A robust, scalable, and cost-efficient approach to whole genome sequencing of RSV directly from clinical samples.

Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections causing significant morbidity and mortality among children and the elderly; two RSV vaccines and a monoclonal antibody have recently been approved. Thus, there is an increasing need for a detailed and continuous genomic surveillance of RSV circulating in resource-rich and resource-limited settings worldwide. However, robust, cost-effective methods for whole genome sequencing of RSV from clinical samples that are amenable to high-throughput are still scarce. We developed Next-RSV-SEQ, an experimental and computational pipeline to generate whole genome sequences of historic and current RSV genotypes by in-solution hybridization capture-based next generation sequencing. We optimized this workflow by automating library preparation and pooling libraries prior to enrichment in order to reduce hands-on time and cost, thereby augmenting scalability. Next-RSV-SEQ yielded near-complete to complete genome sequences for 98% of specimens with Cp values ≤31, at median on-target reads >93%, and mean coverage depths between ~1,000 and >5,000, depending on viral load. Whole genomes were successfully recovered from samples with viral loads as low as 230 copies per microliter RNA. We demonstrate that the method can be expanded to other respiratory viruses like parainfluenza virus and human metapneumovirus. Next-RSV-SEQ produces high-quality RSV genomes directly from culture isolates and, more importantly, clinical specimens of all genotypes in circulation. It is cost-efficient, scalable, and can be extended to other respiratory viruses, thereby opening new perspectives for a future effective and broad genomic surveillance of respiratory viruses. IMPORTANCE: Respiratory syncytial virus (RSV) is a leading cause of severe acute respiratory tract infections in children and the elderly, and its prevention has become an increasing priority. Recently, vaccines and a long-acting monoclonal antibody to protect effectively against severe disease have been approved for the first time. Hence, there is an urgent need for genomic surveillance of RSV at the global scale to monitor virus evolution, especially with an eye toward immune evasion. However, robust, cost-effective methods for RSV whole genome sequencing that are suitable for high-throughput of clinical samples are currently scarce. Therefore, we have developed Next-RSV-SEQ, an experimental and computational pipeline that produces reliably high-quality RSV genomes directly from clinical specimens and isolates.

Considerations and Opportunities for Probe Capture Enrichment Sequencing of Emerging Viruses from Wastewater.

Until recently, wastewater-based monitoring for pathogens of public health concern primarily used PCR-based quantification methods and targeted sequencing for specific pathogens (e.g., SARS-CoV-2). In the past three years, researchers have expanded sequencing to monitor a broad range of pathogens, applying probe capture enrichment to wastewater. The goals of those studies included (1) monitoring and expanding fundamental knowledge of disease dynamics for known pathogens and (2) evaluating the potential for early detection of emerging diseases resulting from zoonotic spillover or novel viral variants. Several studies using off-the-shelf probe panels designed for clinical and environmental surveillance reported that enrichment increased virus relative abundance but did not recover complete genomes for most nonenteric viruses. Based on our experience and recent results reported by others using these panels for wastewater, clinical, and synthetic samples, we discuss challenges and technical factors that affect the rates of false positive and false negative results. We identify trade-offs and opportunities throughout the workflow, including in wastewater sample processing, probe panel design, and bioinformatic analysis. We suggest tailored methods of virus concentration and background removal, carefully designed probe panels, and multithresholded bioinformatics analysis.

Comparison of high-risk HPV detection by the AmpFire® HPV Screening 16/18/HR technique (Atila Biosystems) and the hybrid capture 2 test (Qiagen).

BACKGROUND: Detection of high-risk human papillomaviruses (hrHPV) is widely used at the first line of cervical cancer screening, requiring rigorous validation of the clinical performance of commercial kits designed for this indication. METHODS: Performance of the AmpFire HPV Screening 16/18/HR test (AF, Atila Biosystems) and the Hybrid Capture 2 test (HC2, Qiagen) for detecting hrHPV was cross-compared in 200 cervical samples in our institution. RESULTS: The global percentage of agreement between the 2 techniques was 95.0% (95%CI 92-98%) with a Cohen's kappa coefficient of 0.85 (95%CI 0.75-0.94). Ten samples showed discordant results between the 2 techniques in both directions (5 HC2+/AF- and 5 HC2-/AF+). Among possible explanations for these discrepancies was the detection of HPV66 and HPV53 genotypes in two samples, since these genotypes are targeted by the Ampfire test but not by the HC2 test, as well as intrinsic differences in analytical performance to target specific genotypes. CONCLUSIONS: A high level of agreement was observed between the two techniques, which encourages further testing in order to definitively validate the use of the Ampfire kit for primary cervical cancer screening.

