An outbreak of acute hemorrhagic conjunctivitis due to Coxsackievirus A24 in a residential school, Naharlagun, Arunachal Pradesh: July 2023.

PURPOSE: An acute conjunctivitis outbreak was investigated at a residential school in Naharlagun, Arunachal Pradesh, Northeast India, in July 2023. We aimed to identify the etiological agent and assess any complications in follow-up cases. METHODS: We used a structured questionnaire to record clinical findings and followed up with cases one-month post-conjunctivitis. Sixty-one cases were examined and eight conjunctival and oropharyngeal swab samples were collected after obtaining informed consent from guardians/school authorities. We screened for 33 viral and bacterial pathogens using an IVD-approved Real-time PCR assay. Further, the samples were subjected to nucleic acid sequencing. RESULTS: Among 465 screened students and staff, 80 individuals (approximately 17.2%) showed acute hemorrhagic conjunctivitis symptoms among which 61 cases were available for clinical examination. We identified the Enterovirus responsible by targeted sequencing using next-generation sequencing. The etiological agent was found to be Coxsackievirus A24, a member of Enterovirus C, in seven out of eight samples subjected to sequencing. Common symptoms included conjunctival hyperemia and foreign body sensation (100%), bilateral eye involvement (73.8%), eye pain (70%), watery discharge (49.2%), and eyelid swelling (38%). Only 6.5% had purulent discharge. Most cases resolved within 5-6 days, with only 9.8% reporting abdominal symptoms post-conjunctivitis. No serious complications occurred within one month. Throat swabs aided in diagnosing enterovirus infections alongside eye swabs. CONCLUSIONS: The outbreak of acute conjunctivitis was caused by Coxsackievirus A24, a member of Enterovirus C. Cases resolved spontaneously within 6-7 days, with no severe complications. Collecting oropharyngeal swabs alongside conjunctival swabs could improve enteroviral conjunctivitis diagnosis.

First isolation and characterization of SARS-CoV-2 from COVID-19 patient of North East India.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) responsible for the current pandemic has resulted in over 5 million deaths globally. More than a year has passed, still SARS-CoV-2 panic the public life. Virus isolation is of paramount importance for development of vaccines, in-vitro screening of antiviral compounds, pathogenesis studies, etc., Many cell lines were studied for amplification and replication of SARS-CoV-2 and Vero cells were found to be ideal cell lines for isolation. In May 2020, ICMR-Regional Medical Research Centre, NE region, India, successfully established the SARS-CoV-2 culture system in Vero CCL-81 cell lines. Phylogenetic analyses of the whole genome sequences of the SARS-CoV-2 isolate (EPI_ISL_2501532 | 2020-05-19) showed monophyletic clade G and lineage B.1.1.

Presence of fimH and iss type 1, 2 and 3 genes in uropathogenic Escherichia coli isolates recovered from an apex medical institute in North India.

PURPOSE: To detect the presence of fimH and iss type 1, 2 and 3 genes in uropathogenic Escherichia coli (UPEC) isolates recovered from patients coming to the out patient department (OPD) of our hospital. METHODS: E. coli isolates recovered from patients who had symptoms of urinary tract infection (UTI) were processed for the presence of fimH and iss genes. DNA was extracted using an in house method after which conventional PCR using forward and reverse primers targeting the four genes was carried out. The amplified products were electrophoresed and visualized in a gel documentation imager. Relevant demographic details of the patients were recorded on a pre-designed pro-forma and antimicrobial susceptibility testing of the isolates was done by disc diffusion method. RESULTS: fimH was present in 87.5% of UPEC isolates whereas iss type 1 was seen in 7.3%, type 2 in 4.2% and iss type 3 in 71.9% isolates. Age of the patients ranged from 3 months to 82 â€‹yrs (mean 43.5 SD â€‹± â€‹18.20). UTI was more common in females (60.2%) as compared to males patients (39.8%). Dysuria (66.7%) was the most common symptom in the studied subjects and diabetes mellitus (42.6%) the most common co-morbidity. A total of 56.5% patients gave a history of prior antibiotic intake. The UPEC isolates were resistant to most of the antibiotics tested. However all the isolates were sensitive to polymyxin B and colistin. Fosfomycin resistance was seen in 9.5% of the UPEC isolates harbouring fimH gene. CONCLUSION: This is the first study that highlights the presence of iss type 3 gene in UPEC isolates along with the fimH and iss type 1 and 2 genes. The results of this study can serve as a stepping stone for future in depth research into the significance of the iss genes in causing UTI.

