Heterosexual contact is the predominant mode of HIV-1 transmission in Africa . The quantity of HIV-1 in genital secretions is likely to play an important role in mediating transmission risk [2–4]. In women, antiretroviral therapy (ART) rapidly reduces genital HIV-1 shedding [3,5]. Nonetheless, up to 15% of women on ART continue to have detectable levels of HIV-1 in genital secretions [3,5–7]. The reasons for continued genital HIV-1 shedding among women on ART have not been fully characterized.
Genital infections have been identified as a significant determinant of genital HIV-1 shedding when women are not on ART [8–10]. Conversely, treatment of genital infections reduces the concentration of HIV-1 RNA in genital secretions. For example, in a cohort of ART-naive women, treatment of cervical infections significantly reduced cervical HIV-1 RNA shedding .
With increasing interest in the use of ART to reduce HIV-1 transmission, it will be important to understand the effect of genital infections on HIV-1 shedding. This study examined the effects of cervical infections and their treatment on genital HIV-1 RNA shedding in women receiving ART.
Population and procedures
HIV-1-seropositive women in the Mombasa Cohort and eligible for ART were invited to participate in a prospective study of risk factors for genital HIV-1 shedding. Detailed procedures have been described previously . Briefly, eligible women were counseled on adherence and side-effects before initiating treatment with stavudine or zidovudine, lamivudine, and nevirapine according to the Kenyan guidelines .
At monthly visits, women completed an interview using standardized questionnaires, a physical examination was performed, and samples collected for laboratory diagnosis of genital infections and HIV-1 quantitation. Cervical specimens for HIV-1 quantitation were collected first, by inserting a Dacron swab in the cervical os and rotating two full turns. Swabs for Gram stain and culture for Neisseria gonorrhoeae were collected next. Every 3 months, an additional cervical swab was collected for detection of N. gonorrhoeae and Chlamydia trachomatis by nucleic acid amplification. Blood samples for CD4 lymphocyte count were collected every 3 months.
Cases of cervicitis were identified in women who had incident cervical infections at least 1 month after initiating ART. Each woman served as her own control, with visits before infection and after successful treatment. Symptomatic genital infections were treated syndromically in accordance with Kenyan and WHO guidelines . In addition, women were asked to return for laboratory results 1 week after each examination. At this visit, additional treatment was dispensed if indicated. Neisseria gonorrhoeae was treated with 800 mg of norfloxacin as a single dose. Chlamydia trachomatis was treated with 100 mg of doxycycline twice daily for 7 days. Syndromic treatment for nonspecific cervicitis included norfloxacin with doxycycline. Participants underwent a repeat examination at a test-of-cure visit after 1 month.
Trained study nurses provided free condoms and HIV-1 risk reduction counseling at each visit. Ethical review boards from the University of Washington and Kenya Medical Research Institute approved the study. All participants provided written informed consent.
HIV type-1 screening was performed using ELISA (Detect HIV1/2, BioChem Immunosystems, Montreal Canada). Positive results were confirmed with a second ELISA (Recombigen, Cambridge Biotech, Worchester, Massachusetts, USA or Vironostika HIV-1 Uniform 11 AG/AB, bioMerieux, Marcy l'Etoile, France). Quantitation of CD4 lymphocytes was performed by FACSCount (Becton Dickinson, Forest Lakes, New Jersey, USA).
Nonspecific cervicitis was defined as an average of more than or equal to 30 polymorphonuclear leukocytes in three nonadjacent oil immersion fields on cervical Gram stains. Endocervical secretions were inoculated on modified Thayer–Martin media (Difco BD Diagnostics, Oxford, UK) for isolation of N. gonorrhoeae. A transcription-mediated nucleic acid amplification assay (Aptima Combo2, Gen Probe, San Diego, California, USA) was performed on cervical swabs for detection of N. gonorrhoeae and C. trachomatis.
