Reliable high risk HPV DNA testing by polymerase chain reaction: an intermethod and intramethod comparison [published erratum appears in J Clin Pathol 1999 Oct;52(10):790] (original) (raw)
Related papers
Journal of Clinical Virology, 2006
Infection with human papillomavirus (HPV) is a necessary step in the progression to cervical cancer. Many methods for HPV testing are currently available, most developed to detect pools of HPV types.To evaluate the HPV typing by molecular methods and to compare commercial kits with an established laboratory method.Eighty-four cervical samples found to be positive for HPV DNA by GP5+/6+-polymerase chain reaction-enzyme immunoassay-reverse line blotting (PCR-EIA-RLB) were re-tested with two commercial methods, INNO-LiPA and Amplisense HPV typing, able to identify the HPV type predicted by PCR-EIA-RLB in 76 and 67 samples, respectively.The INNO-LiPA assay revealed HPV DNA in 75/76 samples (98.7%; 95% CI, 0.93–0.99) that would contain HPV types identifiable by this assay. The Amplisense HPV assay revealed HPV DNA in 58/67 samples (86.6%; 95% CI, 0.76–0.93) containing HPV types detectable by this assay. For samples with a single infection, the unweighted kappa for concordance of HPV typing was 0.87 (95% CI, 0.78–0.97) for PCR-EIA-RLB versus INNO-LiPA, 0.94 (95% CI, 0.87–0.99) for INNO-LiPA versus Amplisense HPV, and 0.82 (95% CI, 0.70–0.94) for PCR-EIA-RLB versus Amplisense HPV typing. PCR-EIA-RLB revealed 12 multiple infections, INNO-LiPA revealed 14, and Amplisense HPV revealed 5. The agreement among tests for samples with multiple infections was lower, giving kappa values of 0.44 (95% CI, 0.18–0.70) for PCR-EIA-RLB versus INNO-LiPA, 0.52 (95% CI, 0.19–0.85) for PCR-EIA-RLB versus Amplisense HPV and 0.43 (95% CI, 0.12–0.74) for INNO-LiPA versus Amplisense HPV.In HPV-positive samples, the agreement among tests for HPV typing was high for single infections but markedly lower for infections with multiple HPV types.
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 Virology, 2000
Background: polymerase chain reaction (PCR)-based assays for human papillomavirus (HPV) sequences are in wide use in clinical and epidemiological studies. The reproducibility of these assays is not extensively studied. Objecti6es: to estimate the intra-laboratory reproducibility of generic and type-specific HPV diagnoses by the MY09/MY11/ HMB01 consensus L1 primer-based PCR assay. Study design: systematically collected specimens (n= 207) were masked and retested. Results: when specimens negative in both initial and repeat assays were excluded from analysis, the diagnostic reproducibility was 98.6% for b-globin, 90.7% for generic HPV (any HPV type), and 76.9% for type-specific HPVs. The reproducibility of type-specific diagnosis increased with increase in signal strength in the hybridization reaction of the initial assay. When a specimen contained five or more HPV types in the initial assay, it was rare to identify all of the HPV types in the repeat assay. Conclusions: the degree of reproducibility of the PCR diagnosis should be taken into account in the interpretation of HPV data in clinical and epidemiological studies.
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.
