Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification.

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

Development of Loop-Mediated Isothermal Amplification (LAMP) Assays for the Rapid Detection of Toxigenic Aspergillus flavus and A. carbonarius in Nuts.

Aspergillus species create major postharvest problems due to the food losses caused by their mere presence and the hazardous mycotoxins they produce, such as aflatoxin B1 (AFB1) and ochratoxin A (OTA). These mycotoxins are mainly produced by A. flavus and A. carbonarius, respectively. In this study, we developed a rapid detection method for the two aforementioned species based on loop-mediated isothermal amplification (LAMP). The primers were designed to target genes belonging to the mycotoxin clusters pks and aflT for A. carbonarius and A. flavus, respectively. Result visualization was carried out in real time via the detection of fluorescent signals. The method developed showed high sensitivity and specificity, with detection limits of 0.3 and 0.03 pg/reaction of purified DNA of A. carbonarius and A. flavus, respectively. The assays were further implemented on inoculated nuts, including pistachios and almonds, after one-step crude DNA extraction. These tests revealed a detection level of 0.5 spore/g that shows the effectiveness of LAMP as a rapid method for detecting potentially toxigenic Aspergillus spp. directly in food. The validation of the assays included tests on a larger scale that further confirmed their sensitivity and specificity, as well as enabling the production of ready-to-use LAMP prototype kits. These kits are easy to use and aim to simplify the screening of food samples in order to monitor the presence of specific Aspergillus contaminations.

Rapid and sensitive visual detection of avian leukosis virus by reverse transcription loop-mediated isothermal amplification combined with a lateral flow immunochromatographic strip assay.

Considering that avian leukosis virus (ALV) infection has inflicted massive economic losses on the poultry breeding industry in most countries, its early diagnosis remains an important measure for timely treatment and control of the disease, for which a rapid and sensitive point-of-care test is required. We established a user-friendly, economical, and rapid visualization method for ALV amplification products based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with an immunochromatographic strip in a lateral flow device (LFD). Using the ALVp27 gene as the target, five RT-LAMP primers and one fluorescein-isothiocyanate-labeled probe were designed. After 60 min of RT-LAMP amplification at 64 °C, the products could be visualized directly using the LFD. The detection limit of this assay for ALV detection was 102 RNA copies/µL, and the sensitivity was 100 times that of reverse transcription polymerase chain reaction (RT-PCR), showing high specificity and sensitivity. To verify the clinical practicality of this assay for detecting ALV, the gold standard RT-PCR method was used for comparison, and consistent results were obtained with both assays. Thus, the assay described here can be used for rapid detection of ALV in resource-limited environments.

Diagnóstico por amplificación isotérmica de ácidos nucleicos. Oportunidad para la farmacia comunitaria / Diagnosis by isothermal amplification of nucleic acids. Opportunity for community pharmacy

Esta revisión se centra en describir nuevos sistemas de diagnóstico molecular de tipo POC disponibles en el mercado que pueden implementarse fácilmente en farmacias comunitarias y tienen el potencial de ampliar la cartera de servicios farmacéuticos y hacer una contribución significativa a la mejora de la salud pública.El conocimiento de nuevas técnicas de diagnóstico molecular distintas de la PCR es relativamente desconocido. Sin embargo, las opciones disponibles son diversas y han alcanzado suficiente madurez tecnológica para su uso a gran escala. La pandemia de SARS-CoV-2 ha sacado al mercado pruebas de diagnóstico que, en algunos casos, se han utilizado exclusivamente en investigación durante décadas.La tecnología isotérmica de amplificación de ácidos nucleicos sigue evolucionando y es probable que en los próximos años seamos testigos de un aumento exponencial de su uso, así como del desarrollo de nuevas mejoras que simplifiquen y reduzcan aún más el coste de cada ensayo.Igualmente, no podemos obviar el hecho de que durante la pandemia de COVID-19, el público se ha habituado a autodiagnosticarse a través de canales de distribución masiva como las farmacias comunitarias, lo que puede abrir el sector a otras enfermedades —como las de transmisión sexual o salud animal—, el control de alimentos, la contaminación del agua y del aire (hongos) o la presencia de alérgenos.El conocimiento de estas nuevas tecnologías es esencial estrategia de vigilancia tecnológica e inteligencia competitiva del sector farmacéutico.(AU)

Literature Review of Pathogen Agnostic Molecular Testing of Clinical Specimens From Difficult-to-Diagnose Patients: Implications for Public Health.

