Evaluation of the SPF10-INNO LiPA human papillomavirus (HPV) genotyping test and the roche linear array HPV genotyping test

The need for accurate genotyping of human papillomavirus (HPV) infections is becoming increasingly important, since (i) the oncogenic potential among the high-risk HPV genotypes varies in the pathogenesis of cervical cancer, (ii) monitoring multivalent HPV vaccines is essential to investigate the efficiency of the vaccines, and (iii) genotyping is crucial in epidemiologic studies evaluating HPV infections worldwide. Various genotyping assays have been developed to meet this demand. Comparison of different studies that use various HPV genotyping tests is possible only after a performance assessment of the different assays. In the present study, the SPF(10) LiPA version 1 and the recently launched Roche Linear Array HPV genotyping assays are compared. A total of 573 liquid-based cytology samples were tested for the presence of HPV by a DNA enzyme immunoassay; 210 were found to be positive for HPV DNA and were evaluated using both genotyping assays (163 with normal cytology, 22 with atypical squamous cells of undetermined significance, 20 with mild/moderate dysplasia, and 5 with severe dysplasia). Comparison analysis was limited to the HPV genotype probes common to both assays. Of the 160 samples used for comparison analysis, 129 (80.6%) showed absolute agreement between the assays (concordant), 18 (11.2%) showed correspondence for some but not all genotypes detected on both strips (compatible), and the remaining 13 (8.2%) samples did not show any similarity between the tests (discordant). The overall intertest comparison agreement for all individually detectable genotypes was considered very good (kappa value, 0.79). The genotyping assays were therefore highly comparable and reproducible.     

Detection of high-risk human papillomavirus by two molecular techniques: hybrid capture and linear array

A number of human papillomaviruses (HPV) are the etiological agents of cervical cancer. The present study compared the performance of the hybrid capture method with that of linear array for the detection of high-risk HPV in 218 cervical samples. For the linear array technique, the DNA was extracted using two different procedures, one manual and the other automated. There was no difference in high-risk HPV (HR-HPV) detectability between the two extraction procedures but the automated procedure had the advantages of simplicity, time and efficiency. There was agreement in 199 (91.3%) of the results. The K value for the two assays was 0.81 indicative of "near perfect" agreement. Both methods, hybrid capture and linear array, are sensitive options for detection of HPV in cervical samples. Linear array enables the identification of the genotype present in the sample and the presence of multiple infections.     

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.     

Hybrid capture II, a new sensitive test for human papillomavirus detection. Comparison with hybrid capture I and PCR results in cervical lesions

AIM: To test a new assay for the detection of human papillomavirus (HPV) DNA, hybrid capture II (HC II), compared with the previous commercialized hybrid capture I (HC I) and polymerase chain reaction (PCR) results on cervical scrapes from fresh cone excision biopsy samples. METHODS: The three methods were used on cervical scrapes from 42 fresh cone excision biopsy samples. There were nine metaplastic and inflammatory lesions, five low grade lesions, and 28 high grade lesions. PCR was performed using the general primers GP5+/GP6+. The viral load of high risk HPV DNA was estimated by the ratio of relative light units to positive control values in the samples. RESULTS: The sensitivity of HC I for the detection of high grade lesions was 71.4%, while it was 92.8% for HC II and 96.4% for the PCR. Considering only the absence of detectable cervical in situ neoplasia, the specificity was 88.9% for HC I, 66.7% for HC II, and 66.7% for PCR. With HC II, for a ratio of cervical sample to normal control of > 200, the sensitivity for the detection of high grade lesion was only 34.6% with a specificity of 66.7%. CONCLUSIONS: HPV detection with the HC II assay is more sensitive than the previous HC I and represents a more convenient and easier test than PCR for routine use. Nevertheless the viral load estimated with this test cannot be a reliable predictive indicator of high grade lesions.     

Accuracy and interlaboratory reliability of human papillomavirus DNA testing by hybrid capture.

