Search

Diverse enteric pathogens, transmitted through human and animal feces, can cause gastroenteritis. Enteric viruses, such as human Aichi virus, specifically genotype A (AiV-A), are emerging pathogens that cause illnesses even at low doses and are spreading globally. This research developed a reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay targeting the 3CD junction and a reverse transcription colorimetric loop-mediated isothermal amplification (RT-cLAMP) duplex assay targeting junctions 2BC and 3CD of the AiV-A genome for rapid and sensitive detection of this virus in metropolitan and regional wastewater samples in Queensland, Australia. The performance of these assays was evaluated using control materials and by analyzing wastewater samples. In serially diluted control materials, RT-qPCR provided quantifiable data (mean 1.51 log 10 GC/2 μL of nucleic acid) down to a dilution of 1 × 10 -5  pg/μL. In comparison, the duplex RT-cLAMP assay detected down to 1 × 10 -4  pg/μL, indicating that its sensitivity was one order of magnitude less than that of RT-qPCR. Of the 38 wastewater samples from 38 metropolitan and regional wastewater treatment plants (WWTPs) in Queensland, Australia, 21 (55.3 %) tested positive by RT-qPCR with concentrations ranging from 3.60 to 6.23 log 10 GC/L. In contrast, only 15 (39.5 %) of 38 wastewater samples were positive using the duplex RT-cLAMP assay. The methods demonstrated substantial qualitative agreement (κ = 0.730), with a concordance of 86.5 %, demonstrating the reliability of RT-cLAMP for detecting AiV-A in wastewater samples. The duplex RT-cLAMP assay, despite demonstrating reduced detection sensitivity, has proven effective and holds promise as a supplementary approach, especially in settings with limited resources where rapid and affordable testing is crucial., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Rapid and accurate diagnostic methods are crucial for managing viral gastroenteritis in children, a leading cause of global childhood morbidity and mortality. This study introduces a novel microfluidic-Flap endonuclease 1 (FEN1)-assisted isothermal amplification (MFIA) method for simultaneously detecting major viral pathogens associated with childhood diarrhea-rotavirus, norovirus, and adenovirus. Leveraging the specificity-enhancing properties of FEN1 with a universal dspacer-modified flap probe and the adaptability of microfluidic technology, MFIA demonstrated an exceptional detection limit (5 copies/μL) and specificity in the simultaneous detection of common diarrhea pathogens in clinical samples. Our approach addresses the limitations of current diagnostic techniques by offering a rapid (turn around time <1 h), cost-effective, easy design steps (universal flap design), and excellent detection performance method suitable for multiple applications. The validation of MFIA against the gold-standard PCR method using 150 actual clinical samples showed no statistical difference in the detection performance of the two methods, positioning it as a potential detection tool in pediatric diagnostic virology and public health surveillance. In conclusion, the MFIA method promises to transform pediatric infectious disease diagnostics and contribute significantly to global health efforts combating viral gastroenteritis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: To meet the WHO target of eradicating yaws by 2030, highly sensitive and specific diagnostic tools are needed. A multiplex Treponema pallidum-Haemophilus ducreyi loop-mediated isothermal amplification (TPHD-LAMP) test holds promise as a near-patient diagnostic tool for yaws and H ducreyi. We conducted a prospective evaluation in Cameroon, Côte d'Ivoire, Ghana, and the Republic of the Congo to determine the diagnostic accuracy of the TPHD-LAMP test, as well as to assess its acceptability, feasibility, and cost., Methods: Active case searching within schools and communities was used to locate participants with clinically suspicious laws-like lesions. Individuals with serologically confirmed active yaws provided paired lesion swabs between March, 2021, and April, 2023. For each participant, one swab was tested with the TPHD-LAMP at a local district laboratory and the other with reference quantitative PCR (qPCR) tests conducted at national reference laboratories. The primary outcome was TPHD-LAMP test sensitivity and specificity compared with qPCR. Laboratory technicians were interviewed using a multiple-choice survey to gauge acceptability and feasibility of the TPHD-LAMP test. Costs of each test were calculated., Findings: Of 3085 individuals with at least one suspected yaws lesion, 531 (17%) were serologically confirmed. We enrolled 493 participants with seropositive yaws and a further 32 with negative serology. The sensitivity of the TPHD-LAMP test for detecting T pallidum was 63% (95% CI 56-70) and the specificity was 66% (95% CI 61-71). Sensitivity and specificity for T pallidum improved to 73% (63-82; p=0·0065) and 75% (68-80; p=0·0003), respectively, in H ducreyi-negative samples. Interviews highlighted challenges in user-friendliness and practicality of the TPHD-LAMP test. The cost of the test per sample was one third of that of qPCR, although the TPHD-LAMP test entailed higher costs to establish the assay., Interpretation: This was the first multi-country diagnostic evaluation of a molecular test for yaws. The TPHD-LAMP testing, in its current form, falls short of the WHO target product profile criteria for yaws diagnostics. These findings highlight the importance of assessing new diagnostics in real-world conditions to ensure their suitability for programmatic use., Funding: The EDCTP2 programme supported by the EU., Competing Interests: Declaration of interests SF, MB, and EL are employees of Mast Diagnostica, which produces and sells LAMP products. A patent covering the mediator displacement probe technique, a component of the TPHD-LAMP assay described in this study, has been applied for by the University of Freiburg and Hahn-Schickard. All other authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

