LitCovid-sentences  

PMC:7068162 / 695-12048 JSONTXT 13 Projects

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Id Subject Object Predicate Lexical cue
T7 0-224 Sentence denotes In January 2020, a previously unknown coronavirus – now named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) – was identified as causative agent of a cluster of suspicious pneumonia cases in Wuhan, China [1,2].
T8 225-352 Sentence denotes The World Health Organization (WHO) declared a public health emergency of international concern by the end of January 2020 [3].
T9 353-487 Sentence denotes As at 3 March 2020, more than 90,000 confirmed cases and more than 3,100 fatalities in 13 countries have been attributed to the virus.
T10 488-776 Sentence denotes The ability to quickly confirm or clear suspected cases is crucial during global outbreak scenarios, especially when clinical manifestations are difficult to distinguish from other respiratory infections such as influenza, molecular diagnostics is key for detection of the emerging virus.
T11 777-912 Sentence denotes A variety of suitable assays were made available early on during the course of the outbreak, notably by Corman et al. and others [4,5].
T12 913-1171 Sentence denotes However, their implementation in the diagnostics laboratory usually relies on manual PCR setups requiring a high degree of human interaction for execution and interpretation, thus limiting their capacity to be scaled up for handling large numbers of samples.
T13 1172-1351 Sentence denotes In this study we report the analytical evaluation of a laboratory-developed test for the detection of SARS-CoV-2 using the open channel (utility channel) of the cobas 6800 system.
T14 1353-1390 Sentence denotes SARS-CoV-2 Utility Channel test setup
T15 1391-1535 Sentence denotes A custom-made primer/probe set based on a previously published assay targeting the E gene [4] was optimised for the use on the automated system.
T16 1536-1626 Sentence denotes Primer and probes were ordered from IDT DNA Technologies (Coralville, United States (US)).
T17 1627-1741 Sentence denotes Both primers were modified with 2’-O-methyl bases in their penultimate base to prevent formation of primer dimers.
T18 1742-1829 Sentence denotes The ZEN double-quenched probe (IDT) was used in order to lower background fluorescence.
T19 1830-2164 Sentence denotes The master mix (Mmx) cassette (for 96 tests) is prepared by combining 84 µL forward primer (400 nM, 5´-ACAGGTACGTTAATAGTTAATAGCmGT-3´), 84 µL reverse primer (400 nM, 5´-ATATTGCAGCAGTACGCACAmCA-3´) and 10.5 µL probe (50 nM, 5´-Fam-ACACTAGCC/ZEN/ATCCTTACTGCGCTTCG-Iowa Black FQ-3’)* together with 182 µL water and 5,640 µl Mmx2 mixture.
T20 2165-2228 Sentence denotes After mixing, the 6 mL are transferred to the reagent cassette.
T21 2229-2411 Sentence denotes The Mmx cassette is delivered with a full-process control: it is preloaded with an internal control (IC) RNA together with primers and probe of the IC detection assay by default [6].
T22 2412-2580 Sentence denotes Instrument settings in the cobas mni Utility Channel software (Roche, Los Gatos, US) and the temperature profile used for the RT-PCR reaction are summarised in Table 1.
T23 2581-2709 Sentence denotes Table 1 SARS-CoV-2 PCR cycling conditions and software settings in the Utility Channel software used to create the run template
T24 2710-2756 Sentence denotes Sample type Alcohol-based swab (400 µL input)
T25 2757-2799 Sentence denotes Channels 1 2:SARS-CoV-2 E-gene 3 4 IC
T26 2800-2832 Sentence denotes RFI NA 1.5 NA NA Predefined
T27 2833-2928 Sentence denotes PCR cycling conditions UNG incubation Pre-PCR step 1st measurement 2nd measurement Cooling
T28 2929-2979 Sentence denotes Number of cycles Predefined 1 5 45 Predefined
T29 2980-3004 Sentence denotes Number of steps 3 2 2
T30 3005-3065 Sentence denotes Temperature 55 °C; 60 °C; 65 °C 95 °C; 55 °C 91 °C; 58 °C
T31 3066-3118 Sentence denotes Hold time 120 s; 360 s; 240 s 5 s; 30 s 5 s; 25 s
T32 3119-3179 Sentence denotes Data acquisition None End of each cycle End of each cycle
T33 3180-3289 Sentence denotes IC: internal control; NA: not applicable; RFI: relative fluorescence increase; UNG: uracil-DNA N-glycosylase.
T34 3290-3339 Sentence denotes Assay performance was evaluated for swab samples.
T35 3340-3443 Sentence denotes All clinical specimens used were collected at the University Medical Center Hamburg-Eppendorf (UKE-HH).
T36 3444-3612 Sentence denotes Samples were mixed 1:1 with Roche cobas PCR media (≤ 40% guanidine hydrochloride in Tris-HCL buffer) and incubated for 30 min before loading onto the cobas 6800 system.
T37 3613-3737 Sentence denotes Apart from that sample preparation step, no further manual steps are required during the entire workflow of the novel assay.
