Transmission of drug-resistant HIV-1 has been documented in most countries with access to antiretroviral drugs and across different transmission groups [1–8]. The prevalence of transmitted drug-resistant HIV-1 has been found to vary with location and time. Moreover, a wide variation in temporal trends has been reported [2,3,5,7–12]. Although transmission of resistance is related to therapeutic strategies in the HIV-1-infected population, other factors substantially contribute to measurement of true prevalence of resistance in recently infected individuals, such as the definitions of a recent infection or of drug resistance. In addition, sampling methods can impact on the measured prevalence of transmitted drug resistance, in particular if individuals are selected for resistance testing because of a high risk for resistance transmission. Most published studies completely lack information on whether included individuals truly represent the population of newly infected individuals in a defined geographical region.
The present study analysed the transmission of drug-resistant HIV-1 in a representative population of recently infected individuals from 1996 to 2005 in Switzerland.
Criteria for inclusion in the study were documented acute infection (HIV-1 RNA positive with negative or evolving Western blot) or recent infection (HIV enzyme immunoassay negative within the previous 12 months), and availability of plasma samples with RNA > 1000 copies/ml collected within 1 year of the estimated seroconversion date and before introduction of treatment. The date of seroconversion was estimated as the date of acute infection if known or as the midpoint between the last documented negative HIV-1 enzyme immunoassay and the first positive HIV-1 enzyme immunoassay. Individuals were enrolled from January 1996 to December 2005 in one of the seven HIV outpatient clinics or in private practices participating in the Swiss HIV Cohort Study (SHCS; http://www.SHCS.ch) and in large private laboratories in Switzerland.
Analyses of drug-resistant HIV-1
Population-based sequence analysis of the reverse transcriptase and protease genes was performed by the four laboratories in Switzerland authorized by the Federal Office of Public Health using either commercial assays (Viroseq Vs.1, PE Biosystems, Rotkreuz, Switzerland; Viroseq Vs.2, Abbott AG, Baar, Switzerland; vircoTYPE HIV-1 Assay, Virco Lab., Mechelen, Belgium) or in-house methods . All laboratories participate in the same quality assurance programme [French National Agency for AIDS Research (ANRS)] and comprehensive data validation procedures have been implemented to assure quality of the sequences before they are entered into the SHCS resistance database. Prevalence of mutations associated with transmitted drug-resistant HIV-1 was analysed using the list of mutations for surveillance of transmitted drug resistance . Interpretation of drug resistance was performed using the ANRS (version 14, 2006) algorithm (www.hivfrenchresistance.fr); possible resistance was considered as resistance. Phylogenetic analyses were conducted to investigate laboratory contaminations and HIV-1 subtyping (ClustalX, Phylip packages [14,15]). Resistance data were stored in the SHCS drug resistance database using the HIV Integrated Database Network System (Version 3.3.0, SmartGene, Zug, Switzerland).
Associations of resistance to any drug with baseline characteristics were analysed by univariate and multivariate logistic regression. The following variables were considered: year of infection, sex, presumed mode of HIV transmission, HIV-1 subtype and ethnicity. Tests for trends were conducted using the Cochran–Armitage test for trend, and logistic regressions included the year of infection as continuous covariable. Interactions of year of infection with HIV-1 subtype or the yearly prevalence of non-B subtypes were also investigated.
Data were analysed using SPSS 11.0 and Stata/SE 9.2 (StataCorp, College Station, Texas, USA). All P values were two-sided and the level of significance was set at P < 0.05.
