Single-tube real-time nested polymerase chain reaction for detecting human papillomavirus DNA.

A single-tube real-time nested polymerase chain reaction (PCR) was developed to detect human Papillomavirus (HPV) DNA in a closed tube system. The oligonucleotide primers MY09/MY11 and GP5+/GP6+ were included in contiguous reactions, thus eliminating the need to transfer first round PCR product into a second tube. The sensitivity and specificity of the optimized single-tube nested PCR were comparable with that achieved by two separate reactions on a conventional thermal block system using serial dilutions derived from plasmids containing DNA of 20 HPV types. A minimum of 10 copies of HPV types 11 and 16 DNA could be detected by both systems. In clinical samples, HPV types 1A, 2, 3, 5, 6-8, 10, 11, 14, 16, 17, 18, 20, 31, 33, 35, 39, 45, 49, 50, 52-54, 57, 62, 66, 70, CP8304 and LVX82/MM7 could be detected by both PCR methods. A total of 145 samples collected from patients were tested for the presence of HPV DNA with the two PCR systems; 124 (86.1%) of 144 samples gave concordant results in both assays. The HPV DNA positive PCR amplicons were typed and concordant results were obtained in 47 of 67 positive samples tested in both amplicons. In samples containing multiple HPV types at least one type was common to both amplicons.     

The characteristics of human papillomavirus DNA in head and neck cancers and papillomas

AIM: To determine the prevalence, type, physical state, and viral load of human papillomavirus (HPV) DNA in cases of head and neck cancer and recurrent respiratory papillomatosis (RRP). METHODS: The prevalence and type of HPV DNA was determined in 27 fresh frozen tissue specimens from patients with head and neck cancers and 16 specimens from 10 patients with RRP by MY09/MY11 and GP5+/GP6+ nested polymerase chain reaction (PCR) and subsequent restriction enzyme cleavage. The physical state of HPV DNA was analysed by E1, E2, and E1E2 specific PCRs and Southern blot hybridisation (SBH). RESULTS: HPV DNA was detected in 13 of 27 cancers and 10 of 10 papillomas. Both low risk HPV-6 and HPV-11 and high risk HPV-16 were present in cancers in low copy numbers, whereas papillomas exclusively harboured low risk HPV-6 and HPV-11. E1E2 PCRs failed to determine the physical state of HPV in cancers except one case where HPV-6 DNA was integrated. In contrast to cancers, all papillomas showed the episomal state of HPV DNA and a relatively higher viral load. CONCLUSIONS: Based on the prevalence, type, physical state, and copy number of HPV DNA, cancers and papillomas tend to show a different HPV DNA profile. The 100% positivity rate of low risk HPV types confirms the role of HPV-6 and HPV-11 in the aetiology of RRP.     

Detection of human papillomavirus from archival tissues in cervical cancer patients in Mauritius.

BACKGROUND: Around half a million new cases of cervical cancer are diagnosed worldwide each year, accounting for almost 300,000 deaths. Development of cervical cancer can be multi-factorial, but high-risk human papillomaviruses (HPV) have been associated with the aetiology of cervical cancer. It is believed that HPV DNA integrates into the host DNA causing abnormal cell growth with cells becoming carcinogenic and spreading metastatically. In Mauritius, cervical cancer account for 65% of gynaecological cancers and 3.4% of the cervical cancers are diagnosed at the stage of carcinoma in situ. OBJECTIVES: To determine the prevalence of HPV in histological samples from patients with cervical cancer in Mauritius. STUDY DESIGN: DNA from archival cervical samples from a cohort of 65 patients suffering from cervical cancer and controls from Mauritius were tested for the presence of HPV using MY09/11 and GP5+/6+ primer sets. RESULTS: In a cohort of 65 patients from Mauritius, diagnosed with cervical cancer in the year 2000, 19% of cervical histology sections were found to be positive for the presence of high-grade HPV, exclusively HPV18 using MY09 and MY11 primers. Only 15% of the Mauritian population is over 50 years of age, whereas 66% (35) of the diagnosed cases of cervical cancer were seen in patients above 50 years with 50% (5) affected with HPV. These findings suggest that for an infection with HPV to develop into cancer may take years if not decades. Differences were noted using two different primer sets, MY09/11 and GP5+/6+. The latter produce a much smaller amplicon (150bp) compared to the former ( approximately 450bp). Seven additional positive cases were detected with the GP5+/6+ primer set, resulting in an apparent prevalence of 32% as compared to the 19% seen with the MY09/11 primer set. This may indicate that some degradation of the target DNA has occurred during processing and storage of histological samples. CONCLUSION: Using primer sets MY09/11 and GP5+/6+, only HPV type 18 was found in the Mauritian cohort with a prevalence of 32%.     

