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PubMed2006

Detection of human papillomavirus in cervical cell specimens by hybrid capture and PCR with different primers.

The purpose of this study was to compare hybrid capture assay with PCRs using different primers for the L1, E6-E7 regions for the detection of human papillomavirus (HPV) genome. One hundred twenty-five cervical smears with normal (n=42) and abnormal (n=83) cytology were investigated. Those at high-risk for HPV were studied by hybridization antibody capture assay and PCR with the pU-1M/pU-2R primers. Target DNA from the HPV L1 region was amplified by SPF10 primer set and home-PCR with MY09/MY11 primers. The presence of HPV DNA in cervical smears was detected by SPF10 (in 72% of cases), MY09/MY11 (58%), hybrid capture (55%) and pU-1M/pU-2R (39%). Results obtained with the SPF10 and MY09/MY11 consensus primer sets as well as hybrid capture and pU-1M/pU-2R specific for high-risk types differed significantly (chi2, P<0.0005). The correlation between assays with the use of SPF10 and MY09/MY11 was 86% and between hybrid capture and the pU-1M/pU2R technique--78%. In 49% of samples HPV DNA was detected by the four methods, whereas in 12% only by the SPF10 primers. The most sensitive technique was found to be PCR with the use of SPF10 primers, while the most specific--the MY09/11 PCR method. It seems that home-PCR with MY09/MY11 primers could be applied in screening tests.

Animals↗

PubMed2000

Comparison of human papillomavirus detection and typing by cycle sequencing, line blotting, and hybrid capture.

We compared the results of human papillomavirus (HPV) detection and typing from 781 cervical samples assayed by three methods: L1 consensus PCR followed by cycle sequencing, L1 consensus PCR with biotinylated primers followed by hybridization to a line blot, and Hybrid Capture assay. Both PCR assays used L1 consensus PCR with primers MY09 and MY11. We evaluated the amplification efficiencies of both PCR assays and also compared the specific HPV types detected by each method. The samples positive by the Hybrid Capture assay were compared to the specific types detected by the PCR-based assays. The concordance between the two PCR assays in producing an HPV amplicon visible by gel electrophoresis or in detecting any HPV type was moderate: kappa values were 0.61 (95% confidence interval [CI] = 0.56 to 0.67) and 0.51 (95% CI = 0.46 to 0.58), respectively. The McNemar test for correlated proportions indicated that biotinylated PCR was less likely to produce a band (P = 0.001) and to detect an HPV type (P = 0.001) than the other PCR assay. In comparing the Hybrid Capture assay results with the HPV types detected by the PCR-based assays, we found that positivity by the Hybrid Capture assay for a number of samples may be due to cross-hybridization with HPV types not included in the Hybrid Capture assay probe cocktails.

Biotinylation↗

PubMed1994

Human papillomavirus detection by hybrid capture and its possible clinical use.

AIMS: To determine the sensitivity of the hybrid capture method for human papillomavirus (HPV) detection and potential clinical uses as a screening method for the identification of cervical intraepithelial neoplasia. METHODS: The presence of oncogenic types of HPV was tested for in samples taken from the cervix at colposcopy, and compared with detection by polymerase chain reaction (PCR) in 60 patients. Both sets of results were corrected with the pathology determined by biopsy and smear cytology. RESULTS: Hybrid capture detection showed 86% agreement with PCR. Eighty three percent of CIN 3 lesions, 62% of CIN 2, 59% of CIN 1 and 21% of normal controls were positive for oncogenic HPV types. CONCLUSION: The hybrid capture detection method is reliable, sensitive, and easy to use. The addition of HPV testing to cytological screening would detect a greater proportion of cervical dysplasia with a higher false positive rate.

Cervix Uteri↗

PubMed2004

Hybrid capture 2 viral load and the 2-year cumulative risk of cervical intraepithelial neoplasia grade 3 or cancer.

