Post-transfusion hepatitis C virus infection among ß-thalassaemic individuals with associated clinical parameters.

Background & objectives: Multiple transfusions in ß-thalassaemia patients undergoing regular transfusion regimen are at a risk of developing transfusion transmitted infections, including hepatitis C virus (HCV). The present study was conducted to investigate the association of HCV viraemia and genotype with clinical parameters in HCV seroreactive ß-thalassaemic individuals. Methods: A total of 172 HCV seroreactive ß-thalassaemic individuals aged between 2-35 yr with at least 25 units of blood transfusion were catagorized into four groups (2-12 yr, group 1; 13-19 yr, group 2; 20-29 yr, group 3; 30-35 yr, group 4). Aged matched control samples (n=87; ß-thalassaemics without HCV infection) were also included. HCV RNA was detected by nested reverse transcriptase polymerase chain reaction (RT-PCR) based on 5' UTR of HCV genome, viral load was determined by real-time RT-PCR. Nested RT-PCR amplified partial core region was used for DNA sequencing. Liver function parameters [serum total bilirubin, alanine aminotransferase (ALT) and aspartate aminotransferase (AST)] were also determined. Results: Of the 172 HCV seroreactive individuals, 59.30 per cent (n=102) were HCV RNA positive. HCV viral load ranged from 173 to 32.04×10[5] IU/ml; 87.65 per cent were infected with HCV genotype 3. Liver enzymes, such as ALT, AST and serum total bilirubin were significantly elevated in all age groups compared to control groups. Serum ferritin levels were found to be high in all individuals, but 16.27 per cent of HCV-infected individuals with >10,000 IU/ml viral load also showed high ferritin levels (>1500 µg/l) where the majority of them were infected with HCV genotype 3. Interpretation & conclusions: HCV genotype 3 was the major circulating genotype among ß-thalassaemia patients in this region. Our findings indicated an association between HCV replication and hepatic iron load and also highlighted the need for sensitive quantitative RT-PCR-based detection of HCV RNA in the high risk population.

Interferon λ3 gene (IL28B) is associated with spontaneous or treatment-induced viral clearance in hepatitis C virus-infected multitransfused patients with thalassemia.

BACKGROUND: Hepatitis C virus (HCV) is the major posttransfusion infection in multitransfused individuals in India with thalassemia major. To our knowledge, this study is the first conducted to correlate and comprehend the effects of the host interleukin (IL)28B gene polymorphism at loci rs12979860 and rs8099917 in spontaneous or interferon (IFN)-induced treatment response in the HCV-seroreactive individuals with thalassemia major. STUDY DESIGN AND METHODS: A total of 557 HCV-seroreactive individuals with thalassemia were processed for HCV viral genotyping and host IL28B single-nucleotide polymorphism analysis at loci rs12979860 and rs8099917. RESULTS: Of 557 individuals, 70.92% were found to be HCV RNA positive with Genotype 3 (95.18%) as predominant strain. A favorable CC allele at locus rs2979860 and TT allele at rs8099917 were 75.31 and 77.16%, respectively, which was strongly associated with spontaneous clearance of infection (p < 0.05). Of 85 IFN-treated cases, 56 achieved sustained virologic response (SVR) whereas 27 were relapsed cases. Among these patients who achieved SVR, a favorable CC/TT allele at rs12979860/rs8099917 was found to be predominant with 76.79 and 66.07%, respectively, whereas in the case of relapsed patients, unfavorable CT (55.56%) and TG (59.26%) alleles were found to be predominant. Additionally, low serum ferritin level was significantly associated with SVR. CONCLUSION: CC at rs12979860 and TT at rs8099917 was strongly associated with spontaneous clearance and SVR in the population with thalassemia. Low age group and low serum ferritin level are important cofactors. This allelic pattern will aid clinicians in making an informed decision about prognosis and therapeutic management.

Changing pattern of dengue virus serotypes circulating during 2008-2012 and reappearance of dengue serotype 3 may cause outbreak in Kolkata, India.