A total of 925 individuals with documented HIV-1 seroconversion were identified between January 1996 and December 2005. Samples with HIV-1 RNA above 1000 copies/ml within 1 year of the estimated date of seroconversion and before initiation of antiretroviral therapy were available for 858 individuals. The reverse transcriptase and protease regions were successfully amplified for 822 individuals and included in this analysis. The main reason for exclusion was absence of samples before treatment introduction (54%). Demographic and baseline characteristics of excluded and included individuals were similar (data not shown). Demographic characteristics are reported in Table 1 according to the year of HIV-1 infection. Transmission categories were men having sex with men (MSM; 42%), heterosexual contacts (32%) and injection drug use (20%). Overall, 71% of individuals were infected with subtype B virus. Over 18 different subtypes and circulating recombinant forms (CRF) were identified. The most predominant non-B subtypes were CRF02 (7%), CRF11 (6%), C (3%) and CRF01 (3%). The mean CD4 cell count was 534 cells/μl (SD, 276) and the mean HIV-1 RNA was 5.0 log10 copies/ml (SD, 0.9). From 1996 to 2005, mandatory declarations of confirmed new HIV-1 diagnosis were reported for 7221 individuals in Switzerland; transmission categories were MSM for 25%, heterosexual contact for 54%, intravenous drug use for 15% and others for 6% .
HIV-1 drug resistance
The frequency of mutations associated with drug resistance was first analysed using the Stanford list of mutations for surveillance of transmitted drug resistance. Overall, mutation frequency for resistance to any drug was 7.9%, including 6.0% for nucleoside/nucleotide reverse transcriptase inhibitor (NRTI) mutations, 2.0% for nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations and 2.5% for protease inhibitor (PI) mutations. The most frequent mutation was T215Y/F/C/D/N/S/E/V (4.9%) followed by M41L (3.5%) and M184I/V (1.7%). K65R was not observed, nor were any insertions at codon 69 or Q151M associated with multidrug resistance (Table 2). Among the 49 individuals with mutations associated with resistance to NRTI, 31% harboured one mutation, 39% two mutations and 30% more than two mutations. Among the 20 individuals with mutations associated with PI resistance, 35% harboured one mutation, 30% two mutations and 35% more than two mutations. The mean number of mutations associated with resistance to any drug was 2.9 (range, 1–15).
Using the ANRS resistance interpretation algorithm, the prevalence of transmitted drug resistance during the period 1996 to 2005 was 7.7% (95% confidence interval (CI), 5.9–9.5] for any drug, 5.5% (95% CI, 3.9–7.1) for NRTI, 1.9% (95% CI, 1.0–2.8) for NNRTI and 2.7% (95% CI, 1.6–3.8) for PI. Concerning NRTI resistance, four individuals were not assigned to have acquired drug resistance when compared with the Stanford criteria (three individuals with isolated M41L and one with isolated K70R). For individuals with PI resistance, two individuals with isolated M46L were considered PI resistant by the ANRS algorithm but not by the Stanford list (previous analysis). Five of these individuals were infected with subtype B virus and one with a CRF-02 virus (M41L). The prevalence of drug resistance for individual NRTI ranged widely from 1% (abacavir, didanosine) to 5% (zidovudine, stavudine). Because of the strong cross-resistance between NNRTI, a similar prevalence (2%) was observed for efavirenz and nevirapine, whereas cross-resistance to the investigational new NNRTI TMC-125 was much lower (0.1%). Resistance to PI drugs varied from 0.1% (TMC-114) to 2.3% (indinavir), with prevalence < 1% for the currently approved boosted-PI regimens (lopinavir, atazanavir, fosamprenavir and tipranavir). Among the 18 antiretroviral drugs analysed, the mean number of drugs affected by transmitted resistance was 3.4 (range, 1–16). Resistance to the entire NRTI class was observed in only three individuals; resistance to NNRTI was also detected in two of them. The study did not identify any individuals with resistance to all approved PI drugs.
Figure 1 shows the prevalence of transmitted drug resistance over time according to drug class. No significant changes over time were observed. In 2005, the prevalence of resistance to any antiretroviral drug, NRTI, NNRTI, and PI was 10% (95% CI, 4.1–15.9), 4.0% (95% CI, 0.2–7.9), 6.0% (95% CI, 1.4–10.7) and 4.0% (95% CI, 0.2–7.9), respectively. Overall, most individuals who acquired primary drug resistance harboured viruses resistant to one drug class: 5.7% (95% CI, 4.1–7.3). Dual-class resistance was present to a lesser extent (1.5%; 95% CI, 0.7–2.3) and triple-class resistance was observed very rarely (0.5%; 95% CI, 0–1.0). There was no evidence of a temporal trend in the prevalence of transmitted drug resistance. The only exception was a late increase in NNRTI transmission from 0% in 2004 to 6.0% (95% CI, 1.4–10.7) in 2005.
