Determination of a cut-off value for the serological diagnosis of scrub typhus by detecting anti-Orientia tsutsugamushi immunoglobulin M.

Background & objectives: The diagnosis of scrub typhus (ST) is usually done using enzyme-linked immunosorbent assay (ELISA) due to its ease of performance and reading objectivity. The cut-off value for ELISA needs to be calculated for each geographical location as it depends on zonal endemicity of the disease. This study was, therefore, undertaken to calculate the pan-India cut-off for anti-Orientia tsutsugamushi (OT) immunoglobulin M (IgM) by ELISA. Methods: Samples from cases (cases of ST) and controls (voluntary, consenting, healthy adults) were collected by a network of 29 laboratories across India and tested for anti-OT IgM by immunofluorescence assay (IFA), the considered gold standard test. These samples were retested by ELISA for anti-OT IgM and their optical densities (ODs) were used for cut-off estimation by receiver operating characteristic (ROC) curve. Results: Anti-OT IgM ELISA ODs from 273 controls and 136 cases were used for the cut-off estimation. The ODs of the anti-OT IgM ELISA on healthy individuals and those of confirmed ST cases ranged from 0.1 to 0.75 and 0.5 to 4.718, respectively. ROC curve-based cut-off for ELISA was calculated as 0.554 at a sensitivity of 95.2 per cent and specificity of 95.1 per cent. A value of >1 was noted to have a specificity of 100 per cent in diagnosing ST. Interpretation & conclusions: The cut-off calculated for India was similar to the previous cut-off that was used until now.

Detection and isolation of SARS-CoV-2 Eta variant from the international travelers and local residents of India.

International travel has been the major source for the rapid spread of new SARS-CoV-2 variants across the globe. During SARS-CoV-2 genomic surveillance, a total of 212 SARS-CoV-2 positive clinical specimens were sequenced using next-generation sequencing. A complete SARS-CoV-2 genome could be retrieved from 90 clinical specimens. Of them, 14 sequences belonged to the Eta variant from clinical specimens of international travelers (n = 12) and local residents (n = 2) of India, and 76 belonged to other SARS-CoV-2 variants. Of all the Eta-positive specimens, the virus isolates were obtained from the clinical specimens of six international travelers. Many variants of interest have been found to cause substantial community transmission or cluster infections. The detection of this variant with lethal E484K mutation across the globe and India necessitates persistent genomic surveillance of the SARS-CoV-2 variants, which would aid in taking preventive action.

Expansion of the measles and rubella laboratory network, India.

Objective: To expand the measles and rubella laboratory network of India by integrating new laboratories. Methods: In collaboration with the World Health Organization (WHO), the Indian government developed a 10-step scheme to systematically expand the number of laboratories performing serological and molecular testing for measles and rubella. The Indian Council of Medical Research and WHO identified suitable laboratories based on their geographical location, willingness, preparedness, past performance and adherence to national quality control and quality assurance mechanisms. The 10-step scheme was initiated with training on measles and rubella diagnostic assays followed by testing of both measles and rubella serology and molecular unknown panels, cross-verification with reference laboratories and ended with WHO on-site accreditation. Findings: After extensive training, technical support, funding and monitoring, all six selected laboratories attained passing scores of 90.0% or more in serological and molecular proficiency testing of measles and rubella. Since 2018, the laboratories are a part of the measles and rubella network of India. Within 12 months of initiation of independent reporting, the six laboratories have tested 2287 serum samples and 701 throat or nasopharyngeal swabs or urine samples. Conclusion: The process led to strengthening and expansion of the network. This proficient laboratory network has helped India in scaling up serological and molecular testing of measles and rubella while ensuring high quality testing. The collaborative model developed by the Indian government with WHO can be implemented by other countries for expanding laboratory networks for surveillance of measles and rubella as well as other infectious diseases.

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.

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.

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.

Molecular characterization of influenza A(H1N1)pdm09 viruses circulating at various geographical locations in India, 2017.

