Worldwide, 33.4 million people are infected with HIV type 1 with an estimated 2.7 million new infections yearly.1 Most infections are acquired sexually or perinatally. Higher plasma viral load (PVL) correlates with increased likelihood of sexual and perinatal transmission, although antiretroviral treatment (ART), in particular highly active antiretroviral therapy (HAART), correlates with reduced transmission.2,3,4,5 Presence of HIV in the female genital tract is important in perinatal4 and sexual transmission.6 Increased cervicovaginal HIV level (CV-VL) likely increases the risk of transmucosal HIV passage during penile–vaginal intercourse and vaginal delivery.
The quantity of HIV in the female genital tract correlates with various factors, including PVL,7 immune status,8 cervicovaginal inflammation,9 and ART.10,11 HIV characteristics, including tropism and resistance pattern, can differ markedly in different compartments, including the female genital tract,12,13 male genital tract,14 and central nervous system.15,16 HIV can be detected in cell-free and cell-associated components of cervicovaginal fluid.17 Local conditions in the female genital tract—including inflammation,18 bacterial vaginosis (BV),19 cervical ectopy,20 genital ulceration,18 candidiasis,21 herpes simplex virus (HSV) infection,22 trichomoniasis,20 and other infections—influence cervicovaginal HIV shedding. Menstrual cycle phase23,24 and hormonal contraception25 may affect shedding. ART, in particular HAART, is associated with decreased cervicovaginal HIV shedding.11,26
Discordance between HIV presence in the bloodstream and cervicovaginal shedding is frequent. HIV can be present in the genital tract despite undetectable PVL, suggesting local replication.27,28 Factors that may cause increased cervicovaginal shedding due to local replication include poor penetration of antiretrovirals into genital secretions,29 local resistance,13 and local inflammation.28 Conversely, HIV is sometimes absent in the genital secretions despite high PVL, suggesting that additional factors may be necessary to permit shedding.
Although previous studies have shown association between numerous conditions and cervicovaginal HIV shedding, the mechanisms for the presence and the level of HIV in the cervicovaginal compartment are not well understood.28 Several studies have evaluated the variation in cervicovaginal HIV shedding over various periods, particularly the possible influence of the menstrual cycle.23,24,30,31 However, the frequencies and determinants of different temporal patterns of cervicovaginal HIV shedding have not been fully characterized. The purpose of this study is to evaluate how behavioral, therapeutic, clinical, immunological, and local factors correlate, individually and collectively, with cervicovaginal HIV shedding, at single time points and as a longitudinal pattern.
This substudy was nested within the Women's Interagency HIV Study (WIHS), an ongoing multicenter prospective study of the natural history and treated history of HIV infection in women.32 WIHS participants provided informed consent for their participation in keeping with local, institutional, and national guidelines. All women who had quantitation of HIV-1 in cervicovaginal lavage fluid at least once as part of several WIHS substudies between November 11, 1994, and September 12, 2001, were included in this study.7,33
WIHS methods have been described previously.32 At baseline and each semiannual visit, subject history including health information, medications including antiretrovirals, sexual history, substance use, and other behavioral data were obtained using a standardized interview. Physical and gynecological examinations were performed. Blood and gynecological specimens were collected for local testing and repository storage. Genital tract assessment included visual inspection, speculum, and sometimes bimanual and rectal examination. Cervical lesions (ulcers, vesicles, fissures, or warts), ectopy (“beefy” redness extending from os onto cervix), friability (erythematous tissue that bleeds easily), and exudate (discharge of any type) were ascertained visually. CVL was collected by spraying 10 mL of sterile nonbacteriostatic saline against the cervical os and endocervix and aspirating from the posterior vaginal fornix. Unfractionated CVL was stored at −70°C.
