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Thyroid cancer is the most common type of endocrine malignancy. Papillary thyroid carcinoma (PTC) is its predominant subtype, which is responsible for the vast majority of cases. It is true that PTC is a malignant tumor with a very good prognosis due to effective primary therapeutic approaches such as thyroidectomy and radioiodine (RAI) therapy. However, we are often required to indicate second-line treatments to eradicate the tumor properly. In these scenarios, molecular therapies are promising alternatives, especially if specifically targetable mutations are present. Many of these targetable gene alterations originate from gene fusions, which can be found using molecular diagnostics like next-generation sequencing (NGS). Nonetheless, molecular profiling is far from being a routine procedure in the initial phase of PTC diagnostics. As a result, the mutation status, except for BRAF V600E mutation, is not included in risk classification algorithms either. This study aims to provide a comprehensive analysis of fusion mutations in PTC and their associations with clinicopathological variables in order to underscore certain clinical settings when molecular diagnostics should be considered earlier, and to demonstrate yet unknown molecular–clinicopathological connections. We conducted a retrospective fusion mutation screening in formalin-fixed paraffin-embedded (FFPE) PTC tissue samples of 100 patients. After quality evaluation by an expert pathologist, RNA isolation was performed, and then NGS was applied to detect 23 relevant gene fusions in the tumor samples. Clinicopathological data were collected from medical and histological records. To obtain the most associations from the multivariate dataset, we used the d-correlation method for our principal component analysis (PCA). Further statistical analyses, including Chi-square tests and logistic regressions, were performed to identify additional significant correlations within certain subsets of the data. Fusion mutations were identified in 27% of the PTC samples, involving nine distinct genes: RET, NTRK3, CCDC6, ETV6, MET, ALK, NCOA4, EML4, and SQSTM1. RET and CCDC6 fusions were associated with type of thyroidectomy, RAI therapy, smaller tumor size, and history of Hashimoto's disease. NCOA4 fusion correlated with sex, multifocality, microcarcinoma character, history of goiter, and obstructive pulmonary disease. EML4 fusion was also linked with surgical procedure type and smaller tumor size, as well as the history of hypothyroidism. SQSTM1 fusion was associated with multifocality and a medical history of thyroid/parathyroid adenoma. NTRK3 and ETV6 fusions showed significant associations with Hashimoto's disease, and ETV6, also with endometriosis. Moreover, fusion mutations were linked to younger age at the time of diagnosis, particularly the fusion of ETV6. The frequent occurrence of fusion mutations and their associations with certain clinicopathological metrics highlight the importance of integrating molecular profiling into routine PTC management. Early detection of fusion mutations can inform surgical decisions and therapeutic strategies, potentially improving clinical outcomes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Genetic alterations are well known to be related to the pathogenesis and prognosis of papillary thyroid carcinoma (PTC). Some miRNA expression dysregulations have previously been described in the context of cancer development including thyroid carcinoma. In our study, we performed original molecular diagnostics on tissue samples related to our own patients. We aimed to identify all dysregulated miRNAs in potential association with PTC development via sequencing much higher numbers of control-matched PTC tissue samples and analyzing a wider variety of miRNA types than previous studies. We analyzed the expression levels of 2656 different human miRNAs in the context of 236 thyroid tissue samples (118 tumor and control pairs) related to anonymized PTC cases. Also, KEGG pathway enrichment analysis and GO framework analysis were used to establish the links between miRNA dysregulation and certain biological processes, pathways of signaling, molecular functions, and cellular components. A total of 30 significant differential miRNA expressions with at least ±1 log2 fold change were found related to PTC including, e.g., miR-551b, miR-146b, miR-221, miR-222, and miR-375, among others, being highly upregulated, as well as miR-873 and miR-204 being downregulated. In addition, we identified miRNA patterns in vast databases (KEGG and GO) closely similar to that of PTC including, e.g., miRNA patterns of prostate cancer, HTLV infection, HIF-1 signaling, cellular responses to growth factor stimulus and organic substance, and negative regulation of gene expression. We also found 352 potential associations between certain miRNA expressions and states of clinicopathological variables. Our findings—supported by the largest case number of original matched-control PTC–miRNA relation research—suggest a distinct miRNA expression profile in PTC that could contribute to a deeper understanding of the underlying molecular mechanisms promoting the pathogenesis of the disease. Moreover, significant miRNA expression deviations and their signaling pathways in PTC presented in our study may serve as potential biomarkers for PTC diagnosis and prognosis or even therapeutic targets in the future. [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]

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]

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]

Versatility, sensitivity, and accuracy have made the real-time polymerase chain reaction (qPCR) a crucial tool for research, as well as diagnostic applications. However, for point-of-care (PoC) use, traditional qPCR faces two main challenges: long run times mean results are not available for half an hour or more, and the requisite high-temperature denaturation requires more robust and power-demanding instrumentation. This study addresses both issues and revises primer and probe designs, modified buffers, and low ∆T protocols which, together, speed up qPCR on conventional qPCR instruments and will allow for the development of robust, point-of-care devices. Our approach, called "FlashPCR", uses a protocol involving a 15-second denaturation at 79 °C, followed by repeated cycling for 1 s at 79 °C and 71 °C, together with high Tm primers and specific but simple buffers. It also allows for efficient reverse transcription as part of a one-step RT-qPCR protocol, making it universally applicable for both rapid research and diagnostic applications. [ABSTRACT FROM AUTHOR]

Thyroid cancer is the most common type of endocrine malignancy. Papillary thyroid carcinoma (PTC) is its predominant subtype, which is responsible for the vast majority of cases. It is true that PTC is a malignant tumor with a very good prognosis due to effective primary therapeutic approaches such as thyroidectomy and radioiodine (RAI) therapy. However, we are often required to indicate second-line treatments to eradicate the tumor properly. In these scenarios, molecular therapies are promising alternatives, especially if specifically targetable mutations are present. Many of these targetable gene alterations originate from gene fusions, which can be found using molecular diagnostics like next-generation sequencing (NGS). Nonetheless, molecular profiling is far from being a routine procedure in the initial phase of PTC diagnostics. As a result, the mutation status, except for BRAF V600E mutation, is not included in risk classification algorithms either. This study aims to provide a comprehensive analysis of fusion mutations in PTC and their associations with clinicopathological variables in order to underscore certain clinical settings when molecular diagnostics should be considered earlier, and to demonstrate yet unknown molecular–clinicopathological connections. We conducted a retrospective fusion mutation screening in formalin-fixed paraffin-embedded (FFPE) PTC tissue samples of 100 patients. After quality evaluation by an expert pathologist, RNA isolation was performed, and then NGS was applied to detect 23 relevant gene fusions in the tumor samples. Clinicopathological data were collected from medical and histological records. To obtain the most associations from the multivariate dataset, we used the d-correlation method for our principal component analysis (PCA). Further statistical analyses, including Chi-square tests and logistic regressions, were performed to identify additional significant correlations within certain subsets of the data. Fusion mutations were identified in 27% of the PTC samples, involving nine distinct genes: RET, NTRK3, CCDC6, ETV6, MET, ALK, NCOA4, EML4, and SQSTM1. RET and CCDC6 fusions were associated with type of thyroidectomy, RAI therapy, smaller tumor size, and history of Hashimoto’s disease. NCOA4 fusion correlated with sex, multifocality, microcarcinoma character, history of goiter, and obstructive pulmonary disease. EML4 fusion was also linked with surgical procedure type and smaller tumor size, as well as the history of hypothyroidism. SQSTM1 fusion was associated with multifocality and a medical history of thyroid/parathyroid adenoma. NTRK3 and ETV6 fusions showed significant associations with Hashimoto’s disease, and ETV6, also with endometriosis. Moreover, fusion mutations were linked to younger age at the time of diagnosis, particularly the fusion of ETV6. The frequent occurrence of fusion mutations and their associations with certain clinicopathological metrics highlight the importance of integrating molecular profiling into routine PTC management. Early detection of fusion mutations can inform surgical decisions and therapeutic strategies, potentially improving clinical outcomes.

