|Ahead of print publication
Human papilloma virus infection of uterine cervix and spectrum of cervical pathology in human immunodeficiency virus/AIDS
Bhawna Bhutoria Jain1, Tathagata Adhikary2, Provash C Sadhukhan3, Ayandip Nandi4
1 Department of Pathology, Rampurhat Government Medical College, Birbhum, West Bengal, India
2 MTMTB Hospital, Kolkata Municipal Corporation, Kolkata, West Bengal, India
3 ICMR Virus Unit, National Institute of Cholera and Enteric Diseases, GB4, ID and BG Hospital, Beliaghata Campus, Kolkata, West Bengal, India
4 Department of Pathology, Medical College, Kolkata, West Bengal, India
|Date of Submission||08-Aug-2019|
|Date of Decision||21-May-2020|
|Date of Acceptance||18-Jun-2020|
|Date of Web Publication||10-May-2021|
48/7, Patuapara Lane, Serampore, Hooghly - 712 201, West Bengal
Source of Support: None, Conflict of Interest: None
Background: Human papilloma virus (HPV) is one of the most common causes of sexually transmitted viral diseases worldwide. High-risk HPV types such as HPV16 and 18 are known to cause cervical dysplasia and carcinoma. In human immunodeficiency virus (HIV)-positive individual, chance of HPV coinfection and risk of cervical dysplasia/carcinoma have been found to be significantly more than in HIV-negative individuals.
Aim: In this institution-based, cross-sectional, observational study, we aim to find out the relationship of HPV infection of the uterine cervix with cervical dysplasia and neoplasia in HIV-infected/AIDS patients.
Materials and Methods: Conventional Pap smears were taken from HIV-infected individuals admitted in the department of gynecology and obstetrics and reported by the Bethesda system. A second sample was sent to the virology unit of ICMR for detection and typing of HPV. Control samples were taken from HIV-negative individuals.
Results: Fifty HIV-positive patients were included in this study. On cervical Pap smear examination, 32 cases were cytologically benign and 18 cases showed atypical cytomorphology. Twenty-four cases were HPV positive, among which 16 were cytologically atypical and 8 were benign. HPV 16 was the most common subtype (50%) followed by HPV 18 (37.5%) and others (12.5%) in HIV-positive patients. Chance of cervical dysplasia increased with age independent of HIV infection and with progressive lower CD4 count. Koilocytosis was a significant predictor of HPV infection. Majority of patients were asymptomatic. Peak incidence of HPV infection occurred in reproductive age group (20–40 years). The association between HIV and HPV coinfection (P = 0.002) and between HPV infection and cytology atypia (P < 0.0001) was statistically significant.
Conclusion: Present study highlights the necessity of routine cervical Pap smear screening in HIV infected reproductive age-group women. Early detection enables dysplasia to revert or be effectively managed.
Keywords: Cervical Pap smear, human immunodeficiency virus/AIDS, human papilloma virus isolation, human papilloma virus 16/18, nested polymerase chain reaction
|How to cite this URL:|
Jain BB, Adhikary T, Sadhukhan PC, Nandi A. Human papilloma virus infection of uterine cervix and spectrum of cervical pathology in human immunodeficiency virus/AIDS. J Can Res Ther [Epub ahead of print] [cited 2021 Jun 22]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=315669
| > Introduction|| |
Human papilloma virus (HPV) is one of the most common causes of sexually transmitted viral diseases in both men and women worldwide. The prevalence of HPV infection in the general population is estimated to be between 9% and 13% worldwide and varies between 1.6 and 25.6% in India., More than 200 types of HPV have been recognized on the basis of DNA sequence data showing genomic differences, approximately 40 types among them are associated with lower genital tract infection. Based on their association with cervical cancer and precursor lesions, genital HPVs are grouped in to high-risk (oncogenic HPVs) and low-risk HPV types. Low-risk HPV types include types 6, 11, 42, 43, and 44 and high-risk HPV types include types 16, 18, 31, 33, 34, 35, 39, 45, 51, 52, 56, 58, 59, 66, 73, and 82. The 200 genotypes are defined by nucleotide sequence variation of more than 10% compared with other known HPV types in the E6, E7, and L1 open-reading frames. Those differing by 2%–10% variations are referred to as subtypes, whereas intratype variants may vary up to 2% in the coding region and 5% in the noncoding region when compared to that of prototype. Variations are commonly caused by deletions or insertions of nucleotides. The degree of polymorphism varies from type to type in all HPV types. These variants are generally classified and named according to their geographic relatedness. Interest in HPV variants is growing rapidly, as increasing evidence suggests that HPV variants may differ biologically and etiologically.,
