Publicly available RNA-sequencing data provide a cost-effective springboard for biomarker discovery. However, heterogeneity across studies often complicates analysis. This study presents a modular analytics pipeline that combines publicly available data sets with established open-source tools; standardizes quality control, differential expression analysis, and pathway analysis; and leverages competitive machine learning to unify disparate RNA-sequencing data sets for robust biomarker identification. The workflow is demonstrated across three disease contexts, ranging from small pilot data sets to larger, integrated analyses: i) identifying differentially expressed gene signatures associated with coronavirus disease 2019 (COVID-19) severity, ii) combining differential expression and machine learning techniques to analyze multicohort sepsis data sets, resulting in concise biomarker panels, and iii) using both bulk and single-cell data to examine tissue and cell type specificity of N-acyl-phosphatidylethanolamine phospholipase D in atherosclerosis. These applications demonstrate how an adaptable, modular pipeline using open-source tools can repurpose public data to reduce noise, generate new hypotheses, and reveal meaningful biological insights, thereby establishing a foundation for future research and underlining the importance of public data in exploratory biomarker discovery.

Next-generation RNA sequencing (RNA-seq) enables comprehensive transcriptomic profiling for disease characterization, biomarker discovery, and precision medicine. Despite its potential, RNA-seq has not yet been widely adopted for clinical applications, and a key barrier to its adoption is the variability introduced during processing and analysis. Quality controls (QCs) must be considered through all stages of biomarker discovery. This study describes a comprehensive QC framework for effective RNA-seq biomarker discovery. Multilayered quality metrics were established across preanalytical, analytical, and postanalytical processes. Total RNA-seq was performed by using RNA isolated from whole blood (PAXgene Blood RNA tubes). Bulk RNA controls were incorporated to monitor sequencing batches. This framework was applied to a catalog of prospectively collected or biobanked clinical specimens spanning multiple disease indications. Among all QCs, preanalytical metrics (specimen collection, RNA integrity, and genomic DNA contamination) exhibited the highest failure rates and resulted in the addition of a secondary DNase treatment, which reduced genomic DNA levels. The additional DNase treatment significantly lowered intergenic read alignment and provided sufficient RNA for downstream sequencing and analysis. This end-to-end QC framework for RNA-seq biomarker discovery was developed and implemented to enhance the confidence and reliability of results. To advance the clinical adoption of RNA-seq, developing and implementing standards will improve reliability, accelerate biomarker discovery, and facilitate its translation into clinically actionable diagnostics and therapeutics.

This study aims to identify RNA biomarkers distinguishing neuromyelitis optica (NMO) from relapsingremitting multiple sclerosis (RRMS) and explore potential therapeutic applications leveraging machine learning (ML). An ensemble approach was developed using differential gene expression analysis and competitive ML methods, interrogating total RNA-sequencing data sets from peripheral whole blood of treatment-na & iuml;ve patients with RRMS and NMO and healthy individuals. Pathway analysis of candidate biomarkers informed the biological context of disease, transcription factor activity, and small-molecule therapeutic potential. ML models differentiated between patients with NMO and RRMS, with the performance of certain models exceeding 90% accuracy. RNA biomarkers driving model performance were associated with ribosomal dysfunction and viral infection. Regulatory networks of kinases and transcription factors identified biological associations and identified potential therapeutic targets. Small-molecule candidates capable of reversing perturbed gene expression were uncovered. Mitoxantrone and vorinostat-two identified small molecules with previously reported use in patients with NMO and experimental autoimmune encephalomyelitis-reinforced discovered expression signatures and highlighted the potential to identify new therapeutic candidates. Putative RNA biomarkers were identified that accurately distinguish NMO from RRMS and healthy individuals. The application of multivariate approaches in analysis of RNA-sequencing data further enhances the discovery of unique RNA biomarkers, accelerating the development of new methods for disease detection, monitoring, and therapeutics. Integrating biological understanding further enhances detection of disease-specific signatures and possible therapeutic targets. (J Mol Diagn 2024, 26: 520e529; https://doi.org/10.1016/ j.jmoldx.2024.03.003)

