Exosome-associated microRNA-375 induces cell proliferation by regulating IGFBP4 upon hepatitis C virus infection.

Hepatitis C virus (HCV) infection is one of the most common causes of liver cancer. HCV infection causes chronic disease followed by cirrhosis, which often leads to hepatocellular carcinoma (HCC). In this study, we investigated the roles of exosome-associated miRNAs in HCV-induced disease pathology. Small RNA sequencing was performed to identify miRNAs that are differentially regulated in exosomes isolated from patient sera at two different stages of HCV infection: cirrhosis and hepatocellular carcinoma. Among the differentially expressed miRNAs, miR-375 was found to be significantly upregulated in exosomes isolated from patients with cirrhosis and HCC. A similar upregulation was observed in intracellular and extracellular/exosomal levels of miR-375 in HCV-JFH1 infected Huh7.5 cells. The depletion of miR-375 in infected cells inhibited HCV-induced cell migration and proliferation, suggesting a supportive role for miR-375 in HCV pathogenesis. miR-375, secreted through exosomes derived from HCV-infected cells, could also be transferred to naïve Huh7.5 cells, resulting in an increase in cell proliferation and migration in the recipient cells. Furthermore, we identified Insulin growth factor binding protein 4 (IGFBP4), a gene involved in cell growth and malignancy, as a novel target of miR-375. Our results demonstrate the critical involvement of exosome-associated miR-375 in HCV-induced disease progression.

Dysregulated metabolites and lipids in serum of patients with acute hepatitis E: A longitudinal study.

Hepatitis E is a disease associated with acute inflammation of the liver. It is related to several dysregulated metabolic pathways and alterations in the concentration of several metabolites. However, longitudinal analysis of the alterations in metabolites and lipids is generally lacking. This study investigated the changes in levels of metabolites and lipids over time in sera from men with acute hepatitis E compared to healthy controls similar in age and gender. Untargeted measurement of levels of various metabolites and lipids was done using mass spectrometry on 65 sera sequentially sampled from 14 patients with acute hepatitis E and 25 serum samples from five controls. Temporal changes in intensities of metabolites and lipids were determined over different times at 3-day periods for the hepatitis E virus (HEV) group. In carbohydrate metabolism, glucose levels, fructose 1-6-bisphosphate and ribulose-5-phosphate were increased in the HEV-infected persons compared to the healthy controls. HEV infection is significantly associated with decreased levels of inosine, guanosine, adenosine and urate in purine metabolism and thymine, uracil and ß-aminoisobutyrate in pyrimidine metabolism. Glutamate, alanine and valine levels were significantly lower in the HEV group than in healthy individuals. Homogentisate of tyrosine metabolism and cystathionine of serine metabolism were increased, whereas kynurenate of tryptophan metabolism decreased in the HEV group. Metabolites of the bile acid biosynthesis, urea cycle (arginine and citrulline) and ammonia recycling (urocanate) were significantly altered. Co-enzymes, pantothenate and pyridoxal, and co-factors, lipoamide and FAD, were elevated in the HEV group. The acylcarnitines, sphingomyelins, phosphatidylcholine (PC), phosphatidylethanolamine (PE), lysoPC and lysoPE tended to be lower in the HEV group. In conclusion, acute hepatitis E is associated with altered metabolite and lipid profiles, significantly increased catabolism of carbohydrates, purines/pyrimidines and amino acids, and decreased levels of several glycerophospholipids.

Lactoferrin in breast milk-based powders.

This study aimed to determine lactoferrin (LF) in breast milk-based powders and formulas. Lactoferrin is an important whey protein in all mammalian milks and is responsible in large part for the known antimicrobial effects of human milk in particular. As breast feeding is not always possible, formulas based on cows milk have been developed in order to meet the nutritional needs of the newborn, while more recently human breast milk-based powders have been introduced to offer the biological functionality of human milk to pre-term and critically ill babies. In the present work, the amount of LF in commercial breast milk-based powders was tested by a validated RF-HPLC method for the determination of LF in breast milk in order to examine both the applicability of the method but at a second level the amount of LF in these commercial products. The detection of LF was possible but the complexity of the matrix lead us to the use the standard addition methodology in order to achieve quantification. The results indicated that breast milk-based powders had higher amount of LF than cows milk-based formulas, both non-fortified and fortified.

