One-Pot Assay for Rapid Detection of Stenotrophomonas maltophilia by RPA-CRISPR/Cas12a.

Stenotrophomonas maltophilia (S. maltophilia, SMA) is a common opportunistic pathogen that poses a serious threat to the food industry and human health. Traditional detection methods for SMA are time-consuming, have low detection rates, require complex and expensive equipment and professional technical personnel for operation, and are unsuitable for on-site detection. Therefore, establishing an efficient on-site detection method has great significance in formulating appropriate treatment strategies and ensuring food safety. In the present study, a rapid one-pot detection method was established for SMA using a combination of Recombinase Polymerase Amplification (RPA) and CRISPR/Cas12a, referred to as ORCas12a-SMA (one-pot RPA-CRISPR/Cas12a platform). In the ORCas12a-SMA detection method, all components were added into a single tube simultaneously to achieve one-pot detection and address the problems of nucleic acid cross-contamination and reduced sensitivity caused by frequent cap opening during stepwise detection. The ORCas12a-SMA method could detect at least 3 × 10° copies·µL-1 of SMA genomic DNA within 30 min at 37 °C. Additionally, this method exhibited sensitivity compared to the typical two-step RPA-CRISPR/Cas12a method. Overall, the ORCas12a-SMA detection offered the advantages of rapidity, simplicity, high sensitivity and specificity, and decreased need for complex large-scale instrumentation. This assay is the first application of the one-pot platform based on the combination of RPA and CRISPR/Cas12a in SMA detection and is highly suitable for point-of-care testing. It helps reduce losses in the food industry and provides assistance in formulating timely and appropriate antimicrobial treatment plans.

Detection of Filariid Infections in Mexican Primate Populations Through qPCR.

Filariae are parasitic nematodes of high veterinary and medical importance, responsible for some acute tropical diseases. They are transmitted through the bite of hematophagous vectors such as biting midges and blackflies. Filariae are among the most prevalent vector-borne parasitoses in Neotropical primates in which severe infections can cause inflammatory reactions and tissue damage. Given the location inside the host (peritoneal cavity, bloodstream, and lymphatics), the detection of filariid nematodes is challenging and is mostly postmortem; hence the scarcity of studies on the prevalence of filariae in wild primate populations. Here, we report the prevalence of filariid infections in free-ranging populations of Geoffroy's spider (Ateles geoffroyi) and black howler (Alouatta pigra) monkeys across southern Mexico, using a combination of noninvasive sampling and molecular diagnostic techniques. Fecal samples were screened for filariid DNA by qPCR protocols. A total of 88 samples were examined with an overall prevalence of 26%. Filariae were slightly more common in spider monkeys compared to howler monkeys. This study constitutes the first report of the prevalence of infection of filariid nematodes in populations of wild spider monkey across southern Mexico, and the first reporting of filariae in black howler monkeys, as part of a new era of primate parasitology and the diagnostics of parasite infections in light of the everyday more affordable molecular tools.

Can immuno-PCR (IPCR) transform bacterial disease diagnostics?

INTRODUCTION: Approximately 15 million deaths occur globally each year due to infectious diseases. Timely diagnosis is crucial in promoting cure and preventing disease transmission. Currently, molecular diagnostics have replaced many conventional diagnostic tools due to their inherent limitations. However, the full potential of Immuno Polymerase Chain Reaction (IPCR) remains largely untapped. AREAS COVERED: This review focuses on the use of IPCR in the diagnosis of different bacterial diseases, highlighting its advantages over traditional methods. EXPERT OPINION: Early and accurate diagnosis of infectious diseases is crucial because it enhances treatment effectiveness, reduces morbidity and mortality, helps identify potential causes of sepsis earlier, and reduces the risk of unknowingly spreading the disease to others. IPCR in turn has shown promise for the early diagnosis of bacterial diseases as an alternative to conventional culture-based or serological diagnostic assays leading to delayed diagnosis and treatment. IPCR has the potential to revolutionize the diagnostic field due to its increased sensitivity and specificity. Although efforts are needed to reduce the time of the assay and to reduce background noise, IPCR can be combined with other platforms like lateral flow assay/biosensors/automation to improve its use as a point-of-care assay, especially in resource-limited settings.

Validation of a species-specific probe-based qPCR for detection of Setaria yehi (Filarioidea: Onchocercidae) in Alaskan moose (Alces alces gigas).

