Short Communication: Intermediate Prevalence of HIV Type 1 Primary Antiretroviral Resistance in Ceará State,Northeast Brazil

Abstract

Brazil is a large developing country where almost all FDA-licensed antiretrovirals are made available to more than 200,000 individuals under antiretroviral treatment. General primary HIV-1 resistance in Brazil is assumed to be low, but data are scarce, especially in the Northeast region. To evaluate the prevalence of primary HIV-1 antiretroviral resistance in the state of Ceará, Brazil, a cross-sectional prospective study of antiretroviral-naive HIV-1-infected individuals was performed between May 2008 and May 2009. Genomic sequences of reverse transcriptase and protease regions of the pol gene of HIV-1 using PCR products were obtained. Mutations related to resistance to NRTI, NNRTI, and PI were evaluated according to the WHO mutation list for primary resistance surveillance, which excludes common polymorphisms. Seventy-four individuals were evaluated (50% male) with a median age 30 years; 55.4% were men who have sex with men. Median CD4⁺ T lymphocyte counts were 418 and 960 cells/mm³ and the median viral loads were 4.41 and 4.46 log₁₀ RNA copies/ml for individuals older and younger that 18 years, respectively. Twenty-seven percent of patients were symptomatic. Five patients (6.8%) were recently infected, as detected by the BED test. The mutations 41L, 67N, 215D, 219Q, 101E, and 103N in the RT and 32I, 46I, 54V, 82T, and 90M, in the PR were identified in 9.5% of samples, more frequently in HIV subtype B (85.1%). A significant level of primary HIV resistance was detected in urban Northeast Brazil, a region geographically distant from the more highly populated and wealthier areas of Southeast Brazil, and this emphasizes the need for monitoring resistance in the studied area.

Introduction

B razil is a large developing country where almost all FDA licensed antiretrovirals are made available free of charge to over 200,000 individuals. The prevalence of AIDS in Brazil is higher in large metropolitan areas of the southeast, where the majority of HIV-infected individuals on antiretroviral treatment also live.¹ Primary HIV-1 resistance in Brazil is considered to be generally low, ranging from 5.3% to 8.1%,^2,3 although it may be exceedingly high in some specific areas, such as in Santos, a large coastal city in the southeastern state of São Paulo, where it reaches 36.8%.⁴ The epidemic in Brazil is also rather genetically diverse, with cocirculation of HIV-1 subtypes B, F, and C and a number of unique recombinant forms.⁵

Northeast Brazil is a region comprised of nine states, encompassing a vast and quite heterogeneous area, and within this region HIV primary resistance has been assessed only in the state of Bahia.^3,6 Therefore, we sough to assess the prevalence of HIV-1 primary antiretroviral resistance and to profile HIV-1 subtypes in Ceará, another populous state in Northeast Brazil.

We evaluated the prevalence of HIV-1 primary antiretroviral resistance in the state of Ceará, Brazil.

Materials and Methods

A cross-sectional, prospective study, enrolling HIV-1-infected, antiretroviral-naive individuals, was conducted at the two largest outpatient clinics in the state of Ceará, the José de Alencar Health Center and the São José Infectious Diseases Hospital, from May 2008 to May 2009. The number of patients to be enrolled was calculated using the Epi-Info 6 program, based on the mean annual number of 717 patients who received antiretroviral treatment in the previous 3 years, as informed by the Coordination of Pharmaceutical Assistance of the Secretariat of Health of the State of Ceará. For this calculation, the prevalence of primary resistance was estimated at 10%, admitting 3% as a minimal acceptable value, with a 95% confidence interval.

The sample size was calculated to be 64 patients and the enrollment aimed at 100 patients. Patients of both genders of any age with a confirmed diagnosis of HIV infection at any previous time, without prior treatment with antiretrovirals, even for prophylaxis, who agreed to participate, were enrolled after signing an informed consent form. They were interviewed and a form was filled out with epidemiological and physical data as well as available laboratory results.

