Inter-laboratory testing as a strategy for external quality assessment for qualitative detection of SARS-CoV-2 by real-time RT-PCR testing in India.

To implement the strategy of test, track and treat to tackle the ongoing COVID-19 pandemic, the number of real-time RT-PCR-based testing laboratories was increased for diagnosis of SARS-CoV-2 in the country. To ensure reliability of the laboratory results, the Indian Council of Medical Research initiated external quality assessment (EQA) by deploying inter-laboratory quality control (ILQC) activity for these laboratories by nominating 34 quality control (QC) laboratories. This report presents the results of this activity for a period of September 2020 till November 2020. A total of 597 laboratories participated in this activity and 86 per cent of these scored ≥90 per cent concordance with QC laboratories. This ILQC activity showcased India's preparedness in quality diagnosis of SARS-CoV-2.

Nipah Virus Outbreak in Kerala State, India Amidst of COVID-19 Pandemic.

We report here a Nipah virus (NiV) outbreak in Kozhikode district of Kerala state, India, which had caused fatal encephalitis in a 12-year-old boy and the outbreak response, which led to the successful containment of the disease and the related investigations. Quantitative real-time reverse transcription (RT)-PCR, ELISA-based antibody detection, and whole genome sequencing (WGS) were performed to confirm the NiV infection. Contacts of the index case were traced and isolated based on risk categorization. Bats from the areas near the epicenter of the outbreak were sampled for throat swabs, rectal swabs, and blood samples for NiV screening by real-time RT-PCR and anti-NiV bat immunoglobulin G (IgG) ELISA. A plaque reduction neutralization test was performed for the detection of neutralizing antibodies. Nipah viral RNA could be detected from blood, bronchial wash, endotracheal (ET) secretion, and cerebrospinal fluid (CSF) and anti-NiV immunoglobulin M (IgM) antibodies from the serum sample of the index case. Rapid establishment of an onsite NiV diagnostic facility and contact tracing helped in quick containment of the outbreak. NiV sequences retrieved from the clinical specimen of the index case formed a sub-cluster with the earlier reported Nipah I genotype sequences from India with more than 95% similarity. Anti-NiV IgG positivity could be detected in 21% of Pteropus medius (P. medius) and 37.73% of Rousettus leschenaultia (R. leschenaultia). Neutralizing antibodies against NiV could be detected in P. medius. Stringent surveillance and awareness campaigns need to be implemented in the area to reduce human-bat interactions and minimize spillover events, which can lead to sporadic outbreaks of NiV.

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.

Performance assessment of seven SARS-CoV-2 IgG enzyme-linked immunosorbent assays.

The pandemic of COVID-19 has caused enormous fatalities worldwide. Serological assays are important for detection of asymptomatic or mild cases of COVID-19, and sero-prevalence and vaccine efficacy studies. Here, we evaluated and compared the performance of seven commercially available enzyme-linked immunosorbent assay (ELISA)s for detection of anti-severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) immunoglobulin G (IgG). The ELISAs were evaluated with a characterized panel of 100 serum samples from qRT-PCR confirmed COVID-19 patients, collected 14 days post onset disease, 100 SARS-CoV-2 negative samples and compared the results with that of neutralization assay. Results were analysed by creating the receiver operating characteristic curve of all the assays in reference to the neutralization assay. All kits, were found to be suitable for detection of IgG against SARS-CoV-2 with high accuracy. The DiaPro COVID-19 IgG ELISA showed the highest sensitivity (98%) among the kits. The assays demonstrated high sensitivity and specificity in detecting the IgG antibodies against SARS-CoV-2. However, the presence of IgG antibodies does not always correspond to neutralizing antibodies. Due to their good accuracy indices, these assays can also aid in tracing mild infections, in cohort studies and in pre-vaccine evaluations.

Clinical Characterization and Genomic Analysis of Samples from COVID-19 Breakthrough Infections during the Second Wave among the Various States of India.