Pan-India influenza-like illness (ILI) and Severe acute respiratory infection (SARI) surveillance: epidemiological, clinical and genomic analysis.

Background: Over time, COVID-19 testing has significantly declined across the world. However, it is critical to monitor the virus through surveillance. In late 2020, WHO released interim guidance advising the use of the existing Global Influenza Surveillance and Response System (GISRS) for the integrated surveillance of influenza and SARS-CoV-2. Methods: In July 2021, we initiated a pan-India integrated surveillance for influenza and SARS-CoV-2 through the geographically representative network of Virus Research and Diagnostic Laboratories (VRDLs) across 26 hospital and laboratory sites and 70 community sites. A total of 34,260 cases of influenza-like illness (ILI) and Severe acute respiratory infection (SARI) were enrolled from 4 July 2021 to 31 October 2022. Findings: Influenza A(H3) and B/Victoria dominated during 2021 monsoon season while A(H1N1)pdm09 dominated during 2022 monsoon season. The SARS-CoV-2 "variants of concern" (VoC) Delta and Omicron predominated in 2021 and 2022, respectively. Increased proportion of SARI was seen in extremes of age: 90% cases in < 1 year; 68% in 1 to 5 years and 61% in ≥ 8 years age group. Approximately 40.7% of enrolled cases only partially fulfilled WHO ILI and SARI case definitions. Influenza- and SARS-CoV-2-infected comorbid patients had higher risks of hospitalization, ICU admission, and oxygen requirement. Interpretation: The results depicted the varying strains and transmission dynamics of influenza and SARS-CoV-2 viruses over time, thus emphasizing the need to continue and expand surveillance across countries for improved decision making. The study also describes important information related to clinical outcomes of ILI and SARI patients and highlights the need to review existing WHO ILI and SARI case definitions.

First Report of Rubber Collection Bowls & Plastic and Bamboo Water Containers as the Major Breeding Source of Ae. albopictus with the Indigenous Transmission of Dengue and Chikungunya in Rural Forested Malaria-Endemic Villages of Dhalai District, Tripura, India: The Importance of Molecular Identification.

BACKGROUND: With the reports of indigenous cases of dengue and chikungunya in the forest-covered rural tribal malaria-endemic villages of Dhalai District, Tripura, India, an exploratory study was undertaken to identify the vector breeding sites. METHODS: From June 2021 to August 2022, mosquito larvae were collected from both natural and artificial sources in the villages, house premises, and their nearby forested areas outside of the houses. Other than morphological characterisation, Aedes species were confirmed by polymerase chain reaction targeting both nuclear (ITS2) and mitochondrial genes (COI) followed by bidirectional Sanger sequencing. RESULTS: Aedes albopictus was abundantly found in this area in both natural and artificial containers, whereas Ae. aegypti was absent. Among the breeding sources of molecularly confirmed Ae. albopictus species, rubber collection bowls were found to be a breeding source reported for the first time. Plastic and indigenously made bamboo-polythene containers for storing supply water and harvesting rainwater in the villages with a shortage of water were found to be other major breeding sources, which calls for specific vector control strategies. Natural sources like ponds and rainwater collected on Tectona grandis leaves and Colocasia axil were also found to harbour the breeding, along with other commonly found sources like bamboo stumps and tree holes. No artificial containers as a breeding source were found inside the houses. Mixed breeding was observed in many containers with other Aedes and other mosquito species, necessitating molecular identification. We report six haplotypes in this study, among which two are reported for the first time. However, Aedes aegypti was not found in the area. Additionally, rubber collection bowls, ponds, and water containers also showed the presence of Culex quinquefasciatus and Culex vishnui, known JE vectors from this area, and reported JE cases as well. Different Anopheles vector spp. from this known malaria-endemic area were also found, corroborating this area as a hotbed of several vectors and vector-borne diseases. CONCLUSIONS: This study, for the first time, reports the breeding sources of Aedes albopictus in the forested areas of Tripura, with rubber collection bowls and large water storage containers as major sources. Also, for the first time, this study reports the molecular characterisation of the Ae. albopictus species of Tripura, elucidating the limitations of morphological identification and highlighting the importance of molecular studies for designing appropriate vector control strategies. The study also reports the co-breeding of JE and malaria vectors for the first time in the area reporting these vector-borne diseases.

