The incidence and prevalence of HIV infections in western settings have risen in the era of highly active antiretroviral therapy (HAART), particularly among male-sex-male (MSM) populations [1–3]. Comprehensive surveillance strategies are required to monitor emerging trends in HIV transmission and assess the effectiveness of intervention strategies [4–6].
Sequence data acquired from systematic antiretroviral resistance testing programmes of primary HIV infections can provide a framework for studying transmission dynamics of local epidemics [7–14]. Accumulated HIV pol sequences allow for phylogenetic reconstruction of possible transmission events. Phylogenetic, mathematical, and epidemiological modelling can be combined to better understand factors leading to amplified transmission risk at early/acute disease stages [5–16].
In Quebec, half of early stage infections (n = 481/848) are phylogenetically linked to other primary infections, with 28% (n = 239) forming large clusters, averaging 10 primary HIV infections (PHIs) per cluster . The present study was designed to evaluate the cumulative effects of clustering on forward transmission of viruses containing drug resistance mutations related to nucleoside and nonnucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs), as well as protease inhibitors.
Our PHI study population was drawn from the provincial genotyping program, at either of two Quebec reference laboratories (2001–2007, n = 645) and the Quebec PHI cohort study (1997–2007, n = 249). The inclusion criterion for the genotyping programme, (PHI < 6 months), was based on laboratory requisitions completed by prescribing physicians. These patients have been infected for an average of 4.9 months . The Quebec PHI cohort study provides demographic data and a serologic testing algorithm for recent seroconversions (STARHS) [2,8].
Sequence data were compiled using nonnominative identifiers and cross-identifiers to ensure confidentiality while controlling for repeat sampling. In all, 848 unique PHIs (subtype B infections) were identified. Ethics approval was obtained from individual study sites, the Laboratoire de santé publique du Québec, and the Quebec Ministry of Health committee on confidentiality and access of information.
Genotyping was carried out as previously described to generate sequences spanning the protease and reverse transcriptase regions . All sequences were aligned to consensus HXB2 sequences, removing gaps and cutting to identical sequence lengths using BioEdit software (Sigma-Aldrich, St Louis, Missouri, USA) . Phylogenetic inter-relationships among viral sequences were estimated using neighbour joining trees and maximum likelihood methods using BioEdit and MEGA2 integrated analysis software (Sigma-Aldrich) [18,19]. Clustering was based on the robust statistical criterion of high bootstrap values (>98%) and short genetic distances [8,11,12].
Sequence data identified minor and major resistance mutations . For this study, the standardized list of the HIV-1 protease and reverse transcriptase mutations established by the WHO HIV Drug Resistance Surveillance Programme (V. 07-08-05) was used for comparative analysis of the epidemiology of transmitted resistance . Differences in drug resistance motifs among clustered and nonclustered primary transmissions and chronically infected groups were ascertained using contingency tests .
Cell culture-based phenotypic assays were used to assess drug susceptibilities of selected transmitted resistant variants [23–25]. Four G190A isolates from a large transmission cluster (n = 27), (GenBank accession numbers EU375798-EU375801), were assessed for baseline susceptibility to nevirapine (NVP), efavirenz (EFV), etravirine (ETV), and TMC-120 (dapivirine; Tibotec-Virco, Mechlin, Belgium).
Primary HIV infection study population
Between January 1998 and July 2007, sequence data were available from 848 persons, harbouring subtype B infections having clinical indications of PHI less than 6 months. Temporal changes in baseline demographics, clinical features, cluster dynamics, and transmitted resistance within our PHI cohort are summarized in Table 1. As illustrated, the extent of PHI surveillance improved in 2001 with the introduction of the provincial genotyping programme. Our cohort is representative of infections in Quebec .
Clustering represented a steady approximately 50% of PHI transmission events between 1997 and 2008. This is evident for the MSM population, that is, the male only population that excludes intravenous drug users (Table 1). The accumulation time for clusters averaged 16.5 ± 9.8 months (mean ± SD), using STARSH for the PHI cohort. However, the majority of transmissions within clusters occurred over shorter 6–12-month periods [8,10].
Overall, phylogenetic analysis identified 368 unique PHIs, 89 small clusters (<5 PHI/cluster), and 30 large clusters (≥5 PHI/cluster) representing 43.3, 28.5, and 28.1% of transmission events, respectively. There was an increase in the number and size of existent clusters between December 2005 and July 2007. The numbers of infections in small clusters and large clusters increased from 2.7 ± 0.2 to 3.4 ± 0.2 and from 8.8 ± 1.0 to 10.3 ± 1.2 PHI/cluster, respectively (mean ± SEM).
