High frequency and diversity of parechovirus A in a cohort of Malawian children.

Parechoviruses (PeVs) are highly prevalent viruses worldwide. Over the last decades, several studies have been published on PeV epidemiology in Europe, Asia and North America, while information on other continents is lacking. The aim of this study was to describe PeV circulation in a cohort of children in Malawi, Africa. A total of 749 stool samples obtained from Malawian children aged 6 to 60 months were tested for the presence of PeV by real-time PCR. We performed typing by phylogenetic and Basic Local Alignment Search Tool (BLAST) analysis. PeV was found in 57% of stool samples. Age was significantly associated with PeV positivity (p = 0.01). Typing by phylogenetic analysis resulted in 15 different types, while BLAST typing resulted in 14 different types and several indeterminate strains. In total, six strains showed inconsistencies in typing between the two methods. One strain, P02-4058, remained untypable by all methods, but appeared to belong to the recently reclassified PeV-A19 genotype. PeV-A1, -A2 and -A3 were the most prevalent types (26.8%, 13.8% and 9.8%, respectively). Both the prevalence and genetic diversity found in our study were remarkably high. Our data provide an important contribution to the scarce data available on PeV epidemiology in Africa.

Clinical, virological and epidemiological characteristics of rhinovirus infections in early childhood: A comparison between non-hospitalised and hospitalised children.

BACKGROUND: Several studies have been published regarding the epidemiology and clinical significance of the different rhinovirus (RV) species (-A, -B and -C). However, data on RV types and the associations with clinical outcome in young children are limited. Here, we investigated the clinical, virological and epidemiological characteristics of RV infections in young children with mild or asymptomatic infection (non-hospitalised children) and in symptomatic young children admitted to the hospital. OBJECTIVES: The aim of this study was to evaluate associations between different characteristics of RV infections and clinical outcome in young children. STUDY DESIGN: RV-infected children were retrospectively selected from a Dutch birth cohort (EUROPA-study) and from hospitalised children admitted to the hospital because of respiratory symptoms. In total 120 RV-typed samples could be selected from 65 non-hospitalised and 49 hospitalised children between November 2009 and December 2012. RESULTS: RV-A was the predominant species in both study populations, followed closely by RV-C. RV-B was observed only sporadically. The distribution of the RV species was comparable in non-hospitalised and hospitalised children. In children with respiratory distress who required ICU-admission the distribution of RV species did not differ significantly from the non-hospitalised children. No predominant RV type was present in non-hospitalised nor hospitalised children. However, hospitalised children were younger, had more often an underlying illness, a higher RV load and more frequently a bacterial co-infection. CONCLUSIONS: Clinical outcome of RV infected young children was not related to RV species or types, but may more likely be influenced by multiple (host-specific) factors.

Genetic characterization of multiple hepatitis C virus infections following acute infection in HIV-infected men who have sex with men.

OBJECTIVE: High rates of hepatitis C virus (HCV) reinfections among HIV-infected men who have sex with men (MSM) following clearance of a primary infection suggest absence of protective immunity. Here, we investigated the incidence of HCV super and reinfections in 85 HIV-infected MSM with incident HCV infection. DESIGN AND METHODS: Serial sequencing of a fragment of NS5B and the HCV envelope was used to longitudinally characterize the virus. If the primary genotype was still present at the most recent viremic time point, as indicated by the NS5B sequence analysis, serial envelope 2/hypervariable region 1 (E2/HRV1) sequence analysis was performed to distinguish a new infection with the same genotype (clade switch) from intrahost evolution. Incidence rate and cumulative incidence of secondary infections were estimated, and the effect of the primary genotype (1a versus non1) on the risk of acquiring a second infection with the same genotype was determined using Cox proportional-hazards analysis. RESULTS: Among 85 patients with a median follow-up of 4.8 years, incidence rate of secondary infections was 5.39 cases/100 person-years (95% confidence interval 3.34-8.26). Cumulative incidence of genotype switches was markedly higher than the cumulative incidence of clade switches (26.7 versus 4.8% at 5 years, respectively). In patients with HCV-1a as primary infection, the risk for acquiring another HCV-1a infection was reduced compared to those with a primary non-HCV-1a subsequently acquiring HCV-1a (hazard ratio 0.25, 95% confidence interval 0.07-0.93). CONCLUSION: Risk of acquiring a secondary infection with the primary genotype was strikingly reduced compared with the risk of acquiring a secondary infection with a different genotype.

A comparison of 454 sequencing and clonal sequencing for the characterization of hepatitis C virus NS3 variants.

We compared 454 amplicon sequencing with clonal sequencing for the characterization of intra-host hepatitis C virus (HCV) NS3 variants. Clonal and 454 sequences were obtained from 12 patients enrolled in a clinical phase I study for telaprevir, an NS3-4a protease inhibitor. Thirty-nine datasets were used to compare the consensus sequence, average pairwise distance, normalized Shannon entropy, phylogenetic tree topology and the number and frequency of variants derived from both sequencing techniques. In general, a good concordance was observed between both techniques for the majority of datasets. Discordant results were observed for 5 out of 39 clonal and 454 datasets, which could be attributed to primer-related selective amplification used for clonal sequencing. Both 454 and clonal datasets consisted of a few major variants and a large number of low-frequency variants. Telaprevir resistance-associated variants were observed in low frequencies and were detected more often by 454. We conclude that performance of 454 and clonal sequencing is comparable for the characterization of intra-host virus populations. Not surprisingly, 454 is superior for the detection of low frequency resistance-associated variants. However, despite the greater coverage, 454 failed to detect some low frequency variants detected by clonal sequencing.

