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BACKGROUND: This study characterizes the clinical utility and validity of the Karius test (KT), a plasma microbial cell-free DNA sequencing platform, as an infection surveillance tool among hematopoietic stem cell transplant (HCT) recipients, including monitoring for cytomegalovirus (CMV) and detecting infections relative to standard microbiologic testing (SMT). METHODS: A prospective, observational cohort study was performed among adult HCT recipients as inpatients and outpatients. Serial KTs were performed starting with 1 sample within 14 days before HCT, then weekly from 7-63 days posttransplant then monthly from 3-12 months post-HCT. Diagnostic performance of KT versus CMV polymerase chain reaction was evaluated with positive percent agreement and negative percent agreement. Infectious events (

We herein present a multifunctional self-priming hairpin probe-based isothermal amplification, termed MSH, enabling one-pot detection of target nucleic acids. The sophisticatedly designed multifunctional self-priming hairpin (MSH) probe recognizes the target and rearranges to prime itself, triggering the amplification reaction powered by the continuously repeated extension, nicking, and target recycling. As a consequence, a large number of double-stranded DNA (dsDNA) amplicons are produced that could be monitored in real-time using a dsDNA-intercalating dye. Based on this unique design approach, the nucleocapsid (N) and the open reading frame 1 ab (ORF1ab) genes of SARS-CoV-2 were successfully detected down to 1.664 fM and 0.770 fM, respectively. The practical applicability of our method was validated by accurately diagnosing 60 clinical samples with 93.33% sensitivity and 96.67% specificity. This isothermal one-pot MSH technique holds great promise as a point-of-care testing protocol for the reliable detection of a wide spectrum of pathogens, particularly in resource-limited settings.

INTRODUCTION: As malaria declines, low-density malaria infections (LMIs) represent an increasing proportion of infections and may have negative impacts on child health and cognition, necessitating development of targeted and effective solutions. This trial assesses the health, cognitive and socioeconomic impact of two strategies for detecting and treating LMI in a low transmission setting. METHODS AND ANALYSIS: The study is a 3-arm open-label individually randomised controlled trial enrolling 600 children aged 6 months to 10 years in Bagamoyo district, Tanzania. Children are randomised to one of three arms: active case detection with molecular (ACDm) testing by high volume quantitative PCR (qPCR), passive case detection also with molecular testing (PCDm) and a control of standard PCD using rapid diagnostics tests (RDTs). Over the 2-year trial, ACDm participants receive malaria testing using RDT and qPCR three times annually, and malaria testing by RDT only when presenting with fever. PCDm and PCD participants receive malaria testing by RDT and qPCR or RDT only, respectively, when presenting with fever. RDT or qPCR positive participants with uncomplicated malaria are treated with artemether lumefantrine. The primary outcome is cumulative incidence of all-cause sick visits. Secondary outcomes include fever episodes, clinical failure after fever episodes, adverse events, malaria, non-malarial infection, antibiotic use, anaemia, growth faltering, cognition and attention, school outcomes, immune responses, and socioeconomic effects. Outcomes are assessed through monthly clinical assessments and testing, and baseline and endline neurodevelopmental testing. The trial is expected to provide key evidence and inform policy on health, cognitive and socioeconomic impact of interventions targeting LMI in children. ETHICS AND DISSEMINATION: Study is approved by Tanzania NatHREC and institutional review boards at University of California San Francisco and Ifakara Health Institute. Findings will be reported on ClinicalTrials.gov, in peer-reviewed journals and through stakeholder meetings. TRIAL REGISTRATION NUMBER: NCT05567016.

ABO blood group antigens are critical determinants of immunologic self and non-self and are ubiquitously expressed on all cellular tissues. Antibodies against non-self ABO antigens are naturally present and can mediate pathologic reactions against incompatible transfused blood cells and transplanted tissues. Laboratory testing for ABO antigens and isoagglutinins is essential for safe and effective transfusion and transplantation. Testing for ABO antigens has traditionally depended on serologic testing. However, there is increasing need for evaluation of genetic analysis of ABO antigens, to enable evaluation of ABO blood group in cases where serologic testing may be ambiguous or impossible to accurately perform. The clinical need for ABO genotyping is being addressed by the development of multiple molecular diagnostic approaches. Recent data have clearly demonstrated the potential utility of ABO genotyping in solid organ transplantation, yet widespread implementation has been slow. We propose that this lag is related to practical considerations in laboratory testing, including limited regulatory guidance on the performance and reporting of these assays and the absence of widely available external proficiency testing programs for quality assurance. Here we describe approaches to ABO genotyping, current initiatives in developing ABO genotyping proficiency testing programs, and laboratory quality assurance considerations for ABO genotyping.

Antibody-mediated rejection (AMR) is a major cause of graft failure limiting long-term graft survival after kidney transplantation. Current diagnostic strategy to detect AMR is suboptimal and requires further improvement. Previously suggested treatment regimens for AMR could not demonstrate efficacy, however novel therapeutic agents are currently under investigation. Donor-derived cell-free DNA (dd-cfDNA) is a novel noninvasive biomarker for allograft injury, that has been mainly studied in the context of rejection. Its short-half-life in circulation and injury-dependent release are its key advantages that contribute to its superior diagnostic accuracy, compared to traditional biomarkers. Moreover, previous studies showed that dd-cfDNA-release is well-linked to histological and molecular features of AMR, and thus able to reflect real-time injury. Further observations suggest that dd-cfDNA can be used as a suitable screening tool for early detection of AMR in patients with donor-specific-anti-HLA-antibodies (DSA), as well as for monitoring AMR activity after anti-rejection treatment. The weight of evidence suggests that the integration of dd-cfDNA in the graft surveillance of patients with AMR, or those suspicious of AMR (e.g., due to the presence of donor-specific anti-HLA-antibodies) has an added value and might have a positive impact on outcomes in this specific cohort. [ABSTRACT FROM AUTHOR]

Bactrocera tryoni and Bactrocera neohumeralis are morphologically similar sibling pest fruit fly species that possess different biological attributes, geographic distributions, and host ranges. The need to differentiate between the two species is critical for accurate pest status assessment, management, biosecurity, and maintenance of reference colonies. While morphologically similar, adults may be separated based on subtle characters; however, some characters exhibit intraspecific variability, creating overlap between the two species. Additionally, there is currently no single molecular marker or rapid diagnostic assay that can reliably distinguish between B. neohumeralis and B. tryoni; therefore, ambiguous samples remain undiagnosed. Here we report the first molecular marker that can consistently distinguish between B. tryoni and B. neohumeralis. Our diagnostic region consists of two adjacent single nucleotide polymorphisms (SNPs) within the pangolin (pan) gene region. We confirmed the genotypes of each species are consistent across their distributional range, then developed a tetra‐primer amplification refractory mutation system (ARMS) PCR assay for rapid diagnosis of the species. The assay utilizes four primers in multiplex, with two outer universal primers, and two internal primers: one designed to target two adjacent SNPs (AA) present in B. tryoni and the other targeting adjacent SNPs present in B. neohumeralis (GG). The assay accurately discriminates between the two species, but their SNP genotypes are shared with other nontarget tephritid fruit fly species. Therefore, this assay is most suited to adult diagnostics where species confirmation is necessary in determining ambiguous surveillance trap catches; maintaining pure colony lines; and in Sterile Insect Technique management responses. [ABSTRACT FROM AUTHOR]