Epidemiologists and clinicians wishing to introduce human papillomavirus (HPV) testing into cervical cancer prevention programs need standardized, reliable, and accurate HPV DNA tests that can detect the full spectrum of pathogenic HPV types. The Hybrid Capture System assay from Digene (hybrid capture assay) is a nonradioactive kit designed to detect 14 HPV types in two groups: a mix of 9 high-risk types associated with anogenital cancer (HPV types 16, 18, 31, 33, 35, 45, 51, 52, and 56) and another group of 5 low-risk types associated with condyloma acuminatum (HPV types 6, 11, 42, 43, and 44). The assay yields quantitative data meant to reflect viral concentration. In a study of 199 cervical specimens from women with concurrent Pap smears, we assessed the reliability of the new assay by comparing the hybrid capture assay results from three laboratories. We assessed the accuracy of the hybrid capture assay in comparison with a reference standard of HPV DNA content (multiple testing by several methods in two reference laboratories). We also compared the hybrid capture assay results with the concurrent cytologic diagnoses on the basis of an independent review of each smear by five pathologists. Pairwise interlaboratory agreement rates on HPV positivity for either high-risk or low-risk types ranged from 87 to 94%, and kappa values ranged from 0.61 to 0.83. Among specimens positive for high-risk types (the most important clinical outcome), the interlaboratory correlations of the quantitative data ranged from 0.60 to 0.90.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of human papillomavirus using hybrid capture 2 in oral brushings from patients with oropharyngeal squamous cell carcinoma

Detection of high-risk (HR) human papillomavirus (HPV) in oropharyngeal squamous cell carcinoma (SCC) has important prognostic implications; patients exhibit improved survival compared with patients with HPV- SCC. Oral brushing and rinsing samples were obtained from patients with oropharyngeal, oral cavity, or hypopharyngeal SCC and tested for HR-HPV using Hybrid Capture 2 (HC2; QIAGEN, Valencia, CA). HR-HPV in situ hybridization (ISH) was performed on biopsy tissue samples from the same patients. Oral cytologic samples from 16 SCCs were tested by HC2. Biopsy tissue samples were available for ISH in 11 cases. Five oropharyngeal SCCs were HR-HPV+ by ISH and HC2 (oral brushing). Of the oropharyngeal SCCs, 2 were positive by HC2 (oral brushing) and negative or equivocal by ISH. We found that 2 oral cavity carcinomas and 2 hypopharyngeal carcinomas were negative by HC2. One hypopharyngeal cancer was positive by ISH. All oral rinsing samples were negative by HC2. HC2 may be an effective method of determining HR-HPV status in patients with oropharyngeal SCC.     

Shared and persistent asymptomatic cutaneous human papillomavirus infections in healthy skin

Cutaneous human papillomavirus (HPV) types are commonly found in normal skin, and some of them have been suspected to play a role in the development of non‐melanoma skin cancer. This present study is divided into three sections, the aims of this study were to examine if certain HPV‐types persist over time and if HPV‐types are shared within families. From the first part of the study, swab samples from foreheads were collected for three longitudinal studies from one family with a newborn baby. Five specific HPV‐types were isolated from the family with a newborn, with HPV‐5 and FA67 being found at various time points and prevalence rates in all four members of the family. Part 2 consisted of a followed up study from two families with a 6 years interval. Six of the family members were found to have at least one of the HPV‐types identified in the family 6 years earlier. Many of the HPV‐types identified were shared within the families studied. Part 3 of this study involved weekly samples from four healthy females for 4 months. Among the four healthy individuals, 11%, 65%, and 56% of the weekly samples were HPV‐DNA positive with one individual HPV‐negative. All specimens were tested for HPV‐DNA by PCR using the broad range HPV‐type primer pair FAP59/64. The positive samples were HPV‐type determined by cloning and sequencing. Specific cutaneous HPV‐types persist over long periods of time in healthy skin in most individuals investigated and certain HPVs are shared between family members. J. Med. Virol. 81:1444–1449, 2009. © 2009 Wiley‐Liss, Inc.     

Detection of high risk human papillomavirus in routine cervical smears: strategy for screening.

AIM: To develop a methodology for direct detection of high risk human papillomavirus (HPV) infection in routine cervical smears by non-isotopic in situ hybridisation (NISH) which can be compared with cytopathological assessment of the same cells. METHODS: The methodology was established using cultured cells and routine cervical smears hybridised with digoxigenin labelled probes for HPV, 16, 18, 31, and 33. The technique was applied to the analysis of 53 patients from a sexually transmitted disease clinic. RESULTS: The optimal sensitivity achieved for single HPV detection in cultured cells was 1-2 copies of HPV 16 per cell and that for detection of a cocktail of HPV types in routine cervical smears was 2.5-12 copies per cell. Of parallel smears taken from patients with a normal Papinacolau-stained smear 33.3% (24) contained a HPV 16, 18, 31, and 33 signal indicating an occult HPV infection. The prevalence of these HPV types was similar in women in whom a cytopathological diagnosis of wart virus infection was made (64.7%, 17) and in patients with mild dyskaryosis (75%, 12). CONCLUSIONS: The methodology evolved localises HPV sequences directly to epithelial cell nuclei, which can be morphologically assessed by haematoxylin counterstaining. Sample contamination with exogenous viral sequences can be distinguished from true infection. In this study, a HPV signal was not found in morphologically normal epithelial cells. The methods described will permit the detection of HPV sequences in routinely collected cervical smears and the evaluation of the natural history and potential clinical relevance of HPV infection without changes in clinical practice.     

