Efficacy of combined HBsAg, anti-HBc and anti-HBs screening in minimizing transfusion transmission risk of hepatitis B infection in low resource setting.

Background: Hepatitis B Virus (HBV), and occult Hepatitis B in particular, is a major concern in the transfusion scenario, especially in endemic countries. This study attempted to estimate the prevalence of occult Hepatitis B infection (OBI) among voluntary blood donors in Maharashtra and to evaluate the role of combined screening strategy with implications in minimizing the current transfusion risks of seropositive OBI. Methods: Donor samples were collected from 80 eligible blood banks from various districts of Maharashtra between 2014 and 2017. ELISA based screening of HBsAg, anti-HBc (total and IgM), anti-HBs titres. Real-time quantitative PCR for Hepatitis B Virus DNA (HBV DNA) were performed for all HBsAg and or anti-HBc positive samples. Results: Out of 2398 samples tested, 20 (0.83%) samples were positive for HBsAg, whereas 547 (22.81%) were positive for anti-HBc. Out of 547 samples, 16 (2.92%) were positive for HBV DNA with median level at 247.89 IU/mL (IQR: 126.05-666.67 IU/mL). Anti-HBs levels were positive in 35.83% of OBI cases. ROC curve analysis showed that combined HBsAg, anti-HBc and anti-HBs (>50 mIU/mL) screening can more efficiently detect HBV infection in blood donors than HBsAg alone. Conclusions: A combined HBsAg, anti-HBc and anti-HBs screening for donor samples could be an alternative achievable strategy to minimize the HBV transmission as well as financial burden. In resource limited setup, the proposed combined strategy could be helpful in minimizing the risk of OBI transmission.

Characterization of Circulating Tumor Cells Using Imaging Flow Cytometry in Liver Disease Patients.

Background: Hepatocellular carcinoma (HCC) is asymptomatic at an early stage which delays its timely diagnosis and treatment. Circulating tumor cells (CTCs), derived from a primary or secondary tumor, may help in the management of HCC. Here, we evaluate and characterize CTCs in liver disease patients. Methods: In total, 65 patients, categorized into liver cirrhosis (LC) (n = 30) and HCC (n = 35), were enrolled. Using ImagestreamX MkII imaging flow cytometer, CTCs were detected and characterized using biomarker expression of EpCAM, CK, AFP, CD45, and DRAQ5 in LC and HCC patients. Results: CTCs were detected in 33/35 (94%) HCC patients and in 28/30 (93%) LC patients. In the HCC group, the number of biomarker-positive CTCs was higher in BCLC stage D when compared with others. EpCAM + CK was the most expressed biomarker on CTCs in LC versus HCC (83.3% vs. 77.14%), followed by AFP (80% vs. 65.71%), EpCAM (30% vs. 28.57%), and CK (16.6% vs. 14.28%). The EpCAM cell area was significantly associated (P value = 0.031) with the CTC-positive status. The combination biomarker expression of CTCs cell area (EpCAM, CK, and AFP) performed well with the area under the curve of 0.92, high sensitivity, and specificity in detecting early-stage and AFP-negative HCC as well as in AFP-negative LC cases. Conclusion: Enumeration and cell area of CTCs may be used as a biomarker for early detection of HCC and guiding treatment.

Exploring risk factors and transmission dynamics of Hepatitis B infection among Indian families: Implications and perspective.