Objective: Compare gene expression differences in the peripheral whole blood of patients with multiple sclerosis (MS) versus neuromyelitis optica (NMO), including differences in patients with NMO who are aquaporin-4 (AQP4) antibody positive (AQP4+) versus AQP4 negative NMO (AQP4-). Background: MRI imaging and antibodies against AQP4 have been used to differentiate MS from NMO. However, early in the disease course, some patients do not have imaging or clinical characteristics that distinguish the two disorders, particularly in AQP4 antibody negative patients. Currently, there are no blood-based biomarkers that alone confirm the presence or absence of MS or NMO. Design/Methods: Total RNA-sequencing was used to examine coding and non-coding RNA expression profiles, immune repertoire composition, and biologic pathway activation. Whole blood from relapsing-remitting MS patients (n=105), NMO patients (AQP4+ = 39, AQP4- = 14), and healthy controls (n=70) was collected into PAXgene tubes. MS and NMO patient samples were obtained from the Accelerated Cure Project and were not on disease-modifying treatment. MS and NMO patients were screened for myelin oligodendrocyte glycoprotein (MOG) antibody status. Plasma neurofilament light chain (NfL) was assessed to determine underlying disease activity. Results: Differentially expressed genes, unique immune repertoire, and biologic pathway signatures with the greatest ability to differentiate among MS, NMO, and healthy controls were identified. Activation of pathways associated with antimicrobial responses and neutrophil activation were more prominent in NMO patients. Pathways associated with innate immune responses and type 1 interferons were enriched in the comparison of AQP4+ and AQP4- NMO patients. Conclusions: These findings suggest that RNA-sequencing data derived from peripheral whole blood can identify the characteristic RNAs, immune repertoires, and pathways that distinguish MS from NMO and NMO AQP4+ versus AQP4- individuals. These approaches could lead to novel RNA-based biomarkers to identify patients with demyelinating diseases at their earliest stages and direct therapeutic management. Disclosure: Dr. Wylezinski has received personal compensation for serving as an employee of Decode Health. Dr. Wylezinski has stock in Decode Health. The institution of Dr. Wylezinski has received research support from Decode Health. Dr. Wylezinski has received intellectual property interests from a discovery or technology relating to health care. Mrs. Shaginurova has nothing to disclose. Dr. Grigorenko has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant for Decode Health. Dr. Grigorenko has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for Lifetime Sciences. Dr. Grigorenko has received personal compensation in the range of $5,000-$9,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Pharmozyme. Dr. Wohlgemuth has received personal compensation for serving as an employee of Quest Diagnostics. Dr. Wohlgemuth has stock in Quest Diagnostics. Dr. Cockerill has received personal compensation in the range of $100,000-$499,999 for serving as a Consultant for Quest Diagnostics. Dr. Cockerill has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant for Mainz Biotech. Dr. Cockerill has received personal compensation in the range of $5,000-$9,999 for serving as a Consultant for Imanis Life Sciences. Dr. Cockerill has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant for Scanwell. Dr. Cockerill has received personal compensation in the range of $0-$499 for serving as a Consultant for Visby. Dr. Cockerill has received personal compensation in the range of $0-$499 for serving as a Consultant for Microbiobloc. Dr. Cockerill has received personal compensation in the range of $0-$499 for serving as a Consultant for Decode Health. An immediate family member of Dr. Cockerill has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant for Quest Diagnostics . Dr. Cockerill has received personal compensation in the range of $5,000-$9,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Magnolia Medical Technologies. Dr. Cockerill has received personal compensation in the range of $5,000-$9,999 for serving on a Scientific Advisory or Data Safety Monitoring board for 98point6. Dr. Cockerill has received personal compensation in the range of $0-$499 for serving as an officer or member of the Board of Directors for Decode Health. An immediate family member of Dr. Cockerill has received personal compensation in the range of $0-$499 for serving as an officer or member of the Board of Directors for Online MedED. Dr. Cockerill has stock in ACLIP. Dr. Cockerill has stock in Magnolia Medical Technologies. An immediate family member of Dr. Cockerill has stock in Online Med Ed. Dr. Cockerill has stock in Visby. Dr. Cockerill has stock in 98point6. Dr. Cockerill has stock in Mainz Biomed. Dr. Cockerill has stock in Microbiobloc. Dr. Cockerill has stock in Decode Health. Dr. Cockerill has received intellectual property interests from a discovery or technology relating to health care. Dr. Racke has received personal compensation for serving as an employee of Quest Diagnostics. An immediate family member of Dr. Racke has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Novartis. Dr. Racke has received personal compensation in the range of $10,000-$49,999 for serving as an Editor, Associate Editor, or Editorial Advisory Board Member for Elsevier. An immediate family member of Dr. Racke has received personal compensation in the range of $5,000-$9,999 for serving as an Editor, Associate Editor, or Editorial Advisory Board Member for Elsevier. Dr. Spurlock has received personal compensation for serving as an employee of Decode Health. Dr. Spurlock has received personal compensation for serving as an employee of New York University. Dr. Spurlock has received personal compensation for serving as an employee of IQuity Labs. Dr. Spurlock has stock in Decode Health. Dr. Spurlock has stock in IQuity Labs. The institution of Dr. Spurlock has received research support from National Institutes of Health.