Background: This study aimed to demonstrate that the genomic material of SARS-CoV-2 can be isolated from strips of COVID-19 rapid diagnostic test cassettes. Method: It was a prospective cross-sectional study involving patients admitted to treatment centers and sampling sites in the city of Conakry, Guinea. A total of 121 patients were double sampled, and 9 more patients were tested only for RDT. PCR was conducted according to the protocol of the RunMei kit. Sequencing was performed by using the illumina COVIDSeq protocol. Nine COVID-19 RDTs without nasopharyngeal swabs were in addition tested. Result: Among the 130 COVID-19 RDTs, forty-seven were macroscopically positive, whereas seventy-two were positive according to PCR using RDT strip, while among the 121 VTM swabs, sixty-four were positive. Among eighty-three negative COVID-19 RDTs, twenty-seven were positive by PCR using RDT strip with a geometric mean Ct value of 32.49 cycles. Compared to those of PCR using VTM, the sensitivity and specificity of PCR using RDT strip were estimated to be 100% and 85.96%, respectively, with 93.39% test accuracy. Among the fifteen COVID-19 RDT extracts eligible for sequencing, eleven had sequences identical to those obtained via the standard method, with coverage between 75 and 99.6%. Conclusion: These results show that COVID-19 RDTs can be used as biological material for the genomic surveillance of SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Simple Summary: Old World bollworm is a moth species that causes significant damage to a wide range of agricultural crops. This species was once confined to the eastern hemisphere but has recently spread throughout South America and the Caribbean and has the potential to establish in North America. Molecular detection methods for Old World bollworm have been developed to track its spread and rapidly differentiate it from the native sibling species, corn earworm. Currently, droplet digital PCR (ddPCR) is a preferred method for bulk screening as it is highly accurate and tolerant of PCR inhibitors; however, real-time PCR is less expensive and more widely available in molecular labs. Improvements to DNA extraction yield and purity are crucial for real-time PCR assay optimization, but these improvements must be time- and cost-efficient. In this study, we improve upon previously published bulk DNA extraction methods by reducing bench time and costly materials. Our results indicate that the addition of caffeine and RNase A improves DNA extraction, resulting in higher amounts of target DNA in real-time PCR. Such improvements will enable the use of high throughput screening methods across multiple platforms to improve the probability of detection of Old World bollworm. Helicoverpa armigera is among the most problematic agricultural pests worldwide due to its polyphagy and ability to evolve pesticide resistance. Molecular detection methods for H. armigera have been developed to track its spread, as such methods allow for rapid and accurate differentiation from the native sibling species H. zea. Droplet digital PCR (ddPCR) is a preferred method for bulk screening due to its accuracy and tolerance to PCR inhibitors; however, real-time PCR is less expensive and more widely available in molecular labs. Improvements to DNA extraction yield, purity, and throughput are crucial for real-time PCR assay optimization. Bulk DNA extractions have recently been improved to where real-time PCR sensitivity can equal that of ddPCR, but these new methods require significant time and specialized equipment. In this study, we improve upon previously published bulk DNA extraction methods by reducing bench time and materials. Our results indicate that the addition of caffeine and RNase A improves DNA extraction, resulting in lower Cq values during real-time PCR while reducing the processing time and cost per specimen. Such improvements will enable the use of high throughput screening methods across multiple platforms to improve the probability of detection of H. armigera. [ABSTRACT FROM AUTHOR]

Cystic fibrosis is caused by biallelic pathogenic variants in the CFTR gene, which contains a polymorphic (TG)mTn sequence (the "poly-T/TG tract") in intron 9. While T9 and T7 alleles are benign, T5 alleles with longer TG repeats, e.g., (TG)12T5 and (TG)13T5, are clinically significant. Thus, professional medical societies currently recommend reporting the TG repeat size when T5 is detected. Sanger sequencing is a cost-effective method of genotyping the (TG)mTn tract; however, its polymorphic length substantially complicates data analysis. We developed CFTR-TIPS, a freely available web-based software tool that infers the (TG)mTn genotype from Sanger sequencing data. This tool detects the (TG)mTn tract in the chromatograms, quantifies goodness of fit with expected patterns, and visualizes the results in a graphical user interface. It is broadly compatible with any Sanger chromatogram that contains the (TG)mTn tract ± 15 bp. We evaluated CFTR-TIPS using 835 clinical samples previously analyzed in a CLIA-certified, CAP-accredited laboratory. When operated fully automatically, CFTR-TIPS achieved 99.8% concordance with our clinically validated manual workflow, while generally taking less than 10 s per sample. There were two discordant samples: one due to a co-occurring heterozygous duplication that confounded the tool and the other due to incomplete (TG)mTn tract detection in the reverse chromatogram. No clinically significant misclassifications were observed. CFTR-TIPS is a free, accurate, and rapid tool for CFTR (TG)mTn tract genotyping using cost-effective Sanger sequencing. This tool is suitable both for automated use and as an aid to manual review to enhance accuracy and reduce analysis time. [ABSTRACT FROM AUTHOR]

Background: Since most of the commonly known oral diseases are explained in link with balance of microbial community, an accurate bacterial taxonomy profiling for determining bacterial compositional network is essential. However, compared to intestinal microbiome, research data pool related to oral microbiome is small, and general 16S rRNA screening method has a taxonomy misclassification issue in confirming complex bacterial composition at the species level. Objective: Present study aimed to explore bacterial compositional networks at the species level within saliva of 39 oral disease patients (Dental Caries group: n = 26 and Periodontitis group: n = 13) through comparison with public Korean-specific healthy oral microbiome data. Methods: Here, we applied comprehensive molecular diagnostics based on qRT-PCR and Sanger sequencing methods to complement the technical limitations of NGS-based 16S V3-V4 amplicon sequencing technology. Results: As a result of microbiome profiling at the genus level, relative frequencies of many nitrate-reducing bacteria within each oral disease group were found to be significantly low compared to the healthy group. In addition, the molecular diagnostics-based bacterial identification method allowed the determination of the correct taxonomy of screened primary colonizers (Streptococcus and Actinomyces unclassification clusters) for each oral disease. Finally, as with the results of microbiome profiling at the genus level, many core-species classified within the saliva of each oral disease group were also related to nitrate-reduction, and it was estimated that various pathogens associated with each disease formed a bacterial network with the core-species. Conclusion: Our study introduced a novel approach that can compensate for the difficulty of identifying an accurate bacterial compositional network at the species level due to unclear taxonomy classification by using the convergent approach of NGS-molecular diagnostics. Ultimately, we suggest that our experimental approach and results could be potential reference materials for researchers who intend to prevent oral disease by determining the correlation between oral health and bacterial compositional network according to the changes in the relative frequency for nitrate-reducing species. [ABSTRACT FROM AUTHOR]

Liquid biopsy (LB) represents an advanced, minimally invasive approach that elevates the precision of oncological decision-making by identifying tumor DNA in bodily fluids. However, despite numerous endorsements from international specialty societies and working groups, implementation of LB into routine care is lagging behind due to conceptual and methodological uncertainties. This concise mini review aims to help catalyzing the translation of LB into routine care by exploring key considerations for incorporating circulating tumor DNA (ctDNA) analysis into clinical practice. Addressing eight pertinent questions from the perspective of a molecular oncology laboratory, this review synthesizes insights from the European Society for Medical Oncology (ESMO) recommendations and incorporates the latest findings from relevant literature, offering a comprehensive guide to the implementation of ctDNA assays. [ABSTRACT FROM AUTHOR]

