Moderate Prevalence of Transmitted Drug Resistance Mutations Among Antiretroviral-Naive HIV-Infected Pregnant Women in Rio de Janeiro,Brazil

Abstract

Transmission of drug-resistant HIV-1 strains has been gaining attention and is becoming a growing problem throughout the world. The aim of this study was to determine the prevalence of transmitted drug resistance mutations (TDRM) among antiretroviral (ARV)-naive HIV-infected pregnant women in Rio de Janeiro, Brazil. ARV-naive pregnant women were recruited at Hospital Geral de Nova Iguacu (HGNI), Rio de Janeiro, from 2005 to 2008. HIV genotyping was carried out using ViroSeq (Abbott v. 2.0). TDRM were detected using the Calibrated Population Resistance Tool-CPR v. 6.0.The prevalence of mutations associated with resistance in the protease and reverse transcriptase regions of the HIV genome were assessed in samples collected prior to initiation of ARV prophylaxis or treatment. Among 238 eligible specimens that were collected, 197 samples were successfully amplified using reverse transcription polymerase chain reaction. Eighty-one percent of women were infected with HIV subtype B, 10% with subtype F1 viruses, 1.0% with subtype C virus, and 8.0% with recombinant forms of the virus. The prevalence of HIV TDRM was 5.6% for nucleoside reverse transcriptase inhibitors, 2.0% for nonnucleoside reverse transcriptase inhibitors, and 3.0% for protease inhibitors. The overall prevalence of any drug resistance was 10.7%. There were no multiclass resistant strains identified in the analyzed samples. The prevalence of HIV TDRM among the pregnant women in our cohort was moderate. Resistance testing should be encouraged in Rio de Janeiro, among other locations, for all HIV-infected pregnant women prior to prevention of mother-to-child transmission of HIV.

Introduction

B razil was the first developing country to implement a program that provided universal access to antiretroviral drugs (ARV). Currently, approximately 200,000 patients with HIV/AIDS receive ARV through the Public Health System.¹ The effectiveness of antiretroviral therapy (ART) in this population can be highlighted by the positive impact on survival rates,^2
–4 opportunistic infections rates, and hospitalizations rates.⁵ With widespread and growing access to ART in Brazil, the selection of variants that are resistant to these drugs has been increasing. Thus, transmission of mutant viruses that confer transmitted drug resistance (TDR) has been increasing, and people infected by these viruses may have limited options for future treatment, despite never having received antiretroviral drugs.^6,7

Three million women give birth in Brazil annually. A national surveillance study conducted in 2006 to evaluate the prevalence of HIV infection among pregnant women estimated that 13,000 women are diagnosed with HIV infection each year at the time of delivery.⁶ Universal free access to highly active antiretroviral therapy (HAART) has been available in Brazil for HIV/AIDS treatment since 1996. As the use of ARV drugs for prevention of mother-to-child transmission of HIV (PMTCT) and treatment increases globally, resistance to ARVs will likely become more common.

The prevalence of transmitted drug resistance mutations (TDRM) has increased during the past decade in pregnant women in the United States⁸ and in other countries where ART is widely available.^9,10 However, the prevalence of HIV TDRM in drug-naive pregnant women in Brazil still needs to be better characterized. The aim of this study was to evaluate the prevalence of HIV TDRM in a cohort of ARV-naive HIV-infected pregnant women in Rio de Janeiro, Brazil.

Materials and Methods

Study population

The HIV Family Care Clinic (HHFCC) operates within Hospital Geral de Nova Iguaçu (HGNI), the largest public hospital in an impoverished area adjacent to Rio de Janeiro, Brazil. HHFCC is the main referral center for HIV-infected patients and provides free ARV and services for PMTCT of HIV. A prospective cohort of HIV-infected pregnant women was followed at HHFCC since May 2005. Women were eligible for inclusion in the study if they were pregnant and had a confirmed positive HIV test. Inclusion for the present analysis was limited to the first pregnancy of ARV-naive women and who delivered up to April 2008. Women who had previously received PMTCT were excluded from this cohort.

For the present analysis, patients had their virological and immunological parameters (CD4⁺ T cell, HIV RNA, and genotyping) assessed at the baseline visit, before ARV initiation.