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.

The increased cultivation of Cannabis sativa L. in North America, represented by high Δ9-tetrahydrocannabinol-containing (high-THC) cannabis genotypes and low-THC-containing hemp genotypes, has been impacted by an increasing number of plant pathogens. These include fungi which destroy roots, stems, and leaves, in some cases causing a build-up of populations and mycotoxins in the inflorescences that can negatively impact quality. Viroids and viruses have also increased in prevalence and severity and can reduce plant growth and product quality. Rapid diagnosis of the occurrence and spread of these pathogens is critical. Techniques in the area of molecular diagnostics have been applied to study these pathogens in both cannabis and hemp. These include polymerase chain reaction (PCR)-based technologies, including RT-PCR, multiplex RT-PCR, RT-qPCR, and ddPCR, as well as whole-genome sequencing (NGS) and bioinformatics. In this study, examples of how these technologies have enhanced the rapidity and sensitivity of pathogen diagnosis on cannabis and hemp will be illustrated. These molecular tools have also enabled studies on the diversity and origins of specific pathogens, specifically viruses and viroids, and these will be illustrated. Comparative studies on the genomics and metabolomics of healthy and diseased plants are urgently needed to provide insight into their impact on the quality and composition of cannabis and hemp-derived products. Management of these pathogens will require monitoring of their spread and survival using the appropriate technologies to allow accurate detection, followed by appropriate implementation of disease control measures. [ABSTRACT FROM AUTHOR]

Genetic alterations are well known to be related to the pathogenesis and prognosis of papillary thyroid carcinoma (PTC). Some miRNA expression dysregulations have previously been described in the context of cancer development including thyroid carcinoma. In our study, we performed original molecular diagnostics on tissue samples related to our own patients. We aimed to identify all dysregulated miRNAs in potential association with PTC development via sequencing much higher numbers of control-matched PTC tissue samples and analyzing a wider variety of miRNA types than previous studies. We analyzed the expression levels of 2656 different human miRNAs in the context of 236 thyroid tissue samples (118 tumor and control pairs) related to anonymized PTC cases. Also, KEGG pathway enrichment analysis and GO framework analysis were used to establish the links between miRNA dysregulation and certain biological processes, pathways of signaling, molecular functions, and cellular components. A total of 30 significant differential miRNA expressions with at least ±1 log2 fold change were found related to PTC including, e.g., miR-551b, miR-146b, miR-221, miR-222, and miR-375, among others, being highly upregulated, as well as miR-873 and miR-204 being downregulated. In addition, we identified miRNA patterns in vast databases (KEGG and GO) closely similar to that of PTC including, e.g., miRNA patterns of prostate cancer, HTLV infection, HIF-1 signaling, cellular responses to growth factor stimulus and organic substance, and negative regulation of gene expression. We also found 352 potential associations between certain miRNA expressions and states of clinicopathological variables. Our findings—supported by the largest case number of original matched-control PTC–miRNA relation research—suggest a distinct miRNA expression profile in PTC that could contribute to a deeper understanding of the underlying molecular mechanisms promoting the pathogenesis of the disease. Moreover, significant miRNA expression deviations and their signaling pathways in PTC presented in our study may serve as potential biomarkers for PTC diagnosis and prognosis or even therapeutic targets in the future.

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.

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.

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.

An important approach to molecular diagnostics is integrating organized substances that provide complex molecular level responses to introduced chemical and biological agents with conditions that optimize and distinguish such responses. In this respect, liquid crystal dispersions are attractive components of molecular diagnostic tools. This paper analyzes a colloid system, containing a nematic liquid crystal as a dispersed phase, and aqueous surfactant and polymer solutions as the continuous phases. We applied a microfluidic approach for tuning orientation of liquid crystal molecules in picoliter droplets immobilized on microchannel walls. Introduction of surfactant to the aqueous phase was found to proportionally increase the order parameter of liquid crystal molecules in microdroplets. Infusion of polymer solutions into surfactant-mediated microfluidic liquid crystal dispersions increased the order parameter at much lower surfactant concentrations, while further infusion of surfactant solutions randomized the orientation of liquid crystal molecules. These effects were correlated with the adsorption of surfactant molecules on surfaces of microdroplets, stabilizing the effect of a polymer matrix on bound surfactant ions and the formation of insoluble polymer–colloid aggregates, respectively. The revealed molecular behavior of liquid crystal dispersions may contribute to optimized synthesis of responsive liquid crystal dispersions for in-flow molecular diagnostics of polymers and colloids, and the development of functional laboratory-on-chip prototypes. [ABSTRACT FROM AUTHOR]

Pathogenicity predictors are computational tools that classify genetic variants as benign or pathogenic; this is currently a major challenge in genomic medicine. With more than fifty such predictors available, selecting the most suitable tool for clinical applications like genetic screening, molecular diagnostics, and companion diagnostics has become increasingly challenging. To address this issue, we have developed a cost-based framework that naturally considers the various components of the problem. This framework encodes clinical scenarios using a minimal set of parameters and treats pathogenicity predictors as rejection classifiers, a common practice in clinical applications where low-confidence predictions are routinely rejected. We illustrate our approach in four examples where we compare different numbers of pathogenicity predictors for missense variants. Our results show that no single predictor is optimal for all clinical scenarios and that considering rejection yields a different perspective on classifiers. [ABSTRACT FROM AUTHOR]

