High HIV-1 Diversity and Prevalence of Transmitted Drug Resistance Among Antiretroviral-Naive HIV-Infected Pregnant Women from Rio de Janeiro,Brazil

Abstract

Antiretroviral (ARV) resistance mutations in human immunodeficiency virus type 1 (HIV-1) infection may reduce the efficacy of prophylactic therapy to prevent mother-to-child transmission (PMTCT) and future treatment options. This study evaluated the diversity and the prevalence of transmitted drug resistance (TDR) in protease (PR) and reverse transcriptase (RT) regions of HIV-1 pol gene among 87 ARV-naive HIV-1-infected pregnant women from Rio de Janeiro, Brazil, between 2012 and 2015. The viral diversity comprised HIV-1 subtypes B (67.8%), F1 (17.2%), and C (4.6%); the circulating recombinant forms 12_BF (2.3%), 28/29_BF, 39_BF, 02_AG (1.1% each) and unique recombinants forms (4.5%). The overall prevalence of any TDR was 17.2%, of which 5.7% for nucleoside RT inhibitors, 5.7% for non-nucleoside RT inhibitors, and 8% for PR inhibitors. The TDR prevalence found in this population may affect the virological outcome of the standard PMTCT ARV-regimens, reinforcing the importance of continuous monitoring.

Currently, about 36 million people are living with the human immunodeficiency virus type 1 (HIV-1), the causative agent of acquired immune deficiency syndrome (AIDS).¹ According to estimates of the Brazilian Ministry of Health, ∼734,000 people are HIV infected in Brazil, which corresponds to a prevalence of 0.4%.² Regarding pregnant woman, it is expected that 12,000 cases of HIV infection occur annually in Brazil, however, only 3,700 were reported to the public health system in 2014, of which 8% corresponds to the Rio de Janeiro state.² The HIV-1 epidemic in this state is highly concentrated in its metropolitan region, which is responsible by nearly 90% of the reported HIV infections.²

There is a historic commitment in providing universal free access to highly active antiretroviral therapy for HIV/AIDS treatment in Brazil, including its use for prevention of HIV mother-to-child transmission (PMTCT). Results from the SMART study,³ coupled with strong evidence demonstrating a reduction in the risk of HIV transmission during the use of antiretroviral therapy (ART)⁴ and the clinical benefit from ART among nonpregnant women and men with higher CD4 + T-cell counts,⁵ have led to the evolution of strategies for PMTCT of HIV, and since 2013, the Brazilian Public Health System recommended the immediate start of ART to all HIV-infected individuals, regardless CD4+ T-cell count.⁶ This expanded access to ART and the availability of new antiretroviral (ARV) drugs may lead to a strong impact in morbidity and mortality reduction of HIV-1-infected individuals over time. However, these new treatment guidelines could also favor the emergence of HIV-1 drug-resistant variants due to poor adherence to treatment regimens, combined with the lower genetic barrier of some drugs among other factors, and hence, a large pool of resistant virus might become available to establish new infections. These HIV-1 variants carrying transmitted antiretroviral drug resistance (TDR) mutations may limit availability to future therapeutic options for newly infected people, especially pregnant women, in whom PMTCT of HIV aims to achieve rapid reduction of HIV-1 viral load, with goals to reach levels of HIV-1 RNA below the lower limits of detection. Previous studies conducted with pregnant HIV-infected women from Rio de Janeiro have pointed out a moderate rate (∼10%) of TDR and an epidemic dominated by subtypes B (∼80%), F1 (∼10%), and C (∼2%), as also recombinants between them.^7,8

In this study, we evaluated the viral diversity and the prevalence of TDR among 87 HIV-1-infected, primo-pregnant and ARV-naive women followed at the Hospital Geral de Nova Iguaçu in Rio de Janeiro, Brazil, between January 2012 and June 2015, and compared the results with a similar previous study (2005–2008) conducted by our group at the same location.⁷ This study was approved by the Ethics Committee of the Oswaldo Cruz Foundation/Oswaldo Cruz Institute (Number CAAE: 0067.0.011.316-11), and all women have signed an informed consent form.