The advent of immunotherapy with immune checkpoint inhibitors (ICIs) has revolutionized the management of mismatch repair deficient (MMR-d)/microsatellite instability-high (MSI-H) endometrial cancer (EC). Initially investigated as monotherapy in phase I-II clinical trials for recurrent disease, immunotherapy demonstrated remarkable activity, yielding overall response rates (ORR) ranging from 27% to 58%. Based on these promising findings, phase III trials have explored the integration of immunotherapy into first-line treatment regimens for advanced/recurrent EC in combination with chemotherapy or other agents such as tyrosine kinase inhibitors (TKIs), resulting in improved ORR, progression-free survival, and overall survival compared to the standard chemotherapy regimen of paclitaxel and carboplatin. As a result, the incorporation of ICIs with standard platinum-based chemotherapy is becoming a new standard of care in MMR-d/MSI-H EC. This review synthesizes literature from PubMed, Embase databases, and recent congress abstracts on gynecological cancers. It covers MMR-d/MSI-H EC incidence, molecular diagnostics, clinical trial outcomes, predictive biomarkers for ICIs, patient profiles likely to benefit, resistance mechanisms, and the future of immunotherapy in this setting. By offering a comprehensive overview, this review delineates the pivotal role of ICIs in the management of MMR-d/MSI-H EC. [ABSTRACT FROM AUTHOR]

The emergence of targeted therapies in non-small-cell lung cancer (NSCLC), including inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase, has increased the need for robust companion diagnostic tests. Nowadays, detection of actionable variants in exons 18–21 of the EGFR gene by qPCR and direct DNA sequencing is often replaced by next-generation sequencing (NGS). In this study, we evaluated the diagnostic usefulness of targeted NGS for druggable EGFR variants testing in clinical NSCLC material previously analyzed by the IVD-certified qPCR test with respect to DNA reference material. We tested 59 NSCLC tissue and cytology specimens for EGFR variants using the NGS 'TruSight Tumor 15' assay (Illumina) and the qPCR 'cobas EGFR mutation test v2' (Roche Diagnostics). The sensitivity and specificity of targeted NGS assay were evaluated using the biosynthetic and biological DNA reference material with known allelic frequencies (VAF) of EGFR variants. NGS demonstrated a sufficient lower detection limit for diagnostic applications (VAF < 5%) in DNA reference material; all EGFR variants were correctly identified. NGS showed high repeatability of VAF assessment between runs (CV% from 0.02 to 3.98). In clinical material, the overall concordance between NGS and qPCR was 76.14% (Cohen's Kappa = 0.5933). The majority of discordant results concerned false-positive detection of EGFR exon 20 insertions by qPCR. A total of 9 out of 59 (15%) clinical samples showed discordant results for one or more EGFR variants in both assays. Additionally, we observed TP53 to be a frequently co-mutated gene in EGFR-positive NSCLC patients. In conclusion, targeted NGS showed a number of superior features over qPCR in EGFR variant detection (exact identification of variants, calculation of allelic frequency, high analytical sensitivity), which might enhance the basic diagnostic report. [ABSTRACT FROM AUTHOR]

Abnormal uterine bleeding (AUB) is a common gynecological complaint among premenopausal women, encompassing a wide range of underlying disorders that complicate diagnosis and management. The evolving paradigms in medical science now incorporate advanced imaging techniques, personalized medicine, and molecular diagnostics to improve the accuracy of diagnoses and the effectiveness of treatment plans. This review examines recent advancements in the diagnostic approach, including the use of transvaginal ultrasonography, hysteroscopy, and biomarker analysis, which have significantly refined the identification of endometrial pathologies. Furthermore, we discuss the shift towards individualized treatment strategies that consider patient-specific factors such as age, reproductive plans, and comorbidities, facilitating tailored therapies. Special attention is given to the role of medical therapies ranging from hormonal treatments to novel non-hormonal drugs, as well as the consideration of minimally invasive surgical options as part of a comprehensive management strategy. By integrating current research findings with clinical practice guidelines, this article aims to provide a synthesized view of the dynamic field of AUB management, proposing a multidisciplinary approach to enhance patient outcomes in premenopausal women. [ABSTRACT FROM AUTHOR]

Invasive fungal diseases (IFDs) comprise a growing healthcare burden, especially given the expanding population of immunocompromised hosts. Early diagnosis of IFDs is required to optimise therapy with antifungals, especially in the setting of rising rates of antifungal resistance. Molecular techniques including nucleic acid amplification tests and whole genome sequencing have potential to offer utility in overcoming limitations with traditional phenotypic testing. However, standardisation of methodology and interpretations of these assays is an ongoing undertaking. The utility of targeted Aspergillus detection has been well-defined, with progress in investigations into the role of targeted assays for Candida, Pneumocystis, Cryptococcus, the Mucorales and endemic mycoses. Likewise, whilst broad-range polymerase chain reaction assays have been in use for some time, pathology stewardship and optimising diagnostic yield is a continuing exercise. As costs decrease, there is also now increased access and experience with whole genome sequencing, including metagenomic sequencing, which offers unparalleled resolution especially in the investigations of potential outbreaks. However, their role in routine diagnostic use remains uncommon and standardisation of techniques and workflow are required for wider implementation. [ABSTRACT FROM AUTHOR]

Simple Summary: Head and neck squamous cell carcinoma (HNSCC) remains a global health concern due to the lack of precise early diagnostic biomarkers and often-delayed diagnosis. This study employs machine learning, weighted gene co-expression network analysis, and network biology to identify transcriptomic markers for HNSCC detection. We identified nine genes with significantly differentially expressed activity in samples from HNSCC patients. These gene signatures could greatly improve early HNSCC identification and warrant further investigation to confirm their predictive and therapeutic significance. The transcriptional landscape of platelets in head and neck cancer patients revealed distinct gene expression profiles compared to healthy controls, underscoring the systemic impact of the tumor on blood platelets. Additionally, the study emphasizes the role of tumor-educated platelets (TEPs), which carry RNA signatures indicative of tumor-derived material, offering a non-invasive source for early-detection biomarkers. Although there has been a reduction in head and neck squamous cell carcinoma occurrence, it continues to be a serious global health concern. The lack of precise early diagnostic biomarkers and postponed diagnosis in the later stages are notable constraints that contribute to poor survival rates and emphasize the need for innovative diagnostic methods. In this study, we employed machine learning alongside weighted gene co-expression network analysis (WGCNA) and network biology to investigate the gene expression patterns of blood platelets, identifying transcriptomic markers for HNSCC diagnosis. Our comprehensive examination of publicly available gene expression datasets revealed nine genes with significantly elevated expression in samples from individuals diagnosed with HNSCC. These potential diagnostic markers were further assessed using TCGA and GTEx datasets, demonstrating high accuracy in distinguishing between HNSCC and non-cancerous samples. The findings indicate that these gene signatures could revolutionize early HNSCC identification. Additionally, the study highlights the significance of tumor-educated platelets (TEPs), which carry RNA signatures indicative of tumor-derived material, offering a non-invasive source for early-detection biomarkers. Despite using platelet and tumor samples from different individuals, our results suggest that TEPs reflect the transcriptomic and epigenetic landscape of tumors. Future research should aim to directly correlate tumor and platelet samples from the same patients to further elucidate this relationship. This study underscores the potential of these biomarkers in transforming early diagnosis and personalized treatment strategies for HNSCC, advocating for further research to validate their predictive and therapeutic potential. [ABSTRACT FROM AUTHOR]