Sarcoidosis is a complex inflammatory multisystem disease of unknown etiology that is characterised by epithelioid cell granulomatous lesions affecting various organs, mainly the lungs. In general, sarcoidosis is asymptomatic, but some cases result in severe complications and organ failure. So far, no accurate and validated modelling for clinical and pathohistological manifestations of sarcoidosis is suggested. Moreover, knowledge about disease-specific diagnostic markers for sarcoidosis is scarce. For instance, pulmonary granulomatosis is associated with the upregulated production of proinflammatory molecules: TNF-α, IL-6, CXCL1, CCL2, CCL18, CD163, serum angiotensin-converting enzyme (sACE), lysozyme, neopterin, and serum amyloid A (SAA). Quantum dots (QDs) are widely applied for molecular diagnostics of various diseases. QDs are semiconductor nanoparticles of a few nanometres in size, made from ZnS, CdS, ZnSe, etc., with unique physical and chemical properties that are useful for the labelling and detection in biological experiments. QDs can conjugate with various antibodies or oligonucleotides, allowing for high-sensitivity detection of various targets in organs and cells. Our review describes existing experimental models for sarcoidosis (in vitro, in vivo, and in silico), their advantages and restrictions, as well as the physical properties of quantum dots and their potential applications in the molecular diagnostics of sarcoidosis. The most promising experimental models include mice with TSC2 deletion and an in silico multiscale computational model of sarcoidosis (SarcoidSim), developed using transcriptomics and flow cytometry of human sarcoid biopsies. Both models are most efficient to test different candidate drugs for sarcoidosis. [ABSTRACT FROM AUTHOR]

Researchers active in the field of inflammatory skin diseases from the spectrum of dermatitis and eczema are well aware of a considerable overlap in the clinical pictures and proposed sets of diagnostic criteria for these diseases, which can hardly be overcome through the clinical or epidemiological research. In effect, patients are included in studies based on vague and overlapping criteria, while heterogeneous study populations may, in turn, lead to non-representative outcomes and continued confusion. In this narrative review, a systematics of diseases from the spectrum of dermatitis and eczema is proposed based on the origins of causative factors and the pathomechanisms involved. Difficulties in differentiating between these diseases are discussed, and the extent to which advances in the "omics" sciences might help to overcome them is considered. Of all the "omics" research in this field, more than 90% of the published papers were devoted to atopic dermatitis, with a striking underrepresentation of other diseases from the spectrum of dermatitis and eczema, conditions which collectively exceed the rates of atopic dermatitis by far. A greater "omics" research effort is urgently needed to tackle other dermatitides, like allergic, irritant and protein contact dermatitis, as well as radiation, seborrheic, stasis or autoimmune dermatitis. Atopic dermatitis findings should be validated not only against healthy donors but also other dermatitides. A clinic-oriented approach is proposed for future "omics" studies in the field of dermatitis and eczema. [ABSTRACT FROM AUTHOR]

Versatility, sensitivity, and accuracy have made the real-time polymerase chain reaction (qPCR) a crucial tool for research, as well as diagnostic applications. However, for point-of-care (PoC) use, traditional qPCR faces two main challenges: long run times mean results are not available for half an hour or more, and the requisite high-temperature denaturation requires more robust and power-demanding instrumentation. This study addresses both issues and revises primer and probe designs, modified buffers, and low ∆T protocols which, together, speed up qPCR on conventional qPCR instruments and will allow for the development of robust, point-of-care devices. Our approach, called “FlashPCR”, uses a protocol involving a 15-second denaturation at 79 °C, followed by repeated cycling for 1 s at 79 °C and 71 °C, together with high Tm primers and specific but simple buffers. It also allows for efficient reverse transcription as part of a one-step RT-qPCR protocol, making it universally applicable for both rapid research and diagnostic applications.

The increased cultivation of Cannabis sativa L. in North America, represented by high Δ9-tetrahydrocannabinol-containing (high-THC) cannabis genotypes and low-THC-containing hemp genotypes, has been impacted by an increasing number of plant pathogens. These include fungi which destroy roots, stems, and leaves, in some cases causing a build-up of populations and mycotoxins in the inflorescences that can negatively impact quality. Viroids and viruses have also increased in prevalence and severity and can reduce plant growth and product quality. Rapid diagnosis of the occurrence and spread of these pathogens is critical. Techniques in the area of molecular diagnostics have been applied to study these pathogens in both cannabis and hemp. These include polymerase chain reaction (PCR)-based technologies, including RT-PCR, multiplex RT-PCR, RT-qPCR, and ddPCR, as well as whole-genome sequencing (NGS) and bioinformatics. In this study, examples of how these technologies have enhanced the rapidity and sensitivity of pathogen diagnosis on cannabis and hemp will be illustrated. These molecular tools have also enabled studies on the diversity and origins of specific pathogens, specifically viruses and viroids, and these will be illustrated. Comparative studies on the genomics and metabolomics of healthy and diseased plants are urgently needed to provide insight into their impact on the quality and composition of cannabis and hemp-derived products. Management of these pathogens will require monitoring of their spread and survival using the appropriate technologies to allow accurate detection, followed by appropriate implementation of disease control measures.

The histological diagnosis of sarcoma can be difficult as it sometimes requires the combination of morphological and immunophenotypic analyses with molecular tests. A total of 2705 tissue samples of sarcoma consecutively collected from 2006 until 2020 that had undergone molecular analysis were assessed to evaluate their diagnostic utility compared with histological assessments. A total of 3051 molecular analyses were performed, including 1484 gene fusions tested by c/qRT–PCR, 992 gene rearrangements analysed by FISH, 433 analyses of the gene status of MDM2, 126 mutational analyses and 16 NGS analysis. Of the samples analysed, 68% were from formalin-fixed, paraffin-embedded tissue and 32% were from frozen tissue. C/qRT–PCR and FISH analyses were conclusive on formalin-fixed, paraffin-embedded tissue in 74% and 76% of samples, respectively, but the combination of the two methods gave us conclusive results in 96% and 89% of frozen and formalin-fixed, paraffin-embedded tissues, respectively. We demonstrate the utility of c/qRT–PCR and FISH for sarcoma diagnosis and that each has advantages in specific contexts. We conclude that it is possible to accurately predict the sarcoma subtype using a panel of different subtype-specific FISH probes and c/qRT–PCR assays, thereby greatly facilitating the differential diagnosis of these tumours. [ABSTRACT FROM AUTHOR]