We evaluated the analytical performance of three commercial molecular assays for rapid detection of Clostridioides difficile toxin B in stool samples. The results were compared with results from the BD MAX™ Cdiff assay. We analyzed forty negative and thirty-two positive stool samples with three rapid assays: Roche cobas® Liat® Cdiff, SD Biosensor STANDARD™ M10 C. difficile and Cepheid Xpert® C. difficile BT. The assays demonstrated a sensitivity of 96.9 %, 96.9 % and 100.0 % and a specificity of 100 %, 97.5 % and 97.5 %, respectively. There is limited data available on the analytical performance of the newly introduced STANDARD™ M10 C. difficile assay. In this study, all three rapid assays demonstrated similarly high analytical performance and can be used for detection of toxigenic C. difficile., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Background: Rapid and precise detection of Mycobacterium tuberculosis (MTB) is paramount for effective management and control of tuberculosis. Clustered regularly interspaced short palindromic repeats (CRISPR) technology has emerged as a promising tool for pathogenic diagnosis owing to its specificity and adaptability. This systematic review and meta-analysis aimed to appraise the diagnostic accuracy of CRISPR-based techniques in identifying MTB., Methods: A meticulous search was conducted in Medline, Scopus, Embase, and ISI Web of Science to retrieve relevant studies, adhering to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Quality was assessed using the Joanna Briggs Institute checklist. Data synthesis and analyses, including subgroup analyses, were performed with R v 4.3.1, examining variables like CRISPR variants, gene targets, pre-amplification techniques, and signal readout methods., Results: From 389 identified studies, 14 met the inclusion criteria, encompassing 2175 MTB strains. The pooled sensitivity and specificity of CRISPR-based techniques were 0.93 (95% CI 0.85-0.99) and 0.97 (95% CI 0.94-0.99), respectively. The pooled diagnostic odds ratio was 273.4379 (95% CI 103.3311-723.5794), with an area under the curve of 0.97 for the summary receiver operating characteristic (SROC) curve, denoting excellent diagnostic accuracy. Subgroup analyses illustrated variations in diagnostic metrics based on factors like CRISPR variant utilized, target gene, and pre-amplification methods. For instance, CRISPR-Cas12 exhibited a sensitivity and specificity of 0.93 (95% CI 0.78-0.98) and 0.98 (95% CI 0.93-1), respectively. Moreover, this technology showed a sensitivity of 96% and specificity of 100% in detecting resistant MTB., Conclusion: CRISPR-based methods exhibit substantial diagnostic sensitivity and specificity for detecting MTB, with notable variances across different CRISPR variants and methodological approaches. Further studies must be conducted to optimize CRISPR's potential as a diagnostic tool for MTB in a variety of clinical and research settings., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Neospora caninum (N. caninum) is a protozoan parasite that poses a serious risk to livestock by infecting various domestic and wild animals. Loop-Mediated Isothermal Amplification (LAMP) offers a cost-effective, highly sensitive, and specific method for detecting protozoan parasites. This study aims to develop a precise, rapid, and visually assessable colorimetric LAMP method, improving on traditional techniques. We employed a rigorous screening process to identify the optimal primer set for this experiment. Subsequently, we fine-tuned the LAMP reaction at 65 °C for 40 min with 270 μmol/L neutral red. We then confirmed the specificity of primers for N. caninum through experimental validation. The LAMP method demonstrated a lower detection limit compared to traditional Polymerase Chain Reaction (PCR) techniques. While LAMP offers clear advantages, the prevalence of DNA detected in 89 sheep serum and 59 bovine serum samples using the nested PCR method was 3.37 % (3/89) and 1.69 % (1/59), respectively. In contrast, when the LAMP method was employed, the prevalence of detected DNA rose to 5.61 % (5/89) for sheep and 3.38 % (2 /59) for bovine. A comparison of two molecular assays using the intragroup correlation coefficient (ICC) resulted in a value of 0.999 (95 % CI: 0.993-0.996, p < 0.001), indicating the LAMP method is in the "better" range according to James Lee's categorization. The LAMP technique, optimized with specific primers of N. caninum and neutral red dye, not only exhibited higher sensitivity but also provided convenience over conventional PCR methods, highlighting its potential for on-site applications and cost-effective field detection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Endometriosis, infertility, or recurrent pregnancy loss (RPL) are entities characterised by a decrease in Lactobacillus spp. and an increase in bacterial vaginosis-associated bacteria, (BVAV) according with 16S rRNA sequencing studies. However, the use of nucleic acid amplification tests (NAAT) as a tool for diagnosis algorithms is unknown. Seventy-four patients were included, with a median age of 36.5 years old (IQR: 34-39) including infertility (n=31), endometriosis (n=25), or RPL (n=18), for culturing and NAAT using the Allplex™ Bacterial Vaginosis Plus (ABVP) assay (Seegene Ⓡ ) with endometrial samples. The objective was determining the utility of ABVP assay for diagnosing the entities. Forty-six microorganisms were isolated from 31 out of 74 patients (41.9 %). Twenty-five endometrial samples (33.8 %) were positive for some targets included in the ABVP-assay, with median Ct value ∼37 (IQR: 31.3-37.1) and Qt value 1.43 Log 10 copies/reaction (IQR:1.1-2.6). For Lactobacillus species, sensitivity and specificity were 80 % and 84 %, respectively. Gardnerella vaginalis, 63.6 % and 95.7 %. No significant increase in BVAV was detected in any of the gynaecological entities. The ABVP and culture based algorithm did not show utility as a tool for endometriosis, infertility, or RPL diagnosis., Competing Interests: Declaration of competing interest The authors declare that they have no financial interests or personal relationships that could have influenced the work presented in this article. The authors received no external funding from any of the commercial companies for this study., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The clinical diagnosis of pathogen infectious diseases increasingly requires sensitive and rapid RNA detection technologies. The RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a system has shown immense potential in molecular diagnostics due to its trans-cleavage activity. However, most Cas13a-based detection methods require an amplicon transcription step, and the multi-step open-tube operations are prone to contamination, limiting their widespread application. Here, we propose an ultrasensitive (single-copy range, ∼aM) and rapid (within 40 min) isothermal one-pot RNA detection platform, termed SATCAS (Simultaneous Amplification and Testing platform based on Cas13a). This method effectively distinguishes viable bacteria (0%-100%) under constant total bacterial conditions, demonstrating its robustness and universality. SATCAS excels in identifying single nucleotide polymorphisms (SNPs), particularly detecting 0.5% drug-resistant mutations. We validated SATCAS by detecting infections in biological samples from 68 HBV, 23 EBV, and 48 SARS-CoV-2 patients, achieving 100% sensitivity, 92.86% specificity, and 97.06% accuracy in HBV infection testing. We anticipate that SATCAS has broad application potential in the early diagnosis, subtyping, drug resistance detection, and point-of-care monitoring of pathogen infectious diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Abstract: Prostate cancer (PCa) is the second leading cause of cancer-related death among men. Prostate-specific antigen (PSA) testing is used in screening programs for early detection with a consequent reduction of PCa-specific mortality at the cost of overdiagnosis and overtreatment of the nonaggressive PCa. Recently, several assays have been commercially developed to implement PCa diagnosis, but they have not been included in both screening and diagnosis of PCa. This review aims to describe the actual and novel commercially available molecular biomarkers that can be used in PCa management to implement and tailor the screening and diagnosis of PCa., (Copyright © 2024 Copyright: ©The Author(s)(2024).)

LAMP (Loop-mediated isothermal amplification) is a popular method for the molecular diagnostics of numerous pathogens, specifically useful for point-of-care testing. However, the efficacy and sensitivity of LAMP still need to be maximised for the best performance in clinical settings. Adding a novel fourth primer pair is a promising way to accelerate the LAMP speed. Here, we report PI primers that are part of inner primers and can be used in LAMP without a specific design. PI primers were tested in quantitative LAMP detecting SARS-CoV-2 and MS2. The new primers have increased the speed and sensitivity of quantitative LAMP, RT-LAMP, and duplex LAMP with artificial templates and RNA samples from nasal swabs. Adding PI primers could become a valuable option for LAMP optimisation, especially when a desirable LAMP target is a highly variable DNA sequence with a few conservative sites for primers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Blood culture, the gold-standard method for identifying pathogens in bloodstream infections, is time-consuming and demonstrates low sensitivity. These drawbacks are related to high mortality, especially among pediatric oncology patients presenting febrile neutropenia episodes. Here we describe two novel High-Resolution Melting assays designed for pathogen detection in bloodstream infections. The assays were initially evaluated using five sepsis-associated pathogens. Both assays demonstrated 100 % specificity, detected as low as 100 fg of bacterial DNA, and exhibited reproducibility. Clinical isolates from blood cultures were 100 % identified by both assays. Moreover, blind and direct identification of blood samples from pediatric cancer patients demonstrated sensitivities of 61.5 % and 69.2 % for "Primer Set 1" and "Primer Set 2", respectively. Our study highlights the potential of HRM-based assays as a rapid and efficient diagnostic approach for sepsis-related microorganisms. Further advancements could enhance their clinical utility for better management of febrile neutropenia episodes, especially in pediatric oncology patients., Competing Interests: Declaration of competing interest We wish to confirm that there are no known conflicts of interest associated with this publication., (Copyright © 2024. Published by Elsevier Inc.)

Background: Resource-limited regions face a greater burden of infectious diseases due to limited access to molecular tests, complicating timely diagnosis and management. Current molecular point-of-care tests (POCTs) either come with high costs or lack adequate sensitivity and specificity. To facilitate better prevention and control of infectious diseases in underserved areas, we seek to address the need for molecular POCTs that better align with the World Health Organization (WHO)'s ASSURED criteria-Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free, and Deliverable to end users., Methods: A novel molecular POCT, Pasteur Pipette-assisted isothermal probe amplification (pp-IPA), was developed for malaria detection. Without any microfluidics, this method captures Plasmodium 18S rRNA in a modified Pasteur pipette using tailed genus-specific probes. After washing, the bound tailed probes are ligated to form a template for subsequent novel isothermal probe amplification using a pair of generic primers, bypassing nucleic acid extraction and reverse transcription. The method was assessed using cultured Plasmodium and compared with real-time quantitative reverse transcription PCR (RT-qPCR) or reverse transcription loop-mediated isothermal amplification (RT-LAMP) in clinical blood samples., Results: The entire assay is completed in 60-80 min with minimal hands-on time, using only a Pasteur pipette and a water bath. The pp-IPA's analytical sensitivity is 1.28 × 10 -4  parasites/μl, with 100% specificity against various blood-borne pathogens causing malaria-like symptoms. Additionally, pp-IPA needs only liquid-transfer skill for operation and the cost is around USD 0.25 per test, making it at least 300 times lower than mainstream POCT platforms., Conclusions: Designed to improve the accessibility of molecular detection in resource-limited settings, pp-IPA's simplicity, affordability, high sensitivity/specificity, and minimal equipment requirements make it a promising point-of-care pathogen identification tool in resource-constrained regions., (© 2024. The Author(s).)

Numerous viruses, such as the bovine rotavirus (BRV), the bovine parvovirus (BPV), and the bovine viral diarrhea virus (BVDV), can cause bovine viral diarrhea syndrome. The global livestock industry has been subjected to significant consequences due to this condition. This results in considerable losses and hinders the production of crucial resources such as meat and milk, which are indispensable for sustaining the world's population. It is crucial to develop a quick and precise way of simultaneously detecting BVDV, BRV, and BPV, as they often occur together in mixed infections. A triplex loop-mediated isothermal amplification-lateral flow dipstick (LAMP-LFD) assay that can concurrently detect all three viruses was introduced in this study. The amplification process involved 30 minutes of incubation at 65 °C. The limits of detection (LODs) for BVDV, BRV, and BPV were 2.62 × 10 1 copies per μL, 2.43 × 10 1 copies per μL, and 2.50 × 10 1 copies per μL, respectively. The triplex LAMP-LFD assay was further evaluated in 156 anal swab samples, and the results were in agreement with the results of fluorescence quantitative PCR (qPCR) in more than 99% of the cattle. This assay is expected for the quick identification of triplex viruses in the field because it has high sensitivity and specificity and doesn't depend on laboratory equipment or conditions.