Results of human papillomavirus DNA testing with the hybrid capture 2 assay are reproducible

Reproducibility of the Hybrid Capture 2 Test (HC 2) for human papillomavirus (HPV) DNA detection was evaluated by assaying frozen cervical specimens in 1997 and again in 2001 from 1,775 women with normal cervical cytology. Using a cutoff point of 1.0 pg of HPV DNA/ml between a negative and a positive test result, the result of the kappa test for agreement was 0.72 (a kappa value of >0.60 is considered good agreement). Using cutoff points of 1.0 and 10.0 pg/ml between negative and low positive and between low positive and high positive, respectively, the kappa was 0.68 and the linear-weighted kappa was 0.76. The results of this study indicate that HC 2 testing is reproducible even among cytologically normal women with low test values.     

Detection of integrated high risk human papillomavirus in adenoid cystic carcinoma of the uterine cervix.

AIM: To investigate the role of human papillomavirus (HPV) in adenoid cystic carcinoma of the uterine cervix. METHODS: Eleven archival, paraffin wax embedded specimens were analysed by non-isotopic in situ hybridisation (NISH) for HPV types 6, 11, 16, 18, 31, and 33 using digoxigenin labelled probes. The polymerase chain reaction (PCR) was carried out on each of the cases using consensus primers to HPV. RESULTS: A total of eight adenoid cystic carcinomas harboured the HPV genome by NISH, of which five were PCR positive. Integrated HPV 16 DNA was demonstrated in seven of the eight NISH positive cases. One adenoid cystic carcinoma showed integrated HPV 31. HPV DNA was not detected in the three remaining cases. CONCLUSIONS: Integrated high risk HPV genome, in particular type 16, is associated with this uncommon type of primary cervical cancer.     

Comparative analysis of human papillomavirus infections in cervical scrapes and biopsy specimens by general SPF(10) PCR and HPV genotyping

Human papillomavirus (HPV) can be detected by DNA amplification from clinical samples. The aim of the present study was to compare the HPV status in both cervical scrape and biopsy specimens obtained from 174 patients, using the recently developed broad spectrum SPF(10) PCR-LiPA method. The detection rate of HPV in these materials was determined and the spectrum of HPV genotypes was compared. Cervical scrapes and biopsy specimens were obtained, either on the same day (group I), or with an interval of up to almost 2 years (group II, mean interval 97 days, range 1-469 days). HPV DNA was amplified by SPF(10) PCR and detected in a microtitre plate hybridization assay. Of the HPV-positive cases, the genotype was determined by reverse hybridization of the same SPF(10) amplimer on a line probe assay (LiPA), discriminating between HPV genotypes 6, 11, 16, 18, 31, 33-35, 39, 40, 42-45, 51-54, 56, 58, 59, 66, 68, 70, and 74. The results showed that the detection rate and the spectrum of HPV genotypes in cervical scrapes and the corresponding biopsy specimens were highly comparable in both patient groups, even when multiple genotypes were present. In both groups, multiple HPV genotypes were more frequently detected in cervical scrapes than in the corresponding biopsy specimens. In conclusion, HPV infection can be diagnosed in cervical scrapes and biopsy specimens using the SPF(10) PCR-LiPA system. Analysis of cervical scrapes accurately reflects the spectrum of HPV genotypes in the patient's cervical region, even with a sampling interval between the cervical scrape and the biopsy specimen.     

A novel filtration-based processing method of liquid cytology specimens for human papillomavirus DNA testing by hybrid capture II

We evaluated a more efficient method of processing liquid-based cervical cytology specimens for human papillomavirus (HPV) DNA testing by Hybrid Capture II (HCII). Aliquots were made from 701 specimens in the following sequence: 4.0, 2.0, 1.0, 0.5, and 1.5 mL. The 4.0-mL aliquot was processed by the standard method (STP), and half of the processed material was tested by HCII. Other aliquots were processed with a new, filtration-based processing method (NPM). The 2.0-mL NPM aliquot had HCII test performance most similar to the STP, ie, similar HCII positivity (P = .4) and good test agreement (kappa = 0.85, 95% confidence interval [CI], 0.80-0.89). The 194 cytologic negatives had greater positivity by STP (P = .04) compared with the 2.0-mL aliquot processed by NPM; between-method agreement was modest (kappa = 0.54, 95% CI, 0.36-0.72). A lower positive cut point for the 2.0-mL NPM aliquot partially abrogated this minor difference. In 241 specimens diagnosed as low-grade and 31 as high-grade squamous intraepithelial lesions, there were no significant differences in HPVpositivity (>85% and 90%, respectively) between STP and NPM. NPM reduces specimen handling and decreases total testing time by approximately 33% without significant losses in HCII test performance.     