Helicobacter pylori infections, as one of the most prevalent among humans, are generally acquired during childhood, and are one of the main causes of chronic gastritis and peptic ulcer disease. A bacterial culture from a gastric biopsy is the gold standard and is the only method that has 100% specificity. However, its sensitivity varies, depending on experience of the laboratory staff, applied culture media, specimen transport conditions, biopsy site, and quality of the sample. The same factors compromise all invasive methods and a culture-based H. pylori infection diagnostic, as well as a recent intake of antibiotics, bismuth-containing compounds, and proton pump inhibitors. Molecular methods have been used for clinical microbiology investigation since the beginning of the 21st century. However, their usefulness for H. pylori infections diagnosis remains unclear, especially in pediatric patients. The aim of the study was to assess the incidence of H. pylori infections in a group of 104 pediatric patients and to compare the results of the PCR test with the corresponding histopathological investigation effects. Among the biopsy samples collected from 104 children, 44 (42.3%) were positive in PCR, while 43 (41.3%) and 39 (37.5%) presented histologically-confirmed signs of inflammation and H. pylori colonization, respectively. Moreover, the mean grades of the parameters of the histopathological examination were higher in the group of PCR-positive samples. The compatibility of both research methods was confirmed, emphasizing the usefulness of molecular methods for detecting H. pylori infections in pediatric patients. Considering that the PCR-based method gives reliable results and is less time-consuming and costly, it is worth discussing this method as a new standard in the diagnosis of H. pylori infections, at least among pediatric patients, for which culture-based diagnostics is not sufficient or histopathological examination is negative, while inflammation signs are observed macroscopically. [ABSTRACT FROM AUTHOR]

Heterogeneity of triple-negative breast cancer is well known at clinical, histopathological, and molecular levels. Genomic instability and greater mutation rates, which may result in the creation of neoantigens and enhanced immunogenicity, are additional characteristics of this breast cancer type. Clinical outcome is poor due to early age of onset, high metastatic potential, and increased likelihood of distant recurrence. Consequently, efforts to elucidate molecular mechanisms of breast cancer development, progression, and metastatic spread have been initiated to improve treatment options and improve outcomes for these patients. The extremely complex and heterogeneous tumor immune microenvironment is made up of several cell types and commonly possesses disorganized gene expression. Altered signaling pathways are mainly associated with mutated genes including p53, PIK3CA, and MAPK, and which are positively correlated with genes regulating immune response. Of note, particular immunity-associated genes could be used in prognostic indexes to assess the most effective management. Recent findings highlight the fact that long non-coding RNAs also play an important role in shaping tumor microenvironment formation, and can mediate tumor immune evasion. Identification of molecular signatures, through the use of multi-omics approaches, and effector pathways that drive early stages of the carcinogenic process are important steps in developing new strategies for targeted cancer treatment and prevention. Advances in immunotherapy by remodeling the host immune system to eradicate tumor cells have great promise to lead to novel therapeutic strategies. Current research is focused on combining immune checkpoint inhibition with chemotherapy, PARP inhibitors, cancer vaccines, or natural killer cell therapy. Targeted therapies may improve therapeutic response, eliminate therapeutic resistance, and improve overall patient survival. In the future, these evolving advancements should be implemented for personalized medicine and state-of-art management of cancer patients. [ABSTRACT FROM AUTHOR]

An important approach to molecular diagnostics is integrating organized substances that provide complex molecular level responses to introduced chemical and biological agents with conditions that optimize and distinguish such responses. In this respect, liquid crystal dispersions are attractive components of molecular diagnostic tools. This paper analyzes a colloid system, containing a nematic liquid crystal as a dispersed phase, and aqueous surfactant and polymer solutions as the continuous phases. We applied a microfluidic approach for tuning orientation of liquid crystal molecules in picoliter droplets immobilized on microchannel walls. Introduction of surfactant to the aqueous phase was found to proportionally increase the order parameter of liquid crystal molecules in microdroplets. Infusion of polymer solutions into surfactant-mediated microfluidic liquid crystal dispersions increased the order parameter at much lower surfactant concentrations, while further infusion of surfactant solutions randomized the orientation of liquid crystal molecules. These effects were correlated with the adsorption of surfactant molecules on surfaces of microdroplets, stabilizing the effect of a polymer matrix on bound surfactant ions and the formation of insoluble polymer–colloid aggregates, respectively. The revealed molecular behavior of liquid crystal dispersions may contribute to optimized synthesis of responsive liquid crystal dispersions for in-flow molecular diagnostics of polymers and colloids, and the development of functional laboratory-on-chip prototypes.

Sarcoidosis is a complex inflammatory multisystem disease of unknown etiology that is characterised by epithelioid cell granulomatous lesions affecting various organs, mainly the lungs. In general, sarcoidosis is asymptomatic, but some cases result in severe complications and organ failure. So far, no accurate and validated modelling for clinical and pathohistological manifestations of sarcoidosis is suggested. Moreover, knowledge about disease-specific diagnostic markers for sarcoidosis is scarce. For instance, pulmonary granulomatosis is associated with the upregulated production of proinflammatory molecules: TNF-α, IL-6, CXCL1, CCL2, CCL18, CD163, serum angiotensin-converting enzyme (sACE), lysozyme, neopterin, and serum amyloid A (SAA). Quantum dots (QDs) are widely applied for molecular diagnostics of various diseases. QDs are semiconductor nanoparticles of a few nanometres in size, made from ZnS, CdS, ZnSe, etc., with unique physical and chemical properties that are useful for the labelling and detection in biological experiments. QDs can conjugate with various antibodies or oligonucleotides, allowing for high-sensitivity detection of various targets in organs and cells. Our review describes existing experimental models for sarcoidosis (in vitro, in vivo, and in silico), their advantages and restrictions, as well as the physical properties of quantum dots and their potential applications in the molecular diagnostics of sarcoidosis. The most promising experimental models include mice with TSC2 deletion and an in silico multiscale computational model of sarcoidosis (SarcoidSim), developed using transcriptomics and flow cytometry of human sarcoid biopsies. Both models are most efficient to test different candidate drugs for sarcoidosis.

Pathogenicity predictors are computational tools that classify genetic variants as benign or pathogenic; this is currently a major challenge in genomic medicine. With more than fifty such predictors available, selecting the most suitable tool for clinical applications like genetic screening, molecular diagnostics, and companion diagnostics has become increasingly challenging. To address this issue, we have developed a cost-based framework that naturally considers the various components of the problem. This framework encodes clinical scenarios using a minimal set of parameters and treats pathogenicity predictors as rejection classifiers, a common practice in clinical applications where low-confidence predictions are routinely rejected. We illustrate our approach in four examples where we compare different numbers of pathogenicity predictors for missense variants. Our results show that no single predictor is optimal for all clinical scenarios and that considering rejection yields a different perspective on classifiers.

