Sofosbuvir and Velpatasvir combination is safe and effective in treating chronic hepatitis C in end-stage renal disease on maintenance haemodialysis.

BACKGROUND & AIMS: There is emerging data on the use of Sofosbuvir-based directly acting antiviral (DAA) drug regimens in chronic hepatitis C (CHC) patients with end-stage renal disease (ESRD) on maintenance haemodialysis (MHD). We evaluated the safety and efficacy of Sofosbuvir plus Velpatasvir fixed-dose combination in CHC patients with ESRD on MHD. METHODS: Fifty-one CHC patients with ESRD on MHD were included in a real-life prospective study. All patients irrespective of genotype; presence of cirrhosis; treatment naive or experienced status were treated with full-dose Sofosbuvir (400 mg) plus Velpatasvir (100 mg) fixed-dosed combination given daily for 12 weeks. The efficacy was assessed by the sustained virological response (SVR12) with negative HCV RNA 12 weeks after the end of treatment (ETR). Side effects if any were recorded in all patients. RESULTS: The median HCV RNA level in 51 CHC patients [Males 41 (80.4%), mean age 42.8 ± 14.6 years] was 2.0 × 106 IU/mL. HCV genotype was available in 19 patients with predominant genotype 1 in 15 (79%) patients. Ten (19.6%) patients had evidence of cirrhosis (defined as LSM ≥ 12.5 kPa on Transient Elastography), and 8 (15.6%) patients were treatment experienced. Testing for ETR was done in 36 patients and all 36 (100%) patients achieved ETR, and 49 patients (96%) achieved SVR 12. All 51 patients tolerated the Sofosbuvir + Velpatasvir combination, with none of the patients reporting any serious adverse event. CONCLUSION: Sofosbuvir plus Velpatasvir fixed-dose combination is safe and effective in treating CHC in patients with ESRD on MHD.

Identification of T cell and B cell epitopes against Indian HCV-genotype-3a for vaccine development- An in silico analysis.

Hepatitis C virus (HCV) infects almost 150 million people and is a leading cause of liver disease worldwide. It has been classified into seven genotypes; the most common genotype affecting Indian population is genotype 3 (60-70%). Currently there is no vaccine for any genotype of HCV. In order to develop peptide based vaccine against HCV, it is important to identify the conservancy in the circulating genotypes, along with the Human Leucocyte Antigen (HLA) alleles in the target population. The present study aims to identify conserved CD4 and CD8 T cells and B cell epitopes against Indian HCV-genotype-3a using an in silico analysis. In the present study, 28 promiscuous CD4 T cell epitopes and some CD8 epitopes were identified. The NS4 region was predicted to be the most antigenic with maximum number of conserved and promiscuous CD4 T cell epitopes and CD8 T cell epitopes having strong and intermediate affinity towards a number of HLA alleles prevalent in Indian population. Additionally, some linear B cell epitopes were also identified, which could generate neutralizing antibodies. In order to ascertain the binding pattern of the identified epitopes with HLA alleles, molecular docking analysis was carried out. The authors suggest further experimental validation to investigate the immunogenicity of the identified epitopes.

Detailed investigation of ongoing subclinical hepatitis E virus infections; occurring in outbreak settings of North India.

BACKGROUND & AIMS: Every year globally WHO reports 20 million Hepatitis E virus (HEV) infections. The disease occurs as sporadic cases or focused outbreaks and has potentials to cause massive epidemics. The reservoir of HEV during inter-epidemic period is not well characterized. The sporadic cases usually lack history of contact with clinically overt HEV patients. In the present context we evaluated the occurrence of subclinical HEV as a possible reservoir in endemic region. METHODS: Blood samples were collected from 67 apparently healthy individuals and 10 acute viral hepatitis (AVH) patients during two HEV outbreaks in North India. The serum samples were tested for anti-HEV IgM, IgG, HEV-IgG avidity index, HEV viral load and conventional-PCR followed by sequencing and phylogenetic analysis. RESULTS: A total of 14 (20.89%) apparently healthy individuals showed the presence of anti-HEV IgM and IgG. Of 14 based on HEV-IgG avidity index, 9 (64.28%) had secondary-exposure, 4 (28.57%) had primary exposure, while one patient had intermediate avidity. Subclinical subjects with primary exposure had significantly higher anti-HEV IgM index as compared to secondary-exposure (P = 0.0028). Viral load in clinically jaundiced patients was significantly higher as compared to subclinical subjects (P < 0.0001). Phylogenetic analysis showed HEV sequences retrieved from subclinical individuals clustered along with AVH patients, suggesting matched source. The significantly low viral load in subclinical subjects hints towards the dose dependency for progression of clinical manifestation. CONCLUSION: We document subclinical HEV with low level viremia occurs during outbreak settings and goes un-noticed, which helps maintaining the virus in nature possibly leading to its endemicity.

