A New PCR-Based Mass Spectrometry System for High-Risk HPV, Part I: Methods (original) (raw)
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Journal of Virological Methods, 2009
Knowledge of the central role of high-risk human papillomavirus (HPV) in cervical carcinogenesis, coupled with an emerging need to monitor the efficacy of newly introduced HPV vaccines, warrant development and evaluation of type-specific, quantitative HPV detection methods. In the present study, a prototype PCR and mass spectroscopy (PCR-MS)-based method to detect and quantitate 13 high-risk HPV types is compared to the Hybrid Capture 2 High Risk HPV DNA test (HC2; Digene Corp., Gaithersburg, MD) in 199 cervical scraping samples and to DNA sequencing in 77 cervical tumor samples. High-risk HPV types were detected in 76/77 (98.7%) cervical tumor samples by PCR-MS. Degenerate and type-specific sequencing confirmed the types detected by PCR-MS. In 199 cervical scraping samples, all 13 HPV types were detected by PCR-MS. Eighteen (14.5%) of 124 cervical scraping samples that were positive for high-risk HPV by HC2 were negative by PCR-MS. In all these cases, degenerate DNA sequencing failed to detect any of the 13 high-risk HPV types. Nearly half (46.7%) of the 75 cervical scraping samples that were negative for high-risk HPV by the HC2 assay were positive by PCR-MS. Type-specific sequencing in a subset of these samples confirmed the HPV type detected by PCR-MS. Quantitative PCR-MS results demonstrated that 11/75 (14.7%) samples contained as much HPV copies/cell as HC2-positive samples. These findings suggest that this prototype PCR-MS assay performs at least as well as HC2 for HPV detection, while offering the additional, unique advantages of type-specific identification and quantitation. Further validation work is underway to define clinically meaningful HPV detection thresholds and to evaluate the potential clinical application of future generations of the PCR-MS assay.
A New PCR-Based Mass Spectrometry System for High-Risk HPV, Part II
American Journal of Clinical Pathology, 2011
This was a population-based clinical trial of a polymerase chain reaction-based multiplex high-risk human papillomavirus (HR-HPV) assay using mass spectrometry (MassARRAY [Sequenom, San Diego, CA] matrix-assisted laser desorption/ionization timeof-flight mass spectrometry system [MALDI-TOF]). Participants were 10,000 women between the ages of 25 and 59 years in Guangdong Province, China (SHENCCAST II Study). All women collected a selfsample (tested with Cervista [Hologic, Marlborough, MA] and MALDI-TOF) followed by a cliniciancollected cervical sample (for cytology, Hybrid Capture 2 [HC2; Qiagen, Gaithersburg, MD], Cervista, and MALDI-TOF). Patients with any abnormal result were asked to return for colposcopy and biopsies. This analysis included the data for 8,556 women. The sensitivity values for cervical intraepithelial neoplasia (CIN) 3 or worse for a direct cervical sample were 97.9%, 95.1%, and 94.3 for HC2, Cervista, and MALDI-TOF, respectively (P > .05). The sensitivity for CIN 3 or worse for a self-collected sample tested with MALDI-TOF was also 94.3%, which was similar to a clinician-obtained endocervical sample assayed with the 3 HR-HPV assays. MALDI-TOF combined with a self-collected sample provides a highly sensitive, high-throughput, low-cost-per-case assay for mass screening.
Journal of Clinical Microbiology, 2006
DNA was detected in 433 (82.0%) and 458 (86.7%) samples with PGMY-LB and LA-HPV (P ؍ 0.047), respectively, for an excellent agreement of 93.8% (kappa ؍ 0.76). Of the 17,094 HPV typing results, 16,562 (1,743 positive and 14,819 negative results) were concordant between tests (agreement ؍ 96.9%; kappa ؍ 0.76). The mean agreement between tests for each type was 96.4% ؎ 2.4% (95% confidence interval [CI], 95.6% to 97.2%; range, 86% to 100%), for an excellent mean kappa value of 0.85 ؎ 0.10 (95% CI, 0.82 to 0.87). However, detection rates for most HPV types were greater with LA-HPV. The mean number of types per sample detected by LA-HPV (4.2 ؎ 3.4; 95% CI, 3.9 to 4.5; median, 3.0) was greater than that for PGMY-LB (3.4 ؎ 3.0; 95% CI, 3.1 to 3.6; median, 2.0) (P < 0.001). The number of types detected in excess by LA-HPV in anal samples correlated with the number of types per sample (r ؍ 0.49 ؎ 0.06; P ؍ 0.001) but not with patient age (r ؍ 0.03 ؎ 0.06; P ؍ 0.57), CD4 cell counts (r ؍ 0.06 ؎ 0.06; P ؍ 0.13), or the grade of anal disease (r ؍ ؊0.11 ؎ 0.06; P ؍ 0.07). LA-HPV compared favorably with PGMY-LB but yielded higher detection rates for newer and well-known HPV types.
