Introduction
Since 2000, hepatitis C virus (HCV) has emerged as a sexually transmitted infection (STI) among HIV-positive MSM [1,2]. Sexual transmission of HCV among HIV-positive MSM has been associated with condomless anal sex (CAS); a higher lifetime number of sex partners; recent STI; sexual techniques associated with damage of the anorectal mucosa (e.g. fisting and the use of sex toys); and recreational drugs use before or during sex [3]. Although HIV is no prerequisite for sexual transmission of HCV, HIV-negative MSM remain largely unaffected [4–7].
Why are HIV-positive MSM disproportionally affected by HCV compared with HIV-negative MSM? HIV itself might facilitate sexual transmission of HCV through irreversible immunological damage to the gastrointestinal mucosa – the portal of entry for sexually acquired HCV – and increase host susceptibility to small inoculums of HCV [8]. Transport of HCV across the gastrointestinal mucosa might be further enhanced by HIV-induced activation of Langerhans cells [9]. HIV serosorting (i.e. engaging in sex with partners with the same HIV status) combined with frequent HCV RNA shedding in semen and rectal fluids of HIV coinfected MSM might also (partly) explain why sexual transmission of HCV remains largely confined to HIV-positive MSM [10–13]. An alternative explanation may be that HIV is more easily transmitted through sex than HCV, and therefore often precedes HCV infection. If this last hypothesis is true, increased uptake of preexposure prophylaxis (PrEP), which proved highly successful in preventing HIV infection in MSM at high risk [14], could eventually result in an expanding HCV epidemic, irrespective of HIV status.
The aim of the current study was to determine whether HIV-negative MSM starting PrEP in Amsterdam are at increased risk for HCV infection. Phylogenetic analysis was used to compare HCV strains obtained from HIV-negative MSM with HCV strains circulating among HIV-positive MSM in the Netherlands.
Material and methods
Study population
The Amsterdam PrEP project (AMPrEP) project is a prospective, longitudinal, open label cohort study to assess the uptake and acceptability of daily versus event-driven PrEP. Participants (n = 376) were MSM or transgender persons, at least 18 years, who were HIV-negative but at high-risk for acquiring HIV infection. Eligibility for AMPrEP required at least one of the following during the preceding 6 months: a documented STI (urethral or anal Chlamydia trachomatis or Neisseria gonorrhoeae infection, primary or secondary syphilis); self-reported CAS with casual partners; a course of postexposure prophylaxis after a sexual risk incident; or an HIV-positive partner with unknown or detectable HIV viral load. Inclusion took place from August 2015 to June 2016. Participants were offered daily or event-driven use of tenofovir disoproxil fumarate with emtricitabine and are monitored 3-monthly at the Public Health Service of Amsterdam (GGD) up to June 2018. The study protocol was approved by the ethics committee of the Academic Medical Center in Amsterdam and is available online [15]. All participants provided written informed consent.
Laboratory testing
At baseline, all participants were tested for HIV antigen/HIV antibodies (LIASON XL Murex HIV Ag/Ab; Diasorin, Saluggia, Italy) and HCV antibodies (ARCHITECT anti-HCV; Abbott Laboratories, Mannheim, Germany) with immunoblot confirmation (INNO-LIA HIV I/II and HCV Score; Fujirebio, Ghent, Belgium). Simultaneous nucleic amplification testing (NAT) for HIV-1/2 RNA, hepatitis B virus (HBV) DNA, and HCV RNA was performed on pools of six plasma samples (COBAS Taqscreen MPX v2.0; Roche Diagnostics, Mannheim, Germany). None of the pools tested reactive for HIV-1/2 RNA. If pooled samples were reactive for HBV DNA or HCV RNA, individual NAT testing (CAP-CTM HBV or HCV test v2.0; Roche Diagnostics, Mannheim, Germany) was performed to identify the viremic participant. First, individual NAT testing was performed on participant(s) who tested positive for HBsAg (Architect HBsAg; Abbott Laboratories, Wiesbaden, Germany) or anti-HCV, respectively. If this did not reveal a viremic participant, the remaining serologically negative samples in the pool were tested for HBV DNA or HCV RNA. In addition, all participants were tested for syphilis (LIASON Treponema Screen; Diasorin) as part of routine STI screening, and NAT was performed for C. trachomatis and N. gonorrhoeae (Hologic Gen-Probe Inc., San Diego, California, USA) on urine, anal swabs, and pharyngeal swabs.
