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.

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.

Data on isolation and purification of fibrinolytic enzyme from Pseudomonas baetica SUHU25.

The present dataset provides methodology to isolate and purify fibrinolytic enzyme from microbe isolated from the natural source. The information provided in this data article includes (1) isolation and identification of Pseudomonas baetica SUHU25, (2) optimization of cultural conditions, (3) extraction and purification of fibrinolytic enzyme, (4) protein estimation, (5) assay of fibrinolytic activity, (6) SDS PAGE for purified enzyme protein, (7) effect of pH, temperature and metal ions on fibrinolytic activity of enzyme protein, and (8) In-vitro blood clot dissolution assay.

Higher levels of dengue-virus-specific IgG and IgA during pre-defervescence associated with primary dengue hemorrhagic fever.

Dengue hemorrhagic fever (DHF), although predominantly associated with secondary infections, has also been reported in primary infections. An enhanced immune response including antibodies and cytokines is implicated in the pathogenesis of secondary DHF. However, the factors operating in primary DHF are poorly understood. To understand the role of the antibody response, the relative levels of different antibody isotypes during the acute phase of infection in primary and secondary dengue infections were determined. Levels of DENV-specific IgM, IgG, IgA and IgE were measured in the serum samples of 200 dengue patients and 20 dengue-naïve individuals. Samples were collected within 15 days of onset of illness. The DENV-specific IgM levels were significantly higher in DF cases compared to DHF, which was more evident in secondary infections and in post-defervescence samples. The levels of IgG, IgA and IgE were higher in DHF cases, with greater significance in primary infections. A higher level of IgG in DHF cases was evident in pre-defervescence samples, whilst the IgE level was higher in pre- and post-defervescence samples. There was a significant correlation of IgG titres with platelet counts, with higher titres associated with lower platelet counts. It is speculated that IgG, IgA and IgE produced in response to primary infections may contribute to pathogenesis, whilst IgM produced in response to secondary infections may protect against progression to severe disease.