep and/or rel from different families were identified. Conclusions: Most Efc isolates harboured mosaic plasmids associated with the repA_N family. The influence of particular plasmids or plasmid modules in the success of major Efc lineages and in the distribution of antibiotic resistance genes among different clonal lineages of the same or different enterococcal species remains to be established.
R2360 In vitro fosfomycin activity against extended-spectrum b-lactamase producing Escherichia coli related urinary tract infections T. Demir*, M. Turan, T. Büyükgüçlü (Kirsehir, Karabuk, TR) Objectives: Extended spectrum b-lactamase (ESBL) producing E. coli disseminated worldwide as an important cause of both nosocomial and community-acquired urinary tract infections (UTI). Increasing resistance rates to antimicrobials among these isolates limit the choice of treatment. K. Klesiewicz, N. Szkaradek, A. Gunia, H. Marona, A. Budak (Krakow, PL) Objectives: The aim of the study was to determine anti-H. pylori activity of a series of 14 xanthone derivatives. They were substituted in different position in xanthone structure i.e. 2, 3, 4 and 6, 2 and 6 or 2 and 7. They possessed among other piperazine, pyridine, aminoalkanol, allyl, methylamine, ethylamine, alkoxy, fenoxy or carbamyl moiety, some of them had also chlorine atom(s) in their structure. Methods: Disc diffusion procedure (Kirby-Bauer method) was used for primary screening of susceptibility H. pylori clinical strain isolated from a patient and ATCC 43504 H. pylori strain to the xanthone derivatives (in concentration 10 mg/ml). In addition compounds giving an inhibition zone ranged from 11mm to 45 mm in diameter were chosen to quantitative assay to establish the lowest concentration that inhibits growth of the H. pylori strain (MIC). Bacterial suspension of H. pylori was adjusted to yeld approximately 1.0×10 8 CFU/ml and plated on Mueller Hinton agar with 5% horse blood and NAD plates (Oxoid). A stock solution (10 mg/ml) of each substance was appropriately diluted in DMSO to obtain the required concentrations of 1mg/ml, 200 mgml, 100 mgml and 50 mgml. Filter paper discs were placed on the inoculated agar surfaces and impregnated with 10 ul of each sample solution. Antibacterial activity was expressed as the mean of inhibition diameters (mm) produced by the substances. The lowest concentration required for growth inhibition of H. pylori strain was regarded as MIC.
The results were very similar for two examined strains. Most of the tested compounds were active against both. H. pylori strains at the concentration of 10 mg/ml (stock solution). Two substances exhibited more than 40 mm wide grown inhibition zone, 1 − 39 mm, 2 − from 29 to 30 mm, 7 − from 12 to 20,5 mm. 2 of the presented substances were classified as nonactive. MIC of the xanthones ranged from 10 mg/ml to 50 mgml. Most potent activity with MIC value of 50 mgml was characteristic for 3 compounds. Conclusion: Performed research on anti-H. pylori activity of the series of xanthone derivatives, it could be stated that: chlorine atom as well as allyl moiety are favorable, substitution in the xanthone structure in position 2 is more favorable than in position 3 and 4 and carbonyl group in a side chain is responsible for the partial loss of activity. Further studies will be necessary to investigate the effect of these active compounds. The MRSA dilution of 0.5:106 had very few colonies in culture up to 6 hours but the pellet of 1.5 ml of the broth after 2, 4, and 6 hours incubation there was an increase of 1.7, 3.6 and 4.8 in CT. There was no interference, inhibition or cross-reaction with MSSA in the XMSN assay.
The Copan MRSA-B enrichment broth increases the number of MRSA colonies 27 folds and 11 CT in the XMSN assay after 6 hours incubation. The sensitivity of the XMSN assay is increased when using the pellet from centrifuging 1.0 or 1.5 ml of the MRSA-B enrichment broth. Objectives: The purpose of this study was a research for Methicillinresistant staphylococcus aureus originated from foods based on pheno and genotypic methods. Methods: 93 S. aureus isolates from 913 food samples were investigated for the detection of methicillin resistance using mecA specific PCR, oxacillin agar screen and disk diffusion, penicillin binding protein 2 (PBP2) latex agglutination, b-lactamase production and MIC tests. The MRSA strains were characterized by the antimicrobial susceptibility, production of type A-D staphylococcal enterotoxins (SEs) and toxic shock syndrome toxin1 (TSST1), as well as biotyping and molecular typing based on the x-region of the protein A (spa) and the coagulase (coa) genes. Results: Out of the 93 S. aureus isolates, eight were mecA positive while phenotypic tests showed a discrepancy in comparison with each other. The majority of MRSA isolates were PBP producers and Multi-resistant. All TSST1 producing MRSA were SE producers. Out of the 12 MRSA isolates, eight were belonged to human biotype; three belonged to NHS and one to bovine biotype. Amplification of polymorphic spa and coa genes revealed three and five distinct types, respectively.
Food is an important vector for transmission of antibiotic resistance to human. Some MRSA isolates recovered from different sources showed genetic and phenotypic similarity in the patterns obtained, suggesting that contamination with human origin has been occurred during food handling. The presence of nuc (species confirmation), PVL and toxic shock syndrome toxin 1 (TSST-1) genes were detected by PCR. Molecular typing techniques such as agr typing, spa typing and Multi-Locus Sequence typing (MLST) were performed. Clonal Complexes (CCs) were determined by using eBurst software. Results: All the isolates were confirmed as PVL-positive MSSA. Fourteen isolates were from skin and soft tissue infections (SSTIs), 4 from nasal carrier, 2 from necrotizing pneumonia, one each from an ORL infection, sepsis and osteomielitis. One of these isolates was the strain responsible for an hospital-community outbreak in 2005. Molecular typing revealed that isolates belonged to all the four known agr alleles (from 1 to 4), to 16 different spa type and to 12 different sequence type (ST). Following eBurst analyses, the related STs were grouped in CCs except for ST1209 that was a singleton. The isolates belonged to 9 different genetic lineages. The most common were CC121/agrIV and CC30/agrIII (6 isolates each). The other lineages were: CC5/agrII, ST1209/agrIII and CC22/agrI (2 isolates each) and CC1/agrIII, CC78/agrIII, CC152/agrI and CC8/agrI (1 isolate each). Three strains were TSST-1 positive: both the ST1209/agrIII strains and one CC30/agrIII.
This study showed the great variety of the PVL-positive MSSA clones circulating in Italy, with CC121/agrIV and CC30/agrIII being the most commons. Differently from other countries, we did not find strains belonging to the common CC80/agrIII clone. A new lineage was found (ST1209/agrIII) which also was TSST-1 positive. Further studies should be done to evaluate the circulation and the characteristics of PVL-positive MSSA since it can cause serious infections similarly to its counterpart, PVL-positive MRSA. .7% proved to be GBS positive. 9.5% of the newborns were colonised (ears or throat), and only 0.4% had GBS in the blood. The hypervirulent ST17 clone was detected in 46.2% of GBS+ women and 35.6% of GBS+ newborns. Generally, serotype III was most prevalent (56%), type I was isolated in 18% (mostly from the ears of newborns) and type II in only 7%. The vast majority of the ST17 strains (36/58) were also of type III. The ST17 strains belonged to 4 clearly distinguishable PFGE clones, but were generally closely related to one another. Conclusions: Serotype III was shown to be the most prevalent in neonatal colonisation or asymptomatic carriers also in Hungary. The ST17 clone of GBS identified by multi locus sequence typing (MLST) is recognised as a hypervirulent international clone associated mainly with invasive neonatal infections. ST17 comprised 46.2% of asymptomatic carriers among the pregnant women, and could be transmitted to the newborns at high rates, with a potential of causing severe neonatal infections. These results emphasize the necessity of a regular screening during pregnancy also in Hungary.
K. Laub*, Sz. Kardos, K. Nagy, O. Dobay (Budapest, HU) Objectives: The carriage of pathogenic bacteria such as Staphylococcus aureus in healthy individuals is well known, with a 25−30% prevalence. In this study we surveyed the nasal carriage of students of a medical university, who already attend hospital wards, so they can be a potential source of infection. This is the first study of this kind in Hungary. Methods: Eighty-eight S. aureus isolates were collected from the nasal passages of the students attending Semmelweis University, Budapest, Hungary. The species identity was confirmed by colony morphology, catalase test, Pastorex test (Bio-Rad) and nucA PCR. MRSA strains were screened by mecA PCR. The antibiotic sensitivity was determined by E-test, applying the EUCAST breakpoints. The genetic relatedness of 56 strains was examined by PFGE. Results: Altogether 300 3rd-year students (205 Hungarian and 95 non-Hungarian) were sampled on a voluntary base. The overall S. aureus carriage rate was 29.3%, little higher among the Hungarians (31.7%) than in the non-Hungarian group (24.2%). On one occasion carriage of two unrelated strains was detected. Out of the 88 strains, only 2 carried the mecA gene, but these had oxacillin MICs of only 0.75 and 2 mg/L, respectively. The strains were fully sensitive to gentamicin, ciprofloxacin and vancomycin. There were 9 isolates with high-level (MIC 256 mg/L), and 3 with low-level (MIC = 12−32 mg/L) erythromycin resistance. Only 1 isolate was resistant to clindamycin. Based on the PFGE pattern, 3 clones comprised approximately half of the strains (15, 14 and 6 strains, respectively), but the rest were rather diverse.
Conclusions: The S. aureus carriage rate correlates well with international data. Luckily there were only 2 MRSA strains (2.3%). The one with the higher MIC (the EUCAST breakpoint is >2 mg/L) was highly resistant also to erythromycin, but the other strain was fully sensitive to all tested drugs. The other isolates were generally very sensitive, which is characteristic in the case of carried pathogens. The strains proved to be genetically more diverse than the usual MRSA populations, with only a few smaller clones, indicating the presence of several independent strains. This, and the dissimilarity of isolates within the same groups, indicate that there is no extensive exchange between the students flora. Results: Among the clinical strains, the pilus was present in 24 cases (24.0%). Seven of these derived from invasive infections, and the rest from respiratory specimens. Half of them came from small children (<4,5 y), and half from adults (41−84 y). The majority of the strains was resistant (R) to macrolides, and had elevated MICs to penicillin (0.25−1.5 mg/L). Their serotypes were: 6 (n = 11), 14 (n = 7), 19F (n = 3), 11A (n = 2) and 23F (n = 1). Among the carried isolates, only 15.4% (n = 24) were pilus positive. These were of the following serotypes: 6 (n = 10), 19F (n = 6), 14 (n = 3), 19A (n = 2), 15B (n = 1), 3 (n = 1) and 18C (n = 1). Although the majority of the carried strains was sensitive to antibiotics, nearly half of the pilus + strains was also R to macrolides and non-susceptible to penicillin. Out of the 24 children, only 2 were previously vaccinated with Prevenar.
The rate of pilus positive clinical strains correlates well with international data, but it was significantly lower in the carried strains. This suggests that pili are required more for invasion rather than merely for colonisation. Very probably there is no direct correlation between pili and resistance, but rather we found pili only in certain resistant serotypes. These were almost all (45/48) vaccine-types (with the dominance of serotype 6), except for 3 strains (11A or 15B). The conjugate vaccines seem to be quite effective in preventing colonisation with pilus positive strains, hence preventing subsequent invasion in the body. Kunda, A. Ankirskaya, L. Lubasovskaya, D. Popov, A. Gintsburg (Moscow, RU) Objectives: Coagulase-negative staphylococci (CoNS) are now recognized as the aethiological agents of an important range of infections in humans. Most countries have reported an increase in CoNS infections in hospitalized patients that are resistant to methicillin and other antibiotics. Despite of it information of population structure and global epidemiology of Staphylococcus epidermidis and some other CoNS is scare. The goal of investigation was to analyze collection of CoNS from diverse clinical origins using molecular genotyping. Methods: CoNS isolates were recovered from the blood (36), umbilical wound (10), conjunctivitis (33), urine (1), pulmonary tissue (2) in 2009-2010. Antimicrobial susceptibility was determined by standard methods. Species identification was carried out by amplification and sequencing of tuf gene. S. epidermidis isolates were analyzed by multilocus sequence typing (MLST) by protocol, as described by Thomas J.C. et al. (2007) . MLST of Staphylococcus haemolyticus isolates was performed using original primers sets. Structural components of staphylococcal cassette chromosome mec (SCCmec) were tested by PCR additionally. Results: Identification of CoNS at the species level indicated that S. epidermidis was the most common species with 46 isolates, followed by S. haemolyticus (18), Staphylococcus hominis (10), Staphylococcus warneri (7), Staphylococcus pasteuri (1). 69 (84.2%) isolates were methicillin-resistant. Among S. epidermidis isolates 24 ST were discovered, including 5 new. New alleles for 5 analysing genes were discovered two. ST59 was predominated (41.3%. isolates). S. epidermidis isolates carried SCCmec IV, composite variant SCCmec (mecB+, ccr1,+ccr2) or unusual patterns with two mec gene complexes (class A and class B). Among S. haemolyticus isolates 9 St were recovered. Isolates carried modified SCCmec type IV (ccr2+, comlex mec class B 1kbp), or elements SCCmecV (ccr5+). SCCmec in 42.7% isolates were not typable. The predominant SCCmec type found among S. hominis isolates was a new type with ccr1 and class mec typeA. Conclusion: Only 4 species (S. epidermidis, S haemolyticus, S. hominis, S. warneri were prevalent (98.8%) among tested collection of coagulasenegative staphylococci. High level of genetic diversity of S. epidermidis and S. haemolyticus isolates were discovered. Isolates S. epidermidis ST59 were epidemic in neonatal center and aethiological agent of different forms of infectious process. CoNS may serve as reservoir of new types SCCmec. in intensive care units, and 10 (12%) in surgical wards. According to Friedman's criteria, acquisition was classified as nosocomial in 47 (57%), healthcare-associated in 28 (35%), and community in 7 (9%). The Charlson index was 2 in 65%, and 42% had a Pitt score >2. The most frequent source was an intravascular catheter infection (43%). BSI was considered as complicated in 26 patients (33%); 36 patients (44%) met 1 criteria for therapeutic failure; BSI-related mortality was 31% (25 patients). All 100 isolates were studied. There were 14 different antibiotic resistance patterns, with combined resistance to ciprofloxacin, tobramycin and erythromycin being the most prevalent (29%). By broth microdilution, MIC of vancomycin was <1 and 2 mg/ml for 84% and 6% of the isolates, respectively; by E-test, it was 1, 1.5 and 2 mg/ml in 56%, 39% and 5%, respectively. Regarding daptomycin, only 2% of the isolates showed a MIC >1 mg/ml by microdilution although all the isolates showed a MIC 0.5 mg/ml by E-test. Eighty percent of the isolates were grouped into 5 clusters by PFGE (groups A to E) with the following characteristics: A (9 isolates), spa t008, agrI, and SSCmecIV; B (9 isolates), spa t148 and t3092, agrI y SCCmecIV; clusters C, D and E (61 isolates), predominantly spa t067 and t002, agrII, and SSCmecIV. Acquisition, predisposing features, source of BSI, complicated BSI or mortality were similar among the 5 clusters. Conclusion: Isolates of MRSA causing BSI in our area are grouped into 5 clusters, in which SSCmecIV, agrII and spa t067 predominated. We were unable to find significant epidemiological or clinical differences or trends among the clusters. Objectives: Community-associated MRSA (CA-MRSA) has become a worldwide phenomenon. In this study, we investigated the molecular epidemiology and the antibiotic resistance mechanisms of CA-MRSA selected from clinical specimens according to the CDC criteria. Methods: 34 non-duplicated outpatient isolates were classified as CA-MRSA (July 2009 to July 2010) in our institution. Identification and susceptibility testing was carried out by WIDER ® system and disk diffusion method. All isolates were genotyped by PFGE/SmaI and spa, SCCmec and agr typing. The presence of genes encoding PVL and resistance genes: methicillin (mecA), erythromycin (ER) and clindamycin (CC) (ermA, ermB, ermC and msrA), gentamicin (GM), kanamycin, amikacin and tobramycin (TO) (aac(6 )-Ie-aph(2")-Ia, ant(4 )-Ia and aph (3 ) The msrA gene was found in isolates with macrolide-streptogramine (MS) resistance phenotype and ermC, ermA or msrA+ermC genes in those with MLSBc or MLSBi resistance phenotypes. Aminoglycoside resistance genes detected were ant(4 )-Ia (61.8%), aac(6 )-Ie-aph(2")-Ia (2.9%), aph(3 )-III (2.9%) and aac(6 )-Ie-aph(2")-Ia+aph(3 )-III (5.9%). Five (14.7%) strains were PVL positive. SCCmec type IVc was detected in 27 strains (79.4%), type V in 3 (8.8%) and 4 strains were type I, II, IVa and non-typeable, respectively. The agr type II was identified in 28 strains (82.3%), agr type I in 3 (12%), agr type III in 2 (5.8%) and one strain was non-typeable. The spa-type t067 was the predominant (64.7%) followed by t002 (8.8%). spa type t067 and t002, normally grouped in clonal-complex CC067 and CC5 (sequence-types ST125 and ST228), are responsible for more than half of nosocomial MRSA infections in Spain. Single strains were typed as t008, t019, t024, t116, t127, t314, t548, t1084 and t2220. Conclusion: Five of 34 selected CA-MRSA carried PVL gene and corresponded to spa types classically community-associated: t024 (ST8-USA300), t019 (ST30), t008 (ST8-USA300), t314 (ST121) and t127 (ST1-USA400). The remaining 29 CA-MRSA corresponded to spa types more associated to the hospital environment what suggests the interchange of genetic lineages of MRSA among community and hospital niches. Although Staphylococcus sciuri is only occasionally isolated from humans, less is known about the carriage of this bacterium in humans, especially methicillin-resistant S. sciuri. The aim of the present study was to provide the analysis of carriage of methicillin-resistant S. sciuri in hospitalized patients and healthcare workers (HCWs). Nasal swab were taken from 195 hospitalized and 105 HCWs at the Clinical Center of Serbia in Belgrade. Oxidase positive colonies of staphylococci were further identified as S. sciuri by BD Phoenix Automated Microbiology System. Methicillin resistance was confirmed by PCR for mecA gene. Susceptibility to antibiotics was performed by disk diffusion method in accordance to the CLSI recommendations. Determination of SCCmec types was done by previously described protocol for mec class and ccr type (Kondo et al., 2007) . PFGE was performed as described previously (Bannerman et al., 1995) . Among 195 hospitalized patients and 105 HCWs, 12 (6.1%) and 4 (3.8%) respectively were colonized with methicillin-resistant S. sciuri. All 16 isolates (100%) were resistant or intermediate resistant to two or more antibiotics beside b-lactam antibiotics, i.e. they were multidrug resistant. All tested strains were susceptible to trimethoprim/sulfamethoxazole, vancomycin, linezolid, and pristinamycin, while 15 (93.8%) were resistant to gentamicin, 15 (93.8%) to kanamycin, 15 (93.8%) to tobramycin, 1 (6.2%) to erythromycin, 1 (6.2%) to clindamycin and the remaining 15 (93.8%) were intermediate resistant to clindamycin, 2 (12.5%) to ciprofloxacin, 1 (6.2%) was resistant to rifampin and 1 (6.2%) was intermediate resistant to rifampin, 1 (6.2%) to tetracycline, 4 (25%) to chloramphenicol, 1 (6.2%) to mupirocin, and 12 (75%) were intermediate resistant to fusidic acid. PFGE analysis revealed 11 pulsotypes within the population of methicillin resistant S. sciuri strains. Isolation of methicillin-resistant S. sciuri of the same PFGE cluster B, and E, from different individuals in different hospitals and different wards indicate successfuly spread of this bacterium. All isolated strains had mec type A and ccr type 3. Carriage of methicillin-resistant S. sciuri among hospitalized patients and HCWs was determined to be surprisingly high, 5%,. Carriage was higher in hospitalized patients, 6.1%, than in HCWs, 3.8%. Isolated methicillin-resistant S. sciuri strains has different genotypic and phenotipyc characteristics. to three drugs and 13 (21.3%) strains were resistant to more than four drugs. All strains of CNS resistant to more than four drugs were positive to gene mec A, and two samples carrying the gene were sensitive to the oxacillin disc diffusion method, revealing heteroresistance.
The results from this study shows a high number of Staphylococcus resistant to oxacillin, with a predominance of staphylococcal cassette chromosome type III in CNS and type II in S. aureus, common in hospital strains and inserted in the community explained by being chronic wounds and patients with a history of multiple hospitalizations over of life. There were great similarities between strains of Staphylococcus found and the sensitivity of the drugs tested, when comparing samples found in the same basic health unit, which may indicate the presence of a possible clone of Staphylococcus in several patients. The results also call attention to the high rate of multidrug resistance found in strains isolated these patients with predominance of SCCmec type III which complicates the treatment these infections. The incidence of an orthopedic implantrelated infection is 1.5% to 2.5% for primary knee or hip replacements and 3.2% to 5.6% for revision surgeries. The essential role in development paraprosthetic joint infections (PJI) belongs to Staphylococcus aureus (S. aureus). This bacteria has entered the spotlight as a globally pervasive drug-resistant pathogen. When dealing with prosthetic joint infections (PJI) there is often a need to start empirical antibiotic therapy. The constant monitoring of resistance chosen pathogen allows in good time to correct the schemes an antibiotic therapy that brings about increasing of its efficiency. The present prospective study was performed to evaluate the frequency of detection S. aureus as etiology factor of PJI and conduct the test of its antibiotic resistance. Methods. We examined the records of 938 isolates is chosen from tissue and fluid samples from 618 patients with PJI from 2007 to 2009. Antibiotic sensitivity S. aureus was tested by agar dilution method. Resalt. S. aureus was the most common pathogen in PJI during these years. The frequency of S. aureus was 42.0%, 38. 8% and 38.1% accordingly for 2007, 2008 and 2009 year. The prevalence of methicillinresistant S. aureus (MRSA) almost did not changed (32.8%, 30.3% and 30.4% for each specified year accordingly). Figure 1 summarizes results of dynamic analysis antibiotic resistance S. aureus in PJI. None of these strains was vancomycin and linezolid resistant. More than 90% isolates were sensitive to fuzidic acid, trimethoprim-sulfamethoxazole, rifampicin and fosfomycin. Resistance rates of S. aureus to the antibacterial agents, respectively, were as follows: less than 20% isolates were resistant to erythromycin, lincomycin, more than 20% to ciprofloxacin, gentamycin, amoxicillin/ clavulanate, The highest resistance rate of S. aureus was detected to to penicillin-G (more than 80% Oncology Unit with some stem cell but no solid organ transplantation. Observing a number of significant infections with Enterobacteriaceae sensitive only to amikacin and carbapenems, we prospectively examined first isolates from 24 patients collected over 8 months in 2009. We were concerned about the risk of cross infection and the frequency of such isolates and whether empirical therapy of febrile patients should be with a carbapenem rather than the more conventional piperacillin-tazobactam, ceftazidime or ciprofloxacin with or without gentamicin.
The 24 isolates were identified to species level and underwent detailed susceptibility testing using disc diffusion, agar dilution MIC determination and automated "Walkaway Microscan™" methods. PCR was then used to detect the presence of CTX-M groups, and Escherichia coli isolates underwent PCR for phylogrouping. Results: Samples from outpatients, inpatients, and ICU grew 11 Escherichia coli, 9 Klebsiella spp. and 4 Enterobacter cloacae isolates. 33% of the isolates were from urine, 33% from wounds and sputa, 21% from ICU screening swabs and 13% from blood cultures. 11/24 isolates (3 E. coli, 5 Klebsiella spp, 3 E. cloacae) were from patients with contact with ICU. The patients from whom the 24 strains were isolated originated from the Middle East (13), UK (9) and Nigeria (2). The average interval from admission to detecting the first significant isolate was between 0 and 85 (mean 13, median 2 days). PCR showed that 20 of the 24 isolates belonged to CTX-M group 1, potentially CTX-M-15s by inference of the MICs. Care in ICU was a significant risk factor for the finding of CTX-M ESBL producing pathogens (p < 0.05). Phylogrouping of the 11 E. coli strains revealed 2 group A, 6 group B1, 3 pathogenic group D, and no pathogenic B2 strains. Conclusions: MICs, PCR and phylogrouping of the 24 isolates suggest that despite the profile of sensitivity being consistently to only amikacin and carbapenem, that this profile was created by expression of heterogeneous resistance mechanisms, which would preclude concerns about cross infection. Further work to characterise the CTX-M group 1 ESBL cluster and molecular environment of these genes could clarify the resistance mechanisms and origins of these strains. A. Doudoulakakis, A. Nika, P. Giakkoupi, M. Papadimitriou, A. Papaioannou, J. Kapetanakis, A. Vatopoulos, E. Lebessi* (Athens, GR) Objectives: Enterobacter cloacae has emerged as an important nosocomial pathogen in neonatal units. The aim of this study was to investigate a massive colonization of neonates with ESBL producing E. cloacae in a 30-bed, university-affiliated, level II-IV Neonatal Intensive Care Unit (NICU) at a large pediatric hospital in Athens. Methods: Routine surveillance cultures for the detection of multidrug resistant bacteria at the throat and rectum of the neonates at admission and weekly until discharge is a standard practice in our NICU. Environmental samples are collected during suspected outbreaks. Clinical and environmental samples are processed according to classic microbiologic methods and the susceptibility to antimicrobials are tested according to the CLSI guidelines. ESBLs are detected by phenotypic methods. Molecular typing of isolates is determined by PFGE after digestion of genomic DNA with XbaI. Results: On August 2010 seven neonates were found colonized with ESBL producing E. cloacae as opposed to only 5 neonates found colonized during their NICU stay (NICU-acquired cases), and one neonate colonized upon admission (referred case) during the last 12 months. A nosocomial outbreak due to ESBL(+) E. cloacae was then highly suspected, and a one-day survey was performed on 2/9/2010. Twenty out of 26 neonates were found colonized with ESBL(+) E. cloacae reported. Environmental samples were all found negative.
Three neonates developed bacteremia due to ESBL(+) E. cloacae during the study period, but only one, suffering from serious underlying conditions died. PFGE analysis of 10 epidemic strains plus 4 epidemiologically unrelated E. cloacae isolates proven >85% similarity among the epidemic strains, a fact consistent with nosocomial spread. Strict infection control measures, such as hand hygiene, patient cohorting and shuting down the NICU for new admissions, were established. Continuous surveillance over the next weeks showed a steady decline in the number of colonized neonates, and on 16/11/2010 only 2 out of 18 hospitalized were found colonized. Conclusion: Continuous surveillance was an important tool in early identifying the outbreak, whereas the implementation of proper infection control measures resulted in containment of the outbreak in a short period of time. Objectives: The occurrence of carbapenemase producing Gram-negative bacteria is of high clinical interest as they can hydrolyze most b-lactam agents including carbapenems recognised as antibiotics of choice in ESBL or acquired AmpC producers usually resistant to other antibiotics. The resistance to carbapenems, generally very rare in Klebsiella pneumoniae strains in the Czech Republic, is predominantly caused by an alteration of the cell wall, together with a production of extendedspectrum and/or AmpC b-lactamases. At the end of 2009, the first putative KPC-producing isolates of K. pneumoniae was submitted for confirmation to the National Reference Laboratory for Antibiotics of the National Institute of Public Health in Prague. The isolates were obtained from one patient. Methods: MICs of 24 antibiotics were determined according to EUCAST recommendation. b-lactamases were identified by isoelectric focusing followed by PCR and sequencing of b-lactamase genes. The gene environment of blaKPC-2 was determined by PCR mapping and sequencing of the amplicons. Isolates were compared by PFGE after the restriction with XbaI endonuclease and multilocus sequence typing (MLST). Results: The isolates of K. pneumoniae (ST258) were obtained from a wound swab, decubitus and urine from patient formerly hospitalized in a Greek hospital. The carbapenemase was identified as a KPC-2 with a gene located on Tn4401a traspozon variant. The resistance to colistin developed after 20-days long therapy with this drug, but after this single isolation, the KPC-2 producing strain disappeared. Objectives: A marked increase in the prevalence of the serotype 4,5,12:i-, a monophasic variant of S.Typhimurium, has been noted worldwide in recent years. Our goal was to assess clonal relationships and to characterize antibiotic and/or biocide resistance in Portuguese isolates from different sources. Methods: We studied 132 isolates (2002) (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) from humans (n = 115), food (n = 9), environment (n = 4) and piggeries (n = 4). The serotype was confirmed by PCR (fljA, fljB, hin and fljA-fljB). Antibiotic susceptibility to 10 antibiotics was tested by disk diffusion method (CLSI). Characterization of antibiotic and biocide resistance genes and class 1 integrons were done by PCR, RFLP and sequencing. Clonality was established by MLST in representative isolates. Results: All but 2 isolates (99%) were resistant to antibiotics of which 94% were multidrug-resistant (MDR, 2−8 antibiotics). They expressed resistance to sulfametoxazole (Su, 92%; sul1, sul2 and/or sul3), tetracycline (T, 91%; tetA and/or tetB), streptomycin (S, 88%; aadA and/or strA-strB), ampicillin (A, 67%; blaTEM), chloramphenicol (C, 45%; cmlA1 and/or floR), trimethoprim (Tr, 35%; dfrA12), gentamicin (G, 27%; aac(3)-IV), nalidixic acid (4%) or kanamicin (3%). Three major groups of isolates mostly associated with 3 genotypes could be identified: (i) ASSuT type (n = 48; blaTEM, strA-strB, sul2 and tetB), also carrying the pcoD copper efflux gene and assigned to the ST34 (SLV of ST19); (ii) MDR (3−8 antibiotics) type (n = 45), being more frequent the phenotype ACGSSuTTr (n = 27; blaTEM, cmlA1, aac(3)-IV, aadA, sul1-sul2-sul3, tetA and dfrA12;) carrying an unusual class 1 integron with estX-psp-aadA2-cmlA1-aadA1 associated to qacH, IS440 and sul3, and in addition a class 1 integron of 2000 bp (dfrA12-orfX-aadA2) or an empty one with qacEdelta1 and sul1 in the 3 CS, and belonging to the worldwide spread ST19 and DT104/U302 phagetype. (iii) CSSuTTr type (n = 15; cmlA1, aadA, sul3, tetB and dfrA12), carrying a class 1 integron with the dfrA12-orfF-aadA2-cmlA1-aadA1 gene cassettes associated to an unusual 3 sequence region with qacH, IS440 and sul3 and belonging to ST19. Conclusions: This is the first study characterising the S. enterica serotype 4,5,12:i-in isolates from Portugal. The spread of three MDR genotypes belonging to worldwide spread clonal lineages in association with biocide resistance determinants (pcoD, qacEdelta1 and/or qacH) might account for the recent emergence and success of this serotype. Metallo-b-lactamase (MBL)-producing strains are emerging worldwide, with VIM-2 exhibiting particular relevance in Europe. Dispersion of integrons carrying blaVIM-2, clonal spreading and the association of MBL genes with transposons may contribute to the observed dissemination rates. Our aim was to analyze the population structure of blaVIM-2-carrying P. aeruginosa clinical isolates. Methods: From our collection of carbapenem-resistant P. aeruginosa isolates (2005) (2006) (2007) (2008) , derived from five hospitals in North and Centre of Portugal, all blaVIM-2-producing P. aeruginosa isolates were characterized (n = 12). Antibiotic susceptibility was determined by the agar diffusion method and E-test (CLSI). Class 1 integrons were characterized by PCR and sequencing. Clonal relatedness was studied by PFGE (XbaI and SpeI) and MLST (http://pubmlst.org/paeruginosa). Results: All P. aeruginosa isolates showed a multidrug-resistant phenotype including resistance to imipenem (MIC >32 mg/L), aminoglycosides and ciprofloxacin. They carried blaVIM-2 as a gene cassette inserted in 5 different class 1 integrons: In56, In58, In100, In102 (ORF11, aacA4, aacC1, blaVIM-2) and In103 (aacA7, blaVIM-2, aacA4). In58 was the most frequent, found in 8 isolates. blaVIM-2 gene was found to be chromosomal and/or plasmid located. All isolates were clonally unrelated corresponding to 12 PFGE types. On the other hand, 8 different sequence types (STs) were found: ST111 (n = 2), ST175 (n = 1), ST179 (n = 3), ST235 (n = 1), ST253 (n = 2), ST260 (n = 1), ST282 (n = 1), and ST815 (n = 1). No close relationships (single or double locus variants) were observed among these STs. Most blaVIM-2 producing strains were assigned to widespread STs as ST111, ST179 or ST235, which is the founder of the international Clonal Complex 11 containing MBL isolates.
Conclusions: This is the first report characterizing the population structure of VIM-2-producing P. aeruginosa isolates in Portugal. Our data indicate that these isolates belong to an extremely polyclonal population with no apparent common ancestry. Further studies are imperative to follow-up the relevance of specific clones in the dissemination of blaVIM2 carrying isolates.
M. Demirelli*, T. Sari, F. Temoçin, A. Yazan, C. Bulut, G. Tuncer Ertem, F. Erdinç, B. Oral, N. Eren Tülek (Ankara, TR) Francisella (F.) tularensis, Gram-negative, aerobic, facultative intracellular bacterium have been distinguished to date: F. tularensis subsp. tularensis, holarctica, mediasiatica and novicida. F. tularensis has been detected in more than 200 animal species, with rabbits, hares, small rodents (e.g. voles, field mice) and semi-aquatic animals etc. Tularemia is a re-emerging disease in our country recently. In here, we evaluated the clinico-epidemiological characteristics of our patients with tularemia. Method: Prospective review of clinical records of patients diagnosed with tularemia admitted to our hospital in Ankara, from January to December 2010. Case definition: Patient with suggestive clinical course and epidemiology (coming from the epizootic area) and positive serology (antibodies to Francisella tularensis >1/160). Results: 22 patients (13males, 9 females) with a mean age of 41 years were included to the study. Nine patients were living in endemic areas of turkey and thirteen patients had travelled to the endemic areas. Among 21 cases, contaminated food or water were the most commonly noted exposures (ten patients were consumed spring water), and one had tick bite exposures Thirteen patient reported being exposed to a rat which lived around patients houses. The clinical symptoms period of patients ranged from 10 to 45 days. The final diagnosis of 20 patients was glandular tularemia, whereas two of them were oculoglandular tularemia. 4 had suppurated lymph nodes. Fourteen of the 22 patients were hospitalized (median duration: 4 days [range: 1−27 days]). History of antibiotic use was available for all patients. 18 of patients had been treated with a b-lactam or b-lactam/b-lactamase inhibitors antibiotics those are considered ineffective against tularemia.
Among 3 patients initially treated with streptomycin monotherapy and one of the patient was treated with streptomycin and ciprofloxacin combination. Epidemiological and clinical findings of patients were summarized in Table 1 . Conclusions: Tularaemia should be considered in the differential diagnosis of patients with fever, pharyngitis, conjunctivitis, and cervical lymphadenopathies. Hence, early diagnosis and treatment of tularaemia are important, every clinicians must be aware of diseases. Baranda Camino, J. Galan Ros, L. Robles Fonseca, M. Martinez Serrano, M. Crespo Sanchez (Albacete, ES) Objectives: To determine the epidemiology of urinary tract infections (UTIs) by multiresistant microorganism isolation in hospitalized patients and the community diagnosed at the Hospital Universitario (Albacete, Spain) from 2000 to 2009; to study antimicrobial susceptibility to the drugs commonly used in such infections. Methods: 292,291 urine samples were processed for aerobic culture by standard methods from January 2000 to December 2009. Identification and antimicrobial sensitivity testing were performed with the commercial Wider ® system (Soria Melguizo). The criteria proposed by the CLSI were followed to detect multidrug resistance mechanisms. We considered multidrug-resistant Pseudomonas aeruginosa (MRPA) with resistance to 3 or more antibiotics of the following: ceftazidime, carbapenems, quinolones and aminoglycosides; and multiresistant Acinetobacter baumannii (MRA) when the isolate was sensitive to only tigecycline and colistin.
Of all the urine samples tested, 51,990 (18.0%) were positive. Of these, (1,240; 2.4%) some MDR microorganism was isolated, 1,102 (88.8%) were extended-spectrum b-lactamase-producing Enterobacteria (ESBL), 84 (6.8%) were meticilin-resistant Staphylococcus aureus (MRSA), 28 (2.2%) were MRPA and 26 (2.2%) were MRA. Within the ESBL-producing Enterobacteria we found 996 (90.3%) Escherichia coli, 90 (8.1%) Klebsiella spp. and 12 (1.0%) Proteus spp. Multidrug-resistant isolates increased from 3 in 2000 to 274 in 2009 in both inpatients and the community. Of the 1,240 total patients, 1,188 (95.8%) were adults and 52 (4.2%) were children; 62.4% were women. The antimicrobial resistance rates of ESBL-producing enterobacteria were: nalidixic acid 77.6%, ciprofloxacin 64.8%, cotrimoxazole 60.0%, gentamicin 22.0%, nitrofurantoin 7.8%, fosfomycin 4.2% and amocillinclavulanate 27.6%. No carbapenem resistant strains were found. Conclusions: We observed an increase in the incidence of UTIs through multiresistant pathogens during the study period, both in inpatients and in patients with infections acquired in the community. Among inpatients, the highest incidence corresponded to the Internal Medicine and Geriatric Departments. ESBL-producing Enterobacteria were the most frequently isolated multiresistant organisms. Carbapenems and aminoglycosides could be the best choice for empiric treatement in hospitalized patients. Quinolones and cotrimoxazole should not be recommended due to the high resistance rates observed. R2408 High-throughput DNA sequencing of resistance plasmids: a powerful approach to identify resistance and virulence features L. Villa*, C. Carta, A. García-Fernández, D. Fortini, A. Carattoli (Rome, IT) Introduction: High-throughput DNA sequencing of resistance plasmids allows a very quick determination of plasmid content. By this novel approach it is possible to identify the major characteristics of resistance and virulence and to trace the evolution of specific plasmid families, recognizing their basis for virulence, host range, transferability and stability. We sequenced plasmids identified in Klebsiella pneumoniae (KP) of nosocomial origin that were i) untypable by PCR-Based Replicon Typing (PBRT); ii) relevant being characteristic of epidemic clones. Materials and Methods: A procedure was devised to simultaneously pyrosequence multiple plasmids by the 454 GS-FLX Titanium, using MID-TAG barcoded primers in large volume emPCR (Roche, Italy). Mobile DNA elements associated with resistance genes, regions implicated in replication and plasmid host ranges, maintenance (toxinantitoxin, plasmid partitioning and exclusion properties) and transfer were annotated.
i. The majority of the plasmids carrying blaSHV-12 gene are classified as untypable being negative to the most common replicons of Enterobacteriaceae. One of these plasmids belonged to the novel IncR plasmid family. This plasmid family was characterized by a novel replicase gene that we previously identified in a Salmonella Montevideo from The Netherlands associated to the qnrS1 gene (García-Fernández et al. 2009 ). The novel fully sequenced IncR-SHV-12 plasmid confers resistance to b-lactams and cephalosporines (blaSHV-12, blaTEM-1, blaOXA9), but also to mercuric ions, aminoglycosides (strA-strB, aacA4, aadA1) and sulphonamides and is negative for the conjugative transfer locus, suggesting in trans mobilization mediated by co-resident plasmids. ii. The entire plasmid content of two KP ST258 strains (one multidrug resistant and one susceptible) was determined. The resistant strain was positive for the carbapenemase KPC-3 located on plasmid pKpQIL, previously identified in ST258 from Israel (NC_014016). The susceptible ST258 contained virulence-like plasmids of the FIIK type that likely contributed to the successful spread of this clone, independently by the acquisition of antimicrobial resistance genes.
Since plasmids can be considered as "genomes" within the bacterial genome, a fully sequence approach may help to identify the characteristics enhancing the successful spread of specific plasmid families, but also the ability of plasmids to support the host survival and spread.
A. Novais*, J. Pires, L. Peixe (Porto, PT)
Objectives: Specific UTI-causing E. coli clones have been linked to the emergence and spread of AbR genes but the complete virulence profile of these UPEC clones has only been assessed for a few lineages. Methods: Our collection includes 104 B2-ST131 (n = 29), D-ST393 (n = 12), D-ST69 (n = 10), D-ST405 (n = 11), A-ST10 complex (n = 18), A-ST23 complex (n = 13), B1-ST155 (n = 6) and B1-ST359 (n = 5) isolates, representing isolates from a 19 year period (1991-2010), different origins (62% hospital, 13.6% healthy volunteers, 13.6% animals, 8.7% outpatients and 1% environment) and locations (n = 89, 8 EU countries; n = 4 Korea; n = 11 USA). Of these, n = 46 were UTIisolates and n = 60 were ESBL/AmpC producers. Clonal relatedness was assessed by PFGE/MLST and phylogroups were identified by PCR. Screening for 33 UPEC virulence factors (VFs) including adhesins, toxins, siderophores, polysaccharide coatings and others (PAI, usp, ibeA) was performed by PCR as described. ExPEC isolates were identified by a previously described criterium.
Results: fimH (90%), iutA (78%) and traT (71%) were found within all STs. The number of VFs varied: ST131 or ST393 (n = 18); ST69, ST405 or ST10 (n = 11−14); ST155 or ST359 (n = 7). Common virulence profiles were established for each UPEC clone rather than for UTIcausing isolates. ExPEC isolates (n = 55, 53%) belonged to all ST393, all ST69 and most ST131, which were enriched in kpsMTII, iutA and iha. ExPEC identified in nearly half ST23 and occasionally other STs had pap alleles (papEF, papGIII or papC) and/or kpsMTII besides iutA. ST393 and ST69 were also significantly enriched in pap alleles. Frequency of usp (53%), sat (55%) and fyuA (58%) was high, though with varible distribution: a) fyuA and sat among D and B2-EC and ST23/ST10, respectively; b) usp among ST131, ST10, ST23 and ST359 (54% of these were CTX-M-14 or CTX-M-15 producers). On the other hand, specific VFs were only found in particular STs: i) PAI (31%) mostly within ST131 and ST405; ii) cvaC (12%) was confined to ST23 complex, ST155 and ST359; iii) ibeA (4%) was only identified in ST131 non-CTX-M-15 producers (TEM-4, TEM-24 or CTX-M-1); iv) sfa/doc (3%) was found in ST155 and v) afa/dra (2%) was detected in ST131 isolates. Conclusion: Consistent VFs profiles were found within STs, and differences could not be explained by origin, pathogenesis or AbR profile. A and B1 UPEC profile was linked to the presence of fimH (adhesion), iutA (iron uptake), traT (surface exclusion) colV (colicin) and/or usp.
A. Novais*, C. Rodrigues, R. Branquinho, P. Antunes, G. Ribeiro, L. Peixe (Porto, Coimbra, PT) Objectives: The emergence of carbapenem resistance among ESBLproducing isolates has been increasingly reported worldwide. We aimed to characterize a recent outbreak of ertapenem (ERT)-resistant Enterobacteriaceae isolates from a Portuguese hospital. Methods: ERT-R (n = 15, MIC = 3−>32 mgml, IMI and MER susceptible) and ERT-S (n = 16, presumptive ESBL-producers) K. pneumoniae (KP, n = 21), E. coli (EC, n = 7), E. asburiae (Eas, n = 1), E. aerogenes (Eae, n = 1) and E. cloacae (Ecl, n = 1) isolates were studied (March-August 2010). b-lactamase production was evaluated by phenotypic tests, IEF, PCR (blaKPC, blaNDM, blaVIM, blaIMP, blaOXA, blaGES, blaAmpC, blaTEM, blaSHV and blaCTX-M genes) and sequencing. False positives obtained for carbapenemase production phenotypic assays were confirmed spectrophotometrically. Strain identification and antibiotic susceptibility testing were performed by standard methods. Clonal analysis included PFGE, MLST and identification of E. coli phylogroups. Plasmid content and blaCTX-M-15 location were assessed by S1-PFGE. Presence of class 1 integrons, sul (sul1, sul2 and sul3) and qnr (qnrA, qnrB, qnrS) genes was searched as described. Porins (ompK35, ompK36, ompC, ompF, omp35 and omp36) were investigated by PCR and sequencing, and SDS-PAGE. Results: Seven ERT-non-S clones (3 KP, 1 EC, 1 Eas, 1 Eae and 1 Ecl, MIC 4−>32 mgml), mostly producing ESBL/AmpC were detected. A MDR KP-ST15 epidemic clone (n = 19, 3 PFGE-types, 63% urologytransplant unit, sul2+) was the most frequently identified throughout the whole period and included ERT-S (n = 10, PFGE A and B) and ERT-R (n = 9, PFGE B and C, MIC 3−32 mgml) CTX-M-15 encoding isolates, bla within 50−70Kb plasmids. KP-ST14 and KP-ST45 (n = 1 each, MIC 12 mgml) were sporadic non-ESBL producers. A CTX-M-15-producing EC-B2-ST131 clone [n = 5 isolates/5 PFGE, ERT-R (MIC = 32 mgml) and ERT-S, different units] was detected at the end of the period (06/07.2010), bla located in 130Kb plasmids. One ACT-2 carbapenem-R Eae, one TEM-24-producing Ecl (MIC 4 mgml) and one Eae (MIC >32 mgml) clones were also identified (03/04.2010). Different porin alterations were observed among ERT-R clones. qnrB (1 KP, 1 EC), qnrA (1Ecl) and class 1 integrons (dfrA25, dfrA17-Delta-aadA5 and aacA4) were sporadically detected.
This study reveals a complex allodemic scenario associated with the emergence of ertapenem resistance among different ESBL/AmpC Enterobacteriaceae widespread clones in Portugal.
Objectives: KPC are an important group of b-lactamases given their spreading in nosocomial settings among Enterobacteriaceae. KPC-2 and -3 are the wider disseminated, found mostly on plasmids in K. pneumoniae. KPC-positive K. pneumoniae (KPC-KP) have been reported in many European countries. After the first description of a KPC-KP in Italy in 2009 only few reports has been published. Here we report on the second Italian outbreak, discussing on clinical and molecular features. Methods: 22 K. pneumoniae isolates collected during 2010 at the San Gerardo Hospital in Monza were analyzed as representative of 58 isolates involved in the outbreak. Antibiotic susceptibility tests were performed with Vitek 2. MICs for imipenem and meropenem were confirmed by Etest for isolates that were positive for carbapenemase production (detected by phenyl boronic acid test). Clonal distribution were assessed by rep-PCR, RAPD, PFGE and MLST. Selected isolates were subjected to PCR analysis and sequencing to detect the pattern of resistance determinants.
The 22 investigated isolates showed a MDR phenotype being susceptible only to colistin, gentamicin and tigecycline. Results of genotyping techniques were all concordant and showed that all but one isolates were clonally related and belonged to ST 258. All isolates were confirmed to be blaKPC-2 positive by sequencing. In addition the investigated strains were positive for blaCTX-M-15 and all but one to blaSHV-12 ESBLs. 36 additional isolates showing the same phenotypic pattern were presumed to be related to the epidemic cluster. Conclusion: Before November 2009 at the San Gerardo Hospital no KPC-KP strains has been isolated. During the studied period, 22 clonally related KPC-KP were obtained. Isolates belonged to ST 258 as the first described Italian KPC-KP. This clone is the major responsible of worldwide spreading of these resistance determinants. Molecular analysis demonstrated the presence in all isolates of blaKPC-2, and blaCTX-M-15 and in all but one of blaSHV-12 b-lactamase. The only KPC-KP isolate not clonally related to the outbreak clone, was positive for blaSHV-1 determinant and was obtained from a patient transferred from another Hospital. This different clone represent an exception in the Italian epidemiological scenario where all described KPC-KP belonged to ST258. The rep-PCR approach here described appear to be a rapid and robust technique to investigate clonal relationship of KPC-KP.
L. Papst*, K. Seme, P. Gabrijel, B. Beovic (Ljubljana, SI) Objectives: Detection and isolation of patients colonised with ESBLproducing bacteria is an important measure to prevent transmission of resistant bacteria in hospitals. In a prospective study we wanted to determine the anatomical sites of ESBL-producing Escherichia coli and Klebsiella pneumoniae. Methods: Patients colonised or infected with ESBL-producing bacteria were included in a prospective study from November 2009 to November 2010. Rectal swab, urine culture, and throat swab were performed in each patient. Wound swabs were obtained in patients with wound, and sputum was collected in patients with productive cough. Sample collection was repeated every 3 months. Collected samples were inoculated on chromogenic agar selective for ESBL-producing bacteria. Disc diffusion method was done for K. pneumoniae and/or E. coli isolates to assess antimicrobial susceptibility profile for each isolate. Results: 89 sample collections were done in 42 patients. Patients, 28 males and 14 females, were 24 to 94 years old (62 years on average). 4 patients were on immunosuppressive therapy, 5 had a chronic cardiovascular disease, 3 were diabetics on insulin, 2 patients had chronic kidney failure and 3 had a chronic respiratory illness. 26 patients were colonised with K. pneumoniae, 6 patients with E. coli, in 10 patients both were isolated. At least one sample was positive for ESBL-producing bacteria on 73 occasions. Rectal swab was positive on 68 (93.2%), throat swabs on 22 (30.1%) and urine cultures on 25 (34.2%) occasions. Sputum culture was performed 11 times, 6 (54.6%) were positive. Wound swabs were positive in 50% (9/18 samples taken). On 5 occasions (in 4 patients), in which rectal swab was negative, ESBL-producing K. pneumoniae was isolated only from urine. In 3 out of these 4 patients rectal swabs from initial sample collections were positive, together with urine, throat or wound swabs. 6 to 12 months later, however, urine was the only positive site, in 1 of the patients even on 2 subsequent occasions.
Our results have shown that rectal swabs are positive in most patients colonised with ESBL-producing E. coli and K. pneumoniae. We can assume that rectal swab is an appropriate method for routine screening for colonisation with E. coli and K. pneumoniae producing ESBL. However, in some patients the urine may at least transitory be the only positive site.
Enterobacter cloacae isolates producing the VIM-1 metallob-lactamase in a neonatal unit of a tertiary Greek hospital
Objectives: Enterobacter cloacae isolates, which produce metallo b-lactamases (MBLs) are regarded as an emerging clinical threat. Infections by such pathogens involving neonates remain uncommon. We analyzed clinical characteristics and outcomes of bloodstream infections due to VIM-producing E. cloacae in preterm neonates who were hospitalized in the neonatal unit of a tertiary hospital in Greece.
Methods: During a one-year period (Jan 2010-Dec 2010), neonates presented bloodstream infections due to E. cloacae which exhibited reduced susceptibility to carbapenems were studied. Bacterial isolates were identified by Vitek 2 (bioMérieux). MICs were determined by E-testing (AB Biodisk). Phenotypic testing was performed using the modified Hodge test, the combined EDTA disk test and boronic acid potentiation disk tests. PCR was used to screen for carbapenemase genes (blaVIM, blaIPM and blaKPC) as well as for ESBL (blaCTX-M, blaTEM, blaSHV) and plasmid-mediated AmpC genes. DNA sequencing was used to identify the MBL type gene. Results: During the study, five premature neonates had bloodstream infection due to carbapenem-non-susceptible E. cloacae. The isolates exhibited variable carbapenem MICs ranging for imipenem from 1−4 mg/ml, meropenem 1− 16 mg/ml and ertapenem 0.5− 8 mg/ml. All isolates remained sensitive to tigecycline and colistin, while two were resistant to gentamicin. Phenotypic tests were indicative of MBL production. PCR was used for the verification and identification of the carbapenemase; it confirmed the presence solely of the blaVIM gene in all isolates. DNA sequencing and BLAST search identifying blaVIM-1 gene. Of the 5 neonates, 4 were female and one male, their mean age of gestation was 27.6±2.5 w and their mean weight was 0.974±0.218 kg. Three of the 5 neonates had been exposed to carbapenems during their hospitalization prior to the reported bloodstream infection. Clinical records of these premature neonates were analyzed and the attributable mortality rate was 40%.
The study showed that premature neonates represent a group of patients especially vulnerable to life threatening infections due to MBL-producing E. cloacae isolates. Carbapenem MICs tend to be variable for these strains and early detection remains a challenge for the laboratory. The timely implementation of infection control strategies is an absolute necessity in order to prevent the spread of these MDR bacteria.
Objectives: DHA-1, an inducible acquired AmpC b-lactamase, confers resistance to most b-lactams except cefepime and carbapenems. Since a previous study displaying the genetic context of a well characterized collection of 30 DHA-1 producers showed that donors and transconjugants of two of these isolates of Escherichia coli carried bla(DHA-1) on multiple plasmids as well as on the chromosome, one of these isolates was selected for further analyses to better understand the element mobilizing bla(DHA-1).