PCR detection of human papillomavirus: comparison between MY09/MY11 and GP5+/GP6+ primer systems.

Human papillomavirus (HPV) is an etiologic agent of cervical cancer and is the most common sexually transmitted disease in women. PCR amplification of HPV genomes is the most sensitive method for the detection of cervicovaginal HPV. We have compared the two most commonly used PCR primer sets, MY09/MY11 (MY-PCR) and GP5+/GP6+ (GP+-PCR), for the detection of HPV DNA in cervicovaginal lavage samples from 208 women. Oligonucleotide probes for 39 different HPV types were used. Both primer sets amplified a wide spectrum of HPV genotypes and detected similar overall prevalences of 45% (94 of 208) and 43% (89 of 208), respectively. The MY-PCR system detected 27 of 30 (90%) samples with multiple HPV types, whereas the GP+-PCR system detected 14 of 30 (47%) samples with multiple HPV types. Differences in the detection of HPV types 35, 53, and 61 were noted between the two primer systems. Serial dilution of plasmid templates indicated a 3-log decrease in the amplification of HPV type 35 by MY-PCR and HPV types 53 and 61 by GP+-PCR. These results indicate that although the MY-PCR and GP+-PCR identified nearly equivalent prevalences of HPV in a set of clinical samples, differences in the detection of specific types and infections with multiple types were found. Differences in the sensitivities and characteristics of the PCR systems for the detection of HPV within clinical samples should be considered when comparing data between studies and/or in designing new studies or clinical trials.     

Comparison of five different polymerase chain reaction methods for detection of human papillomavirus in cervical cell specimens

The polymerase chain reaction (PCR) methods enable the detection of large number of human papillomavirus (HPV) genotypes that infect the anogenital tract. In this study, two groups of cervical scrapes with abnormal cytomorphology were analysed. The first group was tested with three sets of consensus primers located within the L1 region of HPV genome, MY09/MY11 (i.e. MY), L1C1/L1C2-1/L1C2-2 (i.e. LC) and pI-1/pI-2 (i.e. pI) primer sets, while the second group of samples, which were all negative with the MY primers, was tested further with the LC primers, as well as with the GP5/GP6 (i.e. GP) primers. The GP primers were used in the nested PCR following amplification with the MY primers (i.e. MY/GP nested PCR). Samples from both groups were also tested with type-specific primers for HPV types 6/11, 16, 18, 31 and 33. In the first study group (N=164) there were 76.2% positive results obtained with at least one set of consensus primers. There were 62.2, 39, 62.2 and 59.1% positive results obtained with the MY, the pI, the LC and the HPV type-specific primer sets, respectively. The best results were obtained when both the MY and the LC primer sets were used, because in combination they detected 75% positive samples compared to 62.2% when used alone. There were 2. 4% samples negative with all consensus primers, but positive with one of the HPV type-specific primers, which increased the overall positivity rate to 78.6%. In the second study group (N=250) there were 8.4, 38.8 and 4% samples positive with the LC primers, the nested MY/GP and the HPV type-specific primer sets, respectively. Thus, the use of the MY/GP nested PCR increased significantly the positivity rate of HPV DNA detection and should be used for samples with a low copy number of HPV DNA. In conclusion, the following diagnostic protocol would be appropriate for detection of cancer-related HPVs: preselection of samples with the MY and the LC primers, additional amplification of the MY- and the LC-negative samples with the MY/GP nested PCR and HPV typing of consensus PCR-positive samples with the HPV type-specific primers.     

