Comparison of the performance of different HPV genotyping methods for detecting genital HPV types
Institute of Medical Biostatistics, Epidemiology and Informatics, Hospital of the University of Mainz, Germany. Journal of Medical Virology
(Impact Factor: 2.35).
07/2008; 80(7):1264-74. DOI: 10.1002/jmv.21191
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 GP5+/6+ 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=0.20) to intermediate (k=0.54) for HPV positivity, but were higher for high-risk type positivity (k=0.31-0.61) and best for recognition of HPV16 (k=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%.
- "Cross-hybridization between related HPV genomes compromises genotyping accuracy[37,64,72,73]. Primers and probes differ with PGMY11/09 for LA, SPF 10 for SPF 10 -LiPA 25 , and GP+ for HPV DNA chip and GP+ reverse line blot, resulting in large differences in sensitivity of detecting HPV infection and individual genotypes among the tests[14,27,28]. With the rapid revolution in technology, NGS applied to HPV genotyping provides high sensitivity in capturing even low frequency types, reflecting unprecedented coverage of viral copies and almost no limit in the number of types detected in a sample by querying reference sequence databases[72,747576. "
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ABSTRACT: Multiple-type human papillomaviruses (HPV) infection presents a greater risk for persistence in asymptomatic individuals and may accelerate cancer development. To extend the scope of HPV types defined by probe-based assays, multiplexing deep sequencing of HPV L1, coupled with an HPV-QUEST genotyping server and a bioinformatic pipeline, was established and applied to survey the diversity of HPV genotypes among a subset of healthy men from the HPV in Men (HIM) Multinational Study. Twenty-one HPV genotypes (12 high-risk and 9 low-risk) were detected in the genital area from 18 asymptomatic individuals. A single HPV type, either HPV16, HPV6b or HPV83, was detected in 7 individuals, while coinfection by 2 to 5 high-risk and/or low-risk genotypes was identified in the other 11 participants. In two individuals studied for over one year, HPV16 persisted, while fluctuations of coinfecting genotypes occurred. HPV L1 regions were generally identical between query and reference sequences, although nonsynonymous and synonymous nucleotide polymorphisms of HPV16, 18, 31, 35h, 59, 70, 73, cand85, 6b, 62, 81, 83, cand89 or JEB2 L1 genotypes, mostly unidentified by linear array, were evident. Deep sequencing coupled with HPV-QUEST provides efficient and unambiguous classification of HPV genotypes in multiple-type HPV infection in host ecosystems.
Available from: Ei Kawahara
- "In the examination of mixed HPVtype plasmids for multiple HPV-type infections, HPV 16 could not be detected in samples that included the minimum detection sensitivity concentration when DNA of other HPV types was added at concentrations greater than 250 × 10 3 copies/test. The reason for this is unknown, but competition for reagents can result in a loss of sensitivity and the detection of viral types present at lower copy numbers (Klug et al., 2008) and may lead even to false-negative results. The cloning and sequencing method, which is considered to obtain the most reliable results, confirmed the validity of the genotypic specificity of the amplified PCR products and confirmed the reliability of a positive result in this assay. "
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ABSTRACT: Regional differences in human papillomavirus (HPV) genotypes and the presence of mixed HPV infections may affect adversely the efficacy of the HPV vaccine. Therefore, a simple and high-throughput HPV genotyping system is required. Recently, a novel HPV genotyping kit (the Mebgen™ HPV kit) was developed. This kit uses multiplex PCR and Luminex xMAP™ technology to detect 13 types of high-risk HPVs and an internal control in a 96-well format. In the present study, the analytical performance of the kit was examined using HPV plasmid DNA. All 13 types of HPVs were detected with a minimum detection sensitivity of 250 