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JAIDS Journal of Acquired Immune Deficiency Syndromes:
doi: 10.1097/QAI.0b013e31826327a0
Brief Report: Clinical Science

Cervical Human Papillomavirus Infection and Shedding of Human Immunodeficiency Virus in Cervicovaginal Fluids: a Cross-Sectional Study

Fornabaio, Chiara MD*; Carvalho, Anna C.C. MD, PhD*; Lillo, Flavia MD; Fiore, José R. MD; Bergamaschi, Viviana MD*; Bigoni, Sara MD*; Puzzi, Petra R. MD*; Cristini, Graziella MD*; Comelli, Mario MSc§; Parisi, Maria R. MD; Matteelli, Alberto MD*

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Author Information

*Institute of Infectious and Tropical Diseases, Spedali Civili Hospital and University of Brescia, Brescia, Italy

Clinical Pathology and Microbiology Laboratory, Hospital Santa Corona, Pietra Ligure, Savona, Italy

Department of Infectious Diseases, University of Foggia, Foggia, Italy

§Department of Health Science, University of Pavia, Pavia, Italy

Department of Infectious Diseases, San Raffaele Hospital, Milan, Italy.

Correspondence to: Alberto Matteelli, MD, Clinic of Infectious and Tropical Diseases, Piazzale Spedali Civili, 1, 25123 Brescia, Italy (e-mail: matteelli@med.unibs.it).

Supported by the fourth Italian program for HIV research Grant D.07. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The authors have no conflicts of interest to disclose.

Received January 27, 2012

Accepted June 5, 2012

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Abstract

Abstract: We evaluated the association between human papillomavirus cervical infection and HIV shedding in cervicovaginal lavage fluid (CVL), studying 89 HIV-infected women recruited at the Department of Infectious Diseases of Brescia (Italy). HIV shedding in CVL was found in a similar proportion of women with (30%; 21/70) and without (31.6%; 6/19) cervical human papillomavirus infection. A statistically significant correlation was found between HIV viral load in serum and CVL among the 27 women with detectable HIV in CVL (r = 0.4; P = 0.04). However, women on highly active antiretroviral therapy were more likely to have detectable HIV-RNA in CVL despite negative viremia (80% vs. 8%; P < 0.005).

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INTRODUCTION

Human papillomavirus (HPV) genital infection is the most common sexually transmitted infection worldwide; lifetime incidence of cervical HPV infection is estimated to be as high as 80%.1 Persistent high-risk HPV type infections are associated with cervical cancer, the second most frequent cancer among women.2

HIV and HPV infections share behavioral and socioeconomic risk factors. Because of the HIV-related immunodeficiency, HIV-infected subjects are more frequently coinfected by HPV, have a higher risk of developing persistent HPV infections, and thus to progress to cervical dysplasia and cancer.3

Plasma HIV viral load has been identified as one of the major determinants of HIV genital shedding. Genital tract infections were also associated with increased HIV viral shedding by causing a local inflammatory reaction with recruitment of lymphocytes.4–7 It has also been demonstrated that genital shedding can be intermittent, variations being related to the menstrual cycle (probably mediated by hormonal changes) with cervical HIV-RNA levels increasing from the mid cycle to the menses and decreasing thereafter.7,8

In vitro studies have demonstrated how HIV promotes HPV replication through the activation of both early and late genes.9 Although it is universally accepted that HPV infection does not elicit a local inflammatory reaction, it has been hypothesized that HPV infection could facilitate HIV transmission through disruption of mucosal integrity and/or altered local immunosurveillance.9,10

The interplay between HPV and HIV at genital mucosa level has been evaluated in a very limited number of studies, which used different methodologies and reached inconsistent results.4,11 The present study aims at estimating whether the presence of cervical HPV infection and HPV viral load at cervical level may alter HIV genital shedding in HIV-seropositive women.