Based on the inclusion criteria, 197 of the original 238 women included in the cohort could be analyzed in terms of the subtype and genotype. Patients who did not have baseline samples available, those who had undetectable HIV RNA levels, and those whose baseline samples could not be amplified and sequenced were excluded from the analysis. The local Institutional Review Board approved this study and informed consent was obtained from all participants prior to study participation.

Sample processing, resistance, and phylogenetic analysis

Whole blood samples were collected in ethylenediaminetetraacetic acid (EDTA). Plasma samples were used for population-based nucleotide sequencing of the entire protease (PR) gene and codons 1–335 of reverse transcriptase (RT) gene using the ViroSeq HIV Genotyping System (version 2.0; Celera Diagnostics, Alameda, CA). The purified PCR products were sequenced with an ABI PRISM 3100 genetic analyzer (Applied Biosystems, Foster City, CA). To determine HIV susceptibility to the various antiretroviral drugs, HIV TDRM were analyzed using the HIVDB sequence analysis program from the Stanford University HIV drug resistance database (available at http://hivdb.stanford.edu/pages/algs/sierra_sequence.html). The Calibrated Population Resistance Tool (CPR) was used to analyze the RT and PR gene sequences (version 6.0 beta; available at http://cpr.stanford.edu/cpr.cgi). TDR was defined as the presence of at least one major mutation from the Surveillance Drug Resistance Mutation (SDRM—2009) list proposed by Gifford et al.¹¹

Subtypes were first screened using the Rega HIV Subtyping Tool (version 2.0; available at http://dbpartners.stanford.edu/RegaSubtyping/) and further confirmed based on phylogenetic tree inferences as implemented in MEGA 5.01.¹² The PR-RT sequences (1,302 bp) were aligned with subtype reference from the Los Alamos HIV Database (www.hiv.lanl.gov). Phylogenetic analyses were performed by the neighbor-joining (NJ) algorithm using the Tamura-Nei nucleotide substitution model, in 100 bootstrap replicate. Recombination profiles were confirmed by bootscanning analysis (sliding window of 350 bp, incremental steps of 10 bases, and the Kimura two-parameter model) using SimPlot 3.5.1 software.¹³ CD4⁺ T cell counts were evaluated using flow cytometry (Trucount; FacScalibur; BD Biosciences Immunocytometry Systems, San Jose, CA). Plasma HIV RNA levels were determined by nucleic acid sequence-based amplification (NASBA QR System Organon Teknika. Durham, NC) and the VERSANT(R) HIV bDNA assay (Bayer 3.0 Quantitative Assay, Berkeley, CA).

Statistical analysis

The primary outcome of interest was the presence of TDRM in the baseline samples of included patients. The prevalence of TDRM was calculated based on the number of women whose baseline sequences had TDRM and based on the total number of women who provided baseline samples that were successfully amplified and sequenced. Data analysis was carried out using the R statistical package (v. 1.9.1), distributed by the “Comprehensive R Archive Network” (CRAN) and available at http://cran.r-project.org.

Results

Characterization of the study population

Between May 2005 and April 2008, 238 HIV-positive ARV-naive pregnant women were included in the cohort. The median age was 26.0 years (IQR, 21.2–29.4) and 71.9% were nonwhite. Around two-thirds of the women reported no more than 8 years of formal education. More than half of the study participants declared that they had either no income or a monthly income equal to or less than the Brazilian minimum wage/month. Cigarette smoking during pregnancy was reported by 22.3% of women. Table 1 summarizes baseline characteristics.

Table 1.

Sociodemographics and Behavioral Data from Pregnant Women Followed at HGNI from May 2005 to April 2008 (N=238)

Baseline characteristics ^a	N	%
Maternal age (N=238)
<20	33	13.7
20–34	188	79.1
≥35	17	7.2
Race (N=238)
White	67	28.1
Nonwhite	171	71.9
Schooling (N=238)
Up to 4 years	60	25.2
5–7 years	79	33.1
More than 8 years	99	41.7
Family income (MS)^b (N=238)
No Income	49	20.7
Up to 2 MS	152	63.8
>2 MS	37	15.5
Married (N=238)
Yes	187	78.7
No	51	21.3
Smoking (current gestation) (N=234)
Yes	56	22.34
No	178	76.06
Without information	4	1.60
Alcohol (current gestation) (N=233)
Yes	51	22.1
No	182	77.9
Without information	5	2.30
Drug use (current gestation) (N=232)
Yes	25	7.98
No	207	89.36
Without information	6	2.66

Numbers vary because of missing information.

