Sjoerd Rebers

Performance Evaluation of the New Roche cobas AmpliPrep/cobas TaqMan HCV Test, Version 2.0, for Detection and Quantification of Hepatitis C Virus RNA

ABSTRACT To evaluate the analytical performance and explore the clinical applicability of the new Roche cobas AmpliPrep/cobas TaqMan HCV test, v2.0 (CAP/CTM v2.0), a platform comparison was performed on panels and diagnostic samples with the Roche cobas AmpliPrep/cobas TaqMan HCV test (CAP/CTM v1.0), the Siemens Versant HCV RNA 3.0 branched DNA (bDNA) test, the Abbott m2000 RealTime HCV assay (Realtime assay), and the Siemens Versant HCV transcription-mediated amplification (TMA) test (TMA assay). The analytical performance of the CAP/CTM v2.0 on WHO and Acrometrix panels and clinical specimens of patients infected with HCV genotype 1, 2, 3, 4, 5, or 6 relative to that of the CAP/CTM v1.0 was significantly improved. In a qualitative comparison of the CAP/CTM v2.0 relative to the TMA assay on genotype 1 to 4 samples, the two tests proved to be almost equally sensitive. Response-guided therapy in one of five HCV genotype 4-infected patients previously tested with the CAP/CTM v1.0 would have significantly changed if tested with the CAP/CTM v2.0. In conclusion, the Roche CAP/CTM v2.0 has significantly better performance characteristics than the former CAP/CTM HCV v1.0 and the bDNA assay and performance characteristics comparable to those of the Realtime assay.

Unexplained diarrhoea in HIV-1 infected individuals

BackgroundGastrointestinal 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.MethodsIn 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).ResultsHIV-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.ConclusionUnexplained 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.

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.

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.

The hepatitis C virus nonstructural protein 3 Q80K polymorphism is frequently detected and transmitted among HIV-infected MSM in the Netherlands.

Objectives: The Q80K polymorphism is a naturally occurring resistance-associated variant in the hepatitis C virus (HCV) nonstructural protein 3 (NS3) region and is likely transmissible between hosts. This study describes the Q80K origin and prevalence among HCV risk groups in the Netherlands and examines whether Q80K is linked to specific transmission networks. Design and methods: Stored blood samples from HCV genotype 1a-infected patients were used for PCR and sequencing to reconstruct the NS3 maximum likelihood phylogeny. The most recent common ancestor was estimated with a coalescent-based model within a Bayesian statistical framework. Results: Study participants (n = 150) were either MSM (39%), people who inject drugs (17%), or patients with other (15%) or unknown/unreported (29%) risk behavior. Overall 45% was coinfected with HIV. Q80K was present in 36% (95% confidence interval 28–44%) of patients throughout the sample collection period (2000–2015) and was most prevalent in MSM (52%, 95% confidence interval 38–65%). Five MSM-specific transmission clusters were identified, of which three exclusively contained sequences with Q80K. The HCV-1a most recent common ancestor in the Netherlands was estimated in 1914 (95% higher posterior density 1879–1944) and Q80K originated in 1957 (95% higher posterior density 1942–1970) within HCV-1a clade I. All Q80K lineages could be traced back to this single origin. Conclusion: Q80K is a highly stable and transmissible resistance-associated variant and was present in a large part of Dutch HIV-coinfected MSM. The introduction and expansion of Q80K variants in this key population suggest a founder effect, potentially jeopardizing future treatment with simeprevir.

Susceptibility of hepatitis B virus to lamivudine restored by resistance to adefovir

Serial monotherapy and add‐on regimes for treatment of chronic hepatitis B virus (HBV) infection may induce the accumulation of viral resistance mutations in patients, reducing the options for ongoing viral suppression. The induction of antiviral resistance by serial application of polymerase inhibitors does not necessarily imply that the subsequent combined use of the drugs will fail. Some HIV strains resistant to one polymerase inhibitor show increased susceptibility to another polymerase inhibitor. After failure of sequential lamivudine and adefovir monotherapy, two patients with hepatitis B changed to treatment with lamivudine plus adefovir and had renewed suppression of HBV. To study the mutational history of resistant HBV subpopulations in the two patients, a part of the HBV polymerase gene was amplified, cloned, sequenced, and analyzed for the presence of mutations, in sequential plasma samples. In both patients serial monotherapy caused the replacement in all HBV clones of wild‐type virus by classical lamivudine resistant mutants (L180M and M204V/I), which were replaced subsequently by adefovir resistant mutants (A181V and N236T). When finally lamivudine was added to adefovir, the A181V adefovir mutation persisted in all clones and lamivudine‐related mutations did not reappear. During 18 months of combination therapy, HBV‐DNA levels decreased 10,000, respectively, 1,000‐fold, despite the earlier resistance to lamivudine and adefovir. Although clinically insufficient, this effect indicates that HBV polymerase resistance mutations may be antagonistic, which is relevant if chronic HBV infection is to be treated by a combination of polymerase inhibitors. J. Med. Virol. 81:413–416, 2009.

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.