To better identify emerging or reemerging pathogens in patients with difficult-to-diagnose infections, it is important to improve access to advanced molecular testing methods. This is particularly relevant for cases where conventional microbiologic testing has been unable to detect the pathogen and the patient's specimens test negative. To assess the availability and utility of such testing for human clinical specimens, a literature review of published biomedical literature was conducted. From a corpus of more than 4,000 articles, a set of 34 reports was reviewed in detail for data on where the testing was being performed, types of clinical specimens tested, pathogen agnostic techniques and methods used, and results in terms of potential pathogens identified. This review assessed the frequency of advanced molecular testing, such as metagenomic next generation sequencing that has been applied to clinical specimens for supporting clinicians in caring for difficult-to-diagnose patients. Specimen types tested were from cerebrospinal fluid, respiratory secretions, and other body tissues and fluids. Publications included case reports and series, and there were several that involved clinical trials, surveillance studies, research programs, or outbreak situations. Testing identified both known human pathogens (sometimes in new sites) and previously unknown human pathogens. During this review, there were no apparent coordinated efforts identified to develop regional or national reports on emerging or reemerging pathogens. Therefore, development of a coordinated sentinel surveillance system that applies advanced molecular methods to clinical specimens which are negative by conventional microbiological diagnostic testing would provide a foundation for systematic characterization of emerging and underdiagnosed pathogens and contribute to national biodefense strategy goals.

Immunological and molecular diagnostic techniques in fish health: present and future prospectus.

Fish health management is critical to aquaculture and fisheries as it directly affects sustainability and productivity. Fish disease diagnosis has taken a massive stride because of advances in immunological and molecular diagnostic tools which provide a sensitive, quick, and accurate means of identifying diseases. This review presents an overview of the main molecular and immunological diagnostic methods for determining the health of fish. The immunological techniques help to diagnose different fish diseases by detecting specific antigens and antibodies. The application of immunological techniques to vaccine development is also examined in this review. The genetic identification of pathogens is made possible by molecular diagnostic techniques that enable the precise identification of bacterial, viral, and parasitic organisms in addition to evaluating host reactions and genetic variation associated with resistance to disease. The combination of molecular and immunological methods has resulted in the creation of novel techniques for thorough evaluation of fish health. These developments improve treatment measures, pathogen identification and provide new information about the variables affecting fish health, such as genetic predispositions and environmental stresses. In the framework of sustainable fish farming and fisheries management, this paper focuses on the importance of these diagnostic techniques that play a crucial role in protecting fish populations and the aquatic habitats. This review also examines the present and potential future directions in immunological and molecular diagnostic techniques in fish health.

TB LAMP assay, a beneficial tool for the diagnosis of Tubercular meningitis in resource-limited settings.

INTRODUCTION: Tubercular meningitis (TBM) is a serious public health problem in developing countries as it leads to significant mortality and residual neurological sequelae. The estimated mortality due to TBM in India is 1.5 per 100,000 population. In resource-limited settings, only the Ziehl-Neelsen (ZN) stain, which has very little sensitivity, is available. The World Health Organization recommended the Loop Mediated Isothermal Amplification (TB LAMP) assay for pulmonary tuberculosis only. We evaluated this test for tubercular meningitis as well. METHODOLOGY: In a cross-sectional study of 2-year duration, we have taken 239 cerebrospinal fluid samples from suspected cases of tubercular meningitis patients. ZN staining along with Mycobacteria Growth Indicator Tube (MGIT) TB culture, Xpert MTB/RIF Ultra assay, and commercial TB LAMP assay were performed for each sample. RESULTS: Out of 239 samples, 40 samples (16.73%) were found TB LAMP assay positive, 48 samples (20.08%) were found Xpert ultra-assay positive, 12 samples (5.02%) were MGIT TB culture positive and acid-fast bacillus smear positive in ten samples (4.18 %). Out of 12 MGIT-positive samples, all samples (100%) were TB LAMP and Xpert ultra positive and one sample (8.33%) was ZN smear positive. In 199 negative samples from the TB LAMP assay, eight samples were positive by Xpert, none by MGIT TB culture and AFB smear. Sensitivity and specificity were found as 100% and 87.66%, respectively, for the TB LAMP assay. CONCLUSION: TB LAMP assay is a rapid, cost-effective, sensitive, and specific test for tubercular meningitis infection in resource-limited settings.

Molecular diagnostics of hepatobiliary and pancreatic neoplasias.