Cervical cancer is the second most common cancer among women worldwide. It evolves through the process of metaplasia and varying dysplasia ultimately leading to invasive cancer. Cytological screening for cervical cancer on Pap smear is based on this progression sequence reflecting various cytological abnormalities. The prevalence of oral, anal, and cervical HPV infection in human immunodeficiency virus (HIV)-positive individuals compared with HIV-negative individuals increases with progressively lower CD4+ levels, as does incident high-grade intraepithelial lesions/dysplasia. Studies have shown that HIV-associated attenuation of HPV-specific immune responses allow for the persistence of high-grade intraepithelial lesion and sufficient time for the accumulation of genetic changes that are important in progression to cancer. Some have also speculated that HIV infection may increase the oncogenicity of high-risk HPV types. HIV also has a negative impact on the recurrence of infection and cervical intraepithelial neoplasia (CIN) after treatment.,
HPV-induced cervical cancer is a multi-step process. HPV DNA in the cervix uteri is frequently detected in women at all ages, most frequently among young women soon after the start of having sexual intercourse. However, in the majority of the women, HPV infection does not lead to a clinical manifestation and is cleared by the host immune system in a relatively short time (6–12 months).,, In a small percentage, HPV infection becomes persistent and induces the development of low-and/or high-grade CIN, which can still regress or progress to an invasive cervical carcinoma after a period of latency. These lesions are usually localized, although why they remain sharply demarcated is not clearly understood. The factors important for the progression to cervical carcinoma are the virulent properties of the virus and the ability to evade the immune system.
HIV-positive men and women are also at the increased risk of anogenital and oral HPV infection. The risks for HPV-associated high-grade intra-epithelial neoplasia and cancer are also increased.
Aims and objective
Our aim was concentrated on the early diagnosis of cervical pathology by Pap smear and to find out the pattern of cervical cytology in HIV-infected women with respect to CD4 T-cell count. We also explore to determine if genital HPV infection is more common in patients with HIV.
| > Materials and Methods|| |
It was an institutional-based, cross-sectional, observational study. The study was conducted mainly in the department of pathology in collaboration with the ICMR Virus Unit and the ART (antiretroviral therapy) Center during the time period of 1½ year. Permission from the Institutional Ethical Committee was obtained.
A total of 50 HIV-infected female patients presenting to gynecology outpatient department (OPD) and indoor patient department were included in this study after taking consent of the patient in the proper consent form. Patient refusing consent, moribund patients, and patients with previous cervical cancer after receiving radiotherapy on follow-up visit were excluded from the study.
HIV-negative female patients presenting for Pap smears screening in gynecology OPD were taken as controls.
Detailed history was taken from each of the patients, including name, age, residential address, religion, contact number (if available), registration number in the hospital registry, followed by chief complaints, menstrual history, obstetric history, significant past history (particularly regarding the age of first intercourse, multiple sex partners, period of affection of HIV, tobacco use), treatment history, drug history, and history of contraceptive use were taken.
Proper clinical examination was done regarding the general health, any comorbid diseases and any local cervical lesions.
Conventional PAP smears from HIV-infected patients were obtained using the combination of a spatula and brush taking universal precaution and laboratory safety measures. A wooden spatula was used first. The spatula was rotated at least 360 degrees. The sample smeared on the slide and fixed in 95% ethyl alcohol and subsequently stained by Papanicolaou stain.
Apart from that, a second sample was taken from the same patient by using the brush inserted in endocervical canal and sent in buffer transport medium maintaining cold chain to referral center for virology for HPV-DNA detection and genotyping.
Pap smear reporting was done as per the Bethesda system.
For the separations of viral DNA, collected specimens were processed for DNA extraction using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's literature. The DNA was finally eluted in 50 μL elution volume and stored in −20°C for further usage.
