Cost estimation of COVID Kavach test developed in 2020: A National Reference Laboratory initiative during early phase of Indian pandemic mitigation.

BACKGROUND OBJECTIVES: A new indigenously developed technology, coronavirus disease (COVID) Kavach, an IgG immunoglobulin-based enzyme-linked immunosorbent assay (ELISA) kit, was developed in 2020 by the Indian Council of Medical Research-National Institute of Virology (ICMR-NIV), Pune, India. The primary objective of this study was to determine the total cost of development of COVID Kavach IgG ELISA and estimate the unit cost (UC) as well. METHODS: The total development cost (TDC) of COVID Kavach and its UC during the early phase of pandemic mitigation were estimated through a micro-costing approach from provider's perspective. An activity-based bottom-up costing approach was used to facilitate data collection from all resources, and analysis was performed using Microsoft Excel version 2016. The micro-costing data were utilized to interpret the breakdown of cost across all inputs and different levels of activity. RESULTS: The TDC of COVID Kavach was estimated to be JOURNAL/ijmer/04.03/02223309-202310000-00007/363FF04/v/2023-11-25T134903Z/r/image-tiff 2,884,032 (US$ 38,265). The UC of providing test results for exposure to severe acute respiratory syndrome corona virus-2 (SARS-CoV-2) was estimated to be JOURNAL/ijmer/04.03/02223309-202310000-00007/363FF04/v/2023-11-25T134903Z/r/image-tiff 300 (US$ 4) during July 2020. The capital and recurrent cost were incurred around 5-10 per cent and 90-95 per cent, respectively, in both the development and UC of COVID Kavach. The major portion of funds (70-80%) was utilized for procurement of laboratory consumables, followed by human resources (8-12%) in the development as well as for UC of COVID Kavach. INTERPRETATION CONCLUSIONS: The estimates from this study can be useful for conducting economic evaluations, which will help in deciding upon the subsidy in government health facilities. The data may be useful to set up laboratory facilities analogous to the National Reference Laboratory located at the ICMR-NIV, Pune and for allotting sufficient budget to develop such assays in government-funded laboratories.

Understanding the resurgence of chikungunya virus during 2020-2021 in Pune, India, based on genomic analyses: A seven year study.

A surge in chikungunya was observed during 2020-21 in Pune district of Maharashtra, India. Whole genome sequencing and phylogenetic analysis of 21 samples/sequences revealed them as Indian ocean lineage of East Central South African genotype. Two distinct sequence clusters were found to circulate during 2020-21; one with E1:K211E and E2:V264A mutations while the other had E1:I317V mutation along with E1:K211E and E2: V264A mutations. The former, the predominant cluster (n = 18), clustered with chikungunya virus (CHIKV) strains of pre 2014 period while the latter (n = 3) clustered with 2016-2018 period Indian strains. Though E1: A226V was not detected in any of the 21 sequences, several unique mutations were detected in the strains which might have played key roles in the enhanced virus transmission during the period. The study highlights parallel evolution, introduction from the neighboring regions and cocirculation of two sequence clusters of CHIKV in Pune. The complete genome data can be useful to determine how the circulating strains differ from candidate vaccines and might help to predict the protective efficacy of chikungunya vaccine candidates.

Outbreaks and sporadic cases of japanese encephalitis in the state of Odisha, India: Outcome of 7 years of laboratory surveillance.

Acute encephalitis syndrome (AES) is a major public health challenge in India. We report here the epidemiology of sporadics and outbreaks of Japanese Encephalitis (JE) in Odisha state during 2012-2018. A total of 4235 AES cases (sporadics - 3394, outbreak cases - 841) recorded including 42 outbreaks; majority (n = 18) of which were during 2016. Overall JE virus (JEV) positivity was 11.78% (outbreak cases - 24.5%, sporadic cases - 8.6%). Age ≤15 years were largely affected during outbreaks, while 16-60 years population was dominant among sporadics. The major outbreak (2016) involved 336 patients from a tribal dominated district, spread over 173 villages. JEV seropositivity was high (43.45%) with 28.57% mortality. Epidemiological linkage with pig rearing was documented through JEV neutralizing antibodies in 50% of pig serum samples. Although the postvaccination period (2017-18) showed increase in AES case reporting but low JE proportion. Ongoing surveillance and preparedness of the health system would be of importance, especially in tribal-dominated districts.

Serum ferritin level as a prognostic biomarker for predicting dengue disease severity: A systematic review and meta-analysis.