Purpose of review: Melanoma in younger individuals has different clinical presentations, histologic characteristics and prognosis from older patients. This review summarizes key differences and important new insights into pediatric and young adult melanoma, as well as recent evolutions in treatment. Recent findings: Molecular techniques have improved the classification of melanocytic neoplasms, and are especially useful in the workup of the diagnostically challenging lesions frequent in this age group. Molecular evaluation highlights differences between melanoma and atypical lesions with Spitz-like morphology, and should routinely be incorporated for diagnosing and classifying Spitzoid melanocytic to guide prognostication and treatment. Once diagnosed, the management of bona fide melanoma in children and young adults is largely similar to older patients, while the optimal management of lesions such as atypical Spitz tumors remains uncertain. Summary: Increased awareness of the presentation and diagnostic characteristics of melanoma in young individuals will allow earlier detection, and improved diagnostic techniques will allow optimum management without over- or under-treatment. [ABSTRACT FROM AUTHOR]

Molecular diagnostics has the potential to revolutionise the field of clinical microbiology. Microbial identification and nomenclature have, for too long, been restricted to phenotypic characterisation. However, this species-level view fails to wholly account for genetic heterogeneity, a result of lateral gene transfer, mediated primarily by mobile genetic elements. This genetic promiscuity has helped to drive virulence development, stress adaptation, and antimicrobial resistance in several important bacterial pathogens, complicating their detection and frustrating our ability to control them. We argue that, as clinical microbiologists at the front line, we must embrace the molecular technologies that allow us to focus specifically on the genetic elements that cause disease rather than the bacterial species that express them. This review focuses on the evolution of microbial taxonomy since the introduction of molecular sequencing, the role of mobile genetic elements in antimicrobial resistance, the current and emerging assays in clinical laboratories, and the comparison of phenotypic versus genotypic analyses. In essence, it is time now to refocus from species to genes as part of a new diagnostic paradigm. [ABSTRACT FROM AUTHOR]

Diagnostic development must occur in parallel with drug development to ensure the longevity of new treatment compounds. Despite an increasing number of novel and repurposed anti-tuberculosis compounds and regimens, there remains a large number of drugs for which no rapid and accurate molecular diagnostic option exists. The lack of rapid drug susceptibility testing for linezolid, bedaquiline, clofazimine, the nitroimidazoles (i.e pretomanid and delamanid) and pyrazinamide at any level of the healthcare system compromises the effectiveness of current tuberculosis and drug-resistant tuberculosis treatment regimens. In the context of current WHO tuberculosis treatment guidelines as well as promising new regimens, we identify the key diagnostic gaps for initial and follow-on tests to diagnose emerging drug resistance and aid in regimen selection. Additionally, we comment on potential gene targets for inclusion in rapid molecular drug susceptibility assays and sequencing assays for novel and repurposed drug compounds currently prioritized in current regimens, and evaluate the feasibility of mutation detection given the design of existing technologies. Based on current knowledge, we also propose design priorities for next generation molecular assays to support triage of tuberculosis patients to appropriate and effective treatment regimens. We encourage assay developers to prioritize development of these key molecular assays and support the continued evolution, uptake, and utility of sequencing to build knowledge of tuberculosis resistance mechanisms and further inform rapid treatment decisions in order to curb resistance to critical drugs in current regimens and achieve End TB targets.Trial registration: ClinicalTrials.gov identifier: NCT05117788..

GeneXpert MTB/RIF, a tool widely used for diagnosing tuberculosis, has limitations for detecting rifampin resistance in certain variants. We report transmission of a pre-extensively drug-resistant variant in Botswana that went undetected by GeneXpert. The public health impact of misdiagnosis emphasizes the need for comprehensive molecular testing to identify resistance and guide treatment.

Objective: To establish Nipah virus diagnostic capabilities at the National Reference Laboratory in Sri Lanka using the NIV Pune real-time PCR kit. Methods: Strict safety precautions were adhered during testing due to the high pathogenicity of the Nipah virus, with all diagnostics conducted in a BSL2+ laboratory at the Medical Research Institute in Sri Lanka. RNA extraction was performed using the QIAamp Viral RNA Mini kit. The NIV Pune in-house real-time PCR kit was employed, following established primer/probe sequences and controls. The assay was validated using the Rotor-Gene Q Series Real-time PCR platform. Results: The validation run of the Nipah virus real-time PCR test demonstrated robust performance, with positive controls consistently detecting Nipah RNA at a Ct value of 21.50±0.01. Negative controls confirmed assay specificity with an external negative control which was also used as an extraction control and showed no interference. The internal control exhibited stable behavior, enhancing confidence in PCR results. The qPCR analysis graph illustrated the successful detection of internal and positive controls, validating the reliability of the assay. Conclusions: Establishing Nipah virus diagnostic capabilities in Sri Lanka signifies a proactive and collaborative response to the persistent global health threat.

Abstract Background This study aimed to demonstrate that the genomic material of SARS-CoV-2 can be isolated from strips of COVID-19 rapid diagnostic test cassettes. Method It was a prospective cross-sectional study involving patients admitted to treatment centers and sampling sites in the city of Conakry, Guinea. A total of 121 patients were double sampled, and 9 more patients were tested only for RDT. PCR was conducted according to the protocol of the RunMei kit. Sequencing was performed by using the illumina COVIDSeq protocol. Nine COVID-19 RDTs without nasopharyngeal swabs were in addition tested. Result Among the 130 COVID-19 RDTs, forty-seven were macroscopically positive, whereas seventy-two were positive according to PCR using RDT strip, while among the 121 VTM swabs, sixty-four were positive. Among eighty-three negative COVID-19 RDTs, twenty-seven were positive by PCR using RDT strip with a geometric mean Ct value of 32.49 cycles. Compared to those of PCR using VTM, the sensitivity and specificity of PCR using RDT strip were estimated to be 100% and 85.96%, respectively, with 93.39% test accuracy. Among the fifteen COVID-19 RDT extracts eligible for sequencing, eleven had sequences identical to those obtained via the standard method, with coverage between 75 and 99.6%. Conclusion These results show that COVID-19 RDTs can be used as biological material for the genomic surveillance of SARS-CoV-2.

The intent of this review is to present basic genetic concepts key to understanding oncogenesis and the role receptor tyrosine kinase (RTK) inhibition plays in the targeted treatment of many cancer types. Oncogenic signaling by RTKs can result from genetic events such as point mutations, chromosomal rearrangements, structural variation, and gene amplification in the cancer genome. The cancer pharmacogenes discussed encode RTKs that exemplify the link between gene variation, the oncogenic process, and the basis of targeted approaches to treatment. Biochemical pathways often involved in oncogenesis and affected by RTK variation are reviewed. Molecular diagnostic testing for the presence of specific gene variants, alterations, and amplifications direct therapy to indicated tyrosine kinase inhibitors and monoclonal antibody drugs. As pharmacists are integral to the selection, preparation, and monitoring of chemotherapy, it is important that they understand the genetic basis for targeted therapies as well as the underlying disease process.

Innovations in infectious disease testing have improved our abilities to detect and understand the microbial world. The 2019 novel coronavirus infectious disease (COVID-19) pandemic introduced new innovations including non-prescription "over the counter" infectious disease tests, mass spectrometry-based detection of COVID-19 host response, and the implementation of artificial intelligence (AI) and machine learning (ML) to identify individuals infected by the severe acute respiratory syndrome - coronavirus - 2 (SARS-CoV-2). As the world recovers from the COVID-19 pandemic; these innovative solutions will give rise to a new era of infectious disease tests extending beyond the detection of SARS-CoV-2. To this end, the purpose of this review is to summarize current trends in infectious disease testing and discuss innovative applications specifically in the areas of POC testing, MS, molecular diagnostics, sample types, and AI/ML.