Northern ungulates contend with Setaria yehi and Rumenfilaria andersoni, filarioid nematodes that are transmitted by ectoparasitic blood-feeding arthropods, which can result in animal and population level impacts. Setaria yehi microfilariae can be detected in fresh blood samples using a modified Knott's test, or by postmortem detection by genetic sampling or through the retrieval of adult specimens in the peritoneal cavity. In this study we validated a novel qPCR for detection of S. yehi DNA in blood samples of moose (Alces alces). Additionally, we compared quantitative values from modified Knott's test to detect both S. yehi and R. andersoni from both fresh and frozen blood samples. Species-specific primers targeting a 121-base pair fragment of the cytochrome oxidase c subunit 1 (cox1) of S. yehi, and a species-specific probe were designed. The qPCR had a detection threshold of 0.157 pg/µL of parasite DNA. We collected 166 blood samples from wild moose captured on the Kenai Peninsula, Alaska from 2019 to 2022. Matching blood aliquots were tested by modified Knott's test and subjected to DNA extraction for subsequent qPCR. Quantitatively, blood samples had an average S. yehi microfilaremia (mf) of 472.2 mf/mL (0-14,490 mf/mL) and R. andersoni of 72.9 mf/mL (0.0-5071.5 mf/mL). Qualitatively, 32.53% (n = 54) of samples tested positive for S. yehi in each of the tests, and 37.35% (n = 62) when both tests were combined, with very good agreement between the results from Knott's test and qPCR (kappa = 0.90). The validation of the qPCR test for S. yehi allows for faster, less labor-intensive diagnosis and epidemiological surveillance of this emerging parasite in moose and other cervid hosts.

Comparing NGS-Based identification of bloodstream infections to traditional culture methods for enhanced ICU care: a comprehensive study.

Background: Accurate identification of infectious diseases using molecular techniques, such as PCR and NGS, is well-established. This study aims to assess the utility of Bactfast and Fungifast in diagnosing bloodstream infections in ICU settings, comparing them against traditional culture methods. The objectives include evaluating sensitivity and specificity and identifying a wide range of pathogens, including non-culturable species. Methods: We collected 500 non-duplicate blood samples from ICU patients between January 2023 and December 2023. Specimens underwent traditional culture, MALDI-TOF, VITEK®2 compact system, and NGS-based Bactfast and Fungifast analyses. Results: Out of the 500 samples, 26.8% (n=134) showed bacterial growth via traditional culture methods, while 4.8% (n=24) were positive for fungal growth. MALDI-TOF and VITEK®2 compact system yielded comparable results, identifying 26.4% (n=132) of specimens with bacterial growth. NGS-based Bactfast detected bacterial presence in 38.2% (n=191) of samples, including non-culturable bacteria missed by traditional methods. However, NGS-based Fungifast showed concordant fungal detection rates with culture methods. Among identified pathogens by culture method included Klebsiella pneumoniae 20.89% (n=28), Enterococcus faecalis 18.65% (n=25), Escherichia coli 15.67% (n=21), Pseudomonas aeruginosa 12.68% (n=17), Acinetobacter baumannii 10.44% (n=14), various Streptococcus species 7.46% (n=10), Mycobacterium tuberculosis 6.71% (n=9), Mycobacterium abscessus 4.47% (n=6), and Salmonella spp 2.98% (n=4). Non-culture-based NGS identified additional (n=33) pathogens, including Klebsiella pneumoniae 27.27% (n=9), Bacteroides fragilis 21.21% (n=7), Aerococcus viridans 15.15% (n=5), Elizabethkingia anopheles 12.12% (n=4), Aeromonas salmonicida 9% (n=3), Clostridium 9% (n=3), and Bacteroides vulgatus 6% (n=2). Candida albicans was reported in 5% (n=24) of samples by both methods. Conclusion: NGS-based Bactfast and Fungifast demonstrate high sensitivity in identifying a wide array of bacterial and fungal pathogens in ICU patients, outperforming traditional culture methods in detecting non-culturable organisms. These molecular assays offer rapid and comprehensive diagnostic capabilities, potentially improving clinical outcomes through timely and accurate pathogen identification.

A Review of the Regulatory Challenges of Personalized Medicine.