Background & objectives: Influenza virological surveillance is an essential tool for the early detection of novel genetic variants of epidemiologic and clinical significance. This study was aimed to genetically characterize A(H1N1)pdm09 virus circulating in 2017 and to compare it with the global data. Methods: The regional/State Viral Research and Diagnostic Laboratories (VRDLs) provided influenza diagnosis for referred clinical samples and shared influenza A(H1N1)pdm09 positives with the Indian Council of Medical Research-National Institute of Virology (ICMR-NIV), Pune, India, for hemagglutinin (HA) gene phylogenetic analysis. Sites at Manipal, Jaipur and Dibrugarh performed the sequencing and shared the sequence data for analysis. The antiviral susceptibility of influenza viruses was assessed for known molecular marker H275Y at the ICMR-NIV, Pune. Results: All the eight VRDLs had well-established influenza diagnostic facilities and showed increased activity of influenza A(H1N1)pdm09 during 2017. Phylogenetic analysis showed that the viruses from the different regions of the country were similar to A/Michigan/45/2015 strain which was the 2017-2018 recommended vaccine strain and were clustered with the globally circulating clade 6B.1 with signature mutations S84N, S162N and I216T. The clade 6B.1 showed further subgrouping with additional mutations S74R, S164T and I295V; however, there was no significant association between the presence of these mutations and severity of disease due to influenza. All the study viruses were sensitive to oseltamivir. Interpretation & conclusions: During the study period, all the study sites reported globally circulating A/Michigan/45/2015 vaccine strain of influenza A(H1N1)pdm09 viruses and remained sensitive to oseltamivir. Further genetic and antigenic characterization of influenza viruses is recommended to address public health concerns.

Geographical distribution of primary & secondary dengue cases in India - 2017: A cross-sectional multicentric study.

Background & objectives: Dengue virus infection is endemic in India with all the four serotypes of dengue virus in circulation. This study was aimed to determine the geographic distribution of the primary and secondary dengue cases in India. Methods: A multicentre cross-sectional study was conducted at Department of Health Research / Indian Council of Medical Research (DHR)/(ICMR) viral research and diagnostic laboratories (VRDLs) and selected ICMR institutes located in India. Only laboratory-confirmed dengue cases with date of onset of illness less than or equal to seven days were included between September and October 2017. Dengue NS1 antigen ELISA and anti-dengue IgM capture ELISA were used to diagnose dengue cases while anti-dengue IgG capture ELISA was used for identifying the secondary dengue cases. Results: Of the 1372 dengue cases, 897 (65%) were classified as primary dengue and 475 (35%) as secondary dengue cases. However, the proportion varied widely geographically, with Theni, Tamil Nadu; Tirupati, Andhra Pradesh and Udupi-Manipal, Karnataka reporting more than 65 per cent secondary dengue cases while Srinagar, Jammu and Kashmir reporting as low as 10 per cent of the same. The median age of primary dengue cases was 25 yr [interquartile range (IQR 17-35] while that of secondary dengue cases was 23 yr (IQR 13.5-34). Secondary dengue was around 50 per cent among the children belonging to the age group 6-10 yr while it ranged between 20-43 per cent among other age groups. Interpretation & conclusions: Our findings showed a wide geographical variation in the distribution of primary and secondary dengue cases in India. It would prove beneficial to include primary and secondary dengue differentiation protocol in the national dengue surveillance programme.

Approach to fever in children.