It should be noted that non-B subtype infections, including subtypes C, A/AG, AG, G, and complex recombinant forms, introduced into Quebec (n = 410) through recent immigration have been excluded from our analysis. Clustering among non-B subtypes was low (13.7%), largely restricted to heterosexual partners, and mostly involved wild-type infections.
Clustering of transmissions harbouring drug resistance
The prevalence of single drug class and multidrug resistance (MDR) in our PHI cohort was 14.9 (n = 126/848) and 2.1% (n = 18/848), respectively. No significant trend in the prevalence of transmitted resistance was obvious (Table 1). The overall incidence of PHI harbouring mutations conferring resistance to NRTIs (n = 58), NNRTIs (n = 65), and protease inhibitors (n = 42) was 6.8, 7.6, and 5.0%, respectively.
The overall frequency of drug-resistant viruses in nonclustered and clustered transmissions was similar, 14.3 and 16.5%, respectively. However, clustering had a significant impact on the relative distribution of resistance mutations for different drug classes. This is illustrated in the phylogenetic tree in Fig. 1a, which includes the 144 cases of transmitted resistance in our treatment-naive, PHI population.
As shown, viral variants harbouring mutations to NRTIs, including revertants at codon 215 (T215C/D/I/N/S), thymidine analogue mutations, and M184V, as well as to protease inhibitors were less frequent in clustered transmissions. In marked contrast, seven transmission clusters (clusters A–G) harboured mutations, for example, K103N, V108I, G190A, associated with resistance to NNRTIs (Fig. 1a). In addition, cluster C represents an MDR transmission network, wherein all four PHIs harboured K103N and three of the four also harboured L10I, I54V, A71V, V82A/I/T, I84I/V, and L90M. These PHIs, dating from October 2002 to May 2003, were resistant to the protease inhibitors available at that time.
Cluster A is noteworthy in that it represents the largest in our cohort, including 24 PHIs and three chronic asymptomatic patients. Clustering was episodic; six patients were infected in 2004, eight patients within a 3-month interval in 2005, and eight within a 6-month interval in 2006. All 27 patients harboured the G190A and A98S mutations. Cell-based phenotypic assays on four isolates revealed more than 100-fold NVP resistance, sensitivity to EFV and ETV, and hypersensitivity to TMC-120. This resistance profile was confirmed for these and two other isolates based on Virco Antivirogram phenotypic analysis (Virco, Mechlin, Belgium).
Effect of clustering on transmissibility of resistance
The overall frequency of viral species harbouring resistance to different classes of drugs in unique and clustered transmissions is summarized in Fig. 1b. There was a marked diminution in the incidence of viruses harbouring NRTI mutations, 215 revertants, and MDR variants in clustered transmissions (X 2 = 15.4, 15.6, and 4.8, P < 0.0001, 0.0001, and 0.05, respectively).
In contrast, clustering led to increased frequencies of NNRTI resistance in large clusters, compared with unique and small cluster transmissions (X 2 = 4.8 and 11.4, P < 0.05 and 0.001, respectively). Such forward transmission of NNRTI resistance in clusters was independent of the G190A mutation (Fig. 1).
There appeared to be no significant impact of clustering on the incidence of viral variants harbouring mutations associated with resistance to protease inhibitors. Most protease inhibitor-transmitted resistance was restricted to infections harbouring single mutations, for example, L90M, V82I, which have limited impact on drug susceptibility and viral replicative capacity. The transmission of complex protease inhibitor mutational patterns, conferring measurable phenotypic resistance, was lower in clustered transmissions (X 2 = 5.2, P < 0.05) (Fig. 1a).
Our findings are consistent with those of other studies [6–8,16] showing that early/PHI infections play a key role in the spread of HIV. Our new study provides additional data that confirm that clustering of early/recent infections in Quebec may contribute to approximately 50% of transmissions . High rates of clustering in our cohort reflect a concentrated MSM epidemic in which persons unaware of their HIV status may engage in high-risk behaviours . Universal access to resistance testing allows for in-depth population sampling. Our results are also consistent with models that suggest that early chronic infection contributes to HIV spread . Universal access to antiretroviral drugs is an obvious factor in transmission of resistant quasispecies.