Nested PCR with the PGMY09/11 and GP5(+)/6(+) primer sets improves detection of HPV DNA in cervical samples

Based on epidemiological and research evidence, HPV has a causal role in cervical carcinogenesis. Several HPV detection methods exist to date; the most commonly used method for detection of genital HPVs consists of nested PCR using the MY09/11 and GP5(+)/6(+) primer sets (MY/GP(+)). Recently, the PGMY09/11 primer set, a modified version of the MY09/11 primer set, was introduced for single PCR and was found to detect a wider range of HPV types. The next logical step was taken and the efficacy of nested PCR using the PGMY09/11 and GP5(+)/6(+) primer sets (PGMY/GP(+)) to detect HPV in cervical samples was evaluated. In this comparative study, nested PCR using the novel PGMY/GP(+) primer set combination was found to be more type sensitive than the nested PCR with the MY/GP(+) primer sets, detecting a wider range of HPV types, low copy HPVs, and better characterizing samples infected with multiple strains of HPV. Standardization and use of the PGMY/GP(+) PCR system could aid physicians in providing more efficient HPV screening and better treatment for patients.     

Detection and typing of human papillomavirus by e6 nested multiplex PCR

A nested multiplex PCR (NMPCR) assay that combines degenerate E6/E7 consensus primers and type-specific primers was evaluated for the detection and typing of human papillomavirus (HPV) genotypes 6/11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 56, 58, 59, 66, and 68 using HPV DNA-containing plasmids and cervical scrapes (n = 1,525). The performance of the NMPCR assay relative to that of conventional PCR with MY09-MY11 and GP5+-GP6+ primers, and nested PCR with these two primer sets (MY/GP) was evaluated in 495 cervical scrapes with corresponding histologic and cytologic findings. HPV prevalence rates determined with the NMPCR assay were 34.7% (102 of 294) in the absence of cervical intraepithelial neoplasia (CIN 0), 94.2% (113 of 120) in the presence of mild or moderate dysplasia (CIN I/II), and 97.8% (44 of 45) in the presence of severe dysplasia (CIN III). The combination of all four HPV detection methods applied in the study was taken as "gold standard": in all three morphological subgroups the NMPCR assay had significantly (P < 0.0001) higher sensitivities than the MY09-MY11 and GP5+-GP6+ assays and sensitivities comparable or equal to those of the MY/GP assay. All 18 HPV genotypes investigated were detected among the clinical samples. The ratio of high- to low-risk HPV genotypes increased from 4:1 (80 of 103) in CIN 0 to 19:1 (149 of 157) in CIN I to III. Multiple infections were detected in 47.9% (124 of 259) of the patients. In conclusion, the novel NMPCR method is a sensitive and useful tool for HPV DNA detection, especially when exact HPV genotyping and the identification of multiple HPV infections are required.     

Classical Molecular Tests Using Urine Samples as a Potential Screening Tool for Human Papillomavirus Detection in Human Immunodeficiency Virus-Infected Women

Human papillomavirus (HPV) is the main risk factor associated with the development of cervical cancer (CC); however, there are other factors, such as immunosuppression caused by the human immunodeficiency virus (HIV), that favor progression of the illness. This study was thus aimed at evaluating the functionality of classical PCR-based molecular tests for the generic identification of HPV DNA (GP5+/GP6+, MY09/MY11, and pU1M/2R primers, individually or in combination) using cervical and urine samples from 194 HIV-positive women. Infected samples were tested with type-specific primers for six high-risk types (HPV-16, -18, -31, -33, -45, and -58) and two low-risk types (HPV-6 and -11). HPV infection prevalence rates were 70.1% for the cervical samples and 63.9% for the urine samples. HPV-16 was the most prevalent viral type in the cervical and urine samples, with higher rates of multiple infections than single infections detected in such samples. HPV DNA detection by PCR (mainly with the pU1M/2R primer set) in urine samples was positively associated with abnormal cytological findings (atypical squamous cells of undetermined significance/squamous intraepithelial lesions [ASCUS/SIL]). It was determined that the operative characteristics for detection of cytological abnormalities were similar for cervical and urine samples. This suggested using PCR for the detection of HPV DNA in urine samples as a potential screening strategy for CC prevention in future prevention and control programs along with currently implemented strategies for reducing the impact of the disease, i.e., urine samples are economical, are easy to collect, have wide acceptability among women, and have operative characteristics similar to those of cervical samples.     