Dengue virus infection is a major cause of morbidity within the endemic tropical and subtropical regions of the world. Dengue virus has four distinct serotypes with specific clinical manifestations. In this study, we observed the changing pattern of dengue serotypes, age-wise dengue infection and useful sero-detection methods needed in a dengue endemic region. We identified dengue serotypes during a period of 5 years among patients with dengue symptoms visiting one of the largest tertiary care infectious disease hospitals of eastern India in Kolkata. A total of 433 dengue RNA positive samples were isolated from 712 acute dengue suspected cases. Age wise distribution highlighted the susceptible age group being >21 years (24.02%) followed by 11-15 years (21.71%) and 5-10 years (21.02%) of the total infected population. Higher numbers of infected cases were found within females as they are involved in more indoor works. The period of study experienced two dengue outbreaks one in 2008 and another in 2012. For early dengue detection, NS1 was found to be more confirmatory than IgM ELISA regarding sensitivity and specificity. DENV-1, 2, and 4 serotypes were the common circulating strains from 2008 until 2010, after which DENV-3 serotype infections rise and led to a massive dengue outbreak in Kolkata with increased numbers of DHF and DSS cases in 2012. The finding within our study emphasizes the public health importance of such prospective surveillance programs with respect to the changing dengue viral etiology and serotypes. J. Med. Virol. 88:1697-1702, 2016. © 2016 Wiley Periodicals, Inc.

Cone-beam computed tomographic evaluation to estimate the prevalence of palatogingival groove in the maxillary anterior teeth and its radiographic characteristics: An institutional retrospective study.

Aim: The purpose of the study was to radiographically evaluate the prevalence of palatogingival groove (PG) in the East Indian population in the maxillary anterior teeth in different genders, its unilateral/bilateral presentation, classified based on its radiographic characteristics, to determine the prevalence of different types, which could help in future treatment planning. Study Design: The design of the study was a retrospective study. Materials and Methods: Analysis of 429 maxillary anterior teeth (144 central incisors, 145 lateral incisors, and 139 canines) in 72 cone-beam computed tomography scans was done (31 males and 41 females, mean age 27.3 ± 7.63). Demographic details of patients and characteristics of PG, i.e. location, extension, depth, and type, were recorded. The presence of alveolar bone loss and periapical pathology was noted. Results: An overall prevalence of PG was found to be 2.33% (n = 10), with PG being detected in 2 (1.388%) central incisors, 8 (5.51%) lateral incisors, and 0 (0%) canines. Eight of the patients had a unilateral presence, while one patient reported with bilateral presence, implying a significantly higher predilection of unilateral occurrence (P = 0.02). The prevalence was found to be higher in females (n = 8). The teeth were categorized as either having Type I (6 teeth), Type II (3 teeth), or Type III (1 teeth). Three of the 10 PGs were present in the mesial, six in the mid-palatal, and one in the distal portion of the palatal surface. Conclusions: The prevalence of PG in the maxillary incisors in this cohort is 2.33%. The maxillary lateral incisors are the most affected teeth. Unilateral presentation is more common.

See-through "C": Case series.

Successful endodontic treatment relies upon a thorough knowledge of root canal anatomical variations along with proper diagnosis, treatment planning, and clinical expertise. One of the difficult root canal configurations that are frequently encountered commonly in mandibular second molars is C-shaped root canal. Due to the intricate root canal configuration, it is often difficult to negotiate, debride, and obturate such canals leading to failure of root canal treatment. Understanding the anatomical variation and adequate visualization will enable the clinician to manage these cases effectively. Advanced irrigation and obturation techniques help in managing such anomalous canal configurations. This article presents the management of two different C-shaped root canal configurations under dental operating microscope using thermoplasticized obturation techniques.

Recent pattern of Co-infection amongst HIV seropositive individuals in tertiary care hospital, Kolkata.

BACKGROUND: Opportunistic Infections (OIs) and co-infections are the major cause of deaths amongst HIV infected individuals and this mostly depends upon the risk factors, type of exposure and geographic region. The commonest types of infections reported are tuberculosis, chronic diarrhoea, oral candidiasis, herpes simplex virus-2, cytomegalovirus, hepatitis B virus and hepatitis C virus. Due to the scarcity of OIs data available from this region, we had designed a study to determine the frequency of different OIs amongst HIV seropositive patients. METHODS: Analysis of the different spectrum of OIs/Co-infections were carried out with 204 HIV sero-positive patients (142 males and 62 females) who visited the HIV/AIDS Apex Clinic in a tertiary care hospital from March 2006 to March 2009. The CD4+ count was estimated using FACS Calibur, the routine smear test, serology, nested RT-PCR and DNA sequencing were carried out to determine the different OIs. RESULTS: In this study, HIV seropositive patients were mostly from middle age group (31-40 yrs) with CD4+ counts in majority of symptomatic AIDS patients below 200 cells/mm3. The common co-infections/opportunistic infections were OC (53.43%), CD (47.05%), HSV-2 (36.76%), TB (35.29%), CMV (26.96%), HBV (15.19%) and HCV (7.35%). Dual infections, like HSV-2 & CMV (15.38%), HSV-2 & TB (14.61%), HSV-2 & oral candidiasis (24.61%) and CMV & oral candidiasis (14.61%) were significant in follow-up patients. Triple infections were also common e.g., TB, CD, OC infection occurring frequently in about 14.21% of the study population. Multiple infections like OC, TB, CD amongst the viral co-infected patients with HSV-2, HCV, CMV and HBV are also reported in this study. The genotyping analysis of the HCV co-infected HIV individuals shows that two belonged to HCV genotype 1 and 8 belonged to genotype 3. CONCLUSIONS: A wide spectrum of OIs were observed amongst HIV-infected patients in the HIV/AIDS Apex Clinic. Oral candidiasis, CD, CMV and HSV-2, were the common OIs in those patients. This study aims to provide a clearer picture regarding infections occurring amongst HIV seropositive individuals so that the scientific findings could be translated into sustainable prevention programmes and improved public health policies. TRIAL REGISTRATION: None.

