Frequent HCV Reinfection and Superinfection in a Cohort of Injecting Drug Users in Amsterdamq

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 seroconverters 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 seroconversion (t = 1), the first visit after seroconversion (t = 1), six months after (t = 2) and one year after (t = 3) seroconversion, 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 superinfected, 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 participating 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 initial 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 llof2 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 diseases 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 80 °C. The moment of HCV serocon- (337 bp, nt 8279–8615). In brief, 25 ll of cDNA was added to 25 ll reac- version was calculated as the midpoint between the last HCV-seroneg- tion mixture containing PCR II Buffer (Applied Biosystems), 200 lmol/ ative visit and the first HCV-seropositive visit. L of each dATP, dCTP and dGTP, plus 400 lmol/L dUTP (Applied Bio- For each HCV seroconverter, samples taken at five time points systems), 0.1 lg/lL bovine serum albumin (Roche Diagnostics), 0.9 lM were selected, if available: the last visit before HCV antibody serocon- sense primer 50-TATGAYACCCGCTGYTTTGACTC-30, 0.9 lM anti- version (t = 1), the first visit with a positive HCV antibody test sense primer 50-TAYCTVGTCATAGCCTCCGTGAA-30, 0.5 U Uracil (t = 1), visits approximately six months later (t = 2) and one year later N Glycolase (Applied Biosystems) and 2.5 U Amplitaq Gold (Applied (t = 3), and the last ACS visit prior to November 2005 (t = 4). A subset T.J.W. van de Laar et al. / Journal of Hepatology 51 (2009) 667–674 669

Biosystems). The final MgCl2 concentration was 2.5 mmol/L. PCR method in Mega version 4.0 [20], using the Tamura–Nei substitution cycling conditions were 2 min at 50 °C and 10 min at 95 °C followed model with c-distribution (a = 0.40). Bootstrap values (n = 1000) were by 45 cycles of: 20 s at 95;°C, 20 s at 55 °C and 60 s at 72 °C, with a final calculated to analyze the robustness of tree topology. incubation of 5 min at 72 °C. The cloning PCR product was cut from the agarose gel, resolved in 900 ll lysis buffer and purified using a shortened Boom-isolation protocol [16]. Purified amplicons were ligated into PCR II vector using the TOPO TA Cloning kit (Invitrogen). The plasmid was 3. Results used to transform competent Escherichia coli cells, which were plated on LB agar plates containing ampicillin (100 lg/ml) and Xgal (5-bromo-4- chloro-3-indolyl-B-galactoside). Based on Xgal selection, 50–100 white 3.1. Study population clones, containing the PCR insert, were picked for each time point and grown overnight at 37 °C on LB agar plates. For each clone, the PCR In the ACS, 456/1259 (36.2%) participating DU were insert was amplified using M13 primers, according to the TOPO TA Cloning kit protocol, and then sequenced. HCV-antibody negative at entry. Their median age was 31 years (IQR, 28–36 years), 70% was male and 11/456 2.6. Sequencing and phylogenetic analysis (2.4%) were HIV-positive. Only 132/456 (28.9%) HCV- antibody negative DU had ever injected, of whom 64 PCR products were sequenced using previously described methods (14.0%) in the last 6 months [13,21]. We identified 59 [18]. Viral genotype was determined after phylogenetic analysis of the DU who seroconverted for HCV during ACS follow- NS5B sequences obtained (GenBank Accession Nos. FJ024088 to FJ024273) along with established GenBank reference sequences [19]. up: 58% was male and their median age at HCV sero- A HCV phylogenetic tree was constructed by the neighbour-joining conversion was 29 years (IQR, 25–34 years). The median