A nested polymerase chain reaction (PCR) amplification technique was used for the amplification of HPV. Forward primer PCO3: 5' ACACAACTGTGTTCACTAGC-3' and reverse primer PCO4: 5' CAACTTCATCCACGTTCACC-3' was used to amplify a 110 bp product of globin gene. The first round PCR was performed in a 25 μL reaction volume comprising of 2.5 μl Taq 10× buffer II, 0.8 mM of dNTPs, 2.5 mM of magnesium chloride, 0.25 μM of forward (MY09) and reverse primer (MY11) and 0.5 U of Taq DNA polymerase (ABI, USA), and 4 μl of the DNA extracted product. The second round amplification was performed using 2 μl of the first round product with 2.5 μl Taq 10× buffer II, 0.8 mM of dNTPs, 2.5 mM of magnesium chloride, 0.25 μM of forward (GP5+) and reverse primer (GP6+) and 0.5 U of Taq DNA polymerase (ABI, USA) in a 25 μL reaction volume. The PCR was performed in a veriti-thermal cycler (ABI, USA) with previously defined PCR conditions.
The HPV-positive specimens provided a clear band at 150 bp in a 1.5% agarose gel stained with ethidium bromide as observed under a gel documentation system (Bio-Rad, USA).
For HPV genotyping, positive samples were further analyzed to search for genotypes 16 and 18. Forward primer 5'-ATTAGTGAGTATAGACATTA-3' and reverse primer 5'-GGCTTTTGACAGTTAATACA-3' were used to amplify HPV-16 DNA, and forward primer 5'-TGGT GTATAGAGACAGTATACCCCA-3' and reverse primer 5'-GCCTCTATAGTGCCCAGGTATGT-3' were used for HPV18. Amplifications were performed with the following cycling profile: Incubation at 94°C for 1 min followed by 35 cycles of 50 s, denaturation at 90°C, 2 min annealing at 54°C for HPV16 and at 58°C for HPV18, and 1 min elongation at 72°C. The last cycle was followed by a final extension of 10 min at 72°C.
The statistical analysis was performed using the Graph pad software.
| > Results and Analysis|| |
Pap smears of total number of 50 HIV-positive patients were included in this study. Control cases were selected from the patients undergoing Pap smear in our gynecology OPD who were HIV negative.
The lowest and highest age of the study population was subsequently 22 years and 52 years; average age was 32.7 years among the HIV-positive patients; most of the HPV infections have occurred between the age groups of 26–37 years of age (62.5%) [Table 1].
|Table 1: Distribution of human papilloma virus infection among human immunodeficiency virus positive women with age|
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Among 50 HIV-positive cases, 32 cases (64%) were cytologically benign NILM (Negative for intraepithelial lesion/malignancy) and 18 cases (36%) showed atypical cytological features which included three cases of atypical squamous cell of undetermined significance (ASCUS), 8 cases of low-grade squamous intraepithelial lesion, 2 cases of high-grade squamous intraepithelial lesion (H-SIL), and 5 cases of squamous cell carcinoma. Among 50 HIV-negative patients, 36 cases (72%) were diagnosed with cytologically benign (NILM) and 14 cases (28%) were atypical (i.e., 1 ASCUS,6 L-SIL,5H-SIL,1SCC and 1 adenocarcinoma) [Figure 1].
|Figure 1: Pie charts showing cytological diagnosis in HIV positive patients and in HIV negative patients|
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Among HIV-positive cases, total 24 cases were shown to be HPV positive, among which 16 cases (66.67%) were cytologically atypical and 8 cases (33.33%) were cytologically benign. The rest of the 26 cases were HPV negative among which 2 cases (7.7%) were cytologically atypical. The two tailed P value between HPV infection and risk of atypical cytology were <0.0001. The association between HPV infection (outcome) and cytological diagnosis was found to be extremely statistically significant.