Blood analyses included complete blood count, lymphocyte subsets, and plasma HIV-RNA (PVL). We determined lymphocyte subsets with standard flow cytometric techniques at local laboratories. Baseline serology for HSV-1 and HSV-2, and syphilis screening were performed with Western blot and rapid plasma reagin test, respectively, in a central laboratory.32 Baseline hepatitis C virus antibody testing was performed by Abbott enzyme immunoassays (version 2.0 or 3.0). Hepatitis C virus RNA levels were measured in a single laboratory (University of Southern California) by polymerase chain reaction (Roche Diagnostics, Indianapolis, IN).34 Baseline hepatitis B profile was also assessed in local laboratories.
We collected whole blood for PVL determination in sodium citrate cell preparation tubes. Plasma HIV quantitation was completed in 4 central laboratories. Initially, PVL was measured with a nucleic acid sequence–based amplification technique (Organon Teknika, Corp, Durham, NC), with a lower threshold of detection of 4000 copies per milliliter. Similar methods with greater sensitivity were used as they became available. Currently, WIHS uses the NucliSens (Organon Teknika, Corp) assay for the quantification of HIV-RNA in plasma with a lower limit of detection (LLD) of 80 copies per milliliter and with 1 mL of sample as input. In general, at the beginning of the study period (visits, 1–7), PVL had an LLD of 4000 copies per milliliter, whereas during visits 7–9, it improved to 400 copies per milliliter, and from visit 10 onward, it improved to 80 copies per milliliter. HIV quantitation in CVL (CV-VL) used the NucliSens assay (LLD of 80 copies/mL). However, these changes occurred in the laboratories at different times.
Cervicovaginal specimens included vaginal swabs for pH, potassium hydroxide preparation, Candida culture, saline preparation for microscopy, Gram stain, swabs for HSV culture if cervical/vaginal ulcer, fissure, or vesicle is present, syphilis screening if ulcer/fissure/vesicle(s) is present, endocervical swabs for gonorrhea/Chlamydia nucleic acid detection tests and Trichomonas culture, and a Papanicolaou (Pap) smear. CVL was tested for microscopic blood and semen. Pap smears were read in a central laboratory (Kyto Diagnostics, New York, NY). Squamous metaplasia (replacement of one normal type of epithelium with another), endocervical cells, inflammation (leukocytes on Pap smear), and inflammation-associated cellular changes (cellular changes found with inflammation including basophilic cytoplasm; enlarged unevenly sized nuclei; enlarged, irregular, or multiple nucleoli; repair) were ascertained from Pap smear. Gram stains were interpreted for BV in a central laboratory (University of Washington) using the Nugent score criteria, with categorization as normal (0–3), intermediate (4–6), or consistent (7–10).35 Trichomoniasis was diagnosed if motile trichomonads were present on wet mount or with positive culture. Candidiasis was diagnosed by the presence of pseudohyphae on potassium hydroxide preparation or positive culture. Polymerase chain reaction to identify 29 types of human papillomavirus (HPV) was performed in central laboratories.36 Separately, we analyzed several high-risk types of HPV (16, 18, 31, 33, and 35).
Demographic covariates included age (categorized as <35, 35–40, 41+ years) and self-identified race/ethnicity (White, African American, Hispanic, Other). HIV exposure category (intravenous drug use, heterosexual risk, transfusion risk, no identified risk, unknown) was specified. Behavioral covariates included current smoking (no, yes), current alcohol consumption (abstainer, <3, 3–13, ≥14 drinks/week), current injection drug use (no, yes), current use of other recreational substances (marijuana, cocaine, heroin, other), number of lifetime male sex partners (0–6, 7–29, 30+), number of male sex partners since last study visit (0, 1, 2+), and vaginal sex with male sex partners in the past 48 hours (no, yes). Therapeutic, immunological, and clinical covariates included the type of ART used since last visit (none, monotherapy, combination therapy, HAART), hormonal contraceptive use in the past 6 months (no, yes), PVL, CD4 cell count (≤200, 201–350, 351–500, and >500 cells/mm3), history of an AIDS-defining illness, hepatitis C status at baseline (antibody negative, antibody positive/RNA negative, antibody positive/RNA positive), and seropositivity for HSV-1 and HSV-2. The definition of HAART was guided by the Department of Health and Human Services/Kaiser Panel 2008.37 Local cervicovaginal covariates included vaginal pH (<4.5, 4.5–5.4, ≥5.5), abnormal Pap (no, yes), BV score,35 and the presence of the following: friability, ectopy, exudate, lesions, candidiasis, Trichomonas vaginalis, endocervical cells, squamous metaplasia, cervicovaginal inflammation, inflammation-associated cellular changes, and HPV (all types, oncogenic types).
