Optimization of human papillomavirus genotype detection in cervical scrapes by a modified filter in situ hybridization test.

Optimal conditions for the screening of cervical scrapes for human papillomavirus (HPV) were investigated by using filter in situ hybridization. Since integrated and episomal HPV can be found, cell lines containing viral DNA in an integrated form (HPV in CaSki) or in an episomal state (BK virus-induced hamster tumor cells) were used for optimization experiments. An increase in sensitivity was achieved by alkaline denaturation and neutralization before the specimens were spotted onto the membrane. This increase was 5-fold for the episomal virus and 16-fold for the integrated virus in the model system, as compared with other methods. To evaluate this method on clinical material, 1,963 cervical scrapes were screened for the presence of HPV 6/11 and HPV 16. Nineteen scrapes were positive for HPV 6/11 or HPV 16; and in 1,810 scrapes, no HPV 6/11 or HPV 16 could be detected by the modified filter in situ hybridization technique. Scrapes from which the interpretation of the modified filter in situ hybridization results were equivocal (n = 71, 3.6%) or in which positivity was detected for both HPV 6/11 and HPV 16 (n = 63, 3.2%) were further analyzed by the DNA dot spot technique. Eight scrapes with an equivocal result and only one scrape showing a double positivity by the modified filter in situ hybridization technique could be confirmed in the dot spot assay. In the total group 12 scrapes were positive for HPV 6/11 DNA, 15 were positive for HPV 16 DNA, and 1 was positive for both HPV 6/11 and HPV 16 DNA. Southern blot analysis on modified filter in situ hybridization-positive and -negative scrapes revealed a 100% correlation.     

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 simple and rapid method of high quantity DNA isolation from cervical scrapes for detection of human papillomavirus infection.

Infection with human papillomavirus (HPV) is an important etiological factor in the development of cervical cancer, and detection of the viral genome is of prognostic importance, particularly for preneoplastic lesions. We developed a simple, easy and efficient non-organic method of DNA extraction from cervical scrapes for reliable detection of HPV DNA sequences. The method involves incubation of cell nuclei in higher concentration of proteinase K at 65 degrees C for 2.5 h. Following prolonged incubation at higher temperature, the enzyme is autoinactivated and the DNA isolated can be used directly for analysis without further purification. The recovery of DNA is more than 95% and it can be easily cleaved by restriction enzymes and is suitable for amplification by the polymerase chain reaction (PCR). The whole procedure is carried out in a single Eppendorf tube and a large number of specimens can be processed at a time without any error of handling. DNA extracted from a single smear sample is sufficient to conduct as many as four different molecular biology tests. This provides an opportunity for verification of sensitivity, specificity and reliability of each test for diagnosis of HPV infection without resorting to biopsy.     

Hybrid Capture 2 vs HPV Consensus kit: comparison of 2 assays for human papillomavirus detection and typing

BACKGROUND: Many laboratories use the DNA Hybrid Capture 2 HPV-high risk assay (Digene) to detect and type oncogenic HPV. The aim of this work was to compare this assay with a new HPV genotyping assay: HPV Consensus kit (Argène). Actually, this assay is not commercially available. MATERIALS AND METHODS: Ninety-four cervical samples were tested with both the routine assay Hybrid Capture 2 and the HPV Consensus kit. Discordant results were analysed by amplification with a nested PCR and sequencing of amplified products. RESULTS: Only 81 results could be analysed concerning the oncogenic risk. The overall concordance was 92,6%. But we find 13 "generic" results with the HPV Consensus kit, the generic probe including high risk and low risk genotypes. CONCLUSION: HPV Consensus kit results showed a better detection sensitivity for this assay than Hybrid Capture 2 assay. Nevertheless, "generic" results give no information about the oncogenic risk of the HPV detected in a sample.     

Evaluation of a new DNA test for detection of carcinogenic human papillomavirus

Using archived specimens, we evaluated a new automated real-time PCR assay (BD Diagnostics) that detects all carcinogenic human papillomaviruses (HPV) and provides HPV genotyping for seven of them, including HPV16 and HPV18, the two most carcinogenic HPV genotypes. We found comparable results with Hybrid Capture 2 (HC2) for detection of carcinogenic HPV (n = 473) and with Linear Array and Line Blot Assay (n = 371) for detection of individual HPV genotypes.     