CD4+ T-cell counts were evaluated using flow cytometry (Trucount; FACSCalibur™; BD Biosciences). Plasmatic HIV-1 RNA levels were determined by the VERSANT^® HIV bDNA (Bayer) and by the Abbott RealTime HIV-1 (Abbott) assays. Viral RNA was extracted from plasma samples using the QIAamp Viral RNA Mini Kit (QIAGEN). The cDNA synthesis input was 10 μl of purified viral RNA in a 20 μl final volume reagent mix containing 1 × First-Strand Buffer, 10 mM of DTT, 40 U of RNaseOUT™ (Life Technologies), 0.5 mM of dNTP mix, 0.1 mM of MMRT6 primer, and 200 U of SuperScript^® III RT enzyme (Life Technologies). Samples were incubated at 37°C/90 min followed by 70°C/15 min for enzyme denaturation.

The protease and reverse transcriptase (PR/RT) fragment of the HIV-1 pol gene was amplified by nested polymerase chain reaction, carried out using 5 μl input template, 1.5 U of Platinum^® Taq DNA Polymerase (Invitrogen, EUA), 1 × PCR Buffer, 2.5 mM of MgCl₂, 0.3 mM of dNTP mix (Invitrogen), and 0.35 μM of each specific primer (forward and reverse) in a 50 μl final volume solution. Supplementary Table S1 (Supplementary Data are available online at www.liebertpub.com/aid) describes the primers used in this study. PCR conditions were one cycle of 94°C/2 min and 35 cycles of 94°C/30 s, 55°C/30 s, 72°C/2 min.

The final PCR products were purified using the Illustra GFX PCR DNA Purification Kit (GE Healthcare) and sequenced using the ABI BigDye Terminator v.3.1 reaction kit (Applied Biosystems). For each sample, eight primers (Table 1) were used to sequencing the entire PR and the first 335 codons of RT (HXB2: 2253–3554), ensuring at least two times coverage by nucleotide position. Chromatograms were assembled and the presence of ambiguous sites was carefully inspected using the SeqMan v7 software, Lasergene package (DNAStar). This protocol is validated by the Virology Quality assessment (VQA) from Rush University, EUA, and is periodically tested for quality control assurance.

Table 1.

PI, NRTI, and NNRTI TDR Mutations Found According to Subtypes

		Mutations			Resistance profile
Sequence ID	Subtype	PI	NRTI	NNRTI	Low	Intermediate	High
GN02	B	M46I	—	—	NFV	—	—
GN03	F1	—	D67N	—	AZT/d4T	—	—
GN12	B	M46L	—	—	NFV	—	—
GN13	B	—	D67N, K219Q	—	AZT/d4T	—	—
GN14	B	M46L	—	—	NFV	—	—
GN16	F1	—	—	K101E, G190A	—	ETR/RPV	EFV/NVP
GN22	B	—	K70R	—	d4T	AZT	—
GN24	B	—	D67N, T69D	Y188L	AZT/d4T/ETR	ddI	EFV/NVP/RPV
GN26	B	M46L	M41L	—	NFV/AZT/d4T	—	—
GN31	B	M46L	—	—	NFV	—	—
GN44	F1	—	—	G190A	ETR/RPV	EFV	NVP
GN48	28/29_BF	D30N, N88D	—	—	—	—	NFV
GN68	B	M46L	—	—	NFV	—	—
GN70	B	—	—	K103N	—	—	EFV/NVP
GN72	B	—	—	K103N	—	—	EFV/NVP

AZT, zidovudine; d4T, stavudine; ddI, didanosine; EFV, efavirenz; ETR, etravirine; NFV, nelfinavir; NNRTI, non-nucleoside reverse transcriptase inhibitors; NRTI, nucleoside reverse transcriptase inhibitors; NVP, nevirapine; PI, protease inhibitor; RPV, rilpivirine; TDR, transmitted drug resistance.

