Molecular Diagnosis Research Articles

Application of cell-free DNA fungal polymerase chain reaction for invasive fungal disease evaluation in pediatric oncology and stem cell transplant patients.

Molecular diagnostics may enable early, noninvasive detection of invasive fungal disease (IFD) in immunocompromised patients. Cell-free deoxyribonucleic acid (cfDNA) fungal polymerase chain reaction (PCR) assays were recently incorporated into institutional prolonged febrile neutropenia pathways. We aimed to evaluate the performance of plasma cfDNA PCR panels (mold and Candida panels) in pediatric oncology and hematopoietic stem cell transplant (HSCT) patients with clinical concern for IFD. This single-center, observational study assessed plasma cfDNA fungal PCR performance for noninvasive IFD detection in hospitalized pediatric oncology and HSCT patients. The primary outcome was IFD diagnosis per published consensus definitions within 1month. Positive and negative agreement between plasma cfDNA fungal PCR and consensus definitions were calculated. We also described test turnaround time and patient survival. From October 2021 to 2022, 54 patients underwent 60 evaluations with 11 proven/probable IFD cases. Comparing plasma cfDNA fungal PCRs to consensus definitions for proven/probable IFD, there was 73% positive agreement and 96% negative agreement. Two proven/probable cases with negative PCRs were caused by organisms not included on either panel. Median time to cfDNA fungal PCR result was 35hours (interquartile range: 19-69) in eight proven/probable cases detected by cfDNA fungal PCR. There were 17 deaths among 54 patients, and IFD contributed to 45% of deaths in patients with proven/probable IFD. Plasma cfDNA fungal PCRs detected relevant molds or yeast in most cases classified as proven/probable IFD. However, this targeted approach missed some cases. More studies are required to determine optimal utilization of molecular diagnostics in pediatric patients.

Innovation and Patenting Activities During COVID-19 and Advancement of Biochemical and Molecular Diagnosis in the Post- COVID-19 Era.

The COVID-19 pandemic is to escalate globally and acquire new mutations quickly, so accurate diagnostic technologies play a vital role in controlling and understanding the epidemiology of the disease. A plethora of technologies acquires diagnosis of individuals and informs clinical management of COVID. Some important biochemical parameters for COVID diagnosis are the elevation of liver enzymes, creatinine, and nonspecific inflammatory markers such as C-reactive protein (CRP) and Interleukin 6 (IL-6). The main progression predictors are lymphopenia, elevated D-dimer, and hyperferritinemia, although it is also necessary to consider LDH, CPK, and troponin in the marker panel of diagnosis. Owing to the greater sensitivity and accuracy, molecular technologies such as conventional polymerase chain reaction (PCR), reverse transcription (RT)-PCR, nested PCR, loop-mediated isothermal amplification (LAMP), and xMAP technology have been extensively used for COVID diagnosis for some time now. To make so many diagnostics accessible to general people, many techniques may be exploited, including point of care (POC), also called bedside testing, which is developing as a portable promising tool in pathogen identification. Some other lateral flow assay (LFA)-centered techniques like SHERLOCK, CRISPR-Cas12a (AIOD-CRISPR), and FNCAS9 editor limited uniform detection assay (FELUDA), etc. have shown auspicious results in the rapid detection of pathogens. More recently, low-cost sequencing and advancements in big data management have resulted in a slow but steady rise of next-generation sequencing (NGS)-based approaches for diagnosis that have potential relevance for clinical purposes and may pave the way toward a better future. Due to the COVID-19 pandemic, various institutions provided free, specialized websites and tools to promote research and access to critically needed advanced solutions by alleviating research and analysis of data within a substantial body of scientific and patent literature regarding biochemical and molecular diagnosis published since January 2020. This circumstance is unquestionably unique and difficult for anyone using patent information to find pertinent disclosures at a specific date in a trustworthy manner.

