PreTect HPV-Proofer: real-time detection and typing of E6/E7 mRNA from carcinogenic human papillomaviruses

Monitoring human papillomavirus (HPV) E6/E7 mRNA expression may provide an accurate and informative diagnostic approach for detection of oncogene activity related to the development of severe dysplasia or cervical carcinoma. A multiplex nucleic acid sequence based amplification (NASBA) assay, utilizing molecular beacon probes for real-time detection was developed for the identification of E6/E7 mRNA from HPV types 16, 18, 31, 33 and 45. The assay is called PreTect HPV-Proofer and this report describes the development and the analytical performance of the assay. The reproducibility of PreTect HPV-Proofer with regard to a positive result was found to be between 96 and 100%, depending on HPV type. The melting temperature for the different molecular beacons was in the range of 48-55 degrees C, indicating conformational stability, i.e. the molecular beacons will not get activated by the 41 degrees C annealing temperature, but will be activated by the annealing to the target itself. The limit of detection for HPV 16 was ten SiHa or CaSki cells and for HPV 18 one HeLa cell. No cross reactivity was observed with E6/E7 mRNA from the other tested HPV types. mRNA from cervical cells was also successfully amplified after more than one year of storage. In conclusion, the PreTect HPV-Proofer assay, individually identifying E6/E7 mRNA expression from five carcinogenic HPV types, is a reproducible assay that may serve as a valuable tool in monitoring HPV infections producing proteins with a transforming potential.     

MassARRAY spectrometry is more sensitive than PreTect HPV-Proofer and consensus PCR for type-specific detection of high-risk oncogenic human papillomavirus genotypes in cervical cancer

Type-specific detection of human papillomavirus (HPV) is indicated for better risk stratification and clinical management of women testing positive for HPV and for epidemiologic surveillance. MassARRAY spectrometry (MassARRAY; Sequenom) is a novel method for type-specific detection of 15 high-risk oncogenic HPV types: HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, -68, and -73. PreTect HPV-Proofer (Proofer; Norchip) is a type-specific assay that detects E6/E7 mRNA from five high-risk oncogenic HPV types: HPV-16, -18, -31, -33, and -45. The performance of these tests for type-specific identification of HPV was assessed with cervical specimens from 192 cases of cervical cancer in comparison with consensus MY09/MY11 PCR followed by nucleotide sequencing (consensus PCR). The overall HPV detection rates were 94.8% (95% confidence interval [CI], 91.7, 97.9), 83.3% (95% CI, 78.1, 88.5), and 86.5% (95% CI, 81.7, 91.3) for MassARRAY, Proofer, and consensus PCR, respectively. All tests were negative in six (3.1%) of the 192 cases. Considering only the specimens that contained at least one of the five types targeted by Proofer, the detection rates were 96.6%, 91.4%, and 86.9% for MassARRAY, Proofer, and consensus PCR, respectively. MassARRAY detected multiple infections in 14.1%, Proofer detected multiple infections in 3.6%, and consensus PCR failed to detect any multiple infections. The agreement was highest at 86.0% (kappa = 0.76) between MassARRAY and Proofer and lowest at 81.8% (kappa = 0.69) between Proofer and consensus PCR. In conclusion, MassARRAY is a highly sensitive and accurate method for type-specific detection of oncogenic HPV in cervical cancer, with Proofer showing impressive performance.     

Isolation of RNA from residual BD SurePath liquid-based cytology specimens and detection of HPV E6/E7 mRNA using the PreTectt HPV-Proofer assay

Infection with high-risk human papillomavirus (HPV) is known to be associated directly with the development of cervical cancer. Recent data suggests that the detection of E6/E7 mRNA from high-risk HPV types may serve as a better diagnostic method for detecting the presence of cervical pre-cancer than HPV DNA testing. This report details a commercially available nucleic acid isolation protocol which can be used to isolate reproducibly RNA from residual BD SurePath liquid-based cytology specimens stored for up to 28 days, and have demonstrated the quality and quantity of mRNA is sufficient for detection with the NorChip PreTect HPV-Proofer assay. Of the 242 specimens tested in this study, 236 (97.5%) tested positive for U1A internal control gene expression. HPV type 16, 18, 31, 33 or 45 mRNA was detected in 16/20 (80%) of the analyzed high-grade squamous intraepithelial lesion (HSIL) specimens, with a low frequency of HPV mRNA detected in the normal lesions (3%). The presence of HPV E6 expression in a subset of HPV positive specimens was also detected by real-time RT-PCR. These findings confirm that RNA of sufficient quality can be isolated from residual BD SurePath cervical cytology specimens for use in downstream NASBA and RT-PCR-based assays.     