Serum ferritin levels serves as biomarkers in many inflammatory and infectious diseases. This current systematic review and meta-analysis evaluated whether serum ferritin levels are associated with severe dengue and its utility as a biomarker of disease severity. Literature searches were conducted in PubMed, Scopus, ScienceDirect, the Cochrane library, and Google Scholar. A total of 18 studies examining the serum ferritin levels in dengue cases in the context of disease severity (nine studies having dengue classification as non-severe vs. severe dengue cases, and nine studies having dengue classification as dengue without warning signs (DwoWS), dengue with warning signs (DwWS), and severe dengue cases) were included and the quality of the studies was assessed using the Quality in Prognostic Factor Studies tool. The meta-analysis was performed using STATA software to calculate the effect size as a standardized mean difference (SMD) or Hedges 'g' for the continuous outcome. Higher serum ferritin levels were found in severe dengue cases compared to non-severe cases [SMD (Hedges 'g') 4.05 (95% C.I. 2.09-6.00), (I2  = 98.8%)]. In the second group, DwWS cases showed high serum ferritin levels compared to DwoWS [SMD 2.01 (95% C.I. 0.92-3.10), (I2  = 97.89%)], and severe dengue cases showed higher levels of serum ferritin compared to DwWS [SMD 2.66 (95% C.I. 1.72-4.48), (I2  = 98.78%)] and DwoWS cases [SMD 6.65 (95% C.I. 1.72-11.59), (I2  = 99.78%]. Subgroup analysis for the country of study (India vs. others), ferritin testing methods, and ferritin measurement day revealed testing method as a significant contributor to heterogeneity. To conclude, the present study suggests serum ferritin as a prognostic marker for dengue disease severity. Multi-centric studies involving a large number of dengue patients with a uniform case definition accounting for all the confounding variables might help in determining a universal cut-off value to discriminate between non-severe and severe dengue.

Genomic Characterization of Mosquito Isolates of Chikungunya Virus (Outbreak Strains 2022) Using Next-Generation Sequencing.

Background: A massive outbreak of dengue-like illness was reported from Pune district of Maharashtra, India during May-June 2022. Isolation and characterization of the etiological agent at genomic level for possible mutations that led to higher transmissibility is the topic of the study. Methods: Entomological investigations were carried out by ICMR-National Institute of Virology (Pune, India); Aedes aegypti mosquitoes were collected and processed for virus detection by molecular techniques. Positive mosquito pools were processed for virus isolation in cell culture. Sanger sequencing and whole-genome sequencing (WGS) using Oxford Nanopore Technology platform were used for genomic characterization. Results: Reverse transcriptase RT-PCR and qRT-PCR analysis detected chikungunya virus (CHIKV) in mosquito samples. Six CHIKV isolates were obtained. WGS revealed four nonsynonymous mutations in the structural polyprotein region, and five in the nonstructural polyprotein encoding region when compared with Yawat-2000 and Shivane-2016 strains. Sixty-four nucleotide changes in the nonstructural polyprotein region and 35 in the structural polyprotein region were detected. One isolate had an exclusive amino acid change, T1123I, in the nsP2 (protease) region. Conclusion: Abundant Ae. aegypti breeding and detection of CHIKV RNA in mosquitoes confirmed it as a chikungunya outbreak. Novel mutations detected in the epidemic strain warrants investigations to address their role in disease severity, transmission, and fitness.

Cost-analysis of real time RT-PCR test performed for COVID-19 diagnosis at India's national reference laboratory during the early stages of pandemic mitigation.