Journal of Clinical Pathology, 1999
Background-The development of a reproducible, sensitive, and standardised human papillomavirus (HPV) polymerase chain reaction (PCR) test is required to implement HPV testing in cervical cancer screening programmes and for triaging women with mild to moderate dysplasia. Aims-To determine the intermethod agreement between diVerent GP5+/6+ and MY09/11 PCR based protocols for the detection and typing of high risk (HR) HPV DNA in cervical smears and to assess the intramethod reproducibility of the GP5+/6+ PCR enzyme immunoassay (EIA) for HR-HPV detection. Methods-For the intermethod comparison, crude aliquots of 20 well characterised cervical smears comprising five HPV negative samples, and six and nine samples containing single and multiple HPV infections, respectively, were coded and sent from reference laboratory (A) to three other laboratories. One of these (laboratory B) used the GP5+/6+ PCR-EIA and was provided with standard protocols. Another laboratory (C) used GP5+/6+ PCR combined with sequence analysis and type specific PCR, whereas two laboratories (D and E) used MY09/11 PCR followed by restriction fragment length polymorphism (RFLP) analysis for the detection and typing of HR-HPV. The intramethod agreement of GP5+/6+ PCR-EIA was analysed in a subsequent study with four other laboratories (F to I) on crude aliquots of 50 well characterised cervical smears, consisting of 32 HR-HPV positive and 18 HPV negative samples. Standardised protocols, primers, and probes were also provided by the reference laboratory for HR-HPV detection. Results-In the intermethod comparison, pairwise agreement of the diVerent laboratories with reference laboratory A for the detection of HR-HPV varied between 75% and 100% ( values: 0.5 to 1). Typing data revealed a broader range in pairwise agreement rates between 32% and 100%. The highest agreement was found between laboratories A and B using standardised protocols and validated reagents. In the intramethod evaluation, pairwise comparison of the laboratories F to I with reference laboratory A revealed excellent agreement rates from 92% to 100% ( values: 0.88 to 1.0) with an overall sensitivity of 97.5% (195/200) and specificity of 99.5% (199/200). Conclusions-The detection of HR-HPV as a group is highly reproducible with GP5+/6+ PCR-EIA provided that standardised protocols and validated reagents are used. (J Clin Pathol 1999;52:498-503) Keywords: human papillomavirus; polymerase chain reaction; intermethod agreement; intramethod agreement J Clin Pathol 1999;52:498-503 498 Reliable high risk HPV DNA testing by PCR 499 group.bmj.com on July 14, 2011 -Published by jcp.bmj.com Downloaded from 1999 52: 498-503 J Clin Pathol M V Jacobs, P J Snijders, F J Voorhorst, et al. and intramethod comparison. polymerase chain reaction: an intermethod Reliable high risk HPV DNA testing by http://jcp.bmj.com/content/52/7/498
Detection and quantitation of HPV in genital and oral tissues and fluids by real time PCR
Virology Journal, 2010
Background: Human papillomaviruses (HPVs) remain a serious world health problem due to their association with anogenital/oral cancers and warts. While over 100 HPV types have been identified, a subset is associated with malignancy. HPV16 and 18 are the most prevalent oncogenic types, while HPV6 and 11 are most commonly responsible for anogenital warts. While other quantitative PCR (qPCR) assays detect oncogenic HPV, there is no single tube assay distinguishing the most frequent oncogenic types and the most common types found in warts. Results: A Sybr Green-based qPCR assay was developed utilizing degenerate primers to the highly conserved HPV E1 theoretically detecting any HPV type. A single tube multiplex qPCR assay was also developed using type-specific primer pairs and TaqMan probes that allowed for detection and quantitation of HPV6,11,16,18. Each HPV type was detected over a range from 2 × 10 1 to 2 × 10 6 copies/reaction providing a reliable method of quantitating typespecific HPV in 140 anogenital/cutaneous/oral benign and malignant specimens. 35 oncogenic and low risk alpha genus HPV types were detected. Concordance was detected in previously typed specimens. Comparisons to the gold standard detected an overall sensitivity of 89% (95% CI: 77% -96%) and specificity of 90% (95%CI: 52% -98%). Conclusion: There was good agreement between the ability of the qPCR assays described here to identify HPV types in malignancies previously typed using standard methods. These novel qPCR assays will allow rapid detection and quantitation of HPVs to assess their role in viral pathogenesis.