Researchers active in the field of inflammatory skin diseases from the spectrum of dermatitis and eczema are well aware of a considerable overlap in the clinical pictures and proposed sets of diagnostic criteria for these diseases, which can hardly be overcome through the clinical or epidemiological research. In effect, patients are included in studies based on vague and overlapping criteria, while heterogeneous study populations may, in turn, lead to non-representative outcomes and continued confusion. In this narrative review, a systematics of diseases from the spectrum of dermatitis and eczema is proposed based on the origins of causative factors and the pathomechanisms involved. Difficulties in differentiating between these diseases are discussed, and the extent to which advances in the “omics” sciences might help to overcome them is considered. Of all the “omics” research in this field, more than 90% of the published papers were devoted to atopic dermatitis, with a striking underrepresentation of other diseases from the spectrum of dermatitis and eczema, conditions which collectively exceed the rates of atopic dermatitis by far. A greater “omics” research effort is urgently needed to tackle other dermatitides, like allergic, irritant and protein contact dermatitis, as well as radiation, seborrheic, stasis or autoimmune dermatitis. Atopic dermatitis findings should be validated not only against healthy donors but also other dermatitides. A clinic-oriented approach is proposed for future “omics” studies in the field of dermatitis and eczema.

Background: The detection rate of viral RNA in tick-borne encephalitis (TBE) is low and variable between studies, and its diagnostic/prognostic potential is not well defined. We attempted to detect RNA of TBE virus (TBEV) in body fluids of TBE patients. Methods: We studied 98 adults and 12 children with TBEV infection, stratified by the disease phase and presentation. EDTA blood and cerebrospinal fluid (CSF) samples were obtained upon hospital admission. RNA was extracted from freshly obtained plasma, concentrated leukocyte-enriched CSF, and whole blood samples, and real time PCR was performed with a Rotor-Gene Q thermocycler. Results: TBEV RNA was detected in (1) plasma of one (of the two studied) adult patients with an abortive infection, (2) plasma of two (of the two studied) adults in the peripheral phase of TBE, and (3) plasma and blood of an adult in the neurologic phase of TBE presenting as meningoencephalomyelitis. No CSF samples were TBEV RNA-positive. Conclusions: The detection of TBEV RNA in blood might be diagnostic in the peripheral phase of TBE. The lack of TBEV RNA in the CSF cellular fraction speaks against TBEV influx into the central nervous system with infiltrating leukocytes and is consistent with a relatively low intrathecal viral burden. [ABSTRACT FROM AUTHOR]

Oncogenic fusion genes have emerged as successful targets in several malignancies, such as chronic myeloid leukemia and lung cancer. Fusion of the fibroblast growth receptor 3 and the transforming acidic coiled coil containing protein—FGFR3-TACC3 fusion—is prevalent in 3–4% of human glioblastoma. The fusion protein leads to the constitutively activated kinase signaling of FGFR3 and thereby promotes cell proliferation and tumor progression. The subgroup of FGFR3-TACC3 fusion-positive glioblastomas presents with recurrent clinical and histomolecular characteristics, defining a distinctive subtype of IDH-wildtype glioblastoma. This review aims to provide an overview of the available literature on FGFR3-TACC3 fusions in glioblastoma and possible implications for actual clinical practice. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic resulted in a universal, immediate, and vast demand for comprehensive molecular diagnostic testing, especially real-time quantitative (qPCR)-based methods. This rapidly triggered a global shortage of testing capacity, equipment, and reagents. Even today, supply times for chemicals from date of order to delivery are often much longer than pre-pandemic. Furthermore, many companies have ratcheted up the price for minimum volumes of reaction master mixes essential for qPCR assays, causing additional problems for academic laboratories often operating on a shoestring. We have validated two strategies that stretch reagent supplies and, whilst particularly applicable in case of scarcity, can readily be incorporated into standard qPCR protocols, with appropriate validation. The first strategy demonstrates equivalent performance of a selection of "past expiry date" and newly purchased master mixes. This approach is valid for both standard and fast qPCR protocols. The second validates the use of these master mixes at less than 1x final concentration without loss of qPCR efficiency or sensitivity. [ABSTRACT FROM AUTHOR]

Polymicrobial sepsis is associated with worse patient outcomes than monomicrobial sepsis. Routinely used culture-dependent microbiological diagnostic techniques have low sensitivity, often leading to missed identification of all causative organisms. To overcome these limitations, culture-independent methods incorporating advanced molecular technologies have recently been explored. However, contamination, assay inhibition and interference from host DNA are issues that must be addressed before these methods can be relied on for routine clinical use. While the host component of the complex sepsis host–pathogen interplay is well described, less is known about the pathogen's role, including pathogen–pathogen interactions in polymicrobial sepsis. This review highlights the clinical significance of polymicrobial sepsis and addresses how promising alternative molecular microbiology methods can be improved to detect polymicrobial infections. It also discusses how the application of shotgun metagenomics can be used to uncover pathogen/pathogen interactions in polymicrobial sepsis cases and their potential role in the clinical course of this condition. [ABSTRACT FROM AUTHOR]

Helicobacter pylori infections, as one of the most prevalent among humans, are generally acquired during childhood, and are one of the main causes of chronic gastritis and peptic ulcer disease. A bacterial culture from a gastric biopsy is the gold standard and is the only method that has 100% specificity. However, its sensitivity varies, depending on experience of the laboratory staff, applied culture media, specimen transport conditions, biopsy site, and quality of the sample. The same factors compromise all invasive methods and a culture-based H. pylori infection diagnostic, as well as a recent intake of antibiotics, bismuth-containing compounds, and proton pump inhibitors. Molecular methods have been used for clinical microbiology investigation since the beginning of the 21st century. However, their usefulness for H. pylori infections diagnosis remains unclear, especially in pediatric patients. The aim of the study was to assess the incidence of H. pylori infections in a group of 104 pediatric patients and to compare the results of the PCR test with the corresponding histopathological investigation effects. Among the biopsy samples collected from 104 children, 44 (42.3%) were positive in PCR, while 43 (41.3%) and 39 (37.5%) presented histologically-confirmed signs of inflammation and H. pylori colonization, respectively. Moreover, the mean grades of the parameters of the histopathological examination were higher in the group of PCR-positive samples. The compatibility of both research methods was confirmed, emphasizing the usefulness of molecular methods for detecting H. pylori infections in pediatric patients. Considering that the PCR-based method gives reliable results and is less time-consuming and costly, it is worth discussing this method as a new standard in the diagnosis of H. pylori infections, at least among pediatric patients, for which culture-based diagnostics is not sufficient or histopathological examination is negative, while inflammation signs are observed macroscopically.