A novel genital human papillomavirus (HPV), HPV type 74, found in immunosuppressed patients.

The genome of a novel human papillomavirus (HPV) type, HPV74, was cloned from an iatrogenically immunosuppressed woman with persisting low-grade vaginal intraepithelial neoplasia. HPV74 was found to be phylogenetically related to the low-risk HPV types 6, 11, 44, and 55. HPV74 or a variant of this type was found in specimens from three additional immunosuppressed women but not in about 3,000 anogenital specimens from immunocompetent patients.     

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%.     

Evaluation of accuracy of three assays for human papillomavirus detection and typing: Hybrid Capture 2, HPV Consensus kit and Amplicor HPV

BACKGROUND: Detection of high-risk human papillomavirus has proved its usefulness in complement of abnormal cervical scrape result. The Hybrid Capture 2 (HC2, Digene) test has proven its efficiency. We have compared this test with HPV Consensus kit (HPVC, Argène) and Amplicor HPV test (AHPV, RocheDiagnostics) on a panel of 88 samples with low HC2 ratios or discordant results between HC2 and cervical scrape. MATERIAL AND METHODS: Cervical samples were tested in parallel by the three methods using a nested amplification of L1 region as reference. RESULTS: Eighty-six samples were suitable for analysis. Results of HC2 and AHPV tests were closely related. The use of a "generic" probe in the HPVC test was responsible for undetermined results, which were not clinically relevant. CONCLUSION: Despite the low viral load of the samples chosen, the hybridization (HC2) and PCR (AHPV or HPVC) methods gave comparable results, with false positive and false negative results for all tests, but a 75% concordance and a high sensibility to detect HPV infection. However, a complementary study on a larger population with ASCUS diagnosis and biopsy under colposcopy would be necessary to valid these assays for a clinical indication.     

Enhanced detection and typing of human papillomavirus (HPV) DNA in anogenital samples with PGMY primers and the Linear array HPV genotyping test

The Roche PGMY primer-based research prototype line blot assay (PGMY-LB) is a convenient tool in epidemiological studies for the detection and typing of human papillomavirus (HPV) DNA. This assay has been optimized and is being commercialized as the Linear Array HPV genotyping test (LA-HPV). We assessed the agreement between LA-HPV and PGMY-LB for detection and typing of 37 HPV genotypes in 528 anogenital samples (236 anal, 146 physician-collected cervical, and 146 self-collected cervicovaginal swabs) obtained from human immunodeficiency virus-seropositive individuals (236 men and 146 women). HPV DNA was detected in 433 (82.0%) and 458 (86.7%) samples with PGMY-LB and LA-HPV (P = 0.047), respectively, for an excellent agreement of 93.8% (kappa = 0.76). Of the 17,094 HPV typing results, 16,562 (1,743 positive and 14,819 negative results) were concordant between tests (agreement = 96.9%; kappa = 0.76). The mean agreement between tests for each type was 96.4% +/- 2.4% (95% confidence interval [CI], 95.6% to 97.2%; range, 86% to 100%), for an excellent mean kappa value of 0.85 +/- 0.10 (95% CI, 0.82 to 0.87). However, detection rates for most HPV types were greater with LA-HPV. The mean number of types per sample detected by LA-HPV (4.2 +/- 3.4; 95% CI, 3.9 to 4.5; median, 3.0) was greater than that for PGMY-LB (3.4 +/- 3.0; 95% CI, 3.1 to 3.6; median, 2.0) (P < 0.001). The number of types detected in excess by LA-HPV in anal samples correlated with the number of types per sample (r = 0.49 +/- 0.06; P = 0.001) but not with patient age (r = 0.03 +/- 0.06; P = 0.57), CD4 cell counts (r = 0.06 +/- 0.06; P = 0.13), or the grade of anal disease (r = -0.11 +/- 0.06; P = 0.07). LA-HPV compared favorably with PGMY-LB but yielded higher detection rates for newer and well-known HPV types.     