In a multivariate logistic model (Table 3), there was no difference in the prevalence of transmitted drug resistance according to gender, exposure category or ethnicity. However, the rate of transmitted resistance was highest among individuals infected with subtype B virus. As shown in Fig. 1c, the prevalence of transmitted drug resistance observed over the last decade appeared to be negatively correlated to the prevalence of HIV-1 non-B subtypes (odds ratio per percentage-point increase in the prevalence of these subtypes, 0.93; 95% CI, 0.88–0.99; logistic regression P = 0.028), thus indicating a potential dilution effect. However, separate analyses of trend for B subtypes and non-B subtypes revealed no change in prevalence over time (Table 4). Baseline HIV-1 RNA level and CD4 cell count were not significantly different in individuals with or without transmitted drug resistance.
We report a prevalence of 7.7% of drug-resistant HIV-1 between 1996 and 2005 in a representative population of newly infected individuals in Switzerland. Most of the individuals diagnosed with primary resistance harboured strains that carried resistance to one drug class only (mostly NRTI); multiple drug resistance was rare (< 2%). This frequency is similar to those reported from other large cohort databases in Europe [5,7], but lower than frequencies reported from the UK  or the USA and Australia . In contrast to most recently published studies [3,5,7,8,12], we did not identify any increase in transmitted drug-resistant HIV-1 over the observation period. This might be related to sampling biases, for example a greater proportion of individuals with drug-resistant HIV-1 being tested in the later years owing to changes in resistance-testing policies.
The main characteristic of our study population is that it is a good representation of the total population of recently HIV-1-infected individuals in Switzerland from 1996 to 2005. In addition to patients of the SHCS, data were also included from four reference laboratories for HIV diagnostic confirmation. The population studied included 90% of the individuals with documented recent HIV-1 infection in the SHCS (http://www.SHCS.ch). Since this study enrolled about 50% of the total HIV-1-infected population in Switzerland, the minimal estimate of the population studied is approximately 57% of the total population recently infected with HIV-1 in Switzerland from 1996 to 2005. The higher proportion of MSM in our study population compared with the general HIV-1-infected population in Switzerland is related to the increase of recent infections in this risk category after 2002 .
We observed fluctuations in the prevalence of transmitted resistance over time. This is related to several factors including relatively moderate numbers of individuals included each year or particular transmission events. For example, in 2000, we detected a cluster of five injection drug users harbouring variants with the M41L and T215D mutations . Phylogenetic analyses showed, however, that most of the resistance transmission observed in this dataset were independent transmission events, since a founder effect was detected only three times: seven injection drug users harbouring M41L, T215D; two harbouring K103N; and two harbouring M41L, L210W, T215D (data not shown). Furthermore, we have shown previously that suboptimal treatment with one or two NRTI was followed by a peak of transmission of drug-resistant virus . More recent antiretroviral regimens also have improved efficacy, leading to less drug resistance , and can control viraemia in an increasing number of patients bearing drug-resistant virus [20–22]. As shown in Fig. 1c, we observed a potential dilution effect by the steady increase in the proportion of non-B subtypes, in whom prevalence of resistance was found to be lower. However, a reassessment of time trends stratified by subtype provided results that did not differ. This dilution effect might explain the aforementioned discrepancies in time trends and prevalence between this study and previously published results.
It is likely that a large proportion of newly infected individuals have been infected by individuals who are unaware of their infection status and consequently have never been exposed to antiviral drugs. Another factor potentially influencing transmission of drug resistance is that fitness of drug-resistant viruses may be lower than for wild-type virus, as has been suggested for the M184V mutation [17,23]. Assessing the relative contributions of all these factors would require very large epidemiological datasets. These elements and the fluctuation of the prevalence of transmitted resistance observed over time indicate that predictions for future trends [24,25] have to be confronted with real datasets using nonbiased recruitment. Also, caution should be exercised when considering the cut-off (> 5% or > 10%) of prevalence recommended for resistance testing in drug-naive individuals [26,27].