The objective of this study was to establish a rapid visual diagnosis method for vancomycin-resistant Enterococcus faecium (VREFm) based on multienzyme isothermal rapid amplification (MIRA) combined with lateral flow strips (LFSs). The MIRA primers and probes were specifically designed to maintain the sequence of the VanA gene of VREFm. We optimized the reaction time and temperature and thoroughly assessed the specificity and sensitivity of the MIRA-LFS system. We also compared the MIRA-LFS method with the polymerase chain reaction (PCR) assay and the disc diffusion method. We then evaluated the MIRA-LFS assay for consistency testing and clinical application. The MIRA-LFS technique completed the amplification process within 30 min, and the results were observed on LFS. The method demonstrated high sensitivity, with a minimum detection limit of 1.066 CFU/µL for VREFm and exhibited specificity without cross-reactivity with other pathogenic bacteria. When applied to the detection of clinical samples, the method exhibited consistency with the PCR and agar dilution methods. The combined use of MIRA and LFS in this study facilitates simplifying the workflow for detecting VREFm, which is of great significance for rapidly detecting the enterococcal infections and preventing and controlling the nosocomial infections., Importance: One of the key approaches to treating and controlling vancomycin-resistant Enterococcus faecium (VREFm) is an accurate and rapid diagnosis. To achieve this goal, a simple and rapid method must be constructed for immediate detection in the field. Multienzyme isothermal rapid amplification (MIRA) is an isothermal rapid amplification method that allows amplification reactions to be completed under room temperature conditions. When combined with lateral flow strips (LFSs), MIRA-LFS enables the rapid detection of pathogenic microorganisms. However, the MIRA method often produces false signals. These false signals are eliminated by using base mismatches introduced in primers and probes. The MIRA-LFS system was constructed with high specificity and sensitivity for the detection of VREfm, without the limitation of sophisticated instruments. This enables the prompt formulation of diagnostic and therapeutic decisions., Competing Interests: The authors declare no conflict of interest.

Background: Toxoplasma gondii, an intracellular parasite, is a significant cause of zoonotic disease, with an estimated one-third of the world's human population believed to be infected. T. gondii is transmitted to humans through the consumption of contaminated water, soil, vegetables, fruits, shellfish or undercooked meat, and can also be passed from human to human through vertical transmission, transplants and blood transfusion. While T. gondii infection typically manifests mild symptoms such as colds among immunocompetent individuals, it can prove lethal for those with weakened immune systems., Methods: To summarize the diagnostic methods for Toxoplasma gondii infection, we performed a literature search on PubMed from 1948 to 2023 using the keywords "T. gondii serological diagnosis" or "T. gondii molecular diagnosis"., Results: Rapid and accurate diagnosis of T. gondii infection is imperative. Although a diagnostic kit is currently commercially available, there are a number of disadvantages to the validation principles applied to each diagnostic kit. Consequently, multiple diagnostic methods are concurrently employed to offset these limitations. Serological methods for diagnosing T. gondii infection include the Dye Test (DT), Agglutination Test (AT), Modified Agglutination Test (MAT), Latex Agglutination Test (LAT), Enzyme-Linked Immunosorbent Assay (ELISA), and Western Blot. Meanwhile, molecular methods such as polymerase chain reaction (PCR), nested PCR, real-time PCR, loop-mediated isothermal amplification (LAMP), multiplex PCR, and PCR-restriction fragment length polymorphism (PCR-RFLP) are also utilized. Each of these methods possess its own set of advantages and disadvantages., Conclusions: By summarizing the advantages and disadvantages of different diagnostic techniques, it is hoped that the epidemiology, prevention, and control of toxoplasmosis will be improved in the future through the use of appropriate technologies., (© 2024. The Author(s).)

Chloroquine is still used as a first-line treatment for uncomplicated Plasmodium vivax malaria in India and resistance to this therapy can act as a major hurdle for malaria elimination. It is difficult to monitor drug-efficacy and drug resistance through in vivo and in vitro studies in case of Plasmodium vivax so analysis of molecular markers serves as an important tool to track resistance. Molecular methods that are currently in use for detecting single nucleotide polymorphisms in resistant genes including Polymerase chain reaction (PCR), Realtime-Polymerase chain reaction require highly sophisticated labs and are time consuming. So, with this background the study has been designed to optimize Loop Mediated Isothermal Amplification Assay to detect single nucleotide polymorphisms in chloroquine resistance gene of Plasmodium vivax in field settings. Eighty-eight Plasmodium vivax positive samples were collected. Pvmdr1 gene was amplified for all the samples and sequenced. Obtained sequences were analyzed for the presence of single nucleotide polymorphisms in the target gene. Further Loop Mediated Isothermal Amplification Assay primer sets were designed for the target mutants and the assay was optimized. Clinical as well as analytical sensitivity and specificity for the assay was calculated. Double mutants with variations at T958M and F1076L were detected in 100% of the Plasmodium vivax clinical isolates with haplotype M958 Y976 Y1028 L1076. Designed primers for Loop Mediated Isothermal Amplification Assay successfully detected both the mutants (T958M and F1076L) in 100% of the isolates and do not show cross-reactivity with other strains. So, the assay was 100% sensitive and specific for detecting single nucleotide polymorphisms in the target Pvmdr1 gene. Limit of detection was found to be 0.9 copies/µl and lowest DNA template concentration detected by designed assay was 1.5 ng/µL. Observed prevalence of single nucleotide polymorphisms in Pvmdr 1 gene is indicating a beginning of trend towards chloroquine resistance in Plasmodium vivax. The present study optimized LAMP for detecting single nucleotide polymorphisms in Plasmodium vivax cases in field settings, thus would help in finding significant hubs of emerging chloroquine drug resistance and ultimately helping in the management of suitable antimalarial drug policy., (© 2024. The Author(s).)

Cost efficient and rapid detection tools to detect mutations especially those linked to drug-resistance are important to address concerns of the rising multi-drug resistance infections. Here we integrated dual probes, namely a calibrator probe and an indicator probe, into isothermal amplification detection system. These two probes are designed to bind distinct regions on the same amplicon to determine the presence or absence of mutation. The calibrator probe signal is used as an internal signal calibrator for indicator probe which detects the presence or absence of the mutation. As an illustrative example, we evaluated the applicability of this dual probe method for detecting mutations associated with rifampicin (RIF) drug resistance at codons 516, 526 and 531 of the rpoB gene in Mycobacterium tuberculosis. In this assessment, we examined 127 artificial samples comprising wild types and mutants with single or multiple mutations. Our results demonstrated 100% accuracy for both wild types and mutants for mutations at codons 526 and 531. As regards to mutations at codon 516, the wild type was identified with 100% accuracy, while the mutants were identified with 95% accuracy. Moreover, when we extended our evaluation to include clinical MTB strains and the Zeptometrix MTB Verification panel, our method achieved 100% accuracy (5 out of 5) in identifying wild-type strains. Additionally, we successfully detected a RIF-resistant strain with mutations at codon 531 of the rpoB gene in Zeptometrix verification panel. Our isothermal mutation detection system, relying on dual probes exhibits a versatile approach. With the capability to identify mutations without prior knowledge of their specific mutation direction, our dual-probe method shows significant promise for applications in drug resistance nucleic acid testing, particularly in resource-limited settings., Competing Interests: The proprietary subjects disclosed herein are property of Revvity, Inc. Although the authors are employed and funded by Revvity, Inc, this should not detract from the objectivity of data generation or interpretation., (Copyright: © 2024 Nandu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Given the ongoing COVID-19 pandemic, there is a need to identify SARS-CoV-2 and to differentiate it from other respiratory viral infections, especially influenza A and B, in various critical settings. Since their introduction, the use of rapid antigen tests has spread worldwide, but there is variability in their diagnostic accuracy. In the present study, we evaluated the clinical performance of the ID NOW™ COVID-19 2.0, a molecular point-of-care test (POCT) based on enzymatic isothermal amplification for the differential diagnosis of SARS-CoV-2 and influenza A/B in a pediatric emergency setting. A cohort of pediatric patients admitted between December 2022 and February 2023 were simultaneously tested with the POCT and standard laboratory molecular assay. Our findings showed high negative agreement of the POCT assay across the different age groups for SARS-CoV-2, influenza A, and influenza B (more than 98.0%), while its positive agreement varied significantly for the abovementioned viral species from 50.0% to 100%. These results highlight the potential of the ID NOW™ COVID-19 2.0 POCT assay as a reliable and rapid tool for excluding SARS-CoV-2 and influenza A/B infections in symptomatic pediatric patients, although its variable positive agreement suggests a need for confirmatory RT-qPCR testing in certain clinical and epidemiological settings in order to ensure accurate diagnosis and appropriate patient management.