Next generation sequencing in lung cancer: An initial experience from India.

INTRODUCTION: Approximately 35% of NSCLC patients in East Asia have EGFR mutations. Next-generation sequencing (NGS) provides a comprehensive mutational profile in lung cancer patients. MATERIAL AND METHOD: Clinicopathologic characteristics and mutational profiling data was analyzed from nonsmall cell lung carcinoma /Adenocarcinoma over a duration of 42 months (October 2014 to March 2018) using next-generation sequencing Ion Ampliseq Cancer Hotspot panel v2 (Ampliseq, Life Technologies) on the Ion torrent PGM platform. RESULTS: A total of 154 cases were processed during this period. The average number of mutations/case varied from one to four 72.07% (111/154), of these cases had minimum one genetic alteration. The most common mutated gene was TP53 gene (37.6%, n = 58) followed by EGFR (32.4%, n = 50), KRAS (18.18%, n = 28), ERBB2 (3.2%, n = 5), BRAF (1.94%, n = 3). EGFR positivity was more in females (43.3%) and non-smokers (52.08%) in comparison to males (26.7%) and smokers (16.1%). CONCLUSION: In this paper, we have described the comprehensive mutational profiling of a large cohort of advanced lung adenocarcinoma patients from the eastern part of India. To the best of our knowledge, this is one of the largest studies from the country describing mutations in BRAF, ERBB2, TP53 genes and their clinicopathologic/histopathologic associations in lung cancers.

MYD88 and CXCR4 Mutation Profiling in Lymphoplasmacytic Lymphoma/Waldenstrom's Macroglobulinaemia.

Recurrent mutations affecting MYD88 and CXCR4 gene nowadays form the basis for the diagnosis, risk stratification and use of inhibitors targeting these signalling pathways in LPL/WM which are rare B cell neoplasms. MYD88 L265P mutation analysis was performed on 33 cases of LPL/WM by AS-PCR (positivity-84.8%, n = 28/33) and by Sanger sequencing (positivity-39.3%, n = 13/33). We had only two cases with CXCR4 non-sense (NS) mutation (p.S338*) using Sanger sequencing. MYD88 (L265P) mutation detection by AS-PCR can form reliable biomarker for the diagnosis of LPL/WM in molecular labs. Although the cohort is small, still the CXCR4 mutation frequency in our study is low as compared to the published literature.

Recombination in hepatitis C virus is not uncommon among people who inject drugs in Kolkata, India.

Recombination in RNA virus is a rare event in the survival and evolution to evade host immune system. This is increasing within high risk group population (HRG) due to super infection that occurs by continuous sharing of common drug equipment by HCV infected or HIV-HCV co-infected recurrent drug users. Recombination causes impediment to vaccine development and therapeutic intervention as standard HCV treatment is still genotype specific. Blood samples of 194 people who inject drugs (PWID) were collected from an Opioid Substitution Therapy Centre in Kolkata, India. HCV sero-reactivity was checked by ELISA. Detection of HCV RNA by nested RT-PCR and genotyping by DNA sequencing were done. Phylogenetic analysis, Simplot, Bootscan plot, Recombination Detection Program were used for recombinant strain identification. Out of 80 HCV sero-reactive samples, 77 were RNA positive (96.25%). Out of 74 HIV mono-infected individuals, 12 HCV sero-nonreactive samples were HCV RNA positive. Out of total 89 RNA positive samples, 64 paired partial core and NS5B region (71.9%) were sequenced by Sanger's method. Two major genotypes (1 and 3), four subtypes and an inter-genotype recombinant strain (3a/1a) with a novel breakpoint in the NS4B coding region were found.

