DIAGNOSIS OF SARS-CoV-2 (COVID-19)
RNA tests can confirm the diagnosis of SARS-CoV-2 (COVID-19) cases with real-time RT-PCR or next-generation sequencing (148, 149, 245, 246). At present, nucleic acid detection techniques, like RT-PCR, are considered an effective method for confirming the diagnosis in clinical cases of COVID-19 (148). Several companies across the world are currently focusing on developing and marketing SARS-CoV-2-specific nucleic acid detection kits. Multiple laboratories are also developing their own in-house RT-PCR. One of them is the SARS-CoV-2 nucleic acid detection kit produced by Shuoshi Biotechnology (double fluorescence PCR method) (150). Up to 30 March 2020, the U.S. Food and Drug Administration (FDA) had granted 22 in vitro diagnostics Emergency Use Authorizations (EUAs), including for the RT-PCR diagnostic panel for the universal detection of SARS-like betacoronaviruses and specific detection of SARS-CoV-2, developed by the U.S. CDC (Table 1) (258, 259).
TABLE 1  FDA-approved in vitro Emergency Use Authorization diagnostics available for SARS-CoV-2 as of 30 March 2020a
Developer  Diagnostic platform
Centers for Disease Control and Prevention (CDC)  CDC 2019-nCoV real-time RT-PCR diagnostic panel
Wadsworth Center, New York State Department of Public Health (CDC)  New York SARS-CoV-2 real-time reverse transcriptase (RT)-PCR diagnostic panel
Roche Molecular Systems, Inc. (RMS)  cobas SARS-CoV-2
Thermo Fisher Scientific, Inc.  TaqPath COVID-19 combo kit
Laboratory Corporation of America (LabCorp)  COVID-19 RT-PCR test
Hologic, Inc.  Panther fusion SARS-CoV-2
Quest Diagnostics Infectious Disease, Inc.  Quest SARS-CoV-2 rRT-PCR
Quidel Corporation  Lyra SARS-CoV-2 assay
Abbott Molecular  Abbott RealTime SARS-CoV-2 assay
GenMark Diagnostics, Inc.  ePlex SARS-CoV-2 test
DiaSorin Molecular, LLC  Simplexa COVID-19 direct assay
Cepheid  Xpert Xpress SARS-CoV-2 test
Primerdesign, Ltd.  COVID-19 Genesig real-time PCR assay
Mesa Biotech, Inc.  Accula SARS-Cov-2 test
BioFire Defense, LLC  BioFire COVID-19 test
PerkinElmer, Inc.  PerkinElmer new coronavirus nucleic acid detection kit
Avellino Lab USA, Inc.  AvellinoCoV2 test
BGI Genomics, Co. Ltd.  Real-time fluorescent RT-PCR kit for detecting SARS-2019-nCoV
Luminex Molecular Diagnostics, Inc.  NxTAG CoV extended panel assay
Abbott Diagnostics Scarborough, Inc.  ID Now COVID-19
Qiagen GmbH  QIAstat-Dx respiratory SARS-CoV-2 panel
NeuMoDx Molecular, Inc.  NeuMoDx SARS-CoV-2 assay
a  Data are from references 258 and 259. Recently, 95 full-length genomic sequences of SARAS-CoV-2 strains available in the National Center for Biotechnology Information and GISAID databases were subjected to multiple-sequence alignment and phylogenetic analyses for studying variations in the viral genome (260). All the viral strains revealed high homology of 99.99% (99.91% to 100%) at the nucleotide level and 99.99% (99.79% to 100%) at the amino acid level. Overall variation was found to be low in ORF regions, with 13 variation sites recognized in 1a, 1b, S, 3a, M, 8, and N regions. Mutation rates of 30.53% (29/95) and 29.47% (28/95) were observed at nt 28144 (ORF8) and nt 8782 (ORF1a) positions, respectively. Owing to such selective mutations, a few specific regions of SARS-CoV-2 should not be considered for designing primers and probes. The SARS-CoV-2 reference sequence could pave the way to study molecular biology and pathobiology, along with developing diagnostics and appropriate prevention and control strategies for countering SARS-CoV-2 (260).