From March to June 2021, India experienced a deadly second wave of COVID-19, with an increased number of post-vaccination breakthrough infections reported across the country. To understand the possible reason for these breakthroughs, we collected 677 clinical samples (throat swab/nasal swabs) of individuals from 17 states/Union Territories of the country who had received two doses (n = 592) and one dose (n = 85) of vaccines and tested positive for COVID-19. These cases were telephonically interviewed and clinical data were analyzed. A total of 511 SARS-CoV-2 genomes were recovered with genome coverage of higher than 98% from both groups. Analysis of both groups determined that 86.69% (n = 443) of them belonged to the Delta variant, along with Alpha, Kappa, Delta AY.1, and Delta AY.2. The Delta variant clustered into four distinct sub-lineages. Sub-lineage I had mutations in ORF1ab A1306S, P2046L, P2287S, V2930L, T3255I, T3446A, G5063S, P5401L, and A6319V, and in N G215C; Sub-lineage II had mutations in ORF1ab P309L, A3209V, V3718A, G5063S, P5401L, and ORF7a L116F; Sub-lineage III had mutations in ORF1ab A3209V, V3718A, T3750I, G5063S, and P5401L and in spike A222V; Sub-lineage IV had mutations in ORF1ab P309L, D2980N, and F3138S and spike K77T. This study indicates that majority of the breakthrough COVID-19 clinical cases were infected with the Delta variant, and only 9.8% cases required hospitalization, while fatality was observed in only 0.4% cases. This clearly suggests that the vaccination does provide reduction in hospital admission and mortality.

An Epidemiological Analysis of SARS-CoV-2 Genomic Sequences from Different Regions of India.

The number of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) cases is increasing in India. This study looks upon the geographic distribution of the virus clades and variants circulating in different parts of India between January and August 2020. The NPS/OPS from representative positive cases from different states and union territories in India were collected every month through the VRDLs in the country and analyzed using next-generation sequencing. Epidemiological analysis of the 689 SARS-CoV-2 clinical samples revealed GH and GR to be the predominant clades circulating in different states in India. The northern part of India largely reported the 'GH' clade, whereas the southern part reported the 'GR', with a few exceptions. These sequences also revealed the presence of single independent mutations-E484Q and N440K-from Maharashtra (first observed in March 2020) and Southern Indian States (first observed in May 2020), respectively. Furthermore, this study indicates that the SARS-CoV-2 variant (VOC, VUI, variant of high consequence and double mutant) was not observed during the early phase of virus transmission (January-August). This increased number of variations observed within a short timeframe across the globe suggests virus evolution, which can be a step towards enhanced host adaptation.

SARS-CoV-2 re-infection: development of an epidemiological definition from India.

The current investigation was conducted with the objective to develop an epidemiological case definition of possible severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) re-infection and assess its magnitude in India. The epidemiological case definition for SARS-CoV-2 re-infection was developed from literature review of data on viral kinetics. For achieving second objective, the individuals who satisfied the developed case definition for SARS-CoV-2 re-infection were contacted telephonically. Taking available evidence into consideration, re-infection with SARS-CoV-2 in our study was defined as any individual who tested positive for SARS-CoV-2 on two separate occasions by either molecular tests or rapid antigen test at an interval of at least 102 days with one negative molecular test in between. In this archive based, telephonic survey, 58 out of 1300 individuals (4.5%) fulfilled the above-mentioned definition; 38 individuals could be contacted with healthcare workers (HCWs) accounting for 31.6% of the cases. A large proportion of participants was asymptomatic and had higher Ct value during the first episode. While SARS-CoV-2 re-infection is still a rare phenomenon, there is a need for epidemiological definition of re-infection for establishing surveillance systems and this study contributes to such a goal.Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) re-infection is an emerging concern and there is a need to define it. Therefore, working epidemiological case definition for re-infection was developed and its magnitude was explored via archive-based, telephonic survey. Re-infection with SARS-CoV-2 was defined as two positive tests at an interval of at least 102 days with one interim negative test. Thirty-eight of the 58 eligible patients could be contacted with 12 (31.6%) being HCWs. Majority of the participants were asymptomatic and had higher Ct value during their first episode. To conclude, a working epidemiological case definition of SARS-CoV-2 re-infection is important to strengthen surveillance. The present investigation contributes to this goal and records reinfection in 4.5% of SARS-CoV-2 infected individuals in India.

