(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 compared 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 Goés, 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 Saõ Paulo, University of Saõ Paulo and the gLaboratory of Retrovirology, Institute Adolfo Lutz, Saõ Paulo, and hCN-DST/AIDS, Ministry of Health, Brası´lia, Brazil. See 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 Fundaõ, 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

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 Saõ 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-

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 SD) 30.7 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). The same type of analysis was performed for the RT When individuals carrying primary mutations in the deduced amino acid sequences. At the positions known RT and PR regions were stratified in different geogra- to be associated with reduced sensitivity to commonly phical areas, there were no clusters of mutation used NRTI or NNRTI [38,39], 8/327 had the V118I

Table 2. Genotypic distribution of primary mutations found.

Epidemiological data

Age Site Sex (years) Risk factors NRTI NNRTI PI

Rio de Janeiro RJ877 F 38 het; parHIV; parARV V106I RJ925 M 29 hom; parHIV; parARV V106I RJ897 M 22 het G48V,I54T,V82A RJ866 F 36 het K103N RJ928 F 36 het; parIDU V32I,M46I,I47V,V82A Rio Grande do Sul RS2032 F 47 het; parHIV M41L;D67N;T215F RS2093 F 39 het I54V,L90M RS2071 F 22 het; parHIV M41L; M184V RS2152 M 37 het; parHIV M46I,I54V,V82A RS2017 M 37 bisex; parHIV G48V;I54T;V82A RS2161 F 37 het; parIDU I84V Parana´ PR1483 M 28 hom V106I,V108I PR1518 M 17 het; parHIV M41L PR1537 F 37 het M184V PR1538 M 24 hom M41L Sa˜o Paulo SP469 M 48 bisex; parHIV V108I SP653 M 54 het M41L V108I SP369 M 37 het D30N Centre North (Para´ + Mato Grosso do Sul) CN28 M 39 het; parHIV M41L CN2398 F 35 mult V106I Northeast (Ceara´ + Bahia) NE269 F 38 het; parHIV V82S NE135 M 26 bisex M41L Genomes with primary mutations 8 (2.31%a) 7 (2.06%a) 8 (2.31%b)

NRTI, nucleoside reverse transcriptase inhibitor; NNRTI, non-nucleoside reverse transcriptase inhibitor; PI, protease inhibitor; F, female; M, male; hom, homosexual; het, heterosexual; bisex, bisexual; mult, multiple risk factors; parHIV, sexual partner HIV positive; parIDU, partner intravenous drug user; parARV, partner taking antiretroviral therapy.aPer- centage based on 339 reverse transcriptases sequenced.bPercentage based on 356 proteases sequenced.

mutation (2.4%), 4/327 had E44D (1.2%), and 4/327 was observed in HIV-1 variants from the HIV-positive had K219R (1.2%). individuals diagnosed in Brazilian voluntary counseling and testing centers in 2001, indicating a low occur- A map of the Brazilian territory showing the distribu- rence (4.42% for NRTI and NNRTI, and 2.24% for tion of subtypes found in our dataset is depicted in Fig. PI) of resistant strains circulating in drug-naive subjects. 1. We have identified 231 (64.9%) and 212 (62.5%) These results contrast with the ones originated from subtype B, 81(22.8%) and 100 (29.5%) subtype C, and samples collected in the country until 1998 [40–43], in 42 (11.8%) and 27 (8%) subtype F viruses based on PR which a low prevalence (2%) of isolates carrying and RT genomic regions, respectively. When both genotypic resistance was found. However, our findings genomic regions were analyzed simultaneously, 42 are in accordance with the proportion of drug-naive sequences showed evidence of subtype divergence subjects infected with drug-resistant viruses identified between two of those subtypes, such as PRC/RTB, in studies from other Latin American countries [44,45] PRB/RT C, PRB/RTF, PRF/RTB, PRC/RTF, and and Africa [23,24]. Many reports suggest no increase PRF/RT C. These divergent genomes together repre- [11,15,20,46,47], while others show increase [12–14, sented 14.48% of the total samples analyzed in both 17,18,44,45] or even a decrease [9] in the prevalence of regions. 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 Discussion subjects with primary HIV infection and in 5 to 17% of therapy-naive subjects. Although the dates of first diag- A low prevalence of primary drug-resistant mutations nosis of HIV infection and sample collection are the

