Increased risk of cervical disease among human immunodeficiency virus–infected women with severe immunosuppression and high human papillomavirus load1

Increased Risk of Cervical Disease Among Human Immunodeficiency Virus–Infected Women With Severe Immunosuppression and High Human Papillomavirus Load ISABELLE HEARD, MD, JEAN-MICHEL TASSIE, MD, VALERIE SCHMITZ, MS, LAURENT MANDELBROT, MD, MICHEL D. KAZATCHKINE, MD, PhD, AND GE´RARD ORTH, PhD Objective: To investigate human papillomavirus (HPV) genotypes, HPV DNA load, and behavioral and sociodemographic factors in a series of human immunodeficiency virus (HIV)-seropositive women, and to correlate HPV infection with cervical disease according to immune status. Methods: Three hundred seven HIV-seropositive women were tested for the presence of HPV DNA by polymerase chain reaction (PCR) and Southern blot hybridization. Cervical disease was assessed using Papanicolaou smears, colposcopy, and biopsies when necessary. Various risk factors for cervical intraepithelial neoplasia (CIN) were tested using multiple logistic regression analysis. Results: Cervical disease was diagnosed in 83 (27.0%) of 307 women and HPV infection in 162 (52.8%). High HPV load (as detectable by Southern blot hybridization) was found in 90 (55.6%) of the 162 infected women. Potentially oncogenic or related genotypes were detected in 74 (82.2%) of these 90 cases. High-load HPV infection was twice as frequent in severely immunosuppressed women (CD4 cell count less than 200/␮L) as in women with higher CD4 cell counts (P ⴝ .002). High-load HPV infection was associated with a high risk of cervical disease (adjusted odds ratio [OR] 16.8; 95% confidence interval [CI] 7.0, 40.3). The risk among severely immunosuppressed women was ten times greater than that among women with CD4 cell counts of at least 200/␮L. Low-load HPV infection (detected by PCR only) was

From the Institut National de la Sante´ et de la Recherche Me´dicale (INSERM) U430 and Unite´ d’Immunologie Clinique, Hoˆpital Broussais; Service d’Obste´trique et de Gyne´cologie, Ho´pital Cochin; INSERM SC4, Faculte´ de Me´decine Saint-Antoine; Unite´ Mixte Institut Pasteur– INSERM U190, Institut Pasteur, Paris, France. This study was supported by INSERM, the Agence Nationale de Recherches sur le SIDA, and SIDACTION, France. The authors are grateful to J.-D. Poveda (Centre de Biologie Me´dicale Spe´cialise´e, Institut Pasteur) for his help with human papillomavirus typing.

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a risk factor for CIN in severely immunosuppressed women only (adjusted OR 7.4; 95% CI 1.3, 43.0). Conclusion: Immunosuppression favors cervical high-load HPV infection with oncogenic genotypes and its clinical expression in HIV-seropositive women. (Obstet Gynecol 2000;96:403–9. © 2000 by The American College of Obstetricians and Gynecologists.)

Infection with specific genotypes of human papillomavirus (HPV) has been shown to be etiologically associated with cervical intraepithelial neoplasia (CIN) and invasive cervical carcinoma.1,2 The major risk factor for CIN is persistent infection with oncogenic HPV genotypes,3–5 particularly with high HPV DNA load.5,6 Systemic and local cell-mediated immunity are major determinants of HPV infection and its clinical expression.2,7,8 Immunodepression secondary to infection with human immunodeficiency virus (HIV) is associated with a high prevalence of CIN and a high rate of persistence and progression of these lesions.8 –12 High prevalence and persistence of oncogenic HPV genotypes are also common features in HIV-seropositive women.13–16 Most epidemiologic studies involving HIV-seropositive women have focused either on demographic, behavioral, and immunologic risk factors for CIN10,12,14 or on risk factors for HPV infection.13,15,16 Only a limited number of studies have dealt with HPV infection as a risk factor for CIN.17–20 This prompted us to undertake an epidemiologic study to evaluate sociodemographic factors, immunosuppression, and viral infection (HPV DNA load and HPV genotypes) as risk factors for CIN in HIV-infected women.