To check if results obtained by the first study were reproducible, we repeated the conjugation assays and picked up 25 independent transconjugant colonies, selected with ceftazidime and rifampin. The plasmid and chromosomal location of bla(DHA-1) in the parental and the 25 transconjugants selected was then investigated by S1 and Xba1 digestion of the entire DNA followed by pulsed field gel electrophoresis (PFGE) and probing with 32P labelled bla(DHA-1) probes.
Results: S1-PFGE and hybridisation analyses with bla(DHA-1) probe among the donor and the 25 transconjugant colonies analysed revealed that bla(DHA-1) genes were carried on plasmids in the donor and six of the 25 transconjugants tested, but also on the chromosome in all cases. Moreover, multiple hybridisation bands were observed when hybridising the Xba1-PFGE with bla(DHA-1) in all transconjugants. Conclusions: Acquired AmpC b-lactamases has recently been described mobilised by integrative conjugative elements (ICEs). These elements are capable to transfer themselves from chromosome to chromosome via conjugation. As bla(DHA-1) was present on the chromosome of the donor and was transferred to the chromosome of all 25 transconjugants, we hypothesize that an ICE could be involved in the mobilisation the bla(DHA-1) genes in these E. coli isolates. Multi Locus Sequence Typing (MLST) is a method to discriminate microbial isolates through the partial sequencing of selected housekeeping genes. The BioNumerics ® software is widely used for the storage and analysis of MLST sequences. With the use of an adapted plugin tool, BioNumerics automatically assembles and processes sequence trace files, connects to the online MLST database, and retrieves corresponding allele numbers, sequence types as well as available clonal complex information.
In the case of Campylobacter jejuni and C. coli (http://pubmlst.org/ campylobacter/) 4378 profiles are available (24/02/2010) linked to 43 clonal complexes.
Using the BioNumerics ® Minimum Spanning Tree (MST) we illustrate the interrelation of these clonal complexes. MST's are well-known in the context of mathematical topology. When a set of distances is given between n samples, a minimum spanning tree connects all samples in such a way that the summed distance of all branches of the tree is minimal. With the principle of maximum parsimony (MP), MST shares the idea that evolution should be explained with as few events as possible. In contrast to MP, a "classical" MST does not allow the creation of hypothetical nodes, relying on all states present in the sampled dataset to explain the relationships and evolution. Therefore, MST is only applicable in studies focusing on a very short evolutionary time frame (micro-evolution) and for complete datasets. The MST algorithm presented here allows the creation of hypothetical nodes when the total distance of the tree is reduced by a user-defined number of events. In the context of MLST, hypothetical nodes are usually missing allelic types for which a number of single locus variants (SLVs) are present in the dataset. Introducing hypothetical allelic types has proven to produce more reliable models in non-complete sampled datasets. Additionally, to select the MST that has the most probable evolutionary interpretation among multiple equivalent solutions (degenerate trees), BioNumerics ® has editable priority rules for tie handling adopted from the BURST program (http://www.mlst.net).
The data flow will be illustrated through the processing of MLST trace files from C. jejuni and C. coli isolates. An UPGMA dendrogram for a mixture of MLST profiles of C. jejuni and C. coli, based on similarities calculated by the categorical coefficient, will be compared to a Minimum Spanning Tree obtained using the same data. Objective: Fast techniques for routine strain typing in the clinical microbiology laboratory are useful tools. We report the results of a comparative study between automated rep-PCR and wholecell matrix-assisted laser desortion ionization-time of flight mass spectrometry (MALDI-TOF MS) for molecular-epidemiological analysis of nososcomial carbapenem-resistant Klebsiella spp. Methods: Thirteen Klebsiella spp (9 K. oxytoca and 4 K. pneumoniae) nosocomial isolates that were VIM-1 metallo-b-lactamase producers were analyzed by automated rep-PCR (DiversiLab System) and MALDI-TOF MS AXIMA. Species identification was performed by Vitek 2 System and by MALDI-TOF. Antimicrobial susceptibility testing of isolated bacteria were assayed using Vitek 2 System and Etest. DNA array was used for detection of KPC and, TEM, SHV and CTX-M extended spectrum b-lactamase. The detection of blaVIM-1 gene was done by PCR and sequencing. Results: All Klebsiella spp isolates except one K. oxytoca had the blaVIM-1 gene and all K. pneumoniae had, also, blaSHV gene associated to ESBL production. Rep-PCR using DiversiLab system showed higher discriminatory power than MALDI-TOF spectra analysis for strain typing.
The combined use of MALDI-TOF for species identification and DiversiLab System for clonal strain typing may be a useful tool for fast and accurate management of nosocomial outbreaks. These trends are mainly driven by fluconazole use, and call for closer monitoring of antimycotic and antifungal use and resistance. Objective: Cefotaxime is a widely used antibiotic in the treatment of neonatal infection. Dosing of b-lactam antibiotics by continuous infusion has been suggested to improve treatment outcome, as compared to intermittent infusion. However, continuous infusion is critical in neonates because of fluid and salt restriction. Glucose 10% is often used as a nutrient supply in neonates and could at the same time serve as a good solvent for cefotaxime. Yet, stability data of cefotaxime in glucose 10% are lacking, restraining pediatricians from continuous infusion of cefotaxime. We investigated the stability of cefotaxime in glucose 10%.
Method: Cefotaxime was dissolved in glucose 10% in a concentration of 80 mg/ml and stored at room temperature. Cefotaxime levels were measured every sixty minutes for eight hours and after 24 hours using a validated High Performance Liquid Chromatography (HPLC) method. All measurements were performed in duplicate. Values estimated for each sample at subsequent time points were calculated as percentages of the initial concentration. Recovery below 90 percent of the initial concentration was defined as significant loss, using worldwide standards. Results: The relative concentration time curve is shown in figure 1. After 24 hours, the cefotaxime concentration declined to 98.1% (SEM 1.65), indicating that cefotaxime is stable for at least 24 hours in glucose 10%. Conclusion: Even after 24 hours, cefotaxime does not decrease to the 90% limit of stability in a concentration of 80 mg/ml when dissolved in glucose 10%. Glucose 10% is a good solvent for cefotaxime allowing continuous infusion with changes of infusion bags only once a day. This facilitates optimal treatment, allows glucose supplementation and reduces nursing staff workload. Objective: Analysis of diagnostic accuracy of the suspected origins of the bacteremias and the prognostic influence of the type of microorganism, adquisition, empirical treatment and origin of bacteremia in the evolution to death. Material and Methods: A retrospective study of bacteremia diagnosed during 2 years in a South Hospital in Madrid, analyzing suspected origin, final origin, acquisition, microorganism, empirical therapy and evolution to death. Results: 326 bacteremia were analyzed. Congruence between diagnosis suspected/definitive origin of nosocomial bacteremia was 80,26% (61/76) with a difference of 19,7% (95% CI 10,7% to 28,6%), Sig: <0,001. Congruence between diagnosis suspected/definitive origin of community bacteremia was of 81,3% (192/236) with a difference of 18,6% (95% CI 15,6% to 30,6%), Sig: <0,001. Bacteremias of respiratory origin had a percent of apropriate empiric diagnosis of 93,5% while that of vascular origin, only 56%. S. pneumoniae had a percent of apropriate empiric diagnosis of 91,6%, while non-fermentative Gram-negative bacilli 60% and coagulase negative Staphylococcus 52,1%. Mortality was 2,95 CI 95% (1,01 to 4,29) fold higher in bacteremic inadequate empirical antibiotic treatment. The mortality in the group receiving inadequate antibiotic therapy was 19,7%. Difference between both groups was 9,1% CI 95% (0,8% to 19%) Sig: 0,042. The difference in mortality between the group with right and wrong suspected diagnosis of origin of bacteremia was 3,45% CI 95% (−0,6% to 13,5%) Sig: 0,47. OR 1,33 (95% CI 0,59 to 2,99).
Clinicians have a high degree of suspicion about correct original focus of bacteremia, higher in the respiratory origin and lowest in those of vascular origin, and these data are consistent with the type of microorganism. The degree of success does not affect the overall mortality of bacteremia but empirical treatment does it.
Objective: To investigate the outcome of patients treated with colistin either by IV or inhalation therapy for nosocomial pneumonia associated with multidrug-resistant (MDR) Pseudomonas aeruginosa. Methods: A retrospective study of 20 intensive care patients with a pneumonia associated with MDR P. aeruginosa receiving colistin sulphomethate sodium (Colistineb ® ) between 2007 and 2009 was performed. A strain was considered multidrug-resistant if it was resistant to at least 6 of the following antibiotics: piperacillin-tazobactam, ceftazidime, cefepime, meropenem, aztreonam, ciprofloxacin, and amikacin. The administration mode, predicted mortality based on the SAPS3 score, SOFA score at onset of the colistin treatment, clinical and microbiological response, and mortality during the episode of the infection were analysed. The non parametric Kruskal-Wallis and Fisher's Exact test were used for statistical analysis of respectively the predicted mortality/SOFA score and mortality rate. Results: Six patients received colistin by inhalation only, 5 were treated only parenterally, and 9 by a combination of both administration modes. All patients received concomitant bètalactam therapy. The mean predicted mortalities were respectively 72%, 68%, and 69% (p = 0.91). SOFA scores at the onset of the treatment were also comparable (p = 0.87). Clinical response was favourable in all patients receiving colistin by inhalation (6/6) and in 40% (2/5) of the patients receiving colistin parenterally (p = 0.06). In the patients with colistin administered both via inhalation and parenterally, clinical response was favourable in 78% of the patients (7/9) (p = 0.27 as compared to the treatment group receiving colistin only parenterally). When all patients with inhalation therapy were compared to the group without inhalation therapy, a favorable clinical response was present in respectively 87% and 40% (p = 0.06). In none of the patients, the Pseudomonas spp. was eradicated from the follow-up cultures. All patients in the parenterally treated group died. None of the patients receiving colistin by inhalation, and 3 of 9 patients of the combination group eventually died (p = 0.002 and p = 0.03 respectively as compared to the group receiving colistin only parenterally). Conclusion: Aerosolized colistin could be beneficial as adjunctive treatment for the management of pneumonia associated with MDR P. aeruginosa. Objectives: Inappropriate use of antimicrobial drugs is associated with increased hospital expenditure, emergence of resistant bacteria and unnecessary side-effects. Concerning this, antibiotic stewardship is among the main concerns of the hospital infection control programmes. A "surgical antibiotic prophylaxis guide" was established in our hospital in 2008. The aim of this study was to investigate the point prevalence of antibiotic prophylaxis in surgery clinics and to evaluate the appropriateness of the antibiotic use.
The study was conducted in the Ankara Training & Research Hospital, a 670-bed tertiary care teaching hospital. The hospital contains 23 operation rooms and over 15.000 operations are performed within a year. On the 16th of December 2010 infectious diseases consultants went to the operation rooms of eight surgery clinics (Plastics & reconstructive surgery, general surgery, ophthalmology, ear-nose-throat, orthopaedics, urology, obstetrics & gynaecology (O&G) and neurosurgery) and investigate the prophylactic antibiotic regimen before the surgeries. The duration of the intended use of antibiotics after the surgeries was also included in the questionnaire. Results: Seventy-two of 196 hospitalized surgery patients were underwent different kind of surgeries. 50 of them (69.4%) were using prophylaxis. Cefazolin (45.8%), sulbactam-ampicillin (11.1%) and ceftriaxone (6.9%) were the most frequent antibiotics used for prophylaxis. In four patients (5.6%) combined antibiotics (metronidazole plus any antibiotics above) were used. 30.6% of the patients did not get any prophylaxis. The mean duration of the antibiotic use was 2.7 (1−22) days and the intended use of the antibiotic prophylaxis after the surgeries was 3.8 (1−10) days. 45 (62.5%) of the antibiotic prophylaxis regimens were inappropriate according to the guidelines. The reasons for the inappropriateness were as follows: wrong antibiotic (8.9%), wrong doses and duration (84.4%) and no antibiotics in spite of the need (6.7%).
The current study showed a high percentage of inappropriate surgical prophylaxis regimens in our hospital. The main problems about the inappropriate prophylactic antibiotic use were the dose and the duration (84.4%). The chosen antibiotic was 91.1% right. As conclusion we suggest that the hospital infection control programmes must include the follow up of the use of established guidelines, just introducing them seems to be not efficient.
Objectives: The first epidemic of tularemia in South-eastern Serbia happened in 1999. During a 10 years period, from 1999. till 2008, 151 patients were hospitalized and treated. The purpose of the study was to analyze and determine the best therapy choices in patients suffered from tularemia. Methods: Before hospitalization 16.5% were not treated, 58.8% were inadequately treated, 10.3% were adequately treated and 14.4% unknown. An average duration of disease before hospitalization was 35 days. Antibiotic monotherapy was administrated to 53 patients: gentamicin (27 patients), ciprofloxacin (11), amikacin (8), streptomycin (5) and doxycycline (2 patients). Combined (polyvalent or successive) antibiotic therapy was administrated to 98 patients: gentamicin and ciprofloxacin (66 patients), gentamicin and doxycycline (18), ciprofloxacin and doxycycline (2), gentamicin, ciprofloxacin and doxycycline (12 patients).
The biggest success in treatment (75% cured) was noticed in patients after combination of gentamicin, ciprofloxacin and doxycycline, then combination of gentamicin and ciprofloxacin (71%), then monotherapy of gentamicin (68%) and combination of gentamicin and doxycycline (67% cured). In comparison to number of treated patients, the biggest success in treatment (71%) had the combination of gentamicin and ciprofloxacin, then monotherapy of gentamicin (68%). Outcome of medicamentosus treatment: 92 patients (61%) of 151 were totally cured. The complications happened in 59 patients (39%): 1. abscessing of lymph nodes with or without fistulisation at 36 patients (23.8), 2. relapse − 12 patients (8%), 3. both complications at 11 patients (7.2%) (graphic 1). The relapses included recidivante lymph node enlargement or intake more than one lymph nodes. Final outome after conservative and radical treatment was total curing of 143 patients (94.7%) and residual persistant lymphadenopathy at 8 patients (5.3%). Conclusion: Reasons for appearance of complications during therapy of tularemic patients: 1. inadequate initial antibiotic treatment 2. late onset of adequate antibiotic therapy 3. possible appearence of resistant species of F. tularensis in the region of South-eastern Serbia. Early, forehand treatment restrains the colliquation of lymph nodes, recurrence of disease and dissemination of infection. Early diagnosis and treatment are crucial for prevention of complication and complete curing.
Objective: There is a variety of methods to improve antimicrobial appropriateness in hospitals. The aim of our study was to investigate the influence of an antimicrobial stewardship program on antibiotic use at the Hospital Sant Joan de Déu de Martorell.
The study was conducted in a 132 bed-hospital. Antibiotic consumption was monitored one year prior (2008)
A. Dinh*, J. Salomon, J. Bru, L. Bernard (Garches, Annecy, Tours, FR) Objective: For several years, antimicrobial resistance rates among bacteria have continued to rise, with limited therapeutic options due to the shortage of new antimicrobial agents. This situation has led to renewed interest in old antibiotics such as fosfomycin. We evaluate efficacy and safety of fosfomycin on resistant bacteria especially in critically ill patients. Method: Retrospective study evaluating the use and efficacy of fosfomycin as the only covering drug for 27 infections due to multi resistant bacteria. Parenteral fosfomycin combined with another antibiotic appears to be safe and effective treatment for severe infections caused by resistant bacteria sensitive only to this antibiotic. A further benefit is its low (~3%) and stable rate of resistance in countries in which it has been in use for a long time. This should be confirmed by further studies to substantiate this finding.
Objectives: To describe the real world clinical experience of the use of daptomycin (DAP) for the treatment of infective endocarditis in the UK. Methods: A retrospective non-interventional review of patients (pts) receiving DAP in 8 UK institutions participating in the European Cubicin ® Outcomes Registry and Experience (EU-CORESM) during the first 2.5 years of the registry. Efficacy was evaluated by the investigator at the end of DAP therapy as cured, improved, failure and non-evaluable. Data were collected on demographics, antibiotic usage, microbiological and clinical outcomes and adverse events from pts treated between January 2006 and August 2009. Patients (pts) were categorised by severity (complicated and uncomplicated) and the anatomical site of the primary infection. All pts included in the registry had received at least one dose of DAP. Results: 51 pts with a mean age of 61 yrs were treated. The majority of pts had significant underlying disease (86%). 55% had left-sided IE. Both sides were involved in 16%, 14% had right-sided and 14% foreign body involvement. Prior antibiotics had been received by 84% pts, with the reason for discontinuation of therapy being loss of susceptibility/ resistance in 46% pts. Cultures were obtained for the primary infection in 94% pts. Staphylococcus aureus was the most frequently isolated organism (31%), followed by and coagulase-negative staphylococci (14%) and Streptococcus spp (8%) Heart valves were replaced in 22% pts, but there was no surgical intervention in the majority of pts (72%). Mean duration of therapy was 21 days and 63% of pts received concomitant antibiotics. DAP 6 mg/kg 24h was the most frequently used dose. Clinical outcomes were success, defined as 'cure plus improved' (73%), failure (4%) and non-evaluable (23%). Adverse events were reported regardless of study drug relationship and were experienced by 29% of pts. The mean time to clinical improvement was 6 days. Therapy was stopped because of an adverse event in 12% pts. Conclusions: Despite the number of prior antibiotic failures and multiple comorbidites in these pts the overall clinical success rate in this UK population was 73%. There is emerging evidence that DAP is being used to treat infections caused by other Gram-positive species that are of increasing importance in IE, particularly where treatment options may be limited. The data from the current series adds to the body of evidence for the efficacy of DAP in left-sided IE. Subjective clinical improvement after NA was described by 12 patients (70.5%) and microbiological eradication of PA was confirmed in 7 patients (41.2%), though there was 1 relapse. NA did not result in clinical improvement in 2 patients, though it was well tolerated. Three patients did not tolerate any of the NA administered (at least COL and TOB). Table 1 shows the reduction in mean number of admissions/year and mean lenght of stay/year in days after NA onset for all patients. A statistically significant reduction is observed when PA is eradicated. On the whole, 8 patients presented side effects at any time during treatment (47%), mainly bronchospasm (60%). Side effects that prompted treatment interruption were observed during 15 NA courses: 6 with COL (40%), 8 with TOB (53.3%) and 1 with GEN.
1. NA resulted in clinical improvement in 70.5% of BQ patients and in eradication of PA in 41.2%. 2. NA reduced number of admissions and length of stay per year in all patients. A statistically significant reduction was observed in patients that achieved PA eradication, in whom NA prevented nearly 2 admissions per year. 3. Side effects were observed in 47% of patients and 17.6% did not tolerate any NA.
Increasing prescription of broad-spectrum antibiotics is regarded to facilitate selection of multiresistant germs. Surveillance of outpatient antibiotic use might contribute to the efforts made for preventing the spread of Methicillin-resistant S. aureus (MRSA) within regional networks. In the EURSAFETY HEALTH-NET, this issue has been addressed by offering training sessions on MRSA and antibiotic awareness to general practitioners in cooperation with the Association of Statutory Health Insurance Physicians Westphalia-Lippe (KVWL) which represents the German part of the EUREGIO and other regions in North Rhine-Westphalia. Furthermore, in 2006, a possibility for reimbursement of MRSA eradication therapy in outpatients has been created. We present data comparing the use of selected antibiotics in outpatient care of medical doctors in the EUREGIO to those in other KVWL regions. Regional data for outpatient prescription of antibiotics (based on Defined Daily Doses (DDD)) were collected by the KVWL and analyzed for the years 2002 to 2009. In order to compare the prescription of different antibiotics like fluoroquinolones or mupirocin in the EUREGIO to the whole KVWL region, we used the Cochrane Armitage Trend Test. Altogether, a total of 12.2 DDD/day per 1,000 inhabitants (DID) were prescribed in 2002, followed by 12.9 DID, 12.9 DID, 14. 4 DID, 13.8 DID, 14.4 DID, 14.7 DID and 14.8 DID from 2003 to 2009 , respectively. From 2002 to 2009 the percentage of prescriptions of all antibiotics and of fluorochinolones decreased significantly in EUREGIO relating to the whole KVWL region. In contrast, the number of mupirocin prescriptions increased significantly more in EUREGIO than in KVWL region. As desirable, the medical doctors in the EUREGIO region tends to more prudent antibiotic use. The increasing number of mupirocin prescription among outpatients reflects the growing demand and, facilitated by new refunding possibilities, the increasing implementation of MRSA eradication therapy in outpatient care. Regional trends which may reflect effects of interventions can be observed in routine data. Objectives: Chronic brucellosis (CB) and chronic fatigue syndrome (CFS) are characterized by a collection of non-specific symptoms and long-lasting fatigue. Of note, there are no objective clinical and diagnostic findings in both CB and CFS. A low-molecular-mass (37 kDa) isoform of RNAse L has been described in peripheral blood mononuclear cell (PBMC) extracts and the ratio of two isoforms of RNAse L (37 kDa/83 kDa) has been proposed as a potential biochemical marker of CFS (Tiev et al., 2003) . To our knowledge, the RNase L has never been analyzed in CB patients. The aim of this study was to determine the ratio of the 37kDa/83KDa isoforms in CB patients and compare the results with healthy wellmatched volunteers.
The CB group consisted of 9 patients (5 women and 4 men; mean standard deviation (SD) age: 49±9 years) that had been diagnosed with brucellosis between five and 35 years prior to the study. Two of the CB patients had focal disease (one spondylitis and one multifocal motor neuropathy). The remaining 7 subjects had non-specific symptoms such as fatigue, malaise, arthralgia and/or myalgia. Five of the 9 CB patients had been diagnosed of CFS. The control group consisted of 7 matched healthy volunteers (4 women and 3 men; mean standard deviation (SD) age: 31±3 years). PBMCs were isolated by densitygradient centrifugation. Target proteins were probed using a Western Blot procedure. Results: PBMC extracts from all subjects give rise to a major 83 kDa and a minor 37 kDa polypeptide band. The ratio of RNase L isoforms (37 kDa/83 kDa) of 0.4 used as a cut-off, allowed discrimination of CB patients from controls with high sensitivity (88.9%), specificity (85.7%), a positive prognostic value (88.9%) and a negative prognostic value (85.7%). The mean amount of the ratio of RNase L isoforms (37 kDa/83 kDa) in chronic brucellosis patients (0.84) was higher than in healthy subjects (0.22) (P < 0.05).
The chronic brucellosis patients are more likely to show a higher ratio of the two isoforms of RNAse L (37 kDa/83 kDa) than the healthy subjects. A high 37 kDa/83 kDa ratio of the RNase L could distinguish CB patients from healthy subjects.
Objectives: Recent publications suggest that 20−30% of surgical site infections are caused by S. aureus, and half of these arise from endogenous flora. This study evaluates rapid molecular identification of S. aureus nasal carriage from patients undergoing urgent surgery.
The study was carried out in Tel-Aviv Medical Center from 9.5-30.11.2010. Patients were selected from different surgical wards: neurosurgery, thoracic, orthopedic, vascular and plastic surgery. Nasal swabs from the patients were collected using a double swab; one swab was used for molecular testing on GeneXpert (Xpert MRSA/SA Nasal Assay − Cepheid) and the other was used for confirmatory culture on solid (chromagar MRSA II and 5% sheep blood) and liquid medium (brain heart Infusion broth). Results: 174 patients were sampled. 54 patients (31%) were found to be cariers of S. aureus by molecular testing: 49 (28%) MSSA, 5 (2.9%) MRSA. Compared to routine culture the sensitivity and specificity of the molecular testing were 94% and 95% respectively; the positive predictive value (ppv) and negative predictive value (npv) were 89% and 98%. 13 patients had invalid results on initial molecular testing. On repeated molecular testing (with the swab used initially for culture) only 3 had invalid results.
Xpert MRSA/SA Nasal Assay is capable of rapid and accurate detection of MSSA and MRSA nasal colonization. Further studies are needed in order to reduce invalid results. The CSF sample was negative for both serology and the PCRQ assay. Three months after completion of the treatment, the patient resumed it for 6 months, because he considered that his clinical improvement was associated with antimicrobial therapy. During the second treatment period 10 samples (5 blood, 5 serum) were analyzed by PCRQ. Only one serum sample was positive with 1125copies/ml. Blood samples were negative. In the last year post-treatment follow-up, we analyzed 12 samples (6 blood, 6 serum) which were all negative by PCRQ. Conventional serology showed no detectable antibody titers and Brucellacapt test showed a titer of 1/40. Nowadays, the patient remains with severe loss of strength in his left hand and right foot and continues receving doses of intravenous IgG monthly. Conclusion: This may be the first report of CB with MMN as neurological complication described, thanks to the high sensitivity and specificity of a PCRQ assay.
Introduction: Pseudomonas aeruginosa is an opportunistic bacterium that has high antibiotic resistance. Immunoprophylaxy and immunotherapy may be considered as desirable ways for control and treatment of Pseudomonas aeruginosa infections. Flagellin is a main virulence factor. There is high homology and cross reaction among flagellin of Pseudomonas aeruginosa strains. N-terminal domain of flagellin plays an important role in attachment to TLR5. It also may play an important role in the induction of protective immune responses. This study was aimed to produce rN-terminal flagellin(1−161). In future, its immunological effect would be evaluated on animal and compared with native flagellin. Materials and Methods: The coding sequence of flagellin N-terminal of Pseudomonas aeruginosa 8821M was isolated by PCR and cloned into pET28a vector. E. coli BL21 (DE3) strain was used as an expression host. The recombinant protein was purified by Ni-Sepharose resin. The Objectives: Chlamydophila pneumoniae and Mycoplasma pneumoniae are important and common causes of community-acquired pneumoniae. The highest incidence of C. pneumoniae and M. pneumoniae infections is among schoolchildren 5 to 14 years old. Symptoms may be mild, nonproductive persistent cough, malaise, and fever, but more severe illness occurs when the lower respiratory tracts is affected, given rise to acute bronchitis and pneumoniae. The agents causing respiratory infections are difficult to distinguish clinically, since many bacterial and viral infection share clinical features, including symptoms. It is therefore important to find a sensitive and effective way to identify these agents and propose the appropriate antibiotic therapy. Currently culture and serological confirmation of the diagnosis of C. pneumoniae and M. pneumoniae are difficult and time-consuming. Real time PCR, sensitive specific and rapid technology, is an effective alternative. We propose a new real-time PCR based diagnsotic tool for C. pneumoniae and M. pneumoniae diagnosis. Methods: Nucleic acids were extracted from nasopharyngeal specimens by using easyMag (bioMérieux) or MagNA Pure Compact (Roche) extraction systems. Purified nucleic acids were added to the ready-touse Chla/Myco pneumo r-gene™ amplification premix. C. pneumoniae and M. pneumoniae were distinguished in a duplex single reaction tube. Amplification was performed on ABI7500 Fast, Bio-Rad CFX96 or Roche LC480 platforms. Results: On QCMD European Proficiency Panel 2010, the 12 positive/negative samples were correctly identified with Chla/Myco pneumo r-gene™. All positives were detected, including weak positives (0,049IFU/100 mL for C. pneumoniae and 50CCU/100 mL for M. pneumoniae). Analytical Sensitivity study on C. pneumoniae and M. pneumoniae samples was performed in respiratory specimens. Specificity study showed no cross reaction with other respiratory bacteria or viruses.
The high quality associated with its compatibility with the major extraction and real time PCR platforms allows an immediate integration of Chla/Myco pneumo r-gene™ in most routine diagnostic laboratories.
G. Vrioni*, I. Daniil, V. Mamali, M. Kimouli, D. Mylona-Petropoulou, K. Themeli-Digalaki, A.
Objectives: Sepsis is a serious medical condition that requires rapidly administered, appropriate antibiotic treatment. The rapid detection of pathogens in blood is critical for a favourable outcome of patients with suspected sepsis. Although blood culture (BC) is considered the criterion standard for diagnosis of bloodstream infection, it take three or more days for final pathogen identification and antimicrobial susceptibility testing. In this observational study, the clinical impact of a commercially available multiplex PCR system in ICU patients with suspected sepsis was been analysed. Methods: Blood samples from patients with presumed sepsis were cultured with the Bactec 9240 system (Becton Dickinson, Heidelberg, Germany) and blood in EDTA from the same patients subjected to analysis with the LightCycler SeptiFast M(grade) Test (LC-SF; Roche Diagnostics, Mannheim, Germany) at two tertiary care centres. LC-SF test is a multiplex, real-time PCR system allowing detection of 16 pathogens at the species level and four groups of pathogens at the genus level (Gram-positive, Gram-negative and fungal microorganisms). For samples with PCR-detected pathogens, the actual impact on clinical management was determined by chart review. Furthermore a comparison between the time to a positive blood culture result and the LC-SF result was made. Results: From 33 patients, 24 (73%) yielded concordant negative and 7 (21%) concordant positive results. In one patient two more pathogens were detected with molecular method (E. coli in blood cultures vs E. coli/S. maltophilia/C. albicans in LC-SF assay) and one patient was LC-SF positive only (negative blood cultures vs P. aeruginosa in LC-SF assay). LC-SF results were obtained in 7−15 h, in contrast to the 24−72 h required for blood culture. According to the LC-SF results, initial therapy was inadequate in five patients, and antibiotic treatment was changed.
Conclusion: This rapid, multiplex pathogen detection LC-SF system complemented traditional culture-based methods and offered some added diagnostic value for the timely detection of causative pathogens having a relevant impact on clinical management for a subset of patients with clinically suspected sepsis.
N. Brankova*, V. Levterova, S. Panaiotov, K. Tankova, T. Kantardjiev (Sofia, BG) Bordetella pertussis, the causative agent of whooping cough is endemic in Bulgaria despite extensive nationwide vaccination since 1950s.
Objectives: Goal of this study was to investigate the incidence of pertussis infection among children and adults in the capital city of Sofia, Bulgaria for a seven years period. Methods: Since seven years the National reference laboratory in molecular microbiology started molecular diagnosis of pertussis. As target PCR marker we selected pertussis toxin gene. 162 bp amplified fragment was detected on agarose gels. DNA was extracted from nasopharyngeal swab samples by automated robot system. A total of 2227 samples were analyzed. Samples from patients clinically suspected for pertussis were analyzed. Results: A total of 2227 samples were analyzed for pertussis by PCR. Of these 496 samples were positive and 1731 negative for pertussis. Positive samples represent 22.3%. The capital city of Sofia has about 1.3 million inhabitants. The incidence of pertussis illness is estimated to be 38.1 patients per 100.000 inhabitants.
Conclusions: Children in Sofia have high immunization coverage. Acellular vaccination is applied since April 2010. Whole cellular vaccine was previously applied. Highest prevalence of pertussis incidence is in group age 0−3 years. In 167 GDH positive and culture positive stool specimens C. difficile was confirmed in 161 cases by PCR (sensitivity 96%). Eight GDH positive stool specimens remained negative when cultured. 152 samples were congruent positive by PCR and tox EIA (culture + direct testing), 17 were congruent negative and 4 only positive by the EIA. Two samples were excluded (sensitivity toxin-PCR 97%, specificity 100%, PPV 100%, NPV 81%). The GenoType ® Cdiff assay was able to identify ribotype O27 in 9 specimens and in 2 specimens 078/126.
The GenoType ® Cdiff assay for the direct detection of Clostridium difficile and major ribotypes from stool shows rapid, sensitive and specific results. The DNA isolation is fully automated. The turnaround time (including hands on time) is approximately 1.5 hours for DNA isolation, 2 hours for amplification and 2 hours for hybridization. The assay provides more information (ribotypes, toxins, binary toxins and Moxifloxacin resistance) as any presently available commercial CD test. Objectives: Implementation of rapid molecular diagnostics in bloodstream infections could significantly improve speed of diagnosis, and thereby outcome. Current molecular tests directly on whole blood samples have suboptimal sensitivity, due to the use of small volumes. Larger volumes show an inhibitory effect of human DNA. The Sepsitest™-assay (Molzym, Germany) incorporates a pre-test enrichment method, which selectively eliminates human cells. This may lead to an increase in input volume and subsequent diagnostic sensitivity. We investigated the use of this assay in a cohort of patient with sepsis on the ICU.
We analysed 55 septicaemic patients on the ICU in whom blood cultures (BC) were taken. Together with the BC (n = 90), an additional blood sample (EDTA) was taken from the same sample site, for analysis with the Sepsitest™-assay (ST). The assay consists of a pre-test-enrichment on 1 ml EDTA-sample, followed by bacterial lysis and DNA-isolation, and subsequent amplification using 16Sbased universal primers for Gram-negative and Gram-positive bacteria.
Amplicons are detected by agar-based gel-electrophoresis. In accordance with instructions of the manufacturer, EDTA-samples were analysed in duplicate, and a sample was considered positive if at least one of the duplicates was positive. Results: Of the 90 BC, 5 (6%) yielded positive results; S. aureus (n = 1), E. faecalis (n = 3), and CNS with E. faecalis (n = 1). Bacteraemia was diagnosed in 3/50 (6%) of the patients. ST was positive in 11/90 (12%) of the samples. Compared to BC, sensitivity was 80% (4/5 positive BC). In total 2/3 patients (66%) with positive BC was positive with ST. Seven patients had positive ST and negative BC. In all of these patients, the clinical suspicion of bacterial infection was high, and 3 of them showed positive BC during septicemia, but at another time point when no EDTA-samples had been taken for this study. These results might therefore represent additional yield of the Sepsitest™-assay. Sequencing of amplicons is currently being performed, to provide species-specific results and control for false-positive results.
-The Sepsitest™-assay can be used in clinical practice for molecular analysis directly on blood samples. -Implementation of the assay may provide additional detection of bacteraemic patients, but results have to be evaluated with sequence results. -Gel-based analysis is applicable, but may be troublesome to implement in the routine workflow. Background and Objectives: Respiratory Infections cause significant morbidity and mortality in both developed and developing countries. Influenza A and B (Flu A&B), RNA viruses of the family Orthomyxoviridae, spreads in regular epidemics resulting in the deaths of more than 250,000 people worldwide annually. Human respiratory syncytial virus (RSV) is a negative single-stranded RNA virus of the family Paramyxoviridae. RSV is the major cause of lower respiratory tract infection and hospital visits during infancy and childhood. Human metapneumovirus (hMPV) is a negative single-stranded RNA virus of the family Paramyxoviridae, and may be the second most common cause (after RSV) of lower respiratory infection in young children. Utilizing proprietary chemistries and processes, we have developed novel, room temperature stable reagents for use in our AmpliVue Taqman ® -based, multiplexed, rt-PCR assays for the detection of Flu A&B or hMPV/RSV. As an added benefit, the AmpliVue™ assays provide a simplified workflow, significantly reducing the number of end-user manipulations required to perform testing. These attributes allow for more widespread and reliable use of molecular diagnostics in the detection of respiratory viruses. In this report, we describe the initial studies performed with these reagents on both the Applied Biosystems ® 7500 FastDx and the Cepheid ® SmartCycler. Methods and Results: Testing was performed on cultured isolates or clinical specimens to establish initial performance of the assays. RNA was extracted on either a NucliSENS ® easyMag ® or Roche MagNA Pure Compact and 5 ul of each sample was added to reconstituted master mix. Each cultured influenza A and B isolate was detected; 19/19 and 14/14, respectively. Clinical specimens analyzed for the presence of either RSV A, RSV B or hMPV were able to detect 10/10 RSV A, 13/13 RSV B, and 26/26 hMPV. Specificity was 100% for all samples evaluated. Initial analytical sensitivity tests for the various viruses indicated detection limits less than 50 TCID50/ml and/or 10vp/mL for each target. Testing with isolates of other common viruses and bacteria confirmed that these reagents are not cross reactive with other common respiratory pathogens.
Results from these studies indicate that our room temperature stable reagents, coupled with the simplified workflow for our AmpliVue molecular assays, provides end-users with sensitive and specific assays for the detection of Flu A&B and hMPV/RSV. and adults (n = 122), immunocompetent (n = 40) and immunosupressed (n = 98), hospitalized with clinical suspicion of CMV infection, in the University Hospital of Ioannina, Greece, were screened for CMV-IgM antibody (AxSYM, Abbott) and CMV-DNA (COBAS AMPLICOR, Roche). Viral load and clinical data was also investigated.
The confirmation of CMV infection by the two methods was obtained in 14 patients. One hundred four patients were positive for CMV-IgM and negative for CMV-DNA. Antibodies for HSV, VZV or EBV were also detected in some of them. By performing PCR, 20 extra cases of active infection were diagnosed, for which no antibodies could be detected. This translates to a 15% rise in the number of diagnoses of active CMV infection as compared with serological approach. The CMV-DNA positive patients were 6 children and 28 adults. The major underlying disease was haematological malignancies (14/34). Pneumonitis was the most common clinical presentation (20/34). Overall, the median viral load was 2255 copies/ml (472-58700), while in them with CMV mononucleosis was 583 copies/ml (472-874). The overall mortality rate was 12% and major cause was respiratory failure. Eighteen of the 34 PCR positive patients received ganciclovir. Treatment led to a marked decrease in CMV DNA copy number. The median time interval necessary to obtain a negative result after implementation of treatment by PCR was 27 days. Conclusion: Quantative PCR CMV assay is rapid, and linear for quantifying CMV viral load, and it seems to be useful in the diagnosis and management of affected patients for predicting disease and monitoring response to antiviral therapy and can serve as surrogate markers for antiviral resistance and clinical relapse in these patients. Compared with traditional serologic assays that detect antibodies to CMV, Q-PCR offers a significant advance through the direct detection of viral DNA, which is independent of a functioning humoral immune system. Amplified DNA Assays were designed to be compatible with transport via the Swab Diluent for the BD ProbeTec CT/GC Q x Amplified DNA Assays (Q x Swab Diluent) and BD UVT or Copan UTM-RT collection devices. This study focused on evaluating performance of the HSV assays using simulated specimen comprising 0.5mL BD UVT medium added to a pre-filled Q x Swab Diluent Tube. Samples were pre-warmed, and then loaded directly onto the BD Viper System for DNA extraction and amplification.
The analytical limits of detection (95% proportion positive) for the HSV1 Q x and HSV2 Q x assays using clean BD UVT medium were estimated to be 24 viral particles (vp)/mL and 88 vp/mL, respectively. The limits of detection for the assays in the presence of external anogenital swab specimen matrix were estimated to be 17 vp/mL and 127 vp/mL for HSV1 Q x and HSV2 Q x , respectively. Both assays were shown to be tolerant to common exogenous and endogenous substances that may be present in external anogenital lesion specimens, including but not limited to blood, mucus, semen, leukocytes and various prescription and over-the-counter medications. In addition, both assays were found not to cross-react with a variety of bacteria, viruses and fungi that could be found in external anogenital lesion specimens. Stability of HSV DNA in simulated external anogenital swab specimens was demonstrated at ambient, refrigerated and frozen temperatures in both the original UVT specimen and once diluted in the Q x Swab Diluent.
The BD ProbeTec Herpes Simplex Viruses (HSV1 & 2) Q x Amplified DNA Assays* offer excellent analytical sensitivity and robust performance for the detection of HSV1 and HSV2 DNA from external anogenital swabs expressed into BD UVT medium. The BD Viper System software allows the user to test both CT/GC specimens and HSV1/HSV2 specimens within the same Viper run, providing workflow flexibility. *Product not for sale, for investigational use only in the US. Background: HCV genotyping is used to predict the response to antiviral therapy and also to optimize the duration of treatment. The 5-untranslated region (5-UTR) is the region of choice for routine genotyping of HCV. However, due to its high level of conservation, the 5-UTR is limited in its ability to discriminate genotype 6 from genotype 1 and subtypes within genotypes 1, 2, 3, 4, and 6. The newly developed Versant HCV genotype assay (LiPA) 2.0 (Versant 2.0) uses sequence information from both the 5-UTR and the core region, allowing distinction between HCV genotype 1 and 6 and between subtypes a and b of genotype 1. Previously, Versant HCV genotype assay (LiPA) 1.0 (Versant 1.0) only used sequence information of 5-UTR. In this study, the results of both genotyping assays were evaluated. Methods: A total of 556 HCV-positive samples (EDTA plasma) were genotyped using the Versant 2.0 according to the manufacturer's instructions. For the comparison study, Versant 1.0 was used. HCV RNA was extracted by using the NucliSENS ® easyMAG™ (BioMèrieux). In each extraction run, positive and negative controls were included. Only interpretable results by both assays were included in the study. Only differences found at the genotype 1 and subtypes 1a and 1b level were taken into account. The Versant 2.0 was considered the reference method. Results: Table 1 gives an overview of the results of the comparison study after testing by both assays. The correlation rate of both tests was 85.8% (477/556). Results from 79 specimens (16.2%) were discordant between the two assays, being the genotype 1a the most difficult to discriminate by the Versant 1.0 (40 samples). Discussion: Versant 2.0 showed an improvement in identifying the correct subtype of genotype 1. This improvement can be attributed to the additional information available from the core region of the HCV genome. As the clinical management of patients infected by genotype 1a and 1b is equal, disagreements among both tests may present epidemiological consequences but at least Versant 1.0 errors do not affect treatment dosage and duration.
The high prevalence observed in women aged 14−17 years is probably due to both the increased susceptibility of this population and an early sexual debut, major risk factor for the acquisition of HPV infection. A characteristic ascending age-specific curve of infection prevalence was observed in the other 3 groups. These data support the existence of an inverse relationship between age and HPV infection prevalence. In addition, vaccination of this whole population would have prevented about 6% of occurred infections.
Objective: The recent introduction of vaccination against HPV in adolescent girls in Italy has focused the attention on the virological surveillance in this age group. Since HPV is one of the major pathogen sexually transmitted worldwide, it will be important to extend the surveillance also to the adolescent males, who could be the next target of the immunization strategy. This study aimed at evaluating the HPV infection prevalence in adolescents both females and males. On purpose, a molecular assay based on urine samples, easy to collect and acceptable for this young individuals, was applied for the detection and genotyping of HPV-DNA. Materials and Methods: 545 urine samples were collected from adolescents (14−17 years) attending community clinics, youth centres of the local sanitary units or day clinics in Northern Italy in the period spanning from September 2009 to July 2010. Analysed subjects were 233 females (mean age 15.3 years) and 312 males (mean age 15.4 years). Samples were centrifuged at 3500xg for 20 to obtain the cell pellet. DNA extraction was carried out using a commercial kit (NucliSENS ® miniMAG ® , BioMérieux, France) and the amplification of a L1 gene fragment (450bp) was performed by degenerated primers. Genotyping was performed by restriction fragment length polymorphism technique using 3 restriction enzymes (RsaI, HaeIII, DdeI, Recombinant Enzyme, BioLabs Inc, New England). Results: HPV-DNA was detected in 2% (11/545) of analysed samples. In particular, in 3.9% (9/233) and in 0.6% (2/312) of samples collected from female and male adolescents, respectively. Both high and lowrisk genotypes were identified: high-risk HPV-16 (11.1%) and HPV-66 (11.1%), low-risk HPV-70 (33.3%), HPV-6 (22.2%), HPV-11 (22.2%) and HPV-87 (11.1%). Among female adolescents, 8 infections were due to a single genotype and 1 was sustained by HPV-16/HPV-70 coinfection. The two infections found in male subjects were sustained by HPV-70. Conclusions: These data show that HPV infection is present in 14−17 years old subjects, more frequently in females than in males. These infections were supported both by high-and/or low-risk genotypes and, among these, 45% (5/11) could now be preventable by the available vaccines that include one or two types found (HPV-6 and HPV-16). Molecular testing on urine sample seems to be a good alternative to those conducted on cervical swab, especially for very young women. Finally, this method seems to be applicable also to the male population. The aim of this study was the evaluation of the often underestimated potential role of herpes viruses in the onset or inauspicious evolution of respiratory pathologies in critical patients. We analyzed 158 bronchoalveolar washes from patients hospitalized with severe acute respiratory pathologies at the intensive care units of some hospitals in Catania, Sicily, Italy.
For the retrospective study we used viral isolation methods and Real-Time PCR, respectively, for viral replication activity and the clinical significance expressed in terms of viral load, correlated with the days the patients were on assisted ventilation until the time of the analysis. In 57.6% (91/158) of the samples DNA was found of at least one, though often two or more, of the herpes viruses (HSV1, VZV, CMV, EBV, HHV7). In particular: 19% (30/158) were HSV1, 10.7% (17/158) CMV, 16% EBV (26/158) and 46% (68/158) HHV7; there was no positivity for VZV. Based on the data relative to the finding of viral nucleic acid and the duration of mechanical ventilation, a statistically significant association was found only for HSV1 DNA with assisted ventilation of more than 7 days (p < 0.05). The increase of the viral load, in some cases, was directly proportional to the days on assisted ventilation reaching a value of 108 gEq/ml for HSV1, compared to 102-104 gEq/ml for CMV and EBV.
The prevalence of herpes viruses, and above all the finding of HSV1, accompanied by a substantial viral load, would confirm the importance that these viruses could have in the onset of respiratory pathologies in immunocompromised subjects. Therefore, the introduction of tests for the detection of the above mentioned viruses in diagnostic protocols would favor early diagnosis and correct therapy that could reduce the rate of mortality in critical long-term patients affected by respiratory pathologies who need assisted ventilation. Objectives: Acute children respiratory infections (ACRI) are a common reason for consulting general practitioners. In most cases the aetiology is unknown, yet most result in an antibiotic prescription. The objective was to study the epidemiology of respiratory virus infections and in particular to determine the virus-specific positivity rates and seasonality in pediatric age for respiratory virus infections over a 15 month period in Pordenone (ITALY), using a multiplex real-time PCR assay to detect multiple viruses in the same reaction. Methods: A total of 195 nasopharyngeal specimens were collected from symptomatic pediatric inpatients (average 3 years) between November 2008 and January 2010. Each specimen was split into two aliquots, one aliquot was processed by using multiplex real-time PCR test and the second was tested for adenovirus using a nested PCR. Total nucleic acid was extracted using the BioMerieux easyMAG. Multiplex real-time PCR test was performed using Dia-ResRNAVir-050 (DIAGENODE) for detection of Influenza A virus (IA) and Influenza B virus (IB), Respiratory Syncytial Virus (RSV), Metapneumovirus (MPV), Rhinovirus (RVs) and Parainfluenza virus (PIV) 1, 2, 3 and 4. Adenovirus was detected by nested PCR (NANOGEN). Results: Of the 195 specimens tested, 158 (81.02%) were positive for at least one respiratory virus including: 64 (32,8%) RSV (A or B), 34 (21,4%) Rhinovirus, 23 (11,8%) Flu A or B, 15 (7.8%) Parainfluenza (1−4), 11 (5,6%) Adenovirus and 11/185 (5,9%) Metapneumovirus. Most pediatric patients (86,6%) was pre-school age. All patients RSV and MPV (38,5%) positive were less than 4 years of age. We have seen a dual respiratory virus infection in 11/195 (5,6%) patients and only one triple virus infection. In terms of seasonal distribution, RVs were distributed across the majority of months. RSV peaks were between December 2008 and March 2009. IA infections were distributed from January and February 2009, with another "atypical" peak between October and December 2009, including Influenza A/H1N1v. IB showed a peach activity in March and April. PIVs peaked in November 2008 and October 2009. MPV peaked between the end of winter and spring months. Adenovirus infections were distributed in winter and spring months. Conclusion: The molecular assay has increased our understanding of the epidemiology of respiratory viral infections and should assist us in the diagnosing the etiology of respiratory tract infections in individual and in outbreak situation. (3,3%) and the remaining 37 were negative (40.7%). All the samples were negative for H3N2 and influenza B. Overall, 54 out of 91 samples (59,3%) were positive for any of the viruses tested. In addition, two different patients were coinfected by A flu -rhinovirus and enterovirusrhinovirus. These coinfections represented 3,7% of the overall positive samples. The average age and range of the infected patients were as follows: for H1N1, 33 years (SD±20.1) and 0−76 years, respectively; for rhinovirus, 28 years (SD±23.7) and 0−69 years respectively; and for enterovirus, 25.7 years (SD±22.4) and 0−42 years, respectively. Conclusions: Our results showed the involvement of other respiratory viruses in patients with influenza-like illness. Thus, a notable proportion of rhinovirus infection was found in a collection of patients diagnosed with influenza during the peak incidence of H1N1 pandemic. These data would suggest a potential indication for rhinovirus detection in acute respiratory diseases. Regarding age and gender distribution of the infected patients, no significant differences were found for all the viruses tested. Results: Eight (n = 8) of the women were excluded from the study because of the incomplete data and a total of 152 women were used for the final analysis. Forty-five patients (29.6%) were found to be infected with a genital HPV. As expected, viral prevalence was higher among women younger than 30 years of age (15.8%) in comparison to those aged 30 or older (13.8%). The rate of o HPV types were as follows: 16 (37.8%), 18 (8.9%) i other (53.3%). Coinfection with multiple HPV types had one woman. The largest number of positive samples was from Herzegovina-Neretva Canton. Conclusion: Our results indicated that HPV infections represent a significant public health concern in Herzegovina. Detailed knowledge of HPV type circulating patterns in specific local geographical areas is essential for appropriate implementation of screening, prevention, and surveillance campaigns.
G. Gioula*, E. Chatzopoulou, M. Exindari, A. Melidou, D. Chatzidimitriou, F. Chatzopoulou, N. Malisiovas (Thessaloniki, GR) Objectives: Influenza viruses, respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are the most common pathogens that cause acute respiratory disease in children. The aim of this study is to present the contribution of the above three pathogens to influenza-like illness (ILI) in children (aged <6 years old) during 2-year (2008-2010) influenza seasons in N. Greece. Materials and Methods: 430 pharyngeal swabs from children younger than 6 years, presented as ILI infections during the last two influenza seasons (2008-2009 and 2009-2010) were examined for influenza A and B, RSV and hMPV, by one step Real-time RT-PCR.
Results: Influenza viruses were detected in 122 (28,3%) of the 430 spesimens, RSV in 45 (10,4%) samples and hMPV in 28 (6,5%). RSV and influenza viruses' co-infections were observed in eight cases, RSV and hMPV co-infections in four cases and hMPV with influenza viruses was found in one case. The majority of the patients (67,7%) were between 3 and 6 years old. Conclusion: Our results demonstrate that influenza viruses, RSV and hMPV contribute to ILI presenting infections at a rate of 45,2% in children younger than 6 years old. Objective: To identify Candida species causing candidaemia by analyzing fungal DNA from blood culture bottles positive for yeasts using a low cost-effectiveness assay. Methods: For DNA extraction an "in house" protocol based on organic solvent extraction was tested. Additional steps of liquid nitrogen incubation followed by mechanical disruption were processed for complete cells lysis. The purity of extracted DNA was evaluated. Fifty blood culture bottles positive for yeasts were processed. PCR assays amplified ITS region of rDNA gene. The amplicons of twenty samples were sequenced and these sequences were submitted for comparison on Genbank database (NCBI) for species identification. Molecular yeast identification was compared to results provided by conventional methods.
The organic solvent extraction protocol showed good reproducibility on the amount of DNA extracted, pure DNA and high PCR sensitivity (10 pg of C. albicans DNA and 90% amplification on PCR assay). Therefore, this method was used to analyze all clinical samples available. The molecular species identification showed 100% concordance to the conventional culture. However, the molecular method could identify one sample with mixed infection caused by C. albicans and C. glabrata, while the conventional culture identified just C. albicans. Moreover, 3 samples identified as C. parapsilosis by the classical method were molecularly identified as C. orthopsilosis, a species belonging to the C. parapsilosis complex. Conclusions: Organic DNA extraction, PCR assay and sequencing could efficiently identify mixed infections, as well as Candida species that only can be molecularly identified. This test can be easily implemented in routine laboratories providing earlier and low cost species identification when compared to the traditional methods. The organic DNA extraction was 4-fold less expensive than protocols using commercial kits. Objectives: Clostridium difficile infections represent significant burden for health care system with small and large outbreaks constantly being present in hospital environment. Some of the PCR ribotypes were lately associated with outbreaks and with increased mortality and morbidity: 027, 078, 017 and 053. It is therefore important to recognize these ribotypes as rapid as possible. Standard typing method in Europe is culturing of C. difficile from faecal sample and subsequent PCR ribotyping. Here we describe for the first time the modification of PCR ribotyping that can be used for direct detection in stool samples and its use during emergence of PCR ribotype 027 in a single hospital. Methods: For direct PCR ribotyping from stool sample we have modified existing primers described by Bidet et al. (1999) to increase specificity for C. difficile. A total of 50 C. difficile culture positive and 43 negative stool samples from the routine laboratory were then used for validation of the method. DNA was isolated from faecal sample and PCR ribotyping was performed with Bidet and with new primers. Additionally, five stool samples from general hospital, which were detected as "C. difficile positive, presumptive 027/NAP1" by Cepheid Xpert C. difficile assay were submitted to the reference laboratory for ribotype 027 confirmations. Results: Direct PCR ribtyping from stool samples was possible in 37 out of 50 C. difficile positive samples using new primers. Other 13 samples were negative (n = 9) or the banding pattern was too weak to be analyzed (n = 4). In 36 out of 37 cases PCR ribotype determined directly from the stool was identical as PCR ribotype of the strain isolated from the same stool sample (sensitivity 0.72−36 out of 50 positive samples). All, but one C. difficile culture negative samples were negative on direct PCR ribotyping with modified primers. In contrast, 30 out of 43 C. difficile negative samples reacted with Bidet primers. All five Cepheid Xpert "C. difficile positive presumptive 027/NAP1" samples were confirmed as ribotype 027 by direct ribotyping as well as conventional ribotyping of the cultured isolate.