Detection and genotyping of human papillomavirus DNA by SPF10 and MY09/11 primers in cervical cells taken from women attending a colposcopy clinic

Diagnosis of erythrovirus B19 (B19) relies on serology and the detection of viral DNA. Recently, a distinct erythrovirus isolate termed V9, markedly different from erythrovirus B19 (> 11% nucleotide disparity), was isolated. Standard B19 PCR assays were inconclusive and serologic tests failed to categorize V9 as an acute B19-like infection. Sequencing, combined with PCR studies, have since demonstrated the need for specific and differentiated techniques when examining samples for possible B19 or V9 viremia. The antigenic properties of the V9 capsid proteins have not been characterized previously. To address this question, V9 VP1 and VP2 open reading frames were cloned and expressed in insect cells using a baculovirus vector. Large quantities of purified recombinant V9 capsid protein were produced and electron micrographs revealed self-assembly of V9 VP1/VP2 and VP2 capsids into empty icosahedral erythrovirus-like particles with a diameter of approximately 23 nm. Screening of a panel of 270 clinical samples for the presence of V9 IgM and IgG antibodies in ELISA showed 100% serologic cross-reactivity between B19 and V9 when comparing V9 VP2 capsids to a commercial B19 VP2 assay. This suggests that both a V9 and a B19 antibody response may be diagnosed equally well by ELISA using either V9 or B19 recombinant capsids as antigen source. Retrospectively, translation of the V9 sequence indicates that despite a significant genetic variation on the DNA level, the majority of the discrepant DNA sequence represents silent mutations leading to an amino acid sequence very similar to the known B19 strains (96-97% homology).     

Analysis of human papillomavirus types in exophytic condylomata acuminata by hybrid capture and Southern blot techniques.

Exophytic condylomata acuminata of the external genitalia of 40 patients were analyzed for human papillomavirus (HPV) DNA by the Southern blot and hybrid capture methods. All lesions were initially analyzed by the Southern blot method by using a mixture of HPV type 6, 11, 16, and 18 whole genomic probes. Southern blots demonstrated characteristic PstI restriction patterns of HPV type 6, 11, or 16 in all but one lesion. HPV 6 subtypes accounted for 28 of 39 HPV-positive lesions. Twenty-seven of these 28 lesions contained HPV type 6a, and 1 lesion contained HPV type 6c. Eight lesions contained HPV type 11 and three contained HPV type 16. Two of the three condylomata acuminata containing HPV type 16 were obtained from solid-organ transplant recipients receiving immunosuppressive medications. The third lesion containing HPV type 16 was a typical exophytic condyloma acuminatum from a woman with previously resected vulvar carcinoma. The hybrid capture assay detected HPV DNAs in all lesions except the Southern blot-negative lesion. Twenty-five lesions were positive for the A probe only (HPV types 6 and 11 and related types). All of these lesions were found to contain HPV type 6 or 11 sequences in the Southern blot assay. The remaining 14 lesions were positive for both the A probe and the B probe (HPV types 16 and 18 and related types). The strongest signal in these 14 lesions by the hybrid capture assay was consistent with the result of the Southern blot assay in all but one case. We conclude that (i) HPV type 6a is the most common type found in these lesions, (ii) HPV type 16 may be present more often in exophytic condylomata acuminata from immunosuppressed individuals, (iii) hybrid capture is a useful tool for documenting the presence of HPV sequences in DNAs from exophytic condylomata acuminata, and (iv) in samples containing multiple HPV types, hybrid capture allows detection of minority HPV types.     

Human papillomavirus detection by the hybrid capture II assay with the liquid cell preservation solution Easyfix

The aim of this study is to validate the liquid cell preservation solution Easyfix for DNA detection of the HPV oncogene using the Hybrid Capture II method. 256 specimens were selected for the cytological study, possible biopsy and HPV oncogene search with the Easyfix fixative fluid and the Cervical Sampler transport medium. The results obtained with both mediums are comparable regardless of the cytological type. The relevance of a cytological study combined with the HPV search is stressed. To conclude, it is possible to put forward that the liquid cell preservation solution Easyfix can be used to detect the HPV oncogene using the Hybrid Capture II method.     