Enhanced fitness of hepatitis C virus increases resistance to direct-acting antivirals.

Drug resistance mutations of hepatitis C virus (HCV) negatively impact viral replicative fitness. RNA viruses are known to change their replication behaviour when subjected to suboptimal selection pressure. Here, we assess whether mutation supply in HCV is sufficiently large to allow the selection of its variants during dual or triple direct-acting antiviral (DAA) treatment associated with augmented virus fitness or impairment. We engineered randomly mutagenized full-genome libraries to create a highly diverse population of replication-competent HCV variants in cell culture. These variants exhibited escape when treated with NS5A/NS5B inhibitors (daclatasvir/sofosbuvir), and relapse on treatment with a combination of NS3/NS5A/NS5B inhibitors (simeprevir or paritaprevir/daclatasvir/sofosbuvir). Analysis of the relationship between virus fitness and drug resistance of JFH1-derived NS5A-5B variants showed a significant positive correlation (P=0.003). At the earliest time points, intracellular RNA levels remain unchanged in both the subgenomic replicon and infection assays, whereas extracellular RNA levels increased upto ten-fold compared to wild-type JFH1. Beneficial substitutions hyperstimulated phosphatidylinositol 4-phosphate during DAA treatment, and showed decreased dependence on cyclophilins during cyclosporine A treatment, indicating an interplay of virus-host molecular mechanisms in beneficial substitution selection that may necessitate infectious virus production. This comprehensive study demonstrates a possible role for HCV fitness of overcoming drug-mediated selection pressure.

LncRNA NEAT1 regulates HCV-induced Hepatocellular carcinoma by modulating the miR-9-BGH3 axis.

Chronic hepatitis C virus (HCV) infection is a leading cause of end-stage liver diseases, such as fibrosis, cirrhosis and hepatocellular carcinoma (HCC). Several cellular entities, including paraspeckles and their related components, are involved in viral pathogenesis and cancer progression. NEAT1 lncRNA is a major component of paraspeckles that has been linked to several malignancies. In this study, analysis of the Cancer Genome Atlas (TCGA) database and validation in HCV-induced HCC tissue and serum samples showed significantly high expression of NEAT1 in patients with liver cancer. Moreover, we found that NEAT1 levels increased upon HCV infection. To further understand the mechanism of NEAT1-induced HCC progression, we selected one of its targets, miR-9-5 p, which regulates BGH3 mRNA levels. Interestingly, miR-9-5 p levels were downregulated upon HCV infection, whereas BGH3 levels were upregulated. Additionally, partial NEAT1 knockdown increased miR-9-5 p levels and decreased BGH3 levels, corroborating our initial results. BGH3 levels were also upregulated in HCV-induced HCC and TCGA tissue samples, which could be directly correlated with NEAT1 levels. As a known oncogene, BGH3 is directly linked to HCC progression mediated by NEAT1. We also found that NEAT1 levels remained upregulated in serum samples from patients treated with direct-acting antivirals (DAA), indicating that NEAT1 might be a molecular trigger that promotes HCC development. Collectively, these findings provide molecular insights into HCV-induced HCC progression via the NEAT1-miR-9-BGH3 axis.

Seroprevalence and burden of hepatitis C virus infection in WHO South-East Asia Region: A systematic review.

BACKGROUND: This systematic review was aimed to estimate hepatitis C virus (HCV) seroprevalence and burden in disease in WHO South East Asia Region (SEAR). METHODS: Electronic databases (PubMed, Scopus, Embase, and Google Scholar) and websites of non-indexed national medical journals, government and international health agencies were searched to identify English language literature published between 1991 and June 2020. We selected the studies reporting HCV seroprevalence in asymptomatic general (low-risk) and high-risk adult populations, that is, persons living with HIV (PLHIV), persons who inject drugs (PWID), sex workers, persons on maintenance hemodialysis (MHD), people in prison, and men sex with men (MSM). Seroprevalence data were combined to estimate weighted pooled prevalence (95% confidence interval) in each group and in each country, using the random-effects model. Estimated pooled seroprevalences were multiplied with estimated populations at risk to estimate the overall HCV burden. RESULTS: The analysis included 538 studies (35 Bangladesh, 6 Bhutan, 2 DPR Korea, 323 India, 43 Indonesia, 2 Maldives, 18 Myanmar, 29 Nepal, 11 Sri Lanka, 67 Thailand, and 2 Timor-Leste). In SEAR, the weighted pooled anti-HCV seroprevalence was estimated as 0.84% (0.56-1.12) in low-risk population and 13.67% (10.95-16.40) in PLHIV, 51.44% (43.67-59.20) in PWID, 25.80% (20.34-32.09) in MHD, 8.39% (5.84-11.51) in prison inmates, 2.69% (1.43-4.13) in people with high-risk sex behavior, and 11.43% (8.61-14.74) in MSM. The total HCV burden in low-risk and high-risk populations in SEAR countries was estimated as 12.45 million and 1.65 million, respectively. CONCLUSION: Our estimates of HCV seroprevalence and burden should help the respective countries in planning their HCV elimination strategies.