Laboratory testing is an important component in the diagnosis of respiratory tract infections such as with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). However, specimen collection not only risks exposure of health care workers and other patients to infection, but also necessitates use of personal protective equipment that may be in short supply during periods of heightened disease activity. Self-collection of nasal or oropharyngeal swabs offers an alternative to address these drawbacks. Although studies in the past decade have demonstrated the utility of this approach for respiratory infections, it has not been widely adopted in routine clinical practice. The rapid spread of coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, has focused attention on the need for safe, convenient, timely, and scalable methods for collecting upper respiratory specimens for testing. The goals of this article are to highlight the literature regarding self-collected nasal or oropharyngeal specimens for respiratory pathogen testing; discuss the role of self-collection in helping prevent the spread of the COVID-19 disease from infected patients and facilitating a shift toward "virtual" medicine or telemedicine; and describe the current and future state of self-collection for infectious agents, and the impacts these approaches can have on population health management and disease diagnosis and prevention.

THE OBJECTIVE OF THE STUDY IS TO DETERMINE THE PREVALENCE OF HIGH-RISK HUMAN PAPILLOMAVIRUS (HRHPV) INFECTION IN TONSILLAR SWABS AND TISSUE: Patients undergoing tonsillectomy for nonmalignant causes were enrolled. A flocked swab and fresh tissue were collected from the left and right tonsil of each patient. Specimens were tested for hrHPV DNA using the Roche cobas test and for the presence of E6/E7 messenger RNA using the Hologic Aptima hrHPV test. Of the 193 patients enrolled, 129 were in the pediatric group (ages 1-12years; median, 5years), and 64 were in the adult group (ages 13-55; median, 22years). All swab and tissue specimens were negative for hrHPV by both methods. Positive, negative, and internal controls performed as expected. We found a 0% rate of infection indicating that detectable hrHPV infection in tonsillar tissue appears to be uncommon in the children and adults in the population sampled.

Yoğun paralel sıralama (MPS) olarak ta adlandırılan yeni nesil sıralama (NGS), hızla klinik laboratuvar testleri ile bütünleştirilmektedir.Mevcut uygulama alanları arasında kalıtsak hastalıklardaki germ sırası değişkenlikleri, kanserlerdeki somatik değişkenler, dolaşımdaki hücresiz DNA

Objectives:(1) Determine incidence of HPV (human papillomavirus) infection in routine tonsillectomy swabs and specimens. (2) Determine if vaccination impacts tonsillar HPV infection.Methods:This was a prospective study conducted from July 2012 to January 2014. Patients ages 1 to 100 years undergoing tonsillectomy for nonmalignant indications were enrolled. Demographic data and immunization status were collected. Specimens collected during surgery included a flocked swab and half of the left and right tonsil. All specimens were tested for HPV DNA using the polymerase chain reaction–based Roche Cobas Amplicor test and for the presence of E6/E7 mRNA using the Gen‐Probe Aptima HPV test.Results:Patients were stratified into 2 groups, pediatric (ages 1‐12 years) and adults (age 13 years and up) based on age of potential sexual maturity. In the pediatric group (n = 130) mean age was 5.2 years, 42% were female, 58% were male, and only 3 patients were fully vaccinated against HPV. In the adult group (n = 64) mean age was 24 years, 76% female, 24% male, and 47% were either fully or partially vaccinated. All specimens were negative for HPV infection via both testing methods. Internal controls confirmed the tests were functioning properly.Conclusions:This is the first study to examine fresh tonsil swabs and tonsillectomy specimens for HPV infection using a Food and Drug Administration–approved method. Interestingly, both adult and pediatric specimens were negative for HPV infection. The role of vaccination in oropharyngeal HPV infection remains unclear.

