Anal human papillomavirus DNA screening by Hybrid Capture II ^TM in human immunodeficiency virus-positive patients with or without anal intercourse

ARTICLE

Auteur(s) : C. DROBACHEFF¹, P. DUPONT¹, C. MOUGIN², Y. BOUREZANE¹, B. CHALLIER³, M. FANTOLI¹, D. BETTINGER², R. LAURENT¹

¹ Service de Dermatologie, CHU, 25000 Besançon, France.
² Laboratoire de Biologie Cellulaire et Moléculaire, CHU Jean Minjoz, Boulevard Fleming, 25030 Besançon cedex, France.
³ Département d’Informatique Médicale, CHU, 25000 Besançon, France.

Article accepted 15/5/03

Patients and methods

The study population consisted of 50 HIV-positive patients from our specialized outpatient clinic (Department of Dermatology, University Hospital of Besançon, France) and 50 HIV-negative patients recruited from a proctologic consultation because of haemorrhoids. All patients gave informed consent before inclusion in the study.
At entry in the study for each HIV-positive patient, the following data were recorded: age, sex, sexual behavior (homosexuality, bisexuality, heterosexuality), intravenous drug use, prior medical history of anogenital warts, anal clinical HPV-associated lesions, CD4 + cell counts, plasma HIV RNA load, CDC stage, antiretroviral drug received. Enrolled HIV-negative patients were matched for age and sex.
At entry, anal swabs were obtained for HPV testing from every patient, followed by anoscopy. Only HIV-positive patients were followed-up every 3 months for one year and at each visit, they had HPV test, anoscopy, and blood collection to assess CD4 + cell counts and plasma HIV RNA load.
Diagnosis of HIV infection was done by means of anti-HIV-1 ELISA and was confirmed by western blot analysis. Measurement of plasma HIV RNA load was performed by the Amplicor HIV-1 Monitor^TM test version 1.5 (Roche Diagnostics System, Meylan, France) with a detection limit of 20 HIV RNA copies/mL. CD4 + T cell count was determined using a flow cytometer (FACS scan cytofluorometer from Beckton Dickinson) and categorized into CD4 + cells = 500/μL and CD4 + cells > 500/μL.
Anal swabs were obtained by anal brushing with the Digene Cervical Sampler^TM (Digene Corporation, Gaithersburg, USA). The cytobrush was then inserted into tubes containing 1 mL of Digene specimen transport medium and samples were frozen at – 20 °C until analysis.
The HPV DNA detection was performed using a chemiluminescent signal amplified hybridization microplate assay, the Hybrid Capture II^TM (HC-II) assay (Digene Corporation) according to the manufacturer’s recommendations. Briefly, 75 μL of the denatured specimens were used for analysis by HC-II. The single-strand DNA was hybridized for 1 h in solutions of two types of RNA probes allowing the detection of low risk HPV types 6, 11, 42, 43, 44 and high risk HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68. The resultant RNA/DNA hybrids were captured on microplate wells that were coated with anti-hybrid antibodies. The detection was performed with a second anti-hybrid antibody conjugated to several alkaline phosphatase molecules and a signal was generated with a chemiluminescent CDP-Star^TM with Emerald II-based substrate. Presence or absence of HPV DNA in the specimen was defined according to the strength in relative light units (RLUs) compared with the 1 pg/mL HPV 16 DNA positive control (PC). The sample was considered positive when the ratio RLU/PC was equal or greater than 1. Values provided an approximate determination of the HPV DNA load as we recently published [17]. A mixed (low risk and high risk HPV) infection was defined when a specimen was positive for both probes.

Statistical analysis

Student’s t test or Mann-Whitney U test was used to compare quantitative data (age, sex, CD4 + cell count, HIV load). Chi-2 test or Fischer’s exact test was used to compare qualitative data (sex, route of HIV transmission, CDC stage, prior medical history of anogenital warts, clinical lesions). Mann-Whitney U test was used to compare the load of HPV DNA detected in the anal sample of HIV-positive patient stratified by CD4 + cell count. To identify risk factors for HPV infection in HIV-positive patients, every available variable was taken into consideration.

Results

Patient characteristics

The main characteristics of the 50 HIV-positive patients at entry in the study are summarized in Table I. This population was representative of the cohort of HIV-infected patients taken in care in our hospital, except for the following: 52 % of patients were homosexual as compared with 38 % in our cohort. Heterosexual patients and intravenous drug users reported never having had receptive anal intercourse. Mean CD4 + cell count was 478/μL (range 55-1,109). Twelve out of 50 patients (24 %) had a CD4 + cell count > 500/μL, 29 patients (58 %) had a CD4 + cell count ≤ 500/μL and > 200/μL, and the remaining 9 patients (18 %) had a CD4 + cell count ≤ 200/μL. Mean HIV load was 1,890 copies/mL (range < 20-33,000). Only 2 patients (4 %) had an HIV load superior to 10,000 copies/mL.

