High-risk (HR) human papillomaviruses (HPVs) are the causal agents of virtually all cervical carcinomas, most anal carcinomas, and a proportion of carcinomas located in the vulva, vagina, penis, and oropharynx.1,2 Natural history studies of cervical HPV infections have clearly demonstrated that persistence of infection by HR types is necessary for the development of invasive cancers.3 Viral and host cofactors influence the transformation process; HPV type, host immune status, and tobacco smoking play important roles.3
Human immunodeficiency virus type 1 (hereafter referred to as HIV) infection is characterized by increasing immune suppression that has been shown to alter the course of anogenital HPV infection. In particular, HIV-infected subjects, in comparison with HIV-negative ones, were shown to have a higher prevalence of both cervical and anal HPV infection4 as well as increased risk of HPV-related cancers, persistent infection, and progression to anogenital dysplasia.5,6
Moreover, patients affected by an HPV-related cancer are at higher risk for developing a second HPV-related neoplasia in a different site.7
Prevalence of oral HPV infection has been shown to be in the range of 1% to 5% in immunocompetent subjects8–10 and in the range of 14% to 45% among HIV-infected individuals.10 A strong association with sexual behavior has been recorded, supporting the sexual transmission of the virus. Studies on natural history of oral HPV infection showed persistence of infection after 6 months in more than 50% of HIV-negative and HIV-positive subjects.11–13 A study14 evaluating oral HPV16 infection in HIV-positive men and women (Human Oral Papillomavirus Etiology study) found that most infections (overall prevalence, 2.8%) were transient. A 2- to 6-fold increased risk of developing oropharyngeal cancer has been shown in persons with HIV/AIDS.11,15
Human herpesvirus-8 (HHV8) is the causal agent of Kaposi sarcoma (KS), an AIDS-defining cancer highly frequent early in the HIV epidemic and markedly decreased after the introduction in 1996 of highly active antiretroviral therapy (HAART).5 Human herpesvirus-8 is acquired through sexual contact during adulthood in both endemic and nonendemic countries, whereas it is mainly transmitted via nonsexual routes during childhood in endemic countries. Saliva plays a central role in the context of both sexual and nonsexual HHV8 transmission, and infectious virus/HHV8 DNA is frequently detected in saliva of HHV8-seropositive patients.16 Prevalence of HHV8 infection among HIV-positive individuals is higher in men who have sex with men (MSM) and is related to a greater number of sexual partners and a history of sexually transmitted diseases in both MSM and heterosexuals.
Because both HPV and HHV8 are sexually transmitted and can be found in saliva, we analyzed 100 oral samples from HIV-positive patients for the presence and typing or load of both viruses.
The aim of the study was to obtain data on the prevalence of type-specific oral HPV infection in Italian HIV-positive individuals, in relation to sex, HIV risk factor, immune function, and HHV8 oral shedding.
MATERIALS AND METHODS
Human immunodeficiency virus–infected individuals at high risk for HHV8 infection were selected for evaluating oral HPV and HHV8 infections; the patients gave informed consent, and the investigations were done following the guidelines required by the involved institutions.
Besides demographic and anatomoclinical data, CD4 cell counts, HIV-RNA viral loads, and antiretroviral (ARV) therapy regimens were recorded, but no specific information on oral sex and oral lesions was obtained.
Unstimulated whole saliva, accumulated in the mouth and then ejected in a sterile vial, was collected (as described in other studies17,18), transported to the laboratory within 6 hours, and centrifuged to separate the cellular compartment (sample volume ranged from 800 to 2000 μL). This sampling method, without brushing or rinsing medium, has been used successfully for detection of DNA viruses.17,18 DNA was extracted from the cells by using the proteinase K/phenol/chloroform procedure.
DNA was analyzed for HPV sequences of low- and HR types by polymerase chain reaction (PCR) with MY09/MY11 consensus primers. Positive samples were typed, as already described,19 by restriction fragment length polymorphism analysis, capable of identifying most low- and HR mucosal HPV types. Samples that were negative by MY PCR were further amplified by nested PCR using biotinylated GP5+/GP6+ consensus primers as the second step, to increase assay sensitivity. Amplimers obtained by nested PCR were typed by direct sequencing, as already described,19 or by reverse Line Blot, using the “Consensus High Risk HPV genotyping kit” (Qiagen, Hilden, Germany), performed according to the manufacturer’s instructions, able to recognize 18 HR/intermediate-risk types: 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82. For statistical analyses, HPV types have been grouped according to the most recent classification,20 as follows:
* HR: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 (groups 1 and 2A)
* Low risk (LR): all the other detected types
* HPV X (unknown): all the remaining untyped samples, that is, positive by direct and/or nested PCR, but negative or undetermined by all typing methods
In case of multiple HPV infection, the sample was considered HR if at least 1 HR genotype was present.