A 10-ml sample of blood was collected at a median time of 14 days postenrollment (ranging from the same day up to 187 days postenrollment) in EDTA and plasma samples were stored at −80°C until genotyping and BED assayed. Following the manufacturer's recommendations, total RNA was purified using a QIAmp kit and the product was subject to amplification by reverse transcriptase polymerase chain reaction (RT-PCR) and sequencing, using the TRUEGENE HIV-1 Genotyping Kit.^7,8 Samples with no detectable amplification and those that did not yield good sequence results were sent to the Retrovirology Laboratory at the Federal University of São Paulo for sequencing using published methods,⁹ as well as for testing by immunoglobulin G-capture BED-enzyme immunoassay (BED-EIA) to detect recent infections (within the last 153 days). BED-EIA was done using the Calypte HIV-1 for IgG capture, according to the manufacturer's recommendations.

Mutations related to resistance to nucleoside/nucleotide reverse-transcriptase inhibitors (NRTIs), nonnucleoside reverse-transcriptase inhibitors (NNRTIs), and protease inhibitors (PIs) were evaluated in accordance with the WHO mutation list for primary resistance surveillance, which excludes common polymorphisms.¹⁰ The Fisher exact test was used in a comparative evaluation of statistical analyses (p ≤ 0.05 considered as significant). This study was approved by the local Internal Review Board.

Results

Seventy-four individuals were analyzed between May 15, 2008 and May 15, 2009. Sixty-three were adults and 11 were younger than 18 years old (Table 1). The majority of patients were asymptomatic, with a high median CD4⁺ lymphocyte count, even considering the great variation observed with this marker. Initial therapy was required for 35 patients (47.3%) because of the presence of AIDS-defining conditions (CDC 1993),¹¹ or ≥10 points by the Rio de Janeiro/Caracas' criteria,¹² or CD4⁺ lymphocyte count <350 cells/mm³.

Table 1.

Demographic and Laboratory Data of Studied Patients ^a

Variable	N (%)	Range
Sex
Male	56 (75.7%)
Female	18 (24.3%)
Median age (years)
All patients	30	1–67
<18 years	5	1–16
≥18 years	31	19–67
Place of residence^b
Capital city^c	62 (83.8%)
Countryside	12 (16.2%)
Place of transmission^d
State of Ceará	58 (78.4%)
Capital city^c	50 (67.6%)
Countryside	8 (10.8%)
Other State	3 (4.1%)
Not known	13 (17.5%)
Risk factor
Sexual	64 (86.5%)
MSM	41 (55.4%)
Heterosexual	23 (31.1%)
MTCT	10 (13.5%)
Median time (months) between diagnosis and enrollment in the study	4.5	0–142
Median baseline CD4⁺ lymphocyte count (cells/mm³) in ≥18 years	418	15–932
Median baselline CD4⁺ lymphocyte count (cells/mm³) in ≥18 years	960	12–2964
Median baseline viral load (log₁₀) in ≥18 years	4.41	2.26–5.7
Median baseline viral load (log₁₀) in <18 years	4.46	3.43–5.9
Clinical status^e
Symptomatic	20 (27%)
Asymptomatic	54 (73%)
AIDS criteria^f
Yes	23 (31.1%)
No	51 (68.9%)
Requirement for immediate treatment^g
Yes	35 (47.3%)
No	39 (52.7%)

MSM, men who have sex with men; MTCT, mother-to-child transmission.

Locale of residence reported by the patient.

Capital city or metropolitan region.

Place where patient is believed to have been infected.

Presence or absence of diarrheal symptoms, loss of weight (>10%), persistent fever, chronic dermatitis, herpes zoster, oropharygeal candidiasis, and tuberculosis.

CD4⁺ < 200 cells/mm³ or the presence of AIDS-defining illness (CDC, 1993) or ≥10 points by the Rio de Janeiro/Caracas criteria.

Presence of AIDS-defining illness (CDC, 1993) or ≥10 points by the Rio de Janeiro/Caracas criteria or CD4 <350 cells/mm³.

Among the adults none reported sexual exposure to partners known to be on antiretrovirals. In only one of the 10 cases of vertical transmission was there previous exposure to antiretroviral therapy. Primary resistance mutations were found in seven patients (9.5%), all of whom were 18 years old or older. In one of these patients resistance mutations were detected both for NRTIs and PIs, two for NRTIs, two for NNRTIs, and two for PIs. The prevalence of primary resistance to NRTIs, PIs, and NNRTIs was 4.1%, 4.1%, and 2.7%, respectively (Table 2). Subtype B was identified both at the reverse transcriptase and protease (PR) regions in 63 samples (85.1%), whereas subtype F was identified in six samples (8.1%), subtype C in four samples (5.4%), and a profile resembling CRF_28 in one sample (1.4%). Four out of seven antiretroviral-resistant samples were of subtype B and three of subtype F (Fisher's exact test, p = 0.009, 95% CI 2.45–29.5). Only five (6.8%) patients were recently infected, as defined by positivity on the BED test, and three of them had resistant virus (Fisher's exact test, p = 0.005, 95% CI 1.93–334.68).

