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).
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 β-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.
We thank Jean-Marc Trépanier and Serge Coté for maintenance of database of the HIPVIRG study and sampling of men. F.C. is a clinical research scholar supported by the Fonds de Recherche en Santé du Québec. E.F. holds a Distinguished Scientist Award from the Canadian Institutes for Health Research.
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Keywords:© 2008 Lippincott Williams & Wilkins, Inc.
PCR; HPV; human papillomavirus; anal cancer; intraepithelial neoplasia; genotyping