Evaluation of Mirasol pathogen reduction system by artificially contaminating platelet concentrates with Staphylococcus epidermidis: A pilot study from India.

BACKGROUND AND OBJECTIVES: This study was conducted to assess the efficacy of Mirasol pathogen reduction system for platelets aimed at preventing bacterial regrowth by spiking buffy coat pooled platelets (BCPP) with clinically relevant load of Staphylococous epidermidis. MATERIALS AND METHODS: BCPP units were prepared using Teruflex BP-kit with Imugard III-S-PL (Terumo BCT, Tokyo, Japan). Two BCPP units were pooled, of which 40 ml of negative control (NC) was removed. The remaining volume of the platelet unit was inoculated with clinically relevant load of bacteria (total of 30 CFU of S. epidermidis in 1 ml); following this the platelet unit was split into two parts. One part served as positive control (PC) and the other part was subjected to pathogen reduction technique (Mirasol PRT, CaridianBCT Biotechnologies, Lakewood, CO, USA). Bacterial detection was performed using BacT/ALERT system, controls after day 1 and day 7 following inoculation of bacteria and on day 7 for Mirasol-treated unit. RESULTS: Of the 32 treatment cycles, 28 were valid and 4 were invalid. No regrowth was observed in 96.4% (27 of 28) after treatment with Mirasol pathogen reduction system. Of four invalid tests, on two instances the NC showed growth, whereas in other 2 no regrowth was detected in 7(th) day PC. Bacterial screening of PCs by BacT/ALERT after 24 h of incubation was 28.6%, whereas the effectiveness increased to 100% when incubated for 7 days. CONCLUSIONS: Mirasol system was effective in inactivating S. epidermidis when it was deliberately inoculated into BCPP at clinically relevant concentrations. Such systems may significantly improve blood safety by inactivating traditional and emerging transfusion-transmitted pathogens.

Current molecular methods for the detection of hepatitis C virus in high risk group population: A systematic review.

Hepatitis C virus (HCV) is an emerging infection worldwide and the numbers of persons infected are increasing every year. Poor blood transfusion methods along with unsafe injection practices are potential sources for the rapid spread of infection. Early detection of HCV is the need of the hour especially in high risk group population as these individuals are severely immunocompromised. Enzyme Immunoassays are the most common detection techniques but they provide no evidence of active viremia or identification of infected individuals in the antibody-negative phase and their efficacy is limited in individuals within high risk group population. Molecular virological techniques have an important role in detecting active infection with utmost specificity and sensitivity. Technologies for assessment of HCV antibody and RNA levels have improved remarkably, as well as our understanding of how to best use these tests in patient management. This review aims to give an overview of the different serological and molecular methods employed in detecting HCV infection used nowadays. Additionally, the review gives an insight in the new molecular techniques that are being developed to improve the detection techniques particularly in High Risk Group population who are severely immunocompromised.

Transmission dynamics of hepatitis C virus among intra venous drug users in the border state of Manipur, India.

Intra venous drug users (IVDUs) are at high risk for hepatitis C virus (HCV) infection owing to their high rate of drug abuses. The north-eastern part of India has a high prevalence of IVDUs with Manipur being the worst hit state. The aim of the study was to document the molecular epidemiology, the patterns of HCV transmission, genomic variation and recombination events within HCV genome among IVDUs of Manipur, India. 91 anti-HCV sero-reactive blood samples were collected from IVDUs in Manipur. The samples were processed for RNA extraction, nested RT-PCR, sequencing and quantitative viral RNA estimation. Phylogeographic analysis of the sequenced core and NS5B regions of HCV genome was performed to determine the probable transmission route and recombinant HCV strains. 83 out of 91 anti-HCV seropositive samples were RNA positive (91.20%) based on 5'UTR of HCV genome by nested RT-PCR. Of the RNA positive samples, 73 paired partial core and NS5B gene were sequenced. Three major genotype and eight subtypes were detected while no recombinant strains were found. Individuals with genotype 1 had the mean viral load (5.94 ± 0.705 log10IU/ml) followed by genotype 3 (4.91 ± 0.49 log10IU/ml) and 6 (3.96 ± 0.32 log10IU/ml). The viral load was statistically significant among the male individuals at 4.822 ± 1.36 log10IU/ml compared to 4.767 ± 0.49 log10IU/ml for females (t=3.249, p<0.005). The phylogeographic results indicated 3b, 6h originated from Vietnam, 1a had Indian origin, 3a, 6k originated from southern China while 1b originated from Myanmar, respectively. The incidence of eight different subtypes in Manipur reflects the transmission of these strains from the "Golden Triangle" drug trafficking regions. Sequence analysis confirmed the transmission routes of HCV, which is linked to China and Vietnam for the newly emergent genotype 6 in north-eastern India.