1-1-2000 DU 12905 N7-6- 1999-23-4-2000? N N 1a 16-1-2004-8-7-20043a 3a 3a 3a 3a

?1a 3a N N DU 16786

DU 16879 N N N 1a 1a 1a

N N31-8- 1999- 19-7-2000 N N16-11-2000 -5-- 32001 ? N N ? N 24-6- 2010N- 14 -10- 2010

DU 16991 1-1-2000

1-1-2000 DU 16994 N 19-1a5-1999-22-8-2000 N N ? 24-5- 2001N- 28-8-2001 N 4-12 -2001 -8-4- 2002 6-8- 2002?-5-11-2002 N N

1-1- 2000 DU 18822 8-4-1996-24-7-1996 N 4d N N10 -12-1997- 6-8- 2003 1a 1a

DU 18917 1a 3-7-1998-14N-6-2001 N 1a* 3a 1a* 1a*

DU 19741 N N1-1-2000 N N 1b 1b 1b

1-1-2000 DU 19753 N N N 3a N

DU 19927 N N N ? 1a 1a 1a 1a 1a 3 years

1-1-2000 DU 12959 N ? 25-23-91--1999-20-2000-22-5-9-20003a 3a? 1a

4d 1-1-2000 4d 4d 1a 1a DU 12962 1b

N 1-1-2000 RNA negative visit DU 12970 N 3a 3a 3a 3a 1a 1a 1a ? genotype untypable

1-1-2000 1a 1a* DU 13925 1a 4d26-9-1998-2-8-20012b 4d 14-114d-2002-12-3-2003 genotype 1a 1b genotype 1b DU 14956 N 1a 1a * ? 1b 1b 1b 1b 2a genotype 2a 2b genotype 2b

1-1-2000 3a 3a* DU 16941 3a 3a* 3a* 3a* 3a* 3a* genotype 3a 4a genotype 4a

10-1-2000 DU 17889 16-3-1999?-1- 1- 2000 1a 3a 4d genotype 4d Follow-up without IDU DU 18781 N 4d 4d 2a Follow-up with IDU

1-1-2000 6-4- 1999- 10-8-2005 HCV seroconversion DU 18787 3a N 3a 3a 3a 3a 1a 3a 3a HIV seroconversion

1-1-2000 DU 18796 1a 3a

1-1-2000 DU 18805 1a 4d 4d 4d 4d

1-1-2000 DU 18898 20-1-1998-3-N3- 2001 1b N 4d 4d 1a 1a12-7-2001-6-3-2003 1a 15 2009 1a N

DU 18934 N 4a 1a 3a 1a* 3 years

Fig. 1. (A) 10 HCV seroconverters with viral clearance and multiple HCV infections. (B) 13 HCV seroconverters without viral clearance and multiple HCV infections. 670 T.J.W. van de Laar et al. / Journal of Hepatology 51 (2009) 667–674 year of HCV seroconversion in the ACS was 1991 [IQR: tent HCV reinfection: DU 18917 already was HIV- 1989–1994], or 2.23 years (IQR: 0.93–6.49 years) after positive before HCV seroconversion and DU 12905 initiation of drug injection. The median ACS follow- developed persistent HCV re- and superinfection up time since HCV seroconversion was 7.06 years around HIV-seroconversion. The other four DU with [IQR, 2.81–12.1]. Only one HCV seroconverter denied persistent HCV reinfection were HIV-negative at the ever injecting. HCV/HIV coinfection occurred in 13/59 time; 1 DU (DU 19927) eventually acquired HIV (22%) HCV seroconverters; HCV infection preceded years after the establishment of persistent HCV rein- HIV infection in 6/13, 5 contracted both viruses during fection. None of the three HCV seroconverters that the same brief period, and two DU were HIV-positive cleared a second or even third viremic episode were before they acquired HCV. coinfected with HIV.