Brazilian minimum wage±US$ 200.

The median gestational age at the time of initiation of prenatal care was 25.0 weeks (IQR, 20.0–30.0). About 70% of women were diagnosed with HIV during the current pregnancy. The median pre-HAART CD4⁺ T cell count and HIV RNA at baseline visit were 517 cells/mm³ (IQR, 379–629) and 3.84 log₁₀ copies (IQR, 3.0–4.4 log₁₀ copies), respectively.

HIV-1 subtypes

The majority of the patients in this study (159/197, 81%) were infected with a subtype B virus, followed in frequency by F at 10% (19/197), C at 1.0% (02/197), and recombinant forms at 8.0% (17/197). Recombinant forms were found as follows: URF_BF (13/197) was present in 6.0% of patients and URF_BC (2/197) in 1%. Two samples (1%) had unique recombinant genomes between subtypes BK and FJK, which will be the object of further full-length sequence analyses to confirm the occurrence of such complex K-like recombinant genomes in the Brazilian AIDS epidemics. Together it is important to point out the occurrence of unique recombinant forms (URF) in 8% of the pregnant women tested in this study.

HIV subtyping and genotyping were not available for 24 women because their HIV RNA levels were below the limit of detection in all study visits. Subtype information was also not available for an additional 12 women because although their HIV RNA levels were above the lower limit of detection, amplification and genotyping were not possible on their blood samples. Five women were excluded from the analysis because there were no baseline samples available.

Prevalence and patterns of resistance

Genotyping was obtained for 82.7% (197/238) of the baseline samples collected in our cohort. Overall, TDRM were detected in 10.7% (21/197) of the women in the study. Table 2 summarizes the prevalence of TDR over time, which remained stable at an intermediate level.

Table 2.

Prevalence of Transmitted Drug Resistance Mutations Among Pregnant Women Followed at HGNI from May 2005 to April 2008

Resistance category	TDR ^a (2005/2006) N=60 (%)	TDR ^b (2006/2007) N=55 (%)	TDR ^c (2007/2008) N=82 (%)	TDR (2005/2008) N=197 (%)
Sequences with any mutation	8 (13.3)	6 (10.9)	7 (8.5)	21 (10.7)
PR sequences with any PI mutation	2 (3.3)	0 (0.0)	4 (4.9)	6 (3.2)
RT sequences with any NRTI mutation	5 (8.3)	3 (5.5)	3 (3.7)	11 (5.6)
RT sequences with any NNRTI mutation	1 (1.7)	3 (5.5)	0 (0.0)	4 (2.0)

May 1, 2005 to April 30, 2006.

May 1, 2006 to April 30, 2007.

May 1, 2007 to April 30, 2008.

TDR, transmitted drug resistance; PR, protease; PI, protease inhibitor; NRTI, nucleoside reverse transcriptase inhibitor; RT, reverse transcriptase; NNRTI, nonnucleoside reverse transcriptase inhibitor.

RT sequences with any nucleoside reverse transcriptase inhibitor (NRTI) mutation present

TDRM for NRTIs were detected in 5.6% (11/197) of the women in the study. A total of 1.0% of these women (2/197) had the M41L mutation, while the D67N mutation was found in 0.5% of women (1/197). Additionally, 0.5% (1/197) of the women had the M184V mutation. The K219R, T215S, T215I, and L74IL mutations were detected in 1.5% (3/197), 1.0% (2/197), 0.5% (1/197), and 0.5% (1/197) of samples, respectively.

RT Sequences with any nonnucleoside reverse transcriptase inhibitor (NNRTI) mutation present

TDRM to NNRTIs were detected in 2.0% (4/197) of the women in the study. A total of 1.5% (3/197) of women had the K103N mutation and in addition to the K103N, the Y181C mutation was also detected in one of these pregnant women, and another woman (0.5%) had the K101E mutation.