OBJECTIVE: The purpose of this study was to determine the clinical value of a semiquantitative measure of human papillomavirus viral load by the hybrid capture 2 assay for stratification of the risk of histologic cervical intraepithelial neoplasia grade 3 or carcinoma. STUDY DESIGN: The Atypical Cells of Unknown Significance and Low-Grade Squamous Intraepithelial Lesions Triage Study was a randomized clinical trial of 5060 women with 2 years of follow-up to evaluate treatment strategies for women with equivocal or mildly abnormal cervical cytologic condition. The usefulness of the continuous hybrid capture 2 output relative light units/positive controls that were above the positive threshold (1.0 relative light units/positive controls), which was a surrogate for human papillomavirus viral load, for distinguishing between hybrid capture 2 positive women who were diagnosed with cervical intraepithelial neoplasia grade 3 or carcinoma during the study from those who were not diagnosed with cervical intraepithelial neoplasia grade 3 or carcinoma was examined with the use of receiver-operator characteristic analyses. RESULTS: Relative light units/positive controls values did not further discriminate between hybrid capture 2 positive women with cervical intraepithelial neoplasia grade 3 or carcinoma from those with less than cervical intraepithelial neoplasia grade 3 or carcinoma. The use of a cervical intraepithelial neoplasia grade 2 or more severe or carcinoma case definition did not alter our findings. CONCLUSION: Among women with atypical cells of unknown significance or low-grade squamous intraepithelial lesion cervical cytologic findings, the hybrid capture 2 viral load measurement did not improve the detection of 2-year cumulative cases of cervical intraepithelial neoplasia grade 3 or carcinoma significantly.

DNA, Viral↗

PubMed1993

Detection of dengue-2 viral RNA by reversible target capture hybridization.

A reversible target capture (RTC) sandwich hybridization technique has been developed for the detection of dengue-2 viral RNA. The RTC is a form of sandwich hybridization that utilizes two probes: a poly(dA)-tailed capture probe and a labeled detector probe. Following hybridization of both probes to the analyte in solution, the poly(dA)-tailed capture probe is used to selectively remove the hybrids by capture on oligo(dT)-coated paramagnetic beads. AFter elution from the beads, the presence of specific hybrids is revealed by detection of the labeled probe. After optimization of all parameters by using 32P-labeled probes, digoxigenin was used as a label to preclude the use of radioisotopes. The sensitivity of the developed RTC procedure was determined. The lowest amount of virus detectable in cell culture lysates by using 32P-labeled probes was 20 PFU, while with digoxigenin-labeled probes, 200 PFU was detectable. The RTC procedure also detected dengue-2 virus in infected mosquitoes, both individually and in pools. The RTC has the advantage of being performed directly on crude samples, eliminating the need for phenol extraction and purification of target nucleic acids. These results indicate that the RTC procedure is sensitive, rapid, and easy to perform and that its use in surveillance programs will allow detection of dengue virus in pools of mosquitoes more rapidly than current procedures.

Animals↗

PubMed1997

Comparison of PCR and hybrid capture methods for detection of human papillomavirus in injection drug-using women at high risk of human immunodeficiency virus infection.

We compared Hybrid Capture, a new technique for detection of human papillomaviruses (HPV), with a PCR assay based on L1 consensus primers. By both methods, the HPV prevalence was higher in human immunodeficiency virus (HIV)-positive women than in HIV-negative women. PCR had a higher sensitivity (0.89 versus 0.48) but lower specificity (0.43 versus 0.93) for detection of Pap smear abnormalities, compared to Hybrid Capture. The higher intensity of hybridization signal by PCR was related to higher estimates of viral load by Hybrid Capture.

DNA, Viral↗

PubMed1995

Human papillomavirus testing by hybrid capture appears to be useful in triaging women with a cytologic diagnosis of atypical squamous cells of undetermined significance.