Norovirus gastroenteritis in children under-five years hospitalized for diarrhea in two cities of northeast India: A retrospective study.

PURPOSE: Norovirus gastroenteritis, known to cause 'winter vomiting disease' is increasingly being identified as a major cause of viral gastroenteritis worldwide. The impact and prevalence of this viral disease are lacking in many parts of India including northeast India. This study aimed to determine the prevalence and association of norovirus gastroenteritis among under-five-year-old hospitalized children in two cities in northeast India (Dibrugarh in Assam & Dimapur in Nagaland). MATERIALS AND METHODS: A retrospective analysis of 407 randomly selected diarrheal stool samples was conducted using a commercial multiplex probed-based real-time RT-PCR assay capable of detecting six-viral gastroenteritis pathogens including Norovirus GI, Norovirus GII, Rotavirus, Human Adenovirus, Human Astrovirus, and Sapovirus. RESULTS: Results showed that norovirus was detected in 18.4% of the samples (75/407; 95% CI: 14.8%-22.5%), with norovirus genogroup II being the predominant group in 97.3% of norovirus cases. A significant association of norovirus diarrhea was found with seasonality, with higher prevalence in colder months compared to warmer months (22.4% vs 9.1%, p-value:0.002). Additionally, 66.7% (50/75) of cases of norovirus gastroenteritis had reported vomiting as the major symptom and had a shorter duration of diarrhea (p-value 0.03). Co-infections with other viral pathogens were seen in 45.9% (187/407) of the cases. The detection of rotavirus was 67.1% (273/407), human adenovirus (45.9%; 187/407), sapovirus and astrovirus (5.9%, 24/407 each), and norovirus GI (0.5%, 2/407) among the cases. CONCLUSION: This study reports the prevalence of norovirus gastroenteritis in northeast India and further highlights that norovirus gastroenteritis is responsible for substantial cases of hospitalization of under-five years children in the region.

Rapid diagnosis of COVID-19 using disposal paper capacitive sensor.

Accurate and rapid detection and isolation become indispensable to restrict the spread of COVID-19. Since the start of COVID-19 pandemic in December 2019, many indisposal diagnostic tools are being developed incessantly. Out of all presently used tools, the gold standard rRT- PCR tool having very high sensitivity and specificity is a time consuming complicated molecular technique having requirements of special expensive equipment. Here, the main focus of this work is to develop rapid disposal paper capacitance sensor having simple and easy detection. We discovered a strong interaction between limonin and Spike-glycoprotein of SARS-COV-2 in comparison to its interaction with other similar viruses such as HCOV-OC43, HCOV-NL63, HCOV-HKU1, Influenza B and A viruses. The antibody free capacitive sensor having comb electrode structure was fabricated on whatman paper with drop coating of limonin (extracted using green method from pomelo seeds) and calibrated with known swab samples. The Blind test with unknown swab samples shows high sensitivity of 91.5% and high specificity of 88.37%. Requiring low sample volume and detection time and using biodegradable materials in the sensor fabrication assure the potential application as a point of care disposal diagnostic tool.

Co-circulation of two Mumps virus genotypes in Assam, India.

