Abundance of Multiple High-Risk Human Papillomavirus (HPV) Infections Found in Cervical Cells Analyzed by Use of an Ultrasensitive HPV Genotyping Assay

Abstract

PCR methods enable the detection of a large variety of human papillomavirus (HPV) genotypes that infect the anogenital tract. However, PCR with consensus primers, general primers, and, to a lesser extent, broadspectrum primers may underrepresent the true prevalence of HPV, especially the true prevalence of multiple infections. We compared the rate of HPV positivity determined by a broad-spectrum PCR with primers BSGP5؉ and BSGP6؉ (BS-PCR) coupled to an established bead-based multiplex HPV genotyping (MPG) assay with the rate of HPV positivity determined by a multiplex PCR with type-specific primers (TS-PCR) coupled to a newly developed MPG assay for 735 selected cervical scraping samples. While the primers used for the BS-PCR are located within the L1 region of the HPV genome, the primers used for the TS-PCR target the E7 gene. The overall rates of positivity for the 19 HPV types included in both assays were 60.9% and 72.2% by the BS-PCR and the TS-PCR, respectively, and the two assays found multiple infections in 34.8% and 58.0% of the specimens, respectively. Both HPV detection assays allowed the semiquantitative detection of HPV types and identified the same dominant HPV type in 66.6% of the multiple infections. In conclusion, the TS-PCR-MPG assay significantly increased the rate of detection of HPV DNA and the number of infections with multiple HPV types detected and demonstrated that the prevalence of low-copy-number HPV infections in the anogenital tract may be strongly underestimated by conventional HPV amplification methods, especially in cases of multiple infections. As a consequence, PCR-TS-MPG appears to be highly suited for analysis of the significance of multiple infections in the development of cervical cancer and for the study the natural history and the latency of HPV.Human papillomaviruses (HPV) are DNA viruses that infect cutaneous and mucosal epithelia. Until now, approximately 100 HPV genotypes have been fully characterized on the basis of the isolation of complete genomes (7), and there is evidence that a larger number exist (1). There are approximately 45 known mucosal HPV types; and these are further divided into three groups on the basis of their epidemiological association with cervical cancer: high-risk HPV (Hr-HPV) types (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82), putative high-risk HPV (pHr-HPV) types (types 26, 53, and 66), and low-risk HPV (Lr-HPV) types (e.g., types 6, 11, 40, 42, 43, 44, and 70) (18). Hr-HPV types are causally associated with several malignant diseases, of which cervical cancer has particular significance, being the second most common cancer in women worldwide and the main cancer of women in most developing countries (18). Hr-HPV type DNA has been detected in 99.7% of cervical cancer tissue specimens (26), and persistent infection with an oncogenic HPV type, particularly HPV type 16 (HPV-16) or HPV-18, is recognized as a necessary cause of cervical cancer. HPV genotyping is of importance for the study of the natural history of infectio...

Biological activity of probable/possible high‐risk human papillomavirus types in cervical cancer

“…The increased sensitivity of the TS-E7-MPG over BS-L1-MPG can be explained by the use of HPV type-specific primers targeting E7 region for reduced number of types (21 over 51 in BS-L1-MPG). 34 However, the majorities (82%) of the infections detected by TS-E7-MPG alone were transcriptionally inactive and thus probably did not contribute to maintenance of the transformed phenotype. The genotyping data of this study provide the first report of the full spectrum of HPV types in Mongolian CxCa tissues.…”

“…In our study, two highly sensitive PCR methods targeting two different regions in the viral genomes (L1 and E7) of all HR and seven pHR types, were used to identify HPV types; [32][33][34] to our knowledge, the most meticulous analysis published so far. Both BS-L1-MPG and TS-E7-MPG showed to be highly suitable and sensitive assays applicable to FFPE materials in our study.…”

“…[32][33][34] Analytic sensitivity of BS-L1-MPG reached 10-1,000 HPV copies per PCR for each HPV type. BS-L1-MPG was performed as described with some modifications.…”

“…34,35 The multiplex type-specific E7 PCR utilizes HPV type-specific primers targeting the E7 region for the detection of 19 HR-/pHR (HPV16, 18,26,31,33,35,39,45, 51, 52, 53, 56, 58, 59, 66, 68a, 68b, 70, 73, 82) and 2 LRtypes (HPV6, 11), with detection limits ranging from 10 to 1,000 copies of the viral genome. 34 Two primers for the amplification of b-globin were also included to provide a positive control for the quality of the template DNA. 35 Additionally, a modified TS-E7-MPG with higher analytical sensitivity was performed with amplicon size of only 90 bp for HPV types 6, 11, 16, 18, 31, 33 and 35 (Gheit et al, unpublished data).…”

Natural variants in the major neutralizing epitope of human papillomavirus minor capsid protein L2

“…These samples were mostly from women with high-grade squamous intraepithelial lesions. HPV genotyping data were generated using the BSGP5þ/6þ-PCR/Multiplex HPV Genotyping (MPG) assay comprising; first, a BSGP5þ/6þ-PCR, which homogenously amplifies all known genital HPV types by generating biotinylated amplimers of~150 bp from the L1 region 20,21 and, second, an MPG assay with bead-based xMAP Luminex suspension array technology, which simultaneously identifies 27 HPV types and the beta-globin gene. 20,22 PCR and L2 epitope sequence analysis Purified DNA isolated from patient samples was amplified using type-specific L2 primers.…”