Type-specific human papillomavirus-DNA load in anal infection in HIV-positive men
Pierangeli, Alessandraa; Scagnolari, Carolinaa; Degener, Anna Martaa; Bucci, Mauroa; Ciardi, Antonioa; Riva, Elisabettab; Indinnimeo, Mariledac; Mancini, Giusepped; D'Ettorre, Gabriellad; Vullo, Vincenzod; Antonelli, Guidoa
aDepartment of Experimental Medicine, Virology Section, Sapienza University, Italy
bVirology Section, University ‘Campus Bio-Medico’, Italy
cChirurgic Department ‘Pietro Valdoni’, Italy
dDepartment of Infectious Diseases, Policlinico Umberto I, Sapienza University; Rome, Italy.
Received 27 March, 2008
Revised 30 June, 2008
Accepted 8 July, 2008
Correspondence to Guido Antonelli, Department of Experimental Medicine – Virology Section, Sapienza University, Viale di Porta Tiburtina 28, 00185 Rome, Italy. Tel: +39 06 4474122; fax: +39 06 44741236; e-mail: firstname.lastname@example.org
Objective: To characterize anal human papillomavirus (HPV) infections in terms of genotype prevalence and type-specific DNA load in HIV-positive men.
Design: HIV-positive men attending the colo-proctological clinic of a University Hospital in Rome were recruited prospectively from November 2004 to July 2007. HIV-negative outpatients attending the same clinic over the same period were used as a control group.
Methods: Anal brushings were tested for HPV-DNA using polymerase chain reactions and direct sequencing; type-specific HPV-DNA copies were measured in most positive samples. HPV data were correlated with patient HIV status and risk factors.
Results: HPV-DNA infection was detected in 81% of HIV-positive men. Almost all homosexual men were HPV-infected. The infection rate in low-risk HPV types was higher than in high-risk types. The spectrum of HPV genotypes was comparable between HIV-positive and HIV-negative men. Numbers of HPV-DNA copies varied greatly between samples but did not differ significantly between HIV-positive and HIV-negative men. In many samples, low-risk (HPV 6, 61, 70, and 74) viral loads were comparable with those of high-risk HPVs.
Conclusion: Type-specific HPV-DNA copies at baseline appear to be independent of patient immune status and of HPV genotype. HPV genotype risk and viral load should be further evaluated for their potential predictive role in persistence and progression.
It is widely accepted that HIV infection increases the risk of human papillomavirus (HPV) acquisition, promotes reactivation of latent HPV infection, favours HPV persistence, and accelerates progression towards anal intraepithelial neoplasia [1–3]. Despite the widespread use of highly active antiretroviral therapy (HAART), no reduction in the incidence of anal cancer has been seen so far . The increased life expectancy of HIV-positive individuals may expose them to a higher risk of carcinomas, indicating a need to identify virological markers that could be used to monitor HPV infection in such patients. It has been shown that HIV-positive men and women are more likely to be infected with high-risk and multiple HPV types than those who are HIV-negative, and that the prevalence of persistent HPV infection is increased due to immune suppression [4,5]. Both innate and antigen-specific host response are thought to be essential for transmission and resolution of HPV infection, with a fundamental role for anogenital mucosa . In this respect, factors determining clearance of HPV or progression are not as clearly identified in men as in women and could be different for different hormonal milieu and other unmeasured risk factors. Anal HPV infection in men often occurs with no initial signs or symptoms of infection, even in the absence of receptive anal intercourse [7,8]. In a recent study , a prevalence of 25% for anal HPV was observed in heterosexual men reporting no signs or symptoms of sexually transmitted diseases, suggesting that HPV may be a common anal infection. However, the actual prevalence of anal infections with specific HPV types and of coinfections with different genotypes, the relative risk of single versus multiple infections, and the rates of persistence and progression remain to be determined. Hence, additional data are needed to determine the actual risk grade of HPV genotypes, to confirm whether or not HPV typing may represent a useful adjunct to anal cytology, and also to predict a possible beneficial use of HPV vaccines in men.
The role of high viral loads in high-risk HPV progression to cervical cancer has been well studied; differences in viral load relevance exist among high-risk HPV genotypes [10–12]. Much less is known about the role of viral loads of specific HPV genotypes in the natural history of anal infections. For anal lesions, quantitative determinations of HPV-DNA copies have been made in only a few studies [13,14], and then only from high-risk genotypes 16, 18, 31, and 33.
To gain new insight into HPV anal infection (i.e. genotype distribution, type-specific viral load), anal brushings were examined from HIV-positive and HIV-negative men attending a colo-proctological clinic. HPV-positive samples were typed by sequencing and HPV type-specific viral loads were determined for most samples. The correlation between HPV results and patient data was then examined.