Identifying Genetic Mutations in Vascular Anomalies Using a Sequencing Panel for Childhood Cancers: A Pilot Study

Introduction: Genetic mutations have been identified in the pathogenesis of vascular anomalies. Due to overlaps in genetic variants causing vascular anomalies and cancer, we used a next-generation sequencing panel for genomic profiling of childhood cancers to detect somatic mutations in children with vascular anomalies. We aim to review the utility of an oncology panel for the molecular diagnosis of vascular anomalies. Methods: Nine patients with histologically confirmed vascular anomalies were included. DNA was extracted from formalin-fixed paraffin-embedded tissue specimens obtained from affected tissue following diagnostic punch biopsies of the skin and core biopsies of the vascular malformation or tumor during sclerotherapy or surgical excision. Molecular analysis of the tissue samples was performed using AmpliSeq for Childhood Cancer DNA Assay Panel. Results: Two patients had antenatally detected vascular anomalies. The median age at diagnosis for the remaining patients was 7.0 years (IQR, 0.6–10.0 years). Seven were diagnosed with vascular malformations, while 2 had vascular tumors. Pathological somatic mutations were identified in 4 patients, leading to a diagnostic yield of 44.4%. Two different PIK3CA mutations were identified in 3 cases: 1 in a case of macrocystic lymphatic malformation, the other in a case of Congenital Lipomatous Overgrowth, Vascular malformations, Epidermal nevus, Spinal/Skeletal anomalies syndrome and Klippel–Trenaunay syndrome. BRAF mutation was identified in a patient with a veno-lymphatic malformation. Conclusion: An oncology next-generation sequencing panel can be used for genetic profiling of vascular anomalies. However, a more customized and sensitive panel may be of better diagnostic yield, as detection of somatic mutations in vascular anomalies is challenging due to tissue mosaicism, low-abundant genetic variants, and specimen limitations.

Clinical, Histopathologic, and Genetic Features of Patients With Myofibrillary and Distal Myopathies: Experience From the Italian Network.

The diagnostic process for myofibrillar myopathies (MFM) and distal myopathies (DM) is particularly complex because of the large number of causative genes, the existence of still molecularly undefined disease entities, and the overlapping features between the 2 categories. This study aimed to characterize a large cohort of patients affected by MFM and DM and identify the most important diagnostic and prognostic aspects of these diseases. Patients with either a myopathological diagnosis of MFM or a clinical diagnosis of DM were included in this retrospective multicentric national study. Demographic, genetic, clinical, and histopathologic data of anonymized patients were collected from the neuromuscular centers of the Italian Association of Myology network. Data regarding 132 patients with MFM (mean age 57.0 ± 15.8 years, 49% female) and 298 patients with DM (mean age 50.7 ± 15.9 years, 40% female) were gathered from 20 neuromuscular centers. 69 patients fulfilled the criteria for both groups (distal myopathies with myofibrillar pathology, DM-MP). Molecular confirmation was achieved in 63% of the patients. Fifty-two percent of the patients with MFM carried pathogenic variants in either DES (n = 30), MYOT (n = 20), or DNAJB6 (n = 18), which were also the most frequent disease-causing genes in DM-MP, while GNE (n = 44) and MYH7 (n = 23) were the genes most commonly carrying pathogenic variants in DM. The mean age at onset varied from <25 years in patients with causative variants in MYH7 and DYSF to 59 years in patients with myotilinopathies. Cardiac involvement was reported in 29% of patients with MFM and 16% of patients with DM, with DES and MYH7 variants significantly associated with the development of cardiomyopathy. Respiratory impairment was more prevalent in patients with TTN and DES variants and rare in other disorders such as GNE myopathy and dysferlinopathies, which were instead associated, together with DNAJB6-related and PLIN4-related myopathies, with the risk of losing ambulation during the disease course. The Italian cohort of patients with MFM and DM recapitulates the phenotypic heterogeneity and the partial overlap between the 2 groups. However, in relative contrast to the encountered phenotypic variability, only 5 genes accounted for most of the molecular diagnoses. Specific genetic entities are associated with significantly increased risk of developing cardiorespiratory complications or loss of ambulation, which has relevant prognostic implications.

