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CLINICAL SCIENCE

Female genital tract shedding of HIV-1 is rare in women with suppressed HIV-1 in plasma

Nelson, Julie A.E.a,b; De Paris, Kristinaa,b; Ramirez, Catalinac; Edmonds, Andrewe; Mollan, Katie R.a,e; Bay, Camden P.a; Compliment, Karaa; Herold, Betsy C.f; Anastos, Kathryng; Minkoff, Howardh; Kassaye, Seblei; Seidman, Dominika L.j; French, Audrey L.k; Golub, Elizabeth T.l; Sheth, Anandi N.m; Ochsenbauer, Christinan; Swanstrom, Ronalda,d; Eron, Joseph J.a,c; Adimora, Adaora A.a,c,e

Author Information
doi: 10.1097/QAD.0000000000002373

Abstract

Introduction

HIV-1 shedding in the female genital tract is the source of virus for female-to-male sexual transmission [1]. Women with plasma viremia shed HIV-1 from the genital tract; studies have detected HIV-1 in the genital tract of 20–80% of women with plasma viremia [2–7]. HIV-1 is more likely to be detected in the genital tract of women with higher plasma viral loads (PVLs) than in women with low PVL [2,5,8–10]. Sexually transmitted infections and genital inflammation also appear to increase genital HIV-1 shedding [5,11]. Even when PVL is undetectable in women on antiretroviral therapy (ART), genital shedding has been detected in 2–34% of samples [4,7,11–14]. The design and methods of these studies have varied substantially with respect to duration and durability of PVL suppression, anatomic location of sampling (cervix versus vagina), and sampling technique.

ART leads to undetectable levels of HIV-1 in the blood and virtually eliminates the risk of sexual transmission [15,16]. Accordingly, public health agencies now promote the message that people whose PVL is undetectable have zero risk of sexually transmitting HIV-1 to others (Undetectable = Untransmittable, U = U) [17]. Questions persist, however, concerning the amount of virus present in the genital tract of women who receive suppressive ART. There are clinical implications for both sexual transmission and perinatal transmission of HIV-1, particularly in the setting of genital tract infection or inflammation. We evaluated the frequency and amount of HIV-1 shedding from the genital tract of women with long-term PVL suppression among participants in the Women's Interagency HIV Study (WIHS), a well characterized cohort of women living with or at risk for HIV infection.

Methods

Recruitment, data collection, and characteristics of WIHS participants have been previously reported [18–20]. Study visits occur every 6 months and include quantitation of PVL, clinical evaluation of genital inflammation, and collection of cervicovaginal lavage (CVL) using 10 ml of saline (0.9% sodium chloride).

Study groups

We selected two different groups of WIHS participants for our present study. Women in Group 1 (n = 294) were selected because they presented with suppressed plasma viremia, had longitudinal PVL and CVL samples available for five consecutive study visits postsuppression, and exhibited signs of clinical inflammation in the vaginal tract during one or more of those five study visits. Clinical inflammation was defined as being positive for at least one of the following seven inflammatory conditions: vaginal pH more than 5.5 (suggestive of bacterial vaginosis), visible cervical lesions, cervical ectopy, cervical friability, cervical exudate, trichomoniasis (diagnosed by wet mount), and inflammation noted on Pap smear (interpreted centrally for the WIHS). Upon identification of these women, one of the five longitudinal CVL samples (usually the third, or midpoint, visit) was selected for testing of genital HIV shedding. The 294 women had PVL suppressed for a median of 7.1 years (range = 0.2–17.0 years, IQR = 3.4–9.8 years). The group 1 samples were collected between 2002 and 2012, during which time the lower limit of quantification (LLQ) for plasma viremia decreased from 80 copies/ml (bioMerieux Nuclisens: bioMerieux, Marcy-l’étoile, France) to 20 copies/ml (Roche Taqman; Roche Diagnostics, Indianapolis, Indiana, USA).

