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Genotyping of Human Papillomavirus DNA in Anal Biopsies and Anal Swabs Collected From HIV-Seropositive Men With Anal Dysplasia [Clinical Science]
  JAIDS Journal of Acquired Immune Deficiency Syndromes:Volume 49(1)1 September 2008pp 32-39
Gohy, Laurent*; Gorska, Isabella PhD*; Rouleau, Danielle MD*; Ghattas, Georges MD*; Pokomandy, Alexandra de MD*; Vezina, Sylvie MD; Cote, Pierre MD; Macleod, John MD; Allaire, Guy MD; Hadjeres, Rachid MD; Kornegay, Janet R PhD; Franco, Eduardo PhD; the HIPVIRG Study Group Francois Coutlee, MD
From the *Departements de Microbiologie et Infectiologie, Gastro-enterologie, Pathologie et Medecine familiale, Laboratoire de Virologie Moleculaire du Centre de Recherche, Laboratoire de pathologie moleculaire, Centre Hospitalier de l'Universite de Montreal, Montreal, Quebec, Canada; Departements de Microbiologie-Immunologie et de Pathologie, Universite de Montreal, Montreal, Quebec, Canada; Division of Cancer Epidemiology and Department of Medicine, McGill University, Montreal, Quebec, Canada; Clinique Medicale l'Actuel, Montreal, Quebec, Canada; Clinique Medicale du Quartier-Latin, Montreal, Quebec, Canada; and _Roche Molecular Systems, Alameda, CA.
Background: Human papillomavirus (HPV) causes anal intraepithelial neoplasia (AIN) in HIV-seropositive men. The detection of HPV genotypes in anal biopsies and swabs was compared.
Methods: HPV DNA was detected in anal swabs and biopsies obtained concurrently from 154 HIV-seropositive men [31 without AIN, 60 low-grade AIN (AIN-1), 62 high-grade AIN (AIN-2,3), and 1 indeterminate AIN] under or eligible to highly active antiretroviral therapy.
Results: HPV DNA was detected in 24.2% of normal biopsies compared with 93.5% with AIN-2,3 (P < 0.001) and 88.3% with AIN-1 (P < 0.001). The proportion of biopsies containing multiple genotypes was greater in AIN-1 (n = 21, 35.0%; P = 0.002) and AIN-2,3 (n = 38, 58%; P < 0.001) than in normal biopsies (n = 2, 6.5%). The most frequent genotypes in order of frequency were in AIN-2,3 biopsies HPV-16, 18, 58, and 45 and were in AIN-1 biopsies HPV-6, 11, 16, and 39. Controlling for age, CD4 count, and smoking, the presence of high-risk HPV DNA in biopsies [odds ratio (OR) = 50.8, 95% confidence interval (CI): 13.0 to 199.5] but not in swabs (OR = 2.0, 95% CI: 0.6 to 7.0) was associated with AIN-2,3.
Conclusions: AIN-2,3 was associated with high-risk HPV infection detected in biopsies but not in swabs in men under or starting highly active antiretroviral therapy, possibly due to the presence of HPV foci outside of the neoplastic lesion.
The incidence of anal cancer has been increasing in the past 2 decades, especially among men who have sex with men.1 The risk of anal cancer is up to 42-fold higher in men infected by HIV than in the general population.1,2 The annual incidence of invasive anal cancer increased from 0 per 100,000 men with AIDS in 1991 to 224 per 100,000 in 2000 in San Diego county in the United States.3 In a cohort study, invasive anal cancer developed mostly in subjects with high-grade anal intraepithelial neoplasia (AIN-2,3) that was detected 13-108 months before invasive cancer.4 More than 50% of HIV-seropositive men having sex with men will develop AIN over 2-4 years.2,5 Anal and cervical cancer share clinical and histological similarities.6 They both arise from precursor intraepithelial neoplastic lesions.6 High-risk human papillomavirus (HR-HPV) genotypes that cause cervical intraepithelial neoplasia (CIN) and cancer are also those that were detected in most AIN-2,3 and anal cancer.1,7-9 There is thus an interest in better understanding the association between HPV infection and AIN and defining the natural history of anal HPV infection and HPV-induced AIN.10
Very few studies examined the prevalence of HPV types in biopsies with AIN (Table 1). Information on the prevalence of the various high-risk types in AIN is of considerable importance. Several investigators used tools that did not identify the full spectrum of genital genotypes, others tested a small number of AIN-2,3 samples or pooled results obtained from individuals with AIN-2,3 and individuals with cancer (Table 1).4,7,11-19 Only one study analyzed more than 30 biopsies with AIN-2,3.20 The concurrent detection of HPV genotypes in anal biopsies and anal swabs has never been assessed.