The histological diagnosis of sarcoma can be difficult as it sometimes requires the combination of morphological and immunophenotypic analyses with molecular tests. A total of 2705 tissue samples of sarcoma consecutively collected from 2006 until 2020 that had undergone molecular analysis were assessed to evaluate their diagnostic utility compared with histological assessments. A total of 3051 molecular analyses were performed, including 1484 gene fusions tested by c/qRT–PCR, 992 gene rearrangements analysed by FISH, 433 analyses of the gene status of MDM2, 126 mutational analyses and 16 NGS analysis. Of the samples analysed, 68% were from formalin-fixed, paraffin-embedded tissue and 32% were from frozen tissue. C/qRT–PCR and FISH analyses were conclusive on formalin-fixed, paraffin-embedded tissue in 74% and 76% of samples, respectively, but the combination of the two methods gave us conclusive results in 96% and 89% of frozen and formalin-fixed, paraffin-embedded tissues, respectively. We demonstrate the utility of c/qRT–PCR and FISH for sarcoma diagnosis and that each has advantages in specific contexts. We conclude that it is possible to accurately predict the sarcoma subtype using a panel of different subtype-specific FISH probes and c/qRT–PCR assays, thereby greatly facilitating the differential diagnosis of these tumours.

Heterogeneity of triple-negative breast cancer is well known at clinical, histopathological, and molecular levels. Genomic instability and greater mutation rates, which may result in the creation of neoantigens and enhanced immunogenicity, are additional characteristics of this breast cancer type. Clinical outcome is poor due to early age of onset, high metastatic potential, and increased likelihood of distant recurrence. Consequently, efforts to elucidate molecular mechanisms of breast cancer development, progression, and metastatic spread have been initiated to improve treatment options and improve outcomes for these patients. The extremely complex and heterogeneous tumor immune microenvironment is made up of several cell types and commonly possesses disorganized gene expression. Altered signaling pathways are mainly associated with mutated genes including p53, PIK3CA, and MAPK, and which are positively correlated with genes regulating immune response. Of note, particular immunity-associated genes could be used in prognostic indexes to assess the most effective management. Recent findings highlight the fact that long non-coding RNAs also play an important role in shaping tumor microenvironment formation, and can mediate tumor immune evasion. Identification of molecular signatures, through the use of multi-omics approaches, and effector pathways that drive early stages of the carcinogenic process are important steps in developing new strategies for targeted cancer treatment and prevention. Advances in immunotherapy by remodeling the host immune system to eradicate tumor cells have great promise to lead to novel therapeutic strategies. Current research is focused on combining immune checkpoint inhibition with chemotherapy, PARP inhibitors, cancer vaccines, or natural killer cell therapy. Targeted therapies may improve therapeutic response, eliminate therapeutic resistance, and improve overall patient survival. In the future, these evolving advancements should be implemented for personalized medicine and state-of-art management of cancer patients.

Background: The detection rate of viral RNA in tick-borne encephalitis (TBE) is low and variable between studies, and its diagnostic/prognostic potential is not well defined. We attempted to detect RNA of TBE virus (TBEV) in body fluids of TBE patients. Methods: We studied 98 adults and 12 children with TBEV infection, stratified by the disease phase and presentation. EDTA blood and cerebrospinal fluid (CSF) samples were obtained upon hospital admission. RNA was extracted from freshly obtained plasma, concentrated leukocyte-enriched CSF, and whole blood samples, and real time PCR was performed with a Rotor-Gene Q thermocycler. Results: TBEV RNA was detected in (1) plasma of one (of the two studied) adult patients with an abortive infection, (2) plasma of two (of the two studied) adults in the peripheral phase of TBE, and (3) plasma and blood of an adult in the neurologic phase of TBE presenting as meningoencephalomyelitis. No CSF samples were TBEV RNA-positive. Conclusions: The detection of TBEV RNA in blood might be diagnostic in the peripheral phase of TBE. The lack of TBEV RNA in the CSF cellular fraction speaks against TBEV influx into the central nervous system with infiltrating leukocytes and is consistent with a relatively low intrathecal viral burden.

The COVID-19 pandemic resulted in a universal, immediate, and vast demand for comprehensive molecular diagnostic testing, especially real-time quantitative (qPCR)-based methods. This rapidly triggered a global shortage of testing capacity, equipment, and reagents. Even today, supply times for chemicals from date of order to delivery are often much longer than pre-pandemic. Furthermore, many companies have ratcheted up the price for minimum volumes of reaction master mixes essential for qPCR assays, causing additional problems for academic laboratories often operating on a shoestring. We have validated two strategies that stretch reagent supplies and, whilst particularly applicable in case of scarcity, can readily be incorporated into standard qPCR protocols, with appropriate validation. The first strategy demonstrates equivalent performance of a selection of “past expiry date” and newly purchased master mixes. This approach is valid for both standard and fast qPCR protocols. The second validates the use of these master mixes at less than 1x final concentration without loss of qPCR efficiency or sensitivity.

Polymicrobial sepsis is associated with worse patient outcomes than monomicrobial sepsis. Routinely used culture-dependent microbiological diagnostic techniques have low sensitivity, often leading to missed identification of all causative organisms. To overcome these limitations, culture-independent methods incorporating advanced molecular technologies have recently been explored. However, contamination, assay inhibition and interference from host DNA are issues that must be addressed before these methods can be relied on for routine clinical use. While the host component of the complex sepsis host–pathogen interplay is well described, less is known about the pathogen’s role, including pathogen–pathogen interactions in polymicrobial sepsis. This review highlights the clinical significance of polymicrobial sepsis and addresses how promising alternative molecular microbiology methods can be improved to detect polymicrobial infections. It also discusses how the application of shotgun metagenomics can be used to uncover pathogen/pathogen interactions in polymicrobial sepsis cases and their potential role in the clinical course of this condition.

Reverse transcription of RNA coupled to amplification of the resulting cDNA by the polymerase chain reaction (RT-PCR) is one of the principal molecular technologies in use today, with applications across all areas of science and medicine. In its real-time, fluorescence-based usage (RT-qPCR), it has long been a core technology driving the accurate, rapid and sensitive laboratory diagnosis of infectious diseases. However, RT-qPCR protocols have changed little over the past 30 years, with the RT step constituting a significant percentage of the time taken to complete a typical RT-qPCR assay. When applied to research investigations, reverse transcription has been evaluated by criteria such as maximum yield, length of transcription, fidelity, and faithful representation of an RNA pool. Crucially, however, these are of less relevance in a diagnostic RT-PCR test, where speed and sensitivity are the prime RT imperatives, with specificity contributed by the PCR component. We propose a paradigm shift that omits the requirement for a separate high-temperature RT step at the beginning of an RT-qPCR assay. This is achieved by means of an innovative protocol that incorporates suitable reagents with a revised primer and amplicon design and we demonstrate a proof of principle that incorporates the RT step as part of the PCR assay setup at room temperature. Use of this modification as part of a diagnostic assay will of course require additional characterisation, validation and optimisation of the PCR step. Combining this revision with our previous development of fast qPCR protocols allows completion of a 40 cycle RT-qPCR run on a suitable commercial instrument in approximately 15 min. Even faster times, in combination with extreme PCR procedures, can be achieved.

