Quality assurance of SARS-CoV-2 testing laboratories during the pandemic period in India - An experience from a designated provider laboratory.

PURPOSE: Indian Council of Medical Research (ICMR) initiated an Inter-Laboratory Quality Control testing (ILQC) program for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) testing. Under this program, SARS-CoV-2 testing laboratories across the country submit specimens to the assigned State Quality Control (SQCs) laboratories for ILQC testing. This study aimed to investigate the performance of public and private SARS-CoV-2 testing laboratories in Delhi and highlights the country's effort in ramping up testing facility with close monitoring of the quality of Covid-19 testing results. METHODS: In the present study, two-years of SARS-CoV-2 testing data is included. During July 2020 through February 2022, a total of 1791 anonymised specimens were received from 56 public and private laboratories. These specimens were processed by reverse transcriptase - polymerase chain reaction (RT-PCR) tests as per National Institute of Virology (NIV) protocol and the results were uploaded on the ICMR quality control/quality assurance (QC/QA) portal without directly conveying the results to respective participating laboratories. This portal generated a final report stating concordance and intimate results to individual laboratories. RESULTS: Among the 1791 specimens, 25 were rejected and the remaining 1766 were tested. Among these specimens 1691 (95.75%) revealed concordance, and 75 (4.24%) were discordant. A total of 29 laboratories had 100% concordance, 21 laboratories had over 90% concordance and six laboratories had over 80% concordance. CONCLUSIONS: The study demonstrates that the establishment of an inter-laboratory comparison program for SARS-CoV-2 testing helped in monitoring quality of SARS-CoV-2 testing in the country.

The Infectious Diseases Society of America Guidelines on the Diagnosis of COVID-19: Molecular Diagnostic Testing (December 2023).

Accurate molecular diagnostic tests are necessary for confirming a diagnosis of coronavirus disease 2019 (COVID-19) and for identifying asymptomatic carriage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The number of available SARS-CoV-2 nucleic acid detection tests continues to increase as does the COVID-19 diagnostic literature. Thus, the Infectious Diseases Society of America (IDSA) developed an evidence-based diagnostic guideline to assist clinicians, clinical laboratorians, patients, and policymakers in decisions related to the optimal use of SARS-CoV-2 nucleic acid amplification tests. In addition, we provide a conceptual framework for understanding molecular diagnostic test performance, discuss nuances of test result interpretation in a variety of practice settings, and highlight important unmet research needs related to COVID-19 diagnostic testing. IDSA convened a multidisciplinary panel of infectious diseases clinicians, clinical microbiologists, and experts in systematic literature review to identify and prioritize clinical questions and outcomes related to the use of SARS-CoV-2 molecular diagnostics. Grading of Recommendations Assessment, Development and Evaluation (GRADE) methodology was used to assess the certainty of evidence and make testing recommendations. The panel agreed on 12 diagnostic recommendations. Access to accurate SARS-CoV-2 nucleic acid testing is critical for patient care, hospital infection prevention, and the public health response to COVID-19 infection. Information on the clinical performance of available tests continues to grow, but the quality of evidence of the current literature to support this updated molecular diagnostic guideline remains moderate to very low. Recognizing these limitations, the IDSA panel weighed available diagnostic evidence and recommends nucleic acid testing for all symptomatic individuals suspected of having COVID-19. In addition, testing is suggested for asymptomatic individuals with known or suspected contact with a COVID-19 case when the results will impact isolation/quarantine/personal protective equipment (PPE) usage decisions. Evidence in support of rapid testing and testing of upper respiratory specimens other than nasopharyngeal swabs, which offer logistical advantages, is sufficient to warrant conditional recommendations in favor of these approaches.

Assessment of thermal and temporal stability of SARS-CoV-2 samples using real-time qRT-PCR.

