Objective: Our objective was to estimate primary resistance in an urban setting in a developing country with a long history of antiretroviral delivery and high coverage levels.
Design: We carried out a resistance surveillance study according to WHO HIV-Resistance Guidelines.
Methods: Blood samples were collected from 323 drug-naive HIV-1 infected individuals diagnosed at two HIV voluntary counselling and testing centers in Buenos Aires. Viral-load, CD4 cell counts and detuned assays were performed on all samples. The pol gene was sequenced and the resistance profile determined. Phylogenetic analysis was performed by neighbor-joining trees and bootscanning analysis.
Results: We found that 12 (4.2%) of the 284 samples sequenced harbored primary resistance mutations, of which K103N, M41L and V108I were most prevalent. Phylogenetic analysis revealed evidence for the transmission of the K103N mutation among the drug-naive population. The proportion of recent infections identified by the detuned assay was 10.1%.
Conclusions: Levels of primary resistance in Buenos Aires are still low, despite a long history of ARV delivery and high coverage levels.
From the aNational Reference Center for AIDS, School of Medicine, University of Buenos Aires, Buenos Aires, Argentina
bServicio de Inmunocomprometidos, Hospital General de Agudos José María Ramos Mejía, Buenos Aires, Argentina
cAsociación Benéfica PRISMA, Lima, Peru
dNexo Non-Governmental Gay Men Organization, Buenos Aires, Argentina.
†Present affiliation: US Military HIV Research Program at the Walter Reed Army Institute of Research, Rockville, Maryland, USA.
Received 14 July, 2006
Revised 13 December, 2006
Accepted 11 January, 2007
Correspondence to Dr. Horacio Salomón, National Reference Center for AIDS, Department of Microbiology, School of Medicine, University of Buenos Aires, Paraguay 2155 piso 11, C1121ABG - Buenos Aires, Argentina. E-mail: firstname.lastname@example.org
Transmission of drug-resistant HIV-1 is well documented and is associated with suboptimal virologic response to a first antiretroviral treatment . In some resource-rich settings, a rise in the prevalence of primary resistance to over 10% [2,3] makes drug-resistance testing recommended not only for casual treatment failure, but also as something to be considered prior to antiretroviral treatment (ART) for HIV infection [4–6]. Recent USA guidelines  recommend resistance testing for all subjects beginning treatment, regardless of evidence of recent infection.
In developing countries, recommendations have been established without sufficient epidemiological data, and the expansion of ART in these settings may be jeopardized by limited data. Increased ART coverage in Latin America in recent years  has caused concern regarding how several health infrastructure-related characteristics of these settings may increase chances for resistance selection . Therefore, context-specific information is required [9,10].
The World Health Organization (WHO) recommends the establishment of HIV drug resistance sentinel suveillance systems in these settings, as in any country in which ART programs have been developed .
Argentina is a developing country with approximately 120 000 HIV-infected individuals. Free access to antiretroviral drugs has been guaranteed since 1992, and Argentina was the first Latin American country to establish an ART program. Buenos Aires, the capital city, is the most populated, containing 71% of all reported HIV cases. Resistance testing is only available for individuals with health insurance, who represent 20% of all infected individuals. A previous study identified low levels of primary resistance (2.3%, ), and a recent study reported an increase of over twofold (7.7%, ); however, no systematic resistance surveillance studies have been carried out.
The objective of this study was to estimate primary resistance among newly diagnosed individuals in Buenos Aires.
Materials and methods
This resistance surveillance study was designed according to WHO HIV resistance guidelines . Briefly, newly HIV-diagnosed individuals without evidence of previous exposure to antiretroviral drugs were sequentially selected in the order in which they appeared for HIV testing at two voluntary and counselling testing centers (VCTs) in Buenos Aires: ‘Hospital de Agudos José María Ramos Mejía’ (HospitalVCT) and ‘Nexo NGO’ (gay men's non-governmental organization, NGOVCT). All clinical records were reviewed to assure that patients were antiretroviral naive and recently diagnosed as HIV positive.
The study population included 5041 individuals presenting themselves for routine HIV testing at both VCTs. Blood samples were collected from 323 drug-naive newly diagnosed HIV-1 individuals. At the HospitalVCT, 3714 individuals were tested between March 2003 and October 2005, of whom 243 (6.5%) subjects were diagnosed as HIV positive. At the NGOVCT, 1327 subjects were tested between March 2004 and October 2005 and 80 (6.0%) were HIV positive.
