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(HIV-Bresnet): a Survey of Chronically Infected Individuals

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(HIV-Bresnet): a Survey of Chronically Infected Individuals CONCISE COMMUNICATION Brazilian Network for HIV Drug Resistance Surveillance (HIV-BResNet): a survey of chronically infected individuals Rodrigo M. Brindeiroa, Ricardo S. Diazb, Ester C. Sabinoc, Mariza G. Morgadod, Ivone L. Pirese,f, Luı´s Brigidog, Maria C. Dantash, Draurio Barreirah, Paulo R. Teixeirah, Amilcar Tanuria and the Brazilian Network for Drug Resistance Surveillance Objective: To study the prevalence of HIV drug resistance mutations and subtype distribution in a Brazilian drug-naive population. Asymptomatic, drug-naive HIV-1- infected individuals were targeted in 13 voluntary counseling and testing centers spread around the country. Methods: Plasma viral RNA was extracted from 535 HIV-1-positive subjects. Protease (PR) and reverse transcriptase (RT) genomic regions were sequenced for subtype determination and analysis of drug resistance mutations. Results: Eight samples (2.24 %) showed primary mutations related to protease inhibitor (PI) resistance, eight (2.36%) to nucleoside reverse transcriptase inhibitors (NRTI) and seven (2.06%) to non-nucleoside reverse transcriptase inhibitors (NNRTI). Accessory mutations were found in the PR gene 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%)]. Mutations known to be associated with reduced sensitivity to NRTI or NNRTI (V118I, E44D, K219R, T69A, and V75L) were found in a low prevalence (0.6–2.4%). A high proportion of the isolates from subtype C was found in the southern states. Subtype F-related viruses were the main non-B variant in the rest of the country. Conclusions: Brazil has a low prevalence of drug-resistant strains circulating among recently diagnosed individuals. However, there was an increase in these rates com- pared with similar studies performed with samples collected in Brazil from 1996 to 1998. Continued surveys are required to detect trends in these rates, but routine genotypic testing in the drug-naive population prior to antiretroviral initiation is not required in Brazil. & 2003 Lippincott Williams & Wilkins AIDS 2003, 17:1063–1069 Keywords: drug resistance, genotyping, HIV surveillance From the aLaboratory of Molecular Virology, the Department of Genetics and the bInstitute of Microbiology Prof. Paulo Go´es, Federal University of Rio de Janeiro, the cDepartment of Imunology, Foundation Institute Oswaldo Cruz, and the dLaboratory of Virology, Institute of Biology do Exe´rcito, Rio de Janeiro, the eLaboratory of Retrovirology, Paulista School of Medicine/UNIFESP,the fFoundation Pro-Sangue, Hemocentro de Sa˜o Paulo, University of Sa˜o Paulo and the gLaboratory of Retrovirology, Institute Adolfo Lutz, Sa˜o Paulo, and hCN-DST/AIDS, Ministry of Health, Brası´lia, Brazil. ÃSee the Appendix for other members of HIV-BResNeT. Requests for reprints to: Dr A. Tanuri, Laborato´rio de Virologia Molecular, Departamento de Gene´tica, Universidade Federal do Rio de Janeiro, CCS–Bloco A–Cidade Universita´ria–Ilha do Funda˜o, 21944-970 Rio de Janeiro, RJ, Brazil. Received: 1 August 2002; revised: 22 November 2002; accepted: 29 November 2002. DOI: 10.1097/01.aids.0000060345.12269.d7 ISSN 0269-9370 & 2003 Lippincott Williams & Wilkins 1063 Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. 1064 AIDS 2003, Vol 17 No 7 Introduction months to avoid resampling. These sites spanned metropolitan regions located in eight different Brazilian The development of drug resistance remains one of the states, Rio Grande do Sul (n ¼ 139), Parana´ (n ¼ 147), most serious obstacles to sustained suppression of HIV Sa˜o Paulo (n ¼ 100), Rio de Janeiro (n ¼ 83), Mato during highly active antiretroviral therapy (HAART) Grosso do Sul (n ¼ 7), Para´ (n ¼ 17) , Bahia (n ¼ 12), [1–7]. and Ceara´ (n ¼ 30), that covered 45% of Brazilian patients taking HAART. None of the subjects has ever In developed countries the prevalence of transmission been exposed to any antiretroviral treatment according of viruses that are resistant to one or more antiretroviral to their written statement. The study was approved by agent has increased in recent years. The range of the Brazilian IRB (project 526–CONEP) as an anony- reported rates among recent seroconverters includes 5– mous unlinked study. 