PR sequences with any protease inhibitor (PI) mutation present

Six (3.0%) women had PI mutations. Of these, four had the M46L mutation, one had the M46I mutation, and another woman had the I85V mutation. Table 3 summarizes PI, NRTI, and NNRTI transmitted drug resistance mutations according to subtype. Almost all women with TDRM were infected with subtype B viruses (20 subtypes B and 1 BF). We did not detect any sequences with both NRTI and NNRTI mutations or any triple class resistance. The resistance pattern found among these ARV-naive women in our study, most of whom had a single resistance mutation, differed from patterns seen in patients who fail to respond to a treatment regimen; these patients generally have multiple resistance mutations.

Table 3.

Protease Inhibitor, Nucleoside Reverse Transcriptase Inhibitor, and Nonnucleoside Reverse Transcriptase Inhibitor Transmitted Drug Resistance Mutations According to Subtypes

Sequence ID	Subtype	PI mutations	NRTI mutations	NNRTI mutations
PG01.0	B	No DRM	M41L	No DRM
PG13.0	B	No DRM	K219R	No DRM
PG36.0	BF	No DRM	D67DN	No DRM
PG40.0	B	I85IV	No DRM	No DRM
PG41.0	B	No DRM	T215S	No DRM
PG57.0	B	No DRM	No DRM	K101EK
PG58.0	B	M46LM	No DRM	No DRM
PG65.0	B	No DRM	T215S	No DRM
PG69.0	B	No DRM	T215IT	No DRM
PG107.0	B	No DRM	K219KR	No DRM
PG110.0	B	No DRM	No DRM	K103N
PG117.0	B	No DRM	No DRM	K103N, Y181C
PG125.0	B	No DRM	M184V	No DRM
PG137.0	B	No DRM	No DRM	K103KN
PG138.0	B	M46L	No DRM	No DRM
PG148.0	B	M46L	No DRM	No DRM
PG149.0	B	No DRM	M41L	No DRM
PG156.0	B	No DRM	K219KR	No DRM
PG169.0	B	M46L	No DRM	No DRM
PG188.0	B	No DRM	L74IL	No DRM
PG268.0	B	M46I	No DRM	No DRM

DRM, drug resistance mutation.

Discussion

Antiretroviral drug resistance is an important cause of treatment failure in the setting of HIV infection. In addition to resistance mutations caused by poor adherence to treatment or inadequate treatment, the transmission of drug-resistant viral strains has been gaining attention and is becoming a growing problem throughout the world. Drug resistance in the setting of PMTCT has been studied most extensively in the setting of single dose nevirapine, given that a single mutation can lead to high level resistance to this drug, as well as to other NNRTIs.¹⁴

Documentation of TDR in ART-naive individuals infected with HIV has been increasing in frequency over the past 5 years.^15,16 Publications from different geographic regions show that the prevalence of resistant virus ranges from 3.3% to 24.1%,^{8, 9,17

–21} depending on the population and region.

The current recommendations of the Brazilian National STD/AIDS Program (MOH) for the treatment of HIV infection do not include screening for resistance with genotyping for individuals that have been recently diagnosed with HIV, who need to start their first antiretroviral regimen, or ART-naive pregnant women. However, guidelines related to this matter published by the American and European groups recommend genotypic resistance testing for all individuals with acute or recent HIV infection. The European HIV Drug Resistance Panel suggests that resistance testing should be strongly considered if suspicion of resistance is high or prevalence of resistance in this population exceeds 10%.²²

A more recent update extended the range of genotyping resistance testing to include all ART-naive individuals with HIV infection, including pregnant women.^23

–26 A study published by Sax and colleagues showed that genotypic testing is cost-effective for ART-naive patients when the prevalence of TDRM in the population is ≥1%.²⁷ In our study, we found a 10.7% (21/197) overall prevalence of TDR among pregnant ARV-naive women from Rio de Janeiro starting HAART for PMTCT. The overall prevalence of TDR remained stable at an intermediate level, but trends of resistance against drug classes differed over time (Table 2). The decrease of NRTI resistance in patients infected with HIV might be related to the introduction of HAART and novel antiretroviral drugs. This prevalence, even though considered of intermediate level by WHO, is higher than that previously reported in other national studies for pregnant women,^28,29 and is also higher than in other ART-naive populations depicting TDR rates up to 8.3% (Brindeiro et al. (6.6%) in 2003, Gonsalez et al. (6.5%) in 2007, Inocencio et al. (8,3%) in 2009, Sprinz et al. (7.0%) in 2009, and Ferreira et al.(5.4%) in 2011).^6,30

–34 The prevalence of TDR in our study was not, however, higher than that described by Sucupira et al. (36.8%) in a study conducted in Santos, a region with a high prevalence of intravenous drug users.³⁵ TDR rates are closely associated with the area that is being surveyed, the local practices regarding ART use, and the group that is being studied. These factors make it very difficult to conduct comparisons between studies.