Neoplasias of the hepatopancreatobiliary tract are growing in numbers, have the poorest prognosis of all major cancer entities, and thus represent a rising clinical problem. Their molecular diagnostic has dramatically improved, contributing to tumor subtyping, definition of malignancy, and uncovering cases with hereditary predisposition. Most of all, predictive molecular testing allows to identify cases amenable to treatment with the rising number of approved targeted drugs, immune-oncological treatment, and clinical trials. In this review, the current state of molecular testing and its contribution to clinical decision-making are outlined.

A handheld isothermal fluorescence detector for duplex visualization of aquatic pathogens via enhanced one-pot LAMP-PfAgo assay.

The expansion of large-scale aquaculture has exacerbated the challenge of aquatic diseases, resulting in substantial economic losses annually. Currently, traditional laboratory-based diagnostic methods are time-consuming and costly, hindering on-site testing for individual farmers. We address this issue by developing a state-of-the-art handheld isothermal nucleic acid amplification device (WeD-1) capable of fluorescence tracking of reactions and integrating it with an enhanced one-pot Prokaryotic Argonaute based nucleic acid detection method, enabling duplex visual detection of aquatic pathogens. WeD-1 is portable, reusable, user-friendly, and cost-effective, offering real-time smartphone interaction and enabling real-time fluorescence observation during the reaction. The enhanced one-pot Loop-Mediated Isothermal Amplification (LAMP)-PfAgo method, incorporating paraffin-encapsulated lyophilized PfAgo protein, achieves precise target-specific cleavage, significantly enhancing multiplex nucleic acid detection. This innovation streamlines on-site testing, negating the need for specialized laboratory conditions while ensuring an aerosol-free system. With newly developed and highly sensitive LAMP primer sets, our compact WeD-1/LAMP-PfAgo nucleic acid rapid testing system exhibits remarkable sensitivity, readily detecting aquatic pathogens with naked eyes from rapidly prepared fish and shrimp samples within 40 min, even when the Ct values are as high as 34.

Evaluation of molecular inhibitors of loop-mediated isothermal amplification (LAMP).

Loop-mediated isothermal amplification (LAMP) is a cost-effective and easy-to-perform assay that enables the direct detection of DNA. Its use in point-of-care diagnostic tests is growing, while it has the potential to be used in presumptive on-the-field forensic tests. Samples are often collected from complex matrices that contain high levels of contaminants. Herein, we evaluate the effect of seven common DNA amplification inhibitors on LAMP - bile salts, calcium chloride, hematin, humic acid, immunoglobulin G, tannic acid and urea. We study the effect of each inhibitor individually in real-time detection systems coupled with end-point measurements to delineate their inhibitory effects from the matrix in which they may be found. Our studies show LAMP inhibitors generally delay the onset of amplicon formation and quench fluorescence at similar or higher concentrations compared to PCR, but that end-point measurements of LAMP amplicons are unaffected. This is important as LAMP amplicons can be detected in non-fluorometric ways thus contributing to the assertions that LAMP is more robust to inhibitors than PCR.

A deep-learning workflow to predict upper tract urothelial carcinoma protein-based subtypes from H&E slides supporting the prioritization of patients for molecular testing.

Upper tract urothelial carcinoma (UTUC) is a rare and aggressive, yet understudied, urothelial carcinoma (UC). The more frequent UC of the bladder comprises several molecular subtypes, associated with different targeted therapies and overlapping with protein-based subtypes. However, if and how these findings extend to UTUC remains unclear. Artificial intelligence-based approaches could help elucidate UTUC's biology and extend access to targeted treatments to a wider patient audience. Here, UTUC protein-based subtypes were identified, and a deep-learning (DL) workflow was developed to predict them directly from routine histopathological H&E slides. Protein-based subtypes in a retrospective cohort of 163 invasive tumors were assigned by hierarchical clustering of the immunohistochemical expression of three luminal (FOXA1, GATA3, and CK20) and three basal (CD44, CK5, and CK14) markers. Cluster analysis identified distinctive luminal (N = 80) and basal (N = 42) subtypes. The luminal subtype mostly included pushing, papillary tumors, whereas the basal subtype diffusely infiltrating, non-papillary tumors. DL model building relied on a transfer-learning approach by fine-tuning a pre-trained ResNet50. Classification performance was measured via three-fold repeated cross-validation. A mean area under the receiver operating characteristic curve of 0.83 (95% CI: 0.67-0.99), 0.8 (95% CI: 0.62-0.99), and 0.81 (95% CI: 0.65-0.96) was reached in the three repetitions. High-confidence DL-based predicted subtypes showed significant associations (p < 0.001) with morphological features, i.e. tumor type, histological subtypes, and infiltration type. Furthermore, a significant association was found with programmed cell death ligand 1 (PD-L1) combined positive score (p < 0.001) and FGFR3 mutational status (p = 0.002), with high-confidence basal predictions containing a higher proportion of PD-L1 positive samples and high-confidence luminal predictions a higher proportion of FGFR3-mutated samples. Testing of the DL model on an independent cohort highlighted the importance to accommodate histological subtypes. Taken together, our DL workflow can predict protein-based UTUC subtypes, associated with the presence of targetable alterations, directly from H&E slides.