Personalized medicine integrates genomics with clinical and familial histories, revolutionizing healthcare by tailoring treatments to individual patient characteristics. At its core, pharmacogenomics enables the customization of medication prescriptions based on genetic profiles, enhancing drug efficacy and safety. This precision medicine approach addresses disease diagnosis, prevention, and treatment, offering targeted therapies for conditions like autoimmune disorders, rheumatoid arthritis, and neoplastic conditions. Examples of pharmacogenomics and personalized medicine include treatment for certain conditions like blood clotting disorders (warfarin (blood thinner), genetic variability, acute lymphoblastic leukemia (ALL), and thiopurine methyltransferase (TPMT) testing) in leukemia treatment. Historically, personalized medicine has evolved from Hippocrates' humoral theories to modern molecular diagnostics. The shift from cellular to molecular-level investigations has led to the current post-genomic era, emphasizing the four chemical components of DNA in understanding and treating disorders. This evolution enhances our ability to predict disease susceptibility, treatment response, and potential adverse reactions, demanding advancements in privacy laws, payment systems, regulatory standards, and education. Personalized healthcare optimizes treatment by considering genetic, environmental, and lifestyle factors, reducing adverse reactions, and improving patient satisfaction. It drives genomic and biotechnological research, fostering the development of targeted therapies and diagnostic tools, and streamlining drug development. Applications in lung cancer, renal carcinoma, and rheumatoid arthritis (RA) illustrate the efficacy of personalized medicine. Targeted therapies, such as tyrosine kinase inhibitors (TKIs) and monoclonal antibodies (mAbs), show significant success in lung cancer. Biomarkers guide renal carcinoma treatments, while gene expression profiles predict rheumatoid arthritis outcomes with tumor necrosis factor-alpha (TNF-α) blockers. The US FDA's increasing approvals of personalized treatments underscore its commitment to precision medicine. However, regulatory challenges persist, necessitating clear processes and policies. Ethical considerations, including equitable access and privacy, are critical for responsible implementation. The FDA's increasing approvals of personalized treatments highlight its commitment to advancing precision medicine, yet regulatory challenges remain, requiring the development of clear processes and policies to manage innovation safely. While these hurdles are being addressed through evolving guidelines and collaborative efforts, ethical considerations, including equitable access and privacy, are critical for responsible implementation. Early examples, such as warfarin and imatinib, demonstrate the role of genetic information in guiding treatment, illustrating the potential of personalized medicine to optimize healthcare outcomes.

Leaving no patient behind! Expert recommendation in the use of innovative technologies for diagnosing rare diseases.

Genetic diagnosis plays a crucial role in rare diseases, particularly with the increasing availability of emerging and accessible treatments. The International Rare Diseases Research Consortium (IRDiRC) has set its primary goal as: "Ensuring that all patients who present with a suspected rare disease receive a diagnosis within one year if their disorder is documented in the medical literature". Despite significant advances in genomic sequencing technologies, more than half of the patients with suspected Mendelian disorders remain undiagnosed. In response, IRDiRC proposes the establishment of "a globally coordinated diagnostic and research pipeline". To help facilitate this, IRDiRC formed the Task Force on Integrating New Technologies for Rare Disease Diagnosis. This multi-stakeholder Task Force aims to provide an overview of the current state of innovative diagnostic technologies for clinicians and researchers, focusing on the patient's diagnostic journey. Herein, we provide an overview of a broad spectrum of emerging diagnostic technologies involving genomics, epigenomics and multi-omics, functional testing and model systems, data sharing, bioinformatics, and Artificial Intelligence (AI), highlighting their advantages, limitations, and the current state of clinical adaption. We provide expert recommendations outlining the stepwise application of these innovative technologies in the diagnostic pathways while considering global differences in accessibility. The importance of FAIR (Findability, Accessibility, Interoperability, and Reusability) and CARE (Collective benefit, Authority to control, Responsibility, and Ethics) data management is emphasized, along with the need for enhanced and continuing education in medical genomics. We provide a perspective on future technological developments in genome diagnostics and their integration into clinical practice. Lastly, we summarize the challenges related to genomic diversity and accessibility, highlighting the significance of innovative diagnostic technologies, global collaboration, and equitable access to diagnosis and treatment for people living with rare disease.

Development of a Multiplex Real-Time PCR Assay for the Simultaneous Detection of Two Fungal Pathogens Causing Pneumonia.