BACKGROUND: As per the guidelines of the Indian Council of Medical Research, nasopharyngeal and oropharyngeal swabs in viral transport medium (VTM) are to be stored at 4 °C for less than 5 days and for more than 5 days at -70 °C. Samples are not transported or stored as per prescribed conditions because of the limitations, resulting in an apprehensive diagnosis. The aim of the study was to test the stability of the SARS-CoV-2 sample stored in VTM at different temperatures. METHODS: In this study, the stability of 21 positive and 9 negative samples for SARS-CoV-2 was evaluated in commercial VTM at different temperatures (-80 °C, -20 °C, 4 °C, and 25 to 30 °C). Stability was checked for up to 50 days in the above storage conditions at different intervals. PathoDetect™ and Hi-PCR® kits were used for the detection of the four genes of SARS-CoV-2. The Cycle Threshold (Ct) value for determining the positivity of samples for PathoDetect™ was < 40 and for Hi-PCR® was < 38. RESULTS: The SARS-CoV-2 confirmatory genes (RdRp and E genes) and the internal housekeeping gene remained detectable even on the 50th day of the study. The Ct of the RdRp and E genes were found to increase with storage duration, but all positive samples remained positive till the end of the study, or the Ct value remained below the cut-off level. The negative samples gave consistent results until the end of the study. When the differences in Ct values were compared between the days in a set of experiments, they were not significantly different except in a few samples. CONCLUSION: The SARS-CoV-2 genetic materials in commercial VTM were stable at room temperature to -80 °C for 50 days.

The effectiveness of COVID-19 testing and contact tracing in a US city.

Although testing, contact tracing, and case isolation programs can mitigate COVID-19 transmission and allow the relaxation of social distancing measures, few countries worldwide have succeeded in scaling such efforts to levels that suppress spread. The efficacy of test-trace-isolate likely depends on the speed and extent of follow-up and the prevalence of SARS-CoV-2 in the community. Here, we use a granular model of COVID-19 transmission to estimate the public health impacts of test-trace-isolate programs across a range of programmatic and epidemiological scenarios, based on testing and contact tracing data collected on a university campus and surrounding community in Austin, TX, between October 1, 2020, and January 1, 2021. The median time between specimen collection from a symptomatic case and quarantine of a traced contact was 2 days (interquartile range [IQR]: 2 to 3) on campus and 5 days (IQR: 3 to 8) in the community. Assuming a reproduction number of 1.2, we found that detection of 40% of all symptomatic cases followed by isolation is expected to avert 39% (IQR: 30% to 45%) of COVID-19 cases. Contact tracing is expected to increase the cases averted to 53% (IQR: 42% to 58%) or 40% (32% to 47%), assuming the 2- and 5-day delays estimated on campus and in the community, respectively. In a tracing-accelerated scenario, in which 75% of contacts are notified the day after specimen collection, cases averted increase to 68% (IQR: 55% to 72%). An accelerated contact tracing program leveraging rapid testing and electronic reporting of test results can significantly curtail local COVID-19 transmission.

A meta-analysis of the diagnostic test accuracy of CT-based radiomics for the prediction of COVID-19 severity.

INTRODUCTION: According to the Chinese Health Commission guidelines, coronavirus disease 2019 (COVID-19) severity is classified as mild, moderate, severe, or critical. The mortality rate of COVID-19 is higher among patients with severe and critical diseases; therefore, early identification of COVID-19 prevents disease progression and improves patient survival. Computed tomography (CT) radiomics, as a machine learning method, provides an objective and mathematical evaluation of COVID-19 pneumonia. As CT-based radiomics research has recently focused on COVID-19 diagnosis and severity analysis, this meta-analysis aimed to investigate the predictive power of a CT-based radiomics model in determining COVID-19 severity. MATERIALS AND METHODS: This study followed the diagnostic version of PRISMA guidelines. PubMed, Embase databases and the Cochrane Central Register of Controlled Trials, and the Cochrane Database of Systematic Reviews were searched to identify relevant articles in the meta-analysis from inception until July 16, 2021. The sensitivity and specificity were analyzed using forest plots. The overall predictive power was calculated using the summary receiver operating characteristic curve. The bias was evaluated using a funnel plot. The quality of the included literature was assessed using the radiomics quality score and quality assessment of diagnostic accuracy studies tool. RESULTS: The radiomics quality scores ranged from 7 to 16 (achievable score: 2212 8 to 36). The pooled sensitivity and specificity were 0.800 (95% confidence interval [CI] 0.662-0.891) and 0.874 (95% CI 0.773-0.934), respectively. The pooled area under the receiver operating characteristic curve was 0.908. The quality assessment tool showed favorable results. CONCLUSION: This meta-analysis demonstrated that CT-based radiomics models might be helpful for predicting the severity of COVID-19 pneumonia.