Mumps is a vaccine-preventable disease, and research on the vaccine's efficacy has recently indicated declining efficacy that has failed to protect against primary infections or reinfections, leading to a global resurgence in nations that use mumps vaccine in their national immunization programmes (NIPs). Lack of reports on its infection, documentation and published studies prevents it from being recognized as a public health issue in India. The waning of immunity is ascribed to the changes between the circulating and vaccine strains. The goal of the current study was to describe the circulating MuV strains in the Dibrugarh district of Assam, India, from 2016 to 2019. Blood samples were examined for IgM antibodies, and throat swab samples were put through Taqman assay for molecular detection. The small hydrophobic (SH) gene was targeted for genotyping through sequencing, and its genetic variations and phylogenetic analysis were carried out. Mumps RNA was found in 42 cases, and Mumps IgM in 14, of which 60% (25/42) of the cases were male and 40% (17/42) were female mostly affecting children between the ages of 6 and 12. Sequence and phylogeny analyses of SH gene revealed Genotypes C (83%) and G (17%) were simultaneously circulating during the study period. The study offers crucial genetic baseline information for the creation of Mumps prevention and control measures. Therefore, based on the research, it is clear that developing an effective vaccination strategy should take into account all currently prevalent genotypes in order to provide better protection against the disease's comeback.

Role of Innate Immune Regulatory Genes, FOXP3 and FOS in Chronic Hepatitis B Infection.

Persistence of hepatitis B virus (HBV) infection leading to chronic infection and its sequalae is responsible for over half a million deaths worldwide. The reason for persistence of chronic hepatitis B (CHB) infection is still not clearly understood. An attempt was made to understand the role of immune regulatory genes in CHB in comparison to spontaneously cleared HBV infection. Relative gene expression of 26 genes involved in innate immunity were studied using Real-Time Polymerase Chain Reaction Array. A total of 679 subjects from three different geographical regions of Northeast India (Assam, Arunachal Pradesh, and Tripura) were included in this case-control study. The cases were subdivided into CHB cases with HBeAg(+)(72), CHB with HBeAg(-)(278), spontaneously cleared controls (88), and healthy controls (228). Overall, 28.3% of the subjects had previous exposure with HBV, while 28.6% had protective antibodies IgG/IgM against HBV. There was a statistically higher number of CHB in men (66.4%) compared to women (33.6%) (p = 0.0001). Proto-oncogene FOS has been found to be moderately upregulated in CHB with HBeAg +ve (2.3-fold) and significantly upregulated (4.1-fold upregulation) in hepatocellular carcinoma. Further, FOXP3 was found to be significantly upregulated (3.0-fold, p = 0.01) in CHB with HBeAg (+) compared to spontaneously cleared HBV infection. In conclusion, CHB with HBeAg positivity was found to have disrupted immune response with upregulation of FOS and FOXP3. Thus, early induction of HBeAg seroconversion with interferon-based therapy or oral nucleos(t)ide analogs along with FOS inhibitors can have important clinical implications in the management of CHB and preventing cirrhosis and HCC.

SARS-CoV-2 and Influenza Virus Co-Infection Cases Identified through ILI/SARI Sentinel Surveillance: A Pan-India Report.

SARS-CoV-2/influenza virus co-infection studies have focused on hospitalized patients who usually had grave sequelae. Here, we report SARS-CoV-2/influenza virus co-infection cases from both community and hospital settings reported through integrated ILI/SARI (Influenza Like Illness/Severe Acute Respiratory Infection) sentinel surveillance established by the Indian Council of Medical Research. We describe the disease progression and outcomes in these cases. Out of 13,467 samples tested from 4 July 2021-31 January 2022, only 5 (0.04%) were of SARS-CoV-2/influenza virus co-infection from 3 different sites in distinct geographic regions. Of these, three patients with extremes of age required hospital admission, but none required ICU admission or mechanical ventilation. No mortality was reported. The other two co-infection cases from community settings were managed at home. This is the first report on SARS-CoV-2/Influenza virus co-infection from community as well as hospital settings in India and shows that influenza viruses are circulating in the community even during COVID-19. The results emphasize the need for continuous surveillance for multiple respiratory pathogens for effective public health management of ILI/SARI cases in line with the WHO (World Health Organization) recommendations.