It was found that HIV-positive patients had higher chance of getting HPV infection compared to HIV-negative patients [Table 2]. The two tailed P value between HIV and HPV positivity was 0.0002. Association between HIV infection and HPV positivity was found to be extremely statistically significant.
|Table 2: Relationship of human immunodeficiency virus and human papilloma virus infection|
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HPV 16 was the most common type of HPV infecting the female genital tract (50%) followed by HPV 18 (37.5%) and others (12.5%) in HIV-positive patients. In HIV-negative cases also, HPV 16 was the most common type (83.33%) followed by HPV 18 (16.66%) in HIV-negative patients [Figure 2].
|Figure 2: Comparison of HPV type distribution among HIV positive and HIV negative female|
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Cervical dysplastic changes increase with age in HIV-positive patients as in <30 years age relative percentage of dysplasia or carcinoma was 33.3% where >30 year age group, the percentage was 38.46%. In HIV-negative population, dysplasia or carcinoma was much lower (4.5%) in <30 years, whereas in >30 years, it was even higher than HIV+ population (46.42%) thus showing age to be an independent variable in cervical cancer.
Presenting symptoms of HIV-positive patients with HPV coinfection varied from per vaginal discharge (21.3%) to menorrhagia (33%), burning sensation in urine (12.5%), and asymptomatic (33%), whereas presenting symptoms of HIV-negative patients with HPV coinfection were mostly asymptomatic (56%).
Koilocytosis was found in 66.66% of HPV-positive patients in comparison to only 16.66% of HPV-negative patients. The P value of the association between HPV and koilocytosis was 0.0606 in our study.
When we compared the cytological diagnosis with CD4 count, we found cytological diagnosis of dysplasia/carcinoma occurred more frequently with declining CD4 count. The statistical P value of the table was 0.5483 [Table 3].
The presence of amplifiable DNA, using primers for HPV 16 and 18 was confirmed in 24 out of the 50 samples [Figure 3]. Out of the positive cases, HPV type 18 was present in 20 cases and HPV type 16 was present in 4 cases. The PCR was carried out using the primers specific to the subtype 16 and 18 and the product sizes were 246 bp (product from L1 region base 379–624) and 162 bp (product from E1 region base 1448 to base 1609), respectively.
| > Discussion|| |
In this study, it was observed that the prevalence of HPV 16 and 18 among HIV-negative control population was 12% which is comparable to the findings from various other studies conducted in India.,,,
The percentage of HPV infection in HIV-positive patients was 48% which was higher than HIV-negative patients (12%). Hence, the study demonstrates significantly higher prevalence of HPV 16/18 among HIV-positive cases compared to HIV-negative controls. The two tailed P value was 0.0002 which is very much comparable with a study by Sarkar et al., Two earlier studies from Western and Southern India and studies conducted in other parts of the world have also documented similar findings.,,
This study has also documented that HIV-positive females are at increased risk of getting infected with oncogenic HPV than the HIV-negative females irrespective of age. In the present study, we have divided the ages of the patients of our study population in two groups. Patients of age group >30 years with cervical dysplasia is more (38.46%) in comparison with the percentage of patients of age ≤30 years (33.33%). Similar finding was demonstrated by studies from South India and African subcontinent.,,
In the present study, it has been found that the age groups: (28–33 years) and (34–39 years) were the two most vulnerable age groups of HPV infection (both sharing seven patients). The reason may be that in these age groups, the people are most sexually active. It has been noted in other studies that HPV prevalence in women shows a peak at younger ages and then declines over the age of 30 years. Even in studies among sex workers, a population which is highly exposed to HPV, Sarkar et al. observed a considerably decreasing trend in HPV prevalence with age in spite of continuous high levels of sexual activity., This is probably due to the development of acquired immunity to HPV infection following repeated exposure.
The cervical dysplasias or carcinomas were found in 36% of HIV-positive patients which is much higher than HIV-negative patients (28%). Hence, it has been concluded that HIV and HPV coinfection is more prone to develop dysplastic lesions in cervix than individually.
In this study, we have also established a relationship between the CD4 T-cell count and cytological diagnoses in Pap smear in HIV-positive patients. It has been noted that 29.41% patients were reported cytologically dysplastic in patients of CD4 T-cell count ≥500, whereas 39.39% patients were reported as cytologically dysplastic in patients of CD4 T-cell count <500. Lower CD4 T-cell count was found to be an important risk factor of carcinomatous changes in cervix. The result is very much comparable with other studies.
The relatively broader distribution of HPV types among HIV-seropositive women with lower CD4 counts suggests reactivation of latent HPV viral infections. Our results are similar to previous studies indicating that HIV seropositive women with CD4 counts ≤200/mm3 have a higher prevalence of any HPV or oncogenic HPV types, when compared to those with CD4 counts >500/mm3.