External quality assessment of COVID-19 real time reverse transcription PCR laboratories in India.

Sudden emergence and rapid spread of COVID-19 created an inevitable need for expansion of the COVID-19 laboratory testing network across the world. The strategy to test-track-treat was advocated for quick detection and containment of the disease. Being the second most populous country in the world, India was challenged to make COVID-19 testing available and accessible in all parts of the country. The molecular laboratory testing network was augmented expeditiously, and number of laboratories was increased from one in January 2020 to 2951 till mid-September, 2021. This rapid expansion warranted the need to have inbuilt systems of quality control/ quality assurance. In addition to the ongoing inter-laboratory quality control (ILQC), India implemented an External Quality Assurance Program (EQAP) with assistance from World Health Organization (WHO) and Royal College of Pathologists, Australasia. Out of the 953 open system rRTPCR laboratories in both public and private sector who participated in the first round of EQAP, 891(93.4%) laboratories obtained a passing score of > = 80%. The satisfactory performance of Indian COVID-19 testing laboratories has boosted the confidence of the public and policy makers in the quality of testing. ILQC and EQAP need to continue to ensure adherence of the testing laboratories to the desired quality standards.

Coupling immuno-magnetic capture with LC-MS/MS(MRM) as a sensitive, reliable, and specific assay for SARS-CoV-2 identification from clinical samples.

Recently, numerous diagnostic approaches from different disciplines have been developed for SARS-CoV-2 diagnosis to monitor and control the COVID-19 pandemic. These include MS-based assays, which provide analytical information on viral proteins. However, their sensitivity is limited, estimated to be 5 × 104 PFU/ml in clinical samples. Here, we present a reliable, specific, and rapid method for the identification of SARS-CoV-2 from nasopharyngeal (NP) specimens, which combines virus capture followed by LC-MS/MS(MRM) analysis of unique peptide markers. The capture of SARS-CoV-2 from the challenging matrix, prior to its tryptic digestion, was accomplished by magnetic beads coated with polyclonal IgG-α-SARS-CoV-2 antibodies, enabling sample concentration while significantly reducing background noise interrupting with LC-MS analysis. A sensitive and specific LC-MS/MS(MRM) analysis method was developed for the identification of selected tryptic peptide markers. The combined assay, which resulted in S/N ratio enhancement, achieved an improved sensitivity of more than 10-fold compared with previously described MS methods. The assay was validated in 29 naive NP specimens, 19 samples were spiked with SARS-CoV-2 and 10 were used as negative controls. Finally, the assay was successfully applied to clinical NP samples (n = 26) pre-determined as either positive or negative by RT-qPCR. This work describes for the first time a combined approach for immuno-magnetic viral isolation coupled with MS analysis. This method is highly reliable, specific, and sensitive; thus, it may potentially serve as a complementary assay to RT-qPCR, the gold standard test. This methodology can be applied to other viruses as well.

The Use of Antigenic SARS-CoV-2 Point-of-Care Test: The Italian Pediatric Real-Life Experience.

In Italy, during the second epidemic wave of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), rapid antigenic (Ag) test at point-of-care (POCT) station were employed to quickly evaluate large numbers of swabs. We collected data of all children who underwent the Ag test in our hospital. All positive patients were recalled to perform reverse transcription polymerase chain reaction. A total of 2133 tests were collected over 1 month. Clinical data of 1941 children (median age = 3.7 years) were analyzed: 1343 (69.2%) patients complained of symptoms, 594 (30.6%) had a history of close contact with SARS-CoV-2-positive individuals. Among symptoms reported, acute rhinitis was the most frequent (67.9%), followed by cough (42.6%) and fever (31.5%). Among all tests, 95.8% resulted negative, 4.2% positive: 37/89 were confirmed. In confirmed cases, fever (56.2% vs 32.2%; P = .041) and gastrointestinal symptoms (18.8% vs 6.25%; P = .041) were significantly more frequent compared with negative children. The use of POCT for Ag test seems appropriate for SARS-CoV-2 screening in the pediatric population. In children, fever and gastrointestinal symptoms may constitute red flags of SARS-CoV-2.