Individual Visit Analysis, Level of Shedding
This analytic approach correlated data from individual person-visits with the level of CV-VL. To accommodate multiple visits per subject, regression models used generalized estimating equations with an exchangeable correlation matrix and an identity link function. The dependent variable for this linear regression model was log10 CV-VL. Visits where the CV-VL was less than the LLD (80 copies/mL) were assigned a value of 0.5 LLD (40). Visit-specific covariates were used from each visit at which CV-VL was measured. Plasma HIV-RNA was analyzed both as categorical variable (≤4000, 4001–9999, 10,000–39,999, 40,000–99,999, ≥100,000 copies/mL), where ≤4000 copies per milliliter was modeled as the referent group, and as a log10-transformed continuous variable, where values less than the LLD for the assay used were assigned a numeric value of 0.5 LLD. Factors were evaluated in unadjusted and multivariate models. The multivariate model retained factors associated with CV-VL in unadjusted analyses at P < 0.10 that remained at P < 0.10 on multivariate analysis. Associations were summarized as β-coefficients with associated standard errors. Two-sided hypotheses were assessed at the 5% significance level. Finally, we stratified person-visits into groups with detectable (≥80 copies/mL) and undetectable (<80 copies/mL) PVL and performed similar analyses on these strata to determine the shedding associations with PVL detectable and undetectable. Person-visits with undetectable PVL when LLD was 400 copies per milliliter (n = 24) and 4000 copies per milliliter (n = 10) were excluded from this analysis.
To better understand the role of cervicovaginal inflammation as a condition in shedding, we devised a tool to assess the presence/absence of any cervicovaginal inflammatory condition associated with shedding. We defined a binary variable, called inflammation summary variable (ISV), to have the value “1” if any local inflammatory factor significantly associated with CV-VL on univariate analysis was present (inflammation-associated cellular changes, cervical ectopy, exudate, friability, lesions, vaginal pH >5.5, intermediate BV score, consistent BV score, trichomoniasis) and “0” if none was present. Using this variable, we used Fisher exact test to evaluate the interaction between PVL detectability and inflammation (by ISV value) in correlation with the presence of shedding (Table 1). We used the generalized estimating equation to test for interaction between cervicovaginal inflammation and PVL in correlation with CV-VL.
Analysis of Longitudinal Shedding Pattern
This analytic approach categorized subjects into 3 groups to evaluate the pattern of cervicovaginal shedding over a series of visits. Categories were defined as follows: “never shedder” if a subject had no visits at which HIV was detected in cervicovaginal secretions; “intermittent shedder” if she had shedding at 1 or 2 visits; “persistent shedder” if she had shedding at 3 or more visits. Shedding category was assigned a 3-level variable (coded 0, 1, and 2) corresponding to never, intermittent, and persistent shedding, respectively. For uniformity, this analysis was restricted to all subjects with at least 3 visits over a 3-year period, where at most, 1 semiannual visit could be missed between evaluable visits. Data included visits from March 31, 1998 to September 12, 2001. Independent variables were the same as in the individual visit analysis but taken from the first evaluable visit. PVL was analyzed as a categoric variable (≤400, 401–3999, 4000–19,999, and ≥20,000 copies/mL) and as a log10-transformed continuous variable, where values less than the LLD for a particular assay were assigned a numeric value of 0.5 LLD. Summary variables were created to characterize the pattern of HAART use (never, intermittent, continuous) and PVL detection (always, sometimes, never) over the evaluated visits. Initial unadjusted analyses were performed using ordinal logistic regression followed by multivariate analyses, using all factors with P < 0.10 in univariate analysis, controlling for the type of ART (none, non-HAART, HAART) and PVL category. Similarly, unadjusted analyses and analyses adjusting for initial PVL were performed on the subgroup of patients with “always detectable PVL” but not on the “sometimes” and “never detectable” groups, due to insufficient cases of persistent shedding (2/50 and 0/24, respectively).