Human papilloma virus in the etiopathogenesis of allergic nasal polyposis: A prospective study.

PURPOSE: To evaluate and compare the prevalence of high-risk HPV and low-risk HPV types in patients with chronic rhinosinusitis with nasal polyposis (CRSwNP) and healthy controls. MATERIALS AND METHODS: A prospective cohort study was conducted in a tertiary care hospital on the patients of CRSwNP undergoing surgical management. All patients underwent preoperative endoscopic evaluation and radiological assessment using NCCT of the nose and paranasal sinuses. The severity of the disease was graded using the Lund-Mackay score on NCCT. All patients underwent endoscopic polypectomy and the sample of tissues was sent for HPV DNA detection using Hybrid Capture II® technique. The clinicopathological characteristics of HPV positive and negative patients were compared. RESULTS: Sixty cases and 20 controls were included in the study. All controls were negative for HPV DNA. 27 patients (45%) had the presence of HPV DNA, out of which 23 had only LR-HPV and 1 had only HR-HPV types. Three patients had both HR-HPV and LR-HPV subtypes. There was a significant difference between the cases and controls for the presence of HPV DNA (p < 0.001). However, the patients with HPV-positive DNA in the nasal specimen did not differ significantly from HPV-negative patients in age, gender, or severity of the disease. CONCLUSIONS: Human papillomaviruses may play a significant role in the etiopathogenesis of CRSwNP, however, do not impact the degree of sinus involvement.

HPV Integration Site Mapping: A Rapid Method of Viral Integration Site (VIS) Analysis and Visualization Using Automated Workflows in CLC Microbial Genomics.

Human papillomavirus (HPV) integration within the host genome may contribute to carcinogenesis through various disruptive mechanisms. With next-generation sequencing (NGS), identification of viral and host genomic breakpoints and chimeric sequences are now possible. However, a simple, streamlined bioinformatics workflow has been non-existent until recently. Here, we tested two new, automated workflows in CLC Microbial Genomics, i.e., Viral Hybrid Capture (VHC) Data Analysis and Viral Integration Site (VIS) Identification for software performance and efficiency. The workflows embedded with HPV and human reference genomes were used to analyze a publicly available NGS dataset derived from pre- and cancerous HPV+ cervical cytology of 21 Gabonese women. The VHC and VIS workflow median runtimes were 19 and 7 min per sample, respectively. The VIS dynamic graphical outputs included read mappings, virus-host genomic breakpoints, and virus-host integration circular plots. Key findings, including disrupted and nearby genes, were summarized in an auto-generated report. Overall, the VHC and VIS workflows proved to be a rapid and accurate means of localizing viral-host integration site(s) and identifying disrupted and neighboring human genes. Applying HPV VIS-mapping to pre- or invasive tumors will advance our understanding of viral oncogenesis and facilitate the discovery of prognostic biomarkers and therapeutic targets.

Comparison of Aptima and hybrid capture-2 HPV tests and Pap test in the referral population in Japan.

