According to the WHO, the rates of HIV-1 acquisition worldwide remain stable, with approximately 2 million new HIV infections per year , with over half acquired in marriage in sub-Saharan Africa [2–4]. Despite the effectiveness of antiretroviral therapy (ART) in the treatment and prevention of HIV acquisition and transmission [5–10], in resource-limited settings, behavioral interventions, such as couples’ voluntary counseling and testing (CVCT), remain the most cost-effective ways to reduce transmission (by up to 75%) in co-habiting couples [11–15]. Moreover, both ART uptake and effectiveness for treatment and prevention are improved when couples are counseled together and made aware of their partner's status [8,16]. Although WHO guidelines support CVCT for HIV prevention , fewer than 10% of African couples have been jointly tested and counseled, highlighting the need for widespread implementation of CVCT .
The prevention clinical trial, HPTN 052, established that transmission could be reduced by up to 96% (approximately 2.5/100 person-years in the delayed treatment control arm vs. <0.5/100 person-years in the early ART arm) [8,9] in HIV-discordant couples who had already benefited from a substantial reduction in transmission (10–11%/year to < 3%/year) following CVCT, when ART was added and viral load in the positive partner was completely suppressed.
Whereas ART can be effective in treating [5,7,10] and preventing HIV-1 transmission , it has become clear that suboptimal adherence, a determinant of ART failure, is a major obstacle to its continued success [19–21]. Indeed, it is estimated that less than half of the HIV-1 population on ART is virologically suppressed , likely due to suboptimal adherence, which is a major driver of drug resistance . In addition, as ART availability increases worldwide, so does the rate of primary drug resistance (PDR) [22–24]. The PharmAccess African Studies to Evaluate Resistance Monitoring (PASER-M) study showed that rates of PDR were significantly higher in sites where there was early widespread access to ART . The increasing risk of acquisition and transmission of drug resistance viral variants suggest ART alone may not be sufficient to tackle this epidemic.
Despite the near 100% effectiveness of ART to prevent transmission in clinical trials, we observe ongoing transmission in HIV-discordant couples reporting ART usage (Wall et al., submitted) in government clinics. Because there is an increased risk for suboptimal adherence in low to lower-middle income countries , we hypothesized that this may be the source of virologic failure and subsequent transmission. As transmission frequency can vary based on viral subtype , we sought to investigate whether this contributed to HIV-1 transmission in both heterosexual Zambian (subtype C) and Rwandan (subtype A) couples where the HIV-positive partner reported ART usage. An analysis of transmission in these two countries allows a comparison in the context of the two most globally common subtypes (C and A) and different healthcare systems. We observed remarkable differences between the two countries. In epidemiologically linked Zambian pairs, we found that, despite the reported use of ART, a majority (7/9) of the transmitting partners had quantifiable viral loads and no antiretrovirals in plasma. In contrast, in seven Rwandan transmission pairs analyzed in parallel, the majority showed quantifiable antiretroviral levels in plasma. In more than half of the Rwandan couples we detected drug resistance mutation (DRM), a majority of which were transmitted to their partners. Identifying the factors associated with HIV-1 transmission, in the context of reported use of antiretrovirals, will inform public health strategies to achieve effective prevention and treatment approaches in the post-ART era.
Materials and methods
Settings and study populations
Heterosexual couples were recruited for CVCT in urban government clinics in Zambia and Rwanda as described in Supplementary Methods (http://links.lww.com/QAD/B283). In Zambia, during the initial visit, couples were tested for HIV-1, and, based on their HIV-1 serostatus, counseled and referred to appropriate services. Serodiscordant couples were asked to return to clinic at 1 month and quarterly after the initial visit for repeat HIV counseling and testing (of the negative partner) and reinforcement of risk reduction counseling messages. If seroconversion in the previously HIV-negative partner was detected, linkage analysis of viruses from both partners was performed . For the current study, we selected couples where the donor partner reported the prior use of ART during their initial counseling visit. The incidence of transmission before CVCT (i.e. where the seronegative partner seroconverted prior to their month 1 visit) in these couples was estimated as approximately 9% (Wall et al., submitted).
Rwandan couples who met the following criteria were chosen for the study: previously undergone CVCT and referral for ART, returning for their quarterly routine follow-up visit in government clinics in Kigali, and the seronegative partner had now seroconverted.
The study protocol was approved by the Institutional Review Boards in Zambia, Rwanda and at Emory registered with the Office of Human Research Protection at the US Department of Health and Human Services; all participants gave written informed consent for participation in the study.