Accurate genetic analysis is essential for the detection of pathogens as it necessitates suitable DNA extraction methods tailored to specific microorganisms such as Gram-positive bacteria. This study examined several commercial and simplified DNA extraction methods for their suitability in isothermal downstream applications. Extracted DNA was assessed using spectrophotometry, electrophoresis, polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) while its stability was inspected after five months of storage. The findings revealed variations in DNA yield, purity and integrity among the extraction methods. While extraction kits demonstrated high yield and purity, the in-house extraction techniques showed incoherent correlation between yield and purity, yet showed promise for a streamlined extraction process. The DNA acquired from all methods yielded positive amplification in PCR and LAMP. DNA extracted by kits exhibits prolonged stability than those obtained via boiling lysis. Both methods offer distinct advantages, with commercial kits providing longer stability and high-quality DNA while boiling lysis stands out for its simplicity, with shorter handling and processing periods. This study emphasizes selecting ideal extraction methods for Streptococcus agalactiae, in the prospect of aquaculture settings. In particular, successful LAMP reaction suggests that boiled extracts are feasible enough for detection, and suited for point-of-care (POC) testing where prompt detection of aquatic pathogens is often critical. Ultimately, the choice of method should be contemplated on a case-by-case basis such as the study goals, intended settings, and type of samples., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Superficial fungal infections caused by dermatophytes are a prevalent global health concern. Rapid and accurate diagnosis of these pathogens through molecular tools would offer a substantial advantage for early detection and effective treatment. The conventional fungal culture presents inherent limitations, including extended result delivery delay and variable sensitivity. This study aimed to evaluate the performance of the multiplex real-time PCR Novaplex dermatophyte assay (Seegene) in comparison to traditional mycological methods including direct examination and culture. A total of 312 nail, skin, and scalp samples collected from patients with suspected superficial fungal infections for mycological diagnosis were retrospectively subjected to the Novaplex dermatophyte assay. Overall, 170 (54.6%) and 186 (59.6%) samples tested positive for dermatophyte culture and dermatophyte PCR, respectively. The concordance between PCR and culture for dermatophyte detection was 87.2%. There were 158 culture-positive/PCR-positive samples, 12 culture-positive/PCR-negative samples, and 28 culture-negative/PCR-positive samples. The sensitivity of PCR against culture varied according to the dermatophyte target, ranging from 90.5% ( Trichophyton mentagrophytes/interdigitale/benhamiae ), 91.2% ( Trichophyton rubrum ), to 100% ( Microsporum spp. and Trichophyton tonsurans ). When considering the final diagnosis using composite criteria, the sensitivity and specificity for the diagnosis of dermatophytosis were 92.9% and 96.6% for PCR, 86.7% and 100% for culture, and 95.4% and 92.2% for direct examination and culture combined, respectively. The Seegene Novaplex dermatophyte assay is an easy-to-use automated one-step extraction-PCR system that offers satisfactory performance for routine diagnosis of dermatophytoses in clinical laboratories, particularly in non-specialized centers. However, it cannot fully replace conventional mycology due to its inability to detect mold infections and to identify dermatophytes at the species level., Competing Interests: The authors declare no conflict of interest.

Rapid detection is crucial for tuberculosis (TB) control. GeneXpert (Cepheid) is a widely used PCR system, known for its simplicity, random access, and point-of-care compatibility. SD BIOSENSOR recently introduced a similar system, STANDARD M10, including a Mycobacterium tuberculosis (MTB) and rifampicin (RIF) and isoniazid resistance (herein, MDR-TB) assay and an MTB/nontuberculous mycobacteria (NTM) assay. We evaluated these assays for the potential to replace the established Xpert MTB/RIF Ultra assay in a low-TB incidence setting. We analyzed 160 clinical respiratory samples (45 MTB-positive and 35 NTM-positive) and further 24 drug-resistant MTB, 30 mycobacterial species (2 MTB, 28 NTM), and 37 non-mycobacterial isolates. Compared with culture, clinical sensitivities and specificities for MTB detection were 88.9% (95% confidence interval [CI] = 76.1-95.6%) and 97.4% (CI = 92.3-99.4%) with Xpert Ultra, 88.9% (95% CI = 76.1-95.6%) and 98.3% (CI = 93.5-99.9%) with M10 MDR-TB, and 84.4% (CI = 70.9-94.4%) and 98.3% (CI = 93.5-99.9%) with M10 MTB/NTM, respectively. For NTM detection, M10 MTB/NTM showed sensitivity and specificity of 65.7% (CI = 49.1-79.2%) and 96.8% (CI = 91.8-99.0%). Compared with phenotypic drug susceptibility testing (DST), sensitivity and specificity for detecting RIF resistance were 100% (CI = 77.3-100%) and 95.6% (CI = 84.4-99.6%) with Xpert Ultra, and 100% (CI = 74.9-100%) and 95.5% (CI = 84.0-99.6%) with M10 MDR-TB. M10 MDR-TB showed 92.3% sensitivity (CI = 74.7-99.0%) and 100% specificity (CI = 87.3-100%) for detecting isoniazid resistance. All discrepancies in DST by PCR were concordant with whole-genome sequencing. While M10 MDR-TB demonstrated great potential as an alternative to Xpert Ultra, M10 MTB/NTM had limitations in NTM screening. Additionally, the M10 sputum pretreatment did not inactivate MTB efficiently, which should be considered in process risk assessment., Importance: The molecular diagnostic STANDARD M10 system is highly analogous to the widely established GeneXpert system, which significantly increases the relevance of this evaluation study in the field of rapid detection of M. tuberculosis . To our knowledge, this is the first clinical evaluation describing the performance of the STANDARD M10 MDR-TB and MTB/NTM assays, including an extensive analytical specificity panel (inclusivity and exclusivity) for the detection of M. tuberculosis , drug resistance, and nontuberculous mycobacteria., Competing Interests: The authors declare no conflict of interest.

We investigated the performance of the targeted next-generation sequencing (tNGS)-based Oxford Nanopore Diagnostics AmPORE TB assay, recently approved by the World Health Organization (WHO) as tuberculosis (TB) diagnostic test for the detection of drug resistance on respiratory specimens. A total of 104 DNA samples from Xpert MTB/RIF-positive TB sputum specimens were tested using the AmPORE TB kit, with the GenoScreen Deeplex Myc-TB as a comparative tNGS assay. For AmPORE TB, DNA samples were divided into five sequencing runs on the MinION device. Data analysis was performed using proprietary software. The WHO catalog of mutations was used for drug resistance interpretation. The assay achieved a high validity rate of 98% (102/104 DNA samples), homogeneous mean reads coverage across TB-positive specimens, and 100% positive and negative agreements for detecting mutations associated with resistance to rifampicin, pyrazinamide, fluoroquinolones, ethambutol, and capreomycin compared with Deeplex Myc-TB. The main discrepancies for the remaining drugs were attributable to the different assay panel designs. The AmPORE TB turnaround time was approximately 5-6 hours from extracted DNA to tNGS reporting for batches of 22 DNA samples. The AmPORE TB assay drastically reduced the time to tNGS reporting from days to hours and showed good performance for drug-resistant TB profiling compared with Deeplex Myc-TB., Importance: Targeted next-generation sequencing (tNGS) of Mycobacterium tuberculosis provides comprehensive resistance predictions matched to new multidrug-resistant/rifampicin-resistant tuberculosis regimens and received World Health Organization approval for clinical use in respiratory samples in 2024. The advanced version of the Oxford Nanopore Diagnostics AmPORE TB tNGS kit was evaluated in this study for the first time and demonstrated good performance, flexibility, and faster turnaround time compared with the existing solutions., Competing Interests: The authors declare no conflict of interest.

Sequencing of plasma microbial cell-free DNA (mcfDNA) has gained increased acceptance as a valuable adjunct to standard-of-care testing for diagnosis of infections throughout the body. Here, we report the analytical and clinical validation of a novel application of mcfDNA sequencing, the non-invasive detection of seven common antimicrobial resistance (AMR) genetic markers in 18 important pathogens. The AMR markers include SCC mec , mecA, mecC , vanA, vanB , bla CTX-M , and bla KPC . The AMR markers were computationally linked to the pathogens detected. Analytical validation showed high reproducibility (100%), inclusivity (54 to 100%), and exclusivity (100%). Clinical accuracy was assessed with 114 unique plasma samples from patients at seven study sites with concordant culture results for target bacteria from a variety of specimen types and correlated with available phenotypic antimicrobial susceptibility test results and genotypic results. The positive percent agreement (PPA), negative percent agreement (NPA), and diagnostic yield (DY) were estimated for each AMR marker. DY was defined as the percentage of tests that yielded an actionable result of either detected or not detected. The results for the combination of SCC mec and mecA for staphylococci were PPA 19/20 (95.0%), NPA 21/22 (95.4%), DY 42/60 (70.0%); vanA for enterococci were PPA 3/3 (100%), NPA 2/2 (100%), DY 5/6 (83.3%); bla CTX-M for gram-negative bacilli were PPA 5/6 (83.3%), NPA 29/29 (100%), DY 35/49 (71.4%); and bla KPC for gram-negative bacilli were PPA 0/2 (0%), NPA: 23/23 (100%), DY 25/44 (56.8%). The addition of AMR capability to plasma mcfDNA sequencing should provide clinicians with an effective new culture-independent tool for optimization of therapy., Importance: This manuscript is ideally suited for the Innovative Diagnostic Methods sections as it reports the analytical and clinical validation of a novel application of plasma microbial cell-free DNA sequencing for direct detection of seven selected antimicrobial resistance markers in 18 target pathogens. Clearly, it has potential clinical utility in optimizing therapy and was incorporated into the Karius test workflow in September 2023. In addition, the workflow could readily be adapted to expand the number of target bacteria and antimicrobial resistance markers as needed., Competing Interests: All employees of Karius, which markets the commercial test evaluated in this study, shared responsibilities for study design, analysis of the data, and writing and editing of the manuscript. John Joseph Farrell (OSF Healthcare), Sanjeet Dadwal (City of Hope), Amy L. Carr (AdventHealth Orlando), Arryn Craney (Orlando Health), Matthew Pike (Carle Foundation Hospital), and James B. Wood (Indiana University School of Medicine) received grant support from Karius to support the collection and curation of orthogonal data that were fundamental to the clinical validation and reviewed the manuscript.

