Cervical cancer remains a major global public health concern, ranking as the fourth most common cancer among women worldwide, with a disproportionately high burden in low- and middle-income countries [1]. India alone contributes nearly one-fifth of the global cervical cancer cases, underscoring persistent gaps in screening coverage, awareness, and access to preventive healthcare services [10]. Despite being largely preventable through early detection and timely treatment, many women in India continue to present with advanced-stage disease due to sociocultural barriers and inadequate screening infrastructure [2]. The natural history of cervical cancer is closely linked to persistent infection with high-risk Human Papillomavirus (HPV) types, which have been firmly established as the primary etiological agents in cervical carcinogenesis [3]. This understanding has shifted global screening guidelines towards HPV-based testing, which is now recommended by the World Health Organization (WHO) as the preferred screening method due to its superior sensitivity and long-term predictive value [1,4]. Currently, three major screening modalities are widely used in India: Visual Inspection with Acetic Acid (VIA), Papanicolaou (Pap) smear cytology, and HPV DNA testing. VIA is a simple, inexpensive, point-of-care test that involves application of acetic acid to the cervix and visual identification of acetowhite lesions. It is especially useful in low-resource settings because it requires minimal infrastructure and provides immediate results, making it conducive to a "screen-and-treat" approach [6,9]. However, its diagnostic performance is heavily operator-dependent, with variable sensitivity and specificity reported across studies. Pap smear cytology has been the cornerstone of cervical cancer screening for decades and has significantly contributed to reduced mortality in countries with established cytology-based screening programs [5]. The test identifies cellular abnormalities such as ASCUS, LSIL, and HSIL, allowing detection of precancerous lesions before malignant transformation. However, Pap smear sensitivity is moderate and depends on proper sampling, slide preparation, and cytotechnologist expertise [9]. HPV DNA testing represents the most advanced and sensitive method for cervical cancer screening, detecting high-risk HPV genotypes that precede morphological abnormalities [4,7]. Numerous studies have demonstrated its superior ability to identify women at risk for high-grade cervical intraepithelial neoplasia (CIN2+), with long-term follow-up confirming its predictive advantage over cytology [7,8]. Although costs and laboratory requirements remain challenges in some regions, the clinical effectiveness of HPV DNA testing makes it increasingly relevant for integration into national screening programs. Given the varying strengths, limitations, and resource implications of these modalities, comparative evaluation is crucial for optimizing cervical cancer screening strategies in the Indian context. The present study aims to compare VIA, Pap smear, and HPV DNA testing over a one-year period at a tertiary care centre in Central India, assessing their diagnostic performance and clinical utility in detecting CIN2+ lesions.

Study Design and Setting This was a cross-sectional, comparative observational study conducted in the Department of Obstetrics and Gynecology at a tertiary care centre in Central India. The study period extended over one year, from February 2024 to January 2025. Study Population and Sample Size A total of 180 women, aged 25-65 years, attending the gynecology outpatient department for routine screening or evaluation of gynecological complaints were included in the study. All selected participants underwent VIA, Pap smear, and high-risk HPV DNA testing. Inclusion Criteria Women were eligible if they: • Were sexually active and aged ≥25 years • Provided written informed consent • Were willing to undergo all three screening tests (VIA, Pap smear, HPV DNA) Exclusion Criteria Women were excluded if they: • Were pregnant • Had active genital infections • Had a previous diagnosis of cervical cancer • Had undergone total hysterectomy • Had participated in cervical screening within the last 6 months Procedure for Screening Tests 1. Visual Inspection with Acetic Acid (VIA) • A sterile Cusco’s speculum was inserted to visualize the cervix. • 5% acetic acid was applied to the cervix using a cotton swab. • The cervix was inspected after 1 minute under adequate light. • VIA was considered positive if distinct, well-defined acetowhite lesions were seen near the squamocolumnar junction. 2. Pap Smear (Conventional Cytology) • Cervical samples were collected using an Ayre’s spatula and endocervical brush. • Smears were fixed immediately in 95% ethanol. • Slides were evaluated by cytopathologists and reported according to The Bethesda System (TBS 2014). • Abnormal cytology was categorized as ASC-US, ASC-H, LSIL, HSIL, AGC, or carcinoma. 3. High-Risk HPV DNA Testing • Cervical samples were collected using a sterile cervical brush and placed in transport medium. • Testing was performed using a validated nucleic acid amplification assay capable of detecting high-risk HPV genotypes (e.g., HPV 16, 18, and other oncogenic types). • Results were reported as positive or negative for high-risk HPV. Colposcopy and Histopathology • All women who tested positive on any of the three screening tests (VIA+, Pap+, or HPV+) underwent: o Colposcopic examination using Reid’s Colposcopic Index o Directed cervical biopsy from suspicious areas • Histopathology was considered the gold standard for confirming cervical intraepithelial neoplasia (CIN) or invasive carcinoma. • CIN was classified as CIN1, CIN2, CIN3, or invasive cancer. Outcome Measures Diagnostic performance of each test was assessed by calculating: • Sensitivity • Specificity • Positive Predictive Value (PPV) • Negative Predictive Value (NPV) CIN2+ lesions (CIN2, CIN3, or carcinoma) were considered clinically significant for evaluating test performance. Statistical Analysis • Data were entered into Microsoft Excel and analyzed using SPSS version 25.0. • Categorical variables were expressed as percentages, continuous variables as mean ± standard deviation. • Diagnostic accuracy of VIA, Pap smear, and HPV DNA was calculated using 2×2 contingency tables with histopathology as the reference standard. • A p-value < 0.05 was considered statistically significant.