3.2. Dominant HCV variant detection 3.4. HCV seroconverters without spontaneous HCV clearance The 59 HCV seroconverters were tested for HCV RNA on a total of 326 visits, varying from 2 to 10 visits Chronic HCV infection without evidence of previous per DU. At 211/326 (65%) of these visits, HCV RNA viral clearance occurred in 35/59 (59%) HCV serocon- was detected using a real- time PCR based on the 50- verters. According to our definitions, 13/35 (37%) sero- UTR; log HCV viral loads varied from 2 to 6.14 IU/ converters had HCV superinfections over time (Fig. 1b); ml. Amplification and sequencing of the HCV NS5B at least two clearly distinct HCV strains were detected in region succeeded in 170/177 (96%) samples with a viral all 13 of them. From 6/13, we isolated three or even four load exceeding 1000 IU/ml and in 15/34 (44%) samples distinct HCV viral strains. In DU 18934, the dominant with a viral load below 1000 IU/ml. Sequencing analysis viral strain switched from HCV genotype 4a to genotype of HCV RNA-positive samples revealed that at least 23/ 1a, subsequently to genotype 3a and eventually to a het- 59 (39%) HCV seroconverters had evidence for multiple erologous strain of genotype 1a (denoted as 1a*). DU HCV infections over time. According to our definitions, 12962 had clear evidence of HCV coinfection. In the last 7 HCV seroconverters had HCV reinfections, 13 were visit predating HCV antibody presence, two distinct superinfected, 2 were reinfected and subsequently super- HCV strains were detected using the NS5B PCR, one infected, and 1 was coinfected and subsequently superin- was HCV subtype 1b and one was HCV subtype 4d. fected. In total, 93 different HCV infections were HCV subtype 4d became the dominant viral strain, how- identified: 59 initial infections, 11 reinfections, 22 super- ever it seems to have submerged after superinfection infections and 1 coinfection. Genotyping and sequenc- with HCV genotype 1a. No particular pattern in the ing succeeded for 74/93 (80%) of HCV infections, switch of HCV strains was observed; for example the finding a genotype distribution of 1a (47%), 3a (32%), dominant HCV strains switched from genotype 1a to 4d (8%), 1b (6%), 2a/b (6%) and 4a (1%). 3a as often as from genotype 3a to 1a.

3.3. HCV seroconverters with spontaneous HCV clearance 3.5. Sequencing and phylogenetic analysis

Spontaneous HCV viral clearance occurred in 24/59 A phylogenetic tree was constructed of all 184 HCV (41%) HCV seroconverters including 2 DU who were sequences obtained from the 59 HCV seroconverters HIV-positive at the moment of HCV seroconversion. (Fig. 2). Minor genetic variations between HCV isolates Within this group of resolvers, 10/24 (42%) DU had evi- obtained from one host, were classified as intrahost dence for multiple HCV infections over time; 7 DU had HCV evolution. However, 5 seroconverters had clear HCV reinfections, 2 DU had HCV reinfection followed evidence of phylogenetically distinct HCV infections by HCV superinfection, and 1 DU had HCV superinfec- with strains of the same genotype (Fig. 2). HCV strains tion but spontaneously resolved both viral strains obtained from 2 other seroconverters were separated by (Fig. 1a). In 2/9 DU with evidence for HCV reinfection bootstrap value >70, and genetic variation exceeded we could confirm the presence of two distinct HCV intrahost evolution observed in other participants strains over time (confirmed reinfection). In the other (Fig. 2). Although reinfection with a very similar HCV 7 DU, however, viral characterisation of either the pri- strain for these 2 seroconverters is plausible, intrahost mary (n = 6) or the reinfecting (n = 1) HCV strain failed genetic evolution cannot be excluded. due to rapid viral clearance of especially primary HCV viremia (probable reinfection). 3.6. Minority HCV variant detection In 6/9 (67%) seroconverters with confirmed or probable HCV reinfection, HCV reinfection persisted Two patients, DU 18898 and DU 18917, were (Fig. 1a). It must be noted that 2/6 seroconverters selected for minority HCV variant detection. DU were coinfected with HIV when they developed persis- 18898 was selected because of two genotype switches T.J.W. van de Laar et al. / Journal of Hepatology 51 (2009) 667–674 671

