The development of drug resistance remains one of the most serious obstacles to sustained suppression of HIV during highly active antiretroviral therapy (HAART) [1–7].
In developed countries the prevalence of transmission of viruses that are resistant to one or more antiretroviral agent has increased in recent years. The range of reported rates among recent seroconverters includes 5–11% in Switzerland [8,9], 10–17% in France [10,11], 13% in German , 14% in the United Kingdom , 15–26% in North America [14–16], 23–26% in Spain [17–19], 5–7% in the Unite States [20,21], and 15.4% in Argentina . Mutations associated with resistance to nucleoside reverse transcriptase inhibitors (NRTI), especially zidovudine and lamivudine, are the most prevalent among drug-naive HIV-infected patients. The prevalence of HIV strains with at least one primary drug- resistant mutation in Africa, South America and the Caribbean is low (less than 7%), and the frequency of accessory mutations is high (up to 90%) [23–26].
Prevalence rates for HIV/AIDS in Brazil are approximately 0.6% of the population (Brazilian Ministry of Health, http://www.aids.gov.br). Brazil has shown a constant change in the character of its HIV-1 epidemic regarding gender infection ratio and risk behavior. Since the beginning of the epidemic, HIV infection patterns have shifted towards women and heterosexuals. The country's HIV-1 subtype profile includes the major circulating subtype B of HIV-1 but other subtypes, such as F, C and B/C and B/F divergent sequences, co-circulate [27–31].
The Brazilian Ministry of Health has been sponsoring a policy of universal access to antiretroviral drugs for AIDS patients since 1996. In order to monitor the transmission of drug-resistant strains and the subtype profile in Brazil, a National Network for Drug Resistance Surveillance (HIV-BResNet) in the drug-naive population was established. This work shows genotypic analysis from asymptomatic, drug-naive HIV-1-infected individuals diagnosed in 13 voluntary counseling and testing centers in different regions of the country in 2001.
Plasma from 535 consecutive HIV-1-positive individuals confirmed by serology was isolated at different voluntary counseling and testing centers of the Brazilian Ministry of Health. The collection period was 3 months to avoid resampling. These sites spanned metropolitan regions located in eight different Brazilian states, Rio Grande do Sul (n = 139), Paraná (n = 147), São Paulo (n = 100), Rio de Janeiro (n = 83), Mato Grosso do Sul (n = 7), Pará (n = 17), Bahia (n = 12), and Ceará (n = 30), that covered 45% of Brazilian patients taking HAART. None of the subjects has ever been exposed to any antiretroviral treatment according to their written statement. The study was approved by the Brazilian IRB (project 526–CONEP) as an anonymous unlinked study.
RNA isolation, amplification and sequencing
Virus RNA was isolated as previously described . Following complimentary DNA generation with random primers, nested polymerase chain reactions (PCR) was conducted for individual amplification of protease (PR, whole region) and reverse transcriptase (RT, nucleotides 105–651) [32,33]. PCR fragments were sequenced in an ABI 310 automated sequencer (Applied Biosystems, Foster City, California, USA). All sequences obtained were subjected to quality control assessments to ensure that there were no sample mix-ups or contamination from other sources .
Phylogenetic and sequence analyses
PR and RT sequences from all samples were submitted to phylogenetic analysis for HIV-1 subtype determination. Sequences were aligned using ClustalW [35,36] against the reference set for subtyping analysis from the Los Alamos database (http://hiv-web.lanl.gov/). Phylogenetic inferences were performed by the neighbor-joining method using the F84 model of substitution implemented in PAUP v. 4.0b2a . In order to study the drug resistance mutations, sequences were translated and aligned. The positions related to HIV drug resistance previously reported [38,39] were manually inspected and scored. The differential incidences of subtypes and resistance, and their association with epidemiological data, were statistically evaluated using a two-tailed Fisher exact test.
Table 1 summarizes all relevant epidemiological data of the individuals analyzed. There were 126 samples (23.6%) that did not generate PCR fragments for either genomic region (PR or RT) and these were excluded from the analysis. The epidemiological data shows nearly an identical sex ratio (male to female ratio of 1.45) among samples.