Nucleic acids of SARS-CoV-2 can be detected from samples (64) such as bronchoalveolar lavage fluid, sputum, nasal swabs, fiber bronchoscope brush biopsy specimen, pharyngeal swabs, feces, blood, and urine, with different levels of diagnostic performance (Table 2) (80, 245, 246). The viral loads of SARS-CoV-2 were measured using N-gene-specific quantitative RT-PCR in throat swab and sputum samples collected from COVID-19-infected individuals. The results indicated that the viral load peaked at around 5 to 6 days following the onset of symptoms, and it ranged from 104 to 107 copies/ml during this time (151). In another study, the viral load was found to be higher in the nasal swabs than the throat swabs obtained from COVID-19 symptomatic patients (82). Although initially it was thought that viral load would be associated with poor outcomes, some case reports have shown asymptomatic individuals with high viral loads (247). Recently, the viral load in nasal and throat swabs of 17 symptomatic patients was determined, and higher viral loads were recorded soon after the onset of symptoms, particularly in the nose compared to the throat. The pattern of viral nucleic acid shedding of SARS-CoV-2-infected patients was similar to that of influenza patients but seemed to be different from that of SARS-CoV patients. The viral load detected in asymptomatic patients resembled that of symptomatic patients as studied in China, which reflects the transmission perspective of asymptomatic or symptomatic patients having minimum signs and symptoms (82). Another study, conducted in South Korea, related to SARS-CoV-2 viral load, opined that SARS-CoV-2 kinetics were significantly different from those of earlier reported CoV infections, including SARS-CoV (253). SARS-CoV-2 transmission can occur early in the viral infection phase; thus, diagnosing cases and isolation attempts for this virus warrant different strategies than those needed to counter SARS-CoV. Studies are required to establish any correlation between SARS-CoV-2 viral load and cultivable virus. Recognizing patients with fewer or no symptoms, along with having modest detectable viral RNA in the oropharynx for 5 days, indicates the requirement of data for assessing SARS-CoV-2 transmission dynamics and updating the screening procedures in the clinics (82).
TABLE 2  Clinical specimens for detection of SARS CoV-2
Sample  Recommendationa
Bronchoalveolar lavage fluid  +++
Sputum  +++
Nasal swabs  +++
Fibrobronchoscope brush biopsy  ++
Pharyngeal swabs  ++
Feces  +
Blood  +
Urine  +
a  Recommendations are based on references 245 and 246. +++, strong; ++, moderate; +, weak. The results of the studies related to SARS-CoV-2 viral loads reflect active replication of this virus in the upper respiratory tract and prolonged viral shedding after symptoms disappear, including via stool. Thus, the current case definition needs to be updated along with a reassessment of the strategies to be adopted for restraining the SARS-CoV-2 outbreak spread (248). In some cases, the viral load studies of SARS-CoV-2 have also been useful to recommend precautionary measures when handling specific samples, e.g., feces. In a recent survey from 17 confirmed cases of SARS-CoV-2 infection with available data (representing days 0 to 13 after onset), stool samples from nine cases (53%; days 0 to 11 after onset) were positive on RT-PCR analysis. Although the viral loads were lower than those of respiratory samples (range, 550 copies per ml to 1.21 × 105 copies per ml), this has essential biosafety implications (151).
The samples from 18 SARS-CoV-2-positive patients in Singapore who had traveled from Wuhan to Singapore showed the presence of viral RNA in stool and whole blood but not in urine by real-time RT-PCR (288). Further, novel SARS-CoV-2 infections have been detected in a variety of clinical specimens, like bronchoalveolar lavage fluid, sputum, nasal swabs, fibrobronchoscope brush biopsy specimens, pharyngeal swabs, feces, and blood (246).
The presence of SARS-CoV-2 in fecal samples has posed grave public health concerns. In addition to the direct transmission mainly occurring via droplets of sneezing and coughing, other routes, such as fecal excretion and environmental and fomite contamination, are contributing to SARS-CoV-2 transmission and spread (249–252). Fecal excretion has also been documented for SARS-CoV and MERS-CoV, along with the potential to stay viable in situations aiding fecal-oral transmission. Thus, SARS-CoV-2 has every possibility to be transmitted through this mode. Fecal-oral transmission of SARS-CoV-2, particularly in regions having low standards of hygiene and poor sanitation, may have grave consequences with regard to the high spread of this virus. Ethanol and disinfectants containing chlorine or bleach are effective against coronaviruses (249–252). Appropriate precautions need to be followed strictly while handling the stools of patients infected with SARS-CoV-2. Biowaste materials and sewage from hospitals must be adequately disinfected, treated, and disposed of properly. The significance of frequent and good hand hygiene and sanitation practices needs to be given due emphasis (249–252). Future explorative research needs to be conducted with regard to the fecal-oral transmission of SARS-CoV-2, along with focusing on environmental investigations to find out if this virus could stay viable in situations and atmospheres facilitating such potent routes of transmission. The correlation of fecal concentrations of viral RNA with disease severity needs to be determined, along with assessing the gastrointestinal symptoms and the possibility of fecal SARS-CoV-2 RNA detection during the COVID-19 incubation period or convalescence phases of the disease (249–252).