T cell responses in symptomatic moderate patients with pandemic 2009 H1N1 influenza A virus infection.

The response of the host immune system should be appropriate to fight against pandemic 2009 H1N1 (pH1N1) influenza A virus without causing damage to its self. T cells play an indispensable role in the fight against the virus, but have the potential to cause host immunopathological changes. A better understanding of the immunoregulation that occurs during pH1N1 infection is necessary for preventing severity of the disease. In this study, we found that a significantly higher percentage of Vδ1+ T cells and increased expression of activation markers in total T cells in patients with moderate pH1N1 infection could lead to its efficient fight against the virus. On the other hand, the percentages of total and CD4+ T cells were decreased along with an increased expression of exhaustion marker-Tim-3 on T cells that might suppress excessive T cell responses in the host. This tuning of T cell responses might be necessary in efficient combat against pH1N1 virus, without aggravating T cell mediated immunopathology in patients with moderate pH1N1-infection. Keywords: pH1N1; T cells; activation; exhaustion; Tim-3.

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.

Characteristics of Circulatory γδ T cells in Patients with Symptomatic Chronic Hepatitis B Infection.

Hepatitis B is a viral infection that can cause serious liver disease. Chronic hepatitis B (CHB) infection places individuals at higher risk of developing cirrhosis of the liver and hepatocellular cancer. Immune dysfunction, including altered distribution and functional status of T cell immunity, is a contributor to hepatitis B virus (HBV) pathogenesis. In this study, we examined the distribution of circulating γδ T cell subpopulations and levels of cell surface expression of suppressive markers on γδ T cells in individuals with CHB infection and clinical liver disease. A significantly higher proportion of terminally differentiated (TEMRA) (CD27-CD45RA+) γδ T cells along with significantly lower percentages of central memory (CD27+CD45RA-) and effector memory (CD27-CD45RA-) γδ T cells were observed in peripheral blood of these individuals. The expression of exhaustion markers-Tim-3 and Lag-3 was elevated in γδ T cells from CHB-infected individuals compared with healthy controls (HC) and blockade of these exhaustion markers resulted in restoration of interferon gamma (IFN-γ) secretion by γδ T cells. In addition, γδ T cells from CHB patients expressed increased levels of CD69, another important regulator of immune responses. Together, these results suggest that CHB patients with clinical sign of liver disease have TEMRA γδ T cells with a potentially exhausted phenotype that may in turn impair their immunoregulatory role and facilitate pathogenesis of CHB disease.

Haemophilus influenzae and SARS-CoV-2: Is there a role for investigation?

During the current pandemic of COVID-19, the authors observed that during screening test for SARS-CoV-2 targeting the E-gene by qRT-PCR, few nasopharyngeal/oropharyngeal samples showed amplification signals at late cycle threshold (CT-value) > 35 despite being negative for other confirmatory target genes. Thirty such samples (taken as cases) showing detectable CT of > 35 cycle in E-gene which were negative for other target genes of SARS-CoV-2 and 30 samples with undetectable fluorescence in E-gene were taken as controls for investigation. An in-vitro diagnostic approved commercial qRT-PCR multiplex kit detecting 33 respiratory pathogens which can also detect Haemophilus influenzae was used for screening the samples. It was observed that out of the 30 samples showing detectable CT> 35 in E-gene, 11 samples were positive for Haemophilus influenzae whereas in the controls only three samples were positive for H. influenzae (p-value: 0.03) which was statistically significant. Further, the probes and primers were screened against H. influenzae for matches in the genome. It was observed that all primers and probes for the E-gene of SARS-CoV-2 had over 13 bp long sequences matching 100% with multiple sites across the H. influenzae genome. This qRT-PCR primer & probes are being used extensively across India, and laboratories using them should be aware of the cross-reactivity of primers & probes with the H. influenzae genome. Further, the authors observed that 95.9% (5415/5642) of COVID-19 positive cases detected in their laboratory were asymptomatic at the time of collection of samples. This warrants further investigations.

