Use of multiple PCR primer sets for optimal detection of human papillomavirus.

Using multiple PCR primer sets, we tried to optimize the detection of human papillomavirus (HPV) in DNA samples isolated from 361 frozen biopsy specimens from patients with invasive cervical carcinomas. The HPVs detected were placed into three distinct groups, including group I/Inex at Telelab (Skien, Norway) and group Ineg and group II at the Norwegian Radium Hospital (Oslo, Norway). The consensus primer sets were Oli-1b-oli-2i, My09-My11, Gp5-Gp6, and Gp(5+)-Gp6+ from the HPV L1 gene and CpI-CpIIG from the E1 gene. Using these consensus primers together with the type-specific primers from E6-E7, we found that 355 patients (98%) were HPV positive. Type-specific primers for HPV types 11, 16, 18, 31, 33, and 35 detected more HPV-infected patients than the most sensitive consensus primer set, while the three consensus primer sets My, Gp/Gp+, and Cp together detected more HPV-positive patients than the type-specific primers. Testing of sensitivity of the PCR with SiHa cells serially diluted in lymphocytes (HPV-negative cells) indicated a detection limit of 6,300 HPV type 16 DNA copies with consensus primers (My, Gp+, and Cp) and 126 original HPV type 16 DNA copies with type-specific primers. Comparison of the amplification results for consensus L1 primers and type-specific E6-E7 primers indicated the presence of L1 deletions in 23 of 56 samples. The conclusion is that in PCR detection systems, multiple consensus primers and type-specific primers should be used in order to detect all patients harboring HPV.     

Comparative analysis of human papillomavirus detection by polymerase chain reaction and Virapap/Viratype kits

Cervical samples from 270 women referred by area physicians were analyzed for the presence of human papillomavirus (HPV) types 6, 11, 16, and 18 by the polymerase chain reaction (PCR) followed by Southern hybridization. Samples from 154 patients were concurrently analyzed by a commercial filter hybridization technique (Virapap and Viratype Kits, Life Technologies, Bethesda Research Labs, Gaithersburg, MD). The sensitivity of the Southern blot procedure combined with PCR was significantly higher than that of the Virapap and Viratype methods. HPV was detected in 67% of women who had positive results for dysplasia by PCR and in 47% by the Virapap method. HPV types 16/18 were found more commonly than types 6/11 in every diagnostic category. More than one HPV type was detected in 12% of HPV-positive patients. The prevalence of HPV in cytologically negative or indefinite patients as measured by PCR was 22% and 40%; in contrast, by the Virapap method, these values were 7% and 10%. These results demonstrate that PCR combined with Southern hybridization provides a higher level of sensitivity than methods that use hybridization without amplification of HPV DNA and also show that the prevalence of HPV is highest in cytologically positive smears.     

The use of nested polymerase chain reaction and restriction fragment length polymorphism for the detection and typing of mucosal human papillomaviruses in samples containing low copy numbers of viral DNA

Mucosal human papillomaviruses (HPVs) that infect the genital area have also been shown to infect the oral cavity. In this study a restriction fragment length polymorphism (RFLP) method was developed on a nested polymerase chain reaction (PCR) product to identify ten high risk HPV types 16, 18, 31, 33, 35, 45, 51, 52, 58 and 59 as well as the low risk HPV 11. HPV DNA was detected in 23/31 (74%) of buccal specimens using a sensitive nested PCR employing degenerate consensus primers (Williamson and Rybicki, 1991). Consensus PCR using the PGMy09/11 primers. was able to detect HPV in only 29% of the specimens that had tested positive using the nested HPV PCR primers. HPV 11 type specific primers detected HPV 11 DNA in only 66% of the specimens showing HPV 11 DNA by means of nested PCR and RFLP. A Genbank search revealed that the PCR primers could detect a wide range of mucosal HPV types including types HPV 70, 72 and 73 which have all been isolated from immunocompromised patients. Of the 23 buccal specimens that were positive for HPV DNA, 13 were single infections, five were dual infections and three were triple infections. The HPV types identified by RFLP were: HPV 11 (18/23), HPV 18 (8/23), HPV 16 (3/23), and HPV 33 (1/23). HPV 13 (2/23) was identified by direct sequencing of the inner amplicon of the PCR product.     