Hepatitis C virus genotyping and phylogenetic analysis
HCV RNA isolation was performed on 400 μl of plasma using QIAmp MinElute Virus Spin Kits (Qiagen, Hilden, Germany). Reverse transcription, PCR, and sequencing were performed as described previously [16]. Transcribed cDNA was used as input for two separate PCR assays targeting the NS5B region, resulting in the amplification of two overlapping NS5B fragments. Fragment 1 (340 nucleotides; nucleotides 8001–8340) and fragment 2 (421 nucleotides; nucleotides 8228–8709) were combined, resulting in an NS5B sequence of a total length of 709 nucleotides. Viral genotype was determined by phylogenetic analysis of NS5B sequences obtained from AMPrEP participants, along with well established GenBank reference sequences [17]. Phylogenetic trees were constructed for each HCV subtype separately, comparing HCV sequences obtained from HIV-negative AMPrEP participants, HIV-positive MSM, and risk groups other than MSM in the Netherlands. HCV strains from HIV-positive MSM were obtained from the MSM Observational Study of Acute Infection with Hepatitis C (MOSAIC) [n = 168, NS5B fragment 1 (F1)] [18,19] and the biannual cross-sectional anonymous surveys of the Amsterdam STI clinic [n = 55, NS5B fragment 2 (F2)] [20]. HCV sequence data from patients other than MSM were obtained from the Los Alamos National Sequence Database and included all available non-MSM HCV sequences from the Netherlands with a sampling date after 1999 (n = 153). We retrieved sequences of the HCV subtypes 1a, 2b, and 4d (Supplementary Table S1 lists all 153 accession numbers with the presumed mode of transmission, https://links.lww.com/QAD/B99). HCV phylogenies were constructed by the maximum-likelihood approach using a general time-reversed substitution model with γ-distribution, assuming a certain fraction of evolutionary invariable sites (GTR + G + I). Bootstrapping (n = 500) was used to analyze the stability of the tree topology (bootstrap values >70 represent robust clusters).
Nucleotide sequence accession numbers
HCV NS5B sequences from AMPrEP participants were submitted to GenBank (accession numbers: KY386877 to KY386891).
Statistical analyses
We calculated HCV prevalence and its corresponding 95% confidence interval (CI) among MSM who started PrEP in Amsterdam. CIs around prevalence were calculated via the binomial exact method. We compared characteristics of HCV-positive and HCV-negative participants using Fisher exact tests for categorical data and rank-sum tests for continuous data. A P value less than 0.05 was considered statistically significant. Analyses were performed using Stata Intercooled 13.1 (StataCorp, College Station, Texas, USA).
Results
Study population, hepatitis C virus prevalence and determinants
From August 2015 to June 2016, 376 participants were enrolled in AMPrEP, of whom 374 were MSM (99.5%) and 2 (0.5%) self-identified as transgender women. For 375 participants, a sample was available for HCV testing. At baseline, 17 of 375 (4.5%) participants tested positive for anti-HCV. HCV RNA was detected in 14 of 17 (82.4%) anti-HCV-positive participants. Pooled NAT screening yielded one additional HCV RNA-positive participant with no detectable anti-HCV, suggestive of recent HCV infection. Overall, 18 of 375 (4.8%, 95% CI 2.9–7.5%) of HIV-negative MSM tested positive for anti-HCV and/or HCV RNA. HCV infection had previously been diagnosed in six (33.3%) HCV-positive participants as a result of lab abnormalities or partner notification. Despite previous HCV diagnosis, three MSM with persisting HCV viremia who started PrEP had not initiated HCV treatment, two had recently started treatment not yet resulting in undetectable HCV RNA, and the remaining person had spontaneously cleared his HCV infection. The remaining 12 (66.6%) participants were not yet aware of their HCV status.
The HCV-positive participants were all MSM, and their median age was 33 years [interquartile range (IQR 28–42)] compared with 40 (IQR 33–48) among participants without HCV infection (P = 0.019) (Table 1). Of the 18 participants with HCV, 16 (88.9%) reported receptive CAS (rCAS) with casual partners, compared with 265 of 357 (74.2%) in those who were HCV-negative at baseline (P = 0.263). In the preceding 3 months, four of 17 (23.5%) HCV-positive participants had injected drugs (one HCV-positive person did not answer this question) compared with 11 of 353 (3.1%) of those without HCV infection (P < 0.005). Chemsex, defined as the use of gamma-hydroxybutyrate/gamma-butyrolactone (GHB/GBL), methamphetamine or mephedrone during sex, was reported by 15 of 18 (83.3%) of the HCV-positive participants, compared with 141 of 352 (40.1%) of the HCV-negative persons (P < 0.005). The choice of PrEP modality (daily versus event-driven) did not significantly differ between participants with and without HCV infection.