Evaluation of a rapid Loop Mediated Isothermal Amplification (LAMP) test for the laboratory diagnosis of sexually transmitted infections.

Sexually transmitted infections (STIs) have seen a considerable increase in the last years and given the health burden they may represent from both a personal and community perspective, they require surveillance and prevention programmes based on a timely and decentralized diagnosis. In this context, user-friendly rapid molecular tests may represent a good trade-off between diagnostic accuracy, accessibility and affordability. In this study we evaluated the diagnostic performance of a new real-time LAMP (Loop Mediated Isothermal Amplification) method for the rapid detection and differentiation of 7 major sexually transmissible pathogens by analysing real clinical samples (genital and extra-genital matrices) from individuals with suspected STIs. The assay showed good overall diagnostic performances in terms of sensitivity, specificity and concordance with a gold-standard PCR-based molecular method. This assay, not requiring specialised laboratory technicians or expensive instrumentation, but nonetheless capable of guaranteeing accurate results, is within the reach of outpatient settings, obstetrics, and gynaecology clinic, hence ensuring on-field access to early diagnosis.

A fluorescence viewer for rapid molecular assay readout in space and low-resource terrestrial environments.

Fluorescence-based assays provide sensitive and adaptable methods for point of care testing, environmental monitoring, studies of protein abundance and activity, and a wide variety of additional applications. Currently, their utility in remote and low-resource environments is limited by the need for technically complicated or expensive instruments to read out fluorescence signal. Here we describe the Genes in Space Fluorescence Viewer (GiS Viewer), a portable, durable viewer for rapid molecular assay readout that can be used to visualize fluorescence in the red and green ranges. The GiS Viewer can be used to visualize any assay run in standard PCR tubes and contains a heating element. Results are visible by eye or can be imaged with a smartphone or tablet for downstream quantification. We demonstrate the capabilities of the GiS Viewer using two case studies-detection of SARS-CoV-2 RNA using RT-LAMP and quantification of drug-induced changes in gene expression via qRT-PCR on Earth and aboard the International Space Station. We show that the GiS Viewer provides a reliable method to visualize fluorescence in space without the need to return samples to Earth and can further be used to assess the results of RT-LAMP and qRT-PCR assays on Earth.

Low-Cost Arduino Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) for Sensitive Nucleic Acid Detection.

This work presents a low-cost transcription loop-mediated isothermal amplification (RT-LAMP) instrument for nucleic acid detection, employing an Arduino Nano microcontroller. The cooling system includes customized printed circuit boards (PCBs) that serve as electrical resistors and incorporate fans. An aluminum block is designed to accommodate eight vials. The system also includes two PCB heaters-one for sample heating and the other for vial lid heating to prevent condensation. The color detection system comprises a TCS3200 color 8-sensor array coupled to one side of the aluminum heater body and a white 8-LED array coupled to the other side, controlled by two Multiplexer/Demultiplexer devices. LED light passes through the sample, reaching the color sensor and conveying color information crucial for detection. The top board is maintained at 110 ± 2 °C, while the bottom board is held at 65 ± 0.5 °C throughout the RT-LAMP assay. Validation tests successfully demonstrated the efficacy of the colorimetric RT-LAMP reactions using SARS-CoV-2 RNA amplification as a sample viability test, achieving 100% sensitivity and 97.3% specificity with 66 clinical samples. Our instrument offers a cost-effective (USD 100) solution with automated result interpretation and superior sensitivity compared to visual inspection. While the prototype was tested with SARS-CoV-2 RNA samples, its versatility extends to detecting other pathogens using alternative primers, showcasing its potential for broader applications in biosensing.

Customizable Nichrome Wire Heaters for Molecular Diagnostic Applications.