Genotyping of human papillomaviruses by a novel one-step typing method with multiplex PCR and clinical applications

We describe here a rapid, high-throughput genotyping procedure that allows the simultaneous detection of 16 high- and low-risk genital human papillomavirus (HPV) types by multiplex PCR in a single reaction tube. Multiplex PCR is based on the amplification of HPV DNA by sets of HPV genotype-specific primers, and the genotypes of HPV are visually identified by the sizes of amplicons after they are separated by capillary electrophoresis. The procedure does not include a hybridization step with HPV-specific probes and is rapid and labor-saving. We detected all 16 HPV genotypes (types 16, 58, 52, 51, 56, 31, 18, 39, 66, 59, 6, 33, 30, 35, 45, and 11) with a high sensitivity and a high degree of reproducibility. By using this newly developed method, we conducted a pilot study to examine the correlation between the prevalence and genotype distributions of HPV and the cytological group classifications for 547 cervical samples. Compared with the group of samples considered normal (14.7%), there was a significant increase in the prevalence of HPV in women with atypical squamous cells of unknown significance (61.3%), low-grade intraepithelial lesions (75.8%), and high-grade intraepithelial lesions (HSILs) (82.2%). The prevalence and distribution of type 58 were correlated with cytological malignancies, with the highest prevalence in women with HSILs. In conclusion, the novel multiplex PCR method described appears to be highly suitable not only for the screening of cervical cancer precursor lesions but also for the characterization of genotype distributions in large-scale epidemiological studies and HPV vaccination trials.     

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.     

Human papillomavirus infection and anal carcinoma. Retrospective analysis by in situ hybridization and the polymerase chain reaction.

To examine the association of human papillomavirus (HPV) infection with anal squamous cell carcinoma, the authors applied the highly sensitive polymerase chain reaction (PCR) and in situ hybridization (ISH) techniques to detect HPV DNA in formalin-fixed, paraffin-embedded tissues from 18 patients. The presence of HPV types 16/18 in 3 (16.7%) of 18 patients with anal carcinoma was found, using a colorimetric ISH technique for HPV types 6, 11, 16, 18, 31, 35, and 51. Results from one of these three patients were also positive for HPV 31, 35, 51 by ISH techniques. When the same series was analyzed using the PCR and consensus primers to the L1 open reading frame of the HPV genomes, the frequency of positive patients rose to 14 (77.8%) of 18. PCR analysis of the 14 lesions containing HPV DNA, using type-specific primers and probes for HPV 6, 11, 16, 18, and 33, showed that 1 contained HPV 6, 1 contained HPV 11, 4 contained HPV 16, 1 contained HPV 18, 1 contained HPV 33, 5 contained HPV of unclassified type(s), and 1 contained a mixture of three HPV types. There was concordance between typing of cases that were positive by ISH and PCR methods. These data agree with the concept that HPV, in particular type 16, is implicated in the pathogenesis of anal cancer.     

Evaluation of high-risk human papillomavirus types PCR detection in paired urine and cervical samples of women with abnormal cytology.

BACKGROUND: During the last decade, increasing efforts have focused on HPV detection in self-obtained samples, to increase the overall proportion of patients participating in cervical cancer screening procedures. OBJECTIVES: A clinical evaluation study of an optimized protocol for PCR detection of high-risk human papillomavirus (HPV) types in urine compared with cervical samples in consecutive women referred to the colposcopy clinic with abnormal cervical cytology. STUDY DESIGN: Paired urine and cervical specimens were collected from 100 consecutive women referred to the colposcopy clinic with abnormal cervical cytology and normal urine parameters. In-house and a commercial PCR method for the detection of HPV types 16 and 18, and a commercial multiplex PCR for HPV types 6, 11, 16, 18, and 33 were performed. All HPV cervix-positive/urine-negative paired urine samples were spiked with serial dilutions of cell lines infected with HPV 16 or 18 to test the sensitivity of HPV detection in these urine samples. RESULTS: In all but two cases HPV type 16 was detected. In cancer cases, the urine/cervix HPV detection sensitivity was 88.8%; in cases with high-grade lesions it was 76.5%; and in cases with low-grade lesions it was 45.5%. In all concordant cases the same HPV type was detected in both samples. The urine/cervix HPV detection sensitivity was higher when urine samples contained two or more epithelial cells per field in urine microscopy. HPV detection in 9 cervix-positive but urine-negative urine samples spiked with serial dilutions of HPV-positive cell lines showed that in these cases urine PCR inhibitors did not affect PCR amplification. CONCLUSIONS: A higher urine/cervix HPV detection sensitivity in cancer and high-grade lesions suggests that urine testing could be used to detect HPV mainly when these lesions are present.     