We found a moderate agreement between anal swabs and biopsies obtained at the same visit for detection of HPV-16 or 18 in a subset of 154 men participating in a cohort study on HPV-induced anal disease. Nearly all participants were infected by HPV in the anal canal. The frequent occurrence of anal HPV infection and multiple-type infections in our participants enabled us to perform this comparison for 36 genotypes. This high prevalence of HPV infection has been reported by others.25,26 Although the number of HPV types detected in anal swabs and biopsies was correlated, it was significantly greater in anal swabs, likely reflecting that HPV types detected in swabs tend to be representative of multiple infection foci in the anal canal.
A majority of HIV-seropositive men had an abnormal cytology smear, and only half of those with a normal cytology had normal findings at high-resolution anoscopy. Because histology is the gold standard for establishing the grade of anal lesion, the distribution of HPV genotypes was described according to grades of histological lesion instead of grade of cytological anomalies. In fact, the sensitivity of cytology smears to detect the presence of AIN-2,3 has been reported to be in the range of 34%-92% when any anomaly was considered significant.20,27,28 Although anoscopy may not detect small AIN-2,3 lesions that lie hidden in a fold of mucosa, we did not find any man with a cytology smear showing high-grade squamous intraepithelial lesions but a normal anoscopy. Biopsies were not performed blindly, explaining in part the small number of normal biopsies evaluated in this study.
HPV DNA has been detected in 70%-100% of anal cancer and 60%-100% of AIN-2,3.7,8,13,18,19,29-32 Only a limited number of studies have identified HPV types implicated in AIN by testing biopsies.4,7,11-19 In our study, which included the greatest number of biopsies with AIN-2,3 tested for HPV up to date, 94% of AIN-2,3 lesions contained HPV DNA. The distribution of HPV types was different in biopsy specimens with AIN-2,3 and AIN-1. HPV-16 was the most frequent genotype in AIN-2,3, whereas type 6 was the most frequent in AIN-1. The prevalence we obtained for HPV-16 was lower in AIN-2,3 than expected from other studies but was similar between AIN-2 and AIN-3. Several studies also reported these findings.4,7,8,15,18,19,29-31 HPV-16 was also the most frequent type detected in anal cancer biopsies in one study8 and was the most frequently detected genotype in anal swab samples in our study, as reported by others.33
The most frequent genotypes detected in anal biopsies with AIN-1 in our study were different from those described in low-grade CIN-1.34 A meta-analysis reported that the most frequent genotypes in CIN-1 were types 16, 51, 56, 53, and 52 in North America, whereas HPV-6 ranked 11th.34 In our subjects with AIN-1, HPV-6 was the most frequent genotype followed by HPV-16 and HPV-11. This difference could be related in part to the frequent occurrence of visible intra-anal condyloma acuminata in our participants with AIN-1. There could also be a difference in the viral etiology of low-grade lesions in the anal canal and cervix. Our findings on the distribution of HPV types in cases of AIN need to be interpreted with caution due to the low number of these cases and should be confirmed in future studies. Our results are in agreement with a study conducted in HIV-seropositive men with anal condyloma.35 In another study including 9 subjects with condyloma/AIN-1, HPV-6 and HPV-11 were the most frequently detected followed by HPV-16 and HPV-61.18 In a third study on 8 AIN-1 biopsies, 6 cases were infected with types 6 and/or 11.19
The higher frequency of detection of each type and higher burden of infection in anal swabs could be due to sampling the entire surface of the anal canal with swabs as opposed to a sampling only a small portion of the transformation zone with biopsies. Similarly, cervicovaginal lavages were also shown to contain more frequently multiple HPV types than biopsies of CIN.36 A recent comparison reported an agreement of 44% for HPV type detection in biopsy and lavage samples in women with cervical disease.36 Cells in cervicovaginal lavages could have exfoliated from the cervix or vagina or from a region of the cervix without disease and may not indicate the HPV type causing the CIN. HPV detection is also hampered in biopsy samples that have been embedded in paraffin. Only ß-globin-positive biopsies were included in the comparison with anal swabs. DNA was extracted in less than 1 month of fixation (data not shown), increasing the likelihood of a successful amplification. The assay used in our work was not quantitative. In future projects, it would be interesting to assess if discordance between results obtained on different types of samples is related to the viral load of types detected. Biopsies with AIN from our participants contained more frequently multiple types than normal biopsies. Infection with multiple HPV types in anal biopsies was common among subjects with AIN-2,3, as reported by others.7,18 HPV types detected in biopsies were not detected in concurrently obtained anal swabs in about 15% of cases. Our results are also in agreement with another publication that also found that there was no association between HPV DNA detection in anal swabs and grade of AIN but lacked HPV DNA data in biopsies.20
In HIV-seropositive men under or eligible for treatment with HAART, infection by HPV in the entire anal canal is not representative of the HPV types causing the actual AIN lesions at the junction of anal and rectal mucosa. Nevertheless, the sensitivity of anal swabs to detect HPV types identified in biopsies with AIN was high. Due to the frequency of detection of HPV and the high number of types detected in swab samples, screening for AIN with HPV testing will probably not be applied as it is for cervical lesions in women.