The direct culture-independent PCR ribotyping of C. difficile is a useful and rapid method for detection of emerging strains and outbreak identification. Results: Eleven studies were identified comparing molecular assays for MRSA over traditional culture and/or chromogenic agars for a total of 22757 patients. 4/6 cost-effectiveness studies found that PCR incurs higher costs but better outcomes while 1/6 found multiplex PCR cost effective among 31 methods when length of ICU stay is <2 days. 1/6 found PCR not cost effective. 3/4 cost-benefit studies found PCR for MRSA cost saving, and 1/4 found PCR not cost saving in an existing search and destroy policy. One cost utility analysis reported that PCR leads to important mortality reduction at a low cost/LYS, due to an under the hour change in empirical antibiotics. Factors that determine cost effectiveness in various settings are the baseline prevalence rate of MRSA, applying screening and pre-emptive isolation to high-risk patients, the availability and cost of isolation and last but not least, the assay characteristics and individual test costs.
The use of PCR instead of culture screening for MRSA is found to be cost effective and/or cost-saving particularly in surgery. The significant benefits are less isolation, less overall hospital stay, more infections avoided, timely and cheaper treatment choices and lower mortality. Although more studies are urgently needed, especially in severe infections diagnosis, infection control teams for MRSA in hospitals can be preliminarily informed about the particulars of the cost effectiveness of molecular assays over traditional techniques according to each hospital's settings.
Objectives: The aim of the study was the evaluation of genomic diversity differences among B. pertussis strains collected from patients before and after the 90-ties in respect to vaccine strains used for production of the DTP vaccine. Objectives: Legionellosis occurs through inhalation of contaminated aerosols expelled by wet cooling systems or hot water distribution systems of healthcare facilities. Despite the prolonged incubation period, culture remains the standard diagnostic method. Alternative methods like antigen detection and molecular techniques are expensive, often unavailable and give no information regarding Legionella pneumophila viability, which can be of minor importance in clinical specimens but of major impact in water samples and therefore in public health security. Considering its high mortality rate and the need of specific treatment, the development of a rapid, sensitive and specific method for L. pneumophila detection is crucial. Also, distinction between viable and non-viable cells in water is warranted. Therefore, we propose a flow cytometry protocol for L. pneumophila detection in clinical and water samples and viability evaluation in the latter. Methods: L. pneumophila, ATCC33155, was used for protocol optimization. Fifty respiratory samples and twenty water samples previously analysed by immunofluorescence were screened by flow cytometry. All samples were stained with a specific antibody MONOFLUO™ Anti-L. pneumophila (BioRad, California) (green-530nm), which reacts with an external membrane protein found in all known L. pneumophila serogroups, and with Propidium iodide (red-FL3) to evaluate bacteria viability in positive water samples, where the analysis was made before and after killing bacteria by heat. Flow cytometry analysis was performed on FACSCalibur Cytometer (BD Biosciences, Sydney). Specificity was studied by mixing L. pneumophila with suspensions of E. coli, S. aureus and C. albicans type ATCC strains, according to the same protocol. Results: L. pneumophila cells displayed an intense green fluorescence, indicating bacteria recognition by specific antibody. Dead cells showed high intensity of fluorescence in both channels, FL1 and FL3. A 100% correlation was achieved between immunofluorescence and flow cytometry assay, in both clinical and water samples. Viability assessment was successful. Conclusion: Flow cytometry proved to be a sensitive and a specific method to detect L. pneumophila and simultaneously to assess its viability. Furthermore, while it has been used in the detection of Legionella in environmental specimens and experimental systems, this is the first time that this technique is applied to detect L. pneumophila in clinical specimens.
Objective: Comparing results of a commercially available selective culture system for isolation of Mycoplasma hominis and Ureaplasma urealyticum with their detection using PCR amplification. Materials and Methods: Two endo-Cervical swabs have been collected from each case of a cohort of 40 infertile females attending fertility centers for management of infertility (group A), and from a control group of 40 multiparus females attending contraception clinic (group B). One swab have been subjected to culture for isolation of urogenital mycoplasmas using a commercially available selective culture system (Bio-Rad, USA) and the other sample was eluted in a buffer for PCR amplification using specific primers for Mycoplasma hominis and Ureaplasma urealyticum.
Results: Using PCR, 19 of group A and 9 of group B were positive for Mycoplasma hominis while 12 of group A and 8 of group B were positive for Ureaplasma urealyticum. According to Duo kit results, 5 cases of groupA and one of control group B were positive for Mycoplasma hominis while 10 and 8 cases were positive to Ureaplasma urealyticum in group A and B respectively. The sensitivity of Duo kit culture system in relation to PCR was found to be 89.47% and the specificity was 90.48. Conclusion: Our study confirm the reliability of Mycoplasma Duo kit culture system for diagnosis of genitourinary colonization of the female genital system with Mycoplasma hominis or Ureaplasma urealyticum. Also we report a higher sensitivity than the PCR.
C. Kalunga* (Lusaka, ZM)
Introduction: The NTP has been conducting quality assurance through the reference laboratory for the past four years to determine quality improvement in Tb control. Quality improvement is a process by which the components of smear microscopy diagnostic services are analyzed with the aim of looking for ways to permanently remove obstacles to success. Direct sputum smear microscopy remains the most cost effective tool for diagnosing patients with infectious tuberculosis and for monitoring their progress on treatment. The case detection rate for Zambia is 58% and falls short of the target of detecting 70% of all infectious cases of TB. (15), Eastern (4), Northern (3) Southern (4),UTH (7) Central (0) and Western (5), Luapula (2) North-Western (2) Methods: A total of 25 serum samples were included from negative controls, acute Q fever patients, and a serial diluted high positive sample. Thirteen laboratories participated: eight from the Dutch Q fever endemic area, two Dutch national laboratories and three reference laboratories from outside The Netherlands. Six labs performed CFA, 9 performed IFA, of which three were in-house assays, and 5 performed ELISA.
Results: IFA, ELISA and CFA values between laboratories using the same methods were within close range and all three methods correctly identified the Q fever patients. In house and commercial IFAs were well comparable quantitatively. In titres reached some differences could be observed. All tests were reasonably specific (92-100%), however the sensitivity showed more variation. IFA was the most sensitive test for both phase 1 and 2, with a sensitivity of 100% if IgG and IgM responses were combined. For phase 2 antibodies the CFA performed well (100%) but the sensitivity for phase 1 was only 61%. The phase 1 IgG ELISA was more sensitive (67%) than phase 1 CFA, while phase 2 IgG and IgM ELISAs were less sensitive than CFA (using the manufacturer's instructions). Discussion: The higher sensitivity of the IFA was supported in the serial dilution, however these observations were based on a limited number of samples and should be refined using a larger set of sera. The IFA appears the method of choice if high sensitivity is required (e.g. early phase of illness). ELISAs can be an alternative or for screening of large sample numbers.
Objective: A characteristic feature of glucose oxidizing acinetobacters is their ability to produce a brown pigment in blood agar. The aim of this study was to evaluate the nature of brown pigment produced by Acinetobacter baumannii. Methods: Two (A/B) A. baumannii strains were isolated from diabetic patient and identified by blaOXA-51-like PCR and restriction analysis of 16S and 23S r-RNA spacer sequences using AluI and NdeII. MICs were estimated by BSAC guidelines. Isosensitest (IST)/M9 glucose broth was used for the growth of strains. Growth of the strains was monitored over 48 hour period using IST broth having gluconic acid concentration range from 0.1 to 4%. The inhibitory activity of the pigment produced by strain A was checked by the ditch plate method.
The MICs for Imipenem, meropenem, ceftazidime and cefepime were 0.5, 0.5, 8 and 4mg/L respectively for both strains. Both the strains were positive for blaoxa-51-like and blaADC but negative for blaoxa-23/40/58 and metallo-b-lactamases. Strain A produced a brown pigment in presence of gluconic acid. It also grew better than strain B over a period of 48 hours in the presence of IST broth containing gluconic acid concentrations ranging from 0.1 to 4%. Neither strain produced pigment in M9 glucose medium. When gluconic acid is added in excess, it increases the pigment in strain A. The brown pigment produced by strain A did not have any inhibitory effect against S. aureus NCTC6571, Ps. aeruginosa NCTC10662, A. baumannii ATCC19606 and E. coli NCTC10418. The glucose dehydrogenase enables both the strains to form 6-phosphogluonate which is free to enter the Entner-Doudouroff pathway hence there is no pigment production seen in both the strains when M9 glucose broth is used. On the other side human blood being enriched with nutrients including gluconic acid helps strain A to survive better by converting gluconic acid to 2,5 diketogluconate which leads to the formation of brown pigmentation.
The survival of A. baumannii A in gluconic acid enriched medium helps it to survive better than A. baumannii B. Excess gluconic acid in strain A leads to brown pigmentation which may offer protection against antioxidant stress. The results show that strain A has multiple routes of metabolism which offers it better chance for survival than strain B.
S. Iyer*, M. Reed, A. Stacey, N. Virgincar (Reading, UK) Objectives: Prompt empirical antimicrobial treatment, after blood culture is recommended for optimal management of sepsis. Rapid pathogen identification (ID) and antimicrobial susceptibility testing (AST) results permit early streamlining of the empirical antimicrobial treatment to the pathogen-directed treatment with potential healthcare benefits. We compared, using the Vitek 2 automated system, ID and AST of Gram-negative bacilli (GNBs) from positive blood culture by 'direct' inoculation from blood culture broths with the 'indirect' inoculation from pure subcultures to improve the turnaround time of microbiological analysis. Methods: Between May-October 2010, 97 consecutive GNBs from 64 monomicrobial positive blood cultures (33 sets in 2/2 and 31 sets in 1/2 bottles) were included in the study. Vitek 2 GN ID and AST N142 (Enterobacteriaceae) cards were 'directly' inoculated with bacterial suspensions, prepared by differential centrifugation of the positive blood culture broth at 160Xg 5min, to separate blood cells and 650Xg 10min to pellet bacteria. Vitek 2 GNID, AST N142 and N 143 (Non-Enterobacteriaceae) were also 'indirectly' inoculated from overnight subcultures, following manufacturer's instructions. The 'indirect' method results were considered the 'gold standard'. API ID system was used if the GNB was not identified by the Vitek 2. Discrepant AST results were interpreted as: a 'very major error' if the result of the direct method was susceptible ("S") and that of the 'Indirect' method was resistant ("R"); a major error was the opposite. All other discrepancies were considered minor errors. Objectives: Seasonal variation in rates of infection with certain microorganisms has already been described with a higher incidence rate during the warmest months than the remainder of the year. The aim of the study was to detect seasonal variation, if any, in the recovery rate of different pathogens from the positive blood cultures obtained from patients of our hospital. Methods: All the bloodstream infections reported during the last five years were included in the study. After incubation in a continuously monitoring blood culture system (BACTEC 9050, Becton Dickinson, USA), positive blood-cultures were inoculated onto appropriate plates for standard aerobic and anaerobic cultures and incubated at 37 0 C for 24h and 48h, respectively. A Gram-stained smear was examined under microscope to obtain valuable information about the types of microorganisms present. The isolated pathogens were identified using the automated system VITEK 2 (BioMerieux, Marcy l'Etoile, France). Spot IF, bioMerieux) for the detection of IgM and IgG antibodies. All serum samples were tested in a 1/40 initial dilution. Intense fluorescence of the Rickettsiae situated in or outside the cells was considered positive reaction. End-point titers were obtained by serial dilution on positive specimens. Sera showing a typical pattern of fluorescence at titers of 1:80 for IgG and/or for IgM antibodies were considered positive. In 64 patients a second serum sample was tested for seroconversion or a fourfold titre increase. Results: Of the total 903 patients, IgM and IgG antibodies to rickettsiae were found in 250 patients. Antibodies to R. conorii were detected in 210 patients (84%), 115 males (54.8%) and 95 females (45.2%). In addition antibodies to R. mooseri were found in 40 patients (16%), 26 males (65%) and 14 females (35%). Difference in prevalence of antibodies to rickettsiae between age groups in men was not significant. However, the highest prevalence of antibodies to rickettsiae was observed in women aged >40 years old. Rickettsial infections are significantly seasonal, with most cases appearing during summer months. Patients from the prefectures of Kavala and Xanthi demonstrated a high prevalence of antibodies to rickettsiae. Conclusions: Data show a wide distribution of R. conorii in Northern Greece and indicate the low frequency of R. mooseri. Men are affected more often than women. Increased incidence of disease occurs during the summer months, especially in Eastern Macedonia and Thrace. Background: Antigen p24 is detected in blood serum at early stages of HIV-infection as a result of virus replication and at later stages of infection, when anti-p24 concentration decreases, and the antigen becomes detectable again. When antibodies to HIV are revealed, antigen p24 is often no longer detectable as a result of formation of a complex between the antigen and antibodies in blood. All existing commercial kits detect only the free p24 antigen, and their sensitivity level unable to determine low concentrations of p24 antigen that is required for early diagnosis of HIV infection. The purpose of the work is the study of diagnostic efficiency of detection of free and bound HIV-1 p24 antigen by the modified kit "DS-EIA-HIV-Ag-screen". An increase the sensitivity at antigen HIV-1 p24 detection is achieved by dissociate the immune complex and detecting total antigen p24 HIV-1 in patients' samples. Materials: Method for the dissociation of immune complex and preserving the free HIV p24 antigen was developed. The samples were treated with glycine hydrochloride to dissociate the immune complexes, followed by neutralization with TRIS-hydrochloric acid. Reagents for immune dissociation designed for use with the kit of the "DS-EIA-HIV-Ag-screen".
The following categories of sera were used in the work: 43 sera positive in EIA and PCR and indeterminate in the immunoblot (IB), 107 well defined and positive in the IB reaction sera from HIV-infected patients. Results: At testing 43 blood sera samples positive in EIA, PCR and indeterminate in the IB, antigen p24 is detected in 31 samples (72%) using the kit "DS-EIA-HIV-Ag-screen" and in 37 samples (86%) using the modified kit. At testing of 107 the samples from HIVinfected patients, confirmed in the IB reaction, antigen p24 is detected in 8 samples (7.48%) using the kit "DS-EIA-HIV-Ag-screen" and additionally in 11 samples after the dissociation of immune complex. The All strains for which the biologist asked complementary identification 1) from May to July 2010 isolates were placed in an Eppendorf tube containing a water-ethanol mixture, then, an extraction was performed before testing on the Ultraflex III. 2) since August, the extraction was eliminated and the bacteria were subcultured the day before testing directly deposited on the target. Results: A total of 170 strains were tested on the Ultraflex III: 94 strains (55%) were identified with a score above 2.00, 37 strains (22%) were identified with a score between 1.70 and 1.99 and for 39 strains (23%) a score below 1.70 was obtained. Finally, an identification was obtained for 77% of the bacterial isolates, avoiding sequencing. Moreover, even if for 23% of strains, no valid identification was obtained (low score), in practice the identifications given by the MALDI-TOF mass spectrometer often helped the biologist in orienting the identification and carrying out additional tests before performing the sequencing.
In conclusion, after 6 months of this protocol, more than 75% of bacteria thus treated were identified and all biologists are convinced of the contribution of spectrometry in clinical bacteriology. Valuable time is saved and in some cases the therapeutic management of patients has been modified and adapted quickly to the results given by mass spectrometry.
Objectives: Rapid antigen detection assays for the respiratory syncytial virus (RSV) antigen are widely available with large differences in performance characteristics, sensitivities varying between 59% and 97% (Henrickson KJ and Hall CB. Pediatr Infect Dis J., 2007 Nov; 26(11 Suppl): S36−40). We evaluated the performance of two commercially available immunochromatographic assays: the BinaxNOW RSV and Clearview RSV (Inverness Medical). Methods: One hundred paediatric nasopharyngeal aspirates were collected and stored during the winters 2007-2010 in the University Hospital of Antwerp and were used for the evaluation of the BinaxNOW; 75 of these specimens were also tested with the Clearview RSV. The results of these rapid assays were compared to the combination of immunofluorescence (IF) directly on the specimens and/or culture on inoculated Hep2 shell vials followed by IF after two days of incubation (VC), which was considered as the gold standard. True positives were defined as positive for either IF and/or VC.
: Of the 100 samples tested, 49 were found positive (49%) for RSV with either IF and/or VC. The sensitivity and specificity of the Clearview and the BinaxNOW assay were 77.1% and 92.5%, 87.8% and 98.0% respectively (table 1) . There was no significant difference in sensitivity (P = 0.3) and specificity (P = 0.3) between the two assays. Conclusion: These easy to perform RSV antigen tests, which have a comparable performance, facilitate urgent testing outside batched runs or outside normal laboratory working hours. In order to increase sensitivity, negative results should be confirmed by IF. Objectives: The diagnosis of infection with human immunodeficiency virus 1 and 2 (HIV-1, HIV-2) is based on the detection of antibodies (Ab) to HIV-1/-2, and, during the stage of primary infection, the detection of p24 antigen or HIV-1 viral RNA. The objectives were to assess the performance of the Access HIV combo assay (Bio-Rad) on Access 2 system (Beckman Coulter) in terms of specificity and sensitivity on serum and plasma samples. This study was performed in three laboratories: Virology Department from Pitié-Salpêtrière hospital, laboratory "CQFD" from the EFS Nord de France (EFS-1) and laboratory "Non-Thérapeutique" from the EFS Normandie (EFS-2 The clinical evaluation of Access HIV combo on Access 2 system showed an excellent specificity for HIV testing for blood donors and hospital patients. All patients infected by HIV-1 or -2, at the chronic stage of the infection, were also identified as positive. These performance are fully suitable for HIV screening in private or hospital laboratory.
Objectives: Campylobacter jejuni is the organism that has most frequently been described in association with Guillain-Barré syndrome (GBS). Although infections with C. jejuni are recognized mainly by culture, serodiagnosis is often useful tool in diagnosis of neurological complications and reactive arthritis occurred after intestinal campylobacteriosis. Many various bacterial antigens have been used for the detection of C. jejuni specific antibodies. In this study we evaluated ELISA with four different antigen preparations for serological diagnosis of C. jejuni infections in patients with GBS. Methods: Sera were obtained from six pediatric patients (age range 4−16 years old; two males and four females) which met the established clinical criteria for GBS. A few weeks before the development of neurological symptoms all the six patients had a diarrhea episodes. Paired serum specimens were obtained from 2 patients. For control of specificity and sensitivity of different ELISA we used sera from culture-positive C. jejuni patients and from blood donors. The serological tests for C. jejuni were performed using one commercial ELISA (Mikrogen) and three home-made ELISA tests, with whole-cell antigen (Virion/Serion), LPS antigen and whole-cell antigen prepared according to method described by Strid and colleagues. Results: The cut-off limit of serum antibodies in home-made ELISA was set at mean antibody titre determined in the sera of 100 blood donors exceeded by three standard deviations. IgG antibodies, in diagnostically significant level, were presented in 4 patients by all four ELISA tests. We observed significant decrease of IgG antibodies titre in serum samples obtained from two patients in chronic phase of disease. ELISA with LPS antigen and ELISA with whole-cell antigen (Virion/Serion) diagnosed the IgM antibodies in four patients. On the other hand the presence of IgA was diagnosed only in serum samples obtained in the acute phase from two patients by commercial ELISA and ELISA with LPS antigen.
The results of our study showed that serological investigation may be a useful tool for identification of C. jejuni infections in patients with post-infectious neurological complications. Therefore, development of a worldwide available, standardized ELISA assay which can be used in serological diagnosis of C. jejuni infections, and its complications such as GBS, is needed. Introduction: Traditionally, clinical samples are manually inoculated and spread onto agar plates by lab technicians using plastic or platinum loops. The most common streaking pattern is the four-quadrant pattern. The aim of this procedure is to isolate discrete colonies for further identification and antibiotic susceptibility testing. KIESTRA Lab Automation (Drachten, The Netherlands) has developed an instrument where the spreading of the samples is performed by beads in an electromagnetic field. The aim of this study was: -to compare the number of discrete colonies obtained by manual spreading with automatic spreading performed by InoqulA ® -to study the variation and reproducibility of discrete colonies created by InoqulA ® Methods: Bacteria from two different species were selected, Enterococcus faecalis ATCC 29212 and Escherichia coli ATCC 25922. Inoculums of each species were prepared by suspending the bacteria in Phosphate Buffered Saline (PBS) to a density of McFarland 0.5 using VITEK ® 2 Densicheck (bioMérieux). The suspension of E. faecalis was used undiluted and the suspension of E. coli was diluted 1/10. The final inoculum spread on each plate were 108 and 107 respectively. Agar plates used was a cromogenic agar called UriSelect 4 (BioRad). Five skilled lab technicians were asked to participate in the study and each lab technician inoculated two plates of each bacterial suspension. The same suspensions were used to inoculate the plates with the InoqulA ® . All plates were inoculated with 10 mL using a pipette. For the manual spreading, plastic loops and the four-quadrant pattern, was used. The plates were incubated in ambient air at 35ºC for 16 hours before discrete colonies were counted. Results: InoqulA ® produced three times as many discrete colonies of E. coli and five times as many discrete colonies of E. faecalis than the manual method (Table 1) . For both E. faecalis ( fig. 1 ) and E. coli ( fig. 2 ) the amount of discrete colonies isolated by manual spreading varied between different lab technicians. There was also a noticeable variation in number of discrete colonies produced by InoqulA ® . Conclusions: -InoqulA ® produced a greater amount of isolated bacterial colonies than manual streaking. -The number of isolated colonies obtained by manual spreading varies between different lab technicians. -Reproducibility and evaluation of different InoqulA ® streaking pattern needs to be further studied.
Introduction: Chronic Abdominal Pain of childhood and adolescent is a common disturbance of patients and their Families. Considering different etiologies of abdominal pain, the role of Helicobacter pylori is unclear.
This study was case-control and prospective from 1387-1388, carried out in Bandar Abbas. 50 patients aged 4−16 years suffering from recurrent abdominal pain (RAP) over 3 months which interfered with their normal life style were selected randomly. 50 healthy preschool and school children with same age were selected as control group. Demographic data were collected in a questionnaire. After physical examination, both groups were checked for Helicobacter pylori stool antigen test (HPSA). Results: 58% (29) patients were female. 14% in case group suffering from RAP and 10% in control group had a positive HPSAg and 1 person (2%) had a borderline result. HPSAg was more positive in males, and not related to age, route of delivery child was born or the family history of peptic ulcer disease. HPSAg result did not differ significantly in case or control group. Initiation of supplemental feeding after 6 months was a strong risk factor for h.pylori infection. Objectives: The prenatal hypoxia is the leading of nervous system patology on one side and infectious processes with tendency of generalization on the other side. We use a tandem of markers in intensive care newborns: PCT − a biomarker of a bacterial infection and S100B − a marker of brain damage. Methods: 31 newborns (3−6 days) in ICU transported from maternity clinic. The gestational age was 28−40 weeks. PCT and S100 serum levels were measured by Elecsys immunoassay. Results: 13 newborns (42%) were with HIE and 18 (58%) with IVH. Patients with IVH was with pre-natal pneumonia in 10 (77%) cases and in 9 (69%) necrotizing coloenteritis. Whereas at patients with HIE an aspiration pneumonia is prevailed (11. 61%). Necrotizing coloenteritis is diagnosed in 4 (22%) cases. PCT level was1.54 ng/ml after hospitalization ( The level of S100B is directly correlate with PCT level (r = 0.51). Subsequent assay was made by 18 patients. PCT was 0.39 (0.192−1.1) ng/ml. In 9 cases PCT have normalized after initially levels more than 2 ng/ml level. In other cases decreased of PCT level before treatment was more than 50%. While S100B remained the same 0.347 (0.192-0.438 Every inoculation was performed in triple on blood agar plates and the experiment was repeated four times. For the implementation of the WASP in the routine microbiology laboratory, results of the inoculation of fifty urine samples and fifty MRSA-screening samples by the WASP were compared with results of manual inoculation. For urine samples, 1 mL was planted on blood agar and McConkey agar plates in a four quadrants streaking pattern. Ten mL of the MRSA screening samples (eSwab) was planted on a chromogenic agar and inoculated in TSB salt enrichment medium. After overnight incubation, enrichment broths were planted onto chromogenic agar plates both by WASP and manually.
The quantitative evaluation of the automated streaking of the 1 mL loop showed that plates inoculated by the WASP had 10 times more colonies growing after overnight incubation as compared to the plates inoculated with 50 mL using the calibrated pipet. This was the same for all three ATCC strains and after a correction was made for the cfu/mL of the initial 0.5 McFarland suspension. Although there were numeric differences between colony count of plates inoculated by WASP and manually, no differences in clinical interpretation was observed.
Testing of the clinical samples will be continued in the next weeks.
Although the WASP is a very promising automated instrument for planting and streaking of bulk samples like urines and MRSA-screening samples in microbiology laboratories, a conscientious validation is recommended before the implementation in daily routine. Objectives: Anaerobic bacteria remain an important group of human pathogens. Most of them are fastidious microorganisms, and their identification by conventional, biochemical methods is frequently tedious and inaccurate. MALDI-TOF mass spectrometry is a fast and reliable technology for microorganism identification, which has been shown useful for microorganisms identification both from culture and from some samples. We have compared the correlation between biochemical identification and MALDI-TOF mass spectrometry (ME) for anaerobic bacteria identification. Sepsis is caused by a variety of different groups of infectious etiologies. Early and adequate antimicrobial therapies correlate with positive clinical outcomes. In recent years matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometry fingerprinting has become a powerful tool in the microbiological diagnostic. The direct identification of microorganisms from a positive blood culture can shorten the diagnostic significantly enabling an earlier specific therapy. Aim of the study was to compare two different methods of sample preparation for the MALDI-TOF directly from a positive blood culture versus the conventional culture, the current gold standard.
In the first part of the study 192 positive blood cultures have been investigated by using tubes with separator gels (BD) to enrich microorganisms in the serum followed by the standard ethanol/formic acid protein extraction for the preparation of the bacterial extracts. In the second part of the study MALDI Sepsityper Kit from Bruker Daltonic has been used for the preparation of the bacterial extracts out from 132 positive blood cultures. 75% of Gram-negative bacteria and 60% of Gram-positive bacteria were correctly identified using the separator tubes compared to the cultures on species level. Using the MALDI Sepsityper Kit from Bruker Daltonic 95% of Gram-negative bacteria and 68% of Gram-positive bacteria were correctly identified. The overall identification Biotyper score in both studies was significantly higher for the Gram-negative (Septityper 2,2 and BD 2,0) compared to the Gram-positive bacteria (Septityper 1,8 and BD 1,7). An alteration of the algorithm for the interpretation of the Biotyper score for Gram-positive bacteria increases the rate of identification of the germ about 12−18%. The hands-on time is higher for the method using the tubes with separator gels. Thus, both protocols are suitable methods for the analysis of blood stream infection with better results observed with Brukers Daltonic Sepsityper Kit. Objectives: Natriuretic peptides compose a family of peptides that present diuretic and vasoconstrictive properties and are associated with left heart ventricle functions. Especially, the amino ending end of brain natriuretic peptide (NT-proBNP) is secreted by heart ventricles due to their dilatation in heart failure. High levels of NT-proBNP are also observed during bacterial infection and sepsis. Procalcitonin (PCT) is a calcitonin propeptide, which levels increase in serious bacterial infection Results: Increased levels of NT-proBNP and CRP were observed in all episodes while increased PCT values were found only in 16 (53%) episodes. The highest levels were observed for NT-proBNP in respiratory truck infections and bacteremias, for PCT in respiratory infections and bacteremias and for CRP in bacteremias and venous catheter infections. Statistically, significant correlation was observed between NT-proBNP and CRP (p < 0.01), while no correlation was observed between NT-proBNP and PCT or between CRP and PCT. No significant correlation was observed between the isolated bacteria and NT-proBNP, PCT or CRP.
In respiratory truck positive cultures it was found statistically significant correlation between NT-proBNP and CRP (p < 0.01) and also between PCT and CRP (p < 0.05).
1. NT-proBNP levels are significantly correlated with CRP levels in severe infections. 2. There is no significant correlation between the isolated bacteria from positive cultures and NT-proBNP, PCT or CRP. Results: On T1 PCT was higher in patients with sepsis, than patients with SIRS due to noninfectious etiology (p < 0.001). PCT levels increased significantly with the severity of illness. The highest levels were observed in patients with septic shock (P < 0.001). Patients who died during the follow up period were grouped as "nonsurvivors". Patients who were alive at the end of 14 days (n = 57) or discharged with cure (n = 8) in this period were grouped as "survivors". A total of 29 patients died in the first 10 days, so PCT levels on T10 for these patients were not available. PCT levels were higher in nonsurvivors on T0, T3 and T7 (p < 0.001, p = 0.001, p= p < 0.019 respectively). The change in PCT levels in time was analyzed. A significant decrease was observed in survivors in whom PCT values on T1, T3, T7, T10 were available (n = 57) (p < 0.001). At a cut-off value of 0.07ng/ml on T0, the negative predictive value (NPV) of PCT for differentiating sepsis from SIRS due to non infectious etiology was 100.0% with 95.7% accuracy. When 17 patients with SIRS were excluded, for 77 patients with sepsis NPV of PCT for mortality were as follows:
On T3, at a cut-off value of 0.7ng/ml, NPV: 91.0% accuracy: 70.6% On T7, at a cut-off value of 3ng/ml, NPV: 97.5%, accuracy: 84.3% Conclusion: We think that PCT is an important marker guiding the clinician in the differential diagnosis of sepsis and prognostication.
K. Behera*, T.K. Mohanty, S.S. Layek, A. Kumaresan, T. Patbandha (Haryana, IN) Objectives: Mastitis continues to be a costly disease in modern dairy farming and detection at subclinical stage is of immense worth both in terms of animal health and economy. The present study was undertaken to evaluate potential value of Acute Phase Proteins (Serum Amyloid A and Haptoglobin) in detecting clinical and subclinical mastitis and their correlation with pH and electro-conductivity in Zebu (Sahiwal) R2492 Performance evaluation of the Access ® HIV combo assay on the UniCel ® DxI 800 E. Roux, N. Groleau, C. Chandelier, F. Margotteau, C. Stoia, D. Duhamel, B. Roussel, C. Bonchamp, V. Potelle, D. Nogues, M. Cottin, R. Falcou-Briatte*, O. Flecheux (Marnes la Coquette, Steenvoorde, FR) Objectives: A new automated HIV combo assay has been developed by Bio-Rad for the qualitative detection of HIV-1 p24 antigen and antibodies to HIV-1 (groups M-N-O) and HIV-2 using the UniCel DxI 800 Immunoassay system (Beckman Coulter). The purpose of this study was to evaluate the performance of this new assay in terms of sensitivity, specificity and precision. Methods: All studies were performed on UniCel DxI 800 immunoassay system. The analytical sensitivity was estimated by dilution study of the AFSSAPS panel and the NIBSC Panel 90/636 (WHO standard). The clinical sensitivity was evaluated by testing 62 subtype and variant samples, 289 commercial positive samples, 199 hospital patient samples and 61 seroconversion panels (including 131 early seroconversion samples). The clinical specificity was studied with 2,552 samples from blood donors, 1,969 selected negative hospital patient samples and 617 non selected hospital patient samples. The precision study has been studied following CLSI EP5A2 guidance by the analysis of 13 samples: a negative sample, 2 low positive samples, a medium positive sample for HIV-1, HIV-2, HIV-1-O and HIV-1 antigen. Results: The specificity was found to be 100% on blood donor samples, 99.85% on selected negative hospitalized patient samples and 100% on non selected hospitalized patient samples.
The analytical sensitivity obtained with the NIBSC 90/636 was equal to 1.1 IU/mL. It was estimated at 20.60 pg/mL with the AFSSAPS panel.
The clinical sensitivity for all positive samples including HIV-1-M, HIV-1-O, HIV-1-N, HIV-2 antibodies and HIV-1 antigen was 100%. The seroconversion sensitivity gave performance in accordance with the state of the art as 131 early seroconversion samples were all detected. Intra-assay and inter-assay precisions were found below 10% with positive samples. Conclusion: The evaluation of the Access HIV combo on the highest throughput UniCel DxI immunoassay system showed excellent performance in terms of global specificity, analytical sensitivity, clinical sensitivity and precision. This new Access HIV combo is fully suited for the screening of HIV, in hospitals or private laboratories. with IB indeterminate and negative results were additionally tested in high sensitive EIA for p24 antigen detection. The majority of these samples (57%) were also evaluated for viral load. Cost-effectiveness of analysis included the calculation of the cost of identifying one case of HIV infection. The reliability of differences was determined by Mann-Whitney's criterion. Results: Out of 4271 analyzed sera with IB indeterminate and negative results, antigen p24 was detected in 4,5% and 2,5% respectively. In p24 positive samples, viral load was detected in 98%. More than in 25% of cases, the viral load exceeds the sensitivity level of the used assay (>750000 copies/ml). A median was more than 100,000 copies/ml in samples with indeterminate IB results and more than 300000 copies/ml in samples with negative IB results. HIV-status was confirmed in 58% of tested patients. The median time until confirmation by IB was 107 days. 40% of patients with indeterminate and negative IB results did not come for the repeat testing. A few of false-positive results (2%) were due to the errors of pre-analytic phase. Objectives: The EUCAST disk diffusion antimicrobial susceptibility testing method for fastidious organisms is based on the Mueller-Hinton fastidious agar (MH-F). In a pilot study, most anaerobic bacteria did not grow well enough on MH-F to permit antimicrobial susceptibility testing. We decided to investigate whether or not the Brucella Blood Agar supplemented with hemin and vitamin K (BBA), recommended for antimicrobial susceptibility testing of anaerobic bacteria with gradient strips, might also be suited for disk diffusion. Methods: Bacteroides fragilis ATCC 25285 and Bacteroides thetaiotaomicron ATCC 29741 were tested with E-test gradient strip (piperacillin/tazobactam, meropenem, metronidazole and clindamycin) on BBA according to the manufacturer's instructions. The corresponding disk (EUCAST disk strength) was included on each plate. Twelve plates were incubated at 37ºC in an anaerobe environment (10% CO 2 , 10% H and 80% N 2 ) for 24 hours with each antimicrobial agent on two different days for intra and inter day variability. E-test results were compared with the acceptable ranges for the two strains (reference agar dilution testing, CLSI guideline M11-A7). Twelve plates with disks only were also incubated at 35ºC.
: All E-test results were within acceptable ranges and the intra and inter day variability was 1 dilution step. Zone diameter mean and range are shown in Table 1 (n = 12). The maximum difference between two means was 2.9 mm and the maximum range was 5.5 mm. Overall, growth was better and zones smaller at 37ºC than at 35ºC. Conclusion: Only a small intra and inter day variability was observed with disk diffusion on BBA with the tested antimicrobial agents. Whether this small variability can be reproduced with clinical isolates and whether resistant isolates can be separated from wild type zone diameter distributions of the Bacteroides fragilis group remains to be investigated. Also the impact of different temperatures needs to be evaluated further. T. Büyükgüçlü, H. Iakar, N. Balaban, D.Çaliskan, B. Yalcin, U. Cinar, E. Guner, M. Eksioglu, N. Atakan, B. Esen (Kirsehir, Ankara, Mersin, Karabuk, TR) Objectives: The heterogenous expression of methicillin resistance in S. aureus make the phenotypic testing difficult and slow. The aim of this study was to compare the results of phenotypic and genotypic methods used for methicillin resistance, determine susceptibilities to antibiotics used for skin and soft tissue infections. Methods: 92 outpatient and 150 inpatient S. aureus strains isolated from skin and soft tissue infection included in the study. The patients were classified as community acquired and hospital acquired by CDC criteria. Methicillin resistance was determined by oxacillin and cefoxitin disk diffusion (DD), then confirmed with oxacillin salt screen agar test, and mecA PCR. Susceptibility test to antimicrobials including clindamycin (CLI), erythromycin (ERY), gentamicin (GEN), penicillin (PEN), mupirocin (MUP), rifampin (RIF), tetracycline (TET), trimethoprimsulphametoxasole (SXT), teicoplanin (TEC) were determined. Data were compared by the chi-square or Fisher's exact test, using SPSS 15.0. Results: mecA was detected in 11 outpatient, 66 inpatient. HA strains showed higher positivity than CA-strains (44%, 12%) (5.79; 2. 85-11.74, p = 0.001 The most ideal spreading pattern to create evenly spread growth and uniform circular inhibition zones was pattern 5 with 1 mm distance between the laps and a full length primary streak. 50 mL of the McF 0,5 suspensions were sufficient for all species except Enterococcus spp which required an inoculum volume of 75 mL. Less inoculum than 50 mL increased the diameter of the inhibition zones. Inoculums with a density of McF < 0,1 yielded larger inhibition zones than inoculums of McF 0,5 density. This had an effect on the SIR-interpretation for some antibioticbacteria combinations.
1. It is possible to use InoqulA ® for inoculation and spreading of agar plates for susceptibility testing according to the new EUCAST guidelines. 2. There is no significant difference in SIR-interpretation of the included species when comparing inhibition zones after manual spreading and after spreading by InoqulA ® 3. Using inoculums of much lower density than recommended increases the size of the inhibition zone, which has an effect on the SIRinterpretation for some antibiotic-bacteria combinations. Bacterial cells were incubated in filtered Muller-Hinton broth until exponential phase and then exposed to 1, 2 and 4 micrograms per milliliter of ciprofloxacin (S-Aldrich) for 0.5, 1 and 2 hours. Several fluorescent dyes were tested in order to obtain the best approach to the subject: Propidium Iodide (S-Aldrich) (FL3-650 nm) a nucleic acid staining, which only penetrates on cells with severe lesion of the membrane i.e. dead cells; Bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4 (3)) (S-Aldrich) (FL1-530 nm), a lipophilic anion able to diffuse across depolarized membranes; and SYBR Green I (Molecular Probes) (FL1-530 nm) fluorescent dye that binds to double stranded DNA. Flow cytometric analysis was performed after staining with those probes in the dark for 30 minutes. Conventional colony-forming units (CFU) assay was performed from the suspensions analyzed by Flow Cytometry. Results: Propidium Iodide and DiBAC4 (3) were not able to discriminate Susceptible and Resistant strains after the tested incubation time. SYBR Green I was able to clearly discriminate between susceptible strains, presenting a decrease in fluorescence intensity in a drug concentration and time dependent manner only on susceptible strains when compared to the viable non-treated bacterial cells even after 0.5 hour treatment. Correlation between conventional CFU assay and Flow Cytometry was successfully achieved. Conclusion: One hour versus 24−48 hours was enough to characterize the susceptibility to ciprofloxacin staining with SYBR Green I. Flow cytometry proved to be an excellent and accurate method representing an alternative approach to evaluate the susceptibility of bacteria to ciprofloxacin.
Objectives: The detection of extended spectrum b-lactamases (ESBLs) in the laboratory has led to a rise in the use of carbapenems worldwide.
Many laboratories now rely on automated systems to perform this task, and the reliability of their results is of crucial importance. Here, we assessed the performance of one such system − the MicroScan WalkAway system (Siemens) and panel NM36 as a screening tool for the detection of ESBL production by a range of enterobacteriaceae. Objective: EUCAST aims to set European breakpoints for antimicrobial resistance and to streamline methods of antimicrobial susceptibility testing (AST) to increase the comparability of results from different laboratories. In our laboratory AST was performed by micro-broth dilution on Phoenix™ and disk diffusion (DD) according to CLSI guidelines. At the moment BD Diagnostics discontinued production of certain non-EUCAST panels, we decided to implement EUCAST-guidelines and breakpoints for DD and automatic testing at the same time. This study describes the draw-backs encountered during the implementation of these guidelines in a routine microbiology laboratory. Besides, breakpoints for several antibiotics tested in our lab are lacking in EUCAST-guidelines e.g. temocillin for Enterobacteriaceae, ceftazidime for Acinetobacter and erythromycin for Enterococci. Practical issues encountered included the unavailability of some of the required disks and plates from some companies at the moment of implementation. Also, Phoenix EUCAST-panels do not all fit completely into EUCAST-strategy as some antibiotic ranges do not include the new breakpoints recommended e.g. erythromycin for enterococci and rifampicin for staphylococci. Due to these issues, a complete quality control testing on 20 consecutive days had to be performed using ATCC strains 25922, 27853, 29213, 29212, 49619 and NCTC strain 8468. Zone diameters were measure daily by 2 trained microbiologists and compared to ranges provide by EUCAST (Tables of QC targets PBP2a™ (BNPBP2a) (Alere) for SA identification and methicillin résistance (MR) detection directly on blood culture bottles in 30 minutes. Methods: Prospective study of CPBP2a: 333 consecutive staphylococcal strains were tested from primary plates. Evaluation and prospective study of BNSA and BNPBP2a: Evaluation: 17 strains representative of MRSA clones circulating through the world were inoculated into artificial blood cultures (BacT/Alert FA and FN with 10 ml fresh human blood, 100 CFU/vial) and incubated in the BacT/Alert automat. Prospective study: 58 clinical blood culture bottles positive for Gram-positive cocci were included. All immunochromatographic results were compared with phenotypic results (Vitek or Phoenix ® ). Any discrepant result was checked by PCR. Results: Test CPBP2a: 329 tests were interpretable. For SA (MSSA, n = 216; MRSA, n = 30), Se, Sp, PPV and NPV were 90%, 100%, 100% and 98.6%, respectively. For coagulase negative staphylococci (CNS) (MSCNS, n = 23; MRCNS, n = 60), Se, Sp, PPV NPV were 70%, 100%, 100%, 56%. No false positive was observed for SA and for CNS. For MRSA and MRCNS isolates not detected on primary culture, the test was positive for all MRSA and for 14 of 18 MRCNS after subculture. Two out of the 4 remaining MRCNS were detected after induction (around cefoxitin disc) and 2 could not be retested. Tests BNSA and BNPBP2a: Evaluation: for BNSA, 1 out of the 34 blood culture bottles (17 pairs aero/ana) remained negative. For BNPBP2a, all strains were positive for PBP2a. Prospective study: for BNSA, Se, Sp, PPV and NPV were 100%, 93.1%, 87.8%, 100%, respectively. All SA were accurately detected while 4 CNS yielded false positive results. For BNPBP2a, Se, Sp, PPV and NPV were 100% for SA (MSSA, n = 27; MRSA, n = 2) while for CNS Se, Sp, PPV NPV were 75%, 90%, 93.8%, 64.3%, respectively. Conclusion: For SA, CPBP2a presents relevant PPV and NPV, enabling early consideration of the MR directly on primary culture. For CNS, the test has an acceptable PPV but a low NPV and improvement would probably require an optimization of the protocol.
For BNSA and BNPBP2a, evaluation study as well as preliminary results from the prospective study appears promising. Objectivity required accepting the fact that infectious diseases and responsibility of them challenge the health system to increase the immunization coverage up to 95%. Material and Methods: During February and July, incl. 2121 cases of measles were registered and 1969 of them were hospitalized in the University Clinic of Infectious Diseases, town of Plovdiv. Laboratory tests were conducted on the standard methodology; virus and serological parameters were investigated in Regional Public Health Institute. The data were statistically processed with SPSS 14 analysis system, using parametric methods in Gaussian distribution and nonparametric when needed. As a significant difference interval was accepted p < 0.05, guaranteeing 95% confidence. Results and Discussion: The highest incidence of measles was repotted during April and May, 34.3% of hospitalized patients. Mainly mediumheavy forms of disease were observed. 65% of treated were children between 1 to 18 years. Measles complicated with pneumonia was found in 504 patients − 25.6%. Pronounced respiratory failure and need of oxygen therapy had 59 fellows. Antibiotics received all complicated cases. X-Ray control was achieved in 74.3% of lung-affected. We observed complications of the nervous system in 7 patients, aged 8 months to 52 years. Measles, complicated with meningitis − two cases, viral encephalitis − 4 and one 8 years old boy with meningomyelitis. Conclusion: Outbreak of measles in Plovdiv and the region in 2010 once again put reasonable challenges of organizational, financial, legal and social-legal aspect to epidemiologists and infectious diseases in particular and the healthcare system in the country as a whole. The neurological complications were rare − in analyzing study 0.36% with benign ending. to 18.8%) had antibody to hepatitis B core antigen, 21 (4.2%) (95% confidence interval 2.8% to 6.4%) were positive to hepatitis B surface antigen, 12 (2.4%) (95% confidence interval 1.8% to 2.6%) were antihepatitis C positive, 9 (1.8%) (95% confidence interval 0.9% to 2.8%) had antibody to hepatitis D and 0 (0%) were positive to antibody to HIV. Conclusions: Although this population theoretically had a low risk for HBV, HCV and HDV infection, these results are higher than expected in accordance with the age range. It has not been found any recruits positive to HIV. Based on the prevalence of serological markers we recommend vaccination against HBV infection after prevaccination screening.
Objectives: To determine the prevalence and predictors of hepatitis C virus (HCV) infection and associated risk factors for this infection among inmates of Bulgarian prisons.
This study was carried out in 2009, among inmates of the fifth biggest Bulgarian prisons (men n = 367 and women n = 131) and a juvenile correctional institution (n = 86). Anonymous cross-sectional data was collected from prisoners who agreed to participate in the study and who were interviewed using a standard questionnaire including demographic, imprisonment history and HCV related risk behaviors items. Thereafter, the blood drawn from the participants were tested for anti-HCV antibodies and HCV-RNA. Discarded serum samples were tested also for a presence of hepatitis B core antibodies (anti-HBc), hepatitis B surface antigen (HBsAg), hepatitis D antibodies (anti-HDV) and antibodies to human immunodeficiency virus (HIV Objectives: Shigatoxin producing Escherichia coli (STEC) are an important cause of foodborne disease. It is transmitted to humans primarily through consumption of contaminated food, such as raw or undercooked ground meat products and raw milk. We report on the first occurrence of STEC O174:H2 in Austria. Methods: Biochemical identification, serotyping and virulence testing were done at the National Reference Centre for Escherichia coli. A descriptive investigation was performed in order to describe the outbreak and to identify the outbreak genesis.
The outbreak involved 7 persons (6 male). The index isolate was from a 46 year old, previously healthy male, who had a fecal specimen taken under intensive care for haemolytic uraemic syndrome (HUS) on April 13 in Graz. The second isolate was from a 46 year old Tyrolean patient (specimen received on April 28); this patient suffered from discharge of slime and bowel irregularities, following watery diarrhea. The patient attended a book fair in Graz 5 days before onset of illness in Innsbruck. He and two working colleagues consumed appetizers (bred with various handmade spreads) at some of the exhibition booths. The two colleagues fell ill with diarrhea (one in Innsbruck, one in Munich, Germany) but had not stool specimens tested. Another stool isolate (June 10) was from a 66 year old male treated for HUS in Vienna. A fourth isolate was from this latter patient's wife (age 63), who developed bloody diarrhea three days after her husbands discharge from hospital. A fifth stool isolate was from a 92 year old patient with diarrhea from Graz (specimen received May 12). All five isolates were sorbitol-fermenting, positive for stx1, stx2 and hlyA, but negative for eae. Analyses by PFGE using XbaI as restriction enzyme yielded patterns indistinguishable from each other, but clearly different from five other STEC O174:H2 provided by the German National Consulting Laboratory on HUS and 48 disease associated Austrian STEC isolates from 2010. Conclusion: Our report emphasizes that stool samples from patients with HUS must be tested by other techniques than sole use of Sorbitol-MacConkey media. Direct person-to-person transmission underlines the importance of instructing family members about the need for post-defecatory handwashing. For five of the six remaining patients, consumption of an unidentified contaminated handmade spread produced and distributed in the Graz area is the likely source of infection. The adherence ability, siderophores, amylase and caseinase were better expressed at 37ºC. At 0% NaCl only amylase and siderophores, at 2% and 3% NaCl the amylase was better expressed and at 6% NaCl, siderophores and caseinase showed the best expression, thereafter, the VF expression gradually decreasing till 10% NaCl. The adherence to HeLa cells was decreased by higher salinities. The tested strains proved high resistance to a broad range of pH from 5 to 9.6. The amylase and caseinase were better expressed at pH 9.6 and siderophores at pH 7. The higher glucose concentrations (3%) inhibited the expression of amylase and caseinase. The incubation conditions exhibited no influence on the VF expression. The molecular detection of VF genes evidenced the Ecp/dA in 82% and EchelD in 85% of the tested strains. The densitometric analysis of the electrophoretic bands obtained in multiplex PCR showed different intensities, suggesting that, in a number of cases, the lipase or DN-ase genes could be lost in a great percent in the bacterial population. Conclusion: Our results proved the high adaptation ability of aquatic enterobacterial strains to different stress conditions and the expression of virulence determinants even in limiting environmental conditions, demonstrating the role of these parameters in the preservation of the virulence gene pool in water.
Objective: Chlamydia trachomatis (CT) detection in both self obtained vaginal swabs (SVS) and first-catch urine (FCU) in two separate assays, results in the highest sensitivity. In most laboratories however, onesample testing is performed for reasons of cost efficiency. To further improve one-sample testing, we assessed the laboratory performance of three different testing approaches to find the most sensitive onesample test procedure: SVS versus FCU versus a combined specimen of FCU/SVS. Methods: All women visiting a STD clinic above the age of 16 were asked to participate in the study. Each client was asked to take a FCU and a SVS with a dual swab. The FCU, SVS and FCU/SVS combination were tested for CT by Strand Displacement Amplification assay (SDA) of Becton Dickinson (ProbeTec ET system, Maryland, USA) or Polymerase Chain Reaction (PCR) by Roche Diagnostics Inc. (Cobas Amplicor system, California, USA). Clients with at least one out of three sample types (SVS, FCU, SVS/FCU combination) tested positive for CT by NAAT, were regarded as CT positive (comparison standard). Results: In total 791 females were included and CT prevalence was 12% (96/791). The CT detection rate for SVS, FCU and SVS/FCU combination were 94%, 90% and 94%, respectively, if results of NAAT by SDA and by PCR were analyzed together. The detection rate was not significantly different between any of the sample types, when tested solely. Discordance in NAAT results within the different sample types was found in 16 out of 96 CT positive results. Conclusion: Our results show that the detection rate of SVS/FCU combination is equal to that of FCU or SVS alone. SVS is an acceptable and feasible specimen for females. Moreover, SVS is the most costeffective sample type for a STD clinic population We can therefore conclude that SVS is the specimen of choice to detect CT in females. Results: Ten patients (8 men, 2 women) were included with matched controls. The mean age was 49.8 (24−77) years. IE (9/10) was the most common distant site infection, one patient developed spondylitis. All patients had some predisposing condition for distant site infection: mitral valve insufficiency (5/10), bicuspid aortic valve (3/10), atrioventricular septal defect (1/10) and discus protrusion (1/10). Five patients had had a preceding dental procedure, which in most cases was removal of dental plaque and calculus (4/10). None of the patients received antibiotic prophylaxis preoperatively. All ten patients had active oral infection foci, most often untreated periodontitis (5/10), likely to cause spontaneous bacteraemia. Patients reported more childhood infections than the controls (p = 0.01). WBC counts (10(3)/mL) on admission were median 8.0 (4.5-10.6) and at maximum 9.9 (5.5-33.6). LOS was 27 days in median (9−75). Conclusion: Patients with predisposing medical conditions seem to be more susceptible for distant site infections caused by S. mutans. The major predisposing factor in our study material was cardiac structural abnormality. All these patients subsequently developed IE as a distant site infection. The results highlight the importance of oral health in patients with cardiac abnormalities and support the use of antibiotic prophylaxis during dental care of oral infection foci in these patients, especially if colonised with S. mutans. Background: Antibiotic resistance is a term that is frequently used by clinicians in their discussions with patients. However, patients and clinicians may not share the same assumptions about the meaning of this term. Objective: We aimed to explore patients' interpretations of the term 'antibiotic resistance' and to consider the implications for strategies to reduce the overuse of antibiotics. Design: Qualitative interview study. Participants: 121 adult patients from nine European cities who had recently consulted a primary care clinician with symptoms of Lower Respiratory Tract Infection (LRTI). Approach: Semi-structured interviews were conducted with participants following their consultation. Data were subject to Framework Analysis.