16710 Apr03 18898 Jun90 Genotype 2 16710 Sep05 97 18898 Jun91 16710 Aug02 18898 Aug96 86 16710 Dec02 DU with 4 different HCV infections 18898 Oct00 18781 Oct95 DU 18934: 4a 1a 3a 1a* 19713 Aug05 19927 Feb92 D00944 2a D10988 2b 94 19927 May98 19927 Oct94 13925 Aug94 DU with 3 different HCV infections 100 19927 Oct93 12851 Feb03 78 DU 12962: 1b/4d 4d 1a 19927 Jun94 12851 Nov01 97 97 18783 Oct92 0.01 12851 Mar02 DU 13925: 1a 4d 2b 4d 18783 Jan93 12851 Aug05 DU 14956: 1a 1a* 1b 18783 Jan94 18783 May97 * DU 18787: 3a 1a 3a* 99 18783 Mar01 18783 Aug05 DU 18898: 1b 4d 1a 18917 Feb96 Genotype 3 79 18917 Sep99 I DU 18917: 1a 1a* 3a 1a* 18917 Sep89 12905 Jul01 81 19674 Nov94 12905 May05 12905 Aug98 18885 Jun87 98 DU with 2 different HCV infections 12905 Jan95 97 18886 Jun99 18886 Mar88 12905 Sep94 DU 12905: 1a 3a 18886 Sep89 12917 Sep88 DU 12959: 3a 1a 18886 Oct88 12917 Apr90 18886 Apr89 D17763 3a DU 12970: 3a 1a 18886 Aug95 12959 Jan88 19702 Jun93 18877 Oct89 DU 16786: 1a 3a 19702 Sep05 99 18877 Nov00 19702 May94 88 18877 May95 DU 16941: 3a 3a* 87 19702 Oct93 77 18877 Feb90 DU 17889: 1a 3a 19702 Mar93 71 18877 May90 18934 Oct05 II 18877 Sep90 DU 18781: 4d 2a 12959 Sep89 12970 Oct90 DU 18796: 1a 3a 18787 Oct95 94 12970 Jun91 19822 Sep89 12970 Jun90 93 DU 18805: 1a 4d 19822 Jan90 12970 Jan91 19822 Aug90 18787 Feb97 99 IV DU 18822: 4d 1a 19909 Oct89 18787 Sep02 11699 Jul05 87 19909 Feb90 19909 Jun89 99 11699 Mar01 19909 Jun90 11699 Aug01 19909 Jun05 11699 Jan02 16994 Mar87 99 12990 Jul96 M62331 1a 12990 Dec96 17889 Jun05 11854 Mar05 16941 Oct91 97 11854 Aug04 V 11854 Oct05 18934 Jun95 13925 Dec93 16652 Jul99 99 16879 Oct99 16652 Feb00 97 16879 Nov00 16652 Oct99 16879 Oct98 18796 Oct91 18917 Jan90 18980 Mar90 71 99 18980 Jul90 18958 Mar91 18990 Oct90 18958 Nov94 18980 Dec95 99 18958 Nov90* 18980 Oct00 18958 Aug90 18980 Feb91 18897 Apr87 18787 Jan93 18796 Jun91 18934 Oct94 18787 Dec93 II 95 18787 Aug94 13952 Jan89 IV 99 18787 Aug93 13952 Jul89 18787 Mar94 13952 Jun92 19868 Jul92 13952 Sep88 99 16941 Jun92 13952 Oct89 80 16941 Mar93 13952 Feb95 16941 Feb92 V 81 14956 Jun90 III 94 16941 Jun96 93 12962 Feb89 95 16941 Oct99 12962 Mar92 16786 Sep94 18775 Dec99 16824 Oct93 19666 Sep04 97 16824 Aug94 19666 May05 99 16824 Feb94 19666 Nov05 19753 Jul94 91 12970 Oct99 16786 Apr96 12970 Jan03 17847 Aug03 12970 Oct95 17889 Oct03 99 17847 Aug04 14956 Nov90 III 70 17847 Jan05 17847 Feb04 98 80 12905 Jan94 18917 Mar88 I 17847 Jul05 18822 Jul04 99 18822 Jul05 0.01 Genotype 1a 19780 May92 19780 Sep92 96 19780 Mar93 19780 Jul91 13925 Apr95 18805 Oct95 99 13925 Jul97 19755 May94 71 90 13925 Apr94 19755 Sep94 0.01 18781 Jun93 19755 Jan95 99 19755 Jan94 18781 Nov93 19755 Aug98 18822 Apr95 19755 Oct01 18898 Feb90 86 19755 Aug04 Genotype 4 18898 Nov89 97 12962 Jan88 Genotype 1b 90 12962 Jun88 14956 Nov91 12962 Oct87A 14956 Dec97 18805 Jan96 98 14956 Nov94 100 18805 Nov97 14956 Sep01 96 18805 Nov96 12962 Oct87B 18805 May97 18898 Mar89 D86537 4d 19741 Jun05 D90208 1b Y11604 4a 18934 Jul94