Overall, 481 women had a total of 976 visits at which genital shedding was evaluated. Baseline demographic and clinical characteristics of the study population are summarized in Table 2. The median number of person-visits was 1, and 31% had 3 or more person-visits. The median age at baseline was 36.3 years. Fifty-two percent of women were African American, and 33% Hispanic. 29.6% had baseline PVL below 4000 copies per milliliter (median = 16,000 copies/mL), while 25.5% had CD4 ≥500 cells per cubic millimeter. Tobacco use was reported at 38% of person-visits. Hormonal contraception was reported at 6% of person-visits. The distribution of risky behaviors included the following: heavy alcohol use, 11%; injection drug use, 36.5%; and >30 lifetime sex partners, 35.9%. Few women had evidence of Chlamydia (0.2%), gonorrhea (0%), or active HSV at baseline (0.2%). HSV seropositivity was common: HSV-1 in 83% and HSV-2 in 77%. 17.2% had a positive screening test for syphilis (rapid plasma reagin). Other cervicovaginal infections present at baseline included trichomoniasis (11.8%), HPV (all types) (52.7%), and BV (Nugent score 7–10) (47.1%). The percentage of participants receiving HAART increased from 0.3% at the initial visit, where shedding was measured, to 70% by the last visit of the study period.
Discordance Between PVL and CV-VL
Subgroups of person-visits stratified with respect to PVL, CV-VL, cervicovaginal inflammation, and antiretroviral therapy are shown in Table 1. Discordance between PVL and CV-VL occurred in 47% of person-visits. In 959 person-visits with measured PVL, 45.3% had detectable PVL/undetectable CV-VL, 32.6% had detectable PVL/detectable CV-VL, 20.4% had undetectable PVL and CV-VL, and 1.6% had undetectable PVL/detectable CVL. Significant inflammation, expressed by the “ISV,” was present in 76.5% and absent in 23.5% of evaluable person-visits (n = 958). CV-VL was detectable in 37.4% of person-visits at which inflammation was present (ISV = 1) and 24% at which it was absent (ISV = 0). When PVL was detectable, CV-VL was detectable in 264 (44.9%) of 587 person-visits with the presence of inflammation and 48 (30%) of 160 person-visits with its absence. In the subgroup with PVL ≥80 (LLD = 80), shedding was significantly more likely with the presence of inflammation (ISV = 1) (P = 0.003).
Factors Associated With Level of Shedding at Individual Person-Visits
Table 3 shows the variables associated with CV-VL level at individual person-visits. In multivariate analysis, higher CV-VL correlated significantly with higher PVL (β = 0.50 per log10 copies/mL; P <0.001), cervical inflammation-associated cellular changes (β = 0.38; P = 0.03), ectopy (β = 0.48; P = 0.009), exudate (β = 0.18; P = 0.005), and trichomoniasis (β = 0.31; P = 0.03). Lower CV-VL correlated with HAART use (β = −0.17; P = 0.01). CV-VL did not correlate with hormonal contraception use. The inflammatory summary variable correlated with increased CV-VL both in univariate analysis (β = 0.29; P = 0.001), and also in a separate multivariate model that included log10 HIV-RNA and antiretroviral therapy (β = 0.16; P = 0.004). PVL had a significant interaction with the ISV in correlating with CV-VL (interaction coefficient, 0.26; P = 0.006), meaning that, when inflammation is present, there is greater PVL/CV-VL correlation than when absent. Multivariate analysis (Table 4) of the PVL-detectable stratum (n = 665) showed strong correlation between higher CV-VL and PVL (for >100,000 copies/mL, β = 0.80; P < 0.001), friability (β = 0.23; P = 0.05), ectopy (β = 0.46; P = 0.02), exudate (β = 0.25; P = 0.001), trichomoniasis (β = 0.33; P = 0.04), and inflammation-associated cellular changes (β = 0.61; P = 0.007). In multivariate analysis of the PVL-undetectable stratum (n = 132 person-visits), cervicovaginal inflammatory conditions did not correlate with CV-VL. As expected, HAART correlated with decreased CV-VL (β = −0.39; P = 0.04).