Recently, several studies focused on the genetics of gliomas. This allowed identifying several germline loci that contribute to individual risk for tumor development, as well as various somatic mutations that are key for disease classification. Unfortunately, none of the germline loci clearly confers increased risk per se. Contrariwise, somatic mutations identified within the glioma tissue define tumor genotype, thus representing valid diagnostic and prognostic markers. Thus, genetic features can be used in glioma classification and guided therapy. Such copious genomic variabilities are screened routinely in glioma diagnosis. In detail, Sanger sequencing or pyrosequencing, fluorescence in-situ hybridization, and microsatellite analyses were added to immunohistochemistry as diagnostic markers. Recently, Next Generation Sequencing was set-up as an all-in-one diagnostic tool aimed at detecting both DNA copy number variations and mutations in gliomas. This approach is widely used also to detect circulating tumorDNAwithin cerebrospinal fluid from patients affected by primary brain tumors. Such an approach is providing an alternative cost-effective strategy to genotype all gliomas, which allows avoiding surgical tissue collection and repeated tumor biopsies. This review summarizes available molecular features that represent solid tools for the genetic diagnosis of gliomas at present or in the next future. [ABSTRACT FROM AUTHOR]

Microsatellite instability (MSI), the spontaneous loss or gain of nucleotides from repetitive DNA tracts, is a diagnostic phenotype for gastrointestinal, endometrial, colorectal, and bladder cancers; yet a landscape of instability events across a wider variety of cancer types is beginning to be discovered. The epigenetic inactivation of the MLH1 gene is often associated with sporadic MSI cancers. Recent next-generation sequencing (NGS)-based analyses have comprehensively characterized MSI-positive (MSI+) cancers, and several approaches to the detection of the MSI phenotype of tumors using NGS have been developed. Bladder cancer (here we refer to transitional carcinoma of the bladder) is a major cause of morbidity and mortality in the Western world. Cystoscopy, a gold standard for the detection of bladder cancer, is invasive and sometimes carries unwanted complications, while its cost is relatively high. Urine cytology is of limited value due to its low sensitivity, particularly to low-grade tumors. Therefore, over the last two decades, several new “molecular assays” for the diagnosis of urothelial cancer have been developed. Here, we provide an update on the development of a microsatellite instability assay (MSA) and the development of MSA associated with bladder cancers, focusing on findings obtained from urine analysis from bladder cancer patients as compared with individuals without bladder cancer. In our review, based on over 18 publications with approximately 900 sample cohorts, we provide the sensitivity (87% to 90%) and specificity (94% to 98%) of MSA. We also provide a comparative analysis between MSA and other assays, as well as discussing the details of four different FDA-approved assays. We conclude that MSA is a potentially powerful test for bladder cancer detection and may improve the quality of life of bladder cancer patients.

Prostate cancer (PCa) in dogs is a highly malignant disease akin to its human counterpart. In contrast to the situation in humans, multi-gene approaches facilitating risk stratification of canine PCa are barely established. The aims of this study were the characterization of the transcriptional landscape of canine PCa and the identification of diagnostic, prognostic and/or therapeutic biomarkers through a multi-step screening approach. RNA-Sequencing of ten malignant tissues and fine-needle aspirations (FNA), and 14 nonmalignant tissues and FNAs was performed to find differentially expressed genes (DEGs) and deregulated pathways. The 4098 observed DEGs were involved in 49 pathways. These 49 pathways could be grouped into five superpathways summarizing the hallmarks of canine PCa: (i) inflammatory response and cytokines; (ii) regulation of the immune system and cell death; (iii) cell surface and PI3K signaling; (iv) cell cycle; and (v) phagosome and autophagy. Among the highly deregulated, moderately to strongly expressed DEGs that were members of one or more superpathways, 169 DEGs were listed in relevant databases and/or the literature and included members of the PCa pathway, oncogenes, prostate-specific genes, and druggable genes. These genes are novel and promising candidate diagnostic, prognostic and/or therapeutic canine PCa biomarkers.

Background and case: An adolescent male presented with a second mediastinal tumor 1.5 years after treatment of a proven malignant germ-cell tumor in that location. The differential diagnosis included a recurrent germ-cell tumor or a non-germ cell malignancy. Serum tumor markers alpha-fetoprotein (AFP) and human chorionic gonadotrophin (HCG) were negative. The first biopsy was not informative, and the second biopsy gave a broad differential diagnosis including secondary non-germ cell malignancy using histology and immunohistochemistry. DNA methylation profiling, RNA sequencing, and targeted microRNA371a-3p profiling was subsequently performed, without a supportive result. After resection of the tumor the definitive diagnosis yielded two secondary non-germ cell malignancies in the form of a leiomyosarcoma and a solitary neuro endocrine carcinoma (NEC). In spite of the differences between the molecular profiles of the initial germ-cell tumor, the leiomyosarcoma and large-cell NEC are clonally related, as determined by the presence of identical chromosomal breakpoints. The copy number profiles suggest an initial polyploidization step, followed by various independent chromosomal gains and losses. This case demonstrates that germ-cell tumors must be evaluated carefully, including molecularly, in which the non-germ cell malignancy is negative for miR-371a-3p, both in tissue as well as in serum, in contrast to the primary tumor. We conclude that the patient presented with a primary type II mediastinal GCT and, a year and a half later, followed by a leiomyosarcoma and a large-cell NEC presenting as two secondary somatic-type malignancies clonally related to the original GCT. Conclusions: Malignant germ-cell tumors are known to recur as a somatic-type malignancy in very rare cases. This case report illustrates the challenges faced in defining the nature and clonality of the secondary somatic-type malignancies.

The increased cultivation of Cannabis sativa L. in North America, represented by high Δ 9 -tetrahydrocannabinol-containing (high-THC) cannabis genotypes and low-THC-containing hemp genotypes, has been impacted by an increasing number of plant pathogens. These include fungi which destroy roots, stems, and leaves, in some cases causing a build-up of populations and mycotoxins in the inflorescences that can negatively impact quality. Viroids and viruses have also increased in prevalence and severity and can reduce plant growth and product quality. Rapid diagnosis of the occurrence and spread of these pathogens is critical. Techniques in the area of molecular diagnostics have been applied to study these pathogens in both cannabis and hemp. These include polymerase chain reaction (PCR)-based technologies, including RT-PCR, multiplex RT-PCR, RT-qPCR, and ddPCR, as well as whole-genome sequencing (NGS) and bioinformatics. In this study, examples of how these technologies have enhanced the rapidity and sensitivity of pathogen diagnosis on cannabis and hemp will be illustrated. These molecular tools have also enabled studies on the diversity and origins of specific pathogens, specifically viruses and viroids, and these will be illustrated. Comparative studies on the genomics and metabolomics of healthy and diseased plants are urgently needed to provide insight into their impact on the quality and composition of cannabis and hemp-derived products. Management of these pathogens will require monitoring of their spread and survival using the appropriate technologies to allow accurate detection, followed by appropriate implementation of disease control measures.