Hepatitis C virus (HCV) antibody dynamics following acute HCV infection and reinfection among HIV-infected men who have sex with men.

BACKGROUND: A decline of hepatitis C virus (HCV) antibody titers (anti-HCV), ultimately resulting in seroreversion, has been reported following clearance of viremia in both acute and chronic HCV infection. However, frequency of seroreversion remains unknown in human immunodeficiency virus (HIV)/HCV-coinfected patients. We describe anti-HCV dynamics among HIV-infected men who have sex with men (MSM) following acute HCV infection and reinfection. METHODS: Primary acute HCV infection was assumed when a subject was anti-HCV negative prior to the first positive HCV RNA test. Anti-HCV was measured at least annually in 63 HIV-infected MSM, with a median follow-up of 4.0 years (interquartile range [IQR], 2.5-5.7 years). Time from HCV infection to seroconversion, and from seroconversion to seroreversion, was estimated using the Kaplan-Meier method. Longitudinal anti-HCV patterns were studied using a random-effects model to adjust for repeated measures. RESULTS: Median time from HCV infection to seroconversion was 74 days (IQR, 47-125 days). Subjects who cleared HCV RNA (n = 36) showed a significant decrease in anti-HCV levels (P < .001). Among 31 subjects with sustained virologic response (SVR), anti-HCV became undetectable during follow-up in 8; cumulative incidence of seroreversion within 3 years after seroconversion was 37% (95% confidence interval, 18%-66%). Eighteen subjects became reinfected during follow-up; this coincided with a subsequent increase in anti-HCV reactivity. CONCLUSIONS: A decline of anti-HCV reactivity was associated with HCV RNA clearance. Seroreversion was very common following SVR. Upon reinfection, anti-HCV levels increased again. Monitoring anti-HCV levels might therefore be an effective alternative for diagnosis of HCV reinfection.

Hepatitis C virus reinfection following treatment among people who use drugs.

Most new cases of hepatitis C virus (HCV) infections in the developed world are associated with injection drug use. However, treatment for people who inject drugs (PWID) is controversial, as successful treatment risks being followed by new infection. Reinfection after sustained virologic response has been reported, but is the risk so great that treatment should be withheld from this large HCV population? Preliminary evidence suggests that the reinfection incidence is low, but studies to date have been limited by small sample size and few cases of reinfection. In this review, we assess data from studies among PWID of HCV reinfection following treatment to give a reasonable estimate on how frequently reinfection appears and try to characterize those most at risk, The observation that spontaneous clearance of HCV reinfection following treatment occurs is suggestive of a partial protective immunity against persistent infection.

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.

Colonial history and contemporary transmission shape the genetic diversity of hepatitis C virus genotype 2 in Amsterdam.

Evolutionary analysis of hepatitis C virus (HCV) genome sequences has provided insights into the epidemic history and transmission of this widespread human pathogen. Here we report an exceptionally diverse set of 178 HCV genotype 2 (HCV-2) isolates from 189 patients in Amsterdam, comprising 8 distinct HCV subtypes and 10 previously not recognized, unclassified lineages. By combining study subjects' demographic information with phylogeographic and molecular clock analyses, we demonstrate for the first time that the trans-Atlantic slave trade and colonial history were the driving forces behind the global dissemination of HCV-2. We detect multiple HCV-2 movements from present-day Ghana/Benin to the Caribbean during the peak years of the slave trade (1700 to 1850) and extensive transfer of HCV-2 among the Netherlands and its former colonies Indonesia and Surinam over the last 150 years. The latter coincides with the bidirectional migration of Javanese workers between Indonesia and Surinam and subsequent immigration to the Netherlands. In addition, our study sheds light on contemporary trends in HCV transmission within the Netherlands. We observe multiple lineages of the epidemic subtypes 2a, 2b, and 2c (together 67% of HCV-2 infections in Amsterdam), which cluster according to their suspected routes of transmission, specifically, injecting drug use (IDU) and contaminated blood/blood products. Understanding the epidemiological processes that generated the global pattern of HCV diversity seen today is critical for exposing associations between populations, risk factors, and specific HCV subtypes and might help HCV screening and prevention campaigns to minimize the future burden of HCV-related liver disease.

Origin and evolution of the unique hepatitis C virus circulating recombinant form 2k/1b.

Since its initial identification in St. Petersburg, Russia, the recombinant hepatitis C virus (HCV) 2k/1b has been isolated from several countries throughout Eurasia. The 2k/1b strain is the only recombinant HCV to have spread widely, raising questions about the epidemiological background in which it first appeared. In order to further understand the circumstances by which HCV recombinants might be formed and spread, we estimated the date of the recombination event that generated the 2k/1b strain using a Bayesian phylogenetic approach. Our study incorporates newly isolated 2k/1b strains from Amsterdam, The Netherlands, and has employed a hierarchical Bayesian framework to combine information from different genomic regions. We estimate that 2k/1b originated sometime between 1923 and 1956, substantially before the first detection of the strain in 1999. The timescale and the geographic spread of 2k/1b suggest that it originated in the former Soviet Union at about the time that the world's first centralized national blood transfusion and storage service was being established. We also reconstructed the epidemic history of 2k/1b using coalescent theory-based methods, matching patterns previously reported for other epidemic HCV subtypes. This study demonstrates the practicality of jointly estimating dates of recombination from flanking regions of the breakpoint and further illustrates that rare genetic-exchange events can be particularly informative about the underlying epidemiological processes.