Testing and Genotyping of High-Risk Human Papillomavirus by the cobas HPV Test and the Hybrid Capture 2 High-Risk HPV DNA Test Using Cervical and Vaginal Samples

The accurate detection and typing of high-risk human papillomavirus (HPV) are critical for cervical cancer screening. The Hybrid Capture 2 (hc2) and cobas HPV tests showed high agreement for cervical samples (94.4%, κ = 0.72, n = 693) and moderate agreement for vaginal samples (κ = 0.62, n = 108). The HPV16 and HPV18 results were highly consistent between the cobas and Linear Array tests (κ ≥ 0.96, n = 197). Three hc2-negative vaginal samples were repeatedly invalid by the cobas test due to β-globin control failures, highlighting amplification control benefits. No cross-contamination was detected in a challenge experiment.     

Comparison of Onclarity Human Papillomavirus (HPV) Assay with Hybrid Capture II HPV DNA Assay for Detection of Cervical Intraepithelial Neoplasia Grade 2 and 3 Lesions

Analytical and clinical performance validation is essential before introduction of a new human papillomavirus (HPV) assay into clinical practice. This study compares the new BD Onclarity HPV assay, which detects E6/E7 DNA from 14 high-risk HPV types, to the Hybrid Capture II (HC2) HPV DNA test, to concurrent cytology and histology results, in order to evaluate its performance in detecting high-grade cervical lesions. A population of 567 women, including 325 with ≥ASCUS (where ASCUS stands for atypical cells of undetermined significance) and any HC2 result and 242 with both negative cytology and negative HC2 results, were prospectively enrolled for the study. The overall agreement between Onclarity and HC2 was 94.6% (95% confidence intervals [CI], 92.3% to 96.2%). In this population with a high prevalence of disease, the relative sensitivities (versus adjudicated cervical intraepithelial neoplasia grades 2 and 3 [CIN2+] histology endpoints) of the Onclarity and HC2 tests were 95.2% (95% CI, 90.7% to 97.5%) and 96.9% (95% CI, 92.9% to 98.7%), respectively, and the relative specificities were 50.3% (95% CI, 43.2% to 57.4%) for BD and 40.8% (95% CI, 33.9%, 48.1%) for HC2. These results indicate that the BD Onclarity HPV assay has sensitivity comparable to that of the HC2 assay, with a trend to an increased specificity. Moreover, as Onclarity gives the chance to discriminate between the different genotypes, we calculated the genotype prevalence and the absolute risk of CIN2+: HPV 16 was the most prevalent genotype (19.8%) with an absolute risk of CIN2+ of 77.1%.     

Comparison of Human Papillomavirus Detections in Urine,Vulvar, and Cervical Samples from Women Attending a Colposcopy Clinic

While urine-based sampling for human papillomavirus (HPV) is being explored as a simple and noninvasive approach for cervical cancer screening, data comparing HPV genotyping in urine and those in cellular sampling of the cervix and vulva, and their correlation with rigorously confirmed cervical disease status, are sparse. We performed HPV genotyping on voided-urine and clinician-collected vulvar and cervical samples from 72 women undergoing colposcopy. Although urine-based HPV carcinogenic HPV detection was lower (58.3%) than cervical (73.6%) and vulvar (72.1%) detection (P = 0.05 and 0.07, respectively), the agreement of urine HPV with cervical and vulvar HPV was moderate (kappa = 0.55) and substantial (kappa = 0.62), respectively. Urine-based carcinogenic HPV detection had a clinical sensitivity of 80.8% (95% confidence interval [CI] = 60.7 to 93.5) and a specificity of 53.3% (95% CI = 37.9 to 68.3) for diagnosing cervical intraepithelial neoplasia grades 2/3 (CIN2/3) on histology; 90.0% of CIN3 was positive for urine HPV. The corresponding sensitivity and specificity values for vulvar sampling were 92% (95% CI = 74 to 99) and 40.5% (95% CI = 25.6 to 56.7), and those for cervical sampling were 96.2% (95% CI = 80.4 to 99.9) and 40% (95% CI = 25.7 to 55.7), respectively. HPV16 was the most common carcinogenic genotype detectable in 25% of urine, 33.8% of vulvar, and 31.9% of cervical samples overall, with prevalence increasing with cervical disease grade, regardless of the sampling method. Stronger cervical HPV PCR signal strengths were associated with increased frequency of urine HPV detection. In summary, the relatively lower detection rates but comparable clinical performance of urine-based HPV sampling underscore the need for larger studies to evaluate urine-based sampling for cervical cancer screening, epidemiologic studies, and postvaccination HPV disease surveillance.     

Comparison of the Abbott RealTime High-Risk Human Papillomavirus (HPV), Roche Cobas HPV, and Hybrid Capture 2 assays to direct sequencing and genotyping of HPV DNA

Infection with high-risk (HR) human papillomavirus (HPV) genotypes is an important risk factor for cervical cancers. We evaluated the clinical performances of two new real-time PCR assays for detecting HR HPVs compared to that of the Hybrid Capture 2 test (HC2). A total of 356 cervical swab specimens, which had been examined for cervical cytology, were assayed by Abbott RealTime HR and Roche Cobas HPV as well as HC2. Sensitivities and specificities of these assays were determined based on the criteria that concordant results among the three assays were regarded as true-positive or -negative and that the results of genotyping and sequencing were considered true findings when the HPV assays presented discrepant results. The overall concordance rate among the results for the three assays was 82.6%, and RealTime HR and Cobas HPV assays agreed with HC2 in 86.1% and 89.9% of cases, respectively. The two real-time PCR assays agreed with each other for 89.6% of the samples, and the concordance rate between them was equal to or greater than 98.0% for detecting HPV type 16 or 18. HC2 demonstrated a sensitivity of 96.6% with a specificity of 89.1% for detecting HR HPVs, while RealTime HR presented a sensitivity of 78.3% with a specificity of 99.2%. The sensitivity and specificity of Cobas HPV for detecting HR HPVs were 91.7% and 97.0%. The new real-time PCR assays exhibited lower sensitivities for detecting HR HPVs than that of HC2. Nevertheless, the newly introduced assays have an advantage of simultaneously identifying HPV types 16 and 18 from clinical samples.     