Artificial intelligence algorithm for neoplastic cell percentage estimation and its application to copy number variation in urinary tract cancer.

Bladder cancer is characterized by frequent mutations, which provide potential therapeutic targets for most patients. The effectiveness of emerging personalized therapies depends on an accurate molecular diagnosis, for which the accurate estimation of the neoplastic cell percentage (NCP) is a crucial initial step. However, the established method for determining the NCP, manual counting by a pathologist, is time-consuming and not easily executable. To address this, artificial intelligence (AI) models were developed to estimate the NCP using nine convolutional neural networks and the scanned images of 39 cases of urinary tract cancer. The performance of the AI models was compared to that of six pathologists for 119 cases in the validation cohort. The ground truth value was obtained through multiplexed immunofluorescence. The AI model was then applied to 41 cases in the application cohort that underwent next-generation sequencing testing, and its impact on the copy number variation (CNV) was analyzed. Each AI model demonstrated high reliability, with intraclass correlation coefficients (ICCs) ranging from 0.82 to 0.88. These values were comparable or better to those of pathologists, whose ICCs ranged from 0.78 to 0.91 in urothelial carcinoma cases, both with and without divergent differentiation/ subtypes. After applying AI-driven NCP, 190 CNV (24.2%) were reclassified with 66 (8.4%) and 78 (9.9%) moved to amplification and loss, respectively, from neutral/minor CNV. The neutral/minor CNV proportion decreased by 6%. These results suggest that AI models could assist human pathologists in repetitive and cumbersome NCP calculations.

Whole genome sequencing enhances molecular diagnosis of primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a genetic disorder affecting motile cilia. Most cases are inherited recessively, due to variants in >50 genes that result in abnormal or absent motile cilia. This leads to chronic upper and lower airway disease, subfertility, and laterality defects. Given overlapping clinical features and genetic heterogeneity, diagnosis can be difficult and often occurs late. Of those tested an estimated 30% of genetically screened PCD patients still lack a molecular diagnosis. A molecular diagnosis allows for appropriate clinical management including prediction of phenotypic features correlated to genotype. Here, we aimed to identify how readily a genetic diagnosis could be made using whole genome sequencing (WGS) to facilitate identification of pathogenic variants in known genes as well as novel PCD candidate genes. WGS was used to screen for pathogenic variants in eight patients with PCD. 7/8 cases had homozygous or biallelic variants in DNAH5, DNAAF4 or DNAH11 classified as pathogenic or likely pathogenic. Three identified variants were deletions, ranging from 3 to 13 kb, for which WGS identified precise breakpoints, permitting confirmation by Sanger sequencing. WGS yielded identification of a de novo variant in a novel PCD gene TUBB4B. Here, WGS uplifted genetic diagnosis of PCD by identifying structural variants and novel modes of inheritance in new candidate genes. WGS could be an important component of the PCD diagnostic toolkit, increasing molecular diagnostic yield from current (70%) levels, and enhancing our understanding of fundamental biology of motile cilia and variants in the noncoding genome.

Harnessing the power of clustered regularly interspaced short palindromic repeats (CRISPR) based microfluidics for next-generation molecular diagnostics.