Comparison of HPV detection technologies: Hybrid capture 2, PreTect HPV-Proofer and analysis of HPV DNA viral load in HPV16, HPV18 and HPV33 E6/E7 mRNA positive specimens

Human papillomavirus (HPV) testing using molecular methods in liquid based cytology (LBC) specimens may be useful as an adjunct to cervical screening by cytology. We compared the positivity rate of the commercially available HPV DNA method hybrid capture 2 (hc2) and the commercially available E6/E7 mRNA method PreTect HPV-Proofer in cytological specimens (n=299). LBC specimens collected (n=299) represented the following cervical cytological disease categories: Normal (n=60), borderline nuclear abnormalities (BNA) (n=34), CIN1 (n=121), CIN2 (n=60), CIN3 (n=24). Overall, 69% (205/299) of the cases were positive by hc2 and 38% (112/299) of the cases were positive by PreTect HPV-Proofer. Concordance rates between the two tests were highest in the high-grade cytology cases (CIN2: 67% and CIN3: 83%) and the normal cytology cases (88%) and lowest in the BNA and CIN1 categories (56% and 52%). HPV DNA viral load analyses were carried out on HPV16 (n=55), HPV18 (n=9) and HPV33 (n=13) samples that were positive by PreTect HPV-Proofer. The sensitivity and specificity of PreTect HPV-Proofer and the hc2 DNA test for the detection of high-grade cytology (i.e. CIN2+) were 71.4% and 75.8% vs 100% and 43.7%, respectively. The relatively low detection rate observed by PreTect HPV-Proofer in the whole range of cytological positive cases, combined with a relatively higher specificity and PPV, suggests that PreTect HPV-Proofer may be more useful than hc2 for triage and in predicting high-grade disease.     

NucliSENS® EasyQ® HPV v1 test – Testing for oncogenic activity of human papillomaviruses

BackgroundAnalytical sensitivity of DNA-based assays to detect infection with human papillomaviruses is very high, but clinical specificity for cervical cancer strongly depends on the age of the patient and case classification. To solve the dilemma between sensitivity and specificity, a new generation of assays focuses on the pathogenic factors that underlie the development of HPV-associated tumors: the expression of the viral oncogenes E6 and E7. Demonstration of persistent expression of these mRNAs or expression in the context of relevant clinical symptoms has a strong positive predictive value for the development of HPV-associated carcinomas and strongly warrants further diagnostic action.ObjectivesThe NucliSENS® EasyQ® HPV v1 test was designed to test cervical scrapes for the expression of the oncogenic E6/E7 mRNA from the five most common carcinogenic HPV types (16, 18, 31, 33 and 45). The test can be used for confirmation and risk stratification of individuals with proven infection with high risk papillomaviruses.Study designIn order to establish performance of the assay, sensitivity, specificity, repeatability, and reproducibility were determined with artificial and clinical specimens. Further, a total of 420 cervical scrapes were tested and the results directly compared to the CE-market device PreTect HPV-Proofer assay (NorChip, Klokkarstua, Norway). For arbitration of discordant clinical results, the specimens were rated according to Pap-classification and the presence of HPV DNA was determined.ResultsThe limit of detection for the five HPV types 16, 18, 31, 33, and 45 ranged from 2.3×10² to 3.0×10⁴ copies/mL on a background of 5×10³ HPV-negative HS67 cells. No cross-reactivity for other viral, bacterial, or fungal agents known to be potentially present in cervical fluids was detected. Repeatability and reproducibility were shown by testing panels of HPV-spiked artificial and clinical samples. A comparative analysis of 420 cervical scrapes demonstrated an overall concordance of >90% between the NucliSENS EasyQ HPV test and the technologically related PreTect HPV-Proofer assay.     

