A Human Papilloma Virus Testing Algorithm Comprising a Combination of the L1 Broad-Spectrum SPF ₁₀ PCR Assay and a Novel E6 High-Risk Multiplex Type-Specific Genotyping PCR Assay

Abstract

Human papillomavirus (HPV) epidemiological and vaccine studies require highly sensitive HPV detection and genotyping systems. To improve HPV detection by PCR, the broad-spectrum L1-based SPF10 PCR DNA enzyme immunoassay (DEIA) LiPA system and a novel E6-based multiplex type-specific system (MPTS123) that uses Luminex xMAP technology were combined into a new testing algorithm. To evaluate this algorithm, cervical swabs (n = 860) and cervical biopsy specimens (n = 355) were tested, with a focus on HPV types detected by the MPTS123 assay (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 6, and 11). Among the HPV-positive samples, identifications of individual HPV genotypes were compared. When all MPTS123 targeted genotypes were considered together, good overall agreement was found (κ = 0.801, 95% confidence interval [CI], 0.784 to 0.818) with identification by SPF10 LiPA, but significantly more genotypes (P < 0.0001) were identified by the MPTS123 PCR Luminex assay, especially for HPV types 16, 35, 39, 45, 58, and 59. An alternative type-specific assay was evaluated that is based on detection of a limited number of HPV genotypes by type-specific PCR and a reverse hybridization assay (MPTS12 RHA). This assay showed results similar to those of the expanded MPTS123 Luminex assay. These results confirm the fact that broad-spectrum PCRs are hampered by type competition when multiple HPV genotypes are present in the same sample. Therefore, a testing algorithm combining the broad-spectrum PCR and a range of type-specific PCRs can offer a highly accurate method for the analysis of HPV infections and diminish the rate of false-negative results and may be particularly useful for epidemiological and vaccine studies.

Clinical Validation of the BD Onclarity? HPV Assay Using a Non-Inferiority Test

“…While L1-based amplification assays have been shown to have clinical performance comparable to clinically validated assays such as HC2 [9,10], there are certain technical limitations to the use of a consensus primer approach, namely, inconsistency in detecting low copy number infections within mixed infections [11][12][13], and the potential risk of missing late stage cancers due to deletion of the L1 target region [14,15]. As an alternative to HPV assays that involve L1 consensus primers, amplification assays that incorporate type specific PCR designs may improve the specificity of detection by returning only positive results where the presence of HPV is above the established, validated cut-off [12,13]. By choosing non L1 regions, any risk of not detecting rare L1 deleted events can be addressed.…”

Current Problems and Recent Advances in the Molecular Diagnosis of Genital Human Papillomavirus Infections

“…Bu nedenle konsensus PCR ile pozitif olarak değerlendirilen örneklerdeki HPV tiplerini tanımlayabilmek için blotlama, ters hibridizasyon, mikrodizi (microarray) hibridizasyon, fl oresan boyalı kapiller elektroforez, lumineks temelli testler ve dizi analizi gibi ek tiplendirme yöntemlerine gereksinim duyulur 7,[10][11][12]17,26 . Ayrıca, konsensus PCR testlerinde çoklu tip içeren örneklerde reaksiyonun bazı tipler üzerinden baskın olarak devam etmesi (tipler arası yarışma), mevcut diğer tip/tiplerin kaçırılmasına yol açabilmektedir 7,36 . Bu nedenle, konsensus PCR testleri ile çoğaltılan amplikonlar üzerinden tiplendirme yapan tanı yöntemlerinin bazı tipleri kaçırabileceği dikkate alınmalıdır 26 .…”

“…Örneğin, MY09/11 ve PGMY09/11 konsensus PCR'nin karşılaştırıldığı bir çalışmada, MY09/11 primerleri test edilen örnekte bulunan bazı tipleri amplifi ye etmede yetersiz kaldığı için, MY09/11 PCR ile saptanan çoklu enfeksiyon oranı (%33.8), PGMY09/11 PCR'ye göre (%40) daha düşük bulunmuştur 7 . Benzer şekilde, E6-bazlı multipleks tipe özgül lumineks temelli bir yöntem ile SPF 10 konsensus PCR (SPF10 LiPA) etkinliğinin karşılaştırıldığı diğer bir çalışmada, çoklu tip varlığında tipler arasında yarışmalı inhibisyon oluştuğu ve bunun sonucunda konsensus PCR etkinliğinin azaldığı gösterilmiştir 36 .…”

Technical Issues Associated With Estimating the Prevalence of HPV-52 in Cervical Intraepithelial Neoplasia

“…51 In contrast to consensus primer pairs or sets, assays based on multiplexed genotypespecific primer pairs are designed to reduce genotype bias by eliminating competition for primer binding. 21,60 All capture and detection probes for genotype identification, regardless of assay type, are designed to be as genotype specific as possible. Some of the earliest and most widely used commercial assays, reverse line probe assays that target part of the L1 gene sequence, experience cross-reactivity in the probe target region of HPV-52.…”