The dominant theme in all networks was that antibiotic resistance arose from having or developing a 'resistant body', where the barrier to antibiotic effectiveness was individual loss of responsiveness. Less commonly, patients correctly conceptualised antibiotic resistance as a property of bacteria. Nevertheless, the over-use of antibiotics was a strong central concept in almost all patients' explanations, whether they viewed resistance as located in either the body or in bacteria.
Most patients were aware of the link between antibiotic use and antibiotic resistance. The identification of the misinterpretation of antibiotic resistance as being located in the body could lead to clinician-patient discussions and public health interventions which are much clearer about the location and mechanism of antibiotic resistance, explaining the transferability and societal relevance rather than focusing on individualised risk, thereby emphasising the public health argument for the prudent use of antibiotics.
Objectives: Diarrhoea remains one of the main sources of morbidity and mortality in the world and enteroaggregative Escherichia coli (EAEC) have been increasingly recognized as an emerging pathogen, causing diarrhoea in both developing and industrialized countries. In Romania, the real contribution of EAEC to diarrhoea disease burden is not known because of the lack of routine EAEC detection protocols in clinical microbiological laboratories. The aim of the present study was to determine the prevalence of faecal carriage of EAEC in Romanian children with sporadic acute diarrhoea.
Methods: E. coli isolates originating in stool samples, collected during diarrheal episodes from 477 children, under five years of age, previously found negative for enteric pathogens such as Salmonella spp., Shigella spp., Yersinia enterocolitica, Campylobacter spp. and other diarrheagenic E. coli pathotypes, were included in the study. From each specimen, one biochemically confirmed E. coli isolate was screened by PCR for the presence of EAEC-associated genes aat, aggR, aap and astA. EAEC isolates were evaluated, using a PCR-based protocol, for their phylogenetic background and tested for antimicrobial susceptibility by the disk diffusion methods.
The molecular approach revealed that 41 (8.6%) E. coli isolates carried at least two of EAEC-associated genes targeted. The most frequently detected genes were aat (40 isolates) and aap (38 isolates). Twenty-nine isolates harboured concurrently aat, aggR, and aap genes. Almost half of these carried also astA gene. Most of the EAEC isolates derived from phylogenetic group A (29 isolates), while the rest belonged to groups B2 (6 isolates), D (4 isolates) and B1 (2 isolates). Twenty-four EAEC isolates expressed resistance, of which five were extended-spectrum b-lactamase producers. All EAEC isolates were fluoroquinolone susceptible.
This study revealed that EAEC pathotype might be a significant cause of sporadic diarrhoea among children in Romania. Our results provide additional support for the reconsideration of the local diagnostic and surveillance strategies.
M. Halánová*, L. Cisláková, P. Juriš, I. Papajová, P. Jarcuška, P. Rudohradská (Košice, SK)
Material dimension of poverty in Roma national minority is particularly noticeable in the sphere of living. Especially, in the segregated settlements with illegal huts (mostly built of wood, iron waste, flat metal stock and other materials obtained from waste dumps or surrounding countryside), devastated environment and with no access to basic infrastructure such as electricity, tap water (they mostly use water wells or streams), sewerage system and waste disposal what in many cases has influence on bad health status and troubles with hygiene. According to the Office of Government Plenipotentiary for Roma issue in Slovakia there is approximately 680 Roma settlements at present. These are often located in rural communities without the necessary basic infrastructure. According to several studies in many settlements, which are often built on loose soils are lacking in their drinking water, sewage, waste pits and landfills, sanitary facilities and lack of garbage disposal. The settlements are concentrated in a small area with a large number of people (about 500 000 people) whose health status is unsatisfactory.
Aim: The aim of this study was to describe and to categorize different clinical pictures of patients with neurobrucellosis in our clinic, and to present demographical and laboratory data about the patients. Material and Methods: Hospital records, between 2003 and 2009, of about 430 patients with brucellosis were followed and retrospectively reviewed in our clinic. Results: Out of 430 patients, 19 (4.4%) had neurobrucellosis. These patients were classified into four groups: Meningitis group (n = 14; 13 cases of subacute-chronic meningitis and one case of acute meningitis), Encephalomyelitis group (n = 3; one case of meningoencephalomyelitis, one case of cerebellar abscess, and one case of transverse myelitis), Polyradicular group (n = 1; one case of Miller Fisher Syndrome), and others (n = 1; one case of intradural abscess). Ten patients (52.6%) were female, and nine patients (47.4%) were male and the mean age of the patients was 48.8 years. About 47.4% of the patients had fever, 26% of the patients had neck stiffness, and 5% of the patients were in an unconscious state. Out of 19 patients, 18 underwent lumbar puncture (LP). According to cerebrospinal fluid (CSF) analyses, the mean leukocyte count was 113/mm3, the mean glucose level was 45 mg/dl, and the mean protein level was 123 mg/dl. Standard tube agglutination test showed brucellosis in all patients who underwent LP. Microorganisms were detected in four patient's blood culture and one patient's CSF culture. There were cranial nerve involvements in five cases. The most frequent was the sixth cranial nerve involvement. Out of 19 patients, 3 recovered with sequels (paraparesis, hearing loss, dementia, and sphincter dysfunction) and 16 patients recovered completely. Conclusion: Although neurobrucellosis is most frequently accompanied by subacute-chronic meningitis, it may have many different clinical pictures. The classical triad of meningitis (fever, neck stiffness, and unconscious state) is rarely seen in brucellosis-related meningitis. Brucellosis should be kept in mind in patients with unexplained neurological findings in regions where brucellosis is endemic. In addition, a classification of brucellosis, which reflects locations of nervous system involvement, clinical picture, and pathogenesis, is needed.
Objectives: Oral candidiasis is an opportunistic infection of the oral cavity and it is common among the elderly. This study was carried out in order to evaluate colonization of the oral cavity with Candida species and oral candidiasis in the institutionalized and non-institutionalized elderly. Methods: A total of 341 elderly were included, 280 institutionalized elderly in nursing home (134 males, 146 females, mean age 72.68±8.43) and 61 non-institutionalized elderly (23 males, 38 females, mean age 70.39±6.16). Specimens were isolated from oral cavity with sterile swabs (Copan, Zagreb, Croatia) from the surface of oral mucosa and were inoculated on a isolation medium, Sabouraud's dextrose agar. Cultures were incubated at 37ºC for 48 hours. In cases of no growth, the plates were considered negative and discarded. The count of colony-forming units was recorded. Oral cavity is considered colonized with Candida spp. in the case of positive microbiological analysis and normal oral mucosal appearance. The diagnosis of oral candidiasis was established by taking account of the clinical appearance (pseudomembranous candidiasis, erythematous candidiasis, denture-related stomatitis or candida-associated lesions) and positive microbiological analysis. The diagnosis was made according to the number of the colonies described by Budtz-Jorgensen. Results: Altogether, 68.92% of institutionalized elderly in our study were colonized with Candida spp. This result was statistically different compared with the non-institutionalized elderly (50.81%; p < 0.001). The results of our investigation also showed an increased number of the institutionalized elderly with oral candidiasis compare with the noninstitutionalized ones (17.14% for institutionalized elderly; 9.83% for non-institutionalized elderly; p = 0.035).
Conclusion: Results of this study showed a higher prevalence of colonization of the oral cavity with Candida spp., as well as oral candidiasis in the institutionalized elderly compared with the noninstitutionalized elderly. Four patients had the diagnosis other than infectious diseases. The duration for the definite diagnosis beginning from the initial symptoms was varied from longer than 4 weeks (n:16) to less than 7 days (n:57) The mean hospitalization day was 9.5 days (5−26 days) Following the consultations, 15 patients were transferred to other departments for the need of other disciplines support. Nine of the patients (5 were in other departments) died.
As the atypical presentations of infectious diseases are very common in elderly patients, the most important problems are delayed or missed diagnosis and long hospitalization periods. Data reveals that neither the lack of fever directs the diagnosis to non-infectious disease nor the unconsciousness is the constant symptom of CNS infection in elderly patients. Detailed investigation of differential diagnosis is very important in geriatric patients.
Objectives: The main aim of the study is to evaluate the role of microbial air contamination on the risk of surgical site infection (SSI) in hip and knee arthroprosthesis controlling for adherence to guidelines for antimicrobial prophylaxis. The project has been funded by the Italian CCM (Centro Controllo Malattie, Ministry of Health). Methods: Hospitals were invited to join the project by GISIO members. SSI surveillance was conducted according to the HELICS protocol (version 9.1, 2004). Microbial air contamination was evaluated at the patient area, once at rest and during each operation, by passive sampling to determine the Index of microbial air contamination (IMA) (Pasquarella et al., 2000) and in some cases also by active sampling to determine the colony forming units (cfu)/m3. Surgical antimicrobial prophylaxis refers to a very brief course of an antimicrobial agent initiated just before an operation begins. A web-based data collection procedure was adopted using three electronic data forms. The two years project started in July 2010, preceded by a three-month pilot study to assess the overall feasibility of the programme.
The project has included so far 8 Hospitals and 20 operating rooms (OR). According to the OR ventilation system in place, 76.5% of OR were with unidirectional airflow, 6.0% with turbulent air ventilation, and 17.6% mixed. A total of 289 surgical procedures, 67.0% hip and 33.0% knee arthroprosthesis, were included until December 2010. Mean duration of operations was 93 minutes for hip and 117 minutes for knee arthroprosthesis. IMA values in OR at rest were as follows: range = 0−8, mean±SD = 0.8±2.1, median = 0; during surgical procedures the following values were registered: range = 0-156, mean±SD = 7.2±14.7, median = 3. A total of 99.6% of patients received antibiotic prophylaxis: 30−60 minutes (36.7%), 1−2 hours (18.5%) and >2 hours (44.8%), before incision. The most frequently administered antimicrobial agents were cefazolin (44.7%), tobramycin (29.1%) and teicoplanin (19.6%).
Although one year of follow-up after implant is requested for SSIs surveillance, the study has depicted the epidemiological scenario in which a complex network of risk factors for SSIs is embedded, already highlighting potential areas for improvement both for air quality and antibiotic prophylaxis.
Brucellosis is a chronic granulomatous infection caused by intracellular bacteria. It is an endemic disease in Mediterranean countries and Turkey. Brucellosis shows the involvement of many systems and it seems to be responsible for the high incidence of relapse.
We identified 14 studies (2 RCTs, 8 case series and 4 case reports) as eligible for inclusion in this review. In 7 studies, boric acid was compared either with nystatin, or azoles (terconazole, flucytosine, itraconazole, clotrimazole, ketoconazole, fluconazole, buconazole and miconazole), while as monotherapy boric acid was studied in 7 studies. The mycological cure rates varied from 40% to 100% in patients treated with boric acid. Four of the 9 included case series reported statistically significant outcomes regarding cure (both mycological and clinical) rates.
None of the included studies reported statistical significant difference in recurrence rates. Regarding the adverse effects caused by boric acid use, vaginal burning sensation (<10% of the cases), water discharge during treatment and vaginal erythema were identified in 7 studies. Conclusion: Our findings suggest that boric acid is a safe, alternative, economic option for women with recurrent and chronic symptoms of vaginitis when conventional treatment fails due to the involvement of non-albicans Candida species or azole-resistant strains.
Objectives: The objective of the present survey was the study of Yersinia enterocolitica infestation in drinking and surface waters in Northwestern Greece. Methods: Over a period of 12 months (November 2008-October 2009) a total number of 180 water samples were examined for the presence of Y. enterocolitica. The water samples were collected from the area of Epirus (Northwestern Greece) and included: 60 samples of drinking water, 60 samples of lake water and 60 samples of marine water. The drinking water samples were tap-water collected from houses from three different municipalities of the area, the lake water samples were collected from two lakes (Pamvotis and Voulkaria) representing two different ecosystems (polluted/urban and rural/unpolluted) both used for recreational activities and the marine water samples were collected from five different point sources of Ionian Sea, including areas of recreational activities. The APHA/AWWA, Standard Methods for the examination of water and waste water were employed for the detection of Y. enterocolitica and other bacterial indicators (total microbial flora, coliforms, fecal coliforms, enterococci). Results: Y. enterocolitica was isolated from two samples of marine water and two samples of lake water. The positive samples were isolated from the most polluted aquatic environments (the urban lake and the Amvrakikos Golf). The isolation of Y. enterocolitica coincided with increased numbers of total coliforms ( 2500 cfu/ml) and enterococci ( 2000 cfu/ml). No Y. enterocolitica strains were isolated from the drinking water samples. Conclusion: The findings of the present survey underline the presence of Y. enterocolitica in marine and lake water as an indicator of microbiological pollution and a potential threat for public health if the contaminated aquatic environment is used for recreational activities. Also, the absence of Y. enterocolitica in the drinking water samples of the examined area proves the efficacy of the chlorination practices.
Objectives: The aim of the present study is to report the event of tick invasion in the premises of the blood transfusion unit (BTU) of a General Hospital following the unusual climatic conditions of spring and summer 2010 in North-western Greece. Methods: During the first week of July 2010, large numbers of ticks appeared in the external and internal walls and inside the premises of the BTU of the "Hatzikosta" General Hospital of Ioannina, causing uneasiness to the unit's medical and paramedical personnel. The BTU is located next to the Microbiology and the Clinical Biochemistry Laboratories and above and below the BTU there are various Clinics. None of the surrounding laboratories and clinics reported any appearance of any ticks, which was also confirmed by in situ Inspection. Inspection of external walls of the BTU building revealed large numbers of ticks, the presence of a number of bird nests and many cracks on the walls. Results: The tick specimen collected by the Microbiology Department of the Medical School of Ioannina were identified to belong to Ixodes spp. The Greek Ornithological society was called and there were identified 80 nests of swallows belonging mostly to the species Delichon urbica, on the BTU building and surrounding hospital buildings. The swallow species Delichon urbica is host of haematophagous ticks belonging to the family of Ixodidae and apparently the nests were the source of the ticks, which astonishingly enough, were moving only towards and into the BTU premises and nowhere else in the Hospital buildings.
The swallow nests have been on the hospital buildings for years and were re-visited every spring by their bird habitats. However, the 2010 appearance of large numbers of ticks is correlated with the earlier spring with high temperatures and heavy rainfalls continued through middle of July. The favorable weather conditions worked well with the cracks on the old building walls resulting in the multiplication and establishment of the ticks on the hospital walls of the BTU.
Purpose: This study has been planned to indicate the clinical course and termination of brucellosis in our region during the recent years, and to compare it to the literature.
The study has been based on a review of the medical records of adult patients older than 14 years, followed with the diagnosis of brucellosis from March 1997 to October 2010. The demographical data, disease diagnosis, course, treatment and termination data of the patients were recorded. Results: 317 patients, including 136 males (43%) with average age 40±17 were included in the analysis. There was a statistically significant relationship between advanced age and development of spondylitis and arthritis (respectively p = 0.000 and p = 0.028). Besides, the frequency of splenomegaly and neurobrucellosis among the young population was found high, which was statistically significant (respectively p = 0.005 and p = 0.001). In cases of spondylitis, the most common (~85%) involvement was of the lumbar vertebrae. Also there was a statistically significant relationship between high ESR and spondylitis, sacroilliitis and visceral abscess (p = 0,001, 0,013 and 0,049 respectively).
The study is sad to see that there haven't been any significant changes in the frequency of the disease and its complications in time. Osteoarticular involvement, and particularly the presence of spondylitis should be searched, and the advanced aged patients should be meticulously scanned for complications. Laboratory parameters, patient's age and duration of symptoms may help to identify complicated cases. From arthropod-borne infections it has been confirmed cases of Lyme disease, Mediterranean spotted fever and Q-fever. Thirty two EUFOR soldiers were tested positive to brucellosis and four confirmed cases of hemorrhagic fever with renal syndrome have been reported. There were a few cases of sexually transmitted diseases and viral hepatitis C but they were not a significant problem during this deployment.
The incidence rate of infectious diseases, during this conflict was relatively low because of combination of factors: the presence of a comprehensive infrastructure of medical care, extensive preventive medicine efforts and several fortuitous circumstances. Nevertheless EUFOR military personnel, because of crowding and unique stressors, were subject to respiratory and diarrheal disease outbreaks. A few diseases (brucellosis, hemorrhagic fever with renal syndrome and Q-fever) caused by potential biological weapon agents were prevalent.
Background: Campylobacter has been recognised as a causative agent of bacterial infectious diarrhoea and remains one of the most prevalent bacterial enteropathogens in the industrial countries. Campylobacter infection is generally foodborne disease. Objectives: The aim of this report is the study of Campylobacter cases recorded during the decade 2001-2010, as well as the susceptibility of Campylobacter isolates to antibiotics. Also the frequency of the disease according to age, sex and season was estimated. Methods: A total of 898 Campylobacter strains were isolated from patients with acute gastroenteritis hospitalized in the Infectious Diseases Hospital of Thessaloniki. The selective Skirrow medium was used. Skirrow medium is blood agar infused with antibiotics: vancomycin, polymixin-B and trimethoprim. The incubation was under microaerophilic conditions at 37ºC and 42ºC. Following isolation on selective media, identification was carried out using the biochemical profile of each Campylobacter strain. Furthermore, Campylobacter strains were tested for susceptibility to antibiotics (ampicillin, gentamicin, tobramycin, cephalothin, ceftriaxone, ciprofloxacin, nalidixic acid, cotrimoxazole, erythromycin) using the Kirby-Bauer method. Results: Of the 898 strains, 846 (94.2%) were C. jejuni and 52 (5,8%) C. coli. From the total strains, 521 (58%) were isolated from males and 377 (42%) from females. Furthermore, 704 strains (78,4%) were isolated from children (47.4% under the age of 2 years) and 194 (21.6%) from adults. In addition, leucocytes were found in 34.4% of the stool specimens. The peak of Campylobacter infection in the study was in the spring months (31.4%) and in the summer months (28. 5% ). An increase in fluoroquinolones and co-trimoxazole resistance (nalidixic acid 23.9%, ciprofloxacin 12% and cotrimoxazole 47.7% respectively) among Campylobacter isolates has been observed. Erythromycin (macrolides) is considered the drug of choice with low resistance. Conclusions: Campylobacter is a major cause of bacterial enteritis, following Salmonella in Northern Greece. Most frequently isolated was C. jejuni. Age specific infection rate was highest in children less than two years old. Campylobacteriosis occurs much more frequently in the spring and summer months. Erythromycin is still an effective antibiotic for the therapy of Campylobacter infection.
Background: The association between Chlamydia pneumoniae and atherosclerosis in hypercholesterolaemia subjects in the absence of other conventional risk factors is unclear. This study was done to determine whether C. pneumoniae seropositivity is associated with increase in intima media thickness (IMT) of the carotid artery, hs-C reactive protein (CRP) and oxidant stress in subjects with hypercholesterolaemia. Methods: Fifty-two hypercholesterolaemic subjects were recruited during health screening programmes organized by our institution. Subject inclusion criteria include baseline LDL-c of 3.4 mmol/l. Subjects with diabetes, hypertension, severe obesity, smoking, primary hypercholesterolemia and presence of acute inflammation (C-reactive protein 10 units) were excluded. IgG and IgA to C. pneumoniae were measured by microimmunofluoresence test. IgG titre 64 and IgA 16 were considered to be seropositive. IMT of the far wall of carotid artery was measured by B-mode ultrasound. hs-CRP was measured by immunoturbidimetric method using an automated analyser. Isoprostanes (an oxidative stress marker) was quantified by liquid chromatography mass spectrometry. Results: Seropositivity for C. pneumoniae was detected in 40.4% of the hypercholesterolaemic subjects. There was no significant difference in the IMT in the hypercholesterolaemic subjects who were C. pneumoniae seropositive compared to the C. pneumoniae seronegative subjects (Mean IMT±SD mm: 0.6±0.7mm vs. 0.6±0.6 mm, p > 0.05). hs-CRP did not show a statistically significant difference between C. pneumoniae seropositives and seronegatives individuals (Mean hs-CRP + SD mg/dl; 1.56±0.29 vs 1.85±0.35; p > 0.05). By contrast, a significant difference was observed when comparing isoprostane, a marker for oxidative stress by serostatus to C. pneumoniae (Mean + SD pg/ml; 46.2±30.6 vs 37.7±20.4; p < 0.05). Conclusion: Our data demonstrates that seropositivity to C. pneumoniae increases the oxidative stress but it is not associated with increase in carotid IMT or in hs-CRP in subjects with hypercholesterolaemia in the absence of other conventional cardiovascular risk factors.
Objectives: The rapid and reliable identification of the three potentially toxigenic Corynebacterium species Corynebacterium diphtheriae, C. ulcerans and C. pseudotuberculosis is usually essential for diagnosis and treatment of diphtheria and diphtheria-like diseases. Classical differentiation of suspected isolates is done by biochemical tests, which are time consuming and may often give unclear results. Recently, matrix assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) was shown to allow identification of isolated microorganisms within 15 minutes and is therefore a fast alternative for species differentiation. Methods: We used matrix assisted laser desorption/ionization timeof-flight mass spectrometry (MALDI-TOF MS) in comparison with classical microbiological (API Coryne) and molecular methods (rpoB sequencing) on 116 Corynebacterium strains. Results: All 90 potentially toxigenic Corynebacterium spp. strains collected by the German National Consiliary Laboratory on Diphtheria in a period of more than ten years were correctly identified by MALDI-TOF MS. In 103/116 strains (88.8%) biochemical identification by API Coryne yielded identical results as rpoB gene sequencing. Twelve strains showing unreliable or ambiguous API results were concordantly identified by rpoB sequencing and MALDI-TOF MS except for one isolate of C. tuberculostearicum. In conclusion, 99.1% of the tested Corynebacteria were correctly identified by MALDI-TOF MS when compared to rpoB gene sequencing. Moreover, both the positive and negative predictive values for identification of potentially toxigenic Corynebacterium species were 100% with MALDI-TOF, respectively. Conclusion: In conclusion, species identification of potentially toxigenic Corynebacterium spp. can be accomplished by MALDI-TOF MS within 15 min. when applied to suspicious colonies. In this scenario, MALDI-TOF MS technology might be used as a rapid screening method helping to decide whether suspicious colonies should be analyzed for the presence of tox by real time PCR. We propose an algorithm for fast and reliable diagnosis of diphtheria incorporating MALDI-TOF MS, real-time tox PCR and Elek testing.
Scrub typhus, which is caused by Orientia tsutsugamushi, is an acute febrile illness characterized by fever, rash, and myalgia. Severe complications are very rare. Recently, cases of scrub typhus with severe complications, such as acute respiratory distress syndrome, septic shock, acute renal failure, myocarditis and meningitis have been increasingly reported. Fulminant myocarditis is characterized by critical illness at presentation. However, if affected patients recover with pharmacologic therapy and mechanical circulatory support, they may have a better long-term prognosis than patients with other forms of myocarditis. An adult patient with scrub typhus presented with normal EKG on admission and no hypotensive period, subsequently developed chest pain on 6 th hospital day. T inversion of EKG and diffuse ventricular dyskinesia of echocardiography appeared on 6th hospital day. We report a cases of acute fulminant myocarditis in adult with scrub typhus. This complication led to severe cardiogenic shock and death.
Streptococcus suis (S. suis) is a swine pathogen that is responsible for meningitis, septicemia, pneumonia and endocarditis. Since the first case report of human infection in Denmark in 1968, human infection with S. suis has been reported in many countries and especially Southeast Asia because of its high density of pigs. We report here on a patient with septic arthritis and bacteremia caused by S. suis. To the best of our knowledge, a case in which S. suis is isolated from joint fluid is very rare and this is first case report of S. suis infection in Korea. This organism was confirmed by phenotypic characterization and 16S rRNA sequence analysis. An 81-year-old Korean female who presented with fever, arthralgia and headache was seen in a secondary referral center in Korea. The aspirated joint fluid and the blood culture revealed growth of S. suis biotype 2, which was identified by the Vitek 2 GPI and API 20 Strep systems (bioMérieux, USA) and this organism was susceptible to penicillin-G and vancomycin. The 16s rRNA sequences of the blood culture isolates showed 99% homology with those of the S. suis subsp. suis reported in GenBank. Although her fever subsided and the subsequent blood and joint cultures were negative after antibiotic therapy, the swelling and pain in the left knee joint persisted. She is planning to receive total knee replacement.
Objectives: Tick-Borne Encephalitis (TBE) is an infectious zoonotic disease present in Central Europe but rare in Italy. Over the last 30 years a continuous increase in the disease morbidity was observed in the endemic countries, where about 13.000 cases occur each year. We report the first paediatric cases described in Italy. Case reports: First: A 7 year old boy came for a remarkable involvement of the Central Nervous System, 2 week after a flu-like syndrome. Non tick-bites were reported, daily consumption of non-pasteurized milk was referred. Lumbar puncture revealed clear cerebrospinal fluid (CSF) with polymorphonuclear pleocytosis, glucose and protein content were normal. The boy received treatment with dexamethasone, ceftriaxone, ampicillin and acyclovir, with regression of symptoms within 24 hours. TBE specific serology in blood and CSF was positive while serology for Borrelia burgdorferi revealed past infection. The diagnosis was a meningeal form of TBE. The boy had a normal neurological outcome. Second: A 12 year old boy came for the onset of walking difficulties, myalgia, leg heaviness, fatigue and nausea, after 4 days of fever and headache. To note the removal of two ticks on the day before the start of symptoms. Clinically he presented only a slight instability in the march on his heels. The blood examination revealed: leuconeutropenia, thrombocytopenia, elevated aminotransferase levels and significant creatine kinase increase. Serology for common viruses and bacteria, also Borrelia burgdorferi, were negative. A first TBE serology was positive only in IgM, after 15 days it was positive in IgM and IgG. There was no need for any treatment because of spontaneous regression of symptoms.
We described the first pediatric TBE cases documented in Italy. Generally the disease was transmitted by a tick of the genus Ixodes, even if there have already been described sporadic cases from non-pasteurized cow's milk, and it manifested by the typical diphasic febrile course (a flu-like syndrome in the first phase, involvement of the central nervous system in the second phase). In our first case no tick bites were known, while the boy often fed nonpasteurized milk and dairy products. The peculiarities of the second case was the short incubation period and the absence of the second phase of infection. These show that in children it is impossible to define a single clinical picture that characterizes the TBE, because of its several clinical forms.
Background: Clostridium sordellii (C. sordellii) is a Gram-positive anaerobic bacillus that has been reported as a rare cause of fatal toxic syndrome after medical abortion. Portugal's legal therapeutic abortion, before 10 weeks of gestation, was approved in 2007. We report a case of a young patient who underwent a medical induced abortion and died of a C. sordellii toxic shock syndrome. Case summary: A 16-year-old women who underwent a medically induced abortion by means of 200 mg of oral mifepristone followed by 800 mg of vaginal misoprostol, presented to the maternity hospital's emergency five days after receiving mifepristone, complaining of lipothimia in the night before and abdominal cramping. On admission, she was conscient, afebrile and hypotense (76/35mmHg). A few hours later she developed a rapid onset-sepsis with marked leukocitosis (83,400 white-cells/mL with 88% of neutrophils), hemoconcentration (hematocrit of 63.4%; hemoglobin of 21.2) and severe metabolic acidosis. The patient underwent a hysterectomy and uterus biopsy cultures and anatomopathological analysis were requested. Patient was transferred to intensive care unit and died 18h after presenting to emergency. At the microbiology laboratory a direct examination by Gram-stain smear of the uterine biopsy showed large, Gram positive rods. The biopsy was inoculated in appropriated agar plates mediums and incubated aerobically and anaerobically. 48 h later only the anaerobic culture was positive and colonies were smeared by Gram stain (showing Gram positive rods) and a C. sordellii was identified by the semi-automated system, Vitek 2 (bioMerieux ® ) with an ANC card (Anaerobes and Corynebacterium card). Discussion: This was a fatal case of pos-abortion C. sordellii sepsis. The distinctive clinical features developed are the same reported in other studies. Gram staining of a uterine biopsy is a good and rapid mean of having a presumptive result and help to diagnose. C. sordellii was identified through uterine biopsy cultures with a semi-automated system which is different from other studies that used anti-clostridium species immunochemical assay and PCR assays performed on formalin fixed uterine tissue pos-autopsy. Conclusion: To improve diagnosis Gram staining and cultures of an endometrial biopsy specimen are a good approach to an earlier recognition of the disease's etiology.
Objectives: The oropharyngeal form of tularemia is known to be common particularly in Eastern European countries including Turkey. The aim of the study was to evaluate the patients with tularemia and to make a point for the disease. Methods: Between February and August 2010, five patients with unilateral cervical mass were admitted to our outpatient clinic with the preliminary diagnosis of tularemia. The diagnosis of tularemia was confirmed by micro-agglutination test and PCR. Results: All of the patients had oropharyngeal tularemia with the suspicion of contaminated water ingestion. Age of the patients ranged from 19−59, three of them were male (60%) and two were female (40%). In initial stage of the disease, all the patients had general symptoms such as fever, headache, malaise and sore throat which lasted about two weeks, after that the general symptoms disappeared but the cervical lymph nodes started swelling in all patients. b-lactam antibiotics were administered to three of them in different centers before admission to our clinic. However there wasn't any response to these treatments. At admission unilateral cervical or submandibular lymphadenopathy and malaise were detected in all patients. One patient (20%) had suppurative lymphadenitis with spontaneous drainage. Leukocytosis was found in one patient (20%), elevated ESR in three (60%) and elevated CRP in three (60%) of patients. Lymph node aspiration was performed when fluctuation was detected but F. tularensis could not be grown in the cultures. However F. tularensis subsp. holarctica DNA was detected in four lymph nodes aspirates by conventional PCR. Micro-agglutination test was positive in four patients (80%) with titres of 1/640 in three patients (60%) and 1/160 in one patient (20%). The patients were treated with a streptomycin and doxycycline or a ciprofloxacin and doxycycline combination for a 2 weeks period due to a delay in initiation of treatment. One patient was received a second coures of antibiotics because of insufficient response to first therapy and continuing suppurative lymphadenitis. No severe complication was observed. Conclusion: Tularemia should be kept in mind in the differential diagnosis of patients with fever, pharyngitis or tonsillitis and cervical lymphadenopathy, especially unresponsive to b-lactam antibiotics in the endemic regions and early treatment with proper antibiotics should be started.
This work evaluated the clinical and parasitic status of malaria as a cause of fever among patients admitted to the Military fever hospitals. Thirty six patients were included twenty already diagnosed as malarial patients, who were recruited from Peace Keeping Mission Forces in Africa and sixteen cases presented with prolonged fever coming from different locations in Egypt.
The results showed that El-Gabal El-Ahmar area (Cairo) was the most extensively infested region (37.4%). This might be due to change of its ecolo-gical pattern since the year 2003 and the environmental conditions favoured by breeding and flaring mosquitoes. El-Sharkia and El-Fayoum Governorates (G.) were next in order (18.7%) and (12.5%) and this might be due to increased rural areas and agricultural projects and reestablishment.
Plasmodium vivax was the main species among locally acquired patients (81.25%), while the imported patients coming back to Egypt from Africa especially (Sudan) had P. falciparum (100%). However, P. falciparum was also present in 6.2% of cases from El Fayoum Governorate while P. ovale and P. malariae were not encountered. Of interest, was a case recruited from Ard-El-Golf, Heliopolis, an area with high social and hygienic standard, and the same condition applied to that from El-Nozha El-Gidida. Such cases included the "runway" or "airport" malaria, in which local transmission of disease has been attributed to an infected mosquito that was transported on a long haul flight. The two locally acquired cases were malaria positive by bone marrow smears and negative by peripheral blood examination. However, the thick blood film was the most sensitive (97.2%). The patients (75%) were clinically and parasit-ologically cured, but one patient died. The best therapeutic response for locally acquired malaria infection was the monotherapy-based one such as Chloro-quine or Mefloquine.
Background: Cat-scratch disease (CSD),caused by Bartonella henselae, is a benign, self-limiting disease in immunocompetent children with history of contact with cats. The organism is commonly found in the blood of cats and other felids. Classic presentation is tender and swollen regional lymph nodes with or without a papule at the site of initial infection. In immunocompromised patients, however, more severe forms of presentation can occur. Diagnosis is usually possible by serology and or histopathology. Case summary: An 14-year-old Kuwaiti girl, previously healthy, was admitted with fever, abdominal pain, nausea and generalized weakness. She had arrived from a trip to Egypt five days earlier. There was no history of diarrhea, vomiting, urinary symptoms or rash. On Examination the patient looked ill & had an oral temperature of 40 ºC with abdominal tenderness and guarding. Initial blood investigations showed a WBC of 14.1×10 9 , ESR 44 mm/h, C-Reactive Protein (CRP) 150 mg/l, urea 3.2 mmol/l and creatinine 39 umol/l. Urinalysis showed presence of RBC & WBC but the culture was negative. A diagnosis of appendicitis was made and appendicectomy was performed. The patient, however, continued to run high grade fever (>40ºC). Therapy with piperacillintazobactam (TAZ) and metronidazole was initiated but no clinical response was observed. CT of the abdomen showed mesenteric and para-aortic lymphadenopathy and multiple splenic lesions. A suspicion of enteric fever prompted changing TAZ to ceftriaxone. Blood culture, brucella agglutination, T spot test, & Widal test were all negative. several antibiotic courses including anti-TB & anti-fungal were tried but none helped to improve her condition. Finally, splenectomy and lymph node biopsy was done and the pathology report concluded multiple splenic granulomas with a picture suggestive of CSD. Serology, however, proved negative (IFA titre: IgG: <1:64; IgM: <1:20). History of contact with cats in Egypt was elicited from the patient after pathology report was available. There was a dramatic improvement in her condition following splenectomy.
To the best of our knowledge this is the first case of CSD being reported from Kuwait. We recommend to consider CSD in the differential diagnosis in children presenting with PUO, lymphadenopathy and splenic involvement. Despite several antibiotic courses, which included those recommended in CSD our patient showed defervescence only after splenectomy was performed.
Objectives: Salmonella is one of the most common causes of bacterial foodborne diseases worldwide. Recently, S. enterica serovar 4,[5],12:i:-emerged and is now among the most common serovars isolated from humans in many countries. This serovar is considered a monophasic variant of serovar Typhimurium (4,[5] ,12:i:1,2). In Greece, this monophasic serovar was firstly recorded in human isolates in 2007 (0.3% of total isolates), increased sharply the next two years and in 2010, it was the 3rd most frequent serotype (9.5% of total isolates).
In Results: As shown in Table 1 : a. Phage typing using the Typhimurium typing phages identified 5 different PTs. The most commonly identified PTs were DT120 (62%) and DT193 (24%), b. Eighty-six percent of the isolates expressed resistance to ampicillin, sulphonamides, streptomycine and tetracycline (R-type ASSuT) with or without additional resistance(s), c. PFGE analysis identified 8 unique profiles (A-H). Eighty-eight percent of strains were represented by three profiles (A,B,C) that shared more than 95% similarity. Conclusion: Combining phenotypic and genotypic characteristics, the most frequent pattern (54%) appeared the one with phage type DT120, R-type ASSuT (plus additional resistances) and the closely related PFGE profiles (A,B,C), followed by the pattern (18%) with phage type DT193, R-type ASSuT (with or without additional resistances) and closely related PFGE profiles (A,B,C). These results are consistent with the possible presence of two different clones of S. enterica serovar 4,[5],12:i:-, that prevail in both human and pig isolates in Greece. Similar data have been recorded in several European countries.
Background: The use of implantable cardiac devices such as permanent pacemakers (PPMs) continues to increase, with infection complicating up to 10% of inserted devices. Management of pacemaker endocarditis generally requires surgical removal of the entire system. Brucella suis is an uncommon cause of endocarditis, not previously reported in association with a PPM.
An 81 year old gentleman from Inuvik (Nunavut, Canada) with a PPM for 4 years was evaluated for syncope at the University of Alberta Hospital (Edmonton, Canada). Investigation revealed evidence of cardiac lead vegetations on a transesophageal echocardiogram. Preoperative blood cultures and intraoperative cultures taken during laser lead extraction became positive for Brucella suis (with MICs of 0.25 mg/L to each of ciprofloxacin, doxycycline, and rifampin). Upon further questioning, the patient a subacute history of constitutional symptoms, and reported hunting, skinning, and butchering Canadian caribou (Rangifer tarandus). The patient's brother, a hunting partner, had been treated for B. suis infection 2 years previously. Our patient was treated with laser lead extraction and combination oral antimicrobial therapy with doxycycline and rifampin for 3 months. Follow up blood cultures were negative. Discussion: Brucella suis type 4 is a known pathogen of Canadian caribou herds. It is hypothesised that cutaneous exposures when while skinning and butchering or ingestion of undercooked meat resulted in zoonotic transmission of the organism to our patient causing bacteraemia and secondary seeding of his pacemaker wires. From our literature search, this is the first reported case of pacemaker endocarditis due to B. suis. It is important that clinicians be aware of the possible zoonotic transmission of B. suis to patients exposed to caribou in the Canadian north and the risk of secondary infection of underlying foreign bodies (such as PPMs). Clinicians are also reminded to alert microbiology labs if Brucella is a suspect pathogen, to allow appropriate biosafety precautions.
Neisseria meningitidis, an agent of bacterial meningitis is classified into 13 different serogroups based on immunologic reactivity of its capsular polysaccarides. Serogroups A, B, C, W135 and Y are among the predominant serogroups. In Turkey, A, B, and C serogroups of N. meningitidis are encountered as the most frequent cause of menigococcal meningitis. A meningococcal vaccine containing serogroups A and C has been used to prevent recruits from this fatal disease till May, 2009. From then on, administration of the quadrivalent vaccine consisting of "A+C+W135+Y" serogroups has been implemented because of the emergence of menigococcal meningitis cases caused by serogroup W135. We present the first known menigococcal meningitis case caused by N. meningitidis serogroup X since the start of the quadrivalent meningococcal vaccine administration to recruits. Serogroup X has emerged as a cause of meningococcal disease in a susceptible population because of suppression of other serogroups not included in the quadrivalent vaccine. The results of mass vaccination campaigns have implied that the mass vaccination programs solely cannot prevent the disease totally and the improvement in environmental conditions especially in risky populations is also essential in prevention of the disease.
Background: Arcanobacterium haemolyticum (AH) is an extremely rare cause of infective endocarditis. We present here an unusual case of superinfection with AH in a primary Staphylococcus aureus (SA) mitral valve endocarditis in a Hepatitis B/C positive intravenous drug user [IVDU] . After a protracted course (with multi-organ impairment) of 3-months the patient was successfully treated. Case study: A 29-year-old IVDU male was transferred from emergency unit of neighbouring hospital to intensive care of this hospital on February 4, with signs of severe sepsis and seizures. CT head revealed small parietal bleed; lumbar puncture was non-conclusive and CTPA was negative for pulmonary embolism, but showed extensive non occlusive thrombus in superior vena cava. Systolic murmur prompted trans-esophageal echo revealing mitral regurgitation with vegetation. Following isolation of meticillin-sensitive SA grown from two sets of blood culture, the patients was treated with Flucloxacillin IV 1g q6h for 4-weeks. After 3 weeks of satisfactory response, the patient started getting intermittent pyrexia, elevation of CRP and WCC; mild renal and hepatic impairment. Blood culture with AH from single bottle was not considered significant and dose of Fluclox was increased to 2g q6h. Further pyrexia, raised inflammatory markers and impairment of renal functions prompted biopsy revealing focal necrotizing glomerulonephritis. Vibrio vulnificus is a human pathogen, frequently transmitted to humans via raw oysters and is isolated from the majority of raw oyster and shellfish harvested from subtropical areas during summer season. A 73year-old woman with diabetes mellitus and liver cirrhosis presented to the emergency department with generalized edema for one month and mental change on a day. She was diagnosed V. vulnificus septicemia and treated ceftazidime and doxicycline. We also isolated V. vulnificus from sea water of outbreak sea and same season (summer). V. vulnificus septicemia was emerged in Jeju island associated with climate change in island. This is the first report about relationship between clinical and environmental isolates according to the climate or environmental change in Jeju island, Korea.
21st ECCMID/27th ICC, Abstracts accepted for publication only observed a radiographic pattern of areas of consolidation often associated with patchy or interstitial infiltration. In fatal cases we observed consolidation with airbronchogram and diffuse alveolar infiltration. Extensive involvement of both lungs, presence of bilateral opacities, interstitial infiltration, lobar consolidation, and patchy infiltration were associated with poor prognosis. Patients with abnormal CT findings had greater risk of intubation, ventilation, ICU admission and death than those with normal CT, but some patients with similar CT findings had various outcomes from improvement and discharge to death. Conclusion: Chest CT findings may be significant in prediction of clinical outcome and management of the disease (e.g., initiation of antibiotics) in patients with influenza infection but CT findings alone does not exactly predict the outcome and other risk factors should be considered. Objectives: G glutamyl transferase (GGT) is an enzyme that is present in the cell membranes of many tissues. High GGT level is used as a prognostic factor for alcoholic liver disease, metabolic syndrome, cardiovascular diseases, liver cancer and liver metastasis of different cancers. Crimean Congo Hemorrhagic Fever has been emerging in Turkey for the last 8 years. Elevation of alanine amino transferase (ALT) and aspartate amino trasnsferase (AST) levels are used both severity criteria and prognostic factor for fatality in the management of CCHF. However, we observed an association between GGT levels and the course of the disease. In this study we aimed to figure out the relation between serum GGT levels and CCHF and its effect on survival, if there is any. Methods: Patients with laboratory-confirmed diagnosis of CCHF were included in to the study. Serum ALA, AST, gama glutamyl trasnferase (GGT) were retrospectively investigated from the records. Two parameters were used for evaluation of relation between CCHF and GGT; serum GGT levels and serum AST-GGT ratio. Because of AST elevation is a prominent feature of CCHF than ALT elevation, AST/GGT ratio was chosen as a marker. Results: Totally 126 patients were included. Of total, 71 (56.3%) were female. The mean age was 50.2 years (15−83 years). The mean hospital stay of the patients was 7.5 days (1−28 days). Remarkable laboratory findings were as follows (mean, min-max); WBC: 2472/mm3 (500-8900). Platelets: 59000/mm3 (7000-361000), AST: 291U/L (17-3060), ALT:133U/L (9-879) and GGT: 91 U/L (6-759). Comparing the serum levels of GGT between fatal and non fatal cases, mean GGT levels were statistically significantly higher in fatal cases on the 2nd and 3rd days of admission. Serum AST levels were also found higher in those days in the fatal group. The mean GGT level continued to increase during the course of the disease (Figure 1 ). When AST/GGT ratio was investigated, the ratio was >1 in the first 7 days of admission but <1 after the 7th day. Higher serum GGT level was the most prominent laboratory finding in all non fatal cases (Figure 2) . A positive correlation was detected between higher AST/GGT ratio and mortality.
Conclusion: Serum GGT levels can be used as a prognostic factor for CCHF. However an increase of GGT level during the recovery period of CCHF has not yet been fully explained. Therefore more studies are needed in this area of research. Background: Crimean Congo hemorrhagic fever is a serious viral hemorrhagic fever. Immunopathogenesis of the disease is not clear enough yet. Interleukin (IL)-12 is a pivotal cytokine that strongly stimulates Th1-associated cellular immunity, and activates humoral immunity to both T-dependent and T-independent antigens. IL-12 has an immunoregulatory functions which may play a role in promoting endogenous protective responses during infections and/or contribute to pathology resulting from unregulated cytokine expression. Pathogen induction of IL-12 elicits interferon-g production by natural killer cells, which contribute to early defense during certain bacterial, parasitic, and viral infections.
S. Tschudin Sutter*, R. Frei, M. Dangel, D. Goldenberger, A.F. Widmer (Basel, CH) Objectives: Actinobaculum schaalii is a Gram-positive, facultatively anaerobic coccoid rod, classified as a new species in 1997, formerly belonging to the species of Actinomyces suis. It grows slowly and therefore is easily overgrown by other bacteria, which are often found concomitantly. Since 1999, Actinobaculum schaalii is routinely investigated at our hospital, whenever its presence is suspected due to the detection of minute grey colonies on blood agar plates and negative reactions for catalsae. The objective of this study was to determine the clinical significance of Actinobaculum schaalii, identified in our microbiology laboratory over the last 11 years. Methods: All consecutive isolates with Actinobaculum schaalii were obtained from the computerized database of the clinical microbiology laboratory and patients whose cultures from any body site yielded this pathogen were analyzed. Observation of tiny colonies of Gram-positive, catalase-negative rods triggered molecular identification based on partial 16S rRNA gene sequencing. An infectious diseases specialist reviewed the medical charts and collected data regarding underlying diseases, clinical manifestations, antibiotic therapy, and clinical outcome.
A. Stavrianou*, V. Kalogeri, E. Koukou, A. Tsipou, S. Pagoni (Athens, GR) Objectives: BNP, PCT and CRP are well known indexes in critically ill patients such as those in sepsis and septic shock. The Purpose of our study was to determine which of these markers's serum levels increase more in sepsis and if they can predict the outcome or be a valuable prognostic marker.
We retrospectively examined 114 patients with febrile bacterial infections admitted to our department over a period of 2 (two) years. No one had a history of established cardiac failure. The diagnostic of the sepsis was based on clinical and laboratory data. We measured BNP, PCT and CRP in blood samples, taken the first 2 (two) days of hospitalization. The outcome was determined as survivors and nonsurvivors. Results: We had 66 female and 48 male patients. Among those patients, 68 were survivors (59,6%) and 46 (40,4%) non-survivors. There was a significant increase of BNP levels in non-survivors patients (1027) vs. BNP levels in survivors patients (432, 55) . Also the PCT mean levels were higher in the non-survivors's group (20,65) vs. the survivors's group (3, 6) . Finally, the mean levels of CRP in survivors was 167,21 and in the non-survivors was 166,4. Conclusions: In our study BNP and PCT levels measured soon after admission were increased significantly in sepsis and they seem to be a valuable prognostic marker in the outcome of these patients. The difference of the CRP mean levels in the 2 (two) groups was not significant. T. Cirkin Guzel*, N. Baykam, S. Menekse Yilmaz, A. Kocagul Celikbas, S. Eren Gok, H. Esener, S. Yaprakci, B. Dokuzoguz (Ankara, TR) Introduction: H1N1 influenza pandemic caused higher morbidity and mortality among pregnant women than in the general population underscores the medical community's urgent need for data regarding the safe and effective use of medications during pregnancy. Although the benefits of treatment appears to outweigh the theoretical risks of antiviral use there is few data about the newborns who borned from mothers who took medications for H1N1 during their pregnancy. In this study we aimed to investigate the clinical characteristics of H1N1 influenza in pregnant women and follow up the health status of newborns of these mothers after delivery. Methods: Twenty pregnant women with clinical symptoms and diagnosis of influenza A (H1N1) were included in this study from October 2009 to January 2010. All the study population were followed up until delivery and newborns were evaluated for any complication associated with infection or medication. Results: Twenty of 276 hospitalized patients with pandemic H1N1 influenza were pregnant (7.2%). The mean age was 24±3 (17−30years). Thirteen (65%) pregnant women were in 2nd trimestre, 5 (25%) were in 3rd trimestre and 2 (10%) were in 1st trimestre when they admitted to hospital. Although 80% of patients had pathologic respiratory auscultation findings, only 4 pregnant patients accepted chest X-ray test and all of them had bilateral patchy alveolar opacities. Two of them required mechanical ventilation. One of the mechanically ventilated pregnant woman died at the second day of hospitalization. Six of the patients didn't accept to get antiviral medication. Only one pregnant women who got antiviral medication (oseltamivir) had premature birth at 36th week. All other deliveries were in term and no complications were detected in newborns up to 4th week of their life. Conclusion: Although early evidence suggests that pregnant women may be at higher risk for severe complications (including stillbirth) from novel H1N1, the benefits of treatment appears to outweigh the theoretical risks of antiviral use. Early anti-viral therapy, may bring clinical benefits to pregnant patients.
F. Carletti*, A. Brozzi, C. Eleni, N. Polici, G. D'alterio, M. Scicluna, C. Castilletti, A. Di Caro, G. Autorino, A. Demetrio, G. Cardeti (Rome, IT) Objective: In July 2009, 5 llamas, farmed in Central Italy, exhibited skin lesions at different sites evolving from nodules to crusts; some of them had a crater morphology typical for pox lesions. On the farm, there are present many species of birds (local and exotic) and mammals (goats, cattle, pigs, donkeys, horses) none of which showed any of the above mentioned symptoms. Methods: From one moribund female which was euthanised samples were collected for laboratory analysis. Skin lesions were processed for electron microscopy techniques. Two mammal cell lines (Vero and BHK21) were used for virus isolation attempts. Total viral DNA was extracted from both homogenized crusts and Vero cells supernatant. Real-time PCR, targeting the crmB gene, sequencing and phylogenetic analysis were performed to identify the virus. Other viral and bacterial diseases were considered as differential diagnosis. Results: Transmission electron microscopy revealed brick particles typical of orthopoxviruses. CPXV-antibodies were detected from llama and human sera (farmers). A Real-Time PCR gave positive results for both crust and cells supernatant. Phylogenetic analysis of two poxvirus genes (HA and crmB) confirmed a Cowpoxvirus infection, homologous to the CPXV-GuWi strain isolated in Germany in 2007.
After this outbreak, no other symptoms have been described among animals and human beings in the farm and no other news about CPXV have been reported in Italy. About the origin of the infection, all the llamas were born in the farm and had had no contact with exotic mammals, as declared by the owner. we can suppose that the virus has been introduced in the farm through bred mice, used as food for birds of prey. Such mice came from a German farm, which had sold infected rats to a zoo were mongooses have recently resulted affected by CPXV.
A. Beltrame*, C. Castilletti, A. Troi, A. Arzese, E. Ndip Nganyuo, D. Lapa, A. Di Caro, B. Del Pin, M. Capobianchi (Udine, Rome, Veneto, IT) Objectives:
R. Tomas*, A. Malet, B. Font, M. Espasa, L. Falgueras, J. Prats, D. Fontanals (Sabadell, ES) Introduction and Objectives: Transrectal ultrasound guided prostate biopsy (TRUSPB) is the technique of choice in the diagnosis of prostate cancer. There is no consensus on antibiotic, dose and duration of the prophylaxis.
The main objectives of the study were: to calculate the incidence of postbiopsy bacteremia (PB), to know the microorganisms and their antibiotic susceptibility and to evaluate the effectiveness of different antibiotic prophylaxis used in our hospital. The PB incidence based on the prophylaxis regimen was: ciprofloxacin − 1.04% (6/575); cotrimoxazol − 2.49% (29/1166) and tobramycin − 3.60% (13/361). The lower incidence of PB in ciprofloxacin's group is statistically significant (p < 0.05 versus cotrimoxazol; p < 0.01 versus tobramycin).
The global incidence of PB was 2.06%. Escherichia coli was isolated in 98% of episodes. Ciprofloxacin prophylaxis was the most effective regimen with lower PB, however susceptibility of E. coli isolates wasn't optimal (73.5%). Taking into account that E. coli susceptibility to ceftriaxone was of 98%, the use of this antibiotic as a new prophylaxis regimen could reduce PB incidence.
Objectives: The objectives of the study were to evaluate healthcare workers' (HCWs') compliance to the hand hygiene guidelines issued by the World Health Organization in 2009, and to assess their knowledge regarding and perceptions toward hand hygiene. We also searched for associations between observed compliance and various factors. Methods: The study was conducted in "Evaggelismos" Hospital, a 950-bed, tertiary care general hospital in Athens, Greece, between March and June 2010. Compliance data collection was based on direct and overt HCW observation during patient care, while anonymous, self-administered questionnaires were used to collect information on knowledge and perceptions. We used the data collection forms and questionnaires provided by WHO. Results: During 41 observation sessions with average duration of 18.6 min, we recorded 587 hand hygiene opportunities. Total average compliance was 13.6%. Compliance was significantly higher among physicians than among nurses [21.6% vs. 9.5% (p < 0.001)]. Professional category (doctor vs. nurse), type of indication (before contact vs. after contact), day of observation (after "on take" day vs. other days) and activity index were found to correlate strongly with compliance. 142/228 knowledge questionnaires were completed. The average number of correct answers to the 25 knowledge questions was 12.38 (SD = 3.40) and differed significantly among age groups, professional categories, departments and wards. Only 4.2% of HCWs answered correctly to more than 19 (75%) questions. 139/228 perception questionnaires were completed. Behavioral beliefs in favor of hand hygiene seemed to be strong and produced a positive attitude toward the behavior. Normative beliefs were also relatively favorable with respect to hand hygiene. As for control beliefs, most HCWs felt that a big effort is required to perform good hand hygiene. According to HCWs' views, the most effective action to improve hand hygiene in the hospital is to make alcohol-based handrub always available at each point of care. Patient empowerment is regarded as the least effective measure. Conclusions: Compliance rates in "Evaggelismos" Hospital are very low compared to those of other studies carried out in Greece or in other countries. Doctors' and nurses' level of knowledge is not satisfactory, while low perceived behavioral control seems to exert strong influence on HCWs' behavior. Action needs to be taken, involving a combination of system change, education and motivation.
