Cobas 4800 platform

VICONEL is a prospective open cohort of people with HIV established in Lesotho, southern Africa, in December 2015. VICONEL includes VL test results from 3 hospitals, 18 health centers, and 2 private clinics in rural districts of Butha-Buthe and Mokhotlong. As part of routine care, blood samples for VL measurement are taken at the participating healthcare facilities per the schedule of the Lesotho National Guidelines. Samples are processed at Butha-Buthe Government Hospital on the Cobas 4800 platform (F. Hoffman-La Roche AG, Basel, Switzerland) with a lower detection limit of 20 copies/mL or, for a small subset, with point-of-care instruments (GeneXpert, Cepheid, Sunnyvale, CA, USA) with a lower detection limit of 40 copies/mL at 3 other participating healthcare facilities. VL results and associated demographic and treatment metadata are uploaded to the secured VICONEL database. Further details on the setup and design of VICONEL have been reported previously [19 (link)]. The Lesotho national rollout of dolutegravir replacing NNRTI as the ART core agent for first-line regimens started in 2019, with most people with HIV in Lesotho transitioned in 2020.

HPV analysis was performed at Karolinska University Hospital (the laboratory of the organized screening program), using the same laboratory and HPV screening platform as used by the organized screening program. This was initially the Cobas 4800 platform (Roche Diagnostics) but was in 2022, following a new tender for the screening program, switched to the BD Onclarity™ HPV Assay that provides an extended genotyping. Samples that had tested positive for “other HPV” on the Cobas platform were re-analyzed using the BD Onclarity™ HPV Assay to ensure the same level of genotyping detail in the entire database. Both assays were fully proficient in the HPVLabNet proficiency testing (limit of detection at least 10 IU/µl for HPV16/18 and 100 IU/µl for the other HPV types)^{15 (link)}.
Results were both available to the women (who could log in to the database online), the screening program and associated health care, and exported to the Swedish National Cervical Screening Registry (http://nkcx.se). HPV-positive women were followed up according to the established national program of care for cervical cancer prevention (available online at https://kunskapsbanken.cancercentrum.se/globalassets/vara-uppdrag/prevention-tidig-upptackt/gynekologisk-cellprovskontroll/vardprogram/nationellt-vardprogram-cervixcancerprevention.pdf, accessed on August 25th, 2023).

This study was conducted at the Department of Pathology, Randers Regional Hospital, Denmark, which is responsible for processing all cervical cytology samples obtained in Central Denmark Region (ie 85, 000‐100, 000 samples annually). In Denmark, cervical cancer screening is organized and free of charge. At present, women aged 23‐59 years are screened with cytology, whereas women aged 60‐64 years undergo HPV‐based screening. At the department, cytology slides are interpreted by cytotechnicians using computer‐assisted microscopy (FocalPoint™ GS Imaging System; BD Diagnostics) and categorized according to the Bethesda 2014 grading system.¹⁸ Human papillomavirus DNA testing is performed using the Cobas 4800 platform (Roche Diagnostics). To ensure high quality of cytology screening, all cytotechnicians in Denmark are recommended, but not required, to pass the Quality Assurance, Training, and Examinations Committee (QUATE) exam provided by European Federation of Cytology Societies (EFCS).¹⁹, ²⁰ Currently, dual staining is not used routinely within the Danish cervical cancer screening program.

HPV DNA testing was performed using the clinically validated cobas^@4800 platform (Roche Diagnostic, USA) [19 (link)] which detects HPV16, HPV18 and a pool of 12 other high-risk HPV genotypes (HPV31/33/35/39/45/51/52/56/58/59/66/68). Liquid-based cytology was performed by introducing a cervical plastic brush into the external cavity and scraping cells from the exocervix and endocervix, and then placed on a smear slide and fixed. Cytology slide results were classified according to the Bethesda grading system (2014) [20 (link)], including no intraepithelial lesions or malignancy (NILM), atypical squamous cells of undetermined significance (ASC-US), low-grade squamous intraepithelial lesion (LSIL), atypical glandular cells of undetermined significance (AGUS), atypical squamous cells cannot exclude high-grade squamous intraepithelial lesion (ASC-H), high-grade squamous intraepithelial lesion (HSIL) or carcinoma.

Vaginal samples were tested on the Cobas 4800 platform (Roche, Pleasanton, California) a DNA-based, FDA-approved assay that detects viral DNA of 12 high risk genotypes with concurrent genotyping of HPV-16 and HPV-18. All samples were tested in a UKAS accredited laboratory (Department of Microbiology, Oxford University Hospitals NHS Foundation Trust, Oxford)

Cervical cell specimens were collected with cervical plastic brush and put into preservation solution ((Tellgen Life Science, Shanghai, China) for both liquid-based cytology (LCT) and HPV DNA tests. Cytology slides were interpreted by experienced cyto-technicians. Results were classified according to the Bethesda grading system (2014) [18 (link)], including no intraepithelial lesions or malignancy (NILM), atypical squamous cells of undetermined significance (ASC-US), low-grade squamous intraepithelial lesion (LSIL), atypical glandular cells of undetermined significance (AG-US), atypical squamous cells cannot exclude high-grade squamous intraepithelial lesion (ASC-H), high-grade squamous intraepithelial lesion (HSIL) or carcinoma. HPV DNA testing was performed on cobas^@4800 platform (Roche Diagnostic, USA) [19 (link)] which detects HPV16, HPV18 and a pool of 12 other high-risk HPV genotypes (HPV31/33/35/39/45/51/52/56/58/59/66/68).