T38 3739-3793 Sentence denotes SARS-CoV-2 Utility Channel test performance evaluation
T39 3794-3912 Sentence denotes Limit of detection (LoD), inter-run variability and cross-reactivity with other respiratory pathogens were determined.
T40 3913-4157 Sentence denotes In-vitro transcribed RNA (IVT RNA) of the E gene of SARS-CoV-2 and purified RNA of SARS-CoV (strain Frankfurt-1) were used as positive controls (obtained via the European virus archive global (EVAg), https://www.european-virus-archive.com) [7].
T41 4158-4476 Sentence denotes Limit of detection of the SARS-CoV-2 UCT was determined by analysing each of eight replicates of a dilution series containing IVT RNA diluted in E-swab medium (Copan, Brescia, Italy; modified liquid Amies medium) and Roche cobas PCR medium (1:1) at 10,000, 1,000, 500, 250 and 125 copies/mL and eight negative samples.
T42 4477-4587 Sentence denotes The LoD was 689.3 copies/mL with 275.72 copies per reaction at 95% detection probability (Figure 1, Figure 2).
T43 4588-4801 Sentence denotes For estimation of inter/intra-run variability, we analysed each of two concentrations (ca 5 × and 10 × LoD spiked IVT SARS-CoV-2 RNA) in five replicates and a negative sample with five replicates in two runs each.
T44 4802-4900 Sentence denotes Minimal deviation was observed with ± 0.5 cycle threshold (Ct) at 10 × LoD and 0.75 Ct at 5 × LoD.
T45 4901-4936 Sentence denotes No false positive results occurred.
T46 4937-5033 Sentence denotes Figure 1 Example for amplification curves of the SARS-CoV-2-UCT SARS-CoV-2 Utility Channel test
T47 5034-5068 Sentence denotes IVT RNA: in-vitro transcribed RNA.
T48 5069-5216 Sentence denotes Dark blue curves: specific SARS-CoV-2 signals of IVT-RNA at 100,000 copies/mL (1), 10,000 copies/ml (2), 1,000 copies/ml (3) and 100 copies/ml (4).
T49 5217-5272 Sentence denotes Light blue curves: signals of the internal control (5).
T50 5273-5391 Sentence denotes Figure 2 Determination of limit of detection of the SARS-CoV-2 Utility Channel test based on in-vitro transcribed RNA
T51 5392-5426 Sentence denotes IVT RNA: in-vitro transcribed RNA.
T52 5427-5511 Sentence denotes Probit analysis revealed a limit of detection of 689.3 copies/mL at 95% probability.
T53 5512-5575 Sentence denotes Blue line: probit curve (dose-response rule); red dotted lines:
T54 5576-5601 Sentence denotes 95% confidence intervals.
T55 5602-5627 Sentence denotes IVT-RNA obtained by EVAg.
T56 5628-5928 Sentence denotes Potential interference of the SARS-CoV-2 UCT with other respiratory pathogens (including other human CoV strains) was evaluated by analysing 88 previously determined clinical samples and an external quality control assessment panel (INSTAND, Düsseldorf, Germany) containing lysates of infected cells.
T57 5929-6112 Sentence denotes None of these organisms were detected by the SARS-CoV-2 UCT assay (Table 2), confirming high specificity of the assay for viruses within the Betacoronavirus subgenus Sarbecovirus [4].
T58 6113-6292 Sentence denotes Table 2 Potential cross-reactivity SARS-CoV-2 Utility Channel test with other respiratory pathogens, evaluated with a panel of organisms typically found in respiratory infections
T59 6293-6343 Sentence denotes Clinical samples with known viruses Number tested
T60 6344-6370 Sentence denotes Coronavirus (not typed) 5
T61 6371-6384 Sentence denotes hCoV HKU-1 2
T62 6385-6397 Sentence denotes hCoV NL63 1
T63 6398-6411 Sentence denotes Adenovirus 2
T64 6412-6424 Sentence denotes Bocavirus 7
T65 6425-6449 Sentence denotes Human metapneumovirus 6
T66 6450-6464 Sentence denotes Influenza A 7
T67 6465-6485 Sentence denotes Influenza A(H1N1) 6
T68 6486-6500 Sentence denotes Influenza B 3
T69 6501-6525 Sentence denotes Parainfluenza 1 virus 3
T70 6526-6550 Sentence denotes Parainfluenza 2 virus 1
T71 6551-6575 Sentence denotes Parainfluenza 3 virus 8
T72 6576-6600 Sentence denotes Parainfluenza 4 virus 3
T73 6601-6638 Sentence denotes Respiratory syncytial virus (A/B) 10
T74 6639-6659 Sentence denotes Rhino/enterovirus 8
T75 6660-6687 Sentence denotes Chlamydophila pneumoniae 4
T76 6688-6712 Sentence denotes Mycoplasma pneumoniae 4
T77 6713-6738 Sentence denotes Legionella pneumophila 3
T78 6739-6762 Sentence denotes Bordetella pertussis 4
T79 6763-6790 Sentence denotes Bordetella parapertussis 1
T80 6791-6828 Sentence denotes Total number of clinical samples 88
T81 6829-6917 Sentence denotes External quality control assessment panel with known viruses (lysates of infected cells)
T82 6918-6930 Sentence denotes hCoV 229E 1
T83 6931-6943 Sentence denotes hCoV NL63 1
T84 6944-6956 Sentence denotes hCoV OC43 3
T85 6957-6968 Sentence denotes MERS-CoV 4
T86 6969-6990 Sentence denotes Coxsackievirus A21 1
T87 6991-7011 Sentence denotes Coxsackievirus B3 1
T88 7012-7029 Sentence denotes Enterovirus 68 1
T89 7030-7043 Sentence denotes Rhinovirus 3
T90 7044-7068 Sentence denotes Human metapneumovirus 3
T91 7069-7086 Sentence denotes Virus-negative 4
T92 7087-7148 Sentence denotes Total number of quality control assessment panel samples 22
T93 7149-7178 Sentence denotes Total number of samples 110
T94 7179-7203 Sentence denotes hCoV: human coronavirus;
T95 7204-7265 Sentence denotes Clinical samples had been pre-analysed by routine diagnostic.