Comparison of the Abbott RealTime High Risk HPV with Genomica HPV Clinical Array for the detection of human papillomavirus DNA

Human papillomavirus (HPV) has been identified as the major cause of cervical cancer worldwide and HPV DNA testing is recommended in primary cervical cancer screening. Several molecular tests for detection/typing of HPV DNA with different sensitivity and specificity are commercially available. The present study compared the performance of the Abbott RealTime High Risk HPV assay and the Genomica HPV Clinical Array CLART2 in 78 specimens (63 cervical smears and 15 rectal/urethral swabs).The typing results of the Genomica assay were in absolute agreement with each of the four possible result categories of the Abbott assay (HPV16, HPV18, Other HR HPV, not detected) in 87.2% (68/78) of the samples, with a Cohen' kappa agreement coefficient for every HR type of 0.62 (95% CI: 0.39–0.85), higher in cervical swabs (k = 0.74, 95% CI: 0.50–0.99) than in rectal/urethral swabs (k = 0.36, 95% CI: 0.00–0.82). There was an excellent agreement of the Genomica results with those of Abbott in cervical samples harbored HPV single infection (100% agreement). Nonetheless, both methods may lose sensitivity for detecting HPV types in multiple infections, giving discordant results (10/78). This underlines the importance of establishing the analytical sensitivity in HPV type detection in single and multiple HPV infections. In rectal/urethral swabs, 5 of 15 (33%) discordant cases were observed, most of which became compatible when the Genomica assay was performed starting from nucleic acid extracted with the Abbott m2000sp system. These results suggest that nucleic extraction based on the magnetic beads technique is suitable for HPV DNA detection in urethral/rectal swabs.     

An improved in situ DNA hybridization protocol for detection of human papillomavirus (HPV) DNA sequences in paraffin-embedded biopsies

In situ DNA hybridization is becoming rapidly an important technique for detection and typing of human papillomaviruses (HPV) in epithelial lesions, some of which (those due to HPV 16, 18 and 31) seem to possess an increased risk for progression into an invasive squamous cell carcinoma. An improved in situ DNA hybridization technique (Technique II) was described, and the results obtained in a series of cervical and penile HPV lesions were compared with those given by the in situ hybridization technique (Technique I) previously used in our laboratory. Special emphasis was made to increase the sensitivity with three basic alterations of the hybridization protocol; omission of the 0.2 N HCl wash, use of increased proteinase K concentration (from 50 micrograms/ml to 1 mg/ml), and elevated denaturation temperature (obtained by a heating block instead of an incubator). Poly-D-lysine as a slide-coating medium was replaced by Kodak Photo-Flo 200 to improve the attachment of the tissue sections on the slides. Identical HPV DNA types were discovered by the two hybridization techniques. The attachment of the tissue sections was equal on the slides coated with either poly-D-lysine or Kodak Photo-Flo 200, and the latter did not interfere with the sensitivity of in situ hybridization. The hybridization signals for HPV DNA were weak or moderate in 15/16 lesions with Technique I, but intense in 10/16 lesions with Technique II (P less than 0.001). Furthermore, the resolution of Technique II seemed to be superior to that of Technique I, while being capable of disclosing HPV DNA in the intermediate cell layers (P less than 0.001) and in basal/parabasal cell layers (P less than 0.025) of both the cervical and penile lesions. The discovery of HPV DNA in the parabasal cells provides important clues to the understanding of the biology of HPV infection in the squamous epithelium, and makes this improved in situ DNA hybridization technique invaluable in assessing the lesions, where low copy numbers of HPV are to be expected.     

Detection of human papillomavirus (HPV) genotypes in cervico-vaginal scrapes of women with normal and abnormal cytology