0029-7844/00/$20.00 PII S0029-7844(00)00948-0

403

Materials and Methods Between June 1993 and February 1996, 307 HIVseropositive women attending the outpatient clinics of Hoˆpital Broussais and the Department of Obstetrics and Gynecology of Hoˆpital Cochin, Paris, were enrolled in a prospective study of cervical disease in HIVseropositive women. The protocol was reviewed and approved by the French National Agency for AIDS Research. All women gave informed consent to participate in the study and were interviewed using a standardized questionnaire with questions about ethnicity, age, marital status, professional activity, tobacco use (smoking more than five cigarettes per day was considered tobacco use), route of HIV infection, history of sexually transmitted diseases (STDs), history of previous HPV-related disease, history of previous cervical disease treatment, age at first intercourse, total number of sexual partners, and obstetric history. The gynecologic examination included a Papanicolaou smear and the obtaining of a cervical cell sample for HPV DNA analysis. For Papanicolaou smears, cells were collected from the endocervix with a cotton swab and from the ectocervix with a wooden spatula. The specimen for HPV detection was obtained by rotating a cytobrush in the cervical os. The specimen was immersed immediately in Eagle’s medium in the presence of antibiotics in a cryotube and stored at ⫺80C. A standardized colposcopic examination of the cervix was performed. Lesions were described in terms of color, margin, vessels, and iodine-staining characteristics. Abnormal colposcopic findings within the transformation zone were divided into minor and major abnormalities. Minor abnormalities were discrete aceto-white areas with irregular borders and a lack of vascular changes. Major abnormalities were thick aceto-white lesions with sharp borders and coarse mosaic or coarse punctuation.21 All patients with major colposcopic abnormalities or high-grade squamous intraepithelial lesions (SILs) underwent biopsies, unless they refused or would not comply with follow-up. Biopsies also were performed in most women with low-grade SILs. All smears and biopsy specimens were interpreted by the same pathologist (Dr. C. Bergeron, IPECA, Laboratoire CERBA, Saint-Ouen-l’Aumoˆne, France). Cytologic categories were normal, atypical squamous cells of undetermined significance, low-grade SILs, and high-grade SILs. Histologic categories were normal, squamous metaplasia, immature metaplasia, low-grade CIN, and high-grade CIN. The CD4⫹ cell counts closest to the time of the gynecologic examination (within 6 months) were obtained from clinical records. Absolute numbers of pe-

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ripheral blood CD4⫹ cells were determined by flow cytometry. For HPV detection, total DNA was extracted from the cell samples and submitted to PCR (1 ␮g per test) using the degenerate MY11/MY09 primers,22 as described.23 The adequacy of DNA preparations for PCR was tested using ␤-globin primers.24 After electrophoresis in an agarose gel, the PCR products were alkali-denatured, transferred to nitrocellulose membranes, and hybridized with 32P-kinased oligonucleotide consensus GP1 and GP2 probes.22 Membranes were exposed overnight to a Kodak X AR film (Eastman Kodak, Rochester, NY). The DNA preparations were analyzed further by Southern blot hybridization, after digestion with Pst I restriction endonuclease, electrophoresis (2.5 ␮g per slot) in triplicate 1% agarose slab gels, denaturation, and transfer to nitrocellulose membranes, as described.23 The membranes were hybridized under nonstringent conditions (40C below the melting temperature of hybrids) with three mixtures of nick-translated 32 P-labeled DNA probes specific for genital HPV types, namely HPV 6, 11, and 42; HPV 16, 18, and 33; or HPV 31, 35, and 39. The membranes were exposed to Hyperfilm-MP films (Amersham Pharmacia Biotech, Little Chalfont, UK) for 2 days, washed under stringent conditions (20C below the melting temperature of hybrids), and re-exposed for 6 days. Human papillomavirus DNA sequences detected by Southern blot hybridization that could not be identified by their cleavage and hybridization patterns were further amplified using MY11/MY0922 or GP5⫹/GP6⫹25 degenerate primers. Universal forward and reverse M13 sequences were added to the five prime ends of the forward and reverse primers, respectively, to allow direct sequencing of the PCR products (a 450 – base pair [bp] fragment and a 145-bp fragment for the MY11/MY09 and GP5⫹/GP6⫹ primers, respectively). Amplification products were sequenced on both strands by the dideoxynucleotide termination method, using fluorescent forward and reverse M13 primers and the ABI Prism Dye-Primer Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) under the conditions described by the manufacturer. Data were generated with an ABI Prism 377 automated DNA sequencer (Applied Biosystems). Nucleotide sequences were compared with the available HPV DNA sequences in Genbank using the FASTA program (Genetic Computer Group, Madison, WI). A sample was considered HPV positive if either PCR or Southern blot hybridization was positive. Twenty-four DNA preparations found to be negative by Southern blot hybridization yielded almost no DNA detectable when the gels were stained with ethidium bromide and were considered nonconclusive. In our experimental conditions, the detection levels of HPV