CRISPR-based (Clustered regularly interspaced short palindromic repeats-based) technologies have revolutionized molecular biology and diagnostics, offering unprecedented precision and versatility. However, challenges remain, such as high costs, demanding technical expertise, and limited quantification capabilities. To overcome these limitations, innovative microfluidic platforms are emerging as powerful tools for enhancing CRISPR diagnostics. This review explores the exciting intersection of CRISPR and microfluidics, highlighting their potential to revolutionize healthcare diagnostics. By integrating CRISPR's specificity with microfluidics' miniaturization and automation, researchers are developing more sensitive and portable diagnostic tools for a range of diseases. These microfluidic devices streamline sample processing, improve diagnostic performance, and enable point-of-care applications, allowing for rapid and accurate detection of pathogens, genetic disorders, and other health conditions. The review discusses various CRISPR/Cas systems, including Cas9, Cas12, and Cas13, and their integration with microfluidic platforms. It also examines the advantages and limitations of these systems, highlighting their potential for detecting DNA and RNA biomarkers. The review also explores the key challenges in developing and implementing CRISPR-driven microfluidic diagnostics, such as ensuring robustness, minimizing cross-contamination, and achieving robust quantification. Finally, it highlights potential future directions for this rapidly evolving field, emphasizing the transformative potential of these technologies for personalized medicine and global health.

Bridging dermatology and hematology: a case of lepromatous leprosy with bone marrow involvement and pancytopenia.

Leprosy, caused by Mycobacterium leprae (M. leprae), primarily manifestswith cutaneous and peripheral nerve involvement. Systemic involvement, particularly in the bone marrow, is exceedingly rare. This report presents a case of lepromatous leprosy with bone marrow involvement, emphasizing the systemic nature of the disease and the importance of comprehensive diagnostic and management approaches. We aim to present a case of lepromatous leprosy with bone marrow involvement, detailing the clinical presentation, diagnostic evaluation, and management approach. A 65-year-old male with lepromatous leprosy and severe erythema nodosum leprosum developed pancytopenia. After undergoing comprehensive clinical evaluation, including history taking, physical examination, and laboratory investigations, bone marrow examination and molecular diagnostics using polymerase chain reaction (PCR) were performed to confirm the presence of M. leprae as an etiology for his pancytopenia. The bone marrow aspirate revealed hypercellularity with erythropoiesis and thrombopoiesis within normal limits. Foamy histiocytes with erythrophagocytosis were observed, along with the presence of M. leprae on Modified Ziehl-Neelsen stain. Molecular analysis confirmed M. leprae DNA in the bone marrow aspirate. Treatment with multi-drug therapy (MDT) and thalidomide resulted in normalization of blood counts and healing of skin lesions. This case underscores the systemic nature of leprosy and the rarity of bone marrow involvement, highlighting the importance of thorough evaluation in cases of persistent symptoms. Comprehensive diagnostic approaches, including bone marrow examination and molecular diagnostics, are essential for accurate diagnosis and timely initiation of appropriate treatment, ultimately improving patient outcomes and minimizing disease complications.

Evaluation of WHO catalog of mutations and five WGS analysis tools for drug resistance prediction of Mycobacterium tuberculosis isolates from China.

The continuous advancement of molecular diagnostic techniques, particularly whole-genome sequencing (WGS), has greatly facilitated the early diagnosis of drug-resistant tuberculosis patients. Nonetheless, the interpretation of results from various types of mutations in drug-resistant-associated genes has become the primary challenge in the field of molecular drug-resistance diagnostics. In this study, our primary objective is to evaluate the diagnosis accuracy of the World Health Organization (WHO) catalog of mutations and five WGS analysis tools (PhyResSE, Mykrobe, TB Profiler, Gen-TB, and SAM-TB) in drug resistance to 10 anti-Mycobacterium tuberculosis (MTB) drugs. We utilized the data of WGS collected between 2014 and 2017 in Zhejiang Province, consisting of 110 MTB isolates as detailed in our previous study. Based on phenotypic drug susceptibility testing (DST) results using the proportion method on Löwenstein-Jensen medium with antibiotics, we evaluated the predictive accuracy of genotypic DST obtained by these tools. The results revealed that the WHO catalog of mutations and five WGS analysis tools exhibit robust predictive capabilities concerning resistance to isoniazid, rifampicin, ethambutol, streptomycin, amikacin, kanamycin, and capreomycin. Notably, Mykrobe, SAM-TB, and TB Profiler demonstrate the most accurate predictions for resistance to pyrazinamide, prothionamide, and para-aminosalicylic acid, respectively. These findings are poised to significantly guide and influence future clinical treatment strategies and resistance monitoring protocols.IMPORTANCEWhole-genome sequencing (WGS) has the potential for the early diagnosis of drug-resistant tuberculosis. However, the interpretation of mutations of drug-resistant-associated genes represents a significant challenge as the amount and complexity of WGS data. We evaluated the accuracy of the World Health Organization catalog of mutations and five WGS analysis tools in predicting drug resistance to first-line and second-line anti-TB drugs. Our results offer clinicians guidance on selecting appropriate WGS analysis tools for predicting resistance to specific anti-TB drugs.

