Unexplained diarrhoea in HIV-1 infected individuals

Background: Gastrointestinal symptoms, in particular diarrhoea, are common in non-treated HIV-1 infected individuals. Although various enteric pathogens have been implicated, the aetiology of diarrhoea remains unexplained in a large proportion of HIV-1 infected patients. Our aim is to identify the cause of diarrhoea for patients that remain negative in routine diagnostics. Methods: In this study stool samples of 196 HIV-1 infected persons, including 29 persons with diarrhoea, were examined for enteropathogens and HIV-1. A search for unknown and unexpected viruses was performed using virus discovery cDNA-AFLP combined with Roche-454 sequencing (VIDISCA-454). Results: HIV-1 RNA was detected in stool of 19 patients with diarrhoea (66%) compared to 75 patients (45%) without diarrhoea. In 19 of the 29 diarrhoea cases a known enteropathogen could be identified (66%). Next to these known causative agents, a range of recently identified viruses was identified via VIDISCA-454: cosavirus, Aichi virus, human gyrovirus, and non-A non-B hepatitis virus. Moreover, a novel virus was detected which was named immunodeficiency-associated stool virus (IASvirus). However, PCR based screening for these viruses showed that none of these novel viruses was associated with diarrhoea. Notably, among the 34% enteropathogen-negative cases, HIV-1 RNA shedding in stool was more frequently observed (80%) compared to enteropathogen-positive cases (47%), indicating that HIV-1 itself is the most likely candidate to be involved in diarrhoea. Conclusion: Unexplained diarrhoea in HIV-1 infected patients is probably not caused by recently described or previously unknown pathogens, but it is more likely that HIV-1 itself plays a role in intestinal mucosal abnormalities which leads to diarrhoea

Hepatitis C virus transmission between eight high-income countries among men who have sex with men: a whole-genome analysis.

BACKGROUND Microelimination of the hepatitis C virus (HCV) among men who have sex with men (MSM) could be complicated by continuous external introductions and the emergence of phylogenetic clusters harbouring clinically significant resistance-associated substitutions (RAS). To investigate international clustering and the prevalence and transmission of RAS, we aimed to analyse whole-genome HCV sequences from MSM with a recently acquired infection who participated in a large, international HCV treatment trial. METHODS For this whole-genome analysis, we obtained HCV sequences from 128 MSM who had acquired HCV within the past 12 months and were participating in the REACT trial. The participants from whom sequences were obtained were recruited at 24 sites in eight countries. We inferred maximum-likelihood phylogenies and identified transmission clusters for HCV genotypes separately. We constructed time-scaled phylogenies to estimate cluster introduction dates and used a Bayesian Skygrid approach to estimate the effective population size over the past 50 years. We calculated the prevalence of RAS and the extent of RAS transmission in the study population. FINDINGS The majority of recent HCV infections were part of international networks that arose in the late 1990s and early 2000s. Sequences obtained in the same country clustered frequently, and in 36% of subclusters since 2015 we found evidence of international transmission. European MSM were more likely than non-European MSM to be in a cluster (odds ratio 11·9 [95% CI 3·6-43·4], p<0·0001). The effective population size decreased rapidly since around 2015 in Europe. RAS associated with substantially diminished cure rates were infrequently detected and transmission of highly resistant viruses was not observed. INTERPRETATION Despite antiviral treatment becoming widely available, international transmission of HCV among MSM has still occurred over the past 8 years, which could complicate microelimination of the virus in this population. RAS-enriched clusters and widespread RAS transmission are currently not a threat to elimination goals. These findings support an international approach for HCV microelimination among MSM. FUNDING National Institutes of Health and Dr. C.J. Vaillant Fonds

Direct-Acting Antiviral Treatment for Hepatitis C Genotypes Uncommon in High-Income Countries:A Dutch Nationwide Cohort Study

Background. The majority of hepatitis C virus (HCV) infections are found in low- and middle-income countries, which harbor many region-specific HCV subtypes. Nevertheless, direct-acting antiviral (DAA) trials have almost exclusively been conducted in high-income countries, where mainly epidemically spread HCV subtypes are present. Recently, several studies have demonstrated suboptimal DAA efficacy for certain nonepidemic subtypes, which could hamper global HCV elimination. Therefore, we aimed to evaluate DAA efficacy in patients treated for a nonepidemic HCV genotype infection in the Netherlands. Methods. We performed a nationwide retrospective study including patients treated with interferon-free DAAs for an HCV genotype other than 1a/1b/2a/2b/3a/4a/4d. The genotype was determined by NS5B region phylogenetic analysis. The primary end point was SVR-12. If stored samples were available, NS5A and NS5B sequences were obtained for resistance-associated substitutions (RAS) evaluation. Results. We included 160 patients, mainly infected with nonepidemic genotype 2 (41%) and 4 (31%) subtypes. Most patients were from Africa (45%) or South America (24%); 51 (32%) were cirrhotic. SVR-12 was achieved in 92% (140/152) of patients with available SVR-12 data. Only 73% (8/11) genotype 3-infected patients achieved SVR-12, the majority being genotype 3b patients with 63% (5/8) SVR. Regardless of SVR, all genotype 3b patients had 30K and 31M RAS. Conclusions. (T)he DAA efficacy we observed in most nonepidemic genotypes in the Netherlands seems reassuring. However, the low SVR-12 rate in subtype 3b infections is alarming, especially as it is common in several HCV-endemic countries. Alongside earlier results, our results indicate that a remaining challenge for global HCV elimination is confirming and monitoring DAA efficacy in nonepidemic genotypes