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DNA were estimated to 150,000 molecules (0.5 copy of HPV genome per cell) by Southern blot hybridization and to 100 –500 molecules (0.5–2.5 copies of HPV DNA per 103 cells) by PCR.23 Thus, in agreement with Ho et al,5 detection of HPV DNA by Southern blot hybridization defined high-load HPV infection, whereas HPV detection by PCR alone defined low-load HPV infection. Statistical analyses were performed using SPSS statistical software (Statistical Package for Social Sciences 8.0; SPSS Inc., Chicago, IL). Differences across groups were tested with the ␹2 test. Risk factors for cervical disease were assessed using multiple logistic regression analysis. Patients with low-grade CIN or high-grade CIN were compared with patients with no evidence of CIN. Patients in whom the presence of CIN was indeterminate were excluded from analysis. The main variables of interest were the presence of HPV and CD4⫹ cell counts. Women were classified into three groups: those with no detectable HPV DNA by PCR and Southern blot hybridization, those with positive PCR results and negative Southern blot hybridization results, and those with positive Southern blot hybridization results, irrespective of PCR results. The CD4 cell count was analyzed, with the value of 200/␮L taken as a dichotomous variable because of the United States Public Health Service/Infectious Diseases Society of America guidelines26 that define persons with fewer than 200 CD4 cells/␮L as severely immunosuppressed and prone to opportunistic infections. In a first step, variables were entered in a logistic regression stepwise model with age (younger than 30 years or 30 years or older), route of HIV infection, ethnic origin (black or Caribbean compared with white or other), age at first intercourse (younger than 16 years or 16 years or older), history of delivery, lifetime number of sexual partners (fewer than 20 or 20 or more), current tobacco use (more than five cigarettes per day or fewer than five cigarettes per day), safe sex practice, and current status with regard to genital warts and other STDs. The remaining risk factors then were analyzed depending on whether the CD4 cell count was less than or at least 200/␮L.

Results The women included in the study were aged between 19 and 72 years, with a median age of 32 years. Two hundred twelve women (69%) were white, 80 (26%) were black or Caribbean, and the remaining 15 had various ethnic backgrounds. The mode of HIV transmission was heterosexual contact in 184 women (60%), intravenous drug use in 90 (29%), transfusion of contaminated blood products in 14 (5%), and unknown in 19 (6%). Seventy-seven women had histories of abnor-

