Worldwide, women constitute nearly half of all individuals living with HIV/AIDS, with a rising proportion of young women affected [1]. Cervical cancer remains the fifth most common cause of cancer-related mortality among women globally [2]. Persistent infection with high-risk human papillomavirus (HPV), particularly HPV-16 and HPV-18, is the most important etiological factor in the development of cervical cancer. HIV infection is a well-recognized co-risk factor for carcinoma of the cervix, and in 1993, the Centers for Disease Control and Prevention (CDC) declared cervical cancer as an AIDS-defining illness [3]. HIV-related immunosuppression is associated with an increased risk of prevalent, incident, and persistent squamous intraepithelial lesions (SILs) of the cervix [4]. HIV-infected women also demonstrate higher rates of cervical HPV infection and persistence [5]. Although highly active antiretroviral therapy (HAART) has significantly reduced HIV viral loads since its introduction in 1995, its impact on HIV-associated cervical cytological abnormalities remains unclear [6,7]. While several viruses such as HPV, HSV, CMV, and Parvovirus are known to induce specific cytomorphological changes, no definitive morphological features have been consistently described for HIV infection. Learmonth G. reported certain subtle cytological alterations in Pap smears of HIV-infected women [8]. Similar subtle changes were also observed by the postgraduate guide of the present study, prompting a systematic evaluation. The present study was undertaken to compare Pap smear findings between HIV-infected and uninfected women and to correlate cytological abnormalities with the duration of HIV infection, immune status (CD4 count), and HAART treatment status [1,8].

This cross-sectional comparative study was conducted in the Department of Pathology, Government Medical College, Bhavnagar, over a period from 1st June 2016 to 30th June 2017. Pap smears of HIV-infected women were collected from patients attending the ART clinic of Sir T. General Hospital, Bhavnagar. The control group consisted of women undergoing routine Pap smear screening at the Gynecology Outpatient Department and screening camps; their HIV status was not known. Study Population A total of 100 Pap smear samples were analyzed, including 50 HIV-positive women (cases) and 50 age-matched controls. Ethical committee and Institutional Review Board approval were obtained prior to the study, and informed consent was taken from all participants. Inclusion Criteria • HIV-positive women aged 21–60 years • Age-matched control women undergoing Pap smear screening Exclusion Criteria • Women outside the specified age group • HIV-positive women with incomplete clinical records Data Collection and Procedure Relevant clinical details including age, symptoms, duration of HIV infection, CD4 count, and HAART status were collected. Pap smears were obtained by the gynecology department, fixed, stained using Papanicolaou stain, and evaluated independently. Cytological features were graded systematically, including cellularity, cytomegaly, nucleomegaly, nuclear membrane thickening, abnormal keratinization, presence of dendritic cells, polymorphs, bacteria, and other infections. Final diagnosis was rendered according to the Bethesda System for Reporting Cervical Cytology 2014. Statistical Analysis Chi-square test was used to compare cytological features between cases and controls. ANOVA was applied to assess associations with CD4 count, duration of HIV infection, and duration of HAART. Results were expressed as frequencies, percentages,