Factors Associated With Pattern of Genital Shedding Over Multiple Visits
Of 481 evaluable women, 136 (31%) had CVL-VL measured at 3 or more visits within a 3-year period with a median of 4 evaluable visits (range, 3–6 visits) (Table 5). The shedding distribution included the following: 40.4% (n = 55) “never,” 44.9% (n = 61) “intermittent,” and 14.7% (n = 20) “persistent.” Ordinal logistic regression adjusted for ART showed that a pattern of higher frequency of shedding over visits (ie, intermittent/persistent vs. never; or persistent vs. intermittent/never) was associated with higher initial PVL [odds ratio (OR) = 2.47 per log10 copies/mL; P < 0.01; Table 5]. With adjustment for ART and PVL, higher shedding frequency also correlated with any alcohol use (OR = 2.20; P = 0.03) and seropositivity for HSV-2 (OR = 3.21; P = 0.009). Never detectable PVL correlated strongly with lower likelihood of higher shedding frequency (OR = 0.10; P < 0.001). Even in the subgroup with always detectable PVL, 18 (29%) of 62 had persistent shedding, but 20 (32%) never shed and 24 (39%) shed intermittently. In this subgroup, higher shedding frequency correlated with alcohol use (OR = 4.92; P = 0.003) and HSV-2 seropositivity (OR = 4.44; P = 0.04) (see Table, Supplemental Digital Content 1, https://links.lww.com/QAI/A269). Additionally, in this subgroup, vaginal candidiasis correlated with a 15-fold increase in the odds of higher shedding frequency (OR = 15.14; P = 0.009).
This study comprehensively assessed demographic, behavioral, clinical, therapeutic, and local factors that correlate individually and collectively with the level and pattern of HIV shedding in the female genital tract both at individual person-visits (cross sectionally) and longitudinally. The 2 analytic approaches are complementary. The person-visit analysis elucidates the association between identified factors and CV-VL at individual time points, whereas the longitudinal analysis clarifies how behaviors and conditions present at the initial visit, as well as summary variables measuring HAART adherence and PVL suppression over all visits, correlate with the temporal pattern of shedding. Subanalyses shed light on the discordance between PVL and cervicovaginal HIV presence, level, and pattern.
As shown previously,11,26,38 at individual person-visits, PVL and HAART are the principal factors associated with CV-VL, correlating with higher and lower levels, respectively. Correlation between PVL and CV-VL may be direct (due to transmigration of cell-free or cell-associated HIV from the bloodstream) indirect (related to local replication responding to the same factors as systemic replication) or both. Similarly, HAART may influence CV-VL directly by reducing local replication, indirectly by reducing HIV bloodstream replication, or both. Consistent with previous studies,18,39,40 local inflammatory conditions (diagnosed clinically) such as exudate, ectopy, friability, and the presence of lesions (microbiologically such as Trichomonas vaginalis and histologically such as inflammation-related cellular changes) correlated significantly with increased shedding in the person-visit analysis.