The definition of transmitted drug resistance is still a matter of debate because of the potential impact of natural polymorphisms. We, therefore, used two definitions of drug resistance: the newly reported list of mutations for surveillance of transmitted drug resistance  and the ANRS algorithm. Both methods led to almost identical prevalence of resistance with the exception of M46L interpretation. It should be noted that the standard population-based genotyping method used in this study detects viral minorities > 20% in a mixed population. Therefore, the prevalence of drug resistance as generally reported might be an underestimation, in particular for the M184V mutation, which can rapidly become undetectable through fitness costs [28–30]. It has been shown recently that measured prevalence of transmitted dug resistance was significantly greater when using more sensitive assays [31,32].
Reports concerning the inferior treatment outcome in patients harbouring transmitted drug resistance have been explored in several studies [2,3,33–35]. However, the precise cost–benefit might be difficult to assess as the knowledge of baseline resistance pattern influences treatment selection and efficacy . As in many other countries, the recent increase in the prevalence of NNRTI resistance observed in Switzerland may potentially increase the risk of first-line virological failure, since antiretroviral treatment containing efavirenz is either the first or second option to initiate treatment . This is probably the strongest argument in favour of resistance testing in all newly infected individuals in countries with good access to antiretroviral drugs. Even more important is the fact that first-line failure with NNRTI-based regimens leads to the emergence of drug resistance against more drug classes than with boosted PI based regimens .
In conclusion, we show that transmitted drug resistance in a well-defined, representative patient population in a country with large access to antiretroviral therapy has remained stable since 1996. More importantly, in contrast to many projections and modelling work, no significant increase of dual- or triple-drug resistance has occurred in Swiss HIV-infected individuals. This suggests that effective antiretroviral treatment by itself contributes to containment of the spread of primary HIV drug resistance in an optimal setting.
We thank the patients for their participation, the physicians and study nurses for excellent patient care, Thomas Junier (Department of Genetic Medicine and Development, University of Geneva Medical School) for help in the phylogenetic analyses, the laboratory technicians of the Swiss resistance laboratories for the quality of the data, and SmartGene for technical support.
Sponsorship: This study has been financed by the HIV Cohort Study (Swiss National Science Foundation grant 3345-062041). Further support was provided by the SNF grant 3247B0-112594/1 (to HFG, SY and BL), and SHCS project 470.
Members of the Swiss HIV Cohort Study: M. Battegay, E. Bernasconi, J. Böni, H. Bucher, Ph. Bürgisser, S. Cattacin, M. Cavassini, R. Dubs, M. Egger, L. Elzi, P. Erb, M. Fischer, M. Flepp, A. Fontana, P. Francioli, H. Furrer, M. Gorgievski, H. Günthard, H. Hirsch, B. Hirschel, I. Hösli, Ch. Kahlert, L. Kaiser, U. Karrer, C. Kind, Th. Klimkait, B. Ledergerber, G. Martinetti, B. Martinez, N. Müller, D. Nadal, M. Opravil, F. Paccaud, G. Pantaleo, M. Rickenbach, C. Rudin, P. Schmid, D. Schultze, J. Schüpbach, R. Speck, P. Taffé, P. Tarr, A. Telenti, A. Trkola, P. Vernazza, R. Weber, S. Yerly.
1. Yerly S, Kaiser L, Race E, Bru JP, Clavel F, Perrin L. Transmission of antiretroviral-drug-resistant HIV-1 variants. Lancet 1999; 354:729–733.
2. Grant RM, Hecht FM, Warmerdam M, Liu L, Liegler T, Petropoulos CJ, et al
. Time trends in primary HIV-1 drug resistance among recently infected persons. JAMA 2002; 288:181–188.
3. Little SJ, Holte S, Routy JP, Daar ES, Markowitz M, Collier AC, et al
. Antiretroviral-drug resistance among patients recently infected with HIV. New Engl J Med 2002; 347:385–394.
