HIV-associated morbidity and mortality have declined among patients with access to antiretroviral therapy (ART) . However, this success was not matched by similar reductions in HIV transmission. Current data even suggest a recent increase in HIV diagnoses in resource-rich settings, particularly among men having sex with men (MSM) [2–4]. Both acute and late stages of HIV-1 infection are associated with high viral loads and, therefore, may disproportionately contribute to the spread of the epidemic [5–7]. In-depth knowledge of the dynamics of HIV-1 transmission within specific transmission groups is fundamental to develop effective prevention strategies [8,9]. Phylogenetic analysis represents one strategy for investigating transmission dynamics between individuals, and occasionally also for forensic use [10–13]. Recent studies have used pol sequences generated for drug resistance genotyping [5,14–16] to reconstruct transmission histories .
Here, we analyzed transmission networks within 111 well documented patients enrolled in the Zurich primary HIV infection (ZPHI) study [18–20] by molecular epidemiological data based on two HIV genes. Furthermore, we studied viral genetic linkage with persons enrolled in the multicenter Swiss HIV Cohort Study (SHCS), which includes data on approximately 50% of all HIV-infected and 75% of all treated individuals in the country and thus reaches an otherwise unmatched representativity [15,21,22]. For the linkage studies, we used pol sequences from the Swiss HIV drug resistance database with additional confirmation through sequencing of clonal HIV env C2–V3–C3 fragments. In particular, we sought to identify transmission events in the viremic phase during acute infection, the aviremic phase during early ART and during the phase of viral rebound after early treatment was stopped.
Our analysis included clinical and genotypic data from the SHCS (for detailed description, see Supplemental Digital Content, http://links.lww.com/QAD/A40), a clinic-based study with continuous enrolment and at least semiannual visits [23,24]. All available HIV pol sequences (12 303) obtained from genotypic resistance tests were used for phylogenetic analysis to select candidates for confirmatory env sequencing.
Patients enrolled in the ZPHI study (http://clinicaltrials.gov: NCT00537966) [18–20,25] presented with documented acute or recent primary HIV-1 infection (for definitions and detailed patient characteristics, see Supplement, http://links.lww.com/QAD/A40). They were offered early ART and could chose after 1 year successful treatment (viral load <50 copies/ml plasma) to stop it. Blood samples were collected at the time of enrolment and sequentially in at least 3-month intervals. The studies complied with the principles of the Declaration of Helsinki and the guidelines of the local ethical committee. Written informed consent was obtained from all participants. Patient data were anonymized for all analyses.
For each patient, a minimal and maximal timepoint of infection was estimated integrating all available information, including patient history relating to known risk situations, occurrence of first symptoms, previous negative test results, avidity assays and western blot. This conjectured time after infection (mean of upper and lower-bound estimates) was positively correlated with env diversity (r2 = 0.07, P = 0.05). Transmission dates were also used to calculate cluster-specific infection rates (for detailed algorithms, see Supplement, http://links.lww.com/QAD/A40).
RNA extraction, amplification, cloning and sequencing of HIV-1 env C2–V3–C3 fragments was performed by modification of previously described methods [26,27] (for detailed description, see Supplement, http://links.lww.com/QAD/A40). Sequences were edited and aligned with SeqMan (DNASTAR Inc., Madison, Wisconsin, USA). Alignments were refined with MAFFT  and manually adjusted. Genetic distance estimates were obtained by Molecular Evolutionary Genetics Analysis software  using the Tamura–Nei model. Neighbor-joining phylogenetic trees were constructed using HXB2 and Non-B strains as references and bootstrapping (1000 replications). Maximum-likelihood trees were inferred by the DNA Maximum Likelihood program using randomized input order, global rearrangements and multiple jumble options (PHYLIP3.6, distributed by J. Felsenstein). The reported env sequences were deposited in GenBank under accession numbers GU471280–GU471687.
Statistical analyses were performed using Prism5 (GraphPad Software, San Diego, California, USA) and Stata10.1 (Stata Corp., College Station, Texas, USA). Nonparametric tests (Mann–Whitney) were used for group comparison.