Comparative Evaluation of Three Commercial Systems for Detection of High-Risk Human Papillomavirus in Cervical and Vaginal ThinPrep PreservCyt Samples and Correlation with Biopsy Results

Genital human papillomavirus (HPV) is the etiologic agent of more than 99% of all cervical cancers worldwide, with 14 genotypes being considered oncogenic or “high risk” because of their association with severe dysplasia and cervical carcinoma. Among these 14 high-risk types, HPV-16 and -18 account for approximately 70% of cervical cancers. The aim of this study was to evaluate three FDA-approved HPV nucleic acid-based tests for the ability to predict high-grade cervical intraepithelial neoplasias (CIN2 or worse) in corresponding tissue biopsy specimens. Residual specimens (total n = 793, cervical n = 743, vaginal n = 50) collected in ThinPrep PreservCyt medium with a cytologic result of ≥atypical squamous cells of undetermined significance were tested by the Hybrid Capture 2 (HC2) assay (Qiagen, Gaithersburg, MD), the cobas HPV test (Roche Diagnostics, Indianapolis, IN), and the APTIMA HPV assay (Hologic, San Diego, CA). Genotyping for HPV-16 and HPV-18 was simultaneously performed by the cobas HPV test. Results were compared to cervical or vaginal biopsy findings, when they were available (n = 350). Among the 350 patients with corresponding biopsy results, 81 (23.1%) showed ≥CIN2 by histopathology. The ≥CIN2 detection sensitivity was 91.4% by the cobas and APTIMA assays and 97.5% by HC2 assay. The specificities of the cobas, APTIMA, and HC2 assays were 31.2, 42.0, and 27.1%, respectively. When considering only positive HPV-16 and/or HPV-18 genotype results, the cobas test showed a sensitivity and a specificity of 51.9 and 86.6%, respectively. While the HC2, cobas, and APTIMA assays showed similar sensitivities for the detection of ≥CIN2 lesions, the specificities of the three tests varied, with the greatest specificity (86.6%) observed when the HPV-16 and/or HPV-18 genotypes were detected.     

Comparison of PCR- and Hybrid Capture-Based Human Papillomavirus Detection Systems Using Multiple Cervical Specimen Collection Strategies

This study compared the performances of three human papillomavirus (HPV) detection tests with specimens collected by three alternative procedures. The HPV tests included the Hybrid Capture Tube test (HCT), the microplate-based Hybrid Capture II test (HC II), and the MY09-MY11 L1 consensus primer PCR-based assay. Initial cervical specimens were collected from study subjects with a broom device, and after Papanicolaou smears were made, residual specimens were placed into PreservCyt (PC), a liquid cytology medium. A second specimen was collected from each subject and placed into Digene Specimen Transport Medium (STM). The device for collection of the second specimen alternated with consecutive subjects between a conical cytology brush and a Dacron swab. At the 1.0-pg/ml cutoff, the results of the HC II agreed well with those of the PCR. Specifically, when PCR data were restricted to the types found by the HC II (HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68), there was greater than 90% agreement between the HC II and PCR results with both STM and PC. At a lower cutoff (0.2 pg/ml), HC II-positive results increased further, especially when the test was applied to the PC specimens. However, false-positive HC II results were more often observed at the 0.2-pg/ml cutoff. HC II yielded the highest HPV positivity with specimens placed into PC, followed by specimens collected with a conical brush and placed into STM and, last, by those collected with a Dacron swab and placed into STM. Our results demonstrate the utility of both the STM and PC specimen collection methods and show good agreement between the HC II and PCR.     

Evaluation of Transported Dry and Wet Cervical Exfoliated Samples for Detection of Human Papillomavirus Infection