Detection of 36 HPV Genotypes in Anal Swabs and Anal Biopsies

The demographic and clinical characteristics of the 154 participants are presented in Table 2. Participants had a mean age of 44 years. A majority of individuals (94%) had a history of receptive anal intercourse, and nearly half of them used condoms at least most of the time. Sixteen percent of men had CD4 cell counts below 200 cells/μL. Only 21% of men did not have AIN on biopsies, reflecting in part the fact that biopsies were not performed blindly in men without visible acetowhite lesions. Only 30% of participants had normal cytology smears. The presence of any anomalies [atypical squamous cells of undetermined significance (ASC-US) grade or above] on a cytology smear was encountered for 47 of the 62 men with AIN-2,3 for a sensitivity of 75.8% (95% CI: 63.8% to 84.8%). All participants with smears showing high-grade squamous intraepithelial lesions had AIN-2,3 on histology.
For each of the 154 participants, HPV genotyping results were obtained on anal swabs and biopsies obtained at the same visit. The distribution of HPV genotypes in biopsies without AIN and with AIN-1 or AIN-2,3 is provided in Table 3, considering the highest grade of AIN on histology for each participant. HPV-16, 18, 58, and 45 were the most frequent genotypes detected in AIN-2,3 biopsies, whereas types 6, 11, 16, 39, and 42 were the most frequently identified in biopsies with AIN-1. HPV-16 was the most prevalent type detected in biopsies present in 2 (7%) of normal, 6 (10%) of AIN-1, and 22 (36%) of AIN-2,3. The proportion of biopsies containing HPV-16 was similar in AIN-2 and AIN-3 (data not shown). HPV DNA was detected in 8 (24.2%) of 31 normal biopsies in contrast to 58 (93.5%) of 62 biopsies with AIN-2,3 and 53 (88.3%) of 60 biopsies with AIN-1 (P < 0.001 for each comparison). There was a significant difference in the proportion of biopsies with AIN-2,3 and those without AIN positive for HPV-16 (P = 0.001), HPV-18 (P = 0.02), or HPV-58 (P = 0.02). We did not study the other high-risk types because the comparison of difference of proportion did not reach significance even if a type was not detected in the 25 normal samples but was detected in AIN-2,3 but in less than 10 biopsies. Controlling for age, CD4 cell count, and smoking by logistic regression, the presence of HPV DNA (OR = 72.1, 95% CI: 13.8 to 376.7), HR-HPV DNA (OR = 50.8, 95% CI: 13.0 to 199.5), or HPV-16 (OR = 8.2, 95% CI: 1.7 to 39.5) in biopsies was significantly associated with AIN-2,3 compared with men without AIN. The comparison of distribution of HPV types between men with CD4 counts above and 200 cells/μL was limited by the small number (n = 25) of men with low CD4 counts but revealed that HPV-16 was the most frequent genotype irrespective of the level of immunosuppression.