The lower respiratory tract sampling techniques, like bronchoalveolar lavage fluid aspirate, are considered the ideal clinical materials, rather than the throat swab, due to their higher positive rate on the nucleic acid test (148). The diagnosis of COVID-19 can be made by using upper-respiratory-tract specimens collected using nasopharyngeal and oropharyngeal swabs. However, these techniques are associated with unnecessary risks to health care workers due to close contact with patients (152). Similarly, a single patient with a high viral load was reported to contaminate an entire endoscopy room by shedding the virus, which may remain viable for at least 3 days and is considered a great risk for uninfected patients and health care workers (289). Recently, it was found that the anal swabs gave more positive results than oral swabs in the later stages of infection (153). Hence, clinicians have to be cautious while discharging any COVID-19-infected patient based on negative oral swab test results due to the possibility of fecal-oral transmission. Even though the viral loads in stool samples were found to be less than those of respiratory samples, strict precautionary measures have to be followed while handling stool samples of COVID-19 suspected or infected patients (151). Children infected with SARS-CoV-2 experience only a mild form of illness and recover immediately after treatment. It was recently found that stool samples of SARS-CoV-2-infected children that gave negative throat swab results were positive within ten days of negative results. This could result in the fecal-oral transmission of SARS-CoV-2 infections, especially in children (290). Hence, to prevent the fecal-oral transmission of SARS-CoV-2, infected COVID-19 patients should only be considered negative when they test negative for SARS-CoV-2 in the stool sample.
A suspected case of COVID-19 infection is said to be confirmed if the respiratory tract aspirate or blood samples test positive for SARS-CoV-2 nucleic acid using RT-PCR or by the identification of SARS-CoV-2 genetic sequence in respiratory tract aspirate or blood samples (80). The patient will be confirmed as cured when two subsequent oral swab results are negative (153). Recently, the live virus was detected in the self-collected saliva of patients infected with COVID-19. These findings were confirmative of using saliva as a noninvasive specimen for the diagnosis of COVID-19 infection in suspected individuals (152). It has also been observed that the initial screening of COVID-19 patients infected with RT-PCR may give negative results even if they have chest CT findings that are suggestive of infection. Hence, for the accurate diagnosis of COVID-19, a combination of repeated swab tests using RT-PCR and CT scanning is required to prevent the possibility of false-negative results during disease screening (154). RT-PCR is the most widely used test for diagnosing COVID-19. However, it has some significant limitations from the clinical perspective, since it will not give any clarity regarding disease progression. Droplet digital PCR (ddPCR) can be used for the quantification of viral load in the samples obtained from lower respiratory tracts. Hence, based on the viral load, we can quickly evaluate the progression of infection (291). In addition to all of the above findings, sequencing and phylogenetics are critical in the correct identification and confirmation of the causative viral agent and useful to establish relationships with previous isolates and sequences, as well as to know, especially during an epidemic, the nucleotide and amino acid mutations and the molecular divergence. The rapid development and implementation of diagnostic tests against emerging novel diseases like COVID-19 pose significant challenges due to the lack of resources and logistical limitations associated with an outbreak (155).
SARS-CoV-2 infection can also be confirmed by isolation and culturing. The human airway epithelial cell culture was found to be useful in isolating SARS-CoV-2 (3). The efficient control of an outbreak depends on the rapid diagnosis of the disease. Recently, in response to the COVID-19 outbreak, 1-step quantitative real-time reverse transcription-PCR assays were developed that detect the ORF1b and N regions of the SARS-CoV-2 genome (156). That assay was found to achieve the rapid detection of SARS-CoV-2. Nucleic acid-based assays offer high accuracy in the diagnosis of SARS-CoV-2, but the current rate of spread limits its use due to the lack of diagnostic assay kits. This will further result in the extensive transmission of COVID-19, since only a portion of suspected cases can be diagnosed. In such situations, conventional serological assays, like enzyme-linked immunosorbent assay (ELISA), that are specific to COVID-19 IgM and IgG antibodies can be used as a high-throughput alternative (149). At present, there is no diagnostic kit available for detecting the SARS-CoV-2 antibody (150). The specific antibody profiles of COVID-19 patients were analyzed, and it was found that the IgM level lasted more than 1 month, indicating a prolonged stage of virus replication in SARS-CoV-2-infected patients. The IgG levels were found to increase only in the later stages of the disease. These findings indicate that the specific antibody profiles of SARS-CoV-2 and SARS-CoV were similar (325). These findings can be utilized for the development of specific diagnostic tests against COVID-19 and can be used for rapid screening. Even though diagnostic test kits are already available that can detect the genetic sequences of SARS-CoV-2 (95), their availability is a concern, as the number of COVID-19 cases is skyrocketing (155, 157). A major problem associated with this diagnostic kit is that it works only when the test subject has an active infection, limiting its use to the earlier stages of infection. Several laboratories around the world are currently developing antibody-based diagnostic tests against SARS-CoV-2 (157).
