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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.

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.

The COVID-19 pandemic sparked rapid development of 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 RT-PCR and MALDI-TOF 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 dataset of 1,262 SARS-CoV-2-positive specimens from Mount Sinai Health System (New York City) from December 2020 through 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.

Signal, probe, and non-PCR target amplification methods have been developed as viable alternatives to PCR testing for infectious agents. These signal amplification methods include hybrid capture and Invader, as well as target amplification methods such as transcription-mediated amplification, strand displacement amplification, loop-mediated isothermal amplification, and helicase-dependent amplification. The principles of these methods and their commercially available FDA-cleared assays are discussed in this chapter.

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.

The superior sensitivity and specificity associated with the use of molecular assays has greatly improved the field of infectious disease diagnostics by providing clinicians with results that are both accurate and rapidly obtained. Herein, we review molecularly based infectious disease diagnostic tests that are Food and Drug Administration approved or cleared and commercially available in the United States as of December 31, 2010. We describe specific assays and their performance, as stated in the Food and Drug Administration's Summary of Safety and Effectiveness Data or the Office of In Vitro Diagnostic Device Evaluation and Safety's decision summaries, product inserts, or peer-reviewed literature. We summarize indications for testing, limitations, and challenges related to implementation in a clinical laboratory setting for a wide variety of common pathogens. The information presented in this review will be particularly useful for laboratories that plan to implement or expand their molecular offerings in the near term.

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.

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.

The introduction of target nucleic acid amplification technologies, such as polymerase chain reaction (PCR), nucleic acid sequence-based amplification (NASBA), and strand displacement amplification (SDA), was accompanied by a plethora of technology patents. This made it very difficult for companies without access to these amplification technologies to compete in the area of assay development for ultrasensitive infectious disease detection and quantification. One way around this intellectual property roadblock was the development of highly sensitive assays that depended not on target amplification, but signal amplification. Signal amplification technologies have one major advantage over target amplification in that the issue of contaminating one test run with previously amplified material from a previous run is not an issue. Also, the three signal amplification technologies that are discussed in this chapter are isothermal, meaning that unlike PCR, thermocycling instrumentation is not required. Lastly, assays can be designed to detect and or quantify specific DNA or RNA targets using each of these methods, and, in the case of RNA, without the need to first convert the RNA target to DNA via reverse transcription. Care must still be taken, however, to minimize cross-contamination between samples being tested due to the enhanced analytical sensitivity inherent in assays using signal amplification technologies. An inherent concern with signal amplification methods is that great care must be taken during the design of the assay to ensure that carryover of the different assay components, from one step to the next, is minimized to reduce background noise, which will degrade the analytical sensitivity of the test in question.

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.