Detection of SARS-CoV-2 in Clinical Samples: Target-specific Analysis of Qualitative Reverse Transcription-Polymerase Chain Reaction(RT-PCR) Diagnostic Kits.

INTRODUCTION: The Coronavirus disease 2019 pandemic caused by Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to the rise of many available modalities for diagnosis. One such modality is the Reverse Transcription-Polymerase Chain Reaction (RT-PCR) kits which require evaluation amongst the many available commercial kits in the market. METHODS: We conducted a performance evaluation of twelve RT-PCR SARS-CoV-2 commercial kits. A total of 75 nasopharyngeal and oropharyngeal clinical samples were selected with their cycling threshold (Ct) values. Inclusion of 5 gene targets: E gene, N gene, S gene, RdRp and ORF1ab were assessed. Data was analyzed using R software version 4.1.1 and Microsoft Excel. RESULTS: We observe that, the positive sample's Ct values differs significantly across the 12 diagnostic kits. However, for gene-specific analysis, we observe that, positive sample's Ct values does not differ significantly across gene targets. There is significant difference in Ct values in Commercial kits targeting all genes except S-gene. All the commercial kits Altona (E and S genes), Thermo (ORF1ab and N genes), Multiplex (E, ORF1ab, RdRdp genes), Meril (N and ORF1ab genes), S D Biosensor (E and ORF1ab genes), Lab Gun (RdRp and N genes) and Lab systems (ORF1ab and E genes) scored a sensitivity of 100%. All other kits scored sensitivity above 95% and lowest sensitivity with the Genes2me (E gene) and Genes2me (RdRp) at 95.08% each. All kits were 100% specific. CONCLUSION: This study provides an accurate comprehensive assessment of the different kits in the detection of SARS-CoV-2 which may promote standardization of testing across laboratories.

Second round statewide sentinel-based population survey for estimation of the burden of active infection and anti-SARS-CoV-2 IgG antibodies in the general population of Karnataka, India, during January-February 2021.

Objective: Demonstrate the feasibility of using the existing sentinel surveillance infrastructure to conduct the second round of the serial cross-sectional sentinel-based population survey. Assess active infection, seroprevalence, and their evolution in the general population across Karnataka. Identify local variations for locally appropriate actions. Additionally, assess the clinical sensitivity of the testing kit used on account of variability of antibody levels in the population. Methods: The cross-sectional study of 41,228 participants across 290 healthcare facilities in all 30 districts of Karnataka was done among three groups of participants (low, moderate, and high-risk). The geographical spread was sufficient to capture local variations. Consenting participants were subjected to real-time reverse transcription-polymerase chain reaction (RT-PCR) testing, and antibody (IgG) testing. Clinical sensitivity was assessed by conducting a longitudinal study among participants identified as COVID-19 positive in the first survey round. Results: Overall weighted adjusted seroprevalence of IgG was 15.6% (95% CI: 14.9-16.3), crude IgG prevalence was 15.0% and crude active infection was 0.5%. Statewide infection fatality rate (IFR) was estimated as 0.11%, and COVID-19 burden estimated between 26.1 to 37.7% (at 90% confidence). Further, Cases-to-infections ratio (CIR) varied 3-35 across units and IFR varied 0.04-0.50% across units. Clinical sensitivity of the IgG ELISA test kit was estimated as ≥38.9%. Conclusion: We demonstrated the feasibility and simplicity of sentinel-based population survey in measuring variations in subnational and local data, useful for locally appropriate actions in different locations. The sentinel-based population survey thus helped identify districts that needed better testing, reporting, and clinical management. The state was far from attaining natural immunity during the survey and hence must step up vaccination coverage and enforce public health measures to prevent the spread of COVD-19.

The burden of active infection and anti-SARS-CoV-2 IgG antibodies in the general population: Results from a statewide sentinel-based population survey in Karnataka, India.

