[Molecular Pathology Diagnosis as Companion Diagnostics].

As genomic medicine advances, opportunities for molecular pathology diagnosis by pathologists to be used as companion diagnostics is increasing. Pathological specimens must be useful not only for pathological diagnosis, but also for genetic testing panel and molecular pathology diagnosis. Companion diagnostics performed by pathologists uses immunohistochemical staining and fluorescence in situ hybridization to determine patient eligibility for molecular target drugs and immune checkpoint inhibitors. By accurately observing a wide variety of diagnostic criteria and performing with high precision, pathological diagnosis will become closer to therapeutic pathology.

Performances of two rapid LAMP-based techniques for the intrapartum detection of Group B Streptococcus vaginal colonization.

PURPOSE: Group B Streptococcus (GBS) is the leading cause of invasive infections in newborns. The prevention of GBS neonatal disease relies on the administration of an intrapartum antibiotic prophylaxis to GBS-colonized women. In recent years, rapid intrapartum detection of GBS vaginal colonization using real-time nucleic acid amplification tests (NAATs) emerged as an alternative to antenatal culture screening methods. METHODS: We compared the performances of two loop-mediated isothermal amplification (LAMP) tests, the Ampliflash® GBS and the PlusLife® GBS tests, to standard culture for GBS detection in vaginal specimens from pregnant women. The study was conducted from April to July 2023 in a French hospital of the Paris area. RESULTS: A total of 303 samples were analyzed, including 85 culture-positive samples (28.1%). The Ampliflash® GBS test and the PlusLife® GBS tests gave a result for 100% and 96.3% tests, respectively. The performances of the tests were as follows: sensitivity 87.1% (95% confidence interval (CI) 78.3-92.6) and 98.7% (95% CI 93.0-99.8), specificity 99.1% (95% CI 96.7-99.8), and 91.9% (95% CI 87.3-95.0), respectively. False negative results of the Ampliflash® GBS test correlated with low-density GBS cultures. Time-to-results correlated with GBS culture density only for the PlusLife® GBS test (p < 0.001). CONCLUSION: Both techniques provide excellent analytical performances with high sensitivity and specificity together with a short turnaround time and results available in 10 to 35 min. Their potential to further reduce the burden of GBS neonatal disease compared with antenatal culture screening needs to be assessed in future clinical studies.

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.

Development of a LAMP-Based Diagnostic for the Detection of Multiple HIV-1 Strains.

Since its first appearance in 1981, HIV-1 has remained a global concern. Current methods for diagnosing HIV-1, while effective, are mostly specific to a given subtype of HIV-1 and often require expensive equipment and highly trained individuals to collect and process the sample. It is necessary to develop a sensitive diagnostic method that can be administered with minimal equipment to provide better care in low-resource settings. Loop-mediated isothermal amplification is a rapid and sensitive method for detecting the presence of specific nucleic acid sequences. Herein we report the development and comparison of two different HIV LAMP assays, integrase and VPR, as well as the comparison between TRIZol and magnetic beads RNA extraction methods for each assay. Our analysis shows that the integrase assay was able to detect the virus from multiple subtypes in under 30 min with a variable limit of detection (LOD) that was dependent on the HIV-1 subtype.

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.

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.

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.

Sampling is decisive to determination of Leishmania (Viannia) species.