Assessment of dried blood samples as an alternative less invasive method for detection of Hepatitis E virus marker in an outbreak setting.

Hepatitis E virus (HEV) is the causative agent of hepatitis E. It can be asymptomatic, associated with acute self-limiting hepatitis or acute liver failure. The conventional diagnosis of HEV infection relies on anti-HEV IgM serology. The collection of blood samples by venepunture for laboratory confirmation is often difficult during an outbreak. Thus, testing the specimens of dried blood spots (DBS) on filter papers can prove to be a feasible alternative. The present study aimed to evaluate the applicability of anti-HEV IgM detection from DBS samples and the stability of anti-HEV IgM detection at varied time interval, at various storage temperatures. Paired blood and DBS sample were collected from 44 jaundiced patients and eight healthy controls during HEV outbreaks. The DBS were tested for anti-HEV IgM by available ELISA kit with in-house modifications. Three cut offs were determined, that is, the CO1: kit cut-off, CO2: mean of negative controls above 3SD and CO3: area under Receiver operating Curve. The sensitivity of anti-HEV IgM detection ranged from 86-91%. The maximum sensitivity (91%) and specificity (100%) was obtained using CO3. Maximum stability of anti-HEV IgM antibodies (100%) was observed till 65 days at 4°C. Storage at 37°C significantly reduced anti-HEV IgM positivity, wherein 42.85% sample became negative by 45 days. DBS showed good sensitivity and specificity for detecting anti-HEV IgM and can be considered an alternate to serum sample. Moreover, anti-HEV IgM was stable at 4°C, which makes DBS a preferred method for storage and transportation of the sample to reference laboratory.

Ribonucleic acid extraction from archival formalin fixed paraffin embedded myocardial tissues for gene expression and pathogen detection.

INTRODUCTION: Archival tissue samples preserved in formalin are a great source of treasure for biomedical research and diagnostics. Formalin, though is a good preservative, causes the modification of nucleic acid limiting the application of fixed tissues. The present study evaluated three methods of RNA extraction for constitutive gene expression and pathogen detection. MATERIAL AND METHODS: Sixteen archival formalin-fixed paraffin-embedded (FFPE) myocardial tissues were subjected to RNA extraction by Trizol, SDS, and RNeasy FFPE kit followed by RT-PCR and Taqman Real-Time PCR to study the expression of housekeeping genes. RESULTS: RNA was extracted from all 16 myocardial tissues (100%) by RNeasy FFPE kit, as compared to 14/16 by Trizol and 8/10 by SDS methods. The expression of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)was observed in RNA extracted by RNeasy FFPE kit and Trizol. High yield of RNA was obtained by RNeasy FFPE kit than Trizol (P = 0.002) and SDS(P = 0.012). Of the three methods, RNeasy FFPE kit was evaluated for Enterovirus RNA detection in 16 other histopathologically confirmed FFPE tissues of dilated cardiomyopathy (DCM) cases and Enterovirus genome was detected in 4/16 (25%) FFPE tissues of DCM cases. The enteroviral sequences of the viral isolates revealed 99% homology with Human coxsackievirus B5. CONCLUSION: The Qiagen RNeasy FFPE kit resulted in significantly high reproducibility of RNA from FFPE myocardial tissues, which are suitable for amplification by Taq-Man Real-Time and RT-PCR. Thus, the results show that these FFPE tissues can be used for gene expression, pathogen detection, and epidemiological studies.

Chronic Hepatitis E With Genotype 1-Masquerading as Allograft Rejection After LiverTransplantation.