A biomarker is defined as a characteristic that is measured as an indicator of a normal biological or pathological process, a response to an exposure or intervention. Biomarkers with a diagnostic approach must identify not only the presence but also the absence of the disease with high precision, so having the biological source of the said marker is of vital importance to ensure precision and accuracy; the aim was to carry out a review of its diagnostic potential. The search strategy was carried out in three databases: PubMed, ScienceDirect, and Scopus. The keywords that were used were as follows: "gingival crevicular fluid", "Biomarker", and "Diagnosis", using the Boolean operator "AND". The filter was used at 10 years. Within the type of molecules most studied, the cytokine family was the most abundant with 25.42% of the studies, followed by metalloproteinases and proteins with 16.9% each one. Studies that included RNA-type genetic material were less frequently found. As has been demonstrated, the use of GCF as a source of biomolecules for diagnostic use has been increasing, both for oral diseases, which reflects the local conditions of the disease; it also has the ability to reflect the development of distant diseases; and this is because GCF is a blood ultrafiltrate., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 María Verónica Cuevas-González et al.)

Background: Cutaneous leishmaniasis (CL) in Ethiopia and some parts of Kenya is predominantly caused by Leishmania aethiopica. While skin-slit (SS) microscopy is routinely used for CL diagnosis, more sensitive molecular tests are available. The Loopamp™ Leishmania detection kit (Loopamp) is a robust loop-mediated isothermal amplification (LAMP) assay with the potential for implementation in primary healthcare facilities. In this study, we comparatively assessed the diagnostic accuracy of four methods currently used to diagnose CL: Loopamp, kinetoplast DNA (kDNA) PCR, spliced leader RNA (SL-RNA) PCR and SS microscopy., Methods: A study on 122 stored tape disc samples of suspected CL patients was conducted in Gondar, northwestern Ethiopia. Routine SS microscopy results were obtained from all patients. Total nucleic acids were extracted from the tapes and subjected to PCR testing targeting kDNA and SL-RNA, and Loopamp. Diagnostic accuracy was calculated with SS microscopy as a reference test. The limit of detection (LoD) of Loopamp and kDNA PCR were determined for cultured L. aethiopica and Leishmania donovani., Results: Of the 122 patients, 64 (52.5%) were identified as CL cases based on SS microscopy. Although the PCR tests showed a sensitivity of 95.3% (95% confidence interval [CI] 91.6-99.1), Loopamp only had 48.4% (95% CI 39.6-57.3) sensitivity and 87.9% (95% CI 82.1-93.7) specificity. The LoD of Loopamp for L. donovani was 100-fold lower (20 fg/µl) than that for L. aethiopica (2 pg/µl)., Conclusions: The Loopamp™ Leishmania detection kit is not suitable for the diagnosis of CL in Ethiopia, presumably due to a primer mismatch with the L. aethiopica 18S rRNA target. Further research is needed to develop a simple and sensitive point-of-care test that allows the decentralization of CL diagnosis in Ethiopia., (© 2024. The Author(s).)

Background: The resurgence of two bed bug species, the common bed bug (Cimex lectularius Linnaeus, 1758) and tropical bed bug (Cimex hemipterus Fabricius, 1803), in the same geographical regions has been frequently reported recently. Consequently, the rapid identification of these species is crucial for implementing targeted capture traps and tailored pyrethroid resistance diagnosis, due to differences in genetic and physiological traits., Methods: To develop molecular diagnostic methods, distinct protocols were established for multiplex PCR and loop-mediated isothermal amplification (LAMP) using species-specific primers based on species-specific segments of internal transcribed spacer 2 sequences. These methods were optimized for rapid and accurate identification of the two bed bug species., Results: Both multiplex PCR and LAMP protocols were effective in simultaneously identifying the two bed bug species, even when utilizing DNA released from dead specimens. Notably, the straightforward procedure and minimal time commitment of LAMP suggest its potential for rapid and accurate diagnosis of bed bugs in the field. The diagnostic accuracy of these methods was validated through a blind test., Conclusions: The multiplex PCR and LAMP protocols lay the foundation for rapid and accurate field identification of bed bug species, enabling the use of appropriate traps and the detection of species-specific pyrethroid resistance mutations. This approach ensures effective management tailored to the unique characteristics of each bed bug species., (© 2024. The Author(s).)

Objectives: This study aims to develop a novel diagnostic approach using the recombinase aided amplification-lateral flow dipstick(RAA-LFD) assay for the distinction of Mycobacterium tuberculosis (MTB) and Mycobacterium avium complex (MAC), enabling rapid and convenient as well as accurate identification of them in clinical samples., Methods: Our study established a duplex RAA-LFD assay capable of discriminating between MTB and MAC. Based on the principles of RAA primer and probe design, specific primers and probes were developed targeting the MTB IS6110 and the MAC DT1 separately. Optimization of reaction time points and temperatures was conducted, followed by an evaluation of specificity, sensitivity, and reproducibility. The established detection method was then applied to clinical samples and compared with smear microscopy, liquid culture, LAMP, and Xpert/MTB RIF in terms of diagnostic performance., Results: The complete workflow allows for the effective amplification of the MTB IS6110 and MAC DT1 target sequences at constant 37°C within 20min, and the amplification products can be visually observed on the LFD test strip. This method exhibits high specificity, showing no cross-reactivity with nucleic acids from M. kansassi , M. abscessus, M. gordonae, M. chelonae, M. fortuitum, M. scrofulaceum, M. malmoense, M. chimaera, M. szulgai and common respiratory pathogens. It also demonstrates high sensitivity, with a detection limit as low as 10 2 CFU/mL. Additionally, the method's Coefficient of Variation (CV) is less than 5%, ensuring excellent repeatability and reliability. Furthermore, clinical performance evaluations, using Xpert/MTB RIF as the gold standard, demonstrated that the duplex RAA-LFD assay achieves a sensitivity of 92.86% and a specificity of 93.75%. It is also noteworthy that the assay exhibits considerable diagnostic efficacy in smear-negative patients., Conclusions: Our study introduces a rapid, specific, and sensitive duplex RAA-LFD assay for the discriminatory diagnosis of MTB and MAC. This method represents a significant advancement in the field of infectious disease diagnostics, offering a valuable tool for rapid detection and management of MTB and MAC infections. The implementation of this approach in point-of-care settings could greatly enhance TB control and prevention efforts, especially in resource-limited environments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Chen, Zhang, Wang, Yi and Fu.)