Viral load testing
HIV-1 viral load determination (in copies/ml) was performed on plasma using the Abbott Real-Time HIV-1 viral load assay (Abbott Molecular; Abbott Park, Illinois, USA). For more details, see Supplemental Methods (http://links.lww.com/QAD/B283).
Liquid chromatography-tandem mass spectroscopy for detection of antiretroviral agents
To detect and quantify antiretroviral levels in patient plasma, a high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach was used similar to previously described methods (see Supplemental Methods, http://links.lww.com/QAD/B283) .
Antiretroviral levels were recorded in ng/ml. Effective concentrations, where 50 or 90% (EC50 or EC90) of the drug's maximal effect was observed, were calculated for each antiretroviral (Supplemental Table 1, http://links.lww.com/QAD/B283).
Viral RNA extraction, PCR amplification, and population sequencing of HIV-1 pol
Viral RNA was extracted from plasma samples and reverse transcribed as previously described . Alternatively, genomic DNA was used as a template for low viral load samples. Nested PCR amplification was performed for 5’-half HIV-1 genomes (∼4.5 kb) with the Q5 High-Fidelty enzyme (New England BioLabs Inc., Ipswich, Massachusetts, USA). Primers are listed in Supplemental Table 2 (http://links.lww.com/QAD/B283).
Positive PCR amplicons were pooled and purified. Pooled populations were sequenced using either Sanger (GenScript; Piscataway, New Jersey, USA) or SMRT technology platform (Pacific Biosciences; Menlo Park, California, USA) based on sample availability. More details are available in Supplemental Methods (http://links.lww.com/QAD/B283).
Multiplex RNA PCR assay for lack of viral load suppression in nontransmitting positive partners
Viral RNA was extracted from plasma samples for 50 nontransmitting positive partners using E.Z.N.A Viral RNA extraction kit (Omega Bio-tech, Norcross, Georgia, USA). As described (Basu et al., manuscript in preparation), RNA was subjected to a multiplexed one-step RT-PCR first round PCR reaction using SuperScript III One-Step RT-PCR System (Invitrogen, Waltham, Massachusetts, USA). Three small regions were multiplexed: gp41 (460 bp), gag (560 bp), and pol (218 bp). Second round amplification for each region was then performed independently.
PAC-BIO library preparation, sequencing, and analysis of HIV-1 pol
A SMRTbell library containing barcoded and nonbarcoded 5’-half genomes was generated, per manufacturer's protocol (Pacific Biosciences Inc., California, USA). Sequencing was performed as previously described . Exported data files were processed using the MDPSeq software pipeline , and output sequences aligned in Geneious (Biomatters Ltd., Auckland, New Zealand).
Drug resistance mutation analysis
Pol sequences for both donor and recipient partners (where available) were submitted for DRM analysis (HIVdb, HIV Drug Resistance database) . Each DRM identified was assigned a drug penalty score, and these scores were added to determine the level of drug resistance to eight antiretrovirals; scores at least 60 were considered high-level resistance.
HIV-1 transmission in Zambian couples reporting antiretroviral use
To understand the factor(s) associated with ongoing HIV-1 transmission in Zambian serodiscordant couples in the context of ART, we identified and recruited nine epidemiologically linked pairs from government clinics where the donor self-reported being on ART. Of the nine couples, all donors had detectable viral loads (Fig. 1a) ranging from 7852 to 444 440 copies/ml (median 47 600 copies/ml). These viral loads were more similar to those of chronically infected ART-naive individuals from our larger heterosexual transmission cohort , Zambia Emory HIV Research Project (ZEHRP), where, for 1652 individuals, the median viral load was 70 000 copies/ml. In contrast, the median viral load for 77 ART-treated individuals in the same cohort was significantly lower (160 copies/ml; P < 0.0001) .
Although the recommended ART regimens for Zambia are tenofovir disoproxil fumarate (TDF)-lamivudine (3TC)/emtricitabine (FTC)-efavirenz (EFV)/nevirapine (NPV), because some individuals may receive other therapies, we quantitated the concentration of a total of 10 antiretrovirals in patient plasma using HPLC-MS/MS (Fig. 1b). Only two donors (MON 3076M and MSH 596F) had detectable antiretrovirals in plasma. MON 3076M had three drugs [FTC, tenofovir (TFV), EFV] above the effective concentration 50 (EC50) and one drug (NVP) below the EC50, which is consistent with first-line ART prescribed in Zambia. For MSH 596F, only zidovudine was detectable.