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Table 1. Prevalence of Cervical Disease According to Cytology, Colposcopic Findings, and Histology Cervical disease

n

%

No evidence of CIN WNL/No colposcopic lesions WNL/No colposcopy/No biopsy* Presence of CIN indeterminate ASCUS, LoSIL, or HiSIL/No colposcopic lesion WNL, ASCUS, or LoSIL/Minor colposcopic lesions/no biopsy WNL ASCUS, LoSIL or HiSIL/No colposcopy (LF-U) LoSIL/Minor colposcopic lesions/Metaplasia Low-grade CIN LoSIL/Minor colposcopic lesions/CIN1 biopsy specimen LoSIL/Major colposcopic lesions/No biopsy WNL/Major colposcopic lesions/No biopsy† High-grade CIN LoSIL/Major colposcopic lesions/CIN3 biopsy specimen HiSIL/Minor colposcopic lesions/CIN3 biopsy specimen HiSIL/Minor colposcopic lesions/No biopsy‡ HiSIL/Major colposcopic lesions/CIN3 biopsy specimen HiSIL/Major colposcopic lesions/CIN1 biopsy specimen Total

148 110 38 76 15

48.2 35.8 12.4 24.8 4.9

24

7.8

34

11.1

3

1.0

42 31

13.7 10.0

2

0.6

9

2.9

41 9

13.3 2.9

8

2.6

1

0.3

18

5.9

5

1.6

307

100

CIN ⫽ cervical intraepithelial neoplasia; WNL ⫽ Papanicolaou smear within normal limits; ASCUS ⫽ atypical squamous cells of undetermined significance; LoSIL ⫽ low-grade squamous intraepithelial lesion; HiSIL ⫽ high-grade squamous intraepithelial lesion; LFU ⫽ lost to follow-up. * Considered as having no CIN on the basis of two normal Papanicolaou smears within 6 months. † Considered as having low-grade CIN because of persistent major colposcopic lesions and a CIN1 biopsy specimen 6 months after enrollment. ‡ Considered as having high-grade CIN because of a CIN3 biopsy specimen 6 months before enrollment.

mal Papanicolaou smears (44 [14.3%] had low-grade SILs and 33 [10.7%] had high-grade SILs); 53 women presented with genital warts. The median number of peripheral blood CD4⫹ cells was 300/␮L (interquartile range 164 – 498), and 93 women (30.5%) had CD4⫹ cell counts less than 200/␮L. The CD4⫹ cell count was not available in the case of two women. Human immunodeficiency virus RNA plasma load was not determined routinely at the time of enrollment (before February 1996), and serum samples were not available for a retrospective analysis. The absence or the presence and degree of severity of cervical disease were determined from the results of the

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Papanicolaou test, colposcopy, and cervical biopsies according to the criteria proposed by Wright et al10 (Table 1). One hundred forty-eight (48.2%) of the 307 women in the cohort had no evidence of CIN. In 76 women (24.8%), the presence of CIN was indeterminate because of incomplete exploration, conflicting results, or patient being lost to follow-up. Eighty-three (27.0%) were classified as having CIN. Cervical intraepithelial neoplasia was low grade in 42 women (13.7%) and high grade in 41 (13.3%). No invasive cervical cancer was detected. At the time of the study, 109 patients (35.5%) were receiving combination antiretroviral therapy with nucleoside analogues alone because protease inhibitors were not yet available. The rate of cervical disease among treated women was higher than among untreated women (43.4% compared with 31.8%) although not significantly different (P ⫽ .07). Human papillomavirus DNA was detected by PCR in 152 (49.5%) of the 307 women. Southern blot hybridization data were available for 283 women. Five of the 24 remaining cases had positive PCR results. One hundred fifty-seven (55.5%) of the 283 women were found to be HPV positive: 67 (42.7% of the positive cases) by PCR only, 80 (50.9%) by both PCR and Southern blot hybridization, and ten (6.4%) by Southern blot hybridization only. Overall, 162 (52.8%) of the 307 women were HPV positive. The same rate of HPV infection was observed among women treated with nucleoside analogues (53.2%) and untreated women (52.5%). Ninety (55.6%) of the infected women were found to be positive by Southern blot hybridization. These patients were considered to have high HPV load because in our experimental conditions, Southern blot hybridization was found to be 300 –1000 times less sensitive than PCR.23 In contrast, women found to be positive by PCR only were considered to have low HPV load.5 The HPV detection rate was higher among women with CD4⫹ cell counts less than 200/␮L than among women with higher CD4⫹ cell counts (65.6% compared with 47.6%) (P ⫽ .003). High HPV load was twice as frequent in the group of women with low CD4⫹ cell counts (less than 200/␮L) as in women with higher CD4⫹ cell counts (43% compared with 24%) (P ⫽ .002). Genotyping was performed in the 90 patients found to be HPV positive by Southern blot hybridization. Evidence for the presence of two distinct HPV genotypes was obtained for 12 (13.3%) of these women. Forty-two of the 102 HPV isolates were identified by their cleavage and hybridization patterns, and 40 by sequencing PCR amplification fragments. Twenty isolates could not be identified and corresponded to HPV sequences related to HPV 16, 18, or 33 (designated Xa), HPV 31, 35, or 39 (Xb), and HPV 6, 11, or 42 (Xc) (Table 2). As a whole, 20 different genotypes were detected.