A total of 105 Pap smears were initially collected during the study period. Five smears (3 controls and 2 HIV-positive cases) were excluded due to poor cellularity. Thus, 50 HIV-positive women (cases) and 50 HIV-negative women (controls) were included for final analysis. None of the HIV-positive participants had undergone prior Pap smear screening. Table 1 compares cytomorphological features between HIV-positive women and HIV-negative controls. Abnormal keratinization, nucleomegaly, and nuclear membrane thickening were significantly more pronounced in HIV-positive women compared to controls (p < 0.05). In contrast, dendritic cells were significantly more abundant in control smears (p = 0.0001). No statistically significant differences were observed between the two groups with respect to cellularity, cytomegaly, polymorphs, cytolysis, or bacterial presence (p > 0.05). Table 1. Comparison of Cytomorphological Features Between HIV-Positive and Control Women Cytological Feature HIV Positive (n=50) Mean ± SD Control (n=50) Mean ± SD p value Cellularity 2.48 ± 1.24 2.64 ± 1.35 0.540 Abnormal keratinization 0.54 ± 0.78 0.16 ± 0.42 0.003 Cytomegaly 0.24 ± 0.59 0.14 ± 0.35 0.306 Nucleomegaly 0.80 ± 0.83 0.48 ± 0.70 0.040 Nuclear membrane thickening 0.66 ± 0.69 0.32 ± 0.51 0.006 Dendritic cells 0.13 ± 0.34 0.94 ± 0.77 0.0001 Polymorphs 1.46 ± 1.47 1.52 ± 1.62 0.847 Cytolysis 0.24 ± 0.59 0.24 ± 0.52 1.000 Bacteria 0.50 ± 1.06 0.44 ± 0.93 0.763 Table 2. Grades of Significant Cytomorphological Features in HIV-Positive and Control Women Feature Group Grade 0 n (%) Grade 1 n (%) Grade 2 n (%) Grade 3 n (%) Cytomegaly HIV +ve 41 (82%) 7 (14%) 1 (2%) 1 (2%) HIV –ve 43 (86%) 7 (14%) 0 (0%) 0 (0%) Nucleomegaly HIV +ve 23 (46%) 14 (28%) 11 (22%) 2 (4%) HIV –ve 31 (62%) 15 (30%) 3 (6%) 1 (2%) Nuclear membrane thickening HIV +ve 22 (44%) 24 (48%) 3 (6%) 1 (2%) HIV –ve 35 (70%) 14 (28%) 1 (2%) 0 (0%) Abnormal keratinization HIV +ve 31 (62%) 12 (24%) 6 (12%) 1 (2%) HIV –ve 43 (86%) 6 (12%) 1 (2%) 0 (0%) Table 2 illustrates the grade-wise distribution of significant cytomorphological features among HIV-positive women and controls. Higher grades of nucleomegaly, nuclear membrane thickening, and abnormal keratinization were more frequently observed in HIV-positive women, whereas most control smears predominantly showed Grade 0 changes. Cytomegaly was largely absent or mild in both groups; however, moderate to severe grades were seen exclusively among HIV-positive women. Overall, the distribution indicates a greater severity of cytological abnormalities in HIV-infected women compared to HIV-negative controls. Table 3. Distribution of Dendritic Cells in HIV-Positive and Control Women Group Grade 0 n (%) Grade 1 n (%) Grade 2 n (%) Grade 3 n (%) HIV +ve (n=47*) 41 (87%) 6 (13%) 0 (0%) 0 (0%) HIV –ve (n=46*) 11 (24%) 30 (65%) 3 (6%) 2 (4%) *Cases with scant cellularity excluded from grading. Table 3 depicts the grade-wise distribution of dendritic cells in HIV-positive women and controls. A markedly higher proportion of HIV-positive women showed complete absence of dendritic cells (Grade 0), whereas most control smears demonstrated mild to moderate presence (Grades 1–3). This distribution indicates a significantly reduced presence of dendritic cells in HIV-positive women, suggesting compromised local immune response compared to HIV-negative controls. Table 4. Association of Cytological Features with CD4 Count, Duration of HIV Infection, and HAART Parameter CD4 Count Duration of HIV Duration of HAART Cytomegaly NS NS NS Nucleomegaly NS NS NS Nuclear membrane thickening NS NS NS Abnormal keratinization NS NS NS No statistically significant association was found between cytomorphological abnormalities and CD4 count, duration of HIV infection, or duration of HAART therapy. Epithelial abnormalities were more frequent in HIV-positive women. A distinct group of Non-Normal Cells (22%) was observed predominantly in HIV-positive cases. Table 5. Comparison of Cervical Cytology According to Bethesda System (2014) Cytological Diagnosis HIV Positive n (%) Control n (%) NILM 14 (28%) 23 (46%) Vaginitis 12 (24%) 9 (18%) Bacterial vaginosis 1 (2%) 3 (6%) Fungal infection 1 (2%) 0 (0%) Reactive changes 5 (10%) 11 (22%) HPV-like changes 1 (2%) 0 (0%) Non-normal cells (NNC) 11 (22%) 1 (2%) ASCUS 4 (8%) 2 (4%) AEC-NOS 1 (2%) 1 (2%) Total 50 50