HIV-1 genetic subtypes were determined by maximum likelihood (ML) phylogenetic and bootscaning analyses with HIV-1 reference sequences (www.hiv.lanl.gov). ML trees were reconstructed using the PhyML 3.0 program,⁹ approximate likelihood ratio test (aLRT) as measure of branch reliability and visualized using the FigTree v1.4.2 program (http://tree.bio.ed.ac.uk/software/figtree/). Bootscaning analyses were performed with Simplot 3.5.1 software,¹⁰ based on 100 bootstrap replicates of 250 nucleotides sliding windows moving in steps of 10 bases. TDR was analyzed using the Calibrated Population Resistance Tool v.6 (CPR; http://cpr.stanford.edu/cpr.cgi) from the Stanford University HIV Drug Resistance Database.¹¹ Descriptive and univariate (two-tailed Fisher's exact test) analyses were performed in GraphPad Prism v6 (GraphPad Software). Statistical significance was defined as an α ≤ 0.05.

All patients analyzed in this study lived in the metropolitan region of the Rio de Janeiro state, Brazil, at the time of analysis and have a median age of 25 years (16–36 years range). The median pre-ART CD4+ T-cell count was 413 cells/mm³ (IQR, 316–589). The median HIV-1 RNA plasma viral load for our study population was 4.04 log₁₀ copies/ml (IQR, 3.35–4.53), which was mostly concentrated (75.4%) between 3 and 5 log₁₀ copies/ml.

The HIV-1 subtype B was the most prevalent clade in our sampling (67.8%, 59/87), followed by subtype F1 (17.2%, 15/87) and subtype C (4.6%, 4/87). Intersubtype recombinant forms represented the third more prevalent HIV-1 group observed in this study (Fig. 1 A, B). Most circulating recombinant forms (CRF) found comprised subtypes B and F1 as follows: CRF12_BF (2.3%, 2/87), CRF28/29_BF (1.1%, 1/87), and CRF39_BF (1.1%, 1/87). One CRF02_AG (1.1%, 1/87) was also found, as well as two URF_BF (2.3%, 2/87), one URF_BC (1.1%, 1/87), and one URF_CF (1.1%, 1/87). The classification of one sequence as CRF28/29_BF was indicated because these two CRFs share a very similar pattern of recombination in PR/RT and formed a unique clade in the ML phylogenetic analysis (Fig. 1B), complicating the genetic distinction between them.

FIG. 1.

Subtype classification of sequences. (A) ML tree of the PR/RT region. HIV-1 reference sequences of pure subtypes (A–D, F–H, J, and K) were included and colored black. The branch support values (aLRT >0.90) are indicated as asterisks at key nodes. Each HIV-1 clade found in the Brazilian samples is colored as indicated in the figure. Horizontal branch lengths are drawn to scale with the bar at the bottom indicating nucleotide substitutions per site. (B) ML tree of the PR/RT region of the intersubtype recombinant samples. HIV-1 reference sequences of pure subtypes and some CRFs (CRF02_AG, CRF12_BF, CRF28_BF, CRF29_BF, CRF31_BC, CRF39_BF, CRF40_BF, CRF70_BF, CRF71_BF, and CRF72_BF) were included and colored black. Each HIV-1 clade found or closely related to the Brazilian samples is colored as indicated in the figure. For visual clarity, some clades are collapsed into triangles and the branch support values are indicated as asterisks (aLRT >0.90). Horizontal branch lengths are drawn to scale with the bar at the bottom indicating nucleotide substitutions per site. Unique recombinant forms are colored in gray and their bootscaning plots are depicted on the right. aLRT, approximate likelihood ratio test; CRF, circulating recombinant form; ML, maximum likelihood; PR/RT, protease and reverse transcriptase. Color images available online at www.liebertpub.com/aid

