The concentration of viral RNA in plasma is the primary risk factor for sexual transmission of HIV-1 [1–3], and reductions in plasma HIV-1 RNA levels due to antiretroviral therapy (ART) result in marked decreases in HIV-1 transmission risk [4,5]. Results from studies of HIV-1 transmission and disease progression may be more difficult to interpret if a substantial proportion of HIV-1 infected partners have low or undetectable viral loads on ART, and thus, ART use at study enrollment is often an exclusion factor.
Recent reports from clinical trial cohorts of HIV-1 transmission in HIV-1 serodiscordant couples have found that nearly a quarter of HIV-1 infected partners had low enrollment plasma HIV-1 RNA levels (<2000 copies/ml) [6,7]. Low levels of plasma HIV-1 RNA in the HIV-1 infected partners, selected for not having transmitted HIV-1 to their partner for studies of candidate interventions to reduce HIV-1 transmission, may reflect natural host control of viral replication. However, an alternative explanation could be unreported ART use. Distinguishing between these potential sources of low viral load is important for studies seeking to understand the biology of HIV-1 transmission. We tested stored samples from a recent HIV-1 prevention clinical trial to determine the frequency of unreported ART use among HIV-1 infected individuals with low plasma HIV-1 RNA levels.
Between November 2004 and April 2007, we enrolled 3408 heterosexual HIV-1 serodiscordant couples from seven African countries in a randomized, double-blind, placebo-controlled clinical trial of herpes simplex virus type 2 (HSV-2) suppressive therapy to reduce HIV-1 transmission (Partners in Prevention HSV/HIV Transmission Study), as previously described [6]. Eligible couples were at least 18 years of age, sexually active, and intending to remain as a couple. All HIV-1 infected partners were HSV-2 seropositive, had CD4 counts ≥250 cells/μL (making them ineligible for antiretroviral therapy under national guidelines of the study countries at that time), not pregnant, and self-reported not currently taking ART. Quarterly plasma and serum samples were collected for up to 24 months and archived at −80°C for subsequent laboratory testing. HSV-2 suppressive therapy did not reduce HIV-1 transmission within the study partnerships [8]. At study screening and all follow-up visits, HIV-1 infected participants were asked if they were currently taking ART.
All participants received HIV-1 primary care, referral for ART according to national guidelines and risk reduction counseling and treatment for sexually transmitted infections during up to 24 months of study follow-up. Written, informed consent was obtained from all participants. The study protocol was approved by the University of Washington Human Subjects Review Committee and ethical review committees at each of the study sites.
All laboratory testing occurred at the end of study follow-up. Plasma HIV-1 RNA levels were quantified using the COBAS Ampliprep/COBAS TaqMan real-time HIV-1 RNA assay, version 1.0 (Roche Diagnostics, Indianapolis, IN), with a lower limit of quantification of 240 copies/mL. For those with plasma HIV-1 RNA <2000 copies/mL at enrollment, high-performance liquid chromatography with ultraviolet detection was used to measure antiretroviral (ART) levels in an archived serum collected at enrollment ([9]. The assay used to measure ART levels was validated for five nucleoside reverse transcriptase inhibitors (abacavir, didanosine, lamivudine, stavudine and zidovudine) and one non-nucleoside reverse transcriptase inhibitor (nevirapine). We considered any quantifiable concentration as indicative of ART use.
Low viral load was stratified into two groups defined as: 1) low detectable (240–2000 copies/mL), or 2) undetectable (<240 copies/mL). We calculated the overall prevalence of unreported ART use at enrollment among HIV-1 infected partners with low plasma HIV-1 RNA (<2000 copies/ml) overall and in each group All analyses were conducted using SAS (v.9.2, Cary, NC).