We determined the feasibility of human papillomavirus (HPV) detection in cervical exfoliated cells collected as dry swab samples. Both dry cervical swab and specimen transport medium (STM) cervical swab samples were collected from 135 patients attending either colposcopy or women''s clinics in Guayaquil, Ecuador, who had a cytology diagnosis within 6 months. HPV was detected by dot blot hybridization and genotyped by the liquid bead microarray assay (LBMA). Overall, 23.1% of dry samples were positive for any high-risk HPV types, and 24.6% of STM samples were positive for any high-risk HPV types. Of 125 paired samples, the type-specific high-risk HPV proportion positive agreement was 60.7% (kappa, 0.69; 95% confidence interval [CI], 0.53 to 0.82). Of six women with cytological evidence of invasive cervical cancer, high-risk HPV DNA was detected in three of their STM samples and in five of their dry samples. Dry samples were more likely to be insufficient for HPV testing than STM samples. Consistent with this observation, the amount of genomic DNA quantitated with the β-actin gene was almost 20 times lower in dry samples than in STM samples when detected by the real-time TaqMan assay; however, HPV DNA viral loads in dry samples were only 1.6 times lower than those in matched STM samples. We concluded that exfoliated cervical cells could be collected as dry swab samples for HPV detection.Human papillomavirus (HPV) infection causes warts and various cancerous and precancerous lesions in men and women. It has been established that high-risk HPV infection is the etiological agent of cervical cancer, which affects almost half a million women worldwide and has a 50% mortality rate (12, 18). In developed countries, cervical cancer control relies on routine cytology screening to detect and treat cervical cancer precursor lesions, and HPV detection has been used for the management of women with equivocal cytology results (1, 4, 14). In resource-poor settings where cytology-based screening is difficult to implement, HPV detection has been proposed as the alternative primary screening test for cervical cancer (4, 17).Currently, HPV detection is performed on cervical samples collected in liquid medium by a trained clinician, which is often impractical or unavailable in remote areas and developing countries. Several approaches have been proposed to simplify the sample collection process for HPV detection. For example, HPV detection in self-collected vaginal swab samples has been proposed as a method that would eliminate the need for clinical visits, and we have shown that HPV detected in self-collected vaginal swab samples stored in specimen transport medium (STM) had sensitivity and specificity similar to those of clinician-collected cervical swab samples stored in STM for detecting cervical neoplasia (16). Currently, cervical exfoliated cells are collected either in phosphate-buffered saline (PBS), which is inexpensive but requires constant refrigeration, or in various liquid media, such as STM or Preservcyt, which preserve HPV DNA at room temperature but are expensive. Additionally, Preservcyt is flammable. Thus, the elimination of the requirement of refrigeration or special media would facilitate the collection, storage, and transport of cervical exfoliated cells in remote areas or resource-poor settings and ultimately reduce cost.A few studies have investigated the feasibility of dry sample collection for HPV detection. For example, dry swab samples could be collected and stored at 4°C before being processed for HPV detection (9, 13). Cervical smear samples could also be collected on filter paper for HPV detection (2, 7, 8). However, no studies have investigated the feasibility of collecting, storing, and shipping dry swab samples at room temperature for HPV detection or the effect of dry sample collection on DNA degradation.In this study, we first determined whether cells of cervical cancer cell lines could be stored at room temperature without significant DNA degradation. We then collected both dry cervical swab and STM cervical swab samples from 135 patients from Guayaquil, Ecuador. HPV detection and genotyping were determined by dot blot hybridization and the liquid bead microarray assay (LBMA), respectively (6). In addition, HPV DNA and the housekeeping gene β-actin were quantitated by using real-time TaqMan assays and 42 paired samples, 32 of which were HPV positive.     

Human Papillomavirus Genotype Specificity of Hybrid Capture 2 Low-Risk Probe Cocktail