0.01 0.02

Fig. 2. HCV NS5B Phylogenetic tree comprising longitudinal samples from 59 drug users with documented HCV seroconversion. Coloured squares, triangles and circles represent HCV seroconverters with phylogenetically distinct HCV infections over time. Seroconverters with multiple infections of the same genotype are marked I–IV. Shaded boxes show intrahost HCV evolution among HCV seroconverters without evidence for HCV reinfection and superinfection. Shaded boxes with an asterisk represent seroconverters who might be superinfected with a very similar HCV strain. 672 T.J.W. van de Laar et al. / Journal of Hepatology 51 (2009) 667–674

(1b ? 4d ? 1a) within a period of 15 months. We imal risk of HCV reinfection. In contrast, studies per- selected, amplified, and sequenced 40–56 clones repre- formed in HCV seroconverter cohorts of young (<30 senting the four HCV RNA-positive visits during this years) actively injecting DU implied absence of partial interval (Fig. 1b). For each time point, all clones were HCV protective immunity based on the fact that the quasispecies of one HCV strain consistent with the dom- incidence of HCV reinfection in these cohorts was simi- inant genotype detected at that time point. Hence, no lar [6] or even higher [10] than the incidence of initial dual infections were detected (data not shown). infection, causing 46–50% of DU who previously cleared DU 18917 was selected because, after resolution of the virus to experience a second viremic episode. the primary HCV 1a infection, we observed reinfection The data in our study are concordant with those of with a different strain of HCV genotype 1a (1a*), subse- other seroconverter cohorts of young actively injecting quent superinfection with HCV 3a, and eventually a DU [6,10]; 42% of previously exposed DU experienced back-switch to the HCV 1a* strain, suggesting 3a/1a* HCV reinfection. Also in our cohort, preliminary superinfection (Fig. 1a). We selected, amplified, and incidence calculations suggest that the incidence of sequenced 35 clones from the first and last HCV RNA HCV reinfection were at least similar to the incidence of sample. Except for variations in quasispecies, all clones naı¨ve HCV infection. The incidence of HCV reinfection belonged to the dominant subtype 1a and heterologous declined from 20.4/100 PY in the period 1985–1995 to 1a*. For the three intermediate time points, 80–100 4.17/100 PY in the period 1995–2005, whereas the inci- clones were sequenced. Evidence of superinfection was dence of initial HCV infection in our cohort dropped from found only in the third intermediate sample; 98/100 27.5/100 PY in the late 1980s to approximately 2.0/100 clones were of subtype 1a* and 2/100 clones were sub- PY in recent years [13]. Based on this incidence rate ratio type 3a (data not shown). it is tempting to imply that partial HCV protective immunity does not exist, but is this justified? As HCV reinfection might be associated with lower peak viremia and 4. Discussion higher rates of spontaneous viral clearance, the real data that address this question are those regarding the In a large longitudinal cohort of DU in Amsterdam, outcome and nature of HCV reinfection. In contrast to we demonstrated that 39% of DU with a documented long-lasting antibody seroconversion, HCV reinfection HCV seroconversion during follow-up, experience mul- requires detection of new HCV viremia [24]. The proba- tiple HCV infections over time. Both HCV reinfection bility of detecting transient HCV reinfection strongly and superinfection were common in this high-risk popu- depends on the frequency and interval of RNA testing lation. Traditional HCV incidence calculations based on [24]. In our study design the relatively long RNA testing HCV antibody seroconversion, with the assumption that intervals will support detection of all persistent HCV rein- HCV seropositive individuals no longer are susceptible, fections, but short episodes of resolved viremia are easily therefore underestimate the true HCV incidence and do missed. Indeed 14/24 (58%) seroconverters resolved initial not properly reflect HCV transmission dynamics within