OBJECTIVE: Our purpose was to determine the clinical value of human papillomavirus deoxyribonucleic acid testing with the hybrid capture test, specifically to examine whether human papillomavirus testing could identify which women with Papanicolaou smears read as atypical squamous cells of undetermined significance were most likely to have histologically confirmed cervical intraepithelial neoplasia. STUDY DESIGN: Hybrid capture testing for 14 human papillomavirus types, repeat Papanicolaou smears, and colposcopically directed biopsies were performed concurrently on 217 women referred to a student health colposcopy clinic with a previous Papanicolaou smear read as atypical squamous cells of undetermined significance. RESULTS: Human papillomavirus deoxyribonucleic acid positivity was associated with an eightfold increased likelihood of histologic confirmation of cervical intraepithelial neoplasia. The sensitivity of hybrid capture for any cervical intraepithelial neoplasia was 86% (43/50) and for grade 2 or 3 was 93% (14/15), whereas the corresponding values for the repeat Papanicolaou smear were 60% (30/50) and 73% (11/15), respectively. Moreover, high viral levels of human papillomavirus types known to be associated with cervical cancer were strongly predictive of high-grade cervical intraepithelial neoplasia. CONCLUSIONS: Testing for human papillomavirus deoxyribonucleic acid with hybrid capture appears to offer an effective means by which patients whose cervical Papanicolaou smears have been read as atypical squamous cells of undetermined significance could be triaged for colposcopy. In particular, sensitivity for high-grade cervical intraepithelial neoplasia could be maintained and specificity markedly improved by referring only those patients who had elevated levels of human papillomavirus deoxyribonucleic acid of cancer-associated viral types.

Adolescent↗

PubMed1996

Analytic sensitivities of hybrid-capture, consensus and type-specific polymerase chain reactions for the detection of human papillomavirus type 16 DNA.

Human papillomavirus type 16 (HPV-16) DNA is detected commonly in cervical carcinomas; in this study, we have determined the analytical sensitivities of Hybrid Capture, HPV-consensus PCR, and three HPV-16-specific polymerase chain reactions (PCRs) for the detection of HPV-16 DNA. Samples investigated included a cervical cancer cell line, cervical scrapes from 20 patients attending colposcopy clinics, and buccal swabs from eight immunosuppressed children. HPV-16 E7 and E5-nested PCRs [Cavuslu et al. (1996): Journal of Virological Methods, in press] produced positive signals from samples containing fewer than ten HPV-16 genomes per reaction. HPV-consensus PCR [Manos et al. (1989): Cancer Cells 7:209-214] and HPV-16 PCR using primers of van den Brule et al. [(1990): Journal of Clinical Microbiology 25:2739-2743] were of intermediate sensitivity (i.e., produced positive signals from samples containing 250 and 2,500 HPV-16 genoms/reaction, respectively) and Hybrid Capture could detect just 50,000 HPV-16 genomes/reaction. Highest rates of positivity for cervical samples were detected with HPV-16 E7 or E5-nested PCRs [50% (10 of 20 samples) and 60% (12 of 20 samples) positive, respectively], intermediate rates with HPV-consensus PCR and PCRs using the primers of van den Brule et al. [both 35% (7 of 20 samples)], and lowest rates of positivity [25% (5 of 20 samples)] with Hybrid Capture. None of eight buccal swab samples from immunosuppressed children were positive by Hybrid Capture, yet three (37.5%) were positive by HPV-16 E5-nested PCR. These data indicate that HPV-16 type-specific PCRs should be used for the investigation of specimens that may contain low amounts of HPV-16 DNA.

Adolescent↗

PubMed2000

Discrimination between Atlantic and Pacific Subspecies of Northern Bluefin Tuna (Thunnus thynnus) by Magnetic-Capture Hybridization Using Bacterial Magnetic Particles.

