Journal of Hepatology 51 (2009) 667–674 www.elsevier.com/locate/jhep Frequent HCV reinfection and superinfection in a cohort of injecting drug users in Amsterdamq Thijs J.W. van de Laar1,3,*, Richard Molenkamp2, Charlotte van den Berg1,3, Janke Schinkel2, Marcel G.H.M. Beld2, Maria Prins1,3, Roel A. Coutinho1,3,4, Sylvia M. Bruisten1,3 1Cluster of Infectious Diseases, Public Health Service, Nieuwe Achtergracht 100, 1018 WT Amsterdam, The Netherlands 2Department of Medical Microbiology, Academic Medical Centre, Amsterdam, The Netherlands 3Department of Internal Medicine, Centre for Infection and Immunity Amsterdam (CINIMA), Academic Medical Centre, Amsterdam, The Netherlands 4Centre for Infectious Disease Control, National Institute of Public Health and the Environment, Bilthoven, The Netherlands See Editorial, pages 628–631 Background/Aims: This study investigates the occurrence of HCV reinfection and superinfection among HCV serocon- verters participating in the Amsterdam Cohort Studies among drug users from 1985 through 2005. Methods: HCV seroconverters (n = 59) were tested for HCV RNA at five different time points: the last visit before sero- conversion (t = À1), the first visit after seroconversion (t = 1), six months after (t = 2) and one year after (t = 3) serocon- version, and the last visit prior to November 2005 (t = 4). If HCV RNA was present, part of the NS5B region was amplified and sequenced. Additional phylogenetic analysis and cloning was performed to establish HCV reinfection and superinfection. Results: Multiple HCV infections were detected in 23 /59 (39%) seroconverters; 7 had HCV reinfections, 14 were super- infected, and 2 had reinfection followed by superinfection. At the moment of HCV reinfection, 7/9 seroconverters were HIV-negative: persistent HCV reinfection developed in both HIV-positive cases but also in 4/7 HIV-negative cases. In total, we identified 93 different HCV infections, varying from 1 to 4 infections per seroconverter. Multiple HCV infections were observed in 10/24 seroconverters with spontaneous HCV clearance (11 reinfections, 3 superinfections) and in 13/35 seroconverters without viral clearance (20 superinfections). Conclusions: HCV reinfection and superinfection are common among actively injecting drug users. This might further complicate the development of an effective HCV vaccine. Ó 2009 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. Keywords: HCV; Reinfection; Superinfection; Epidemiology; Phylogeny; Injecting drug use; Immune protection Received 9 September 2008; received in revised form 23 April 2009; 1. Introduction accepted 5 May 2009; available online 18 June 2009 Associate Editor: J.G. McHutchison q The authors who have taken part in this study declared that they do Injecting drug users (DU) are at high risk for hepati- not have anything to disclose regarding funding from industry or tis C virus (HCV) infection through the shared use of conflict of interest with respect to this manuscript. needles and injection equipment. The reported HCV * Corresponding author. Tel.: +31 20 5555506; fax: +31 20 5555533. seroprevalence among injecting DU ranges from 30 to E-mail address: [email protected] (T.J.W. van de Laar). Abbreviations: ACS, Amsterdam cohort studies; DU, drug users; 90% in Europe, North-America, and Australia [1–3]. HCV, hepatitis C virus; HIV, human immunodeficiency virus; PCR, Over decades, persistent HCV viremia can cause chronic polymerase chain reaction; PY, person years. hepatitis, liver cirrhosis, and hepatocellular carcinoma 0168-8278/$36.00 Ó 2009 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2009.05.027 668 T.J.W. van de Laar et al. / Journal of Hepatology 51 (2009) 667–674 [4]. Spontaneous resolution of the virus occurs in of 19 HCV seroconverters from the ACS identified in an earlier study 15–40% of those infected [5]. However, both HCV had more than five samples available [15]. reinfection and HCV superinfection have been docu- 2.2. Definitions: reinfection, coinfection and superinfection mented among individuals with ongoing risk behaviour [6,7], suggesting that neither viral clearance nor ongoing Spontaneous viral clearance was assumed if two or more consecu- HCV infection consistently protect against new HCV tive visits, at least 4 months apart, showed HCV antibodies without infection. Evidence for partial protective immunity detectable HCV RNA. During our study period, none of the partici- pating DU had received HCV antiviral treatment. against HCV infection is derived largely from chimpan- HCV reinfection refers to a situation in which a primary HCV zee studies. Chimpanzees that previously cleared HCV infection is spontaneously cleared prior to subsequent infection with and were rechallenged with homologous HCV strains, a HCV strain of a homologous or heterologous lineage. All subjects who had two or more consecutive anti-HCV positive visits without generally showed