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Table 1: Baseline characteristics including sexually transmitted infections of hepatitis C virus-positive and hepatitis C virus-negative MSM starting preexposure prophylaxis in 2015/2016 in the Netherlands.
Hepatitis C virus genotyping and phylogenetic analysis
HCV genotyping was successful for all 15 HCV RNA-positive participants. HIV-negative MSM were predominantly infected with HCV subtype 1a (n = 11; 73%), followed by subtype 4d (n = 3; 20%) and 2b (n = 1; 7%). Phylogenetic trees were constructed separately for HCV subtypes 1a, 4d, and 2b. Most HCV phylogenetic studies in HIV-positive MSM in the Netherlands have sequenced either the NS5B F1 or the NS5B F2, but not both fragments. For the 15 HCV RNA-positive MSM who started PrEP, we obtained 15 of 15 (100%) NS5B F2 and 12 of 15 (80%) NS5B F1 sequences. Figure 1 shows the NS5B F2 phylogenies of HCV-1a, HCV-4d, and HCV-2b containing HCV sequences from our 15 HCV RNA-positive AMPrEP participants and 55 HCV sequences from HIV-positive MSM and 147 HCV sequences from risk groups other than MSM (predominantly people who inject drugs) in the Netherlands. HCV sequences obtained from HIV-negative MSM starting PrEP are highly interspersed with HCV sequences obtained from HIV-positive MSM: 13 of 15 (87%) of AMPrEP participants were part of six robust MSM-specific HCV clades, including four HCV-1a clusters, one HCV-4d cluster, and one HCV-2b pair. Figure S1 (Supplementary information, https://links.lww.com/QAD/B99) shows the NS5B F1 phylogenies of HCV-1a, HCV-4d, and HCV-2b containing 12 of 15 HCV RNA-positive AMPrEP participants along with 168 HCV sequences obtained from HIV-positive MSM and 117 HCV sequences from risk groups other than MSM in the Netherlands. Although phylogenetic clustering for NS5B F1 is less robust than for NS5B F2 due to shorter fragment length (340 bp for F1 versus 421 bp for F2), the NS5B F1 and NS5B F2 phylogenies are highly similar. The NS5B F1 phylogenies, however, include more HCV isolates from HIV positive MSM (n = 168 versus 55), and all 168 HIV-positive MSM in the NS5B F1 trees were diagnosed with acute HCV infection in the period 2008–2015. Despite its less robust clustering results, the NS5B F1 phylogeny contains more and more recent HCV isolates from HIV-positive MSM and suggests that in fact all 15 HIV-negative MSM with HCV who started PrEP were part of MSM-specific HCV clusters (six HCV-1a clusters, one HCV-4d cluster, and one HCV-2b cluster).
Fig. 1: Hepatitis C virus NS5B fragment 2 phylogenetic trees for hepatitis C virus subtypes 1a, 2b, and 4d comparing hepatitis C virus sequences from HIV-negative MSM starting preexposure prophylaxis (red branches and red stars) with hepatitis C virus sequences obtained from HIV-positive MSM (blue branches and blue dots) and unrelated hepatitis C virus-positive people other than MSM (black branches) in the Netherlands.
Discussion
The HCV prevalence in our cohort of 375 HIV-negative MSM and transgender persons who started PrEP in Amsterdam was 4.8%, including one HCV RNA-positive anti-HCV-negative MSM, probably a case of recent HCV infection. Similar to HIV-positive MSM in the Netherlands, HIV-negative MSM were mostly infected with HCV genotypes 1a (73%) and 4d (20%) [18,19]. HCV sequences of HIV-negative MSM starting PrEP were highly interspersed with HCV sequences obtained from HIV-positive MSM; all HCV mono-infected MSM were part of robust MSM-specific HCV clusters containing predominantly MSM with HIV infection. HCV infection among HIV-negative MSM starting PrEP was associated with younger age, more partners with whom rCAS was reported, an STI in the preceding 6 months, recent injecting drug use (IDU), and use of GHB/GBL, mephedrone and/or methamphetamine during sex.