Background   Certain types of human papillomavirus (HPV) are associated with cervical intraepithelial neoplasia (CIN) and invasive cervical carcinoma. The study addressed the expression and detection of HPV genotypes in cervical and vaginal specimens of women with normal and abnormal cytology by polymerase chain reaction (PCR), each woman serving as her own control.
Methods   Study participants (127) were subgrouped into CIN-positive and CIN-negative, based on cytology screening, and endocervical and vaginal scrapes were collected by a gynecologist and placed immediately in saline. HPV DNA was assessed by PCR, and HPV genotypes were determined by hybridization of PCR products with type-specific biotinylated probes.
Results   Of the 127 participants, 55 tested positive and 72 tested negative for HPV DNA. While there was no difference between the two groups with regards to age or to number of pregnancies, higher numbers of smokers and of women with multiple sexual partners and abnormal cytology were seen in the HPV-positive group ( P  < 0.001). HPV DNA was detected in the vaginal scrapes of all HPV-positive, but in none of the HPV-negative women (sensitivity and specificity = 1.0). Furthermore, the HPV genotype was the same in vaginal and endocervical specimens in all the HPV-positive women.
Conclusion   HPV detection by PCR, using endocervical or vaginal sampling, is a sensitive and highly specific test for the identification of HPV infection, in particular in women with cytomorphologically normal cervices.     

Comparison of HPV detection in parallel biopsies and cervical scrapes by PCR.

PCR was used to detect HPV DNA in cervical scrapes of 23 women with cervical cancer. Compared with PCR-assisted HPV DNA detection in parallel biopsies, the sensitivity was 81% and the specificity 100%. We conclude that cervical scrapes can be used for detection of HPV DNA in women with cervical cancer.     

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.     

Rapid detection of human papillomavirus in cervical scrapes by combined general primer-mediated and type-specific polymerase chain reaction.

A two-step polymerase chain reaction (PCR) procedure was used as a new screening strategy for the detection of human papillomavirus (HPV) genotypes in cervical scrapes omitting prior DNA extraction. Sample preparation consisted of a freeze-thaw step followed by boiling the cells before the PCR mixture was added. This pretreatment was as efficient and reproducible for HPV DNA amplification as DNA purification. By using crude cell suspensions, a prescreening of the samples with the general primer-mediated PCR method (GP-PCR) was performed to detect a broad spectrum of sequenced and still unsequenced HPV types at the subpicogram level. HPV-containing scrapes by GP-PCR were subjected to HPV 6, 11, 16, 18, 31, and 33 type-specific PCR (TS-PCR) to identify the sequenced HPV types. This direct GP/TS-PCR method was tested on a large group of cervical scrapes (n = 459) from women visiting a gynecologic outpatient clinic. The results were compared with HPV data obtained by a method using modified filter in situ hybridization and TS-PCR in which the PCR was mainly used to confirm HPV positivity. A substantially higher HPV prevalence rate was found by direct GP/TS-PCR strategy. The results indicate that GP/TS-PCR is a rapid, sensitive, and reliable detection method for HPV in cervical scrapes. The easy performance on crude cell suspensions makes this strategy applicable for large HPV-screening programs.     

Serum antibodies to human papillomavirus (HPV) pseudovirions correlate with natural infection for 13 genital HPV types

BackgroundSerology for human papillomaviruses (HPV) types -16 and -18 is established as an important tool for studies of HPV vaccinology and epidemiology. However, as there are a large number of oncogenic genital types of HPV there is a need for development of high-throughput, validated HPV serological assays that can be used for more comprehensive seroepidemiological studies and for research on multivalent HPV vaccines.ObjectivesTo develop a multiplexed pseudovirion-based serological assay (PsV-Luminex) encompassing 21 HPV types and validate the method by correlating the serology with the presence of type specific HPV DNA in cervical samples.Study designCervical swabs from 3,291 unvaccinated women attending organized cervical screening in Slovenia were tested with 3 different HPV DNA detection methods and presence of HPV DNA compared to presence of serum antibodies to pseudovirions from 15 genital HPV types (HPV-6,-11,-16,-18,-31,-33,-35,-39,-45,-52,-56,-58,-59,-68,-73).ResultsOn average 51% of the HPV DNA positive women were seropositive for the same HPV type that was detected in the cervical specimen. We found a strong correlation with presence of HPV DNA and antibodies to the same HPV type for 13/15 genital HPV types (median OR = 5.7, CI 95% = 2.4–12.9). HPV-52 serology failed the validation and HPV-11 serology could not be validated because only a single woman was positive for HPV-11 DNA. The correlation between serology and HPV DNA status tended to be stronger among women infected with single HPV type (median OR = 10.5, CI 95% = 2.4–48.4) than among women with multiple HPV infections (median OR = 4.6, CI 95% = 1.8–11.7).ConclusionsA multiplexed HPV PsV-Luminex assay has been developed and validated to correlate with natural HPV infection for 13 HPV types, thus enabling more comprehensive studies in HPV epidemiology and vaccine research.     

