• Users Online: 196
  • Print this page
  • Email this page

 Table of Contents  
Year : 2022  |  Volume : 12  |  Issue : 4  |  Page : 152-156

Anal cytological abnormalities in human immunodeficiency virus-infected men and prevalence of high-risk human papillomavirus co-infection

1 Department of Medicine, Sir Ganga Ram Hospital, New Delhi, India
2 Department of Cytopathology, Sir Ganga Ram Hospital, New Delhi, India

Date of Submission26-Nov-2021
Date of Acceptance19-Jul-2022
Date of Web Publication30-Aug-2022

Correspondence Address:
Dr. Atul Kakar
Department of Medicine, Sir Ganga Ram Hospital, New Delhi
Login to access the Email id

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/cmrp.cmrp_114_21

Rights and Permissions

Background: Human immunodeficiency virus (HIV) infects cells of the immune system, leading to a compromised and depleted immune system. Progressive failure of the immune system predisposes an individual to many life-threatening opportunistic infections and malignancies. As compared to the general population, the incidence of anal squamous cell carcinoma (ASCC) is substantially higher in HIV-infected individuals. Majority of ASCC are related to infections caused by high-risk strains of human papillomavirus (HPV).
Aims: We conducted an observational study on HIV-positive men who had a history of homosexual contact. The aim of this study was to assess the spectrum of cytological abnormalities on anal smear cytology in individuals with HIV infection, and also estimate the prevalence of anal infection with high-risk HPV strains.
Materials and Methods: We enrolled 56 individuals for this pilot study. This study involved the collection of specimens from the anal canal of the patients by using cytobrush in liquid-based cytology (LBC) vial. The sample was simultaneously tested for cytological abnormalities by LBC (Sure Path, BD) and for 13 high-risk strains by Hybrid Capture II technique (Qiagen) based on antibody capture and chemiluminescent signal detection. Anal smear cytology was reported as per guidelines of The Bethesda System of reporting anal cytology, 2014.
Results: The prevalence of high-risk HPV infection was seen in 41.07% of individuals and low-grade squamous intraepithelial lesion and atypical squamous cells of undetermined significance were seen in 12.5% and 16.07% individuals, respectively. Cytology was useful in 7% of cases to diagnose opportunistic infections. The latter is a field yet to be tapped.
Conclusion: We would recommend LBC in HIV-positive patients, for screening of cytological abnormalities and HPV status. This would also give an opportunity to screen for opportunistic infections, which have otherwise not been diagnosed.

Keywords: Anal squamous cell carcinoma, human immunodeficiency virus/acquired immunodeficiency syndrome, human papilloma virus, liquid-based cytology

How to cite this article:
Kakar A, Bakshi P, Tripathi S, Gogia A. Anal cytological abnormalities in human immunodeficiency virus-infected men and prevalence of high-risk human papillomavirus co-infection. Curr Med Res Pract 2022;12:152-6

How to cite this URL:
Kakar A, Bakshi P, Tripathi S, Gogia A. Anal cytological abnormalities in human immunodeficiency virus-infected men and prevalence of high-risk human papillomavirus co-infection. Curr Med Res Pract [serial online] 2022 [cited 2022 Sep 27];12:152-6. Available from: http://www.cmrpjournal.org/text.asp?2022/12/4/152/355197

  Introduction Top

The human immunodeficiency virus (HIV) is a type of retrovirus that causes acquired immunodeficiency syndrome (AIDS) over a period of time.[1] The virus is of two types HIV 1 and HIV 2. It impairs the immune system and predisposes an individual to life-threatening opportunistic infections and malignancies that are rare in people with normal immune system. With the availability of combination antiretroviral therapy patients with HIV/AIDS are now living longer. The focus of attention is now shifting from opportunistic infections to malignancies in these patients. The common malignancies in HIV/AIDs patients are lymphomas, Kaposi sarcoma and cervical and anal carcinoma.

In the Indian population, incidence of HIV infection is higher than the world average. Majority of the cases are among high-risk population which includes commercial sex workers and men who have sex with men (MSM).[2] Human papillomavirus (HPV) is a small-sized virus, with double-stranded DNA. It commonly involves skin or cells of the mucosa.[3] The commonest sexually transmitted infections (STIs) in HIV-infected individuals is by HPV.