Although an interruption in ART likely contributed to the majority of transmission events in the nine Zambian couples, to determine if DRM contributed to transmission, we amplified and sequenced the pol gene from both donor and recipient in each couple. In one couple, MON 3076, we detected six DRMs that together conferred high-level resistance to six antiretrovirals in the donor partner (Fig. 2a), including the four (FTC, TFV, EFV, NVP) detected in donor plasma (Fig. 1b). All but one DRM (V106M) was detected in the recipient (Fig. 2b). Although both partners exceeded the threshold for high-level resistance to EFV and NVP (cumulative penalty scores of 60 and above), the donor's level of resistance was higher (140 for EFV and NVP) than the recipient (85 for EFV and 135 for NVP).
Viral load and antiretroviral detection in nontransmitting Zambian partners reporting ART
To confirm that a majority of individuals in Zambian government clinics who reported antiretroviral use understood their ART status and correctly answered the question, we randomly selected 50 Zambian serodiscordant couples from the same clinics and where the HIV-positive partner (25M, 25F) self-reported antiretroviral use. These individuals had not transmitted to their partners. Forty out of the fifty nontransmitters (80%) had no trace of viral RNA in plasma samples in an in-house multiplex qualitative RNA PCR assay (Basu et al., manuscript in preparation) in which we amplified three genomic regions of HIV-1 (gag, pol, gp41). We were able to detect viral RNA (for at least one genomic region) in the remaining 10 individuals. The viral loads in these individuals ranged from 264 to 194 928 copies/ml, with a median viral load of 5466 copies/ml (Fig. 1a). As a control, we quantified the viral load of eight randomly selected individuals from the 40 that were undetectable to confirm the absence of viral RNA/viral replication. Consistent with the qualitative results, all viral loads were undetectable (Fig. 1a).
Of the 10 viral RNA-positive individuals (Fig. 1c), 4 had no drug present in their plasma, five had at least one drug above its EC50, and one had only one drug (below the EC50) detected. Plasma from the eight RNA-negative control individuals all contained three drugs with plasma concentrations near or above the EC50. Thus, for those positive partners reporting antiretroviral use in government clinics, ART was consistent with their response to the questionnaire, and overall adherence in the population was high.
HIV-1 transmission in Rwandan couples reporting antiretroviral therapy use
Rwanda differs from Zambia in that CVCT is deployed country-wide, and a majority of couples know their partner's HIV status. We hypothesized that consistent adherence, therefore, would be better than in Zambia. We identified seven acute transmission pairs from Rwandan government clinics, where the donor partner reported antiretroviral usage, and determined viral load and antiretroviral levels for these couples. The plasma viral load in the donors at the time of sampling ranged from undetectable (<160 copies/ml) to 914 000 copies/ml, with a median of 3658 copies/ml (Fig. 3a). Although this value was lower than the median viral load for a set of untreated Rwandans for whom viral loads were available (median 17 244 copies/ml), it was not significantly different (P = 0.085). In contrast, a set of ART-treated individuals from the Projet San Francisco (PSF) transmission cohort had a median viral load of 160, which was significantly lower (P < 0.0001).
To confirm ART status, we screened plasma samples for the presence of 10 antiretrovirals (Supplementary Table 1, http://links.lww.com/QAD/B283; Fig. 3b). In contrast to what we observed in Zambia, a total of four of seven ART-reporting donors (57%) had at least three antiretrovirals with concentrations above the EC50, whereas two of seven (29%) had only one antiretroviral present. The remaining individual (14%) had no detectable antiretrovirals. The triple therapies detected were again consistent with WHO-recommended first-line regimens (TDF/3TC/EFV). Due to the detection of antiretrovirals in 86% of these donors, we hypothesized that drug resistance rather than an interruption in ART may contribute to transmission in this subset. Indeed, we were able to determine or infer the presence of DRM in four of the seven couples; of these couples, there was evidence for transmitted drug resistance in three pairs (75%). In the BGO 003 pair, we detected one non-nucleoside reverse transcriptase inhibitor (NNRTI) DRM, V106M, in the donor, rendering the virus highly resistant (penalty score 60) to NVP and EFV treatment (Fig. 4a; left); however, this DRM was not detected in the partner (Fig. 4a; right), although reversion between transmission and sampling is possible.