Among 3371 HIV-1 infected partners who had results for enrollment plasma HIV-1 RNA tested, 798 (23.7%) had plasma HIV-1 RNA <2000 copies/mL, including 443/798 (13.1%) with low plasma HIV-1 RNA levels (240–2000 copies/mL) and 355/798 (10.5%) with undetectable RNA (<240 copies/mL). Those with enrollment plasma HIV-1 RNA <2000 copies/mL were more likely to be female compared to those with plasma HIV-1 levels >2000 copies/mL (78.1% vs. 64.1%, p<0.05); all other characteristics were similar for those with higher versus lower plasma HIV-1 RNA (Table).
Table.
Enrollment characteristics and type of antiretroviral (ART) detected among HIV-1 infected partners
| ≥ 2000 copies/mL, N=2573 | <2000 copies/mL, N=443 | Undetectable, N=355 | |
|---|---|---|---|
| Demographic characteristics | |||
| Female gender | 1650 (64.1%) | 335 (75.6%) | 288 (81.1%) |
| Age, years | 32 (27–39) | 31 (26–37) | 32 (28–37) |
| Education, years | 8 (6–11) | 8 (7–12) | 8 (6–11) |
| Has monthly income | 940 (36.3%) | 159 (35.9%) | 120 (33.8%) |
| Couple characteristics | |||
| Married and/or cohabiting | 2340 (90.9%) | 399 (90.1%) | 324 (91.3%) |
| Duration of partnership, years | 5.3 (2.3–10.4) | 4.8 (2.2–9.6) | 6.2 (2.6–10.6) |
| Number of children | 1 (0–2) | 1 (0–2) | 1 (0–3) |
| Sex with outside partner, prior 30 days | 92 (3.6%) | 16 (3.6%) | 7 (2.0%) |
| Any unprotected sex, prior 30 days | 725 (28.2%) | 120 (27.1%) | 117 (33.0%) |
| Clinical characteristics | |||
| CD4 count, cells/mm3 | 458 (345–626) | 471 (352–640) | 467 (348–650) |
| Any sexually transmitted infection* | 380 (14.8%) | 56 (12.6%) | 51 (14.4%) |
| Antiretroviral (ART) tested | N=430 | N=341 | |
| NRTIs | |||
| Abacavir (ABC) | 0 (0%) | 1 (0.3%) | |
| Zidovudine (AZT) | 0 (0%) | 9 (2.6%) | |
| Lamivudine (3TC) | 12 (2.8%) | 148 (43.4%) | |
| Stavudine (D4T) | 3 (0.7%) | 43 (12.6%) | |
| Didanosine (DDI) | 0 (0%) | 1 (0.3%) | |
| NNRTIs | |||
| Nevirapine (NVP) | 11 (2.6%) | 126 (37.0%) | |
| Any ART detected | 14 (3.3%) | 157 (46.0%) |
Including Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis
ART testing was performed on 771 (96.6%) of the persons with plasma HIV-1 RNA <2000 copies/mL where specimens were available. Antiretrovirals were detected in 171/771 (22.2%): 157/341 (46.0%) in those with undetectable plasma HIV-1 RNA (<240 copies/mL) and 14/430 (3.3%) in those with low detectable plasma HIV-1 RNA (240–2000 copies/mL). The most common ARTs detected were lamivudine (20.8%) and nevirapine (17.8%). Most, (83.6%), of the 171 participants with detectable ARTs had evidence of multiple drugs, specifically the combinations nevirapine/lamivudine (52.0%), nevirapine/lamivudine/stavudine (21.6%), stavudine/lamivudine (5.3%), and zidovudine/lamivudine with or without nevirapine (4.1%). Differences in ART detection were found among the study sites, but there were no difference in ART detection between men and women.
We have previously reported that nearly a quarter (23.7%) of HIV-1 infected partners in HIV-1 serodiscordant partnerships for the Partners in Prevention HSV/HIV Transmission Study had plasma HIV-1 levels <2000 copies/mL at baseline, in the absence of reported use of ART [6]. Our analysis here demonstrates that 22% of those, and nearly half of the subset with undetectable plasma HIV-1, had evidence of unreported ART use. Thus, undetectable plasma HIV-1 RNA is a potential marker of unreported ART use in HIV-1 infected partners. This finding could be significant for studies focused on describing host factors associated with natural viral control, since a large proportion of individuals with low plasma HIV-1 levels had pharmacologic and not immunologically induced viral suppression. For example, in genetic studies of elite controllers, the inclusion of subjects with ART-induced viral suppression would undermine the ability of the study to identify any potentially valuable genetic markers.