The distribution of HPV types in anal swabs collected concurrently with biopsy is provided in Table 4. HPV-16 was also the most frequently detected type in anal swabs being identified in 59 (38.3%) of 154 participants. In opposite to anal biopsies, most swab samples contained more than one type, and nearly all swab specimens were positive for HPV. Anal swabs were negative for HPV in only 3 (2.0%) men (2 normal and 1 AIN-1). In opposite to biopsy samples, the presence of HPV DNA (OR = 0.8, 95% CI: 0.1 to 4.6) or HR-HPV (OR = 2.0, 95% CI: 0.6 to 7.0) in swab samples was not associated with AIN-2,3, controlling for age, CD4 cell count, and smoking by logistic regression. The presence of HPV-16 in anal swabs was, however, significantly associated with the presence of AIN-2,3 (OR = 5.1, 95% CI: 1.8 to 14.1).
We then investigated if HPV DNA testing on anal swab could help triage men with cytologies showing ASC-US to anoscopy. Of the 41 men with ASC-US, 24 had AIN-1, 16 had AIN-2,3, and 1 had AIN of undetermined grade. In men with a cytology showing ASC-US, there was no difference in the proportion of swabs positive for HR-HPV DNA between men with AIN-1 (20 of 24) and those with AIN-2,3 (14 of 16) on histology. HPV DNA testing was thus not useful to select men with ASC-US at the highest risk for AIN-2,3 in opposite to women with ASC-US for cervical high-grade intraepithelial neoplasia.
Comparison of HPV Detection in Anal Swabs and Biopsies
The proportion of samples positive for HPV DNA in 154 anal swab and anal biopsy pairs obtained at the same visit was compared in Table 5. This comparison was limited to the most frequent HR-HPV genotypes as a group to avoid rarer types for which most samples were negative. There was a poor agreement between anal biopsies and anal swabs with very low kappa values below 0.50. Agreement for the presence of high-risk and low-risk types was 64.7% and 36.5%, respectively (Table 5). The low level of agreement was mostly caused by an important number in each situation of HPV-positive swabs only.
Burden of HPV Infection in Swabs and Biopsies
The proportion of biopsies containing multiple HPV types was greater in AIN-1 (21 of 60, 35.0%; P = 0.002) and AIN-2,3 (38 of 62, 61.3%; P < 0.001) than in biopsies without AIN (2 of 31, 6.5%). The number of types detected in swabs was significantly greater than the number of types detected in biopsy samples. A mean of 0.9 high-risk types (95% CI: 0.7 to 1.0, median 1, range 0-4) was detected in biopsies compared with 3.0 (95% CI: 2.7 to 3.3, median 3, range 0-11) in anal swabs (P < 0.001). Similarly, 0.4 low-risk types (95% CI: 0.3 to 0.5, median 0, range 0-3) were detected per biopsy compared with 1.8 types (95% CI: 1.6 to 2.1, median 2, range 0-6) per swab specimen (P < 0.001). Nevertheless, we found a significant correlation in 154 paired samples of the total number of types (r = 0.39, P < 0.001), high-risk types (r = 0.49, P < 0.001), and low-risk types (r = 0.20, P = 0.01).

Paired anal biopsies and anal swabs from the same visit were obtained from 154 men participating in the HIPVIRG cohort conducted in Montreal, Canada. Because more than 1 biopsy could be obtained at each visit, the data that will be presented will reflect results of the highest grade lesion detected during the first 3 visits. All participants gave written informed consent to participate. This cohort study was approved by the ethics committees of all participating institutions. HIV-seropositive men were eligible for enrollment if they were 18-65 years old, had a history of anal sex in the last year, had a nadir CD4 cell count <500/mm3, had been treated for at most 2 years with HAART, or were eligible to start a new HAART regimen irrespective of previous treatments. Men with a nadir CD4 count >500/mm3 were not selected because they were not eligible for HAART and the objective of this study was to assess the effect of HAART on AIN incidence and progression. From January 2002 to January 2005, a total of 249 men accepted to participate. At baseline and follow-up visits at every 6 months, subjects completed a self-administered questionnaire and had blood CD4+ counts and plasma HIV RNA loads measured.