Use of PGMY primers in L1 consensus PCR improves detection of human papillomavirus DNA in genital samples

The novel PGMY L1 consensus primer pair is more sensitive than the MY09 and MY11 primer mix for detection and typing with PCR of human papillomavirus (HPV) DNA in genital specimens. We assessed the diagnostic yield of PGMY primers for the detection and typing of HPV by comparing the results obtained with PGMY09/PGMY11 and MY09/MY11/HMB01 on 299 genital samples. Amplicons generated with PGMY primers were typed with the line blot assay (PGMY-line blot), while HPV amplicons obtained with the degenerate primer pool MY09/MY11/HMB01 were detected with type-specific radiolabeled probes in a dot blot assay (standard consensus PCR test). Cervicovaginal lavage samples (N = 272) and cervical scrape samples (N = 27) were tested in parallel with both PCR tests. The PGMY-line blot test detected the presence of HPV DNA more frequently than the standard consensus PCR assay. The concordance for HPV typing between the two assays was 84.3% (214 of 255 samples), for a good kappa value of 0.69. Of the 177 samples containing HPV DNA by at least one method, 40 samples contained at least one HPV type detected only with PGMY-line blot, whereas positivity exclusively with the standard consensus PCR test was found for only 7 samples (P < 0.001). HPV types 45 and 52 were especially more frequently detected with PGMY than MY primers. However, most HPV types were better amplified with PGMY primers, including HPV-16. Samples with discordant results between the two PCR assays more frequently contained multiple HPV types. Studies using PGMY instead of MY primers have the potential to report higher detection rates of HPV infection not only for newer HPV types but also for well-known genital types.     

A two-tier polymerase chain reaction direct sequencing method for detecting and typing human papillomaviruses in pathological specimens.

An in-house polymerase chain reaction direct sequencing (PCR-DS) approach for HPV detection and typing was developed, taking advantage of two widely used pairs of human papillomavirus (HPV)-specific PCR primers, MY09/MY11 and GP5/GP6, and 33P-labeled dideoxynucleotides. In this study, 105 pathological specimens were examined: 89% were diagnosed as cervical intraepithelial neoplasia (CIN) grade I-III, 76.2% were HPV-positive by PCR-DS. The PCR using GP5/GP6 (first tier) and MY09/MY11 primers (second tier for the GP5/GP6-negative samples) detected additional 15%-25% HPV-positive samples compared with each pair used separately. Direct sequencing was then used to type the HPV. A readout of a sequence as short as 34 nucleotides within a specific region in the L1 gene is sufficient to type known or novel sequences. Because of its high sensitivity and cost-effectiveness, the two-tier PCR-DS was adopted by the authors as the current method of choice for HPV diagnosis with ultimate sequence precision.     

Routine genotyping of human papillomavirus samples in Denmark

In order to examine a sensitive unbiased consensus PCR with routine sequencing for HPV typing, we analysed Danish male and female patients suspected of having an HPV infection. We used the well-characterised nested PCR setting with MY09/MY11 and GP5+/GP6+ primers, followed by routine cycle sequencing. Of 1283 clinical samples from female patients based on suspected HPV infection, we found 379 (29%) negatives and 894 (70%) positives. Samples containing >5000 HPV copies/ml were genotyped by sequencing. Of the 552 HPV genotyped samples from women >15 years of age, 398 were characterised as high-risk types and the remaining 154 as low-risk types. The most commonly found high-risk types were HPV-16, HPV-31, HPV-33, HPV-18, HPV-58, and HPV-52, and the most commonly found low-risk types were HPV-6, HPV-53 and HPV-11. In addition, we observed that other typing assays could not perform as sensitively or accurately as the nested PCR/cycle sequencing method used in this study. For instance, 87 out of 552 genotyped samples could not have been typed correctly in the Hybrid Capture II assay. Of these 87 samples, 46 (53%) were considered as high-risk types.     