Cervical swabs for HIV-1 RNA quantitation were stored in freezing media at −80°C and then shipped to Seattle, USA, on dry ice. HIV-1 RNA quantitation was performed using the Gen Probe HIV-1 viral load assay (San Diego, California, USA). The lower limit of linear quantitation was 100 copies/ml, which corresponded to 100 copies per swab, as swabs were placed in 1 ml of freezing medium.
Most women had HIV-1 RNA levels below the limit of linear quantitation. Therefore, we used generalized estimating equations with a binomial outcome distribution, logit link, and exchangeable correlation matrix to compare the presence of detectable HIV-1 RNA (above versus below the threshold for quantitation). The precervical infection visit was compared with visits during infection and after successful treatment. The primary cervicitis end point combined N. gonorrhoeae, C. trachomatis, and nonspecific cervicitis. We included visits with concurrent vaginal infections, as these have not affected cervical HIV-1 shedding in our earlier studies [9,11].
Potential confounding factors were identified as variables that differed significantly between visits or were significantly related to the outcome (P < 0.10). Our analysis plan included a priori adjustment for number of months since ART initiation, as longer duration of treatment may result in greater HIV-1 suppression. The final multivariate model included hormonal contraceptive use, menstrual cycle stage, and duration on ART. Analyses were performed using SPSS 16.0 (SPSS Inc., Chicago, Illinois, USA) and Stata 9 (StataCorp LP, College Station, Texas, USA).
Of 147 women on ART followed between March 2004 and December 2008, 30 participants contributed a total of 31 successfully treated episodes of cervicitis. These included 13 (41.9%) with nonspecific cervicitis, 17 (54.8%) with N. gonorrhoeae, and one (3.2%) with C. trachomatis. One woman contributed two infections (one episode of C. trachomatis and one of N. gonorrhoeae). The median age of the participants was 36 years [interquartile range (IQR) 31–38]. Oral contraceptive pills (N = 1, 3.2%) and depot medroxyprogesterone acetate (N = 7, 22.6%) were used by a minority of participants. The median duration on ART was 15 months (IQR 8–21) and participants had high adherence by pill count (median 100%, IQR 96.5–100). Twenty-nine (96.7%) were taking the standard first-line ART regimen. One woman (3.3%) was on a second-line regimen including a protease inhibitor. The median CD4 lymphocyte count at the preinfection visit was 308 cells/μl (IQR 238–395).
HIV-1 RNA concentrations above 100 copies per swab were detected in cervical secretions before, during, and after cervicitis at one (3.2%), five (16.1%), and three (9.7%) visits, respectively. Compared with baseline, there was a trend (P < 0.10) for increased HIV-1 RNA detection with cervical infections (Table 1). This result was similar, but became statistically significant, after additional adjustment for contraceptive use and menstrual cycle stage (adjusted odds ratio 5.7, 95% confidence interval 1.0–30.3, P = 0.04). Following successful treatment of cervicitis, detection of HIV-1 RNA was less frequent and no longer significantly increased relative to baseline.
Figure 1 presents HIV-1 RNA levels by visit among women with detectable cervical shedding. The one sample with detectable HIV-1 RNA at the preinfection visit had 407 copies per swab. At the infection visit, five women had detectable HIV-1 RNA (median 115, range 100–820 copies per swab). Following successful treatment, the median HIV-1 RNA level was 657 copies per swab (range 110–1770 copies per swab) in the three women with detectable virus. Two participants 3001 and 6813 had near identical patterns of shedding and are superimposed on one another. Detection of 1770 copies per swab at a posttreatment visit was the highest level of HIV-1 shedding observed. Reported pill-count adherence for this visit was 95.3%, so the reason for increased genital HIV-1 shedding was not immediately evident. We cannot rule out explanations such as measurement error, rather than a true biological effect.
This study demonstrated that cervical infections may increase detection of HIV-1 RNA in cervical secretions. However, even when HIV-1 RNA was detected, most cervical HIV-1 RNA concentrations remained near the threshold for quantitation (100 copies per swab). Higher levels of HIV-1 RNA shedding are generally seen in ART-naive women even in the absence of cervical infections. For example, in an earlier study of cervicitis among untreated HIV-1-seropositive women, median cervical HIV-1 RNA was 11 220 copies per swab at diagnosis. This was reduced to a median of 1738 HIV-1 RNA copies per swab after successful treatment . In contrast, the present study of ART-treated women found that the majority have undetectable cervical HIV-1 RNA even in the presence of cervical infections.