The Aptima human papillomavirus (HPV) test (APTIMA) detects E6-E7 mRNA in abnormal cells in the uterine cervix. To investigate the accuracy of APTIMA for cervical cancer screening in Japan, 423 subjects, mostly referrals with abnormal cytology or being followed up for cervical intraepithelial neoplasia (CIN)1, were screened using two HPV tests, hybrid capture 2 (HC2) and APTIMA, and by the Pap test. Colposcopy was conducted in all subjects with a positive result in either test type. HPV genotyping was performed by Genosearch-31. A result of atypical squamous cells-undetermined significance (ASC-US) or worse on the HC2 test (ASC-US-HC2), and low-grade squamous intraepithelial lesion (LSIL) or worse (LSIL+) on the Pap test, was regarded as positive. APTIMA (97.5%) was more sensitive than LSIL+ (85.1%) for detecting CIN2 or worse (CIN2+) (McNemar test; p = .0003), and more sensitive (98.6%) than ASC-US-HC2 (92.7%) for detecting CIN3+. APTIMA and HC2 had similar sensitivities. HPV genotyping revealed that CIN2/3 with high-risk HPV (HR-HPV) was overlooked in five cases by ASC-US-HC2, and in four cases by HC2, while no such lesions were missed by APTIMA. Thus, APTIMA might be superior to HC2 for primary HPV screening in Japan. One cancer case positive for HPV67 (potentially high risk, [pHR]) was overlooked by Pap test and both HPV tests, suggesting a need for a new HPV test able to detect pHR-HPV types.

Agreement between careHPV and hybrid capture 2 in detecting high-risk HPV in women in Tanzania.

INTRODUCTION: Visual inspection of the cervix with acetic acid is used to control the burden of cervical cancer in low- and middle-income countries. This method has some limitations and HPV DNA testing may be an alternative, but it is expensive and requires a laboratory setup. Cheaper and faster HPV tests have been developed. This study describe the agreement between a fast HPV test (careHPV) and hybrid capture 2 (HC2) in detection of high-risk HPV among Tanzanian women. MATERIAL AND METHODS: The study involved women attending routine cervical cancer screening at the Ocean Road Cancer Institute and Kilimanjaro Christian Medical Centre in Tanzania. The women were offered HIV testing. Two cervical samples were subsequently obtained; the first sample was processed at the clinics using careHPV and the second sample was transported to Denmark and Germany for cytology and HC2 analysis. Kappa statistic was calculated to assess the agreement between careHPV and HC2. The sensitivity, specificity and predictive values of careHPV were calculated using HC2 as reference. The analyses were done for the overall study population and stratified by testing site and HIV status. RESULTS: A total of 4080 women were enrolled, with 437 being excluded due to invalid information, lack of careHPV or HC2 results. Overall agreement between the tests was substantial with a kappa value of 0.69 (95% confidence interval [CI] 0.66-0.72). The sensitivity and specificity of careHPV was 90.7% (95% CI 89.6-91.8) and 84.2% (95% CI 81.2-86.8), respectively. The agreement was similar in the stratified analyses where the kappa values were 0.75 (95% CI 0.70-0.79) in women aged 25-34, 0.66 (95% CI 0.62-0.70) in women aged 35-60, 0.73 (95% CI 0.70-0.77) at the Ocean Road Cancer Institute, 0.64 (95% CI 0.60-0.69) at the Kilimanjaro Christian Medical Center, 0.73 (95% CI 0.68-0.79) in HIV-positive and 0.66 (95% CI 0.63-0.70) in HIV-negative women. The kappa value of 0.64 (95% CI 0.39-0.88) for cervical high-grade lesions indicates a substantial agreement between careHPV and HC2 in detecting HPV among women with cervical high-grade lesions. CONCLUSIONS: A substantial agreement was found between careHPV and HC2 in detecting HPV overall as well as detecting HPV among women with cervical high-grade lesions. However, given the limited resources available in low and middle-income countries, the HPV testing assay should be weighed against the cost-effectiveness of the test.

Hybrid capture of 964 nuclear genes resolves evolutionary relationships in the mimosoid legumes and reveals the polytomous origins of a large pantropical radiation.