TSP-based PCR for rapid identification of L and S type strains of SARS-CoV-2.

BACKGROUND: In the initial few months of the COVID-19 pandemic, two distinct strains of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) were identified (L and S strain) based on a tightly linked SNP between two widely separated nucleotides at location 8782 (ORF1ab T8517C) and position 28,144 (ORF8: C251T, codon S84L). MATERIALS AND METHODS: A Type Specific Primer based one step RT-PCR (TSP-PCR) test to distinguish the L and S type strains of SARS-CoV-2 without the need for viral genome sequencing, was developed. The study also analyzed 18,221 whole genome sequences (WGS) available up to April 2020 to know the prevalence of L and S type of strains. Phylogenetic and recombination analysis of SARS-CoV-2 genome with nearest animal and human coronaviruses were analyzed using MEGA X and SimPlot version 3.5.1 software respectively. RESULTS: The rapid TSP-PCR distinguished the L and S type strains of SARS-CoV-2 by amplifying a specific 326 bp and 256 bp fragment of the L and S type strain respectively. The test was used to analyzed 120 random SARS-CoV-2 positive samples from Assam, India among which 118 were found to be of L-type strains only. On analysis of 18,221 WGS, it was found that L type was the predominant strain with an overall prevalence ∼90%. However, pockets of high prevalence of S-type strains (>35%) were still in circulation in Washington region in April 2020. The study did not detect any significant recombination events between closely related coronavirus and SARS-CoV-2. CONCLUSION: TSP-based PCR for identification of circulating strains of SARS-CoV-2, will add in rapid identification of strains of COVID-19 pandemic to understand the spread of the virus, its transmissibility and adaptation into human population. Though, the S-type strains have decreased drastically across the globe since April 2020, the role of TSP-PCR in geographical niches where such strains are still prevalent may help in rapidly distinguishing the strains and study its evolution.

Epidemiology of rotavirus diarrhea among children less than 5 years hospitalized with acute gastroenteritis prior to rotavirus vaccine introduction in India.

BACKGROUND: Rotavirus is an important cause of severe diarrhea requiring hospitalization, accounting for approximately 78,000 deaths annually in Indian children below 5 years of age. We present epidemiological data on severe rotavirus disease collected during hospital-based surveillance in India before the introduction of the oral rotavirus vaccine into the national immunization schedule. METHODS: The National Rotavirus Surveillance Network was created involving 28 hospital sites and 11 laboratories across the four geographical regions of India. From September 2012 to August 2016 children less than 5 years of age hospitalized for diarrhea for at least 6 h, were enrolled. After recording clinical details, a stool sample was collected from each enrolled child, which was tested for rotavirus antigen using enzyme immunoassay (EIA). Nearly 2/3rd of EIA positive samples were genotyped using reverse transcription polymerase chain reaction to identify the G and P types. RESULTS: Of the 21,421 children enrolled during the 4 years surveillance, 36.3% were positive for rotavirus. The eastern region had the highest proportion of rotavirus associated diarrhea (39.8%), while the southern region had the lowest (33.8%). Rotavirus detection rates were the highest in children aged 6-23 months (41.8%), and 24.7% in children aged < 6 months. Although rotavirus associated diarrhea was seen throughout the year, the highest positivity was documented between December and February across all the regions. The most common rotavirus genotype was G1P[8] (52.9%), followed by G9P4 (8.7%) and G2P4 (8.4%). CONCLUSIONS: There is high burden of rotavirus gastroenteritis among Indian children below 5 years of age hospitalized for acute diarrhea thereby highlighting the need for introduction of rotavirus vaccine into the national immunization program and also for monitoring circulating genotypes.

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.