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Table 2. Prevalence of Human Papillomavirus Genotypes as Detected by Southern Blot Hybridization HPV genotype* High risk 16 31 18 35 52 56, 58, 66 33, 39 45, 51, 70 Xa Xb Low risk 42 6, 53 11 CP8304 40 MM7 Xc

No. of positive cases†

Infected women (%) (n ⫽ 90)

17 (3) 11 (3) 8 (2) 5 4 (1) 3‡ 2‡ 1‡ 11 (5) 6 (2)

18.8 12.2 8.9 5.6 4.4 3.3‡ 2.2‡ 1.1‡ 12.2 6.7

5 (4) 4‡ (1) 3 3 (1) 1 1 3 (2)

5.6 4.4‡ 3.3 3.3 1.1 1.1 3.3

HPV ⫽ human papillomavirus. * High-risk HPVs correspond to genotypes found in invasive cervical carcinoma1 as well as to unidentified isolates found to be related to HPV 16, 18, and 33 (Xa) or to HPV 31, 35, and 39 (Xb). Low-risk HPVs represent genotypes seldom or not found in invasive cancers1 or unidentified isolates found to be related to HPV 6, 11, and 42 (Xc). † Values in parentheses are numbers of double infections. Double infections were detected in 12 women: 16 ⫹ 42, 31 ⫹ 42, 52 ⫹ 53, 16 ⫹ Xa, 16 ⫹ Xb, 18 ⫹ Xc, 18 ⫹ Xa, 31 ⫹ Xc, 31 ⫹ Xa, 42 ⫹ Xa, 42 ⫹ Xa, and CP8304 ⫹ Xb. ‡ Number of positive cases or percentage of infected women for each of the HPV genotypes indicated.

Human papillomavirus 16 was the most prevalent. High-risk HPV types were identified in 61 (67.8%) of the 90 women. In addition, HPVs related to high-risk genotypes (Xa and Xb) were found in 17 women, including four women also infected with another oncogenic type (Table 2). Thus, of the 90 women with high HPV load, potentially oncogenic or related genotypes were detected in 74 (82.2%) women. This prevalence was higher among women with CD4⫹ cell counts less than 200/␮L than among women with higher CD4⫹ cell counts (93% compared with 74%) (P ⫽ .02). Among the 83 women with cervical disease, the prevalence of HPV infection was 85.5%, as determined by either PCR or Southern blot hybridization. It was significantly lower among the 76 women (46.1%) in whom the presence of CIN was indeterminate and among the 148 women (41.2%) with no evidence of CIN (P ⬍ .001). Southern blot hybridization data were available for 78 (94.0%) of 83 women with cervical disease (37 with low-grade CIN and 41 with high-grade CIN) and 137 (92.6%) of 148 women with no evidence of CIN. Fifty-five (70.5%) women with cervical disease, but only

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Table 3. Risk Factors for Cervical Intraepithelial Neoplasia as Calculated by Multiple Logistic Regression Analysis* All women (n ⫽ 231)†

CD4⫹ cell count ⬍200/␮L (n ⫽ 69)