Persistence of NS5B-S282T, a sofosbuvir resistance-associated substitution, in a HIV/HCV-coinfected MSM with risk of onward transmission

To the Editor: Treatment with direct-acting antiviral agents (DAAs) for chronic hepatitis C virus (HCV) infection is very effective and well tolerated. Nevertheless, resistance-associated substitutions (RAS) have been described, including the nonstructural protein 5B (NS5B) S282T substitution which confers major resistance to sofosbuvir in vitro. The presence of this substitution in treatment-naïve patients has never been described, presumably because it severely impairs viral replicative fitness and therefore usually does not persist after treatment cessation. Herein, we report a case of S282T persistence for 21 weeks upon DAA treatment failure. A 66-year-old man who has sex with men (MSM) was diagnosed with a human-immunodeficiency virus-1 (HIV) infection in 1996, and has since then been successfully treated with antiretroviral therapy. He reported an active sex life with condomless anal intercourse and active and passive fisting with several different male partners at different locations across Europe and the USA. His medical history included three acute HCV infections, which were all successfully treated with diverse antiviral regimens shortly following acute infection (Fig. 1). In September 2016, he acquired his fourth HCV infection (genotype 4d), for which treatment was initiated in November 2016 with sofosbuvir and ledipasvir for 12 weeks. At treatment initiation (t0 in Fig. 1), Sanger sequencing showed no RAS in NS5B (nucleotide 7953–9293 from start of H77 reference genome), but P/T58L in NS5A. Shortly following treatment initiation, HCV RNA (COBAS AmpliPrep/COBAS TaqMan HCV Test v2.0 [CAP/CTM; Roche Diagnostics, Mannheim, Germany]) decreased (Fig. 1). However, in February 2017, twelve weeks after treatment initiation (t1 in Fig. 1), HCV RNA load was 7,570,000 IU/mL, and sequence analysis showed the S282T substitution in NS5B (Fig. 1). In NS5A, 58L was still present. Hereafter, HCV RNA levels declined rapidly, but increased again over time (t2 in Fig. 1), which in retrospect could be explained by the fact that the patient, as he later reported, had decided to interrupt sofosbuvir/ledipasvir at week 8 and restarted two to four weeks afterwards. Remarkably, even when HCV RNA levels were high, the S282T substitution persisted over time (Fig. 1). While analyzing persistence of the NS5B-S282T substitution by Sanger sequencing, a genotype 1a superinfection was found. This superinfection was confirmed with clonal analysis of this NS5B fragment. This genotype was not detectable in earlier samples, using a subtype specific NS5A-PCR. Sequence analysis did not show any RAS in genotype 1a. In July 2017, the patient initiated treatment with sofosbuvir, elbasvir/grazoprevir and ribavirin for 24 weeks. HCV RNA levels decreased rapidly and

Respiratory Viruses in Invasively Ventilated Critically Ill Patients-A Prospective Multicenter Observational Study

Objectives: The presence of respiratory viruses and the association with outcomes were assessed in invasively ventilated ICU patients, stratified by admission diagnosis. Design: Prospective observational study. Setting: Five ICUs in the Netherlands. Patients: Between September 1, 2013, and April 30, 2014, 1,407 acutely admitted and invasively ventilated patients were included. Interventions: None. Measurements and Main Results: Nasopharyngeal swabs and tracheobronchial aspirates were collected upon intubation and tested for 14 respiratory viruses. Out of 1,407 patients, 156 were admitted because of a severe acute respiratory infection and 1,251 for other reasons (non–severe acute respiratory infection). Respiratory viruses were detected in 28.8% of severe acute respiratory infection patients and 17.0% in non–severe acute respiratory infection (p < 0.001). In one third, viruses were exclusively detected in tracheobronchial aspirates. Rhinovirus and human metapneumovirus were more prevalent in severe acute respiratory infection patients (9.6% and 2.6% vs 4.5 and 0.2%; p = 0.006 and p < 0.001). In both groups, there were no associations between the presence of viruses and the number of ICU-free days at day 28, crude mortality, and mortality in multivariate regression analyses. Conclusions: Respiratory viruses are frequently detected in acutely admitted and invasively ventilated patients. Rhinovirus and human metapneumovirus are more frequently found in severe acute respiratory infection patients. Detection of respiratory viruses is not associated with worse clinically relevant outcomes in the studied cohort of patients.

Course â Prevalence, Clinical Outcomes and Viral Shedding Patternsduring Viral Respiratory Tract Infections in Intubated Intensive CareUnitâÂÂpatients: Design and Protocol

Background: There is uncertainty about the prevalence of viral respiratory tract infections in intensive care unit (ICU) patients, and whether these infections contribute to disease severity and final outcome. Furthermore, the pattern of viral shedding during influenza infections in critically ill patients is largely unknown. Objectives: This study will assess prevalence of viral respiratory tract infections in intubated and ventilated ICU patients. Secondary objectives are to assess risk factors for and burden of viral respiratory tract infections, to describe the proportion of these infections missed in routine care, and to describe viral shedding patterns during influenza infection. Design: This is an investigator–initiated national multicenter prospective observational study conducted during one influenza season. Consecutive intubated and ventilated adult critically ill patients are included, regardless of admission diagnosis. Nasopharyngeal swabs and tracheobronchial aspirates are collected daily till patients are weaned of mechanical ventilation. Samples will be tested using multiplex real–time polymerase chain reaction (RT–PCR) for respiratory viruses. In influenza–positive patients, subsequent daily samples will be tested for influenza via RT–PCR until viral clearance. Influenza RT–PCR positive samples will be cultured and influenza subtyping will be performed. The primary endpoint is prevalence of viral respiratory tract infections in ICU patients. Discussion: COURSE will provide insight into the prevalence of, risk factors for, burden of and shedding patterns in viral respiratory tract infections in intubated and ventilated ICU patients during one winter season in the Netherlands. It is the largest prospective observational study thus far, with simultaneous sampling of both upper and lower respiratory tract of consecutive acutely admitted intubated and ventilated ICU patients, regardless of the admission diagnosis. Results of COURSE may guide future allocation and methods of viral diagnostic testing, quarantine practices, and duration of treatment with anti–viral drugs in ICU patients.