Anal cytology is now the standard of care according to many guidelines for screening in HIV/AIDS patients.[4],[5],[6] Worldwide, several studies have discussed the HPV strains causing anal squamous cell carcinoma (ASCC). Thus, we planned to conduct this study, to evaluate the prevalence of high-risk HPV infections and anal cytological abnormalities in HIV-infected homosexual men.


The aim of this study was to assess the spectrum of cytological abnormalities on anal smear cytology in the HIV-positive men who had sex with men, and also estimate the prevalence of anal infection with high-risk HPV strains.

  Materials and Methods Top

This study was conducted in the departments of Internal Medicine and Cytopathology in a tertiary care centre. The inclusion criteria included all consenting adult male individuals who were more than 18 years of age and were known cases of HIV and who gave a history of at least one homosexual contact with their partners. We excluded the patients if they had any active lower gastrointestinal bleeding, or, if the individual had undergone any recent anal or perianal surgery within 4 weeks of the day of enrolment. Individuals who were genetic females or had had sex change surgery or had any known psychiatric illness were also excluded from the study. Any patients who had received enema before sampling, or, if the individual had had anal intercourse an hour before the sampling, they were also excluded.

This was a prospective observational type of study. The study duration was from December 2018 to November 2019. The study was cleared by the Institutional Ethical Committee of Sir Ganga Ram Hospital, New Delhi. EC No.: EC/01/19/1484. Date of Approval: 15th February 2019.

Anal  Pap smear More Details sampling

After taking due consent the subject was explained the procedure, and after taking all aseptic precautions the procedure was performed. Cytobrush was used for sample collection. The sampling target extended from the distal rectal vault (proximal) to the anal verge (distal). It included the anal transformation zone and the non-keratinised and keratinised squamous epithelium of the anal canal. The tip of the cytobrush was moistened with water. It was inserted approximately 3.5–5 cm into the anal canal. Post-insertion deep into the anus (to collect both rectal columnar and anal squamous cells), the brush was pulled out, while applying some pressure to the wall of the anus, and rotating it spirally five times in clockwise motion. The cells collected on cytobrush were thoroughly rinsed and swirled in the preservative fluid in the liquid-based cytology (LBC) vial. The cytobrush was left in the LBC vial. Vial was properly labelled and sent to the cytology laboratory accompanied by a laboratory request form for anal smear cytology and HPV DNA testing on the same sample. The sample in the LBC vial was processed by the fully automated BD Sure Path system. One smear was prepared for each case and was stained with Papanicolaou stain. The same sample was used to test for the 13 high-risk strains of HPV by the Hybrid Capture II technique (Qiagen) based on antibody capture and chemiluminescent signal detection. Single cytopathologists reported all the specimens as per The Bethesda System (TBS) of reporting anal cytology, 2014 guidelines. If the cytology reported the sample to be insufficient, we repeated sampling, if the patient was willing and consented for the same.

Definitions used in the study

Anal cytological abnormalities were defined and reported as per TBS for reporting anal cytology, 2014.[7]

  1. Negative for intraepithelial lesion or malignancy: Adequate number of nucleates squamous cells were sampled and these were normal in appearance. There is no evidence of HPV-related changes
  2. Atypical squamous cells of undetermined significance (ASC-US): The squamous cells show evidence of atypia. Cells showing some abnormal changes are not sufficient to classify as a squamous intraepithelial lesion, and could even be due to inflammation
  3. Atypical squamous cells – cannot exclude HSIL (ASC-H): Another form of atypia. Cells are abnormal, but not enough to be classified as high-grade squamous intraepithelial lesion (HSIL)
  4. Low-grade squamous intraepithelial lesion (LSIL): This report indicates mild dysplasia (Also known as an anal intraepithelial neoplasm, AIN)
  5. HSIL: Very abnormal cells that have dysplastic characteristics. Often described as moderate (AIN-2) or severe (AIN-3) dysplasia. This is considered precancerous. Invasive characteristics are not seen
  6. Squamous cell carcinoma (SCC): This is invasive anal cancer
  7. Atypical glandular cells: Changes are found in glandular cells that raise concern for the presence of pre-cancer or cancer.

  Results Top

The study was done in a tertiary care centre on HIV-positive patients. The study population consisted of 56 patients. The anal opening was normal in 53 patients, had warts in two patients and fissure was in one patient. Anal smear cytology was done in 52 patients, while HPV-HR DNA testing was done in all 56 patients. The four cases, for whom the cytology was not done, were all negative for HPV and did not want to get cytology done.

