Performance of Commercial Reverse Line Blot Assays for Human Papillomavirus Genotyping

Abstract

The performance of three line blot assays (LBAs), the Linear Array HPV genotyping assay (LA) (Roche Diagnostics), INNO-LiPA HPV Genotyping Extra (LiPA) (Innogenetics), and the reverse hybridization assay (RH) (Qiagen), was evaluated using quantitated whole genomic human papillomavirus (HPV) plasmids ( T he introduction of human papillomavirus (HPV) vaccines has highlighted the need for standardized accurate HPV genotyping assays to ensure comparability of results in laboratories worldwide, both before and after vaccine introduction. A large variety of assays, using both in-house methods and commercial kits, are available for HPV detection and typing (6), and more can be anticipated. International proficiency testing organized by the WHO HPV LabNet has documented significant differences in sensitivity, specificity, and reproducibility between different laboratories using a variety of testing platforms (2). Variation in performance for laboratories using the same assay implicates laboratory practice; however, the results also indicated differences between assays. The WHO HPV LabNet has further recognized the need for HPV typing assays that can easily be standardized, require minimal equipment for assay performance and interpretation, and can be used in a variety of laboratory settings. Following its meeting on the standardization of HPV assays and the role of the WHO HPV LabNet in supporting vaccine introduction (WHO, Geneva, Switzerland, 23 to 25 January 2008 [http://www.who.int/biologicals /publications/meetings/areas/vaccines/human_papillomavirus /HPV%20Jan%20meeting%20report_20080909%20_Clean _.pdf]), it was agreed that commercial assay kits should be evaluated in collaborative studies for proficiency.Reverse line blot assays (LBAs), based on consensus amplification of conserved regions of HPV followed by hybridization to type-specific probes on line blot strips, have been widely used. Available LBAs differ in the primer set (1, 3, 4) used in the amplification phase and in the number and sequences of the detection probes. Beyond thermocyclers for target amplifications, only temperature control water baths and visual inspection are required to carry out these assays. Therefore, the LBA platforms have the potential to meet the needs of the WHO for high-performance genotyping that does not involve expensive equipment.Studies have been conducted previously to compare and evaluate the performance of these HPV typing assays. Typically, these were restricted to DNA extracts from patient-collected anogenital specimens, which cannot be validated independently and limit the analysis to relative comparisons and assessments of type prevalence found by the individual assays (5,7,10). The use of plasmid standards offers clear advantages for more objective evaluations beyond that level. Cloned, full-length HPV genomic DNA (gDNA) provides complete control over genotype identity as well as copy number input and eliminates the need for a gold standard test.We selected three commercial LBAs using different primer sets and subjec...

Global Improvement in Genotyping of Human Papillomavirus DNA: the 2011 HPV LabNet International Proficiency Study

“…The genotyping assays used today differ in their analytical performances with regard to type-specific sensitivities and specificities. Several studies have compared different HPV typing assays to assess their performances in screening and epidemiology using clinical samples (3)(4)(5). However, evaluations of assay performance in different laboratories need to be performed in a standardized manner, such that different assay performance measures can be evaluated and the results can be compared over time against a known and accepted standard (4).…”

Molecular Triage of Premalignant Lesions in Liquid-Based Cervical Cytology and Circulating Cell-Free DNA from Urine, Using a Panel of Methylated Human Papilloma Virus and Host Genes

“…Urine samples tested by the TrDNA HPV capillary electrophoresis test had high concordance with corresponding cervical and urine samples tested by the widely used linear array HPV genotyping test (51). However, the TrDNA capillary electrophoresis HPV test is not quantitative, can only detect the presence or absence of hrHPV and, similar to previously published urinebased HPV tests, has limited specificity.…”

“…To examine the hrHPV genome in urine ccfDNA, we developed custom dualsequence capture baits to enrich samples for hrHPV DNA from 12 high-risk types (HPV16, 18,31,33,35,39,45,51,52,56,58, and 59). We used liquid-based sequence capture baits to measure the hrHPV genome in urine ccfDNA from women with and without cervical premalignant lesions.…”

HPV Genotyping Linear Assay Test Comparison in Cervical Cancer Patients: Implications for HPV Prevalence and Molecular Epidemiology in a Limited-resource Area in Bandung, Indonesia

“…The commercial tests are too expensive though those two kits have plus and minus points. Several studies around the world has showed that linear assay 1 we used has a good performance and is sensitive (Wong et al, 2010;Steinau et al, 2012), hence this test is costly and need a especial PCR machine which is gold plated to get a good reliable result. In low-and medium-resourced countries where the PCR machines are scarce this assay cannot be performed.…”

“…GF and LA showed significant discrepancy in detecting HPVgenotypes 39, 52, 26, 66, and 11. More sensitive detection of HPV52 by GF offers an advantage in regions where HPV52 is more prevalent (Wong et al, 2012), hence, linear assay 1 has an advantage for internationally comparable genotyping studies (Steinau et al, 2012).…”