The diagnosis of neuromuscular diseases (NMDs) has been progressively evolving from the grouping of clinical symptoms and signs towards the molecular definition. Optimal clinical, biochemical, electrophysiological, electrophysiological, and histopathological characterization is very helpful to achieve molecular diagnosis, which is essential for establishing prognosis, treatment and genetic counselling. Currently, the genetic approach includes both the gene-targeted analysis in specific clinically recognizable diseases, as well as genomic analysis based on next-generation sequencing, analyzing either the clinical exome/genome or the whole exome or genome. However, as of today, there are still many patients in whom the causative genetic variant cannot be definitely established and variants of uncertain significance are often found. In this review, we address these drawbacks by incorporating two additional biological omics approaches into the molecular diagnostic process of NMDs. First, functional genomics by introducing experimental cell and molecular biology to analyze and validate the variant for its biological effect in an in-house translational diagnostic program, and second, incorporating a multi-omics approach including RNA-seq, metabolomics, and proteomics in the molecular diagnosis of neuromuscular disease. Both translational diagnostics programs and omics are being implemented as part of the diagnostic process in academic centers and referral hospitals and, therefore, an increase in the proportion of neuromuscular patients with a molecular diagnosis is expected. This improvement in the process and diagnostic performance of patients will allow solving aspects of their health problems in a precise way and will allow them and their families to take a step forward in their lives.

A rapid, sensitive and simple point-of-care (POC) nucleic acid diagnostic test is needed to prevent spread of infectious diseases. Paper-based toehold reaction, a recently emerged colorimetric POC nucleic acid diagnostic test, has been widely used for pathogen detection and microbiome profiling. Here, we introduce an amplification method called reverse transcription loop-mediated amplification (RT-LAMP) prior to the toehold reaction and modify it to enable more sensitive and faster colorimetric detection of RNA viruses. We show that incorporating the modified RT-LAMP to the toehold reaction detects as few as 120 copies of coronavirus RNA in 70 min. Cross-reactivity test against other coronaviruses indicates this toehold reaction with the modified RT-LAMP is highly specific to the target RNA. Overall, the paper-based toehold switch sensors with the modified RT-LAMP allow fast, sensitive, specific and colorimetric coronavirus detection.

Although molecular testing, and RT-qPCR in particular, has been an indispensable component in the scientific armoury targeting SARS-CoV-2, there are numerous falsehoods, misconceptions, assumptions and exaggerated expectations with regards to capability, performance and usefulness of the technology. It is essential that the true strengths and limitations, although publicised for at least twenty years, are restated in the context of the current COVID-19 epidemic. The main objective of this commentary is to address and help stop the unfounded and debilitating speculation surrounding its use.

Riboswitches and toehold switches are considered to have potential for implementation in various fields, i.e., biosensing, metabolic engineering, and molecular diagnostics. The specific binding, programmability, and manipulability of these RNA-based molecules enable their intensive deployments in molecular detection as biosensors for regulating gene expressions, tracking metabolites, or detecting RNA sequences of pathogenic microorganisms. In this review, we will focus on the development of riboswitches and toehold switches in biosensing and molecular diagnostics. This review introduces the operating principles and the notable design features of riboswitches as well as toehold switches. Moreover, we will describe the advances and future directions of riboswitches and toehold switches in biosensing and molecular diagnostics.

Inter-patient molecular heterogeneity is the major declared driver of an expanding variety of anticancer drugs and personalizing their prescriptions. Here, we compared interpatient molecular heterogeneities of tumors and repertoires of drugs or their molecular targets currently in use in clinical oncology. We estimated molecular heterogeneity using genomic (whole exome sequencing) and transcriptomic (RNA sequencing) data for 4890 tumors taken from The Cancer Genome Atlas database. For thirteen major cancer types, we compared heterogeneities at the levels of mutations and gene expression with the repertoires of targeted therapeutics and their molecular targets accepted by the current guidelines in oncology. Totally, 85 drugs were investigated, collectively covering 82 individual molecular targets. For the first time, we showed that the repertoires of molecular targets of accepted drugs did not correlate with molecular heterogeneities of different cancer types. On the other hand, we found that the clinical recommendations for the available cancer drugs were strongly congruent with the gene expression but not gene mutation patterns. We detected the best match among the drugs usage recommendations and molecular patterns for the kidney, stomach, bladder, ovarian and endometrial cancers. In contrast, brain tumors, prostate and colorectal cancers showed the lowest match. These findings provide a theoretical basis for reconsidering usage of targeted therapeutics and intensifying drug repurposing efforts.

Recently, several studies focused on the genetics of gliomas. This allowed identifying several germline loci that contribute to individual risk for tumor development, as well as various somatic mutations that are key for disease classification. Unfortunately, none of the germline loci clearly confers increased risk per se. Contrariwise, somatic mutations identified within the glioma tissue define tumor genotype, thus representing valid diagnostic and prognostic markers. Thus, genetic features can be used in glioma classification and guided therapy. Such copious genomic variabilities are screened routinely in glioma diagnosis. In detail, Sanger sequencing or pyrosequencing, fluorescence in-situ hybridization, and microsatellite analyses were added to immunohistochemistry as diagnostic markers. Recently, Next Generation Sequencing was set-up as an all-in-one diagnostic tool aimed at detecting both DNA copy number variations and mutations in gliomas. This approach is widely used also to detect circulating tumor DNA within cerebrospinal fluid from patients affected by primary brain tumors. Such an approach is providing an alternative cost-effective strategy to genotype all gliomas, which allows avoiding surgical tissue collection and repeated tumor biopsies. This review summarizes available molecular features that represent solid tools for the genetic diagnosis of gliomas at present or in the next future.

Isothermal nucleic-acid amplification methods such as Loop-Mediated isothermal AMPlification (LAMP) are increasingly appealing alternatives to PCR for use in portable diagnostic system due to the low cost, weight, and power requirements of the instrumentation. As such, interest in developing new probes and other functionality based on the LAMP reaction has been intense. Here, we report on the development of duplexed LAMP assays for pathogen detection using spectrally unique Assimilating Probes. As proof of principle, we used a reaction for Salmonella enterica as a model coupled with a reaction for λ-phage DNA as an internal control, as well as a duplexed assay to sub-type specific quarantine strains of the bacterial wilt pathogen Ralstonia solanacearum. Detection limits for bacterial DNA analyzed in individual reactions was less than 100 genomic equivalents in all cases, and increased by one to two orders of magnitude when reactions were coupled in duplexed formats. Even so, due to the more robust activity of newly available strand-displacing polymerases, the duplexed assays reported here were more powerful than analogous individual reactions reported only a few years ago, and represent a significant advance for incorporation of internal controls to validate assay results in the field.