How local inflammatory conditions lead to increased cervicovaginal HIV shedding is not well understood. There may be several mechanisms, and their order of importance may vary depending on circumstances. Cervicovaginal inflammation may increase vascular permeability, allowing HIV transmigration from bloodstream to cervicovaginal compartment.18 Local inflammation may directly stimulate HIV replication or lead to recruitment of HIV-producing leukocytes from adjacent lymphoid tissue.22,39 When HIV is undetectable in the bloodstream, local replication (allowed by inadequate antiretroviral levels or resistance) may lead to detectable HIV in cervicovaginal secretions. Some authorities suggest that the source of most inflammation-associated cervicovaginal HIV is local replication.18,39
There was significant discordance between PVL and CV-VL both at individual person-visits and as a pattern. Similar to previous studies,27,28 CV-VL was detectable at 7.6% (15/211) of person-visits when PVL was undetectable. Surprisingly, CV-VL was undetectable at 58% (436/748) of person-visits when PVL was detectable. With detectable PVL, CV-VL correlated significantly with inflammatory conditions and PVL. Indeed, presence of inflammation (ISV = 1) led to increased correlation between PVL and CV-VL. This suggests that when local inflammatory conditions are present, a significant amount of HIV in cervicovaginal secretions is due to transmigration from the bloodstream, rather than local replication, likely due to a compromised bloodstream–tissue barrier. However, even in the absence of cervicovaginal inflammation, CV-VL was sometimes detectable. Conversely, in the undetectable PVL stratum, increased shedding did not correlate with local inflammatory conditions. This suggests that when antiretroviral suppression is effective, cervicovaginal inflammatory conditions are insufficient to cause shedding. HAART correlated with decreased CV-VL in the entire group and was the only factor to correlate with decreased CV-VL in the PVL-undetectable stratum. Although the main protective effect of HAART stems from PVL suppression, an additional protective effect may be suppression of cervicovaginal HIV replication, particularly when PVL is already low or undetectable. However, the number of person-visits with undetectable PVL was insufficient to draw reliable conclusions about all but very strong associations in this stratum.
In longitudinal analysis, even with always undetectable PVL, intermittent CVL shedding was sometimes present (5/24 subjects). Conversely, many subjects with always detectable PVL never had CVL HIV shedding (20/62 subjects). Persistent shedding in this group was associated with inflammatory factors such as HSV-2 seropositivity and vaginal candidiasis, as well as alcohol use. Thus, the longitudinal pattern of shedding correlates not only with HIV levels in the bloodstream but also with factors leading to cervicovaginal inflammation.
Interestingly, we noted an association between alcohol use and shedding persistence. Association between alcohol use and cervicovaginal shedding was demonstrated in one previous study.41 This is plausible, as alcohol affects HIV replication and susceptibility, causing increased Simian Immunodeficiency Virus replication in animal models42 and increased HIV replication in peripheral blood mononuclear cells and susceptibility of CD4 lymphocytes to HIV infection in vitro.43 No other behavior, including recent sexual intercourse, was associated with increased cervicovaginal shedding. Similar to previous studies,44 HSV-2 seropositivity without overt lesions correlated with shedding persistence. This may be due to inflammation from low-grade HSV replication.
Our study has several limitations. As an observational study, it cannot determine causation. During the study period, there were major changes in HIV quantitation and treatment and the resultant population health status. HIV levels in cervicovaginal secretions were measured in CVL, a semiquantitative method, rather than more precise means, such as cervical wick or swab. In addition, the presence of intracellular integrated HIV-provirus was not evaluated, possibly leading to underestimation of HIV quantity in CVL.
Undetectable PVL due to effective HAART is strongly associated with reduced CV-VL but does not assure shedding absence.28 Conversely, cervicovaginal HIV shedding may be undetectable without antiretroviral therapy and with high PVL. When HIV is present in the bloodstream, “permissive” factors, conditions, or behaviors associated with cervicovaginal inflammation are associated with increased PVL/CV-VL correlation, and thus correlate with increased shedding. “Protective” factors include HAART and control of such conditions or behaviors. Therefore, the prediction of cervicovaginal HIV shedding solely on the basis of ART and PVL is unreliable. As a practical matter, HIV-infected women should be counseled that cervicovaginal inflammatory conditions may increase the risk of sexual transmission of HIV, and medical providers should be advised to diagnose and treat such conditions as a means of reducing HIV transmission. Serodiscordant couples with perfect HAART adherence and consistently undetectable PVL in the infected partner should be advised that although sexual transmission is unlikely, consistent condom use combined with HAART remains the most reliable means of prevention.2,45 Further studies are needed to determine the source of HIV in cervicovaginal secretions and factors that lead to shedding despite the control of systemic replication.
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