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

Abstract

Compared to high-risk human papillomavirus (HPV) DNA detection, detection of E6/E7 transcripts from high-risk HPV genotypes (1) has been proposed as a more specific tool for cervical cancer triage (2). Moreover, highly sensitive transcription-mediated amplification (TMA)-based detection of functional RNA transcripts can theoretically ascribe precursor oncogenicity to HPV-specific nucleic acid. These molecular profiles may precede cellular changes observed in routine cervical cytology. Early assessments of TMA-based detection of E6/E7 transcripts have largely been performed with a digene HC2 high-risk DNA test (HC2; Qiagen, Gaithersburg, MD) predicate device. Reports have described equivalent sensitivity between the two detection modalities, yet improved specificity of E6/E7 detection assays (3-6). In this report, the utility of TMA-based APTIMA HPV (APTIMA; Hologic/Gen-Probe, Incorporated, San Diego, CA) is compared to Cervista HPV HR (Cervista; Hologic, Madison, WI) in the triage of women with underlying cervical intraepithelial neoplasia grade 2ϩ (CIN2ϩ) lesions. Analogous to comparisons with HC2 (3-6), improved specificity was noted with APTIMA, yet our data characterize a phenomenon inherent to Cervista that contributes to deficits in specificity.(Results of this work were previously presented, in part, at the 112th General Meeting of the American Society for Microbiology, San Francisco, CA, 16 to 19 June 2012.)In a study approved by the Wheaton Franciscan Healthcare Institutional Review Board, residual fluid from 4,056 liquid-based cytology specimens (ThinPrep; Hologic, Marlborough, MA) was forwarded for tandem molecular analysis. Inclusion criteria consisted of the following: age of woman of Ն20 years, processing of ThinPrep specimen within 21 days of collection, and presence of sufficient residual fluid for both molecular assays. Glacial acetic acid (GAA)-treated specimens were excluded from analysis, as previous data (7) describe a deleterious effect of GAA on Cervista performance.Matched cytological diagnoses were classified as high-grade squamous intraepithelial lesions (HSIL), low-grade squamous intraepithelial lesions (LSIL), atypical squamous cells of undetermined significance (ASC-US), atypical squamous cells but cannot exclude HSIL (ASC-H), or negative for intraepithelial lesions and malignancy (NILM) (8). When an endocervical biopsy was indicated, histological results were delineated into categories of CIN2ϩ or results of lesser significance.Two-milliliter ThinPrep aliquots were subjected to Cervistabased detection of high-risk HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 (9). Relative genomic DNA content (gDNA) was assessed by a human histone gene-specific oligonucleotide. DNA extracts were assayed in triplicate; individual luminescence-labeled oligonucleotides were distributed throughout three oligonucleotide mixtures. HPV-specific luminescence from one or two oligonucleotide mixtures was required to equal or exceed a 1.525 ratio to background for a positive result. HPV...

Immediate histopathologic follow‐up of cervista and aptima high‐risk HPV assays in women with LSIL cytology

“…To the best of our knowledge, there are very few studies to date comparing the clinical performance of Cervista and Aptima. Munson et al examined 4056 cytology specimens and found an overall concordance between the Cervista and Aptima test results of only 88.7% and that the Cervista test had a much higher number of false‐positive results. In addition, the study from Nolte et al also demonstrated a similar result.…”

Effect of Preanalytical Processing of ThinPrep Specimens on Detection of High-Risk Human Papillomavirus by the Aptima HPV Assay

“…The mean HPV-specific luminescence-to-background ratio of the HPVpositive vials (5.209) was approximately 240% greater than the cutoff value required for a positive result (data not illustrated). The aforementioned 96.7% concordance rate between Aptima HPV/Cervista results in the carryover portion of this investigation was higher than the 88.7% concordance rate between the two assays previously described in a study set of 4,056 specimens (5). In addition to the smaller study set utilized in the current investigation, preselection of (Cervista) strongly HPV-positive ThinPrep vials would inherently lend itself to a higher concordance rate than would a prospective analysis.…”

“…1), the possibility existed for discordance between the Cervista and Aptima HPV data. Explanations for this have ranged from increased sensitivity of TMA-based testing versus that of DNA hybridization (17) to a recent characterization of false-positive results inherent to Cervista (5). Furthermore, specimens may have contained DNA or RNA, but not both.…”

“…Transcription-mediated amplification (TMA) of these targets (Aptima HPV; Hologic/Gen-Probe, San Diego, CA) performs in an equivalent fashion to high-risk HPV DNA hybridization assays, such as the Digene HC2 high-risk HPV DNA test (Qiagen, Gaithersburg, MD) (1-4) and the Cervista HPV HR (Cervista; Hologic, Madison, WI) (5), for the detection of indolent cervical intraepithelial neoplasia (CIN) 2ϩ. The aforementioned studies also document improved specificity of Aptima HPV over that of DNA hybridization in the context of atypical squamous cells of undetermined significance (ASC-US) or nonsignificant cytology diagnoses and in cases of non-CIN 2ϩ biopsy results.…”

Future microfluidic and nanofluidic modular platforms for nucleic acid liquid biopsy in precision medicine

“…27 Recently, FDA has approved several assays and platforms for detection of genomic and messenger viral RNA, summarized in Table I. 26,[28][29][30][31][32][33][34] These platforms offer a limit of detection (LOD) of 10-200 copies/ml, and linear quantification up to 10 À8 copies/ml. Such new point-of care diagnostic assays capable of achieving comparable or lower limits of detection than current ones would allow better patient management with antiviral therapies and maybe even allow higher cure rates.…”