Rickettsiosis includes a diversity of culture-negative non-specific systemic infections. Laboratory diagnosis of rickettsiosis is often not easy. In this 12-month study, six patients with a variety of rickettsia infections of the spotted fever group, typhus group and scrub typhus were diagnosed directly or indirectly by metagenomic next-generation sequencing (mNGS). The patient with Japanese spotted fever was rapidly made when mNGS analysis of the patient's blood revealed Rickettsia japonica sequences. For the two patients with Rickettsia felis chest infections, the bacterium was detected in the bronchoalveolar lavage of one case and lung biopsy of the other. Both patients had underlying malignancies, carcinoma of the breast and carcinoma of the lung respectively, and were on chemotherapy with immunosuppressive effect. For the remaining three patients who presented over a period of 13 weeks, all had fever, headache and the typical eschar. They also had increased serum transaminases and responded promptly to doxycycline. However, the Weil-Felix test results of all three patients were negative. Since we considered the three cases typical of rickettsiosis, we submitted their serum samples for mNGS analysis. Results showed that Orientia tsutsugamushi sequences were present in the serum of one case. In view of the positive mNGS results, we repeated the Weil-Felix test for the residual sera of all three patients and it revealed that those of the other two cases showed OX-19 titers of 1:640 and 1:160 respectively, inferring that these two patients probably had rickettsiosis of the typhus group. As for the patient positive for O. tsutsugamushi sequences, we also detected IgM for O. tsutsugamushi in the serum, which double confirmed that it was a case of scrub typhus. mNGS is an important molecular tool and can complement serology for laboratory diagnosis of rickettsiosis., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: PCYW has provided scientific advisory/laboratory services for Gilead Sciences, Incorporated; International Health Management Associates, Incorporated; Merck & Corporation, Incorporated; Micología Molecular S.L. and Pfizer, Incorporated. The other authors report no conflict of interest. The authors alone are responsible for the content and the writing of the manuscript., (Copyright: © 2024 Xing et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Listeria monocytogenes, a zoonotic foodborne pathogen, presents a significant threat to global public health. Therefore, rapid and sensitive detection methods are crucial in mitigating the spread of L. monocytogenes induced diseases. This study introduced a loop-mediated isothermal amplification (LAMP) lyophilized powder detection reagent specifically designed for identifying Listeria monocytogenes. The reagent is user-friendly, quick, and can be easily transported and stored at room temperature. It exhibits no cross-reactivity with eight other types of bacteria and boasts a sensitivity of 10 1  CFU/mL. In a comparative study of 30 samples, the LAMP lyophilized powder detection reagent demonstrated higher sensitivity than the commercial Listeria monocytogenes qPCR detection kit. Additionally, the experimental time was reduced by approximately 30 min, making it highly suitable for rapid diagnosis. Preparation of lyophilized LAMP reagents may facilitate large-scale deployment, particularly in endemic areas or regions facing rapid outbreaks. This could greatly aid in controlling the transmission of pathogens, especially those transmitted through food., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Streptococcus equi subspecies equi , commonly referred to as "strangles", poses a significant biosecurity challenge across equine farms worldwide. The continuous prevalence and highly transmissibility of strangles necessitates a rapid and accurate diagnostic procedure. However, current "gold-standard" techniques, such as cultures and quantitative polymerase chain reaction (qPCR), are unreliable or inaccessible, and require lengthy periods between sample collection and results. Moreover, the lack of a standardized detection protocol can lead to variations in results. This study aimed to develop a reproducible and field-deployable diagnostic assay to detect strangles in real-time. Utilising the rapid technique loop-mediated isothermal amplification (LAMP), we developed an assay targeting a conserved region of the S. equi- specific M gene (SeM). Additionally, we optimised our assay with guanidine hydrochloride (GuHCl) to enhance the assay's performance and detection capabilities. The Str-LAMP was able to detect S. equi within 13 minutes and 20 seconds for both synthetic DNA and clinical isolates, with a limit of detection (LOD) of  53 copies/µl. Our assay demonstrated high repeatability with the inter-coefficient of variation ranging from 0.17% to 3.93%. Furthermore, the clinical sensitivity and specificity was calculated at 91.3% and 93.3%, respectively, with a correct classification rate of 91.8%. The implementation of this newly developed strangles assay can be employed as an efficient aid for in-field surveillance programs. The assay's reproducibility can allow for equine managers to undertake routine self-surveillance on their properties, without the requirement of specialised training. The Str-LAMP assay has the potential to be a valuable tool to help mitigate potential strangles outbreaks., Competing Interests: Travis Beddoe is an Academic Editor for PeerJ., (©2024 Knox and Beddoe.)

Avian Leukosis Virus (ALV) is a retrovirus that induces immunosuppression and tumor formation in poultry, posing a significant threat to the poultry industry. Currently, there are no effective vaccines or treatments for ALV. Therefore, the early diagnosis of infected flocks and farm sanitation are crucial for controlling outbreaks of this disease. To address the limitations of traditional diagnostic methods, which require sophisticated equipment and skilled personnel, a dual-tube detection method for ALV-J based on reverse transcription isothermal amplification (RAA) and the CRISPR-Cas13a system has been developed. This method offers the advantages of high sensitivity, specificity, and rapidity; it is capable of detecting virus concentrations as low as 5.4 × 10 0 copies/μL without cross-reactivity with other avian viruses, with a total testing time not exceeding 85 min. The system was applied to 429 clinical samples, resulting in a positivity rate of 15.2% for CRISPR-Cas13a, which was higher than the 14.7% detected by PCR and 14.2% by ELISA, indicating superior detection capability and consistency. Furthermore, the dual-tube RAA-CRISPR detection system provides visually interpretable results, making it suitable for on-site diagnosis in remote farms lacking laboratory facilities. In conclusion, the proposed ALV-J detection method, characterized by its high sensitivity, specificity, and convenience, is expected to be a vital technology for purification efforts against ALV-J., Competing Interests: The authors declare no conflict of interest.

Respiratory pathogen infections are seasonally prevalent and are likely to cause co-infections or serial infections during peak periods of infection. Since they often cause similar symptoms, simultaneous and on-site detection of respiratory pathogens is essential for accurate diagnosis and efficient treatment of these infectious diseases. However, molecular diagnostic techniques for multiple pathogens in this field are lacking. Herein, we developed a microfluidic LAMP and real-time fluorescence assay for rapid detection of multiple respiratory pathogens using a ten-channel microfluidic chip with pathogen primers pre-embedded in the chip reaction well. The microfluidic chip provided a closed reaction environment, effectively preventing aerosol contamination and improving the accuracy of the detection results. Its corresponding detection instrument could automatically collect and display the fluorescence curve in real time, which was more conducive to the interpretation of results. The results showed that the developed method could specifically recognize the nucleic acid of influenza A(H1N1), Mycoplasma pneumoniae , respiratory syncytial virus type A, and SARS-CoV-2 with low detection limits of 10 4 copies per mL or 10 3 copies per mL. The test results on clinical samples demonstrated that the developed method has high sensitivity (92.00%) and high specificity (100.00%) and even has the capability to differentiate mixed-infection samples. With simple operation and high detection efficiency, the present portable and simultaneous detection assay could significantly improve the efficiency of on-site detection of respiratory infectious diseases and promote the accurate treatment, efficient prevention and control of the diseases.

American plum line pattern virus (APLPV) is the most infrequently reported Ilarvirus infecting stone fruit trees and is of sufficient severity to be classified as an EPPO quarantine A1 pathogen. In late spring, yellow line pattern symptoms were observed on leaves in a few flowering cherries ( Prunus serrulata Lindl.) grown in a public garden in Northwest Italy. RNA extracts from twenty flowering cherries were submitted to Ilarvirus multiplex and APLPV-specific RT-PCR assays already reported or developed in this study. One flowering cherry (T22) with mixed prunus necrotic ringspot virus (PNRSV) and prune dwarf virus (PDV) infection also showed infection with APLPV. Blastn analysis of PCR products of the full coat protein ( CP ) and movement protein ( MP ) genes obtained from flowering cherry T22 showed 98.23% and 98.34% nucleotide identity with reference APLPV isolate NC&#95;003453.1 from the USA. Then, a LAMP-specific assay was designed to facilitate the fast and low-cost identification of this virus either in the laboratory or directly in the field. The developed assay allowed not only the confirmation of APLPV (PSer22IT isolate) infection in the T22 flowering cherry but also the identification of APLPV in an asymptomatic flowering cherry tree (TL1). The LAMP assay successfully worked with crude flowering cherry extracts, obtained after manually shaking a single plant extract in the ELISA extraction buffer for 3-5 min. The developed rapid, specific and economic LAMP assay was able to detect APLPV using crude plant extracts rather that RNA preparation in less than 20 min, making it suitable for in-field detection. Moreover, the LAMP assay proved to be more sensitive in APLPV detection in flowering cherry compared to the specific one-step RT-PCR assay. The new LAMP assay will permit the estimation of APLPV geographic spread in the territory, paying particular attention to surrounding gardens and propagated flowering cherries in ornamental nurseries.

Loop-Mediated Isothermal Amplification (LAMP) represents a valuable technique for DNA/RNA detection, known for its exceptional sensitivity, specificity, speed, accuracy, and affordability. This study focused on optimizing a LAMP-based method to detect early signs of Plasmopara halstedii, the casual pathogen of sunflower downy mildew, a severe threat to sunflower crops. Specifically, a set of six LAMP primers (two outer, two inner, and two loop) were designed from P. halstedii genomic DNA, targeting the ribosomal Large Subunit (LSU). These primers were verified by in silico analysis and experimental validation using both target and non-target species' DNAs. Optimizations encompassing reaction conditions (temperature, time) and component concentrations (magnesium, Bst DNA polymerase, primers, and dNTP) were determined. Validation of these optimizations was performed by agarose gel electrophoresis. Furthermore, various colorimetric chemicals (Neutral Red, Hydroxynaphthol Blue, SYBR Safe, Thiazole Green) were evaluated to facilitate method analysis, and the real-time analysis has been optimized, presenting multiple approaches for detecting sunflower downy mildew using the LAMP technique. The analytical sensitivity of the method was confirmed by detecting P. halstedii DNA concentrations as low as 0.5 pg/μl. This pioneering study, establishing P. halstedii detection through the LAMP method, stands as unique in its field. The precision, robustness, and practicality of the LAMP protocol make it an ideal choice for studies focusing on sunflower mildew, emphasizing its recommended use due to its operational ease and reliability., (© 2024. The Author(s).)

