Detection by PCR of human papillomavirus in Colombia: Comparison of GP5+/6+ and MY09/11 primer sets

The aims of this study were to determine the prevalence of HPV infection and evaluate the concordance and performance of two primer sets for detecting single and multiple viral infections. A total of 1810 Colombian women were enrolled in the study, and molecular, cytological and epidemiological analyses were performed. Both concordance and performance of two different PCR amplification primer sets (GP5+/6+ and MY09/11) were assessed. The results showed that 60.2% of females with positive HPV DNA were infected by more than one viral type. The OR for multiple infections was 18.2 when using the MY09/11 primer set and 6.52 with the GP5+/6+ primer set. The results also showed an association between GP5+/6+ positivity and the severity of the disease regarding the cytological findings. It was also found that using a single primer set led to underestimating the prevalence for HPV infection. The simultaneous use of these primer sets is an important tool for the detection of HPV DNA, being equally relevant for identifying multiple infections and low viral DNA copies. This study highlights the importance of suitable assessment of HPV epidemiological profiles; screening programs must also be strengthened to broaden the coverage of the most vulnerable populations.     

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.     

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.     

Cervical cancer and human papillomavirus among slum dwellers in India

Almost half a million new cases of cervical cancer are diagnosed each year worldwide. Human papillomavirus is recognised as one of the leading causes and is associated with 90% of cases. However, other risk factors (e.g., age of first sexual contact, number of sexual partners, multiparity, diet, genetic predisposition and environment) are also associated with cervical cancer. The present retrospective study is performed on a cohort of women from the slums of a major Indian city. The patients are aged between 38 and 68 years (mean: 49.3 years) and are multiparous (mean number of children: 3.4). In this group, 61% have a history of miscarriages. Histological sections from cone biopsy are tested for the presence of high-grade human papillomavirus (HPV) using GP5+/GP6+ and MY09/MY11 primers and a set of beta3-globin primers. Only 33% of the cancer patients studied were positive for high-grade HPV DNA, suggesting that predisposition to cervical cancer in this cohort is not highly associated with HPV, and that other risk factors may increase the risk of cervical cancer.     

Human papillomavirus associated with oesophageal cancer

AIM: To study the prevalence and the different types of human papillomavirus (HPV) in patients with oesophageal cancer from a high risk area of South Africa (Transkei). METHODS: DNA samples from 50 paraffin wax embedded tissue sections were analysed by nested polymerase chain reaction (PCR) using the degenerate HPV L1 consensus primer pairs MY09/MY11 and GP5+/GP6+. Positive PCR samples were subjected to DNA sequence analysis. RESULTS: HPV DNA was detected in 23 of the 50 samples. Sequence analysis revealed that most patients (11) harboured DNA to HPV type 11, whereas other types included DNA HPV type 39 (seven patients), type 16 (two patients), and type 52 (one patient). HPV type 39 has not previously been shown to be associated with oesophageal cancer. In contrast to earlier studies that have found HPV type 16 to be more frequently associated with oesophageal cancer, HPV type 11 was the predominant subtype in this study. CONCLUSIONS: The high frequency of occurrence of HPV in oesophageal tumours (23 of 50 patients; 46%) implicates HPV as one of the possible aetiological factors in this disease. The finding that the low risk HPV subtypes predominate indicates that transformation may be effected via the E6 and E7 proteins.     

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.     

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.     

The diagnostic performance of classical molecular tests used for detecting human papillomavirus

Cervical samples were evaluated for human papillomavirus (HPV) presence using the hybrid capture-2 (HC2) assay and the polymerase chain reaction (PCR) with three different primer sets (GP5+/6+, MY09/11 and pU1M/2R). PCR results were compared to HC2 and results of all assays were compared to cytological and colposcopy findings. Post-test probability was assessed in individual assays and test combinations. HPV-DNA prevalence was 36.5% with HC2 and 55.2% with PCR. MY09/11 detected HPV-DNA in 38% of samples, GP5+/6+ in 19.1% and pU1M/2R in 16.4%. pU1M/2R and HC2 had the highest concordance (75.31%, k=0.39 in the whole population; 74.1%, k=0.5 in women with abnormal cytology). pU1M/2R had the best diagnostic performance, including optimal post-test probabilities and cervical abnormality detection (individually or in a panel of tests). Women positive for pU1M/2R may be at higher risk of disease progression; the assay performance when combined with a Pap smear in cervical cancer screening programs should be evaluated.     

