High Prevalence of Cervical High-Grade Lesions and High-Risk Human Papillomavirus Infections in Women Living with HIV: A Case for Prioritizing Cervical Screening in This Vulnerable Group

Cancer of the uterine cervix accounted for 604,127 new cancer cases and 341,831 deaths worldwide in 2020 [1]. In India, cervical cancer constitutes the second most common malignancy in females after breast cancer [1]. A large proportion of cervical cancers are caused by human papillomavirus (HPV) infection, especially types 16 and 18. In India, about 5% of women with normal cervical cytology are found to have HPV16/18 in cervical samples [2].

Since both HIV and HPV are transmitted via sexual contact and compromised immune status in women living with HIVs (WLHIVs) allows for persistence of HPV infections, it has been postulated that prevalence of high-risk cervical HPV and precancerous lesions were likely to be higher among WLHIV. This has been confirmed by studies conducted in various countries and different settings [3‒5]. With increasing use of highly active antiretroviral therapy leading to improved life expectancy, the incidence of cervical precancerous lesions and cervical cancer in WLHIVs is increasing. The Centre for Disease Control and Agency for Health Care Policy and Research have recommended cervical smears in the initial evaluation of WLHIVs [6]. For resource-limited settings, the Federation of Obstetrics and Gynaecologic Societies of India also recommends cervical cancer screening as soon as HIV is diagnosed followed by screening every 3 years [7]. However, appropriate screening of WLHIVs is still not undertaken widely.

Considering the global public health problem of cervical cancer, the World Health Organization and World Health Assembly devised a “Global strategy for cervical cancer elimination” in 2020 urging countries to reduce the incidence rate of cervical cancer to below four per 100,000 women by 2030 [8]. One of the targets of this strategy is ensuring at least 70% coverage of women for cervical cancer screening using a high-performance test, as per the cost-effectiveness for the concerned country. In resource-constrained countries like ours, a population-based cancer screening program is yet to be rolled out. In this scenario, effective screening of a highly vulnerable population such as WLHIVs for common cancers is still far from attained. Studies conducted in India have demonstrated the higher prevalence of cervical precancerous lesions and/or cervical HPV infection in WLHIVs [3, 4, 9]. However, majority of these studies did not include a control group of HIV-negative women that could highlight the difference in prevalence of cervical lesions or HPV in WLHIVs versus general population.

The present study was aimed at assessing the prevalence of cervical epithelial abnormalities and cervical HPV in WLHIVs and comparing these with HIV-negative subjects recruited from women undergoing opportunistic screening. In addition, clinic-demographic factors associated with cervical precancerous lesions and HPV in WLHIVs were also examined to duly guide the care providers of this vulnerable population.

This was a cross-sectional exploratory study of WLHIV and HIV-negative women recruited between October 2019 and October 2021. WLHIVs were recruited from Integrated Testing and Counselling Centre of a tertiary care hospital and the linked ART center. HIV-negative participants were enrolled from among the women undergoing opportunistic cervical cancer screening at the health promotion clinic in our Institute.

Participants (WLHIVs and HIV-negative women) in the reproductive age group with intact cervix, no prior history of cytological high-grade/histologic CIN2+ lesions, and giving informed consent were included in the study. Women who had been previously treated for a cervical precancerous lesion or received pelvic radiation, undergone hysterectomy for a benign/malignant lesion, any disease that precluded pelvic examination, or did not consent for cervical sample collection were excluded.

For all included participants, a semi-structured questionnaire was used to elicit demographic and clinical information including obstetric and sexual history. All the enrolled participants underwent cervical smear collection using cytobrush and extended tip Ayre’s spatula, along with a sample for HPV DNA detection by Hybrid capture 2 technique. In addition, blood was collected from WLHIVs for CD4+ counts, and data on antiretroviral therapy (ART) use and its duration were collected.

Conventional Papanicolaou-stained smears were prepared from cervical samples for cytological screening. The smears were reported in accordance with the Bethesda system for reporting cervicovaginal cytology (2014) [10]. Cases with unsatisfactory smears were excluded from the study.

HR-HPV DNA detection was performed through the HC2 high-risk HPV DNA test using the manufacturer’s instructions (Digene Corporation, Gaithersburg, MD, USA). Samples with R/CO values of >1 for high-risk probes were categorized as HR-HPV positive, while those with values less than 1 as negative.