4. Brenner B, Wainberg MA, Salomon H, Rouleau D, Dascal A, Spira B, et al
. Resistance to antiretroviral drugs in patients with primary HIV-1 infection. Investigators of the Quebec Primary Infection Study. Int J Antimicrob Agents 2000; 16:429–434.
5. Wensing AM, van de Vijver DA, Angarano G, Asjo B, Balotta C, Boeri E, et al
. Prevalence of drug-resistant HIV-1 variants in untreated individuals in Europe: implications for clinical management. J Infect Dis 2005; 192:958–966.
6. Descamps D, Chaix ML, Andre P, Brodard V, Cottalorda J, Deveau C, et al
. French national sentinel survey of antiretroviral drug resistance in patients with HIV-1 primary infection and in antiretroviral-naive chronically infected patients in 2001–2002. J Acquir Immune Defic Syndr 2005; 38:545–552.
7. Masquelier B, Bhaskaran K, Pillay D, Gifford R, Balestre E, Jorgensen LB, et al
. Prevalence of transmitted HIV-1 drug resistance and the role of resistance algorithms: data from seroconverters in the CASCADE collaboration from 1987 to 2003. J Acquir Immune Defic Syndr 2005; 40:505–511.
8. Chaix ML, Descamps D, Harzic M, Schneider V, Deveau C, Tamalet C, et al
. Stable prevalence of genotypic drug resistance mutations but increase in non-B virus among patients with primary HIV-1 infection in France. AIDS 2003; 17:2635–2643.
9. Cane P, Chrystie I, Dunn D, Evans B, Geretti AM, Green H, et al
. Time trends in primary resistance to HIV drugs in the United Kingdom: multicentre observational study. BMJ 2005; 331:1368.
10. Bezemer D, Jurriaans S, Prins M, van der Hoek L, Prins JM, de Wolf F, et al
. Declining trend in transmission of drug-resistant HIV-1 in Amsterdam. AIDS 2004; 18:1571–1577.
11. Routy JP, Machouf N, Edwardes MD, Brenner BG, Thomas R, Trottier B, et al
. Factors associated with a decrease in the prevalence of drug resistance in newly HIV-1 infected individuals in Montreal. AIDS 2004; 18:2305–2312.
12. Simon V, Vanderhoeven J, Hurley A, Ramratnam B, Louie M, Dawson K, et al
. Evolving patterns of HIV-1 resistance to antiretroviral agents in newly infected individuals. AIDS 2002; 16:1511–1519.
13. Shafer RW, Rhee SY, Pillay D, Dalwani A, Miller V, Sandstrom P, et al
. HIV-1 protease and reverse transcriptase mutations for drug resistance surveillance. AIDS 2007; 21:215–223.
14. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucl Acids Res 1994; 22:4673–4680.
15. Felsenstein J. PHYLIP Inference package version 3. 5. Seattle: Department of Genetics, University of Washington; 1993.
16. Federal Department of Public Health. Tableaux Trimestriels
. Bern: Federal Department of Public Health; 2007. http://www.bag.admin.ch/hiv_aids/
17. Yerly S, Jost S, Telenti A, Flepp M, Kaiser L, Chave JP, et al
. Infrequent transmission of HIV-1 drug-resistant variants. Antivir Ther 2004; 9:375–384.
18. Yerly S, Vora S, Rizzardi P, Chave JP, Vernazza PL, Flepp M, et al
. Acute HIV infection: impact on the spread of HIV and transmission of drug resistance. AIDS 2001; 15:2287–2292.
19. von Wyl V, Yerly S, Böni J, Bürgisser P, Klimkait T, Battegay M, et al
. Emergence of HIV-1 drug-resistance in previously untreated cART initiators: A comparison of different regimen types. Arch Int Med 2007; 167:1782–1790.
20. Lalezari JP, Henry K, O'Hearn M, Montaner JS, Piliero PJ, Trottier B, et al
. Enfuvirtide, an HIV-1 fusion inhibitor, for drug-resistant HIV infection in North and South America. N Engl J Med 2003; 348:2175–2185.