Development and clinical evaluation of a highly sensitive PCR-reverse hybridization line probe assay for detection and identification of anogenital human papillomavirus.

Human papillomavirus (HPV) can be detected by amplification of viral DNA. A novel PCR primer set generating a short PCR fragment (SPF PCR) was used for amplification of a fragment of only 65 bp from the L1 region and permitted ultrasensitive detection of a broad spectrum of HPV genotypes. The intra- and intertypic sequence variations of the 22-bp interprimer region of this amplimer were studied. Among 238 HPV sequences from GenBank and clinical specimens, HPV genotypes were correctly identified based on the 22-bp sequence in 232 cases (97.2%). Genotype-specific probes for HPV genotypes 6, 11, 16, 18, 31, 33 to 35, 39, 40, 42 to 45, 51 to 54, 56, 58, 59, 66, 68, 70, and 74 were selected, and a reverse hybridization line probe assay (LiPA) (the INNO-LiPA HPV prototype research assay) was developed. This LiPA permits the use of amplimers generated by the SPF as well as the MY 09/11 primers. The assay was evaluated with a total of 1, 354 clinical specimens, comprising cervical scrapes (classifications ranging from normal cytology to severe dyskaryosis) and formalin-fixed, paraffin-embedded cervical carcinoma samples. LiPA results were highly concordant with sequence analysis of the SPF amplimer, genotype-specific PCR, and sequence analysis of amplimers generated by MY 09/11 primers. The sensitivity of the SPF primers was higher than that of the GP5(+)/6(+) primers over a broad range of HPV types, especially when multiple HPV genotypes were present. In conclusion, the SPF LiPA method allows extremely sensitive detection of HPV DNA as well as reliable identification of HPV genotypes in both cervical smears and paraffin-embedded materials.     

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.     

Development of a sensitive and specific assay combining multiplex PCR and DNA microarray primer extension to detect high-risk mucosal human papillomavirus types

The importance of assays for the detection and typing of human papillomaviruses (HPVs) in clinical and epidemiological studies has been well demonstrated. Several accurate methods for HPV detection and typing have been developed. However, comparative studies showed that several assays have different sensitivities for the detection of specific HPV types, particularly in the case of multiple infections. Here, we describe a novel one-shot method for the detection and typing of 19 mucosal high-risk (HR) HPV types (types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, and 82). This assay combines two different techniques: multiplex PCR with HPV type-specific primers for amplification of viral DNA and array primer extension (APEX) for typing. This novel method has been validated with artificial mixtures of HPV DNAs and clinical samples that were already analyzed for the presence of mucosal HPV types by a different consensus PCR method, i.e., GP5+/GP6+. Our data showed a very good agreement between the results from the multiplex PCR/APEX assay and those from the GP5+/GP6+ PCR (overall rates of HPV positivity, 63.0 and 60.9%, respectively). Whereas the GP5+/GP6+ PCR was slightly more sensitive for the detection of HPV type 16 (HPV-16), multiplex PCR-APEX found a higher number of infections with HPV-33, HPV-53, and multiple HPV types. These favorable features and the high-throughput potential make our present novel assay ideal for large-scale clinical and epidemiological studies aimed at determining the spectrum of mucosal HR HPV types in cervical specimens.     

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 the linear array HPV genotyping PCR assay and influence of DNA extraction method on HPV detection

Real-time human papillomavirus (HPV) type-specific multiplex PCR assays were developed to detect HPV DNA in specimens collected for the efficacy determination of the quadrivalent HPV (type 6, 11, 16, and 18) L1 virus-like particle (VLP) vaccine (Gardasil). We evaluated the concordance between type-specific multiplex HPV PCR and the widely used, commercially available Roche Linear Array genotyping PCR assay. Female genital swab specimens were tested for the presence of L1, E6, and E7 sequences of HPV type 6 (HPV6), HPV11, HPV16, HPV18, HPV31, HPV45, HPV52, and HPV58 and E6 and E7 sequences of HPV33, HPV35, HPV39, HPV51, HPV56, and HPV59 in type- and gene-specific real-time multiplex PCR assays. Specimens were also tested for the presence of L1 sequences using two versions of the Roche Linear Array genotyping assay. Measures of concordance of a modified version of the Linear Array and the standard Linear Array PCR assay were evaluated. With specimen DNA extraction using the Qiagen Spin blood kit held as the constant, multiplex PCR assays detect more HPV-positive specimens for the 14 HPV types common to both than either version of the Linear Array HPV genotyping assay. Type-specific agreements between the assays were good, at least 0.838, but were often driven by negative agreement in HPV types with low prevalence, as evidenced by reduced proportions of positive agreement. Overall HPV status agreements ranged from 0.615 for multiplex PCR and standard Linear Array to 0.881 for multiplex PCR and modified Linear Array. An alternate DNA extraction technique, that used by the Qiagen MinElute kit, impacted subsequent HPV detection in both the multiplex PCR and Linear Array assays.     