INTRODUCTION: Hepatitis B virus (HBV) is global health problem. Family members of HBV infected people are considered as high-risk groups due to frequent household transmission of HBV among contacts of HBsAg carriers. The present study aimed to investigate the intrafamilial transmission of HBV among family members of HBV-infected persons and to identify the risk factors for viral transmission in these setting. METHODS: 361 index cases and their 1083 family contacts were tested for markers of Hepatitis B, viz. HBsAg and HBcAb using commercial ELISA. The demographic details and risk factors for acquiring HBV infection among the family members were recorded using a structured questionnaire. RESULTS: The median (IQR) age of index cases and family members was 37 (27 - 48) and 26 (14 - 38) years, respectively. Among the screened family members, 9.23% (n = 100) members were positive for HBsAg and 32.75% (n = 355) were positive for HBcAb. At least one member of the family was affected in 229/361 (63.43%) index cases. Significantly lower percent of household contacts (9.23%, n = 100)were vaccinated against HBV.HBV transmission risk was significantly higher in families with more than four members(p < 0.0001). Multinomial logistics regression analysis for familial risk factors for transmission of HBV such asclose contact with carrier (aOR overt: 1.172, aOR occult: 1.173), sharing of bed/bedding (aOR overt: 1.258, aOR occult:1.264), personal hygiene items (aOR overt:1.260, aOR occult: 1.451), and eating in common utensils (aOR overt: 2.182, aOR occult: 1.307)were significantly associated with the transmission of HBV (p < 0.05). DISCUSSION: Close contact with carrier, sharing of bed/bedding or personal hygiene items and eating in common utensils were significantly associated with the transmission of HBV. Increasing awareness about Hepatitis B infection and vaccination of family members in close contact with carrier is essential to prevent Hepatitis B transmission.

Antibody profile in post-vaccinated & SARS-CoV-2 infected individuals.

Background & objectives: During the COVID-19 pandemic it was important to assess the antibody profile in individuals vaccinated with Covaxin (BBV152) and Covishield (ChAdOx1 nCoV-19) with both 28 and 84 days gaps between two doses, those infected with SARS-CoV-2 and post-COVID-19-infected individuals vaccinated with only one dose of either of the vaccines. The present study was aimed to assess these objectives. Methods: Fifty real time reverse transcription-polymerase chain reaction (qRT-PCR)-confirmed COVID-19-infected individuals, along with 90 COVID-19-naïve (BBV152 and ChAdOx1 nCov-19)-vaccinated individuals, were included in the study. Individuals who received a single dose of either vaccine with a confirmed past diagnosis of SARS-CoV-2 infection (n=15) were also included. Blood samples were collected strictly between the 4th and 5th wk after development of symptoms for SARS-CoV-2 infected individuals and after the first/second vaccination dose. Antibody profile assessment was done using whole-virus, spike-receptor binding domain (RBD) and nucleocapsid-specific ELISA kits along with neutralizing antibody kit. Results: There was an overall 97.7 per cent seropositivity rate in vaccinated individuals, and a strong correlation (R2=0.8, P<0.001) between neutralizing and spike-RBD antibodies. Among individuals who received two standard doses of ChAdOx1 nCoV-19 vaccine, the spike antibody levels developed were of higher titre with a longer prime boost interval than in those with shorter intervals (P<0.01). Individuals vaccinated with two doses as well as only one dose post-SARS-CoV-2 infection had high neutralizing and spike-specific antibodies. Interpretation & conclusions: High neutralizing and spike-specific antibodies were developed in individuals vaccinated only with one dose of either vaccine post-SARS-CoV-2 infection. With the main priority being vaccinating majority of the population in our country, single-dose administration to such individuals would be a sensible way to make the most of the limited supplies. Furthermore, neutralizing antibody levels observed in COVID-19-naïve vaccinees imply the need for booster vaccination.

Serosurvey for Health-Care Workers Provides Supportive Evidence for the Effectiveness of Hydroxychloroquine Prophylaxis against SARS-CoV-2 Infection.