The presence of HHV8 sequences was assayed by nested PCR using 2 sets of primers (ORF25 and ORF26) for 2 nonoverlapping regions of HHV8 genome, and positive samples were quantified by real-time PCR, as previously described.21,22
DNA extraction, PCR setup, and the addition of template were performed in separate rooms to minimize cross-contamination. Barrier pipette tips were used for all procedures; no template controls were included in all PCRs.
When available, subsequent samples from patients positive for oral HPV infection were also analyzed.
The association between independent variables was evaluated by χ2 and logistic regression tests, as appropriate, using the STATA 10.0 and R software. P < 0.05 was considered statistically significant. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated for risk factors for oral HPV and HHV8 infections. Logistic generalized linear models (GLMs) were also estimated, including sex, age, ARV therapy use, CD4+ cell counts, and HIV viral load.
A total of 100 saliva samples collected in 2007 to 2010 from HIV-infected subjects belonging to 3 different risk groups (38 MSM, 24 non-MSM men, and 38 women) were analyzed for HPV and HHV8 sequences. Most patients were of white origin, and 6 men (3 of group 1 and 3 of group 2) had a history of KS; relevant data are summarized in Table 1.
Overall, HPV sequences were detected in 37% and HHV8 in 46% of the samples. As summarized in Table 1, their prevalence was different in the 3 groups. The highest HPV (16/38; 42.1%) and HHV8 (21/38; 55.3%) prevalences were recorded among MSM, despite better immune function (higher percentage of CD4 > 500/μL) and better HIV control (higher percentage of viral load <50 copies/mL). The lowest (31.6% and 36.8%, respectively) prevalences were observed among heterosexual women, whereas among the women who acquired HIV infection through intravenous drug use, 3 of 5 had HPV infection (not shown). Human herpesvirus-8 infection had a lower prevalence and a lower viral load in women than in men.
High-risk HPV types were detected in 13 samples (9 single infections, 2 mixed HR, 2 mixed with LR) and LR or untyped HPV sequences in 24; an amplification band was visible after single-step MY09/MY11 PCR in 15 cases and only after GP5+/GP6+ nested PCR in 22 cases, indicative of a lower viral load. Infection with multiple types was present in 6 specimens. The specific HPV type(s) involved could be determined in 34 cases (single HPV X in 3 samples). The prevalence of HR types was almost 10 times lower in women than in men (P = 0.05; Table 1). Overall, 22 different types were found (Table 2); the most frequently detected were HPV6, HPV10, HPV16, HPV61, HPV66, and HPV83 (3 cases each). Analysis of the distribution of oral HPV types in relation to HHV8 shedding at baseline showed that HPV infection was predominantly found in HHV8-negative samples (26/37; 70.3%). Moreover, although single and multiple infections with LR HPV types were almost equally distributed among samples with detectable or undetectable HHV8, single and multiple infections with only HR or unknown types were mainly found in oral samples with undetectable HHV8.
Odds ratios of different variables for oral HPV and HHV8 infections are reported in Table 3; a statistically significant inverse correlation was observed between concurrent oral HPV and HHV8 shedding (OR, 0.34; P = 0.007). The logistic GLM for oral HPV infection shows a statistically significant correlation with oral HHV8 infection and HIV viral load (result shown also by an OR of 0.43, closed to statistical significance), whereas the GLM for HHV8 infection shows a statistically significant correlation with oral HPV infection only.
Among the 37 patients with oral HPV infection, a follow-up specimen could be obtained from 24 (65%) of them at a median of 7 months (range, 2–16 months) after the positive index sample; a follow-up sample was available for a significantly higher number of men (21/25; 84%) than women (3/12; 25%). No HPV sequences were found in the follow-up sample of 11 patients at a median of 11 months (range, 2–16 months); persistent infection with the same type(s) was recorded in 10 cases at a median of 6 months (range, 2–13 months); HPV DNA of different types was present in 3 cases at a median of 9 months (range, 8–15 months). The probability of persistence was higher for infections with higher viral loads, as indicated by a positive result with single-step PCR, recorded in 6 of 10 persistent infections versus 4 of 11 transient infections (data not shown).
We performed a study on Italian HIV-positive male and female patients, grouped according to sex and risk factors, to investigate oral HPV and HHV8 infections. Men who have sex with men represent more than 60% of the male group, and the female group is mostly composed of women with sexually acquired HIV infection. Therefore, our study population cannot be considered representative of all HIV-infected individuals; nonetheless, this allows inferences on concomitant different sexually transmitted infections.
Overall, we found 37% and 46% prevalence rates for oral HPV and HHV8 infections, respectively; for both viruses, MSM showed the highest (42% and 55%, respectively) and women the lowest values (32% and 37%, respectively). High-risk HPV types were detected much less frequently in women (2.6%) than in men (19%), whereas nonsignificant differences (17%–29%) were observed for LR types. Human herpesvirus-8 viral shedding was more frequent and with higher viral loads in men than in women.