Group 2 constituted 38 HIV-positive women who were selected if their CVL specimens were available at the visit prior to ART initiation and at the 1-year visit after PVL suppression (approximately 1.5 years after the first sample). These samples were collected from women between 1995 and 2013, during which the LLQ for PVL testing decreased from 4000 copies/ml (NASBA) to 20 copies/ml (Roche TaqMan). Group 2 women were further selected to be in four subgroups based on the number of clinical inflammation markers at the pre-ART and 1-year-suppressed visits, having: at least one clinical inflammation marker at both visits (Inflammation/Inflammation or I/I; n = 10), at least one marker at only the pre-ART visit (Inflammation/No inflammation or I/N; n = 10), at least one marker at only the 1-year-suppressed visit (No inflammation/Inflammation or N/I; n = 10), or no markers at either visit (No inflammation/No inflammation or N/N; n = 8). These inflammation criteria and CVL availability constrained the number of women who could be included, so we did not expand the number of women in group 2 beyond these 38. Unfractionated CVL samples from both time points were tested for genital shedding of HIV.

HIV-1 RNA quantitation in cervicovaginal lavage

The optimization of HIV-1 RNA quantitation in unfractionated CVL samples is described in the Supplemental Materials, http://links.lww.com/QAD/B529, with results of the optimization shown in Supplemental Table S1. CVL specimens were tested for HIV-1 RNA levels with the Abbott RealTime HIV-1 Assay after pretreatment with proteinase K. Specifically, 0.06 ml Abbott Proteinase K (catalog# 03L78–060) was added to 0.74 ml CVL and manually mixed by pipetting, incubated at 53 °C for 20 min, vortexed briefly, and spun at 3200g for 5 min. The mixture was run on the Abbott m2000sp with the 0.6 ml plasma program, and quantified on the Abbott m2000rt. Results were corrected for the small dilution, so the LLQ was 42 copies/ml.

Biomarker testing

Inflammation biomarkers were quantified in CVL from group 2 women using a Milliplex 17-plex kit (MilliporeSigma, Burlington Massachusetts, USA) run on Luminex MagPix (GM-CSF, IFN-γ, IL-10, IL-12p40, IL-12p70, IL-15, IL-1RA, IL-1α, IL-2, IL-4, IL-6, IL-8, IP10, MCP-1, MIP-1β, RANTES, and TNFα). Five additional biomarkers were tested by ELISA: CD163 (R&D Systems, Minneapolis, Minnesota, USA), SLPI (R&D Systems), IFN-α (PBL Assay Science, Piscataway, New Jersey, USA), and beta-defensins two and three (Assay Biotechnology Company, Sunnyvale, California, USA).

Statistical analysis

The unadjusted association between log10 PVL and detectable viral load in CVL was analyzed using logistic regression to estimate an odds ratio (OR), and by Spearman rank correlation. Likewise, logistic regression was used to assess the association between clinical inflammation marker category (0, 1, or 2+ markers) and detectable viral load in CVL; a three-category functional form was selected using visual assessment of the model fit. Associations between the number of clinical markers (range: 0--5) and biomarker levels were evaluated using Kendall's tau-b correlation coefficient at the pre-ART and post-ART time points, separately. To account for conducting 42 statistical tests (21 evaluated biomarkers × two time points), a Benjamini--Hochberg false discovery rate (FDR) P-value adjustment was applied. At the pre-ART visit, biomarker levels were compared by viral shedding status (detectable vs. not detectable) using a Wilcoxon rank-sum test and a Hodges--Lehmann 95% confidence interval (CI) for location shift; a Benjamini--Hochberg FDR P-value adjustment was applied. Left and right censoring of biomarker levels was handled by setting values below or above the cutoffs to the lowest or highest rank, respectively. IFNα was removed from the analysis because of a lack of variability.