[Table 1] summarises the cytological findings. In 7/56 (12.5%) individuals, the cytology was reported as LSIL, and 9/56 (16.07%) had ASC-US. Sigmoidoscopy was ordered in 9 patients of ASC-US, of which only seven patients consented, and were reported normal. 8/56 (14.2%) patients received an unsatisfactory result in the cytological study, where, 'unsatisfactory' was defined as 'inadequate number of nucleated squamous cells present in the smear for the pathologist to make an opinion'. No patient was reported to have anal cancer. In the study, HPV was positive in 23 (41.07%) patients. The thirteen high risk strains of HPV tested were 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. Moreover, 4/56 patients showed the incidence of opportunistic infections, where 1 finding was of tuberculosis, 2 of HSV (herpes simplex virus) and 1 of a fungal infection.
Table 1: Anal cytological findings

Click here to view

  Discussion Top

To the best of our knowledge, studies regarding cytological abnormalities in anal pap smear in HIV-infected Indian population are not available, and our study is a pilot project in this field. In 2007, Anderson et al. performed a study on HIV-infected people with CD4 cell count >300 cells/microliter for prevalence of abnormal anal cytology and high-risk HPV infection. They enrolled 126 HIV-infected patients with a median age of 45 years. Spectrum of cytological changes reported were: ASCUS - 32 (25%), normal cytology - 30 (24%), LSIL - 24 (19%), HSIL - 16 (13%) and ASC-H - 13 (10%). They concluded that abnormal cytology was strongly associated with high-risk HPV infection.[5] Our study had a comparable mean age of individuals and similar methods of anal pap smear sampling. Abnormal cytology was found in 28.57% which was lower than the aforementioned report, which could be explained by the fact that Anderson et al. included high-risk MSM individuals only, while our study included besides MSM individuals, any patient who gave a history of even a single homosexual anal encounter. A -cross-sectional study was done in Korea on 201 HIV-infected men irrespective of their sexual behaviour. The study enrolled 133 MSM and 68 men who had sex with women (MSW). MSM had abnormal anal cytology more commonly than MSW, 42.9% and 19.1%, respectively.[8] Our study reported an abnormal cytology in 28.57% of cases which was around the mean value of their findings. Arora et al. conducted a study on MSM (both HIV-positive and -negative individuals) for screening anal intraepithelial neoplasia and found that cytological abnormality was demonstrated in 27.7% of HIV-positive MSM individuals.[9]

In 2001, Bethesda System  Atlas More Details first included anal cytology.[7] It has now gained acceptance as an important screening tool for anal cancer.[8],[10] TBS 2001 includes guidance on anal sample collection, its adequacy, use of Bethesda Terminology for reporting of anal cytological abnormalities and their important morphologic characteristics. The 2014 update expanded the information on the performance of anal cytology and also included the role of HPV testing and biomarkers. The majority of ASCC is attributable to persistent HPV infection.

Anal cytology is used as a screening test for anal squamous intraepithelial lesions, similar to the use of Pap tests in cervical cancer screening. Both conventional smears and liquid-based cytologic preparations have been used. Some authors have reported comparable performance of both[11] while others have reported some advantages of LBC, like increased cell yield and decreased compromising factors such as obscuring faecal material, air drying and mechanical artefacts.[12],[13] Lindsey et al. performed a study to validate anal Papanicolau (Pap) smears as a screening test for ASCC. They concluded that anal Pap smear is a valuable procedure that provides the opportunity for early detection of cytological abnormalities associated with HPV infection and guides appropriate follow-up and intervention.[14] In a meta-analysis of anal cytology for HSIL, LBC's sensitivity was found to be ranging from 69% to 93% and specificity ranging from 32% to 59% which was comparable to that of Pap tests.[15]

Worldwide studies have been conducted which have found the varying prevalence to HPV/HIV Co-infection, which is summarised in [Table 2]. In our study, the prevalence was 41.07%, which was significantly lower than the previously conducted studies. The reason for the same might be that in the previous studies, the eligibility criteria were very stringent and they only included individuals with an extensive history of MSM-type homosexual contact, while in our study we included HIV-positive MSM individuals and also any subject who gave a history of even a single encounter of homosexual anal contact. It has been noted that in developed countries, the incidence of anal HPV infection and anal cancer is highest in HIV-positive men who have had homosexual contact with other men (MSM).
Table 2: Prevalence of human papilloma virus/human immunodeficiency virus co-infection