Amino acid variation at VP1-145 of enterovirus A71 determines the viral infectivity and receptor usage in a primary human intestinal model

Enterovirus A71 (EV-A71) can elicit a wide variety of human diseases such as hand, foot, and mouth disease and severe or fatal neurological complications. It is not clearly understood what determines the virulence and fitness of EV-A71. It has been observed that amino acid changes in the receptor binding protein, VP1, resulting in viral binding to heparan sulfate proteoglycans (HSPGs) may be important for the ability of EV-A71 to infect neuronal tissue. In this study, we identified that the presence of glutamine, as opposed to glutamic acid, at VP1-145 is key for viral infection in a 2D human fetal intestinal model, consistent with previous findings in an airway organoid model. Moreover, pre-treatment of EV-A71 particles with low molecular weight heparin to block HSPG-binding significantly reduced the infectivity of two clinical EV-A71 isolates and viral mutants carrying glutamine at VP1-145. Our data indicates that mutations in VP1 leading to HSPG-binding enhances viral replication in the human gut. These mutations resulting in increased production of viral particles at the primary replication site could lead to a higher risk of subsequent neuroinfection.ImportanceWith the near eradication of polio worldwide, polio-like illness (as is increasingly caused by EV-A71 infections) is of emerging concern. EV-A71 is indeed the most neurotropic enterovirus that poses a major threat globally to public health and specifically in infants and young children. Our findings will contribute to the understanding of the virulence and the pathogenicity of this virus. Further, our data also supports the identification of potential therapeutic targets against severe EV-A71 infection especially among infants and young children. Furthermore, our work highlights the key role of HSPG-binding mutations in the disease outcome of EV-A71. Additionally, EV-A71 is not able to infect the gut (the primary replication site in humans) in traditionally used animal models. Thus, our research highlights the need for human-based models to study human viral infections.Graphical Abstrac

Performance of the New Bayer VERSANT HCV RNA 3.0 Assay for Quantitation of Hepatitis C Virus RNA in Plasma and Serum: Conversion to International Units and Comparison with the Roche COBAS Amplicor HCV Monitor, Version 2.0, Assay

We have evaluated the VERSANT HCV RNA 3.0. Assay (HCV 3.0 bDNA assay) (Bayer Diagnostics, Berkeley, Calif.), which is an improved signal amplification procedure for the HCV 2.0 bDNA assay for the quantitation of hepatitis C virus (HCV) RNA in serum or plasma of HCV-infected individuals. The HCV 3.0 bDNA assay has a linear dynamic range of 2.5 × 10(3) to 4.0 × 10(7) HCV RNA copies per ml (c/ml). The performance of the HCV 3.0 bDNA assay was evaluated using three different test panels. An overall specificity of 96.8% relative to the detection limit of the HCV 3.0 bDNA assay was found. The intra- and interrun reproducibilities for both the dilution panel and the NAP (AcroMetrix, Benicia, Calif.) panel were consistent with coefficients of variation of less than 9%. Quantitation with the HCV 3.0 bDNA assay was linear over the entire range of both panels (ranges of 4.4 × 10(3) to 3.5 × 10(6) c/ml and 5 × 10(3) to 2 × 10(6) IU/ml, respectively), with correlation coefficients of 0.999, slopes close to one, and intercepts close to zero. The regression equation indicated that 1 IU corresponded to about 4.8 copies of HCV RNA. A correlation coefficient of 0.941 was found for HCV RNA values (in international units per milliliter) obtained from the HCV 3.0 bDNA assay and the HCV Monitor version 2.0 assay (HCV Monitor 2.0 assay) (Roche Diagnostic Systems, Branchburg, N.J.). Quantitative results obtained close to the lower limit of the HCV 3.0 bDNA assay might imply that its lower limit should be reconsidered and raised, if necessary. It appeared that quantitation values obtained from the HCV Monitor 2.0 assay of between 5 × 10(2) and 10(5) IU/ml were in general higher than those obtained from the HCV 3.0 bDNA assay, whereas values obtained from the HCV Monitor 2.0 assay were underestimated for samples with HCV RNA levels above 10(5) IU/ml

Dried blood spot self-sampling at home is a feasible technique for hepatitis C RNA detection