Chest CT is an ideal diagnostic tool for identifying viral pneumonia. The sensitivity of chest CT is far superior to that of X-ray screening. The chest CT findings associated with COVID-19-infected patients include characteristic patchy infiltration that later progresses to ground-glass opacities (158). Early manifestations of COVID-19 pneumonia might not be evident in X-ray chest radiography. In such situations, a chest CT examination can be performed, as it is considered highly specific for COVID-19 pneumonia (118). Those patients having COVID-19 pneumonia will exhibit the typical ground-glass opacity in their chest CT images (154). The patients infected with COVID-19 had elevated plasma angiotensin 2 levels. The level of angiotensin 2 was found to be linearly associated with viral load and lung injury, indicating its potential as a diagnostic biomarker (121). The chest CT imaging abnormalities associated with COVID-19 pneumonia have also been observed even in asymptomatic patients. These abnormalities progress from the initial focal unilateral to diffuse bilateral ground-glass opacities and will further progress to or coexist with lung consolidation changes within 1 to 3 weeks (159). The role played by radiologists in the current scenario is very important. Radiologists can help in the early diagnosis of lung abnormalities associated with COVID-19 pneumonia. They can also help in the evaluation of disease severity, identifying its progression to acute respiratory distress syndrome and the presence of secondary bacterial infections (160). Even though chest CT is considered an essential diagnostic tool for COVID-19, the extensive use of CT for screening purposes in the suspected individuals might be associated with a disproportionate risk-benefit ratio due to increased radiation exposure as well as increased risk of cross-infection. Hence, the use of CT for early diagnosis of SARS-CoV-2 infection in high-risk groups should be done with great caution (292).
More recently, other advanced diagnostics have been designed and developed for the detection of SARS-CoV-2 (345, 347, 350–352). A reverse transcriptional loop-mediated isothermal amplification (RT-LAMP), namely, iLACO, has been developed for rapid and colorimetric detection of this virus (354). RT-LAMP serves as a simple, rapid, and sensitive diagnostic method that does not require sophisticated equipment or skilled personnel (349). An interactive web-based dashboard for tracking SARS-CoV-2 in a real-time mode has been designed (238). A smartphone-integrated home-based point-of-care testing (POCT) tool, a paper-based POCT combined with LAMP, is a useful point-of-care diagnostic (353). An Abbott ID Now COVID-19 molecular POCT-based test, using isothermal nucleic acid amplification technology, has been designed as a point-of-care test for very rapid detection of SARS-CoV-2 in just 5 min (344). A CRISPR-based SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) diagnostic for rapid detection of SARS-CoV-2 without the requirement of specialized instrumentation has been reported to be very useful in the clinical diagnosis of COVID-19 (360). A CRISPR-Cas12-based lateral flow assay also has been developed for rapid detection of SARS-CoV-2 (346). Artificial intelligence, by means of a three-dimensional deep-learning model, has been developed for sensitive and specific diagnosis of COVID-19 via CT images (332).
Tracking and mapping of the rising incidence rates, disease outbreaks, community spread, clustered transmission events, hot spots, and superspreader potential of SARS-CoV-2/COVID warrant full exploitation of real-time disease mapping by employing geographical information systems (GIS), such as the GIS software Kosmo 3.1, web-based real-time tools and dashboards, apps, and advances in information technology (356–359). Researchers have also developed a few prediction tools/models, such as the prediction model risk of bias assessment tool (PROBAST) and critical appraisal and data extraction for systematic reviews of prediction modeling studies (CHARMS), which could aid in assessing the possibility of getting infection and estimating the prognosis in patients; however, such models may suffer from bias issues and, hence, cannot be considered completely trustworthy, which necessitates the development of new and reliable predictors (360).