Detuned assay to identify recent infections
Plasma samples were tested using a modified version of the HIV-1 enzyme immunoassay (Vironostika HIV-1EIA; bioMerieux, Durham, North Carolina, USA)  in order to classify samples as potential recent infections (time of infection less than 4–6 months prior to sample collection). The Serologic Testing Algorithm for Recent HIV Seroconversions (STAHRS) strategy was performed .
HIV-1 RNA isolation and sequencing
RNA was isolated from plasma by the QIAamp viral extraction Kit (QIAGEN GmbH, Hilden, Germany). The pol gene was amplified between positions 2142 to 3798 (reference strain HXB2 numbering ) by reverse transcriptase-polymerase chain reaction (RT-PCR), as previously described , and sequenced by ABI Prism 3100/3100-Avant equipment (Applied Biosystems, Foster City, California, USA). [GeneBank accession numbers EF120074-EF120356]. Twenty-one samples were also amplified and sequenced in the vpu gene between positions 5967 to 6570  [GeneBank accession numbers EF119856, EF119871, EF119880, EF119902, EF119904, EF119931, EF119939, EF119974, EF119983, EF120007, EF120013, EF120015, EF120021, EF120023, EF120027, EF120028, EF120030, EF120034, EF120046, EF120057, EF120070].
Viral load and CD4 testing
Plasma viral load (VL) was assessed by branched DNA (b-DNA) technology, with a detection limit of 50 HIV-1 RNA copies/ml (Versant HIV-1 RNA 3.0; Bayer Co., Tarrytown, New York, USA). CD4+ cells from peripheral blood were enumerated by cytometry (Coulter XL; Coulter Co., Hialeah, Florida, USA).
Sequences were analyzed as previously described  to identify mutations associated with reduced susceptibility to protease and RT inhibitors, as reported by International AIDS Society-USA in 2005 : RT – M41L, A62V, K65R, D67N, T69D, 69 insert, K70R, L74V, V75I, F77L, L100I, K103N, V106A, V106M, V108I, Y115F, F116Y, Q151M, Y181C, Y181I, M184V, M184I, Y188C, Y188L, Y188H, Y188C, G190A, G190S, L210W, T215Y, T215F, K219Q, K219E, P225H, M230L, and P236L; protease – D30N, M46I, M46L, G48V, I50V, V82A, V82S, V82F, V82T, I84V and L90M.
Sequence alignment was performed by CLUSTAL W (BioEdit 188.8.131.52 sequence alignment editor ). Neighbor-joining (NJ) trees were constructed under the Kimura 2-parameter model with the MEGA3 program . Sequences were individually analyzed by Simplot 2.5  and recombination analysis was then performed by bootscanning analysis .
Chi-square test and Fisher's exact test were used to compare proportions of resistance mutations and trend analysis was performed by chi-squared test. A Mann-Whitney test was performed for non-parametric analysis.
A total of 323 newly HIV-1 diagnosed individuals were evaluated. Of these, 39 samples could not be successfully sequenced and were excluded from the analysis, resulting in a total of 284 samples. Samples that could not be sequenced had a significantly lower levels of viral load (P < 0.05), which may explain the inability to amplify them. The characteristics of the study population are shown in Table 1. We identified 10.1% of recent infections by detuned assay. Phylogenetic analysis showed that 147 (51.8%) samples were inter-subtype BF recombinants, 128 (45.1%) were subtype B and nine (3.2%) were ‘nonB-nonBF’ variants.
Twelve individuals (4.2%) were found to harbor primary resistance mutations (Table 2). These mutations were more prevalent in individuals with probable recent infection detected by detuned assay (7.1 versus 4.0% in those not recently infected), although this difference was not statistically significant. There were no significant differences in prevalence of resistance mutations according to risk group (Table 1). Analysis of primary resistance mutations by year showed no significant variations (2.6% in 2003, 5.9% in 2004 and 2.2% in 2005, chi-squared for trend, P = 0.549).