Click here to view

Over 100 types of HPV strains have been classified. Strains that affect the anogenital region are divided into two groups, based on their oncogenic potential – high-risk strains and low-risk strains. The low-risk types of strains are non-oncogenic and can cause genital warts and benign cellular changes. The high-risk strains are associated with carcinoma of the cervix, anal canal, vagina, vulva, penis and oropharynx.[3]

HPV is associated as a causative agent with various cancers: cervical (100%), anal (90%), penile (50%), vulval (40%), vaginal (70%) and oropharyngeal (20%–60%). Amongst these, the incidence of anal carcinoma is 40–80 fold greater in HIV-infected people as compared to the general population.[6] SCC comprises 80%–90% of all anal cancer. More than 90% of it are caused by co-infection with high-risk HPV strains.[11] Infection with HPV, sexual habits and smoking are the major risk factors for anal cancer in HIV-infected men. Although, no incidence of anal cancer was noted in any of our individuals. Patients with a precancerous lesion or anal cancer may be asymptomatic or may present with a wide range of complaints. They might complain of pain in the anal or pelvic region and anal bleeding (approximately half of patients). Approximately 30% of the patients complain of a sensation of a rectal mass. Local wetness, irritation and prolapse of tissue are also reported. Other major complaints include incontinence of flatus or liquid or solid stool and obstipation.[23]

Another important finding in our study was the occurrence of opportunistic infections (OI) in HPV positive HIV-positive patients, detected in anal pap smear testing. It was reported positively in 7.14% of the anal pap smears studies. HSV infection was reported in two cases and tuberculosis and fungal infection were reported in one case each. Such OI have previously been reported in pap smears in cervical cancer screening; however, they have not been reported in anal cytology before our study.[24] It shall be an interesting area to extend our horizon to in the future.

One of the main limitations of this study can be recognised as the lack of HSIL cases, as it would have been good, from the research point of view, to have been able to analyse and discuss those findings.