R2572 Does oral care with chlorhexidine influence bacterial epidemiology or incidence of nosocomial pneumonia in the intensive care unit? Z. T. Yang*, Y. Chen, Y. Cai, Y.J. Yang, S.Y. Zhao, Z.Q. Che, C. Zhu, W.J. Zhou, M. Zhong, F. Jing, E.Z. Chen (Shanghai, CN) Objective: To investigate the effect of oral hygiene with chlorhexidine influences bacterial epidemiology or incidence of nosocomial pneumonia in intensive care unit. Methods: Patients admitted and stayed for more than 48h to our 18bed polyvalent ICU of a Chinese tertiary care teaching hospital were enrolled in our study. From May 2008 to April 2009, during preintervention period, protocol of oral hygiene consisted of metronidazole in the morning and boiled water in the evening and has been changed to 0.2% chlorhexidine gluconate twice daily since May 2009. Bacterial epidemiology of nosocomial pneumonia (NP) and ventilator associated pneumonia (VAP) was studied. Statistical analysis was performed by SAS 8.0. Results: Five hundred and forty-two patients admitted in our ICU from May 2008 to April 2010, of which four hundred and eighty-seven patients were screened, 234 patients, 103 episodes of NP, of which 44 VAP (21/33 for patients intubated) for 1070 device-day, occurred during the pre-intervention period; while 253 patients, 93 NP, of which 42 VAP (28/43 for patients intubated) for 1205 device-day during the intervention period. There was no significant difference between the intubated patients for sex (60.6% vs. 78.7%, p = 0.078), age (62.6±21.3 vs. 62.2±17.7, p = 0.968), median ICU stay (37d vs. 29d, p = 0.259) and ventilator-day (1070d vs. 1205d, p = 0.496). Overall rate of catheter related pneumonia (VAP) and rate of NP was 37.8 (episodes per 1000 ventilator-day) and 20.0 (episode per 1000 patient-day), 41.1 and 34.9; 24.3 and 16.7 for preintervention and intervention period, respectively. Among 143 bacteria isolated for the first year, 32 (22.4%) were G+, of which were18 MRSA, 111 (77.6%) were G-, of which 63.1% were non-fermentive bacteria. During the intervention period, among 113 bacteria, 28 (24.8%) were G+ of which were 19 MRSA, 85 (75.2%) were G-, of which 72.9% were non-fermentive bacteria. For MRSA and non fermentive bacteria distribution, there was no significant difference between 2 studied periods for VAP. Conclusions: Oral hygiene with 0.2% chlorhexidine twice daily changed little bacterial distribution of NP in ICU compared with pre-intervention period. However, the slightly decreased 15% (41.1 to 34.9) incidence of VAP by episodes per 1000 catheter-day and 31% (24.3 to 16.7) incidence of NP by 1000 patient-day were observed during the intervention period suggests a possible benefit of oral hygiene for ventilated patients in ICU. (1987.9-1990 .2), period B (1990.3-1997.8), period C (1997.9-1999.2), period D (1999.3-2004.10), and period E (2004.11-2010−8) , and compared the periods. In period B, Cefazolin and cefotiam were administered as prophylaxis. The dosing continued for 4 days, including the day of surgery. Moreover, nonscreening pre-emptive isolation and cohorting (NSPEI&C) were applied to the patients undergoing endotracheal intubation or tracheotomy who were admitted to single rooms (isolation) and multiple patients with similar conditions in one room (cohorting), regardless of whether or not MRSA was isolated. Since August 1997, however, the idea of evidencebased medicine (EBM) has spread over Japan so that we had no other choice than stopping NSPEI&C because NSPEI&C had no confirmed evidence. Since then, MRSA become isolated from postoperative patients even more often. Then, NSPEI&C was strictly instituted again, and the dosing period of prophylactic antibiotics for postoperative infection being shortened or extended, the isolation rate of MRSA after digestive surgeries could be significantly reduced. We report herein the method to prevent the isolation of MRSA for two decades. However, NSPEI&C was stopped in period C, but was implemented again, and prophylactic antibiotics were administered only on the day of surgery in period D. In period E, prophylactic antibiotics were administered for 3 days. Objectives: Infections of prostheses and surrounding tissue are one of the most serious complications in orthopedic surgery. Most, these infections caused by staphylococci, especially Staphylococcus aureus. Nowadays S. aureus often has a complete resistance against b-lactams (methicillin-resistant S. aureus (MRSA)), mostly connected with a resistance against other antibiotics, e.g. lincosamides, quinolones. Exchange of the implant and temporary implantation of antibioticcontaining spacers is the mode of choice in treatment of these biofilmassociated infections. Our in vitro-study was aimed to determine the antimicrobial activity of antibiotic-containing specimens over a time period of up to three months. Methods: Specimens were prepared from commercially available bone cements (without antibiotics, with 12.5 mg gentamicin/g, with 25 mg gentamicin and 25 mg clindamycin/g cement). Additionally the cement containing 12.5 mg gentamycin was supplemented with 25 mg and 100 mg/g vancomycin. Specimens were placed in nutrient broth with five test strains (2 methicillin-susceptible S. aureus (MSSA), 2 MRSA, 1 S. epidermidis). If bacterial growth was visible in the broth specimens were removed and bacteria attached to the surface of the specimens were counted. Further, specimens were soaked into nutrient broth and phosphate buffered saline; eluates were taken for determination of antimicrobial activity by means of agar diffusion test. Results: Cements containing gentamicin and clindamycin showed a higher antimicrobial activity against MSSA and S. epidermidis in comparison to cement containing only gentamicin. All specimens were infected 15 d after starting the experiments. Specimens with gentamicin and clindamycin inhibited biofilm formation and acted antimicrobial against MSSA strains and S. epidermidis strain up to 84 days. Cements with gentamicin also combined with clindamycin did not have any effect on MRSA strains. Only supplementation of vancomycin was able to inhibit growth of MRSA strains; concentration of 100 mg was more active up to 25 days.
Microbiological diagnosis should exclude as fast as possible an infection by MRSA. If infection is caused by an MSSA, combination of gentamicin and clindamycin can be recommended. For treatment of MRSA, application of an vancomycin-containing cement seems to be necessary.
Pseudomonas aeruginosa and Acinetobacter baumannii using the supersonic wave and plasma irradiation Y. Nakano*, S. Fujimura, T. Sato, H. Takane, A. Watanabe (Sendai, JP) Objectives: The nosocomial infection caused by Acinetobacter baumannii and Pseudomonas aeruginosa has been problem worldwide. It is known that these bacteria contaminate the water supply environments such as faucet, sink and bath of the hospital for a long time. And it is extremely difficult to sanitize strains on these environments. The aim of the present study is to evaluate the bactericidal effect against biofilmformed these strains using the supersonic wave or plasma irradiation.
The clinical isolate of A. baumannii and the standard strain of P. aeruginosa PAO1were used in this study. To form biofilm, the 5cm square stainless steel plate was added in TSB of each bacterial suspension of McFarland No.0.5 and incubated at 37ºC for 24hr under anaerobic conditions. These stainless plates were added in water tank and irradiated a supersonic wave of 38 kHz for 5, 10 or 30 minutes. Whereas, a plasma flow generated by a dielectric barrier discharge in air at 12 kVpp with 2 k Hz was irradiated to another plates with each biofilm-formed bacteria for 1, 10 or 30 minutes. After irradiation, each plate was cultured on the MH agar plate at 37 oC for 24hr. Bactericidal effect was observed using the scanning electron microscope (SEM) and the above-mentioned cultural method. Results: Both strains of A. baumannii and P. aeruginosa except biofilmformed strains were sterilized by the supersonic wave for 5 minutes. However, biofilm-formed both strains were not sterilized by that of 30 minutes. On the other hand, all strains were sterilized within 10 minutes by the plasma irradiation. With or without biofilm, the bursting of bacteria by the plasma irradiation was confirmed using the SEM.
This study results showed that the plasma irradiation is effective for sterilization of the hospital environmental contamination by P. aeruginosa and A. baumannii.
A. Babak*, S.M. Zahraei, Z. Pezeshky, Z. Nokhodian, B. Ataei, M. Ataie (Isfahan, IR) Objectives: Nosocomial infections are those which are created during hospitalization or as its result. The health care workers' hands are the main route of microorganism transmission. Hand washing is the most important measure to prevent spread of this infection. Compliance of hand hygiene (HH) among healthcare workers is generally low (less than 50%). This study aimed to determine the acceptance level of hand hygiene in Isfahan hospitals, 2010 to design interventions to increase awareness, motivation and performance among Healthcare workers. Methods: This cross sectional study was conducted in three large hospitals in Isfahan (a public training medical center, a public medical center, and a private medical center) and a total of seven trained observers for different working shifts at three hospitals were selected. 7 working days during two different weeks and every day, eight 20-minute sessions was considered. During these sessions, observers monitored healthcare workers during their routine activities through a check list. Five indications (before touching a patient, before a procedure, after a procedure or body fluid exposure risk, after touching a patient, after touching a patient surroundings) were observed based on the World Health Organization guideline. Four different professional groups (physician, nursing, student, others) were considered for this study. HH compliance was calculated as "(number of positive hand hygiene actions/number of total hand hygiene indications) × 100" based on each indication and professional group. Results: From the total 3097 observed indications, in general, the HH compliance rate was 5.6% and statistically significant between three different hospitals (7.4%, 4.1%, and 1.4% respectively). The most rates were observed among the nursing group (7.3%), and then the student group (6.5%), (p-value less than 0.001). Among the various indications, the highest rate of compliance was observed after a procedure or body fluid exposure risk (9.4%), (p-value less than 0.001). Conclusion: In this study, the hand hygiene compliance rate in hospitals studied was low and needs to plan for improve. Phadebact monoclonal antibody typing (Boule Diagnostic AB, Hudding, Sweden) was performed on these isolates. Penicillinase production was performed using nitrocephin method. Results: All 36 isolates were WII/WIII (IB serovars), yielded 100% invitro susceptibility to ceftriaxone (MIC 0.5mg/l) and spectinomycin (MIC 128mg/l). Three isolates (8.3%) were resistance to penicillin (MIC 2mg/l) and were penicillinase producing strains. Twenty eight isolates (77.8%) yielded reduced susceptibility to penicillin (MIC 0.12−1mg/l). Thirty one isolates (86%) were resistance to penicillin or yielded reduced susceptibility to penicillin. Five isolates (13.9%) were resistant to ciprofloxacin (QRNG) (MIC 1mg/l) of these five isolates three yielded penicillin reduced susceptibility pattern while two were resistant to penicillin. Six (16.7%) were resistant to tetracycline (MIC 2mg/l) (TRNG). All the ciprofloxacin resistant isolates were from male subjects. Seven distinct antimicrobial susceptibility patterns were observed. A 0.74% isolation rate was obtained in this study population. Conclusion: Ciprofloxacin and penicillin resistance is increasing. Cephalosporin resistance was not observed but, local continuous antimicrobial surveillance is paramount and should be ongoing. There was no distinct link between the phadebact monoclonal typing of these isolates and their antimicrobial susceptibility pattern.
J. Wojkowska-Mach, D. Romaniszyn*, A. Chmielarczyk, B. Wojcik-Stojek, B. Gryglewska, T. Grodzicki, M. Pobiega, P.B. Heczko (Cracow, PL) Nosocomial infections have been a very well-know public health problem with many consequences like medical, economical, ethical etc. Information about epidemiology and microbiology is used as a basis for legislation of special programs for infection prevention and control in hospitals and long term facilities (LTCF). Unfortunately, is not known situation in polish LTCF.
The aim of this work was to analyze the epidemiology of infections and multidrug resistance organisms among 108 residents in one LTCF. Results: 108 residents and 33 968 resident days (pds) were included in the studied period. 61,6% of studied residents had wheelchair disability or were bedridden, 10,6% were disoriented in time and/or space, the average age was: 76,3. Prospective epidemiological surveillance was carried out, with trained professionals using McGeer definitions. The incidence density per 1000 pds was 3,6. Objectives: Foreign body (external fixator, prosthesis) infections cause a significant morbidity and mortality in orthopedic surgery. Silver ion coating of metal external fixators can reduce bacterial colonization consequently infections. Methods: In the study, 66 metal external fixators were used. Titanium (22 TiA14V) external fixator was coated with silver ion containing calcium phosphate based ceramic powder by using electrospray method. 22 implants were coated with hydroxyapatite in the same way, and the remaining 22 external fixators were without any coating.
To induce experimental infection, Staphylococcus epidermidis clinical isolate which have slime factor, was used. In the study, 1×10 4 CFU/mL suspension of the clinical isolate in tryptic soy broth was prepared. Following 24 h incubation, quantitative culture of bacteria and determination of silver ion by atomic absorption were performed on the broth. Quantitative culture of bacteria on the external fixators was performed in addition to microscopic examination and the possible antibacterial efficacy of implants due to silver ion coating was investigated for 8 weeks (day 2, week 2, 4, 6 and 8). Accordingly, the implants in glass tubes (diameter 0.4 cm) were placed in shaking incubator at 35 ºC/80 rpm. Implants were rinsed with distilled water in aseptic conditions once a week. Results: The bacterial growth was statistically higher in broth containing titanium external fixators compared to broth media containing silver ioncoated and hydroxyapatite-coated external fixators at 24 hours (p = 0.036 and p = 0.009, respectively). The release of bacteria from silver ioncoated fixators was statistically less compared to hydroxyapatite-and titanium-coated fixators (p = 0.039 and p = 0.002, respectively). MIC levels for 5% silver ion powder was 8 mg/mL for CNS. No free silver ions were detected in broth media using this method which has a detection threshold as low as 0.02 ppm. In electron microscopy, it was observed that less bacteria adhered to silver ion-coated external fixators.
We observed that there was no bacteria release from silver ion-coated external fixator to surrounding liquid. As a result of silver ion coating of metal external fixators, bacterial colonization reduces significantly. Such effect may help preventing post-operative infections caused by external fixators commonly used in orthopedic operations.
In-vivo evaluation of this effect is warranted. -the IPSE Core Curriculum for Infection Control Practitioner was submitted to the national representatives, who were asked to score(?or grade?) each item of the curriculum: -in a meeting held in Udine, results, graduation and future endorsement strategies were discussed; -after the meeting a coordination group elaborated the final document; -this document was sent again to all NCPICT for their final approval. All 33 NCPICT participated in the process and approved the document. Results: The proposed European Core Competencies aim to be helpful in: -standardizing Infection Control/Hospital Hygiene Professionals (IC/HHPs) competencies in Europe; -designing and implementing training courses according to different national contexts and facilitating the mutual recognition of competence across Europe; -providing an opportunity for IC/HHPs to review their own performance and to plan their professional development; -providing an opportunity for health care institutions and organizations to evaluate their needs in terms of professional human resources. The document is organized into three main areas: Programme Management, Quality Improvement and Infection Control. Each consists of different professional tasks common to infection control doctors and nurses (except one: related to antibiotic prescribing). For each professional task, competencies were classified into foundation practice (this applies to newly appointed IC/HHP staff with little or no previous experience and expert practice (for a IC/HHP confident and experienced in all competencies). Conclusions: We have achieved a consensus document on European Infection Control/Hospital Hygiene Core Competencies. However, to ensure that they will make ae a positive impact it will be necessary to spread and endorse them with all the relevant stakeholders.
Objectives: To assess the influence of a new carbapenem-based deescalating strategy of antimicrobial therapy in treatment of infectious complications of severe burn injury by resistance of nosocomial Gramnegative bacteria. Methods: A retrospective analysis of antibiograms from burn patients admitted to our burn care unit (BCU) of the sensitivities to common Gram-negative pathogens before (2007)
The implementation of a de-escalation strategy of treating infectious complications of thermal burns in our burn care unit increased the susceptibility of multidrug-resistance to Gram-negative pathogens.
There is a potential benefit in reducing the overall costs of treatment, due to direct costs of antibiotic treatment and shortening the length of stay in the hospital without increasing the mortality rate.
Background: This is the first large-scale study that focused on the prevalence of LTBI in the laboratory personnel, especially in the intermediate incidence setting. Methods: We recruited 173 laboratory personnel and performed QuantiFERON-TB Gold In-Tube test (QFT-G) and tuberculin skin test (TST). Agreement between QFT-G and TST was analyzed, and risk factors for the positivity of each test were assessed. Results: QFT-G was positive in 21.4% of the enrolled laboratory personnel, and TST was positive in 33.3% with a cut-off of 10 mm. The agreement between the two tests was fair (kappa = 0.234; 95% confidence interval = 0.054-0.414). Age and duration of health care profession were significantly associated with positive QFT-G and TST results by univariate analyses. With multivariate logistic regression analyses, household contact to TB patients (P = 0.013), the laboratory sections of microbiology (P = 0.045) and chemistry/immunology (P = 0.014) were significantly associated with positive QFT-G results. Conclusions: Our data shows the high prevalence of LTBI in the laboratory personnel, and emphasizes the importance of LTBI screening for the health care workers. QFT-G seems to be superior to TST for the LTBI screening. T. Panayea, G. Petrikkos, A. Armaganidis, H. Giamarellou (Chaidari, Athens, GR) Objectives: Copper has been shown to have antimicrobial properties and when used on hospital surfaces it minimized environmental contamination by pathogenic bacteria. We investigated the effects of two copper alloys on the survival of VIM and/or KPC-producing multi-drug resistant (MDR) Enterobacteriaceae. Methods: MDR clinical isolates were screened for the production of VIM or KPC carbapenemase using the EDTA-imipenem or the boronic acid-meropenem synergy test, respectively. The genes were confirmed by PCR with specific primers. To assess the antimicrobial properties of copper, coupons (1 cm × 1 cm) of copper alloys (A and B containing Cu99% and Cu63%Zn37%, respectively) were sterilized, inoculated with 20 ml of bacterial suspension (10 8 cfu/ml) and incubated at room temperature for 0, 1, 3, 5, 6 and 24 hours. Then coupons were placed in sterile neutralizing broth (Difco) and vortexed. The broth was serially diluted and quantitatively cultured for recovery of viable bacteria. The lower limit of detection was 2.6 log10 cfu/ml. Stainless steel (C) and polyvinylchloride (D) coupons were used as controls. Mean viable counts (log10 cfu/ml) after incubation at each time interval with each coupon were compared for statistical analysis. Reduction of viable counts by 3 log10 from starting inoculum was characterized as bactericidal activity. Results: Thirteen isolates including K. pneumoniae (7), E. coli (4) and Enterobacter spp. (2) producing either VIM (2), KPC (10) or both (1) were tested. At 3 and 5 hours, mean viable bacterial counts on coupon A decreased by 1.2 and 3.4 log10, respectively while the reduction produced by coupon B at 5 and 6 hours was 2.1 and 3.2 log10, respectively. Controls C and D reduced bacterial counts by 2.1 log10 at 6 and by 2.3 log10 at 24 hours, respectively. A bactericidal effect was detected by coupons A and B at 5 and 6 hours, respectively. Viable bacteria could not be recovered from 10/13 tested strains incubated on coupon A after 5 hours and from 8/13 strains after 6 hours of incubation on coupon B.
Copper alloys reduced the number of viable carbapenemase-producing bacteria significantly at 3 hours and produced a bactericidal effect at 5 hours. Cu99% was more effective than CuZn. These data suggest that the use of copper as surface material in the hospital could aid in diminishing the environmental reservoir of MDR Gram-negative pathogens.
R. Guner, I. Hasanoglu, S. Keske, M.A. Tasyaran* (Ankara, TR) Objectives: Central venous catheter (CVC) usage is becoming more and more frequent in intensive care units (ICU). Besides its mechanical complications, catheter related bloodstream infections (CRBSI) are frequent, preventable and costly with high mortality and morbidity. Although it is essential for an ICU patient, urinary catheterisation also brings the risk of catheter related urinary tract infection (CRUTI). We aimed to decrease our CRBSI and CRUTI rates with observation, education, feedback of infection rates, and reward.
We performed an observational intervention study in a 550bed education and training hospital in Ankara, Turkey with twelve neurology-neurosurgery, ten coronary, six cardiovascular surgery and nine general ICU beds. The study was carried out in three phases, as preintervention, intervention, and postintervention. After six months of preintervention period, we performed education meetings to all ICU doctors and nurses. Education program highlighted prevention bundle of urinary catheter (UC) and CVC related infections. We observed CVC and UC with checklists and follow-up charts. CRBSI and CRUTI diagnosis were performed according to CDC definitions. Six months after the education, all staffs of the departments which decreased their infection rates were promised to be awarded with 10% pay raise. Results: During the study period 462 CVCs and 1258 UCs were inserted to 1075 patients and catheter days were 4313 and 6918, respectively. Most observed indications of CVC are peripheral intravenous access problem (41%) and haemodialysis (29%). Insertion sites were 41% jugular and 41% subclavian. 20% of CVCs were inserted in emergency conditions. Mean lenght of catheterisation was 11 days. Compliance to maximum barrier precautions was 41%, but this rate was correlated with doctors' education years. Most common displacement reasons for CVC were exitus (50%) and discontinuity of indication (32%). Compliance to prevention bundle was 13%. Mean lenght of catheterisation was 13.25 days. There was a relationship between the clinic which inserted the catheter and compliance to maximum barrier precautions for CVCs and UCs. We decreased our CRBSI rates from 8.2 to 3.19 (61%) and CRUTI rates from 3.7 to 2.19 (40.8%).
Our study showed that education, feedback of infection rates, and reward are effective for reducing CRBSI and CRUTI rates but continuity of education is essential for maintaining low infection rates.
C. Carrilho*, J.C. Pascual Garcia, R.A. Belei, N.S. Paiva, N.J. Cornetta, R.L. Oricolli, C. Grion, M. Pelisson, S.F. Costa (Londrina, BR) Objectives: Report an outbreak of carbapenem-resistant enterobacteria in a university hospital, control measures and mortality associated with these pathogens. Conclusions: Carbapenem-resistant enterobacteria are a great challenge concerning control and treatment. These outbreaks take place frequently in the Intensive Care Units, nearly half the patients manifest infections and mortality among infected patients is high.
C. Fariñas-Alvarez*, P. Rodriguez-Cundin, M.L. Fernandez-Nuñez, O. Gonzalez-Martinez, I. De-Benito, A.B. Campo-Esquisabel, R. Teira, G. Marcano (Santander, ES) Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most important multidrug-resistant bacteria. Its surveillance and control must be a priority in a hospital setting. The aim of this study is to describe the incidence of MRSA in our hospital after the implementation of a prevention and control programme.
The programme of prevention and control was implanted in 2003 after a nosocomial MRSA outbreak, with an incidence of 0.24% in 2002. It is based in a bundle of recommendations: identification, isolation and control of the incidental cases, early detection of the condition of carrier and record of incidental cases in a database. This computing record is linked to the hospital information system (HIS) that allows us to detect the hospital readmissions of colonized/infected patients. These patients are isolated immediately. Results: After the programme implementation, a gradual increase of the incidence was observed in the last six years at expense of communityassociated MRSA (0.08% in 2003, 0.09% in 2004, 0.13% in 2005, 0.15% in 2006, 0.16% in 2007, 0.16% in 2008 and 0.25% in 2009 ) while the incidence of nosocomial MRSA has kept around 0.1% (0.15% in 2003, 0.10% in 2004, 0.11% in 2005, 0.07% in 2006, 0.14% in 2007, 0.12% in 2008 and 0.12% in 2009) . The most frequent location of infection for nosocomial MRSA during these years has been always the low respiratory tract infection with or without pulmonary condensation (25.0%) followed primary bacteraemia (11, 8%) , and for communityassociated MRSA the cutaneous infections (72,7%). All patients were isolated after detecting the MRSA: the daily average was 5 isolated patients which represented 2.5% of hospital bed occupancy.
We have not detected any nosocomial MRSA outbreak in our hospital from 2003. The incidence of nosocomial MRSA is kept in rates around 0.1%. It is important to emphasize the decrease of the proportion of nosocomial MRSA isolations, whereas communityassociated MRSA have increased. A dynamic record connected to the hospital information system allows us to identify and control the colonized/infected patients before any entry to the hospital.
Objectives: Retrospective evaluation of the results in the detection of MRSA of a commercial PCR (Genohm ® , Becton Dickinson) compared to culture (chromID™ MRSA, Biomérieux) after one year use (2009) in two geriatric wards (60 beds) and an intensive care unit (8 beds) of a 415 bed community hospital. Any impact on MRSA epidemiology is also evaluated. Methods: All patients were screened on admission in the geriatric wards and in the intensive care unit. Swabs taken from nose and perineum were pooled, tested by PCR and inoculated on chromogenic agar to detect MRSA. No enrichment in broth culture was made. Samples arriving on Friday evening and in the weekend were not examined by PCR. Patients were isolated as soon as MRSA positive results of PCR or culture were known.
In our hands the Genohm ® PCR test had a rather low sensitivity and positive predictive value compared to culture even if culture was not ideal (only two sites, no enrichment). Introduction of the PCR was meant to reduce transmission of MRSA and hence reduction of infection by faster isolation because of the speed of the test. We can not deduce from our data that this policy has had any impact on the decrease of nosocomial MRSA infection.
A. Guleri*, R. Palmer, L. Moorhouse, S. Staff, N. Harper, J. Lickiss (Blackpool, UK) Background: Blackpool Victoria, a large district teaching foundation trust hospital in NW England, operates a very successful comprehensive CDI containment programme. There is special emphasis on antibiotic stewardship and real time programme monitoring initiatives like root cause analysis of all CDIs in community/acute trust, multidisciplinary audit and surveillance. We present findings from 1st 6-months of an innovative pilot project "multidisciplinary CDI performance management meetings [MCPM]" aimed to spearhead the trust CDI programme in future. The aims of MCPM include team working, problem solving, real time "shop-floor" troubleshooting, raising profile and awareness in a friendly interactive session. The results have been used to inform a regularly monitored strategic action plan. In 2008 we recorded one P. aeruginosa PAN-R strain, sensible only to colistin. In May-September 2008, an unexpected high incidence of VRE was reported. AFLP analysis of these VRE strains documented a genetic relationship between two different groups of isolates: 2 isolates show similar AFLP patterns but different from those of the other 4 genetically related isolates. These data suggest a possible person to person transmission. After corrective measures no other case of VRE was documented. No Gram-negative outbreak was recorded in the same period.
Our results showed a new increase of Gram-negative infections and a progressive antimicrobial resistance. The high discriminatory AFLP method was fast enough to allow a 'real-time' monitoring of the outbreak, permitting additional preventive measures.
R2593 Clostridium difficile detection: identification of colonisation, subclinical and overt disease F.M. Awadel-Kariem*, C. Malone, H. Gough, H. O'Connor (Stevenage, UK) Background: Detection of C difficile or its toxins has been observed to be problematic. False negative results have meant that patients were tested repeatedly when symptomatic. Among the reasons for poor negative predictive value are the labile nature of the toxins and the limitations of available technologies. Newer and more sensitive technologies were introduced such as PCR. These technologies have reduced the rates of false negatives and eliminated the need for multiple samples. In recent years, and through a combination of infection control measures, CDAD rates were reduced dramatically. However, these successes brought to the front the significance of high false positive rates especially with the ultra-sensitive new technologies. It is wellestablished that toxin-producing C. difficile can colonise young children and older patients with history of repeated and prolonged hospitalisation. There is a need for a new approach to determine the clinical value of a positive result. Objectives: This study was designed to assess the concordance between different toxin assays and clinical presentation in Antigen positive/Toxin negative patients. Methods: Twenty six Antigen positive/toxin negative stool samples (Techlab EIA for both GDH Ag and toxin) were included in the study. Patients with multiple samples were identified. Samples were then tested using VIDAS EIA, culture and ribotyping, CEPHEID GeneXPert PCR and cytotoxicity assay. Clinical presentation and follow up data were then analysed to assess clinical significance of each result. Results: Patients who had alternative explanations for their transient diarrhoea (e.g. laxatives), were found to be toxin-negative by EIA. However, 86% of these samples were positive by PCR and all produced cytotoxic effects following culture. Isolates were from a number of ribotypes (001, 002, 015 and 054). Conclusions: These results suggest that combined clinical/laboratory interpretation is of more value at the current epidemiological setting. Highly sensitive technologies, such as PCR, may detect the presence of toxin genes in cases of colonisation or subclinical presence of C. difficile. Our data suggest that samples found to be Antigen positive but toxin negative by EIA, in the presence of alternative clinical causes for diarrhoea, should be treated as colonisation. However, these patients should be monitored closely and should be isolated, while having diarrhoea, to limit environmental contamination.
P. Nikou*, E. Giannitsioti, S. Athanasia, A. Drakou, E. Koratzanis, V. Sakka, K. Kouvelas, S. Kanellaki, A. Fragkou, G.M. Gourgoulis, G. Koukos, P. Panagopoulos, E. Paramythiotou, D. Plachouras, V. Spyropoulos, H. Giamarellou, A. Papadopoulos, K. Kanellakopoulou (Athens, GR) Objectives: Chronic foot osteomyelitis (CFO) is a diagnostic and therapeutic challenge for appropriate regimen and length of antimicrobial therapy. Most CFO occurred on the ground of diabetic foot (DF). This study aims to evaluate factors influencing successful outcome in patients (pts) with CFO with or without DF. Patients and Methods: Pts with CFO were retrieved from a data-base of 700 pts with bone and joint infections followed in our reference clinic from 1999 to 2009. Demographics, DF, foot vascular and neurologic complications, charcot joint and gangrene were assessed following standard definitions. Diagnosis of CFO was based upon clinical, imaging and laboratory/microbiological evaluation. A completely restoration of all the above parameters was considered as cure. Chi-square and Mann Whitney tests were applied for categorical and continuous variables respectively. A logistic regression analysis of factors predictive of outcome was performed. Results: DFO pts (n = 137), were male (59%) with median age + IQR(25−75) = 60 (52−70) and comorbidities (n = 76) including diabetes mellitus-DM (n = 64, insulin dependent, n = 60), rheumatic arthritis (n = 3), non DM vascular disease (n = 6), vascular (n = 44), neurological (n = 27) DM complications, charcot joint (n = 22) and gangrene (n = 24) with amputations (n = 25) and orthopedic device (n = 2). HbA1C was above 7 in32 pts. Median IQR(25−75) time from infection onset to treatment was 5 (2−12) months. Sinus tract and ulcer but no fever were found in 71 and 49 pts respectively. Microbiological documentation of DFO was based on intra-operative cultures (n = 83), prone to the bone or aspiration samples (n = 21). Gram positive bacteria (86%) included MRSA (32.5%), MRSE (17%) while Gram negative bacteria accounted for 49%. Among 84 polymicrobial cases, 16.6% were S. aureus plus P. aeruginosa. ESR/CRP baseline were abnormal in 90% of pts. Combined antimicrobial therapy was given to 117/137 pts including fluoroquinolones (n = 92,67%). Only 16 fully reversible adverse events were noted. Median IQR (25−75) treatment duration was 6 (6−8) months. However, 34% were treated for up to 3 months, 42% up to 6 months and 24% more than 6 months. Median follow up was 12 months. Cure Analysis demonstrated that only HbA1C more than 7 was independently related to adverse outcome (p = 0.0001). Conclusions: Only normal HBA1c and n were predictive of the pts' outcome. Strict glycemic control is mandatory in the management of pts with DFO.
Objectives: Rapid routine screening of patients for methicillinresistant Staphylococcus aureus − MRSA is very important. Improved chromogenic media can detect MRSA within 18−24 hours. The purpuse of this study was to evaluate the abillity of two chromogenic media Oxoid Brilliance™MRSA II Agar (Oxoid, Ltd, Basingstoke, UK) and ChromID™MRSA (bioMérieux, Marcy-l'Etoile, France), for detection of MRSA from surveillance specimens. Methods: Brilliance and ChromID were tested using six strains of MRSA (ATCC 43300), hVISA (ATCC 700698) and hGISA (ATCC 700699), two strains of MSSA (ATCC 29213) and a strain of E. faecalis and E. coli. The rest were strains isolated from patients (laboratory strain collection). One to two colonies of the test strain from the third subcultivation were inoculated on Brilliance and ChromID. 55 patient samples were processed. Skin, nose or throat swabs were sent in an Amies transport medium. Each sample was inoculated into Todd-Hewitt enrichment broth (THBS), blood agar (BA), Brilliance and ChromID. The first 28 samples were inoculated in the following order: 1. THBS 2. BA, 3. Brilliance, 4. ChromID. The next 27 samples were inoculated in a different order: 1. THBS 2. BA, 3. ChromID, 4. Brilliance. The plates were inspected after 18−21h of incubation at 35ºC. The plates were inspected after 18−21h of incubation at 35ºC. All denim-blue colonies on Brilliance and green colonies on ChromID were processed according to CLSI guidelines. Antimicrobial susceptibility testing was performed for all DNAse and coagulase positive strains with positive result of methicillin-resistance screening (MRSA strains) according to CLSI guidelines. Denim-blue and green colonies that were DNAse and coagulase negative (regardless of the result of methicillin-resistance screening) were considered false positive (CNS); DNAse and coagulase positive and oxascreen negative colonies were considered false positive (MSSA). Results: All MRSA strains produced denim-blue or green colonies following 18−24 hours of incubation (Table 1) . One discrepant result was obtained from a patient sample, the colonies denim-blue were on Brilliance, and yellow on ChromID. MRSA was confirmed in both cases. Strains of MSSA, E. faecalis and E. coli failed to grow on both chromogenic media. Conclusion: Both agars are fast, easy to use and reliable. They require no additional costs and are accessible to every microbiological laboratory in comparison with rapid PCR methods.
Aim: The compartmental immune response of ascitic fluid seems to be important, especially in spontaneous bacterial peritonitis (SBP). The aim of our study was to determine the pattern of cytokine synthesis in the ascitic fluid (AF) of cirrhotic patients, with or without SBP. Patients and Methods: We prospectively studied 13 cirrhotic patients with ascitis, who were admitted at the University Hospital of Patras from May 2006 to December 2007. Patients were separated into two groups: (a) group 1: patients with SBP (b) group 2: patients without SBP. Cirrhosis was diagnosed on the basis of typical clinical, laboratory, ultrasonographic findings and/or liver biopsy. Upon admission a paracentesis of ascetic fluid was performed. Ascitic levels of IL-1b, IL-1ra, IL-6, IL-10, TNFa, sTNFRI and sTNFRII were measured by using an ELISA method. Data are presented as mean±standard deviation and were compared using t test. A P value <0.05 was considered significant. Results: Characteristics of patients with SBP VS those without SBP are: age 69±12 vs 62±15, male/female ratio 5/2 vs 5/1, ethanol use 5 vs 4, viral hepatitis 1 vs 2, cryptogenic 1 vs noone. The ascitic concentrations of individual cytokines in group 1 vs group 2 are the following: IL-10: 160±142 vs 80±74, IL-6: 2413±3183 vs 2257±2478, TGF-a: 11±20 vs 0.5±1, sTNFRI: 8191±3077 vs 5618±2384, sTNFRII: 5259±2354 vs 2883±932, IL1-ra: 946±703 vs 131±84, TGF-1b: 279±681 vs 378±671. Multivariate analysis showed significant (P < 0,05) differences in the levels of sTNFRII and IL-1ra between the two groups. Ascitic levels of IL-10, IL-6, IL-1ra, TNF-a, STNFRII and STNFRI were higher, while TGF-b1 levels were lower in the ascitic fluid of patients with SBP (differences not significant). It is remarkable that IL-1b was not expressed in patients either with or without spontaneous bacterial peritonitis.
We demonstrated an increased cytokine production in ascitic fluid of cirrhotic patients, while the levels of anti-inflammatory cytokines sTNFRII and IL-1ra are significantly increased in patients with SBP. It seems, therefore, that an ascitic fluid anti-inflammatory response is characteristic in SBP,and this might compromise the final outocome.
Enterobacteriaceae: which sites?
Introduction: Extended-spectrum b-lactamases (ESBL)-producing organisms belong to the most important multiresistant pathogens in hospitals. More recently, ESBLs are also spreading in the community worldwide. Carriers of ESBL-producing Enterobacteriaceae remain colonized for prolonged periods, possibly representing an important source of spread. Knowledge on the body sites most commonly colonized with these pathogens is therefore of great importance in order to develop appropriate screening schemes. The aim of this study therefore, was to determine the frequency of colonization for each body site. Conclusions: Urine and the rectum are the sites most commonly colonized with ESBL-producing Enterobacteriaceae (61.5%) followed by the inguinal fold. These three sites should therefore be considered when screening for ESBL carriage is performed. Throat swabs and wound samples were positive in less than 5% of all specimens, suggesting that these sites are not useful for screening, however should be considered in the individual patient if a decolonization regimen is performed. A. Pérez-Parra, M. Sánchez-Luna, E. Zamora, A. Cuenca, A. Bustinza,Á. Carrillo, E. Gómez, B. Padilla, P. Martín-Rabadán, E. Bouza (Madrid, ES) Assessment of knowledge of CDC guidelines for the prevention of central venous catheter (CVC)-related infection is recommended for healthcare workers (HCWs). Information on this knowledge in pediatric and neonatal intensive care units (P-ICU, N-ICU) is very scarce, and there are no comparative data on knowledge between physicians, nurses, and medical/nursing students. Objectives: We compared different professional groups in order to assess HCWs knowledge of guidelines for preventing CVC-related infection in the P-ICU and N-ICU of a large teaching institution.
We distributed a 12-question multiple-choice questionnaire to HCWs in both units between November 2009 and May 2010. We also recorded participants' demographic data. Each correct answer was scored as 1 and incorrect answers as 0. We created individual and grouped scores of adequate responses ranging from 0 to 12. Results: We collected 174 questionnaires from both the P-ICU and the N-ICU (91 and 83, respectively). Of these, 6 (3.4%) were returned by medical staff, 2 (1.1%) by medical residents, 99 (56.9%) by nurse staff, 18 (10.3%) by replacement nurses, 8 (4.6%) by nursing students, and 41 (23.6%) by nursing assistants. The overall mean score was 7.05. As for professional category, there were statistically significant differences between mean scores for nursing assistants (5.9) and medical/nursing staff (8.7 and 7.6). Moreover, years of experience were associated with better scores (>10y, 8.1 vs. <1y, 6.9 and 1−5 y, 6.7) and those participants who received training sessions in the last year had better scores than those who did not (7.7 vs. 6.6). Conclusions: Our results show that there is room for improvement in HCWs knowledge of prevention of CVC infection. Simple, easyto-obtain scores can help to evaluate the impact of educational and interventional bundles.
Clinical epidemiology of nosocomial infections (POWI, VAP, UTI, BSI, . . . )
Background: Aim of this study was to investigate the risk factors associated with high antibiotic resistance rates belong to Gram positive and negative organisms isolated in 2010 using epidemiological analysis. Methods: A case-control study of 72 patients with Extended Spectrum b Lactamase producing organisms and 33 patients with Amp C producing organisms, 222 patients with non-ESBL organisms and 75 patients with Gram positive organisms was conducted in a subsidial care hospital ofÇanakkale province. Demographics, co-morbidities, antibiotics usage were analysed for all patients.
Results: ESBL and AmpC (+) producing microorganisms were more isolated from patients that belong to over 65-year age and 0−1 age groups (p:0,005). The risk factors including hospitalisation (OR:3,1; 1,19−8,1; p:0,018), co-morbidity (OR:2,1; 1,12−7,3, p:0,024), were found higher in ESBL (+) group compared to ESBL(−) group. Hospitalisation at last three months was found as a risk factor for resistance to any 3rd generation cephalosporins (OR:2,4; 0,9−6,3; p:0,04). There was a correlation between ESBL (+) production and resistant to many antibiotics that are generally used for ampiric treatment (p < 0,05, Pearson correlation r: 0,78), except for piperacillintazobactam (p > 0,05). ESBL production was significantly higher in K. pneumoniae (p: 0,0005), E. cloacae (p:0,001), E. sakazakii (p:0,001) and A. baumannii (p:0,005) strains more than other Gram negative strains revealed in Table- 1. Hospitalisation was single significant risk factor for patients with MRSA (OR:1,8, 0,7−1,2, p:0,005; Table-1) . Vancomycin resistant Enterococcus was not isolated. Discussion: Hospitalisation and co-morbidity causing to colonisation and impairment on immunity are the most important risk factors for ESBL producing microorganisms related infections as our study revealed. Although antibiotic usage including 2nd, 3rd generation cephalosporins and quinolones was cited as a risk factor for ESBL related infections, it was not found significant statistically in our study. Piperacillin-tazobactam could be thought as an option to treat the resistant Gram negative infections associated with K. pneumoniae, E. cloacae, E. sakazakii and A. baumannii whose ESBL rates are frequently higher in our local settings but carbapenems, should be opted for serious patients with co-morbidites. Consequently, immunity and infection control programs are important factors against increasing antibiotic resistance rates. Objective: Infections in patients with traumatic brain injury (TBI) are associated with prolonged hospitalisation and adverse outcomes. The acknowledgement of the pre-disposing risk factors may help decrease the morbidity and mortality. We conducted a retrospective cohort study to determine the incidence, bacteriology and risk factors for development of infection after TBI.
The records of patients >18 years old, admitted with head injury in Crete Univ Med Ctr between 1999 and 2005 were abstracted. Data were analysed with SPSS. Results: 760 patients (75% men, median age 41) were analyzed. Two hundred fifty eight patients (33% of the admissions) underwent 342 surgical procedures. Burr-hole was the most common procedure (29.2%). The median duration of surgery was 2 hours. In 23% of the patients there was some kind of drain inserted. Two hundred fourteen infections were observed. The majority were infections of the lower respiratory tract (47%), mainly ventilator associated pneumonia (VAP) (33%), followed by surgical site infections (SSI) (17%). Fifty three of the admissions (6.3%) were complicated with at least one SSI, superficial or deep. The most common SSI was wound infection (2.2% of the cohort). The patients who underwent neurosurgery had a lower Glasgow Coma scale (GCS) on admission, they were more prone to be admitted to the Intensive Care Unit and bear drains. They were also more prone to develop meningitis, SSI and had increased mortality. Multivariate analysis showed that SSI development was independently associated with performance of >2 procedures (p < 0.001), presence of concomitant infections, namely VAP (p = 0.004) and urinary tract infections (p = 0.001), insertion of lumbar (p = 0.002) and ventricular drains (p = 0.050) and cerebrospinal fluid (CSF) leak (p = 0.050).
Meningitis was independently associated with prolonged hospitalisation (p = 0.001) and insertion of lumbar (p < 0.001) and ventricular drains (p = 0.0017).
There was a predominance of Acinetobacter spp as a VAP pathogen (38%) whereas Gram(+) organisms (especially coagulasenegative staphylococci) remained the most prevalent in SSI (53%). Conclusions: Respiratory tract infections were the most common among patients with head injury. Device-related communication of the CSF with the environment and prolonged hospitalisation were independent risk factors for SSI and meningitis. The prevalence of the pathogens must be determined upon institutional basis for the establishment of proper treatment of these life threatening infections.
Objectives: Rates of hospital-acquired infections have varied between 5 and 10 episodes per 1000 hospital admissions and continue to be a major problem, causing high morbidity, mortality and significantly increasing the length of hospitalization and cost of treatment. The purpose of this study was to evaluate the hospital-acquired infections at Duzce University Hospital, in Turkey.
The study was carried out at the Duzce University Hospital (350-bed). 2009 prevalence of hospital-acquired infections in our hospital was prospectively evaluated. A total of 11333 inpatients were searched.
During one year follow up period, 384-hospital-acquired infection episodes were detected in hospitalized patients (n = 11333). The prevalence rates of patients with hospital-acquired infections were 3.4%. The incidence density was 5.2/1000 patient days. The most common hospital-acquired infections by primary site were pneumonia (47.1%), followed by urinary tract infection (19.5%), surgical site infections (16.9%), bacteremia (14.8%), and other infections (1.6%). Hospitalacquired infections were seen frequently in the department of neurology (7.7%), internal medicine (3.3%), neurosurgery (2.9%), general surgery (2.5%), urology (2.4%), orthopedics (1.9%), infectious disease (0.5%), pediatrics (0.5%). Hospital-acquired infection rate in intensive care units was 26.2%. The most prevalent microorganisms were Pseudomonas spp. (33.5%), Escherichia coli (14.5%), Staphylococcus aureus (12.6%), Acinetobacter spp. (9.5%), coagulase-negative staphylococci (8.1%), Enterobacter spp ( Objectives: Greece has been participating in the ARTEMIS global surveillance programme since 1997 through October 2009, in order to study the epidemiology of Candida strains in our hospital and monitor their susceptibility to fluconazole and voriconazole using a low cost, reproducible and accurate standard in vitro test. Methods: Strains isolated from patients with severe Candida infections from all body sites and all hospital locations were collected, identified and analyzed. Susceptibility was investigated with disk diffusion testing according to CLSI M44 method. Test results were read, interpreted and recorded with the Biomic plate reader system. Results: A total of 1461 Candida strains were recorded. Most prominent species was C. albicans (63.6%), with a year-frequency ranging from 52 to 75%, followed by C. glabrata (14.5%, range 6.9-23.5%) and C. parapsilosis (7.2%, range 0.9−9.6%, except for the years 2007 and 2009 in which a 24.1% frequency was observed). All other species together had a frequency of 5−24%.
The sensitivity of C. albicans to fluconazole equalled that to voriconazole (92.5% versus 92.6% of strains respectively). For the C. glabrata, the respective percentages were 72.4 and 75 and for the C. parapsilosis 85.7 and 92.8. Most of the resistant strains of all species were isolated from miscellaneous body fluids, the upper respiratory tract and blood and derived from the ICU and the surgical wards. During the years 2006-2009, the C. albicans strains resistant to fluconazole were 13.1% and to voriconazole 14.3%. In the previous years only 5% and 4.4% were resistant, respectively. A comparable increase in resistant strains was noted also for C. glabrata (47.8% versus 33.4% in previous years to fluconazole and 43.9% versus 16.9% to voriconazole) and C. parapsilosis (11.2% versus 6.6% and 4.6% versus 2.3% respectively). The remaining species did not show increasing rates. Conclusions: Of the Candida species isolated in our hospital, the most prominent was C. albicans, followed by C. glabrata and C. parapsilosis. The frequency of isolation of these strains varied from year to year without a clear trend. However, an increase in strains resistant to fluconazole and voriconazole was observed in the last four years. These results confirm the need for monitoring the local epidemiology and antifungal susceptibility of Candida strains in order to optimize the empirical or targeted therapy, especially in critically ill patients.
Introduction and Aim: Hydatid cyst is an infection of Echinococci and still represents a serious problem in endemic regions, especially in the Middle East, Mediterranean countries and Australia. Rupture of a hydatid cyst commonly gives rise to allergic phenomena, including anaphylactic shock. Rupture of cyst can occur spontaneously or during surgery as well as due to trauma. We describe a patient who after trauma presented to the emergency department with scant physical signs. Case report: A 17-year-old girl was admitted to our emergency department after falling down of motorcycle, 15 to 20 minutes previously. She was only complained of pain of her head and she had two minor traumas on her face. On presentation, her Glasgow Coma Scale was 15, blood pressure 120/80 mmHg, pulse rate 110 beats per minute, temperature was 36.8ºC and her physical examination was normal except for mild abdominal tenderness. There was no evidence of any other injury and the secondary survey was otherwise normal. White blood cell count was 16,400/mm 3 , eosinophilia was absent and the other laboratory tests were within normal limits. She underwent head and neck computed tomography without sciagraphic fluid and during examination general urticaria developed and it was progressive. The examination stopped and fluid resuscitation, methyloprednisolone and diphenylhydramine were administered intravenously. Once her condition had stabilized she underwent abdomen computed tomography to determine the cause of the abdomen pain. There was no free fluid which usually excludes intraperitoneal bleeding. There was one ruptured cyst on the right lobe of the liver and another cyst measuring 104 mm with thin wall on the left lobe of the liver. The patient turned to have a rupture of a hydatid cyst and she was immediately taken to the operation room by the general surgeons. Drainage and capitonage of the cysts were performed. The patient was treated with albendazole for three months and at six months' follow-up she remained well without any complications. Conclusion: Rupture of hydatid cyst is rare and can occur spontaneously following serious injuries or even minor trauma. Anaphylaxis is the most frequent cause of death in cases of hydatid cyst rupture. Echinococcus liver cysts should be suspected in cases of anaphylaxis of uncertain etiology. Acute vascular collapse, generalized cutaneous erythema, urticaria and edema are suggestive of anaphylaxis from hydatidosis, especially in endemic areas. A. Rodriguez Guardado*, G. Martin, L. Casado, F. Perez, M. Rodriguez, V. Carcaba, J. Carton (Oviedo, ES) Introduction: Travelers visiting friends or relatives (VFRs) typically demonstrate travel and behavioural patterns which render them at high risk for acquisition of preventable infection. Pre-travel services are rarely sought by VFRs, whereas misconceptions that they possess life-long immunity against malaria make them less likely to receive or adhere to antimalarial chemoprophylaxis recommendations. Objective: We analyzed the adherence of this group against these measures of prophylaxis and health problems resulting from the travel. Methods: All VFRs travellers attended in a tropical medicine referral unit between 2007-2010 were interviewed with a questionnaire investigating use of malaria prophylaxis, vaccination, and knowledge of dietary and hygiene measures before and during the travel. VFR was defined as all immigrants permanently established in Spain who temporarily travelling to their place of origin.
We analyzed 61 immigrants (mean time of travel: 91 days). They travelled to Senegal (33%) Equatorial Guinea (25%), Ecuador (18%), Bolivia (8%), Venezuela (5%), Brazil, Cameroon and Colombia (3% each) and Ethiopia (2%). Only 23% patients received health's advice before travel, 72% of travellers didn't received any vaccine and 78% did not carry out antimalaria chemoprophylaxis. Only 25% used safe water. No one use repellents. Twenty-eight patients (46%) were to be illness to return home. Twenty-six patients had diarrhea during the trip and 20% fever, being the most frequent reasons for consultation. In 32% of cases the diarrhea was due to parasitic infections. Eight patients had malaria, in 6 cases due to P. falciparum and two to P. vivax. Conclusions: VFRs are a group of high risk for imported disease due to poor adherence to vaccination and prophylaxis measures. At present, immigrants visiting friends and relatives (VFRs) constitute the most significant group of travellers for malaria importation in developed countries, with sub-Saharan Africa destinations carrying the highest risk. There is an urgent need to increase health's travel advice in this group of travellers. Statistical data analysis used descriptive and and analytic methods − Mann-Whitney U test, Wilcoxon signed-rank test and Spearman's rank correlation coefficient. The value of p < 0.05 was considered statistically significant. Results: Within 5 years 44 patients were treated with trihinellosis. Around 2/3 (68.18%) of them were men, average age being 41.18±14.51, mostly from urban region (65.9%). Source of infection was known in 95% of the cases, out of which in 90% the cause was found to be with domestic animals. Duration of the disease before making diagnosis was on the average 11.5±6.55 days, and duration the hospitalisation lasted 16.5±8.1 days. There were no lethal outcomes, the disease have ended well with 39 patients, while 5 patients (11.36%) had a disease complications-two of them hepatitis, one neurotrichinellosis with neurological consequences, while two patients had myocarditis (one of them developed deep venous thrombosis). Comparing groups of patients with or without complications, the statistically significant difference was shown in time between the intake of food and the appearance of symptoms (p = 0,461). Duration of the disease before the diagnostic and treatment had no effect on the frequency of complications (p = 0,643), while there was statistically significant correlation between the duration of the sickness before the diagnosis was made and duration of the hospitalisation (p < 0.05). Conclusion: Although trihinellosis is a rare disease, it still represents a significant medical problem which we need to be reminded about.
Timely diagnosis and beginning of treatment affects the shortening of the hospitalisation and time of recovery, but has no influence on the frequency of complications.
Spain could be a potential area in Europe for the development and spread of emerging diseases from the tropics due to its characteristics, and their important migratory flows We analyzed clinical-epidemiological characteristics of infectious diseases imported by immigrants from developing countries in the north of Spain.