Eight samples (2.24 %) showed primary mutations related to PI resistance, eight (2.36%) to NRTI, and seven (2.06%) to non-nucleoside reverse transcriptase inhibitors (NNRTI). No individuals were found carrying primary mutations for more than one class of antiretroviral drugs, except for sample SP653 (Table 2). When individuals carrying primary mutations in the RT and PR regions were stratified in different geographical areas, there were no clusters of mutation prevalence in any particular area, except for PI, which was not found in the northern states. There was no significant association between the presence of resistant genotypes in the individuals studied and the sexual partner's reported HIV serostatus (P = 0.4552) or use of antiretroviral drugs (P = 0.2696).
In contrast to the low prevalence of primary mutations to drug resistance, many accessory mutations were found in PR gene, with high prevalence at the following positions: L63P/V/T/A/I [153/345 (44.3%)], M36I/L [149/345 (43.2%)], L10I/F/V [82/345 (23.8%)], V77I [60/345 (17.4%)], A71V/T [11/345 (3.2%)], K20M/R [10/345 (2.9%)], and V82I [4/345 (1.2%)] There was a clear association of accessory substitutions L63P and M36I with subtype assignment. The first one was associated with B subtype whereas the second was clearly associated with subtypes F and C.
The same type of analysis was performed for the RT deduced amino acid sequences. At the positions known to be associated with reduced sensitivity to commonly used NRTI or NNRTI [38,39], 8/327 had the V118I mutation (2.4%), 4/327 had E44D (1.2%), and 4/327 had K219R (1.2%).
A map of the Brazilian territory showing the distribution of subtypes found in our dataset is depicted in Fig. 1. We have identified 231 (64.9%) and 212 (62.5%) subtype B, 81(22.8%) and 100 (29.5%) subtype C, and 42 (11.8%) and 27 (8%) subtype F viruses based on PR and RT genomic regions, respectively. When both genomic regions were analyzed simultaneously, 42 sequences showed evidence of subtype divergence between two of those subtypes, such as PR C/RT B, PR B/RT C, PR B/RT F, PR F/RT B, PR C/RT F, and PR F/RT C. These divergent genomes together represented 14.48% of the total samples analyzed in both regions.
A low prevalence of primary drug-resistant mutations was observed in HIV-1 variants from the HIV-positive individuals diagnosed in Brazilian voluntary counseling and testing centers in 2001, indicating a low occurrence (4.42% for NRTI and NNRTI, and 2.24% for PI) of resistant strains circulating in drug-naive subjects. These results contrast with the ones originated from samples collected in the country until 1998 [40–43], in which a low prevalence (2%) of isolates carrying genotypic resistance was found. However, our findings are in accordance with the proportion of drug-naive subjects infected with drug-resistant viruses identified in studies from other Latin American countries [44,45] and Africa [23,24]. Many reports suggest no increase [11,15,20,46,47], while others show increase [12–14, 17,18,44,45] or even a decrease  in the prevalence of transmission of these resistant variants. However, studies conducted in Europe [8–14,16,18,48] and in North America [14–16,47,49–51] have reported the transmission of drug-resistant variants in up to 10 to 25% of subjects with primary HIV infection and in 5 to 17% of therapy-naive subjects. Although the dates of first diagnosis of HIV infection and sample collection are the same in our study, we cannot exclude the possible reversion of transmitted drug-resistant mutants to wild-type viruses since primary infection. However, although our data may underestimate the true prevalence of primary antiretroviral drug resistance, the persistence of transmitted or acquired drug-resistant mutations in the absence of selective drug pressure has been demonstrated [23,25,44,45,47,52].