High-risk human papillomavirus (hrHPV) E6/E7 mRNA testing by PreTect HPV-Proofer for detection of cervical high-grade intraepithelial neoplasia and cancer among hrHPV DNA-positive women with normal cytology

Our aim was to investigate whether high-risk HPV (hrHPV) mRNA detection by PreTect HPV-Proofer can be used to stratify hrHPV DNA-positive women of different cytology classes for risk of high-grade cervical intraepithelial neoplasia or worse (cervical precancer or cancer, i.e., cervical intraepithelial neoplasia grade 2 or higher [≥ CIN2]). A total of 375 women participating in population-based screening, with a GP5+/6+-PCR hrHPV DNA-positive cervical scrape with normal cytology (n = 202), borderline or mild dyskaryosis (BMD) (n = 88), or moderate dyskaryosis or worse (>BMD) (n = 85), were enrolled. Cervical scrapes were additionally subjected to HPV16/18/31/33/45 E6/E7 mRNA analysis by PreTect HPV-Proofer (mRNA test). Referral and follow-up policies were based on cytology, hrHPV DNA, and mRNA testing. The primary study endpoint was the number of ≥CIN2 detected within 3 years of follow-up. The mRNA positivity increased with the severity of cytological abnormality, ranging from 32% (64/202) in hrHPV DNA-positive women with normal cytology to 47% (41/88) in BMD and 68% (58/85) in >BMD groups (P < 0.01). Women with ≥ CIN2 were more likely to test positive by mRNA test (63%) than women without evidence of ≥ CIN2 (32%; P < 0.01). A positive mRNA test result conferred an increased ≥ CIN2 risk in hrHPV DNA-positive women with normal cytology, i.e., 0.55 (95% confidence interval [95% CI], 0.34 to 0.76) in mRNA-positive versus 0.20 (95% CI, 0.07 to 0.33) in mRNA-negative women. In hrHPV DNA-positive women with BMD or >BMD, the result of the mRNA test did not influence the ≥ CIN2 risk. In conclusion, mRNA testing by PreTect HPV-Proofer might be of value to select hrHPV DNA-positive women with normal cytology in need of immediate referral for colposcopy.     

Comparison of real-time multiplex human papillomavirus (HPV) PCR assays with INNO-LiPA HPV genotyping extra assay

Real-time type-specific multiplex human papillomavirus (HPV) PCR assays were developed to detect HPV DNA in samples collected for the efficacy determination of the quadrivalent HPV (type 6, 11, 16, and 18) L1 virus-like particle (VLP) vaccine (Gardasil). Additional multiplex (L1, E6, and E7 open reading frame [ORF]) or duplex (E6 and E7 ORF) HPV PCR assays were developed to detect high-risk HPV types, including HPV type 31 (HPV31), HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, and HPV59. Here, we evaluated clinical specimen concordance and compared the limits of detection (LODs) between multiplex HPV PCR assays and the INNO-LiPA HPV Genotyping Extra assay, which detects 28 types, for the 14 HPV types common to both of these methods. Overall HPV detection agreement rates were >90% for swabs and >95% for thin sections. Statistically significant differences in detection were observed for HPV6, HPV16, HPV18, HPV35, HPV39, HPV45, HPV56, HPV58, and HPV59 in swabs and for HPV45, HPV58, and HPV59 in thin sections. Where P was <0.05, discordance was due to detection of more HPV-positive samples by the multiplex HPV PCR assays. LODs were similar for eight HPV types, significantly lower in multiplex assays for five HPV types, and lower in INNO-LiPA for HPV6 only. LODs were under 50 copies for all HPV types, with the exception of HPV39, HPV58, and HPV59 in the INNO-LiPA assay. The overall percent agreement for detection of 14 HPV types between the type-specific multiplex HPV PCR and INNO-LiPA genotyping assays was good. The differences in positive sample detection favored multiplex HPV PCR, suggesting increased sensitivity of HPV DNA detection by type-specific multiplex HPV PCR assays.     

Identification of multiple genital HPV types and sequence variants by consensus and nested type-specific PCR coupled with cycle sequencing

Consensus and type-specific HPV primers were employed for PCR and cycle sequencing of genital HPVs in scrapings and colposcopically directed biopsies of the cervix from a cohort of 188 female sex workers. A total of 27 individuals tested positive for a broad spectrum of HPV types, including HPVs 6b, 16, 18, 31, 33, 34, 35, 45, 56 and 58, as well as a new HPV type, with seven individuals displaying dual infections. Good correlation between the results of individually paired samples was observed. A HPV 16 primer biotinylated at the 5' end was also used as a probe, which could successfully detect amplified products of HPV 16 but not other HPV types tested by an automated ELISA detection system. DNA sequence analysis revealed several HPV sequence variants that harbored mutations, especially in the E6 gene, many of which culminated in non-conservative amino acid substitutions in the transforming E6 oncoprotein. Such an approach of coupling PCR with cycle sequencing permits the determination of many known and even novel HPV types associated with varying degrees of risk to cervical carcinogenesis, and enables the identification of HPV sequence variants of putative biological and clinical significance, thus justifying its utility as an adjunct tool to complement cervical cytology and colposcopy. This study also emphasises the need for educational, interventional and behavioral modification to minimise HPV transmission, such as through consistent condom usage among sex workers.     