Methods: A retrospective descriptive study of 341 immigrants from developing countries who live in Spain was conducted. Demographic data, details of country of origin, time on Spain, clinical syndromes, and diagnoses were analyzed. Results: The countries of origin were Ecuatorial Guinea (28%), Senegal (21%), Ecuador (10%), Bolivia (7%), Nigeria (6%), Guinea-Conakry (4%), Marruecos (4%), Brazil (3%). The mean time of Spain was 910 days. Most common syndromes were abdominal pain (25%), diarrhea (21%), cutaneous syndrome (10%) and fever (7%). Most frequent diagnoses were intestinal parasites (48%), filariasis (17%), Chagas disease (5,5%), malaria (4,5%) . and schistosomiasis (3.5%). The most frequent intestinal parasites were: Entamoeba histolytica (22%), Strongyloides spp (12.6%), Trichuris trichiura (10%) and Uncinaria spp (5%). 70% of patients had one parasite, 17% two parasites, 9% three parasites and 4% 4 or more parasites. 69,8% of patients were from Subsaharian Africa. All patients diagnosed of filariasis were from Ecuatorial Guinea. The most frequent were M. perstans (70%), filariasis Loa loa (20%) and Onchocerca volvulus (10%). Conclusions: Increased migratory flows is a key factor for the development and spread of emerging pathogens. Information on these diseases is essential to early diagnostic and treatment. Intestinal parasites were the most frequent disease following to filariasis, malaria and Chagas disease.
Objectives: World Health Organization (WHO) has determined that Leishmaniasis is considered one of the most important zoonotic diseases.
Turkey. Transmission of L. infantum is from dog to vector to human. The percentage of positive dogs of our study is in accordance with previous studies performed in Turkey. Since a high proportion of infected dogs are asymtomatic, diagnosis of Leishmania infections is an serious problem. The parasitological methods present complex interferences, such as NNN culture has a low sensitivity and seeing the parasites fixed in glass slides after Giemsa staining is difficult. Therefore Real Time PCR is effective for especially differential diagnosis of sub-clinical and asymptomatic Dogs.
S. Cho*, J. Chung, J. Ahn, K. Chang, M. You, J. Chai, Y. Kang, S. Kim, H. Jeoung, D. Cheon, A. Jeoung, E. Choi (Seoul, KR) The morbidity of travelers' diarrhea (TD) is still high. This study examined the incidence of common pathogens and characteristics of TD among Korean travelers who visited South-East Asian countries. We performed a prospective study involving 479 Korean travelers with diarrheal disease from February 2009 to April 2009 and stool samples were examined and questionnaires were done after arrival. Enterotoxigenic Escherichia coli (ETEC) was found in 36.0% of TD cases, as were the following: Enteroaggregative Escherichia coli (EAEC) in 27.0%, Vibrio parahaemolyticus in 13.1%, and Norovirus in 11.5%. The detected rate of classic TD was higher in men, in patients who had a shorter duration trip and in patients who drank more than 1 liter of water per day (P = 0.007, P = 0.023, and P = 0.037, respectively). Positive stool culture rates were higher in men, in hospitalized patients, and in those who consumed impure water or raw foods (P = 0.005, P = 0.013, and P = 0.033, respectively). A higher severity of disease corresponded to a significantly higher culture positivity rate (P = 0.029). We should consider the possibility of other pathogens in addition to ETEC in patients with TD who visit South-East Asia and travelers need to educate about risk factors associated with TD.
I. Marincu*, L. Negrutiu, I. Iacobiciu, M. Mares, A. Neghina, L. Marincu, R. Neghina (Timisoara, Iasi, RO) Objectives: Diarrhea is an important cause of morbidity and mortality worldwide, although its etiology remains unknown in many cases. The present study aimed at establishing the etiological spectrum of pathogens causing diarrhea in a group of refugee arrivals to Center for Immigrants from Timisoara, Romania. Methods: There were retrospectively analyzed the medical records of 68 immigrants hospitalized with diarrhea at Clinic of Infectious Diseases in Timisoara during 2008-2009. Diagnosis was established based on the epidemiological aspects (the acute onset following collective or individual consumption of food with inadequate physical and organoleptic properties, stored in poor conditions or even expired), physical examination (fever, repeated shivers, nausea, vomiting, headache, multiple watery diarrhea, fetid stools, tenesmus, abdominal colic, pyrosis, dizziness, paresthesias of lower limbs), laboratory tests (erythrocyte sedimentation rate, blood counts, fibrinogen, C reactive protein, glycemia, blood cultures, lingual swabs, throat swabs, stool cultures, stool parasitological examinations, natremia, kalemia, calcaemia) and other medical explorations (abdominal ultrasound, electrocardiography). Data of the epidemiological survey were collected from the Institute of Public Health from Timisoara. The data was statistically processed using Epi Info 3 software. (analgesics, antipyretics, antispasmodics, antacids) . Twelve cases received antibiotics therapy prior to hospital admission, thus complicating the detection of the etiologic agent. All patients had favorable outcomes. Conclusion: Knowledge of the etiology of diarrhea among immigrants patients allows the proper implementation of targeted therapy and prophylaxis measures in order to limit diarrheal morbidity in persons at risk.
I. Marincu*, L. Negrutiu, I. Iacobiciu, M. Mares, A. Neghina, L. Marincu, R. Neghina (Timisoara, Iasi, RO) Objectives: Scabies is a highly contagious parasitic dermatosis with a worldwide distribution that may cause significant morbidity and large nosocomial outbreaks. The present study aimed at analyzing the clinical and epidemiological characteristics of scabies in hospitalized patients with infectious diseases. Methods: Data were retrospectively collected from the medical charts of 62 patients found with scabies lesions at Clinic of Infectious Diseases, Timisoara during 2009. The positive diagnosis was established based on the epidemiological aspects (recent travels, family or collective outbreaks, sex with multiple partners), physical examination (lesions such as papules, pustules, burrows, nodules, and occasionally urticarial papules and plaques with patognomonic distribution: interdigital web spaces of the hands, perimalleolar and periumbilical region, ribs, horizontal gluteal crease, radiocarpal joint) and intense generalized skin itching at night. The diagnosis was confirmed either by microscopic identification of mites in skin scrapings or by dermoscopy. Patients followed treatment with precipitated sulfur apply topically to entire trunk and extremities every 24 hours for 3 days. Disinfection was carried out at all linen and personal items. Results: Of the study group, 40 (64.5%) patients were rural inhabitants and 22 (35.5%, p = 0.002) lived in urban areas. Six familial outbreaks occured in rural regions. Scabies was mainly associated with respiratory diseases (22.6%), liver diaseases (19.4%), intestinal diseases (12.9%), psychiatric disorders (16.1%), urinary infections (9.7%). Scabies lesions had the following patterns: edematous and urticariform papules (26 cases), papulo-vesicular lesions (22 cases), escoriations due to scratching (48 cases), and scratching related superinfected lesions with streptococcal or staphylococcal bacteria (35 cases). Common form of scabies was evidenced in 24 patients (38.7%) whereas 38 cases (61.3%) had atypical course of the disease (p = 0.02). All patients had favourable outcomes following therapy with precipitated sulfur associated with symptomatic medication (calcium, antihistamincs, antibiotics in bacterial superinfections), individual hygiene and disinfection.
The study of clinical and epidemiological characteristics of scabies in hospitalized patients with infectious diseases allows early diagnosis of this parasitosis as well as timely implementation of effective prophylactic measures. Background: Cryptosporidium sp taxonomy has gone through a number of changes in the last three decades. Initially a single species, C parvum − with a number of clonal genotypes loosely corresponding to specific vertebrate hosts, had been proposed. The current nomenclature suggests, for all cryptosporidial organisms with an oocyst size of 4−6 um, a number of cryptic species that are identified based on molecular genotyping. This is believed to facilitate better understanding of transmission and other epidemiological factors.
Objectives: This study was designed to assess the value, to the clinician and the epidemiologist, of the new cryptic species and whether this information would improve practice. Methods: Isolates that were positive for Cryptosporidium oocysts by auramine staining were typed by PCR. The resulting molcular speciation was then analysed with clinical data (age, travel history, contact with animals, sporadic or part of an outbreak). Results: In this study 27 isolates were typed, 13 C. parum, 12 C. hominis, 1 C. meleagridis and 1 C. felis. There were some trends. Two isolates from Egypt and 1 from Greece were identified as C. hominis, while isolates from Pakistan and St Lucia were identified as C. parvum. This may represent epidemiological differences between the various travel desinations. One patient has both C. parvum and Shigella sonnei isolated, this would be strongly suggestive of a contaminated water source of infection. A second patient had both C. felis and Campylobacter sp isolated. This patient had recently acquired a kitten. It is possible that both pathogens contributed to the diarrhoea, or that the Campylobacter was the cause of the diarrhoea while the presence C. felis oocysts (a primary pathogen of feline hosts) may represent oocysts passing through the patient's gut rather than tissue invasion. Conclusions: Identification of cryptosporidial isolates either by genotype or as one of the cryptic species allows better understanding of potential source. Further studies are needed to confirm the trends seen in this study. Objectives: The purpose of this study was to compare the Neo-Sensitabs method with the CLSI reference broth microdilution (document M27-A3, S3) method for testing the susceptibility of 98 Candida spp. isolates to five antifungal agents (amphotericin B, fluconazole, itraconazole, ketoconazole, and voriconazole). Methods: In this study, the broth microdilution method was performed according to the CLSI recommendations. The Neo-Sensitabs method was performed according to the manufacturer's instructions (Neo-Sensitabs; A/S Rosco Diagnostica, Taastrup, Denmark). C. parapsilosis ATCC 22019 was used as quality control strain. Results: A total of 98 isolates of Candida spp.[C. albicans (n = 56), C. tropicalis (n = 22), C. parapsilosis (n = 6), C. glabrata (n = 4), C. dubliniensis (n = 3), C. krusei (n = 3), C. famata (n = 2), C. lusitaniae (n = 1), C. pelliculosa (n = 1)] were evaluated by both of the methods. MIC ranges were found as 0.03−8.0 mg/ml, 0.05−64 mg/ml, 0.03−0.5 mg/ml, 0.03−1.0 mg/ml, and 0.03-0.12 mg/ml for amphotericin B, fluconazole, itraconazole, ketoconazole, and voriconazole for the microdilution method respectively. All isolates were susceptible to voriconazole by both of the methods. Ninety one (93%) of all isolates were determined as susceptible, two (2%) (C. albicans) were found susceptible dose dependent (SDD), and five (5%) [C. krusei (n = 3), C. glabrata (n = 1), C. albicans (n = 1)] were found resistant to fluconazole by both of the methods. Ninety two (94%) of all isolates were determined as susceptible, six (6%) [C. krusei (n = 3), C. albicans (n = 3)] were found SDD to itraconazole by both of the methods. There was 100% agreement between two of the methods for these three antifungal agents in this study. Two (2%; C. albicans, C. glabrata) and eight [8%; C. albicans(n = 3), C. krusei(n = 3), C. glabrata, C. tropicalis] isolates were determined as resistant to amphotericin B and ketoconazole by both of the methods respectively. One (C. albicans) and three [C. albicans (n = 2), C. glabrata] isolates were intermediate to amphotericin B and ketoconazole by Neo-Sensitabs method, but the same strains were susceptible to these antifungals by standard method. In this situation minor errors were calculated 0.01% for amphotericin B, and 0.03% for ketoconazole by Neo-Sensitabs method.
The Neo-Sensitabs method may be an alternative and easy method for the clinical laboratories to determine the susceptibility of yeast isolates.
C. Tascini*, A. Leonildi, I. Ciullo, E. Tagliaferri, F. Menichetti (Pisa, IT) Objectives: To study the activity of antifungal agents against Candida spp strains isolated from invasive candidiasis and to assess the fungicidal activity of amphotericin B, caspofungin and anidulafungin. Fungicidal activity of azoles was not studied because they are considered fungistatic. Methods: All the strains of Candida spp isolated from invasive candidiasis in Pisa Hospital in the 2009-2010 period were studied. MICs of all strains were performed with the Sensititre method under manufacturer instructions. All the wells without growth of yeast were sub-cultured in order to measure the fungicidal activity of amphotericin B, caspofungin and anidulafungin. Minimal fungicidal concentration (MFC) of these drugs was defined by a 99,9% reduction of the inoculum. Results: Thirty-three Candida spp isolates were included in this study. Thirty strains were isolated from blood, 1 from liver abscess, 1 from vertebral osteomyelitis, 1 from peritonitis. Among these strains, C. albicans were 17 strains and Candida non-albicans 16 strains (7 C. parapsilosis, 4 C. glabrata, 3 C. tropicalis and 2 C. krusei). MIC 50 and MIC 90 for all strains are shown in table 1. All antifungal agents, except amphotericin B have an increase of MIC 90 for Candida nonalbicans strains, with an increase of several dilutions with respect to those of C. albicans strains. MFC50 and MFC90 are shown in table 1. MFC50 and MFC90 of amphotericin B were 2 mg/L and between 4 and 8 mg/L, instead values for echinocandins were superior especially for Candida non-albicans. Conclusion: Candida non-albicans strains have elevated MICs for all antifungal agents. Amphotericin B fungicidal activity is superior to that of echinocandins, especially among Candida non-albicans strains. If this activity might be used in the clinical ground has to be studied. H. Bernhardt*, M. Knoke, G. Schwesinger, J. Bufler, J. Bernhardt (Greifswald, Berlin, Rostock, DE) Objectives: We investigated the antifungal effect of anidulafungin (AND) or voriconazole (VRC) on biofilms and MIC values of different Candida species in a small tube system of continuous flow culture. Methods: We carried out 21 experiments with 23 strains. Strains of C. albicans (Ca), C. glabrata (Cgl), C. tropicalis (Ct), C. krusei (Ck), C. parapsilosis (Cp), C. orthopsilosis (Co), C. metapsilosis (Cm), C. lusitaniae (Cl) and C. guilliermondii (Cgui) from blood culture isolates were grown in small tubes (V = 0.48 ml). Peptone-yeast extract (0.5%/0.2%) + 50 mM glucose±AND (8 mg/l) or VRC (16 mg/l) was supplied at very low flow rates (1.3 ml/h). The flow in the tube was 63-times within 24 h. Biomass production, glucose concentration as metabolic activity, pH and planktonic CFUs were measured. MIC testing was performed by Etest ® on RPMI. Morphology of adherent fungal cells was assessed microscopically after staining with lactophenol cotton blue. Results: In the tube system the development of biofilm varied from species and strains and took place after 2 or 3 days. After continuous input of AND the production stopped in case of sensible species like Ca or Ct, but the metabolic activity measured by glucose concentration did seldom reach the beginning concentration. This could partly be shown after continuous input of VRC. Different strains of Cp showed different results against AND, but there was no increase of resistance in these long term trials up to 10 days. Biofilm of strains with a low susceptibility against AND like some Cp strains or candidaemia strains of Cl or Cgui stopped growth after input of VRC as also metabolic activity did so. Conclusion: The small tube system is a model to get some information from biofilms of different Candida strains under conditions of long term cultivation with supply of low nutrition more similar to real conditions than batch culture. -3 protocol) . Results: During the study period a total of 142 episodes of candidemia occurred in 129 patients, accounting for a global incidence of 12.1 episodes/10,000 in-patients. Median age of the patients was 56 years and 58% of them were males. The most common species isolated was C. albicans (79 episodes, 55.6%), followed by C. parapsilosis (26 episodes, 18.3%), C. glabrata (18 episodes,12.7%), C. tropicalis (10 episodes, 7.0%), C. guilliermondii (4 episodes, 2.8%), C. krusei (3 episodes, 2.1%), C. dubliniensis and C. norvegensis (1 episode, 0.8%). During the study period the incidence of candidemia increased from 7.1 to 18.5 episodes/10,000 in-patients (p < 0,01). C. glabrata and C. krusei fungemia occurred more frequently in patients admitted to intensive care units (p = 0.04). All the isolates resulted susceptible to voriconazole, posaonazole, amphotericin B and to the three echinocandins The susceptibility rate of fluconazole and itraconazole was 99.3 and 72.5%, respectively. The antifungal agents tested had the following MICs90: fluconazole 8 mg/dl, itraconazole 2 mg/dl, mg/dl voriconazole 0.125, mg/dl posaconazole 0.5, amphotericin B 1 mg/dl, caspofungin 0.5 mg/dl, anidulafungin 2 mg/dl, and micafungin 2 mg/dl. Conclusions: Candidemia is still a frequent complication among hospitalized patients. We observed a progressive increase during the study period; this trend could be due to the increase of immunocompromised patients admitted to our institution, in particular hematological patients, solid organ transplant recipients and surgical patients admitted to intensive care units. Nevertheless, C. albicans remained the prevalent species and the resistance rates to antifungal agents were very low.
K. Serefhanoglu, F. Timurkaynak*, F. Can,Ö. Kurt Azap, H. Arslan (Istanbul, Ankara, TR) Objectives: The objective of this prospective, observational, multicenter study was to describe factors associated with bloodstream infections (BSIs) with Candida non-albicans (NAC) species, compared with Candida albicans BSIs, and antifungal susceptibility patterns in intensive care unit (ICU) patients.
The study was conducted in adult medical/surgical ICUs at two university hospitals. All adult patients with ICU-acquired BSI due to Candida, excluding patients with neutropenia, malignancy or AIDS, between 2007-2009 were included. Potential factors occurring up to 30 days before candidemia, including demographic characteristics, comorbidities, exposure to antibiotics and antifungals, ICU-related factors (i.e TPN, invasive procedures) and outcome were determined. Antifungal susceptibility testing was performed using broth microdilution assay method described by the Clinical Laboratory Standards Institute. Results: Clinical characteristics of the patients were presented in the Table. Seventy-six cases (59,4%) of candidemia were due to C. albicans and 52 (40,6%) to NAC spp. Distribution of the first three NAC spp. was C. tropicalis (22/52, 42,3%), C. glabrata (12/52, 23,1%) and C. parapsilosis (9/52, 17,3%). Multivariate logistic regression analysis of the factors showed that presence of glucocorticosteroid treatment and a central venous catheter were variables independently associated with BSI due to NAC, compared with BSI due to C. albicans (P:0,001, OR: 7,18, 95% CI: 2,22−23,22; and P0,001, OR: 20,13, 95% CI: 3,64-111,18, respectively). A total of 65 patients died within 30 days of the diagnosis of candidemia. The mortality rate was higher in those with NAC than C. albicans BSI (61,5% vs 43.4%) and candidemia due to NAC spp. was independently associated with death (P: 0.04). Except for one C. lusitaniae strain, which was resistant to amphotericin B and four C. glabrata strains, which were fluconazole susceptible dose dependent, all Candida species were susceptible to fluconazole, caspofungin, voriconazole and amphotericin B. Conclusion: Presence of a central venous catheter and glucocorticosteroid treatment were significantly associated with BSI due to NAC. BSI due to NAC was significantly associated with death, compared with BSI due to C. albicans. The in-vitro activity of fluconazole is encouraging, and this agent, an efficacious, inexpensive and safe drug, can continue to play an important role in the management of invasive candidiasis.
D. Kofteridis, D. Dimopoulou*, A. Valachis, S. Maraki, A. Thanasia, V. Theodorakopoulou, I. Aristeidou, N. Spernovasilis, G. Samonis (Heraklion Crete, GR) Objectives: Candiduria is encountered in cancer patients (pts) but its significance is not defined. However, it may be indicator of urinary tract infection (UTI) or invasive infection. The study aimed to determine the significance of candiduria in neoplastic pts. Patients and Methods: Adult pts with neoplasia and positive urine cultures for Candida, admitted to the Oncology or Haematology department of the University Hospital of Heraklion, Greece from 2005 to 2010 were retrospectively reviewed. Results: Seventy one pts with candiduria were identified. Of them 48 (68%) were males. The median age was 71 (range 40−88). The underlying disease was solid tumor in 51 (72%) and hematologic malignancy in 20 (28%). The most frequent risk factor was use of urinary catheter or nephrostomy (69%), followed by antimicrobial treatment (60%), corticosteroids (42%), anatomic malformations of the urinary tract (29%), neutropenia (24%) and diabetes mellitus (21%). Fever was present on admission in 59 (83%). Nineteen pts (27%) had dysuria. Flank pain and vomiting were prominent clinical features in 7 (10%) and 5 (7%) pts respectively. UTI by established criteria was present in 51 (72%). Among the 71 positive urine cultures, C. albicans was isolated in 44 (62%), and non-albicans in 27 (38%) [C. tropicalis 9 out of 27 (33%), C. parapsilosis 7 (26%) C. glabrata 6 (22%), C. famata 3 (11%), C. guilliermondii 1 (4%) and C. lusitaniae 1 (4%)]. Blood cultures were obtained from 59 (83%) and were positive in 15 (21%). However, only 4 pts (6%) had Candida in their blood. Death occurred in 26 (37%) pts with candiduria. Factors associated with death included disease progression in 7 (27%) and sepsis syndrome in 19 (73%). Candida spp. has been present in the blood of 2 pts who died. All pts who died had advanced neoplasia.
Conclusions: Factors predisposing to candiduria, included urinary catheter or nephrostomy, prior use of antimicrobial agents, corticosteroids, anatomic malformations of the urinary tract and diabetes mellitus. Candiduria was rarely associated with candidemia, however, has been associated with high mortality, probably because it occurred in pts with advanced neoplasia. , 2003 -2010 Objectives: Dermatophytoses are considered as one of the major public health problems in many parts of the world which distribution varies in different countries and geographical areas depending on several factors. This study was conducted to investigate the aetiological agents and the clinical variants of dermatophytoses, in Western Greece during a 8-year period (2003) (2004) (2005) (2006) (2007) (2008) (2009) (2010) . Methods: A total of 3210 samples (skin scales, nail and hair fragments) obtained from 2616 patients with suspected dermatomycoses referred to the laboratory of Clinical Microbiology at the University Hospital of Ioannina, Greece. The causative agents were identified by direct microscopy and culture. Results: Dermatophytes were isolated from 524 patients (20.03%), mainly adults. The most common type of infection was onychomycosis (34.6%) followed by tinea pedis (28.5%), tinea corporis (18.5%), tinea cruris (8.5%), tinea capitis (4.6%), tinea manum (4.0%) and tinea facei (1.3%). In forty seven patients the same dermatophyte was isolated from two different sites of infection. The frequency for all types of tineas was higher in males than in females, while males exceeded females in cases of tinea corporis and tinea facei. Trichophyton rubrum was the most prevalent dermatophyte species isolated from 51.5% of the 607 positive samples, followed by T. mentagrophytes (20.3%), Microsporum canis (8.2%), Epidermophyton floccosum (5.4%), T. violaceum (3.0%), Trichophyton spp (1.8%) and M. gypseum (1.7%). Other species as T. tonsurans, T. verrucosum and T. schoenleinii were occasionally isolated. Conclusion: In our region, Trichophyton rubrum is the most common aetiological agent in all types of dermatophytoses except of tinea capitis and tinea facei. The epidemiological data collected would serve as reference for future research while many factors that contribute to the change of prevalence of dermatophytoses alter to the pass of time. Background: Voriconazole (VRZ) remains the first line therapy for invasive aspergillosis. Because some Aspergillus species are resistant to azoles and because the prognosis or aspergillosis remains poor; antifungal combinations could be of interest. We tested here the in vitro combination of Anidulafungin (ANI) and VRZ against azole susceptible or resistant Aspergillus spp by two different methods. Methods: Thirty clinical isolates of Aspergillus spp from five various Aspergillus species identified by b-tubulin sequencing were tested. The ANI-VRZ combination was evaluated using checkerboard (CK) based on the CLSI broth microdilution method M38-A3 and using an agar diffusion technique (Etest). For CK, final inoculums were 10 4 /mL. After 48h at 35ºC, both MIC and MEC were recorded visually. The fractional inhibitory concentration index (FICI) was interpreted as synergic if FICI 0.5, no interaction if 0.5 < FICI < 4 and antagonistic if FICI 4. Tests were run in duplicates. For Etest, MIC of VRZ and ANI were determined alone as well as in combination after 48h at 35ºC. In combination, ANI strip was placed on agar for 1h, removed and then VRZ strip was applied over demarcation left from previous strip. Synergy and antagonism were defined respectively as a decrease or an increase of 3 dilutions of the resultant MIC. Results: Using CK method, there was no interaction between VRZ and ANI with MEC as endpoint (table) except for 1 A. nidulans isolate for which an antagonism was shown. Using Etest, no interaction was observed in 27 isolates. According to the endpoint used, an antagonism or a synergism was observed for 2 and 1 A. calidoustus isolates, respectively. Conclusions: Overall no in vitro interaction was shown between VRZ and ANI against most of the azole susceptible or resistant Aspergillus isolates by using different techniques. Confirmation of these results in vivo is warranted.
M. Cavling Arendrup*, S. Sulim, A. Holm, L. Nielsen, S.D. Nielsen, J.D. Knudsen, N.E. Drenck, J.J. Christensen, H.K. Johansen (Copenhagen,Århus, Odense, Herlev, Hvidovre, Køge, DK) Objectives: This study investigated diagnostic issues, underlying host factors, management and outcome factors for Danish fungaemia patients.
Methods: Isolates and clinical information were collected at six centres. 335 isolates from 319 episodes in 306 patients were included corresponding to 2/3 of the national episodes. Results: Species distribution varied by age, prior antifungal treatment (more isolates with intrinsic reduced fluconazole susceptibility P = 0.007) and clinical specialty (61% C. glabrata and C. krusei at haematology wards versus 29%, P = 0.002). Colonisation samples were not predictive for the invasive species in 11/100 cases with species identification. Blood culture positivity varied by system, species and procedure. Thus, cultures drawn via arterial lines or with C. glabrata needed longer incubation and cases with concomitant bacteraemia were less common using BacT/ALERT compared to the BACTEC system (9% (11/124) versus 28% (53/192); P < 0.0001). 56% of the patients had undergone surgery, 51% were ICU patients and 33% had malignant disease. Mortality varied by age (increasing by age, P = 0.009), species (numerically highest for C. krusei 36%, lowest for C. parapsilosis 25% and other Candida species 14%), severity of underlying disease (47% in ICU patients versus 24%; P = 0.0001), and choice but not timing of initial therapy (12% versus 48% in patients with C. glabrata receiving caspofungin versus fluconazole, P = 0.023). Initial antifungal choice was deemed suboptimal upon species identification in 15% of the cases, which would have been 6.5% had current guidelines been followed. Conclusion: A high proportion of Danish fungaemia patients were severely ill and received sub-optimal initial antifungal treatment.
Optimisation of diagnosis and therapy is possible.
Aim: To describe the role of prepared voriconazole (2%) eye drop in the management of fungal keratitis. Methods: It is a retrospective observational case series involving patients of culture proven fungal keratitis from April 2008 to March 2010 attending to the cornea clinic of a tertiary care eye hospital in eastern India. Among the 144 fungal keratitis cases 66 (45.83%) cases were identified for analysis. The ulcer size, organism, treatment modalities and MIC value were analyzed. Voriconazole was used either topically (2%), or in AC wash (50 mg/0.1 ml),) and Intrastomally (50 mg/0.1 ml). Results: Among 66 cases, 39 showed healing with corneal scar formation while 27 underwent therapeutic keratoplasty. 25 cases (64.10%) required topical use, 9 cases (23.07%) required intracameral use and 5 (12.82%) cases required intrastomal administration. Among filamentous fungi Aspergillus sp responded well (25/66;37.87%) followed by Fusarium sp (5/66;7.57%),unidentified sp (3/66;4.54%), and equal no of Penicillium sp, Scedosporium sp, and Cladosporium sp (2/66;3.03%). Among the cases undergoing therapeutic keratoplasty Candida keratitis was maximum with 11 cases followed by 7 cases of Fusarium sp, 5 cases of Aspergillus sp and 2 case each of Alternaria sp and Penicillium sp. In relation with ulcer size response to voriconazole therapy obtained in (20/39;51.28%) where ulcer size is <6 mm and remaining (19/39;48.71%) cases where ulcer size is >6 mm. The minimal inhibitory concentration of voriconazole was 0.125 mg/ml against Aspergillus sp and 0.5 mg/ml against Fusarium sp. Candida sp showed resistance activity upto 2% of voriconazole. Conclusion: Voriconazole has promising activity against filamentous fungi and may prove useful in the management of fungal keratitis but it shows no response in Candida keratitis from our centre.
T. Narkwiboonwong*, K. Trakulhun, K. Watanakijthavonkul, P. Paocharern, A. Singsakul, A. Wongsa, N. Woracharoensri, N. Worasilchai, A. Chindamporn, A. Panoi, T. Methipisit, P. Sithinamsuwan (Bangkok, TH) We here reported a thalassemic patient suffering from cerebral pythiosis (left common carotid pythiosis arterial aneurysms, septic embolisms evolving to brain abscess over the left anterior cerebral artery territory). This was the first case in the world of Pythium insidiosum involving left common carotid artery and producing septic emboli, causing pythiosis brain abscess.
A 27-year-old man, developed nasal congestion with whitish discharge from the left nostril. Three weeks before admission, he developed high grade fever. One week later, he complained of occipital headache, followed by a focal seizure starting at right hand, and then he developed secondarily generalized tonic-conic seizures. Computed tomography scan of the brain was performed. A 5x6 cm hypodensity lesion was noted over the left frontoparietal region. Intravenous ceftriaxone 4 gm/day and metronidazole 1,500 mg/day were prescribed. One week later, fever was still persisted and right-sided weakness was worsened. Personal and past medical history: This patient was diagnosed with b-thalassemia haemoglobin E disease. The pertinent physical findings were high grade fever, significant left carotid bruit, right facial palsy and spastic right hemiparesis. MRI brain showed a large rim-enhancing brain lesion over the left hemisphere. MRA showed multiple aneurysms at left common and internal carotid arteries. His serum was tested for antibody against pythiosis using 3 different techniques, which were ELISA, immunodiffusion and Western blot. The results of all the tests were positive. We also prescribed itraconazole, terbinafine and P. insidiosum antigen immunotherapy, but the patient eventually died from brain herniation. Autopsy revealed gross pus at left frontal area. The left common and internal carotid arteries also found 2 cm in diameter of aneurysm. Pus Wright's stains showed hyphae with infrequently septate and branching. Tissue culture from his brain was confirmed the diagnosis of pythiosis by isolation the organism and proof by Polymerase chain reaction (PCR) with specific primers in the COX II region. Discussion: We couldn't eradicate infected brain tissue as well as infected carotid arteries because of the area of brain and carotid artery involvements were harmful to resection. Cerebral pythiosis should be the differential diagnoses of brain abscess in thalassemic patients. Early recognition and prompt surgical treatment should be considered to reduce mortality or morbidity.
We bronchoscoped 3 patients: ABPA, prior IA and COPD and Aspergillus bronchitis. We compared (1) the UK standard method for processing respiratory cultures (BSOP57) (modified to plate 10uL instead of 1uL) with (2) high volume culture (range 20 mL − 2.3mL, avg 0.5mL) (100% plated) on Sabouraud dextrose agar (37ºC) and 3) real time PCR (MycAssay Aspergillus) preceded by DNA extraction using the MycXtra kit. The sensitivity of this assay is <1 genome, following a >10% extraction efficiency. Sputum samples were obtained before and after the bronchoscopy procedure. Material obtained was split into 'highly mucoid' material and more liquid material, if possible. Approximately equal volumes of material (33% each) were used for the PCR and 2 culture methods. Results: 21 samples were cultured. All (100%) were Aspergillus negative by routine culture and 14 of 21 (67%) negative by high volume culture.
Only 2 (10%) were negative by PCR, 3 were below clinical cutoff (Ct <36) and 16 (76%) positive (Ct values 28.9-35.7). Of the 6 sputum samples (2 split), all were positive by PCR and 5 of 8 (63%) were positive by high volume culture (1−6 CFU). BAL samples were all Aspergillus culture negative, and 8 of 10 samples (80%) were PCR positive. In 2 patients the highest PCR yield was the initial bronchoscopy trap aspiration (often discarded as contains lignocaine), but not in one patient; hyphae and Charcot-Leyden crystals were visible in this sample from patient with ABPA and 19 CFU were grown in high volume culture.
The UK standard method for culture is grossly sub-optimal for Aspergillus spp. and needs revision. Improved culture methods may be of value for sputum but are inferior to real time PCR using the MycXtra DNA extraction system and MycAssay Aspergillus assay. Bronchoscopy sampling has considerable variability in diagnostic yield; sputum may be superior.
A. Spiliopoulou*, C. Bartzavali, E.D. Anastassiou, M. Christofidou (Patras, GR) Objectives: To evaluate the performance of a multiplex PCR (m-PCR) method for detection of dermatophytes, in general, and specifically T. rubrum as compared to conventional methods (microscopy and culture) for the diagnosis of tinea unguium. Methods: A total of 65 nail samples from clinically suspected tinea unguium were examined by direct microscopic examination and culture, as well as, by an m-PCR method (Dermatophyte PCR Kit − SSI Diagnostica − Denmark). DNA from a clinical T. rubrum isolate was used as a positive control. m-PCR includes a two step extraction, a primer mix containing two primer pairs directed toward genes encoding chitin synthetase 1 (chs1) for detection of dermatophytes in general (PCR product = 366bp) and internal transcribed spacer 2 (its2) for detection of T. rubrum (PCR product = 203 bp), an internal control (PCR product = 660bp), and two positive controls (Dermatophyte and T. rubrum genomic DNA We describe a case of disseminated Sporothrix schenckii infection in a man with underlying hairy cell leukemia.
A 44-year-old man, who was initially treated with immune suppression for a presumed autoimmune inflammatory disorder, developed disseminated sporotrichosis with involvement of his skin, lungs and eyes. His immmunity was further impaired by a previously unrecognised hematological malignancy, hairy cell leukemia. The degree of dissemination was so severe that the organism, S. schenckii, was isolated from blood cultures while using standard automated culture techniques. His infection was initially refractory to antifungal therapy with traditional amphotericin B, in part due to drug toxicity. Treatment with liposomal amphotericin B and oral posaconazole, along with treatment of his underlying leukemia, resulted in dramatic clinical improvement with no residual evidence of infection. The immunological defects associated with this malignancy, as well as the management of refractory sporotrichosis are reviewed. All of them were grown in Sabouraud dextrose agar (SDA) for 24 and 48 hours at 30ºC. We looked for macroscopic morphology in SDA and microscopic morphology in Corn Meal agar. We differenced saccharomyces cerevisiae from other yeast by chromogenic medium (CHROMagar™ Candida) and by test of assimilation of sugars (auxacolor™, BIO-RAD). Risk factors in this study: previous use of antibiotic/antifungal, probiotics based in S. cerevisiae, hospitalization and immunosuppression. We followed clinical and microbiological criterias to prove if we were in an infection or colonization. Results: Risks factor weren't brake down into their different headings due to the difficulty of ensuring that patients received Saccharomyces cerevisiae (Ultralevura ® ). Only we found that only patients admitted with chronic diarrhea and elderly in hospital received probiotic. In community was impossible to know. Vaginitis and cervicitis, due to S. cerevisiae, were the main infection after using of azoles or antibiotics to treat other infections. We had not isolated in blood or sterile samples.
We have not had any invasive infection. We should unify criteria to decide which patients can be given or not Ultralevura ® to prevent infections. We should study phenotypic and genotypic factors of yeast related to its ability to invade the host and antifungal test to establish epidemiological criteria for treatment. It is complicated to know the origin of S. cerevisiae.
P. Mehta*, R. Mehta, M.V. Raghavendra Rao, M. Balaramiah, A. Iban Abdallah (Sirte, LY) Objective: Fungal keratitis is one of the major causes of the ulcerative and sight threatening infection of the cornea but its incidence is usually underestimated. Such a study has never been carried in this part of the world. The objectives of this study was to determine its incidence and associated etiological risk factors in this region. Conclusion: Contrary to popular perception of low incidence of fungal keratitis in this region, high incidence has been found in this study. It can be attributed to increasing trend towards farming activity in this region which predisposes the people to vegetative injury. Seasonal variations throughout the year in this region leads to altered tear film which again could be a major predisposing factor. Increasing use of the contact lenses also predisposes the users to contact lens related infection. Diabetes mellitus was found in some patients which can be a predisposing factor.
Prevalence of individual fungi T. Dantin-Delafoulhouze, S. Lucas-Daver, S. Dumas, T. Fosse, R. Collomp (Nice, FR) Candidaemia are an important cause of morbidity and mortality in hospitals, especially among severe patients. Over the past decade, the epidemiology and antifungal therapies have evolved. Our university hospital is committed since 2005 in a multidisciplinary approach to help diagnosis and treatment of invasive fungal infections (IFI) initially focused on costly treatments. Our objectif was to study trends in epidemiology, susceptibility, and antifungal use in our hospital. Methods: All patients with a blood culture yielding yeast between January 2007 and December 2009 were included. Patient data, epidemiology, susceptibility, treatments and interventions of our antifungal team were collected from computerized patient records, the Mycology Laboratory and the Pharmacy. Results: 74 patients were included. During this period a diversification of yeast species was observed, with a significant reduction of Candida albicans (25% in 2009 vs 52% in 2007) . The rate of resistance to fluconazole has not exceeded 6% among strains of Candida and the first strain of Candida parapsilosis resistant to caspofungin was isolated in 2009. First line antifungal treatments have been established according to local recommendations (96%). The antifungal team has intervened for nearly 80% of patients in 2009, to continue the initial antifungal treatment or to propose a de-escalation therapy (20%). In 2009, 45% of patients (11/24) died, underlyng the severity of those IFI. Conclusion: This work has underlined our local specificity both in terms of epidemiology (most important decrease in Candida albicans compared the litterature), and in terms of multidisciplinary management of IFI. Among the proposals from this study include the systematic monitoring of Candidaemia during our antifungal team meetings wathever the drug and the search for a possible nosocomial cause of these severe infections.
A. Navascués*, I. Rodríguez, J. Repáraz, S. Salvo, A. Gil-Setas, J.M. Martínez-Peñuela, C. Ezpeleta (Pamplona, Zaragoza, ES) Objective: The increased presence of immigrants from Latin America and increased travel to that continent has increased the incidence S790 21st ECCMID/27th ICC, Abstracts accepted for publication only of Histoplasmosis in Spain. Our aim was to review the cases of histoplasmosis diagnosed in our hospital. Methods: Review the diagnostic methods employed and clinical characteristics of cases of histoplasmosis diagnosed during the last six years. Case reports: We diagnosed 4 cases, three of whom were HIV positive (male, 28−43 years) and one patient (female 50 years) treated with immunosuppressive drugs. All of them were from South America. The spectrum of disease in HIV positive patients was one liver disease, one pulmonary and one gastrointestinal. Two had a good outcome but the other, in spite of treatment with amphotericin B, had a rapidly progress to a multiorganic failure and died. The female had a liver histoplasmosis and she also had a good clinical response. The treatment in all cases was amphotericin B 5 mg/kg/day (two weeks) followed by itraconazole 200 mg/12h. The laboratory diagnosis was carried out by histological (PAS and PAS-Diastase Stain) and microbiological study (culture and PCR directly): in three cases, we isolated H. capsulatum var. capsulatum and in the other, the microbiological diagnosis was thanks a direct PCR in the tissue. Conclusion: Most infected people by histoplasmosis remain asymptomatic, but they can develop serious clinical forms depending on the level of exposure and the patient's immune status. Usually, severe forms are seen in HIV positive patients, but as has occurred in our series of cases it's also possible in patients with immunosuppressive therapy. In our country, histoplasmosis is an imported infection and because of this it is necessary to have a high index of suspicion and perform a detailed history to get a diagnosis. It is an infection to be considered in the differential diagnosis in immunosuppressed patients, both HIV positive and immunosuppressive therapy, which originate from endemic areas or who have a history of stay in them.
S.N. Khostelidi, Y.V. Borzova, R.A. Araviyskiy, T.S. Bogomolova, V.A. Zinserling, I.S. Zjuzgin, N.V. Vasilyeva, N.N. Klimko* (Saint Petersburg, RU) Objectives:
O. Kaya*, S. Alp-Cavus,Ö. Turhan, M. Tasbakan, H. Pullukcu, B. Ertugrul, S. Senol, B. Cetin, B.Özhak-Baysan, S. Sayin-Kutlu, D. Metin, M. Avci, G. Mermut, V. Avkan-Oguz, N
Objectives: The incidence of Zygomycosis has increased due to immunosuppression in recent years. We aimed to evaluate 16 cases of Zygomycosis from eight centers retrospectively. Methods: Zygomycosis patients were collected between 2004 and 2010. Zygomycosis was diagnosed by culture positivity and/or histopathological findings in addition to clinical findings. The clinical forms of zygomycosis have been described on the basis of organ involvement: rhinoorbito-cerebral, cutaneous, pulmonary, gastrointestinal and disseminated. Disseminated infection was defined as infection at 2 non-contiguous sites.
Results: There were 11 female and five male subjects. The mean age was 52.50±14.55 (22−68) years. The majority of cases (15 cases, 94%) were immunocompromised patients, mainly diabetes mellitus (10 cases, 62.5%). Seven patients had received corticosteroid treatment. The most common symptoms and clinical signs in patients was fever (n = 9), retroorbital pain (n = 7). The most common form was rhino-orbitocerebral. R2632 Fungemia in the intensive care unit: a five-year study in two centres E. Paramythiotou *, F. Frantzeskaki, A. Antoniadou, G. Kallitsi, M. Haritidou, F. Klouva, E. Trika, H. Giamarellou, D. Plahouras, T. Panagea, L. Zerva, G. Petrikkos, A. Armaganidis (Athens, GR) Objectives: Candidemia is frequently encountered in the nosocomial setting, particularly in the Intensive Care Unit (ICU) causing considerable morbidity and mortality. The increasing incidence of non-albicans Candida species could also be important. The aim of the present study was to record the epidemiology, risk factors, mortality, strains susceptibility to antifungal drugs. This is a clinical and microbiological retrospective study of all fungemia episodes registered in two medicalsurgical ICUs between 1/1/2005 and 31/12/2009. The records of the research laboratory of the 4th department of Internal Medicine, the microbiology department of Attikon university hospital and the microbiology department of Thriassion Hospital were used in order to identify patients. Medical records were then retrieved. Special forms were completed for each patient including demographic information, concomitant conditions, Apache II and Sofa severity scores the day of ICU admission, the risk factors within the preceding 10 days, data of colonization and candidemia related information. Results: Attikon hospital is a 640-bed teaching tertiary care hospital with a 25-bed medical and surgical ICU and Thriassion hospital is a 433-bed tertiary care hospital with an ICU with 8 beds. During the study period a total of 1663 pts were hospitalized in both ICUs. Among them 67 patients developed fungemia. Median patients' age was 56 years. Median ICU length of stay was 37 days. Medical cause of admission was present in 31 cases. Species isolated were C. albicans (38.8%), C. parapsilosis, C. tropicalis (8,9%), C.spp, C. glabrata, and non-albicans spp. Median time elapsed between ICU admission and candidemia was 19 days. Mean Apache II score was 19 on the day of admission and overall mortality was 50.7%. Attributable mortality was 55,9%. Ostrosky prediction rule was positive in 41 patients. Urine or lung colonization was present in 20.5%,and multiple site colonization in 29%. Fifteen pts were submitted to an intraabdominal operation. Fifteen pts received TPN prior to candidemia episode. Caspofungin was the most commonly introduced treatment. Conclusions: Compared to other blood infections fungemias are not common among our patients but they are often lethal. A high Apache II score at admission, multiple site colonization in combination with abdominal surgery should raise a high suspicion index and a prophylactic therapy should start. Non-albicans species are on the rise. In 2009 the most isolations were in May (8.53%) and August (5.00%) and in 2010 were in July (5.44%) and in October (8.23%) as cause of onychomycosis. No dermatophytes were isolated more than these. Conclusions: Confirmation of the diagnosis should be considered as essential. A bad treatment predispose to advanced invasion with dermatophytes and/or other molds. We should assay sensibility to antifungals in our area.
A. Nazli *, M. Tasbakan, H. Pullukcu, O. Sipahi, T. Yamazhan, B. Arda (Izmir, TR) Objectives: In this study, it was aimed to review systematically the published mucormycosis cases from Turkey. Method: Published mucormycosis cases from Turkey in national and international medical literature in the last fifteen years were retrieved from three national (Ulakbim, Turkish Medical Literature and Medline Plexus) and two international databases [Pubmed and Science Citation Index Expended (SCI)]. As keywords "mukor", "mukormikoz" were used in national databases and "mucor", "mucormycosis" adding "Turkey" in international databases. Data related to age, gender, comorbidities, signs, diagnostic tools, therapeutic modalities, and mortality were analysed from the retrieved articles.
Results: Data for a total of 100 mucormycosis patients (47 female, 53 male, aged 46.1) with definitive diagnosis of invasive fungal infections according to criteria of European Organization for Research and Treatment of Cancer (EORTC) were obtained from 48 reports (22 international, 26 national). We could not achieve the full texts of two reports published in international databases and three reports in national databases. In terms of common clinical findings, the most common symptom was swelling of eye and face (64%) followed by headache (56%), fever (54%), opthtalmoplegia (38%), loss of vision (31%) and other neurological signs (%27). The most common comorbidity was diabetes (53%) followed by, hematological malignancies and corticosteroid usage (33% . Sequence-analysis of the infecting fungal strains showed two mutations in the Cyp51Agene and a tandem repeat in the gene promoter. During treatment, posaconazole trough levels were 0.6 microg/ml. Blood and postmortem tissue posaconazole concentrations indicated AUC/MIC ratios of 30 to 60. Table 1 . Culture results and posaconazole drug concentrations in blood and tissue samples obtained at autopsy.
The efficacious AUC/MIC for posaconazole is probably above 200, but this could not be achieved in our patient. Plasma levels >4 microg/ml would have been required to achieve the pharmacodynamic target for these A. fumigatus strains with posaconazole MIC of 0.25 and 0.5, which was impossible to achieve with the current posaconazole formulation. Posaconazole should be used caution in invasive aspergillosis caused by strains with attenuated posaconazole susceptibility, as drug exposure may be inadequate resulting in therapeutic failure.
M.J. Muñoz-Davila, A. Gomez-Lopez, O. Cores, J.L. Rodriguez-Tudela, M. Cuenca-Estrella* (Murcia, Majadahonda, ES) Background: The list of uncommon fungal species causing human infections is growing. Conventional methods of classification based on morphological, biochemical and physiological features have proven ineffective in accurately identifying such species. Candida haemulonii has been reported among uncommon yeasts with decreased susceptibility to antifungal agents causing human disease. We have reviewed the identification and antifungal susceptibility results of a collection of clinical isolates of C. haemulonii. Methods: A total of 10 isolates, received in our institution over 10-year period (2001-2010) , were evaluated. One of them was isolated from blood and the other nine strains were recovered from superficial sites. They were identified by morphology and biochemical tests. In addition, molecular identification was done by sequencing of ribosomal DNA (ITS domain). Susceptibility testing followed the recommendations proposed by the EUCAST. Results: A total of 90% (9/10) isolates were not discriminated by phenotyping and strains were classified as unidentifiable according to their biochemical profile. One strain (10%) was misidentified as Candida sake. Objectives: Candida spp. is the fourth most common pathogen causing nosocomial blood stream infections and invasive infections in immunocompromised and seriously ill patients. Methods: Two retrospective data sets each with 100 patients from the time periods 1985-1990 (period 1) and 2001-2005 (period 2) respectively were evaluated. All patients whose data were included into this study have been diagnosed with candidemia at the University Hospital of Vienna, a 2200-bed referral centre. The aim of the study was to identify changes in the epidemiology, the clinical manifestation and the outcome in these two groups. The demographic dissemination in terms of age, gender etc. was analysed as well as differences in underlying diseases in both periods. The duration of hospitalisation and time until Candida albicans isolation from blood culture were determined. The progress of candidemia, the success of therapy and the mortality rate in these two periods were evaluated. Results: During period 1 significantly more patients with proven candidemia were found at Intensive Care Units, in period 2 significantly more at medical wards. The main hospitalisation duration until isolation of candida was 23 days in period 1 and 27,9 days in period 2.
In both periods two third each of the patients were diagnosed as immunocompromised.
In period 1 60 patients died: 20 in consequence of candidemia, 10 later but from the persistent fungal infection, 16 patients from their underlying disease and 12 from bacterial sepsis. Thirty-seven patients received antifungal therapy, 23 remained untreated. Fifty-four percent of the treated patients died, as opposed 74 percent of the patients without therapy.
In period 2 51 patients died: 23 in consequence of candidemia, 2 later but from the persistent fungal infection, 20 from their underlying disease and 6 patients from bacterial sepsis. Forty-five of the 51 deceased patients received antifungal therapy, 6 remained untreated. Fifty-four percent died in the treated group, 50 percent in the group without treatment. Conclusion: Although much progress has been made in the treatment of Candida albicans, mortality of patients is still high. For a further decrease in candida infections better tools for diagnosis, guidelines for management and more awareness of this pathogen are absolutely essential.
A.M. Tortorano*, A. Prigitano, C. Ossi, A. Grancini, E. Casari, G. Viola, G. Giuliani, M. Mella, M. Longo, R. Passerini, M. Facchini, M. Tejada, M. Passera, G. Delvecchio, E. Sala, C. Sturla, A. Ceraminiello, C. Cavanna, M. Re, C. Bezzi, D. Longo, M. Arghittu, L. Re, M.R. Sala, P. Clerici, A. Grossi, P. Pedroni, G. Munafò, C. Bonetti, G. Brigante and remained associated mainly, even if at a reduced rate, to surgery (45% of the cases vs 56% in the previous study) and intensive care treatments (35% vs 45%). A shift of the species causing fungaemia was demonstrated by the comparison of the two periods: while the proportion due to C. albicans decreased from 58 to 52%, an increase of C. glabrata (from 13 to 20%) and C. tropicalis (from 6 to 8%) was noted. The proportion of C. parapsilosis remained unchanged (14.5%). A decreased of the crude mortality at day 30 from 35 to 32% was observed. The highest mortality rate was detected in patients with C. tropicalis (41%) and C. albicans (33%) bloodstream infections.
The present study revealed an increasing incidence of candidaemia, mainly in aged subjects, and an increasing proportion of isolates with decreased susceptibility to fluconazole.
C. Lacroix, A. Gicquel, F. Morio, J. Lambert, I. Accoceberry, E. Bailly, G. Desoubeaux, E. Collin, L. Feghoul, N. François, F. Gabriel, F. Gay Andrieu, J. Guitard, C. Hennequin, C. Kauffmann Lacroix, D. Lauzin, B. Sendid, O. Lortholary, M.E. Bougnoux* (Paris, Nantes, Bordeaux, Tours, Lille, Poitiers, FR) Objectives: A prospective, multicentre observational study was conducted in France to compare the epidemiology of Candida spp. isolated in high risk patients from intensive care units (ICU), haematology units (HU) and renal transplantation units (RTU). Methods: From January to February 2010, all Candida spp. isolated from patients hospitalized in medical and surgical ICU, adults and paediatrics HU and RTU from 8 French university hospitals were collected. C. albicans was identified using chromogenic agar media, and screened with BichroDubli ® (Sofibel) to identify C. dubliniensis. C. krusei and C. glabrata were identified with KruseiColor ® (Sofibel) and Glabrata RTT ® (Sofibel), respectively. All other Candida spp. were identified using ID32C ® (BioMérieux). Unequivocal identification using rDNA sequencing was done for all C. glabrata, C. parapsilosis, C. kefyr, C. inconspicua/norvegensis, and those with ID32C identification score <95%. In vitro antifungal susceptibility was determined by E-test ® (BioMérieux). First isolate of each Candida sp. from each different body site was tested in each patient. were C. glabrata. Isolates with non-susceptibility to caspofungin were C. guilliermondii (n = 2) and C. parapsilosis (n = 1). Conclusion: This epidemiological study conducted on a short period of time allowed us to point out differences in Candida spp. isolated from different clinical units and body sites. Local epidemiology must be considered for an appropriate empirical antifungal therapy.
M. Mahelova*, F. Ruzicka, V. Hola, M. Votava (Brno, CZ) The most commonly isolated yeats Candida albicans as well as other yeasts can colonize the gastrointestinal tract of humans and be a reservoir for the development of infections at other body sites. In 1995, a new species of the Candida genus was identified and classified as Candida dubliniensis. This species shares many phenotypic similarities with C. albicans, which leads to its misidentification and underestimation. It was primarily isolated and identified from the samples of HIVpositive individuals and AIDS patients suffering from oral candidiasis, and many other publications reported its prevalence in the oropharynx.