Women with cervical cytology results of ASC-US and above or HR-HPV positivity were considered as screen-positive. Women with cervical ASC-US underwent a repeat cervical cytology 6 months after the initial smear, while those with low-grade squamous intraepithelial lesion (LSIL) + lesion or HR-HPV positivity were recalled for colposcopy, and a directed biopsy was taken if indicated. The biopsies were taken within 3 months of cytological diagnosis. Histologically confirmed cervical intraepithelial neoplasia (grades 1–3) that were eligible for ablative treatment were managed through thermal ablation. Women with lesions that were not eligible for ablation were referred to a tertiary care center for conization or loop electrosurgical excision of transformation zone, as indicated.

Contingency table analysis was performed to compare the prevalence of cervical intraepithelial neoplasia and HR-HPV positivity between WLHIV and HIV-negative women. For WLHIV, association between various participant characteristics and cervical cytological abnormalities and HR-HPV positivity was assessed through χ² test. Multivariate logistic regression analysis was performed to detect association of relevant characteristics with either of the output variables (abnormal cervical cytology and cervical HR-HPV). p values of <0.05 were considered as significant. All statistical analyses were performed using open-access Python language in Jupyter software, downloadable from https://www.anaconda.com/products/individual. The study was approved by the Institutional Ethics Committee, and all participants gave written informed consent for inclusion in the study.

This study included 141 WLHIVs and 161 HIV-negative women. The demographic characteristics of WLHIV and HIV-negative women in the study are summarized in Table 1. All the women enrolled in the study (WLHIVs and HIV-negative) underwent cervical cancer screening for the first time as a part of this study.

As seen in Table 1, there was no significant difference among WLHIVs and HIV-negative women for parameters such as age, education status, past history or current STI, and cervical examination findings. Gynecological complaints at the time of enrolment were similar among WLHIVs and HIV-negative women. The most frequent symptom was lower backache followed by discharge per vaginum, menstrual irregularities, perivulvar itching, and pain in the lower abdomen.

Of the WLHIVs, six smears (4.2%) were found to be unsatisfactory and were excluded from the study. One case from HIV-negative women was excluded due to unsatisfactory smear. Hence, 135 WLHIVs and 160 HIV-negative women were included for analysis of cervical cytological abnormalities and HR-HPV positivity.

On cervical smear examination, specific organisms were seen in 16.8% (22 of 135) smears among WLHIVs and 18.6% (30 of 160) HIV-negative women with no statistical difference between them (p = 0.69). The most commonly detected organism in both the groups was bacterial vaginosis, followed by fungal species consistent with Candida and Trichomonas vaginalis. The cytological findings as per TBS 2014 and HR-HPV detection in the two groups are tabulated in Table 2.

As shown in Table 2, the prevalence of both cervical cytological abnormalities and HR-HPV positivity were significantly higher among WLHIVs compared to HIV-negative women. All the cytological abnormalities (five cases, 3.1% of 160 participants) in HIV-negative women were low-grade (ASC-US in 4 and LSIL in one) in the present study, and no high-grade lesions were detected in this group. Epithelial cell abnormalities were detected in 19 WLHIVs (14.1% of 135). Of these 19 cases, ASC-US was seen in seven (5.2% of 135), LSIL in eight (5.9%), ASC-H in one (0.9%), and HSIL in 3 patients (2.2%). There was no case of squamous cell carcinoma in either of the groups. High-grade lesions (ASC-H/HSIL) were found in five cases among WLHIVs (Fig. 1). These lesions comprised 3.7% of all smears from WLHIVs and 26.3% of all ASC-US + lesions in this group of women. In WLHIVs, an increasing trend of HR-HPV positivity was seen in worsening grades of cervical cytological abnormalities. HR-HPV was detected in only 14.2% of ASC-US and 83.3% of LSIL compared to 100% of ASC-H and HSIL.

All the screen-positive women (on cytology or HPV testing) were rigorously followed-up. Women (both WLHIV and HIV-negative) with cervical cytological diagnosis of ASC-US were recalled for a repeat cervical smear after 6 months. Repeat smears were reported as NILM in six of seven WLHIV, while one had persistent ASC-US on repeat smear; her HR-HPV test was negative, and she has been advised follow-up with repeat cytology after 1 year. All cases reported as ASC-US in HIV-negative women reverted to normalcy on repeat smear examination after 6 months.