The HCV prevalence of 4.8% found in our study is substantially higher than has been previously reported among HIV-negative MSM. Biannual cross-sectional surveys during the period 2007–2010 among HIV-negative MSM visiting the Amsterdam STI clinic showed a low and stable anti-HCV prevalence of about 0.6% (varying between 0 and 1.7%) [20]. Likewise in other western countries, the HCV prevalence among HIV-negative MSM was low (0.2–1.2%) [4,21] or strongly associated with IDU [22]. Does this mean that the HCV-epidemic is expanding to HIV-negative MSM? We have to keep in mind that HIV-negative MSM who start PrEP are not representative of the general HIV-negative MSM population. As the MSM who started PrEP in Amsterdam were at high-risk for HIV, they may have also been at increased risk for HCV. Recent Dutch studies suggest that the anti-HCV prevalence among the larger population of HIV-negative MSM remained low. Among 370 HIV-negative MSM visiting the Amsterdam STI clinic in November 2015 and 584 HIV-negative MSM participating in the Amsterdam Cohort Studies between January to June 2016 the anti-HCV prevalence was 1.4 and 0.34%, respectively (unpublished data). In contrast, the estimated HCV-prevalence (antibodies or HCV RNA) among HIV-positive MSM in HIV care in the Netherlands is 12% [23]. Although the anti-HCV prevalence in the larger population of HIV-negative MSM has remained low and HCV incidence among MSM seems to have stabilized in our region [6,23], we cannot exclude that we are observing the start of an HCV-epidemic among HIV-negative MSM with high-risk sexual behavior, with the potential of HCV spread to the larger HIV-negative MSM population.
Most guidelines on the use of PrEP are not specific on whether or not to test for HCV [24–26]. Previous PrEP trials and demonstration projects excluded MSM with HCV [27], tested only a subset of the participants at baseline [28], or did not report on HCV prevalence [29–31]. In three PrEP studies, the HCV incidence rate was 0.7–1.3 per 100 person-years [7,27,32]. If PrEP use leads to risk compensation (e.g. increased sexual risk-taking due to less concern for HIV), this may result in a substantial number of incident HCV infections among HIV-negative MSM on PrEP, especially in our setting where the baseline HCV prevalence was already much higher than previously reported. The indications for treatment of chronic HCV monoinfection and HIV/HCV coinfection do not differ and include treatment of people at risk for transmitting HCV, such as MSM with high-risk sexual behavior [33]. In the Netherlands, well tolerated and highly effective interferon-free direct-acting antivirals (DAAs) are available for all patients with chronic HCV infection since the end of 2015. Of the AMPrEP participants, one of three study participants who had not yet initiated treatment for HCV infection was enrolled before the availability of DAAs for all patients; it is unclear why treatment was not initiated by the other two HCV-infected patients. Further spread of HCV in the Netherlands may be limited by the fast uptake of DAAs among HIV/HCV coinfected and monoinfected MSM [34]. However, DAAs are not registered for use during acute infection. Modeling studies suggest that despite high treatment uptake among MSM, further HCV treatment scale-up and behavioral interventions are required to curb the epidemic [35,36].
All HCV-positive AMPrEP participants were part of robust MSM-specific HCV clusters that predominantly contained HIV-positive MSM. Indeed, 62% of HIV-negative MSM in AMPrEP reported HIV-positive sexual partners (data not shown). The overlap between sexual networks of HIV-positive and HIV-negative MSM might further increase, especially if the concern for HIV and HCV infections decreases with the availability of PrEP, combination antiretroviral therapy for HIV and DAAs for HCV. In Amsterdam, sex parties that were previously restricted to HIV-positive MSM are now open for men on PrEP as well [37]. These developments might facilitate introduction of HCV into the HIV-negative population and eventually result in an expanding HCV epidemic, irrespective of HIV status. This stresses the need for routine HCV testing in HIV-positive MSM and in MSM starting with or on PrEP, and treatment of those diagnosed with HCV.
In our study, only 17% of participants had no detectable HCV RNA in the presence of HCV antibodies, suggestive of spontaneous HCV clearance. This is lower than previously reported in sexually acquired acute HCV infections among MSM [5,38]. However, a systemic review by Micallef et al.[39] suggests a broad range in spontaneous clearance rates of HCV (0–80% with a mean of 26%, 95% CI 22–29%) with lower rates in men (20%) compared with women. Along with homozygosity for rs12979860 CC, female sex is a known predictor for spontaneous clearance of HCV infection. In addition, some of the men might have been recently infected and not yet cleared their infection, as the median time from acute infection to viral clearance is estimated at 16 weeks [40]. Since we took a cross-sectional sample among a highly sexually active group of MSM, we cannot exclude that some of our participants were recently infected and therefore had not yet cleared HCV infection.