A genotyping study of 285 Hybrid Capture 2 low-risk probe cocktail-positive specimens showed cross-reactivity with several untargeted human papillomavirus genotypes. Cross-reactivity was often clinically beneficial due to the detection of untargeted low-risk genotypes. A total of 8.4% of positive results, usually weak, were due to cross-reactivity with high-risk genotypes. Establishment of a gray zone is recommended.Low-risk alpha-human papillomaviruses (HPV) have never been at the fore in HPV research, due to their connection with benign neoplasm only. However, interest in these genotypes has increased substantially in recent years, due to the fact that quadrivalent HPV vaccine contains, in addition to a “cervical cancer component” (against HPV16 and HPV18), virus-like particles of the two most important low-risk alpha-HPV genotypes: HPV6 and HPV11. These two closely related HPV genotypes are the etiological agents of at least 90% of genital warts and laryngeal papillomas (1, 8, 10, 20) and at least 12.1% of cervical low-risk squamous intraepithelial lesions (5). In phase III clinical trials, this quadrivalent prophylactic HPV vaccine was shown to be highly effective against genital warts, e.g., reducing the burden of genital warts by 99% (95% confidence interval = 93.7 to 100%) among HPV-naïve vaccinated women aged 16 to 26 years (11). The quadrivalent HPV vaccine is currently licensed in more than 105 countries and has already been included in national vaccination programs in several countries. The widespread use of this vaccine has created an immediate need for a very specific detection tool for low-risk alpha-HPV.The Hybrid Capture 2 HPV DNA test (hc2), originally developed by Digene Corporation (Gaithersburg, MD) and currently marketed by Qiagen (Hilden, Germany), is the most widely used molecular method for the detection of a subset of clinically important HPV genotypes (14-16, 18). In this assay, exfoliated cells are first treated with alkali-denaturing reagent, and the processed samples are hybridized under high-stringency conditions with two mixtures of unlabeled full-genomic-length RNA probes, one specific for 13 high-risk HPV genotypes (HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, and HPV68) and one for 5 low-risk HPV genotypes (HPV6, HPV11, HPV42, HPV43, and HPV44). Positive specimens are detected by binding the hybridization complexes onto the surface of a microplate well coated with monoclonal antibodies specific to RNA-DNA hybrids. Immobilized hybrids are detected by the addition of an alkaline phosphatase-conjugated antibody to RNA-DNA hybrids, followed by the addition of a chemiluminiscent substrate. The emission of light is measured as relative light units (RLU) in a luminometer. Thus, hc2 does not allow the exact determination of HPV genotype(s) present in a clinical specimen but rather expresses the results of tested high-risk or low-risk HPV genotypes as positive or negative. The hc2 high-risk cocktail is very reliable for the routine detection of clinically important HPV infection and is, at present, the only commercially available HPV DNA assay with sufficient scientific data to support its performance in a clinical setting. However, several studies have shown significant analytical inaccuracy of the high-risk cocktail, mainly due to cross-reactivity with several untargeted HPV genotypes (2-4, 6, 9, 16, 19, 21-26, 27). This phenomenon certainly reduces the analytical specificity of the high-risk cocktail, but cross-reactivity with some HPV genotypes has proven to be clinically beneficial (4, 19).The U.S. version of hc2, containing the high-risk probe cocktail only, is approved by the U.S. Food and Drug Administration (FDA) for triage (in cases of equivocal cytology results showing the presence of atypical squamous cells of undetermined significance) to determine which patients should be referred to physicians for a colposcopy and as a screening test for use in addition to cytology screening for women 30 years of age and older (15). Although the use of the hc2 low-risk probe cocktail is not recommended in the U.S. due to lack of FDA approval, the “Conformité Européene” (CE)-certified version of hc2, containing both high-risk and low-risk probe cocktails, is currently used in at least 40% of laboratories outside the U.S., mainly for individuals with clinically suspected low-risk HPV infection or as a reflex test for women with atypical squamous cells of undetermined significance who tested negative for high-risk HPVs. In contrast to the established cross-reactivity of the high-risk probe cocktail with several untargeted HPV genotypes, the specificity of the hc2 low-risk cocktail has never been studied in detail. According to the data presented in the hc2 package insert, the only recognized cross-reactivity of the hc2 low-risk cocktail is with HPV13, a genotype commonly detected in lip lesions of certain ethnic groups but never in anogenital lesions (17). In the present study, therefore, we have for the first time systematically examined the analytical specificity of the hc2 low-risk cocktail by determining the exact HPV genotype(s) present in 285 consecutive samples recognized using the hc2 low-risk probe cocktail as HPV DNA positive.To determine the specificity and accuracy of hc2 in the detection of the five HPV genotypes (HPV6, HPV11, HPV42, HPV43, and HPV44) included in the low-risk probe cocktail, 285 consecutive cervical specimens obtained from the same number of women and recognized as HPV positive using the hc2 low-risk probe cocktail were included in the study. Fifty-six out of 285 samples were positive using both low-risk and high-risk probe cocktails. The specimens included in the study were collected between June 2007 and May 2008, using a DNAPaP cervical sampler and specimen transport medium (STM; Digene Corporation, Gaithersburg, MD), from Croatian and Slovenian women undergoing routine gynecological examination. hc2 testing was performed not later than 5 days after the collection of specimens, strictly following the manufacturer''s instructions. According to the manufacturer''s interpretation criteria, the specimens with a RLU-per-cutoff (RLU/CO) value higher than 1.0 were considered positive for one or more low-risk HPV genotypes included in the cocktail, and the specimens with a RLU/CO value of <1.0 were considered negative for the five low-risk HPV genotypes tested.The presence of the five targeted low-risk genotypes, as well as other, untargeted low-risk and high-risk HPV genotypes, in the hc2 low-risk cocktail-positive samples was determined by using seven different PCR-based genotyping methods. To keep the number of tests to a minimum, the seven methods were used consecutively, e.g., if the first genotyping method reliably detected at least one of the five targeted HPV genotypes in the particular hc2 low-risk cocktail-positive sample, this sample was excluded from further testing. All seven genotyping tests were performed on the same sample used for hc2 testing, i.e., 500 μl of STM was removed before the addition of hc2 denaturing reagent solution (first step of hc2 testing) and kept at −70°C until genotyping. After the isolation of DNA from 200 μl of nondenatured STM using a QIAamp DNA mini kit (Qiagen, Hilden, Germany), all 285 samples were first tested using a recently developed real-time PCR (RT-PCR) assay that allows very specific detection and reliable differentiation of HPV6 and HPV11 (13). The sensitivity of the test, assessed by probit analysis at a 95% detection level, is 42.9, 43.4, and 25.3 DNA copies per assay for prototypic and nonprototypic HPV6 variants and HPV11, respectively (13). Sixty-six samples were found to be positive for HPV6, and 6 samples contained HPV11 (Table ​(Table1).1). The remaining 213 samples were then tested using HPV42 genotype-specific RT-PCR, and 105 were found to contain this low-risk HPV. The remaining 108 samples were then tested using HPV43 genotype-specific RT-PCR, followed by testing of all negative samples using HPV44/55 genotype-specific RT-PCR. By using the two RT-PCR tests, the presence of HPV43 and HPV44/55 was identified in 8 and 17 samples, respectively (Table ​(Table1).1). The HPV42, HPV43, and HPV44/55 genotype-specific primers and probes were designed according to the L1 long control region genomic sequences of the prototype isolates (GenBank accession nos. {"type":"entrez-nucleotide","attrs":{"text":"M73236","term_id":"333211","term_text":"M73236"}}M73236, {"type":"entrez-nucleotide","attrs":{"text":"AJ620205","term_id":"40804474","term_text":"AJ620205"}}AJ620205, {"type":"entrez-nucleotide","attrs":{"text":"U31788","term_id":"1020242","term_text":"U31788"}}U31788, and {"type":"entrez-nucleotide","attrs":{"text":"U31791","term_id":"1020266","term_text":"U31791"}}U31791, respectively). As shown in Table ​Table2,2, two primers and two hybridization (fluorescent resonance energy transfer) probes were selected for each HPV genotype studied. A common probe was designed for HPV44 and HPV55, which has recently been recognized as a subtype of HPV44 and is no longer considered a separate genotype (7). A BLAST search of the GenBank nr database was performed for each sequence in order to verify HPV genotype specificity. The assays were set up on a LightCycler 2.0 real-time system (Roche Diagnostics GmbH, Mannheim, Germany) and performed using a QuantiTect probe PCR kit (Qiagen, Hilden, Germany) according to the manufacturer''s instructions.