PREMISE: Targeted enrichment methods facilitate sequencing of hundreds of nuclear loci to enhance phylogenetic resolution and elucidate why some parts of the "tree of life" are difficult (if not impossible) to resolve. The mimosoid legumes are a prominent pantropical clade of ~3300 species of woody angiosperms for which previous phylogenies have shown extensive lack of resolution, especially among the species-rich and taxonomically challenging ingoids. METHODS: We generated transcriptomes to select low-copy nuclear genes, enrich these via hybrid capture for representative species of most mimosoid genera, and analyze the resulting data using de novo assembly and various phylogenomic tools for species tree inference. We also evaluate gene tree support and conflict for key internodes and use phylogenetic network analysis to investigate phylogenetic signal across the ingoids. RESULTS: Our selection of 964 nuclear genes greatly improves phylogenetic resolution across the mimosoid phylogeny and shows that the ingoid clade can be resolved into several well-supported clades. However, nearly all loci show lack of phylogenetic signal for some of the deeper internodes within the ingoids. CONCLUSIONS: Lack of resolution in the ingoid clade is most likely the result of hyperfast diversification, potentially causing a hard polytomy of six or seven lineages. The gene set for targeted sequencing presented here offers great potential to further enhance the phylogeny of mimosoids and the wider Caesalpinioideae with denser taxon sampling, to provide a framework for taxonomic reclassification, and to study the ingoid radiation.

Hybrid Capture-Based Genomic Profiling Identifies BRAF V600 and Non-V600 Alterations in Melanoma Samples Negative by Prior Testing.

BACKGROUND: BRAF and MEK inhibitors are approved for BRAF V600-mutated advanced melanoma, with response rates of up to 70%. Responses to targeted therapies have also been observed for diverse non-V600 BRAF alterations. Thus, sensitive, accurate, and broad detection of BRAF alterations is critical to match patients with available targeted therapies. MATERIALS AND METHODS: Pathology reports were reviewed for 385 consecutive melanoma cases with BRAF mutations or rearrangements identified using a hybrid capture-based next-generation sequencing comprehensive genomic profiling (CGP) assay during the course of clinical care. RESULTS: Records of prior BRAF molecular testing were available for 79 (21%) cases. Of cases with BRAF V600 mutations, 11/57 (19%) with available data were negative by prior BRAF testing. Prior negative BRAF results were also identified in 16/20 (80%) cases with non-V600 mutations, 2 of which harbored multiple BRAF alterations, and in 2/2 (100%) cases with activating BRAF fusions. Clinical outcomes for a subset of patients are presented. CONCLUSION: CGP identifies diverse activating BRAF alterations in a significant fraction of cases with prior negative testing. Given the proven clinical benefit of BRAF/MEK inhibitors in BRAF-mutated melanoma, CGP should be considered for patients with metastatic melanoma, particularly if other testing is negative. IMPLICATIONS FOR PRACTICE: Published guidelines for melanoma treatment recommend BRAF mutational analysis, but little guidance is provided as to selection criteria for testing methodologies, or as to clinical implications for non-V600 alterations. This study found that hybrid capture-based next-generation sequencing can detect BRAF alterations in samples from a significant fraction of patients with advanced melanoma with prior negative BRAF results. This study highlights the need for oncologists and pathologists to be critically aware of coverage and sensitivity limitations of various assays, particularly regarding non-V600E alterations, of which many are potentially targetable.

A novel approach to improving hybrid capture sequencing targeting efficiency.

At present, approaches to hybrid capture sequencing have many limitations, from their significant complexity and labor requirements, to their low enrichment efficiency, resulting in their limited utilization. In an effort to overcome these drawbacks, we have developed a novel method that relied upon direct genomic DNA hybridization and single-stranded DNA library preparation. Using this novel protocol, we were able to achieve a targeting efficiency as high as 75%, and we found this approach to overall be an efficient and simple approach to DNA library construction.

Hybrid Capture-Based Comprehensive Genomic Profiling Identifies Lung Cancer Patients with Well-Characterized Sensitizing Epidermal Growth Factor Receptor Point Mutations That Were Not Detected by Standard of Care Testing.