It is important to note that while unreported ARV detection was strongly associated with plasma HIV-1 level (48% for undetectable versus only 3% for detectable but <2000 copies/mL), the proportion of individuals in the overall study cohort with unreported ART use and detectable ARVs was very small (171/3408, 5%) and equally distributed between the randomization arms in the clinical trial. For these reasons, it is unlikely that unreported ART use would have an important impact on the overall outcomes of this clinical trial, or other randomized clinical trials of this kind.
Since ART use was an exclusion criterion in this study and thus by definition was not reported, inferences about reasons or circumstances underlying this finding are largely speculative. Women had a significantly higher proportion of plasma HIV-1 RNA <2000, suggesting women may have been more likely to have received ART, possibly clinically indicated for prevention of mother-to-child transmission (PMTCT) for which nevirapine and lamivudine are included in recommended regimens and also prominently represented among the ARVs detected in this analysis [10–11]. However, in our analysis, we did not find significant difference in ART by gender and do not have evidence that women were more likely to have unreported ART detected. Without knowing specific timing of recent doses, we cannot make any determination on whether or not persons with detected ARV discontinued drug use prior to study screening or what drug dose was taken. Nevirapine can be detected in women more than two weeks after receiving single-dose nevirapine for PMTCT [12–13], while most nucleoside reverse transcriptase inhibitors such as lamivudine and stavudine can be detected, at most, for a few days after discontinuation [14].
In resource-limited communities, services and other benefits offered through clinical trial participation could provide an incentive to not disclose ART use, which would have made them ineligible for the trial [15]. Couples enrolled in the Partners in Prevention HSV/HIV Transmission Study did not receive a monetary incentive for participation but did receive benefits including free counseling, screening and treatment for sexually transmitted infections, condoms and travel reimbursement. One prior study of participation in a large-scale HIV-1 prevention trial in South Africa concluded that the level of reimbursement could be a motivating factor for some participants to misreport information during enrollment screening [16]. In multiple studies of willingness to participate in HIV-1 clinical trials in different settings, the majority of participants reported primarily altruistic motivations for participants, although a minority or respondents stated monetary incentives and access to health care as the primary motivator for participation [17–19]. We recommend further research to understand nondisclosure of ART by participants enrolling in an HIV-1 prevention study.
In summary, we found unreported use of ART to be prevalent among HIV-1 infected individuals with undetectable plasma virus. For randomized control trials of HIV-1 prevention interventions in HIV-1 serodiscordant couples or of novel HIV-1 treatments, assessing viral load may improve the efficiency of the study, by excluding those with low viral loads who would be unlikely to transmit or have a substantial virologic response to treatment. More importantly, for observational studies of pathogenesis and transmission, it may be critical to understand the etiology of undetectable viral loads, and thus particularly important to identify unreported ART use. Studies recruiting HIV-1 infected participants with low levels of plasma HIV-1 RNA, should consider laboratory testing for ART.
Acknowledgments
We gratefully acknowledge the contributions of couples that participated in this study and teams at the study sites and at the University of Washington.