DNA-EIA to detect high and low risk HPV genotypes in cervical lesions with E6/E7 primer mediated multiplex PCR.

BACKGROUND: Oncogenicity of human papillomavirus (HPV) DNA in premalignant and malignant uterine cervical diseases is mainly induced by E6/E7 open reading frame (ORF). The presence of an oncogenic HPV DNA may be a diagnostic marker for the detection of cytologically negative smears. AIMS: To evaluate an original polymerase chain reaction enzyme immunoassay (PCR-EIA) for the detection and typing of oncogenic and non-oncogenic HPV types. METHODS: The test was an original multiplex labelled PCR-EIA for the detection and typing of oncogenic and non-oncogenic HPV using three consensus sequence primers within the oncogenic E6/E7 ORF. One primer was dinitrophenyl (DNP) labelled and the DNP labelled amplimers could be further hybridised with specific biotinylated oligoprobes mixed in only two cocktails: oncogenic (16, 18, 31, 33, 35, 52, and 58) and non-oncogenic (6 and 11) HPV types in only two wells; then biotinylated oligoprobes were deposited in streptavidin-coated microplates. The PCR-EIA was validated on HPV plasmids (types 6, 11, 16, 18, 31, 35, 52, and 58) and used to evaluate cervical scrapes from 181 patients (median age 32 years) at high risk for cervical cancer. RESULTS: HPV were detected in the cervical scrapes of 88 of 181 patients (48.6%); nine with non-oncogenic HPV (5.0%) and 79 with oncogenic HPV (43.6%) including 29 coinfections with oncogenic and non-oncogenic HPV. The number of oncogenic HPV infections increased with the presence of high grade lesions: 95.8% of the cervical scrapes from patients with high grade lesions contained oncogenic HPV compared with 32.1% of the specimens from patients without any lesions detectable by colposcopy and/or by cytological examination of the cervical smears. Moreover, 60% of cervical scrapes exhibiting low grade lesions contained oncogenic HPV. CONCLUSIONS: This test is simple, specific, sensitive, safe, fast, reproducible, and easy to use in routine practice. Thus, it is possible to detect simultaneously on a simple cervical scrape, two kinds of HPV--oncogenic and non-oncogenic--in just two microplate wells with non-isotopic oligoprobes.     

Multiple HPV genotypes in cervical carcinomas: improved DNA detection and typing in archival tissues.

BACKGROUND: Human papillomaviruses (HPV) have been considered to be the necessary and central agents of cervical carcinoma. OBJECTIVE: The aim of this study was to determine the prevalence and genotypes of HPV in archival cervical carcinomas. STUDY DESIGN: The study included 152 paraffin-embedded, formaldehyde-fixed cervical carcinoma specimens. To improve the detection and typing of HPV in archival tissues, we conducted a comprehensive study in which, polymerase chain reaction (PCR)-based methods using E7 type-specific (TS) and L1 modified general primers (MY11/GP6+ and GP5+/GP6+) were employed. RESULTS: Overall HPV prevalence was 98% in the cervical carcinomas. HPV 16 was detected in 66% of the tumors, HPV 18 in 22%, HPV 31 in 13%, HPV 33 in 9%, and HPV 58 in 9%. Notably, multiple HPV types were present in 44 (28.9%) of the 152 cervical carcinomas. The most common co-infections were HPV types 16/18 (12 cases), followed by HPV types 16/31 (7 cases). Additionally, HPV 18 was more frequent in adenocarcinomas and adenosquamous carcinomas (86%) than in squamous cell carcinomas (15.8%) (P = 0.0002). CONCLUSIONS: The combination of L1 general primers and E7 type-specific primers can be of use in detecting HPV DNA in archival tissues. The present study showed a high frequency of multiple HPV infections in cervical carcinomas. Hence, relevant HPV typing information in cervical carcinoma is very important for further HPV vaccine design and application.     