BACKGROUND: Fever in children is one of the most common reasons for outpatient visits as well as in-patient evaluation, often causing anxiety among parents and caregivers. Fever can be a standalone feature or be associated with other localising symptoms and signs like rash, lymphadenopathy, or any other organ system involvement with or without a focus of infection. The etiologies of fever vary depending on the clinical setting and epidemiology. India being a tropical country, sees a distinct spectrum of tropical infections. Physicians need to stay updated on the prevalent diseases in their region and the unique factors that may influence the clinical presentations and course of fever in the cohort of children they manage. The challenge lies in balancing the benefit of early treatment for severe diseases versus the harms of unnecessary investigations and treatment for self-resolving illnesses. OBJECTIVES: This review aims to provide a comprehensive overview of fever in children, covering its etiology, clinical features, and management strategies. This review offers an algorithmic approach to fever tailored to the Indian setting to guide physicians in identifying the disease based on clinical symptoms and signs, ordering essential laboratory investigations, and initiating appropriate management promptly. CONTENT: The review categorises fever into various segments like fever with localising signs like rash, lymphadenopathy, fever due to infection localised to a particular organ system, and fever without a focus including fever of unknown origin. It delves into the diverse etiological factors contributing to fever in each of these categories, encompassing infectious and non-infectious origins. It gives pointers to identify the etiology from history, examination, and confirm them with judicious use of diagnostic investigations with emphasis on identifying the red flag signs that require immediate attention, especially in vulnerable groups like neonates and young infants.

Approach towards surveillance-based diagnosis of acute respiratory illness in India: Expert recommendations.

BACKGROUND: Emerging infectious diseases, often zoonotic, demand a collaborative "One-Health" surveillance approach due to human activities. The need for standardized diagnostic and surveillance algorithms is emphasized to address the difficulty in clinical differentiation and curb antimicrobial resistance. OBJECTIVE: The present recommendations are comprehensive diagnostic and surveillance algorithm for ARIs, developed by the Indian Council of Medical Research (ICMR), which aims to enhance early detection and treatment with improved surveillance. This algorithm shall be serving as a blueprint for respiratory infections landscape in the country and early detection of surge of respiratory infections in the country. CONTENT: The ICMR has risen up to the threat of emerging and re-emerging infections. Here, we seek to recommend a structured approach for diagnosing respiratory illnesses. The recommendations emphasize the significance of prioritizing respiratory pathogens based on factors such as the frequency of occurrence (seasonal or geographical), disease severity, ease of diagnosis and public health importance. The proposed surveillance-based diagnostic algorithm for ARI relies on a combination of gold-standard conventional methods, innovative serological and molecular techniques, as well as radiological approaches, which collectively contribute to the detection of various causative agents. The diagnostic part of the integrated algorithm can be dealt at the local microbiology laboratory of the healthcare facility with the few positive and negative specimens shipped to linked viral disease research laboratories (VRDLs) and other ICMR designated laboratories for genome characterisation, cluster identification and identification of novel agents.

An overview of transducers as platform for the rapid detection of foodborne pathogens.

The driving advent of portable, integrated biosensing ways for pathogen detection methods offers increased sensitivity and specificity over traditional microbiological techniques. The miniaturization and automation of integrated detection systems present a significant advantage for rapid, portable detection of foodborne microbes. In this review, we have highlighted current developments and directions in foodborne pathogen detection systems. Recent progress in the biosensor protocols toward the detection of specific microbes has been elaborated in detail. It also includes strategies and challenges for the implementation of a portable platform toward rapid foodborne sensing systems.

External quality assurance of serological diagnosis of dengue, chikungunya and Japanese encephalitis virus infection.

Introduction: Dengue, chikungunya and Japanese encephalitis are the most common arthropod-borne viral diseases in India. Due to overlapping clinical symptoms, accurate, high-quality and timely laboratory-based differential diagnosis is essential for control and containment of outbreaks. This is most commonly done by detection of IgM antibodies in serum using enzyme-linked immunosorbent assays. The Resource Centre for Virus Research and Diagnostic Laboratories (VRDLs) in Pune, India organized an external quality assurance (EQA) study to check the accuracy of serological diagnostics in the VRDL network. Methods: Three panels, one each for anti-dengue virus, anti-chikungunya virus and anti-Japanese encephalitis virus IgM antibodies, comprising six human serum samples (two positive and four negative) were distributed to test the sensitivity, specificity and reproducibility of serological testing in 124 VRDLs across India in 2018-19 and 2019-20. Results: Among the 124 VRDLs, the average concordance for both 2018-19 and 2019-20 was 98%. In 2018-19, 78.33%, 13.33% and 6.66% of VRDLs reported 100% concordance, 91-99% concordance and 81-90% concordance with the reference results, respectively, and 1.66% of VRDLs had concordance <80%. In 2019-20, 79.68%, 14.06% and 4.68% of VRDLs reported 100% concordance, 91-99% concordance and 81-90% concordance with the reference results, respectively, and 1.56% of VRDLs had concordance <80%. Conclusion: The EQA programme was beneficial for assessing and understanding the performance of the VRDLs. The study data indicate good proficiency in serological diagnosis of dengue, chikungunya and Japanese encephalitis in the VRDL network laboratories. Further expansion of the EQA programme to cover other viruses of public health importance will increase confidence among the VRDL network, and generate evidence of high-quality testing.