BACKGROUND: In our recent study, of cases positive for epidermal growth factor receptor (EGFR) exon 19 deletions using comprehensive genomic profiling (CGP), 17/77 (22%) patients with prior standard of care (SOC) EGFR testing results available were previously negative for exon 19 deletion. Our aim was to compare the detection rates of CGP versus SOC testing for well-characterized sensitizing EGFR point mutations (pm) in our 6,832-patient cohort. MATERIALS AND METHODS: DNA was extracted from 40 microns of formalin-fixed paraffin-embedded sections from 6,832 consecutive cases of non-small cell lung cancer (NSCLC) of various histologies (2012-2015). CGP was performed using a hybrid capture, adaptor ligation-based next-generation sequencing assay to a mean coverage depth of 576×. Genomic alterations (pm, small indels, copy number changes and rearrangements) involving EGFR were recorded for each case and compared with prior testing results if available. RESULTS: Overall, there were 482 instances of EGFR exon 21 L858R (359) and L861Q (20), exon 18 G719X (73) and exon 20 S768I (30) pm, of which 103 unique cases had prior EGFR testing results that were available for review. Of these 103 cases, CGP identified 22 patients (21%) with sensitizing EGFR pm that were not detected by SOC testing, including 9/75 (12%) patients with L858R, 4/7 (57%) patients with L861Q, 8/20 (40%) patients with G719X, and 4/7 (57%) patients with S768I pm (some patients had multiple EGFR pm). In cases with available clinical data, benefit from small molecule inhibitor therapy was observed. CONCLUSION: CGP, even when applied to low tumor purity clinical-grade specimens, can detect well-known EGFR pm in NSCLC patients that would otherwise not be detected by SOC testing. Taken together with EGFR exon 19 deletions, over 20% of patients who are positive for EGFR-activating mutations using CGP are previously negative by SOC EGFR mutation testing, suggesting that thousands of such patients per year in the U.S. alone could experience improved clinical outcomes when hybrid capture-based CGP is used to inform therapeutic decisions. IMPLICATIONS FOR PRACTICE: This study points out that genomic profiling, as based on hybrid capture next-generation sequencing, can identify lung cancer patients with point mutation in epidermal growth factor receptor (EGFR) missed by standard molecular testing who can likely benefit from anti-EGFR targeted therapy. Beyond the specific findings regarding false-negative point mutation testing for EGFR, this study highlights the need for oncologists and pathologists to be cognizant of the performance characteristics of testing deployed and the importance of clinical intuition in questioning the results of laboratory testing.

Clinical and Analytical Evaluation of the Anyplex II HPV HR Detection Assay within the VALGENT-3 Framework.

In 2012, VALidation of human papillomavirus (HPV) GENotyping Tests (VALGENT) was initiated to provide a formalized and uniform study framework for comparison and validation of HPV assays with genotyping capability. In VALGENT-3, the clinical and analytical performance of Anyplex II HPV HR detection (Anyplex) was compared to that of the Hybrid Capture 2 HPV DNA test (hc2) and the cobas 4800 HPV test (cobas). The panel comprises 1,300 stored samples that were obtained from women 25 to 64 years old who participated in the Slovenian cancer screening program, enriched with 300 samples from women with abnormal cervical cytology. The sensitivity and specificity of Anyplex were noninferior to those of hc2, with a relative sensitivity of 1.01 (95% confidence interval [CI], 0.97 to 1.04) for cervical intraepithelial neoplasia grade 2 or worse (CIN2+) and 1.01 (95% CI, 0.97 to 1.06) for CIN3+ and relative specificity of 1.02 (95% CI, 1.00 to 1.03) for a CIN grade of ≤1. The clinical sensitivity of Anyplex for CIN2+ and CIN3+ was comparable to that of hc2 (P values for McNemar test [pMcN] of 0.655 and 0.564, respectively), but its specificity was significantly higher (pMcN = 0.008). The sensitivity and specificity of Anyplex were also noninferior to those of cobas, with relative sensitivity of 1.01 (95% CI, 0.98 to 1.04) for CIN2+ and 1.01 (95% CI, 0.99 to 1.04) for CIN3+ and relative specificity of 1.00 (95% CI, 0.99 to 1.01) (pMcN value of >0.05 in all cases). Regardless of the clinical outcome (CIN2+ or CIN3+), age restriction (women ≥30 years old), or comparator test used, Anyplex consistently showed excellent clinical performance and can be considered validated for primary cervical cancer screening.

Dense infraspecific sampling reveals rapid and independent trajectories of plastome degradation in a heterotrophic orchid complex.