The presence of multiple PR accessory mutations can reduce the genetic barrier and yield a faster selection of resistant strains when compared with the wild type isolates. Recent data suggest that some of the polymorphic mutations at positions 10, 36, 63, 71, and 77 in PR can be associated with therapeutic failure in naive or therapy-experienced patients . We could also find some individuals carrying viruses showing mutations such as V106I and V108I, previously implicated in intermediate levels of phenotypic resistance to NNRTI drugs [38,39]. Phenotypic resistance to NNRTI from 4- to 10-fold to wild-type reference strains were not associated with the virological outcome of therapeutic regimens containing these drugs .
A large proportion of HIV-1 subtype C in the south of Brazil was observed. Previous surveys in Brazil have shown frequencies of around 3% for subtype C viruses in the country [27,55]. We have found that almost 30% of the viruses circulating in southern Brazil are of subtype C. We can speculate an increase in the prevalence of C subtype in southern cities. This high prevalence of subtype C samples decreases northbound: the prevalence was 24% in Paraná state and 3% in São Paulo state. Subtype F variant is spread all over the country and corresponds approximately to 10% of the samples analyzed in Rio de Janeiro and São Paulo cities. Recently, several groups have reported the appearance of recombinant forms of subtype B and F. In fact, in our dataset, the prototypic F strains are in low numbers compared with the divergent subtype genome counterparts carrying F sequences . Our data support previous findings showing a wide geographical spread of prototypic and divergent subtype forms of subtype F isolates. Clade C-related strains decrease northbound, clade F-related viruses increase towards the north and the center of the country (Fig. 1).
The isolates’ subtype assignment could not be correlated with risk factors or primary mutations. However, a significant correlation was observed between the subtype C isolates and gender in the southern region sites. These variants were found preferentially in women (P < 0.0026), with a sex ratio (male to female) 0.89; this compares with a ratio of 2.45 for subtype B viruses. This observation can be linked to the recent introduction of this subtype in this geographic region concomitant with the feminization of the epidemic in Brazil. Similarly, there was a tendency to find subtype C isolates in individuals reporting heterosexual practice as a risk factor (P < 0.112).
Although recent reports suggest that genotypic resistance testing has a significant benefit in predicting virological response to alternative therapy for patients showing virological failure , no data exist to support the routine use of resistance testing in drug-naive patients beginning therapy. The findings reported in our study do not suggest the need for routine genotypic testing in the drug-naive population. However, epidemiological monitoring of the prevalence of primary resistance must be implemented on a routine basis in countries with widespread availability of HAART.
All members of HIV-BResNet equally contributed to this study.
Sponsorship: This study was supported by the AIDS/STD National Program, Brazilian Ministry of Health, the State Science Foundation of Rio de Janeiro Grant E-26/151.970/00, the Brazilian Council for Scientific and Technologic Development Grant 462394/00-0, and Fundação de Amparo e Pesquisa de São Paulo Grant 98/14381-4.
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Other members of HIV-BResNet: M. A. Soares, M. Arruda, P. A. Brindeiro, A. Afonso, E. Soares (UFRJ, Rio de Janeiro); E. Caride (BioManguinhos, FIOCRUZ), F. A. B. Speranza, S. Ishii (IBEX, Rio de Janeiro); S. Chequer-Fernandez, J. C. Couto-Fernandez (FIOCRUZ, Rio de Janeiro); E. Cavalieri, L. Costa (EPM/UNIFESP, São Paulo); A. S. Nishiya N. Gaburo (Fundação Pró-Sangue, São Paulo); R. Rodriges, R. Custódio (Instituto Adolfo Lutz, São Paulo); C. Lauria (Lab Ac. Nucleicos, HPE, UERJ, Rio de Janeiro). Contributors from voluntary counseling and testing centers in alphabetical order: Cristina Caliento, Ana M. S. Cavalcanti, Vera M. S. Costa, Maria A. Daher, Maria I. Espineli, Rosa M. C. Ferreira, Cecília K. Kanashiro, Maria E. F. P. Magalhães, Jaciléia M. Pereira, Cristiane M. S. Quadros, Célia F. R. Queiroz, Carlos A. Silva, Marta da-S. Soares, Maria A. A. S. C. Soidan, Isete M. Stela, Jahiel M. de-S. Tavares, Paulete S. Zular.