Diagnostic Molecular Pathology, 2010
The demonstration of human papillomavirus (HPV) in 99.7% of cervical carcinoma surgical specimens from around the world required investigations by multiple alternative polymerase chain reaction (PCR) assays. A similar approach may therefore be necessary to best characterize HPV prevalence and genotype distribution among cervical cytology samples. In an earlier study, 752 of 799 (94.1%) abnormal and 82 of 300 (27.3%) normal cytology specimens tested HPV positive after PCR using GP5+/6+primers. This study has reinvestigated the ''HPV negative'' abnormal samples (20 atypical squamous cells of undetermined significance, 5 low-grade squamous intraepithelial lesion, 14 atypical squamous cells, cannot exclude HSIL, 6 highgrade squamous intraepithelial lesion) and an age-matched cohort of ''HPV negative'' normal (negative for an intraepithelial lesion or malignancy) samples by PCR using PGMY09/11, FAP59/64, and LCR-E7 primers. PGMY09/11-GP5+/6+ nested PCR was performed on samples that were HPV negative by PGMY09/11 PCR. After the first 3 assays, HPV was detected in 41 of 45 (91.1%) abnormal and in 10 of 47 (21.3%) normal samples (P<0.0001). Eighteen HPV genotypes were detected and in some samples the genotype that was identified differed between the tests. The nondetection of common HPV genotypes (eg, HPVs 6, 11, 16, and 18) was notable. High-grade histopathology was found for 2 patients with HPV52-positive cytopathology. Combined with our earlier study, HPV (40 different genotypes) is shown in 99.5% of abnormal samples (99.8% inclusive of the nested PCR data). These findings show that HPV genotype and prevalence estimates are dependent on the method(s) of detection and indicate that suboptimal analytical sensitivity for one or more of the less common high-risk HPV genotypes could lead to impaired clinical sensitivity. HPV may be causal in almost every instance of abnormal cervical cytology; however, passenger HPV that is incidental to an abnormality may also have been detected.
International Journal of Cancer, 1999
More than 90% of high-grade cervical intraepithelial neoplasia (CIN 2/3) and cervical cancers are associated with high-risk (HR) human papillomavirus (HPV) types. HPV tests applicable for population screening have to detect all HR HPV types in a simple manner. It is likely that HPV testing will augment cytological investigations in screening programs . In addition, women with the cytologic diagnosis of atypical squamous cells of undetermined significance (ASCUS) can be triaged on the basis of HPV positivity . Persistent HR HPV infections are a marker for progressive CIN disease , indicating the importance of HPV detection methods. In view of such broad applications, it is essential that HPV detection assays are standardized with respect to sensitivity and specificity. We designed a study to assess inter-method variations according to HR HPV detection and HR HPV typing in cervical scrapes. Three different polymerase chain reaction (PCR)-based methods using 2 different PCR primer pairs to generate either a 150-bp or a 450-bp PCR fragment were used in 5 laboratories.
Journal of Clinical Virology, 2009
Background: Cervical screening detects precancerous cells and routine screening could be improved by testing for Human Papillomavirus (HPV), the virus that causes cervical cancer. HPV infection is common and the benefit of HPV testing would be identification of women who are HPV negative and at low risk of developing cancer. Study design: The aim of this study was to evaluate the Greiner Bio-one PapilloCheck ® micro-array assay (PapilloCheck) for detection of HPV in comparison with Hybrid Capture II (hc2) and PCR-enzyme immunoassay (PCR-EIA) using the GP5/6+ primers. Results: Samples from a cytologically defined population (n = 878) were analysed and 187 samples also had histology information. Overall, 674 out of 878 samples gave a consistent result (76.8%; 95% CI 73.83-79.52%) on all three platforms. The genotype results obtained by PapilloCheck and PCR-EIA were compared and 94% were consistent (95% CI 92.1-96.4%). The main difference was the poor Kappa agreement for detection of high risk (HR) type 35 (Kappa = 0.190) with all inconsistent results being HR positive by PCR-EIA assay but negative on the PapilloCheck platform. There was no statistically significant difference between the performance of each assay when HR HPV positive samples were linked with clinical result (cytology and histology grade). PapilloCheck detected the highest number of HR HPV infections in samples with histology confirmed as CIN1, CIN2 and CIN 3 (76.6%, 85% and 91.7%, respectively). Conclusions: Overall, PapilloCheck proved to be a sensitive, reproducible, robust molecular assay for HPV genotyping with the potential for high throughput of specimens in a clinical setting.