Heterotrophic plants provide excellent opportunities to study the effects of altered selective regimes on genome evolution. Plastid genome (plastome) studies in heterotrophic plants are often based on one or a few highly divergent species or sequences as representatives of an entire lineage, thus missing important evolutionary-transitory events. Here, we present the first infraspecific analysis of plastome evolution in any heterotrophic plant. By combining genome skimming and targeted sequence capture, we address hypotheses on the degree and rate of plastome degradation in a complex of leafless orchids (Corallorhiza striata) across its geographic range. Plastomes provide strong support for relationships and evidence of reciprocal monophyly between C. involuta and the endangered C. bentleyi. Plastome degradation is extensive, occurring rapidly over a few million years, with evidence of differing rates of genomic change among the two principal clades of the complex. Genome skimming and targeted sequence capture differ widely in coverage depth overall, with depth in targeted sequence capture datasets varying immensely across the plastome as a function of GC content. These findings will help to fill a knowledge gap in models of heterotrophic plastid genome evolution, and have implications for future studies in heterotrophs.

Comparison of the digene hybrid capture 2 and Roche cobas 4800 HPV tests for detection of CIN2+ in a referral population in Japan.

To examine validity of the hybrid capture-2 and cobas 4800 HPV tests, 396 women including 188 women visiting for cancer screening, and 208 referral cases were examined with both HPV tests and the liquid-based cervical Pap test. Concordant results between the HPV assays were observed in 333 cases (coincident rates; 84.1%, kappa value; 0.682). The sensitivity for CIN2+ was 98.6% (69/70) and 82.9% (58/70) for HC2 and cobas 4800 (McNemar's test; P = 0.0026). The sensitivity for CIN3+ was 97.2% (35/36) and 83.3% (30/36) (Not significant, P = 0.0736). The specificities for CIN2+ or CIN3+ did not differ between the tests. The HPV16, 52, 18, 31, and 58 were the most common types in CIN2+ cases. Reasonable sensitivity for HPV52, and cross-hybridization with some probable high-risk HPV type such as HPV82 explain the higher sensitivity of HC2 than cobas 4800 in detection of CIN2+ in a referral population in Japan.

Comparison of AdvanSure HPV GenoBlot and Hybrid Capture 2 assays for detection of HPV infection.

BACKGROUND: We sought to compare the performance of the AdvanSure assay to the Hybrid Capture (HC) 2 for the detection of high-risk human papillomavirus (HR HPV). METHODS: A total of 855 cervical swab specimens were obtained. We submitted all specimens for HR HPV detection with HC2 and the AdvanSure assay. We subsequently analyzed discordant results and specimens that were positive on both assays using restriction fragment mass polymorphism (RFMP) genotyping analysis. RESULTS: HC2 yielded positive results in 12.0% of specimens, while the AdvanSure assay detected one of 13 HR HPV types in 11.5% of specimens. The overall agreement rate between the assays was 98.5% with a kappa coefficient of 0.928. Discordant results between these two assays were observed in 12 cases, seven were positive only on HC2 and five were positive only on AdvanSure. RFMP analysis of the 12 discordant cases revealed three false-positive results using HC2, and one false-positive and five false-negative results using AdvanSure. CONCLUSIONS: Considering the high agreement rate with HC2 and the ability to differentiate 35 HPV genotypes including HPV 16/18, the AdvanSure assay could be used as a laboratory testing method for HPV infection screening.

Clinical Evaluation of INNO-LiPA HPV Genotyping EXTRA II Assay Using the VALGENT Framework.

