Development and clinical evaluation of a highly sensitive PCR-reverse hybridization line probe assay for detection and identification of anogenital human papillomavirus.

Human papillomavirus (HPV) can be detected by amplification of viral DNA. A novel PCR primer set generating a short PCR fragment (SPF PCR) was used for amplification of a fragment of only 65 bp from the L1 region and permitted ultrasensitive detection of a broad spectrum of HPV genotypes. The intra- and intertypic sequence variations of the 22-bp interprimer region of this amplimer were studied. Among 238 HPV sequences from GenBank and clinical specimens, HPV genotypes were correctly identified based on the 22-bp sequence in 232 cases (97.2%). Genotype-specific probes for HPV genotypes 6, 11, 16, 18, 31, 33 to 35, 39, 40, 42 to 45, 51 to 54, 56, 58, 59, 66, 68, 70, and 74 were selected, and a reverse hybridization line probe assay (LiPA) (the INNO-LiPA HPV prototype research assay) was developed. This LiPA permits the use of amplimers generated by the SPF as well as the MY 09/11 primers. The assay was evaluated with a total of 1, 354 clinical specimens, comprising cervical scrapes (classifications ranging from normal cytology to severe dyskaryosis) and formalin-fixed, paraffin-embedded cervical carcinoma samples. LiPA results were highly concordant with sequence analysis of the SPF amplimer, genotype-specific PCR, and sequence analysis of amplimers generated by MY 09/11 primers. The sensitivity of the SPF primers was higher than that of the GP5(+)/6(+) primers over a broad range of HPV types, especially when multiple HPV genotypes were present. In conclusion, the SPF LiPA method allows extremely sensitive detection of HPV DNA as well as reliable identification of HPV genotypes in both cervical smears and paraffin-embedded materials.

Novel short-fragment PCR assay for highly sensitive broad-spectrum detection of anogenital human papillomaviruses.

A novel set of polymerase chain reaction (PCR) primers, designated SPF1 and SPF2 and located in the L1 region, was developed for universal detection of human papillomavirus (HPV). A short PCR fragment (SPF) of only 65 pb was synthesized. SPF amplimers were detected in a microtiter-based hybridization system, using a mixture of oligonucleotide probes. The SPF system allowed detection of at least 43 different HPV genotypes. The clinical performance of the novel SPF system was assessed in three different patient groups. 1) Analysis of 534 cervical scrapes, obtained from treated patients, showed that the detection rate in 447 (83.7%) scrapes with normal cytology was significantly higher using the SPF system as compared with the universal primer set GP5+/6+ (P < 0.001). 2) The SPF assay detected HPV DNA in 299 (98.4%) of 304 scrapes with cytological dyskaryosis. 3) The SPF system detected HPV DNA in 100% of 184 formalin-fixed, paraffin-embedded cervical carcinoma specimens. In conclusion, the novel SPF system permitted universal and highly sensitive detection of HPV DNA in diverse clinical materials and may improve the molecular diagnosis and epidemiology of this important virus.

Genotyping human papillomavirus type 16 isolates from persistently infected promiscuous individuals and cervical neoplasia patients.

Nucleotide sequence variation in the noncoding region of the genome of human papillomavirus type 16 (HPV16) was determined by direct sequencing and single-strand conformation polymorphism analysis of DNA fragments amplified by PCR. Individuals of diverse sexual promiscuity and/or cervicopathology were studied. In a group of 14 healthy, monogamous HPV16-positive females, only two HPV16 sequence variants could be documented. Among 17 females and 3 males with multiple sex partners and living in the same geographical region, nine sequence variants were found, whereas among 7 patients with cervical neoplasia from another region, five variants were detected. Although numbers are limited, in the group of individuals at high risk of acquiring a sexually transmitted disease or with cervical neoplasia, a larger number of HPV16 sequence variants was encountered (two types among 14 individuals versus nine types among 20; Fisher's exact test, P = 0.07). Seven of the individuals were sampled repeatedly over time. For these persistently infected women, no differences in HPV16 sequences were detected, irrespective of promiscuity, and persistence of a single viral variant, spread over multiple anatomic sites, for more than 2 years could be demonstrated. This indicates that viral persistence may be a common feature and that successful superinfection with a new variant may be rare, despite a potentially high frequency of viral reinoculation.

Absence of human papillomavirus type 16 E6 transcripts in HPV 16-infected, cytologically normal cervical scrapings.

By a combination of reversed transcription and subsequent polymerase chain reaction (RNA-PCR), 23 cytologically normal cervical scrapings, positive for the presence of human papillomavirus type 16 (HPV 16) DNA, were analyzed for the presence of transcripts originating from the E6 region of the viral genome. This region is thought to be involved in transformational, tumorigenic events. No mRNAs of the E6 region were detectable using the most sensitive PCR-mediated procedure currently available. Since it was previously shown that in cytological abnormal cervical scrapings about one-half of the samples positive for HPV 16 DNA express mRNAs of the E6 region, a difference between normal and abnormal cervical scrapings, when the HPV 16 is present, exists. The observed difference between cytologically normal and abnormal, HPV-DNA-positive cervical scrapes may eventually be used as a prognostic marker for screening of women at risk for the development of cervical carcinoma. However, firm establishment of the putative correlation between tumor progression and the presence of E6 transcripts requires extensive follow-up analysis of HPV-positive patients.