Date Published: January 15, 2013
Publisher: Hindawi Publishing Corporation
Author(s): Joao Leandro Paula Ferreira, Rosangela Rodrigues, Andre Minhoto Lança, Valeria Correia de Almeida, Simone Queiroz Rocha, Taisa Grotta Ragazzo, Denise Lotufo Estevam, Luis Fernando de Macedo Brigido.
Human immunodeficiency virus type 1 (HIV-1) transmitted drug resistance (TDR) is an important public health issue. In Brazil, low to intermediate resistance levels have been described. We assessed 225 HIV-1 infected, antiretroviral naïve individuals, from HIV Reference Centers at two major metropolitan areas of Sao Paulo (Sao Paulo and Campinas), the state that concentrates most of the Brazilian Aids cases. TDR was analyzed by Stanford Calibrated Population Resistance criteria (CPR), and mutations were observed in 17 individuals (7.6%, 95% CI: 4.5%–11.9%). Seventy-six percent of genomes (13/17) with TDR carried a nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance mutation, mostly K103N/S (9/13, 69%), potentially compromising the preferential first-line therapy suggested by the Brazilian HIV Treatment Guideline that recommends efavirenz-based combinations. Moreover, 6/17 (35%) had multiple mutations associated with resistance to one or more classes. HIV-1 B was the prevalent subtype (80%); other subtypes include HIV-1 F and C, mosaics BC, BF, and single cases of subtype A1 and CRF02_AG. The HIV Reference Center of Campinas presented more cases with TDR, with a significant association of TDR with clade B infection (P < 0.05).
Access to free antiretroviral therapy (ART) is part of the Brazilian response to the Aids epidemic and transmitted drug resistance (TDR); it has been a concern since the introduction of highly active antiretroviral therapy (HAART) in the late 1990s . TDR surveillance is an important strategy to monitor the emergence of genetic resistance as it may impact ART efficacy . This issue was especially sensible in Brazil that deployed a free ARV program in the late 90s amidst a suboptimal health care system. This initiative could boost the emergence of transmitted drug resistance variants and jeopardize Human immunodeficiency virus (HIV-1) treatment . However, most studies in Brazil have shown TDR prevalence similar to that observed among developed countries. Two recent Brazilian national surveys had accessed this issue [4, 5] but included a small representation of São Paulo metropolitan areas. We and others have analyzed mutations in treatment-naive individuals [6–12]; but to trace trends for TDR prevalence, continual monitoring is necessary.
People living with HIV, asymptomatic and naïve to ART were recruited at outpatient Clinic or Voluntary Counseling and Testing (VCT) at two metropolitan areas, the HIV State Reference Center at the city of Sao Paulo and the Municipal HIV Reference Center at the city of Campinas. Volunteers’ selection was conducted by their primary physicians’ or by VCT counseling personnel, with additional revision to confirm ARV exposure history. Individuals that agreed to participate were interviewed by clinical staff to access risk, review of potential previous exposures to ART (e.g., MTCT or postexposure prophylaxis), and document the knowledge of a partnership (sexual or sharing of drug paraphernalia) with individuals using ARV. Blood samples were collected from May 2008 to November 2009. Briefly, HIV-1 RNA was extracted with QIAamp viral RNA mini kit (Qiagen, Germany) and reverse transcribed with random primers and Superscript III enzyme (Invitrogen, USA). In samples of low HIV-1 RNA viral load or negative plasma detection, DNA from peripheral blood mononuclear cells (PBMCs) was extracted (Qiagen, Germany). Nested PCR products were sequenced using Big Dye terminators at an ABI 3100 Genetic Analyzer (ABI, USA) to evaluate protease (PR, codons 1 to 99) and partial reverse transcriptase (RT, codons 1 to 235) genes as previously described . Sequences were manually edited using Sequencher 4.7 software (Gene Codes, USA). Ambiguous DNA bases (mixtures) at resistance associated codons were considered at sequence edition. HIV genotyping resistance test results were reported to the HIV Reference Centers. TDR was defined according to the Calibrated Population Resistance Version 6.0 (CPR, Stanford Database, SDRM 2009), an algorithm specifically designed for the epidemiologic surveillance of HIV-1 transmitted drug resistance mutations (DRMs) . International Antiviral Society (IAS) 2011 resistance list  was additionally considered to evaluate the impact of resistance in ART response, which considers all mutations that impact ARV susceptibility. To contribute to HIV molecular epidemiology surveillance, HIV-1 subtyping was performed at NCBI genotyping and REGA HIV subtyping tools and confirmed by phylogenetic methods (PAUP* 4.10b), using evolution model selected by ModelTest3.7. Sequences are available at GenBank with accession numbers: HM533970 to HM534205; HQ015155 to HQ015157.
Of the 243 HIV-1 infected individuals enrolled in the study, partial HIV-1 pol sequences from 230 (95%) individuals were successfully sequenced (96% from plasma and 4% from PBMC). The inclusion criterion of no previous exposure to ART was met by 225 individuals and included in the analysis. Among the study individuals, six females had previous exposure to MTCT prophylaxis, documented at the interview. Most of the sequences analyzed from these women (3/4) exhibited one or more DRM but were not included in TDR prevalence estimate that was generated from the 225 ART naive individuals. Also, one case with unknown information of exposure to ART, but with history of undetectable viremia in previous years, had several resistance mutations and was excluded from analysis. Unprotected exposure among men who have sex with men (MSM) was the most frequent transmission route. Table 1 depicts demographic and laboratorial data from study volunteers at Campinas and São Paulo sites.
Among this population of antiretroviral naïve HIV-1-infected individuals attending the Campinas and São Paulo Reference Centers, transmitted drug resistance was detected overall in 7.6% of individuals. This TDR prevalence was similar to that reported in other regions of Brazil that used Stanford CPR criteria for these estimates, [4–12, 15]. The reasons for this stabilization, or even decrease of TDR prevalence in most surveys are unclear, but there may well be a plateau where the circulation of mutated isolates may come to some equilibrium, depending on multiple factors as the ARV therapy usage, therapy combinations, adherence, and social networking, among others. The followup of the small but consistent increase in TDR in Africa after the introduction of treatment programs  will allow verifying this hypothesis.
We observed low to intermediate levels of transmitted drug resistance mutations (7.6%), with most of them impairing susceptibility to efavirenz, a preferential ARV in first-line therapy at Brazil, confirming previous findings in the country. It is important both to monitor transmitted resistance trends and to define algorithms that might subsidize treatment alternatives where access to genotype test prior to therapy initiation to all individuals may be unrealistic. On the other hand, targeting genotypic test to population segments most susceptible to TDR may be cost effective. This would have both the potential benefit in HIV treatment response and an impact in reducing the drug resistance transmission of to the overall population.