Short course of low-dose pregabalin is effective for the treatment of restless leg syndrome in patients with cirrhosis: A pilot study.

BACKGROUND AND AIM: Restless leg syndrome (RLS) is common in patients with cirrhosis, but its treatment in such patients remains unclear. This pilot study assessed the clinical effectiveness of intravenous iron and a 6-week course of low-dose (75 mg/day) pregabalin for the treatment of RLS in patients with cirrhosis. METHODS: It was a prospective, interventional study that included adult patients with cirrhosis and RLS. The participants underwent serum ferritin measurement. Patients with low serum ferritin (< 75 µg/dL) were treated with intravenous iron. Those with normal ferritin levels and those with low levels whose RLS symptoms failed to respond to iron replacement were treated with oral pregabalin, initially 75 mg/day for 6 weeks, followed by 150 mg/day for 6 weeks if there was no response. Recurrence of symptoms was assessed at 6-12 weeks after stopping pregabalin. RESULTS: Of the 50 patients (male patients 52%; median age 48 [interquartile range: 21-65] years; median Child-Pugh-Turcotte score 8 [5-13] and median Model for End-Stage Liver disease score 17 [12-20]) studied, 29 (58%) had low ferritin; of them, 14 (48%) responded to intravenous iron alone. Eleven of 15 (38%) patients with low ferritin and nonresponse to iron, and 16 of 21 (76%) with normal ferritin levels had a response with low-dose pregabalin. Of the nine nonresponders who received 150 mg/day of pregabalin, four had to discontinue it because of adverse effects. CONCLUSION: A short course of low-dose (75 mg/day) pregabalin was effective (82%) in alleviating RLS in patients with cirrhosis. (CTRI/2019/02/017642).

Prevalence and Time Trend of SARS-CoV-2 Infection in Puducherry, India, August-October 2020.

We conducted 3 population-based cross-sectional surveys, at 1-month intervals, to estimate the prevalence and time-trend of severe acute respiratory syndrome coronavirus 2 infection in Puducherry, India. Seropositivity rate increased from 4.9% to 34.5% over 2 months and was 20-fold higher than the number of diagnosed cases of infection.

Genome-Wide Mutagenesis of Hepatitis C Virus Reveals Ability of Genome To Overcome Detrimental Mutations.

To gain insight into the impact of mutations on the viability of the hepatitis C virus (HCV) genome, we created a set of full-genome mutant libraries, differing from the parent sequence as well as each other, by using a random mutagenesis approach; the proportion of mutations increased across these libraries with declining template amount or dATP concentration. The replication efficiencies of full-genome mutant libraries ranged between 71 and 329 focus-forming units (FFU) per 105 Huh7.5 cells. Mutant libraries with low proportions of mutations demonstrated low replication capabilities, whereas those with high proportions of mutations had their replication capabilities restored. Hepatoma cells transfected with selected mutant libraries, with low (4 mutations per 10,000 bp copied), moderate (33 mutations), and high (66 mutations) proportions of mutations, and their progeny were subjected to serial passage. Predominant virus variants (mutants) from these mutant libraries (Mutantl, Mutantm, and Mutanth, respectively) were evaluated for changes in growth kinetics and particle-to-FFU unit ratio, virus protein expression, and modulation of host cell protein synthesis. Mutantm and Mutantl variants produced >3.0-log-higher extracellular progeny per ml than the parent, and Mutanth produced progeny at a rate 1.0-log lower. More than 80% of the mutations were in a nonstructural part of the mutant genomes, the majority were nonsynonymous, and a moderate to large proportion were in the conserved regions. Our results suggest that the HCV genome has the ability to overcome lethal/deleterious mutations because of the high reproduction rate but highly selects for random, beneficial mutations.IMPORTANCE Hepatitis C virus (HCV) in vivo displays high genetic heterogeneity, which is partly due to the high reproduction and random substitutions during error-prone genome replication. It is difficult to introduce random substitutions in vitro because of limitations in inducing mutagenesis from the 5' end to the 3' end of the genome. Our study has overcome this limitation. We synthesized full-length genomes with few to several random mutations in the background of an HCV clone that can recapitulate all steps of the life cycle. Our study provides evidence of the capability of the HCV genome to overcome deleterious mutations and remain viable. Mutants that emerged from the libraries had diverse phenotype profiles compared to the parent, and putative adaptive mutations mapped to segments of the conserved nonstructural genome. We demonstrate the potential utility of our system for the study of sequence variation that ensures the survival and adaptation of HCV.