According to drug class, mutations associated with resistance to non-nucleoside reverse transcriptase inhibitors (NNRTI) were the most prevalent and were found in six (2.1%) individuals. Mutations conferring low levels of resistance to NNRTIs were found in seven (2.5%) individuals as follows: A98G (n = 1), V179D (n = 5) and V179E (n = 1). Nucleoside reverse transcriptase inhibitor (NRTI) mutations were found in four (1.4%) individuals. Primary mutations associated with resistance to protease inhibitors (PI) were found in four individuals (1.4%). Secondary mutations associated with resistance to this class of drugs were present in 249 (87.7%) sequences. A total of 160 (56.3%) sequences had two or more of these class mutations.
Overall, the most prevalent resistance mutations were K103N, M41L and V108I. Each of these three mutations was found in three of the 12 individuals with primary resistance. As shown in Table 2, the three individuals infected with the mutant K103N were men, one of them was a 54-year-old heterosexual man enrolled at the HospitalVCT, and the other two were young men who have sex with men (MSM) enrolled at the NGOVCT. The latter two patients shared the same primary and secondary mutations. Phylogenetic analysis of both pol (containing the K103N mutation) and vpu genes independently revealed a high relationship between both viral strains (data not shown). These samples were collected in 2004 from individuals who were diagnosed as HIV-1 positive 3 weeks before sampling. One of them was identified as a recent infection by detuned assay (Table 2); epidemiological data obtained from clinical records allowed us to confirm that their transmissions were linked.
One of the three individuals harboring M41L was a 32-year-old woman enrolled at the HospitalVCT in 2004 with no evidence of recent infection. The other two were MSM enrolled in 2004: one was a 36-year-old from the HospitalVCT with evidence of recent infection by detuned assay, and the other was a 28-year-old MSM enrolled at NGOVCT. No relationship between these viral sequences was found by phylogenetic analysis.
Based on our results, 4.2% of newly diagnosed individuals in Buenos Aires harbor resistance mutations associated with reduced susceptibility to antiretroviral drugs. This figure is lower than in North America and Europe, suggesting that primary resistance may not yet be a serious problem with regard to public health in Buenos Aires. Similar results were recently reported in Brazil .
However, Argentina has a lower proportion of HIV-infected individuals who know their serologic status than do more developed countries (40%, as reported by the Argentinean Ministry of Health , compared with 80% in the USA ). This is consistent with the low proportion of recent infections among newly diagnosed individuals in our study (10.1% by detuned assay). The inability to detect infected individuals may have limited access to ART, resulting in a decreased selective pressure for the maintenance of resistance. This may explain why countries such as Argentina and Brazil have lower levels of primary resistance than do developed countries, despite having similar ART coverage over the population diagnosed.
The K103N and M41L mutations were the most prevalent in our study, similar to Europe and the USA [2,3], perhaps due to these mutations having a lower impact on viral fitness and a greater chance to persist in the absence of drug pressure . Other resistance mutations not found in our study may have been selected and transmitted but eventually lost due to a reduced population-level selective pressure and/or back mutation and overgrowth by wild-type virus.
In our study, we found evidence that K103N is spreading and persisting in the absence of drug pressure. Together with recent reports , our results suggest that K103N may increase in prevalence in Argentina concomitant with increased use of NNRTI-based therapy [26,27].
Mutation V108I was also found in three individuals. The fact that all three individuals harboring V108I lacked other resistance mutations suggests that V108I may be a natural polymorphism, as it is associated only with low-level resistance to nevirapine and efavirenz. However, V108I (like all primary mutations in our study) meets the criteria for resistance mutations as defined by Bennett et al. . Evidence regarding the linking of V108I with resistance was previously reported .
It is possible that our study underestimates primary resistance, due to the low proportion of recent infections. However, our findings are consistent with those of others .
In summary, our results argue in favor of extending access to ART in developing countries. Characterization of newly diagnosed HIV-positive population may represent a practical tool for the surveillance of primary resistance in these settings, a subject of public health importance in both wealthy countries and those with limited resources. Analysis performed by detuned assay may allow for the assessment of the proportion of individuals diagnosed in early stages of infections, which in turn may impact primary resistance levels.
We thank Dr. Mark A. Wainberg for his useful comments and manuscript revision.
The authors do not have any commercial or other association that might pose a conflict of interest.
The information has not been presented at any meeting.
Sponsorship: This study was supported by Agencia Nacional de Promoción Científica y Tecnológica (Código Proyecto 14104.PICT 2003.) and M005, Programación Científica UBACYT 2004-2007.
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