Study population and data collection
HIV-positive men, outpatients attending the colo-proctological clinic of the Department of Infectious Diseases, Policlinico Umberto I, Sapienza University of Rome, were recruited prospectively over the period November 2004 to July 2007. Exclusion criteria were abnormal anal cytology, a history of AIN, and being already diagnosed or treated for HPV/anal condyloma. The study was approved by the institution's Ethics Committee. Patients were enrolled after informed consent had been obtained. HIV-positive men answered a questionnaire on sociodemographic characteristics and sexual behaviour, and provided recent data on HIV viral load and CD4 T-cell count. HIV-negative men referred to the same colo-proctological clinic by a general physician over the same period were selected as a control group with the same exclusion criteria.
Anal examination and brushing
All patients underwent proctological examination and brushing of the anal canal with an Endobrush (Biogyn, Mirandola, Italy) for detection of HPV-DNA and anal cytology. The Endobrush was inserted into the anal canal up to the dentate line (1.5–2.0 cm from the anal margin) and used to scrape along the anal walls by rotating it three times clockwise and three times anticlockwise. Anal cytology reports of koilocytosis, hyperkeratosis, and presence of condyloma were categorized as HPV infection.
HPV-DNA detection and typing
Anal brushings collected in 1 ml phosphate buffered saline (PBS) were centrifuged at low speed; the cell pellets then underwent DNA extraction using a QIAamp Blood kit (Qiagen, Hilden, Germany). To assess the quality of the target DNA, human leukocyte antigen-specific primers were used in a PCR analysis as described previously . A 450 bp fragment from the L1 region of HPV-DNA was amplified using the consensus primers MY09/11 . To increase sensitivity, with detection of a broader range of HPV genotypes, a second PCR assay was performed on sample DNA; the complete E6 gene and part of the E7 gene were amplified using four couples of degenerated consensus primers able to detect 36 HPV types .
PCR products corresponding to proper fragments were purified, and sequencing of the two genomic regions was performed using an automatic DNA sequencer (model 370A; Applied Biosystems, Foster City, California, USA). Sequence homology was determined as previously described . A sample was considered to be infected with a genotype if at least one PCR and a related sequence gave a positive result. If both L1 and E6/7 gave weak positive signals, a sample was considered positive for HPV and not typed; if only one weak PCR signal was obtained, a second specimen was tested. If the L1 and E6/7 sequence gave discordant results, the patient was considered to be infected with two different HPV genotypes.
HPV-DNA load was measured using a quantitative real-time PCR fluorogenic assay (TaqMan PCR, Applied Biosystems). The total amount of DNA in each sample was calculated as OD260, and 5-μl samples containing 20–100 ng were used in the PCR reaction. Type-specific TaqMan probes and primers were designed to anneal in the E6 gene following the general rules outlined in the Primer Express software (version 2.0, Applied Biosystems) and deduced by multiple alignments of HPV type sequences available from GenBank, as described previously .
The reactions were carried out in 25 μl samples using Universal Master Mix (Applied Biosystems), 300 nmol/l for each forward and reverse type-specific primer, and a 100 nmol/l double-labelled (6-carboxy-fluorescein [FAM], and 6-carboxy-tetramethyl-rhodamine [TAMRA], at 5′ and 3′ ends, respectively), type-specific probe. All samples were tested in triplicate along with positive (standard template) and negative (DNA from negative samples and no DNA) controls. Amplification of PCR products was undertaken using the default method on the ABI Prism 7000 Sequence Detection System instrument (Applied Biosystems). PCR data were collected and analysed using the proprietary Sequence Detector software (version 1.7). Copy numbers were calculated by means of an external standard curve generated by amplifying serial 10-fold dilutions (10–108 copies) of a DNA plasmid containing the E6/7 fragment of each genotype, as described previously . These type-specific standards were generated by cloning E6/E7 fragments in Topo TA vector (Invitrogen, San Diego, California, USA) .
Coefficients of variation (CV) were also calculated between replicas, and samples with CV of about 50% were tested twice in triplicate from independent DNA extractions. The lower limit of sensitivity of the assay is about 10 copies/ng of total DNA.
The HPV type-specific viral load determined in each sample was expressed as the number of HPV-DNA copies/ng of total DNA in the sample.
A χ2-test was applied to compare the distribution of two unpaired groups, and the Mann–Whitney non-parametric test was used for independent measurement in samples. Significance was fixed at the 5% level. The analysis was undertaken using SPSS v.13.0 for Windows (SPSS Inc., Chicago, Illinois, USA).
Human papillomavirus genotype distribution
The characteristics of 36 HIV-positive patients enrolled in the study are described in Table 1; HPV coinfected patients did not differ significantly from non-HPV-infected in terms of median age, median CD4 cell count at the time of proctologic visit and at nadir, HIV-RNA plasma level and other clinical data. On the contrary, there was a statistically significant difference between groups in HIV transmission risk factors (HPV coinfection in 94% homosexuals versus 60% in bisexual men/injecting drug users, P < 0.05) and in median numbers of sex partners between HPV-positive and HPV-negative (10 versus four partners over lifetime and five versus one over the past 6 months, P < 0.05).