Acute hepatitis C treatment in advanced renal failure using 8 weeks of pan-genotypic daclatasvir and reduced-dose sofosbuvir.

BACKGROUND: Sofosbuvir is not recommended in persons with estimated glomerular filtration rate (eGFR) <30 mL/min. We report the results of treatment with an off-label 8-week regimen of daclatasvir and half-dose sofosbuvir in patients with acute infection with hepatitis C virus ( HCV) and eGFR <30 mL/min. METHODS: Clinic records were searched to identify treatment-naïve, noncirrhotic adults with acute hepatitis C (HCV viremia and a ≥10-fold elevation of serum alanine aminotransferase activity) and eGFR <30 mL/min, who had been treated with a sofosbuvir-based regimen. Treatment response was assessed using serum HCV RNA testing at 4 weeks of treatment, end of the 8-week treatment and 12 weeks after stopping treatment. RESULTS: Of the 31 patients with acute hepatitis C, 27 [median age (range): 36 (18-74) years; 20 (74%) male] were started on treatment with 200 mg sofosbuvir and 60 mg daclatasvir daily for 8 weeks, irrespective of HCV genotype. All the 27 completed the planned 8-week treatment. One patient died 10 weeks after completing the treatment of an unrelated cause. All the 27 patients had undetectable HCV RNA after 4 weeks of and at the end of treatment. At 12 weeks after completion of treatment, only one tested HCV RNA positive and 25 were negative, with sustained virological response rate of 25/27 (92.6%) and 25/26 (96.2%) on intention-to-treat and per-protocol basis, respectively. CONCLUSION: Eight-week course of daclatasvir and half-dose sofosbuvir is effective for acute hepatitis C in patients with eGFR <30 mL/min and could be a useful alternative to costly, kidney-safe anti-HCV oral drugs in resource-constrained settings.

Temporal profile of HEV RNA concentration in blood and stool from patients with acute uncomplicated hepatitis E in a region with genotype 1 predominance.

Acute hepatitis E virus (HEV) is associated with viremia and faecal excretion of the virus. The information on duration and temporal pattern of viremia and faecal shedding in HEV infection is important, but is not available. Serial serum and stool specimens were collected from patients with acute hepatitis E (typical clinical picture, serum alanine aminotransferase levels > 5-folds the upper limit of normal and presence of IgM anti-HEV), beginning from within 7 days of the onset of symptoms. HEV RNA concentrations were measured in sera and 10% stool suspensions, using a real-time Taqman-based nucleic acid amplification assay. Seventeen patients (median age 25 [range 19-61] years; all men) were enrolled within a median of 5 (range 3-8) days of the onset of the first symptom and provided 113 serum specimens and 71 stool specimens. The median (range) highest levels of serum bilirubin, alanine aminotransferase and aspartate aminotransferase in the patients were 10.3 (5.9-43.4) mg/dL, 1817 (442-4642) IU/L and 1016 (88-4561) IU/L, respectively. All the 17 patients had demonstrable viremia, and 12 of the 13 patients who were tested had faecal excretion at one or more time points. The HEV RNA titres were the highest in the early phase of disease and declined rapidly with time, becoming nondetectable in the serum by day 20 and in the stool by day 21. In most of the patients with acute uncomplicated acute hepatitis E, the degree of viremia and faecal shedding decline quickly after the onset of clinical illness and rapidly disappear in parallel with each other.

Prevalence of hepatitis E viraemia among blood donors: a systematic review.