A retrospective analysis of 386,706 specimens representing a variety of matrix types used in qualitative real-time PCR assays determined the overall inhibition rate to be 0.87% when the inhibition control was added preextraction to 5,613 specimens and 0.01% when the inhibition control was added postextraction but preamplification in 381,093 specimens. Inhibition rates of ≤ 1% were found for all specimen matrix types except urine and formalin-fixed, paraffin-embedded tissue.

Microbiological diagnosis is pivotal to the appropriate management and treatment of infective endocarditis. We evaluated PCR-electrospray ionization mass spectrometry (PCR/ESI-MS) for bacterial and candidal detection using 83 formalin-fixed paraffin-embedded heart valves from subjects with endocarditis who had positive valve and/or blood cultures, 63 of whom had positive valvular Gram stains. PCR/ESI-MS yielded 55% positivity with concordant microbiology at the genus/species or organism group level (e. g., viridans group streptococci), 11% positivity with discordant microbiology, and 34% with no detection. PCR/ESI-MS detected all antimicrobial resistance encoded by mecA or vanA/B and identified a case of Tropheryma whipplei endocarditis not previously recognized.

ABSTRACTThe Bruker Biotyper and Vitek MS matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) instruments were evaluated for the identification of nonfermenting Gram-negative bacilli (NFGNB) by a blinded comparison to conventional biochemical or molecular methods. Two hundred NFGNB that were recovered from cultures from cystic fibrosis patients in the University of Iowa Health Care (UIHC) Microbiology Laboratory between 1 January 2006 and 31 October 2010 were sent to Mayo Clinic for analysis with the Bruker Biotyper (software version 3.0) and to bioMérieux for testing with Vitek MS (SARAMIS database version 3.62). If two attempts at direct colony testing failed to provide an acceptable MALDI-TOF identification, an extraction procedure was performed. The MS identifications from both of these systems were provided to UIHC for comparison to the biochemical or molecular identification that had been reported in the patient record. Isolates with discordant results were analyzed by 16S rRNA gene sequencing at UIHC. After discrepancy testing, the Bruker Biotyper result agreed with the biochemical or molecular method, with 72.5% of isolates to the species level, 5.5% to the complex level, and 19% to the genus level (3% not identified). The level of agreement for Vitek MS was 80% species, 3.5% complex, 6% genus, and 3.5% family (7% not identified). Both MS systems provided rapid (≤3 min per isolate) and reliable identifications. The agreement of combined species/complex/genus-level identification with the reference method was higher for the Bruker Biotyper (97% versus 89.5%,P= 0.004) but required an extraction step more often. Species-level agreement with the reference method was similar for both MS systems (72.5% and 80%,P= 0.099).

Objective: To compare the effectiveness of self-collected and health care worker (HCW)-collected nasal swabs for detection of influenza viruses and determine the patients' preference for type of collection.Patients and Methods: We enrolled adult patients presenting with influenzalike illness to the Emergency Department at Mayo Clinic, Rochester, Minnesota, from January 28, 2011, through April 30, 2011. Patients self-collected a midturbinate nasal flocked swab from their right nostril following written instructions. A second swab was then collected by an HCW from the left nostril. Swabs were tested for influenza A and B viruses by real-time reverse transcription-polymerase chain reaction, and percent concordance between collection methods was determined.Results: Of the 72 paired specimens analyzed, 25 were positive for influenza A or B RNA by at least one of the collection methods (34.7% positivity rate). When the 14 patients who had prior health care training were excluded, the qualitative agreement between collection methods was 94.8% (55 of 58). Two of the 58 specimens (3.4%) from patients without health care training were positive only by HCW collection, and 1 of 58 (1.7%) was positive only by patient self-collection. A total of 53.4% of patients (31 of 58) preferred the self-collection method over the HCW collection, and 25.9% (15 of 58) had no preference.Conclusion: Self-collected midturbinate nasal swabs provide a reliable alternative to HCW collection for influenza A and B virus real-time reverse transcription-polymerase chain reaction. (C) 2012 Mayo Foundation for Medical Education and Research Mayo Clin Proc. 2012; 87(6): 548-554