BACKGROUND: Accuracy of molecular tools for the identification of parasites that cause human cutaneous leishmaniasis (CL) could largely depend on the sampling method. Non-invasive or less-invasive sampling methods such as filter paper imprints and cotton swabs are preferred over punch biopsies and lancet scrapings for detection methods of Leishmania based on polymerase chain reaction (PCR) because they are painless, simple, and inexpensive, and of benefit to military and civilian patients to ensure timely treatment. However, different types of samples can generate false negatives and there is a clear need to demonstrate which sample is more proper for molecular assays. METHODOLOGY: Here, we compared the sensitivity of molecular identification of different Leishmania (Viannia) species from Peru, using three types of sampling: punch biopsy, filter paper imprint and lancet scraping. Different composite reference standards and latent class models allowed to evaluate the accuracy of the molecular tools. Additionally, a quantitative PCR assessed variations in the results and parasite load in each type of sample. PRINCIPAL FINDINGS: Different composite reference standards and latent class models determined higher sensitivity when lancet scrapings were used for sampling in the identification and determination of Leishmania (Viannia) species through PCR-based assays. This was consistent for genus identification through kinetoplastid DNA-PCR and for the determination of species using FRET probes-based Nested Real-Time PCR. Lack of species identification in some samples correlated with the low intensity of the PCR electrophoretic band, which reflects the low parasite load in samples. CONCLUSIONS: The type of clinical sample can directly influence the detection and identification of Leishmania (Viannia) species. Here, we demonstrated that lancet scraping samples consistently allowed the identification of more leishmaniasis cases compared to filter paper imprints or biopsies. This procedure is inexpensive, painless, and easy to implement at the point of care and avoids the need for anesthesia, surgery, and hospitalization and therefore could be used in resource limited settings for both military and civilian populations.

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)

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.

Direct TAMRA-dUTP labeling of M. tuberculosis genes using loop-mediated isothermal amplification (LAMP).

Fluorescent molecule-based direct labeling of amplified DNA is a sensitive method employed across diverse DNA detection and diagnostics systems. However, using pre-labeled primers only allows for the attachment of a single fluorophore to each DNA strand and any modifications of the system are less flexible, requiring new sets of primers. As an alternative, direct labeling of amplified products with modified nucleotides is available, but still poorly characterized. To address these limitations, we sought a direct and adaptable approach to label amplicons produced through Loop-mediated isothermal amplification (LAMP), using labeled nucleotides (dUTPs) rather than primers. The focus of this study was the development and examination of a direct labeling technique of specific genes, including those associated with drug resistance in Mycobacterium tuberculosis. We used 5-(3-Aminoallyl)-2'-deoxyuridine-5'triphosphate, tagged with 5/6-TAMRA (TAMRA-dUTP) for labeling LAMP amplicons during the amplification process and characterized amplification and incorporation efficiency. The optimal TAMRA-dUTP concentration was first determined based on amplification efficiency (0.5% to total dNTPs). Higher concentrations of modified nucleotides reduced or completely inhibited the amplification yield. Target size also showed to be determinant to the success of amplification, as longer sequences showed lower amplification rates, thus less TAMRA incorporated amplicons. Finally, we were able to successfully amplify all four M. tuberculosis target genes using LAMP and TAMRA-modified dUTPs.

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.

Development of a closed-tube, calcein-based loop-mediated isothermal amplification assay to detect Salmonella spp. in raw meat samples.

Foodborne pathogens compromise food safety and public health, and Salmonella spp. are among the major pathogenic bacteria that cause outbreaks worldwide. Proper surveillance through timely and cost-effective detection methods across the food animal production chain is crucial to prevent Salmonella outbreaks and agricultural losses. Traditional culture methods are labor- and resource-intensive, with lengthy turnaround times. Meanwhile, conventional molecular tools, such as PCR and qPCR, are expensive and require technical skills and equipment. Loop-mediated isothermal amplification (LAMP) is a simple, rapid, inexpensive, highly sensitive, and specific molecular assay that does not require expensive equipment. Hence, this study developed and optimized a closed-tube, calcein-based LAMP assay to detect Salmonella using the invA gene and performed evaluation and validation against conventional PCR. The LAMP assay showed high specificity and sensitivity. It showed 10-fold higher sensitivity than conventional PCR, at <1 ng/µL DNA concentrations. Meanwhile, for CFU/mL, LAMP assay showed 1000-fold higher sensitivity than conventional PCR at 4.8 × 103 cells/mL than 4.8 × 107 cells/mL, respectively. For parallel testing of 341 raw meat samples, after conventional culture enrichment (until Rappaport-Vassiliadis broth), the optimized LAMP assay showed 100% detection on all samples while conventional PCR showed 100%, 99.04%, and 96.64% for raw chicken, beef, and pork samples, respectively. Meanwhile, a shortened enrichment protocol involving 3-h incubation in buffered peptone water only, showed lower accuracy in tandem with the optimized LAMP assay ranging from 55 to 75% positivity rates among samples. These suggest that the optimized LAMP assay possesses higher sensitivity over conventional PCR for invA gene detection when coupled with conventional enrichment culture methods. Hence, this assay has potential as a powerful complementary or alternative Salmonella detection method to increase surveillance capacity and protect consumer food safety and public health worldwide.