In a prospective cohort study conducted in Argentina, the Bahamas, Brazil, and Mexico (NISDI Perinatal Study), TDRM occurred among 16% of HIV-1-infected mothers from Latin American and Caribbean countries receiving MTCT prophylaxis³⁶; however, most of the pregnant women included in this analysis were ART-exposed. Some other international studies with pregnant women showed lower a prevalence of TDR, ranging from 1% to 7%.^37

–40

The prevalence of TDRM in our study is also in accordance with the results from multiple international studies focusing on ART-naive individuals; a large prospective study conducted in 17 European countries, which found an overall prevalence of 9.1%¹⁸; other European cohorts^9,17,21; and the SPREAD study cohort (which was comprised of patients from multiple European countries and reported a prevalence of 9.1%).²⁰

Our study describes the prevalence of drug resistance in newly diagnosed pregnant women, a population that is screened for HIV infection routinely. In our analysis, we used the list of mutations proposed by Gifford et al.¹¹ as markers of HIV TDRM. The 10.7% overall prevalence of TDRM found is significantly greater than 5% (p<0.001), the limit at which the WHO has suggested that it becomes cost effective⁴¹ to perform genotypic resistance testing in individuals newly diagnosed with HIV infection.

Mutations to NRTI were the most prevalent in our population of pregnant women. This may be a reflection of the use of NRTI monotherapy (zidovudine and d4T) and NRTI dual therapy before the HAART era in Brazil. The low impact that analog mutations (TAMs) and codon 215 mutations have on the replicative capacity of the virus in ART-naive patients allows these genetic changes to remain for a long period of time in untreated patients, thereby increasing the likelihood of transmission of the resistant virus.⁴² In our study six individuals had either TAMs or revertant mutations in codon 215 and they did not coexist with other mutations. About one out of 10 ARV-naive pregnant women newly diagnosed with HIV infection had resistance mutations in her pretreatment blood sample. The use of molecular approaches to detect minor populations with resistance mutations to ARV in future studies will help to better define the prevalence of TDR in this population.

When we applied the Stanford algorithm to the sequences of the samples that had TDR for evaluation of viruses with decreased sensitivity to the currently available ARVs, we observed that 50% (10/21) of the pregnant women with TDRM in their initial pre-HAART sample did demonstrate reduced sensitivity to ARV combinations containing either two NRTIs plus an NNRTI or two NRTIs plus a PI, which are the first-line treatment regimens proposed by the Ministry of Health,¹ and could impair the response to the first line ART and subsequent virologic failure.

We have to acknowledge that the frequency of TDRM detected in this study population may be an overestimate since we were not able to amplify DNA samples from all women included in the study.

Conclusions

In settings in which the prevalence of TDRM is moderate to high, it may be advisable to screen patients for resistance prior to initiation of ART to reduce the chances that drug resistance will impair the response to first-line ART. Moreover, TDRM persist over years,^43,44 and individuals who have not yet been treated may be infected with drug-resistant viruses through potential transmittal.⁴⁵

Our study provides evidence to support the idea that genotypic resistance testing should be routinely considered in Brazil for all HIV-infected pregnant women to optimize the success of chemoprophylaxis, prevent the development of further resistance mutations, and prevent transmission of resistant viruses to other individuals. Continued surveillance of TDR in this population of women is warranted in Brazil to explore the patterns and factors contributing to the transmission of TDRM.

Footnotes

Acknowledgments

We thank the laboratory and clinical staff from the Hospital Geral de Nova Iguaçu, Rio de Janeiro, and the laboratory team from the AIDS and Molecular Immunology Laboratory/Instituto Oswaldo Cruz/FIOCRUZ, Rio de Janeiro, Brazil, for their hard work processing and analyzing the blood samples.

Financial support was provided by the Brazilian Ministry of Health (BMOH).

Author Disclosure Statement

J.C. has received research grant support for UCLA for clinical trials from Merck and Company and Tibotec Therapeutics in the past 48 months.

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