11% in Switzerland [8,9], 10–17% in France [10,11], 13% in German [12], 14% in the United Kingdom RNA isolation, amplification and sequencing [13], 15–26% in North America [14–16], 23–26% in Virus RNA was isolated as previously described [32]. Spain [17–19], 5–7% in the Unite States [20,21], and Following complimentary DNA generation with ran- 15.4% in Argentina [22]. Mutations associated with dom primers, nested polymerase chain reactions (PCR) resistance to nucleoside reverse transcriptase inhibitors was conducted for individual amplification of protease (NRTI), especially zidovudine and lamivudine, are the (PR, whole region) and reverse transcriptase (RT, most prevalent among drug-naive HIV-infected pa- nucleotides 105–651) [32,33]. PCR fragments were tients. The prevalence of HIV strains with at least one sequenced in an ABI 310 automated sequencer (Ap- primary drug- resistant mutation in Africa, South plied Biosystems, Foster City, California, USA). All America and the Caribbean is low (less than 7%), and sequences obtained were subjected to quality control the frequency of accessory mutations is high (up to assessments to ensure that there were no sample mix- 90%) [23–26]. ups or contamination from other sources [34]. Prevalence rates for HIV/AIDS in Brazil are approxi- Phylogenetic and sequence analyses mately 0.6% of the population (Brazilian Ministry of PR and RT sequences from all samples were submitted Health, http://www.aids.gov.br). Brazil has shown a to phylogenetic analysis for HIV-1 subtype determina- constant change in the character of its HIV-1 epidemic tion. Sequences were aligned using ClustalW [35,36] regarding gender infection ratio and risk behavior. against the reference set for subtyping analysis from the Since the beginning of the epidemic, HIV infection Los Alamos database (http://hiv-web.lanl.gov/). Phylo- patterns have shifted towards women and heterosexuals. genetic inferences were performed by the neighbor- The country’s HIV-1 subtype profile includes the joining method using the F84 model of substitution major circulating subtype B of HIV-1 but other implemented in PAUP v. 4.0b2a [37]. In order to subtypes, such as F, C and B/C and B/F divergent study the drug resistance mutations, sequences were sequences, co-circulate [27–31]. translated and aligned. The positions related to HIV drug resistance previously reported [38,39] were manu- The Brazilian Ministry of Health has been sponsoring a ally inspected and scored. The differential incidences of policy of universal access to antiretroviral drugs for subtypes and resistance, and their association with epi- AIDS patients since 1996. In order to monitor the demiological data, were statistically evaluated using a transmission of drug-resistant strains and the subtype two-tailed Fisher exact test. profile in Brazil, a National Network for Drug Re- sistance Surveillance (HIV-BResNet) in the drug-naive population was established. This work shows genotypic analysis from asymptomatic, drug-naive HIV-1-infected Results individuals diagnosed in 13 voluntary counseling and testing centers in different regions of the country in Table 1 summarizes all relevant epidemiological data of 2001. 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 Methods nearly an identical sex ratio (male to female ratio of 1.45) among samples. Samples Plasma from 535 consecutive HIV-1-positive indivi- Eight samples (2.24 %) showed primary mutations duals confirmed by serology was isolated at different related to PI resistance, eight (2.36%) to NRTI, and voluntary counseling and testing centers of the Brazi- seven (2.06%) to non-nucleoside reverse transcriptase lian Ministry of Health. The collection period was 3 inhibitors (NNRTI). No individuals were found carry- Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. HIV drug resistance surveillance in Brazil Brindeiro et al. 1065 Table 1. Epidemiological data. prevalence in any particular area, except for PI, which was not found in the northern states. There was no Characteristics Number (%) significant association between the presence of resistant Gender: male 225 (59.2) genotypes in the individuals studied and the sexual Age (years, mean Æ SD) 30.7 Æ 9.1 partner’s reported HIV serostatus (P ¼ 0.4552) or use Risk factor for HIVa Homosexual 55 (19.71) of antiretroviral drugs (P ¼ 0.2696). Heterosexual 172 (61.65) Intravenous drug user 14 (5.02) In contrast to the low prevalence of primary mutations Bisexual 20 (7.17) Other/multiple 18 (6.45) to drug resistance, many accessory mutations were Partner found in PR gene, with high prevalence at the following HIV positiveb 145 (39.61) positions: L63P/V/T/A/I [153/345 (44.3%)], M36I/L HIV positive and using antiretroviral 44 (30.34) c [149/345 (43.2%)], L10I/F/V [82/345 (23.8%)], V77I therapy [60/345 (17.4%)], A71V/T [11/345 (3.2%)], K20M/R aCalculation based on 279 individuals who respond to this item of [10/345 (2.9%)], and V82I [4/345 (1.2%)] There was a the questionnaire. bCalculation based on 366 individuals who re- clear association of accessory substitutions L63P and spond to this item of the questionnaire. cCalculation based on 145 individuals who respond to this item of the questionnaire. M36I with subtype assignment. The first one was associated with B subtype whereas the second was clearly associated with subtypes F and C. ing primary mutations for more than one class of antiretroviral drugs, except for sample SP653 (Table 2).
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