Table 2.

Primary Resistance Mutations Found in Strains of HIV-1 in Ceará (2008–2009) ^a

Sample code	Subtype	NRTI mutations	NNRTI mutations	PI mutations
16CE081007	B	M41L, D67N, T215D		V32I, M46I, I54V, V82T, L90M
16CE081022R	B		K101E
16CE081050	B			M46I
16CE081058	F		K103N
16CE091075R	B	D67N, K219Q
16CE091083	F	M41L
16CE091070R	F			M46I

NRTI, nucleoside analog reverse transcriptase inhibitor; NNRTI, nonnucleoside analog reverse transcriptase inhibitor; PI, protease inhibitor.

No statistically significant differences in primary resistance were detected between genders, routes of transmission, residence area, prior residence in another city, city where transmission most probably occurred, time of diagnosis (more or less than 12 months), need for immediate treatment, or the presence of overt AIDS.

Discussion

The prevalence of HIV infection in the state of Ceará is 0.6%,¹ and 74.6% of the HIV-infected individuals live in the capital city (Fortaleza) and metropolitan area. The ratio of HIV-infected men/women for the state of Ceará is 2:1, which is similar to the 3:1 ratio found in this study. However, the percentage of individuals younger than 18 years in the present study (14.9%) differs from the statewide frequency of HIV infection for this age group, which is approximately 4.8%.¹³

In Brazil, HIV resistance testing in the public health system is offered prior to treatment only to pregnant women and children, and this facilitated the enrollment of children in the study. The high proportion of individuals requiring therapy is in keeping with previously published results.¹⁴ The 9.5% rate of HIV primary drug resistance found in this study is higher than that obtained in a recently conducted national study, which found less than 5% for the same geographic region, but another sampling method was used.²

When samples from the five geographic regions of the country were tested in 2001, the frequency of HIV primary resistance in the Northeast region was 4.8%, whereas in the country as a whole it was 5.3%.³ In another study done with samples collected mostly in the South and Southeast regions, following the resistance guidelines established by the International AIDS Society, United States, primary resistance was rated at 7%,¹⁵ similar to the 7.7% found in Argentina among patients recently infected.¹⁶ In other countries in South America HIV resistance rates were 3.3% in Peru,¹⁷ 5.2% in Cuba,¹⁸ and 8.1% in Venezuela.¹⁹

Although only five patients in this study were identified as recently infected by the BED test, three of them had viruses with mutations for resistance, which was found to be statistically significant within the constraints of a small sample size. Similar frequencies of primary resistance in recently infected patients have been found in other studies.^20,21 The overall frequency of 9.5% of HIV primary drug resistance may indicate that it is cost effective to carry on resistance testing before starting antiretroviral therapy, as previously suggested by others.^22,23

The distribution of resistance was similar within the three antiretroviral classes. Interestingly, the M46I mutation was present in HIV from all three individuals harboring virus with PI mutations, possibly reflecting long-term exposure of the HIV population to indinavir, the first available PI in the country. The mutations found in the PR and RT regions were in agreement with the most recent findings from Brazil,³ except for one sample that was resistant to two classes of antiretrovirals. The M184V mutation commonly found in secondary resistance was not detected in this study, perhaps revealing its tendency to disappear in the absence of the selective pressure of antiretrovirals, in contrast to what is seen with other primary resistance mutations.²⁴

The prevalence of subtype B (85.1%) in this study is similar to what has been previously determined in Ceará, whereas the prevalence of subtype F seems to be a little higher than previously determined.^25,26 Somewhat surprisingly for Brazil, the prevalence of primary resistance was higher with HIV subtype F as compared to B, but this finding should be viewed with caution until confirmed by larger studies. Moreover, the genome sequencing was limited to the RT and PR regions, and that would not allow the detection of recombinant forms between HIV subtypes.