A total of 600 women were included in the study, and all participants successfully underwent VIA, Pap smear, and HPV DNA testing. The overall demographic and clinical characteristics of the study population, as well as the performance of the three screening methods, are summarized in the tables below. Table 1. Baseline Characteristics of Study Participants (n = 180) Characteristics Number (%) Mean age (years) 38.6 ± 9.4 Parity ≥2 72% Rural background 63% Use of contraception 48% History of irregular bleeding 21% Table 1 describes the demographic and reproductive characteristics of the study participants, highlighting the population profile relevant to cervical cancer risk assessment. Table 2: Test Positivity Rates Screening Test Positive (%) VIA 18.5% Pap smear 12.2% HPV DNA test 22.7% Table 2 presents the proportion of women who tested positive by each screening method, indicating variation in detection yield across modalities. Table 3: Diagnostic Accuracy Compared with Histopathology Test Sensitivity (%) Specificity (%) PPV (%) NPV (%) VIA 62.7 81.4 38.2 92.3 Pap smear 78.3 93.5 69.1 95.8 HPV DNA 92.4 89.1 72.4 97.1 Table 3 shows the diagnostic performance of each screening test against the histopathological gold standard, including key parameters such as sensitivity, specificity, PPV, and NPV. Table 4: CIN2+ Detection Rates Test CIN2+ Detected (%) VIA 61% Pap smear 78% HPV DNA test 94% Table 4 compares the ability of VIA, Pap smear, and HPV DNA testing to detect clinically significant lesions (CIN2+), demonstrating differences in diagnostic yield. Figure 1. Comparison of the sensitivity of VIA, Pap smear, and HPV DNA testing. The graph illustrates the superior sensitivity of HPV DNA testing compared to Pap smear and VIA, highlighting its effectiveness in detecting cervical precancerous lesions.

Cervical cancer continues to remain a major public health concern in low- and middle-income countries, including India, where inadequate screening coverage and late diagnosis contribute significantly to disease burden [1,10,11]. Persistent infection with high-risk HPV types has been unequivocally established as the primary etiological factor in cervical carcinogenesis, making HPV detection a critical component of modern screening strategies [3,12]. As global guidelines increasingly shift toward HPV-based primary screening due to its superior sensitivity and negative predictive value, comparative evaluations such as the present study are essential to inform context-appropriate screening policies [1,4,7,13]. In our study, HPV DNA testing demonstrated the highest sensitivity and CIN2+ detection, findings that align with large-scale randomized trials and meta-analyses showing that HPV testing outperforms cytology and VIA in identifying high-grade lesions earlier in the disease trajectory [2,4,7,8,14]. The strong performance of HPV testing is supported by extensive evidence proving that HPV negativity confers long-term protection, allowing extended screening intervals and reducing lifetime screening burden [7,12,15]. Data from Indian cohorts have similarly reported that HPV screening significantly reduces cervical cancer incidence and mortality when compared with cytology-based programs [2,11]. These consistent findings reinforce the potential of HPV DNA testing as the preferred method for early detection and prevention. Pap smear cytology, although historically central to cervical cancer screening programs, demonstrated moderate sensitivity but high specificity in our cohort. This matches earlier evidence that cytology can miss a proportion of early CIN lesions due to sampling error and interpretational variability [5,9,16]. Nonetheless, cytology continues to hold value in settings where HPV testing is not universally accessible, serving effectively as a triage test after HPV positivity or as an alternative when cost constraints limit molecular testing [5,9,13]. Several studies from India and other low-resource settings have observed similar diagnostic performance patterns, emphasizing cytology’s continued relevance in hybrid screening algorithms [9,11,17]. VIA, which is widely promoted in low-resource regions due to its simplicity and cost-effectiveness, showed the lowest sensitivity and specificity in this study-findings consistent with international reports highlighting wide operator-dependent variability [6,9,18]. Despite these limitations, VIA remains crucial in many rural and resource-constrained environments because it allows immediate results and facilitates “screen-and-treat” approaches, which are critical where follow-up rates are low [1,6,10,18]. Studies in sub-Saharan Africa and South Asia have demonstrated that VIA-based programs, when implemented with adequate training and quality assurance, can still provide meaningful reductions in cervical cancer burden [6,17]. The substantial difference in diagnostic accuracy between HPV DNA testing and VIA observed in this study further supports WHO’s recommendation advocating a shift toward HPV-based primary screening as resources permit [1]. However, in real-world Indian settings, challenges such as cost, laboratory infrastructure, and logistical constraints remain significant barriers to universal molecular screening [10,11,13]. Thus, several authors recommend hybrid algorithms, such as HPV testing followed by cytology or VIA triage, to optimize both accuracy and feasibility [4,7,8,14,16]. These strategies help minimize unnecessary referrals, improve risk stratification, and ensure rational utilization of limited resources-an essential consideration for widespread screening implementation in India. Overall, the present study reinforces the complementary roles of all three screening modalities: HPV DNA testing provides maximal sensitivity and long-term risk prediction; Pap smear offers high specificity and well-established clinical utility; and VIA remains an indispensable tool in low-resource field settings. The integration of these methods-tailored to population needs, infrastructure variability, and economic considerations-represents a practical and evidence-based pathway toward reducing the burden of cervical cancer in Central India and similar settings globally [1-18].