Hairy cell leukemia (HCL) is an uncommon mature B-cell malignancy characterized by a typical morphology, immunophenotype, and clinical profile. The vast majority of HCL patients harbor the canonical BRAF V600E mutation which has become a rationalized target of the subsequently deregulated RAS-RAF-MEK-MAPK signaling pathway in HCL patients who have relapsed or who are refractory to front-line therapy. However, several HCL patients with a classical phenotype display non-canonical BRAF mutations or rearrangements. These include sequence variants within alternative exons and an oncogenic fusion with the IGH gene. Care must be taken in the molecular diagnostic work-up of patients with typical HCL but without the BRAF V600E to include investigation of these uncommon mechanisms. Identification, functional characterization, and reporting of further such patients is likely to provide insights into the pathogenesis of HCL and enable rational selection of targeted inhibitors in such patients if required. [ABSTRACT FROM AUTHOR]

Relevance. The problem of diagnosing hereditary neuromuscular diseases is one of the most difficult in the medical specialists’ practice. Molecular genetic diagnostics is one of the fundamental aspects in the classification and subsequent approaches to the treatment and prevention of hereditary diseases. Pathogenic variants identification leads to the formation of separate subtypes and phenotypically identical diseases syndromes. This review examines modern diagnostic methods and algorithmization of patients with neuromuscular diseases. Despite enormous research and clinical efforts, the molecular causes remain unknown for almost half of patients with neuromuscular diseases due to genetic heterogeneity and molecular diagnostics based on a gene-by-gene approach. Next-generation sequencing (NGS) is an effective and cost-effective strategy for accelerating patient diagnosis. However, the diagnostic value of conducting and prescribing whole- exome or whole- genome sequencing is largely dependent on the clinical picture of the disease and the professional competence of the doctor. Hereditary neuromuscular diseases have similar initial symptoms, and molecular genetic diagnostics can pinpoint the cause and pathogenesis of the observed disorders in the patient. Conclusion . The molecular diagnostics algorithm is based on sequential analysis, starting with the search for the most common pathogenic variants using inexpensive and rapid methods, and progressing to the search for rare, previously undescribed pathogenic variants using whole-g enome/whole-exome studies. The phasing allows science and medicine to uncover previously unknown causes of severe disease in patients with neuromuscular diseases, which often leading to disability or premature death. Earlier genetic diagnosis should provide more effective treatment of the disease and better genetic counseling for families and will also allow access to pathogenetic therapy for neuromuscular diseases.

Abstract Background Development of sequential changes of mucous leading to gastric cancer and familial cases of gastric cancer of intestinal type is widely connected with Helicobacter pylori infections. In this study we analysed variants of genes involved in cancerogenesis and inflammatory processes of intestines in patients infected with H.pylori. Our goal was to test whether mutations in these genes predestinate to development of gastric cancer, and whether there is a genetic factor that makes it more likely for infections with H.pylori to cause gastric cancer. As infections with H. pylori are relatively common, discovering such genetic predispositions could be used for establishing risk-groups and for planning treatments. Methods Our studies cover analysis of variants in genes involved in cancerogenesis: TP53 (rs11540652, rs587782329, COSM10771), MSH2 (rs193922376), MLH1 (rs63750217), and inflammatory processes of intestine: NOD2 (rs2066847, rs2066842), IL1A (rs1800587) and IL1B (rs1143634) from H.pylori-infected patients. Results Mutations were more common in the group of patients with gastric cancer of intestinal type and familial cases of gastric cancer in comparison with patients with chronic gastritis, chronic atrophic gastritis, intestinal metaplasia, dysplasia or gastric cancer (p-value = 0.00824), with the prevalence of p53 mutations in patients with familial gastric cancer vs. patients with other changes of mucosa (p-value = 0.000049). Additionally, gastric cancer patients have mainly genotype TT or CT of the rs2066842 variant of the NOD2 gene. Conclusions The lack of statistically significant changes of other interleukin genes involved in inflammatory processes may suggest the presence of H.pylori infection as a potential trigger for the development of the inflammatory process of the mucosa, leading through microbiota dysbiosis to the development of enteric gastric cancer. Mutations in analysed genes correlated with more severe mucosal changes, with a much more frequent presence of TP53 gene mutations, with a limited presence of other mutations in the familial history of gastric cancer.

Alzheimer’s disease (AD) is the world’s most common neurodegenerative disease, expected to affect up to one-third of the elderly population in the near future. Among the major challenges in combating AD are the inability to reverse the damage caused by the disease, expensive diagnostic tools, and the lack of specific markers for the early detection of AD. This paper highlights promising research directions for molecular markers in AD diagnosis, including the diagnostic potential of microRNAs. The latest molecular methods for diagnosing AD are discussed, with particular emphasis on diagnostic techniques prior to the appearance of full AD symptoms and markers detectable in human body fluids. A collection of recent studies demonstrates the promising potential of molecular methods in AD diagnosis, using miRNAs as biomarkers. Up- or downregulation in neurodegenerative diseases may not only provide a new diagnostic tool but also serve as a marker for differentiating neurodegenerative diseases. However, further research in this direction is needed.

AbstractFusarium and Neocosmospora are two fungal genera recently recognized in the list of fungal priority pathogens. They cause a wide range of diseases that affect humans, animals, and plants. In clinical laboratories, there is increasing concern about diagnosis due to limitations in sample collection and morphological identification. Despite the advances in molecular diagnosis, due to the cost, some countries cannot implement these methodologies. However, recent changes in taxonomy and intrinsic resistance to antifungals reveal the necessity of accurate species-level identification. In this review, we discuss the current phenotypic and molecular tools available for diagnosis in clinical laboratory settings and their advantages and disadvantages. [ABSTRACT FROM AUTHOR]

Brain tumors and genomics have a long-standing history given that glioblastoma was the first cancer studied by the cancer genome atlas. The numerous and continuous advances through the decades in sequencing technologies have aided in the advanced molecular characterization of brain tumors for diagnosis, prognosis, and treatment. Since the implementation of molecular biomarkers by the WHO CNS in 2016, the genomics of brain tumors has been integrated into diagnostic criteria. Long-read sequencing, also known as third generation sequencing, is an emerging technique that allows for the sequencing of longer DNA segments leading to improved detection of structural variants and epigenetics. These capabilities are opening a way for better characterization of brain tumors. Here, we present a comprehensive summary of the state of the art of third-generation sequencing in the application for brain tumor diagnosis, prognosis, and treatment. We discuss the advantages and potential new implementations of long-read sequencing into clinical paradigms for neurooncology patients. [ABSTRACT FROM AUTHOR]