Infectious diseases caused by fungal sources are of great interest owing to their increasing prevalence. Invasive fungal infections, including invasive pulmonary aspergillosis caused by Aspergillus fumigatus, and Pneumocystis pneumonia caused by Pneumocystis jirovecii, are significant causes of morbidity and mortality among immunocompromised patients. The accurate and timely detection of these pathogens in this high-risk population is crucial for effective patient management. We developed a multiplex real-time polymerase chain reaction (PCR) assay, RF2 mRT-PCR, specifically designed to detect two respiratory fungi, P. jirovecii and A. fumigatus, and evaluated its performance in specimens of patients with lower respiratory tract infection. The performance was evaluated using 731 clinical samples, 55 reference species, and one synthetic DNA. The reproducibility test yielded a probit curve with a lower limit of detection of 19.82 copies/reaction for P. jirovecii and 64.20 copies/reaction for A. fumigatus. The RF2 mRT-PCR assay did not cross-react with non-A. fumigatus Aspergillus species or other common bacterial and viral species, and showed 100% in vitro sensitivity and specificity with reference assays. Additionally, it simultaneously detected A. fumigatus and P. jirovecii in co-infected samples. Therefore, the RF2 mRT-PCR assay is an efficient and reliable tool for in vitro diagnosis of A. fumigatus and P. jirovecii pulmonary infections.

Diagnostic Accuracy of Five Molecular Assays for the Detection of Dengue Virus.

Background and Objectives: The steady spread of dengue virus (DENV) poses a profound public health threat worldwide. Reverse transcription real-time polymerase chain reaction (RT2-PCR) has been increasingly recognized as a reference method for the diagnosis of acute dengue infection. The goal of this study was to assess the diagnostic accuracy of five different RT2-PCR kits for the detection of DENV in a historically processed set of sera samples. Materials and Methods: In this retrospective study, 25 sera samples from routinely processed unique adult patients with a known DENV status (previously tested in both molecular and serological assays) were tested in parallel using four conventional (RealStar Dengue PCR Kit 3.0, Clonit'ngo Zika, Dengue & Chikungunya, BioPerfectus Zika Virus/Dengue Virus/Chikungunya Virus Real Time PCR Kit and Novaplex Tropical fever virus) and one sample-to-result (STANDARD M10 Arbovirus Panel) RT2-PCR assays. Additionally, an end-point dilution analysis was conducted in quintuplicate on six serial dilutions of an RNA preparation obtained from a culture-grown DENV serotype 1 strain for a total of 150 tests. Results: The overall accuracy of the evaluated tests ranged from 84% to 100%. In particular, the sensitivity of three conventional RT2-PCR assays (RealStar, Clonit'ngo and Novaplex) was 100% (95% CI: 79.6-100%), while it was lower (73.3%; 95% CI: 48.1-89.1%) for the BioPerfectus kit. The sample-to-result STANDARD M10 panel performed comparatively well, showing a sensitivity of 92.9% (95% CI: 68.5-98.7%). No false positive results were registered in any assay. The end-point dilution analysis suggested that the RealStar kit had the lowest limit of detection. Conclusions: Available RT2-PCR kits for the detection of DENV are highly specific and generally sensitive and, therefore, their implementation in diagnostic pathways is advisable.

Detection of the BRAFV600E Mutation in Circulating Free Nucleic Acids as a Biomarker of Thyroid Cancer: A Review.

Background: Liquid biopsy is a method that could potentially improve the management of thyroid cancer (TC) by enabling the detection of circulating tumor DNA and RNA (ctDNA, ctRNA). The BRAFV600E mutation appears to be the most representative example of a biomarker in liquid biopsy, as it is the most specific mutation for TC and a target for molecular therapeutics. The aim of this review is to summarize the available data on the detection of the BRAFV600E mutation in liquid biopsy in patients with TC. Methods: A comprehensive analysis of the available literature on the detection of the BRAFV600E mutation in liquid biopsy in TC was performed. Thirty-three papers meeting the inclusion criteria were selected after full-text evaluation. Results: Eleven papers discussed correlations between BRAF mutation and clinicopathological characteristics. Nine studies tested the utility of BRAFV600E detection in the assessment of residual or recurrent disease. Seven studies investigated BRAF-mutated circulating tumor nucleic acids (ctNA) as a marker of response to targeted therapy. In seven studies the method did not detect the BRAFV600E mutation. Conclusions: This review shows the potential of BRAFV600E-mutated ctNA detection in monitoring disease progression, particularly in advanced TC. The diagnostic value of BRAFV600E-mutated ctNA detection appears to be limited to advanced TC. The choice of the molecular method (quantitative PCR [qPCR], droplet digital polymerase chain reaction [ddPCR], and next-generation sequencing [NGS]) should be made based on the turnaround time, sensitivity of the test, and the clinical indications. Despite the promising outcomes of some studies, there is a need to verify these results on larger cohorts and to unify the molecular methods.