On biopsy, six cases with cytological LSIL in WLHIV showed features of CIN-1 (Fig. 2), while one of LSIL cases showed CIN-3 lesion. The five cases with ASC-H/HSIL, all showed CIN-3 without evidence of invasion (Fig. 3). Hence, histologically confirmed CIN2+ lesions were seen in 4.4% of 135 WLHIVs included in this study and 31.5% of all ASC-US + lesions detected in this group. In HIV-negative women, one case of LSIL on cytology was reported as chronic cervicitis on biopsy. Cytohistological correlation, as per the American Society of Cytopathology 2017 guidelines [11], was seen in 84.6% (11 of 13 cases that underwent biopsy), while one each (7.7% each) were categorized as minor undercall (cytological LSIL and biopsy report of CIN-3) and overcall (LSIL on cervical cytology and chronic cervicitis on biopsy), respectively.

Twenty-four (24) of the 28 WLHIVs and eight of 13 HIV-negative women with negative cervical cytology and positive HR-HPV test underwent colposcopy. No significant lesion was seen on colposcopy in these women. All these women were advised to visit for annual follow-up with cervical cytology and/or colposcopy.

Bivariate analysis with various socio-demographic parameters revealed that among WLHIV, cervical cytological abnormalities were associated with current STI in the participant (p = 0.03), having ≥1 sexual partner (p < 0.001), history of tobacco use (smoked or smokeless) by the participant (p = 0.02), and nadir low CD4 count <200/µL (p = 0.02). On the other hand, factors such as education status, past history of STI, multiparity (≥3 deliveries), and intake of ART did not show any significant association with cervical cytological abnormalities among WLHIVs (Table 3). Further analysis showed that high-grade cervical lesions were more common in WLHIVs using tobacco (p = 0.01) and HR-HPV infection (p = 0.001) than those with low-grade lesions. However, education level, parity, history of STI, or having multiple sexual partners did not show any association with high-grade lesions (data not shown).

Cervical HR-HPV positivity in WLHIVs showed significant association with current STI (p = 0.001), history of tobacco use (p = 0.002), nadir CD4 counts less than 200/µL (p = 0.03) and negative association with ART intake (p = 0.004). Cervical HR-HPV positivity was higher among WLHIVs with abnormal cervical smears (p < 0.001). No significant association was found between cervical HR-HPV positivity and education status of the participant, past history of STI, having multiple sexual partners, and multiparity (Table 4).

The parameters of age at enrolment, age at first delivery, and age at last delivery demonstrated multicollinearity and hence, were excluded from analysis. Logistic regression analysis revealed a significant association of cervical cytological abnormalities with having multiple sexual partners (p < 0.001), as shown in Table 3. Cervical HR-HPV, on the other hand, showed a positive association with current STI (p = 0.01), nadir CD4 count <200/µL (p = 0.004), abnormal cervical cytology (p = 0.002) and a negative association with ART intake (p = 0.03), as depicted in Table 4.

In WLHIV, cervical cancer has been categorized as an AIDS-defining illness by the Centre for Disease Control and Prevention, while cervical intraepithelial neoplasia is classified as indicative of early symptomatic HIV infection [12]. In view of the immunosuppression, WLHIV are at a higher risk of persistent HPV infection leading to a two to six-times increased incidence of cervical lesions compared to HIV-negative women [13].

The present cross-sectional study examined the cervical cytological abnormalities and HR-HPV infection in WLHIV and compared the same with HIV-negative women recruited from the general population rather than women attending STI clinic. The socio-demographic characteristics of WLHIV in the present study were similar to those reported earlier in studies from India [3, 4]. The previous studies did not include a control group of HIV-negative women, unlike the present study. No significant difference was noted between the two groups with regards to age, education status, past history or current STIs, and cervical examination findings. The prevalence of specific infections detected in cervical smear was also similar among WLHIV and HIV-negative women in the present study.