We analyzed patients enrolled in the ZPHI study before December 2007. One hundred eleven patients were followed over 3.3 (median, range 0.5–6.7) years (for details see, Table S1, http://links.lww.com/QAD/A40). Ninety-three patients underwent early ART that was stopped in 51% of them (n = 47) after approximately 1 year of viral suppression. Baseline blood samples were available within 5 (2–13) weeks after the estimated timepoint of infection in acute and within 13.5 (7–24) weeks in recently infected patients. To investigate transmission dynamics within the ZPHI study and to identify transmission networks 6500 pol sequences from 4276 well characterized SHCS patients were used along with those from the ZPHI patients to generate a neighbor-joining phylogenetic tree. Clusters, containing at least two ZPHI patients, with bootstrap values above 98% and genetic distances below 1.5% (consistent with [5,17]) were preselected. Then, in an independent analysis exploiting the genetic information inherent to the highly variable envelope V3 region, we confirmed the suggested transmission clusters by constructing maximum likelihood trees with clonal env sequences of the 111 ZPHI patients as well as from the SHCS patients clustering to ZPHI patients within the pol phylogeny.
Six clusters containing 20 ZPHI patients (18%) and eight SHCS patients were identified (Fig. 1). No dual infections and no transmitted major drug resistance mutations  were observed in these populations. Transmission of minor populations carrying resistance mutations M184V and K103N have been observed in 13.9% of patients in the ZPHI study . The individual transmission events were studied among these patients within a median follow-up time of 4.0 (0.5–7.6) years (Fig. 2, Table S2, http://links.lww.com/QAD/A40). The extent of observed transmissions may be underestimated, as at the time of analysis some potential transmitters have not yet been diagnosed. Therefore, we performed a second phylogenetic analysis in May 2009 including 12 300 pol sequences of the growing SHCS dataset from 8837 patients.
In cluster A, ZPHI-A1 and ZPHI-A2 formed a serodiscordant couple living in monogamous relationship. ZPHI-A2 has been infected 304 days after ZPHI-A1 stopped continuing early ART. Transmission occurred during the chronic phase of the index partner while his viral load was 33 478 copies/ml.
In cluster B, ZPHI-B2 has been infected most likely by passive anal intercourse during the chronic phase of ZPHI-B1 136 days after discontinuing early ART. The viral load at the estimated timepoint of infection was 930 copies/ml.
ZPHI-C1 and ZPHI-C2 together consulted our outpatient clinic because they suspected HIV infection of ZPHI-C2 due to symptoms of an acute retroviral syndrome. Actually, ZPHI-C1 infected ZPHI-C2 146 days after stopping ART (viral load 2237 copies/ml) during chronic phase. Because they formed a monogamous relationship at that time the two additional SHCS patients within this cluster could be excluded as potential transmitters to patient ZPHI-C2. ZPHI-C1 was infected approximately 2 years earlier, possibly by SHCS-C4.
ZPHI-D1 appeared phylogenetically linked to ZPHI-D2 (genetic distance 1.26% in pol, 0.92% in env). However, ZPHI-D1 was aviremic (viral load <50 copies/ml) at the timepoint of infection of ZPHI-D2 due to successful ART. This transmission pair, therefore, seems unlikely, as proven transmissions with undetectable plasma viremia have never been reported . Moreover, in the extended second analysis, three other potential transmitters were found.
In cluster E, ZPHI-E1 may have infected ZPHI-E2 8 days after having been infected. The viral load at this potential transmission event was 1 690 000 copies/ml. Both, E1 or E2 may have infected ZPHI-E3 during their recent phase (mean 132 days after infection; max viral load 1039 copies/ml). ZPHI-E4 and ZPHI-E5 likely have been infected by either ZPHI-E2 or ZPHI-E3 after stopping early ART but not by ZPHI-E1 who continued suppressive therapy (Table S2, http://links.lww.com/QAD/A40).
Although the complexity in cluster F with six SHCS patients and seven ZPHI patients prohibited analysis of the transmission dynamics, sources of new infections seem to be untreated chronically infected rather than acutely infected patients. Chronically infected patients SHCS-F8, F9 and F10 did not receive treatment during several years, when ZPHI-F1, ZPHI-F3, ZPHI-F6, ZPHI-F4, ZPHI-F2, ZPHI-F5 and ZPHI-F7 were infected sequentially. All of the latter ones reached undetectable viral load during most of the timepoints when those new infection events happened because they received early ART.
Taken together, these patients demonstrate the importance of using clinical, laboratory and epidemiological data to supplement phylogenetic analysis in the assessment of putative transmission chains . Surprisingly, only in one example transmission may have happened during the acute phase (ZPHI-E1, ZPHI-E2) and in one patient within the recent phase (ZPHI-E3) of the possible source. However, in five other patients, transmission presumably took place while the index patient was already in the chronic stage of infection 109–425 days after interruption of early ART. Notably, under treatment, all patients showed undetectable viral load (except few blips) indicating that adherence to therapy was generally very good. Taking into account the overall time when patients were under virologically suppressive treatment, we estimate that 3.5 [95% confidence interval (CI) 0.9–13.5] infection events per person-year occurred prior to treatment initiation and 1.8 (95% CI 0.5–5.8) events per person-year after cessation of the initial treatment.