Evaluation of the Abbott BinaxNOW COVID-19 Test Ag Card for rapid detection of SARS-CoV-2 infection by a local public health district with a rural population.

SARS-CoV-2 RT-PCR, the gold standard for diagnostic testing, may not be readily available or logistically applicable for routine COVID-19 testing in many rural communities in the United States. In this validation study, we compared the BinaxNOW™ COVID-19 Test Ag Card with SARS-CoV-2 RT-PCR in 214 participants who sought COVID-19 testing from a local public health district in Idaho, USA. The median age of participants was 35 and 82.7% were symptomatic. Thirty-seven participants (17.3%) had positive RT-PCR results. Results between the two tests were 94.4% concordant. The sensitivity of the BinaxNOW™ COVID-19 Test Ag Card was 67.6% (95% CI: 50.2-81.9%), and the specificity was 100.0% (95% CI: 97.9-100.0%). The positive predictive value (PPV) for the BinaxNOW™ COVID-19 Test Ag Card was 100.0% (95% CI: 86.2-100.0%), and the negative predictive value (NPV) was 93.6% (95% CI: 89.1-96.6%). Although the sensitivity of BinaxNOW™ COVID-19 Test Ag Card was lower than RT-PCR, rapid results and high specificity support its use for early detection of COVID-19, especially in settings where SARS-CoV-2 RT-PCR testing is not readily available. Rapid antigen tests, such as the BinaxNOW™ COVID-19 Test Ag Card, may be a more convenient tool in quickly identifying and preventing COVID-19 transmission, especially in rural settings.

Temporal stability and detection sensitivity of the dry swab-based diagnosis of SARS-CoV-2.

The rapid spread and evolution of various strains of SARS-CoV-2, the virus responsible for COVID-19, continues to challenge the disease controlling measures globally. Alarming concern is, the number of second wave infections surpassed the first wave and the onset of severe symptoms manifesting rapidly. In this scenario, testing of maximum population in less time and minimum cost with existing diagnostic amenities is the only possible way to control the spread of the virus. The previously described RNA extraction-free methods using dry swab have been shown to be advantageous in these critical times by different studies. In this work, we show the temporal stability and performance of the dry swab viral detection method at two different temperatures. Contrived dry swabs holding serially diluted SARS-CoV-2 strains A2a and A3i at 25°C (room temperature; RT) and 4°C were subjected to direct RT-PCR and compared with standard VTM-RNA based method. The results clearly indicate that dry swab method of RNA detection is as efficient as VTM-RNA-based method in both strains, when checked for up to 72 h. The lesser CT values of dry swab samples in comparison to that of the VTM-RNA samples suggest better sensitivity of the method within 48 h of time. The results collectively suggest that dry swab samples are stable at RT for 24 h and the detection of SARS-CoV-2 RNA by RT-PCR do not show variance from VTM-RNA. This extraction free, direct RT-PCR method holds phenomenal standing in the present life-threatening circumstances due to SARS-CoV-2.

SARS-CoV-2 Testing Before International Airline Travel, December 2020 to May 2021.

Although there have been several case reports and simulation models of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission associated with air travel, there are limited data to guide testing strategy to minimize the risk of SARS-CoV-2 exposure and transmission onboard commercial aircraft. Among 9853 passengers with a negative SARS-CoV-2 polymerase chain reaction test performed within 72 hours of departure from December 2020 through May 2021, five (0.05%) passengers with active SARS-CoV-2 infection were identified with rapid antigen tests and confirmed with rapid molecular test performed before and after an international flight from the United States to Italy. This translates to a case detection rate of 1 per 1970 travelers during a time of high prevalence of active infection in the United States. A negative molecular test for SARS-CoV-2 within 72 hours of international airline departure results in a low probability of active infection identified on antigen testing during commercial airline flight.