Background: During the coronavirus disease 19 (COVID-19) pandemic, diagnostic testing of the general population proved challenging due to limitations of the gold-standard diagnostic procedure using reverse transcription real-time polymerase chain reaction (RT-qPCR) for large-scale testing on the centralised model, especially in low-resource areas., Objectives: To address this, a point-of-care (PoC) diagnostic protocol for COVID-19 was developed, providing fast, reliable, and affordable testing, particularly for low-mid develop areas., Methods: The PoC diagnostic process combines a simple paper-based RNA extraction method housed within a 3D-printed plastic device with a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay. Nasopharyngeal/oropharyngeal swabs (NOS) and saliva samples were tested between 2020 and 2021, with the assistance of Santa Catarina's State Health Secretary, Brazil., Findings: The developed diagnostic protocol showed a limit of detection of 9,900 copies and an overall diagnostic specificity of 98% for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from 1,348 clinical analysed samples. The diagnostic sensitivity was 95% for NOS samples, 85% for early morning saliva, and 69% for indiscriminate saliva., Main Conclusions: In conclusion, the developed device successfully extracted SARS-CoV-2 viral RNA from swabs and saliva clinical samples. When combined with colorimetric RT-LAMP, it provides results within 45 min using minimal resources, thus delivering a diagnostic kit protocol that is applicable in large-scale sampling.

Salmonella enterica serovar Typhimurium (ST) is a predominant serovar causing foodborne illnesses worldwide. Traditional detection methods often face challenges, including the need for specialized equipment, skilled operators, and lengthy procedures. To address these limitations, we developed a rapid, sensitive, and specific ST detection method by integrating loop-mediated isothermal amplification (LAMP) with the clustered regularly interspaced short palindromic repeats and associated protein 12b (CRISPR/Cas12b) system, all within a single tube. Our results indicate that the LAMP-CRISPR/Cas12b reaction can be completed isothermally in under 1 h without requiring specialized instruments. The platform's limit of detection (LoD) is 12.5 copies per reaction. Additionally, the system demonstrated 100% inclusivity and exclusivity when tested against 30 reference strains, highlighting its specificity. In practical applications, the LoDs for ST in pure nucleic acid and contaminated fecal samples were 2.32 and 23.2 CFU/mL, respectively, with higher sensitivity observed in pure nucleic acid samples. Overall, our findings underscore the potential of the one-tube LAMP-CRISPR/Cas12b platform as a rapid, sensitive, and specific tool for ST detection, particularly in resource-limited settings., Importance: Here, we have provided a novel one-step method for Salmonella Typhimurium detection in one pot by integrating the LAMP assay with the CRISPR/Cas12b system, offering significant advantages in terms of simplicity, speed, and accuracy., Competing Interests: The authors declare no conflict of interest.

We evaluated the Xpert MTB/Rif Ultra assay performance for Mycobacterium tuberculosis (MTB) detection in formalin-fixed paraffin-embedded tissue (FFPET) compared to mycobacterial culture or laboratory-developed MTB PCR test (LDT). FFPET samples with histological features suggestive of tuberculosis from 2018 to 2023 were selected. Five hundred microlitres of tissue lysis buffer was added to FFPET scrolls and incubated at 75 °C for 5 min. After adding 50 µl of proteinase K and overnight incubation at 56 °C, sample aliquots were processed as per the manufacturer's instructions. MTB culture or LDT assay results were used as a reference for sensitivity and specificity calculations. Of 51 eligible FFPET, 32 were positive for MTB either by culture or LDT PCR on FFPET. Xpert MTB/Rif Ultra detected MTB in 23/32 positive specimens [71.9%, 95% confidence interval (CI) 54.6-84.4%]. Of nine discordant specimens, seven were MTB positive by culture and two were identified by LDT MTB PCR only, as no specimen was submitted for MTB culture. Of 19 negative samples, 100% specificity (95% CI 83.2-100.0%) was attained via Xpert MTB/Rif Ultra. Implementation of Xpert MTB/Rif Ultra on FFPET within clinical laboratories is promising, given its improved turnaround time compared to MTB culture and ability to detect MTB in cases where no tissue is available for culture.

Cervical cancer screening is a cornerstone of cervical cancer elimination. Detection of high-risk human papillomavirus (hrHPV) is recommended as the first step in screening provided that the assay used has been adequately validated. The Sansure® Human Papillomavirus DNA Diagnostic Kit is a new assay designed to detect HPV16, HPV18 and 13 other HPV in aggregate. The study aimed to evaluate the intra- and interlaboratory reproducibility of the assay according to international guidelines. Five hundred and fifty cervical residual cell samples from women attending cervical cancer screening were selected from the biobank of the HPV National Reference Centre in Belgium and used in this study. After DNA extraction, HPV was tested using the Sansure® Human Papillomavirus DNA Diagnostic Kit. The lower 95% confidence limit around the general reproducibility of this assay should be greater than or equal to 87%, with κ ≥ 0.50. Five hundred and thirty-three samples had valid results. The Sansure® Human Papillomavirus DNA Diagnostic Kit demonstrated an excellent intra-laboratory reproducibility of 93.8% (95% confidence interval [CI]: 91.4-95.7, κ = 0.85). The interlaboratory reproducibility was 93.4 (95% CI: 91.0-95.4, κ = 0.84). Intra and interlaboratory reproducibility were also excellent at the genotype level. Excluding HPV53 single infection samples from the analyses also resulted in excellent agreement. These data show that the Sansure® Human Papillomavirus DNA Diagnostic Kit is highly reproducible., (© 2024 The Author(s). Journal of Medical Virology published by Wiley Periodicals LLC.)

Background: Some lower-grade gliomas (LGG) are difficult to distinguish morphologically from glial cell proliferation or inflammatory changes during surgery, leading to a high risk of incorrect diagnosis. It is crucial to differentiate between the two for making surgical decisions. We define these critical cases as "ultra early stage lower-grade gliomas (UES-LGG)"., Methods: We analyzed 11 out of 13 cases diagnosed with "gliosis" or "inflammatory changes" during surgery who tested positive for isocitrate dehydrogenase (IDH). Additionally, we conducted qRT-PCR detection on 35 samples diagnosed with LGG during surgery and analyzed their DNA content within an effective circulating threshold range to infer the critical value between UES-LGG and LGG. We conducted experiments using five standardized samples to infer the limited range of accurate detection of UES-LGG during surgery., Results: In the comparative analysis of 11 samples and 35 samples, it was found that while there was no significant difference in the average DNA detection concentration between the two groups (159.36 ± 83.3 ng/μL and 146.83 ± 122.43 ng/μL), there was a notable statistical variance in the detection threshold for positive mutations (31.78 ± 1.14 and 26.14 ± 2.69, respectively). This suggests that the IDH mutation rate may serve as an indicator for differentiation between the two groups. Subsequently, DNA was extracted from standardized IDH mutant samples and subjected to gradient dilution for detection purposes. The results indicated a consistent increase in detection threshold as detection concentration decreased. When the detection concentration fell below <0.1 ng/μL, it became impossible to carry out effective threshold range detections. To further identify the precise detection interval, we conducted gradient division once again and sought to simulate the functional relationship between DNA copy number and cycle threshold within this interval. The research revealed that when the minimum detection concentration exceeded 250 copies/μL, a 100% detection rate could be achieved., Conclusions: This article defines UES-LGG as a tumor type easily misdiagnosed in clinical practice due to its extremely low positivity rate during surgery. The popularization of qRT-PCR based intraoperative molecular diagnosis greatly reduces errors caused by manual detection and improves disease detection rates during surgery. It provides a theoretical basis for more accurate surgical plans for surgeons., (© 2024 The Author(s). CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

Purpose of Review: Viruses are the most common etiological agents of diarrhea in children. Despite rotavirus vaccine introduction, rotavirus remains as the leading cause of death globally, followed by norovirus, which represents a diagnostic challenge. Here, we describe new advances in the diagnosis and management of viral diarrheas., Recent Findings: Although immunoassays are widely used for their fast turnaround time and low cost, molecular techniques have become the most reliable diagnostic method due to their high sensitivity and capacity to analyze multiple pathogens in gastrointestinal panels. Isothermal nucleic acid amplification assays (LAMP and RPA) are promising techniques since they do not require sophisticated equipment and can be used as point-of-care testing. CRISPR/Cas nucleic acid detection systems are new diagnostic methods with great potential. Several recent published articles describe the role of human intestinal enteroids to characterize norovirus infection, to test new drugs, and for vaccine development. The interaction between the human gut microbiota and gastrointestinal viral infections has been extensively reviewed and offers some innovative mechanisms for therapeutic and preventive measures., Summary: Although important advances have been made, more research is needed to address remaining challenges and further improve diagnostic capabilities and better management strategies for this critical infectious disease., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Carbapenem resistance in Acinetobacter baumannii is a critical global health threat attributed to transferrable carbapenemase genes. Carbapenemase genotyping using polymerase chain reaction (PCR) presents a challenge in resource-limited settings because of its technical requirements. This study designed new loop-mediated isothermal amplification (LAMP) primers using multiple sequence alignment-based workflows, validated the primer performance against multiple target variants in silico , and developed novel LAMP assays (LAntRN-OXA23 and LAntRN-ISAba1) to detect the transferable bla OXA-23-like carbapenemase genes and ISAba1 elements in pure cultures and A. baumannii -spiked serum samples. The designed LAMP primers bind to the conserved regions of their highly polymorphic targets, with their in silico performance comparable with other published primers. The in vitro LAMP assays (using 30 PCR-profiled A. baumannii and 10 standard multidrug-resistant gram-negative isolates) have 100% concordance with the PCR-positive clinical samples, limits of detection as low as 1 pg/µL (200 copies/µL), and specificities of 57.89-100%. Both assays produced positive results when testing DNA samples (extracted using a commercial kit) from bla OXA-23-like and ISAba1-bla OXA-51-like PCR-positive A. baumannii -spiked normal human sera (five set-ups per target). In summary, the LAMP assays accurately detected the target genes and have applications in infection management, control, and point-of-care testing in resource-limited healthcare settings.