BACKGROUND: Hepatitis E virus (HEV) is usually transmitted by faecal-oral route. Recent reports have documented HEV viraemia in donated blood units and HEV transmission through blood transfusion. This systematic review summarizes the available data on prevalence of HEV viraemia in blood donors. METHODS: Electronic databases were searched on 17 December 2018 to identify full-text English papers reporting original data on prevalence of HEV RNA in donated blood units. Two authors independently extracted the relevant data, which were pooled using simple aggregation as well as a random-effects meta-analysis; heterogeneity was assessed using the I2 method. RESULTS: In all, 59 data sets from 28 countries were identified. The available data showed marked heterogeneity. Of a total of 2 127 832 units studied, 561 (263·6 [95% confidence intervals = 242·7-286·4] per million units) tested positive for HEV RNA. On random-effects meta-analysis, the pooled prevalence was 60·9 [6·7-155·4] per million units. In the viraemic units, HEV RNA titre varied by nearly one million-fold, and most had genotype 3 HEV. The prevalence was higher in blood units with anti-HEV antibodies or elevated alanine aminotransferase. Only nearly one-fourth of viraemic units had anti-HEV antibodies. CONCLUSIONS: The prevalence of HEV viraemia among healthy blood donors is low, though the available data had limited geographical representation and marked heterogeneity. There is a need for further data on HEV viraemia in blood donors from areas with non-3 HEV genotype preponderance.

Construction & assessment of a unified curated reference database for improving the taxonomic classification of bacteria using 16S rRNA sequence data.

Background & objectives: For bacterial community analysis, 16S rRNA sequences are subjected to taxonomic classification through comparison with one of the three commonly used databases [Greengenes, SILVA and Ribosomal Database Project (RDP)]. It was hypothesized that a unified database containing fully annotated, non-redundant sequences from all the three databases, might provide better taxonomic classification during analysis of 16S rRNA sequence data. Hence, a unified 16S rRNA database was constructed and its performance was assessed by using it with four different taxonomic assignment methods, and for data from various hypervariable regions (HVRs) of 16S rRNA gene. Methods: We constructed a unified 16S rRNA database (16S-UDb) by merging non-ambiguous, fully annotated, full-length 16S rRNA sequences from the three databases and compared its performance in taxonomy assignment with that of three original databases. This was done using four different taxonomy assignment methods [mothur Naïve Bayesian Classifier (mothur-nbc), RDP Naïve Bayesian Classifier (rdp-nbc), UCLUST, SortMeRNA] and data from 13 regions of 16S rRNA [seven hypervariable regions (HVR) (V2-V8) and six pairs of adjacent HVRs]. Results: Our unified 16S rRNA database contained 13,078 full-length, fully annotated 16S rRNA sequences. It could assign genus and species to larger proportions (90.05 and 46.82%, respectively, when used with mothur-nbc classifier and the V2+V3 region) of sequences in the test database than the three original 16S rRNA databases (70.88-87.20% and 10.23-24.28%, respectively, with the same classifier and region). Interpretation & conclusions: Our results indicate that for analysis of bacterial mixtures, sequencing of V2-V3 region of 16S rRNA followed by analysis of the data using the mothur-nbc classifier and our 16S-UDb database may be preferred.

Novel Synthesis and Phenotypic Analysis of Mutant Clouds for Hepatitis E Virus Genotype 1.

Many RNA viruses exist as an ensemble of genetically diverse, replicating populations known as a mutant cloud. The genetic diversity (cloud size) and composition of this mutant cloud may influence several important phenotypic features of the virus, including its replication capacity. We applied a straightforward, bacterium-free approach using error-prone PCR coupled with reverse genetics to generate infectious mutant RNA clouds with various levels of genetic diversity from a genotype 1 strain of hepatitis E virus (HEV). Cloning and sequencing of a genomic fragment encompassing 70% of open reading frame 1 (ORF1) or of the full genome from variants in the resultant clouds showed the occurrence of nucleotide mutations at a frequency on the order of 10-3 per nucleotide copied and the existence of marked genetic diversity, with a high normalized Shannon entropy value. The mutant clouds showed transient replication in cell culture, while wild-type HEV did not. Cross-sectional data from these cell cultures supported the existence of differential effects of clouds of various sizes and compositions on phenotypic characteristics, such as the replication level of (+)-RNA progeny, the amounts of double-stranded RNA (a surrogate for the rate of viral replication) and ORF1 protein, and the expression of interferon-stimulated genes. Since mutant cloud size and composition influenced the viral phenotypic properties, a better understanding of this relationship may help to provide further insights into virus evolution and prediction of emerging viral diseases.IMPORTANCE Several biological or practical limitations currently prevent the study of phenotypic behavior of a mutant cloud in vitro We developed a simple and rapid method for synthesizing mutant clouds of hepatitis E virus (HEV), a single-stranded (+)-RNA [ss(+) RNA] virus, with various and controllable levels of genetic diversity, which could then be used in a cell culture system to study the effects of cloud size and composition on viral phenotype. In a cross-sectional analysis, we demonstrated that a particular mutant cloud which had an extremely high genetic diversity had a replication rate exceeding that of wild-type HEV. This method should thus provide a useful model for understanding the phenotypic behavior of ss(+) RNA viruses.