The occurrence of C. dubliniensis in the human feces samples has been tested only rarely. C. dubliniensis is not routinely identified in most of clinical laboratories in the Czech Republic. The aim of this study was to evaluate the incidence of C. dubliniensis in human clinical material, especially in stool samples. Three common phenotypic methods were used for discriminating C. dubliniensis from C. albicans (the color of colonies on CHROMagar Candida, the inability to grow at 42 ºC and the colony morphology on Staib medium). The identification of C. dubliniensis was verified by polymerase chain reaction with the species-specific primer pair and universal primer pair. The experiments took place from December 2007 to December 2010. We have collected almost 500 samples from the gastrointestinal tract and about 1500 isolates from the airways and oropharynx, all of which were originally identified in the laboratories as C. albicans. Two to three % of these isolates were identified as C. dubliniensis. The incidence in the oropharynx and airways (3%) was significantly higher than in the stool samples (2%). The significance of the finding lies primarily in the fact that only a small number of publications deals with this particular clinical specimen and that not many publications about the incidence of C. dubliniensis in any other clinical materials have been published in Czech republic. Acknowledgement: This work was supported by The Ministry of Education, Youth and Sports, INGO-LA09032
A. Candoni*, E. Simeone, M. Caira, M. Mazzucco, R. Fanin, L. Pagano (Udine, Rome, IT) Background: Acute Myeloid Leukemia (AML) patients are at high risk of Invasive Fungal Diseases (IFDs). We report our real-life experience with POS prophylaxis in AML. We also compare the performance of POS prophylaxis with an historical, well matched, control group of AML pts who received prophylaxis with Fluconazole (FLUCO) or Itraconazole (ITRA). Patients and Results: Fifty-five unselected and consecutive AML pts received POS prophylaxis (600 mg daily) between Jan 2009 and Oct 2010. Median age of this population was 47 yrs (range 18−69). All cases were given chemotherapy with anthracyclines and cytarabine. The POS was started when neutrophil (PMN) count was less than 1000 mL and was stopped at PMN recovery. The median duration of severe neutropenia (PMN lower than 500 mL) was 15 days (range 7−41); 10/55 (18%) of cases had an oral mucositis grade II-III CTC (common toxicity criteria) and 73% (40/55) of these pts received a proton pump inhibitor. An active diagnostic work up was made in all cases with Galactomannan assay, standard chest X-ray and thoracic CT scan in case of fever (FUO) lasting over 48 hours. The median duration of POS prophylaxis was 15 days (range 7 to 41). Only 4/55 (7%) of pts required parenteral empiric or pre-emptive antimycotic therapy and only 2/55 (4%) experienced a proven IFDs (Fusarium solani fungemia and Aspergillus sp pneumonia). Mortality IFDs related was 0%. POS was well tolerated and only 9% (5/55) of pts experienced mild drug related side effects. No cases of POS discontinuation, due to the side effects or intolerance, were reported. When we compare the 55 pts who received POS with an historical control group of 55 AML pts who received FLUCO (45/55) or ITRA (10/55)prophylaxis, between Jan 2008 and Jun 2009, no significant differences were observed for underling disease status, age, IFDs risk factors, days of severe neutropenia and days of prophylaxis. Instead, there were significant differences in breakthrough IFDs (4% in POS group vs 16% in control group; P = 0,02), and in days of parenteral antymicotic therapyn (37 vs 163).
Conclusions: This real-life experience confirms that POS prophylaxis is feasible, safe, well tolerated and effective (prevention of IFDs) in unselected AML patients. Only 7% of these high risk pts required parenteral antimycotic therapy and only 4% experienced breakthrough IFDs. We also confirm that POS is more effective than FLUCO or ITRA as antifungal prophylaxis in AML pts. Y. Toyoda, K. Cieply, M. Nguyen (Pittsburgh, US) Background: The diagnosis of invasive asperigllosis (IA) is limited by poor sensitivity and specificity of microbiologic cultures, and the inability of conventional histopathologic tests to distinguish IA from invasive fungal infections (IFI) caused by non-Aspergillus moulds. Aspergillus-specific immunohistochemistry (IHC) and in situ hybridization (ISH) could facilitate direct diagnosis of IA within tissue. Methods: IHC and ISH were performed on deparaffinized, formalinfixed sections from previously diagnosed cases of proven IFI, and slides interpreted by pathologist blinded to diagnosis. IHC: Primary Aspergillus antibody (Abcam, MA) and secondary anti-rabbit antibody were detected with Avidin Biotin Complex and visualized using liquid DAB. ISH: Commercially synthesized Locked Nucleic Acid probe for Aspergillus was detected using antiflourescein AP. Poly T stained tissue section was used to determine the area of tissue to be studied. Results: 8 tissue sections were available from patients with IA (A. fumigatus = 5; A. niger = 1; non-speciated = 2, and 12 sections were available from patients with IFI due to other fungi (Zygomyces = 6; Candida = 5; Dactylaria gallopava = 1). IA samples were obtained from 6 tissue biopsies (3 lungs, 2 upper airways, 1 vocal cord), and 2 autopsy samples. 2 serial sections from one patient with IA due to A. fumigatus were excluded from the study because positive control stains were negative. The sensitivity of IHC and ISH for IA was 83% (5/6) and 100% (6/6), respectively. IHC was falsely negative in 1 pt with IA due to A. fumigatus. The specificity of IHC and ISH was 100% (12/12), as the tests were each negative in all sections associated with IFI due to non-Aspergillus fungi. IHC was associated with significant levels of non-specific background staining in 33% of the sections that could be analyzed, compared to 0% for ISH. Conclusion: Data from this pilot study suggest that Aspergillus-specific IHC and ISH will facilitate diagnosis of IA and exclude IFI due to non-Aspergillus fungi. ISH was superior to IHC by limiting false negative results and non-specific background staining. We are currently assessing IHC and ISH prospectively on non-fixed tissues. Objectives: Invasive pulmonary aspergillosis (IPA) among patients with chronic obstructive pulmonary disease (COPD) is increasing in frequency and associated with mortality exceeding 70% in some series. We conducted this study to find out the approximate incidence of IPA in patients hospitalized with acute exacerbation of COPD (AECOPD), its true mortality and to identify potential factors associated with mortality.
We retrospectively included all patients admitted with AECOPD and isolation of Aspergillus over the last 3 years. IPA was defined according to Bulpa criteria. Charts were retrospectively reviewed and patients were classified into probable IPA and colonization. Results: We identified 68 patients admitted in the last 3 years with AECOPD and aspergillus isolation. Thirty-seven (54%) had COPD stage III-IV. Forty-six (68%) were male and median age was 69 (55−90). Fifty-five patients (81%) had increased dyspnoea and 52 (76%) had radiological alterations, mostly new infiltrates in chest x-ray. Thirty-nine (57%) received >700 mg of steroids during admittance and 16 (24%) had received >20 mg/day during the past 3 months. Twenty patients (28%) had more than one aspergillus isolation. Twenty four patients (35%) received voriconazole for a median of 19 days (1−50 days). Crude mortality was 21% (14/68). Patients with COPD stage III-IV were older (71 vs 63 years, p = 0,02) and had higher mortality, 13/37 (35%) vs 3/32 (9%) (p = 0, 01, OR: 3, 9 CI95: 1, (8) (9) (10) (11) (12) (13) (14) (15) . Similarly, patients with COPD stage III-IV received more frequently high dose steroids 28/37 (76%) vs 11/32 (34%), p < 0,001, and had more frequently increased dyspnoea during admittance, 36/37 (97%) vs 20/32 (62%), p < 0,001. Neither daily dose of steroids nor voiconazole therapy were associated with increased mortality. In multivariate analysis, only increase dyspnoea was significantly associated with mortality. Conclusion: COPD patients stage III-IV had increased mortality because of IPA although it is lower than described by other authors. Neither previous nor high steroid doses during admittance were associated with mortality.
Objectives: The diagnosis of Pneumocystis jirovecii pneumonia (PCP), which affects various types of immunocompromised patients, can be at times problematic. We sought to evaluate the diagnostic accuracy of (1?3)-b-D-glucan (BDG) for the diagnosis of PCP.
We did a meta-analysis of relevant studies, identified through PubMed and Scopus. Eligible studies were those that reported BDG diagnostic data in cases with documented PCP and controls with other conditions. We excluded cases of invasive fungal infections or healthy controls. We performed a bivariate meta-analysis of sensitivity and specificity and constructed a hierarchical summary receiver operating characteristics (HSROC) curve. Results: Twelve studies were included in the meta-analysis. BDG data were provided for 334 PCP cases and 1663 controls. The pooled (95% confidence interval) sensitivity and specificity of BDG were 94.7% (90.2-97.2%) and 86.6% (81.5-90.5%), respectively. The positive and negative likelihood ratios were 7.1 (5.1−9.9) and 0.06 (0.03-0.12), respectively. The area under the HSROC curve was 0.964 (0.944-0.977 Objectives: This study assessed a pilot antifungal prevention strategy based on itraconazole levels in consecutive high risk patients with the aim of reducing empirical treatment. The patients underwent chemotherapy or allogeneic stem cell transplantation for haematological malignancies and received itraconazole suspension prophylaxis. The study is ongoing. Methods: 48 neutropenic febrile episodes, 25 of which were refractory at 4 days to broad spectrum antibiotics initiated on day one, were studied. All patients had serum galactomannan assays (EIA) twice a week and trough itraconazole levels measured on day one of fever. If they were still febrile after 4 days, HRCT scan chest was performed. Antifungal treatment was required to be given only when well defined clinical, microbiological and radiological criteria were present. Invasive Fungal Disease ( Conclusions: The incidence of probable or proven IFD was 2%. Three patients with possible IFD died (all had received iv antifungal therapy). 20% of the patients overall had subtherapeutic itraconazole levels. Our strategy reduced the rate of antifungal use by 50%.
L. Chishimba *, R.N. Niven, J. Cooley, D.W. Denning (Manchester, UK) Background and Objectives: Allergic bronchopulmonary aspergillosis (ABPA) and severe asthma with fungal sensitisation (SAFS) are progressive allergic fungal lung diseases. Current treatment with itraconazole (itra) is associated with a 40% failure rate and adverse events (AEs). Very little is known about the response rates or appropriateness of treatment with voriconazole (vori) or posaconazole (posa). This study assessed the effect of vori or posa as second and third line therapy.
We conducted a retrospective audit of 27 adult asthmatic patients who fulfilled diagnostic criteria for either ABPA or SAFS. All patients had previously received itraconazole. Vori (300-600 mg/day) or posa (800 mg/day) (adjusted by TDM) was given for at least 6 months if tolerated. Clinical, radiological and immunological evaluation was used to assess response. We defined response as improvement in symptoms and either fungal serology or radiological abnormalities. The rates of clinical response or failure and their adverse effects (AEs) after 3, 6, and 12 mos of treatment were analyzed. Co-existing diagnoses, Aspergillus antibody titre, vori and posa levels and lung function were used as covariates. Results: There were 27 patients, ABPA (n = 22) or SAFS (n = 5), 11 males, median age = 59 yrs. All patients had failed itra (n = 11) or developed AEs (n = 12), had low serum concs (n = 2) or itra resistance (n = 2). There were 34 courses of therapy analysed, 25 with vori and 9 with posa; only 2 posa courses were not preceded by vori (resistance). Clinical response to voriconazole was observed in 17/25 (68%) at 3 mos, 15/20 (75%) at 6 mos and 12/16 (75%) at 12 mos, compared to 7/9 (78%) at 3, 6 and 12 mos for posa. 6/25 (24%) vori pts had AEs requiring discontinuation before 6 mos compared to 0/9 posa patients. Vori AEs included GI upset (7), skin photosensitivity (11), blistering (4), visual light flashes (10), insomnia (2), visual hallucinations (2), depression (1), adrenal suppression (2), peripheral neuropathy (4), eye irritation (2), vivid dreams (1), dizziness (1) and headache (1) but most of them were transient and mild. Posa AEs included insomnia (2), GI upset and mild liver impairment. Among those who discontinued, 4 relapsed (one at 3 mo, 3 at 12 months). Conclusion: Both voriconazole and posaconazole are safe and effective treatment options as second line antifungal therapy for SAFS and ABPA. Larger prospective studies are required.
A . Capetti, N. Zanchetta*, S. Melzi, P. Zucchi, L. Carenzi, M. Cossu, M. Gismondo, G. Rizzardini (Milano, IT) Background: The new methods of measuring HIV-1 RNA have lowered the levels of detection, and not all those patients whose viremia was classified as undetectable have levels below the new cut-offs. In this study we tried to assess some of the factors related to deeper HIV-1 RNA suppression among subjects on rescue therapy. Methods: The VERSANT HIV-1 RNA 1.0 Assay kit (kPCR, Siemens) is a procedure of kinetic polymerase chain reaction (kPCR) with reverse transcription (RT) for the direct quantification of HIV-1 RNA, with a range of detectability between 37 and 11.000.000 copies/mL. Below 37 copies, the system can still see some signal (SS) or no signal at all (NS), suggesting that the virus concentration in the sample in this case is extremely low. We selected our adherent patients on salvage therapy with darunavir/raltegravir-based regimens (the deep salvage patients, A) and matched each with patients on late lopinavir-based regimens (the experienced patients, B).
The two populations (A, n = 33, and B, n = 32) were homogeneous by age (48, A, vs 45.1 years, B, P = 0,13), baseline CD4+ T-cells (292 vs 338/mmc, P = 0,4) HIV-1 RNA log10 copies/mL (4,6 vs 4,8, P = 0,6) , and time on viral suppression (112,6 vs 115,1 weeks, P = 0,77). However, group A was on average in 11th treatment line with a mean GSS = 1,88, while group B was scattered around the 5th line, having a GSS = 2,83, P < 0,001. 21/33 subjects in group A and 16/32 in group B reached NS viremia, p = 0.32. Reaching NS viremia does not seem to be correlated with lower baseline viral load (4,6 in NS vs 2,9 log copies/mL in SS, P = 0,69), nor even with baseline GSS (2,3 vs 2,4, P = 0,81). Also the CD4-T cell nadir (158 vs 149, P = 0,77) and zenith HIV-1 RNA log log10 copies/mL (5,2 vs. 5,6, P = 0,21) were not predictive of NS response.
Overall, a fair proportion of subjects in rescue therapy had NS viral loads (57%), and late treatment lines were not associated to worse virologic outcome. The relationship between extreme viral load suppression (NS) and the latently infected T-cell pool will be further investigated. in three cases a low AI was detected. All other sera from long-standing HIV infection had a high AI (average: 1.01+0.08), with no differences between B and non-B genotypes. Conclusions: These results may have relevant implication in understanding the complex mechanism of maturation of the immune response to HIV. We will expand our study to other patients with recent HIV-1 infection and to treated patients in order to confirm this preliminary data. 23.3% of controls were on chronic steroid therapy for various reasons, mainly COPD. Two of the controls were obese. Conclusions: Although the HIV associated immunosuppression may initially predict a more severe clinical course of influenza, our results suggest that evolution does not differ from not HIV-infected people. We found a higher frequency of smoking among HIV patients, with an odds ratio of 2.4. It is striking also the high occurrence of COPD in both groups, without differences between them. If we exclude HCV coinfected patients with liver disease, none of the HIV patients had other chronic diseases, unlike controls. A.I. Papadopoulos, B. Ferwerda, V. Sakka*, L. Galani, A. Antoniadou, D. Kavatha, P. Panagopoulos, G. Poulakou, K. Protopapas, J.W. van der Meer, M. Netea, E.J. Giamarellos-Bourboulis (Athens, GR; Nijmegen, NL) Objectives: MyD88 adaptor-like (Mal/TIRAP) is an adaptor protein bridging activation of Toll-like receptors 2 and 4 after stimulation by exogenous and endogenous ligands. We investigated the association between the presence of the S180L SNP of Mal and the risk of severe infection in individuals with human immunodeficiency virus (HIV)-1 infection.
The SNP S180L was determined in a cohort of 179 HIV-1 infected Greek patients. Analysis of the prevalence of this SNP in relation to the infectious complications was evaluated.
One hundred and thirty two (73.3%) patients were bearing the wild type (WT) haplotype, 43 (24%) were heterozygous (HT) for the SNP, and four (2.2%) were homozygous (HO) for the variant allele. 39 patients carrying the WT haplotype experienced an infection (29.5%) compared to 12 HT patients (27.3%) and one HT patient (25%) (p ns). The individuals with a nadir CD4 count <200 cells/mm 3 who carried the S180L variant demonstrated a 4-fold decrease in the odds ratio (OR) for any serious infection compared with those who carried the wild-type 180S genotype (OR 0.58 vs OR 2.6, p = 0.016). Six patients developed B-cell non-Hodgkin lymphomas: two among patients bearing WT haplotypes (1.5%); and four among patients bearing the HT haplotype (9.3%, p = 0.040). Conclusions: This study suggests a protection effect of the Mal S180L SNP against serious infections in HIV-1 infected individuals with low CD4 cell counts.
H. Lederer*, Y. Achermann, M. Tinguely, F. Stenner, J. Fehr (Zurich, CH) In February 2010, a 55 year-old man with human immunodeficiency virus (HIV) infection was referred to our hospital with fever, weakness and diarrhea. The past three months he has been suffering from several febrile episodes. Extensive work-up during previous hospitalisations revealed no cause. HIV was diagnosed in 2007. Despite an excellent virological response to antiretroviral treatment with an undetectable plasma viral load the patient remained severely immunosuppressed with 107 CD4 cells/ml (5%).
On admission he was in reduced general condition, febrile (39.5ºC), had a normal heart rate, a low blood pressure, multiple marginally enlarged lymph nodes and an enlarged spleen. Laboratory tests showed pancytopenia, elevated C-reactive protein and acute renal failure. Vast examinations for bacteria, mycobacteria, helminths, protozoas remained all negative. Upper and lower endoscopic bowel examination with tissue biopsies showed no evidence of disease. Fluorodeoxyglucose (FDG)positron emission tomography scan revealed a pathological FDG-uptake in lymphatic tissue. Immunohistochemical examination was positive for human herpes virus type 8 (HHV-8) latency-associated nuclear antigen ( Figure) , which allowed the diagnosis of multicentric Castleman's disease (MCD). A treatment with rituximab, etoposide and valganciclovir for six cycles was started. Ten months later, the patient was free from new febrile episodes, was working full-time and CT scan revealed a decreased size and number of lymph nodes. MCD is a HHV-8 associated lymphoproliferative disease and should be considered as a possible cause of episodic fever. Disease presentation varies widely and the optimal treatment-regime is not yet established. Improvement of the immune system and treatment of the underlying disease is crucial. Antiviral agents have been successfully investigated. A promising approach for treatment in selected cases is rituximab. Antineoplastic agents such as etoposide or vinblastine are highly active in preventing the evolution of MCD towards lymphoma. Because of the unspecific findings, MCD can be difficult to diagnose in the setting of episodic fever and malaise.
We present a case of MCD for which it was extremely challenging to establish the diagnosis and assume that MCD is still underestimated even though it is more important than ever to have MCD diagnosed timely as new very promising therapeutical approaches exist. Objectives: Hepatitis B virus (HBV) infection is more frequent and severe in HIV-infected patients than in the general population. We assessed the impact of a 3.5-year nurse intervention to improve immunity against HBV in patients from the Swiss HIV Cohort Study (SHCS). The intervention was conducted in one center (intervention center) and 6 other centers were used as a comparator (control centers). Methods: SHCS participants who were seronegative for HbsAg and anti-HBc in January 2007 were included in the study groups and followed up until June 2010. Non immune patients (anti-HBs <10 IU/L) and patients with unknown immunity were eligible for nurse intervention, consisting of (1) documenting HBV serostatus in patients with previously missing information, (2) providing vaccination (3 doses with a minimal interval of 1 and 6 months) to non-immune patients, (3) measuring vaccination effectiveness 1 month after the 3rd dose and (4) providing a second course of vaccination to non-responders. Results: A total of 238 and 2712 patients seronegative for HbsAg and anti-HBc were included in the intervention and control centers, respectively ( Figure 1 ). Between 2007 and 2010, the number of patients with absent immunity decreased from 124 (52%) to 56 (24%) in the intervention center, and from 1658 (61%) to 1505 (55%) in the control centers (P < 0.001). The number of patients with undocumented immunity decreased from 76 (32%) to 0 (0%) in the intervention center, and from 174 (6%) to 131 (5%) in the control centers (P < 0.001).
Conclusions: HBV immunity in the HIV population is insufficient in Switzerland and can be significantly improved by nurse intervention. (12%) were HIV antibody positive. Sixty six patients (65%) had HBsAg checked but only one was positive. Fifty nine (58%) patients had a viral load measured and 31 (30%) had the virus genotype checked. The AST was measured in 75 (74%) patients. 99 patients were HBsAg positive. These tests were requested by above specialist teams in 43 (43%) patients. HIV serology was checked in 63 (64%) patients and this was positive in 6 (10%). 52 (53%) had HCV antibody checked. 49 (49%) patients had a HBV viral load checked and AST was measured in 77 (78%) patients. One patient was positive for HBV, HCV and HIV. Conclusions: HIV testing in hepatitis B and C patients is far from universal, however the same applies to testing for a second blood borne hepatitis virus in these patients. The laboratory currently recommends referral to gastroenterology services in cases of positive viral hepatitis B & C markers on the reports. This should be extended to include considering testing for other blood borne viruses including HIV. The results suggest that a substantial number of requests did not come from the specialist teams in the hospital suggesting they may not have been involved with all patients. Further investigation is warranted to establish the targets for further education.
The fusion process of the Human Immunodeficiency Virus type 1 (HIV-1), is mediated by the gp120 surface protein and the gp41 transmembrane protein.
To facilitate viral entry, the gp120 glycoprotein must bind to cell-surface CD4, alter its conformation to reveal a site for co-receptor attachment, and trigger conformational rearrangements in the gp41 glycoprotein to mediate fusion of viral and host cell membranes. Therefore the gp120-CD4 interaction is critical for virus-cell fusion. The gp120 region that binds CD4 is the target of the broadly neutralizing antibody B12. The B12 antibody targets gp120 and recognizes a highly conserved epitope overlapping the CD4-binding region of gp120. This antibody is one of the four known human monoclonal antibodies identified that can efficiently neutralize a broad array of primary isolates of HIV-1 in vitro and can protect against viral challenge in vivo. The goal of this project is to evaluate the potential of CD4 targeting and virus-cell fusion inhibition of HIV-1 by a single domain antibody grafted with the gp120 highly conserved epitope recognized by the B12 antibody. This will be done using the 23 amino acids loop of gp120, grafted at the CDR1 of a highly stable rabbit single domain VL antibody, and designated VL-B12. The potential of this VL-B12 construct has been tested for CD4 binding by ELISA assay against soluble CD4 and by FACS analysis using HeLa, HeLa-P4, 293T and Jurkat cell lines. VL-B12 is highly specific and exhibits a high binding to CD4 in the conditions tested. Preliminary inhibition assays performed in Jurkat cells, a Human T lymphocyte cell line, in the presence of the VL-B12 construct show no apparent HIV-1NL4−3 inhibition, indicating that VL-B12-CD4 binding alone is not sufficient to block virus-cell fusion and that a steric effect due to the presence of a larger molecule may be necessary for HIV-1 fusion inhibition. The VL-B12 is being tested in Cell-to-cell inhibition assays and standard Jurkat cell line inhibition assays in interaction with different molecules to further evaluate its HIV-1 inhibition potential.
In conclusion, this VL-B12 appears to be very promising for CD4 targeting and a valuable mediator of biopharmaceuticals. To fill these gaps, the CCH Fever project proposes to create a multidisciplinary collaborative research environment by bringing together selected competitive advantages such as: operative capacity with appropriate high security research facilities, reference centers and clinical samples from endemic areas and an international network of e xperienced researchers. This multidisciplinary research consortium will facilitate the progress in several key research areas of the field. This program will mainly focus on (i) developing sensitive and biosafe stateof-art diagnostic tools for CCHFV, (ii) gathering the forces and resources in Europe to build a Biobank of clinical samples, (iii) building a comprehensive database consisting in clinical, laboratory and surveillance data, (iv) taking advantage of unique and state-of-art tools to progress towards vaccine candidates and specific antivirals against this bio-treat and (v) disseminating the appropriate knowledge to the health care workers in endemic regions and contributing to capacity building. These achievements will provide tools for local and European public health authorities to prevent or counter future outbreaks and monitor the spread of the disease thanks to the established novel and unique tools and resources. All patients were examined by gynecologist. There was drawn the biopsy material from cervical canal of uterus, the blood serum in each patient. At the time of relapse a PCR method and a fluorescence immunoassay were used for detection of HSV2 in biopsy material and immunoglobulin G to HSV2 in serum respectively. Also urinalysis and urine culture were done. All patients were treated by acyclovir. Results: All patients had a history of more than 4−8 episodes of LUTI per year. The most common complaints were frequent and painful urination, itch and burning. There was observed the relationship of LUTI relapses with menstrual cycle in each woman. The gynecological examination was displayed no abnormal changes in each patient. In the urinalysis there was revealed the increased amount of epitheliocytes only. All urine cultures were negative. All biopsy materials from cervical canal of uterus were negative for HSV2, but about 75% (45/60) patients with LUTI symptoms were found high level of immunoglobulin G to HSV2 (1:1600−1:3200) in blood serum. Three weeks later the antibody G titer increased twice. The patients were treated by acyclovir 400 mg three times a day for five days. Then the suppressive therapy with acyclovir 400 mg twice a day was given for 3−5 months. After the full course of treatment no one patient complained of LUTI.
In the patients with frequent episodes (more than 4 per year) of LUTI symptoms and normal urinalysis herpes genitalis should be suspected. The fluorescence immunoassay may be used in case of receiving negative PCR results. Results: In our study, 27 patients were enrolled due to M. marinum infections. Among thirty isolates, three were repetitively isolated from two patients. According to the criteria of clinical outcomes mentioned in methods, 18 patient's outcomes were successful, eight were failed, and one was lost to follow-up. Statistically, 8 failed and 1 lost to followup were pooled into "not successful" group. In "successful" group, the mean (±SD) age is 50.1 (±21.6). In "not successful" group, the mean (±SD) age is 45.8 (±19.3). Twelve patients had either fish tank contact or fishing hobby. Two had shrimp contact, and one played in a swimming pool. One received intra-articular steroid injection. Three patients got minor or superficial trauma on their extremities. Eight had no documents about their contact history. In successful group, 9 patients had ever received surgical debridement; in not-successful group, 1 patient had received surgical debridement (2sided Fisher's exact p = 0.0912). Conclusion: In Taiwan, this is the first study to unravel the relationship between clinical outcome and susceptibility testing of bacterial strain. Optimal anti-mycobacterial regimens have not well been established, all drugs had been reported as successful agents or failed agents. In our study, we cannot identify the risk factors of treatment failure, but the duration of prescribing clarithromycin, rifampicin, ethambutol, and doxycycline were different between "successful" group and "notsuccessful" group. Pulmonary NTM infection appears not to be a uniform disease. The clinical and radiological presentation varies, but seems independent of mycobacteria species involved. None of our patients progressed or deteriorated due to NTM-infection, four patients are cured. In our opinion this result was achieved because of thorough interdisciplinary discussions in the ID-board considering the clinical presentations, co-morbidities and expected drug-toxicities which led finally to a favourable outcome of our patients. Comparison between test in vivo (TST) and in-vitro (IGRA PPD) demostrated that IGRA performes better than TST when CD4 cells are below 500 cells/ml; TST is not useful in detecting immunological response to tubercular infection in case of immunodeficiency. L. Gkaravela*, A. Foka, M. Sevdali, F. Kolonitsiou, A. Spiliopoulou, E.D. Anastassiou, I. Spiliopoulou (Patras, GR) Objective: An increase in the number of tuberculosis cases in Southwestern Greece is observed. The re-emergence of disease combined with isolation of multidrug-resistant strains has intensified the need for rapid diagnostic methods for mycobacteria. The rate of mycobacteria isolation with the Bactec/9000MB system and the Löwenstein-Jensen Barry, A. Al-Somily, F. Buba, U. Yusuf, N. Al Anazi (Riyadh, SA) Objectives: 1. Compare the baseline and post treatment values of CD4 and CD8 of patients with tuberculosis and controls. 2. To analyze the sequential CD4 counts during and after treatment. Subjects and Methods: Twenty eight (28) consecutive adult patients diagnosed with different clinical forms of tuberculosis were recruited. Eligible patients were enrolled based on compatible symptoms of TB and positive Mycobacterium tuberculosis based on Ziel-Nielsen smear and/or culture of relevant specimens as determined by the Bactec system and/or Lowenstein-Jensen culture methods. All controls were selected relying on the absence of history suggestive of tuberculosis and negative tuberculin tests. Both subjects and controls were screened for HIV and ensured negative using Enzyme-linked immunosorbent assay (ELISA) and Recombinant immunoblot assay (RIBA). Flow cytometry study was done as per protocol. Patients and controls were excluded if they have the following conditions: any form of immunodeficiency syndromes, diabetes mellitus, chronic kidney disease and concurrent use of immunosuppressant medications. Informed consent was sought from both subjects and controls before enrollment.
Results: Twenty eight consecutive (28) patients with varied forms of tuberculosis were enrolled. The baseline CD4 counts of patients (mean ±SD of 556.8±297.8) were significantly reduced as compared with matched controls (1132.3±259.9) at a p value of 0.000. Further the pretreatment and post treatment values of patients were significantly different as recorded as follows: 556.8±297.8 versus 954.3±210.9 with a p value 0.000. The baseline CD8 counts were not significantly reduced (p value 0.013) as the values were 1136.0±512.1 and 1461.9±367.0 for patients and controls respectively. However, there were improvement of the CD8 counts after treatment; baseline counts of 1136.0±512.1 versus 1316.5±286.2 after treatment (P value, 0.002).
The study showed significantly lower baseline CD4 counts among patients with tuberculosis as compared with the controls. Further, the counts significantly rose towards normalization at the end of treatment. We therefore conclude that tuberculosis is associated with CD4 lymphoenia independent of other notable causes. The counts are reversible and may indicate a success of treatment. Objectives: To compare the use of tuberculin skin test (TST) and Interferon-g (IFN-g) Release Assay using three specific antigens (ESAT-6, CFP-10 and TB7.7) (QuantiFERON ® -TB Gold in tube) for the diagnosis of tuberculosis infection (TBI) and indication of treatment in health care workers. Methods: We conducted a prospective transversal study of health personnel who came for routine health care study (may 2007 to june 2010). All were screened with TST and QFT (Cellestis, Australia) and risk factors were registered in a questionnaire. Patients with a positive result (TST or QFT) were screened also with chest X-ray. TST was performed by Mantoux method and a positive test was defined as an induration 5mm in non-vaccinated and 15 mm in vaccinated people. QTF was made according to the manufacturer specifications. We considered as vaccinated persons those presenting with a suggestive scar. CDC recommendations were followed for the interpretation of the QFT. Agreement between TST and QFT was assessed by the Cohen kappa coefficient. Results: We studied 316 health care workers (72.2% women) from the General Hospital of Jerez. Average age was 43.4 years (SD: 8.8), 76.4% had been vaccinated with BCG. TST was not done in 104 (32.9%) persons because of a previous positive TST. TST was positive in 50 (68.5%) and QFT in 30 (41.1%) non-vaccinated people. Whereas, for vaccinated people TST was positive in 93 (39.1%) and QFT was positive in 53 (22.2%). Agreement between the TST and QFT was 64.4% (Kappa 0.33, CI (0.15-0.51)) among the non vaccinated group; when we defined positive test for TST as an 10 mm induration, agreement was 71.2% (Kappa 0.43, CI (0.23-0.63). Agreement was 64% (Kappa 0.18, CI (0.06-0.30)) for the vaccinated group. Two indetermined results were detected by QFT. The indication of TBI treatment made by TST and risk situation was modified in 70% of cases according to QFT test. We prescribed treatment of TBI by QFT in 13% of the patients that did not have this indication according to the TST.
1. Agreement between TST and QFT was low in vaccinated and nonvaccinated people. 2. QFT was better than TST for recent tuberculosis infection diagnosis in health care workers because of its high specificity and no interference of booster 3. QFT was a better indicator for treatment of tuberculosis infection. Further studies addressing IFN-g sero-conversion and -reversion in health care workers for the follow-up of health care personnel are needed. 2) and came for screening of tuberculosis infection. All were screened with chest X-ray, TST, QFT (Cellestis, Australia) and risk factors were registered in a questionnaire. TST was performed by Mantoux method and a positive test was defined as an induration 5mm in non-vaccinated and 15 mm in vaccinated people. QFT was made according to the manufacturer specifications. We considered as vaccinated persons those presenting with a suggestive scar. CDC recommendations were followed for the interpretation of the QFT and the treatment of the TBI. Agreement between TST and QFT was assessed by the Cohen kappa coefficient. Results: Agreement between TST and the QFT among non-vaccinated patients was 84.2% (Kappa 0.69, CI (0.56-0.82)). When we redefined positive test for TST as an induration 10 mm, agreement rose to 86.8% (Kappa 0.74, CI 0.61-0.86)). Agreement for vaccinated people was 70.7% (Kappa 0.38, CI (0.26-0.50)). QFT (−)/TST (+) results was the most frequent disagreement in non-vaccinated people. QFT was indeterminated for 5 patients, 4 of these were negative for TST. We prescribed treatment of TBI by QFT in 14% of the patients that did not have indication according to the TST. The indication of TBI treatment made by TST and risk situation was modified in 46% of cases according to QFT test.
1. Agreement between TST and QFT was good in non vaccinated intravenous drug users. 2. Agreement between TST and QFT was low in vaccinated intravenous drug users even if TST was considered positive with the 15 mm criterion. 3. QFT test is a better tool to identify infected individuals and to reduce the number of unnecessary TBI treatment.
Background: Global control of tuberculosis is hampered by slow, insensitive diagnostic methods, particularly for the detection of drugresistant forms and in immunocompramised patient's Early detection of tuberculosis is essential to reduce the overall mortality and morbidity as well as elimination of disease transmission, but the complexity and infrastructure needs of sensitive methods limit their accessibility and effect. R2690 Risk factors for long-term treatment in lymph node tuberculosis J.P. Lanoix*, T. Guimard, N. Ettahar, A. Grannec, C. Flateau, C. Chapuzet, P. Tattevin, J.L. Schmit (Amiens, Rennes, Tourcoing, Caen, Lille, Rouen, FR) Introduction: Lymph node tuberculosis (LNTB) is the most frequent extrapumonary tuberculosis (TB), but some authors reported huge differences between duration of treatment instead of WHO 2003 recommendations for 6 to 9 months of antiTB therapy. We conducted a retrospective multiple centre study in the aim to describe the clinical factors for prolongation of antiTB drugs. Material and Method: We included retrospectively all patients who presented with at least LNTB during 1998-2010 periods from seven hospitals in North of France. TB was diagnosed either by culture or histology or clinical suspicion improving with antitubercular drugs. Five universities hospitals participated. We excluded from treatment duration analysis patients with bone and neurological involvement. Results: 148 patients were included, 57 of them were men (38.5%), and 58 (41.1%) were Africa born. Median age was 43.2 years (extremes 13.2-91.3). There were 21 (16.2%) HIV infected patients, 13 of them (54.2%) were diagnosed HIV positive at the same time at the TB diagnosis. Mean CD4 cell was 193.7/mm3±151. Forty two patients (28.8%) had another TB localisation (of whom 4 had bone or neurological involvement), 91 (61.9%) had superficial lymph nodes, 73 (49.6%) had cervical localisation only. Four (2.7%) patients died, 13 (8.8%) were lost of follow up. Duration treatment analysis was undertaken on 126 patients: median duration was 9 months (extremes 2−30). Treatment was significantly longer in HIV positive patients (p < 0.01), in patients with other TB localisation than lymph node (p < 0.01), in patients with loss of weight (p = 0.02), and if patient had presented new lymph node during treatment (p = 0.023). The treatment was significantly shorter when no complication occurred, and the main cause for treatment prolongation was non favourable evolution. Seven patients presented relapse, 2 have been treated for 9 months, 2 for 6 months, 2 for 4 months, one for 30 months before relapse. Conclusion: LNTB is treated much longer than WHO recommendations. Only 58% of treatment length is in accordance with 2003 recommendations, and 23% with 2009 recommendations which suggest that 6 months are sufficient for all extrapulmonary TB except for bone and neurological TB. Factors associated with treatment duration are HIV infection, loss of weight and other tuberculosis localisation.
R2691 Prevalence and aetiological pathogens of asymptomatic bacteriuria in type 2 diabetic patients with and without microalbuminuria I. Daniil*, A. Papazafiropoulou, S. Konstantopoulou, A. Sotiropoulos, E. Balampani, A. Kokolaki, S. Bousboulas, S. Pappas, D. Petropoulou (Piraeus, GR) Objectives: The prevalence of asymptomatic bacteriuria (ASB) in diabetic patients is high, especially in women. The aim of this study was to evaluate the prevalence and to identify the aetiological pathogens of ASB in patients with type 2 diabetes mellitus (T2D) with and without microalbuminuria (MA). Methods: A total of 200 patients with T2D (100 with MA and 100 without MA) were recruited in the study. Patients with overt diabetic nephropathy or nephropathy from other causes, with symptoms of urinary track infection or use of antimicrobial drugs in the last 14 days were excluded by the study. Microalbuminuria was diagnosed measuring the albumin excretion rate (AER) by radioimmunoassay (RIA) method (Pharmacia, Pharmacia and Upjon Diagnostics AB, Uppsala, Sweden). Midstream clean voiding urinary specimens were collected for urinalysis, examined by Gram stain and cultured on blood and MacConkey agar for detection of uropathogens. Any isolated pathogen (after 24−48hours incubation) was identified using BBL™ Enterotube™ II (BD Diagnostic Systems, Germany), Api System and Vitek 2 Compact (Biomerieux, France). Results: The prevalence of ASB was increased in diabetic patients with MA compared to diabetic patients without MA (21% versus 8%, P < 0.001, respectively). Escherichia coli was the most prevalent pathogen isolated in diabetic patients with and without MA (12% versus 3.0%, P = 0.01, respectively) followed by Proteus mirabilis (6% versus 5%, P = 0.75, respectively) and Klebsiella spp (5% versus 1%, P = 0.09, respectively). Conclusion: ASB was more prevalent among T2D patients with MA and the main causative agent was E. coli. Screening for ASB is warranted in diabetic patients especially if pyuria is detected in urine analysis since ASB has been found to be a risk factor for developing symptomatic urinary tract infection. Results: Kp strains revealed various susceptibility to imipenem and meropenem. All strains were resistant to ertapenem, but most of them were susceptible to gentamicin, tetracycline, tigecycline and colistin. All strains were KPC-2 producers and belonged to the same clone. Patients' mean age was 70 years (range 25−81). Clinical diagnosis were: nine acute leukemia, one chronic lemphogenic leukemia, one aplastic anemia and one non Hodgkin lymphoma. The duration of patients' hospitalization was 0−24 days, before the isolation of Kp strains. In three patients, a simultaneous isolation of KPC strains from urine cultures was observed. Two patients with bacteremia were found to be colonized in the intestine with with KPC strains, while four patients developed first the bacteremia and intestine colonization followed. Neutropenia was observed in eight patients, with an absolute number of neutrophils 0-400/ml. Before the isolation of the KPC strains, all patients were treated with a combination of a b-lactam antibiotic and an aminoglycoside, while after the isolation the treatment changed to colistin, tigecycline and/or gentamycin. The response was poor and ten patients out of twelve (83%) died because of the KPC infection. Conclusions: Bacteremia from KPC-2 producers Kp, in hematological patients, has high mortality and consists a serious medical-nursery problem, in addition with the already existing high bacteria resistance. T. Damci (Istanbul, TR) Objectives: To identify risk factors contributing treatment responses and prognoses in severe diabetic foot infections. Material and Method: Between April 2008 and October 2010, a prospective study was conducted by Diabetic Foot Study Group on severe diabetic foot infections. Results: A total of 62 patients were included in this study, the mean age was 64.09 (range 37 to 87) years and forty three (69.4%) patients were male. Eighty patients were diabetic for more than ten years. 56.5% of the patient had previous hospital stay and 83.9% had previous antibiotic use within the last three months. The duration of wound infections were more than four weeks in 84% of the patients and 56.5% of them had purulent discharge. Leucocytosis was found in 26 (46.8%) patients and 41 (66.1%) patients had elevated CRP levels more than five fold. 14.5% of patients were classified as Wagner stage 4 and 8% were Wagner stage 5. Thirty three (53.2%) of the patients were presented with osteomyelitis. Etiology was identified in 41 (66.1%) patients. Gram negative bacilli were isolated in 19 (46.3%), Gram positive cocci were isolated in 10 (24.4%) and 12 (29.2%) grew mixed bacteria. Ten of 41 microorganisms isolated were ESBL producing Gram negative bacilli, three were MRSA, nine were MRSE, seven were multidrug resistant (MDR) P. aeruginosa and three were MDR A. baumannii. The mean duration of hospitalization was 38.4 days; 37 (59.7%) patients underwent debridement and amputation was performed in 30 (48.4%) patients. Major amputation was reqüired in 9% of cases. Conclusion: Complete clinical improvement was observed in 26 (41.9%) patients. Duration of diabetes over ten years (p = 0.017), higher fever ( 38.5ºC) (p = 0.004), existance of purulent discharge (p = 0.020), higher Wagner stage of the wound (p = 0.000), elevated CRP levels (>5 fold) (p = 0.01), infection with MDR bacteria (p = 0.066), requirement of amputation (p = 0.000) and prolonged hospital stay (p = 0.01) were negative predictors of treatment success and prognosis. Methods: 103 CMV seropositive patients over the age of 18 − and whose donors were also seropositive − who had undergone renal transplantation between January 2007 and June 2010 were included in the study. The data on these patients were recorded retrospectively using follow-up forms. The form included the following information: age, gender, primary disease, cold ischemia time, type of transplantation, immunosuppressive treatment scheme, duration of valganciclovir use, adverse effects, CMV antigenemia level and whether CMV infection/ disease developed after transplantation or not. In the prophylaxis group VGC (n = 29) was administered 450mg/day. For the other group (n = 26) VGC was not administered only short time IV gansiklovir was administered. If the patient received antithymocyte globulin (ATG) treatment, IV ganciclovir was administered only during the treatment. The patients were followed using CMV antigenemia tests (1−3 times/ month). All patients were followed for 6−30 months after transplantation. The data were evaluated using chi-square test and t test for independent groups on SPSS 15 software.
The demographic characteristics of the RTr who did or did not receive VGC prophylaxis were similar. Total ATG dose was significantly higher in the prophylaxis group than in the other group. It was determined that, although not significant, the rate of CMV infection development was lower in the patients to whom prophylaxis was administered ( Results: 2 cases of HMPV pneumonia were identified in an Australian haematology unit in spring, 2010. The cases were in separate but adjacent rooms raising the possibility of nosocomial transmission.
Lymphopenia was present in both as a complication of chemotherapy for anaplastic large cell lymphoma (case one) and after autologous peripheral stem cell transplant for multiple myeloma (case two). MRSA and HMPV were identified in bronchoalveolar lavage (BAL) fluid. Respiratory intubation, ventilation and intensive care management was required for progressive respiratory failure whilst on maximal therapy for MRSA infection. Bilateral ground glass infiltrates progressed to nodular, confluent parenchymal changes. Salvage treatment with ribavirin was started more than a week after symptom onset. Ribavirin 25mg/kg/day (intravenous) was followed by 15mg/kg/day (intravenous) for a total of 7 days in both cases. One patient cleared virus from respiratory tract, but progress was complicated by a cerebrovascular accident. The second patient developed extensive air space consolidation and cavitation. HMPV was detected by PCR at 6 weeks after treatment with ribavirin.
Conclusion: HMPV infection is associated with significant morbidity and poor outcomes. Lymphopenia is a risk factor for infection with HMPV in adult patients receiving treatment for hematological malignancy. Co-infection with MRSA occurs. Neutrophil recovery and hypogammaglobulinaemia may contribute to severity of infection. Ribavirin was well tolerated. Late treatment with intravenous ribavirin for seven days did not eradicate viral shedding in one patient, and may have contributed to clinical and virological cure in the other. HMPV may be detected in respiratory secretions for greater than 6 weeks in adult patients with haemopoeitic malignancy, with implications for infection control measures. was an independent risk factor for PBSI, while prior blood transfusion was revealed as protective factor (OR 3, 9; 95% CI, 5) . Empirical inappropriate antimicrobial treatment has been given to 20 patients (59%) with PBSIs, and 7 (21%) with MBSIs (p = 0.003). The infectionrelated mortality was 29% in patients with PBSI and 12% in those with MBSI (p = 0.072). No differences in duration of hospitalization or overall mortality were observed. Conclusion: PBSIs represent a significant percentage of BSIs among patients with malignancy. Although inappropriate empirical antimicrobial treatment has been given in higher percentage of patients with PBSI than those with MBSIs, no differences in duration of hospitalization or overall mortality have been observed. Recognition of the risk factors for PBSIs and knowledge of their microbiology are important for the selection of appropriate empirical antimicrobial treatment that may result in improved outcome. Results: Sixty-one UTI episodes were diagnosed in 51 patients. Sixtynine percent of the patients were female. The range of the ages were 18−60 years. More than one UTI episodes were seen in 8 patients. The three leading uropathogens were E. coli, Klebsiella spp and enterococci. E. coli was detected in 41 (67%) episodes, Klebsiella spp. was detected in 11 (18%) episodes and enterococci was detected in 5 (8%) episodes. Twenty-one of the E. coli isolates (51%) were ESBL positive. The antimicrobial resistance rates of ESBL negative and ESBL positive E. coli isolates were shown in the Table. The risk factors determined are hospitalization at the time of diagnosis or hospitalization within 1 month, recent (within 1 month) antibiotic use, urinary catheterization. Conclusion: Risk factors for urinary tract infections caused by ESBL producing bacteria among renal transplant recipients are similar with the other patient groups but nearly half of the uropathogen E. coli strains are ESBL positive in renal transplant recipients. This high percentage of resistant pathogens causes difficulties in the management of these patients. Multidrug resistant bacteria are increasingly isolated from transplant recipients and cause high morbidity, mortality. The aim of this study is to determine the disribution and etiologic agents of bacterial infections seen in the early period after liver transplantation.
A retrospective study was conducted on 90 patients who underwent orthotopic liver transplantation consecutively from January 2008 to October 2010 at Baskent University Hospital. Microbiologically documented bacterial infections seen during the first month after liver transplantation were included in this study. A total of ninety liver transplantations were performed during the study period. Twenty eight bacterial infection episodes were diagnosed in 20 patients within 30 days after transplantation operation. Twelve (13%) of the infections were intraabdominal (9 Gram negative and 3 Gram positive), 8 (9%) were catheter-related (4 Gram negative, 4 Gram positive), 6 (6%) urinary tract infection (5 Gram negative, 1 Gram positive), 2 ventilator associated pneumonia (1 Gram negative, 1 Gram positive). A total of 19 episodes were caused by Gram negative bacteria and the remaining 9 were caused by Gram positive bacteria. Strains susceptible only to colistin and tigecycline were defined as extensively drug resistant (XDR). Seven of the isolated Gram negative strains were Klebsiella pneumoniae (2 ESBL positive, 2 XDR strains), 6 were Escherichia coli (4 ESBL positive strains), 4 Acinetobacter baumannii (3 XDR strains) , 1 Pseudomonas aeruginosa. The distribution of the Gram positive pathogens were 4 Enterococcus faecium (2 were vancomycin resistant), 3 methicillin resistant coagulase negative staphylococci, 1 methicillin sensitive Staphylococcus aureus and 1 methicillin resistant Staphylococcus aureus. A total of 20 bacteremia episodes were detected of which 12 (60%) were secondary and 8 (40%) were primary. The incidence of colonization and infection with multi-drug resistant bacteria particularly Gram negative strains has been increasing throughout the world. Data regarding the transplantation patients are limited but common usage of extended spectrum antibiotics among these patients both during the preoperative and postoperative phases undoubtedly predispose to difficult-to treat infections. The infection rates seen in the early postoperative period obtained in this study are comparable with the previous ones but the high percentages of multidrug resistant strain is the alarming issue.
Community-acquired infections including CAP, sepsis, STD, . . . C. trachomatis was significantly most frequent isolated in: >1 sexual partner (17.4% vs 5.8%), discharge (18.5% vs 9.3%), dysuria (18.9% vs 9.1%) and leukocytes in GRAM stain (21.6% vs 10.1%). U. urealyticum had significant difference for age (28.6± 7 vs 33.6±10) and we did not find significant difference for the rest of issues studied. We did not find significant differences for the rest of isolated microorganisms. Conclusions: Urethritis was most frequent in young men, with >1 sexual partner, with discharge and leukocytes in GRAM stain. N. gonorrhoeae was most frequently isolated in HSH, with >1 sexual partner, with pain and discharge like symptoms and leukocytes in GRAM stain. C. trachomatis was isolated in men with >1 sexual partner, dysuria and leukocytes in GRAM stain and U. urealyticum was most frequent in youngest men. Microbial etiology of peritoneal dialysis (PD) related peritonitis seems to vary widely in children and data for adults are scarce. The objective of this study was to determine the microbial etiology, correlation between leucocyte (wbc) count and culture results as well as the concordance between culture and the Gram stain of PD related cases of peritonitis in both children and adults. We reviewed the records of all patients whose peritoneal dialysate (n = 1285) was sent to the lab for microbiology of the Antwerp University Hospital between January 2005 and August 2009. Microbial etiology, wbc count, Gram stain and antibiotic treatment were analyzed per episode. 142 adults and 22 children were included. From each episode, microbial etiology was based on culture results of the first sample. Gram stain was performed when the leucocyte count exceeded 200 per mm3. Logistical and linear regression were used in addition to a mixed effects model with repeated patient effect to determine the degree of correlation between the wbc count and positive cultures.
In 158/39 episodes of adult/pediatric peritonitis, 70.7/70.0% of samples were culture positive (+): 72.1/52.4% of isolates were Gram positives (G+), 21.2/38.1% Gram negatives (G−), 1.9/0.0% anaerobes and 4.8/9.5% yeasts. In 29.3/30.0% no etiology was found: 32.6/22.0% of the culture − were taken during antimicrobial treatment. Gram stain showed predominant morphotypes concordant with culture results in 30.5/16.7%. In adults, the G+ were mainly coagulase negative staphylococci (CNS) (70.7%), S. aureus (8.0%) and viridans streptococci (8%); in children, predominant pathogens were E. faecalis (27.3%), CNS (27.3%), Coryneforms (18.2%) and S. aureus (9.1%). The most prevalent G− pathogens in adults were E. coli (22.7%), E. cloacae (18.2%), K. pneumoniae (18.2%), P. aeruginosa (13.6%) and other non-fermenters (13.6%). In children, the most frequent G− were E. cloacae (25.0%), P. aeruginosa (12.5%) and other non-fermenters (25%). A significant difference in wbc count was found between culture+ and culture− samples in adults (p = 0.001).
In conclusion, a microbial etiology was found in the majority of cases. Gram positive related cases of peritonitis are more frequent in adults, Gram negatives in children and the distribution of pathogens also differs. The concordance between Gram stain and culture appears low. Finally, there are significantly less leucocytes in culture negative dialysates compared to culture positive dialysates.
R2703 International PMEN clones equal or exceed the fitness of other strains despite the accumulation of antibiotic resistance D. Rudolf, N. Michaylov, M. van der Linden, L. Hoy, K.P. Klugman, T. Welte, M.W
Objectives: A small number of global pneumococcal clones defined by the Pneumococcal Molecular Epidemiology Network (PMEN) dominate the population of antibiotic-resistant pneumococci. It remains unclear why PMEN clones spread so successfully despite the scientific paradigm that a loss in biological fitness is the price for acquisition of resistance. The aim of this study was to detect PMEN clone related clinical isolates from adult patients with community-acquired pneumonia (CAP) collected by the German CAPNETZ surveillance study during [2002] [2003] [2004] [2005] [2006] and to compare them to unrelated clones in terms of antibioticresistance, biological fitness and clinical parameters. Methods: Multi-locus sequence typing (MLST) data were used to define relatedness between PMEN clones and clinical pneumococcal isolates. Relatedness was defined by the sharing of alleles at 5 of 7 loci. Fitness was determined by the measurement of growth curves. The bacterial growth was measured by a microplate reader at optical density of 600 nm at intervals of five minutes over a period of 16 hours. To compare bacterial growth we analysed the maximum slope of each curve and the experiment was repeated nine times. Statistical analysis were performed by the chi-square test or Fisher's exact test for categorial variables and the t-test or analysis of variances (ANOVA) for continuous variables. Results: We analysed 154 pneumococcal isolates and 46 (30%) isolates showed a close relationship to the global PMEN clones. These isolates were equal or exceeded the fitness of isolates without relationship to PMEN clones (1.48±0.73 vs. 1.18±0.54; P = 0.015) and constituted 80% of antibiotic-resistant isolates. The survey of clinical parameters showed no prominent significant difference between both groups. Conclusion: The success of international PMEN clones is based on the combination of resistance and fitness and may result in the endurance of these strains despite a reduction of antibiotic usage. New vaccines can interrupt the transmission of resistant strains, but continued attention to the replacement of nonvaccine serotypes and development of vaccines with a broader coverage will be necessary. Objective: The aim of this study was to compare the occurrence of genital Chlamydia trachomatis, genital mycoplasmas and ureaplasmas in semen samples of fertile and infertile men in Kuwait.