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.

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.

Isolation and characterization of SARS-CoV-2 Beta variant from UAE travelers.

BACKGROUND: The emergence of SARS-CoV-2 variants in places where the virus is uncontained poses a global threat from the perspective of public health and vaccine efficacy. Travel has been important factor for the easy spread of SARS-CoV-2 variants worldwide. India has also observed the importation of SARS-CoV-2 variants through international travelers. METHODS: In this study, we have collected the oropharyngeal and nasopharyngeal swab specimens from 58 individuals with travel history from United Arab Emirates (UAE), East, West and South Africa, Qatar, Ukraine and Saudi Arabia arrived in India during February-March 2021. The clinical specimens were initially screened for SARS-CoV-2 using Real time RT-PCR. All the specimens were inoculated on to Vero CCL-81 cells for virus isolation. The viral isolates were further sequenced using Next-Generation Sequencing. RESULTS: All 58 cases were tested positive for SARS-CoV-2 using Real time RT-PCR. Four specimens showed progressive infectivity with fusion of the infected cells with neighboring cells leading to large mass of cells. Replication competent virus was confirmed from culture supernatant of the passage 2 using Real time RT-PCR. Two plaque purified SARS-CoV-2 isolates demonstrated high viral RNA load of 3.8-7.5 × 1011 and 1.1-1.6 × 1011 at passage 4 and 5 respectively. Nucleotide variations along with amino acid changes were also observed among these two isolates at passage 2-5. All four cases were male with no symptoms and co-morbidity. The sequence analysis has shown two different clusters, first cluster with nucleotide deletions in the ORF1ab and the spike, while second cluster with deletions in spike region. The viral isolates demonstrated 99.88-99.96% nucleotide identity with the representative sequences of Beta variant (B.1.351). CONCLUSION: These findings suggest easier transmission of SARS-CoV-2 variants with human mobility through international travel. The isolated Beta variant would be useful to determine the protective efficacy of the currently available and upcoming COVID-19 vaccines in India.

Zika a Vector Borne Disease Detected in Newer States of India Amidst the COVID-19 Pandemic.

Background: During the second wave of the COVID-19 pandemic, outbreaks of Zika were reported from Kerala, Uttar Pradesh, and Maharashtra, India in 2021. The Dengue and Chikungunya negative samples were retrospectively screened to determine the presence of the Zika virus from different geographical regions of India. Methods: During May to October 2021, the clinical samples of 1475 patients, across 13 states and a union territory of India were screened and re-tested for Dengue, Chikungunya and Zika by CDC Trioplex Real time RT-PCR. The Zika rRTPCR positive samples were further screened with anti-Zika IgM and Plaque Reduction Neutralization Test. Next generation sequencing was used for further molecular characterization. Results: The positivity was observed for Zika (67), Dengue (121), and Chikungunya (10) amongst screened cases. The co-infections of Dengue/Chikungunya, Dengue/Zika, and Dengue/Chikungunya/Zika were also observed. All Zika cases were symptomatic with fever (84%) and rash (78%) as major presenting symptoms. Of them, four patients had respiratory distress, one presented with seizures, and one with suspected microcephaly at birth. The Asian Lineage of Zika and all four serotypes of Dengue were found in circulation. Conclusion: Our study indicates the spread of the Zika virus to several states of India and an urgent need to strengthen its surveillance.