The purpose of this study was to assess the added diagnostic value of whole genome sequencing (WGS) for patients with inherited retinal diseases (IRDs) who remained undiagnosed after whole exome sequencing (WES). WGS was performed for index patients in 66 families. The datasets were analyzed according to GATK's guidelines. Additionally, DeepVariant was complemented by GATK's workflow, and a novel structural variant pipeline was developed. Overall, a molecular diagnosis was established in 19/66 (28.8%) index patients. Pathogenic deletions and one deep-intronic variant contributed to the diagnostic yield in 4/19 and 1/19 index patients, respectively. The remaining diagnoses (14/19) were attributed to exonic variants that were missed during WES analysis due to bioinformatic limitations, newly described loci, or unclear pathogenicity. The added diagnostic value of WGS equals 5/66 (9.6%) for our cohort, which is comparable to previous studies. This figure would decrease further to 1/66 (1.5%) with a standardized and reliable copy number variant workflow during WES analysis. Given the higher costs and limited added value, the implementation of WGS as a first-tier assay for inherited eye disorders in a diagnostic laboratory remains untimely. Instead, progress in bioinformatic tools and communication between diagnostic and clinical teams have the potential to ameliorate diagnostic yields. [ABSTRACT FROM AUTHOR]

Background: Development of sequential changes of mucous leading to gastric cancer and familial cases of gastric cancer of intestinal type is widely connected with Helicobacter pylori infections. In this study we analysed variants of genes involved in cancerogenesis and inflammatory processes of intestines in patients infected with H.pylori. Our goal was to test whether mutations in these genes predestinate to development of gastric cancer, and whether there is a genetic factor that makes it more likely for infections with H.pylori to cause gastric cancer. As infections with H. pylori are relatively common, discovering such genetic predispositions could be used for establishing risk-groups and for planning treatments. Methods: Our studies cover analysis of variants in genes involved in cancerogenesis: TP53 (rs11540652, rs587782329, COSM10771), MSH2 (rs193922376), MLH1 (rs63750217), and inflammatory processes of intestine: NOD2 (rs2066847, rs2066842), IL1A (rs1800587) and IL1B (rs1143634) from H.pylori-infected patients. Results: Mutations were more common in the group of patients with gastric cancer of intestinal type and familial cases of gastric cancer in comparison with patients with chronic gastritis, chronic atrophic gastritis, intestinal metaplasia, dysplasia or gastric cancer (p-value = 0.00824), with the prevalence of p53 mutations in patients with familial gastric cancer vs. patients with other changes of mucosa (p-value = 0.000049). Additionally, gastric cancer patients have mainly genotype TT or CT of the rs2066842 variant of the NOD2 gene. Conclusions: The lack of statistically significant changes of other interleukin genes involved in inflammatory processes may suggest the presence of H.pylori infection as a potential trigger for the development of the inflammatory process of the mucosa, leading through microbiota dysbiosis to the development of enteric gastric cancer. Mutations in analysed genes correlated with more severe mucosal changes, with a much more frequent presence of TP53 gene mutations, with a limited presence of other mutations in the familial history of gastric cancer. [ABSTRACT FROM AUTHOR]

Digital PCR (dPCR) holds immense potential for precisely detecting nucleic acid markers essential for personalized medicine. However, its broader application is hindered by high consumable costs, complex procedures, and restricted multiplexing capabilities. To address these challenges, an all‐in‐one dPCR system is introduced that eliminates the need for microfabricated chips, offering fully automated operations and enhanced multiplexing capabilities. Using this innovative oscillation‐induced droplet generation technique, OsciDrop, this system supports a comprehensive dPCR workflow, including precise liquid handling, pipette‐based droplet printing, in situ thermocycling, multicolor fluorescence imaging, and machine learning‐driven analysis. The system's reliability is demonstrated by quantifying reference materials and evaluating HER2 copy number variation in breast cancer. Its multiplexing capability is showcased with a quadruplex dPCR assay that detects key EGFR mutations, including 19Del, L858R, and T790M in lung cancer. Moreover, the digital stepwise melting analysis (dSMA) technique is introduced, enabling high‐multiplex profiling of seven major EGFR variants spanning 35 subtypes. This innovative dPCR system presents a cost‐effective and versatile alternative, overcoming existing limitations and paving the way for transformative advances in precision diagnostics. [ABSTRACT FROM AUTHOR]

The development and performance of two mass spectrometry (MS) workflows for the intraoperative diagnosis of isocitrate dehydrogenase (IDH) mutations in glioma is implemented by independent teams at Mayo Clinic, Jacksonville, and Huashan Hospital, Shanghai. The infiltrative nature of gliomas makes rapid diagnosis necessary to guide the extent of surgical resection of central nervous system (CNS) tumors. The combination of tissue biopsy and MS analysis used here satisfies this requirement. The key feature of both described methods is the use of tandem MS to measure the oncometabolite 2-hydroxyglutarate (2HG) relative to endogenous glutamate (Glu) to characterize the presence of mutant tumor. The experiments i) provide IDH mutation status for individual patients and ii) demonstrate a strong correlation of 2HG signals with tumor infiltration. The measured ratio of 2HG to Glu correlates with IDH-mutant (IDH-mut) glioma (P < 0.0001) in the tumor core data of both teams. Despite using different ionization methods and different mass spectrometers, comparable performance in determining IDH mutations from core tumor biopsies was achieved with sensitivities, specificities, and accuracies all at 100%. None of the 31 patients at Mayo Clinic or the 74 patients at Huashan Hospital were misclassified when analyzing tumor core biopsies. Robustness of the methodology was evaluated by postoperative re-examination of samples. Both teams noted the presence of high concentrations of 2HG at surgical margins, supporting future use of intraoperative MS to monitor for clean surgical margins. The power of MS diagnostics is shown in resolving contradictory clinical features, e.g., in distinguishing gliosis from IDH-mut glioma. [ABSTRACT FROM AUTHOR]

Molecular diagnostics is an excellent tool used by healthcare management for infectious diseases to detect and manage infections. As such, many researchers have addressed the ideas surrounding the molecular diagnosis. This review explores the role of molecular diagnostics in managing and diagnosing infectious diseases, highlighting its potential to prevent catastrophic outcomes. The literature surrounding the historical aspects and the importance of molecular diagnostics in modern healthcare can help explain the role of the tool and how it can improve the patient's outcome and help the therapeutic decisions. In this literature review, databases are searched using relevant keywords to gather information on the role of molecular diagnostics in managing and diagnosing infectious diseases. The review highlights the significance of molecular diagnostics in detecting resistant strains and guiding evidence-based antimicrobial treatment to combat antimicrobial resistance. Furthermore, the review discusses how adopting new diagnostic approaches as standard practice in clinical departments introduces various subjects. The findings demonstrate that implementing molecular diagnostics in clinical settings reduces mortality rates, improves healthcare efficiency, and enhances public surveillance. [ABSTRACT FROM AUTHOR]

Xylella fastidiosa is a plant‐pathogenic bacterium that poses a serious threat to the production of economically important plant species including grapes, almonds, olives and a broad range of amenity plants, causing significant economic losses worldwide. While multiple molecular detection assays have been developed for X. fastidiosa, there is a lack of molecular tools available for detection and differentiation of the closely related pear pathogen, Xylella taiwanensis. In this study, we present a novel conventional PCR assay with primers that can amplify both Xylella species. The amplified product could be sequenced and used for discrimination between the two species and the subspecies within the fastidiosa species. This PCR assay was designed using a genome‐informed approach to target the ComEC/Rec2 gene of both Xylella species, ensuring a higher specificity than other previously developed PCR assays. A test performance study across five national plant diagnostic laboratories in Australia and New Zealand demonstrated this assay's high sensitivity and specificity to all known species and subspecies within the Xylella genus. This PCR assay can be used for Xylella identification at the species and subspecies level and is compatible with Sanger sequencing and nanopore sequencing for rapid turnaround time. The newly developed conventional PCR assay presented here offers rapid detection and accurate identification of both Xylella species from plant, insect vector or bacterial samples, enabling timely implementation of biosecurity measures or disease management responses. [ABSTRACT FROM AUTHOR]

