Improved cervical screening using HPV type restriction and cycle threshold limit setting with the AmpFire assay: A prospective screening cohort of women with and without HIV in Botswana.

Objective: The aim of this study was to evaluate the performance of HPV type restriction and cycle threshold (Ct)-limit setting to optimize detection of cervical intraepithelial neoplasia (CIN) with primary HPV testing.

Methods: Baseline cervical screening at time of entry into a prospective longitudinal cohort of women with and without HIV was conducted from February 2021 to July 2022 in Botswana. All women underwent HPV testing of 15 individual types using the AmpFire assay; all HPV-positive and a random subset of HPV negative had histopathology collected. Performance parameters of HPV type restriction groupings were calculated, and sensitivity by individual HPV type Ct-value limits were plotted.

Results: Among 2964 women who underwent primary HPV screening, 1293 (43.6%) tested HPV-positive. Among women with HIV (WWH), HPV types 16/18/33 were associated with the greatest burden of CIN2+/CIN3+ (53%/56%). In WWH, grouping by HPV types separately reported in commercial assays (16/18/45) had low sensitivity (44% [CI: 36%-52%]) but high specificity (86% [CI: 84%-88%]) for CIN2+; 8-type HPV restriction (16/18/31/33/35/45/52/58) improved sensitivity (79% [CI: 72%-86%]) and maintained reasonable specificity (67% [CI: 65%-70%]) for CIN2+. Similar results were seen in women without HIV. Ct-limit setting for medium oncogenic HPV types (31,33,35,52,58) maintained a sensitivity of 72% in WWH while reducing over-detection of non-pathogenic HPV.

Conclusion: Eight-type HPV restriction and Ct-limit setting are promising strategies for improving the performance of primary HPV screening. A potential strategy to improve 8-type HPV restriction would be to treat all with HPV 16/18/45; treat HPV 31/33/35/52/58 if below the type-specific Ct limit and repeat HPV testing in 1-year for other positive HPV results.