Funding statement: This study was supported by research grants from the Bill and Melinda Gates Foundation (grant ID #26469), the US National Institutes of Health (R01 MH095507), University of Washington Center for AIDS Research Clinical Retrovirology Core (UW CFAR, NIH AI-27757) and the University of North Carolina Center for AIDS Research (UNC CFAR P30-A150410)
Partners in Prevention HSV/HIV Transmission Study Team
University of Washington Coordinating Center and Central Laboratories, Seattle, USA: Connie Celum (principal investigator), Anna Wald (protocol co-chair), Jairam Lingappa (medical director), Jared M. Baeten, Mary Campbell, Lawrence Corey, Robert W. Coombs, James P. Hughes, Amalia Magaret, M. Juliana McElrath, Rhoda Morrow, James I. Mullins
Study sites and site principal investigators: Cape Town, South Africa (University of Cape Town): David Coetzee; Eldoret, Kenya (Moi University, Indiana University): Kenneth Fife, Edwin Were; Gaborone, Botswana (Botswana Harvard Partnership): Max Essex, Joseph Makhema; Kampala, Uganda (Infectious Disease Institute, Makerere University): Elly Katabira, Allan Ronald; Kigali, Rwanda (Rwanda Zambia HIV Research Group, and Emory University): Susan Allen, Kayitesi Kayitenkore, Etienne Karita; Kisumu, Kenya (Kenya Medical Research Institute, University of California San Francisco): Elizabeth Bukusi, Craig Cohen; Kitwe, Zambia (Rwanda Zambia HIV Research Group, and Emory University): Susan Allen, William Kanweka; Lusaka, Zambia (Rwanda Zambia HIV Research Group, and Emory University): Susan Allen, Bellington Vwalika; Moshi, Tanzania (Kilimanjaro Christian Medical College, Harvard University): Saidi Kapiga, Rachel Manongi; Nairobi, Kenya (University of Nairobi, University of Washington): Carey Farquhar, Grace John-Stewart, James Kiarie; Ndola, Zambia (Rwanda Zambia HIV Research Group, and Emory University): Susan Allen, Mubiana Inambao; Orange Farm, South Africa (Wits Reproductive Health & HIV Institute, University of the Witwatersrand): Sinead Delany-Moretlwe, Helen Rees; Soweto, South Africa (Perinatal HIV Research Unit, University of the Witwatersrand): Guy de Bruyn, Glenda Gray, James McIntyre; Thika, Kenya (University of Nairobi, University of Washington): Nelly Rwamba Mugo
Footnotes
Conflict of Interest Statement: None of the authors have commercial or other conflicts of interest related to the contents of this manuscript
Previous Presentation: This work was presented in part at the 2012 International Microbicides Conferences, Sydney, Australia, April 15-18 2012. Abstract 347.
References
- 1.Quinn TC, Wawer MJ, Sewankambo N, Serwadda D, Li C, Wabwire-Mangen F, Meehan MO, Lutalo T, Gray RH. Viral load and heterosexual transmission of human immunodeficiency virus type 1. Rakai Project Study Group. N Engl J Med. 2000 Mar 30;342(13):921–929. doi: 10.1056/NEJM200003303421303. [DOI] [PubMed] [Google Scholar]
- 2.Gray RJ, Wawer MJ, Brookmeyer R, et al. Probability of HIV-1 transmission per coital act in monogamous, heterosexual, HIV-1-discordant couples in Rakai, Uganda. Lancent. 2001;357(9263):1149–1153. doi: 10.1016/S0140-6736(00)04331-2. [DOI] [PubMed] [Google Scholar]
- 3.Lingappa JR, Hughes JP, Wang RS, et al. Estimating the impact of HIV-1 RNA reductions on heterosexual HIV-1 transmission risk. PLoS One. 2010 Sep 13;5(9):e12598. doi: 10.1371/journal.pone.0012598. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Cohen MS, Chen YQ, McCauley M, et al. Prevention of HIV-1 infection with early antiretroviral therapy. N Engl J Med. 2011 Aug 11;365(6):493–505. doi: 10.1056/NEJMoa1105243. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Donnell D, Baeten JM, Kiarie J, et al. Heterosexual HIV-1 transmission after initiation of antiretroviral therapy: a prospective cohort analysis. Lancet. 