The present study illustrates the potential benefits of phylogenetics in understanding factors that govern HIV transmission and the spread of drug resistance. The implications of PHI/early infection and viral fitness in transmission networks of drug resistance are underscored by the distribution of resistant drug classes in early and chronic infection [23,29–33]. The frequencies of mutations among Quebec patients failing HAART are NRTI (64%) > protease inhibitor (42%) > NNRTI (38%) . The reverse order is observed in transmitted resistance, that is, NNRTI (11%/7%) > protease inhibitor (5%/5%) > NRTI (2%/8%) in clustered and nonclustered transmissions, respectively.
Viruses harbouring NRTI and MDR resistance may be replicatively unfit, creating a bottleneck for forward transmission of such variants [29,30,32]. In contrast, clustering may facilitate spread of viruses harbouring NNRTI resistance mutations. This finding is highlighted by a transmission chain of 27 G190A-containing infections and a transmission network of NNRTI/protease inhibitor dual resistant infections (n = 4). In general, NNRTI mutations do not impact on viral replicative fitness to the same extent as either NRTI or protease inhibitor mutations [29,32,33]. The elevated frequency of acquired NNRTI resistance reported here is consistent with data from other PHI cohorts [34,35]. In contrast, clustering did not impact on the frequency of transmitted drug-resistant infections in the Swiss cohort .
Our findings further underscore the recommendation that all newly infected persons undergo drug resistance testing [37,38]. Resistance to antiretroviral drugs is present 10–25% of the time in PHI [8,34–36]. Transmitted drug resistance persists over time, can affect disease course in drug-naive patients, and limit strategies for antiretroviral therapy [39–44]. Although MDR variants may be less replicatively fit than wild-type archival species, the generation of resistant and MDR strains that may circumvent replicative transmission barriers is of growing concern . Observations of superinfection and coinfections with resistant and MDR viruses show that resistant viruses may become more virulent and transmissible over time [33,39]. Public health strategies that target early/PHI stage infection, including the introduction of rapid testing and routine genotyping, may be of significant benefit towards reducing the incidence and spread of HIV, including that of drug-resistant variants [6–8,39,44].
We thank the patients and attending staff from all centres participating in the Quebec genotyping programme and the Montreal PHI cohort studies. Members of the Quebec Genotyping Programme/Montreal HIV Prevention Study Group: INSQ (Joe Cox, Gilles Lambert, M. Couillard), R. Rousseau (Séro-Zéro), J. Flores, R. Lavoie (COCQ), G. Emond (Concordia University); J. Otis (UQAM). Members of the Quebec PHI Study Group: M. Legault (PHI cohort coordinator); R. Leblanc, R.G. Lalonde, N. Gilmore, M. Klein, J. MacLeod, G. Smith, J. Allan, C. Tsoukas, M. Potter, J. Falutz, J. Cox (McGill University Health Center); D. Rouleau, C. Tremblay, J. Bruneau, C. Fortin, A. de Pokomandy (CHUM); R. Thomas, B. Trottier, F. Asselin, M. Boissonnault, L. Charest, H. Dion, S. Lavoie, D. Legault, D. Longpré, P.J. Maziade, M.E. Morin, D. Murphy, V.K. Nguyen, R. O'Brien, S. Vézina (Clinique médicale l'Actuel); J.G. Baril, P. Côté, S. Dufresne, P. Junod, F. Laplante, D. Poirier, Y. Parent, M.A. Charron, B. Lessard, D. Tessier, É Sasseville, A. Talbot, M.S. Joyal (Clinique médicale du Quartier Latin); N. Lapointe (Hôpital Ste-Justine); A. Dascal (Jewish General Hospital); M. Munoz (CLSC Cote des Neiges).
Sponsorship: Canadian Institutes for Health Research and the Fonds de la Recherche en Santé du Québec-Réseau SIDA (FRSQ-SIDA).
Roles of authors: B.G.B. wrote the article and coordinated all aspects of this study. M.R. and M.W. direct sites for genotypic and phylogenetic analysis for the Quebec PHI cohort and provincial genotyping program, and reviewed and revised the article. D.M., I.H. and R.T. provided phylogenetic (D.M.), genotypic (D.M. and I.H.), and database (D.M. and R.T.) analysis. M.O. performed viral isolation and phenotypic analysis. H.C. and J.-P.R. are the coordinators of the HIV genotypic programme and the Quebec PHI cohort study, respectively, and also reviewed the article.
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