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PATIENTS AND METHODS

Study Population

HIV-infected women enrolled in the prospective DOSETHIV cohort (DOnne Sieropositive E Trasmissione dell'HIV), followed at the Department of Infectious Diseases of the University of Brescia between July 2003 and August 2007 were included in this cross-sectional analysis. Eligibility criteria required HIV infection, age between 18 and 45 years, no previous ablative cervical or uterine surgery, and the absence of gynecological symptoms or pregnancy at the time of enrollment. After written informed consent, the participants answered to a standardized questionnaire regarding history of sexually transmitted diseases and genital disorders, risk behavior for acquisition of HIV infection, and antiretroviral therapy. Pelvic examination was performed and cervicovaginal samples were obtained for screening of genital infections (Trichomonas vaginalis and Chlamydia trachomatis) and genital disorders (yeasts and anaerobic bacteria). Every woman had a Pap-smear done.

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Samples

Paired peripheral blood sample and cervicovaginal lavage (CVL) were taken on the same day or within 1 month from the date of enrollment. CVLs were obtained by irrigating the vaginal walls and the cervical area with 3 mL of sterile 10 mM LiCl phosphate–buffered saline solution; after 60 seconds, the fluid was collected from the posterior vaginal fornix and processed as previously described.5,12

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HIV-RNA Load in Plasma and CVL

Blood samples were analyzed for lymphocyte subset determination and plasma HIV-1 RNA by branched-DNA with a cutoff value of 50 copies per milliliter [VERSANT HIV-1 RNA 3.0 Assay (bDNA), Siemens Healthcare Diagnostics, Tarrytown, NY]. The branched-DNA method was also used on CVL supernatants to evaluate and measure the presence of cell-free HIV-RNA; the cutoff was in this case 500 copies per milliliter, as we used a 1:10 dilution of the CVL sample.

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Determination of Cervical HPV-DNA

The presence and quantity of cervical HPV was determined on specimens collected by eso-endocervical brushing suspended in a TEN (Tris–HCl, EDTA, NaCl) solution; the samples were stored at −80°C till analytical determination.

Samples were first tested for the presence, quality, and quantity of amplifiable human DNA by a Real-time polymerase chain reaction (TaqMan 7900 HT Sequence Detection System, Applied Biosystems, Foster City, CA) in the house keeping human telomerase gene (2 copies per cell). The human DNA concentration was then used to normalize HPV load toward a fixed amount of cells (HPV copies per 105 cells).

HPV presence was first screened by qualitative in-house MY09-11 and GP5-GP6+ consensus polymerase chain reaction in the L1 capsid region. Positive samples were then tested by a consensus L1 Real Time amplification (SPF10 primers) to measure the total amount of HPV DNA and for the presence of HPV-16 and HPV-18 types by specific amplification.

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Statistical Analysis

Univariable and multivariable analysis by logistic regression were done to evaluate the variables (CD4+ T-cell count, plasma HIV viremia, use of antiretroviral therapy, time from HIV diagnosis, HIV exposure category, the presence of genital dysplasia, and genital infections) associated with the presence of HIV-RNA in the CVL. The linear regression model was used to evaluate the correlation of log10 cervicovaginal HIV-RNA with log10 HPV viral load and plasmatic HIV-RNA. The chosen level of significance was 5%, and the P values reported are 2-tailed.

Patient clinical and laboratory data were imported from the software NetCare v.1.05.11 (Healthware SpA, Salerno, Italia) and entered into a specific database file (Microsoft Access 2003 and Excel 2003). SPSS 12.0 for Windows (SPSS, Inc, Chicago, IL) and R version 2.8.1 were used for statistical analyses.

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RESULTS

Of 126 HIV-seropositive women included in the DOSETHIV cohort, 89 (71%) were included in the present analysis (Table 1). Thirty-seven women were excluded because cervicovaginal samples were not available for determination of quantitative HPV and/or HIV at enrollment. The sociodemographic and clinical characteristics of included and excluded women were similar (data not shown). The study population was mainly represented by Italian (80%), followed by African (19%), and East European (1%) women. The median age at enrollment was 37 years (range, 20—46), 58% of the women had no history of parity. Sexual exposure was slightly more frequent as risk factor for HIV acquisition (55%) than drug addition (45%). The median time since first HIV diagnosis was 93 months (range, 3—262), and the proportion of women with an AIDS diagnosis at enrollment was low (9%). Sexual intercourse in the 6 months before sampling was reported by 49% of the women and 42% had a stable partner. Condom use during last sexual intercourse was reported by 68% of the women.