During the last decade, molecular diagnostics has become increasingly important, especially during the SARS‐CoV‐2 pandemic. Advances in droplet‐based microfluidics, which divides samples into thousands of microdroplets, are responsible for high‐throughput data provision, increased analysis sensitivity, and improved performance to overcome challenges in controlling diseases and early diagnosis. Different targets, such as nucleic acids, proteins, or single cells, can be detected and analyzed in terms of their characteristics in a much more extensive and precise manner with the integration of droplet microfluidics into various molecular diagnostic techniques. In this review, we explore recent advances in the development of droplet microfluidics‐based molecular diagnostic techniques and future perspectives in the field. [ABSTRACT FROM AUTHOR]

Young onset breast cancer (YOBC) is an increasing demographic with unique biology, limited screening, and poor outcomes. Further, women with postpartum breast cancers (PPBCs), cancers occurring up to 10 years after childbirth, have worse outcomes than other young breast cancer patients matched for tumor stage and subtype. Early-stage detection of YOBC is critical for improving outcomes. However, most young women (under 45) do not meet current age guidelines for routine mammographic screening and are thus an underserved population. Other challenges to early detection in this population include reduced performance of standard of care mammography and reduced awareness. Women often face significant barriers in accessing health care during the postpartum period and disadvantaged communities face compounding barriers due to systemic health care inequities. Blood tests and liquid biopsies targeting early detection may provide an attractive option to help address these challenges. Test development in this area includes understanding of the unique biology involved in YOBC and in particular PPBCs that tend to be more aggressive and deadly. In this review, we will present the status of breast cancer screening and detection in young women, provide a summary of some unique biological features of YOBC, and discuss the potential for blood tests and liquid biopsy platforms to address current shortcomings in timely, equitable detection. [ABSTRACT FROM AUTHOR]

Introduction: Tissue-based broad molecular profiling of guideline-recommended biomarkers is advised for the therapeutic management of patients with non-small cell lung cancer (NSCLC). However, practice variation can affect whether all indicated biomarkers are tested. We aimed to evaluate the impact of common single-gene testing (SGT) on subsequent comprehensive genomic profiling (CGP) test outcomes and results in NSCLC. Methods: Oncologists who ordered SGT for guideline-recommended biomarkers in NSCLC patients were prospectively contacted (May–December 2022) and offered CGP (DNA and RNA sequencing), either following receipt of negative SGT findings, or instead of SGT for each patient. We describe SGT patterns and compare CGP completion rates, turnaround time, and recommended biomarker detection for NSCLC patients with and without prior negative SGT results. Results: Oncologists in > 80 community practices ordered CGP for 561 NSCLC patients; 135 patients (27%) first had negative results from 30 different SGT combinations; 84% included ALK, EGFR and PD-L1, while only 3% of orders included all available SGTs for guideline-recommended genes. Among patients with negative SGT results, CGP was attempted using the same tissue specimen 90% of the time. There were also significantly more CGP order cancellations due to tissue insufficiency (17% vs. 7%), DNA sequencing failures (13% vs. 8%), and turnaround time > 14 days (62% vs. 29%) than among patients who only had CGP. Forty-six percent of patients with negative prior SGT had positive CGP results for recommended biomarkers, including targetable genomic variants in genes beyond ALK and EGFR, such as ERBB2, KRAS (non-G12C), MET (exon 14 skipping), NTRK2/3, and RET. Conclusion: For patients with NSCLC, initial use of SGT increases subsequent CGP test cancellations, turnaround time, and the likelihood of incomplete molecular profiling for guideline-recommended biomarkers due to tissue insufficiency. Plain Language Summary: Patients with non-small cell lung cancer (NSCLC) should have their tumor tissue tested for all recommended biomarkers that can help identify their best treatment options. Traditional tests look at gene biomarkers one by one (single-gene testing), and doctors can order some or all these tests individually or in a group. However, some recommended biomarkers cannot be tested by traditional single-gene tests at all. Newer technology (next-generation sequencing) covers all current recommended treatment biomarkers in one test (comprehensive genomic profiling), but this testing is more expensive and can take more time. Our study shows that NSCLC patients do not get all recommended treatment biomarkers tested when a single-gene testing approach is taken. Single-gene testing also used up some patients' tumor tissue entirely, such that further testing by comprehensive genomic profiling could not be done at all (17% vs. 7% for patients with no prior single-gene tests), resulted in more sequencing failures (13% vs. 8%), and had turnaround time for results greater than 14 days for more patients (62% vs. 29%). When comprehensive genomic profiling was completed, 46% of patients with negative results from prior single-gene testing had positive results for recommended treatment biomarkers that were not included in the initial single-gene tests. To ensure that NSCLC patients receive testing for all recommended biomarkers, comprehensive genomic profiling must be performed first. [ABSTRACT FROM AUTHOR]

Fasciola hepatica has a complex lifecycle with multiple intermediate and definitive hosts and influenced by environmental factors. The disease causes significant morbidity in children and its prevalent worldwide. There is lack of data about distribution and burden of the disease in endemic regions, owing to poor efficacy of the different diagnostic methods used. A novel PCR-based test was developed by using a portable mini-PCR® platform to detect Fasciola sp. DNA and interpret the results via a fluorescence viewer and smartphone image analyzer application. Human stool, snail tissue, and water samples were used to extract DNA. Primers targeting the ITS-1 of the 18S rDNA gene of Fasciola sp. were used. The limit of detection of the mini-PCR test was 1 fg/μL for DNA samples diluted in water, 10 fg/μL for Fasciola/snail DNA scramble, and 100 fg/μL for Fasciola/stool DNA scramble. The product detection by agarose gel, direct visualization, and image analyses showed the same sensitivity. The Fh mini-PCR had a sensitivity and specificity equivalent to real-time PCR using the same specimens. Testing was also done on infected human stool and snail tissue successfully. These experiments demonstrated that Fh mini-PCR is as sensitive and specific as real time PCR but without the use of expensive equipment and laboratory facilities. Further testing of multiple specimens with natural infection will provide evidence for feasibility of deployment to resource constrained laboratories. [ABSTRACT FROM AUTHOR]

Persistent infection with high‐risk human papillomavirus (HPV) is recognized as the main cause for the development of anogenital cancers. This study prospectively evaluated the diagnostic performance of the novel Allplex‐HPV28 assay with the Anyplex‐II‐HPV28 to detect and genotype HPV in 234 consecutive swabs and 32 biopsies of the anogenital tract from 265 patients with atypical findings in cytomorphological screening. Agreement in HPV‐DNA detection between the Anyplex‐II and Allplex‐HPV28 assays was 99%. There was a notable diversity in the HPV‐virome, with the most prevalent high‐risk HPV types being 16, 53, 66, and 68. The agreement rates for detecting these genotypes exceeded 93% between the Anyplex‐II and Allplex‐HPV28 assays. Discrepancies in test results were solely noted for Anyplex‐II‐HPV28 results with a low signal intensity of "+", and for Allplex‐HPV28 results with cycle thresholds of ≥36. The semi‐quantitative analysis of HPV‐DNA loads showed significant agreement between the Anyplex‐II‐HPV28 and Allplex‐HPV28 assays (p < 0.001). Furthermore, HPV‐DNA detection rates and mean HPV‐DNA loads significantly correlated with the grade of abnormal changes identified in cytopathological assessment, being highest in cases of HSIL, condyloma accuminatum, and squamous cell carcinoma. Overall agreement rates for detecting specific HPV‐types among the Anyplex‐II and Allplex‐HPV28 assays exceeded 99.5% in cases of atypical squamous cells, condyloma accuminatum, and squamous cell carcinoma. The novel Allplex‐HPV28 assay shows good diagnostic performance in detecting and genotyping HPV commonly associated with anogenital cancers. Consequently, this assay could offer substantial potential for incorporation into future molecular screening programs for anogenital cancers in clinical settings. [ABSTRACT FROM AUTHOR]

