Linear array hpv genotyping test

Specimen that were positive with the Cobas® HPV test and typed as ‘Other HPV‘but negative with HPVIR were genotyped using the Roche LINEAR ARRAY^® HPV genotyping test (Roche molecular systems, 4300 Hacienda Dr., Pleasanton, CA 94588, USA). To this end, 5 mL ThinPrep LBC cervical cell material were centrifuged at 5000 g for 30 min in 4 °C, and the cell pellet was resuspended in 400 μL phosphate-buffered saline. DNA was extracted from resuspended cells using MagNA Pure Compact (Roche) and the MagNA Pure Compact Nucleic Acid Isolation Kit (Roche). HPV genotyping was then performed using the Roche Linear Array HPV genotyping test which identifies 37 different high- and low-risk types (HPV6, 11, 16, 18, 26, 31, 33,35, 39, 40, 42, 44, 45, 51, 52, 53, 54, 56,58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, 83, 84, 89 and IS39).

All samples were subjected to detect hrHPV (14 genotypes) DNA using the Cobas 4800 HPV test (Roche Molecular Systems, USA), a fully-automated platform based on real-time PCR technique. Samples with hrHPV positivity were subsequently genotyped by using the Linear Array (LA) HPV genotyping test (Roche Molecular Systems, Pleasanton, CA).

HPV testing was performed at the STI Section, Centre for HIV and STIs, National Institute for Communicable Diseases (NICD), in Johannesburg. Three archived specimens for each of the 200 enrolled MSM participants were analysed, which included a first-void urine specimen, an oro-pharyngeal swab and an anal swab, which were all previously stored at −70 °C at the STI Section, Centre for HIV and STIs, NICD.
Genomic DNA was extracted from urine and swab specimens using the MagNAPure Compact Automated DNA Extractor (Roche) and used for HPV genotyping. The Linear Array (LA) HPV Genotyping Test (Roche Molecular Systems, Inc., Branchburg, NJ, USA) was used to determine the HPV genotype distribution in these 600 specimens. The LA test amplified the target HPV DNA for 37 anogenital HPV genotypes, which included 24 low risk HPV types (6, 11, 26, 40, 42, 53, 54, 55, 61, 62, 64, 66, 67, 69, 70, 71, 72, 73, 81, 82, 83, 84, IS39 and CP6108) and 13 high risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68). HPV-52 was only recorded positive in the absence of HPV types 33, 35 and 58 due to the combined probe used for these 4 types in the LA assay. The β-globin gene was amplified as a control for cell adequacy, extraction and amplification. Samples with a negative β-globin result and a positive HPV DNA result were considered valid and adequate for analyses.