We would recommend LBC in HIV individuals for screening of cytological abnormalities and HPV status. This would also give an opportunity to screen for opportunistic infections, which have otherwise not been diagnosed.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Douek DC, Roederer M, Koup RA. Emerging concepts in the immunopathogenesis of AIDS. Annu Rev Med 2009;60:471-84.  Back to cited text no. 1
Paranjape RS, Challacombe SJ. HIV/AIDS in India: An overview of the Indian epidemic. Oral Dis 2016;22 Suppl 1:10-4.  Back to cited text no. 2
Human Papillomavirus (HPV) | in Patients with HIV [Internet]. AIDS Institute Clinical Guidelines. [cited 2019 Dec 24]. Available from: https://www.hivguidelines.org/sti-care/hpv-infection/.  Back to cited text no. 3
Moore HG, Guillem JG. Anal neoplasms. Surg Clin North Am 2002;82:1233-51.  Back to cited text no. 4
Anderson J, Hoy J, Hillman R, Gittleson C, Hartel G, Medley G, et al. Abnormal anal cytology in high-risk human papilloma virus infection in HIV-infected Australians. Sex Transm Infect 2008;84:94-6.  Back to cited text no. 5
Marchetti G, Comi L, Bini T, Rovati M, Bai F, Cassani B, et al. HPV infection in a cohort of HIV-positive men and women: Prevalence of oncogenic genotypes and predictors of mucosal damage at genital and oral sites. J Sex Transm Dis 2013;2013:915169.  Back to cited text no. 6
Teresa MD, Joel MP. Anal cytology. In: Nayar R, Wilbur DC, editors. The Bethesda System for Reporting Cervical Cytology. 3rd ed. Switzerland: Springer International Publishing; 2015. p. 263-85.  Back to cited text no. 7
Lee CH, Lee SH, Lee S, Cho H, Kim KH, Lee JE, et al. Anal human papillomavirus infection among HIV-infected men in korea. PLoS One 2016;11:e0161460.  Back to cited text no. 8
Palefsky JM, Holly EA, Hogeboom CJ, Berry JM, Jay N, Darragh TM. Anal cytology as a screening tool for anal squamous intraepithelial lesions. J Acquir Immune Defic Syndr Hum Retrovirol 1997;14:415-22.  Back to cited text no. 9
Arora R, Pandhi D, Mishra K, Bhattacharya SN, Yhome VA. Anal cytology and p16 immunostaining for screening anal intraepithelial neoplasia in HIV-positive and HIV-negative men who have sex with men: A cross-sectional study. Int J STD AIDS 2014;25:726-33.  Back to cited text no. 10
Maia LB, Marinho LC, Wanderley Paes Barbosa T, Batalha Filho ES, Ribeiro Velasco LF, Garcia Costa PG, et al. A comparative study between conventional and liquid-based cytology in screening for anal intraepithelial lesions in HIV-positive patients. Diagn Cytopathol 2014;42:840-5.  Back to cited text no. 11
Darragh TM, Jay N, Tupkelewicz BA, Hogeboom CJ, Holly EA, Palefsky JM. Comparison of conventional cytologic smears and ThinPrep preparations from the anal canal. Acta Cytol 1997;41:1167-70.  Back to cited text no. 12
Sherman ME, Friedman HB, Busseniers AE, Kelly WF, Carner TC, Saah AJ. Cytologic diagnosis of anal intraepithelial neoplasia using smears and cytyc thin-preps. Mod Pathol 1995;8:270-4.  Back to cited text no. 13
Lindsey K, DeCristofaro C, James J. Anal pap smears: Should we be doing them? J Am Acad Nurse Pract 2009;21:437-43.  Back to cited text no. 14
Bean SM, Chhieng DC. Anal-rectal cytology: A review. Diagn Cytopathol 2010;38:538-46.  Back to cited text no. 15
Ong JJ, Chen M, Tabrizi SN, Cornall A, Garland SM, Jin F, et al. Anal HPV detection in men who have sex with men living with HIV who report no recent anal sexual behaviours: Baseline analysis of the Anal Cancer Examination (ACE) study. Sex Transm Infect 2016;92:368-70.  Back to cited text no. 16
Hernandez AL, Karthik R, Sivasubramanian M, Raghavendran A, Gnanamony M, Lensing S, et al. Prevalence of Anal HPV infection among HIV-positive men who have sex with men in India. J Acquir Immune Defic Syndr 2016;71:437-43.  Back to cited text no. 17
Daling JR, Madeleine MM, Johnson LG, Schwartz SM, Shera KA, Wurscher MA, et al. Human papillomavirus, smoking, and sexual practices in the etiology of anal cancer. Cancer 2004;101:270-80.  Back to cited text no. 18
Davis KG, Orangio GR. Basic science, epidemiology, and screening for anal intraepithelial neoplasia and its relationship to anal squamous cell cancer. Clin Colon Rectal Surg 2018;31:368-78.  Back to cited text no. 19
Lin CC, Hsieh MC, Hung HC, Tsao SM, Chen SC, Yang HJ, et al. Human papillomavirus prevalence and behavioral risk factors among HIV-infected and HIV-uninfected men who have sex with men in Taiwan. Medicine (Baltimore) 2018;97:e13201.  Back to cited text no. 20
Donà MG, Benevolo M, Vocaturo A, Palamara G, Latini A, Giglio A, et al. Anal cytological abnormalities and epidemiological correlates among men who have sex with men at risk for HIV-1 infection. BMC Cancer 2012;12:476.  Back to cited text no. 21
Wilkin TJ, Palmer S, Brudney KF, Chiasson MA, Wright TC. Anal intraepithelial neoplasia in heterosexual and homosexual HIV-positive men with access to antiretroviral therapy. J Infect Dis 2004;190:1685-91.  Back to cited text no. 22
Kakar A, Gogia A, Ganwani A. Prevention of anal carcinoma in HIV/AIDS patients. ARC J AIDS 2018;3:3-14.  Back to cited text no. 23
Seth A, Kudesia M, Gupta K, Pant L, Mathur A. Cytodiagnosis and pitfalls of genital tuberculosis: A report of two cases. J Cytol 2011;28:141-3.  Back to cited text no. 24
[PUBMED]  [Full text]  


  [Table 1], [Table 2]


Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

  In this article
Materials and Me...
Article Tables

 Article Access Statistics
    PDF Downloaded58    
    Comments [Add]    

Recommend this journal