Although acquisition of a cervical infection was associated with a statistically significant increase in cervical HIV-1 RNA, it is interesting to note that the prevalence of detectable cervical HIV-1 RNA at the final visit did not return to the precervicitis baseline. This may simply reflect variation in detection in a study with a modest sample size, or could represent a gradual decline in genital HIV-1 shedding following infection. A similar finding has been observed in men treated for urethritis , with progressive reductions in seminal HIV-1 at 1 and 2 weeks posttreatment. At completion of follow-up, seminal HIV-1 RNA remained higher than in a control group without urethritis.
Our study used genital HIV-1 RNA as a surrogate marker for infectivity. Recent studies among HIV-1-serodiscordant couples have demonstrated that higher genital HIV-1 levels are associated with increased transmission risk . This association was present even after adjustment for plasma HIV-1 RNA concentration, suggesting that genital HIV-1 RNA level is a useful surrogate marker for infectivity.
Recently, there has been interest in the use of ART to reduce HIV-1 transmission. A systematic review found that the overall risk of transmission was reduced by 92% in HIV-1-serodiscordant couples on ART compared with couples in whom the index case was untreated . Our results further highlight the potential benefits of ART as a prevention strategy. Nonetheless, it should be noted that even low concentrations of genital HIV-1 RNA could present some risk of transmission.
This study had several strengths. Women were followed prospectively. Therefore, it was possible to compare genital HIV-1 RNA concentrations before, during, and after successful treatment of cervicitis. With prolonged follow-up of this cohort, we accrued 31 cervical infections for analysis. High ART adherence provided an opportunity to determine the effect of cervicitis on HIV-1 shedding under near-optimal conditions. With lower ART adherence, cervicitis could have a greater impact on genital HIV-1 shedding.
There were limitations to this study. Because of the modest sample size, we did not have adequate power to evaluate each cervical infection separately. Cervical HIV-1 RNA was below the limit for quantitation at 71% of visits, limiting the power to detect changes in cervical HIV-1 shedding. This study did not evaluate shedding of cell-associated HIV-1 proviral DNA, which may provide a better measure of the potential for cell–cell transmission. Risk factors for HIV-1 RNA and proviral DNA shedding may differ , and it is not known which of these markers is most closely associated with transmission risk .
In conclusion, even in the setting of cervicitis, cervical HIV-1 RNA concentrations remain low in the majority of women who are adherent to ART. Nonetheless, increases in cervical HIV-1 RNA occur in a minority of women during cervicitis and could increase transmission risk. Identification and treatment of cervical infections may help to optimize the secondary HIV-1 prevention benefits of ART.
This research was supported by National Institutes of Health grant AI58698. R.W.G., S.M.G., and L.N.M. were supported by Fogarty International Center grant 5D43 TW000007. Additional support for the Mombasa Field Site was received from the University of Washington Center for AIDS Research (CFAR), an NIH funded program (P30 AI027757) which is supported by the following NIH Institutes and Centers: NIAID, NCI, NIMH, NIDA, NICHD, NHLBI, NCCAM.
The authors would like to acknowledge the invaluable contributions of the clinical, laboratory, and administrative staff to this study. The authors thank the Mombasa Municipal Council for providing clinical space and the Coast Provincial General Hospital for providing laboratory space and also thank all the women who participated in this study. This manuscript was approved for publication by the Director of the Kenya Medical Research Institute.
Data presented previously in part at 17th Conference on Retroviruses and Opportunistic Infections (CROI 2010); 16–19 February 2010; San Francisco (poster 482).
1. Vernazza PL, Eron JJ, Fiscus SA, Cohen MS. Sexual transmission of HIV: infectiousness and prevention. AIDS 1999; 13:155–166.