Neurodevelopmental profiles of 14 individuals with phosphomannomutase deficiency (PMM2-CDG).

PMM2-CDG (formerly CDG-1a), the most common type of congenital disorders of glycosylation, is inherited in an autosomal recessive pattern. PMM2-CDG frequently presents in infancy with multisystemic clinical involvement, and it has been diagnosed in over 1000 people worldwide. There have been few natural history studies reporting neurodevelopmental characterization of PMM2-CDG. Thus, a prospective study was conducted that included neurodevelopmental assessments as part of deep phenotyping. This study, Clinical and Basic Investigations into Known and Suspected Congenital Disorders of Glycosylation (NCT02089789), included 14 participants (8 males and 6 females ages 2-33 years) with a confirmed molecular diagnosis of PMM2-CDG. Clinical features of PMM2-CDG in this cohort were neurodevelopmental disorders, faltering growth, hypotonia, cerebellar atrophy, peripheral neuropathy, movement disorders, ophthalmological abnormalities, and auditory function differences. All PMM2-CDG participants met criteria for intellectual disability (or global developmental delay if younger than age 5). The majority never attained certain gross motor and language milestones. Only two participants were ambulatory, and almost all were considered minimally verbal. Overall, individuals with PMM2-CDG present with a complex neurodevelopmental profile characterized by intellectual disability and multisystemic presentations. This systematic quantification of the neurodevelopmental profile of PMM2-CDG expands our understanding of the range in impairments associated with PMM2-CDG and will help guide management strategies.

Integration of concentration gradient generator with competitive PCR in a droplet for rapid nucleic acid quantification

Competitive PCR is an efficient way for the measurement of nucleic acids without requiring a standard curve. It uses a competitor template mimicking the target sequence for direct comparison and quantification during amplification. However, manually preparing various concentrations for competitive PCR is labor-intensive. Additionally, target and competitor DNA bands are often inadequately separated on gels due to their similar sizes. This leads to inaccurate distinction and imprecise measurement of bandwidths and intensities, resulting in high standard deviations in quantification. Thus, this study presents an alternative way to combine droplet competitive PCR with the concentration gradient generator in a microfluidic device that simplifies competitive PCR preparation and provides precise control over reaction conditions and sample handling. Generating a wide range of competitor-to-target ratios in a single experiment enhances statistical reliability and isolating individual reactions in droplets reduces background noise and increases sensitivity. Additionally, it eliminates gel electrophoresis, improves consistency and reproducibility, requires smaller sample and reagent volumes, and allows direct sample quantification through a calibration curve for fast, simple quantification. We envision that our platform can offer a promising alternative to digital PCR, addressing its challenges and driving progress in molecular biology and diagnostics.

Identification of CUBN variants in triplets with a 20-year history of proteinuria.