Results

Absence of HIV-1 shedding in cervicovaginal lavage in women on suppressive antiretroviral therapy for a median of 7 years

The women in group 1 were aged 25–74 years, with a median of 45 years (IQR = 40–51 years); 52% were premenopausal, 12% perimenopausal, and 36% postmenopausal (all self-reported) and had suppressed PVL for a median of 7.1 years (IQR = 3.4–9.8 years). HIV-1 shedding has been associated with genital tract inflammation [5,11], and, therefore, we purposely biased our sample selection toward women on suppressive ART who showed clinical evidence of inflammation. Two-thirds of the group 1 women (193 of 294, 66%) presented with at least one of the seven clinical markers of inflammation (see Methods) at the tested visit (108 with exudate, 79 with vaginal pH less than 5.5, 47 with inflammation noted on Pap, 26 with cervical friability, nine with cervical ectopy, eight with trichomoniasis, and six with cervical lesions). However, HIV-1 shedding was not detected in any of the CVL specimens among the group 1 women (95% CI: 0–1.2%). When we tested CVL collected at the preceding visit from 30 randomly chosen women among those with inflammation at the initially tested visit, HIV-1 was again not detectable. The absence of genital HIV shedding was confirmed by spiking with HIV-1 duplicate CVL samples from nine randomly chosen women from group 1 to demonstrate that HIV-1 could be detected if present (1907 copies/ml were detected in the spiked PBS control and 415–1557 copies/ml were detected in the nine spiked CVL samples).

Transition from shedding in the absence of antiretroviral therapy to no shedding on suppressive antiretroviral therapy

To assess the potential impact of inflammation on HIV-1 shedding in the genital tract around the time of ART initiation, we selected a second group of WIHS participants for whom two CVL specimens were available that were collected just prior to ART initiation and at approximately 1 year after onset of PVL suppression (Table 1). We hypothesized that 1 year of suppression was a time when most women should have stopped shedding, but likely close enough to ART initiation to evaluate the impact of inflammation. Close to 50% (18 of 38; 47%) of the women in group 2 had detectable HIV-1 RNA in their pre-ART CVL samples. CVL virus load ranged from 44 to 4776 copies/ml with a median value of 668 copies/ml (IQR = 235–2305 copies/ml), and was correlated with the magnitude of pre-ART plasma viremia (n = 37 evaluable, Fig. 1). A 1.0 log10 increase in PVL corresponded to 2.9-times the odds of detectable CVL viral load (estimated OR = 2.9, 95% CI 1.2--6.9, P = 0.01; Supplemental Figure S1, http://links.lww.com/QAD/B529). This result confirmed that the failure to detect shedding virus in women with long-term suppression was not because of an inability to detect virus when it is present.

Table 1
Table 1:
HIV-1 quantitation in cervicovaginal lavage and plasma among 38 participants in the Women's Interagency HIV Study before antiretroviral therapy initiation and 1 year after achievement of viral suppression.
Fig. 1
Fig. 1:
HIV-1 RNA levels in plasma and cervicovaginal from 37 Women's Interagency HIV Study participants before antiretroviral therapy initiation.CVL with detectable viral loads are shown in gray circles and CVL with undetectable viral loads are shown in white circles. CVL, cervicovaginal,

There was no clear association between detectable CVL HIV-1 RNA and clinical inflammation, although only one woman in the N/N group (no clinical inflammation markers at either visit) had detectable viral load in her CVL at the pre-ART visit, and this was the sample that was detectable below the quantitation limit of the assay (listed as less than 42 in Table 1). Overall, 55% of CVL samples from visits with clinical inflammation markers had detectable HIV-1 RNA, compared with 39% of samples from visits without clinical inflammation markers.

After 1 year of suppressed PVL on ART, one CVL sample from group 2 had detectable CVL HIV-1 RNA (770 copies/ml). This woman had no markers of clinical inflammation at the second visit, but had one inflammation marker (vaginal pH more than 5.5) at her pre-ART visit, when viral RNA was not detectable in her CVL specimen. The group 2 women (at the latter visits) were aged 29–65 years, with a median of 47 years (IQR = 36–53 years); 66% were premenopausal, 8% perimenopausal, and 26% postmenopausal (all self-reported). In summary, only 1 of the 38 CVL samples collected after 1 year of suppressed PVL had detectable HIV-1 (2.6%, 95% CI 0.067--14%), whereas HIV-1 genital shedding was detected in 47% (18 of 38, 95% CI 31--64%) of the women before initiating therapy.