BACKGROUND: The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic has resulted in occupational exposure among Healthcare Workers (HCWs) and a high risk of nosocomial transmission. Asymptomatic infection and transmission of infection before the development of symptoms are well-recognized factors contributing to the spread of infection. We conducted a cross-sectional observational study to understand the seroprevalence of SARS-CoV-2 infection among HCWs and to verify the appropriateness of infection control measures, particularly Hydroxychloroquine (HCQ) prophylaxis. METHODS: A cross-sectional sero-surveillance study was conducted among 500 HCWs in Dombivli and surrounding Mumbai Metropolitan area (Maharashtra, India) between 21st July and 3rd August 2020. The vulnerability of the study participants to SARS-CoV-2 infection was ascertained through a history of (i) involvement in direct care, (ii) exposure to aerosol-generating procedures, (iii) co-morbidities, (iv) Personal Protective Equipment (PPE) use, and (v) HCQ prophylaxis. SARS-CoV-2 IgG antibodies were tested using COVID KAVACH anti-SARS-CoV-2 IgG antibody detection enzyme-linked immunosorbent assay (ELISA) from Zydus Cadila. A systematic analysis of the correlation between the development of antibodies and factors affecting vulnerability to infection was performed. RESULTS: The overall SARS-CoV-2 seroprevalence in the study population was 11%. Providing direct care to COVID-19 patients (Adjusted OR 16.4, 95% CI 3.3-126.9, p = 0.002) for long hours and irregular use of PPE (Adjusted OR 3.78, 95% CI 1.1-11.9, p = 0.02) were associated with an increased incidence of seropositivity. Prophylaxis with HCQ may have a role in reducing the vulnerability to infection as depicted by univariate and multivariate analysis (Adjusted OR 0.55, 95% CI 0.3-0.9, p = 0.047). It was also noted that those not on HCQ prophylaxis were threefold more prone to infection and developed severe disease as compared to those on HCQ prophylaxis. CONCLUSION: Prophylaxis with HCQ may have a role in mitigating the incidence and severity of SARS-CoV-2 infection. Although vaccination is the most robust strategy to safeguard against COVID-19, it will be months before vaccination percolates to the masses. In the face of the second wave of COVID-19, the use of HCQ prophylaxis in combination with use of face-masks regularly may be considered as a cost-effective measure for population dense areas like urban slums where social distancing is not possible.

Innovative Betulin Nanosuspension exhibits enhanced anticancer activity in a Triple Negative Breast Cancer Cell line and Zebrafish angiogenesis model.

We present a nanosuspension of betulin, a BCS class II anticancer drug, particularly effective against resistant breast cancer. As anticancer efficacy of betulin is hampered by poor aqueous solubility, a nanosuspension with surface area was considered to enhance efficacy. An innovative approach wherein the betulin nanosuspension is generated instantaneously in situ, by adding a betulin preconcentrate (BeTPC) comprising drug and excipients, to aqueous medium, is successfully demonstrated. The optimal BeTPC when added to isotonic dextrose solution instantaneously generated an in situ nanosuspension (BeTNS-15) with high precipitation efficiency (92.7 ± 1.21%), average particle size (383.74 ± 7.24 nm) and good stability as per ICH guidelines. TEM revealed elongated particles while DSC and XRD indicated partial amorphization. Significantly higher cytotoxicity of BeTNS-15 (IC50 38.44 µg/ml) compared to betulin (BetS) (IC50 69.54 µg/ml) in the resistant triple negative human breast cancer cell line MDA-MB-231, was attributed to high intracellular uptake confirmed by HPLC and Imaging Flow cytometry (IFC). IFC confirmed superior anti-cancer efficacy of BeTNS-15 mediated by mitochondrial membrane disruption and inhibition of the G0/G1 phase. BeTNS-15 also exhibited significantly greater anti-angiogenic efficacy (p < 0.05) in the zebrafish model confirming superior efficacy. Simplicity of the innovative in situ approach coupled with superior efficacy proposes BeTNS as an innovative and highly promising anticancer formulation.

Prevalence of Vitamin D Deficiency Amongst Indian Orthopaedic Surgeons.