Fungi-sensitized individuals have unique profiles where Alt a 1 dominates promoting response to grass, ragweed and cat allergens.

Introduction: The aim of our work was to determine comprehensively the sensitization profile of patients hypersensitive to fungal allergenic components in the Ukrainian population, identifying features of their co-sensitization to allergens of other groups and establishing potential relationships between causative allergens and their ability to provoke this hypersensitivity. Methods: A set of programs was developed using Python and R programming languages, implementing the K-means++ clustering method. Bayesian networks were constructed based on the created clusters, allowing for the assessment of the probabilistic interplay of allergen molecules in the sensitization process of patients. Results and discussion: It was found that patients sensitive to fungi are polysensitized, with 84.77% of them having unique allergological profiles, comprising from 2 to several dozen allergens from different groups. The immune response to Alt a 1 may act as the primary trigger for sensitization to other allergens and may contribute to a high probability of developing sensitivity to grasses (primarily to Phl p 2), ragweed extract, and the Amb a 1 pectate lyase, as well as to pectate lyase Cry j 1 and cat allergen Fel d 1. Individuals polysensitized to molecular components of fungi were often sensitive to such cross-reactive molecules as lipocalins Fel d 4 and Can f 6, as well. Sensitivity to Ambrosia extract which dominated in the development of sensitization to ragweed pollen indicating the importance of different allergenic components of this plant's pollen. This hypothesis, along with the assumption that Phl p 2 may be the main trigger for sensitivity to grasses in patients with Alternaria allergy, requires further clinical investigation.

Minimal residual disease testing for classical Hodgkin lymphoma: A comprehensive review.

Classical Hodgkin lymphoma (cHL) is a common lymphoma that affects young patients. Fortunately, the disease is highly curable as it is susceptible to the currently available treatment modalities. Disease monitoring with Positron Emission Tomography and Computed Tomography (PET/ CT) is an integral part of managing these patients. PET guided protocols are currently used to adjust treatment according to the response. The pivotal idea behind the use of response-adapted approaches is to preserve efficacy while decreasing the toxicity. It also helps to intensify therapy in patients in need because of suboptimal response. However, imaging techniques are limited by their sensitivity and specificity. Minimal Residual Disease (MRD) assessment is a newly emerging concept in many hematologic malignancies. It utilizes various molecular techniques such as polymerase chain reaction (PCR), and next-generation sequencing (NGS) as well as flow cytometry, to detect disease traces. This review looks into MRD detection techniques, its current applications, and the evidence in the literature for its use in cHL.

Rapid real-time quantitative colorimetric LAMP methodology for field detection of Verticillium dahliae in crude olive-plant samples.

BACKGROUND: Verticilium dahliae is the most important wilt pathogen of olive trees with a broad host range causing devastating diseases currently without any effective chemical control. Traditional detection methodologies are based on symptoms-observation or lab-detection using time consuming culturing or molecular techniques. Therefore, there is an increasing need for portable tools that can detect rapidly V. dahliae in the field. RESULTS: In this work, we report the development of a novel method for the rapid, reliable and on-site detection of V. dahliae using a newly designed isothermal LAMP assay and crude extracts of olive wood. For the detection of the fungus, LAMP primers were designed targeting the internal transcribed spacer (ITS) region of the rRNA gene. The above assay was combined with a purpose-built prototype portable device which allowed real time quantitative colorimetric detection of V. dahliae in 35 min. The limit of detection of our assay was found to be 0.8 fg/µl reaction and the specificity 100% as indicated by zero cross-reactivity to common pathogens found in olive trees. Moreover, detection of V. dahliae in purified DNA gave a sensitivity of 100% (Ct < 30) and 80% (Ct > 30) while the detection of the fungus in unpurified crude wood extracts showed a sensitivity of 80% when multisampling was implemented. The superiority of the LAMP methodology regarding robustness and sensitivity was demonstrated when only LAMP was able to detect V. dahliae in crude samples from naturally infected trees with very low infection levels, while nested PCR and SYBR qPCR failed to detect the pathogen in an unpurified form. CONCLUSIONS: This study describes the development of a new real time LAMP assay, targeting the ITS region of the rRNA gene of V. dahliae in olive trees combined with a 3D-printed portable device for field testing using a tablet. The assay is characterized by high sensitivity and specificity as well as ability to operate using directly crude samples such as woody tissue or petioles. The reported methodology is setting the basis for the development of an on-site detection methodology for V. dahliae in olive trees, but also for other plant pathogens.

