Transmission clusters and HIV-1 resistance
Transmission clusters were more frequently identified in newly diagnosed individuals infected with drug-resistant strains than in individuals infected with sensitive ones (59.3 vs. 32.6%, respectively; P < 0.0001). Among the newly diagnosed individuals with TDR, 27 of 32 (84.4%) were part of transmission clusters composed only of newly diagnosed individuals, two (6.3%) of clusters composed of chronic infection individuals and at least two newly diagnosed individuals, and only three (9.4%) were part of clusters consisting only of individuals with chronic infection (Fig. 3b). The transmission cluster size was larger in individuals infected with drug-resistant strains when compared with those infected with sensitive strains, with 36% and 18% of clusters including three or more individuals, respectively.
Consistent with the earlier-mentioned observations, the prevalence of TDR was significantly higher in individuals who were part of clustered transmissions than in nonclustered ones (14.4 vs. 5.3%, respectively; P < 0.0001). Higher resistance rates in clustered transmissions were observed for NRTIs (9.9 vs. 4.3%, respectively; P = 0.006), NNRTIs (5.4 vs. 2.4%, respectively; P = 0.048), and protease inhibitors (4.1% vs. 0.7%, respectively; P = 0.005).
Several predictive factors that might affect the risk to belong to a transmission cluster were explored. Table 2 presents the results of the univariate and multivariate logistic regression model assessing the likelihood of belonging to a clustered transmission group. The final model containing all predictors of interest was statistically significant (chi-squared = 38.9; P < 0.0001) and explained as a whole between 7.1 and 9.7% of the variance in transmission clusters, and correctly classified 68.2% of cases. As shown in Table 2, the strongest predictor of the likelihood to belong to transmission clusters was the detection of drug-resistant strains with an odds ratio of 3.02 (1.54–5.92). The others significant predictors were a high CD4 cell count (odds ratio, 2.37) and a high viral load (odds ratio, 1.83).
Mutations associated with resistance were compared between all individuals of each transmission cluster. Complete concordance was found for seven of 14 clusters including 19 individuals. In three cases, discrepancies were related to one individual without resistance mutations in a cluster containing three, four and seven newly diagnosed individuals with resistance mutations (184V, 215D and 103N, respectively). In the latter, the mutation 103N was not detected at the time of diagnosis, but available follow-up resistance testing revealed this mutation 1 year later (see below). The remaining four discrepancies were related to one newly diagnosed individual with resistance mutation (103N in three, and 41L and 184V in one) included in clusters of chronic infection without detected resistance mutations at the time of sample analysis.
Large clusters of transmitted drug resistance
On the basis of the present analysis, two large clusters of TDR containing only newly diagnosed individuals were identified. The first cluster included five newly diagnosed individuals, all men who had sex with men. Of these, recent infections were documented in four within a short period of time (13 months). Mutations associated with resistance-included reverse transcriptase 69 insertion (69I), 70R and protease 90M. This subtype B strain is considered multiresistant according to reference algorithms [http://www.hivfrenchresistance.org]. A complete concordance of resistance mutations was found between all individuals.
The second cluster included seven newly diagnosed intravenous drug users; recent infections were documented in four within an 8-month period. The 103N mutation and a 35insertion (T) in protease were detected on this subtype C strain. Although not detected in the initial sample, the index case later developed the 103N mutation while on NNRTI therapy. This allowed a more precise timing of the first transmission event in this cluster. This particular 35insertion in protease has been rarely reported and the phenotypic consequences still need to be fully characterized. Interestingly, a blast search of this protease sequence on the NCBI database revealed a sequence with 98.6% homology reported in Portugal in 2002 where the index case was reported to be infected.
We performed a phylogenetic analysis of HIV strains representing almost all newly infected individuals in a restricted geographical area. Thirty-five per cent of newly diagnosed individuals, and more than 50% of recently acquired HIV infections, can be linked to individuals infected with a closely related strain. Two-thirds of these linked infections were part of clusters composed only of newly diagnosed cases. In comparison, only 10% of chronically infected individuals were clustered. These results suggest that many new infections were due to recently HIV-infected individuals, still unaware of their infection, which are an unexpected source of drug-resistant strains, given that nearly half of TDR was linked to transmission clusters composed only of newly diagnosed individuals.
Overall, 8.5% of all newly infected patients had HIV strains with mutations conferring antiretroviral resistance to at least one drug, although they had not been previously exposed to antiretroviral treatment. Transmitted resistance increased significantly over the 8-year study period. Most importantly, the average resistance rate was even higher (15%) in the subgroup of those in whom the HIV infection could be dated as having occurred within the preceding year, a consequence of the frequent chains of transmissions observed in this population. Although the prevalence of newly transmitted drug-resistant HIV-1 can vary widely with location, risk behavior, sampling time after infection, and years of infection [11–15,25–27], the prevalence of TDR observed in our cohort is consistent with recent reports in developed countries .
It is generally assumed that treated individuals with chronic infection are the main source of TDR. Advances in treatment, including improved virological control and rapid management of treatment failure, are expected to lower rates of resistance and, consequently, diminish TDR. Some studies have correlated the rate of treatment failure with the subsequent rate of TDR [1,7,29–31]. Other studies have linked more precisely the TDR rate to the patterns of drug resistance in the population exposed to antiretroviral therapy [2,32]. These observations have been supported by mathematical models, predicting significant associations between the TDR rate, treatment guidelines, and the replication fitness of drug-resistant strains [33,34].