Comparison of the performance of different HPV genotyping methods for detecting genital HPV types
Journal of Medical Virology, 2008
Classification of high-risk HPV types for cervical cancer screening depends on epidemiological studies defining HPV type-specific risk. The genotyping tests that are used, are however, not uniform with regard to type-specific detection rates making comparisons between different studies difficult. To overcome the lack of a ''gold standard'' four tests were evaluated crosswise using 824 cervical smears pretested by HC2. The tests evaluated were the L1-PCR-based assays PGMY09/11 LBA, HPV DNA Chip and SPF LiPA and an E1 consensus PCR followed by cycle sequencing (E1-PCR). A subset of 265 samples was tested in addition with the GP5þ/6þ reverse line blot assay. Differences were noted in the sensitivity and range for specific HPV types, e.g. with detection rates for HPV53 ranging from 2.3% to 11.6%. HPV16 was the most prevalent type detected by all tests except for the SPF-10 LiPa, which detected HPV31 more often. Kappa values calculated ranged from poor (k ¼ 0.20) to intermediate (k ¼ 0.54) for HPV positivity, but were higher for high-risk type positivity (k ¼ 0.31-0.61) and best for recognition of HPV16 (k ¼ 0.53-0.72). The analytical sensitivity of the tests ranged between 15% and 97% for individual types and specificity was highly dependent on which test system was used as ''gold standard'' for the analysis. The results of histology were used for calculation of clinical sensitivity and specificity. E1-PCR, PGMY09/11 LBA and SPF-10 LiPA had a high clinical sensitivity (>95%) for the detection of cervical intraepithelial neoplasia 2 or higher, whereas the HPV DNA Chip reached only 84.1%.
Journal of Clinical Microbiology, 2007
The development of cervical cancer is strongly associated with the presence of persistent high-risk (HR) human papillomavirus (HPV) infection. Recently, the commercially manufactured PCR-based Roche AMPLICOR (AMP) and LINEAR ARRAY (LA) HPV tests have become available for HPV detection. However, knowledge of their clinical performance compared to the U.S. Food and Drug Administration-approved Hybrid Capture 2 (HC2) assay is limited. This study evaluated the concordance between the HC2, AMP, and LA tests in detecting HR-HPV among a cohort of 1,679 women with previous abnormal Pap smear results. Overall, 1,393 specimens (81.3%) generated concordant results for HR-HPV presence or absence by the three assays. The concordance levels were substantial between the HC2 and AMP tests (84.4%, ؍ 0.6419) and between the HC2 and LA tests (84.0%, ؍ 0.6341) and nearly perfect between the AMP and LA tests (97.8%, ؍ 0.9441). HR-HPV prevalence, as detected by the AMP or LA tests, was significantly higher among women with cytological or histological high-grade disease (CIN2 or greater) than that detected by HC2 (P < 0.0001). The AMP and LA tests exhibited greater sensitivity, but lower specificity, than HC2 for detecting HR-HPV among this cohort of women with underlying cervical abnormalities, particularly among subjects with histologically proven high-grade disease. Both PCR-based HPV tests may be valuable in the management of care for women with underlying cervical abnormalities, in predicting treatment success, and in studying the clearance or acquisition of new infections.
A New PCR-Based Mass Spectrometry System for High-Risk HPV, Part I: Methods
American Journal of Clinical Pathology, 2011
Infection with high-risk (HR) human papillomaviruses (HPVs) has been confirmed as the necessary cause of cervical cancer. There are many studies that have established and confirmed the relationship of specific HPV types and the risk of invasive cervical cancer. We have developed a novel genotyping method for detecting 14 HR-HPV genotypes simultaneously with MassARRAY (Sequenom, San Diego, CA) technique based on the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). All 14 HPVs showed high specificities and high sensitivities in the plasmid test; lower detection limits for each genotype were from 10 to 100 copies. Furthermore, the MS system has highthroughput capacities, capable of processing, with type-specific output, 4,500 samples in 24 hours. The MS HPV assay is a sensitive and useful tool for HPV genotyping. It has the potential to be suitable for largescale epidemiologic studies and routine diagnostic clinical applications owing to its high-throughput capacity, high sensitivity, and low cost per case.