In this diagnostic test validation study, we assessed the clinical accuracy and HPV genotyping performance of the INNO-LiPA HPV Genotyping Extra II (INNO-LiPA) within the VALGENT-3 framework. VALGENT is designed to assess the analytical and clinical performance of HPV tests with genotyping capacity. The VALGENT-3 panel comprised 1300 consecutive cervical cell specimens enriched with 300 samples with abnormal cytology obtained from women attending the Slovenian cervical cancer screening programme. The INNO-LiPA allows type-specific detection of 32 HPV types; however, for the clinical accuracy assessment, we considered it as high-risk (hr)HPV positive when at least one of the following HPV types was present: HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, and HPV68. Clinical accuracy for detection of cervical intraepithelial neoplasia grade 2 or worse (CIN2+) was compared between INNO-LiPA and Hybrid Capture 2 (HC2), which is a standard comparator test for HPV tests used in cervical cancer screening. In addition, hrHPV and type-specific detection HPV types were compared between INNO-LiPA and Linear Array HPV Genotyping Test (Linear Array). The prevalence of hrHPV determined by INNO-LiPA was 17.1% (95% CI, 15.0⁻19.2%) in the screening population. HrHPV testing with INNO-LiPA had a sensitivity for CIN2+ of 96.9% (95% CI, 92.1⁻99.1%) which was non-inferior to HC2 (relative sensitivity of 1.01; 95% CI, 0.97⁻1.04; pn.inf = 0.0002) and a specificity for ≤CIN1 of 85.3% (95% CI, 83.2⁻87.3%) which was inferior to HC2 (relative specificity of 0.95; 95% CI, 0.93⁻0.97; pn.inf = 0.9998). Genotyping agreement between INNO-LiPA and Linear Array was excellent for hrHPV, HPV16, HPV18, HPV35, HPV45, HPV58 and HPV59, but good or fair for other HPV types. To conclude, INNO-LiPA demonstrated non-inferior clinical sensitivity but lower specificity compared to HC2 in addition to excellent concordance compared to Linear Array for hrHPV and some genotypes.

Risk of high-grade precancerous lesions and invasive cancers in high-risk HPV-positive women with normal cervix or CIN 1 at baseline-A population-based cohort study.

Infection with high-risk human papillomavirus (HR-HPV) is transient and clears on its own in majority of the women. Only a few women who have persistent infection may finally develop cervical intraepithelial neoplasia (CIN) or cervical cancer in later years. The risk of progression in the HR-HPV-positive women with normal cervix or low-grade lesion on colposcopy and histopathology at baseline is less studied. We performed a longitudinal study on 650 HR-HPV-positive women with colposcopy and/or histopathology-proved normal or CIN1 diagnosis at baseline to assess the cumulative risk of development of high-grade CIN. After a mean follow-up of 2.1 person years of observation (PYO) (range 0.1-5.1), the cumulative incidence of CIN2+ (6.4%; 3.0/100 PYO) was significantly higher in women who had persistent HR-HPV infection compared to those who cleared the infection (adjusted HR 6.28; 95% CI 2.87-13.73). The risk of viral persistence in women aged 50-60 years was two times higher compared to women aged 40-49 years and three times higher compared to women aged 30-39 years. The probability of having persistent infection increased progressively with higher viral load at baseline (adjusted HR 3.29, 95% CI 2.21-4.90 for RLU ≥100; adjusted HR 2.69, 95% CI 1.71-4.22 for RLU 10-100). Women with increasing viral load at follow-up had four times higher risk of developing CIN2 or worse lesions as compared to those with decreasing load (20.9% vs 4.8%; p < 0.001). In the context of developing countries where cytology or genotyping triaging is not feasible, colposcopy referral of HR-HPV-positive women with advancing age, viral persistence, and increasing viral load may be considered.

Evaluation of the Clinical Performance of the HPV-Risk Assay Using the VALGENT-3 Panel.