Table I. Main characteristics of the 50 HIV-infected patients at entry in the study

Enrolled HIV-negative patients comprised 34 men and 16 women with a mean age of 40.4 years (range 24-65 yrs). Neither past history of anogenital warts nor present HPV- associated lesions was noted in this population.

Anal HPV infection characteristics

At enrolment, HPV DNA was detected by the HC-II test in the anal canal of 29 (58 %) of 50 HIV-positive patients and 3 (6 %) of 50 HIV-negative subjects (RR: 9.67 [95 % CI: 3.15-29.69]) (p < 0.00001). Among the 50 HIV-negative individuals, one (2 %) was harbouring low-risk HPV DNA and 2 (4 %) were infected with high-risk HPV alone.
Among the 50 HIV-positive patients, low risk HPV DNA alone was detected in 9 patients (18 %) and high-risk HPV DNA was detected in 20 patients (40 %) either alone in 6 patients or associated with low risk HPV DNA in 14 patients. Anal HPV DNA was detected as frequently in patients without anal intercourse (15/24 with high risk HPV in 10 patients) as in homosexual men (14/26 with high risk HPV in 10 patients). Surprisingly, anal high risk HPV DNA was frequently found in women (9/14). However, we had no consistent information regarding potential anal sexual intercourse. Moreover, the mean load of high risk HPV DNA was higher in patients with CD4 + cells = 500/μL (177.3 pg/mL) than in patients with CD4 + cells > 500/μL (11.2 pg/mL) (p < 0.04).
In the light of research which has shown that persistence of high risk HPV is a risk factor for development of premalignant and malignant changes in the cervix, we tested for HPV infection outcome only in HIV-positive patients. Interestingly, the rate of viral DNA detection progressively decreased during the one-year follow-up. With the use of HC-II assay, we observed that 5 patients out of the 20 who harboured high risk HPV at entry (month 0: M0) were repeatedly HPV positive at each visit (M3, M6, M9 and M12). At M12, the mean HPV load was 375 pg/mL.
At enrolment, clinical examination revealed that among the 50 HIV-positive patients, one woman and one man had current lesions of the anal mucosa, which were associated with the presence of high risk HPV DNA. In the other patients, anoscopy did not reveal presence of anal lesions, neither at entry in the study nor over the 12 month follow-up period. Two other women demonstrated vulvar and cervical warts.

Risk factors for anal HPV infection among HIV-positive pat1ients

At entry in the study, the following factors: age, sex, route of HIV transmission, HPV-related clinical lesions, plasma HIV RNA load, were not significantly associated with the detection of anal HPV among HIV-positive patients.
On the other hand, the degree of immunosuppression was a risk factor for anal HPV infection. Indeed, HIV-infected patients with a CD4 + cell count less than 500/μL were at greater risk for anal oncogenic HPV infection than patients with CD4 + cells > 500/μL (RR: 2.13 [95 % CI: 1.0-4.7]) (p = 0.02). Of the 20 patients harbouring high risk HPV DNA, 15 (75 %) had CD4 + = 500/μL while 5 (25 %) had CD4 + > 500/μL. Interestingly, the 5 patients who were high risk HPV positive throughout the follow-up always had CD4 + cell count = 500/μL.
With respect to stage of HIV disease (based on the Centers for Diseases Control and Prevention criteria), we observed that among HPV-negative patients, 11 (52.4 %) were in stage A, 6 (28.6 %) in stage B and 4 (19 %) in stage C; among HPV-positive patients, 7 (24.1 %) were in stage A, 15 (51.8 %) in stage B and 7 (24.1 %) in stage C. Although, HIV-positive patients harbouring HPV DNA in the anal canal were more numerous in stage B and C compared with patients who were HPV negative, the tests for trend were not significant. A previous history of anogenital warts was also a risk factor for oncogenic HPV infection (RR: 2.36 [95 % CI: 1.2-4.6]) (p = 0.03). Among HIV-positive patients with history of anogenital warts, HPV DNA was detected 2 times more frequently (59 %) than in patients without previous lesions (30 %).