Table 1
Table 1
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Gynaecological examination was performed at a median time of 17 days from the last menstruation (range, 4–90 days). Five women (6%) had an abnormal Pap smear: 4 had low-grade squamous intraepithelial lesion and 1 had ASCUS (atypical squamous cells of undetermined significance).13 The prevalence of genital infections and disorders was 3% for Trichomonas vaginalis, 8% for Chlamydia trachomatis, 11% for Candida spp, and 27% for bacterial vaginosis. The median CD4+ T-cell count was 492 cells per microliter (range, 120–890) and most women (57%) had detectable HIV viremia, with a median viral load of 3.72 log10 copies per milliliter (range 1.7–5.7). Fifty-three percent of the women (47/89) were on highly active antiretroviral therapy (HAART), equally distributed between protease inhibitor (n = 21) and nonnucleosidic reverse transcriptase inhibitors (n = 22), with 4 women being on triple nuke therapy. Among women on HAART, 25.5% (12/47) had detectable plasma viral load. No association was found between type of HAART and HIV-RNA detection in plasma.

HPV-DNA was detected in the endocervix of 70 (79%) HIV-infected women, with a median viral load of 2.03 log10 copies. Twenty (29%) of the 70 HPV-positive women had a type 16 and/or 18 infection. Cervicovaginal HIV shedding was detected in 27 (30%) women, with a median load of 3.61 log10 HIV-RNA copies per milliliter. HIV shedding in CVL was found in a similar proportion of women with (30.0%; 21/70) and without (31.6%; 6/19) cervical HPV infection. No statistically significant correlation was found between quantitative HPV and HIV copies in cervicovaginal fluids, neither in the 70 HPV-positive women group (r = −0.08; P = 0.52) (Fig. 1A) nor in the 21 HPV-positive women with genital HIV shedding (r = 0.19; P = 0.41)(Fig. 1B).

Figure 1
Figure 1
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Figure 2 shows the distribution of women according to HAART and HIV-RNA positivity in plasma and in the CVL. Almost half of the women with genital HIV shedding (13/27; 48%) had undetectable plasma viral load. Women on HAART were more likely to have detectable HIV-RNA in CVL despite negative viremia compared with those not on HAART (12/15, 80% vs. 1/12, 8%; P < 0.005), although they had slightly lower HIV-RNA load in CVL (3.44 vs. 3.87 log10 HIV-RNA copies per milliliter; P = 0.02). Among the 27 women with detectable HIV-RNA in CVL, a modest correlation was found between cervicovaginal and plasma HIV-RNA (r = 0.4; P = 0.04). No association was found between the type of HAART and the presence of detectable HIV RNA in CVL.

Figure 2
Figure 2
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In the univariable analysis, none of the considered variables showed a statistically significant association with the presence of HIV genital shedding. Women with sexual acquisition of HIV had a trend toward a higher probability of vaginal HIV shedding, but this association did not reach statistical significance in the multivariable logistic regression analysis (P = 0.06).