TABLE 1.

Summary of genotyping results of 285 consecutive samples recognized as HPV DNA positive using the hc2 low-risk probe cocktail

Comparison of the Novel Human Papillomavirus 4 Auto-capillary Electrophoresis Test with the Hybrid Capture 2 Assay and with the PCR HPV Typing Set Test in the Detection of High-Risk HPV Including HPV 16 and 18 Genotypes in Cervical Specimens

The aim of this study was to compare the novel human papillomavirus (HPV) detection method, the HPV 4 Auto-capillary Electrophoresis (ACE) test with the hybrid capture (HC) 2 assay for the detection of high-risk HPVs. In addition, we compared the HPV 4 ACE test with the polymerase chain reaction HPV Typing Set test for the detection of HPV 16 and HPV 18 genotypes. One hundred ninety-nine cervical swab samples obtained from women with previous abnormal Pap smears were subjected to testing with the three HPV tests. The HPV 4 ACE test and the HC 2 assay showed substantial agreement for detection of high-risk HPVs (85.4%, kappa=0.71). The HPV 4 ACE test also showed substantial agreement with the PCR HPV Typing Set test in the detection of HPV 16 and HP V 18 genotypes (89.9%, kappa=0.65). In correlation with cytologic results, the sensitivities and specificities of the HPV 4 ACE test and HC 2 assay were 92.9% vs. 92.9% and 48.1% vs. 50.8%, respectively, when high-grade squamous intraepithelial lesions were regarded as abnormal cytologies. The novel HPV 4 ACE test is a valuable tool for the detection of high-risk HPVs and for genotyping of HPV 16 and HPV 18.     

Study Comparing Human Papillomavirus (HPV) Real-Time Multiplex PCR and Hybrid Capture II INNO-LiPA v2 HPV Genotyping PCR Assays

Human papillomavirus (HPV) DNA genotyping is an essential test to establish efficacy in HPV vaccine clinical trials and HPV prevalence in natural history studies. A number of HPV DNA genotyping methods have been cited in the literature, but the comparability of the outcomes from the different methods has not been well characterized. Clinically, cytology is used to establish possible HPV infection. We evaluated the sensitivity and specificity of HPV multiplex PCR assays compared to those of the testing scheme of the Hybrid Capture II (HCII) assay followed by an HPV PCR/line hybridization assay (HCII-LiPA v2). SurePath residual samples were split into two aliquots. One aliquot was subjected to HCII testing followed by DNA extraction and LiPA v2 genotyping. The second aliquot was shipped to a second laboratory, where DNA was extracted and HPV multiplex PCR testing was performed. Comparisons were evaluated for 15 HPV types common in both assays. A slightly higher proportion of samples tested positive by the HPV multiplex PCR than by the HCII-LiPA v2 assay. The sensitivities of the multiplex PCR assay relative to those of the HCII-LiPA v2 assay for HPV types 6, 11, 16, and 18, for example, were 0.806, 0.646, 0.920, and 0.860, respectively; the specificities were 0.986, 0.998, 0.960, and 0.986, respectively. The overall comparability of detection of the 15 HPV types was quite high. Analyses of DNA genotype testing compared to cytology results demonstrated a significant discordance between cytology-negative (normal) and HPV DNA-positive results. This demonstrates the challenges of cytological diagnosis and the possibility that a significant number of HPV-infected cells may appear cytologically normal.Cervical cancer is the second most common cause of cancer death among women globally. An estimated 510,000 new cases will be diagnosed around the world this year, and 288,000 women will die from the disease (15). In developing countries, cervical cancer can be the most common cancer among women. There is a strong causal relationship between infection with human papillomavirus (HPV) and cervical cancer, as the prevalence of the virus in cervical cancers has been estimated to be as high as 99.7% (14).Reliable, sensitive, and accurate detection of specific HPV types is critical for the study of cervical cancer causation as well as the support of vaccine clinical trials. Several PCR-based methodologies have been developed to amplify and detect HPV DNA (1, 4, 7, 8, 9, 12, 13). Many of these methodologies exploit the high homology found within specific open reading frames (ORFs) across different HPV types through the use of consensus or degenerate primer pairs that are capable of PCR amplifying numerous HPV types present in a sample. The resultant amplicons are detected and genotyped by utilizing HPV type-specific probes and colorimetric precipitation. While a greater number of HPV types can be detected by these methodologies, additional experimentation beyond PCR cycling is required to identify specific HPV types. Concerns arise over the increased contamination risk with postamplification experimentation and equivalent optimization between HPV types using single-amplification and single-hybridization conditions.For efficacy determinations of the recently FDA-approved quadrivalent HPV (types 6, 11, 16, 18) L1 VLP vaccine (Gardasil; Merck & Co., Inc.) (2, 3), highly sensitive and specific HPV multiplex PCR assays were developed (5, 10, 11) and used, which simultaneously detect the L1 and/or E6 and E7 ORFs of a specific HPV type using the ABI Prism 7700 sequence detection system instrument (Applied Biosystems, Foster City, CA). In this system, DNA is purified from multiple anogenital swab samples per patient by utilizing spin column chemistry under very stringent conditions. All samples are then screened for HPV DNA using real-time HPV multiplex PCR assays. Studies of the direct comparability of HPV multiplex PCR assays and other HPV genotyping methods will begin to clarify the HPV prevalence inconsistencies reported in the literature, teasing out potential assay testing differences from true geographical prevalence differences. In addition, comparisons between HPV genotyping and the cytological diagnosis of a sample will begin to provide insight into the limitations of the various tests.Herein we compare real-time HPV multiplex PCR assays to a method which involves a two-step process of initial screening with the Hybrid Capture II (HCII; Digene Corp., Gaithersburg, MD) assay followed by genotyping of HCII-positive samples using the INNO-LiPA v2 assay, an HPV PCR line hybridization assay (Innogenetics, Ghent, Belgium). Cross-tabulation analysis was performed between the HPV multiplex PCR assay high-risk HPV composite and the HCII-LiPA v2 high-risk HPV composite by SurePath cytology diagnosis. A post hoc analysis was also performed to evaluate the comparability of the HPV multiplex PCR assay to the INNO-LiPA v2 assay directly.(Data presented herein were presented in part at the 23rd Annual International Papillomavirus Conference and Clinical Workshop, Prague, Czech Republic, 1 to 7 September 2006.)     