Impact of host IL28B rs12979860, rs8099917 in interferon responsiveness and advanced liver disease in chronic genotype 3 hepatitis C patients.

BACKGROUND AND AIMS: Genetic polymorphisms near interleukin 28B gene are associated with spontaneous and treatment induced clearance of hepatitis C virus (HCV). Our objective was to evaluate the impact of interleukin 28B single nucleotide polymorphism (rs12979860, rs8099917) variability in HCV genotype 3 infected populations. METHODS: 400 hepatitis C seroreactive patients from different population groups in Eastern and North Eastern part of India were assessed for host and viral genotypic analysis. 83 HCV genotype 3 infected patients were administered pegylated interferon- ribavirin therapy. Viral genotyping was performed using nested reverse transcriptase-PCR followed by direct sequencing methods. Host interleukin 28B genotyping was performed using real-time PCR based single nucleotide polymorphism analysis. RESULTS: Out of 400 hepatitis C seroreactive individuals, 73.25% were found to be RNA positive. HCV genotype 3 (65.87%) was found to be the major circulating strain in this region followed by genotype 1 (32.08%). rs12979860 CC genotype was significantly associated with sustained virological response in HCV genotype 3 infected population. In patients achieving rapid virological response, favourable CC/TT allele at rs12979860, rs8099917 was found to be predominant at both the alleles at 77%, 73.2% respectively; whereas in case of patients with relapsed HCV infection CT, TG alleles were found to be predominant. Additionally, CC genotypes at rs12979860 were found to be associated with sustained virological response in patients with high viral load (OR = 6.75, 0.05

Modulation of TLR 3, 7 and 8 expressions in HCV genotype 3 infected individuals: potential correlations of pathogenesis and spontaneous clearance.

BACKGROUND: Hepatitis C virus is the major cause of chronic hepatitis worldwide which finally leads to the development of hepatocellular carcinoma. Toll like receptors (TLRs) play an important role in the course of many viral infections, but the role of TLRs in HCV pathogenesis has not been well elucidated so far. OBJECTIVE: The aim of this study was to analyse the mRNA expression of TLRs 3, 7, and 8 in different stages of HCV infection including chronic, cirrhosis, interferon treated resolved, and relapsed cases. METHODOLOGY: Total RNA from whole blood was extracted and mRNA expression of TLRs 3, 7, and 8 genes was analyzed by quantitative real-time RT-PCR using ß-Actin gene as an internal control. RESULTS: This study consisted of 100 HCV infected individuals and twenty healthy controls. TLR 3 expression was found to be significantly elevated in individuals who had spontaneously cleared the virus (p < 0.001), whereas TLR 7 was found to be 3.26 times more elevated in patients with cirrhosis of liver. In IFN induced individuals, TLR 8 expression levels were found to be 2.28-fold elevated as compared to control population. CONCLUSION: TLRs 3, 7, and 8 are prime biomarker candidates for HCV infection mRNA expression analysis which might improve current therapeutic approaches.

Development of rapid, sensitive one-tube duplex RT-PCR assay for specific and differential diagnosis of Chikungunya and dengue.

Chikungunya and dengue, two arboviral infections are common in South-East Asia and their early clinical manifestations are very similar hence it is important to discriminate between them as early as possible for better clinical management. The aim of this study was to design a rapid, sensitive and specific method for the differential diagnosis of these two viruses simultaneously. A rapid one-tube duplex RT-PCR assay was developed that requires 110 min including RNA extraction, RT-PCR and agarose gel electrophoresis by using a novel Taq polymerase with high processivity. This one-tube duplex RT-PCR system with primers designed from the conserved regions of the genome allowed discrimination between the two viral groups. Bioinformatics analysis of the DNA sequences from PCR amplified products confirmed that this method was very specific and accurate. The time required for this duplex RT-PCR was comparable to the standard IgM capture ELISA method. This novel approach would help to diagnose specifically and accurately these two closely related arboviruses and enable early detection from blood. This method could be applied in resource limited settings, for surveillance in endemic regions or for routine epidemiological screening.