Discussion

Years ago, it was shown that impaired cellular immunity enhanced susceptibility to viral infection and malignant diseases [18]. Increased risk for HPV-associated anogenital diseases has been confirmed in HIV-infected men and women and HPVs have emerged as opportunistic complications in this population [19, 20]. Indeed, in the study conducted by Piketty et al. [21], anal HPV infection was identified in 85 % of HIV-positive men having sex with men.
To date, prevalence of HPV and SIL has not been frequently evaluated in HIV-infected patients in the absence of receptive anal intercourse, such as in intravenous drug users and women [21].
In the present study, we have described the prevalence and characteristics of HPV infection in a series of 50 HIV infected patients, a representative series of our registered cohort. The proportion of homosexual men is however slightly higher than that observed in the hospital cohort (52 % vs 38 %). Our series also appears to under-represent women (28 %) but sex was not found to be a risk factor for HPV infection.
The overall prevalence of HPV in the anal canal of HIV-positive patients was significantly higher (58 %) than that observed in HIV-negative patients (6 %) and confirmed previous observations [4, 8, 13, 21-25]. In addition, our results showed that in HIV-positive patients, the rate of oncogenic HPV genotypes was high and the occurrence of mixed infections (low risk and high risk HPV) was very common, according to numerous studies [21, 23-27].
Hybrid Capture II assay gives slightly lower sensitivity than PCR for HPV DNA detection [28, 29], but it represents a more convenient and easier test for routine applications [30]. Some reports have suggested that type specific persistence of HPV DNA [31] or HPV load [17, 32-34] are determinants for development of cervical intraepithelial lesions. Although HC-II results do not permit us to quantify a specific HPV genotype, they can however be considered as a reflection of HPV DNA load in the specimen [7, 17, 35]. Here, we have reported that HPV DNA loads detected in HIV-infected patients were related to CD4 + cell counts, patients with low CD4 + cell counts having significantly higher HPV load than patients with CD4 + cell counts > 500/ μL, which is consistent with previous data [7]. Moreover, the 5 patients with repeated presence of oncogenic HPV had high viral load throughout the study and were less likely to clear their HPV infection than those with low levels of HPV DNA, as already reported for cervical infection in women [17, 36]. In that case, it has been considered that HPV load could be a sensitive indicator for progression to cervical high grade lesions [17, 32-34]. Similarly, the high viral load might be a risk factor for anal SIL and anal cancer in immunosuppressed patients.
Interestingly, we did not find any difference in the rate of anal HPV infection according to the route of HIV transmission. Although it might be assumed that homosexual men would be at higher risk of anal HPV infection than heterosexual men and women, recent studies have shown that anal infection is as frequent in heterosexual men, women and intravenous drug users as in homosexual men [37]. In HIV-infected women, the anal infection is even more common than cervical HPV infection [27, 38]. In a study that enrolled 200 women, HPV DNA was detected in 79 % of anal and 53 % of cervical samples from HIV-positive women; HPV DNA was also found in 43 % of anal and 24 % of cervical samples from high risk HIV-negative women [39]. It has been thus suggested that anal HPV infection was not associated with a history of receptive anal intercourse, but rather with low CD4 + cell counts [27, 39]. HIV-positive homosexual men and HIV-positive women with the same CD4 + cell counts were similarly infected by HPV [39]. Despite the relatively small sample size, we similarly noted that the prevalence of HPV was related to the number of CD4 + cell counts. Risk factors also included history of genital warts which has already been described recently [40]. Although, Holly et al. [40] and Piketty et al. [21] found an elevated risk for HPV-associated anal lesions with the RNA HIV load, our results support the predominance of the CD4 marker over plasma HIV load for predicting HPV infection outcome.
Studies from the era prior to the introduction of HAART have shown that the prevalence of anal HPV-associated lesions in homosexual men was 36 % to 60 % [41, 42]. In 1999, Voltz et al. found clinical HPV lesions in 19 % of homosexual men [25]. Here, we observed a low prevalence of clinical or subclinical anal lesions that might be related to the characteristics of the cohort. Nevertheless, Piketty et al. recently reported low grade SIL in 49 % of HIV-infected homosexual males and in 16 % of HIV-infected intravenous drug users who never have had receptive anal intercourse; high grade SIL was diagnosed at the same rate (18 %) in both groups [21]. Our study has at least 2 limitations. The follow-up period might be too short for development of anal lesions. Anal cytology was not evaluated and biopsy was performed only in patients with a suspect anoscopy. Thus, the true prevalence of anal lesions might be underestimated.
In conclusion, HPV infection is highly prevalent in HIV-infected patients not only in homosexual males but also in patients without history of receptive anal intercourse. A close follow-up of this cohort will permit us to establish which factors would be the most predictive of anal cancer onset. We should anticipate the possibility of such an HIV-related cancer, since HIV-infected patients live longer because of HAART and the risk of anal malignancies may increase due to longer latency periods. Therefore, our results suggest that it might be worthwhile to integrate anal screening with cytology, colposcopy and HPV detection in HIV-positive patients, especially in those with severe immunodeficiency, regardless of history of anal intercourse.

Acknowledgements. We thank D. Devred, F. Pelletier, S. Cairey-Remonay and C. Chervet for their participation and care of patients, L Madoz for technical assistance and L. Rose for English suggestions. < 

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