Hepatitis E is one of the leading causes of acute viral hepatitis worldwide. Chronic infection with hepatitis E is less common and limited to immunosuppressed patients and is usually due to genotype 3 of the virus. Genotype 1, the most prevalent strain in the South Asian region, is seldom known to be associated with chronic hepatitis. Here we describe a case of chronic hepatitis E with genotype 1 in a post-liver transplant setting. In the index case, previously compensated cryptogenic cirrhosis was decompensated by an acute hepatitis E infection, which necessitated liver transplantation because of acute chronic liver failure. This later progressed to chronicity. This case may have significant implications in management, especially in the post-liver transplant setting.

Institutional outbreak of rubella in a healthcare center in Chandigarh, North India.

Rubella is traditionally considered a childhood disease but it has the potential to cause outbreaks in closed communities when a susceptible population accumulates. The present study reports an outbreak of rubella among healthcare workers in the pediatric center of a tertiary care North Indian hospital. The cases of rubella were identified by clinical features and confirmed by the detection of anti-rubella IgM antibodies in blood by enzyme linked immunosorbent assay. A total of 23 cases of rubella occurred over a period of one and a half month, out of which 9 (39%) were males. All the patients were in the age group of 21-35 years. None of the patients gave a history of rubella vaccination. This outbreak of rubella occurred due to the accumulation of a susceptible population in a closed hospital environment. There is need for the introduction of rubella vaccination in healthcare workers to prevent outbreaks at work place.

Effect of Acyclovir Solid Lipid Nanoparticles for the Treatment of Herpes Simplex Virus (HSV) Infection in an Animal Model of HSV-1 Infection.

BACKGROUND: Acyclovir use is limited by a high frequency of administration of five times a day and low bioavailability. This leads to poor patient compliance. OBJECTIVES: To overcome the problem of frequent dosing, we used nanotechnology platform to evaluate the proof of concept of substituting multiple daily doses of acyclovir with a single dose. METHODS: Acyclovir was formulated as solid lipid nanoparticles (SLN). The nanoparticles were characterized for particle size, surface charge and morphology and in vitro drug release. The pharmacokinetic and pharmacodynamic of SLN acyclovir were compared with conventional acyclovir in a mouse model. RESULTS: SLN showed drug loading of 90.22% with 67.44% encapsulation efficiency. Particle size was found to be of 131 ± 41.41 nm. In vitro drug release showed 100% release in SIF in 7 days. AUC0-∞ (119.43 ± 28.74 µg/ml h), AUMC0-∞ (14469 ± 4261.16 µg/ml h) and MRT (120.10 ± 9.21 h) were significantly higher for ACV SLN as compared to ACV AUC0-∞ (12.22 ± 2.47 µg/ml h), AUMC0-∞ (28.78 ± 30.16 µg/ml h) and MRT (2.07 ± 1.77 h), respectively (p<0.05). In mouse model, a single dose of ACV SLN was found to be equivalent to ACV administered as 400mg TID for 5 days in respect to lesion score and time of healing. CONCLUSION: The proof of concept of sustained-release acyclovir enabling administration as a single dose was thus demonstrated.

Clinical profile and neurodevelopmental outcome of new-onset acute symptomatic seizures in children.

PURPOSE: To study clinical profile, neurodevelopmental outcome and its predictors in children with acute symptomatic seizures (ASS). METHODS: Short-term neurodevelopmental outcome and predictors of poor outcomes were prospectively assessed in 105 consecutive children with ASS aged 3 months-12 years RESULTS: Mean age was 51.2+42.2months (3-144 months); 67.2% were males. Central nervous system (CNS) infection in 82%, status epilepticus in 15.2%, abnormal neuroimaging in 62.8% and abnormal electroencephalography in 22.3% were noted. At discharge, 27.6% had poor outcome including death (13%); CNS infections were significantly associated with poor outcome compared to ASS of other aetiologies (32.6% vs 5.2%, p=0.02). Low GCS (OR 4.9, 95%CI 1.2-20.7), abnormal electroencephalograph (OR 4.3, 95%CI 1-16.9) and neuroimaging (OR 12.1, 95%CI 1.4-105.2) were independent predictors of poor outcome. After 6 months, 16% children had delayed neurodevelopment and cognition; 6% had seizure recurrences. Abnormal electroencephalograph (p=0.002; OR 6.8, 95%CI 2.0-23.1), abnormal neuroimaging (p=0.015; OR 9.47, 95%CI 1.18-75.8),>1 anti-epileptic (p=0.00; OR 9.9, 95%CI 2.88-33.9), intubation (p=0.004; OR 6.25, 95%CI 1.79-21.7) and poor outcome at discharge (p=0.02; OR 4.44, 95%CI1.38-14.2) predicted abnormal neurodevelopment. CONCLUSIONS: CNS infections are the most common cause of ASS in children from developing countries. Abnormal neurodevelopment and seizure recurrences on short-term follow-up are seen in a minority of children.