Sample Collection
At each visit, a saline-moistened Dacron swab was inserted 3 cm into the anal canal, retrieved with a twirling motion and gentle pressure, and rolled on slides and immediately fixed with a cytospray for Pap smears. Smears were classified in accordance with the Bethesda system. A second anal sampling with a swab was obtained. The swab was agitated in 1.5 mL of PreservCyt (Cytyc Corporation, Boxborough, MA).21 High-resolution anoscopy after application of 3% acetic acid was performed at baseline and yearly thereafter for those without AIN or with AIN-1 and every 6 months for individuals with AIN-2,3 at previous visits. Acetowhite areas with different aspects were biopsied with forceps. Classical exophytic condylomas were not systematically biopsied. Biopsies were fixed in 10% formalin and reviewed by the same pathologist.
Biopsy and swab samples were obtained concurrently in at least 1 of the first 3 visits from 154 (61.8%) of the 249 men recruited in the cohort. The remaining 95 subjects did not provide paired specimens because high-resolution anoscopy was normal at the first 3 visits (n = 21), high-resolution anoscopy disclosed only the presence of exophytic condyloma acuminate at any of the first 3 visits (n = 10), residual biopsy material was not available for HPV analysis (n = 47; 19 normal, 22 condyloma, and 7 AIN-2,3), or available biopsies tested negative for ß-Globin (n = 16; 9 normal, 4 AIN-1, and 3 AIN-2,3). The latter biopsies may have tested negative for ß-Globin because of the small amount of residual material or fragmentation of DNA during processing for histopathology. Overall, 177 biopsies were obtained concurrently with anal swabs from 154 men during the first 3 visits. Only the biopsy with the highest grade lesion per participant was considered for the analysis of HPV genotypes. HPV results from all biopsies obtained at the same visit were pooled for the comparison between anal biopsies and swabs for the detection of HPV genotypes, which was included in the comparison study, for a total of 154 biopsy-anal swab pairs.
Biopsy and Anal Swab Sample Preparation
After centrifugation at 13,000g for 15 minutes at 22°C of samples in PreservCyt, the supernatant was discarded and the cell pellet was left to dry and resuspended in 300 μL of 20 mM Tris buffer, pH 8.3. DNA was purified with Master pure (Epicentre, Madison, WI).21 After section of formalin-fixed paraffin-embedded blocks of anal biopsies with a disposable microtome blade, DNA was purified as described previously with xylene treatment, proteinase K digestion at 58°C overnight, and heat inactivation by boiling for 8 minutes.22 Five hundred nanograms of DNA extracted from biopsies was tested with PCR.
PGMY-Line Blot Assay
ß-Globin was amplified with PC04 and GH20 as described previously.23 Samples negative for ß-Globin were considered inadequate. Samples testing positive for ß-Globin were tested for HPV with PGMY09/PGMY11 primers without further coamplification for ß-globin. HPV genotyping for 36 genital HPV types was performed with the reverse line blot detection system as previously described.21,24 Samples that were not positive for any of these types were considered HPV negative. The following procedures were implemented to avoid contamination. Samples were processed with disposable pipettes. PCR mixtures were prepared in a biosafety cabinet using pipettors with filter pipette tips dedicated exclusively for this purpose. All reagents were aliquoted before use. Post-PCR products were handled with different pipettors in a separate room.
Statistical Methods
Because more than 1 biopsy could be obtained at each visit, the data that will be presented will reflect the results of the highest grade lesion detected during the first 3 visits. The crude percent agreement between anal biopsies and swabs was the percentage of paired samples with equivalent results considering positivity for HPV DNA irrespective of types detected, positivity for HR-HPV types as a group (types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, and 82), and positivity for HPV-18 and for HPV-16, each considered individually. The modified Wald method was used to calculate 95% confidence intervals (CIs) around binomial proportions. The unweighted kappa statistic was calculated to adjust for chance agreement between HPV detection methods. The significance of differences in proportions of HPV-16 DNA-positive or multiple-type infections was assessed using the Fisher exact test. The nonparametric Sign test was used to test the significance of differences between swabs and biopsies for the number of types detected. The relative correlation between the number of types identified in swab samples and biopsy samples was assessed by the Spearman rank-order correlation coefficient. The magnitude of the association between HPV DNA and AIN-2,3 was assessed by calculating odds ratios (ORs) and respective 95% CIs by multiple logistic regression while controlling for the confounding effects of age, CD4 counts as categories (<200 and ≥200 cells/μL of blood), and smoking. Statistical analyses were performed with STATISTICA version 6 software (StatSoft, Tulsa, OK).
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