copies/test, and highly specific signals were observed. HPV 16 plasmid was detected in samples containing mixtures with other HPV-type plasmids in ratios ranging from 1:1 to 1:1,000. No cross reactivity was observed with DNA from 27 types of other infectious microbes. A clinical evaluation was carried out using cervical samples from 356 patients with persistent abnormal smears diagnosed at mass public health screenings for cervical cancer. The samples were preserved in Tacas™ medium until analysis. HPV was detected in 162 (45.5%) samples including 110 (67.9%) with single infections and 52 (32.1%) with multiple infections. The type distribution of the 13 high-risk HPVs was as follows: 28.4% HPV 16, 11.7% HPV 18, 6.8% HPV 31, 3.1% HPV 33, 3.7% HPV 35, 9.3% HPV 39, 1.9% HPV 45, 8.6% HPV 51, 37.0% HPV 52, 9.3% HPV 56, 16.7% HPV 58, 3.7% HPV 59, and 1.9% HPV 68. To evaluate sample stability over time, changes in the detection of HPV DNA derived from HeLa and SiHa cells were measured in 3 types of liquid-based cytology media. HPV DNA was detected in Tacas and Thinprep™ samples after storage at 4°C or 30°C for 4 weeks and within 1 week of collection in Surepath™ samples. These results suggest that this newly developed HPV genotyping kit is suitable for use in both clinical applications and large-scale epidemiological studies.
Available from: Alexander Luyten
- "All samples that tested positive for HR-HPV with HC2 and 10% (every tenth) of all HC2-negative samples underwent HPV genotyping. HPV genotyping was performed as described previously using SPF-10-PCR, followed by Reverse Line Probe Assay LiPA Extra (SPF-10-PCR) . Briefly, total DNA was isolated from the cervical samples with the use of a MagNAPure device (Roche, Indianapolis, IN) and analyzed with INNO-LiPA Extra HPV prototype assay (Innogenetics, Inc, Gent, Belgium) according to the manufacturer’s instructions. "
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High-risk human papilloma virus (HR-HPV) infection is associated with the development of cervical cancer. HPV vaccination reduces the risk of developing malignant lesions and is expected to change the dynamics of HPV transmission. Data from non-vaccinated women may provide an important benchmark to allow the impact of HPV vaccination programs to be assessed.This study was designed to prospectively determine the changing dynamics of HR-HPV infection and associated genital diseases in young women, most of whom were non-vaccinated.
Data from a population-based cohort study, comprising women of two predefined birth cohorts (women born in 1983/84 or 1988/89), were analyzed between 19 October 2009 and 31 December 2010 to determine risk factors for high-risk HPV infection and the association between specific HR-HPV types and atypical Pap smear test results. HPV status was determined by Hybrid Capture 2 (HC2) assay and genotyping.
The prevalence of HR-HPV was 22.8% in the 1983/84 cohort (150/659) and 23.7% in the 1988/99 cohort (142/599). Only the number of sexual partners was a significant risk factor for HPV infection (odds ratios 22.687 and 6.124 for more than five versus one partner 84 cohort,/84 and 1988/89 cohorts, respectively) in multivariate analysis. HPV16 positive-women were significantly more likely to have abnormal Pap smears of any degree than HPV16-negative women (22.0% versus 3.61%, p < 0.0001 for the 1983/84 cohort and 9.09% versus 2.52%, p = 0.0482 for the 1988/89 cohort). CIN3 was diagnosed in six women 84 cohort,/84 cohort and two in the 1988/89 cohort. All women with CIN3 tested positive for HC2-HR and all six CIN3 cases 84 cohort,/84 cohort tested positive for HPV16. In the 1988/89 cohort, the rate of HPV16 infection was significantly lower in vaccinated than non-vaccinated women (1.59% versus 8.88%; p = 0.003).
HR-HPV infection was highly prevalent in both cohorts and associated with an increased risk of abnormal Pap smears and biopsy proven CIN2+. HPV16 infection was associated with a high risk of clinically relevant lesions. HPV vaccination significantly decreased the risk of HPV16 infection.
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