Classification of tumors of the head and neck has evolved in recent decades including a widespread application of molecular testing in tumors of the sinonasal tract, salivary glands, and soft tissues with a predilection for the head and neck. The availability of new molecular techniques has allowed for the definition of multiple novel tumor types unique to head and neck sites. Moreover, an expanding spectrum of immunohistochemical markers specific to genetic alterations facilitates rapid identification of diagnostic molecular abnormalities. As such, it is currently possible for head and neck pathologists to benefit from a molecularly defined tumor classification while making diagnoses that are still based largely on histopathology and immunohistochemistry. This review covers the principal molecular alterations in sinonasal malignancies, such as alterations in DEK, AFF2, NUTM1, IDH1-2, and SWI/SNF genes in particular, that are important from a practical standpoint for diagnosis, prognosis, and prediction of response to treatment. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Pediatric Infection / Çocuk Enfeksiyon Dergisi is the property of Journal of Pediatric Infection / Cocuk Enfeksiyon Dergisi and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Die Dermatologie is the property of Springer Nature and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Die Dermatologie is the property of Springer Nature and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Background: Ukraine remains a high World Health Organization priority country for drug-resistant tuberculosis (TB). Rifampicin-resistant TB (RR-TB) has a more protracted, more complicated, and more expensive treatment. In 2021, Ukraine reported 4025 RR-TB cases – 5.4 times more (751) than all 30 European Union/ European Economic Area countries together. Objectives: The objective of the study was to determine the diagnostic accuracy of line probe assay (LPA), AID Autoimmun Diagnostika GmbH, for detecting resistance to anti-TB drugs and its clinical application for selecting treatment regimens. Design: A prospective observational cohort study. Methods: From May 2019 to June 2020, we consecutively enrolled patients with active TB hospitalized at the Regional Phthisiopulmonology Center (Vinnytsia, Ukraine), aged between 18 and 82 years. The LPA was performed in the Genetic Research Laboratory at National Pirogov Memorial Medical University, Vinnytsia, Ukraine. Results: A total of 84 clinical specimens and 97 culture isolates from 126 TB patients were tested during the study. Accuracy (95% confidence interval) of LPA for clinical samples in comparison with phenotypic drug susceptibility test (DST) was 80.1 (68.5–89.0) for isoniazid (H), 74.7 (62.4–84.6) for rifampicin (R), 74.4 (62.5–84.1) for ethambutol, 71.4 (41.9–91.6) for streptomycin, 84.6 (62.4–96.5) for prothionamide/ethionamide, and 84.6 (73.6–92.3) for levofloxacin (Lfx), respectively. We found a significantly higher sensitivity of LPA for H, R, and Lfx for the culture isolates compared to clinical specimens (p < 0.05). LPA detected different mutations in 6 out of 17 (35.5%) patients susceptible to R by Xpert. A shorter treatment regimen with an injectable agent demonstrated a low suitability rate of 5% (8/156) in a cohort of RR-TB patients from Ukraine. Conclusion: Initial LPA testing accurately identifies resistance to anti-TB drugs and facilitates the selection of an appropriate treatment regimen, minimizing exposure to empirical therapy. Plain language summary: Study about the impact of rapid resistance detection on the treatment of patients with tuberculosis in Ukraine written by healthcare and biomedical professionals to better understand how we can improve the results of treatment and to prevent spreading of resistant bacteria Why was the study done? Ukraine has over 4000 patients with tuberculosis (TB) resistant to at least one drug (rifampicin) - five times that of all 30 European Union/European Economic Area countries combined. Unfortunately, only about 60% of such patients have been successfully treated in 2019. At that time, the majority of people suffering from tuberculosis in Ukraine, after checking resistance to rifampicin, initially received standard combinations of the first-line or second-line anti-TB medicines before the result of traditionally used tests (usually few weeks later) became available to individualize the treatment. Alternatively, the sputum could be transported to some overloaded reference laboratories located hundreds of km away from the treatment places. What did the researchers do? The INNOVA4TB team implemented rapid diagnostics of drug resistance in routine practice, guiding key antibiotics use in TB patients. A total of 181 samples from 126 individuals were tested during 2019-2020. What did the researchers find? This new diagnostic technology accurately detected resistance to 9 anti-TB drugs in sputum samples. It could be helpful to select appropriate TB treatment regimens, reducing time for decision from 1 month up to 2 days. Recommended at the study time 9-month shorter standardized treatment regimen with injectable agent was suitable only for 5% of patients for whom it was indicated in Vinnytsia region of Ukraine. What do the findings mean? The study has demonstrated successful implementation of the new molecular diagnostic technology from scratch in a country with restricted resources and limited TB laboratory capacity. This test can facilitate optimal distribution of available wards among patients with different profiles of resistance and correct choice between treatment options. [ABSTRACT FROM AUTHOR]

ImmunoCAP ISAC E112i (ISAC) and Allergy Explorer 2 (ALEX2) detect specific immunoglobulin E (IgE) reactivity. Both multiplex assays contain molecular allergens and ALEX2 additionally includes allergen extracts and inhibitors that block the binding of IgE to cross-reacting carbohydrate determinants (CCDs). This study aimed to compare the performance of ISAC and ALEX2 by determining the IgE reactivity against allergen extracts and/or allergen components and by using qualitative, semiquantitative, and quantitative analyses of all comparable allergen components in sera from 216 participants recruited in South Tyrol/Italy. For extract sensitization in ALEX2, the analysis revealed negative corresponding allergen components in 18.4% and at least one positive corresponding allergen component in 81.6% of all cases. For ISAC, the corresponding results were 23.5% and 76.5% of cases, respectively. The ALEX2 CCD inhibitor eliminated CCD-positive signals detected by ISAC in 88.5% of cases. Based on sensitization values of 0.3–14.9 ISU or kUA/L, there was good agreement between ALEX2 and ISAC, although ALEX2 showed higher values than ISAC. The addition of allergen-extract tests in ALEX2 resulted in the detection of more sensitizations than with corresponding allergen components alone. In the range of <15 ISU or kUA/L, ALEX2 may be more effective in detecting sensitizations. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) elimination is an important global public health goal. However, the United States is not on track to meet the World Health Organization's 2030 targets for HCV elimination. Recently, the White House proposed an HCV elimination plan that includes point-of-care (POC) HCV RNA testing, which is currently in use in many countries but is not approved in the United States. POC HCV RNA testing is crucial for implementing community-based testing and for enabling test-and-treat programs, assessing cure, and monitoring for reinfection. Here, we review the status of POC HCV RNA testing in the United States, discuss factors that are needed for successful implementation, and issue specific public health and policy recommendations that would allow for the use of POC HCV RNA testing to support HCV elimination. [ABSTRACT FROM AUTHOR]