2. Baeten JM, Overbaugh J. Measuring the infectiousness of persons with HIV-1: opportunities for preventing sexual HIV-1 transmission. Curr HIV Res 2003; 1:69–86.
3. Graham SM, Holte SE, Peshu NM, Richardson BA, Panteleeff DD, Jaoko WG, et al
. Initiation of antiretroviral therapy leads to a rapid decline in cervical and vaginal HIV-1 shedding. AIDS 2007; 21:501–507.
4. Nunnari G, Sullivan J, Xu Y, Nyirjesy P, Kulkosky J, Cavert W, et al
. HIV type 1 cervicovaginal reservoirs in the era of HAART. AIDS Res Hum Retroviruses 2005; 21:714–718.
5. Cu-Uvin S, Caliendo AM, Reinert S, Chang A, Juliano-Remollino C, Flanigan TP, et al
. Effect of highly active antiretroviral therapy on cervicovaginal HIV-1 RNA. AIDS 2000; 14:415–421.
6. Neely MN, Benning L, Xu J, Strickler HD, Greenblatt RM, Minkoff H, et al
. Cervical shedding of HIV-1 RNA among women with low levels of viremia while receiving highly active antiretroviral therapy. J Acquir Immune Defic Syndr 2007; 44:38–42.
7. Shen L, Siliciano RF. Viral reservoirs, residual viremia, and the potential of highly active antiretroviral therapy to eradicate HIV infection. J Allergy Clin Immunol 2008; 122:22–28.
8. Johnson LF, Lewis DA. The effect of genital tract infections on HIV-1 shedding in the genital tract: a systematic review and meta-analysis. Sex Transm Dis 2008; 35:946–959.
9. Mostad SB, Overbaugh J, DeVange DM, Welch MJ, Chohan B, Mandaliya K, et al
. Hormonal contraception, vitamin A deficiency, and other risk factors for shedding of HIV-1 infected cells from the cervix and vagina. Lancet 1997; 350:922–927.
10. Rotchford K, Strum AW, Wilkinson D. Effect of coinfection with STDs and of STD treatment on HIV shedding in genital-tract secretions: systematic review and data synthesis. Sex Transm Dis 2000; 27:243–248.
11. McClelland RS, Wang CC, Mandaliya K, Overbaugh J, Reiner MT, Panteleeff DD, et al
. Treatment of cervicitis is associated with decreased cervical shedding of HIV-1. AIDS 2001; 15:105–110.
12. World Health Organization. Scaling up antiretroviral therapy in resource-limited settings: treatment guidelines for a public health approach
. Revision 2003. Geneva: WHO Press; 2004. pp. 1–44.
13. World Health Organization. Guidelines for the management of sexually transmitted infections
. Geneva: WHO Press; 2003. pp. 1–91. http://www.who.int/hiv/pub/sti/en/STIGuidelines2003.pdf
14. Cohen MS, Hoffman IF, Royce RA, Kazembe P, Dyer JR, Daly CC, et al
. Reduction of concentration of HIV-1 in semen after treatment of urethritis: implications for prevention of sexual transmission of HIV-1. AIDSCAP Malawi Research Group. Lancet 1997; 349:1868–1873.
15. Baeten JM, Kahle E, Lingappa JR, Coombs RW, Donnell D, Wald A, et al. Genital HIV-1 RNA concentrations and heterosexual HIV-1 transmission risk: ‘higher genital HIV levels increase HIV sex transmission risk’
[abstract LBPEA07]. In: Proceedings of 5th IAS Conference on HIV Pathogenesis, Treatment, and Prevention
; 19–22 July 2009; Cape Town, South Africa.
16. Attia S, Egger M, Muller M, Zwahlen M, Low N. Sexual transmission of HIV according to viral load and antiretroviral therapy: systematic review and meta-analysis. AIDS 2009; 23:1397–1404.
17. Overbaugh J, Kreiss J, Poss M, Lewis P, Mostad S, John G, et al
. Studies of human immunodeficiency virus type 1 mucosal viral shedding and transmission in Kenya. J Infect Dis 1999; 179(Suppl 3):S401–S404.