Percentage (proportion) Percentage (proportion) with CIN AOR (95% CI) with CIN CD4 cell count ⬍200/␮L ⱖ200/␮L HPV Negative PCR⫹/SBH⫺ SBH⫹ Current smoking No Yes

56.5 (39/69) 27.2 (44/162)

4.1 (1.8, 9.1) 1

11.5 (12/104) 29.1 (16/55) 76.4 (55/72)

1 2.5 (1.0, 6.1) 16.8 (7.0, 40.3)

9.5 (2/21) 43.8 (7/16) 93.8 (30/32)

28.0 (28/100) 42.0 (55/131)

1 2.26 (1.1, 4.9)

51.5 (17/33) 61.1 (22/36)

AOR (95% CI)

CD4⫹ cell count ⱖ200/␮L (n ⫽ 162) Percentage (proportion) with CIN AOR (95% CI)

1 7.4 (1.3, 43.0) 134 (18.5, 1098) 1

12.0 (10/83) 23.1 (9/39) 62.5 (25/40)

1 1.9 (0.7, 5.4) 12.6 (4.9, 32.7)

16.4 (11/67) 34.7 (33/95)

1 3.0 (1.2, 7.2)

AOR ⫽ adjusted odds ratio; CI ⫽ confidence interval; HPV ⫽ human papillomavirus; PCR ⫽ polymerase chain reaction; SBH ⫽ Southern blot hybridization. Other abbreviation as in Table 1. * Adjusted odds ratio are those calculated for the model selected by logistic regression analysis. † Women in whom the presence of CIN was indeterminate were not included.

17 (12.4%) women without cervical disease, had high HPV load (P ⬍ .001). High-load infection with an oncogenic HPV genotype or an uncharacterized related genotype was found in 19 women (51.4%) with lowgrade disease and 27 women (65.9%) with high-grade disease, with a 4.5-fold– higher prevalence of HPV 16 (24.4% compared with 5.4%) in this latter group. Thirty-five percent of the 162 HPV-positive women showed no evidence of cytologic, colposcopic, or histologic abnormalities, indicating that they were infected latently. The proportion of seropositive women with high-load HPV infection and no cervical disease was six-fold higher (37.5% compared with 6.2%) when CD4 cell counts were at least 200/␮L. Seventy-three (34.4%) of the 212 women with high CD4⫹ cell counts and 19 (20.4%) of the 93 women with low CD4⫹ cell counts were HPV DNA–negative and showed no evidence of CIN (P ⫽ .001). The analysis of risk factors involved the 231 women with no evidence of cervical disease or with CIN. Using stepwise multivariate analysis, we found no relationship between cervical disease and age, ethnic origin, age at first intercourse, obstetric history, total number of sexual partners, safe sex practice, and current status with regard to genital warts. Cervical intraepithelial neoplasia was associated with smoking in women with CD4 cell counts of at least 200/␮L (adjusted odds ratio [OR] 3.0; 95% confidence interval [CI] 1.2, 7.2) but not in severely immunocompromised women (Table 3). The presence of HPV DNA and CD4 cell counts were strongly associated with the risk of cervical disease and there was a significant interaction between these two variables. High-load HPV infection was associated with a high risk of cervical disease (adjusted OR 16.8; 95% CI 7.0, 40.3). This risk was found to differ depending on the number of CD4 cells. In women with CD4 cell counts of

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at least 200/␮L, only high HPV load was associated with an increased risk of cervical disease (adjusted OR 12.6; 95% CI 4.9, 32.7) (Table 3). In women with CD4 cell counts less than 200/␮L, high HPV load was associated with a ten-fold increase in this risk (adjusted OR 134; 95% CI 18.5, 1098). In these severely immunosuppressed women, however, even a low HPV load was a marker for increased risk of cervical disease (adjusted OR 7.4; 95% CI 1.3, 43.0). Thirty of the 32 women with high HPV load were infected with cancer-associated HPV genotypes. For this reason, analysis according to genotypes was not performed. Results were similar when we excluded women with nonconclusive Southern blot hybridization results (16 patients).

