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.

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.

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.

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.

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.

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.

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.

Neutralizing antibody responses to SARS-CoV-2 in COVID-19 patients.

BACKGROUND & OBJECTIVES: The global pandemic caused by SARS-CoV-2 virus has challenged public health system worldwide due to the unavailability of approved preventive and therapeutic options. Identification of neutralizing antibodies (NAb) and understanding their role is important. However, the data on kinetics of NAb response among COVID-19 patients are unclear. To understand the NAb response in COVID-19 patients, we compared the findings of microneutralization test (MNT) and plaque reduction neutralization test (PRNT) for the SARS-CoV-2. Further, the kinetics of NAb response among COVID-19 patients was assessed. METHODS: A total of 343 blood samples (89 positive, 58 negative for SARS-CoV-2 and 17 cross-reactive and 179 serum from healthy individuals) were collected and tested by MNT and PRNT. SARS-CoV-2 virus was prepared by propagating the virus in Vero CCL-81 cells. The intra-class correlation was calculated to assess the correlation between MNT and PRNT. The neutralizing endpoint as the reduction in the number of plaque count by 90 per cent (PRNT90) was also calculated. RESULTS: The analysis of MNT and PRNT quantitative results indicated that the intra-class correlation was 0.520. Of the 89 confirmed COVID-19 patients, 64 (71.9%) showed NAb response. INTERPRETATION & CONCLUSIONS: The results of MNT and PRNT were specific with no cross-reactivity. In the early stages of infection, the NAb response was observed with variable antibody kinetics. The neutralization assays can be used for titration of NAb in recovered/vaccinated or infected COVID-19 patients.

Development of indigenous IgG ELISA for the detection of anti-SARS-CoV-2 IgG.

BACKGROUND & OBJECTIVES: Since the beginning of the year 2020, the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) impacted humankind adversely in almost all spheres of life. The virus belongs to the genus Betacoronavirus of the family Coronaviridae. SARS-CoV-2 causes the disease known as coronavirus disease 2019 (COVID-19) with mild-to-severe respiratory illness. The currently available diagnostic tools for the diagnosis of COVID-19 are mainly based on molecular assays. Real-time reverse transcription-polymerase chain reaction is the only diagnostic method currently recommended by the World Health Organization for COVID-19. With the rapid spread of SARS-CoV-2, it is necessary to utilize other tests, which would determine the burden of the disease as well as the spread of the outbreak. Considering the need for the development of such a screening test, an attempt was made to develop and evaluate an IgG-based ELISA for COVID-19. METHODS: A total of 513 blood samples (131 positive, 382 negative for SARS-CoV-2) were collected and tested by microneutralization test (MNT). Antigen stock of SARS-CoV-2 was prepared by propagating the virus in Vero CCL-81 cells. An IgG capture ELISA was developed for serological detection of anti-SARS-CoV-2 IgG in serum samples. The end point cut-off values were determined by using receiver operating characteristic (ROC) curve. Inter-assay variability was determined. RESULTS: The developed ELISA was found to be 92.37 per cent sensitive, 97.9 per cent specific, robust and reproducible. The positive and negative predictive values were 94.44 and 98.14 per cent, respectively. INTERPRETATION & CONCLUSIONS: This indigenously developed IgG ELISA was found to be sensitive and specific for the detection of anti-SARS-CoV-2 IgG in human serum samples. This assay may be used for determining seroprevalence of SARS-CoV-2 in a population exposed to the virus.