Accurate sample heating is vital for nucleic acid extraction and amplification, requiring a sophisticated thermal cycling process in nucleic acid detection. Traditional molecular detection systems with heating capability are bulky, expensive, and primarily designed for lab settings. Consequently, their use is limited where lab systems are unavailable. This study introduces a technique for performing the heating process required in molecular diagnostics applicable for point-of-care testing (POCT), by presenting a method for crafting customized heaters using freely patterned nichrome (NiCr) wire. This technique, fabricating heaters by arranging protrusions on a carbon black-polydimethylsiloxane (PDMS) cast and patterning NiCr wire, utilizes cost-effective materials and is not constrained by shape, thereby enabling customized fabrication in both two-dimensional (2D) and three-dimensional (3D). To illustrate its versatility and practicality, a 2D heater with three temperature zones was developed for a portable device capable of automatic thermocycling for polymerase chain reaction (PCR) to detect Escherichia coli (E. coli) O157:H7 pathogen DNA. Furthermore, the detection of the same pathogen was demonstrated using a customized 3D heater surrounding a microtube for loop-mediated isothermal amplification (LAMP). Successful DNA amplification using the proposed heater suggests that the heating technique introduced in this study can be effectively applied to POCT.

Barriers and Unequal Access to Timely Molecular Testing Results: Addressing the Inequities in Cancer Care Delays across Canada.

Genomic medicine is a powerful tool to improve diagnosis and outcomes for cancer patients by facilitating the delivery of the right drug at the right dose at the right time for the right patient. In 2023, a Canadian conference brought together leaders with expertise in different tumor types. The objective was to identify challenges and opportunities for change in terms of equitable and timely access to biomarker testing and reporting at the education, delivery, laboratory, patient, and health-system levels in Canada. Challenges identified included: limited patient and clinician awareness of genomic medicine options with need for formal education strategies; failure by clinicians to discuss genomic medicine with patients; delays in or no access to hereditary testing; lack of timely reporting of results; intra- and inter-provincial disparities in access; lack of funding for patients to access testing and for laboratories to provide testing; lack of standardized testing; and impact of social determinants of health. Canada must standardize its approach to biomarker testing across the country, with a view to addressing current inequities, and prioritize access to advanced molecular testing to ensure systems are in place to quickly bring innovation and evidence-based treatments to Canadian cancer patients, regardless of their place of residence or socioeconomic status.

[Molecular testing of prostate cancer: timing, methods and consequences]. / Molekulare Testung des Prostatakarzinoms: wann, wie und mit welcher Konsequenz?

Metastatic prostate cancer is a heterogeneous disease. To date, however, treatment decisions are often based on the extent and symptom burden of the tumour, concomitant diseases, and the patient's wishes. Molecular pathology aspects are rarely taken into account. Declining costs and the increasing use of next-generation sequencing (NGS) have led to an increase in molecular testing and a better understanding of the significance of molecular alterations for the development and spread of prostate cancer. More consistent germline testing reveals hereditary predispositions. Following the approval of olaparib for the treatment of BRCA1/2 mutated, castration-resistant prostate cancer, further targeted therapeutic approaches are currently under development. In our review article, we provide an overview of current molecular testing in prostate cancer and discuss possible consequences.

Effects of single and multiple nucleotide mutations on loop-mediated isothermal amplification.

Testing is pivotal for early identification of disease and subsequent infection control. Pathogens' nucleic acid sequence can change due to naturally-occurring genetic drift or intentional modification. Because of the reliance on molecular assays for human, animal, and plant disease diagnosis, we must understand how nucleotide mutations affect test accuracy. Primers designed against original lineages of a pathogen may be less efficient at detecting variants with genetic changes in priming regions. Here, we made single- and multi-point mutations in priming regions of a model SARS-CoV-2 template that was used as input for a loop-mediated isothermal amplification (LAMP) assay. We found that many of the modifications impacted assay sensitivity, amplification speed, or both. Further research exploring mutations at every position in each of the eight priming regions should be conducted to evaluate trends and determine generalizability.

FGFR inhibitors in urothelial cancer: Contextualizing THOR within the new treatment landscape.

As frontline treatment of advanced urothelial cancer (UC) evolves, optimal sequencing of subsequent therapies remains unclear. The phase 3 THOR trial compared the efficacy of erdafitinib to chemotherapy or immunotherapy in FGFR3/2-altered advanced UC. THOR offers valuable data informing sequencing strategies, reinforcing the need for molecular testing in UC.