It has been noted that HPV16 was the most common type specific HPV virus in HIV nonreactive persons (83.33%), whereas the prevalence of HPV 18 was slightly high (37.5%) in HIV-infected cases. However, Sarkar et al. showed that among HIV-positive subjects, HPV 18 had the highest prevalence followed by HPV16.,
We have selected cervical Papsmear as a screening method of cervical cancer detection. We have also followed up few of the patients with histopathological findings whose Pap smear revealed dysplastic changes. Cervical Pap preferably by liquid-based preparation followed by HPV testing remains the gold standard worldwide for early detection of cervical carcinoma.
In our study, the most of the patients both in study and control group were asymptomatic or with the presenting symptom of vaginal discharge which proved the necessity of cervical Pap smear examination at regular intervals for the early detection of cervical dysplastic lesions. The morphologic hallmark of HPV infection of the cervical squamous epithelium is koilocytotic atypia (term coined by Leopold Koss). This change is thought to be related to expression of the viral E4 protein and the disruption that this causes in the cytoplasmic keratin matrix. In the present study also the koilocytic changes were found in 16 cases among 24 HPV-positive patients.
| > Conclusion|| |
HIV infection increases the risk of getting HPV infection and chance of progression of cervical dysplasia/carcinoma. Increasing age, lower CD4 count, and koilocytosis are the important predictors.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| > References|| |
Clifford GM, Gallus S, Herrero R, Muñoz N, Snijders PJ, Vaccarella S, et al
. Worldwide distribution of human Papilloma virus types in cytologically normal women in the International Agency for Research on Cancer HPV Prevalence surveys: A pooled analysis. Lancet 2005;366:991-8.
Clifford GM, Smith JS, Plummer M, Muñoz1 N, Frances chi S. Human Papilloma virus types in invasive cervical cancer worldwide: A meta-analysis. Br J Cancer 2003;88:63-73.
Huh K, Zhou X, Hayakawa H, Cho JY, Libermann TA, Jin J, et al
. Human Papilloma virus type 16 E7 oncoprotein Associates with the cullin 2 ubiquitin ligase complex, which contributes to degradation of the retinoblastoma tumour suppressor. J Virol 2007;81:9737-47.
Folashade O, Steven YH, Bruce K, Gregory CT, Anthony CE. Human Papilloma virus infections in primary care. Clin Med Res 2007;5:210-7.
Pande S, Jain N, Prusty BK, Bhambhani S, Gupta S, Sharma R, et al
. Human Papilloma virus type 16 variant analysis of E6, E7, and L1 Genes and long control region in biopsy samples from cervical cancer patients in North India. J Clin Microbiol 2008;46:1060-6.
Aho J, Hankins C, Tremblay C, Lang F, Forest P, Pourreaux K, et al
. Molecular analysis of human papillomavirus type 52 isolates detected in the genital tract of human immunodeficiency virus-seropositive and-seronegative women. J Infect Dis 2003;188:1517-27.
Franco EL. Chapter 13: Primary screening of cervical cancer with human papillomavirus tests. J Natl Cancer Inst Monogr 2003;(31):89-96. doi:10.1093/oxfordjournals.jncimonographs.a003488.
Franco EL, Villa LL, Sobrinho JP, Prado JM, Rousseau MC, Désy M, et al
. Epidemiology of acquisition and clearance of cervical human papillomavirus infection in women from a high-risk area for cervical cancer. J Infect Dis 1999;180:1415-23.
Suzanne DV, Elizabeth RU, Margaret AP, Sibailly TS, Stefan ZW, Ghys PD, et al
. HIV and human Papilloma virus as independent risk factors for cervical neoplasia in women with high or low numbers of sex partners. Sex Transm Inf 1999;75:258-60.
Bernard HU, Burk RD, Chen Z, van Doorslaer K, zur Hausen H, de Villiers EM. Classification of Papilloma viruses (PVs) based on 189 PV types and proposal of taxonomic amendments. Virology 2010;401:70-9.