HPV DNA testing emerged as the most sensitive and reliable method for detecting cervical precancerous lesions, while Pap smear demonstrated high specificity and remains a valuable screening tool where molecular testing is limited. VIA, though less accurate, offers a practical and cost-effective option in low-resource settings. Adopting a combined or tiered screening approach may enhance early detection and help reduce the cervical cancer burden in Central India.

1. World Health Organization. WHO guidelines for screening and treatment of cervical pre-cancer lesions for cervical cancer prevention. Geneva: WHO; 2021. 2. Sankaranarayanan R, Nene BM, Shastri SS, et al. HPV screening for cervical cancer in rural India. N Engl J Med. 2009;360(14):1385-1394. 3. Schiffman M, Castle PE. The role of human papillomavirus in cervical cancer. Lancet. 2003;361(9376):1227-1234. 4. Arbyn M, Ronco G, Anttila A, et al. Evidence regarding HPV testing in primary cervical cancer screening. Lancet Oncol. 2012;13(11):104-116. 5. Solomon D, Davey D, Kurman R, et al. The 2001 Bethesda System for Reporting Cervical Cytology. JAMA. 2002;287(16):2114-2120. 6. Denny L, Quinn M, Sankaranarayanan R. Screening for cervical cancer in developing countries. Vaccine. 2006;24 Suppl 3:S3/71-77. 7. Ronco G, Dillner J, Elfström KM, et al. Efficacy of HPV-based screening vs cytology-based screening: long-term follow-up of European trials. Lancet. 2014;383(9916):524-532. 8. Cuzick J, Clavel C, Petry KU, et al. Overview of HPV DNA testing in primary screening. Int J Cancer. 2006;119(5):1095-1101. 9. Nanda K, McCrory DC, Myers ER, et al. Accuracy of the Papanicolaou test in screening for cervical cancer: a systematic review. Obstet Gynecol. 2000;96(4):653-659. 10. Bhatla N, Aoki D, Sharma DN, et al. Cancer of the cervix uteri: epidemiology, prevention, and screening in India. Int J Gynecol Obstet. 2018;143(S2):13-22. 11. Sankaranarayanan R, Wesley RS. A practical manual on visual screening for cervical neoplasia. IARC Press; 2003. 12. Walboomers JMM, Jacobs MV, Manos MM, et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol. 1999;189(1):12-19. 13. Perkins RB, Guido RS, Castle PE, et al. 2019 ASCCP Risk-Based Management Consensus Guidelines. J Low Genit Tract Dis. 2020;24(2):102-131. 14. Koliopoulos G, Nyaga VN, Santesso N, et al. Cytology versus HPV testing for cervical cancer screening: systematic review & meta-analysis. BMJ. 2017;359:j3883. 15. Kitchener HC, Canfell K, Gilham C, et al. The long-term protective effect of HPV primary screening: ARTISTIC trial. Health Technol Assess. 2014;18(23):1-196. 16. Fahey MT, Irwig L, Macaskill P. Meta-analysis of Pap test accuracy. Lancet. 1995;345(8961):153-157. 17. Sahasrabuddhe VV, Parham GP, Mwanahamuntu MH, et al. VIA screening in resource-limited settings: outcomes from community programs. Cancer Detect Prev. 2006;30(3):199-204. 18. Blumenthal PD, Gaffikin L, Deganus S, et al. Cervical cancer prevention with VIA and cryotherapy: feasibility studies. J Womens Health. 2007;16(9):1341-1349.