Resistance analysis of PR and RT sequences from the 87 samples showed that at least one drug resistance mutation was observed in 15 samples, leading to an overall TDR prevalence of 17.2%. TDR mutations for protease inhibitors (PI) were detected in 8.0% (7/87) of the patients. Of these, five had the M46L mutation (5.7%, 5/87) and one had the M46I mutation (1.1%, 1/87). The other mutations found were D30N and N88D, both harbored by the same individual. TDR mutations for nucleoside reverse transcriptase inhibitors (NRTI) were detected in 5.7% (5/87) of the women in the study. The mutation M41L was found in one patient (1.1%, 1/87), while the D67N mutation was found in three (3.4%, 3/87). Furthermore, the mutations T69D and K219Q were detected in one individual each (1.1%, 1/87) and both also exhibited the D67N mutation. TDR mutations for non-nucleoside reverse transcriptase inhibitors (NNRTI) were detected in 5.7% (5/87) of the patients analyzed. The frequency of K103N mutation was 2.3% (2/87). The same frequency was observed for the G190A mutation (2.3%, 2/87). The mutation K101E was found in one patient (1.1%, 1/87) who also had the G190A mutation. One sequence carrying both NRTI and NNRTI mutations was detected, however, no sequence with triple class resistance was found. Table 1 summarizes PI, NRTI, and NNRTI transmitted drug resistance (TDR) mutations according to the sample HIV-1 subtype. Almost all TDR mutations were found in women infected with HIV-1 subtype B viruses (73%, 11/15), the remaining were infected with subtype F1 (20%, 3/15) and CRF28/29_BF (7%, 1/15).

This TDR and HIV-1 diversity survey was performed in pregnant women from a populous and impoverished area located in the metropolitan region of the Rio de Janeiro state in Brazil. The 17.2% overall prevalence of TDR found in this study places the region analyzed in the high-level stratum of TDR prevalence (>15%) according to the WHO criteria.¹² Reaching this level, WHO has suggested that it becomes cost-effective to perform genotypic resistance testing to all individuals with newly diagnosed HIV infection, before the beginning of ART. However, our findings are limited by the small sample size, as well as by the use of different criteria of those recommended by the WHO HIV drug resistance surveillance guidelines that consider the thresholds of 25 years of age and CD4+ T-cell count above 500 cells/mm³ as inclusion criteria.¹² One strength of our study is that the population sampling for the period of 2012–2015 pertains to the same clinical research site (CRS) we have data for the period of 2005–2008, enabling the evaluation of how TDR rates evolved in this location among pregnant women recently diagnosed with HIV infection between these two time points.

In Brazil, previous national cross-sectional surveys pointed out a moderate level of HIV-1 TDR (8%–12%).^13,14 The TDR prevalence found during the period of time analyzed in our study (17.2%) was higher than that described in other studies conducted with HIV-1-infected pregnant woman from Rio de Janeiro, depicting TDR rates of 9.8% and 10.7%.^7,8 This TDR rate increase becomes more relevant when one considers that the work of Pilotto et al. ⁷ was conducted in a population of pregnant women enrolled with the same inclusion criteria and at the same CRS of present study in a previous period (2005–2008). However, this sudden increase in the overall prevalence of TDR (from 10.7% to 17.2%) was not statistically significant (p = .1271), indicating that this trend could possibly be an artifact due to the reduced sample size and no changes in TDR might have occurred between these periods, despite the adoption of new practices regarding ART use in Brazil initiated in 2013.⁶ More TDR surveillance studies, including newly HIV-infected patients, are needed to definitively understand whether the TDR rate is increasing in this population of women and the impact of the new ART practices in the TDR emergence.