Comparative Evaluation of First- and Second-Generation Digene Hybrid Capture Assays for Detection of Human Papillomaviruses Associated with High or Intermediate Risk for Cervical Cancer

In the present study we comparatively evaluated the first- and second-generation Digene Hybrid Capture assays for detection of human papillomaviruses (HPV) associated with high or intermediate risk for cervical cancer in cervical specimens. Concordant results were obtained with 468 of 483 (96.8%) specimens. All 15 specimens which gave repeatedly discordant results were positive by the second-generation test only, and 14 of them tested PCR positive. The enhanced sensitivity of the second-generation assay is mainly a result of the reformulation of hybridization reagents and, to a lesser extent, a result of the addition of new HPV probes.     

Human Papillomavirus DNA Detection in Menstrual Blood from Patients with Cervical Intraepithelial Neoplasia and Condyloma Acuminatum

The Papanicolaou test generates pain and embarrassment, and cytology screening has limited sensitivity for detection of cervical neoplasia. These factors urge the use of another screening test that can overcome these limitations. We explore a completely noninvasive method using detection of human papillomavirus (HPV) DNA in women''s menstrual blood (MB). The participants were divided into 3 cohorts: (i) 235 patients with cervical intraepithelial neoplasia 3 (CIN 3) (n = 48), CIN 2 (n = 60), CIN 1 (n = 58), or condyloma acuminatum (CAC) (n = 69) before treatment or remission; (ii) from the first cohort of patients, 108 CIN 3 or CIN 2 patients after treatment and 62 CIN 1 or CAC patients after remission; and (iii) 323 apparently normal subjects (ANS) without any cervical disease. The HPV genotypes of the infected patients were confirmed by direct sequencing. Quantitative real-time PCR (QRT-PCR) was used to measure the MB HPV16 load for 15 infected patients. Results showed that the sensitivity, specificity, and positive and negative predictive values for detection of MB HPV DNA in samples from patients with CIN or CAC were 82.8%, 93.1%, 90.0%, and 87.9%, respectively. Moreover, MB HPV DNA was found in samples from 22.2% of CIN 3 or CIN 2 patients after treatment, 0.0% of CIN 1 or CAC patients after remission, and 8.1% of ANS, 4 of whom were found to have CIN 1 or CAC. Furthermore, QRT-PCR showed that the normalized MB HPV16 DNA copy numbers in samples from patients with CIN 1 to CIN 3 were significantly increased. These preliminary results suggested that MB HPV DNA is a potential noninvasive marker for these premalignant cervical diseases.Cervical cancer (CC) is the second most common malignancy and cause of cancer-related death in women worldwide (17). The well-defined premalignant phase of this cancer has contributed to the success of the cytology-based Papanicolaou (Pap) screening test; the incidence of CC in resource-rich countries has been dramatically reduced by the use of this test (11). However, the Pap test has several limitations: (i) the collection of cervical cells can create discomfort and embarrassment (7); (ii) evaluation of test results in cytology screening involves subjective assessments with high susceptibility to intraindividual and interindividual variability (11); (iii) the test exhibits low (around 51%) sensitivity for detection of high-grade (HG) cervical intraepithelial neoplasia (HGCIN) (11); and (iv) cytology-trained personnel and special equipment with excellent quality control are needed, which involves a huge amount of resources (3). Therefore, there is a need for another cervical screening test that can overcome these limitations. Recent studies have shown that the human papillomavirus (HPV) DNA test is more sensitive than the Pap test in detecting HGCIN (11-13). However, the sample collection method, using a cytobrush, is still invasive and unpleasant, which would affect a woman''s decision whether to take the Pap test. We hypothesized that menstrual blood (MB) collected in sanitary napkins would contain HPV-infected cervical cells from patients with CIN or condyloma acuminatum (CAC), as HPV is known to be the etiological agent for both conditions (10). In this study, we explored the possibility of detecting and typing HPV DNA in MB collected in sanitary napkins from patients with CIN or CAC. As a high HPV16 DNA load has been reported to be associated with a higher grade of CIN or a higher risk of development of HGCIN during follow-up (5, 19), it would be interesting to explore whether HPV16 DNA loads can be measured using quantitative real-time PCR (QRT-PCR) with MB specimens. The information obtained would be important for enhancing understanding of the diagnostic and prognostic potential of MB HPV DNA for patients with CIN and CAC, and possibly for determining whether MB HPV DNA can be used as the basis for a noninvasive screening test for detection of these premalignant cervical diseases (PCD).     