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.     

Improved amplification of genital human papillomaviruses

Genital human papillomaviruses (HPVs) are commonly detected from clinical samples by consensus PCR methods. Two commonly used primer systems, the MY09-MY11 (MY09/11) primers and the GP5+-GP6+ (GP5+/6+) primers, amplify a broad spectrum of HPV genotypes, but with various levels of sensitivity among the HPV types. Analysis of the primer-target sequence homology for the MY09/11 primers showed an association between inefficient amplification of HPV types and the number and position of mismatches, despite accommodation of sequence variation by inclusion of degenerate base sites. The MY09/11 primers were redesigned to increase the sensitivity of amplification across the type spectrum by using the same primer binding regions in the L1 open reading frame. Sequence heterogeneity was accommodated by designing multiple primer sequences that were combined into an upstream pool of 5 oligonucleotides (PGMY11) and a downstream pool of 13 oligonucleotides (PGMY09), thereby avoiding use of degenerate bases that yield irreproducible primer syntheses. The performance of the PGMY09-PGMY11 (PGMY09/11) primer system relative to that of the standard MY09/11 system was evaluated with a set of 262 cervicovaginal lavage specimens. There was a 91.5% overall agreement between the two systems (kappa = 0.83; P < 0.001). The PGMY09/11 system appeared to be significantly more sensitive than the MY09/11 system, detecting an additional 20 HPV-positive specimens, for a prevalence of 62.8% versus a prevalence of 55.1% with the MY09/11 system (McNemar's chi(2) = 17.2; P < 0.001). The proportion of multiple infections detected increased with the PGMY09/11 system (40. 0 versus 33.8% of positive infections). HPV types 26, 35, 42, 45, 52, 54, 55, 59, 66, 73, and MM7 were detected at least 25% more often with the PGMY09/11 system. The PGMY09/11 primer system affords an increase in type-specific amplification sensitivity over that of the standard MY09/11 primer system. This new primer system will be useful in assessing the natural history of HPV infections, particularly when the analysis requires HPV typing.     

Detection and typing of human papillomaviruses in mucosal and cutaneous biopsies from immunosuppressed and immunocompetent patients and patients with epidermodysplasia verruciformis: a unified diagnostic approach.

AIM: To develop a unified diagnostic approach for the detection of human papillomavirus (HPV) DNA in skin and mucosal biopsies from both immunocompetent and immunosuppressed individuals using a degenerate polymerase chain reaction (PCR) method. METHODS: The sensitivity and specificity of three published degenerate primer sets (HVP2/B5 and F14/B15; MY09/MY11; CP62/69 outer and CP65/68 nested primer pairs) were evaluated in PCR reactions with serial dilutions of 12 representative cloned HPV types. This combination of primers was then used to detect HPV DNA in 49 benign and malignant lesions of cutaneous and mucosal origin from immunosuppressed, immunocompetent, and epidermodysplasia verruciformis (EV) patients, and compared with detection rates using single primer sets alone. RESULTS: The observed sensitivity of MY09/MY11 and CP62/69 + CP65/68 was high for mucosal and EV HPV types, respectively. The sensitivity of all primer sets for cutaneous types was low, but nonetheless the use of this combination of primers allowed HPV DNA detection in all of the benign warts analysed. Several mixed infections were also identified. A high prevalence of HPV DNA was similarly detected in squamous cell carcinomas from immunocompromised patients; the HPV types found were exclusively EV related. CONCLUSIONS: The use of a combined degenerate primer PCR approach considerably improves HPV DNA detection over individual primer sets and allows detection of mixed infections. The findings may help explain the discrepancies in published reports relating to HPV DNA detection in benign and malignant skin lesions. Further modifications to this method are in progress which should significantly improve comprehensive HPV detection and typing for diagnostic purposes.     