Real-time reverse transcription polymerase chain reaction (rRT-PCR) is one of the most accurate and extensively used laboratory procedures for diagnosing COVID-19. This molecular test has high diagnostic accuracy (sensitivity and specificity) and is considered as the gold standard for COVID-19 diagnosis. During COVID-19 surge in India, rRT-PCR service was encouraged and supported by the government of India through existing healthcare setup at various levels of healthcare facilities. The primary purpose of this research was to determine the per-unit cost of providing COVID-19 rRT-PCR services at the national reference laboratory at ICMR-National Institute of Virology in Pune during the early phase of COVID-19 pandemic mitigation, from the provider's perspective. The monthly cost for rRT-PCR testing as well as an estimated annual average unit cost for testing that takes account of peaks and troughs in pandemic were investigated. The time frame used to estimate unit cost was one year (July 2020-June 2021). For data collection on all resources spent during the early phase of pandemic, a conventional activity-based bottom-up costing technique was used. Capital costs were discounted and annualized over the estimated life of the item. Apportioning statistics were selected for cost heads like human resources, capital, and equipment based on time allocation, sharing of services, and utilization data. The data was also used to understand the breakdown of costs across inputs and over time and different levels of testing activity. During the initial phase of pandemic mitigation, the per unit cost of providing the COVID-19 rRT-PCR test was estimated to be ₹566 ($7.5) in the month of July 2020, where the total 56318 COVID-19 rRT-PCR tests was performed. The major proportion (87%) of funds was utilized for procuring laboratory consumables, followed by HR (10%), and it was least for stationary & allied items (0.02%). Unit cost was found to be the most sensitive to price variations in lab consumables (21.7%), followed by the number of samples tested (3.9%), salaries paid to HR (2.6%), price of equipment (0.23%), and building rental price (0.14%) in a univariate sensitivity analysis. The unit cost varies over the period of the pandemic in proportion with the prices of consumables and inversely proportional with number of tests performed. Our study would help the Government to understand the value for money they invested for laboratory diagnosis of COVID-19, budget allocation, integration and decentralization of laboratory services so as to help for achieving universal health coverage.

Evaluation of immunogenicity post two doses of inactivated SARS-CoV-2 vaccine, Covaxin after six months.

We have evaluated the immunogenicity of two dose of Covaxin given at a one-month interval to two adult populations, i.e. COVID-19 naïve-vaccinated individuals (n = 118) and COVID-19 recovered individuals (n = 128) with the vaccination. The immune response in the study population were assessed at three follow-ups, namely at one month post first dose, one and six months after the second dose. The persistence of S1RBD IgG and neutralizing antibodies for six months post vaccination was observed at different time intervals. The enhanced immune response was observed in both the participant groups. The study emphasizes the need for a booster dose post six months of vaccination.

Understanding the dynamics of IgM & IgG antibodies in COVID-19-positive patients.

Background & objectives: The pandemic caused by the SARS-CoV-2 has been a threat to humankind due to the rapid spread of infection and appearance of multiple new variants. In the present study, we report the dynamics and persistence of immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies in asymptomatic and symptomatic COVID-19 patients by chemiluminescent assay. Methods: A total of 463 serum samples from 218 SARS-CoV-2 PCR-positive patients were collected over a period of 124 days post-onset of disease (POD). Antibody levels were measured by chemiluminescence bioanalyzer. Neutralizing antibody titres were assessed by plaque reduction neutralization test (PRNT) for SARS-CoV-2. Results: Both IgM and IgG started appearing from day five post-infection in symptomatic and asymptomatic patients. IgM antibody response peaked around day 35 POD and rapidly diminished thereafter, with the last IgM-positive sample observed at 90 days POD. IgG antibody response peaked around 45 days POD and persisted till 124 days. The chemiluminescence immunoassay (CLIA) results showed a moderate correlation (R=0.5846, P<0.001) compared with PRNT. Additional analysis indicated a neutralizing titre of 250 corresponded to 12.948 AU/ml of YHLO iFlash SARS-CoV-2 IgG units. Interpretation & conclusions: Both symptomatic and asymptomatic COVID-19 patients seem to initiate production of antibody responses from day five of onset of disease. Although the CLIA gives high sensitivity and specificity and also its binding IgG antibody titres may correlate moderately with protective immunity, our results indicate that the values of binding antibody alone may not be a perfect guide to represent virus neutralization titre during donor selection for plasma therapy. However, IgM and IgG antibody detection may help in monitoring the status of disease progression and burden in the community.

Peripheral lymphocyte subset alteration in patients with COVID-19 having differential clinical manifestations.

