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.

Replication of SARS-CoV-2 in cell lines used in public health surveillance programmes with special emphasis on biosafety

Background & objectives: Polio, measles, rubella, influenza and rotavirus surveillance programmes are of great public health importance globally. Virus isolation using cell culture is an integral part of such programmes. Possibility of unintended isolation of SARS-CoV-2 from clinical specimens processed in biosafety level-2 (BSL-2) laboratories during the above-mentioned surveillance programmes, cannot be ruled out. The present study was conducted to assess the susceptibility of different cell lines to SARS- CoV-2 used in these programmes. Methods: Replication of SARS-CoV-2 was studied in RD and L20B, Vero/hSLAM, MA-104 and Madin–Darby Canine Kidney (MDCK) cell lines, used for the isolation of polio, measles, rubella, rotavirus and influenza viruses, respectively. SARS-CoV-2 at 0.01 multiplicity of infection was inoculated and the viral growth was assessed by observation of cytopathic effects followed by real-time reverse transcription–polymerase chain reaction (qRT-PCR). Vero CCL-81 cell line was used as a positive control. Results: SARS-CoV-2 replicated in Vero/hSLAM, and MA-104 cells, whereas it did not replicate in L20B, RD and MDCK cells. Vero/hSLAM, and Vero CCL-81 showed rounding, degeneration and detachment of cells; MA-104 cells also showed syncytia formation. In qRT-PCR, Vero/hSLAM and MA-104 showed 106 and Vero CCL-81 showed 107 viral RNA copies per ?l. The 50 per cent tissue culture infectious dose titres of Vero/hSLAM, MA-104 and Vero CCL-81 were 105.54, 105.29 and 106.45/ml, respectively. Interpretation & conclusions: Replication of SARS-CoV-2 in Vero/hSLAM and MA-104 underscores the possibility of its unintended isolation during surveillance procedures aiming to isolate measles, rubella and rotavirus. This could result in accidental exposure to high titres of SARS-CoV-2, which can result in laboratory acquired infections and community risk, highlighting the need for revisiting biosafety measures in public health laboratories

MCI guidelines on publications for academic Promotions.

We read with interest the editorial by Aggarwal et al. 1 regarding the revised guidelines of the Medical Council of India (MCI) for publications for academic promotions.2 We agree with the authors that the new guidelines raise several important issues. The primary among them is the restriction of acceptable publications to original research with raw data. We do feel that systematic reviews, meta-analyses (including Cochrane reviews), brief communications (often because the journal will accept an original article in only this format), and case reports in journals with high impact factor should also be acceptable. The various indexing databases suggested do not include Science Citation Index and Indmed that are definitely more acceptable than Index Copernicus, which contains some journals of poor merit. We suggest that authorship in a high impact factor journal should be given more credit than one in a low impact factor journal. Finally, the first, second and last author should be given credit, rather than only the first and second. Issues of lack of adequate credit for the senior author in collaborative projects and ‘gift authorship’ are concerns with both extremes.

Evaluation of a programme for prevention of vertical transmission of hepatitis B in a rural block in southern India.

Background & objectives: This study was undertaken to evaluate a community based programme of antenatal screening for hepatitis B surface antigen (HBsAg) and selective immunization of children commencing at birth, at a secondary care hospital in south India. The primary objective was to assess immunization coverage among children born to HBsAg positive women; secondary objectives were to study the prevalence of HBsAg among antenatal women, prevalence of HBsAg among immunized children (to estimate vaccine efficacy), seroconversion rate and relationship of maternal hepatitis B e antigen (HBeAg) to hepatitis infection. Methods: The prevalence of hepatitis B antigen among antenatal women and immunization coverage achieved with hepatitis B vaccine in a rural block in Vellore, Tamil Nadu were assessed through examination of records. Children born between May 2002 and December 2007 to hepatitis B positive women were followed up for a serological evaluation, based on which vaccine efficacy and the effect of maternal hepatitis B e antigen (HBeAg) on breakthrough infection was estimated. Results: The prevalence of hepatitis B surface antigen among antenatal women was 1.58 % (95% CI: 1.35-1.81%). Vaccine coverage for three doses as per a recommended schedule (including a birth dose) was 70 per cent, while 82.4 per cent eventually received three doses (including a birth dose). Estimated vaccine efficacy was 68 per cent and seroconversion 92.4 per cent in children aged 6-24 months. Maternal HBeAg was significantly associated with either anti-HBc or HBsAg in immunized children, RR=5.89 (95% CI: 1.21-28.52%). Interpretation & conclusions: The prevalence of hepatitis B among antenatal women in this region was low and a programme of selective immunization was found to be feasible, achieving a high coverage for three doses of the vaccine including a birth dose.