T96 7266-7423 Sentence denotes External quality control assessment panel (INSTAND) samples contained inactivated lysates of virus infected cells including four virus-negative cell lysates.
T97 7425-7435 Sentence denotes Discussion
T98 7436-7556 Sentence denotes Outbreaks of novel pathogens, such as the ongoing SARS-CoV-2 situation, represent a challenge for molecular diagnostics.
T99 7557-7752 Sentence denotes However, identification of the agent, sequencing and publication of specific PCR assays for general use, a process that took several weeks 15 years ago, can now be accomplished in a few days [8].
T100 7753-7979 Sentence denotes With this rapid pace in identification and sharing of information comes the responsibility of local laboratories to be able to implement available assays and provide tests for the outbreak strain in an equally short timeframe.
T101 7980-8154 Sentence denotes Furthermore, demand can be unpredictable and may suddenly spike, even outside the primary endemic areas, putting testing capacity under strain and potentially causing delays.
T102 8155-8383 Sentence denotes For example, in February 2020, an entire cruise ship carrying almost 4,000 passengers and crew was quarantined off the port of Yokohama, Japan, and hundreds of people had to be tested for the virus in a short period of time [9].
T103 8384-8534 Sentence denotes Automated solutions for molecular diagnostics can help handle large numbers of samples and can be scaled to keep pace with fluctuating demand [10-12].
T104 8535-8648 Sentence denotes The system used in this study fully automates nucleic acid extraction, purification, amplification and detection.
T105 8649-8814 Sentence denotes We, among others, have previously demonstrated that laboratory-developed tests can be adapted for fully automated PCR platforms such as the cobas 6800 system [6,13].
T106 8815-9170 Sentence denotes After brief preparation, clinical samples can be loaded directly into the device; required hands-on time and manual steps are reduced by up to 60% (manual steps reduced from 33 to 14, hands-on time reduced from 74 min to 14 min) compared with conventional workflows which usually involve automated extraction and PCR performed as separate procedures [13].
T107 9171-9348 Sentence denotes The inclusion of a full-process control for each reaction further facilitates the handling of results, allowing interpretation by personnel not familiar with RT-PCR diagnostics.
T108 9349-9594 Sentence denotes The system has passed clinical evaluation for a variety of viral and bacterial targets [14-17] and is also used for blood safety testing, another diagnostic field in which large numbers of samples have to be cleared by PCR-based screening tests.
T109 9595-9860 Sentence denotes In this study, we demonstrated good analytical performance of an adapted SARS-CoV-2 assay on swab samples with an LoD of 689.3 copies/mL (e.g. 275.72 copies/process) at 95% detection probability, which is roughly in line with results published by Corman et al. [4].
T110 9861-10024 Sentence denotes They report an LoD for SARS-CoV-2 RNA of 5.2 copies per reaction at 95% detection probability, which corresponds to 208 copies/mL based on a 25 µL reaction volume.
T111 10025-10344 Sentence denotes It has to be noted that manual determination of LoD usually involves adding purified target RNA directly into the reagent mix for amplification, whereas in our study, control material (purified RNA) was spiked into samples and underwent the full workflow of the cobas 6800 device, including extraction and purification.
T112 10345-10421 Sentence denotes Therefore, differences in nominal analytical performance are to be expected.
T113 10422-10600 Sentence denotes One important limitation of the study is that we had to rely on spiked-in material and could not validate the performance of the assay using clinical SARS-CoV-2-positive samples.
T114 10601-10733 Sentence denotes However, we believe that our assay designed for high-throughput molecular testing could be useful in the ongoing outbreak situation.
T115 10734-10928 Sentence denotes It demonstrates how commercial PCR platforms can enhance outbreak readiness for emerging pathogens, allowing for large numbers of patients to be screened in a reasonable timeframe, if necessary.
T116 10929-11110 Sentence denotes It has to be noted that by its nature as a screening test targeting only a single viral gene, positive results should always be confirmed with an independent PCR as recommended [4].
T117 11111-11353 Sentence denotes Finally, we want to stress the importance of closely coordinating with local reference centres and public health authorities for determining clinical indications for testing, as well as the handling of confirmed cases and contact precautions.