In Table 2, data on anal HPV infection in 36 HIV-positive patients are compared with data from 25 HIV-negative patients; groups were similar in age but differed at a statistically significant level in their sexual orientation and numbers of partners. The rates of HPV infection in HIV-positive patients were comparable with those in HIV-negative men (81% versus 68%, P > 0.05). The infection rate of low-risk and high-risk HPV types was also not significantly different between HIV-positive and HIV-negative men (46% high-risk in HIV-positive men versus 36% high-risk in HIV-negative men). The spectrum of HPV genotypes was comparable between the two groups, with the possible exception of HPV 53 infecting three HIV-positive and no HIV-negative men. Three HIV-positive patients had two different HPV infections, corresponding to 12% of total HPV genotypes characterized among this group; one patient had two high-risk genotypes (HPV 53 and 59), another had two low-risk genotypes (HPV 11 and 61), and a third had both low-risk and high-risk HPV (HPV 58 and 83). The latter was considered to be among the high-risk infected patients.
There were 52 samples for which anal cytology was available or adequate; cytology results and results of the HPV-DNA tests are reported in Table 3. Agreement of cytology results with results of HPV-DNA test (both HPV-positive or both negative) was good: 76.5% in HIV-positive and 72% in HIV-negative men. HPV-DNA tests detected HPV infections with greater sensitivity than cytology (79% HPV-positive with HPV-DNA test versus 67% HPV-positive from cytology in HIV-positive men; 79% versus 63% in HIV-negative men).
Human papillomavirus viral load
HPV type-specific viral load was determined for genotypes 6, 11, 16, 31, 33, 53, 58, 59, 61, 66, 70, and 74 that infected 22 HIV-positive and 13 HIV-negative men. HPV load, expressed as DNA copy number per nanogram of total DNA in each sample, was extremely variable in the same genotype (CV > 100%); single copy number data are presented logarithmically in Fig. 1. The cumulative mean values of DNA copies of high-risk HPV were approximately 10 times higher than low-risk loads but this difference was not statistically significant because of the elevated variability of the measurements (CV > 100%; Table 4). Determinations from samples infected with low-risk HPVs (copy range 101–2 × 105) were comparable with those with high-risk HPV (copy range 4.2 × 101–1.2 × 106), with the possible exception of HPV 11, for which the copy number did not go beyond 1.5 × 103 copies in the four samples tested (Fig. 1). The mean number of copies of HPV 6 measured in seven HIV coinfected samples did not differ significantly from the values measured in the same number of samples from HIV-negative men (Fig. 1). In HIV-positive men, samples with cytological evidence of HPV infection had higher mean viral loads than those with negative cytological reports but the difference did not achieve statistical significance (Table 4).
In this study, anal brushings were examined for HPV-DNA, the spectrum of HPV genotypes involved, and type-specific viral load to show, at baseline, determinants of HPV infection in HIV-positive individuals that could constitute potential risk factors for progression.
Previous studies [19–21] had found rates of anal HPV infections similar to those reported here but, unlike in this study, they found a predominance of high-risk genotypes among HIV-positive men. Conversely, low-risk genotypes have been found in higher proportions in heterosexual men with no other sexually transmitted disease in mostly asymptomatic anal infections .
The highest rate of HPV infection detected among HIV-positive homosexual men (94%) and the greater number of sex partners in those who were HPV-positive rather than HPV-negative in this study (Table 1) confirmed the concept that receptive anal sex is strongly associated with HPV detection in the anal canal [19,23].
Reported rates of HPV multiple infections in several studies [2,8,21] are much higher than those reported here, in which only 12% of typed anal brushings from HIV-positive patients presented more than one genotype compared with none from HIV-negative individuals. Different coinfection rates between groups could be due to the numbers of sex partners, which has been associated with an increased risk of multiple HPV types . Indeed, in the present study, numbers of sexual partners over lifetime and during the past 6 months were significantly higher for HIV-positive than for HIV-negative men (Table 2). In particular, a recent study from Italy  reported that 85% of HIV-positive men referred to a sexually transmitted disease unit had anal HPV infections; 57.6% had a high-risk genotype and 61.8% had more than one HPV type. Discrepancies with the present study could, in part, be explained by the different HPV genotyping methods used. In fact, PCR amplification and sequencing precisely detects the more abundant (and probably more clinically relevant) types and underestimates coinfections; untyped samples are likely to be mixed infections that cannot be resolved by sequencing but should be resolved by cloning PCR fragments before sequencing. This method had been used by Sanclemente et al.  who found, in HIV-positive patients, a majority of low-risk HPV and 27% anal samples coinfected with two HPV genotypes. Conversely, hybridization-based methods and restriction fragment length polymorphisms tend to overestimate coinfections, thus also overestimating the high-risk infection rate.