The prevalence of cytologically detected cervical epithelial abnormalities was found to be significantly higher in WLHIVs compared to the HIV-negative women (14.1% vs. 3.1%, respectively) in the present study. The prevalence of abnormal cervical smears among WLHIV was similar to those reported by Isaakidis et al. [3] but lower than that seen by Chakravarty et al. [14], Mane et al. [4], and Branca et al. [15]. The distribution of various grades of cervical lesions was however similar to the earlier reports with low-grade squamous epithelial lesions being the most frequent among WLHIVs. Cytological high-grade lesions (ASC-H/HSIL) were detected in 3.7% of WLHIVs in the present study. This prevalence is more or less similar to the 5% HSIL detected in the study by Isaakidis et al. [3] and 2.7% seen by Mane et al. [4] but lower than the 6.9% seen by Chakravarty et al. [14]. On the other hand, histologically confirmed CIN2+ lesions were seen in 4.4% of all WLHIVs in our study. This compared favorably with 7% CIN2+ in the study by Isaakidis et al. [3] but lower than 10.8% reported by Mane et al. [4]. Chakravarty et al. did not perform colposcopic and histologic confirmation of the cytologically detected cases in their study [14]. Isaakidis et al. [3] and Mane et al. [4] employed colposcopic evaluation of all their WLHIVs, while in the present study, colposcopy was performed for women with cytological report of LSIL + or with HR-HPV positivity and normal cytology. Performing colposcopy in all WLHIVs in the present study was not considered due to cost constraints. In the present study, no high-grade lesion was detected in the 160 HIV-negative women recruited from the women undergoing opportunistic screening at our Institute. In contrast to the earlier reports, the inclusion of control group of HIV-negative women in the present study and the significantly higher prevalence of abnormal cervical cytology in WLHIV serve to emphasize the importance of cervical cancer screening in this highly vulnerable group. Majority of the earlier reports with an HIV-negative group recruited the controls from high-risk women. Though the risk of squamous epithelial lesions was higher among WLHIVs compared to HIV-negative women, the prevalence of HPV DNA was high in both the groups in the study by Branca et al. [15].

Cervical HPV infection has been reported to vary between 25 and 45% in WLHIVs [14‒17]. This variation can partly be explained by the difference in the techniques employed for HPV detection in the various studies. A recent meta-analysis reported a 2.6-times higher risk of HPV acquisition among WLHIVs compared to HIV-negative women [18]. The present study utilized Hybrid capture 2, a US-FDA-approved method for HPV detection in cervical samples and found HR-HPV positivity in 28.9% of the samples from WLHIVs compared to 9.3% in HIV-negative women, the difference being statistically significant (p < 0.001). The distribution of HR-HPV positivity in various grades of cervical epithelial lesions in the present study was in accordance with the earlier reports [4, 14] of a trend of increasing prevalence of HPV in severe grades of lesions. Similar to the reports by Mane et al. [4] and Chakravarty et al. [14], 25.8% of WLHIVs with cytology report of NILM had HR-HPV. Follow-up of these cases with colposcopy and repeat cervical smear after 6 months of initial recruitment did not yield any cervical abnormality. Isaakidis et al. did not report any additional cervical epithelial lesions in the 12-month follow-up in their study [3]. However, a longer duration of follow-up is required in WLHIVs with negative cytology and positive HR-HPV to elucidate the risk of development of cervical precancerous lesions in such patients.

In the present study, detection of cervical cytological abnormalities among WLHIVs was associated with current STI in the participant (p = 0.03), having multiple sexual partners (p < 0.001), and tobacco use (p = 0.02) on bivariate analysis. Earlier studies have reported various factors associated with detection of abnormal cervical smears in WLHIVs. Delmas et al. reported a positive association of cervical ECAs with CD4 cell count, prior history of squamous intraepithelial lesion, genital warts, and site of recruitment. However, no association was seen with factors such as ART intake, past history of STI, sexual history, or contraceptive history [19]. The study by Chakravarty et al. [14] reported WHO stage and CD4 count <350/µL as determinants for abnormal cervical cytology [14], while Mane et al. [4] showed association of SIL with cervical HPV but not with low CD4 counts [4]. A recent meta-analysis from Sub-Saharan Africa demonstrated significant association of cervical precancerous lesions with having multiple sexual partners, history of STI, multiparity, and low CD4 counts <200/µL [20]. Another analysis by Liu et al. [18] reported lower progression from normal to LSIL in WLHIVs on ART. Thus, HIV infection in itself is a risk factor for higher prevalence of cervical epithelial abnormalities. Among WLHIVs, increasing immunosuppression (evidenced by low CD4 counts) and certain clinic-demographic factors such as having multiple sexual partners, multiparity, tobacco use are also associated with abnormal cervical smears. Hence, eliciting a detailed history and motivation of WLHIVs to come forward for cervical cancer screening seem imperative to reduce the morbidity and mortality due to cervical cancer in these women.

