RT-PCR/MALDI-TOF Diagnostic Target Performance Reflects Circulating SARS-CoV-2 Variant Diversity in New York City.

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to circulate, multiple variants of concern have emerged. New variants pose challenges for diagnostic platforms because sequence diversity can alter primer/probe-binding sites (PBSs), causing false-negative results. The MassARRAY SARS-CoV-2 Panel (Agena Bioscience) uses RT-PCR and mass spectrometry to detect five multiplex targets across N and ORF1ab genes. Herein, we use a data set of 256 SARS-CoV-2-positive specimens collected between April 11, 2021, and August 28, 2021, to evaluate target performance with paired sequencing data. During this time frame, two targets in the N gene (N2 and N3) were subject to the greatest sequence diversity. In specimens with N3 dropout, 69% harbored the Alpha-specific A28095U polymorphism that introduces a 3'-mismatch to the N3 forward PBS and increases risk of target dropout relative to specimens with 28095A (relative risk, 20.02; 95% CI, 11.36 to 35.72; P < 0.0001). Furthermore, among specimens with N2 dropout, 90% harbored the Delta-specific G28916U polymorphism that creates a 3'-mismatch to the N2 probe PBS and increases target dropout risk (relative risk, 11.92; 95% CI, 8.17 to 14.06; P < 0.0001). These findings highlight the robust capability of MassARRAY SARS-CoV-2 Panel target results to reveal circulating virus diversity, and they underscore the power of multitarget design to capture variants of concern.

Robust clinical detection of SARS-CoV-2 variants by RT-PCR/MALDI-TOF multitarget approach.

The coronavirus disease 2019 (COVID-19) pandemic has sparked the rapid development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostics. However, emerging variants pose the risk for target dropout and false-negative results secondary to primer/probe binding site (PBS) mismatches. The Agena MassARRAY® SARS-CoV-2 Panel combines reverse-transcription polymerase chain reaction and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry to probe for five targets across N and ORF1ab genes, which provides a robust platform to accommodate PBS mismatches in divergent viruses. Herein, we utilize a deidentified data set of 1262 SARS-CoV-2-positive specimens from Mount Sinai Health System (New York City) from December 2020 to April 2021 to evaluate target results and corresponding sequencing data. Overall, the level of PBS mismatches was greater in specimens with target dropout. Of specimens with N3 target dropout, 57% harbored an A28095T substitution that is highly specific for the Alpha (B.1.1.7) variant of concern. These data highlight the benefit of redundancy in target design and the potential for target performance to illuminate the dynamics of circulating SARS-CoV-2 variants.

Multicenter Performance Evaluation of the Simplexa Bordetella Direct Kit in Nasopharyngeal Swab Specimens.

Detection of Bordetella pertussis and Bordetella parapertussis using molecular methods is sensitive and specific with a short turnaround time compared to other diagnostic methods. In this multicenter study, we compared the performance of the Simplexa Bordetella Direct kit to those of other molecular assays in detecting and differentiating B. pertussis and B. parapertussis in nasopharyngeal swab specimens. The limits of detection (LODs) were 150 CFU/ml or 3 fg/µl of DNA for B. pertussis and 1,500 CFU/ml or 10 fg/µl of DNA for B. parapertussis A total of 1,103 fresh and residual frozen specimens from eight clinical sites were tested. Combining the data from individual clinical sites using different comparative assays, the overall positive percent agreement (PPA) and negative percent agreement (NPA) for B. pertussis were 98.7% and 97.3%, respectively. The overall PPA and NPA for B. parapertussis were 96.7% and 100%, respectively. For prospective fresh specimens, the overall PPA and NPA for both targets were 97.7% and 99.3%, respectively. For retrospective frozen specimens, the overall PPA and NPA for both targets were 92.6% and 93.2%, respectively. The percentage of invalid results was 1.0%. A cross-reactivity study using 74 non-Bordetella bacterial species and five yeast species revealed that the Simplexa Bordetella Direct kit was 100% specific. The hands-on time and assay run time of the Simplexa Bordetella Direct kit are favorable compared to those of other commercial and laboratory-developed tests. In summary, the Simplexa Bordetella Direct kit has a performance comparable to those of other molecular assays for the detection of B. pertussis and B. parapertussis.

Evaluation of the Luminex ARIES® system for the detection and quantification of BK virus (BKV) DNA in plasma samples from kidney transplant recipients.

BK virus (BKV) nephropathy is a serious complication in renal transplant recipients due to the need for immunosuppression. Nearly 50% of renal transplant patients with BKV nephropathy experience a significant loss of function of the transplanted kidney. It is routine practice to screen renal transplant recipients regularly for BK viremia. In this study, we compared the performance of BKV quantitative polymerase chain reaction analyte specific reagents by ELITech and Luminex for measuring BKV viral load in plasma using the Roche Cobas® z480 instrument and the Luminex ARIES® platform, respectively. A total of 34 patients previously tested on the z480, with results spanning the test's linear range, were analyzed on the ARIES®. The BKV DNA copy number correlation between the 2 methods was very good, with an R2 value of 0.96. The average difference in log copy number between the 2 methods was -0.3, indicating that the ARIES® method may have slightly greater analytical sensitivity. BKV quantification results were closely matched between the 2 different methods. The workflow with the ARIES® System is greatly simplified by elimination of DNA extraction and most hands-on steps. The high degree of automation allows samples to be tested as they arrive into the laboratory, resulting in enhanced patient care due to more rapid turnaround time for results back to the ordering physician.