In our study population, as recognized, anal cytology had a lower sensitivity than PCR-based tests for HPV infection, and negative cytology did not exclude the presence of high-risk HPV genotypes. Accordingly, the HPV-DNA test seems to be a useful adjunct to cytology, revealing additional cases of infection and determining the HPV genotype.
To our knowledge, these are the first determinations of type-specific quantitative viral loads from low-risk HPV and from a number of high-risk HPV (HPV 53, 58, 59, 66) in male anal samples. The HPV copy numbers in samples were expressed per nanogram of total input DNA, to take into account the variable amounts of cells taken during brushing of the anal canal. There were wide variations between determinations, consistent with previous results from cervical samples [11,18]. Interestingly, in many samples, low-risk HPV had quite high DNA copy numbers, similar to high-risk HPV. This finding could be explained by increased HPV persistency in anal versus cervical infections, as stated in a recent report in HIV-infected women . Indeed, high viral load has been related to HPV persistency in cervical lesions and to an increased risk of progression towards cervical cancer precursors [10–12]; however, only the HPV 16 viral load seems to be predictive of future squamous abnormalities at a stage when a Papanicolaou smear tested negative .
The elevated level of HPV-DNA also found in low-risk HPVs in anal samples is in keeping with papers reporting that infections with low-risk HPVs were an independent risk factor for anal intraepithelial neoplasia [27–29]. Moreover, HPV copy number mean values did not differ significantly between HIV-positive and HIV-negative patients, even though the high variability in the measurements could have hidden a trend. Taken together, these observations apparently contrast with the classic view of HIV infection as a high-risk condition for developing HPV-related malignancies; however, they are in keeping with reports stating that restoration of immunity during HAART did not lower the relative risk of developing high-grade lesions and cancer [2,30]. Indeed, the role of the immune system seems to be exerted at different sites and stages during HPV's natural history and, in recent years, the local milieu of anogenital mucosa has been recognized as playing a pivotal role in HPV infections . As seen in HIV-seropositive women, immune functions in epithelial tissues are not fully accounted for by circulating CD4 T-cell counts and HIV-RNA levels [31,32] and are very complex to monitor. Several studies have reported somewhat conflicting data when measuring inflammatory markers, numbers of CD4 and CD8 T cells, macrophages, Langerhans cells in the cervix of HIV-infected women, either HPV coinfected or not [33–35]; no such studies have been undertaken in anal mucosa. Other possible explanations for the diminished capability of HIV-infected individuals to resolve HPV infections could be a lack of immune response to specific HPV antigens  or differential regulation of HPV-DNA expression due to HIV proteins [37,38].
Apparently benign perianal warts could, in HIV-positive patients, correspond to low-grade anal disease, which could progress to high-grade or to cancer; hence, these patients should be monitored frequently. In the light of the extreme variability in the numbers of HPV-DNA copies found in samples, a high viral load could be regarded as a marker of persistency. Certainly, such issues point to a need for more prospective studies with precise determination of type-specific HPV-DNA copies at baseline and at follow-up visits, in order to obtain a better understanding of the role of HPV load in persistence and progression towards AIN.
In conclusion, HPV-DNA tests, and eventually HPV load measurements, should also be performed in university clinics in the absence of dysplastic lesions in anal canal cells, at least in HIV-positive homosexual men. Information on HPV genotypes in subsequent tests can discriminate between persistent and reinfections and may lead to the identification of the high-risk and low-risk genotypes in anal infections in men.
The present study was supported by grants to G.A. from ISS 2006 # 30G.43 and ANLAIDS 2006 # 455.
Role of each of the authors in the study: Alessandra Pierangeli was responsible for design of the study, execution of the HPV-DNA experiments and writing of the manuscript; Carolina Scagnolari for execution of the TaqMan experiments, analysis of the data and revising of the manuscript; Anna Marta Degener for design of the HPV-DNA test and analysis of the sequencing data; Mauro Bucci for execution of the HPV-DNA tests; Antonio Ciardi for execution of the cytological tests; Elisabetta Riva for design and execution of the HPV plasmids for the TaqMan experiments; Marileda Indinnimeo for performing anal brushings and collecting HIV-negative patient data; Giuseppe Mancini for performing HIV test and analysing of the HIV-positive patient data; Gabriella D'Ettorre for contributing to design of the study, to selection of HIV patients, and to revise the manuscript; Vincenzo Vullo for selection of HIV patients, analysis of clinical data, and revising of the manuscript; and Guido Antonelli for design of the study, writing of the manuscript, and grants owner.
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