Role of clinical markers of inflammation in predicting HIV-1 shedding in the genital tract

Among the women in group 2 with clinical inflammation markers before starting ART and/or after 1 year of PVL suppression, the most common marker was high vaginal pH (more than 5.5). The presence and distribution of the clinical inflammation markers by group and visit is summarized in Table 2. To evaluate more closely the relationship between genital tract HIV-1 shedding and the presence of specific clinical inflammation markers in women with HIV-positive CVL samples (n = 18 women pre-ART and n = 1 post-PVL suppression), we examined whether the type of clinical marker or a combination of markers was associated with shedding. Although the small sample size (n = 38) limited the sensitivity of this analysis, we did not find an association between clinical marker category and the odds of detectable CVL viral load [OR = 1.2 (95% CI 0.24--6.1) P = 0.81] for 1 vs. 0 clinical markers, and OR = 4.3 [95% CI 0.80--23, P = 0.09] for at least 2 vs. 0 clinical markers. These results implied that genital tract HIV-1 shedding in these WIHS participants was not associated with inflammation based on clinical inflammation markers (Supplemental Figure S2, http://links.lww.com/QAD/B529). To further strengthen this conclusion, we examined whether known immune biomarkers of inflammation were correlated with clinical markers of inflammation. We measured 21 biomarkers in both the pre-ART visit and 1-year suppressed ART time point in group 2 CVL samples (Supplemental Figure S3, http://links.lww.com/QAD/B529). Nine of the biomarkers (GM-CSF, IL-8, IL-10, IL-4, IL-1α, MIP-1β, RANTES, CD163, IL-12p40) were positively correlated with the number of clinical inflammation markers found pre-ART and/or postsuppression, with Kendall tau-b correlation coefficients between 0.22 and 0.42 and unadjusted P-values less than 0.10 (Fig. 2). However, after false discovery rate adjustment, only the GM-CSF posttreatment correlation remained significant (P = 0.04) in this small number of samples. Further, among women in group 2 with clinical inflammation, no major differences in biomarker levels were detected at the pre-ART visit between women with detectable (n = 18) and women with nondetectable (n = 20) CVL HIV-1 RNA.

Table 2
Table 2:
Clinical markers of genital inflammation in 38 Women's Interagency HIV Study participants before antiretroviral therapy initiation and 1 year after achievement of viral suppressiona.
Fig. 2
Fig. 2:
Immune biomarker associations with number of clinical inflammation markers at pre-antiretroviral therapy initiation and postvirologic suppression visits of Women's Interagency HIV Study participants.Kendall tau-b values plotted for pre-ART (n = 37) and postsuppression (n = 38) for each of the 21 biomarkers [GM-CSF, IFN-γ, IL-10, IL-12p40, IL-12p70, IL-15, IL-1RA, IL-1α, IL-2, IL-4, IL-6, IL-8, IP10, MCP-1, MIP-1β, RANTES, TNFα, CD163, SLPI, IFN-α, beta-defensin two (BD2) and beta-defensin three (BD3)]. Clinical inflammation markers included vaginal pH more than 5.5, visible cervical lesions, cervical ectopy, cervical friability, cervical exudate, trichomoniasis, and inflammation noted on Pap smear. ART, antiretroviral therapy.

Discussion

The goal of this study was to assess the association between local inflammation in the female genital tract and HIV-1 shedding in women with suppressive ART. We selected two groups of women from the well characterized WIHS cohort, with women in group 1 being virally suppressed in plasma for a median of 7.1 years, whereas women in group 2 were suppressed for 1 year. Our results indicate that suppression of viremia, even in the presence of clinical inflammation, prevents genital tract HIV-1 shedding, further supporting the message that ‘undetectable is untransmittable’.