BACKGROUND: Vitamin D deficiency is a widely prevalent condition with patients in both symptomatic and asymptomatic spectrum. With the lack of routine screening there exists an unknown population of Indian Orthopaedic surgeons who are deficient in Vitamin D and lead to an unexplained loss of quality of work and increased susceptibility to various other diseases. The easiest access to resources for supplementation is available to this group of treating physicians however its use for their personal cure is rarely recognised. This study aims to highlight this endemic disease and to find out its correlation with other parameters. METHODS: It is a prospective observational study including 150 practicing orthopaedic surgeons from entire India who visited our centre during 3 months duration for various educational meetings. Venous sample was collected after due informed consent and analysed at a single laboratory for 25-OH Cholecalciferol levels by a chemiluminescent assay. All the samples were analysed and a questionnaire was sent to the participants via google forms regarding various parameters under study. RESULTS: The mean serum Vitamin D levels were 18.6 ± 9.67 ng/ml in the sample studied. 17 out of 150 participants (11.3%) were found to have sufficient serum levels of 25(OH) Cholecalciferol. 105 participants (70%) were having deficient levels and 28 (18.7%) had insufficient levels of Vitamin D. Overall 88.7% participants had Vitamin D deficiency among the sample studied. CONCLUSION: This widespread prevalence of Vitamin D deficiency warrants frequent screening and routine supplementation of Vitamin D in orthopaedic surgeons thereby providing a low cost solution to improve the troublesome situation among healthcare providers.

Applications of imaging flow cytometry in the diagnostic assessment of red cell membrane disorders.

BACKGROUND: Red cell membranopathies refers to phenotypically and morphologically heterogeneous disorders. High throughput imaging flow cytometry (IFC) combines the speed, sensitivity, and phenotyping abilities of flow cytometry with the detailed imagery and functional insights of microscopy to produce high content image analysis with quantitative analysis. We have evaluated the applications of IFC to examine both the morphology as well as fluorescence signal intensity in red cell membranopathies. METHODS: Fluorescence intensity of eosin-5-maleimide (EMA) labeled red cells was measured for diagnosis of RBC membrane protein defect on Amnis ImageStreamX followed by Image analysis on IDEAS software to study features such as circularity and shape ratio. RESULTS: The hereditary spherocytosis (HS) group showed significantly decreased MFI (52,800 ± 9,100) than normal controls (81,100 ± 4,700) (p < .05) whereas non-HS showed 78,300 ± 9,900. The shape ratio of hereditary elliptocytosis (HE) was significantly higher (43.8%) than normal controls (14.6%). The circularity score is higher in HS (64.15%) than the normal controls (44.3%) whereas the circularity score was very less in HE (10%) due to the presence of elliptocytes. CONCLUSIONS: The advantages of the IFC over standard flow cytometry is its ability to provide high-content image analysis and measurement of parameters such as circularity and shape ratio allow discriminating red cell membranopathies (HS and HE) due to variations in shape and size. It could be a single, effective, and rapid IFC test for detection and differentiation of red cell membrane disorders in hematology laboratories where an IFC is available.

Differentially expressed serum host proteins in hepatitis B and C viral infections.

Hepatitis B virus (HBV) and Hepatitis C virus (HCV) infection often lead to hepatocellular carcinoma (HCC), which is mostly detected in advanced stage. Hence, its early detection is of paramount importance using a biomarker having sensitivity and specificity both. The present study highlights differentially expressed host proteins in response to HBV/HCV infection at different stages. Comparative proteomic study was done by two-dimensional gel electrophoresis followed by mass spectrometry. Sera from each of chronically infected, liver cirrhosis and HCC in HBV or HCV infection along with controls were selected. Analysis of functional association between differentially expressed proteins with viral hepatitis was extensively carried out. Forty-three differentially expressed spots (≥ 1.5 fold; P < 0.05) on two-dimensional gel electrophoresis were corresponded to 28 proteins by mass spectrometry in variable liver diseases. Haptoglobin protein levels were decreased upon disease progression to HCC due to HBV infection. The other proteins expressed differentially are ceruloplasmin, serum paraoxonase 1, retinol binding protein and leucine rich alpha 2 proteins in plasma maybe associated to HBV HCC. Whereas, upregulation of C4a/C4b showed it as a reliable marker in patients with end stage liver disease related to HCV infection. ApolipoproteinA1 levels in liver diseases in both HBV and HCV infection corresponding to healthy controls may be a common marker for early diagnosis and disease monitoring. Protein interaction studies by extensive pathway analysis using bioinformatics tools such as EnrichNet application and STRING revealed significant associations with specific infections.