The previously developed magnetic-capture hybridization technique employing bacterial magnetic particles was applied to discriminate between Atlantic and Pacific subspecies of the northern bluefin tuna (Thunnus thynnus) using specific DNA sequences. Nucleotide sequences of a 925-bp fragment (ATCO) flanking the mitochondrial ATPase and cytochrome oxidase subunit III genes in these two subspecies were compared. Two regions having single-nucleotide and three-nucleotide differences between the subspecies were adopted to design DNA probes (NR1, 21-mer; NR2, 29-mer), and two internal primer sets were designed to amplify DNA fragments containing these regions. The DNA probes were immobilized on bacterial magnetic particles via streptavidin-biotin conjugation and subjected to magnetic-capture hybridization with the digoxigenin-labeled fragments amplified using the internal primers. The luminescence intensities of DNA on bacterial magnetic particles obtained by hybridization between the probes and the complementary fragments were higher than those obtained by hybridization with noncomplementary fragments. These data suggest that this system employing DNA on bacterial magnetic particles may be useful for discrimination of these two subspecies by recognizing a single-nucleotide difference.

Journal Article↗

PubMed2001

Hybrid capture as a means of detecting human papillomavirus DNA from liquid-based cytology specimens: a preliminary evaluation.

The connection between human papillomavirus (HPV) and cervical cancer is well documented, as is the benefit of an adjunctive, standardised objective test to complement cervical screening. Here, 197 liquid-based cytology (LBC) specimens previously processed for cytological examination (75 negative, 108 borderline/mild dyskaryosis, and 14 moderate/severe dyskaryosis) are examined for the presence of HPV using a hybrid capture assay. Of the 197 specimens tested for HPV DNA, 97 (49.2%) were positive using the combined high/low-risk probe, and 59 (29.9%) were negative cytologically and with the hybrid capture HPV test; however, 16/175 (21.3%) specimens classified as negative cytologically with LBC proved positive for HPV by hybrid capture. Of the 122 samples that showed abnormal cytology, 81 (66.4%) were positive for HPV. Although the number of samples tested is small, the results show that the use of LBC technology for both cervical cytological screening and the hybrid capture test has the potential to reduce both false-negative and -positive rates associated with cytological screening alone.

Adolescent↗

PubMed2005

[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.

DNA Primers↗

PubMed2001

Detection of Salmonella and simultaneous detection of Salmonella and Shiga-like toxin-producing Escherichia coli using the magnetic capture hybridization polymerase chain reaction.

The magnetic capture hybridization polymerase chain reaction (MCH-PCR) was used to detect Salmonella and also to simultaneously detect Salmonella and Shiga-like toxin-producing Escherichia coli (SLTEC). Fifty-seven Salmonella and 41 SLTEC were included in the study. Salmonella were detected either individually by a single MCH-PCR assay targeting the inv gene or simultaneously with SLTEC by a multiplex MCH-PCR in which SLTEC were detected using primers for the slt genes. Both single and multiplex assays were found to be specific for tested pathogens. The results indicate that MCH-PCR can be used as means of detecting single or multiple bacterial pathogen(s).

Adhesins, Bacterial↗

PubMed2002

Optimizing the hybrid capture II human papillomavirus test to detect cervical intraepithelial neoplasia.

OBJECTIVE: To estimate the optimal relative light unit ratio, and correspondingly viral load, of the hybrid capture II oncogenic human papillomavirus deoxyribonucleic acid test for detecting cervical intraepithelial neoplasia (CIN). METHODS: Women with abnormal cytology were referred for colposcopy, and a cervical swab or brush specimen was obtained for human papillomavirus testing. Sensitivities, specificities, and likelihood ratios of different relative light unit ratio cutoffs were calculated using a reference standard of colposcopy or biopsy of either CIN II+ (CIN II, III, or carcinoma), or CIN I+ (CIN I, CIN II+). The receiver operating characteristic curve was used to estimate optimal test-positive cutoff points for the hybrid capture II test. RESULTS: CIN II+ was found in 18.7% (98 of 524) and CIN I in 10.5% (55 of 524) of the women. The optimal relative light unit ratio was 15.56, giving a sensitivity and specificity of 82.7% and 73.2% for CIN II+, and 74.2% and 77.8% for CIN I+. In a stratified analysis, a higher relative light unit cutoff (15.19) optimized sensitivity and specificity for CIN II+ (sensitivity 81.8%, specificity 51.5%) for women with low-grade squamous intraepithelial lesions cytology, whereas the optimal cutoff was 2.36 (sensitivity 100%, specificity 73.0%) for women with atypical squamous cells of undetermined significance, yielding referral rates of 53.3% and 28.7%, respectively. Use of a swab or brush was not associated with the level of human papillomavirus detected (P >.05). CONCLUSION: Use of a higher cutoff for the relative light unit ratio (higher viral load) of the hybrid capture II test may improve the management of women, especially those with low-grade squamous intraepithelial lesions cytology.