On the basis of previous studies [5,11–13,21,22], including those that reported the contribution of inflammation to genital tract HIV-1 shedding, we had expected 2–34% of the CVL samples to have detectable HIV-1 while on suppressive ART. However, within the limits of the sensitivity of our optimized HIV-1 RNA assay in CVL samples, none of the women in group 1, who were virologically suppressed for a median of 7.1 years, had detectable genital tract HIV-1 shedding. This result was consistent with a prior small-scale study by our group in which we also found no genital tract shedding of HIV-1 when we analyzed multiple specimen types in a group of women with suppressed plasma viremia [23].

The difference in the frequency of genital shedding during PVL suppression between our study (0% for group 1) and other studies (2–34%) may be because of differences in the duration of PVL suppression. Some studies did not specify the length of time on suppressive ART [5,11,21], whereas others followed women for up to 2 years after starting ART [12,13], but did not evaluate risk of shedding with longer time on ART. Another study reported that risk of HIV-1 transmission persists during the first 6 months after initiation of ART, likely due in part to incomplete HIV-1 suppression in genital compartments [24].

The latter findings are indirectly supported by our results in the women in group 2. Pre-ART, 47% of CVL samples of group 2 women tested HIV-1 RNA-positive, but only 1 woman had evidence of genital tract HIV-1 shedding after ART suppression for 1 year. These results confirm that ART markedly reduces genital tract HIV-1 shedding among women in the WIHS cohort, consistent with the absence of transmission in clinical studies [15,16].

In the current study, the detection of HIV-1 RNA in pre-ART CVL samples was correlated with plasma viremia. Thus, one could hypothesize that the kinetics of suppression in plasma viremia may directly influence the kinetics of suppression of genital tract shedding. Another variable that could impact the time to suppression might be the type of ART; evaluation of the relationship between different ART regimens and genital tract HIV-1 shedding was outside the scope of the current study.

The difference in frequency of genital shedding between our study and others is unlikely to be because of the use of different assays or different assay sensitivity. In fact, we identified a similar proportion of genital tract HIV-1 shedding among women who were not on ART (47%) as reported in other studies [5,11–13,21]. The odds of genital tract HIV-1 shedding were associated with PVL level at these pre-ART visits, with 2.9 times the odds of detectable shedding for each 10-fold increase in PVL (95% CI 1.2--6.9), which is similar to the odds ratio of 6.1 (95% CI 2.9--12.9) reported by Kovacs et al.[2] among WIHS participants who were younger (18–45 years) than those in our study. These results are also similar to those reported by Homans et al.[3] of approximately 2.5 times the odds of detectable shedding for every 10-fold increase in initial PVL, although their study included detection of genital shedding of HIV-1 at multiple visits.

All of the WIHS participants studied here were selected based on the presence or absence of seven specific clinical inflammation markers documented in the WIHS cohort. Previous studies showed that genital inflammation, evidenced by either clinical inflammation markers or measurement of cytokines, was associated with higher risk of genital tract HIV-1 shedding in women both on and off ART [3,5,6,25–30]. As most of the women in our study (213 of 332) had at least one clinical inflammation marker while on suppressive therapy and nearly all (331 of 332 women in groups 1 and 2 combined) had undetectable genital tract HIV-1 shedding post-ART, our results suggest that genital inflammation does not lead to detectable shedding if sufficiently suppressive ART therapy is present. This result appears to be in contrast to other studies [3,7,11,26], including those with the WIHS cohort [3,5], that demonstrated an association between genital inflammation and the odds of genital tract HIV-1 shedding. However, as some immune biomarkers correlated with the presence of clinical inflammation, the lack of a statistically significant association between genital tract HIV-1 shedding and clinical inflammation observed in the current study was unlikely because of under-diagnosis or over-diagnosis of inflammation, but is more likely because of the small size (n = 38) of the group of participants we studied.