Background & objectives: The COVID-19 disease profile in Indian patients has been found to be different from the Western world. Changes in lymphocyte compartment have been correlated with disease course, illness severity and clinical outcome. This study was aimed to assess the peripheral lymphocyte phenotype and subset distribution in patients with COVID-19 disease from India with differential clinical manifestations. Methods: Percentages of peripheral lymphocyte subsets were measured by flow cytometry in hospitalized asymptomatic (n=53), mild symptomatic (n=36), moderate and severe (n=30) patients with SARS-CoV-2 infection, recovered individuals (n=40) and uninfected controls (n=56) from Pune, Maharashtra, India. Results: Percentages of CD4+Th cells were significantly high in asymptomatic, mild symptomatic, moderate and severe patients and recovered individuals compared to controls. Percentages of Th memory (CD3+CD4+CD45RO+), Tc memory (CD3+CD8+CD45RO+) and B memory (CD19+CD27+) cells were significantly higher in the recovered group compared to both asymptomatic, mild symptomatic patient and uninfected control groups. NK cell (CD56+CD3-) percentages were comparable among moderate +severe patient and uninfected control groups. Interpretation & conclusions: The observed lower CD4+Th cells in moderate+severe group requiring oxygen support compared to asymptomatic+mild symptomatic group not requiring oxygen support could be indicative of poor prognosis. Higher Th memory, Tc memory and B memory cells in the recovered group compared to mild symptomatic patient groups might be markers of recovery from mild infection; however, it remains to be established if the persistence of any of these cells could be considered as a correlate of protection.

First case of Zika virus infection during an outbreak of chikungunya in a rural region of Maharashtra state, India.

BACKGROUND: In July 2021, an outbreak of chikungunya virus (CHIKV) was reported in a rural region of Maharashtra state, India. METHODS: Serum samples of symptomatic cases (n=33) were screened for dengue virus (DENV), CHIKV and Zika virus (ZIKV) by molecular and serological assays. RESULTS: The first case of ZIKV infection from Maharashtra was detected and confirmed by molecular and serological assays. Complete genome sequencing revealed that the ZIKV sequence belongs to the Asian genotype and had a closer homology with pre-epidemic strains present before 2007. CONCLUSIONS: ZIKV surveillance needs to be strengthened in the regions experiencing dengue and chikungunya outbreaks.

Economic evaluation of hepatitis A vaccines by income level of the country: A systematic review.

Background & objectives: Although several reviews of economic evaluation (EE) studies on hepatitis A virus (HAV) vaccine exist, there remains a need to corroborate such data from time to time. This study aimed to systematically review the literature for reports on EE of HAV vaccination by type of population, characteristics of intervention and income level of the country. Methods: PubMed and Scopus were searched to identify relevant studies from inception up to May 2021 using topic-specific key words in various combinaiton. Full EE studies comparing HAV vaccination to no vaccine or immunoglobulin were included. The risk of bias was assessed by using the ECOBIAS checklist. Results: Among the 1984 identified studies, 43 were found eligible. Of these, 27 were from high-income countries (HICs), 15 from middle-income countries (MICs), and one from low income country. Majority of the studies used Markov model and/or decision tree (n=26). Eight studies used a dynamic model. The discount rate, perspective and time horizon varied across the studies. Universal HAV vaccination without screening was cost-effective among children (14/16, 87.5%) and adolescents (1/5, 20%) but not in adults (0/4, 0%). Analysis by the level of income found that universal HAV vaccination among children without screening was cost-effective in 81.8 per cent of the studies conducted in MICs (9/11) as compared to 66.7 per cent in HICs (4/6). About one-third of the studies conducted among children found that screening and HAV vaccination were cost-effective compared to no vaccination. Interpretation & conclusions: The finding of this review suggest that universal vaccination of children without screening was likely to be cost-effective, especially in MICs. Nevertheless, it should be noted that the methodology varied across studies. Several aspects should also be considered in transferring the EE results across jurisdictions.

Longitudinal clinico-serological analysis of anti-nucleocapsid and anti-receptor binding domain of spike protein antibodies against SARS-CoV-2.

OBJECTIVES: Monitoring the antibody responses to SARS-CoV-2 infection and its correlation to clinical spectrum of disease is critical in understanding the disease progression and protection against re-infection. We assessed the nucleocapsid (N) and receptor-binding-domain of spike (SRBD) protein specific IgG and neutralizing antibody (NAb) responses in COVID-19 patients up to 8 months and its correlation with diverse disease spectrum. METHODS: During the first wave of the SARS-CoV-2 pandemic, from 284 COVID-19 patients, 608 samples were collected up to 8 months post infection. The patients were categorized as asymptomatic, symptomatic and severe. The N and SRBD IgG and NAb titers were evaluated and correlated with clinical data. RESULTS: A steep increase in antigen specific antibody titers was observed till 40 days post onset of the disease (POD), followed by a partial decline till 240 days. Severe disease was associated with a stronger SRBD IgG response and higher NAb titers. The persistence of antibody response was observed in 76% against N, 80% against SRBD and 80% for NAbs of cases up to 8 months POD. CONCLUSION: RBD and N protein specific IgG persisted till 240 days POD which correlated with NAb response, irrespective of individual`s symptomatic status indicating overall robust protection against re-infection.