Enhancing multiplex detection capabilities of the Cas12a/blocker DNA system.

In the field of molecular diagnostics, the demand for multiplex detection, aimed at reducing overall analysis costs and streamlining procedures, is on the rise, prompting ongoing developments in various technologies. In this study, we developed a novel system, the split T7 promoter-based three-way junction-transcription, coupled with Cas12a/Blocker DNA (T3-CaB), for the multiplex detection of target nucleic acids. The T3-CaB system builds upon the foundation of the T3 system, generating numerous RNA transcripts upon encountering target nucleic acids. Subsequently, these RNA transcripts displace the blocker DNA from reporter DNA, allowing active Cas12a to engage in efficient trans-cleavage reaction on the reporter DNA, resulting in a strong fluorescence signal. Importantly, the proposed system operates at the isothermal condition (37 °C), with the entire analysis completed within 90 min. Further, the detection performance of the proposed system surpasses that of the preceding Cas12a/Blocker DNA system. Model targets, namely the 16S rRNA of Staphylococcus aureus and Escherichia coli, were selected, and a successful demonstration of multiplex detection was achieved. This technology holds promise for broadening the applicability of CRISPR/Cas-based diagnostics, especially in settings necessitating multiplex detection capabilities.

Commercial Opportunity or Addressing Unmet Needs-Loop-Mediated Isothermal Amplification (LAMP) as the Future of Rapid Diagnostic Testing?

Loop-Mediated Isothermal Amplification (LAMP) technology is emerging as a rapid pathogen testing method, potentially challenging the RT-PCR "gold standard". Despite recent advancements, LAMP's widespread adoption remains limited. This study provides a comprehensive market overview and assesses future growth prospects to aid stakeholders in strategic decision-making and policy formulation. Using a dataset of 1134 LAMP patent documents, we analyzed lifecycle and geographic distribution, applicant profiles, CPC code classifications, and patent claims. Additionally, we examined clinical developments from 21 curated clinical trials, focusing on trends, geographic engagement, sponsor types, and the conditions and pathogens investigated. Our analysis highlights LAMP's potential as a promising rapid pathogen testing alternative, especially in resource-limited areas. It also reveals a gap between clinical research, which targets bacterial and parasitic diseases like malaria, leishmaniasis, and tuberculosis, and basic research and commercial efforts that prioritize viral diseases such as SARS-CoV-2 and influenza. European stakeholders emphasize the societal impact of addressing unmet needs in resource-limited areas, while American and Asian organizations focus more on research, innovation, and commercialization.

Use of a synthetic oligonucleotide to detect false positives caused by cross-contamination in nested PCR.

Nested PCR is a useful tool for identifying low-abundance target sequences of pathogens and avoiding false negatives. However, it carries an increased risk of cross-contamination, especially with its positive control. Here, we propose using customized synthetic oligonucleotides to detect false positives due to cross-contamination.

Diagnosis of bacteria from the CMNR group in farm animals.

The CMNR group comprises bacteria of the genera Corynebacterium, Mycobacterium, Nocardia, and Rhodococcus and share cell wall and DNA content characteristics. Many pathogenic CMNR bacteria cause diseases such as mastitis, lymphadenitis, and pneumonia in farmed animals, which cause economic losses for breeders and represent a threat to public health. Traditional diagnosis in CMNR involves isolating target bacteria on general or selective media and conducting metabolic analyses with the assistance of laboratory biochemical identification systems. Advanced mass spectrometry may also support diagnosing these bacteria in the clinic's daily routine despite some challenges, such as the need for isolated bacteria. In difficult identification among some CMNR members, molecular methods using polymerase chain reaction (PCR) emerge as reliable options for correct specification that is sometimes achieved directly from clinical samples such as tracheobronchial aspirates and feces. On the other hand, immunological diagnostics such as the skin test or Enzyme-Linked Immunosorbent Assay (ELISA) for Mycobacterium tuberculosis yield promising results in subclinical infections with no bacterial growth involved. In this review, we present the methods most commonly used to diagnose pathogenic CMNR bacteria and discuss their advantages and limitations, as well as challenges and perspectives on adopting new technologies in diagnostics.