Tjalma WA, Van Waes TR, Van den Eeden LE, Bogers JJ. Role of human papillomavirus in the carcinogenesis of squamous cell carcinoma and adenocarcinoma of the cervix. Best Pract Res Clin Obstet Gynaecol 2005;19:469-83.
Giuliano AR, Harris R, Sedjo RL, Baldwin S, Roe D, Papenfuss MR, et al
. Incidence, prevalence, and clearance of type-specific human Papilloma virus infections: The young women's health study. J Infect Dis 2002;186:462-9.
Goodman MT, Shvetsov YB, McDuffie K, Wilkens LR, Zhu X, Thompson PJ, et al
. Prevalence, acquisition, and clearance of cervical human papillomavirus infection among women with normal cytology: Hawaii Human Papillomavirus Cohort Study. Cancer Res 2008;68:8813-24.
Reich O, Pickel H, Regauer S. Why do human papillomavirus infections induce sharply demarcated lesions of the cervix? J Low Genit Tract Dis 2008;12:8-10.
Asiaf A, Ahmad ST, Zargar Md A, Mufti S, Mir SH. Prevalence of human papilloma virus infection in a Kashmiri Ethnic Female Population. Genet Test Mol Biomarkers 2012;16: 904-9.
Carvalho Nde O, del Castillo DM, Perone C, Januário JN, Melo VH, Brasileiro Filho G. Comparison of HPV genotyping by type-specific PCR and sequencing. Mem Inst Oswaldo Cruz 2010;105:73-8.
Ghosh SK, Choudhury B, Hansa J, Mondal R, Singh M, Singh M, et al
. Human papillomavirus testing for suspected cervical cancer patients from Southern Assam by fast-PCR. Asian Pac J Cancer Prev 2011;12:749-51.
Sarkar K, Pal R, Bal B, Saha B, Bhattacharya S, Sengupta S, et al
. Oncogenic HPV among HIV infected female population in West Bengal, India. BMC Infect Dis 2011;11:72.
Sarkar K, Bhattacharya S, Bhattacharyya S, Chatterjee S, Mallick AH, Chakraborti S, et al
. Oncogenic human papilloma virus and cervical pre-cancerous lesions in brothel-based sex workers in India. J Infect Public Health 2008;1:121-8.
Sankaranarayanan R, Bhatla N, Gravitt PE, Basu P, Esmy PO, Ashrafunnessa KS, et al
. Human papillomavirus infection and cervical cancer prevention in India, Bangladesh, Sri Lanka and Nepal. Vaccine 2008;26 Suppl 12: M43-52.
Joshi SN, Gopalkrishna V, Kumar BK, Dutta S, Nyaynirgune P, Thakar M, et al
. Cervical squamous intra-epithelial changes and human papillomavirus infection in women infected with human immunodeficiency virus in Pune, India. J Med Virol 2005;76:470-5.
Peedicayil A, Thiyagarajan K, Gnanamony M, Pulimood SA, Jeyaseelan V, Kannangai R, et al
. Prevalence and risk factors for human Papilloma virus and cervical intraepithelial neoplasia among HIV-positive women at a tertiary level hospital in India. J Low Genit Tract Dis 2009;13:159-64.
Firnhaber C, Van Le H, Pettifor A, Schulze D, Michelow P, Sanne IM, et al
. Association between cervical dysplasia and human papillomavirus in HIV seropositive women from Johannesburg South Africa. Cancer Causes Control 2010;21:433-43.
Schuman P, Ohmit SE, Klein RS, Duerr A, Cu-Uvin S, Jamieson DJ, et al
. Longitudinal study of cervical squamous intraepithelial lesions in human immunodeficiency virus (HIV)-seropositive and at-risk HIV-seronegative women. J Infect Dis 2003;188:128-36.
Palefsky JM, Minkoff H, Kalish LA, Levine A, Sacks HS, Garcia P, et al
. Cervicovaginal human papillomavirus infection in human immunodeficiency virus-1 (HIV)-positive and high-risk HIV-negative women. J Natl Cancer Inst 1999;91:226-36.
Conley LJ, Ellerbrock TV, Bush TJ, Chiasson MA, Sawo D, Wright TC. HIV-1 infection and risk of vulvovaginal and perianal condylomata acuminata and intraepithelial neoplasia: A prospective cohort study. Lancet 2002;359:108-13.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]