Detection of high-risk subtypes of human papillomavirus in cervical swabs: routine use of the Digene Hybrid Capture assay and polymerase chain reaction analysis

Human papillomaviruses (HPVs) are major causative agents in the pathogenesis of cervical cancer, and more than twenty types are associated with its development. With the introduction of liquid-based preparation systems, it is envisaged that large-scale HPV testing will be established in the near future. Preliminary studies demonstrate the accessibility of these samples for DNA testing using both the Digene Hybrid Capture assay (DHCA) and polymerase chain reaction (PCR) techniques. This study aims to assess the validity and sensitivity of the DHCA system to detect high-risk HPV DNA, using two sets of HPV consensus primers (Gp5+/Gp6+ and MY09/MY11) in tandem with routine assessment of cervical smear and biopsy samples. Results indicate that the combination of DHCA and PCR detects more high-grade lesions than does the DHCA alone. DHCA-negative cases were categorised by subsequent PCR amplification into low-grade HPV-negative (12/16) cervical lesions and high-grade HPV-positive (7/9) cervical lesions. Gp5+/Gp6+ primers were less sensitive in detecting HPV-positive samples than was the MY09/MY11 primer set. These results support the use of high-risk HPV testing by DHCA, with subsequent analysis of DHCA-negative samples by PCR using the MY09/MY11 primers.     

Reliable high risk HPV DNA testing by polymerase chain reaction: an intermethod and intramethod comparison

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 different 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 different laboratories with reference laboratory A for the detection of HR-HPV varied between 75% and 100% (kappa 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% (kappa 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.     

PCR detection of human papillomavirus of the mucosa: comparison between MY09/11 and GP5+/6+ primer sets

BACKGROUND AND OBJECTIVES: Human papillomaviruses (HPV) are the causal agent for the development of carcinomas in the cervix uteri and further pathological changes of the skin including mucosa, particularly warts, condylomas and dysplasias. Therefore, we investigated the efficacy of different consensus primers pairs for HPV detection by PCR using brushed samples from the oral cavity in comparison with samples from the cervix uteri. STUDY DESIGN: In the present study, we used two well-established sets of PCR primers in different combinations for the detection of HPV DNA in 106 non-invasive brush biopsy samples of the oral mucosa and 56 samples from the cervix uteri. Direct sequencing of PCR products in all cases determined HPV genotype and specificity. RESULTS: Overall, HPV was detected in 69 of 106 oral mucosa samples. HPV specific amplicons were obtained in 35.8% (N = 38) when using GP5+/6+ primers. The positivity rate was increased to 65.1% in a GP5+/6+ auto-nested PCR approach. In contrast, MY9/11 PCR and nested PCR with MY9/11 outer followed by GP5+/6+ inner primers yielded 2.2% and 16.1%, respectively. In gynaecological samples, PCR results were similar independent of the primer combination used. Thus, DNA quality and DNA content could be additional factors influencing the rate of positivity. CONCLUSION: For oral mucosa samples, auto-nested GP5+/6+ PCR is in our hands the most suitable approach for epidemiological studies because of its high sensitivity, high reliability and reproducibility as well as its relatively simple laboratory procedure.     

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.     

Detection of human papillomavirus DNA in cervical lavage specimens by a nonisotopic consensus PCR assay.