In the RUG 001 pair, we identified two NNRTI DRM in the donor virus (Fig. 4b; left), K103N and K238T, which contributed to high-level resistance and were detected in the partner (Fig. 4b; right). Although we were unable to directly sequence the COR 140 donor, we detected two distinct drug resistance viral populations in the recipient's plasma, suggesting multivariant transmission. Viral variant 1 contained three NNRTI and one nucleoside reverse transcriptase inhibitor (NRTI) DRM, which confer high-level resistance to both classes of drug (Fig. 4c; left). Viral variant 2 contained six DRMs, three of which were present in variant 1 (Fig. 4c; right). This variant encoded two additional NNRTI and one additional NRTI DRM (Fig. 4c). In the COR 150 pair, we detected nine DRMs in the donor virus (Fig. 4d; left). Three mutations contributed to high-level NNRTI resistance, whereas two rendered the virus highly resistant to several NRTIs. Only five DRMs were detected in the partner (Fig. 4d; right). Finally, viral sequences obtained from the KIN 001 and KIM 168 couples showed no evidence of DRM.
These data show that, in contrast with Zambia, at least half of the ART-reporting transmitting couples had evidence of DRM, indicating drug resistance was strongly associated with HIV-1 transmission in these Rwandan heterosexual transmission pairs.
In HIV discordant couples, CVCT followed by ART remains the only effective intervention to prevent HIV-1 transmission [8,9]. Moreover, in developing countries where the current infrastructure may prohibit the successful deployment of resources necessary to achieve full virologic suppression [33,34], CVCT is still a key component of prevention efforts. The goal of this study was to understand the basis of ongoing HIV-1 transmission in the context of ART in discordant African couples, where the disease burden is the greatest.
HIV-1 transmission in heterosexual Zambian couples
Many factors, including socioeconomic status, ART availability, adherence, duration of treatment, and acquired or transmitted drug resistance, can affect the efficacy of ART regimens [19,22,23,25,35]. Of these factors, virologic suppression is largely dependent on strict adherence . Previous work assessing ART adherence in sub-Saharan Africa has focused mainly on identifying barriers to adherence and the consequences of non or suboptimal adherence on the individual [19,36]. However, limited data are available on how lack of adherence and drug resistance impact HIV-1 transmission in couples in African settings .
To address this, we analyzed nine cases of acute transmission identified in couples attending CVCT in government clinics where transmitting partners reported ART use prior to the time of transmission. We measured viral load and screened for antiretrovirals in plasma samples of the transmitting partners as these measures help gauge adherence and ART efficacy. In Zambia, all nine transmitting partners had detectable viral loads with a median similar to that of ART-naïve Zambians from a larger cohort, demonstrating that none of these individuals were on HIV-1-suppressive therapy. Indeed, a majority (7/9) of donors had no detectable antiretrovirals in their plasma, providing unambiguous evidence for why these individuals had quantifiable viral loads. However, despite measurable levels of antiretrovirals in two donors, both had plasma viral loads above 10 000 copies/ml. These data suggested that treatment failure may have allowed transmission in these two couples. We detected six DRMs in one donor that contributed to high-level drug resistance to several antiretrovirals including those present in donor plasma. As the likelihood of developing drug resistance in ART-treated individuals is relatively low when adherence is high [22,23,36,38,39], the presence of DRM in the donor suggests suboptimal adherence at some point in time. Although our study was limited by sample size and lacked longitudinal follow-up, these data suggest that HIV-1 transmission in this group was largely associated with nonadherence; however, a larger prospective study with longitudinal sampling is required to establish a causal relationship.
The lack of adherence at the time of sampling observed in these transmitting couples was not simply due to a lack of understanding of the question during CVCT, because in a group of 50 nontransmitting HIV-positive partners tested in the same government clinics in Lusaka and Ndola, a majority (40 of 50) had undetectable viral loads indicating that adherence in the overall population was high.
HIV-1 heterosexual transmission in Rwandan couples
Naturally occurring polymorphisms that differentiate viral subtypes have been shown to alter the structure of drug targets resulting in differential responses to antiretrovirals [40,41]. Thus, factor(s) contributing to HIV-1 transmission in the face of reported antiretroviral use could be different in Zambia (subtype C) versus Rwanda (mainly subtype A). Moreover, because CVCT is deployed nationally in Rwanda and both partners know their partner's HIV status, we predicted that adherence may be better than in Zambia. Indeed, in a similar analysis of seven Rwandan heterosexual transmission pairs, where the transmitting partner reported being on ART, we detected antiretrovirals in six of seven donors (although only four were on fully therapeutic regimens of a triple therapy). Over half of the transmitting partners (4/7) had DRM that yielded intermediate to high-level drug resistance. In addition, of these four couples, three transmitted DRM to their acutely infected partner. The frequency with which DRMs were detected in the seroconverting partner may be an underestimate, because sampling at 3 months could allow reversion to wild-type, and may explain why fewer DRMs were identified in the seroconvertor. These data indicate that a majority of transmissions in this limited number of Rwandan pairs was associated DRM.