We acknowledge that patient selection not using a population-based model is a limitation of the present study. In addition, the percentage of children was higher than expected according to the HIV epidemic profile for the region, and this may also reflect some selection bias. However, the patients studied represent approximately 10% of all new instances of HIV infection in which the patients were started on antiretroviral therapy in the state of Ceará in the study period, what indicates that the data are representative of the region. In addition, this study indicates for the first time that the frequency of primary drug resistance in HIV in the state of Ceará is higher than expected.

In conclusion, significant levels of primary HIV resistance occur in the State of Ceará, a place geographically distant from the more highly populated and wealthier areas of Brazil. This emphasizes the need for dedicated monitoring of the prevalence of primary resistance in this area and the need for an analysis of cost effectiveness when considering the adoption of resistance testing before the initiation of antiretroviral therapy. Relationships between different HIV subtypes and primary resistance need to be carefully evaluated in further studies.

Footnotes

Acknowledgment

The authors would like to thank Lilian Amaral Inoscêcio, from Unidade de Laboratório do Departamento Nacional de DST/Aids e Hepatites Virais do Ministério da Saúde, for supporting this study by providing the reagents for genotyping.

Author Disclosure Statement

No competing financial interests exist.

References

Brasil. Ministério da Saúde, Secretaria de Vigilância em Saúde, Programa Nacional de DST e Aids. Boletim Epidemiológico, Aids e DST: Ano V, no. 1, 27^a– 52^a, semanas epidemiológicas, julho a dezembro de 2007 e Ano V, no. 1, 01^a– 26^a, semanas epidemiológicas, janeiro a junho de 2008. Ministério da Saúde, Brasília, 2008. http://www.aids.gov.br/data/documents/storedDocuments/%7BB8EF5DAF-23AE-4891-AD36-1903553A3174%7D/%7B31A56BC6-307D-4C88-922D-6F52338D0BF4%7D/Boletim2008_vers%E3o1_6.pdf. 2010 July 16.

Brindeiro

, Diaz

, Sabino

, Morgado

, Pires

, Brigido

et al. Brazilian Network for HIV Drug Resistance Surveillance (HIV-BResNet): A survey of chronically infected individuals. AIDS, 2003; 17:1063–1069.

Inocencio

, Pereira

, Sucupira

MAC

, Fernandez

JCC

, Jorge

, Souza

DFC

et al. Brazilian Network for HIV Drug Resistance Surveillance: A survey of individuals recently diagnosed with HIV. J Int AIDS Soc, 2009; 12:20.

Sucupira

MCA

, Caseiro

, Alves

, Tescarollo , Janini

, Sabino

et al. High levels of primary antiretroviral resistance genotypic mutations and B/F recombinants in Santos, Brazil. AIDS Patient Care STDs, 2007; 21,2:116–128.

Bello

, Guimaraes

, Morgado

. Evolutionary history of HIV-1 subtype B and F infections in Brazil. AIDS, 2006; 20:763–768.

Pedroso

, Queiroz

ATL

, Alcântara

, Drexler

, Diaz

, Wey

et al. High prevalence of primary antiretroviral resistance among HIV-1-infected adults and children in Bahia, a Northeast State of Brazil. J Acquir Immune Defic Syndr, 2007; 45,2:251–252.

QIAamp Viral RNA Mini Handbook 2nd December 2005. Quiagen, Inc.: Duesseldorf, Germany.

Instruções de Uso. Trugene HIV-1 Genotyping Kit: Para uso com o Opengene DNA Sequencing System. Rev. C., October 2004. Siemens Medical Solutions Diagnostics: Berkeley, CA.

Sucupira

, Souza

, Costa

, Scheinberg

, Diaz

. Antiretroviral treatment failure and HIV-1 genotypic resistance in Sao Paulo, Brazil. Antivir Ther, 2001; 6,4:263–264.

10.

Bennett

, Camacho

, Otelea

, Kuritzkes

, Fleury

, Kiuc

et al. Drug resistance mutations for surveillance of transmitted HIV-1 drug-resistance: 2009 update. PLoS ONE, 2009; 4,3:e4724, 10.1371/journal.pone.

11.

CDC. 1993. revised classification system for HIV infection and expanded surveillance case definition for AIDS among adolescents and adults. MMWR, 1992; 41,RR-17.

12.