OBJECTIVE: To estimate the burden of active infection and anti-SARS-CoV-2 IgG antibodies in Karnataka, India, and to assess variation across geographical regions and risk groups. METHODS: A cross-sectional survey of 16,416 people covering three risk groups was conducted between 3-16 September 2020 using the state of Karnataka's infrastructure of 290 healthcare facilities across all 30 districts. Participants were further classified into risk subgroups and sampled using stratified sampling. All participants were subjected to simultaneous detection of SARS-CoV-2 IgG using a commercial ELISA kit, SARS-CoV-2 antigen using a rapid antigen detection test (RAT) and reverse transcription-polymerase chain reaction (RT-PCR) for RNA detection. Maximum-likelihood estimation was used for joint estimation of the adjusted IgG, active and total prevalence (either IgG or active or both), while multinomial regression identified predictors. RESULTS: The overall adjusted total prevalence of COVID-19 in Karnataka was 27.7% (95% CI 26.1-29.3), IgG 16.8% (15.5-18.1) and active infection fraction 12.6% (11.5-13.8). The case-to-infection ratio was 1:40 and the infection fatality rate was 0.05%. Influenza-like symptoms or contact with a COVID-19-positive patient were good predictors of active infection. RAT kits had higher sensitivity (68%) in symptomatic people compared with 47% in asymptomatic people. CONCLUSION: This sentinel-based population survey was the first comprehensive survey in India to provide accurate estimates of the COVID-19 burden. The findings provide a reasonable approximation of the population immunity threshold levels. Using existing surveillance platforms coupled with a syndromic approach and sampling framework enabled this model to be replicable.

Performance evaluation of Truenat™ Beta CoV & Truenat™ SARS-CoV-2 point-of-care assays for coronavirus disease 2019.

Background & objectives: The rapid diagnosis of coronavirus disease 2019 (COVID-19) is a significant step towards the containment of the virus. The surge of COVID-19 cases in India and across the globe necessitates a rapid and sensitive molecular assay. Rapid point-of-care (PoC) assays (Truenat Beta CoV and Truenat SARS-CoV-2 assays) for the diagnosis of COVID-19 have been developed which are expected to shorten the turnaround time of reporting of results and also can be used for field investigations of COVID-19. The objectives of the study were to validate the performance of Truenat Beta CoV and Truenat SARS-CoV-2 PoC assays for the detection of SARS-CoV-2 infected cases with reference to analytical sensitivity, precision/inter-machine variation, clinical sensitivity and clinical specificity. Methods: The rapid PoC screening and confirmatory assays were prospectively validated at the State Level Virus Research and Diagnostic Laboratory at Bangalore Medical College and Research Institute, Bengaluru, under technical supervision by the Indian Council of Medical Research-National Institute of Virology (ICMR-NIV), Pune. Real-time reverse transcription-polymerase chain reaction (rRT-PCR)was considered as the reference standard against which the rapid assays were validated for all samples tested based on analytical sensitivity, precision/inter-machine variation, clinical sensitivity and clinical specificity. Results: Truenat Beta CoV and Truenat SARS-CoV-2 assays showed concordant results when compared with the reference standard rRT-PCR. These PoC assays exhibited 100 per cent sensitivity, specificity, positive predictive value and negative predictive value. Interpretation & conclusions: Truenat Beta CoV and Truenat SARS-CoV-2 assays showed concordance with the reference standard assay and may be recommended for screening and confirmation of SARS-CoV-2 in the field settings.

Presence of viral RNA of SARS-CoV-2 in conjunctival swab specimens of COVID-19 patients.

Purpose: To detect the presence of viral RNA of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in conjunctival swab specimens of coronavirus disease-19 (COVID-19) patients. Methods: Forty-five COVID-19 patients positive for real-time reverse transcription-polymerase chain reaction (RT-PCR) for SARS-CoV-2 in nasopharyngeal swab with or without ocular manifestations were included in the study. The conjunctival swab of each patient was collected by an ophthalmologist posted for COVID duty. Results: Out of 45 patients, 35 (77.77%) were males and the rest were females. The mean age was 31.26 ± 12.81 years. None of the patients had any ocular manifestations. One (2.23%) out of 45 patients was positive for RT-PCR SARS-CoV-2 in the conjunctival swab. Conclusion: This study shows that SARS-CoV-2 can be detected in conjunctival swabs of confirmed cases of COVID-19 patients. Though the positivity rate of detecting SARS-CoV-2 in conjunctival swabs is very less, care should be exercised during the ocular examination of patients of COVID-19.