CUBN encodes cubilin, which plays a role in the reabsorption of glomerular-filtered albumin in the proximal tubule. CUBN-related proteinuria was recently established as a new disease concept and may be present in proteinuric cases that were previously undiagnosed either genetically or histologically. We herein report a case of triplets diagnosed with chronic benign proteinuria due to CUBN variants 20years after its onset. The proband, the first child of triplets, tested positive for urinary protein several times during the neonatal period. A urine screening test at 3years old was positive. Proteinuria persisted for years within a non-nephrotic range. Kidney biopsy at 8years old revealed minor glomerular abnormalities. Renin-angiotensin system inhibitors were started for albumin-based proteinuria but were ineffective. Since the two other triplets had similar courses, analyses of the NPHS1/2 and WT1 genes were performed but revealed no abnormalities. The triplets transitioned to adult care at 15years old. CUBN-related proteinuria was reported in 2020; therefore, we re-analyzed their DNA samples and identified compound heterozygous variants in CUBN in all three triplets. The molecular diagnosis of CUBN-related proteinuria will save patients from unnecessary treatments and concerns about renal prognosis.

The utility of next-generation sequencing in challenging liver FNA biopsies.

Fine-needle aspiration (FNA) biopsy is increasingly used for the diagnosis of hepatocellular masses. Because distinguishing well differentiated hepatocellular carcinoma (HCC) from other well differentiated hepatocellular lesions (e.g., large regenerative nodules or focal nodular hyperplasia) requires an assessment of architectural features, this may be challenging on FNA when intact tissue fragments are not sampled. Poorly differentiated HCC and intrahepatic cholangiocarcinoma (ICC) may exhibit overlapping pathologic features. Molecular testing can be helpful, because mutations in TERT promoter and CTNNB1 (β-catenin) are characteristic of HCC, whereas mutations in BAP1, IDH1/IDH2, and PBRM1 may favor ICC. The goal of this study was to assess the role of next-generation sequencing (NGS) in further subclassifying indeterminate liver lesions sampled by FNA. A retrospective review of liver cytology cases with NGS on cell block material was performed. Age, radiologic features, background hepatic disease and treatment, outcome, and NGS data were obtained from the electronic medical record. Twelve FNA biopsies that had cell blocks from clinically suspected primary hepatic masses were identified. The presence of a TERT promoter mutation supported a diagnosis of HCC for one well differentiated neoplasm. For three patients, the presence of mutations, such as IDH1, CDKN2A/CDKN2B, and BRAF, supported a diagnosis of ICC. Of the eight poorly differentiated carcinomas, NGS helped refine the diagnosis in six of eight cases, with one HCC, three ICCs, and two that had combined HCC-ICC, with two cases remaining unclassified. Molecular diagnostics can be helpful to distinguish HCC and ICC on FNA specimens, although a subset of primary hepatic tumors may remain unclassifiable.

Three-way junction-mediated three-letter coded SDA cascade CRISPR/Cas12a system for circRNA detection

Circular RNAs (circRNAs), a special type of non-coding RNAs with covalently closed circular structure, have emerged as promising liquid biopsy biomarker. However, the precise detection of circRNAs is severely hampered by interference from homologous linear RNAs, posing a significant obstacle for their applications in molecular diagnostics. Herein, a strategy named three-way junction (TWJ)-mediated three-letter coded strand displacement amplification (SDA) cascade CRISPR/Cas12a system (TTSC) has been devised to achieve precise detection of circRNAs. Three-letter coded SDA (TC-SDA) has been originally designed in this study for eliminating undesired interference from linear RNA and performing dual functionality in both discrimination and amplification. With this design, the proposed strategy elegantly distinguishes between circRNA and homologous linear RNA through proximity effect of TWJ structure and non-specific amplification blocking ability of the three-letter code. Ultimately, this strategy successfully identified circRNA down to 0.1 % abundance. In addition, benefiting from the synergistically accelerated multiple signal amplification in one step, TTSC strategy exhibited good linear relationship of 0.5–1000 pM with a detection limit of 80.6 fM. Notably, the recovery test indicated that the proposed strategy has good prospects for clinical applications. Therefore, TTSC is expected to provide an ideal platform for circRNA detection in biotechnology research and molecular diagnostics.