21. Lazzarin A, Clotet B, Cooper D, Reynes J, Arasteh K, Nelson M, et al
. Efficacy of enfuvirtide in patients infected with drug-resistant HIV-1 in Europe and Australia. N Engl J Med 2003; 348:2186–2195.
22. Gathe J, Cooper DA, Farthing C, Jayaweera D, Norris D, Pierone G Jr, et al
. Efficacy of the protease inhibitors tipranavir plus ritonavir in treatment-experienced patients: 24-week analysis from the RESIST-1 trial. Clin Infect Dis 2006; 43:1337–1346.
23. Brown AJL, Frost SDW, Matthews WC, Dawson K, Hellmann NS, Daar ES, et al
. Transmission fitness of drug-resistant human immmunodeficiency virus and the prevalence of resistance in the antiretroviral-treated population. J Infect Dis 2003; 187:683–686.
24. Blower SM, Aschenbach AN, Gershengorn HB, Kahn JO. Predicting the unpredictable: transmission of drug-resistant HIV. Nat Med 2001; 7:1016–1020.
25. Blower S, Bodine E, Kahn J, McFarland W. The antiretroviral rollout and drug-resistant HIV in Africa: insights from empirical data and theoretical models. AIDS 2005; 19:1–14.
26. Vandamme AM, Sonnerborg A, Ait-Khaled M, Albert J, Asjo B, Bacheler L, et al
. Updated European recommendations for the clinical use of HIV drug resistance testing. Antiviral Ther 2004; 9:829–848.
27. Hammer SM, Saag MS, Schechter M, Montaner JS, Schooley RT, Jacobsen DM, et al
. Treatment for adult HIV infection: 2006 recommendations of the International AIDS Society-USA Panel. JAMA 2006; 296:827–843.
28. Back NK, Nijhuis M, Keulen W, Boucher CA, Oude Essink BO, van Kuilenburg AB, et al
. Reduced replication of 3TC-resistant HIV-1 variants in primary cells due to a processivity defect of the reverse transcriptase enzyme. EMBO J 1996; 15:4040–4049.
29. Wei X, Liang C, Gotte M, Wainberg MA. Negative effect of the M184V mutation in HIV-1 reverse transcriptase on initiation of viral DNA synthesis. Virology 2003; 311:202–212.
30. Barbour JD, Hecht FM, Wrin T, Liegler TJ, Ramstead CA, Busch MP, et al
. Persistence of primary drug resistance among recently HIV-1 infected adults. AIDS 2004; 18:1683–1689.
31. Metzner KJ, Rauch P, Walter H, Boesecke C, Zollner B, Jessen H, et al
. Detection of minor populations of drug-resistant HIV-1 in acute seroconverters. AIDS 2005; 19:1819–1825.
32. Johnson JA, Li J-F, Wei X, Craig C, Stone C, Horton JH, et al
. Baseline detection of low-frequency drug resistance-associated mutations is strongly associated with virological failure in previously antiretroviral-naive HIV-1 infected persons. Antiviral Ther 2006; 11:S79.
33. Violin M, Cozzi-Lepri A, Velleca R, Vincenti A, D'Elia S, Chiodo F, et al
. Risk of failure in patients with 215 HIV-1 revertants starting their first thymidine analog-containing highly active antiretroviral therapy. AIDS 2004; 18:227–235.
34. Chaix ML, Descamps D, Deveau C, Schneider V, Harzic M, Tamalet C, et al
. Antiretroviral resistance, molecular epidemiology and response to initial therapy among patients with HIV-1 primary infection in 1999–2000 in France. Antiviral Ther 2002; 7:S180.
35. Pillay D, Bhaskaran K, Jurriaans S, Prins M, Masquelier B, Dabis F, et al
. The impact of transmitted drug resistance on the natural history of HIV infection and response to first-line therapy. AIDS 2006; 20:21–28.
36. Oette M, Kaiser R, Daumer M, Petch R, Fatkenheuer G, Carls H, et al
. Primary HIV drug resistance and efficacy of first-line antiretroviral therapy guided by resistance testing. J Acquir Immune Defic Syndr 2006; 41:573–581.