Burden of hepatitis C virus infection in India: A systematic review and meta-analysis.

BACKGROUND AND AIM: Burden of hepatitis C in India is not known. We therefore conducted a systematic review of the available data on anti-hepatitis C virus (HCV) seroprevalence in the Indian population. METHODS: We searched several publication databases for English language papers that reported data on anti-HCV seroprevalence from India and also identified other unpublished sources of such data. Data on groups likely to represent seroprevalence in general population and in selected high-risk groups were extracted and subjected to meta-analysis. RESULTS: Of the 3995 published papers and 94 additional data sources identified, 327 were selected; these provided 414 anti-HCV seroprevalence data points. Pooled anti-HCV seroprevalence rates in community-based studies, blood donors, and pregnant women were 0.85% (95% confidence interval: 0.00-3.98%), 0.44% (0.40-0.49), and 0.88% (0.21-1.90), respectively. Among groups considered at high risk of HCV, pooled anti-HCV seroprevalence rates were as follows: people living with HIV (40 studies from 17 states: 3.51% [2.43-4.76]), persons on maintenance hemodialysis (37, 13; 19.23% [13.52-25.65]), people who inject drugs (46, 14; 44.71% [37.50-52.03]), multi-transfused persons (38, 12; 24.06% [20.00-28.36]), persons with sexually transmitted diseases (7, 5; 4.10% [0.98-9.04]), and those with high-risk sex behavior (6, 5; 4.06% [1.79-7.10]). CONCLUSIONS: Community-based data on HCV seroprevalence in India were limited. Large amount of data on blood donors and pregnant women were identified, with pooled anti-HCV seroprevalence rates of 0.44% and 0.88%, respectively. Among high-risk groups, anti-HCV prevalence was higher among people living with HIV, those with sexually transmitted diseases, high-risk sex behavior or injection drug use, and those receiving hemodialysis or frequent transfusions.

Daclatasvir and reduced-dose sofosbuvir: An effective and pangenotypic treatment for hepatitis C in patients with estimated glomerular filtration rate <30 mL/min.

AIM: Sofosbuvir is a key agent for HCV treatment. It is not recommended for patients with chronic kidney disease (CKD) and estimated glomerular filtration rate (eGFR) <30 mL/min. We report real-life experience of treating a cohort of CKD patients with eGFR <30 mL/min using daclatasvir and half-daily dose of sofosbuvir. METHODS: Adults patients who (i) had eGFR<30 mL/min and detectable HCV RNA and (ii) were treated with interferon and ribavirin free, DAA based regimens were included. All patients were treated with daily doses of daclatasvir 60 mg and sofosbuvir 200 mg. The planned duration of treatment was 12 weeks, except for 24 weeks in those with either clinical evidence of cirrhosis or on immunosuppressive drugs. The end-points of the study were: (i) 12 weeks of follow-up after treatment completion, (ii) treatment discontinuation, or (iii) death or loss to follow-up. RESULTS: Thirty-six (88%) among 41 included patients (median [range] age: 48 [19-75] years; 25 [61%] male; genotype 1/3/4 were 17/ 22/2; cirrhosis 5) completed the treatment, two discontinued and three died during treatment. On an intention-to-treat basis, HCV RNA were undetectable at 4 weeks of treatment, treatment completion and after 12 weeks of follow-up in 40/41 (97.6%), 37/41 (90.2%) and 37/41 (90.2%), respectively. None of the patients had a relapse. CONCLUSIONS: Daclatasvir and half-daily dose of sofosbuvir was effective against genotype 1 and 3 HCV infection in patients with eGFR <30 mL/min. This combination could be a pangenotypic treatment option for such patients.