Detection of multiple types of human papillomavirus in a giant condyloma from a grafted patient. Analysis by immunohistochemistry, in situ hybridisation, Southern blot and polymerase chain reaction.

Immunosuppressed patients such as transplant recipients are known to develop multiple lesions suggestive of human papillomavirus (HPV) infection. A giant anal condyloma was obtained from a transplant patient; several fragments taken from different areas were examined for the presence of HPV DNA using in situ hybridisation, polymerase chain reaction (PCR) and Southern blot. Typical koilocytes were seen in routinely stained tissue sections, suggesting an HPV infection; furthermore, group specific HPV antigen was detected in one of four frozen fragments. Different results were obtained by in situ hybridisation according to the fragment tested. HPV types 6/11 were detected in each of the five fragments, frozen or fixed in Bouin's or formalin solutions. However, the number of HPV DNA positive cells and the intensity of the reaction greatly varied with the specimen. HPV 16 and 18 probes also reacted positively with the sample fixed in formalin; a stronger signal was observed with HPV 18 in one large focus than with HPV 16. HPV type 5 was detected in a few isolated cells of two frozen fragments. With the Southern blot technique, the profile of an HPV 6/11 was seen only in one of two frozen fragments; in this case, the bands were intense. A slight positive reaction was also obtained in one frozen fragment with HPV 16 probe. Four frozen fragments were analyzed with PCR: HPV 6/11 was detected in each fragment; HPV 18 was detected in the four samples but with different intensities; HPV types 5 and 16 did not show any positive signal. In conclusion, the lesion is an example of infection with several HPV types, demonstrated by three different techniques. This suggests the need for careful dermatological or colposcopic follow-up of transplant recipients, in order to prevent possible malignant transformation of anogenital lesions.     

A general primer pair for amplification and detection of genital human papillomavirus types.

A general primer pair localized in the E7 and E1 regions was identified and used for the detection of genital human papillomaviruses (HPVs). The genital HPV types 6b, 11, 16, 18, 31 and 33 were amplified and detected by the polymerase chain reaction (PCR) performed at a high stringency annealing temperature (60 degrees C). HPV-2, -3, -7, -13 and -30 were amplified only at lower temperatures. Twelve biopsies from women with invasive cancer in the cervix were analysed with the general primer pair. The amplification product specific for the general primer pair was detected in 11 of the 12 biopsies. The eleven HPV DNA positive specimens were shown to contain HPV-6b, HPV-16 and/or HPV-18 by Southern blot hybridization of the PCR products. The general primers were also used for analysis of 57 cervical scrapes from women with normal cytology, condyloma or CIN. By ethidium bromide staining after agarose gel electrophoresis we could detect 21 positives. Slot-blot analysis of the amplification products from all 57 scrapes confirmed the specificity of the 21 positives and revealed 5 additional positives. Among the 57 scrapes, 15/21 CIN scrapes, 10/21 condyloma scrapes and 1/15 normal scrapes contained HPV DNA. Eight different HPV types were detected. The general primer pair from the E7/E1 region is thus a powerful tool for the detection of HPV in clinical samples. The amplimer obtained offers a possibility for further typing by slot-blot hybridization using HPV-type specific probes.     

Prevalence of human papillomavirus in sinonasal papillomas: a study using polymerase chain reaction and in situ hybridization.

