To the Editor:
Infection with human papillomavirus (HPV) is recognized as the major determinant of anogenital cancers.1-3 Most research has focused on HPV infections in women, but HPV malignancies other than cervical cancer have increasingly been reported in recent years among HIV-infected or uninfected men.4-6 Highly oncogenic HPV-16 and HPV-18 genotypes are reported as the major cause of anogenital cancers in women and men, but a broad spectrum of HPV genotypes has been associated with cancer development.7,8 Severely immunosuppressed HIV-infected patients are at significantly higher risk of viral persistence, multiple HPV infections, low- or high-grade squamous intraepithelial lesions, and progression to invasive cancers than immunocompetent individuals. HPV-16 is generally reported as the most common high-risk type in HIV-infected women and HIV-infected men,9-11 but the high rate of multiple infections prevents determination of the role of each genotype in carcinogenesis.7
This study focuses on the spectrum of HPV types among HIV-infected men and their relation to cytologic/histologic lesions.
From June 2003 to March 2005, consenting HIV-infected male patients referred to the sexually transmitted disease (STD) Unit (II Department of Infectious Disease, L Sacco Hospital Milan, Italy) were evaluated for the point prevalence of HPV genotypes and related dysplastic lesions. Demographic, epidemiologic, and immunovirologic parameters and current antiretroviral regimen information were evaluated through the clinical record of the patients.
Patients underwent cytologic brushing of the anal canal (Cervex brush; Cytobrush Plus-Medscand Medical AB, Sweden) for cytologic evaluation (according to the 2001 Bethesda System for cervical cytologic reporting) using the Hybrid Capture 2 HPV test (HC2; Digene, Gaithersburg, MD) and for HPV DNA detection (QIAamp DNA Mini Kit; QIAGEN GmbH, Hilden, Germany). HPV DNA-positive samples were typed by the restriction fragment length polymorphism (RFLP) technique with the Rsa I, Dde I, and HaeIII digestion systems (New England Biolabs, Ipswich, MA). The pattern of the fragments obtained from the 3 digestive processes allowed identification of 31 different HPV types. High-resolution anoscopy was performed as the primary screening procedure in all patients, and biopsies, performed in case of acetic white lesions, warts, unsatisfactory cytologic results, atypical squamous cells of undetermined significance (ASCU-US) or atypical squamous cells cannot exclude high-grade squamous intraepithelial lesion (ACS-H), or a positive high risk (HR) or HR/low risk (LR) genotype on a hybrid capture test within 1 month from the Papanicolaou smear, have been considered for this study.
A total of 253 HIV-positive men agreed to participate and were included in this study. HIV infection was acquired for unprotected homosexual contacts in 177 (69.96%), unprotected heterosexual contacts in 17 (6.72%), drug addiction in 11 (4.35%), and unknown or unreported in 48 (18.97%). The median age was 34 (interquartile range [IQR] = 30 to 42) years, median CD4 count was 468 (IQR = 329.5 to 639) cells/μL, and median HIV RNA log10 level was 2.43 (IQR = 1.69 to 4.055). Fifty-three patients (20.95%) were off highly active antiretroviral therapy (HAART), with 36 not needing treatment according to national guidelines for antiretroviral treatment of HIV-infected patients and 17 being recently diagnosed as HIV-infected at the time of their visit to the STD Unit.
Of 233 stored samples, 30 were negative on polymerase chain reaction (PCR) amplification and 203 were subsequently analyzed for the HPV typing. In 4 cases, no HPV genotype could be identified, and in the remaining 199 samples, 30 different HPV genotypes were isolated. HPV-16 was isolated in 85 samples, HPV-6 in 63, HPV-11 in 62, and HPV-30 in 20. HR-HPV genotypes account for 57.6% of all the specific HPV isolated.
Anal lesions evaluated on a composite cytologic/histologic examination in these 199 samples were as follows: 24 negative for intraepithelial lesion (NIL), 3 ASC-US or ASC-H, 139 low-grade squamous intraepithelial lesion (L-SIL)/anal intraepithelial lesion (AIN), 32 high-grade squamous intraepithelial lesion (H-SIL)/AIN-2 to 3, and 1 invasive cancer. Cytologic/histologic abnormalities were variably distributed according to HPV oncogenic risk genotypes, but no specific association with single HPV genotypes could be demonstrated in this point assessment study. In 61.8% (123 of 199) of the cases, infections with multiple HPV genotypes were identified: 2 genotypes were isolated in 76 patients (38.19%), 3 genotypes in 32 patients (16.08%), and ≥4 genotypes in 13 patients (6.53%).