Two novel genital human papillomavirus (HPV) types, HPV68 and HPV70, related to the potentially oncogenic HPV39.

The genomes of two novel human papillomavirus (HPV) types, HPV68 and HPV70, were cloned from a low-grade cervical intraepithelial neoplasia and a vulvar papilloma, respectively, and partially sequenced. Both types are related to HPV39, a potentially oncogenic virus. HPV68 and HPV70 were also detected in genital intraepithelial neoplasia from three patients and one patient, respectively. Comparison with sequence data in the literature indicates that the subgenomic ME180-HPV DNA fragment, cloned from a carcinoma cell line, corresponds to an HPV68 subtype and that several HPV DNA fragments amplified by PCR from genital neoplasia represent worldwide distributed variants of HPV68 and HPV70.     

Use of Cervista HPV HR assay for detection of human papillomavirus in samples with hybrid capture borderline negative results

Galan‐Sanchez F, Rodriguez‐Iglesias MA. Use of Cervista HPV HR assay for detection of human papillomavirus in samples with hybrid capture borderline negative results. APMIS 2010; 118: 681–4. We have evaluated Cervista HPV HR for papillomavirus detection in 65 samples previously borderline negative by the hybrid capture method (Digene), using InnoLipa and sequencing as confirmatory techniques. Nine samples were found to be positive by Cervista HPV HR, of which five (7.6%) were confirmed by InnoLipa. Four samples (6.1%) were false positive, of which three samples were reactive for A9 probes. The Cervista HPV HR assay can detect HPV‐positive samples from those with hybrid capture borderline results but can produce false‐positive results when tested for reactivity with A9 probes.     

Human papillomavirus (HPV) E6/E7 mRNA as a triage test after detection of HPV 16 and HPV 18 DNA

High‐risk human papillomavirus (HPV) DNA detection provides high sensitivity but low specificity for moderate‐grade cervical intraepithelial neoplasia or worse histological identification. A prospective study evaluated mRNA testing efficacy for predicting this histological diagnosis in case of HPV 16 and/or 18 DNA detection. A total of 165 endocervical samples harboring HPV 16 and/or 18 DNA were tested with NucliSENS‐EasyQ® HPV E6/E7‐mRNA‐assay (Biomerieux, Marcy l´Etoile, France). Women with cytological alterations were referred to colposcopy (n = 111). Moderate‐grade cervical intraepithelial neoplasia or worse was diagnosed in 25.8% of women presenting atypical squamous cells of undetermined significance or low‐grade squamous intraepithelial lesions and in 89.8% of women with high‐grade squamous intraepithelial lesions. mRNA sensitivity was 81.3% and 84.1%, respectively. Specificity was 52.2%, and 80.0%, respectively. Negative predictive value (NPV) was 88.9% in undetermined or low‐grade squamous lesions. Positive predictive value (PPV) was 97.4% in high‐grade squamous lesions. mRNA reduced colposcopies by 44.3% in undetermined or low‐grade squamous lesions. Direct treatment of mRNA‐positive cases reduced 77.5% of colposcopies in high‐grade squamous lesions. Women without cytological alterations were followed for 18 months (n = 35), and moderate‐grade cervical intraepithelial neoplasia or worse was diagnosed in 34.3%; mRNA sensitivity and specificity were 83.3% and 86.9%, respectively. PPV and NPV were 76.9% and 90.9%, respectively for predicting moderate‐grade cervical intraepithelial neoplasia or worse in 18 months. mRNA reduced the number of visits for follow‐up in 62.2%. In conclusion, NucliSENS‐EasyQ® HPV E6/E7‐mRNA‐assay (Biomerieux) can serve as a triage test in case of HPV 16 and/or 18 DNA detection. J. Med. Virol. 85: 1063–1068, 2013. © 2013 Wiley Periodicals, Inc.