To facilitate HCV diagnosis, we developed an HCV-RNA testing service, which involved home-sampled dried blood spots (DBS). The main objective of this study was to evaluate the feasibility of self-sampling at home. Furthermore, to optimise the processing of DBS samples for RNA detection, we evaluated two elution buffers: phosphate-buffered saline (PBS) and L6-buffer. 27 HCV-RNA and 12 HIV-1 RNA positive patients were included. Laboratory spotted DBS (LabDBS) were made by a technician from blood samples drawn at inclusion. Patients received a DBS home-sampling kit and were requested to return their self-sampled DBS (ssDBS) by mail. We compared the RNA load of PBS and L6-eluted labDBS, and of L6-eluted ssDBS, L6-eluted labDBS and plasma. LabDBS load measurements were repeated after 7-13 and 14-21 days to evaluate RNA stability. All 39 plasma samples provided quantifiable RNA loads. In 1/39 labDBS sample, RNA could not be detected (plasma HCV load: 2.98 log10 IU/ml). L6-eluted samples gave a 0.7 log10 and 0.6 log10 higher viral load for HCV and HIV-1 respectively, compared to PBS-eluted samples. Strong correlations were found between labDBS and ssDBS HCV RNA (r = 0.833; mean difference 0.3 log10 IU/mL) and HIV-1 RNA results (r = 0.857; mean difference 0.1 log10 copies/mL). Correlations between labDBS and plasma values were high for HCV (r = 0.958) and HIV-1 (r = 0.844). RNA loads in DBS remained stable over 21 days. Our study demonstrates that self-sampling dried blood spots at home is a feasible strategy for the detection of HCV and HIV-1 RNA. This could facilitate one-step diagnostics and treatment monitoring in communities with high HCV prevalence

Detection and Quantitation of Hepatitis C Virus RNA in Feces of Chronically Infected Individuals

Hepatitis C virus (HCV) RNA was detected and quantified in human fecal specimens with the Roche COBAS AMPLICOR system adapted by us for fecal specimens. HCV RNA could be detected in the feces of four of six (67%) patients chronically infected with HCV, with loads up to about 2.8 × 10(5) copies/ml of feces. The same HCV genotypes were observed in feces and plasma as determined by direct sequencing of the 5′ untranslated region

Human Parechovirus 1, 3 and 4 Neutralizing Antibodies in Dutch Mothers and Infants and Their Role in Protection Against Disease.

Human parechoviruses (HPeVs) are common pathogens in young children, and in the Netherlands, HPeV1, HPeV3 and HPeV4 are the most frequently detected genotypes. HPeV3 in particular has been associated with severe disease in young infants below 3 months of age while the other genotypes more often infect older children and elicit mild symptoms. We investigated if maternal neutralizing antibodies (nAbs) against HPeV1, HPeV3 and HPeV4 protect young Dutch infants from severe disease related to HPeV infection. We conducted a prospective case-control study of Dutch mother-infant pairs. Thirty-eight HPeV-infected infants and their mothers were included as cases, and 65 HPeV-negative children and their mothers as controls. In control infants, we observed nAb seropositivity rates of 41.4%, 33.3% and 27.6%, with median nAb titers of 1:16, 1:12 and 1:8, against HPeV1, HPeV3 and HPeV4, respectively. In control mothers, nAb seropositivity rates were 84.6%, 55.4% and 60.0% with median nAb titers of 1:128, 1:32 and 1:45 against HPeV1, HPeV3 and HPeV4, respectively. The HPeV3 nAb seroprevalence was significantly lower in HPeV3-infected infants and their mothers (0.0% with P < 0.05 and 10.0% with P < 0.001, respectively). In contrast, no differences in nAb seroprevalence against HPeV1 or HPeV4 could be detected between case and control infants or mothers. Our results suggest that young Dutch infants are protected against severe disease related to HPeV1 and HPeV4 by maternal nAbs, but less so against HPeV3 explaining the distinct age distributions and disease severity profiles of children infected with these HPeV genotypes

Evaluation of persistence of resistant variants with ultra-deep pyrosequencing in chronic hepatitis C patients treated with telaprevir.

BACKGROUND & AIMS: Telaprevir, a hepatitis C virus NS3/4A protease inhibitor has significantly improved sustained viral response rates when given in combination with pegylated interferon alfa-2a and ribavirin, compared with current standard of care in hepatitis C virus genotype 1 infected patients. In patients with a failed sustained response, the emergence of drug-resistant variants during treatment has been reported. It is unclear to what extent these variants persist in untreated patients. The aim of this study was to assess using ultra-deep pyrosequencing, whether after 4 years follow-up, the frequency of resistant variants is increased compared to pre-treatment frequencies following 14 days of telaprevir treatment. METHODS: Fifteen patients from 2 previous telaprevir phase 1 clinical studies (VX04-950-101 and VX05-950-103) were included. These patients all received telaprevir monotherapy for 14 days, and 2 patients subsequently received standard of care. Variants at previously well-characterized NS3 protease positions V36, T54, R155 and A156 were assessed at baseline and after a follow-up of 4±1.2 years by ultra-deep pyrosequencing. The prevalence of resistant variants at follow-up was compared to baseline. RESULTS: Resistance associated mutations were detectable at low frequency at baseline. In general, prevalence of resistance mutations at follow-up was not increased compared to baseline. Only one patient had a small, but statistically significant, increase in the number of V36M and T54S variants 4 years after telaprevir-dosing. CONCLUSION: In patients treated for 14 days with telaprevir monotherapy, ultra-deep pyrosequencing indicates that long-term persistence of resistant variants is rare