Chronic Myeloid Leukemia in Bulgaria in the New Millennium: Identification of Directions for Improvement in Management and Outcomes Reporting

Background: In the last two decades, tyrosine kinase inhibitors (TKIs) and advances in molecular diagnostics have revolutionized management and long-term clinical outcomes in chronic myeloid leukemia (CML). Real-world data from different countries allow for the identification of country-specific issues in the clinical management and development of specific plans for improvement. Here, we aimed to analyze the trend in overall survival in Bulgarian CML patients since 2000. Methods: We retrieved publicly available Bulgarian CML data from several sources such as the Bulgarian National Cancer Registry, Bulgarian National Statistical Institute, and National Health Insurance Fund since 2000. We used the retrieved data of a total of 1513 Bulgarian CML patients to describe the trends in overall survival (OS), conditional overall survival, life expectancy, and life years lost over five time periods. We also described the trends in healthcare expenditures for TKIs and CML patients’ coverage with TKIs since 2014. Results: In both uni- and multivariate models, we found a constant increase in OS over the three 5-year periods until 2014. The period 2015–2019 was not associated with an additional increase in OS. Identical dynamics in the improvement in life expectancy (LE) and in life years lost (LYLs) was observed. Additionally, conditional 5-year survival did not improve during 2015–2019 in comparison to 2010–2014. Population-level data did not show consistent changes in the documented number of deaths due to CML since 2013. The period after 2013 is marked by a constant increase in the annual expenditures for TKIs, reaching to about 2.0 EUR/capita. The number of patients who received at least one TKI also increased during that period. Conclusions: After the initial significant improvement in the clinical outcomes for Bulgarian CML patients until 2014, subsequent periods did not bring further benefit in spite of the improved coverage with second- and third-line TKIs. Multiple factors may contribute to these suboptimal outcomes. Therefore, one can propose several additional measures at the country level, which could lead to additional improvement in the OS of Bulgarian CML patients.

Visualizing DNA/RNA, Proteins, and Small Molecule Metabolites within Live Cells.

Live cell imaging is essential for obtaining spatial and temporal insights into dynamic molecular events within heterogeneous individual cells, in situ intracellular networks, and in vivo organisms. Molecular tracking in live cells is also a critical and general requirement for studying dynamic physiological processes in cell biology, cancer, developmental biology, and neuroscience. Alongside this context, this review provides a comprehensive overview of recent research progress in live-cell imaging of RNAs, DNAs, proteins, and small-molecule metabolites, as well as their applications in molecular diagnosis, immunodiagnosis, and biochemical diagnosis. A series of advanced live-cell imaging techniques have been introduced and summarized, including high-precision live-cell imaging, high-resolution imaging, low-abundance imaging, multidimensional imaging, multipath imaging, rapid imaging, and computationally driven live-cell imaging methods, all of which offer valuable insights for disease prevention, diagnosis, and treatment. This review article also addresses the current challenges, potential solutions, and future development prospects in this field.

Comparison of microbiological and molecular diagnosis for identification of respiratory secondary infections in COVID-19 patients and their antimicrobial resistance patterns

We report the use of a new multiplex Real-Time PCR platform to simultaneously identify 24 pathogens and 3 antimicrobial-resistance genes directly from respiratory samples of COVID-19 patients. Results were compared to culture-based diagnosis.Secondary infections were detected in 60% of COVID-19 patients by molecular analysis and 73% by microbiological assays, with no significant differences in accuracy, indicating Gram-negative bacteria as the predominant species. Among fungal superinfections, Aspergillus spp. were detected by both methods in more than 7% of COVID-19 patients. Oxacillin-resistant S. aureus and carbapenem-resistant K. pneumoniae were highlighted by both methods.Secondary microbial infections in SARS-CoV-2 patients are associated with poor outcomes and an increased risk of death. Since PCR-based tests significantly reduce the turnaround time to 4 hours and 30 minutes (compared to 48 hours for microbial culture), we strongly support the routine use of molecular techniques, in conjunction with microbiological analysis, to identify co/secondary infections.