A pilot study on the distribution of human papillomavirus genotypes and HPV-16 variants in cervical neoplastic lesions from Ecuadorian women

Human papillomaviruses (HPVs) are the cause of cervical intraepithelial neoplasia and invasive carcinomas of the uterine cervix. The distribution of specific HPV genotypes varies greatly across populations and HPV surveys have been performed in different geographical regions in order to apply appropriate vaccine strategies. The aim of this study was to determine the spectrum of HPV genotypes and HPV-16 variants among women with cervical lesions living in Ecuador. A total of 71 cases have been analyzed, including 32 chronic cervicitis, 29 cervical intraepithelial neoplasia grade 1, and 10 cervical intraepithelial neoplasia grade 2-3. HPV sequences were detected by broad spectrum consensus-primer-pairs MY09/MY11 and GP5+/GP6+-based polymerase chain reaction and characterized by nucleotide sequence analysis. Overall, 31 (43.7%) cases were HPV positive with prevalence rates of 37.5%, 44.8%, and 60% in patients with chronic cervicitis, cervical intraepithelial neoplasia grade 1 and cervical intraepithelial neoplasia grade 2-3, respectively. Among the positive cases, the most common genotypes were HPV 16 (64.5%) and HPV 81 (29%) followed by HPV 31, 53, 56, and 58, in descending order of prevalence. Seventeen (85%) HPV-16 isolates were classified as European and three (15%) as African-1 variant on the basis of nucleotide signature present within the MY09/MY11 L1 sequence. The results suggest that HPV 16 has a very high prevalence among women with cervical lesions in Ecuador; therefore, an effective HPV-16 based vaccine should prevent the development of cervical cancer in a large proportion of Ecuadorian women.     

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.     

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.     

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.     

Comprehensive study of several general and type-specific primer pairs for detection of human papillomavirus DNA by PCR in paraffin-embedded cervical carcinomas.

We have compared the efficacies of three general primer pairs for the detection of human papillomavirus (HPV) DNA in formaldehyde-fixed paraffin-embedded carcinomas. The use of these primer pairs leads to underestimates of the HPV prevalence (GP5/6, 61.1%; CPI/IIG, 57.4%; MY09/11, 46.9%; combined, 72.8%). The efficacy of each primer pair seemed to be inversely correlated to the length of the amplimer produced. By using newly developed type-specific primer pairs (amplimer length, approximately 100 bp), an increase in HPV DNA detection (87.6%) was found.     

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.     

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.     

Intermethod variation in detection of human papillomavirus DNA in cervical smears.

In order to investigate the reliability of detection of human papillomavirus (HPV) DNA in cervical smears, we have compared the performance of two HPV PCR systems, the CPI/IIG and MY09/11 primer-mediated PCRs and the Hybrid Capture System HPV DNA detection test (hybrid capture assay), in detecting HPV DNA in cervical smears. We also included in our study the MY09/11B PCR plus SHARP (solution hybridization assay for PCR products) Signal System. This SHARP Signal System was recently developed to detect MY09/11B-generated biotinylated PCR products. The detection rate of the hybrid capture assay was lower than those of the CPI/IIG and MY09/11 PCRs and the MY09/11B PCR plus SHARP Signal System. The detection rates of the CPI/IIG PCR and the MY09/11B PCR plus SHARP Signal System were similar and higher than that of the conventional MY09/11 PCR system. The agreement beyond chance of the PCR methods was nearly perfect (kappa value between 0.82 and 0.84). The agreement beyond chance of the hybrid capture assay and the PCR methods was fair to good (kappa value between 0.64 and 0.70). The systems detected HPV DNA in different but overlapping sets of smears. Our results indicate that each of the detection methods alone underestimates the prevalence of HPV.