Comparison of Commercial Hybridization and Automated Transcription-Mediated Amplification Modalities for Detection of High-Risk Human Papillomavirus Nucleic Acid

Assessment of 4,056 cytology collections by Cervista HPV HR and APTIMA HPV yielded 88.7% concordance, with increased detection by Cervista in patients with atypical squamous cells of undetermined significance (ASC-US) and patients negative for intraepithelial lesions and malignancy (NILM) (P ≤ 0.02). Both assays detected ≥91.7% of cervical intraepithelial neoplasia grade 2+ (CIN2+) lesions. A total of 262 specimens demonstrated luminescence within all three Cervista oligonucleotide mixtures (triple positive). APTIMA HPV and PCR-based microarray confirmed triple-positive results at rates of 7.3% and 5.9%, respectively.     

Classical Molecular Tests Using Urine Samples as a Potential Screening Tool for Human Papillomavirus Detection in Human Immunodeficiency Virus-Infected Women

Human papillomavirus (HPV) is the main risk factor associated with the development of cervical cancer (CC); however, there are other factors, such as immunosuppression caused by the human immunodeficiency virus (HIV), that favor progression of the illness. This study was thus aimed at evaluating the functionality of classical PCR-based molecular tests for the generic identification of HPV DNA (GP5+/GP6+, MY09/MY11, and pU1M/2R primers, individually or in combination) using cervical and urine samples from 194 HIV-positive women. Infected samples were tested with type-specific primers for six high-risk types (HPV-16, -18, -31, -33, -45, and -58) and two low-risk types (HPV-6 and -11). HPV infection prevalence rates were 70.1% for the cervical samples and 63.9% for the urine samples. HPV-16 was the most prevalent viral type in the cervical and urine samples, with higher rates of multiple infections than single infections detected in such samples. HPV DNA detection by PCR (mainly with the pU1M/2R primer set) in urine samples was positively associated with abnormal cytological findings (atypical squamous cells of undetermined significance/squamous intraepithelial lesions [ASCUS/SIL]). It was determined that the operative characteristics for detection of cytological abnormalities were similar for cervical and urine samples. This suggested using PCR for the detection of HPV DNA in urine samples as a potential screening strategy for CC prevention in future prevention and control programs along with currently implemented strategies for reducing the impact of the disease, i.e., urine samples are economical, are easy to collect, have wide acceptability among women, and have operative characteristics similar to those of cervical samples.     

LcrV Capture Enzyme-Linked Immunosorbent Assay for Detection of Yersinia pestis from Human Samples

In the United States, there is currently a major gap in the diagnostic capabilities with regard to plague. To address this, we developed an antigen capture assay using an essential virulence factor secreted by Yersinia spp., LcrV, as the target antigen. We generated anti-LcrV monoclonal antibodies (MAbs) and screened them for the ability to bind bacterially secreted native Yersinia pestis LcrV. Anti-LcrV MAb 19.31 was used as a capture antibody, and biotinylated MAb 40.1 was used for detection. The detection limit of this highly sensitive Yersinia LcrV capture enzyme-linked immunosorbent assay is 0.1 ng/ml. The assay detected LcrV from human sputum and blood samples treated with concentrations as low as 0.5 ng/ml of bacterially secreted native Y. pestis LcrV. This assay could be used as a tool to help confirm the diagnosis of plague in patients presenting with pneumonia.     

Nonisotopic Detection and Typing of Human Papillomavirus DNA in Genital Samples by the Line Blot Assay

The line blot assay, a gene amplification method that combines PCR with nonisotopic detection of amplified DNA, was evaluated for its ability to detect human papillomavirus (HPV) DNA in genital specimens. Processed samples were amplified with biotin-labeled primers for HPV detection (primers MY09, MY11, and HMB01) and for beta-globin detection (primers PC03 and PC04). Amplified DNA products were hybridized by a reverse blot method with oligonucleotide probe mixtures fixed on a strip that allowed the identification of 27 HPV genotypes. The line blot assay was compared to a standard consensus PCR test in which HPV amplicons were detected with radiolabeled probes in a dot blot assay. Two hundred fifty-five cervicovaginal lavage specimens and cervical scrapings were tested in parallel by both PCR tests. The line blot assay consistently detected 25 copies of HPV type 18 per run. The overall positivity for the DNA of HPV types detectable by both methods was 37.7% (96 of 255 samples) by the line blot assay, whereas it was 43. 5% (111 of 255 samples) by the standard consensus PCR assay. The sensitivity and specificity of the line blot assay reached 84.7% (94 of 111 samples) and 98.6% (142 of 144 samples), respectively. The agreement for HPV typing between the two PCR assays reached 83.9% (214 of 255 samples). Of the 37 samples with discrepant results, 33 (89%) were resolved by avoiding coamplification of beta-globin and modifying the amplification parameters. With these modifications, the line blot assay compared favorably to an assay that used radiolabeled probes. Its convenience allows the faster analysis of samples for large-scale epidemiological studies. Also, the increased probe spectrum in this single hybridization assay permits more complete type discrimination.     

