CVL supernatant fluid added to U1 cell cultures increased HIV-1 viral expression compared with controls, as measured by p24 antigen in the cell culture supernatant. Antigen levels were significantly greater with unheated CVL fluid (median = 872.0 pg/mL, range: 0-22,311) than with heated CVL fluid (median = 154.5 pg/mL, range: 0-19,145.0; P < 0.0001). The quantitative results for unheated and heated HIF activity were strongly correlated (r = 0.63, P < 0.001). Quantitative HIF was strongly associated with BV by Nugent criteria assessing unheated (P < 0.0001) and heated (P < 0.0001) CVL fluid.
Using a cutoff value of 600 pg/mL of p24 antigen, equivalent to 3 SD above the control mean, HIF was present in unheated CVL fluid samples from 160 (54%) of 295 women and in heated CVL fluid samples from 66 (22%) of 295 women. Using this definition, HIF in unheated CVL fluid was present in 47 (42%) of 111 women with normal vaginal flora, in 10 (30%) of 33 women with intermediate flora (OR compared with normal flora = 0.59, 95% CI: 0.26, 1.36), and in 69 (77%) of 90 women with BV (OR compared with normal flora = 4.47, 95% CI: 2.42, 8.30).
Three different categories of HIF could be distinguished in heated CVL fluid (Fig. 1): activity that persisted in samples from 62 women (21%) and was called heat-stable HIF, activity that decreased below the cutoff in 98 women (33%) and was called heat-labile HIF, and activity that appeared for the first time in 4 women (1%) and was called heat-induced HIF.
Heat-stable HIF was detected in samples from 1 (1%) of 111 women without BV, 5 (15%) of 33 women with intermediate Nugent scores (OR = 19.64, 95% CI: 2.21, 174.94), and 43 (48%) of 90 women with BV (OR = 100.64, 95% CI: 13.46, 752.47) (P for trend <0.0001). Heat-labile HIF had an inverse association with intermediate flora (OR = 0.25, 95% CI: 0.09, 0.70) and no association with BV (OR = 0.57, 95% CI: 0.32, 1.04).
In unadjusted analysis, heat-stable HIF was also associated with vaginal discharge, trichomoniasis, and elevated vaginal pH but not with other genital tract conditions (see Table 2). Including all these genital conditions in the full logistic model for heated HIF, only intermediate flora and BV remained significant (see Table 2). Using backward elimination to create a reduced model containing only variables associated with heated HIF, only intermediate flora and BV remained associated (see Table 2). There were no significant associations between heat-labile HIF and any other genital tract conditions in unadjusted analysis, nor was heat-labile HIF associated with BV or other conditions using adjusted logistic regression or backward elimination (data not shown).
Additional analyses of HIF as a categorical or continuous variable in unheated and heated CVL fluid gave similar results. In bivariate analysis, the (categorical) presence of HIF in unheated CVL fluid was associated with BV, a high pH, and squamous intraepithelial lesions (SILs) on cytology; in the final logistic regression models, unheated HIF was associated only with BV (OR = 3.97, 95% CI: 1.28, 12.32) but not with intermediate flora. The analysis of heated HIF gave results essentially identical to those for heat-stable HIF (the group with heated HIF included 62 women with heat-stable HIF and 4 women with heat-induced HIF). In bivariate analysis of unheated and heated quantitative HIF (p24 antigen in supernatant as a continuous variable), there were strongly significant associations with BV, vaginal fluid pH, and trichomoniasis but not with the other variables. In the full multivariate gamma model, only BV remained significantly associated with unheated (P < 0.0001) or heated (P < 0.0001) HIF. Using backward elimination, the model for unheated quantitative HIF reduced to 2 variables: BV (P < 0.0001) and pH (P = 0.02). For heated quantitative HIF, the model also reduced to 2 variables: BV (P < 0.0001) and a low but not high level of red blood cells (RBCs) in CVL fluid (P = 0.004).
In unadjusted logistic regression models, any genital tract HIV-1 shedding was associated with heat-stable HIF activity (OR = 2.48, 95% CI: 1.21, 5.08; P = 0.01; Table 3). After adjustment for plasma HIV-1 RNA level, there was no significant association between HIF and genital tract HIV-1 shedding. Finally, in Weibull survival models, there were no statistically significant associations between quantitative HIV-1 RNA measures in CVL fluid or endocervical samples with heated or unheated HIF after adjusting for plasma HIV-1 RNA (data not shown).
In CVL fluid of HIV-1-infected women, HIF is associated with BV, confirming earlier observations made on a small number of women.15 This association is attributable to HIF activity that is present in samples before and after heating CVL fluid (heat-stable). HIF activity that is eliminated by heating (heat-labile) is not associated with BV.