HCV viremia but lacked evidence for HCV reinfection, a network of high-risk individuals [22]. implying either rapid spontaneous viral clearance, protec- As the occurrence of HCV reinfection after initial tion against HCV reinfection or cessation of HCV risk viral clearance has been previously described [6,10– behaviour. Of them, 7/14 (50%) were at minimal risk for 12,23], there is no question that in humans HCV protec- HCV reinfection as they quit injection within 6 months tive immunity is not even close to being complete. The after HCV seroconversion, the remaining 7 DU had no existence of partial protective HCV immunity in detectable HCV viremia despite ongoing risk behaviour. humans, however, remains controversial. Previous stud- Additionally, 3/9 seroconverters with documented HCV ies among anti-HCV positive DU showed that HCV reinfection resolved a second or even third viremic epi- infection occurred at lower rate in previously exposed sode. Besides infrequent sampling, data on persistence individuals than in naı¨ve individuals [11,12,24]. Despite of HCV reinfection are often skewed by HIV/HCV coin- reinfection rates of 3–12%, this suggests that some level fection. HIV-coinfection is associated with increased of immunity is being induced from past exposure. How- rates of HCV persistence (up to 95%); lack of robust ever, these studies were conducted in DU with prevalent CD4+ T cell responses may contribute to the failure of HCV infection; the period of high-risk behaviour which early viral control of HCV [25,26]. HCV reinfection per- led to anti-HCV seroconversion most likely preceded the sisted in the two DU coinfected with HIV, but also in 4/ study period by several years. As a result, previously 7 DU without HIV. HCV-infected DU were generally older (median age Our study has several limitations. Despite our clear- 41–47 years) and had less active drug use compared to cut definitions of HCV reinfection, coinfection and HCV-uninfected DU [24]. In fact, 35–58% of partici- superinfection, the unpredictable course of HCV infec- pants in those studies had quit injecting before the tion makes it difficult to distinguish them as such. Mos- actual start of the study period, being therefore at min- ley et al. [27] showed that intercalating HCV RNA T.J.W. van de Laar et al. / Journal of Hepatology 51 (2009) 667–674 673 positivity among those who eventually clear infection demic Medical Center of the University of Amsterdam, and intercalating HCV RNA negativity among those Sanquin Blood Supply Foundation and the University who eventually develop persistent infection, are com- Medical Center Utrecht, are part of the Netherlands mon especially during the initial phase of HCV infection HIV monitoring Foundation and financially supported [27]. It cannot be excluded that in our study DU with by the Netherlands National Institute for Public Health ‘probable reinfections’ actually have fluctuating levels and the Environment. Website: http://www.amster- of low viremia, which would have led us to overestimate damcohortstudies.org. Additional funding was provided the rate of HCV reinfection. To minimise the risk of by The Netherlands Organisation for Health Research such misclassification, we defined viral clearance as and development (Grant 912-03-005). two consecutive HCV RNA-negative visits at least 4 months apart. Underestimation of the rate of HCV reinfection was also very likely for three reasons. First, HCV References reinfection in humans and chimpanzees has been associ- [1] Roy K, Hay G, Andragetti R, Taylor A, Goldberg D, Wiessing L. ated with short episodes of low viremia [9,12], therefore Monitoring hepatitis C virus infection among injecting drug users reinfections might have been missed as a result of too in the European Union: a review of the literature. Epidemiol low frequency of sampling and RNA levels below detec- Infect 2002;129:577–585. tion limit. Second, in the context of a steady injection [2] Amon JJ, Garfein RS, Ahdieh-Grant L, Armstrong GL, Ouellet partner, reinfection with a HCV strain almost identical LJ, Latka MH, et al. 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