Human immunodeficiency virus–infected individuals are known to be at higher risk of viral infections and virus-related lesions. In particular, KS, Epstein-Barr virus–related lymphomas, and HPV-related invasive cervical carcinomas are more prevalent in HIV-positive than in the general population and represent AIDS-defining conditions.5 This higher prevalence of infections and associated lesions is a consequence of both higher virus transmission and impaired immune response. The advent of HAART in industrialized countries in 1996 determined a decline in the occurrence of KS and lymphomas but not of cervical cancer.5 Indeed, the prevalence of HPV infection and related diseases in anogenital and upper aerodigestive tracts is increased in HIV-infected persons, and the longer survival obtained by HAART may be associated with increased risk of HPV-associated cancers.6 The different anatomical sites (i.e., cervix, anus, and oropharynx) display differences in frequency of infection, proportion of HPV-associated tumors, and, possibly, the natural history of HPV infection. In particular, oral HPV infections are less frequent than cervical and anal infections in both immunocompetent and immunocompromised individuals,11 and the proportion of HPV-positive carcinomas is lower in the oropharynx.1,2 Moreover, important geographical variations for oropharyngeal cases exist, with rates in the range of 35% to 70% in North America and 10% to 80% in Europe2; in our region we have a prevalence of approximately 30%23 (unpublished data). These differences are probably relative to different proportions of tumors associated with alcohol and tobacco consumption.
Our data on higher HPV prevalence among MSM than among heterosexual women are in line with previous observations.24–26 Because a higher proportion of MSM in our study were assuming ARV therapy and showed a better control of HIV infection (as indicated by higher CD4 cell counts and lower HIV-RNA loads) than the other 2 groups, sexual behavior seems to be more important than immune function for transmission of both viruses, posing MSM at highest risk.
Among our patients, the most frequent HR type was HPV16, but oral HPV infections were most frequently caused by LR types, mainly HPV types 6, 10, 61, 66, and 83. Human papillomavirus types 10 and 32, previously described in oral papillomatous lesions and oral condylomas in HIV-infected patients,27 were detected in 6 of our patients. Some differences in HPV type distribution emerged from other studies,24,26,28 probably due to differences in genotyping methodology and HPV type (low/high risk) classification. Detection of oral HPV infection can be affected also by sample collection and DNA extraction methods.29 In our study, we sampled oral cells by collecting unstimulated whole saliva, as described in other studies.17,18 Although other sampling techniques such as oral rinse, gargle, or brushing are used in studies on oral HPV infection, the DNA of spontaneously exfoliated mucosal cells (that represent the main human DNA source of whole saliva) was shown to be more easily amplified, genotyped, and sequenced than the DNA of artificially removed cells.30
To gain some information on the natural history of oral HPV infection, we obtained saliva follow-up samples from 24 (65%) of 37 HPV-positive subjects at a median of 7 months (range, 2–16 months) after the positive index sample (no follow-up samples were asked to HPV-negative subjects); persistence with the same type(s) was detected in 10 (42%) patients. Previous studies12,13 on natural history of oral HPV infection recorded persistence in 53% to 55% of HIV-positive persons and detected a significantly higher cumulative prevalence with a 2-week than a 6-month sampling interval. It has to be pointed out that in our study, only a limited number of follow-up samples, obtained at variable and longer time points, were analyzed; as a consequence, we might have underestimated the frequency of HPV persistence.
The rate of salivary shedding of HHV8 DNA at baseline among our HIV-infected patients was 46%; interestingly, we observed that the presence of HPV and HHV8 DNA in the cell-associated fraction of unstimulated whole saliva samples was inversely correlated (OR, 0.34; P = 0.007). Although this finding needs to be further investigated, it might indicate that infection of the oral epithelium with HHV8 or HPV may somehow modify the permissiveness of the mucosal epithelial cells to the other virus, by limiting concomitant infection, spreading, and/or expression. Thus, although HIV might contribute to the dissemination of HHV8 to mucosal epithelial cells, subepithelial lymphocytes, and macrophages, HHV8 infection might, in turn, restrict HPV spread to the mucosal epithelium and vice versa. Single and multiple infections with only LR or unknown types were predominantly found in oral samples with undetectable HHV8, suggesting that these HPV types may be more frequently involved in a protective role against HHV8 infection. Interestingly, it was shown that HPV-infected women are less likely to develop neoplastic cervical lesions if adeno-associated virus infection is present, and the authors hypothesized that adeno-associated virus may interact and reduce the risk for HPV-driven cervical carcinogenesis.31 Because HIV-infected subjects are susceptible to acquire several viral infections that persist because of their immune impairment, a better understanding of viral interactions will shed light on carcinogenesis and guide clinical management in case of concomitant multiple infections.
In conclusion, our data indicate a high prevalence of oral HPV and HHV8 infections in men with sexually acquired HIV infection. Men who have sex with men are at particularly high risk for HPV and HHV8 infections, despite good control of their HIV infection. They could therefore benefit from HPV prophylactic vaccination.
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