Molecular pathology curriculum for medical laboratory scientists: A report of the association for molecular pathology training and education committee.

Molecular diagnostics is a rapidly growing specialty in the clinical laboratory assessment of pathology. Educational programs in medical laboratory science and specialized programs in molecular diagnostics must address the training of clinical scientists in molecular diagnostics, but the educational curriculum for this field is not well defined. Moreover, our understanding of underlying genetic contributions to specific diseases and the technologies used in molecular diagnostics laboratories change rapidly, challenging providers of training programs in molecular diagnostics to keep their curriculum current and relevant. In this article, we provide curriculum recommendations to molecular diagnostics training providers at both the baccalaureate and master's level of education. We base our recommendations on several factors. First, we considered National Accrediting Agency for Clinical Laboratory Sciences guidelines for accreditation of molecular diagnostics programs, because educational programs in clinical laboratory science should obtain its accreditation. Second, the guidelines of several of the best known certifying agencies for clinical laboratory scientists were incorporated into our recommendations. Finally, we relied on feedback from current employers of molecular diagnostics scientists, regarding the skills and knowledge that they believe are essential for clinical scientists who will be performing molecular testing in their laboratories. We have compiled these data into recommendations for a molecular diagnostics curriculum at both the baccalaureate and master's level of education.

Assessment of clinical and analytical performance characteristics of an HPV genotyping test.

Human papillomavirus (HPV), the known cause of cervical cancer, is found in essentially all cervical cancer specimens. Infection with high-risk HPV genotypes carries the greatest risk of viral persistence and the potential to develop precancerous lesions or cervical cancer. Identifying women infected with HPV 16 and/or 18, the two genotypes most commonly found in cervical cancer, helps further stratify women for either immediate referral to colposcopy or repeat cytological and HPV DNA testing in 12 months. Genotyping additional, less carcinogenic HPV types may be of limited clinical utility. Detection of multiple individual genotypes may capture a greater percentage of those with the potential to develop cervical disease. However, the advantage of individual detection must be weighed against the potential error of multiple measurements. In order for genotyping tests to have clinical benefit in patient management, the Centers of Medicare and Medicaid Services Divisions of Laboratory Services have set forth a rigorous clinical validation procedure. This procedure addresses the quality process, characteristics and specifications of the assay, comparison of the test/device to existing tests/devices, technical aspects, and other assurances that guarantee the test/device will meet all performance requirements of the US Food and Drug Administration (FDA). Currently, only the Cervista® HPV 16/18 genotyping test is approved by the FDA and recommended for clinical use in published guidelines. This manuscript reviews the current best practices for the development and use of HPV genotyping tests.

A comparative review of laboratory-developed tests utilizing Invader HPV analyte-specific reagents for the detection of high-risk human papillomavirus.

BACKGROUND: Testing for human papillomavirus (HPV) has emerged as an important component of cervical cancer screening and disease management. Analytic performance (sensitivity, specificity, positive predictive value, negative predictive value, accuracy) and clinical relevance are important criteria by which any new test must be reviewed. OBJECTIVE: This paper compares laboratory-developed tests, utilizing Invader analyte-specific reagents (ASRs) for the detection of oncogenic HPV DNA, to the Digene Hybrid Capture 2 test (HC2). In addition to accurately identifying the presence or absence of HPV, genotyping has emerged as an important consideration for risk stratification and patient management. This paper reviews all current relevant Invader HPV literature to explain how these results shape our current understanding of the topic.

Detection of high-risk papillomavirus DNA with commercial invader-technology-based analyte-specific reagents following automated extraction of DNA from cervical brushings in ThinPrep media.

We compared the performances of the Third Wave Technology Invader method and the Digene Hybrid Capture 2 assay to detect high-risk human papillomaviruses in 87 cervical brushing specimens submitted in Cytyc ThinPrep media. Two different methods for the extraction of DNA from squamous epithelial cells were also evaluated.

Comparative evaluation of three commercially available methodologies for hepatitis C virus genotyping.

We compared the performances of three hepatitis C virus genotyping methodologies supplied by Bayer, Abbott, and Third Wave Technologies. Genotypes were determined for 136 of 137 specimens by the Bayer method, 121 of 137 specimens by the Invader assay, and only 77 of 137 specimens by the Abbott assay. All reported genotypes were concordant by all three methods.