Pooled testing for COVID-19 diagnosis by real-time RT-PCR: A multi-site comparative evaluation of 5- & 10-sample pooling.

BACKGROUND & OBJECTIVES: Public health and diagnostic laboratories are facing huge sample loads for COVID-19 diagnosis by real-time reverse transcription-polymerase chain reaction (RT-PCR). High sensitivity of optimized real-time RT-PCR assays makes pooled testing a potentially efficient strategy for resource utilization when positivity rates for particular regions or groups of individuals are low. We report here a comparative analysis of pooled testing for 5- and 10-sample pools by real-time RT-PCR across 10 COVID-19 testing laboratories in India. METHODS: Ten virus research and diagnostic laboratories (VRDLs) testing for COVID-19 by real-time RT-PCR participated in this evaluation. At each laboratory, 100 nasopharyngeal swab samples including 10 positive samples were used to create 5- and 10-sample pools with one positive sample in each pool. RNA extraction and real-time RT-PCR for SARS-CoV-2-specific E gene target were performed for individual positive samples as well as pooled samples. Concordance between individual sample testing and testing in the 5- or 10-sample pools was calculated, and the variation across sites and by sample cycle threshold (Ct) values was analyzed. RESULTS: A total of 110 each of 5- and 10-sample pools were evaluated. Concordance between the 5-sample pool and individual sample testing was 100 per cent in the Ct value ≤30 cycles and 95.5 per cent for Ctvalues ≤33 cycles. Overall concordance between the 5-sample pooled and individual sample testing was 88 per cent while that between 10-sample pool and individual sample testing was 66 per cent. Although the concordance rates for both the 5- and 10-sample pooled testing varied across laboratories, yet for samples with Ct values ≤33 cycles, the concordance was ≥90 per cent across all laboratories for the 5-sample pools. INTERPRETATION & CONCLUSIONS: Results from this multi-site assessment suggest that pooling five samples for SARS-CoV-2 detection by real-time RT-PCR may be an acceptable strategy without much loss of sensitivity even for low viral loads, while with 10-sample pools, there may be considerably higher numbers of false negatives. However, testing laboratories should perform validations with the specific RNA extraction and RT-PCR kits in use at their centres before initiating pooled testing.

Laboratory preparedness for SARS-CoV-2 testing in India: Harnessing a network of Virus Research & Diagnostic Laboratories.

Background & objectives: An outbreak of respiratory illness of unknown aetiology was reported from Hubei province of Wuhan, People's Republic of China, in December 2019. The outbreak was attributed to a novel coronavirus (CoV), named as severe acute respiratory syndrome (SARS)-CoV-2 and the disease as COVID-19. Within one month, cases were reported from 25 countries. In view of the novel viral strain with reported high morbidity, establishing early countrywide diagnosis to detect imported cases became critical. Here we describe the role of a countrywide network of VRDLs in early diagnosis of COVID-19. Methods: The Indian Council of Medical Research (ICMR)-National Institute of Virology (NIV), Pune, established screening as well as confirmatory assays for SARS-CoV-2. A total of 13 VRDLs were provided with the E gene screening real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay. VRDLs were selected on the basis of their presence near an international airport/seaport and their past performance. The case definition for testing included all individuals with travel history to Wuhan and symptomatic individuals with travel history to other parts of China. This was later expanded to include symptomatic individuals returning from Singapore, Japan, Hong Kong, Thailand and South Korea. Results: Within a week of standardization of the test at NIV, all VRDLs could initiate testing for SARS-CoV-2. Till February 29, 2020, a total of 2,913 samples were tested. This included both 654 individuals quarantined in the two camps and others fitting within the case definition. The quarantined individuals were tested twice - at days 0 and 14. All tested negative on both occasions. Only three individuals belonging to different districts in Kerala were found to be positive. Interpretation & conclusions: Sudden emergence of SARS-CoV-2 and its potential to cause a pandemic posed an unsurmountable challenge to the public health system of India. However, concerted efforts of various arms of the Government of India resulted in a well-coordinated action at each level. India has successfully demonstrated its ability to establish quick diagnosis of SARS-CoV-2 at NIV, Pune, and the testing VRDLs.