This chapter discusses general fluorescence resonance energy transfer (FRET) probe technology and the use of FRET with single and dual hybridization probes in microbiology. It focuses on hydrolysis and hybridization FRET probes and the many variations used for detection of amplification products in PCR amplification. True multiplex assays simultaneously amplify different nucleic acid targets and result in multiple unique PCR products. These types of assays often have two or more sets of primers and probes and are most commonly used with genomic targets in which the amount of target nucleic acid is normally large and the ratio of the two target nucleic acids is close to unity. Recent applications of FRET probes in bacteriology include assays for Vibrio parahaemolyticus, Clostridium difficile, Brucella, Francisella tularensis, Clostridium perfringens, Listeria monocytogenes, Burkholderia cepacia complex, Campylobacter jejuni and Campylobacter coli, and penA and ponA genotypes in Neisseria gonorrhoeae. The use of homogeneous FRET probe technology for detection of PCR products provides an opportunity for microbiologists to use molecular detection in a closed system. The necessary specificity and sensitivity of many microbiology tests are achievable using PCR with FRET detection. The technology has become widely available, and configurations of instrumentation and FRET design are available for many applications. Hybridization FRET probes provide great sensitivity and specificity to real-time PCR with the benefit of sensitive detection of nucleic acid sequences with unexpected polymorphisms. The hybridization FRET probes also enable multiple formats for robust multiplexing reactions often with just a single set of primers and probes.

ABSTRACT Using data from 23,313 patients, we assessed whether two blood culture sets of three bottles per set would detect more pathogens than two sets of two bottles per set and achieve similar sensitivity to collecting three sets of two bottles per set. We also compared the yield of aerobic and anaerobic bottles. Thirty milliliters of blood was distributed to one anaerobic and two aerobic bottles. Among 26,855 collections of ≥60 ml within 30 min, 1,379 (5.1%) were positive for a pathogen not requiring detection in more than one set to be considered a pathogen, with 72 additional distinct pathogens detected using two 30-ml compared to two 20-ml sets of one aerobic and one anaerobic bottle (increased yield, 7.9%; 95% confidence interval [CI], 6.2 to 9.8%). For conditional pathogens requiring detection in at least two positive blood cultures for classification as pathogens (i.e., otherwise classified as contaminants), there were 162 positive detections with two 30-ml sets, of which 16 would not have been detected by two 20-ml sets (increased yield, 11.0% [95% CI, 6.4 to 17.2%]). Among 134 subjects who had three sets of 30 ml each within a 30-min interval, there was complete concordance between 60 ml of blood drawn in the first two sets of 30 ml and three 20-ml sets (P = 1.0). One aerobic bottle plus one anaerobic bottle yielded more pathogens than two aerobic bottles for organisms requiring a single (P < 0.001) and two (P = 0.04) positive sets to be defined as pathogens. In conclusion, we showed that collection of two aerobic and one anaerobic blood culture bottles per set results in improved yield compared to two bottles per set. We also confirmed that an anaerobic bottle should be included in blood culture sets.

This chapter contains sections titled: Introduction to Real-Time PCR Real-Time PCR Instrumentation PCR Detection Chemistries Primer and Probe Design Assay Optimization Quality Control and Quality Assurance Real-Time PCR in the Clinical Microbiology Laboratory Costs of Real-Time PCR in the Clinical Microbiology Laboratory Summary References

Data on the burden of disease from tuberculosis (TB) in Filipino households are limited. To determine the magnitude of undiagnosed TB in TB households, and the demographic and socio-economic factors associated with TB in the Philippines, household contacts of adult smear-positive TB patients seen from July 2001 to June 2003 were assessed based on interview, chest X-ray, tuberculin skin test and sputum examination. History of TB and older age were independently associated with TB disease, and age and duration of cohabitation with TB infection. TB and TB infection are highly prevalent in TB households in the Philippines.