2010;375(9731):2092–2098. doi: 10.1016/S0140-6736(10)60705-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Lingappa JR, Kahle E, Mugo N, et al. Characteristics of HIV-1 discordant couples enrolled in a trial of HSV-2 suppression to reduce HIV-1 transmission: The Partners Study. PLoS One. 2009;4(4):e5272. doi: 10.1371/journal.pone.0005272. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Mujugira A, Baeten JM, Donnell D, et al. Characteristics of HIV-1 serodiscordant couples enrolled in a clinical trial of antiretroviral pre-exposure prophylaxis for HIV-1 prevention. PLoS One. 2011;6(10):e25828. doi: 10.1371/journal.pone.0025828. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Celum C, Wald A, Lingappa JR, et al. Acyclovir and transmission of HIV-1 from persons infected with HIV-1 and HSV-2. N Engl J Med. 2010;362(5):427–439. doi: 10.1056/NEJMoa0904849. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Rezk NL, Tidwell RR, Kashuba AD. Simultaneous determination of six HIV nucleoside analogue reverse transcriptase inhibitors and nevirapine by liquid chromatography with ultraviolet absorbance detection. J Chromatogr B Analyt Technol Biomed Life Sci. 2000 Jul 5;791(1–2):137–147. doi: 10.1016/s1570-0232(03)00224-1. [DOI] [PubMed] [Google Scholar]
- 10.WHO. HIV/AIDS Programme. World Health Organization; Geneva, Switzerland: 2006. Antiretroviral therapy for HIV infection in adults and adolescents: recommendations for a public health approach, 2006 revision. [Google Scholar]
- 11.WHO. Rapid advice: Use of antiretroviral drugs for treating pregnant women and preventing HIV infection in infants. World Health Organization; Geneva, Switzerland: Jun, 2010. version 2. [PMC free article] [PubMed] [Google Scholar]
- 12.Muro E, Droste JA, Hofstede HT, et al. Nevirapine plasma concentrations are still detectable after more than 2 weeks in the majority of women receiving single-dose nevirapine: implication for intervention studies. J Acquir Immune Defic Syndr. 2005 Aug 1;39(4):419–421. doi: 10.1097/01.qai.0000167154.37357.f9. [DOI] [PubMed] [Google Scholar]
- 13.Mackie NE, Fidler S, Tamm N, et al. Clinical implications of stopping nevirapine-based antiretroviral therapy: relative pharmacokinetics and avoidance of drug resistance. HIV Med. 2004 May;5(3):180–184. doi: 10.1111/j.1468-1293.2004.00208.x. [DOI] [PubMed] [Google Scholar]
- 14.Piliero PJ. Pharmacokinetic properties of nucleoside/nucleotide reverse transcriptase inhibitors. J Acquir Immune Defic Syndr. 2004 Sep 1;37(S1):S2–S12. doi: 10.1097/01.qai.0000137001.40505.56. [DOI] [PubMed] [Google Scholar]
- 15.Grady C. Payment of clinical research subjects. J Clin Invest. 2005 Jul;115(7):1681–1687. doi: 10.1172/JCI25694. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Ramjee G, Coumi N, Dladla-Qwabe N, et al. Experiences in conducting multiple community-based HIV prevention trials among women in KwaZulu-Natal, South Africa. AIDS Res Ther. 2010 Apr 23;7:10. doi: 10.1186/1742-6405-7-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Ruzagira E, Wandiembe S, Bufumbo L, et al. Willingness to participate in preventive HIV vaccine trials in a community-based cohort in south western Uganda. Trop Med Int Health. 2009 Feb;14(2):196–203. doi: 10.1111/j.1365-3156.2008.02200.x. [DOI] [PubMed] [Google Scholar]
- 18.Colfax G, Buchbinder S, Vamshidar G, et al. Motivations for participating in an HIV vaccine efficacy trial. J Acquire Immune Defic Syndr. 2005 Jul 1;39(3):359–364. doi: 10.1097/01.qai.0000152039.88422.ec. [DOI] [PubMed] [Google Scholar]
- 19.Bentley JP, Thacker PG. The influence of risk and monetary payment on the research participation decision making process. J Med Ethics. 2004 Jun;130(3):293–298. doi: 10.1136/jme.2002.001594. [DOI] [PMC free article] [PubMed] [Google Scholar]