We found a significant increase in heat-stable HIF with increasing alterations in vaginal flora. Heat-stable HIF is strongly associated with Nugent scores diagnostic of BV (range: 7-10) and is less strongly associated with intermediate Nugent scores (range: 4-6) when either is compared with normal scores (range: 0-3). Previously published evidence suggests that bacterial products have in vitro HIV-1-inducing activity and that this activity is heat-stable or even enhanced by heat treatment.15,21-23 Other in vitro studies suggest that a cytokine, myeloid-related protein (MRP-8), is a source of HIF.24,25 Our results support the interpretation that products of BV-associated bacteria are the source of heat-stable HIF, whereas MRP-8 may be the source of heat-labile HIF.
We explored possible associations of HIF with other vaginal conditions. In multivariate logistic regression and the reduced logistic model, heat-stable HIF remained associated only with BV and intermediate flora. In the reduced logistic regression model, heat-labile HIF was associated neither with Nugent scores nor with any other vaginal condition. In a reduced gamma model, unheated quantitative HIF was associated with BV and pH. If the association with pH were real, it could be a result of the increased vaginal pH from altered vaginal flora and BV. Trichomoniasis, which may raise vaginal pH, and which we may have underdiagnosed (DATRI 009 relied on microscopy rather than on culture), might also have contributed. Trichomonas infection itself has been associated with abnormal vaginal flora and BV in HIV-1-infected women.10,26 Heated quantitative HIF was associated with BV and RBCs in a reduced gamma model. The statistical association with RBCs is not likely of biologic importance. It was present in only one of many models, and the effect was noted for a low level of CVL fluid RBCs but not for a high level of RBCs when both were compared with the absence of CVL fluid RBCs. Hence, the strong and specific associations of heat-stable and/or heated HIF with BV and intermediate flora are unequivocal. A possible association of unheated HIF with pH may also be an effect of alterations in vaginal bacteria not fulfilling the Nugent criteria for intermediate flora or BV. No other vaginal condition is associated with HIF.
We had hypothesized that the presence of HIF could increase genital tract HIV-1 shedding, which could lead to more efficient transmission of HIV-1 from women with BV. Although heat-stable HIF was associated with genital tract HIV-1 shedding in unadjusted analysis, it was not significantly associated after controlling for plasma HIV-1 RNA levels in this cohort of women. In DATRI 009, the parent study, no association was noted between BV and cervicovaginal HIV-1 shedding.16 Analyzing 123 samples from HIV-1-infected women, Cu-Uvin et al27 found a statistically significant association between BV and HIV-1 RNA by RNA PCR in CVL fluid (OR = 5.9, 95% CI: 1.4, 25.0) after controlling for plasma HIV-1 RNA, antiretroviral therapy, and CD4 lymphocyte count. In a study of 122 HIV-1-infected women, Spinillo et al28 found that BV was associated with cell-associated (OR = 3.58, 95% CI: 1.22, 10.54) and cell-free (OR = 2.94, 95% CI: 1.0, 8.7) HIV-1 RNA in CVL fluid after controlling for plasma viremia. Most recently, Sha et al29 presented data demonstrating an association of quantitative PCR of BV-associated bacteria with genital tract HIV-1 shedding among women with detectable plasma HIV-1 RNA levels. Our results may differ from the observations of Sha and her colleagues because a large proportion of the women in our study had undetectable plasma and genital tract HIV-1 RNA and the study by Sha and her colleagues analyzed a larger number of women with BV and intermediate flora using more specific quantitative measures of vaginal bacteria.
In conducting this substudy, we performed a large number of biologic and statistical analyses on a large number of specimens obtained for different, although related, purposes. Our investigation was designed while study visits for DATRI 009 were in progress and preceded laboratory and statistical analysis within DATRI 009. All our laboratory assays were performed without knowledge of participants' clinical status or the results from other laboratory procedures. All in vitro work was performed in quality-assured laboratories using standard methods, except for the HIF assays that were performed according to previously published methods.24 In prior results, HIF activity was found in CVL fluid from HIV-1-infected and uninfected women and was demonstrated with clinical HIV-1 isolates in primary human-derived cell lines as well as in laboratory-adapted cells and virus.14 HIF was not associated with the use of antiretroviral therapy in our study (data not shown). Finally, statistical analyses of our data using varied approaches provided consistent results.
In conclusion, we found a strong association, and a dose effect, between heat-stable HIF and alterations in vaginal flora, including BV. Further laboratory studies to characterize the contribution of bacterial products and MRP-8 to HIF are underway. The association between BV-associated bacterial products and genital HIV-1 shedding are being further investigated. A more sophisticated understanding of the influence of cervicovaginal cytokines and vaginal microbial flora on genital HIV-1 quantity and dynamics should help to develop new strategies, such as topical microbicides, to reduce heterosexual HIV transmission.
The authors thank Anna Soloviov and Michael Parloglou of Westat, Rockville, MD, for their contributions to this manuscript. They also thank the women enrolled at the University of California-San Francisco, University of Southern California, Georgetown University, and The Cook County Hospital whose specimens were used in this study.
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Keywords:© 2005 Lippincott Williams & Wilkins, Inc.
HIV-1; bacterial vaginosis; vaginitis; cervicovaginal lavage; sexually transmitted diseases; MRP-8 protein