Primary Pap Smear Cytological Diagnosis of ASC-US: HR-HPV Genotyping and Follow-Up of HPV-Positive Patients

ASC-US refers to cytological abnormalities suggestive of squamous intraepithelial lesions (SILs) that are qualitatively or quantitatively insufficient for a definitive diagnosis. In addition to HPV infection, which is the etiological agent of preneoplastic and neoplastic cervical lesions, several other conditions may induce cytological changes classified as ASC-US, including inflammation, atrophy with degeneration, hormonal effects, and technical artifacts. The aim of this study was to evaluate the incidence of primary Pap tests diagnosed as ASC-US that also tested positive for HR-HPV genotypes. In this retrospective study, 6181 primary Pap tests collected between 21 July 2021 and 9 January 2024 from the Roma 2 Local Health District were reviewed. ThinPrep containers (Hologic) were used for liquid-based cytology, allowing both cytological examination and molecular testing for HR-HPV detection. The Anyplex™ II HR HPV Detection assay (Seegene) was used for the identification, by real-time PCR, of 14 HR-HPV genotypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68). Among the 6181 Pap tests analyzed, 202 cases (3.3%) were diagnosed as ASC-US and subsequently underwent HR-HPV molecular testing. Of these, 107 cases (53.0%) were negative, while 95 (47.0%) were positive for HR-HPV. In HPV-positive samples, single-genotype infections were detected in 65 cases (68.4%), while multiple genotypes were identified in 30 cases (31.6%). A total of 135 HR-HPV genotypes were detected. HPV 16 was identified in nine cases (6.7%), HPV 18 in six (4.4%), HPV 31 in nine (6.7%), HPV 33 in five (3.7%), HPV 35 in two (1.5%), HPV 39 in 10 (7.4%), HPV 45 in four (3.0%), HPV 51 in 20 (14.8%), HPV 52 in seven (5.2%), HPV 56 in 15 (11.1%), HPV 58 in eight (5.9%), HPV 59 in 17 (12.6%), HPV 66 in 12 (8.9%), and HPV 68 in 11 cases (8.1%). Our findings indicate that approximately half of ASC-US diagnoses are associated with HR-HPV infection. Interestingly, the detected viral genotypes mainly belonged to less common HR-HPV strains. HR-HPV testing following an ASC-US cytological diagnosis is essential for colposcopy triage, while extended genotyping enables the monitoring of genotype persistence over time, a key factor in the development of cervical dysplastic and neoplastic lesions.