lower levels of HCV viremia and detectable HCV RNA, at least 4 months apart, prior to an HCV self-limited infection [8,9]. Whether humans previously RNA-positive visit were defined as reinfected. This includes (i) HCV infected with HCV gain protection against a second seroconverters that had HCV RNA positive visits, separated by at least two RNA negative visits, but also (ii) HCV seroconverters that HCV infection remains controversial [6,10–12]. remained HCV RNA negative at their first anti-HCV positive test The incidence rate ratio of naı¨ve HCV infection ver- (t = 1), then returned at least one more consecutive RNA negative visit sus HCV reinfection in a high-risk population, is an prior to an RNA-positive visit. HCV coinfection and HCV superinfection both refer to HCV dual indirect but frequently used tool to gain insight into infections. Coinfection is defined as infection with two or more heter- the presence or absence of partial HCV immunity ologous HCV strains simultaneously or within a window period too [6,10–12]. Clearly, if HCV infection occurs at a lower narrow for the first HCV infection to have resulted in detectable HCV antibodies. Superinfection is defined as a subsequent infection rate in previously exposed individuals compared to with a heterologous HCV strain in the presence of a previous HCV naı¨ve individuals it is reasonable to assume some level strain and the antibodies that it has generated. All changes in HCV of immunity is being induced from past exposure to strains over time without a demonstrated spontaneous clearance in between were considered as superinfections. HCV. Alternatively, if HCV reinfection occurs at similar or higher rates, this means that at least some individuals 2.3. HCV RNA quantification despite having the immunological capacity to clear ini- tial HCV infection, fail to elicit an immune response that HCV serum RNA was quantified by an in-house real-time PCR assay 0 provides sufficient protection against HCV reinfection based on the 5 -UTR region of the HCV genome [15]. In brief, RNA extraction was performed using the Boom method [16], in which 200 ll [12]. Insight into HCV protective immunity after previ- of serum and 15 ll of internal control were added to 900 ll of lysis buffer ous exposure contributes to vaccine development and and 20 ll of size-fractioned coarse silica particles. RNA was eluted in a also provides health guidance for preventive strategies volume of 100 ll and transcribed to cDNA as detailed elsewhere [17]. Real-time PCR mixes (25 ll total volume) contained 12.5 llof2Â in populations at risk. Therefore, we studied the occur- LC480 probes master, 0.6 lM of forward and reverse primers (HCV47F: rence of HCV reinfection and superinfection in a well- 50-GTGAGGAACTACTGTCTTCACG-30, HCV312R: 50-ACTCGC 0 defined cohort of injecting DU from the moment of AAGCACCCTATCAGG-3 ), and 0.2 lM of labeled HCV and IC taq- man probes (HCV-P129: 50-FAM-CTCCCGGGAGAGCCATAGTG HCV seroconversion until the end of follow-up. This GTCTGCG-MGB-NFQ-30, HCV-IC: 50-VIC-ATGGCCACAGCGCC study uniquely combines longitudinal data from pro- GCGGTGTTAGTGC-MGB-NFQ-30). Real-time PCR was performed spectively identified HCV seroconverters with an epide- on a Roche LC480 using the following cycling conditions: 2 min at 50 °C and 10 min at 95 °C followed by 50 cycles of: 20 s at 95 °C, 20 s at 55 °C, miologic and phylogenetic approach. and 1 min at 72 °C. Quantification of viral RNA was performed by using standard curves which were produced by linear regression analysis of dilution series of plasmid DNA. 2. Materials and methods 2.4. Dominant HCV variant detection: the NS5B PCR 2.1. Study population and sample selection HCV RNA-positive samples for each DU were selected to docu- ment changes in the dominant HCV viral variant in individuals over This study comprises all DU participants (n = 59) who serocon- time. From each sample, 3 ll cDNA was used as input for a nested verted for HCV during follow-up in the Amsterdam Cohort Studies multiplex PCR which amplifies 449 nucleotides of the HCV NS5B (ACS) between December 1985 and November 2005 [13]. The ACS region (nt 8546–8994). Except for some modifications made to use is an open, prospective cohort study of both injecting and non-inject- cDNA instead of RNA as PCR input, conditions and primers of the ing DU, initiated in December 1985 [14]. Recruitment is still ongoing NS5B PCR were those we described earlier [18]. and takes place via local methadone posts, sexually transmitted dis- eases clinics, and by word of mouth. At ACS visits every 4–6 months, 2.5. Minority HCV variant detection: the cloning PCR participants complete a standardised questionnaire about personal health, risk behaviour and socio-economic situation; blood is drawn The cloning PCR is a single round PCR targeting the NS5B region for HIV-testing and storage at À80 °C. The moment of HCV serocon- (337 bp, nt 8279–8615).