Differences between Zambian and Rwandan couples
Although subtype could offer a biological basis for some of the observed differences between the two groups, these disparities are more likely linked to differences in infrastructure including national CVCT and treatment of the HIV-positive partner in Rwanda.
As a part of routine government CVCT visits, HIV-positive individuals are asked whether or not they are on ART. The transmission pairs included in this study were selected based on reported ART use prior to counseling by the chronically infected transmitting partner, yet the majority of Zambians (8/9) and a little over 40% of the Rwandans were not taking therapeutic levels of antiretrovirals at the time of sampling, consistent with a previous report that self-report is not the most reliable measure of adherence .
Several studies have reported that the risk of HIV-1 transmission varies based on plasma viral load of the HIV-positive partner with transmission being negligible for individuals with viral loads below 1500 copies/ml [43,44]. While in Zambia, all the transmitting partners had viral load above 8000 copies/ml, almost half of the Rwandan transmitting partners had viral load below 1500 copies/ml. Although these Rwandan couples were selected because the transmitting partner reported ART use prior to transmission, we cannot rule out a brief period of nonadherence where viral load increased sufficiently to allow transmission. While there is evidence to suggest a correlation between blood and genital fluid viral loads, ART can lead to differences in these compartments due to differential penetration of tissues . Thus, it is also possible that insufficient penetration of antiretrovirals in the genital tract could explain how these donors with extremely low plasma viral loads (<500 copies/ml) still transmitted to their partners. Evaluation of viral load in the genital compartments may be required to firmly establish the cause of transmission in couples with such low viral loads.
In conclusion, although CVCT is effective at preventing HIV-1 infection and ART in discordant couples is additive in its preventive impact , it is unlikely that ART alone will end the AIDS epidemic. In this study, our data suggest that nonadherence and drug resistance are both associated with ongoing transmission in the context of more widely available ART in Zambia and Rwanda, respectively, and that these factors differentially impacted both cohorts. We noted that the development of drug resistance not only led to treatment failure in the donor, but also subsequent transmission of DRM to the recipient partners, compromising future treatment options for that individual. Because viral load testing is either limited or not available in Rwanda and Zambia, respectively, early detection of problems with adherence or drug resistance is not occurring. Together, these data suggest that public health interventions, such as early partner involvement through CVCT, which reinforce the importance of adherence, and also the development and widespread deployment of cost-effective viral load and drug resistance testing are essential to achieving the maximal benefit from ART.
Author roles: E.W., A.G., C.M., D.B., S.T., and Y.J. performed the experiments. E.W., W.K., and E.H. wrote the manuscript. W.K., E.K., and S.A. directed the fieldwork and sample collection. S.A., D.B., E.W., and E.H. conceived the study. All authors reviewed and edited the manuscript.
The investigators thank all the volunteers in Zambia and Rwanda who participated in this study and all the staff at the ZEHRP in Lusaka and PSF in Kigali, respectively, who made this study possible. The investigators would like to thank Jon Allen, Sheng Luo, and Paul Farmer for technical assistance, sample management, and database management. This study was funded by R01 MH095503-05 (S.A.), R37 AI51231 and R01 AI64060 (E.H.). This work was also supported, in part, by the Virology Core at the Emory Center for AIDS Research by performing viral load determinations (Grant P30 AI050409); the Yerkes National Primate Research Center base grant through the Office of Research Infrastructure Programs/OD P51OD11132. This study is supported in part by IAVI (S.A.), whose work is made possible by generous support from many donors including: the Bill & Melinda Gates Foundation; the Ministry of Foreign Affairs of Denmark; Irish Aid; the Ministry of Finance of Japan; the Ministry of Foreign Affairs of the Netherlands; the Norwegian Agency for Development Cooperation (NORAD); the United Kingdom Department for International Development (DFID), and the United States Agency for International Development (USAID). The full list of IAVI donors is available www.iavi.org. The contents are the responsibility of the study authors and do not necessarily reflect the views of USAID or the United States Government. D.B. was in receipt of postdoctoral training support from NIH Training Grant R25 TW009337. E.H. is a Georgia Research Alliance Eminent Scholar.
Conflicts of interest
There are no conflicts of interest.
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