Human papillomavirus (HPV) testing is increasingly being incorporated into cervical cancer screening. The Validation of HPV Genotyping Tests (VALGENT) is a framework designed to evaluate the clinical performance of various HPV tests relative to that of the validated and accepted comparator test in a formalized and uniform manner. The aim of this study was to evaluate the clinical performance of the HPV-Risk assay with samples from the VALGENT-3 panel and to compare its performance to that of the clinically validated Hybrid Capture 2 assay (HC2). The VALGENT-3 panel comprises 1,300 consecutive samples from women participating in routine cervical cancer screening and is enriched with 300 samples from women with abnormal cytology. DNA was extracted from original ThinPrep PreservCyt medium aliquots, and HPV testing was performed using the HPV-Risk assay by investigators blind to the clinical data. HPV prevalence was analyzed, and the clinical performance of the HPV-Risk assay for the detection of cervical intraepithelial neoplasia grade 3 or worse (CIN3+) and CIN2 or worse (CIN2+) relative to the performance of HC2 was assessed. The sensitivity of the HPV-Risk assay for the detection of CIN3+ was similar to that of HC2 (relative sensitivity, 1.00; 95% confidence interval [CI], 0.95 to 1.05; P = 1.000), but the specificity of the HPV-Risk assay was significantly higher than that of HC2 (relative specificity, 1.02; 95% CI, 1.01 to 1.04; P < 0.001). For the detection of CIN2+, similar results were obtained, with the relative sensitivity being 0.98 (95% CI, 0.93 to 1.02; P = 0.257) and the relative specificity being 1.02 (95% CI, 1.01 to 1.03; P < 0.001). The performance of the HPV-Risk assay for the detection of CIN3+ and CIN2+ was noninferior to that of HC2, with all P values being ≤0.006. In conclusion, the HPV-Risk assay demonstrated noninferiority to the clinically validated HC2 by the use of samples from the VALGENT-3 panel for test validation and comparison.

Variant call concordance between two laboratory-developed, solid tumor targeted genomic profiling assays using distinct workflows and sequencing instruments.

Targeted genomic profiling (TGP) using massively parallel DNA sequencing is becoming the standard methodology in clinical laboratories for detecting somatic variants in solid tumors. The variety of methodologies and sequencing platforms in the marketplace for TGP has resulted in a variety of clinical TGP laboratory developed tests (LDT). The variability of LDTs is a challenge for test-to-test and laboratory-to-laboratory reliability. At the University of Vermont Medical Center (UVMMC), we validated a TGP assay for solid tumors which utilizes DNA hybridization capture and complete exon and selected intron sequencing of 29 clinically actionable genes. The validation samples were run on the Illumina MiSeq platform. Clinical specificity and sensitivity were evaluated by testing samples harboring genomic variants previously identified in CLIA-approved, CAP accredited laboratories with clinically validated molecular assays. The Molecular Laboratory at Dartmouth Hitchcock Medical Center (DHMC) provided 11 FFPE specimens that had been analyzed on AmpliSeq Cancer Hotspot Panel version 2 (CHPv2) and run on the Ion Torrent PGM. A Venn diagram of the gene lists from the two institutions is shown. This provided an excellent opportunity to compare the inter-laboratory reliability using two different target sequencing methods and sequencing platforms. Our data demonstrated an exceptionally high level of concordance with respect to the sensitivity and specificity of the analyses. All clinically-actionable SNV and InDel variant calls in genes covered by both panels (n=17) were identified by both laboratories. This data supports the proposal that distinct gene panel designs and sequencing workflows are capable of making consistent variant calls in solid tumor FFPE-derived samples.

Genetic Structure and Phylogeography of the Leopard Cat (Prionailurus bengalensis) Inferred from Mitochondrial Genomes.

The Leopard cat Prionailurus bengalensis is a habitat generalist that is widely distributed across Southeast Asia. Based on morphological traits, this species has been subdivided into 12 subspecies. Thus far, there have been few molecular studies investigating intraspecific variation, and those had been limited in geographic scope. For this reason, we aimed to study the genetic structure and evolutionary history of this species across its very large distribution range in Asia. We employed both PCR-based (short mtDNA fragments, 94 samples) and high throughput sequencing based methods (whole mitochondrial genomes, 52 samples) on archival, noninvasively collected and fresh samples to investigate the distribution of intraspecific genetic variation. Our comprehensive sampling coupled with the improved resolution of a mitochondrial genome analyses provided strong support for a deep split between Mainland and Sundaic Leopard cats. Although we identified multiple haplogroups within the species' distribution, we found no matrilineal evidence for the distinction of 12 subspecies. In the context of Leopard cat biogeography, we cautiously recommend a revision of the Prionailurus bengalensis subspecific taxonomy: namely, a reduction to 4 subspecies (2 mainland and 2 Sundaic forms).