Clinical applicability of various dengue diagnostic tests in resource-limited endemic settings.

INTRODUCTION: Dengue is one of the most important arboviral infections caused by one of the four dengue serotypes, 1-4. OBJECTIVE: To study the applicability of different diagnostic methods in diagnosis of dengue viral infection. MATERIALS AND METHODS: A total of 2101 blood samples were collected for confirmation of dengue viral infection. All the samples were tested by dengue-specific IgM ELISA, of which 111 were also tested for NS1 antigen detection and 27 acute samples (≤5 days) were further subjected for viral RNA detection by RT-PCR and isolation in C6/36 cell line. To detect the sensitivity of NS1 antigen for different dengue virus serotypes, four dengue serotype 1 and 12 dengue 3 were subjected for the NS1 antigen assay. RESULTS: Most common age group affected was 16-45 years, with male to female ratio of 2.8:1. During first 3 days of illness virus isolation and RT-PCR were the most sensitive (83%) followed by NS1 antigen detection (75%) and IgM detection (37.5%). The positivity of IgM detection was found to be significantly higher as compared to NS1 detection during 4 to 5 days and also after 5 days of illness (P < 0.05). Dengue serotypes 1 and 3 were found to be co-circulated, dengue 1 being the predominant serotype. CONCLUSION: Virus isolation and RT-PCR were the most sensitive tests during the early period of illness whereas beyond third day, IgM antibody detection was found to be the most sensitive method of dengue diagnosis.

Rubella seronegativity among health care workers in a tertiary care north Indian hospital: implications for immunization policy.

BACKGROUND: Rubella is traditionally considered a childhood disease but has the potential to cause outbreaks in hospital set ups. It is important to know the susceptibility status of health care workers (HCWs) as to frame guidelines for their immunization and thus prevent hospital outbreaks. PARTICIPANTS: The rubella susceptibility status of 313 HCWs working in the institute was assessed. This study was initiated after we reported an outbreak due to rubella among HCWs of our institute. MATERIALS AND METHODS: The serum samples were tested to determine Rubella IgG titres by enzyme linked immunosorbent assay (ELISA). RESULTS: Overall, 48 (15.3%) subjects were found to be negative, thereby indicating their susceptibility to infection. Out of them, 29 (60.5%) were in contact with pregnant women during the course of their employment. There is a risk of nosocomial transmission of rubella from affected HCWs to their contacts especially pregnant women as many of the rubella infections are asymptomatic. CONCLUSION: Hence, we stress the need for vaccinating the HCWs at the start of their employment to contain the spread of infection and also to reduce the risk of outbreaks in work place.

Immunogenicity and safety of two schedules of Hepatitis B vaccination during pregnancy.

AIM: To test the immunogenicity and safety of HepatitisB (HB) vaccination during pregnancy and passive transfer of antibodies to the newborn. METHODS: Ninety-nine HBsAg-negative pregnant women were divided randomly in two groups; group I was given two, and group II was given three doses of recombinant Hepatitis B vaccine and followed up until 4 months after delivery. RESULTS: The vaccination was safe. The maternal anti-HB antibody levels at delivery were significantly higher in three dose schedule as compared to two dose schedule. Similarly, the antibody levels in the newborns were higher in the three dose schedule. The maternal antibody levels were higher at 2 and 4 months post-delivery in group II but were observed to be declining in the newborns. CONCLUSION: Hepatitis B vaccination during pregnancy is safe and highly immunogenic, and there is passive transfer of antibodies to the newborns.