Comparison between prototype hybrid capture 3 and hybrid capture 2 human papillomavirus DNA assays for detection of high-grade cervical intraepithelial neoplasia and cancer

We compared the performance of a prototype version of the Hybrid Capture 3 (HC3) human papillomavirus (HPV) DNA assay to the current generation Hybrid Capture 2 (HC2) assay, both of which target 13 oncogenic HPV types, for the detection of cervical intraepithelial neoplasia grade 3 and cancer (CIN3+) with cervicovaginal lavage specimens collected at enrollment into a 10-year cohort study at Kaiser Permanente (Portland, Oreg.). HC3 results for a risk-stratified sample (n = 4,364) were compared to HC2 results for the entire cohort (n = 20,810) with receiver operating characteristics curves, and the optimal cut points for both tests (relative light units [RLU]/positive control [PC]) for the detection of CIN3+ were determined. Specimens were also tested for HPV16 and HPV18 with separate HC3 type-specific probes. The optimal cut point for detecting CIN3+ was 1.0 RLU/PC for HC2, as previously shown, and was 0.6 RLU/PC for HC3. At the optimal cut points, HC3 and HC2 had similar screening performance characteristics for CIN3+ diagnosed at the enrollment visit. In analyses that included cases CIN3+ at enrollment and those diagnosed during early follow-up, HC3 had nonsignificantly higher sensitivity and equal specificity for the detection of CIN3+ compared to HC2; this increase in sensitivity was primarily the result of increased detection of CIN3+ in women who were 30 years of age or older and were cytologically negative (P = 0.006). We also compared the performance of the hybrid capture tests to MY09/11 L1 consensus primer PCR results (n = 1,247). HC3 was less likely than HC2 to test positive for specimens that tested positive by PCR for any untargeted types (P < 0.001). HC3 was less likely than HC2 to test positive for untargeted PCR-detected single infections with HPV53 (P = 0.001) and HPV66 (P = 0.01). There was good agreement between test positivity by PCR and by single type-specific HC3 probes for HPV16 (kappa = 0.76; 95% confidence interval [CI] = 0.71 to 0.82) and for HPV18 (kappa = 0.73; 95% CI = 0.68 to 0.79). In conclusion, we suggest that HC3 (>/=0.6 RLU/PC) may be slightly more sensitive than and equally specific test as HC2 (>/=1.0 RLU/PC) for the detection of CIN3+ over the duration of typical screening intervals.     

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.     

Clinical performance of the CLART human papillomavirus 2 assay compared with the hybrid capture 2 test

Persistent infection by high‐risk human papillomavirus (HR‐HPV) is a cause of cervical cancer. The use of HPV detection in cervical screening programs may improve the ability to identify women at risk of cervical cancer. Therefore, the development of appropriate methods for the detection of HR‐HPV is essential. The aim of this study was to evaluate the clinical performance of the CLART Human Papillomavirus 2 assay (CLART) in comparison with the Hybrid Capture 2 test (HC2), using a clinical cut‐off of cervical intraepithelial neoplasia grade 2 or worse. Discrepant results were analyzed further by the PapilloCheck HPV genotyping system. In the 425 studied women, HR‐HPV positivity rates were similar by both tests (CLART‐13 HR‐HPV: 63.1%; CLART‐17 HR‐HPV: 64.7%; HC2: 64.5%). Agreement between CLART‐13 HR‐HPV (κ = 0.969; concordance level 98.6%), CLART‐17 HR‐HPV (κ = 0.974; concordance level 98.8%), and HC2 were very good. When 13 HR‐HPV types were considered, the two tests showed a clinical sensitivity of 96% (95% CI: 92.6–97.9). The clinical specificity of CLART‐13 HR‐HPV was 73.6% (95% CI: 66.7–79.5) for cervical intraepithelial neoplasia grade 2 or worse, which was comparable to HC2 (71.4%; 95% CI: 64.3–77.5). When all 17 HR‐HPV types were considered, CLART showed a clinical sensitivity of 96.9% (95% CI: 93.8–98.5) and a clinical specificity of 71.9% (95% CI: 64.9–78.0). In conclusion, the CLART assay is efficient, sensitive, reproducible, and has a similar performance to HC2 for cervical intraepithelial neoplasia grade 2 or worse. Furthermore, this assay has the advantage of detecting and genotyping 35 HPV types by a single test, which can provide additional information on the predictive value of infection with HR‐HPV. J. Med. Virol. 83:272–276, 2011. © 2010 Wiley‐Liss, Inc.