Adolescent↗

PubMed2000

Contribution of human papillomavirus testing by hybrid capture in the triage of women with repeated abnormal pap smears before colposcopy referral.

OBJECTIVE: The purpose of this work was to evaluate the ability of testing for high-risk human papillomavirus (HPV) types using the hybrid capture technique to predict the presence of cervical intraepithelial neoplasia (CIN) II,III in patients with repeated atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesion (LGSIL) on Pap smears. METHODS: Hybrid capture testing and tissue biopsy were performed on 503 consecutive women with ASCUS or LGSIL on repeated Pap smears who were referred for colposcopy. RESULTS: A highly significant association (P < 0.0001) was found between a positive test for high-risk HPV types and CIN II,III, with an 87.0% positive predictive value and a 95.7% negative predictive value. In 226 women with ASCUS on repeated Pap smears, a positive test for high-risk HPV types had a 85.7% sensitivity and a 97% specificity for CIN II,III. In 277 patients with LGSIL on repeated Pap smears, a positive test for high-risk HPV types had an 88.2% sensitivity and a 94.7% specificity for CIN I,II. Reserving colposcopy examination for women who were positive for high-risk HPV types would have reduced the number of referrals for colposcopy to 24.6% and maintained a sensitivity of 87.0% for CIN II,III. CONCLUSIONS: A positive hybrid capture test for high-risk HPV types was highly sensitive and specific for the presence of CIN II,III in patients with ASCUS and LGSIL on repeated Pap smears. We believe that improved methodology will eventually enable more selective colposcopy referrals without affecting patient safety among these women.

Adolescent↗

PubMed2004

Optimal cutoff of the hybrid capture II human papillomavirus test for self-collected vaginal, vulvar, and urine specimens in a colposcopy referral population.

OBJECTIVE: To estimate the optimal relative light unit ratio, as a measure of viral load, of the Hybrid Capture II human papillomavirus (HPV) test in self-collected specimens for detecting cervical intraepithelial neoplasia (CIN). METHODS: Two hundred women referred for colposcopy with abnormal cytologic, self-collected vaginal and vulvar swabs and urine for HPV testing. The receiver operating characteristic (ROC) curve method was used to estimate optimal cutoffs for the Hybrid Capture II test. The reference standard was colposcopy, with directed biopsy as required. RESULTS: The estimated optimal cutoffs of the relative light unit ratio for detecting CIN 2 or higher for urine, vulvar, and vaginal samples gave sensitivities of 72.4%, 82.8%, and 89.0% and specificities of 57.0%, 52.1%, and 55.9%, respectively. At the manufacturer's recommended 1.0 cutoff, sensitivities were 44.8%, 62.1%, and 86.2% for urine, vulvar, and vaginal samples, with specificities of 69.7%, 62.7%, and 53.5%, respectively. The likelihood ratios (likelihood of being truly positive after a positive test result) were similar for the optimal and the 1.0 cutoff. CONCLUSIONS: The ROC curve methods did not improve the overall diagnostic accuracy of the Hybrid Capture II test compared with the 1.0 relative light unit ratio cutoff.