A gene amplification method that combines PCR with an enzyme immunoassay (PCR-EIA) for quantitation of amplified DNA was developed for the detection of human papillomavirus (HPV). Samples were amplified with consensus primers MY09 and MY11. Amplified DNA products were reacted in solution with type-specific nested RNA probes labelled with digoxigenin-11-UTP. Hybrids were captured on a microtiter plate coated with an antidigoxigenin antibody. Bound DNA-RNA hybrids were quantitated by the addition of an alkaline phosphatase-labelled monoclonal antibody directed against DNA-RNA hybrids and a fluorogenic substrate. The detection limit of PCR-EIA was six copies of HPV type 18 DNA in the original specimen. The assay was used to assess HPV infection of the uterine cervixes of 65 women referred to a colposcopy clinic. In 66 cervicovaginal lavage specimens, all 23 HPV strains detected by a standard isotopic PCR assay were also detected by the PCR-EIA (sensitivity, 100%; 95% confidence interval, 85.2 to 100%). Forty-two of the 43 samples that did not contain HPV types 6/11, 16, 18, 31, 33, 35, and 45 were also negative by PCR-EIA, for a specificity of 97.7%. Low-level cross-reactivity was encountered between HPV types 18 and 45 as well as between types 33 and 58. PCR-EIA provides a convenient means of objectively measuring PCR-amplified HPV DNA from common genital HPV types.     

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.     

Detection of human papillomavirus in large cell neuroendocrine carcinoma of the uterine cervix: a study of 12 cases

AIM: To investigate the role of human papillomavirus (HPV) in large cell neuroendocrine carcinoma (LCNEC) of the uterine cervix. METHODS: Twelve archival, immunohistochemically and/or electron microscopically confirmed cases of cervical LCNEC were studied. Non-isotopic in situ hybridisation (NISH) was performed on the formalin fixed, paraffin wax embedded biopsies using digoxigenin labelled probes to HPV types 6, 11, 16, 18, 31, and 33. The tumours were then subjected to polymerase chain reaction (PCR) analysis using GP5+/GP6+ consensus primers to the HPV L1 gene, in addition to type specific primers to the E6 and E6/E7 genes. RESULTS: HPV-16 was detected by NISH and/or PCR in seven of the 12 carcinomas. Two additional tumours were HPV-18 positive by NISH and/or PCR. HPV DNA was not detected in the three remaining cases. CONCLUSION: Integration of high risk HPV, in particular type 16 and to a lesser extent type 18, is associated with this uncommon variant of cervical carcinoma.     

Human papillomavirus (HPV) study of 691 pathological specimens from Quebec by PCR-direct sequencing approach

Human papillomaviruses (HPV) are etiological agents of cervical cancer. In order to address clinical demand for HPV detection and sequence typing, mostly in pre-cancerous cervical lesions, we applied our two-tier PCR-direct sequencing (PCR-DS) approach based on the use of both MY09/MY11 and GP5 + /GP6 + sets of primers. We tested 691 pathological specimens, all of which were biopsies, 75% of which were diagnosed histologically as cervical intraepithelial neoplasia (CIN) grades I-III. In total, 484 samples (70%) tested HPV-positive, yielding 531 HPV sequences from 47 HPV types, including two novel types. Four most frequently found HPV types accounted for 52.9% of all isolates: HPV6, 16, 11, and 31 (21.5%, 20.0%, 7.0%, and 4.5%, respectively). Some interesting results are the following: all currently known high-risk HPV (14 types) and low-risk HPV (6 types) were detected; HPV18 was not the 1st or 2nd but rather the 4th-5th most frequent high-risk HPV type; the highest detection rate for HPV (86%) among samples suspected to be HPV-infected was found in the youngest age group (0-10 years old), including 70% (44/63) "genital" HPV types; HPV types of undetermined cervical cancer risk represented 19% and of the total HPV isolates but were strongly increased in co-infections (36.5% of all isolates). To our knowledge, this is the largest sequencing-based study of HPV. The HPV types of unknown cancer risk, representing the majority of the known HPV types, 27 of the 47 types detected in this study, are not likely to play a major role in cervical cancer because their prevalence in CIN-I, II, and III declines from 16% to 8% to 2.5%. The two-tier PCR-DS method provides greater sensitivity than cycle sequencing using only one pair of primers. It could be used in a simple laboratory setting for quick and reliable typing of known and novel HPV from clinical specimens with fine sequence precision. It could also be applied to anti-cancer vaccine development.