Quantitation of hepatitis B virus DNA in plasma using a sensitive cost-effective "in-house" real-time PCR assay.

BACKGROUND: Sensitive nucleic acid testing for the detection and accurate quantitation of hepatitis B virus (HBV) is necessary to reduce transmission through blood and blood products and for monitoring patients on antiviral therapy. The aim of this study is to standardize an "in-house" real-time HBV polymerase chain reaction (PCR) for accurate quantitation and screening of HBV. MATERIALS AND METHODS: The "in-house" real-time assay was compared with a commercial assay using 30 chronically infected individuals and 70 blood donors who are negative for hepatitis B surface antigen, hepatitis C virus (HCV) antibody and human immunodeficiency virus (HIV) antibody. Further, 30 HBV-genotyped samples were tested to evaluate the "in-house" assay's capacity to detect genotypes prevalent among individuals attending this tertiary care hospital. RESULTS: The lower limit of detection of this "in-house" HBV real-time PCR was assessed against the WHO international standard and found to be 50 IU/mL. The interassay and intra-assay coefficient of variation (CV) of this "in-house" assay ranged from 1.4% to 9.4% and 0.0% to 2.3%, respectively. Virus loads as estimated with this "in-house" HBV real-time assay correlated well with the commercial artus HBV RG PCR assay ( r = 0.95, P < 0.0001). CONCLUSION: This assay can be used for the detection and accurate quantitation of HBV viral loads in plasma samples. This assay can be employed for the screening of blood donations and can potentially be adapted to a multiplex format for simultaneous detection of HBV, HIV and HCV to reduce the cost of testing in blood banks.

Clinicopathological features and genotype distribution in patients with hepatitis C virus chronic liver disease.

Background and Objective Hepatitis C virus (HCV) genotype influences the severity of disease and response to therapy. This retrospective study examined the clinical and histological features and the genotype distribution in biopsied patients with HCV related chronic liver disease. Methods Of 105 biopsies from patients with HCV infection, 96 from patients with chronic liver disease were reviewed. The Ishak scoring system was used for histological analysis. Results Genotype 3 was most common accounting for 77.1%, and genotype 1 for 9.4% of cases. There was no significant association of transaminase levels, viral load or necroinflammatory activity score with genotype. A severe degree of fibrosis was seen in 77.8% cases of genotype 1 and in 63.5% of genotype 3 (p=0.76). Variable degrees of steatosis were noted in 68.8% of cases. However, severe steatosis was noted only in genotype 3 (7 cases). Serum transaminase levels did not correlate with either histological activity (p=0.43) or degree of fibrosis (p=0.72). Severe fibrosis / cirrhosis was seen in 74.24% of patients above 40 years of age as compared to 33.3% of patients below 40 years (p=0.001). The frequency of Mallory hyaline was significantly different between genotypes 1 and 3 infection (P<0.001). Conclusions This study confirms the preponderance of genotype 3 in Indian patients with HCV related chronic liver disease. Severe steatosis was seen only in genotype 3 and Mallory hyaline was very common in genotype 1. The small numbers of patients in non genotype 3 could be a reason for the apparent lack of histological differences between different HCV genotypes. Severe fibrosis seen in older age groups confirms that HCV infection is progressive and major acceleration of the disease process occurs after 40 years of age.

Correlation between hepatitis B genotypes, 1896 precore mutation, virus loads and liver dysfunction in an Indian population.

BACKGROUND/OBJECTIVES: Hepatitis B virus (HBV) genotypes may differ in pathogenicity. However, the interplay between different virus characteristics such as genotypes, mutants and virus loads has not been well studied . We investigated the association between HBV genotype, presence of 1896 precore mutation and HBV viral loads in patients with HBV-related liver disease. METHODS: One hundred and sixteen HBV DNA-seropositive patients attending a gastroenterology outpatient clinic and 107 HBV DNA-seropositive blood donors were recruited. The subjects were stratified as those with normal (Group I, n=164) and elevated (Group II, n=59) ALT levels. The HBV genotype and the presence of the 1896 precore mutation were determined, and plasma HBV DNA levels measured. RESULTS: Genotype C was more common in Group II than in Group I (10 (17%) vs. 4 (2.4%); p< 0.005). There was no relationship between the 1896 precore mutation and the HBV DNA levels. Subjects with genotype C (n=14) had higher HBV DNA levels than those with genotypes A (n=33) or D (n=158). CONCLUSIONS: The infecting genotype, but not the presence of 1896 precore mutation, correlates with HBV load. The association of genotype C with higher virus loads and with elevated ALT may point to a greater pathogenicity of this genotype.