Advances in the Diagnosis and Treatment of Follicular Thyroid Carcinoma: A Comprehensive Review.

Follicular thyroid carcinoma (FTC) is the second most common type of thyroid cancer, presenting unique diagnostic and therapeutic challenges. This review provides a comprehensive analysis of the recent advancements in the diagnosis and treatment of FTC, emphasizing the significance of these developments in improving patient outcomes. We discuss the evolution of diagnostic techniques, including advancements in imaging modalities, fine needle aspiration biopsy, and molecular diagnostics, which have enhanced the accuracy of FTC detection and differentiation from benign conditions. The review also evaluates current treatment strategies, including surgical interventions, radioactive iodine therapy, and targeted therapies, examining their effectiveness and impact on patient prognosis. Additionally, we address ongoing challenges in FTC management, such as variability in treatment guidelines and disparities in care. Finally, the review explores emerging therapies and future research directions, highlighting innovations that may further optimize FTC management. By synthesizing current knowledge and identifying future research opportunities, this review aims to contribute to refining diagnostic and therapeutic approaches for FTC.

Non-canonical BRAF variants and rearrangements in hairy cell leukemia.

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.

Analysis of miRNA Expression Profiles in Traumatic Brain Injury (TBI) and Their Correlation with Survival and Severity of Injury.

Traumatic brain injury (TBI) is the leading cause of traumatic death worldwide and is a public health problem associated with high mortality and morbidity rates, with a significant socioeconomic burden. The diagnosis of brain injury may be difficult in some cases or may leave diagnostic doubts, especially in mild trauma with insignificant pathological brain changes or in cases where instrumental tests are negative. Therefore, in recent years, an important area of research has been directed towards the study of new biomarkers, such as micro-RNAs (miRNAs), which can assist clinicians in the diagnosis, staging, and prognostic evaluation of TBI, as well as forensic pathologists in the assessment of TBI and in the estimation of additional relevant data, such as survival time. The aim of this study is to investigate the expression profiles (down- and upregulation) of a panel of miRNAs in subjects deceased with TBI in order to assess, verify, and define the role played by non-coding RNA molecules in the different pathophysiological mechanisms of brain damage. This study also aims to correlate the detected expression profiles with survival time, defined as the time elapsed between the traumatic event and death, and with the severity of the trauma. This study was conducted on 40 cases of subjects deceased with TBI (study group) and 10 cases of subjects deceased suddenly from non-traumatic causes (control group). The study group was stratified according to the survival time and the severity of the trauma. The selection of miRNAs to be examined was based on a thorough literature review. Analyses were performed on formalin-fixed, paraffin-embedded (FFPE) brain tissue samples, with a first step of total RNA extraction and a second step of quantification of the selected miRNAs of interest. This study showed higher expression levels in cases compared to controls for miR-16, miR-21, miR-130a, and miR-155. In contrast, lower expression levels were found in cases compared to controls for miR-23a-3p. There were no statistically significant differences in the expression levels between cases and controls for miR-19a. In cases with short survival, the expression levels of miR-16-5p and miR-21-5p were significantly higher. In cases with long survival, miR-21-5p was significantly lower. The expression levels of miR-130a were significantly higher in TBI cases with short and middle survival. In relation to TBI severity, miR-16-5p and miR-21-5p expression levels were significantly higher in the critical-fatal TBI subgroup. Conclusions: This study provides evidence for the potential of the investigated miRNAs as predictive biomarkers to discriminate between TBI cases and controls. These miRNAs could improve the postmortem diagnosis of TBI and also offer the possibility to define the survival time and the severity of the trauma. The analysis of miRNAs could become a key tool in forensic investigations, providing more precise and detailed information on the nature and extent of TBI and helping to define the circumstances of death.