In a recent study from the Swiss HIV Cohort Study, the prevalence of drug resistance among antiretroviral therapy-exposed individuals decreased from 50% in 1999 to 37% in 2007 . This needs to be related to the increased rate of TDR observed in our study, similar to other reports [12,13,27,35], which supports the view that drug-exposed individuals with chronic infection are not the only ones responsible for TDR. Reconstruction of the HIV transmission networks using phylogenetic analysis reveals that nearly 60% of newly diagnosed individuals with TDR belong to transmission clusters, and that 84% of these clusters include only newly diagnosed individuals. This demonstrates that newly diagnosed, untreated individuals are a significant source of resistant strains, thus suggesting an important self-fueling mechanism for TDR. These results are in contrast with a Canadian study on recently infected individuals showing a lower prevalence of mutations associated with resistance to NRTIs and protease inhibitors in clustered transmissions as compared with nonclustered infections . However, in a recent analysis of the same cohort, the authors found that clustering was associated with an increased transmission of NNRTI resistance . Indeed, the population studied differs and the low number of TDR observed precludes the possibility of drawing any strong conclusion. Similar to other reports [36,37], our data highlight that drug-resistant variants do not harbor a reduced transmissibility.
Unlike acquired drug resistance in chronically drug-experienced individuals, transmitted drug-resistant mutations can persist over time in the absence of drug pressure [38,39]. We found a high concordance of resistance mutations in most transmission clusters composed of newly diagnosed individuals, and a detailed analysis of each large cluster revealed that the same strain caused multiple transmission events occurring years apart. The discrepancies in resistance mutations observed within a cluster may have been related to the potential reversion of a particular mutation (that is 184V, 215D) in some individuals with an infection of unknown duration. Thus, transmission of resistance from untreated individuals who may not be aware of their infection remains a cause for concern. This implies that prevention measures should be targeted to these individuals and that the widespread use of HIV screening might be beneficial in this respect.
Several limitations of the study should be acknowledged. First, the representativeness of the populations studied is crucial. Although the present study included approximately 90% of the chronically infected population with detectable HIV RNA during 2000–2008, many persons remain undiagnosed and unaware of their HIV status; this population was estimated at around 25% by the Swiss Federal Office of Public Health . In addition, chronically infected individuals with undetectable HIV RNA during the 2000–2008 period of time could not be genotyped and are therefore not included in our analysis. Nonetheless, some of these individuals may have been the source of infection before 2000. Second, despite its ability to detect relationships between HIV strains, phylogenetic analysis alone cannot be considered as a substitute for other epidemiological information, such as sexual behavior.
In conclusion, our findings highlight the role of newly diagnosed individuals, not yet exposed to antiretroviral drugs, in replenishing the pool of individuals infected with drug-resistant HIV.
We thank the patients for their participation, the physicians for patient care, and Chantal Gaille and Wanda Caveng for their excellent technical contribution.
The members of the Swiss HIV Cohort Study and the Swiss Mother and Child HIV Study are: C. Aebi, M. Battegay, E. Bernasconi, J. Böni, P. Brazzola, HC Bucher, Ph. Bürgisser, A. Calmy, S. Cattacin, M. Cavassini, J.-J. Cheseaux, G. Drack, R. Dubs, M. Egger, L. Elzi, M. Fischer, M. Flepp, A. Fontana, P. Francioli (President of the SHCS, Centre Hospitalier Universitaire Vaudois, CH-1011- Lausanne), HJ. Furrer, C. Fux, A. Gayet-Ageron, S. Gerber, M. Gorgievski, H. Günthard, Th. Gyr, H. Hirsch, B. Hirschel, I. Hösli, M. Hüsler, L. Kaiser, Ch. Kahlert, U. Karrer, C. Kind, Th. Klimkait, B. Ledergerber, G. Martinetti, B. Martinez, N. Müller, D. Nadal, F. Paccaud, G. Pantaleo, L. Raio, A. Rauch, S. Regenass, M. Rickenbach, C. Rudin (Chairman of the MoChiV Substudy, Basel UKBB, Römergasse 8, CH-4058 Basel), P. Schmid, D. Schultze, J. Schüpbach, R. Speck, P. Taffé, A. Telenti, A. Trkola, P. Vernazza, R. Weber, C.-A. Wyler, S. Yerly.
S.Y. designed and conducted the study, analyzed the data and drafted the manuscript. T.J. performed phylogenetic analyses under the supervision of E.Z., B.H. and L.K. designed the study and drafted the manuscript. A.G.-A. performed the statistical analysis. E.B.ElA. collected clinical data. H.F.G. and V.vW. collected sequences data and drafted the manuscript. All authors have reviewed the latest version of the manuscript and have approved its content.
This study was financed by the Swiss HIV Cohort Study (SHCS) (Swiss National Science Foundation (SBF) grant #3345-062041). Further support was provided by the Fond Benoit from Geneva's University Hospitals, the SNF grant #3247B0-112594/1, SHCS projects 470, 528 and 569, the SHCS Research Foundation, and a research grant of the Union Bank of Switzerland in the name of a donor (H.F.G.).
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Keywords:© 2009 Lippincott Williams & Wilkins, Inc.
HIV; phylogeny; primary drug resistance; recent infection; transmission