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. Based on cervical cancer case-control studies, 15 human papillomavirus (HPV) types, HPV-16,-18,-31,-33,-35,-39,-45,-51,-52,-56,-58,-59,-68,-73, and-82, are classified as high-risk oncogenic types, and an additional three types, HPV-26,-53, and-66, are classified as probable oncogenic types associated with cervical cancer (6, 18). A recent report from the International Agency for Cancer Research lists 12 of these types, HPV-16,-18,-31,-33,-35,-39,-45,-51,-52,-56,-58, and-59, as having sufficient evidence for causing cervical cancer (3). Regardless, just eight of these types, HPV-16,-18,-31,-33,-35,-45,-52, and-58, account for 95% of HPV-positive cervical cancers worldwide with HPV-16 and-18 accounting for about 70% of cervical cancers (2, 18). In recent years, evidence has mounted for the type-specific identification of HPV-16 and-18 to aid risk stratification and better clinical management of women testing positive for HPV, as these two types are associated with persistent infection and lesion progression, thus conferring a significantly higher risk for cervical cancer than other oncogenic types even in the absence of cytological abnormalities (8, 14). Further, type-specific detection of HPV could increase the overall specificity in routine screening and posttreatment follow-up (13). However, the currently available HPV tests generally do not provide information on individual genotypes (9, 11, 12). MassARRAY spectrometry (MassARRAY, Sequenom Inc.) is a novel method for type specific detection of 15 high-risk onco-genic types, HPV-16,-18,-31,-33,-35,-39,-45,-51,-52,-56,-58,-59,-66,-68, and-73. The test utilizes a three-step process composed of competitive PCR, primer extension, and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) separation of products on a matrix-loaded silicon chip array. MassARRAY is a fully automated high-throughput method which can process up to 2,000 samples in about 11 h. In addition, this test incorporates an internal oligomer standard for quality assurance which also allows for viral load quantitation (24). The latter option has not been previously available in commercial kits and may have useful clinical application. While these features are appealing, the efficacy and clinical utilization of MassARRAY have not been fully assessed. PreTect HPV-Proofer (Proofer; Norchip) is a unique type-specific test in that it is the only test of its kind which detects E6/E7 mRNA from the five most common oncogenic types, HPV-16,-18,-31,-33, and-45, individually. Several studies have shown this test to be significantly more specific than other
Comparison of Two PCR-Based Human Papillomavirus Genotyping Methods
2008
We compared two consensus primer PCR human papillomavirus (HPV) genotyping methods for the detection of individual HPV genotypes and carcinogenic HPV genotypes as a group, using a stratified sample of enrollment cervical specimens from sexually active women participating in the NCI/Costa Rica HPV16/18 Vaccine Efficacy Trial. For the SPF 10 method, DNA was extracted from 0.1% of the cervical specimen by using a MagNA Pure LC instrument, a 65-bp region of the HPV L1 gene was targeted for PCR amplification by using SPF 10 primers, and 25 genotypes were detected by reverse-line blot hybridization of the amplicons. For the Linear Array (LA) method, DNA was extracted from 0.5% of the cervical specimen by using an MDx robot, a 450-bp region of the HPV L1 gene was targeted for PCR amplification by using PGMY09/11 L1 primers, and 37 genotypes were detected by reverse-line blot hybridization of the amplicons. Specimens (n ؍ 1,427) for testing by the LA method were randomly selected from strata defined on the basis of enrollment test results from the SPF 10 method, cytology, and Hybrid Capture 2. LA results were extrapolated to the trial cohort (n ؍ 5,659). The LA and SPF 10 methods detected 21 genotypes in common; HPV16and -73 were considered the carcinogenic HPV genotypes. There was no difference in the overall results for grouped detection of carcinogenic HPV by the SPF 10 and LA methods (35.3% versus 35.9%, respectively; P ؍ 0.5), with a 91.8% overall agreement and a kappa value of 0.82. In comparisons of individual HPV genotypes, the LA method detected significantly more HPV16, HPV18, HPV39, HPV58, HPV59, HPV66, and HPV68/73 and less HPV31 and HPV52 than the SPF 10 method; inclusion of genotype-specific testing for HPV16 and HPV18 for those specimens testing positive for HPV by the SPF 10 method but for which no individual HPV genotype was detected abrogated any differences between the LA and SPF 10 methods. The LA method detected more carcinogenic-HPV-genotype infections per specimen than the SPF 10 method (P < 0.001). In conclusion, the LA method and the SPF 10 method with HPV16 and HPV18 genotype-specific detection among ungenotyped HPV-positive specimens were comparable for detection of HPV16 and HPV18, the two HPV genotypes targeted by current prophylactic HPV vaccines. Both approaches are suitable for monitoring the impact of HPV16/18 vaccines in clinical trials.