Principios y aplicaciones de la reacción en cadena de la polimerasa / Principle and application of the chain reaction of the polymerase

El descubrimiento de la reacción en cadena de la polimerasa (PCR), técnica para la amplificación de ácidos nucleicos, ha tenido un enorme impacto sobre áreas diversas, tanto de la investigación básica como de la investigación clínica. Desde su descubrimiento en 1985, los informes sobre una gran variedad de aplicaciones de la PCR han recibido mucha atención en la literatura médica y científica. Esta tecnología ha demostrado tener una gran aplicabilidad para el diagnóstico de enfermedades humanas, incluyendo áreas tan diversas como enfermedades infecciosas, desórdenes genéticos y cáncer. Este artículo presenta una amplia revisión de los principios de la PCR, que incluyen conceptos genéricos que deben manejarse cuando se use o se diseñe un ensayo basado en la PCR. También se discute la aplicación de tales ensayos para el diagnóstico de desórdenes genéticos y para el estudio de enfermedades infecciosas. En los últimos 10 años ha aumentado, de manera notable, la aplicación de herramientas de la biología molecular para el diagnóstico de las enfermedades humanas. Los ensayos con sondas de ADN están, ahora, disponibles comercialmente para la detección e identificación de una gran variedad de patógenos humanos, así como para el diagnóstico de desórdenes genéticos humanos. Una manifestación del rápido crecimiento de la tecnología del ADN, ha sido el desarrollo de técnicas para amplificar secuencias específicas de ácidos nucleicos. En la literatura, han sido descritos muchos métodos, que se enumeran en la tabla i. Una comparación de estos métodos ha sido el tema de una revisión frecuente. Entre ellos se destacan la reacción en cadena de la polimerasa, como la de mayor impacto, tanto como herramienta de investigación como de diagnóstico. El objetivo de este artículo es presentar una breve revisión de los principios y aplicaciones de la amplificación de PCR, que incluyen una discusión de la selección correcta de las secuencias en blanco, el diseño de los "primer" y los medios necesarios para llevar a cabo la técnica

Principios y aplicaciones de la reacción en cadena de la polimerasa / Principle and application of the chain reaction of the polymerase

El descubrimiento de la reacción en cadena de la polimerasa (PCR), técnica para la amplificación de ácidos nucleicos, ha tenido un enorme impacto sobre áreas diversas, tanto de la investigación básica como de la investigación clínica. Desde su descubrimiento en 1985, los informes sobre una gran variedad de aplicaciones de la PCR han recibido mucha atención en la literatura médica y científica. Esta tecnología ha demostrado tener una gran aplicabilidad para el diagnóstico de enfermedades humanas, incluyendo áreas tan diversas como enfermedades infecciosas, desórdenes genéticos y cáncer. Este artículo presenta una amplia revisión de los principios de la PCR, que incluyen conceptos genéricos que deben manejarse cuando se use o se diseñe un ensayo basado en la PCR. También se discute la aplicación de tales ensayos para el diagnóstico de desórdenes genéticos y para el estudio de enfermedades infecciosas. En los últimos 10 años ha aumentado, de manera notable, la aplicación de herramientas de la biología molecular para el diagnóstico de las enfermedades humanas. Los ensayos con sondas de ADN están, ahora, disponibles comercialmente para la detección e identificación de una gran variedad de patógenos humanos, así como para el diagnóstico de desórdenes genéticos humanos. Una manifestación del rápido crecimiento de la tecnología del ADN, ha sido el desarrollo de técnicas para amplificar secuencias específicas de ácidos nucleicos. En la literatura, han sido descritos muchos métodos, que se enumeran en la tabla i. Una comparación de estos métodos ha sido el tema de una revisión frecuente. Entre ellos se destacan la reacción en cadena de la polimerasa, como la de mayor impacto, tanto como herramienta de investigación como de diagnóstico. El objetivo de este artículo es presentar una breve revisión de los principios y aplicaciones de la amplificación de PCR, que incluyen una discusión de la selección correcta de las secuencias en blanco, el diseño de los "primer" y los medios necesarios para llevar a cabo la técnica (AU)

Principios e aplicaçoes da reaçao da cadeia de polimerase / Principles and applications of the polymerase chain reaction

A invençao da tecnica da reaçao da cadeia de polimerase(PCR) para a amplificaçao de acido nucleico tem tido um impacto maior sobre diversas areas tanto da pesquisa basica como da clinica. Desde seu inicio em 1985, comunicaçoes sobre uma grande variedade de aplicaçoes da PCR foram recebidas com grande atençao na literatura cientifica e medica. Esta tecnologia tem mostrado grande aplicabilidade ao diagnostico de doenças humanas, incluindo areas tao diversas como doenças infecciosas, desordens geneticas e cancer. O presente artigo apresenta uma ampla revisao dos principios da PCR incluindo consideraçoes geneticas que devem ser observadas no uso ou no planejamento de ensaios baseados na PCR. A aplicaçao destes ensaios com base na PCR para o diagnostico de desordens geneticas e para triagem de doenças infecciosas sao tambem discutidas