Molecular Detection of Respiratory Tract Viruses in Chickens at the Point of Need by Loop-Mediated Isothermal Amplification (LAMP)

Accurate and timely molecular diagnosis of respiratory diseases in chickens is essential for implementing effective control measures, preventing the spread of diseases within poultry flocks, minimizing economic loss, and guarding food security. Traditional molecular diagnostic methods like polymerase chain reaction (PCR) require expensive equipment and trained personnel, limiting their use to centralized labs with a significant delay between sample collection and results. Loop-mediated isothermal amplification (LAMP) of nucleic acids offers an attractive alternative for detecting respiratory viruses in broiler chickens with sensitivity comparable to that of PCR. LAMP’s main advantages over PCR are its constant incubation temperature (∼65 °C), high amplification efficiency, and contaminant tolerance, which reduce equipment complexity, cost, and power consumption and enable instrument-free tests. This review highlights effective LAMP methods and variants that have been developed for detecting respiratory viruses in chickens at the point of need.

Rapid detection of SARS-CoV-2 with a mobile device based on pulse controlled amplification

In the past, vast research has been conducted on biosensors and point-of-care (PoC) diagnostics. Despite rapid advances especially during the SARS-CoV-2 pandemic in this research field a low-cost molecular biosensor exhibiting the user-friendliness of a rapid antigen test, and also the sensitivity and specificity of a PCR test, has not been developed yet. To this end we developed a novel microfluidics based and handheld PoC device, that facilitates viral detection at PCR sensitivity and specificity in less than 40 min, including 15 min sample preparation. This was attained by incorporation of pulse controlled amplification (PCA), a method which uses short electrical pulses to rapidly increase the temperature of a small fraction of the sample volume. In this work, we present a low-cost PCA device with a microfluidic consumable intended for the use in a decentralized or home-setting. We used finite element analysis (FEA) simulations to display the fundamental principle and highlight the critical parameter dependency of PCA, such as pulse length and resistor shape. Furthermore, we integrated a simple and fast workflow for sample preparation and evaluated the limit of detection (LoD) for SARS-CoV-2 viral RNA, which is 0.88 copies/μL (=44 copies/reaction), and thus, comparable to conventional RT-qPCR. Additionally, target specificity of the device was validated. Our device and PCA approach enables cost-effective, rapid and mobile molecular diagnostics while remaining highly sensitive and specific.

Performance of a Rapid Digital PCR Test for the Detection of Non-Small Cell Lung Cancer (NSCLC) Variants.

Next-generation sequencing is widely used for comprehensive molecular profiling for many cancers including lung cancer. However, the complex workflows and long turnaround times limit its access and utility. ChromaCode's High Definition PCR Non-Small Cell Lung Cancer Panel (HDPCR™ NSCLC Panel) is a low-cost, rapid turnaround, digital polymerase chain reaction assay that is designed to detect variants in nine NSCLC genes listed in National Comprehensive Cancer Network guidelines. This assay uses TaqMan® probe limiting chemistry and proprietary analysis software to enable multi-target detection within a single-color channel. We compared the performance of the HDPCR™ NSCLC Panel against an in-house, laboratory-developed, targeted next-generation sequencing panel used in the Molecular Diagnostics Laboratory at the University of Minnesota Medical Center to detect biomarkers for NSCLC. The overall accuracy of the HDPCR panel was 99.48% (95% confidence interval 99.01-99.76) with a sensitivity of 95.35% (95% confidence interval 88.52-98.72) and a specificity of 99.69% (95% confidence interval 99.29-99.90). The HDPCR wet lab workflow was 4 h, and the time to generate variant calls from raw data using the ChromaCode Cloud was 2 minutes. We demonstrated that the HDPCR™ NSCLC Panel provides timely, comprehensive, and sensitive mutation detection in NSCLC samples with results in less than 24 h.