In this study, we took advantage of our well characterized ZPHI cohort [18–20] and the sequence database linked to the clinical data of the SHCS . In addition to phylogenetic analysis of pol sequences confirmed by sequencing of clonal env fragments, we estimated the timepoint of infection and followed 111 patients longitudinally over years. Eighteen percent of the ZPHI patients formed transmission clusters. We further dissected eight transmission events in five phylogenetic clusters; only one transmission occurred during acute and another one during the recent phase. However, five transmissions occurred during chronic stage of the presumed transmitters, more than 3 months after stopping early ART. This was unexpected and worrisome because it shows the limitations of prevention measures in this sexually active MSM cohort. Infectiousness during chronic infection was quite high in this population also at relatively low viral load in some cases (range 314–1 690 000 copies/ml).
In previous studies using phylogenetic reconstruction, patients were categorized as acutely or chronically infected according to their stage at diagnosis but not when transmission actually occurred [5,32]. This probably led to overestimation of transmission frequencies during acute/early infection. Phylogenetic analyses have limitations, as one can never rule out transmissions potentially originating from other index cases not known to the investigator. This effect is attenuated in our setting because the primary HIV infection patients enrolled represent approximately 55% of all newly HIV-infected MSM patients in the canton of Zurich and the likelihood that HIV-infected patients are enrolled in the SHCS is high [21,22]. In contrast to other phylogenetic studies assessing transmission dynamics [5,15,33,34], we used two different samples and analyzed two different genetic regions to increase genetic information and to exclude laboratory and database errors . Moreover, plausibility of the transmission clusters was controlled by longitudinal viral load data.
This study demonstrates that in our intensely studied sexually active MSM, collective preventive safer-sex counselling was insufficient as documented by the high number of new infections originating from patients who stopped early ART according to study protocol. Of note, the same individuals were very adherent to ART. Furthermore, we detected a remarkable proportion of new infections originating from index patients being already in their chronic phase, sometimes with low viral loads. These findings argue strongly for early, continuous ART in sexually active HIV-1-infected MSM. This strategy, most likely, will have a profound impact to reduce the further spread of HIV-1.
This study has been financed in the framework of the SHCS, supported by the Swiss National Science Foundation (SNF #33CSC0-108787). Further support was provided by the SNF grant #3247B0-112594 and #320000-116035 (to H.F.G.), the Union Bank of Switzerland in the name of a donor to H.F.G., unrestricted research grant from Tibotec, Switzerland, the SHCS research foundation and SHCS projects #470 and #528.
We are grateful to all the patients participating in the ZPHI Study and in the SHCS; Barbara Hasse, Urs Karrer, Rolf Oberholzer, Elisabeth Presterl, Reto Laffer, Ulrich von Both, Klara Thierfelder, Dominique Braun, Markus Flepp and Thomas Frey for their dedicated patient care; Friederike Burgener and Dominique Klimpel for excellent laboratory assistance; Christine Vögtli and Ingrid Nievergelt for administrative support. We also would like to thank the UZH IT services for giving us access to the high-performance computer of the University of Zurich. Furthermore, we thank all the staff of the SHCS clinical centers, the data center and all resistance laboratories for their great work and Joseph K. Wong for critical review of the manuscript.
Günthard conceptualized, designed and supervised the study. Data acquisition was done by Rieder, Joos, von Wyl, Kuster, Grube, Leemann, Böni, Yerly, Klimkait, Bürgisser and Günthard. Analysis of the manuscript was done by Rieder, Joos, Günthard, and von Wyl. Critical revision of this article was done by Fischer, Weber, Kuster, Bürgisser, Yerly, Böni and Klimkait.
H.F.G. has been an adviser, consultant or both for the following companies: GlaxoSmithKline, Abbott, Novartis, Boehringer-Ingelheim, Roche, Tibotec and Bristol-Myers Squibb, and has received unrestricted research and educational grants from Roche, Abbott, Bristol-Myers Squibb, GlaxoSmithKline, Tibotec and Merck Sharp & Dohme. S.Y. has participated in advisory boards of Bristol-Myers Squibb and Tibotec, and has received travel grants from GlaxoSmithKline and Merck Sharp & Dohme. V.V.W., P.R., H.K., C.G., C.L., J.B., T.K., P.B., R.W. and M.F. have no conflicts of interest.
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