Comparing triage algorithms using HPV DNA genotyping,HPV E7 mRNA detection and cytology in high-risk HPV DNA-positive women

BackgroundHigh-risk human papillomavirus (hrHPV) DNA positive women require triage testing to identify those with high-grade cervical intraepithelial neoplasia or cancer (≥CIN2).ObjectiveComparing three triage algorithms (1) E7 mRNA testing following HPV16/18/31/33/45/52/58 genotyping (E7 mRNA test), (2) HPV16/18 DNA genotyping and (3) cytology, for ≥CIN2 detection in hrHPV DNA-positive women.Study designhrHPV DNA-positive women aged 18–63 years visiting gynecology outpatient clinics were included in a prospective observational cohort study. From these women a cervical scrape and colposcopy-directed biopsies were obtained. Cervical scrapes were evaluated by cytology, HPV DNA genotyping by bead-based multiplex genotyping of GP5+6+-PCR-products, and presence of HPV16/18/31/33/45/52/58 E7 mRNA using nucleic acid sequence-based amplification (NASBA) in DNA positive women for respective HPV types. Sensitivities and specificities for ≥CIN2 were compared between E7 mRNA test and HPV16/18 DNA genotyping in the total group (n = 348), and E7 mRNA test and cytology in a subgroup of women referred for non-cervix-related gynecological complaints (n = 133).ResultsSensitivity for ≥CIN2 of the E7 mRNA test was slightly higher than that of HPV16/18 DNA genotyping (66.9% versus 60.9%; ratio 1.10, 95% CI: 1.0002–1.21), at similar specificity (54.8% versus 52.3%; ratio 1.05, 95% CI: 0.93–1.18). Neither sensitivity nor specificity of the E7 mRNA test differed significantly from that of cytology (sensitivity: 68.8% versus 75.0%; ratio 0.92, 95% CI: 0.72–1.17; specificity: 59.4% versus 65.3%; ratio 0.91, 95% CI: 0.75–1.10).ConclusionFor detection of ≥CIN2 in hrHPV DNA-positive women, an algorithm including E7 mRNA testing following HPV16/18/31/33/45/52/58 DNA genotyping performs similar to HPV16/18 DNA genotyping or cytology.     

A Human Papilloma Virus Testing Algorithm Comprising a Combination of the L1 Broad-Spectrum SPF10 PCR Assay and a Novel E6 High-Risk Multiplex Type-Specific Genotyping PCR Assay

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.     

Use of multiple PCR primer sets for optimal detection of human papillomavirus.

Using multiple PCR primer sets, we tried to optimize the detection of human papillomavirus (HPV) in DNA samples isolated from 361 frozen biopsy specimens from patients with invasive cervical carcinomas. The HPVs detected were placed into three distinct groups, including group I/Inex at Telelab (Skien, Norway) and group Ineg and group II at the Norwegian Radium Hospital (Oslo, Norway). The consensus primer sets were Oli-1b-oli-2i, My09-My11, Gp5-Gp6, and Gp(5+)-Gp6+ from the HPV L1 gene and CpI-CpIIG from the E1 gene. Using these consensus primers together with the type-specific primers from E6-E7, we found that 355 patients (98%) were HPV positive. Type-specific primers for HPV types 11, 16, 18, 31, 33, and 35 detected more HPV-infected patients than the most sensitive consensus primer set, while the three consensus primer sets My, Gp/Gp+, and Cp together detected more HPV-positive patients than the type-specific primers. Testing of sensitivity of the PCR with SiHa cells serially diluted in lymphocytes (HPV-negative cells) indicated a detection limit of 6,300 HPV type 16 DNA copies with consensus primers (My, Gp+, and Cp) and 126 original HPV type 16 DNA copies with type-specific primers. Comparison of the amplification results for consensus L1 primers and type-specific E6-E7 primers indicated the presence of L1 deletions in 23 of 56 samples. The conclusion is that in PCR detection systems, multiple consensus primers and type-specific primers should be used in order to detect all patients harboring HPV.     

Sensitivity, specificity, and clinical value of human papillomavirus (HPV) E6/E7 mRNA assay as a triage test for cervical cytology and HPV DNA test