Journal of Molecular Diagnostics, 2010
Infection with a high-risk carcinogenic type of human papillomavirus (HPV) is necessary for the development of cervical cancer. The digene HC2 HPV Test (HC2) is an important screening tool but lacks genotyping capability. To address this issue , we developed an assay for the rapid genotyping of HPV in cervical specimens. The three steps of this assay include Hybrid Capture target enrichment , whole-genome amplification , and Luminex XMAP detection. The assay includes the simultaneous detection of two genomic regions from each of 17 high-risk and two low-risk HPV types most associated with disease. The assay performance was tested on HPV plasmids as well as clinical specimens. An analytical limit of detection of 100 copies or less was demonstrated for linear, circular, and integrated HPV DNA. This finding is at least 1 log lower than the HC2 assay limit of detection. There was no cross-reactivity among the HPV types up to 1,000,000 copies. There was also no substantial assay interference from substances in cervical specimens. Although the clinical performance of the assay was not formally tested, the assay had good agreement (Cohen's kappa equal to 0.72) with both a PCR-based HPV genotyping assay (n ؍ 131) and the HC2 assay (n ؍ 502) using representative cervical specimens. This assay may be easy to automate and could be applied for the detection of other targets in future studies. (J Mol Diagn 2010,
Journal of clinical Virology, 2002
Background: Hybrid Capture II HPV Test (HCII) (Digene Corporation, Gaithersburg, MD) is a signal amplified hybridization microplate-based assay designated to detect 18 human papillomavirus (HPV) genotypes using two probe cocktails, for high-risk HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 11, 42, 43 and 44. At present, HCII is the only commercially available HPV assay with sufficient scientific data to support its performance in the clinical setting. Objectives: To determine the specificity and accuracy of HCII high-risk probe cocktail for detection of 13 HPV genotypes included in the high-risk probe cocktail. Study design: Cervical samples obtained from 325 women recognized as HPV positive using the HCII high-risk probe cocktail were included in the study. HPV genotypes were determined by restriction fragment analysis of PGMY09/PGMY11 polymerase chain reaction (PCR) products using seven restriction endonucleases. Results: A 450 bp fragment of HPV L1 gene was successfully amplified from 312 out of 325 samples. Of the 312 PCR-positive samples, 280 samples were associated with the expected high-risk HPV genotypes and 32 samples with the HPV genotypes not included in the HCII high-risk probe cocktail. Thus, HPV53 was detected in 8 samples, HPV66 in 4 samples, HPV54 in 3 samples, HPV6, HPV26, HPV70 each in 2 samples, and HPV11, HPV40, HPV42, HPV61, HPV73, HPV81, MM4, IS39, CP6108 each in 1 sample. In 2 samples, we were not able to determine the HPV genotype. Conclusions: Our study shows that HCII highrisk probe cocktail detects at least 15 HPV genotypes not included in the current HCII high-risk probe cocktail. The potential impact of HCII high-risk probe cocktail cross-reactivity with phylogenetically related and unrelated HPV genotypes, including genotypes currently considered to be low-risk HPVs, remains to be determined. #