Discussion In this series of 307 HIV-seropositive women, a high prevalence (27%) of biopsy-confirmed cervical disease was observed, in agreement with previous reports.8,10 A proportion of the women (18.2%) could not undergo biopsies despite abnormal Papanicolaou smears and/or minor colposcopic lesions, suggesting that the overall prevalence of CIN may have been underestimated. A high prevalence of HPV infection (52.8%) was noted in our study population when PCR and Southern blot hybridization data were combined. In previous studies, infection rates have ranged between 19%27 and 90%28 depending on the study group, the mode of cell sampling, and the test used for HPV DNA detection.13,15,16,29 A wide spectrum of HPV genotypes was disclosed in our study group, in agreement with other reports,13,15,16 and the oncogenic HPV types 16, 18, and 31 were the most commonly detected genotypes. We found a strong association (P ⬍ .001) between CD4 cell counts and the detection of oncogenic types, as reported

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in other studies.13,16,30 The prevalence of infection with multiple HPV types (13.3% as determined by Southern blot hybridization) was lower than the prevalences reported by Palefsky et al13 (37%) and Sun et al14 (51%). This may be due to the techniques used for cell sampling13,14 (use of a cervical brush instead of cervicovaginal lavage) and for HPV detection (Southern blot hybridization instead of restriction fragment length polymorphism analysis14 or hybridization of PCR products with type-specific probes13). In our study, CIN was found to be associated with smoking in women with CD4 cell counts of at least 200/␮L, in contrast with other reports.10,17 However, variables including age, ethnic origin, age at first intercourse, obstetric history, total number of sexual partners, safe sex practice, and status with regard to genital warts and other STDs were not found to be independent predictors of CIN in a multivariate analysis, in agreement with other studies involving HIVseropositive women.10,17,31 Genital HPV infection and severe immunosuppression previously were found to be independent risk factors for CIN.9,17,18 Risk of CIN was increased significantly among women with high HPV load.17,18,32 In our study, a significant interaction was observed between HPV infection and the level of immunosuppression, leading us to stratify analysis for the level of CD4 cell count (less than or at least 200/␮L).26 Low HPV DNA load was a significant risk factor for cervical disease only in severely immunosuppressed women. High HPV load always was associated with an increased risk of CIN, but a tenfold increase of the risk was observed in highly immunocompromised women. The proportion of seropositive women with no cervical disease but high HPV load was six-fold higher when CD4 cell counts were at least 200/␮L. In a previous study, we observed that in women receiving highly active antiretroviral therapy, a significant proportion of cervical lesions might revert to normality or to a lower grade, despite persistence of high loads of oncogenic HPV types.33 These data point to the major role of cell-mediated immunity in the control of the clinical expression of HPV infection but suggest that additional mechanisms are involved in the control of HPV infection. Direct molecular interactions between HIV and HPV appear unlikely, because HIV infection of cervical or vaginal epithelial cells has never been observed.34 –36 Aberrant expression of cytokines or chemokines by HPV-infected keratinocytes may modulate local immune responses.37,38 Conversely, cytokines produced by HIV-infected macrophages34 may influence the level of expression of HPV E6 and E7 oncoproteins, which are involved in keratinocyte growth stimulation, immunogenicity of tumor cells, and the HPV life cy-

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cle.38,39 Thus, the transition between silent high-load HPV infection and cervical disease could depend on a balance between systemic and/or local immune surveillance and levels of viral oncoproteins.

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Address reprint requests to:

Isabelle Heard, MD Unite´ INSERM 430 Immunopathologie Humaine Hoˆpital Broussais, 96 rue Didot 75674 Paris Cedex 14 France E-mail: [email protected]

Received October 7, 1999. Received in revised form March 13, 2000. Accepted March 24, 2000. Copyright © 2000 by The American College of Obstetricians and Gynecologists. Published by Elsevier Science Inc.

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