The present study highlights distinct cytomorphological alterations in Pap smears of HIV-infected women when compared with HIV-negative controls. Although no high-grade squamous intraepithelial lesions were detected, HIV-positive women demonstrated a significantly higher frequency of subtle nuclear and cytoplasmic abnormalities, underscoring the impact of HIV-related immunosuppression on cervical epithelial morphology. In this study, abnormal cervical cytology was observed in 10% of HIV-positive women and 6% of controls. These findings are comparable with Indian studies by Kusuman et al. and Madan et al., which reported abnormal cytology in 12% of HIV-infected women [9,10], but lower than figures reported from African and European cohorts where prevalence ranged from 15% to 36% [11-15]. The relatively lower prevalence in the present study may be attributed to regional variations in HPV prevalence, differences in sexual behavior, and effective immune reconstitution due to antiretroviral therapy. A key observation was the significantly increased occurrence of Nucleomegaly, nuclear membrane thickening, and abnormal keratinization in HIV-positive women. Similar subtle cytological alterations have been described by Learmonth G., who suggested that these changes may result from micronutrient malabsorption, particularly vitamin A and vitamin B12 deficiency, commonly observed in HIV-infected individuals [8]. The presence of higher-grade nucleomegaly and nuclear membrane thickening in HIV-positive smears in the present study supports this hypothesis and suggests early epithelial instability preceding overt dysplasia. An important and unique finding of this study was the identification of a group of epithelial cells showing nuclear enlargement insufficient to meet ASCUS criteria and without associated inflammation. These changes were classified as Non-Normal Cells (NNC) and were observed predominantly in HIV-positive women (22%). Such borderline cytological abnormalities have not been widely reported in earlier studies and may represent early HIV-related epithelial alterations. Similar observations have been suggested in studies emphasizing expanded cytomorphological evaluation beyond conventional Bethesda categories [8]. Contrary to several studies that reported a higher prevalence of squamous intraepithelial lesions with declining CD4 counts [11,16,17], the present study did not demonstrate a statistically significant association between cytological abnormalities and CD4 count, duration of HIV infection, or duration of HAART. This finding is consistent with studies by Vafaei et al. and Enbe et al., which also failed to establish a strong correlation between immune status and cytological abnormalities [14,18]. This may reflect immune reconstitution following HAART or the absence of persistent oncogenic HPV infection in the study population. The significantly lower presence of dendritic cells in HIV-positive smears is another notable observation. Dendritic cells play a critical role in local immune surveillance and antigen presentation. Their reduced presence in HIV-positive women suggests impaired mucosal immunity, which may contribute to increased susceptibility to cervical epithelial abnormalities [19]. Similar findings have been reported in immunocompromised populations and reinforce the role of local immune dysfunction in HIV-associated cervical pathology [19-20]. Despite the absence of high-grade lesions such as LSIL, HSIL, or squamous cell carcinoma, the presence of subtle yet statistically significant cytomorphological alterations emphasizes the need for regular cervical screening in HIV-infected women. Early detection of these changes may allow closer surveillance and timely intervention before progression to overt neoplasia. The study is limited by a relatively small sample size and the lack of HPV DNA testing, which could have further clarified the role of oncogenic HPV in cytological abnormalities. Additionally, HIV status of control subjects was not confirmed serologically, which may have led to misclassification bias.

The present study demonstrates that HIV-infected women exhibit a higher frequency and greater severity of subtle cytomorphological abnormalities on Pap smear examination compared to HIV-negative controls. Features such as abnormal keratinization, nucleomegaly, and nuclear membrane thickening were significantly more common in HIV-positive women, while dendritic cells were markedly reduced, indicating impaired local immune surveillance. Although high-grade squamous intraepithelial lesions were not observed, the presence of these early cytological alterations underscores the importance of regular cervical cytology screening in HIV-infected women. Early identification of such changes may facilitate closer follow-up and timely intervention, thereby contributing to the prevention of progression to overt cervical neoplasia.

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