Patients with multiple infections (≥3 HPV types) had significantly lower CD4 cell counts than patients with single HPV infections, and higher HIV viral loads were observed in patients with ≥4 HPV genotype infections (Fig. 1).
The odds of multiple infections was 22.24 (95% confidence interval [CI]: 8.93 to 55.39) times greater in patients with HPV-16 or HPV-18 and 6.42 (95% CI: 2.62 to 15.76) times greater in patients with HR-HPV other than HPV-16 or HPV-18 compared with patients with only LR genotypes. The odds of multiple infection were also increased in patients with detectable HIV RNA >5000 copies/mL (odds ratio [OR] = 2.196, 95% CI: 0.99 to 4.86) and in patients with CD4 counts <200 cells/mL (OR = 4.85, 95% CI: 0.99 to 23.68). There was a trend for an association of increased risk of multiple HPV infections for decreasing CD4 cell counts (P = 0.037, χ2 test for trend). Middle-aged patients had a lower probability of multiple infections (OR = 0.40, 95% CI: 0.20 to 0.80) than younger patients (<30 years of age; P < 0.01), but no differences in risk of multiple infections were observed between the younger and older (≥50 years of age) groups.
The longer survival of HIV-infected patients attributable to the widespread use of HAART allows progression of HPV preneoplastic diseases to overt cancer, whereas improvement of immune response does not affect the incidence of infections, which are more related to risk behaviors than to an effective immune response. We found a high point prevalence of anal HPV infection by HC2 (92.17%) and of HPV-related cancer precursor lesions by cytology (83.48%) in patients despite a high CD4 cell count.
The broad spectrum of HPV genotypes and the high prevalence of multiple infections found in this series are points of concern. As in other studies on HIV-infected patients, we found HPV-16 to be the most prevalent genotype isolated, whereas we found a low rate of HPV-18 (5.5%) compared with other epidemiologic studies on HIV-infected women7,9 and men.5 Dunne et al,5 in a systematic review of literature on HPV infections in men, report that the low- and high-risk genotypes included in the quadrivalent and bivalent vaccines (HPV-6, HPV-11, HPV-16, and HPV-18) are less prevalent in men than in women and that age, site of brush collections, methods of analysis, HIV serostatus, and geographic variables can contribute in determining the wide variety of prevalence reported and the differences in specific genotypes isolated.
Coinfection with multiple genotypes, reported in 61.8% of our patients and at rates as high as 88% in other studies,10 is related to low CD4 cell counts, high HIV viral loads, and young age (<30 years of age), but we did not find an age-related decrease in HPV prevalence and infections from multiple HPV genotypes in the older age group in this study. In the EXPLORE study, Chin-Hong et al11,12 found no protective immunity to re-exposure and no cross-protection with phylogenetically related genotypes in men who have sex with men (MSM) in repeated infections, suggesting that naturally acquired protective immunity to specific genotypes is not long lasting. The increased rate of multiple infections found in this study in the senior age group could be explained by this mechanism.
Multiple infections are closely related to the genotypes classically associated with cancer development. If we cluster the patients on the basis of the HPV isolates, we found that patients with the higher risk cluster (HPV-16/18) have a probability of multiple infection 22 times higher than those with the LR-HPV cluster. A similar positive association was found for the HR-HPV cluster (excluding the HPV-16 to HPV-18 genotypes), which has a 6.4-fold higher probability of multiple infections than the LR-HPV cluster.
HAART seems not to prevent multiple infections. Patients treated with several antiretroviral regimens have the same probability of multiple HPV infections as patients who are off HAART. The finding of a 6.3 times greater risk of AIN 2 or 3 in men treated with HAART regimens compared with patients who were not currently taking antiretrovirals, as described by Palefsky et al,10 increases the concern about the paradoxic effects of antiretrovirals in the natural history of genital HPV infections in HIV-infected men.
Several aspects should be addressed when interpreting our results. Point assessment of genital dysplastic lesions could be related to transient infections rather than to established and progressive lesions. A prospective study with follow-up to define the evolution is thus necessary to evaluate the real risk of progression for single genotypes and for multiple infections. Larger epidemiologic screening studies are needed to assess the epidemiologic risk of less represented HPV types so as to understand interactions and clinical meanings of multiple infections and to verify the role of anti-HPV vaccination programs in the general target population and/or special at-risk subgroups.
Orlando Giovanna, MD
Tanzi Elisabetta, PhD
Beretta Rosangela, MD
Amendola Antonella, PhD
Fasolo M. Michela, MD
Bianchi Silvia, PhD
Cellerino Paola, MD
Mazza Francesca, PhD
Zappa Alessandra, PhD
Rizzardini Giuliano, MD
STD Unit, II Department of Infectious Diseases L Sacco Hospital Milan, Italy
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