High-Grade Anal Intraepithelial Neoplasia Among HIV-1-Infected Men Screening for a Multicenter Clinical Trial of a Human Papillomavirus Vaccine

Abstract

Purpose: High-grade anal intraepithelial neoplasia (HGAIN) is the precursor lesion to invasive anal cancer. Human papillomavirus (HPV) vaccination holds great promise for preventing anal cancer. Methods: We examined 235 HIV-1-infected men screening for participation in a multisite clinical trial of a quadrivalent HPV vaccine. All participants had anal swabs obtained for HPV testing and cytology and high-resolution anoscopy with biopsies of visible lesions to assess for HGAIN. Results: HPV types 16 and 18 were detected in 23% and 10%, respectively; abnormal anal cytology was found in 56% and HGAIN in 30%. HGAIN prevalence was significantly higher in those with HPV16 detection compared to those without (38% vs 17%; P = .01). Use of antiretroviral therapy and nadir and current CD4+ cell count were not associated with abnormal anal cytology or HGAIN. Conclusion: HGAIN is highly prevalent in HIV-infected men. Further studies are needed on treatment and prevention of HGAIN.     

Performance of a Polymer-Based DNA Chip Platform in Detection and Genotyping of Human Papillomavirus in Clinical Samples

Human papillomavirus (HPV) plays a key role in the development of cervical and laryngeal cancers. The aim of our study was to compare the performance of a new hydrogel-based HPV genotyping biochip assay (Biochip) to a commercially available and CE-marked conventional PCR followed by reverse hybridization (GenID-PCR). One hundred twenty-three samples were available for the study. Of these samples, 101/123 were gynecological swabs, 8/123 were swabs or biopsy samples of genital warts, 7/123 were biopsy samples of otorhinolaryngeal lesions, 5/123 were samples of skin warts, and 2/123 were samples of orolabial abnormalities. These molecular methods for HPV genotyping showed comparable sensitivity and specificity. However, 19/123 of the results were discrepant. Specifically, Biochip showed better performance in the detection of multiple infections, especially when more than one high-risk genotype was present. Due to the different probe configurations used in the two assays, GenID-PCR achieves only group-specific detection of many HPV genotypes, whereas Biochip allows for specific identification. Overall, the newly developed HPV chip system (Biochip) proved to be a suitable tool for HPV detection and genotyping; it also proved to be superior for establishing HPV genotyping methods.Persistent infection with human papillomavirus (HPV) is now well known to play the key role in the development of cervical carcinoma, which is the second most common malignancy in women worldwide. Certain HPV genotypes have been shown to be associated with a high risk for carcinogenesis. HPV type 16 (HPV-16) and HPV-18 are responsible for more than 70% of cervical carcinomas, whereas other high-risk genotypes (HPV-31, -33, -45, -52, -58, and others) are found less frequently (34, 38, 39).Due to the high negative predictive value of a negative HPV test result combined with negative cytology results, it is expected that, in the future, DNA testing will complement cytology in routine gynecological screening, thus enabling the development of a more cost-efficient screening program with expanded intervals between patient visits (11). For women with low-grade squamous intraepithelial lesions or atypical squamous cells of undetermined significance, HPV testing is an important requirement for triage strategies (3, 32). Since a significant proportion of HPV infections, including high-risk genotypes HPV-16 and -18, are cleared within several months by the immune system, single detection of HPV DNA alone is not sufficient to predict cancer development, especially in younger women (5). Consequently, the ability to genotype at least the most common high-risk HPV strains, i.e., HPV-16 and -18, in order to distinguish persistent infections with the same genotype from subsequent infections with other HPV genotypes is an important feature of modern HPV assays (16, 33).In the last 2 years, we have seen a breakthrough in the prevention of HPV infections. Much of the progress is the result of immunological studies that have paved the way for development of the first effective preventive vaccines (9, 25, 35, 37). Since the vaccines presently being marketed are considered effective only in a preventive and not in a therapeutic setting, a highly sensitive and specific diagnostic tool for HPV genotyping is required to exclude active HPV infection before vaccination (10).Furthermore, a versatile tool for HPV genotyping will be useful for monitoring epidemiologic consequences following broad clinical use of HPV vaccination, i.e., long-term protection against genotypes included in the vaccine, cross-protection against certain other genotypes, and a potential shift in the prevalence of genotypes not covered by direct or cross-protection.Traditional methods for HPV detection, e.g., cytological and immunological methods, show low specificity and sensitivity. Currently, molecular methods, including nucleic acid hybridization and PCR, are most frequently used for HPV detection (18, 30). However, there is an inherent danger of false-positive results due to unspecific amplification, as well as false-negative results due to variations in primer-binding sites in the target region of the virus, which decrease the amplification efficiency of some HPV genotype sequences. In addition, HPV genotyping requires either genotype-specific PCR or subsequent labor-intensive procedures, such as sequencing and hybridization. These methods exhibit difficulties in identifying all HPV genotypes present in multiple infections (36).Recently, the first studies have been reported involving the genotyping of HPV with genotype-specific oligonucleotides and DNA microarray analysis (1, 2, 7, 12, 13, 14, 15, 19, 20, 22, 23, 24, 27, 28, 31). HPV chip technology offers advantages both in the detection of multiple infections and in sensitivity (T. Brandstetter, S. Böhmer, O. Prucker, E. Bissé, A. zur Hausen, J. Alt-Mörbe, and J. Rühe, submitted for publication).In this study, we compared a new polymer-based DNA biochip assay (Biochip) for HPV detection and genotyping (Brandstetter et al., submitted) to a CE-marked commercially available PCR kit that includes reverse hybridization of PCR amplicons on nitrocellulose membrane strips (GenID-PCR), using clinical samples (cervical swabs, tissue biopsy samples, and others). Genetic sequencing served as a reference standard in case of divergent results from the two methods.