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DISCUSSION

Our data suggest that cervical HPV infection does not interfere with the probability of HIV shedding in cervical fluid in HIV-infected women: the proportion of genital HIV shedding was similar between women with or without cervical HPV infection. Moreover, cervical HPV viral load had no significant influence on the amount of cell-free HIV-RNA detected in CVL in women with genital HIV shedding. These findings are in contrast with previously published data from 2 cross-sectional studies that analyzed the factors associated with HIV genital shedding.4,11 In these studies, however, the study population was characterized by a high prevalence of other genital infections and cytological abnormalities at the Pap-smear, both factors that can contribute to an increased HIV cervicovaginal shedding.6,7,14,15 The lack of association between cervical HPV infection and HIV genital shedding observed in our study is consistent with the fact that HPV does not elicit a local inflammatory reaction.1

In the study of Smith-McCune et al,16 an increased risk of HIV acquisition associated with recent or concurrent HPV genital infection was described, particularly for some specific HPV types, considered to be a risk factor for HIV infection. Our data do not support this hypothesis because we did not observe any association between the 2 viruses at a genital level, where they seem to act independently. As HIV and HPV infections share behavioral and socioeconomic risk factor, it is often difficult to draw conclusions on a causative relationship.

Our data confirm a high prevalence of HPV infection (79%) among HIV-positive women. HPV 16 and 18, the types responsible for most cervical cancer cases, were not frequently found in our sample (30% prevalence). This finding is consistent with the observed very low prevalence of high-grade cervical dysplasia in our sample.

In the logistic regression analysis, sexual acquisition of HIV was the only variable associated with HIV genital shedding (P = 0.06), suggesting that women who have acquired HIV infection through sexual exposure had a higher probability of shedding HIV in their cervicovaginal fluids, independently from other possible risk factors. This finding might be explained by viral compartmentalization of HIV that could lead to the persistence of the original viral clones in the primary site of infection17 or by the hypothesis that viral types with a particular genital tropism can more easily be transmitted by sexual intercourse.18,19

Although plasma HIV viral load is the major determinant of genital HIV shedding,4,5,10 48% of the women in our sample had detectable cervicovaginal HIV-RNA despite undetectable plasma HIV viral load, independent from HAART (Fig. 2). This finding is consistent with previous studies showing HIV shedding in CVL in a significant proportion (20%–35%) of HIV-infected women with undetectable HIV in plasma.4,5,7,15,19 In our study, the probability of HIV shedding despite negative viremia was associated with HAART. These data might be justified by factors related to the viral compartmentalization and the role played by the immune status. In fact, some studies suggest that the immune response influences the development of viral genotypes in some compartments and that women with higher CD4+ T-cell counts are more likely to have compartmentalized virus shed in their genital tract.20,21 It has also been demonstrated that some antiretrovirals do not penetrate well in the cervicovaginal fluids, thus potentially leading to a selective pressure and to the emergence of a reservoir of drug-resistant virus in the genital tract.22–24

Our study has limitations: first, it is a negative study with a small sample size that cannot rule out completely the existence of an association between HPV infection and HIV shedding; however, the differences in HIV shedding between HPV-positive and -negative women are so small that such association is very unlikely. Second, the cross-sectional study design precludes inferences on HIV plasma load dynamic and HIV genital shedding over time.

In summary, we did not observe any association between cervical HPV infection and HIV genital shedding. The finding of HIV cervicovaginal shedding in a group of women with undetectable plasmatic HIV-RNA supports the hypothesis of a viral compartmentalization between plasma and female genital tract, thus indicating that an undetectable plasma viral load in subjects on HAART cannot be taken as an indicator of absence of risk for sexual transmission of HIV.

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REFERENCES

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16. Smith-McCune KK, Shiboski S, Chirenje MZ, et al.. Type-specific cervico-vaginal human papillomavirus infection increases risk of HIV acquisition independent of other sexually transmitted infections. PLoS One. 2010;5:e10094.

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19. Cu-Uvin S, DeLong AK, Venkatesh KK, et al.. Genital tract HIV-1 RNA shedding among women with below detectable plasma viral load. AIDS. 2010;24:2489–2497.

20. Sullivan ST, Mandava U, Evans-Strickfaden T, et al.. Diversity, divergence, and evolution of cell-free human immunodeficiency virus type 1 in vaginal secretions and blood of chronically infected women: associations with immune status. J Virol. 2005;79:9799–9809.

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Keywords:

cervical HPV; cervicovaginal HIV; HIV shedding; STI

© 2012 Lippincott Williams & Wilkins, Inc.

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