RORAIMA divergent AMAPÁ F 9.09% 4.55%

AMAZONAS MARANHÃO PARA CEARÁ B R. G. DO NORTE 86.36% PARAÍBA PIAUI Northeastern - 7.9% ACRE PERNANBUCO

ALAGOAS

RONDONIA SERGIPE MATO GROSSO BAHIA divergent 8.57% Centre-North - 6.1% GOIÁS F 5.71% divergent 5.88% C MINAS GERAIS MATO GROSSO 5.71% F E. SANTO DO SUL B 23.53% SÁO PAULO 80.00% B RIO DE JANEIRO 70.59% PARANA Rio de Janeiro - 12.6% São Paulo - 11% divergent S. CATARINA 12.90% Paraná - 16.9% RIO GRANDE C Rio Grande do Sul - 45.5% DO SUL 3.23% divergent F 2.13% B divergent 4.26% 83.87% 22.05% B 29.92% C F 29.79% 3.15% B 63.83%

C 44.88% Fig. 1. Distribution of subtypes by geographical site. Brazilian map showing the distribution of subtype based on PR and RT genomic regions. The samples showing discordant subtype call between PR and RT genes are counted as divergent genomes in graphic areas. The graphic areas are proportional to the sample size of each site analyzed.

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. HIV drug resistance surveillance in Brazil Brindeiro et al. 1067 same in our study, we cannot exclude the possible Brazil. Similarly, there was a tendency to find subtype reversion of transmitted drug-resistant mutants to wild- C isolates in individuals reporting heterosexual practice type viruses since primary infection. However, as a risk factor (P , 0.112). although our data may underestimate the true prevalence of primary antiretroviral drug resistance, the Although recent reports suggest that genotypic resis- persistence of transmitted or acquired drug-resistant tance testing has a significant benefit in predicting mutations in the absence of selective drug pressure has virological response to alternative therapy for patients been demonstrated [23,25,44,45,47,52]. showing virological failure [56], no data exist to support the routine use of resistance testing in drug- The presence of multiple PR accessory mutations can naive patients beginning therapy. The findings reported reduce the genetic barrier and yield a faster selection of in our study do not suggest the need for routine resistant strains when compared with the wild type genotypic testing in the drug-naive population. How- isolates. Recent data suggest that some of the poly- ever, epidemiological monitoring of the prevalence of morphic mutations at positions 10, 36, 63, 71, and 77 primary resistance must be implemented on a routine in PR can be associated with therapeutic failure in basis in countries with widespread availability of naive or therapy-experienced patients [53]. We could HAART. also find some individuals carrying viruses showing mutations such as V106I and V108I, previously im- plicated in intermediate levels of phenotypic resistance to NNRTI drugs [38,39]. Phenotypic resistance to NNRTI from 4- to 10-fold to wild-type reference Acknowledgements strains were not associated with the virological out- All members of HIV-BResNet equally contributed to come of therapeutic regimens containing these drugs this study. [54]. Sponsorship: This study was supported by the AIDS/STD A large proportion of HIV-1 subtype C in the south of National Program, Brazilian Ministry of Health, the State Brazil was observed. Previous surveys in Brazil have Science Foundation of Rio de Janeiro Grant E-26/ shown frequencies of around 3% for subtype C viruses 151.970/00, the Brazilian Council for Scientific and in the country [27,55]. We have found that almost 30% Technologic Development Grant 462394/00-0, and Fun- of the viruses circulating in southern Brazil are of daçaõ de Amparo e Pesquisa de Saõ Paulo Grant 98/ subtype C. We can speculate an increase in the 14381-4. prevalence of C subtype in southern cities. This high prevalence of subtype C samples decreases northbound: the prevalence was 24% in Parana´ state and 3% in Saõ Paulo state. Subtype F variant is spread all over the References country and corresponds approximately to 10% of the 1. Carpenter CCJ, Fischl MA, Hammer SM, Hirsch MS, Jacobsen samples analyzed in Rio de Janeiro and Saõ Paulo DM, Katzenstein DA, et al. Antiretroviral therapy for HIV cities. Recently, several groups have reported the infection in 1998. 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