In conclusion, our results are consistent with studies that have documented a decline in genital tract shedding of HIV-1 at a similar rate to that in plasma after ART initiation [4,31,32]. These data indicate that successful suppression of PVL by ART reduces female genital tract shedding to undetectable levels, in accordance with studies that show suppressive ART reduces transmission risk to near zero (untransmittable) [16,33]. The duration of HIV-1 suppression may be an important factor in the relationship between viral suppression and absence of transmission. These data should further strengthen providers’ messages as they counsel women and their sexual partners that ‘Undetectable is untransmittable.’

Acknowledgements

J.A.E.N., K.D.P., C.R., A.E., R.S., J.J.E., and A.A. conceived of and designed the study. C.R., A.E., B.C.H., K.A., H.M., S.K., R.M.G., A.L.F., E.T.G., A.N.S., and C.O. collected and/or managed the data. K.C. performed CVL viral load testing. J.A.E.N. performed immune biomarker testing. K.R.M, and C.P.B. analyzed the data with input from J.A.E.N and K.D.P. J.A.E.N. and K.D.P. drafted the manuscript. All authors revised the manuscript and gave final approval.

This study was funded by the National Institutes of Health through the WIHS (U01 AI103390), the UNC Center for AIDS Research (P01 AI050410), and the other WIHS sites (U01-AI-103401, U01-AI-103408, U01-AI-035004, U01-AI-031834, U01-AI-034993, U01-AI-034994, U01-AI-103397, U01-AI-034989, U01-AI-042590, U01-HD-032632). WIHS data collection is also supported by NIH grants UL1-TR000004, UL1-TR000454, P30-AI-050410, and P30-AI-027767.

Data in this manuscript were collected by the Women's Interagency HIV Study (WIHS). The contents of this publication are solely the responsibility of the authors and do not represent the official views of the National Institutes of Health (NIH). WIHS (Principal Investigators): UAB-MS WIHS (Mirjam-Colette Kempf and Deborah Konkle-Parker), U01-AI-103401; Atlanta WIHS (Ighovwerha Ofotokun, Anandi Sheth, and Gina Wingood), U01-AI-103408; Bronx WIHS (Kathryn Anastos and Anjali Sharma), U01-AI-035004; Brooklyn WIHS (Deborah Gustafson and Tracey Wilson), U01-AI-031834; Chicago WIHS (Mardge Cohen and Audrey French), U01-AI-034993; Metropolitan Washington WIHS (Seble Kassaye and Daniel Merenstein), U01-AI-034994; Miami WIHS (Maria Alcaide, Margaret Fischl, and Deborah Jones), U01-AI-103397; UNC WIHS (Adaora Adimora), U01-AI-103390; Connie Wofsy Women's HIV Study, Northern California (Bradley Aouizerat and Phyllis Tien), U01-AI-034989; WIHS Data Management and Analysis Center (Stephen Gange and Elizabeth Golub), U01-AI-042590; Southern California WIHS (Joel Milam), U01-HD-032632 (WIHS I – WIHS IV). The WIHS is funded primarily by the National Institute of Allergy and Infectious Diseases (NIAID), with additional co-funding from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), the National Cancer Institute (NCI), the National Institute on Drug Abuse (NIDA), and the National Institute on Mental Health (NIMH). Targeted supplemental funding for specific projects is also provided by the National Institute of Dental and Craniofacial Research (NIDCR), the National Institute on Alcohol Abuse and Alcoholism (NIAAA), the National Institute on Deafness and other Communication Disorders (NIDCD), and the NIH Office of Research on Women's Health. WIHS data collection is also supported by UL1-TR000004 (UCSF CTSA), UL1-TR000454 (Atlanta CTSA), P30-AI-050410 (UNC CFAR), and P30-AI-027767 (UAB CFAR).

Conflicts of interest

There are no conflicts of interest.

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Keywords:

cervicovaginal lavage; genital HIV-1 shedding; genital inflammation; inflammation biomarkers; suppressed; Women's Interagency HIV Study; women

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