Spectrum of genomic variations in Indian patients with progressive familial intrahepatic cholestasis.

BACKGROUND: Progressive familial intrahepatic cholestasis (PFIC) is caused by variations in ATP8B1, ABCB11 or ABCB4 genes. Data on genetic variations in Indian patients with PFIC are lacking. METHODS: Coding and splice regions of the three genes were sequenced in unrelated Indian children with PFIC phenotype. The variations identified were looked for in parents, 30 healthy persons and several variation databases, and their effect was assessed in-silico. RESULTS: Among 25 children (aged 1-144 months), nine (36%) had unique major genomic variations (ATP8B1: 4, ABCB11: 3 and ABCB4: 2). Seven had homozygous variations, which were assessed as 'pathogenic' or 'likely pathogenic'. These included: (i) four amino acid substitutions (ATP8B1: c.1660G > A/p.Asp554Asn and c.2941G > A/p.Glu981Lys; ABCB11: c.548 T > C/p.Met183Thr; ABCB4: c.431G > A/p.Arg144Gln); (ii) one 3-nucleotide deletion causing an amino acid deletion (ATP8B1: c.1587_1589delCTT/p.Phe529del); (iii) one single-nucleotide deletion leading to frame-shift and premature termination (ABCB11: c.1360delG/p.Val454Ter); and (iv) a complex inversion of 4 nucleotides with a single-nucleotide insertion leading to frame-shift and premature termination (ATP8B1: c.[589_592inv;592_593insA]/p.Gly197LeufsTer10). Two variations were found in heterozygous form: (i) a splice-site variation likely to cause abnormal splicing (ABCB11: c.784 + 1G > C), and (ii) a nucleotide substitution that created a premature stop codon (ABCB4: c.475C > T/p.Arg159Ter); these were considered as variations of uncertain significance. Three of the nine variations were novel. CONCLUSIONS: Nine major genomic variations, including three novel ones, were identified in nearly one-third  of Indian children with PFIC. No variation was identified in nearly two-thirds of patients, who may have been related to variations in promoter or intronic regions of the three PFIC genes, or in other bile-salt transport genes.

Effect of administration of a probiotic preparation on gut microbiota and immune response in healthy women in India: an open-label, single-arm pilot study.

BACKGROUND: Probiotics have been shown to be useful for the treatment of many disease conditions. These beneficial effects are believed to be mediated by change in the composition of gut microbiota and modulation of the host immune responses. However, the available data on the effect of probiotics on these parameters are quite limited. METHODS: We studied the composition of fecal microbiota, using 16S rRNA sequencing, and host immune responses in peripheral blood (plasma cytokine levels, T cell subsets and in vitro cytokine production after stimulation with anti-CD3/CD28 antibody or lipopolysaccharide) in a group of 14 healthy women at three time-points - before and after administration of a probiotic preparation (a capsule of VSL#3, each containing 112.5 billion freeze-dried bacterial cells belonging to 8 species, twice a day for 4 weeks), and 4-weeks after discontinuation of the probiotic administration. RESULTS: There was no change in the abundance of various bacterial taxa as well as in the alpha diversity of gut microbiota following administration of the probiotic, or following its discontinuation. Probiotic administration led to a reduction in the relative frequency of circulating Th17 cells, and in vitro production of cytokines in whole-blood cultures in response to lipopolysaccharide stimulation. However, it had no effect on the relative frequencies of Th1, Th2 and T regulatory cells among circulating peripheral blood mononuclear cells, on plasma cytokine levels and on in vitro production of cytokines by T cells. CONCLUSIONS: We found that VSL#3 administration did not lead to any changes in gut flora, but led to a reduction in the frequency of Th17 cells and in the production of pro-inflammatory cytokine on lipopolysaccharide stimulation. These findings suggest that the beneficial anti-inflammatory effect of this preparation in patients with autoimmune and allergic disorders may be related to reduced production of monocyte-derived cytokines rather than to changes in the composition of gut microbiota. TRIAL REGISTRATION: NCT03330678 , Date of registration 30th October 2017. Retrospectively registered.