A total of non-duplicated 315 semen samples collected from 127 infertile and 188 control men seen at the infertility clinics in Maternity hospital were studied after informed consent. Semen analysis was performed according to the guidelines of World Health Organization. The specimens were examined for the presence of Ureaplasma urealyticum, Mycoplasma hominis, Mycoplasma genitalium and Chlamydia trachomatis by PCR using published specific primers. Biodata, such as age, nationalities, and marital status were carefully recorded.
Results: The frequency of genital U. urealyticum, M. hominis, M. genitalium and C. trachomatis in all semen samples was respectively 26% (82/315), 27% (86/315), 5.4% (17/315) and 8.3% (26/315). Mixed infections were detected in 14% (44/315). The infertile participants positive for U. urealyticum and M. hominis, respectively had semen samples that showed statistically significant difference in the mean of sperm concentration, vitality percentage, total progressive and rapid progressive motility in comparison to control fertile participants (P < 0.001). Similar statistical significance difference was noted for those infertile and fertile men positive for M. genitalium and C. trachomatis (P < 0.001). Infections in infertile men who had been married for less than 5 years were significantly higher than in infected fertile men of the same length of marriage.
Infections caused by these urogenital pathogens were more common among infertile men than fertile men and may possibly play a role, in part, in the etiology of male infertility in this part of the world. Genital mycoplasmas and chlamydial infections may negatively influence semen quality. Objective: Pleural empyema is a serious infection characterized by accumulation of pus in the pleural space. In the US, 65,000 patients suffer from empyema each year, with 30-day mortality up to 15%. Many studies addressing risk factors and outcome of empyema rely on hospital discharge diagnosis codes recorded in health care databases. We validated the ICD-10 diagnosis of pleural empyema in the Danish National Registry of Patients (DNRP).
We randomly selected hospitalized inpatients in the North Denmark Region with a first-time discharge diagnosis of pleural empyema between 1995 and 2009. We retrieved and reviewed medical records and estimated the positive predictive value (PPV) of the empyema diagnosis. Definite empyema was defined by frank pus aspirated from the pleural space, a positive Gram stain/subculture for pathogenic micro-organisms in the pleural fluid, and/or an autopsy diagnosis of empyema. Patients with clinical symptoms suggestive of empyema in association with compatible radiographic features (e.g. pleural thickening, loculated and/or septated pleural effusions) who did not meet the criteria for definite empyema were classified as having probable empyema. Results: We could retrieve the medical records of 224/225 sampled patients with empyema (99.6%). Of those, we classified 182 (81.3%) as being definite empyema cases. Another 21 patients were classified as probable empyema cases. In 21 patients whose empyema diagnosis was rejected, eight had pneumonia, one had a pulmonary abscess, one pulmonary tuberculosis, one had sarcoidosis, two had emphysema. The overall PPV of the empyema diagnosis was 90.1% (95% CI 86.0-94.1). The PPV was 81.3% (95% CI 81.3-86.1) when including definite cases only. PPVs of empyema diagnosed in 1995-1999, 2000-2004, and 2005-2009 were 90.7% (95% CI 81.7-96.2), 94.6% (95% CI 86.7-98.5), and 86.7% (95% CI 76.8-93.4), respectively, indicating that no major changes in coding validity occurred over the 15-year study period. The PPV decreased slightly from 95.7% in patients aged 15−39 years to 87.5% in patients aged 80 years and over but was uniformly high regardless of study period, hospital or department type, or cause of empyema.
The high overall PPV indicated good agreement between ICD-10 codes for pleural empyema and medical records. Registry-based discharge codes may be a suitable source of data on pleural empyema for epidemiological research. Introduction: Nocardia is a rare pathogen causing predominantly pulmonary and/or skin and soft tissue infections. Since immunosuppression is one main predisposing factor, nociardial infections may increase because of the rising number of patients on immunosuppressive therapy (e.g. solid organ or bone marrow transplantation). Data on futher risk factors, clinical course and treatment of nocardial infections are limited to case reports and small case series. We aim to establish a register for nocardia infections in Germany, Switzerland and the Netherlands. Methods: Retrospective analysis of all microbiologically proven pulmonary Nocardia spp. infections in 4 hospitals in Germany and the Netherlands between 1999 and 2009, defined as detection of Nocardia spp. in respiratory samples + radiolagical changes and signs and symptoms of pulmonary infection. Results: Twenty-one cases of pulmonary nocardiosis could be identified (18 male, 3 female; mean age 54.7±18.1 years). In addition to pulmonary involvement, disseminated disease was detected in 3 patients (all with cerebral abscess formation). Eleven of 21 patients had pulmonary comorbidities (n = 4 COPD; n = 3 bronchiektasis, n = 2 cystic fibrosis, n = 1 asthma, n = 1 post-TB). Twelve of 18 patients had identifiable causes of immunosuppression (hematological dieseases, malignancies, drug induced immunosuppression, diabetes mellitus). All patients tested (16/21) were HIV negative. Time from sampling to availability of microbiological results was 9.1 days. All cases were proven by culture and in 15 patients additional PCR-sequencing was performed (N. farcinica n = 4; N. abscessus n = 3; N. asteroides n = 3; N. nova, N. carnea, N. cyriacigeorgica, N. otitidiscaviarum and N. paucivorans each n = 1) The pathogen was isolated from BAL (n = 9), sputum (n = 7), biopsy (n = 3), tracheobronchial aspirate (n = 1), and abscess aspirate (n = 1). Most patients received cotrimoxazol treatment (15/21), mean duration of treatment was 12.2 weeks. One patient died. Conclusion: Pulmonary nocardiosis remains a rare disease. Although immunosuppression (e.g. drug induced immunosuppression or malignancies) is its major risk factor, it can also occur in patients without obviously impaired immunocompetence. These patients most frequently suffer from chronic pulmonary diseases such as COPD. The delayed in vitro growth of Nocardia spp. may lead to misdiagnosis or underestimation of nocardiosis in patients without typical risk factors.
R2708 A prospective study on predictors for early death from bacteraemia D.C. Lye*, S. Pada, T. Ng, R. Llorin, D. Kee, T. Lee, P. Krishnan, T. Barkham, B. Ang (Singapore, SG) Objective: Inactive empiric antibiotic occurred in 33% of patients with bacteraemia, with mortality of 27% at discharge in 2006. A blood culture service started in April 2007 to ensure active antibiotic within 2 days of blood culture collection. A prospective bacteraemia study from February to June 2009 noted mortality at discharge decreased to 9%. Death occurred in 2% before positive blood culture was notified. We aim to study patients with early death for clinical predictors. Methods: Patients who died before (early death) and after (late death) positive blood culture was notified were compared, so was early death with survivors. Univariate and multivariate analysis were performed for independent predictors of early death. Results: Of 452 patients with 467 cases of bacteraemia, 42 patients died, and 9 were early death. Male comprised 60%, median age was 78 years and Charlson's score 5. Staphylococcus aureus occurred in 10 (7 was methicillin-resistant [MRSA]), Escherichia coli and Klebsiella pneumoniae 5 each (6 carried extended-spectrum b-lactamase), Proteus mirabilis 3 and Candida species 2. On univariate analysis of early death vs. late death, age 75 years (odds ratio [OR] 9.14, confidence interval , and ventilatory support (OR 30.29, ). On multivariate analysis, the independent predictors of early death vs. survivors were: pneumonia (AOR 18.61, CI 2.68-129.53), inactive empiric antibiotic (AOR 24.40, CI 1.57-378−23), hypotension (AOR 17.01, , and ventilatory support (AOR 59.59, CI 1.27-2786.41).
Our study showed that hypotensive patients needing ventilatory support for pneumonia with inactive empiric antibiotic were more likely to die before blood culture could be notified. It is crucial to ensure adequate empiric antibiotic in this cohort.
Cobas ® Amplicor™ CT/GC test and SiemensVersant ® CT/GC DNA 1.0 assay (kPCR) for the detection of Chlamydia trachomatis/Neisseria gonorrhoeae
Objectives: Abbott RealTime m2000 (AR) method was compared to Roche Cobas Amplicor (RCA) for detection of Chlamydia trachomatis (Ct) and Neisseria gonorrhoeae (Gc) in urine samples. Siemens Versant (SV) was the third test for the minor series of the urine samples. AR and SV are automated systems with short hands-on-time. Tecan miniprep was used for DNA extraction for RCA. Methods: Ct was tested in 297 urine samples and Gc in 200 urine samples in RCA and AR. The samples were taken in the years 2008−10, tested with RCA within 1−2 days after the collection and frozen. The minor selection included 75 frozen urine samples for AR. Also these were tested within 1−2 days after the collection with RCA-and SVmethods.
Results: AR detected 170 Ct-positive while RCA 176 among 297 samples with the correlation of 0,966 for Ct-positive and of 1,05 for negative results. In Gc-testing, RCA found 13 (confirmed) positive samples while AR 12. AR detected as Ct-positive 45 out of 75 samples while RCA 47 with the correlation of 0,957 and SV 49 with 1,042, respectively. All the tests gave the same 20 samples as Ct-negative and 40 as Ct-positive. RCA and SV detected the same samples negative and SV 2 positive more than RCA, while RCA compared to AR, AR had 5 negatives and 1 positive more than RCA. SV and AR tested the same samples Ct-negative but SV 4 Ct-positive samples more. And, RCA detected the other 3 Ct-negative samples more than AR-and RCA. All 3 methods detected the same 4 Gc-samples as positive.
Conclusion: RCA used the DNA extractor more sensitive for contamination than the other two. The manual pipetting may have been the risk for false-positivity, too. There is also possibility of false-negative samples, because of inability to detect the new variant of Ct (nvCt) unlike AR and SV. Sample storing as frozen may have some effect on results. AR needed sample volume less than SV. AR offers separated units for DNA extraction and PCR itself. Gc-positive samples detected by AR (target the opaA gene) need no confirmation because of high specificity. These make AR a suitable system for clinical microbiology laboratories. The prevalence of nvCt needs to be tested.
S. Cho*, J. Chung, S. Choi, Y. Kwak (Seoul, Gyeonggi, KR) Objectives: Actinomycosis is a chronic infection caused by anaerobic or microaerophilic, Gram-positive bacteria, Actinomyces spp. classically it involves cervicofacial (55%), abdominopelvic (20%), thoracic (15%), and mixed organs (10%), including skin, brain, pericardium, and extremities. But recent studies reveal change about types of actinomycosis. This retrospective study describes the clinical manifestations of patients with actinomycosis.
this study, the duration of IV antibiotic treatment ranged from 10 to 30 days. 2 patients received oral antibiotic therapy without IV antibiotics. The duration of oral antibiotic treatment ranged from 67 to 150 days. One patient received surgical treatment only without antibiotics. Conclusion: Over the last three decades, the incidence of actinomycosis has declined markedly because of better oral hygiene and more extensive use of antibiotics and clinical pictures have changed. The incidence of cervicofacial type is declined and thoracic type is increased. The treatments of actinomycosis are also changed, short-course chemotherapy has recently been reported to be successful.
M. Renko*, H. Kukkola, H. Kauma, T. Tapiainen, M. Uhari (Oulu, FI) Background: Streptococcus pneumoniae is a common cause of community acquired invasive bacterial infections both in children and adults and infects both previously healthy and diseased subjects. Incidence of invasive pneumococcal diseases in different age groups is well known but the effect of age on the clinical outcome of invasive pneumococcal diseases has not been documented. We wanted to analyze whether our clinical impression that children recover more quickly from invasive pneumococcal diseases than adults is true. Methods: We reviewed the medical records of all blood culture positive community acquired cases of pneumococcal diseases admitted to University Hospital of Oulu in years 2000-2007. Data on clinical symptoms, laboratory values, imaging studies, medications and outcome were collected and compared between children and adults. During the study period vaccination against pneumococcal diseases was recommended only to specific risk groups (patients with immune deficiencies and people older than 65 years). Results: There were 56 paediatric (<18 years) and 229 adult cases. One hundred and seven of the patients (26% of adults and 84% of children) were previously healthy. None of the children died while there was a 15% (35/229) mortality among the adults (difference 15%, 95% confidence interval, CI, 8−21%). The median length of stay at the hospital (LOS) was 2 days in children and 9 days in adults (difference of the medians 6 days, CI 5−7). When taking account only the patients with underlying conditions the median LOS was 2 days in children and 8 days in adults (P < 0.001).
The course and outcome of invasive pneumococcal infections are far more severe in adult patients compared to children. This may explain the differences in the attitudes towards pneumococcal vaccination among physicians taking care of either children or adults.
N. Borovkova, J. Stsepetova, H. Oopkaup, P. Korrovits, M. Punab, R. Mändar* (Tartu, EE) Under physiological conditions, the vaginal microflora (VMF) contains high numbers of lactic acid bacteria which provide the colonization resistance. At the same time VMF is an open ecosystem that can be significantly affected by sexual intercourse. Our aim was to clarify the influence of sexual intercourse on partner's vaginal lactoflora. Methods: Study group included 17 women with mean age 29.9 (21−39) years. Two self-collected vaginal samples were taken in the follicular phase, before intercourse and 8−12 hours after intercourse. VMF was assessed by Nugent method. Lactobacilli were cultured on MRS agar, typed by AP-PCR method and identified by sequencing. Results: According to the Nugent scores, normal vaginal flora (score 0−3) was found in 9 women in both specimens. Intermediate score (4−6) was found in 3 women and bacterial vaginosis (score 7) in 1 woman. In 4 women normal microflora was found in the first sample but intermediate microflora in post-intercourse sample. In addition, the score increased in two more women, and in total, the mean score was higher after intercourse (1.94 vs 2.71).
Culturable lactobacilli were found from 15 out of 17 women. The mean proportion of lactobacilli in total microflora was somewhat lower in afterintercourse sample (49.7±33.8% vs 34.6±26.2%). All isolated strains were identified as Lactobacillus jensenii (in 67% of women), L. crispatus (58%) and L. gasseri (25%). In one third of women more than 1 species was isolated. AP-PCR allowed us to confirm the persistence of the same strains in all cases though in a quarter of women strains of the same species but different fingerprints were revealed. Conclusions: Lactobacillus species composition in Estonian women coincides with that of described earlier. Sexual intercourse causes shifts in vaginal lactoflora as revealed by increased Nugent score and decreased lactobacillus proportion in VMF.
J. Blocher*, I. Eckert, J. Elster, J. Wiefek, H. Eiffert, H. Schmidt (Göttingen, DE) Objectives: Aquaporins (AQP) are proteins that facilitate water transport through cell membranes. Due to the main localization of AQP1 in the plexus choroideus and of AQP4 in the perivascular endfeet of astrocytes in the brain, these channels are supposed to play a pivotal role in oedema formation. Cerebral oedema formation is one of the main contributors of neuronal damage in bacterial meningitis (BM).
Our study aimed to determine whether AQP1 and AQP4 are present in cerebrospinal fluid (CSF) and if BM induced an increase of these proteins in the CSF, and whether these concentrations correlated with routine CSF parameters of inflammation. ; p = 0.092). AQP1 and AQP4 concentrations in the CSF of BM patients were inversely inter-correlated (R = −0.47, p = 0.004) but we could not find any other correlation between the concentrations of AQP1 or AQP4 with routine CSF parameters (leukocytes, lactate, protein, Qalbumin), age, a prediction-score, outcome-score or GCS at admission. Conclusion: Bacterial meningitis causes an increased release of AQP1 and AQP4 into the CSF. Whether the significant elevation of AQP1 is due to a higher expression on intact choroidal cells or due to the destruction of glial cells needs to be determined.
M. Coen*, S. Passerini, R. Terzi, P. Zucchi, G. Rizzardini (Milan, IT) Objectives: The aims of our study were: 1) to evaluate the outcome of prosthetic joint infections (PJIs) due to different microorganisms and treated with different options 2) to analyze the economical impact of PJIs. Methods: We consider retrospectively patients with PJIs from 2004 to 2009. The isolation of microorganisms was obtained from blood culture and/or deep samples and/or intraoperatory samples. We defined as cured patients without signs of local infection and negative inflammatory index >6 months after antimicrobial therapy interruption. In order to quantify the costs of the treatment we consider the average weekly costs of antimicrobial therapy. Results: 50 PJIs were included in the analysis. Only in 27 (54%) we obtained the isolation of the microrganism ( in 61% of cases. Furthermore, we evaluated the importance of the choice of the therapeutical option: with prosthetic removal and consequent substitution we cured 89% vs 69% of patients who underwent debridement and retention and 64% of patients treated only with longterm suppressive antimicrobial treatment. The mean weekly cost of PJIs in our analysis was 278€ but when the microrganism involved were MRSA/MRSE was increased to 404€. Conclusion: In our analysis. there is a significant proportion of PJIs where the pathogen remains unidentified. As previously described, the two stages substitution of the prosthesis resulted to be the most successful option. Taken as a whole, our data suggest the importance of a multidisciplinary approach to the management of prosthetic joint infections at all stages, allowing a timely identification of the microorganism whenever is possible and a medical and surgical approach that allows more favourable outcome. MRSA/MRSE continue to be a serious challenge in patients with prosthetic infections, being associated to have a worse outcome and greater costs when compared to infections caused by other microorganisms.
H. Y. Kim*, N.S. Lee, O. Kwon, Y.K. Kim, Y. Uh, J. Lee (Wonju, Chungju, KR) Objectives: To evaluate the clinical significance of delta neutrophil index (DNI), which corresponds to the immature granulocytes in the peripheral blood, in patients with sepsis. Methods: We reviewed medical and laboratory records of the consecutive 116 hospitalized adult patients with sepsis from May 2007 to June 2010 in one tertiary university hospital. DNI was measured by blood cell analyzer (ADVIA 120, Siemens, Inc.). Results: Of a total 116 patients with sepsis, mean age was 69.3±12.0 years and mean DNI value was 5.5±7.4% (range 0-34.6%) and mean APACHE II score was 17.8±6.9 (range 6−42). DNI values of sepsis (n = 43), severe sepsis (n = 25) and septic shock (n = 48) were 4.2±4.3%, 6.5±8.9% and 6.0±8.6% respectively (p = 0.34). DNI value of bacteremic patients (n = 45) was higher than non-bacteremic patients (n = 71) (7.9±8.6% vs 4.0±6.0%, p = 0.01). DNI values were no significant difference in survivors (n = 98) and non-survivors (n = 18) (5.0±6.8% vs 7.9±9.8%, p = 0.24). But, DNI values in bacteremic patients were significantly higher in non-survivor (n = 7) than survivor (n = 38) (14.2±12.1% vs 6.7±7.5%, p = 0.03).
The present findings indicate that DNI may be useful prognostic marker of bacteremic sepsis.
O. Gorbich*, G. Chistenko, Y. Gorbich (Minsk, BY) Objective: To evaluate epidemiology of community-acquired pneumonia (CAP) in hospitalized Belarusian children. Methods: All consecutive immunocompetent children hospitalized in Main city children's infectious diseases hospital with radiographically confirmed CAP were evaluated retrospectively from January 2009 through December 2009. We analyzed age, past history, clinical manifestations, length of hospital stay, the severity of the disease and the influence of repeated cases of CAP in the same child. Results: 743 patients were studied during 12 months. Ages ranged from 1 month to 17 years old (55% were boys). The majority of pneumonia cases (34%) were registered in two-years-old children. The second place was occupied by one-year-old children (19%), and the third place − by three-years-old children (13%). Children younger than 1 year of age had pneumonia only in 9% of cases. The majority of patients were hospitalized during the first week of their illness (mostly on 2−4 days). In only 32.23% of cases children were admitted with initial diagnosis of pneumonia.
Pneumonia had moderate severity level in 90% of hospitalized children. While 10% of patients had severe CAP. The mean length of hospitalization was 10.5±0.34 days (range 1−38 days). 686 and 183 children had the history of the acute upper respiratory tract infection and bronchitis, respectively. 68 patients experienced one or more CAP episodes in their past history. Influenza was registered in the anamnesis of 53 patients. 60% of studied patients attended pre-school and school organizations. The analysis of the repeated cases of pneumonia among studied patients hasn't revealed the statistically significant interrelation between the severity of the disease and the presence of the pneumonia episode in the patient's history (odds ratio: 1.3; 95% confidence interval 0.6−2.8, p = 0.695). CAP cases were registered round the year. The morbidity increased in September then slightly decreased in December. Another increase was recorded in January. The maximum levels were noted in November (101 cases) and in April (100 cases). Conclusion: Children's CAP is the significant problem for Belarusian Health System. The main group associated with CAP was children aged from 1 to 3 years old. In the future, immunization with the use of licensed pneumococcal and influenza vaccines may reduce the frequency of pneumonia. year old man, father of the first patient, IgG= 140IU/ml, IgM= 1.08IU/ml. They hosted stray cats at their garden and had close and long-term contact with them. 40 year old woman, IgG= 300IU/ml, IgM= 6.60IU/ml, infection is attributed to eating raw meat. Conclusions: 20.31% of women tested proved to be seropositive, a percentage which comes in total compliance with other studies conducted in Northern Greece (20% among women of reproductive age). Comparing our results to previous studies we can clearly observe a decrease in seropositivity rates during the last decades (1984: 35.6%, 1994: 25.6%) . 79.69% of women tested had no previous contact with T. gondii, and therfore are susceptible. Possible sources of infection for susceptible individuals, taking into account people's habits in Drama Province: Eating habits: low consumption of undercooked meat, high consumption of lamp, goat, sheep, smoked pork sausages, and salads consisting of raw and wild vegetables and fruits (well documented sources of T. gondii) Great number of stray cats unknown responses, was the primary efficacy endpoint. Microbiologic response was defined as eradication or presumed eradication. Secondary endpoints were global response at end of IV therapy (EOIVT) and 2 and 6 weeks after EOT, time to negative blood culture, survival at day 90 and adverse events (AEs). Results: 216 pts received study drug and 170 were included in the MITT population. Most had candidemia (71%) and C albicans only was the most common causative pathogen (56%). Common C/IC risk factors included broad-spectrum antibiotics (90% of pts), central venous catheter (87%), prior surgery (67%), and total parenteral nutrition (58%). Most tested baseline isolates (153/167) were fully susceptible to all of ANI/FLU/VOR, with the exception of: 2/96 C albicans to both FLU and VOR; 4/27C glabrata to FLU; 1/21 C parapsilosis to ANI and 5/21 to FLU; and 2/2 C krusei to FLU. Overall MIC 90 for ANI, FLU and VOR was 0.5 mgmL, 8 mgmL and 0.5 mgmL, respectively. Microbiologic and global response at different time points, across the selected subpopulations/baseline pathogens and by infection site are shown in the table. Mean time to first negative blood culture was 4 days and 90-day survival was 54%. 33/216 (15%) of patients had treatmentrelated AEs, which led to study discontinuation in 5 pts. Infusion-related AEs occurred in 6 pts.
In these selected ICU populations, ANI was effective, safe and well tolerated as first-line therapy of C/IC. EOT outcomes, incl. microbiologic responses, were similar across baseline pathogens, infection sites and patient populations.
pylori-positive patients with duodenal ulcer N. Zakharova*, V. Simanenkov, A. Suvorov, G. Belov (Saint Petersburg, RU) Background: The Helicobacter pylori (H. pylori) eradication rate following standard triple therapies is decreasing worldwide. The lowest incidence of side-effects to standard therapy has been observed when probiotics were combined with antibiotics. Because of the low eradication rates and lack of treatment regimens alternative approaches for H. pylori have been explored to assess the efficacy and safety of synbiotic compared with prebiotic for H. pylori eradication in duodenal ulcer (DU) patients. Methods: Randomized, double-blind, placebo-controlled trial lasting 8 weeks. Each of pills contained either specific synbiotic (E. faecium L-3 10 6−7 CFU/g, pectin and soy protein hydrolyzate, Laminaria saccharina) or prebiotic (pectin and soy protein hydrolyzate, Laminaria saccharina) or placebo. Pills were manufactured to conduct the trial. 81 H. pylori-positive patients in remission of DU were randomly assigned to three groups: Group A received synbiotic, group B received prebiotic, group C received placebo. All patients took three pills three times daily. Gastroscopy was performed before and 6 weeks after the end of treatment for DU activity evaluation. H. pylori detection was carried out by rapid urease test and polymerase chain reaction (PCR) with the primers vacA, ureaB and ureaC in gastric biopsies. Differences between eradication frequency were evaluated by the chi-square test.
Both intention-to-treat (ITT) and per-protocol (PP) analyses were used for the assessment of the eradication rates of H. pylori. Results: H. pylori eradication rates for the groups A, B, and C were 38% (11 of 29 patients), 8.7% (2 of 23 patients) and 10% (3 of 25 patients), respectively according to PP and ITT. Synbiotic showed a higher eradication rate than prebiotic (p = 0.0156) or placebo (p = 0.0317). Clinical or endoscopic relapses of DU or chronic gastritis were observed in 14 patients in group A, 6 patients in group B and 8 patients in group C. Though H. pylori was eradicated, relapses still occurred in some cases. Attributive relapse risk was 0.48, 0.26 and 0.28, respectively. Relapse risk ratio was 2.45 (synbiotic vs. placebo). No cases of enterococcus colonization of gastric mucosa were revealed by PCR with E. faecium specific primers. Conclusion: Monotherapy with synbiotic based on E. faecium is more effective then prebiotic or placebo for H. pylori eradication. Monotherapy with synbiotic increases the risk of DU relapse, indicating H. pylori is the major cause of DU but not the only one.
R2726 Phase 2 prospective randomised study investigating daptomycin 6 and 8 mg/kg versus standard antibiotic therapy in the treatment of staphylococcal prosthetic joint infections I. Byren, A. Wheeler*, E. Campanaro, S. Yankelev, D. Anastasiou, G. Kuropatkin, R. Evans, D. Osmon (Oxford, UK; Lexington, US; Samara, RU; Little Rock, Rochester, US) Objectives: Evolving resistance of Gram-positive organisms support the investigation of higher doses of daptomycin in the management of prosthetic joint infections (PJI).
Prospective, open-label study of 75 subjects randomised to daptomycin (DAP) 6 or 8 mg/kg, or comparator (vancomycin, teicoplanin, or semi-synthetic penicillin) undergoing 2-stage replacement for a hip or knee PJI. After removal of the prosthesis, subjects received 6 weeks treatment and a 2−6 week antibiotic-free period before implanting a new prosthesis. Test of cure (TOC) was 1−2 weeks after re-implantation. The primary objective was evaluation were measured to compare the prevalence of IDA and H. pylori infection in the groups. Results: 52.5% of cases were girls (53.1% of cases and 51.4% of control group) and 47.8% were boys (46.9% of cases group and 48.6% of controls). H. pylori serology was positive in 46.3% of cases and 19% of control group were infected. There were a meaningful difference (p-value >0001). Conclusion: Our results support the proposal that H. pylori infection is associated with IDA in children. A. Herrera-Martinez*, Y. Herrera-Martinez, M. Limper (Barquisimeto, VE; Amsterdam, NL) Background: Sexually transmitted diseases (STDs) during pregnancy pose a major risk to the fetus due to vertical transmission. Congenital disease still represents a significant public health problem worldwide, particularly in developing countries. Methods and Materials: A retrospective investigation was performed comprising all pregnant women with HIV and/or syphilis infections admitted at the central hospital cof two Western Cities of Venezuela, during January 2007-September 2010; pregnant women without STDs served as control group. Epidemiological characteristics were reported, anthropometrical variables in newborns were considered. Statistical significance was defined as p < 0.05. Statistical analyses were performed on SPSS v.17 ® . Results: 76 pregnant HIV patients and 77 patients with syphilis infection were identified, three of them being coinfected. 87 pregnant women without STD served as controls. Mean age of infected mothers (HIV/syphilis) was 26 years (range 14−42 yrs) with a mean of 3 pregnancies (range 1−12). In the control group, pregnancies of 38±1 weeks were observed; newborns had a mean birth weight of 3,220±524 and a mean height of 51±3cm. In HIV infected patients, mean gestation was 36 weeks (range 26−41 weeks). Mean birth weight of newborns was 2,829±686 g (range 510−3,900 g); 22,6% were low birth weight newborns; mean birth height was 49±4 cm (range 30−56). In syphilis infected patients, mean gestation was 37 weeks (range 22−41 weeks). Mean birth weight of newborns was 3,159±649 g (range 700−4,240 g); 11,5% were low birth weight newborns, mean birth height was 51±4 cm (range 33−59 cm). Maternal VDRL titers were strongly associated with birth weight; higher mother VDRL titers correlated with lower birth weight. Cephalic, thoracic and abdominal circumference did not show considerable differences between groups. Conclusion: STDs cause considerable morbidity in women during the gestational period. Congenital and perinatal infection of the newborn, miscarriage and low birth weight have been described. In this study, both HIV and syphilis infections resulted in lower birth weight, particularly in newborns from HIV infected patients. Treatment of the etiologic agent is considered effective for prevention of vertical transmission and is recommended for STDs.
A. Kyratsa, I. Grafakos, T. Guahardo, P. Giannopoulou, F. Lykou, A. Zabou, I. Varzakakos, A. Voyatzi, E. Trikka* (Athens, GR) Parvo B19 is a DNA virus responsible for erythema infectiosum or fifth disease, an infectious benign rash, common in childhood. Pregnant women may also develop Parvo B19 infection with severe conjenital malformation. Objectives: To study the epidemiologic profile of ParvoB19 infection in hospitalized paediatric population of Athens. Methods: During two years period (2007) (2008) IgM and IgG antibodies against Parvo B19 virus were detected in 287 blood samples from hospitalized children, aged 2 months till 15 years, with fever and suspicion of parvoviral infection. Antibody titre was detected with immunoassay and reconfirmed with indirect immunofluorescence (Bios, Germany). Results: From 287 clinical samples from equal number of patients, ParvoB19 acute infection was diagnosed in 94 patients (32,7%). IgG antibodies were detected in 27.2% of children. A slight difference was observed in the incidence of antibody detection between sexes. The incidence of IgG antibody detection in children aged less than 4 years (27,3%) was inferior in comparison with incidence in children aged above 10 years (34,7%). Increased incidence of infection was observed during winter and spring months. Conclusions: Early and accurate diagnosis of Parvo B19 infection based on IgM antibody detection or an increase of IgG antibody titre is crucial in hospitalized children with infectious rash and leukopenia or chronic haemolytic anaemia.
population of Athens T. Guahardo, I. Grafakos, A. Kyratsa, F. Lykou, P. Giannopoulou, T. Athanasopoulos, I. Varzakakos, E. Trikka*, A. Voyatzi (Athens, GR) Toxoplasmosis is a worldwide public health disease, nowdays. Incidence varies among different countries, depending on living standards, dietary habits and socioeconomic status of population. Objectives: To determine the incidence of toxoplasmosis in children with cervical or generalized lymphadenopathy, by detection of IgM and IgG antibodies, during five years (2004) (2005) (2006) (2007) (2008) (2009) . Methods: Determination of IgM and IgG antibody titre against Toxoplasma gondii was performed with immunoassay in 1254 sera from children with lymphadenopathy, aged 1 till 14 years, who were hospitalized or admitted at the emergency department. In every child two blood samples were drawn with a distance period of 15 days and examined for TORCH virulent agents. Reconfirmation of acute infection was performed by indirect immunofluorescence as well as by observation of fourth time rise of IgG antibody titre. Results: From 1254 children examined 85 (6.8%) were positive for IgG and 25 (1.9%) for IgM antibodies against Toxoplasma gondii 19 (1.5%) children were positive for both IgG and IgM antibodies. Rise of seropositivity was observed according the increase of age. Higher incidence of infection was observed in the age group between 8 and 14yrs. There was no difference observed in the incidence of antibody detection between sexes. Conclusions: Toxoplasma gondii is an important cause of lymphadenopathy during childhood in Greece, with low incidence, probably due to the habit of eating well-cooked meat and the good socioeconomic status of the study population. Objectives: Isolation of Candida spp. in cerebrospinal fluid (CSF) in preterm neonates is rare. We describe the clinical characteristics and diagnosis of two cases of hospitalized premature neonates in whose Candida albicans isolated from CSF. Methods: The first case was about a set of female dizygotic twins, who were born at 32 w gestation. Twin a died within few hours, while twin b was transferred to neonatal intensive care unit (NICU). Their mother was treated for cervical incompetence and vaginal candidiasis during the 2nd trimester of her pregnancy. Neonate b was started on empiric antibiotic therapy, because of elevated CRP, and low CSF glucose level. Blood and CSF cultures were negative. On day 8, the neonate looked slightly unwell and lumbar puncture (LP) was repeated. The second case was about a male neonate, who was born at 28 w gestation. The neonate presented respiratory distress syndrome and neonatal jaundice, and transferred to NICU. Empiric antibiotic therapy was started, although
In univariate binary logistic regression models, the impact of neonatal and maternal factors on sepsis risk was estimated in terms of odd ratio (OR) with 95% confidence interval (CI).
Results: This study revealed the impact of bacterial pathogens, neonatal and maternal predisposing factors on sepsis as follows. Maternal factors: PROM affect the sepsis risk to more than 3-fold (OR = 3.8; 95% CI:1.37-10.56; P < 0.05). Neonatal factors: the mean age of neonate ±SD of whom with early-onset sepsis (1.56±0.88) was lower than that of with late-onset sepsis (10.40±5.50) and this difference was statistically significant (P < 0.05). Low birth weight (LBW) <2500 g increase the risk of sepsis to more than 2-fold (OR = 2.9, 95% CI:1.17-9.86; P < 0.01). Gestation age (GA) <29 weeks was significantly associated with sepsis (P < 0.01). The septicemia in turn, increase the risk of death up to more than 5-fold (OR = 5.5; 95% CI:1.98-15.3; P < 0.01). More than half of septic neonates had positive result for CRP whereas only 1.9% of neonates with sepsis were CRP negative and this difference was statistically significant (P < 0.001). T. Tashiro, A. Ueda*, N. Kohda, S. Kohno (Otsu, Nagasaki, JP) Objectives: The fatalities caused by Streptococcus pneumoniae (S. pneumoniae) infection are high in the young children and the elderly. Host-defense mechanisms against these pathogenic infections involve both innate and acquired immune systems. In particular secretory immunoglobulin A (IgA) and innate immunities in the airway mucosa play a principal role in preventing these infections. Lactobacillus pentosus ONRICb0240 (b240) has been screened as the excellent IgAinducing bacterium in vitro. The present study aimed to investigate whether nonviable b240 can prevent S. pneumoniae infection in mice. Methods: CBA/J mice were administered with b240 for 3 weeks prior to lethal S. pneumoniae infection. The survival and body weight of the mice were monitored daily for 2 weeks after infection. Then, isolated lungs were evaluated the severity of pneumonia. The number of S. pneumoniae, concentration of inflammatory cytokines, and expression of toll-like receptor 2 in lung homogenate on 2days after infection were determined.
In the pneumococcal pneumonia model, oral intake of b240 led to the prolongation of survival time and significantly inhibited of body weight loss, as well as reduced bronchitis. There were a significant decrease in the secretion level of inflammatory cytokines and toll-like receptor 2 expression was promoted in b240-treated mice. These results were exhibited in accordance with the alleviated pathological severity of pneumonia. Conclusion: These results suggest that Lactobacillus pentosus ON-RICb0240 intake can facilitate protection against S. pneumoniae infection via the modulation of innate immunities.
T. Matsumoto*, H. Ishikawa, E. Kutsukake, T. Fukui, I. Sato, T. Shirai, T. Kurihara, N. Okada, H. Danbara, M. Toba, Y. Maeda, N. Kohda (Tokyo, Otsu, Uenohara, JP) Objectives: One of the beneficial effects of probiotic bacteria is protection of the host against infection by various enteric pathogens. Viable lactobacilli are used in most probiotic studies, while few studies using heat-killed probiotic bacteria have been reported. The purpose of this study was to investigate the effects of heat-killed Lactobacillus pentosus ONRICb0240 (b240) on systemic infection by Salmonella enterica serovar Typhimurium (S.Typhimurium) and to determine the mechanism by which b240 protects against infection.
To determine whether oral administration of heat-killed b240 would affect the survival of mice after S. Typhimurium infection, mice were orally administered b240 or saline daily for 3 weeks. S. Typhimurium was orally inoculated and survival of the mice was monitored until 20 days after inoculation. To study the effect of b240 on bacterial translocation of S. Typhimurium, mice were treated orally with b240 or saline daily for 3 weeks. Various organs were removed 6 days after S. Typhimurium inoculation and viable numbers of bacteria were evaluated. We also examined the effects of b240 on adhesion and invasion of S. Typhimurium into HeLa cells by in vitro assay.
The mice treated with b240 were significantly protected against S.Typhimurium as compared to those fed saline. Moreover, viable numbers of S.Typhimurium in each organ in b240-treated mice tended to be less than in the control mice. In vitro study demonstrated the inhibitory effect of b240 on binding and invasion of S.Typhimurium into cells.
The oral administration of Lactobacillus pentosus ON-RICb0240 protected mice from S.Typhimurium infection through the inhibition of adhesion and invasion of S.Typhimurium to intestinal epithelial cells in association with a reduction of bacterial systemic infection. Our results suggest that nonviable lactic acid bacteria also play an important role in preventing infection by enteric pathogens.
R2746 Immunogenicity of the pneumococcal conjugate 7-valent vaccine and impact on pneumococcal carriage in infants from Venezuela with sickle cell disease or HIV infection I.A. Rivera-Olivero*, B. del Nogal, M.C. Sisco, M. Fuentes, R. Cortez, D. Bogaert, P.W. Hermans, J.H. de Waard (Caracas, VE; Utrecht, Nijmegen, NL) Objectives: To evaluate immunogenicity of the pneumococcal 7-valent conjugate vaccine (PCV7) and the impact on pneumococcal carriage in infants with sickle cell disease (SCD) or HIV infection. Methods: Children with SCD (n = 25) and HIV (n = 12) listed at the Children Hospital in Caracas, Venezuela were enrolled in this study and vaccinated according to their age with the 7-valent conjugate vaccine followed by a booster dose of a 23-valent pneumococcal polysaccharide vaccine (PS-23). Blood samples and nasopharyngeal swabs for the determination of antibody concentrations for vaccine serotypes and for the isolation of S. pneumoniae respectively were obtained immediately before and 1 month after the PS-23 booster. Results: Of the 37 infants enrolled in this study, pneumococcal carriage prior the first immunization was found in 32% (n = 12) of the children with 67% (n = 8) carrying non-vaccine serotypes. After boosting 100% of the subjects had antibody titers >0.35 mg/mL for all the 7 vaccine serotypes, ranging from 0.42 mg/mL (serotype 6B) to 62.21 mg/mL (serotype 14). One month after completion of the vaccination scheme pneumococcal carriage was found in 27% (n = 10) of the children with 80% (n = 8) carrying non vaccine serotypes. Conclusions: High antibody titers for the serotypes of the 7-valent conjugate vaccine were obtained with this vaccination scheme in our children. However, no significant effect on carriage rate in general or carriage rates for vaccine serotypes was observed.
In group 1, consisting of healthy S. aureus carriers, mean levels of serum cytokines amounted as follows: IL-1b 0.31±0.3 pg/ml, TNF-a 0.56±0.22 pg/ml, IFN-g 0.21±0.095 pg/ml. In group 2 at the first sampling levels of the cytokines were as follows: IL-1b 0.28±0.16 pg/ml, TNF-a 0.54±0.09 pg/ml, IFN-g 2.37±1.38 pg/ml, and only the level of IFN-g was significantly higher (p < 0.0001) than mean level in group 1. In turn, on the second sampling mean level of IFN-g was significantly higher (p = 0.0152) than the level noted upon the first sampling and amounted to 4.41±2.6 pg/ml. Serum levels of IL-1b and TNF-a increased in 10 patients (2.77±2.45 pg/ml, p = 0.0005 and 1.67±0.69 pg/ml, p < 0.0001, respectively) and remained unaltered in the 6 remaining patients. At the same time in 10 patients presence of S. aureus could not be detected in throat smears obtained at the time of the second sampling. Conclusion: The effective immune response against S. aureus seems to depend on levels of circulating cytokines IL-1b and TNF-a, the production of which may be stimulated by S. aureus autovaccine. Background: Some evidence has shown a relationship between human cytomegalovirus (CMV) infection and pregnancy loss. However, whether recurrent or latent CMV infection or altered immune response to CMV may relate to recurrent pregnancy loss (RPL) is unclear and few data are available in this regard. We evaluated CMV infection and humoral immunological response to CMV in women with RPL and compared it to women without any history of abortion.
This cross-sectional, observational study was conducted in Clinical Immunology outpatient clinic, Alzahra University Hospital, Isfahan, Iran, between 2008 and 2010. Cases were 43 women with RPL referred by Obstetric and Gynecology outpatient clinic and controls were randomly selected from healthy age match multiparous women without history of abortion. Inclusion criteria were at least 3 recurrent spontaneous abortions, and no history of any diagnosed underlying diseases as etiology of RP L. Blood samples were obtained from patients and controls to evaluate CMV IgG and IgM antibodies and IgG avidity index by the Enzyme Linked Immunosorbant Assay Method (ELISA). Data were analyzed with SPSS version 16.0, using Student's t-test, chi-squared test, and multivariate analyses. Results: One case (2.3%) of positive IgM in each group of RPL's and controls was detected. Also, there were 39 (90.6%) and 30 (69.8%) cases of positive IgG in RPL's and controls, P < 0.05. Patients and controls were similar regarding serum IgM titer (P > 0.05). But, IgG titer was significantly higher in RPL group, P < 0.05. No differences were found between the two groups in IgG avidity index (P > 0.05). In multivariate analyses, only IgG positivity was related to RPL (P < 0.05) and avidity was not related to RPL (P = 0.277). Also, IgG positivity (P = 0.159), IgM positivity (P = 0.856), or avidity index (P = 0.440) were not related to the number of abortions in RPL patients. Conclusions: Previous exposure to CMV detected by a positive IgG antibody is significantly related to RPL in the present study. However, we found no relationship between IgG avidity index and RPL. Whether Latent CMV infection starting an indirect process of autoimmune etiology in RPL's or women with RPL had recurrent or reactivation of CMV infection but avidity index is not the best index to detect it, mostly because of altered immune function to CMV in RPL patients need further investigations.
A. Atrasheuskaya*, I.A. Karpov, G.M. Ignatyev (Minsk, BY) Mumps virus-neutralizing antibodies are believed to be the most predictable surrogate marker of protective immunity. The remote history of vaccination also suggests that waning immunity could have contributed to the vaccinees' susceptibility. Mumps outbreaks in vaccinated populations are usually caused by the phylogenetically distinct from the vaccine strains mumps viruses (MuV).
To examine these issues, the kinetics of titer, avidity and neutralizing activity level and spectrum of the specific IgG were studied in 3-year follow-up serum samples from the healthy volunteers (n = 60) with no vaccination and mumps in the past, after the one vaccine dose contained "Leningrad-3" (L-3) MuV strain. Full seroconversion, confirmed by ELISA and PRN against the vaccine MuV strain, was registered in all volunteers 3 months after vaccination. Antibodies induced by the vaccine strain effectively neutralized five heterologous MuV strains (genotype A, B, D, and those earlier isolated during the local mumps outbreaks, C and H) starting from month 6 after vaccination at all time points tested thereafter, albeit at levels lower than those seen against the vaccine strain (>2-fold difference). The specific IgG titers measured in ELISA demonstrated an "early" decrease after 9 months post vaccination. At the same time, the specific IgG functional activity characterized by their avidity, level and spectrum of neutralizing activity reached its maximum within month 18 after vaccination. Antibodies capable of neutralizing the heterologous MuV genotypes in the absence of antigenic boosting demonstrated a decrease to the critical levels ( 1:8) in a part of vaccinees after the third year post vaccination, while the specific IgG neutralizing activity against the vaccine strain still demonstrated a titer previously associated with protection against MuV infection ( 1:8).
The current study possessing several limitations demonstrated for the first time a broad-spectrum neutralizing activity of the specific IgG in recipients of one dose vaccine containing the L-3 MuV strain within 3 years after vaccination.
K. Hamuro*, Y. Kotani, S. Inoue, Y. Ogasawara, S. Yamahira, T. Saito, M. Toba, N. Kohda (Otsu, JP) Objective: The foreign substances, such as pathogenic microorganisms, usually invade internal body via mucosal surface with ingestion or breathing. Thus, activation of mucosal immune function is essential for maintaining and better health. Secretory immunoglobulin A (SIgA) play a pivotal role in mucosal immune system by the multiple protective roles. This study objective was to find out lactic acid bacteria (LAB) for promoting IgA.
Methods: First, we tested 150 LAB strains for IgA production capability in vitro using a murine Peyer's patch cell culture system and selected Lactobacillus pentosus ONRICb0240 (b240). In addition, comparison of cytokine profile between b240 and low IgA inducible LAB was examined. Next, we administrated heat-killed b240 to the mice for 3 weeks and investigated the intestinal IgA enhancement. Moreover, we attempted an ex vivo study to assess the reactivity of murine Peyer's patch cells against b240. Results: We found out b240 to be exhibiting excellent IgA producing activities in 150 strains of LAB. Interestingly, plant derived LAB including b240 tended to show higher activity than dairy LAB. After 3weeks of b240 administration, intestinal IgA significantly increased with first vaccination at 2, 8 and 16 weeks of age, respectively. Vaccine induced immune response, mainly the Th1 type cytokine secretion, was evaluated in different age groups following booster doses at equal time intervals. Preliminary results show higher antigen specific IFN-g levels in response to heat shock protein and ESAT-6 family member protein antigens. It was observed that there was no effect of age on the IFN-g producing capacity of the animals in the different age groups after stimulation of whole blood with SEB. However, animals in the older age group responded well to the MAP multi-antigens and might need only one booster compared to the younger animals. Findings from this work could be interesting to determine the appropriate age of vaccination so as to generate the memory T cell pool and for MAP vaccine challenge experiments. Smallpox, caused by Variola virus, is considered as a dreadful scourge for human. Smallpox vaccination, using strains of Vaccinia virus (VACV) has leaded to it eradication in 1980. However, adverse reaction could result, so it was abandoned. Current bioterrorism attack threat are feared the disease re-emergence, then new smallpox vaccines more safe but less effective in animals have been developed. Attenuation of our candidates have been measured in Swiss Nude or SCID mice. Mice have been vaccinated with 10 5 pfu of each viruses by tail scarification. Signs of disease and mortality were followed over an 8 week period. Protection of BALB/c mice against a lethal challenge have been performed, 28 days after vaccination, with 2.10 6 DICT50 of cowpoxvirus by intranasal route (i.n). VACV neutralizing antibodies (Abs) in serum samples were titrated by plaque assay. Serum samples were first incubated at 56ºC for 30 minutes then submitted to serial 2-fold dilutions. The samples were mixed with an equal volume of VACV containing 35 PFU in 0.1ml for one hour at 37ºC and added to Vero cell monolayers. Two days later virus plaques were counted. To measure T lymphocyte responses the percentage of CD4+ and CD8+ lymphocytes expressing intracellular IFN-g was measured by flow cytometry. Mature bone marrow dendritic cells from uninfected BALB/c mice were infected with VACV then incubated with spleen cell suspensions from infected animals for six hours. Brefeldin A (5 mg/ml) was added for the last four hours to block cytokine secretion. Cells were stained with FITCcoupled anti-CD8b2 and APC-coupled anti-CD4 monoclonalAbs, fixed and permeabilised. IFN-g was stained with a PE-coupled anti-IFN-g mAb and cells were fixed in 2% formaldehyde. These vaccine candidates have been shown as protective as the traditional vaccine (1G) against an i.n cowpox challenge in mice, and they have been closely as safe as MVA strain in NUDE mice. The neutralizing antibodies titration showed that every deleted viruses induced a humoral response as strong as 1G. Finally intracellular cytokine staining showed a similar specific CD4+ response to that of 1G and a slightly lower specific CD8+ response than that of 1G. Our results show that several deleted vaccines could advantageously complete the range of the third generation vaccines already developed. The utilisation of one of these candidates as a viral vector for a vaccine against Ebola virus is under investigation in our laboratory.
S. Luque*, S. Grau, C. Serra, P. Pi-Sunyer, J.P. Horcajada, N. Berenguer, O. Ferrández, O. Urbina, M. Espona, E. Salas (Barcelona, ES) Objectives: Several strategies have been designed to increase adherence to vaccination programs aimed at health-care professionals, though the results were not always satisfactory.
The differences between adherence to seasonal and pandemic influenza vaccination after the implementation of a vaccination program between health-care workers were assessed and compared by age, gender, professional category and workplace. Two different prognostic models for predicting adherence to seasonal and pandemic influenza were developed. In statistical analysis, univariate logistic regression was performed to identify risk factors associated with adherence to vaccinations. Multiple regression analysis backwards, stepwise variable selection was used to identify independent risk factors for adherence. The internal validation of the predictive models was evaluated by measures of calibration and discrimination power. Results: 7.6% of professionals were vaccinated against pandemic influenza, and 33.7% against seasonal influenza. Statistically significant differences were observed for both vaccines when comparing vaccinated to unvaccinated professionals as for age, professional category and workplace, while sex differences were only related to pandemic influenza. The predictive model of adherence to pandemic H1N1 vaccine, which showed a good discriminatory power (Area under ROC curve: 0.843), included the variables age, professional category, workplace and previous vaccination against seasonal influenza as independent predicting factors. On the contrary, the predictive model of adherence to seasonal vaccine, which showed a poor discriminatory power (Area under ROC curve: 0.567), included the variables age, gender and workplace. Both models showed a good calibration (table 1) . Conclusions: Adherence to pandemic H1N1 and seasonal influenza vaccination program was very low, which suggests the need to implement new strategies into vaccination programs aimed at healthcare professionals. A good model for predicting adherence to pandemic vaccine was developed when compared with the poor discriminating power of the predictive model for seasonal influenza vaccination.
Objective: Adenovirus are responsible for various diseases to humans and many species of animals & birds. There are no antiviral drugs to treat adenoviral infections. The use of immunoinformatics has greatly revolutionized the field of vaccine research, discovery and development. Hence approach has been made to identify the relevant B-cell & T-cell epitopes to design vaccine against adenovirus using bioinformatics.
fatality rates between 21% and 34%; and age-specific estimates from 2% to 35% among adults aged <65 years, and 30% to 43% among older adults.
The epidemiologic burden of IPD is greater among older adults. These synthesized estimates may therefore serve as a baseline measure against which to evaluate the cost-effectiveness of new strategies for preventing pneumococcal infection. Resistance of BPZE1-challenged MDDC to apoptosis was assessed by AnnexinV/Propidium Iodide staining. Chemokine-driven chemotaxis was also evaluated. Co-culture experiments with T lymphocytes were performed to assess antigen-presenting activity, T-helper cell polarizing ability and induction of functional suppressor T cells. Results: BPZE1 is able to induce phenotypic maturation of human MDDC which are resistant to apoptosis. BPZE1-primed MDDC produce a broad spectrum of proinflammatory and regulatory cytokines, and very efficiently migrate in vitro in response to the lymphatic chemokine CCL21, due to the inactivation of the pertussis toxin enzymatic activity. BPZE1-primed MDDC acquire antigen-presenting capacity, drive a mixed Th1/Th17 polarization, and induce functional suppressor T cells. Suppressing activity requires cell contact rather than the production of soluble factors. Conclusion: Our findings support the potential of BPZE1 as a novel live attenuated pertussis vaccine strongly activating the maturation of DC with full-blown activity. BPZE1-challenged DC acquire the capacity to survive death signals and migrate from the site of infection to the lymph nodes. BPZE1-committed DC have the ability to orchestrate a broad spectrum of Th1/Th17 responses, protective in experimental B. pertussis infection, and regulatory T cell responses, likely balancing each other to restore local homeostasis. Background: Patients receiving cytotoxic therapy for solid tumors are at increased risk of severe influenza, Data on flu vaccine immunogenicity are needed in those patients to improve the vaccine coverage. Methods: In a multicentric prospective study, 25 patients with breast (n = 13) or prostate (n = 12) cancer received one dose of a trivalent inactivated influenza vaccine the same day that the IV administration of docetaxel. Serum hemagglutination-inhibition (HI) antibody response was assessed 21 days after vaccination.
The median age of the study population was 65 years (min-max, 33−87); 52% were female. At baseline, the percentages of patients with titers of vaccine strain-specific HI antibodies 1:50 were 100%, 72%, and 100% against H1N1, H3N2 and B, respectively. Immunogenicity results at day 21 show that this vaccine is poorly immunogenic (see Table 1 ). No serious adverse events (AE) have been reported during the first 3 weeks after vaccination. All the reported AE were from mild to moderate intensity.
The results show that the trivalent inactivated influenza vaccine triggers a low immunogenicity in adults receiving docetaxel for solid tumors, although it demonstrated a good safety profile. Strategies using more immunogenic influenza vaccines have to be evaluated in patients with cancer.
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