Hepatitis C virus infection in a tertiary care hospital in Mumbai, India: Identification of a mixed and novel genotype.

PURPOSE: Hepatitis C virus (HCV) is a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC). HCV being a ribonucleic acid virus has considerable sequence variability. Assessment of viral load and genotype is necessary for designing treatment strategies and monitoring for viral resistance among HCV-infected cases. HCC is the most common form of liver cancer, often occurring in people with chronic hepatitis B or C. We undertook this study to observe genotype distribution of the virus in HCV patients from Mumbai. MATERIALS AND METHODS: Between January 2017 and December 2017, the study was conducted on 120 chronic hepatitis outpatients from a tertiary care hospital, Mumbai, after obtaining ethics approval. All these diagnosed cases of HCV were subjected to molecular diagnosis in a research institute, Mumbai, by real-time polymerase chain reaction-based techniques. RESULTS: Males were more preponderant than females with HCV infection, and the highest number of HCV-infected cases was observed in the age group of 41-50 years. Genotype 3 (n = 70; 58.3%) accounted for the highest number of cases followed by genotypes 1b (n = 29; 24.2%) and then 1a (n = 14; 11.7%). Mixed genotypes 1b + 3 and individual genotype 4 were found in two cases each (1.7%). A total of three samples (2.5%) were found with untypeable genotype. CONCLUSION: The major HCV genotype observed was 3 which is difficult to treat with direct-acting antivirals, owing to the more rapid progression of liver disease, increased rates of steatosis (non-alcoholic fatty liver disease), a higher risk for cancer (HCC). We believe this study is the first one to address the prevalence of mixed genotypes and untypeable genotype from India.

Individualization of antiretroviral therapy--pharmacogenomic aspect.

Combination therapy with three drug regimens for human immunodeficiency virus (HIV) infection significantly suppresses the viral replication. However, this therapeutic impact is restricted by adverse drug events and response in terms of short and long term efficacy. There are multiple factors involved in different responses to antiretrovirals (ARVs) such as age, body weight, disease status, diet and heredity. Pharmacogenomics deals with individual genetic make-up and its role in drug efficacy and toxicity. In depth genetic research has provided evidence to predict the risk of developing certain toxicities for which personalized screening and surveillance protocols may be developed to prevent side effects. Here we describe the use of pharmacogenomics for optimal use of HAART (highly active antiretroviral therapy).

Role of anti-human lymphocyte culture cytotoxic antibodies in recurrent spontaneous pregnancy loss women.

BACKGROUND: Recurrent spontaneous pregnancy (RSA) is defined as a sequence of three or more consecutive spontaneous abortions. One of the major causes of RSA is immunological where alloimmune antibodies develop towards human leucocyte antigen (HLA) antigens. Earlier research had suggested that anti-HLA antibodies are produced in normal women; studies have been reported that normal pregnant women develop anti-HLA antibodies, mostly after 20-28 weeks of gestation. AIM: To evaluate the role of anti-HLA antibodies in RSA patients MATERIALS AND METHODS: A total of 80 randomly selected couples with unexplained three or more RSA and control group of 50 normal pregnant women were screened for anti-HLA A and B antibodies. The anti-HLA antibodies were analyzed following the standard two-stage NIH microlymphocytotoxicity assay. RESULTS: In our study group a high frequency of anti-HLA antibodies among women with RSA (26.25%) was detected compared to normal pregnant women (8.0%). Most of the sera showed HLA-A and HLA-B antibodies which had high titer, up to a dilution of 1: 4096. CONCLUSION: This incidence of high anti-HLA antibodies in RSA women during early weeks of gestation may explain the recurrent pregnancy loss.