Brasil. Ministério da Saúde. Secretaria de Vigilância em Saúde: Programa Nacional de DST e Aids. Critérios de Definição de Casos de Aids em Adultos e Crianças. PN-DST/Aids. Ministério da saúde, Brasília, 2003. http://www.aids.gov.br/data/documents/storedDocuments/%7BB8EF5DAF-23AE-4891-AD36-1903553A3174%7D/%7B6C99F8D6-CC7B-4D09-BECD-1AA2C7E7B587%7D/criterios_aids_2004.pdf. 2010 July 16.

13.

Ceará. Secretaria da Saúde: Coordenadoria de Promoção e Proteção à Saúde. Núcleo de Epidemiologia. Informe Epidemiológico, AIDS. Maio. Fortaleza, 2010. http://www.saude.ce.gov.br/site/index.php?option=com_phocadownload&view=category&id=9:boletins&Itemid=247. 2010 July 16.

14.

Medeiros

, Diaz

, Castelo

. Estimating the length of the first antiretroviral therapy regimen durability in Sao Paulo, Brazil. Braz J Infect Dis, 2002; 6,6:298–304.

15.

Sprinz

, Netto

, Lima

MPJS

, Furtado

, Eira

, Zajdenverg

et al. Primary antiretroviral drug resistance among HIV type 1-infected individuals in Brazil. Aids Res Hum Retroviruses, 2009; 25,9:861–867.

16.

Petroni

, Deluchi

, Pryluka

, Rotryng

, Bortolozzi

, Lopardo

et al. Update on primary HIV-1 resistance in Argentina: Emergence of mutations conferring high-level resistance to nucleoside reverse transcriptase inhibitors in drug-naïve patients. J Acquir Immune Defic Syndr, 2006; 42:506–510.

17.

Lama

, Sanchez

, Suarez

, Caballero

, Laguna

, Sanchez

et al. Linking HIV and antiretroviral drug resistance surveilance in Peru: A model for a third generation sentinel surveillance. J Acquir Immune Defic Syndr, 2006; 42:501–505.

18.

Pérez

, Álvarez

, Carmona

, Aragonés

, Delgado

, Thomson

et al. Genotypic resistance to antiretroviral drugs in patients infected with several HIV type 1 genetic forms in Cuba. Aids Res Human Retroviruses, 2007; 23,3:407–414.

19.

Castillo

, Comegna

, Quijada

, Jauvin

, Pinson

, Masquelier

et al. Surveillance of HIV type 1 drug resistance among naive patients from Venezuela. Aids Res Human Retroviruses, 2009; 25,12:1329–1333.

20.

Viani

, Peralta

, Aldrovandi

, Kapogiannis

, Mitchell

, Spector

et al. Prevalence of primary HIV-1 drug resistance among recently infected adolescents: A Multicenter Adolescent Medicine Trials Network for HIV/Aids Interventions Study. J Infect Dis, 2006; 194,11:1505–1509.

21.

Booth

, Garcia-Diaz

, Youle

, Johnson

, Phillips

, Geretti

. Prevalence and predictors of drug resistance in newly diagnosed HIV-1 infection. J Antimicrob Chemother, 2007; 59,3:517–524.

22.

Hecht

, Grant

. Resistance testing in drug-naïve HIV-infected patients: Is it time? Clin Infect Dis, 2005; 41:1324–1325.

23.

Sax

, Islam

, Wanlesky

, Losina

, Weinstein

, Goldie

et al. Should resistance testing be performed for treatment-naive HIV-infected patients? A cost-effectiveness analysis. Clin Infect Dis, 2005; 41:1316–1323.

24.

Jain

, Sucupira

, Deeks

, Liegler

, Pilcher

, Grant

et al. Persistence of transmitted drug-resistance mutations in patients with acute HIV. 16th Conference on Retroviruses and Opportunistic Infections, February February 8 11, 2009, Montreal, CanadaPoster #672.

25.

Gadelha

, Shindo

, Cruz

JNM

, Morgado

, Galvão-Castro

. Molecular epidemiology of human immunodeficiency vírus-1 in the State of Ceará, Northeast, Brazil. Mem Inst Oswaldo Cruz, 2003; 98,4:461–464.

26.

Cavalcanti

AMS

, Lacerda

, Brito

, Pereira

, Medeiros

, Oliveira

. Antiretroviral resistance in individuals presenting therapeutic failure and subtypes of the human immunodeficiency virus type 1 in the Northeast region of Brazil. Mem Inst Oswaldo Cruz, 2007; 102,7:785–792.