Molecular Testing for Bethesda III Thyroid Nodules: Trends in Implementation, Cytopathology Call Rates, Surgery Rates, and Malignancy Yield at a Single Institution.

Background: Molecular testing (MT) has become standard practice to more accurately rule out malignancy in indeterminate Bethesda III (BIII) thyroid lesions. We sought to assess the adoption of this technology and its impact on cytology reporting, malignancy yield, and rates of surgery across community and academic sites affiliated with a tertiary medical center. Methods: We performed a retrospective cross-sectional study including all fine-needle aspirations (FNAs) analyzed at our institution from 2017 to 2021. We analyzed trends in MT utilization by platform and by community or academic site. We compared BIII call rates, MT utilization rates, rates of subsequent surgery, and malignancy yield on final pathology before and after MT became readily available using chi-square analysis and linear regression. Results: A total of 8960 FNAs were analyzed at our institution from 2017 to 2021. There was broad adoption of MT across both community and academic sites. There was a significant increase in both the BIII rate and the utilization of MT between the pre- and post-MT periods (p < 0.001 and p < 0.001). There was no significant change in the the malignancy yield on final pathology (57.1% vs. 50.0%, p = 0.347), while the positive predictive value of MT decreased from 85% to 50% (p = 0.008 [confidence interval 9.5-52.5% decrease]). Conclusions: The use of MT increased across the institution over the study period, with the largest increase seen after a dedicated pass for MT was routinely collected. This increased availability of MT may have led to an unintended increase in the rates of BIII lesions, MT utilization, and surgery for benign nodules. Physicians who use MT should be aware of potential consequences of its adoption to appropriately counsel patients.

Determination of the cycle threshold value of the Xpert Xpress SARS-CoV-2/Flu/RSV test that corresponds to the presence of infectious SARS-CoV-2 in anterior nasal swabs.

Despite having high analytical sensitivities and specificities, qualitative SARS-CoV-2 nucleic acid amplification tests (NAATs) cannot distinguish infectious from non-infectious virus in clinical samples. In this study, we determined the highest cycle threshold (Ct) value of the SARS-CoV-2 targets in the Xpert Xpress SARS-CoV-2/Flu/RSV (Xpert 4plex) test that corresponded to the presence of detectable infectious SARS-CoV-2 in anterior nasal swab samples. A total of 111 individuals with nasopharyngeal swab specimens that were initially tested by the Xpert Xpress SARS-CoV-2 test were enrolled. A healthcare worker subsequently collected anterior nasal swabs from all SARS-CoV-2-positive individuals, and those specimens were tested by the Xpert 4plex test, viral culture, and laboratory-developed assays for SARS-CoV-2 replication intermediates. SARS-CoV-2 Ct values from the Xpert 4plex test were correlated with data from culture and replication intermediate testing to determine the Xpert 4plex assay Ct value that corresponded to the presence of infectious virus. Ninety-eight of the 111 (88.3%) individuals initially tested positive by the Xpert Xpress SARS-CoV-2 test. An anterior nasal swab specimen collected from positive individuals a median of 2 days later (range, 0-9 days) tested positive for SARS-CoV-2 by the Xpert 4plex test in 39.8% (39/98) of cases. Of these samples, 13 (33.3%) were considered to contain infectious virus based on the presence of cultivable virus and replication intermediates, and the highest Ct value observed for the Xpert 4plex test in these instances was 26.3. Specimens that yielded Ct values of ≤26.3 when tested by the Xpert 4plex test had a likelihood of containing infectious SARS-CoV-2; however, no infectious virus was detected in specimens with higher Ct values.IMPORTANCEUnderstanding the correlation between real-time PCR test results and the presence of infectious SARS-CoV-2 may be useful for informing patient management and workforce return-to-work or -duty. Further studies in different patient populations are needed to correlate Ct values or other biomarkers of viral replication along with the presence of infectious virus in clinical samples.