Childhood encephalitis hospitalizations associated with virus agents in medium-endemic states in India.

BACKGROUND: Japanese encephalitis (JE) is the leading cause of childhood acute encephalitis syndrome (AES) in India. We enhanced the AES surveillance in sentinel hospitals to determine trends and virus etiologies in central India. METHODS: The neurological hospitalizations among children ≤15 years were tracked by using the AES case definition implemented by the national program. Acute and convalescent sera along with cerebrospinal fluid (CSF) specimens were collected and tested at the strengthened site hospital laboratories for anti-JE, anti-Dengue and anti-Chikungunya virus by IgM ELISA; along with Chandipura virus RT-PCR. Herpes simplex and enterovirus testing was undertaken at the reference laboratory. RESULTS: Among 1619 pediatric neurological hospitalizations reported during 2015-16, AES case definition was fulfilled in 332 (20.5%) cases. After excluding 52 non-AES cases, 280 AES cases resident from study districts were considered eligible for study. The treating physicians diagnosed non-viral causes in 90 cases, therefore 190 (67.9%) of 280 AES cases were suspected with viral etiologies. We enrolled 140 (73.7%) of 190 eligible AES cases. Viral etiologies were confirmed in 31 (22.1%) of 140 enrolled AES cases. JE (n = 22) was the leading cause. Additional non-JE viral agents included Chikungunya (5), Dengue (2) and Chandipura (2). However, only 21 (9.4%) of 222 additional AES cases referred from peripheral hospitals were confirmed as JE. CONCLUSIONS: Japanese encephalitis virus continues to be the leading cause of childhood acute encephalitis syndrome in central India despite vaccination program. Surveillance needs to be intensified for assessing the true disease burden of Japanese encephalitis following vaccination program implementation.

Pro-inflammatory CXCL-10, TNF-α, IL-1ß, and IL-6: biomarkers of SARS-CoV-2 infection.

Currently, the world is witnessing the pandemic of COVID-19, a disease caused by the novel coronavirus SARS-CoV-2. Reported differences in clinical manifestations and outcomes in SARS-CoV-2 infection could be attributed to factors such as virus replication, infiltration of inflammatory cells, and altered cytokine production. Virus-induced aberrant and excessive cytokine production has been linked to the morbidity and mortality of several viral infections. Using a Luminex platform, we investigated plasma cytokine and chemokine levels of 27 analytes from hospitalized asymptomatic (n = 39) and mildly symptomatic (n = 35) SARS-CoV-2-infected patients (in the early phase of infection), recovered individuals (45-60 days postinfection) (n = 40), and uninfected controls (n = 36) from the city of Pune located in the state of Maharashtra in India. Levels of the pro-inflammatory cytokines IL-1ß, IL-6, and TNF-α and the chemokine CXCL-10 were significantly higher, while those of the antiviral cytokines IFN-γ and IL-12 p70 were significantly lower in both asymptomatic and mildly symptomatic patients than in controls. Comparison among the patient categories revealed no difference in the levels of the cytokines/chemokines except for CXCL-10 being significantly higher and IL-17, IL-4, and VEGF being significantly lower in the mildly symptomatic patients. Interestingly, levels of all key analytes were significantly lower in recovered individuals than in those in both patient categories. Nevertheless, the level of CXCL10 was significantly higher in the recovered patients than in the controls, indicating that the immune system of SARS-CoV-2 patients may take a longer time to normalize. Our data suggest that IL-6, IL-1ß, TNF-α, CXCL-10, and reduced antiviral cytokines could be used as biomarkers of SARS-CoV-2 infection.

Epidemiological characterization of COVID-19 - Pune, 2020-2021.

The present study describes the epidemiological characteristics of 3,08,259 suspected cases of COVID-19 from the Pune district, India. The samples were referred for COVID-19 testing between January 24, 2020 and April 30, 2021. Demographic and clinical data were extracted from the ICMR-portal as a single dataset and analyzed. Of the 3,08,259 samples tested, 2,63,833 (85.6%) were asymptomatic. Symptomatic cases ratio in the first and the second COVID-19 wave was 1:2. Among symptomatic cases, cough was the most common complaint, followed by fever. Among the COVID-19 positives, one-fifth were asymptomatic, highlighting the necessity for close contact tracing even among apparently healthy contacts. The second wave of COVID-19 had double the per cent of symptomatic individuals as compared to the first wave.

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.