There is evidence that testing for human papillomavirus (HPV) E6/E7 mRNA is more specific than testing for HPV DNA. A retrospective study was carried out to evaluate the performance of the PreTect HPV-Proofer E6/E7 mRNA assay (Norchip) as a triage test for cytology and HPV DNA testing. This study analyzed 1,201 women, 688 of whom had a colposcopy follow-up and 195 of whom had histology-confirmed high-grade intraepithelial neoplasia or worse (CIN2+). The proportion of positive results and the sensitivity and specificity for CIN2+ were determined for HPV mRNA in comparison to HPV DNA and cytology. All data were adjusted for follow-up completeness. Stratified by cytological grades, the HPV mRNA sensitivity was 83% (95% confidence interval [CI] = 63 to 94%) in ASC-US (atypical squamous cells of undetermined significance), 62% (95% CI = 47 to 75%) in L-SIL (low-grade squamous intraepithelial lesion), and 67% (95% CI = 57 to 76%) in H-SIL (high-grade squamous intraepithelial lesion). The corresponding figures were 99, 91, and 96%, respectively, for HPV DNA. The specificities were 82, 76, and 45%, respectively, for HPV mRNA and 29, 13, and 4%, respectively, for HPV DNA. Used as a triage test for ASC-US and L-SIL, mRNA reduced colposcopies by 79% (95% CI = 74 to 83%) and 69% (95% CI = 65 to 74%), respectively, while HPV DNA reduced colposcopies by 38% (95% CI = 32 to 44%) and by 15% (95% CI = 12 to 19%), respectively. As a HPV DNA positivity triage test, mRNA reduced colposcopies by 63% (95% CI = 60 to 66%), having 68% sensitivity (95% CI = 61 to 75%), whereas cytology at the ASC-US+ threshold reduced colposcopies by 23% (95% CI = 20 to 26%), showing 92% sensitivity (95% CI = 87 to 95%). In conclusion, PreTect HPV-Proofer mRNA can serve as a better triage test than HPV DNA to reduce colposcopy referral in both ASC-US and L-SIL. It is also more efficient than cytology for the triage of HPV DNA-positive women. Nevertheless, its low sensitivity demands a strict follow-up of HPV DNA positive-mRNA negative cases.     

Oligonucleotide primers for DNA amplification of the early regions 1, 6, and 7 from human papillomavirus types 6, 11, 16, 18, 31, and 33

Summary Human papillomavirus (HPV) type-specific sequences required for polymerase chain reaction (PCR) mediated amplification of HPV DNA sequences are presented. One primer pair within the E1 open reading frame (ORF) was shared by HPV 6, HPV 11, HPV 16, and HPV 31, whereas the other primer pair within the E1 ORF was specific for HPV 16. Eight primer pairs from the E6 and E7 ORFs specifically detected HPV 6, HPV 16, HPV 18, and HPV 33 sequences. This system has been used for detection of HPV DNA in biopsies, cytological smears and sections of formalin-fixed tissues.     

Post Hoc Analysis of the PATRICIA Randomized Trial of the Efficacy of Human Papillomavirus Type 16 (HPV-16)/HPV-18 AS04-Adjuvanted Vaccine against Incident and Persistent Infection with Nonvaccine Oncogenic HPV Types Using an Alternative Multiplex Type-Specific PCR Assay for HPV DNA

The efficacy of the human papillomavirus type 16 (HPV-16)/HPV-18 AS04-adjuvanted vaccine against cervical infections with HPV in the Papilloma Trial against Cancer in Young Adults (PATRICIA) was evaluated using a combination of the broad-spectrum L1-based SPF₁₀ PCR-DNA enzyme immunoassay (DEIA)/line probe assay (LiPA₂₅) system with type-specific PCRs for HPV-16 and -18. Broad-spectrum PCR assays may underestimate the presence of HPV genotypes present at relatively low concentrations in multiple infections, due to competition between genotypes. Therefore, samples were retrospectively reanalyzed using a testing algorithm incorporating the SPF₁₀ PCR-DEIA/LiPA₂₅ plus a novel E6-based multiplex type-specific PCR and reverse hybridization assay (MPTS12 RHA), which permits detection of a panel of nine oncogenic HPV genotypes (types 16, 18, 31, 33, 35, 45, 52, 58, and 59). For the vaccine against HPV types 16 and 18, there was no major impact on estimates of vaccine efficacy (VE) for incident or 6-month or 12-month persistent infections when the MPTS12 RHA was included in the testing algorithm versus estimates with the protocol-specified algorithm. However, the alternative testing algorithm showed greater sensitivity than the protocol-specified algorithm for detection of some nonvaccine oncogenic HPV types. More cases were gained in the control group than in the vaccine group, leading to higher point estimates of VE for 6-month and 12-month persistent infections for the nonvaccine oncogenic types included in the MPTS12 RHA assay (types 31, 33, 35, 45, 52, 58, and 59). This post hoc analysis indicates that the per-protocol testing algorithm used in PATRICIA underestimated the VE against some nonvaccine oncogenic HPV types and that the choice of the HPV DNA testing methodology is important for the evaluation of VE in clinical trials. (This study has been registered at ClinicalTrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT00122681","term_id":"NCT00122681"}}NCT00122681.)