Inverted and fungiform papillomas of the sinonasal cavity share a common origin from the Schneiderian membrane, but they differ widely in their rates of recurrence and progression to carcinoma. To determine the role of human papillomavirus in the etiology of these lesions, 15 inverted papillomas, five fungiform papillomas, and two squamous cell carcinomas associated with inverted papilloma were examined for the presence of HPV by in situ hybridization (ISH) and polymerase chain reaction (PCR). ISH was carried out on formalin-fixed, paraffin-embedded material using HPV types 6/11, 16/18, and 31/33/35 DNA probes. Tissue DNA was amplified by PCR with HPV L1 consensus primers, and the product was detected by gel electrophoresis, Southern blotting, and hybridization with type specific probes (HPV types 6/11, 16, 18). Three of 15 inverted papillomas and two of five fungiform papillomas were positive for HPV 6/11 by ISH, whereas PCR detected HPV 6/11 sequences in two of 15 inverted and three of five fungiform papillomas. Biopsies from two patients who had serial resections contained HPV 6/11 in the original lesions and all recurrences. No HPV was detected in the carcinomas by ISH, whereas PCR detected HPV 16 in one carcinoma. These findings confirm the presence of HPV DNA sequences in both inverted and fungiform sinonasal papillomas as well as in an associated squamous carcinoma. This would suggest a role for HPV in the pathogenesis of Schneiderian membrane lesions. Furthermore, our data indicate that ISH and PCR are equally sensitive in detecting HPV in sinonasal papillomas.     

Detection of specific types of human papillomavirus in cervical scrapes, anal scrapes, and anogenital biopsies by DNA hybridization

Specific varieties of human papillomavirus (HPV) infecting the anogenital region were detected in clinical samples by use of a filter hybridization technique suitable for rapid screening of cervical and anal scrapes. In this way possibly benign types (HPV6 and HPV11) could be differentiated from types thought to be capable of malignant transformation (HPV 16 and HPV 18). Cervical or anal canal cells were applied directly to nylon filters and fixed by u.v. irradiation before hybridization with mixed viral DNA probes under both low- and high-stringency conditions. In addition, probe for the human Alu-repeated DNA sequence was used to assess the relative amount of total nucleic acids in each sample applied to the filter. HPV DNA was detected in 3 of 19 cervical scrapes from patients with no past or present history of wart virus infection or cervical dysplasia. Within a positive study group totalling 71 patients, HPV (6/11 or 16/18) was detected in cervical scrapes from 24% of 41 patients who did not have visible genital dysplasia, 30% of 27 patients with visible genital dysplasia or cervical intraepithelial neoplasia (CIN) I, and in 1 of 3 patients with past CIN II/III. In addition, HPV6/11 or 16/18 DNA was detected in anal scrapes from 3 of 6 male patients and in 85% of genital biopsies. A notably high proportion (4/6) of vaginal condylomata were positive with both the HPV6/11 and the HPV16/18 mixed viral DNA probes. Of the biopsies prepared for histopathology and positive for HPV DNA, the HPV group-specific antigen could be detected in only 60%.     

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.     

Nonisotopic detection of human papillomavirus DNA in clinical specimens using a consensus PCR and a generic probe mix in an enzyme-linked immunosorbent assay format

We assessed the value of a new digoxigenin (DIG)-labeled generic probe mix in a PCR-enzyme-linked immunosorbent assay format to screen for the presence of human papillomavirus (HPV) DNA amplified from clinical specimens. After screening with this new generic assay is performed, HPV DNA-positive samples can be directly genotyped using a reverse blotting method with product from the same PCR amplification. DNA from 287 genital specimens was amplified via PCR using biotin-labeled consensus primers directed to the L1 gene. HPV amplicons were captured on a streptavidin-coated microwell plate (MWP) and detected with a DIG-labeled HPV generic probe mix consisting of nested L1 fragments from types 11, 16, 18, and 51. Coamplification and detection of human DNA with biotinylated beta-globin primers served as a control for both sample adequacy and PCR amplification. All specimens were genotyped using a reverse line blot assay (13). Results for the generic assay using MWPs and a DIG-labeled HPV generic probe mix (DIG-MWP generic probe assay) were compared with results from a previous analysis using dot blots with a radiolabeled nested generic probe mix and type-specific probes for genotyping. The DIG-MWP generic probe assay resulted in high intralaboratory concordance in genotyping results (88% versus 73% agreement using traditional methods). There were 207 HPV-positive results using the DIG-MWP method and 196 positives using the radiolabeled generic probe technique, suggesting slightly improved sensitivity. Only one sample failed to test positive with the DIG-MWP generic probe assay in spite of a positive genotyping result. Concordance between the two laboratories was nearly 87%. Approximately 6% of samples that were positive or borderline when tested with the DIG-MWP generic probe assay were not detected with the HPV type-specific panel, perhaps representing very rare or novel HPV types. This new method is easier to perform than traditional generic probe techniques and uses more objective interpretation criteria, making it useful in studies of HPV natural history.     

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.