Cervical HPV infection among WLHIVs has earlier been shown to be higher in rural and illiterate women in the study by Chakravarty et al. [14], while Mane et al. [4] reported association of “carcinogenic” HPV genotypes with multiple sexual partners or lower CD4 counts [4, 14]. Similarly, Isaakidis et al. [3] reported higher HPV infection in WLHIVs with nadir CD4 counts <200/µL, ART intake of less than 12 months, and ≥2 previous pregnancies. In the present study, cervical HR-HPV positivity showed a positive association with current STI in the woman, tobacco use, nadir CD4 counts less than 200/µL and a negative association with ART therapy. Short-term follow-up of WLHIVs with negative cytology but positive cervical HR-HPV in the present study did not yield any significant abnormality. However, the duration of follow-up was relatively short in our study. Longer follow-up duration is required to elucidate the risk and possible progression from normal cytology to cervical epithelial lesions in WLHIVs with positive HR-HPV test results. The issue of effect of ART on HPV acquisition or persistence or the risk of progression of epithelial changes is still debatable with conflicting reports [21‒23]. Well-designed longitudinal follow-up studies are imperative to settle this controversy.

The World Health Organization, in 2020, announced a global call for action to eliminate cervical cancer as a public health problem. In this appeal, the Organization laid down targets including 90% of the girls to be vaccinated against HPV, 70% of eligible women to be covered by a cervical cancer screening program, and 90% of the screen-detected be given access to treatment and palliative care. These goals are to be achieved by the signing countries by 2030 [24]. However, many low-resource settings like ours are still in the process of rolling out a population-based cervical cancer screening program while depending on opportunistic screening. In view of the high prevalence of high-grade cervical epithelial abnormalities in the vulnerable populations such as WLHIV compared to the general population, it would be only prudent to prioritize and undertake cervical screening in such women by integrating it into their routine investigative and follow-up procedures, to reduce the morbidity and mortality from cervical cancer in them.

The main strength of this study was the inclusion of a control group of HIV-negative women recruited from the opportunistic screening activities rather than a hospital-based population, thus providing an almost true comparison of the prevalence of cervical cytological abnormalities and HR-HPV prevalence between WLHIVs and HIV-negative women. The screen-positive women were followed-up rigorously and recalled for colposcopic evaluation to detect, provide histologic confirmation of the epithelial lesions, thus reducing the chances of missing any high-grade lesions.

A few limitations of the study deserve mention. First, the duration of follow-up in our study was limited due to which the progression of cytological abnormalities or development of new changes in women having a baseline negative cytology could not be evaluated. Due to the cross-sectional nature of our study design, another shortcoming is the non-availability of follow-up data of women with baseline HR-HPV positivity at this point of time, thereby limiting the assessment of resolution or persistence of HPV and its outcomes on cytology/histology. However, all the women with HR-HPV positivity are under follow-up as per the standard protocol. Also, we could not undertake HPV genotyping in the present study due to paucity of available funds.

In conclusion, the present study reaffirms the heightened risk of cervical cytological abnormalities (including high-grade lesions) and HR-HPV infection in WLHIV compared to the HIV-negative women. Hence, regular cervical cancer screening through cytology or HR-HPV testing should be included in the routine investigative protocols for these vulnerable women, especially in low-resource settings lacking an organized cervical cancer screening program.

The authors wish to acknowledge Dr. Anurag Bhargava, Chief Medical Superintendent; staff of the ART Centre, MMG Hospital, Ghaziabad; and staff of the Love Life Society (a non-governmental organization) involved in care of individuals living with HIV. We are also extremely grateful to all the participants who gave consent to be a part of this important study.

All participants gave their written informed consent for inclusion in the study. The study protocol was approved by the Ethics Committee, ICMR-NICPR (registered with Department of Health Research, Govt. of India) vide letter no. NICPR/Dir/EC/2016. The study protocol was approved by the Scientific Advisory Committee of ICMR-NICPR vide a meeting held on March 11, 2016.

The authors have no conflicts of interest to declare.

The study was funded through research grant from the Indian Council of Medical Research, India (Grant ID: 2017-0105).

Ruchika Gupta: conceptualization, study design, literature review, data collection and analysis, drafting, and revision of manuscript. Showket Hussain: HPV DNA testing and data collection and analysis. Roopa Hariprasad and Kavitha Dhanasekaran: subject recruitment, management of screen-detected cases, and review of script. Sheel Verma: ART Incharge, subject recruitment, data sharing and collection, and review. Vineeta Agarwal: subject recruitment, management of screen-positive cases, and review. Sandeep: subject recruitment, data collection, and sample processing. Shahana Perveen and Avineet Kaur: subject motivation and recruitment, sample collection, and data management. Chandresh Pragya Verma, Amita, and Reena Dwivedi: subject recruitment and sample collection. Sompal Singh: data analysis and presentation andcritical review. Sanjay Gupta: conceptualization, study design, literature review, data collection, and critical review.

The data that support the findings of this study are not publicly available due to their containing information that could compromise the privacy of research participants but are available from the corresponding author (S.G.).