Rethinking COVID-19 test sensitivity-a strategy for improving the detection limit.

Numerous genetic tests for the detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, including those based on the ever-popular real-time polymerase chain reaction (RT-qPCR) technique, have been reported. These diagnostic tests give false negatives particularly during the early and late stages of COVID-19 clearly indicating inadequate test sensitivity. The entire COVID-19 diagnostic workflow is often overlooked and given very little attention. Herein, we propose that volumetric modifications to COVID-19 workflows would significantly improve detection limits. We would therefore encourage researchers to adopt a holistic approach, in which all the steps of a COVID-19 diagnostic workflow, are carefully scrutinised, particularly those upstream factors at the viral sampling and pre-analytical stages.

Improvement of Sensitivity of Pooling Strategies for COVID-19.

Group testing (or pool testing), for example, Dorfman's method or grid method, has been validated for COVID-19 RT-PCR tests and implemented widely by most laboratories in many countries. These methods take advantages since they reduce resources, time, and overall costs required for a large number of samples. However, these methods could have more false negative cases and lower sensitivity. In order to maintain both accuracy and efficiency for different prevalence, we provide a novel pooling strategy based on the grid method with an extra pool set and an optimized rule inspired by the idea of error-correcting codes. The mathematical analysis shows that (i) the proposed method has the best sensitivity among all the methods we compared, if the false negative rate (FNR) of an individual test is in the range [1%, 20%] and the FNR of a pool test is closed to that of an individual test, and (ii) the proposed method is efficient when the prevalence is below 10%. Numerical simulations are also performed to confirm the theoretical derivations. In summary, the proposed method is shown to be felicitous under the above conditions in the epidemic.

Rapid diagnosis of COVID-19 in the first year of the pandemic: A systematic review.

BACKGROUND: COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global health threat and remains a challenge for modern medicine. Rapid and accurate diagnosis of COVID-19 is vital for proper disease and outbreak management. Our review aimed to analyze scientific articles published in the literature addressing the rapid tests available for COVID-19 diagnosis at the first year of the pandemic. METHODS: A systematic review was performed from October 22 to 27, 2020, searching data published in PubMed and Google Scholar databases, using subject headings or keywords related to point of care and rapid test diagnostic for SARS-CoV-2 and COVID-19. RESULTS: The first survey identified 403 articles, but only 23 met the defined criteria for the systematic analysis. The sensitivity and specificity parameters were assessed in 19 studies, and the data suggested that there was lower sensitivity in the period 1 to 7 days after the emergence of symptoms (∼38%) higher sensitivity at 8 to 14 days (∼90%), and the highest at 15 to 39 days (∼98%). Accuracy was reported in six studies, reporting values above 50%. Only three studies reported a possible cross-reaction. CONCLUSIONS: Our findings indicate that the rapid tests used in the first year of the pandemic were tested with a small number of samples and not adequately validated. And the studies that described them were conducted with little scientific rigor.

SARS-CoV-2 screening of asymptomatic health care workers: experience of a General hospital.

Health care workers (HCWs) are at major risk to be infected by SARS-CoV-2 and transmit the virus to the patients. Furthermore, travels are a major factor in the diffusion of the virus. We report our experience regarding the screening of asymptomatic HCWs returning from holidays, following the issue of a national guideline on 08/20/2020. The organization of the occupational health department and the clinical laboratory was adapted in order to start the screening on August, 24, 2020. All HCWs tested for SARS-CoV-2 the week before and 4 weeks after the implementation of the screening were included. The mean number of tests was analyzed per working day and working week. Overall, 502 (31.4%) HCWs were tested for SARS-CoV-2 during the study period. The mean number of HCWs tested per working day was 27.1. HCWs accounted for 36.9% (n = 167) and 11.2% (n = 84) of the tests performed in the 1st and the 4th week following the implementation of the guidelines. The number of tests performed each week in HCWs increased by at least 20-fold after the implementation of the guidelines. No asymptomatic HCW was tested positive. Screening of asymptomatic HCWs was poorly effective in the context of low circulation of the virus. We suggest giving priority to infection prevention and control measures and screening of symptomatic subjects and asymptomatic contacts.