Hepatitis C virus genotype 6 infection in India.

We report two patients with chronic liver disease--a 46-year-old man and a 52-year-old woman, both from eastern India--who were found to be infected with hepatitis C virus genotype 6 strains. These strains have been previously reported only from Hong Kong and Southeast Asia.

Is dengue emerging as a major public health problem?

BACKGROUND & OBJECTIVES: Diagnosis of dengue infection is easily and best accomplished by demonstration of specific IgM antibodies in blood. We analyzed retrospectively the dengue IgM seropositivity available for samples obtained over a period of five year (1999-2003) from patients with suspected dengue fever (DF)-like illness to investigate whether there was an overall increase in the dengue IgM prevalence over this period. METHODS: Serum samples from a total of 1426 individuals (suspected dengue cases) obtained over five year were tested for dengue specific IgM antibodies. Of the 1426 patients, 693 were adults (>15 yr) and 694 children (<15 yr) (excluding 39 individuals whose age was not known). There were 807 males and 610 females (excluding 9 individuals whose status on sex was unknown). RESULTS: A total of 423 (29.7%) samples were positive for dengue IgM over the five year period. Overall, there was a significant increase in the percentage of dengue IgM positive individuals over the this period (P<0.001). When the individuals were grouped into children (<15 yr) and adults (>15 yr), a significant increase in the number of dengue IgM positive individuals was noticed only in children (P<0.001) and not in adults. When the individuals were grouped into males and females, a significant increase in the number of dengue IgM positive individuals was noticed in both the sexes (P<0.03). Month-wise analysis of the dengue IgM positivity rates indicated the year-wide occurrence of dengue. A total of 158 (41%) of the dengue IgM positive individuals showed positivity for dengue IgG also suggestive of a secondary heterotypic infection. INTERPRETATION & CONCLUSION: The overall significant increase in dengue IgM seropositivity among the suspected cases indicates an increase in dengue virus activity, raising the question whether dengue is emerging/re-emerging as a major health problem in southern India. Increase in probable secondary infection (as evidenced by dual positivity for dengue IgM and IgG) seen in this study is also a point of concern. Such an increase especially in a country like ours where multiple serotypes are prevalent, raises concern over probable increase in the incidences of the more serious DHF/DSS. As this report could well be an underestimate of true incidence, the alarming increase observed in 2003, may be a warning/indication of epidemics to come soon that merits serious consideration.

Granulocyte macrophage colony stimulating factor augmented hepatitis B vaccine protocol for rapid seroprotection in voluntary kidney donors.

BACKGROUND & OBJECTIVES: Conventional hepatitis B vaccine protocols do not provide rapid seroprotection against hepatitis B. This randomized controlled trial was carried out to investigate the efficacy of granulocyte macrophage-colony stimulating factor (GM-CSF) augmented double-dose vaccine protocol in voluntary kidney donors prior to donor nephrectomy. METHODS: A total of 54 kidney donors, who had no history of hepatitis B infection, hepatitis B vaccination and tested negative for anti-HBs and anti-HBc antibodies were randomly allocated to the control or test groups. GM-CSF (300 microg) was administered subcutaneously on day 0, followed by 40 microg of recombinant hepatitis B vaccine intramuscularly on the same deltoid on day 1. The control group received only 40 microg of intramuscular hepatitis B vaccine. Anti-HBs titres were measured at the end of 4 wk. RESULTS: Of the 54 donors studied, there was a significant (P<0.003) seroconversion in the GM-CSF group (82%) compared to the control group (37%), after a single immunization with double-dose recombinant hepatitis B vaccine by 4 wk. Minor side effects such as fever in four patients and myalgia in three were noticed. INTERPRETATION & CONCLUSION: GM-CSF augmented double-dose hepatitis B vaccine could be used in unvaccinated patients when a rapid response is desired.

Hepatitis C virus genotypes: special reference to the Indian scene.

The heterogeneity observed between different isolates of the hepatitis C virus has led to stratification of these isolates into different genotypes. Clinically the genetic diversity of the virus has important implications as genotypes respond variably to antiviral therapy and can influence disease prognosis. This article attempts to summarize current knowledge about the different biological properties of HCV genotypes, and their geographical distribution, with special reference to the Indian scenario.