Mutations to NRTI were the most prevalent (5.6%) in pregnant women sampled in the previous study (2005–2008), which were associated with the use of NRTI mono and dual therapy before the HAART era in Brazil.⁷ In this study, the TDR mutations to PIs were the most prevalent, with a frequency of 8.0%, which is higher than the previous observations (3.0%), although the difference was not statistically significant (p = .0721). This high TDR rate for PIs may be explained by the effect of poor patient adherence to PI-based regimens, frequently caused by high pill burden and low tolerance.¹⁵ This could explain the resistance mutations found in PR (M46I/L, N88D and D30N), which are responsible for variable resistance levels to nelfinavir (NFV).¹⁶ NFV-containing regimens initially used in Brazil imposed a high pill burden (five pills twice a day), becoming one of the major PIs used in the country, until its replacement by newly developed PIs, such as lopinavir/r and atazanavir.¹⁷ Among the resistance mutations in RT, thymidine analogue-associated mutation-related mutations D67N and K219Q were found. These mutations are associated to low-level resistance to zidovudine (AZT) and stavudine.¹⁶ Two patients exhibited the mutation K103N that causes high-level resistance to efavirenz and nevirapine (NVP). The other mutations found (K101E, Y188L, and G190A) were responsible for variable levels of resistance to all NNRTIs, but all confer high-level resistance to NVP.¹⁶ Taken together, the TDR mutation profile found can abrogate the first-line treatment regimen and PMTCT prophylaxis commonly used in Brazil, which recommends the use of two NRTI (AZT and lamivudine) plus a PI, or alternatively an NNRTI (specifically NVP).¹⁸ These findings enforce the need to promote routine and universal genotype ARV resistance susceptibility testing to all Brazilian HIV-infected pregnant women for PMTCT purposes.

Regarding HIV-1 diversity, the predominance of subtype B followed by subtype F1 and subtype C is in accordance with previous descriptions of the HIV diversity found in the Rio de Janeiro state and with the previous study of pregnant women recruited from 2005 to 2008 at the same CRS.^7,8 However, a statistically significant increase in the prevalence of nonsubtype B viruses between the two periods was observed (p = .0330), indicating a change over the years toward a marked increase in the circulation of non-B strains in Rio de Janeiro, driven mainly by intersubtype recombinants. One CRF02_AG strain was detected among the recombinant forms described in this study. In a previous study from our group, this CRF02_AG strain was described as participating in an autochthonous transmission network, which has been disseminated locally in the Rio de Janeiro state over the last 30 years.¹⁹ It is also noteworthy that two CRF12_BF strains were found in our study, since this CRF is highly prevalent in Argentina and Uruguay, but rarely detected in Brazil.²⁰ These findings demonstrate the role of the HIV epidemic in the Rio de Janeiro metropolitan area (a major touristic destination and an important commercial hub) as a hot spot for the introduction and dissemination of new viral clades in the Brazilian epidemic.

The results presented here indicate an increase in the HIV-1 molecular diversity and an upward trend in the TDR prevalence in Rio de Janeiro between the periods of 2005–2008 and 2012–2015 among ARV-naive pregnant women. The HIV-1 TDR mutations found in this population could affect the virological outcome of the standard ARV regimens used for PMTCT of HIV and also sexual transmission of HIV-resistant strains. Our data add to the growing body of evidence about the need of continuous monitoring of the HIV-1 genetic diversity and TDR prevalence in Brazil for all HIV-infected pregnant women.

Sequence Data

Sequences were deposited in GenBank under accession numbers KX357219 to KX357305.

Footnotes

Acknowledgments

E.D. was supported by Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ). We thank all the patients and the staff of the Department of Sexually Transmitted Diseases (HGNI) who participated in this study. We also thank Dr. Vera Bongertz for the grammar review of the article. The authors are grateful to the Plataforma Genômica–Sequenciamento de DNA-RPT01A-PDTIS/FIOCRUZ.

Author Disclosure Statement

No competing financial interests exist.

References

UNAIDS. The Gap Report. Geneva, Switzerland, 2014.

Brasil. Ministério de Saúde, Departamento de DST, Aids e Hepatites Virais. Boletim Epidemiológico Aids e DST. Brasília, DF Brasil, 2014.

Strategies for Management of Antiretroviral Therapy (SMART) Study Group, Lundgren

, Babiker

, El-Sadr

, Emery

, Grund

, et al. Inferior clinical outcome of the CD4+ cell count-guided antiretroviral treatment interruption strategy in the SMART study: Role of CD4+ cell counts and HIV RNA levels during follow-up. J Infect Dis, 2008; 197:1145–1155.

Cohen

, Chen

, McCauley

, Gamble

, Hosseinipour

, Kumarasamy

, et al.: Prevention of HIV-1 infection with early antiretroviral therapy. N Engl J Med, 2011; 365:493–505.

