Comparison of Two Widely Used Human Papillomavirus Detection and Genotyping Methods, GP5+/6+-Based PCR Followed by Reverse Line Blot Hybridization and Multiplex Type-Specific E7-Based PCR

Abstract

f GP5؉/6؉-based PCR followed by reverse line blot hybridization (GP5؉/6؉RLB) and multiplex type-specific PCR (E7-MPG) are two human papillomavirus (HPV) genotyping methodologies widely applied in epidemiological research. We investigated their relative analytical performance in 4,662 samples derived from five studies in Bhutan, Rwanda, and Mongolia coordinated by the International Agency for Research on Cancer (IARC). A total of 630 samples were positive by E7-MPG only (13.5%), 24 were positive by GP5؉/6؉RLB only (0.5%), and 1,014 were positive (21.8%) by both methods. Ratios of HPV type-specific positivity of the two tests (E7-MPG:GP5؉/6؉RLB ratio) were calculated among 1,668 samples that were HPV positive by one or both tests. E7-MPG:GP5؉/6؉RLB ratios were >1 for all types and highly reproducible across populations and sample types. E7-MPG:GP5؉/6؉RLB ratios were highest for HPV53 (7.5) and HPV68 (7.1). HPV16 (1.6) and HPV18 (1.7) had lower than average E7-MPG:GP5؉/6؉RLB ratios. Among E7-MPG positive infections, median mean fluorescence intensity (MFI; a semiquantitative measure of viral load) tended to be higher among samples positive for the same virus type by GP5؉/6؉RLB than for those negative for the same type by GP5؉/6؉RLB. Exceptions, however, included HPV53, -59, and -82, for which the chances of being undetected by GP5؉/6؉RLB appeared to be MFI independent. Furthermore, the probability of detecting an additional type by E7-MPG was higher when another type was already detected by GP5؉/6؉RLB, suggesting the existence of masking effects due to competition for GP5؉/6؉ PCR primers. In conclusion, this analysis is not an evaluation of clinical performance but may inform choices for HPV genotyping methods in epidemiological studies, when the relative merits and dangers of sensitivity versus specificity for individual types should be considered, as well as the potential to unmask nonvaccine types following HPV vaccination. Human papillomavirus (HPV) DNA detection and genotyping are central to molecular epidemiological studies of HPV natural history and carcinogenicity, as well as to evaluations of HPV vaccine efficacy and effectiveness. There are various PCR-based methods for HPV DNA detection and genotyping, of which one of the most commonly used in epidemiological studies is the GP5ϩ/6ϩ primer set targeting conserved sequences within the L1 gene, thus allowing detection of a broad range of mucosal HPV types in a single reaction. The HPV genotype can subsequently be identified by various methods, of which the most commonly used is hybridization with type-specific probes using a reverse line blot hybridization assay (henceforth referred to as GP5ϩ/6ϩRLB) (1). The GP5/6 primers were initially developed to maximize detection of HPV6, and were subsequently elongated at their 3= ends to generate the primers GP5ϩ and GP6ϩ to improve the detection of a broader range of HPV types (3). This methodology has been widely validated against clinical outcomes in large population-based screening programs and shows favorable c...

Evaluation of the performance of Human Papillomavirus testing in paired urine and clinician-collected cervical samples among women aged over 30 years in Bhutan

“…A type specific E7 PCR bead-based multiplex genotyping assay (E7-MPG) was performed at IARC, Lyon using a Luminex bead-based platform [14]. The assay detects DNA from 21 HPV types (6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73 and 82).…”

“…Two β-globin primers are included to control DNA quality. This assay is known to be more sensitive than GP5+/6+ in detecting low viral copy numbers and, in particular, in detecting individual HPV types in multiple-type infections [14]. …”

“…We aimed to estimate the performance of the above mentioned urine sampling protocol directly against paired cervical cytological samples, and using two HPV assays of differing sensitivity, namely GP5+/6+RLB, an assay developed for clinical specificity in HPV-based cervical screening and E7-MPG, which has greater analytical sensitivity [14]. This was done in women undergoing colposcopy, with histological confirmation of lesions, in a cohort of well-characterised women in Bhutan.…”

Oral human papillomavirus infection incidence and clearance: a systematic review of the literature

“…Both methods detected 22 % HPV-positive samples, and 14 % were positive with the E7-MPG method only and 0.5 % positive with the GP5+/6+ RLB method only. Nested and GP5+/6+ PCR was used by all studies included in our analysis, but this method of HPV detection was not evaluated [29]. The nested MY09/11 and GP5+/6+ PCR has been found to be more type-sensitive, detecting a wider range of HPV types, low-copy-number HPV infections and more infections with multiple HPV types than MY09/11 PCR or GP5+/6+ PCR only [30].…”

“…A recent publication [29] compared the performance of the commonly used HPV detection and typing methods GP5+/6+ PCR followed by reverse dot blot hybridization (GP5+/6+ RLB) and multiplex type-specific E7-based bead-based multiplex HPV genotyping (MPG) assay (E7-MPG) on almost 5000 cervical cell and urine samples. Both methods detected 22 % HPV-positive samples, and 14 % were positive with the E7-MPG method only and 0.5 % positive with the GP5+/6+ RLB method only.…”