Of particular concern is the report of a rise in IDU (referred to as ‘slamming’) and possible needle-sharing among MSM at sex parties in the United Kingdom, which might provide a second and more efficient route of HCV transmission among HIV-negative MSM [41,42]. Indeed, HCV-positive AMPrEP participants were more likely to report IDU than their negative counterparts, as has been observed among HIV-positive men [20]. Nevertheless, IDU did not explain HCV infection in more than half of the HIV-negative MSM who started PrEP.
A limitation of this study is that we may have missed acute HCV infections, as we tested HCV RNA in pools, followed by individual NAT testing starting with those who were already anti-HCV-positive. Hence, if a positive pool included both an acute and chronic case of HCV infection, we credited the antibody-positive participant without further testing, thereby possibly missing acute cases. Hence our findings might be an underestimation, and the true HCV prevalence might be even higher. After 12, 18, and 24 months following the PrEP start visit, all participants will be individually tested for anti-HCV and if positive, for HCV RNA. Another limitation is that, not anticipating such a high HCV prevalence, we collected only data on recent risk behavior and not on traditional risk factors (e.g. blood transfusion before 1992). Further research is needed to identify which risk factors are most relevant among HIV-negative MSM. Finally, the HCV NS5B region used in this study is suitable for diagnostic HCV genotyping, but its phylogenetic signal for detailed epidemiological purposes is limited [43]. However, none of the NS5B sequences obtained from risk groups other than MSM were part of MSM-specific clusters, which suggests that these clusters represent actual sexual transmission networks of MSM. In addition, the phylogenetic robustness of MSM-specific NS5B clusters has been confirmed by sequencing alternative HCV genomic regions, including NS3, E2, and the hypervariable region (HVR) [18,44], as well as by sequencing longer NS5B fragments [16]. The fact that all HCV-sequences obtained from HIV-negative MSM enrolled in AMPrEP were part of MSM-specific NS5B clusters containing mostly HIV-positive MSM suggests overlap between the sexual networks of HIV-positive and HIV-negative MSM. To better characterize the extent and nature of this overlap, more detailed information on transmission dynamics and perhaps even individual transmission events within these MSM-specific NS5B clusters could be obtained by choosing a less conserved HCV genomic region such as the HVR.
Based on our findings, we feel it is important to offer routine HCV testing to MSM at PrEP start and follow-up visits. In addition to education and targeted behavioral interventions, continued monitoring of HCV infection among HIV-negative MSM is recommended for timely detection of potential HCV spread to the larger population of HIV-negative MSM.
Acknowledgements
The authors wish to thank the following persons for their support to this study: Godelieve de Bree, Sylvia Bruisten, Yvonne van Duijnhoven, Kees de Jong, Paul Oostvogel, Ilya Peters, Lucy Phillips, Peter Reiss, Gerard Sonder, teams of clinic for STIs and research department of the GGD Amsterdam, and all of those who contribute to the H-team (Supplementary information Annex 1, https://links.lww.com/QAD/B99).
The AMPrEP project received funding as part of the H-team initiative, from ZonMw (grant number: 522002003), the National Institute for Public Health and the Environment and internal GGD research funds. The study drug is provided by Gilead Sciences. The H-TEAM initiative is being supported by the Aidsfonds Netherlands (grant number: 2013169). Amsterdam Dinner Foundation, Gilead Sciences Europe Ltd (grant number: PA-HIV-PREP-16-0024), Gilead Sciences (protocol numbers: CO-NL-276-4222, CO-US-276-1712), Janssen Pharmaceutica (reference number: PHNL/JAN/0714/0005b/1912fde), M.A.C. AIDS Fund, and ViiV Healthcare (PO numbers: 3000268822 and 3000747780). The MOSAIC study was financially supported by the Aidsfonds Netherlands (grant numbers 2008.026 and 2013.037).
Author's contributions: Study concept and design: E.H., T.J.W.v.d.L., and M.P. Acquisition, analysis or interpretation of the data: E.H., T.J.W.v.d.L., R.C.A.A., M.P., J.S., H.J.C.d.V., M.F.S.v.d.L., U.D., and A.H. Drafting of the article: E.H. and T.J.W.v.d.L. Critical revision of the article: all authors.
Conflicts of interest
E.H.: my institute received financial reimbursement for time spent serving on advisory boards of Gilead Sciences. J.S.: my institute received funding for improvement in HCV care from Gilead, MSD, Abbvie, and Janssen pharmaceuticals.
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