Grinsztejn

, Hosseinipour

, Ribaudo

, Swindells

, Eron

, Chen

, et al.: Effects of early versus delayed initiation of antiretroviral treatment on clinical outcomes of HIV-1 infection: Results from the phase 3 HPTN 052 randomised controlled trial. Lancet Infect Dis, 2014; 14:281–290.

Brasil. Ministério de Saúde, Departamento de DST, Aids e Hepatites Virais. Protocolo Clínico e Diretrizes Terapêuticas para Manejo da Infecção pelo HIV em Adultos. Brasília, DF Brasil, 2013.

Pilotto

, Grinsztejn

, Veloso

, Velasque

, Friedman

, Moreira

, et al.: Moderate prevalence of transmitted drug resistance mutations among antiretroviral-naive HIV-infected pregnant women in Rio de Janeiro, Brazil. AIDS Res Hum Retroviruses, 2013; 29:681–686.

de Lourdes Teixeira

, Nafea

, Yeganeh

, Santos

, Isabel Gouvea

, Joao

, et al.: High rates of baseline antiretroviral resistance among HIV-infected pregnant women in an HIV referral centre in Rio de Janeiro, Brazil. Int J STD AIDS, 2015; 26:922–928.

Guindon

, Dufayard

J-F

, Lefort

, Anisimova

, Hordijk

, Gascuel

: New algorithms and methods to estimate maximum-likelihood phylogenies: Assessing the performance of PhyML 3.0. Syst Biol, 2010; 59:307–321.

10.

Lole

, Bollinger

, Paranjape

, Gadkari

, Kulkarni

, Novak

, et al.: Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination. J Virol, 1999; 73:152–160.

11.

Gifford

, Liu

, Rhee

, Kiuchi

, Hue

, Pillay

, et al.: The calibrated population resistance tool: Standardized genotypic estimation of transmitted HIV-1 drug resistance. Bioinformatics, 2009; 25:1197–1198.

12.

World Health Organization. World Health Organization global strategy for the surveillance and monitoring of HIV drug resistance: An update. Geneva, Switzerland, 2012.

13.

Inocencio

, Pereira

, Sucupira

MCA

, 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; 12:1–6.

14.

Alencar

, Sabino

, Carvalho

SMF

, Leao

, Carneiro-Proietti

, Capuani

, et al.: HIV genotypes and primary drug resistance among HIV-seropositive blood donors in Brazil. JAIDS J Acquir Immune Defic Syndr, 2013; 63:387–392.

15.

Nachega

, Parienti

, Uthman

, Gross

, Dowdy

, Sax

, et al.: Lower pill burden and once-daily antiretroviral treatment regimens for HIV infection: A meta-analysis of randomized controlled trials. Clin Infect Dis, 2014; 58:1297–1307.

16.

Wensing

, Calvez

, Günthard

, Johnson

, Paredes

, Pillay

, et al.: 2014 update of the drug resistance mutations in HIV-1. Top Antivir Med, 2014; 22:642–650.

17.

Nunn

, Fonseca

, Bastos

, Gruskin

, Salomon

: Evolution of antiretroviral drug costs in Brazil in the context of free and universal access to AIDS treatment. PLoS Med, 2007; 4:1804–1817.

18.

Brasil. Ministério de Saúde, Departamento de DST, Aids e Hepatites Virais. Recomendações para Profilaxia da Transmisão Vertical do HIV e Terapia Antirretroviral em Gestantes. Brasília, DF Brasil, 2010.

19.

Delatorre

, Velasco-De-Castro

, Pilotto

, Couto-Fernandez

, Bello

, Morgado

: Reassessing the origin of the HIV-1 CRF02_AG lineages circulating in Brazil. AIDS Res Hum Retroviruses, 2015; 31:1230–1237.

20.

Bello

, Soares

, Schrago

: The use of bioinformatics for studying HIV evolutionary and epidemiological history in South America. AIDS Res Treat, 2011; 2011:154945.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.02 MB