Journal Article↗

PubMed2004

[Investigation of human papillomavirus DNA in cervical specimens by hybrid capture assay].

The aim of this study was to investigate the presence of human papillomavirus DNA (HPV-DNA) in cervical specimens of those randomly selected women, age between 18-62 years and applied to Gynecology and Obstetric Clinics in a six months period. Two hundred and six women were enrolled during the study. HPV-DNA in the cervical specimens were investigated with Hybrid Capture System (Digene Hybrid Capture System, UK) and also the cytologic specimens were examined simultaneously. HPV-DNA positivity was 4.9% overall. HPV-DNA positivity was found to be higher among younger population (30-39 age group: 7.3%), women with lower education level (6.9 %) and with lower income level (11.8%). Additionally, HPV-DNA positivity rate was found higher among women who experienced their first sexual intercourse at lower ages (15-19 age group: 6.3%) and who use oral contraceptives (7.7%). HPV-DNA positivity was found to be 2.1% in women with normal cervical cytology and 42.9% in women with epithelial cell abnormalities, and this difference was statistically significant (p=0.000). The presence of HPV together with high rates of epithelial cell abnormalities leading to cervical cancer, supported the association between HPV and malignancy. Thus, HPV-DNA testing should be included in the cervical cancer screening programmes.

Adolescent↗

PubMed2003

[Performance of cervical cytology with review by different observers and hybrid capture II in the diagnosis of cervical intraepithelial neoplasia grades 2 and 3].

UNLABELLED: To evaluate the performance of initial cervical cytology and that collected at the reference service with a review conducted by different observers and techniques, as well as hybrid capture II, in the diagnosis of high-grade cervical intraepithelial neoplasia (CIN), 105 women attended from August 2000 to June 2001 for preneoplastic atypia upon cervical cytology were included. A new cervical cytology and hybrid capture II for DNA-HPV were conducted in all the patients. Cervical biopsies were taken in 91 women. Performance of the investigative procedures was described by estimating the sensitivity, specificity, and positive likelihood ratio (PLR), with a 95% confidence interval (95% CI), considering histological diagnosis the gold standard. RESULTS: initial cervical cytology showed sensitivity and specificity of 57% and 82% for diagnosis of CIN grades 2 and 3, with a PLR of 3.2 (95% CI: 1.5-6.8). Referral cervical cytology showed a sensitivity and specificity for CIN 2 and 3 of 79% and 84%, respectively, with a PLR of 5.0 (95% CI: 2.5-10.0). Sensitivity (86%), specificity (80%), and PLR (4.3) were similar when a second observer (using a routine technique) reviewed the slide. Using rapid revision by a third observer, the sensitivity was significantly lower (64%). Hybrid capture II showed a high sensitivity (100%), low specificity (43%), and low PLR (1.7, 95% CI: 1.4-2.2).

Colposcopy↗

PubMedDate not supplied

[Simultaneous detection of HBV-DNA in hepatitis B surface antigen positive serum samples by "in-house" polymerase chain reaction and hybrid capture methods].

In this study, a total of 136 serum samples positive for hepatitis B surface antigen (HBsAg), have been screened for the presence of HBV DNA, by using in-house polymerase chain reaction (PCR) and hybrid capture (HC) assay (Digene Hybrid Capture System, UK), and the results have been compared by means of their performances. Of the serum samples tested, 53 (38.9%) and 34 (25%) were found positive for HBV DNA by PCR and HC assays, respectively. Accordingly, 19 (13.9%) PCR positive samples, were found negative with HC assay (p < 0.001). None of PCR negative samples were positive with HC assay. Eighty three (61%) of the 136 specimens were found negative by both of the methods. As a result, it was concluded that, in-house PCR was significantly more sensitive than HC assay for the detection of HBV DNA and may be used for the detection of HBV DNA, while HC assay may be useful especially for monitoring and predicting the response to antiviral therapy.

DNA, Viral↗