Purpose of Review: Emerging technologies are revolutionizing parasitology diagnostics and challenging traditional methods reliant on microscopic analysis or serological confirmation, which are known for their limitations in sensitivity and specificity. This article sheds light on the transformative potential of artificial intelligence and molecular assays in the field, promising more accurate and efficient detection methods., Recent Findings: Artificial intelligence has emerged as a promising tool for blood and stool parasite review, when paired with comprehensive databases and expert oversight result in heightened specificity and sensitivity of diagnoses while also increasing efficiency. Significant strides have been made in nucleic acid testing for multiplex panels for enteric pathogen. Both multiplex and single target panels for Plasmodium , Babesia , filaria, and kinetoplastids have been developed and garnered regulatory approval, notably for blood donor screening in the United States. Additional technologies such as MALDI-TOF, metagenomics, flow cytometry, and CRISPR-Cas are under investigation for their diagnostic utility and are currently in the preliminary stages of research and feasibility assessment., Summary: Recent implementation of artificial intelligence and digital microscopy has enabled swift smear screening and diagnosis, although widespread implementation remains limited. Simultaneously, molecular assays - both targeted and multiplex panels are promising and have demonstrated promise in numerous studies with some assays securing regulatory approval recently. Additional technologies are under investigation for their diagnostic utility and are compelling avenues for future proof-of-concept diagnostics., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

The inherited disease community in Sri Lanka has been widely neglected. This article aimed to present accumulated knowledge in establishing a pro bono cost-effective national, island-wide, free-of-charge molecular diagnostic service, suggesting a model for other developing countries. The project provided 637 molecular diagnostic tests and reports free of charge to a nation with limited resources. We pioneered the implementation of mobile clinics and home visits, where the research team acted as barefoot doctors with the concept of the doctor and the researcher at the patient's doorstep. Establishing pro bono, cost-effective molecular diagnostics is feasible in developing countries with limited resources and state funding through the effort of dedicated postgraduate students. This service could provide an accurate molecular diagnosis of Duchenne muscular dystrophy, Huntington's disease, Spinocerebellar ataxia, and Spinal muscular atrophy, a diagnostic yield of 54% (343/637), of which 43% (147/343) of the patients identified as amenable for available gene therapies. Initiated human resource development by double doctoral degree opportunities with international collaborations. Established a neurobiobank and a national registry in Sri Lanka, a rich and unique repository, wealth creation for translational collaborative research and sharing of information in neurological diseases, as well as a lodestar for aspiring initiatives from other developing countries., (© 2024. The Author(s).)

This study aimed to design and evaluate a universal primer for Polymerase Chain Reaction (PCR)- based detection of mucormycosis-causing fungi by targeting their Internal Transcribed Spacer (ITS) sequences. In-silico analysis was conducted to assess primer suitability. Using Clustal Omega and Primer-BLAST, ITS sequences of 32 fungi species causing mucormycosis were aligned and subjected to primer design. Generated primers were sorted and in silico PCR simulations were performed to identify primers capable of amplifying all fungal species. Instead of identifying one pair of universal primer, in silico PCR analysis identified a panel of 14 primer pairs designed from full-length ITS sequences, and a panel of 12 primer pairs designed from conserved regions, that could detect 31 species. The study recommends a panel of 12 primers for multiplex-PCR to detect mucormycosis-causing fungi instead of a long list of PCR analyses for each fungus in diagnosing mucormycosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Bacterial blight caused by Xanthomonas phaseoli pv. manihotis (Xpm) is considered the main bacterial disease that affects cassava, causing significant losses when not properly managed. In the present study, a fast, sensitive, and easy-to-apply method to detect Xpm via colorimetric loop-mediated isothermal amplification (LAMP) was developed. To ensure the use of a unique-to-the-target pathovar core region for primer design, 74 complete genomic sequences of Xpm together with different bacterial species and pathovars were used for comparative genomics. A total of 42 unique genes were used to design 27 LAMP primer sets, from which nine primers were synthesized, and only one (Xpm&#95;Lp1 primer set) showed sufficient efficiency in preliminary tests. The sensitivity, assessed by a serial dilution of the type strain (IBSBF 278) DNA, yielded high sensitivity, detecting up to 100 fg. The LAMP primers showed high specificity, did not cross-react with other bacterial species or other pathovars tested, and amplified only the Xpm isolates. Tests confirmed the high efficiency of the protocol using infected or inoculated macerated cassava leaves without the need for additional sample treatment. The LAMP test developed in this study was able to detect Xpm in a fast, simple, and sensitive way, and it can be used to monitor the disease under laboratory and field conditions., Competing Interests: The author(s) declare no conflict of interest.

Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV) and transmissible gastroenteritis virus (TGEV) are three clinically common coronaviruses causing diarrhea in pigs, with indistinguishable clinical signs and pathological changes. Rapid, portable and reliable differential diagnosis of these three pathogens is crucial for the prompt implementation of appropriate control measures. In this study, we developed a triplex nucleic acid assay that combines reverse transcription recombinase-aided amplification (RT-RAA) with lateral flow assay (LFA) by targeting the most conserved genomic region in the ORF1b genes of PEDV, PDCoV and TGEV. The entire detection process of the triplex RT-RAA-LFA assay included 10-min nucleic acid amplification at 42 °C and 5-min visual LFA readout at room temperature. The assay could specifically differentiate PEDV, PDCoV and TGEV without cross-reaction with any other major swine pathogens. Sensitivity analysis showed that the triplex RT-RAA-LFA assay was able to detect the viral RNA extracted from the spiked fecal samples with the minimum of 1 × 10 0 TCID 50 PEDV, 1 × 10 4 TCID 50 PDCoV, and 1 × 10 2 TCID 50 TGEV per reaction, respectively. Further analysis showed that the 95 % detection limit (LOD) of triplex RT-RAA-LFA for PEDV, PDCoV, and TGEV were 22, 478, and 205 copies of recombinant plasmids per reaction, respectively. The diagnostic performance of triplex RT-RAA-LFA was compared with that of PEDV, PDCoV and TGEV respective commercial real-time RT-PCR kits by testing 114 clinical rectal swab samples in parallel. The total diagnostic coincidence rates of triplex RT-RAA-LFA with real-time RT-PCR kits of PEDV, PDCoV and TGEV were 100 %, 99.1 % and 99.1 %, respectively, and their Kappa values were 1.00, 0.958 and 0.936, respectively. Collectively, the RT-RAA-LFA assay is a powerful tool for the rapid, portable, visual, and synchronous differential diagnosis of PEDV, PDCoV, and TGEV., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Molecular tests have an inherent limit of detection (LOD) and, therefore, require samples with sufficiently high percentages of neoplastic cells. Many laboratories use tissue dissection; however, optimal procedures for dissection and quality assurance measures have not been established. In this study, several modifications to tissue dissection procedures and workflow were introduced over 4 years. Each modification resulted in a significant improvement in one or more quality assurance measures. The review of materials following dissection resulted in a 90% reduction in KRAS mutations below the stated LOD (P = 0.004). Mutation allele frequencies correlated best with estimated tumor percentages for pathologists with more experience in this process. The direct marking of unstained slides, use of a stereomicroscope, validation of extraction from diagnostic slides, and use of a robust, targeted next-generation sequencing platform all resulted in reduction of quantity not sufficient specimens from 20% to 25% to nearly 0%, without a significant increase in test failures or mutations below the LOD. These data indicate that post-dissection review of unstained slides and monitoring quantity not sufficient rate, test failure rate, and mutation allele frequencies are important tumor dissection quality assurance measures that should be considered by laboratories performing tissue dissections. The amendments to tissue dissection procedures enacted during this study resulted in a measurable improvement in the quality and reliability of this process based on these metrics., Competing Interests: Disclosure Statement None declared., (Copyright © 2024 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Dollar spot is a major fungal disease affecting turfgrass worldwide and can quickly destroy turfgrass swards. An assimilating probe-based loop-mediated isothermal amplification (LAMP) assay was developed to detect Clarireedia monteithiana and C. jacksonii , the causal agents of dollar spot within the continental United States. Five LAMP primers were designed to target the calmodulin gene with the addition of a 6-carboxyl-fluorescein florescent assimilating probe, and the temperature amplification was optimized for C. jacksonii and C. monteithiana identification. The minimum amount of purified DNA needed for detection was 0.05 ng μl -1 . Specificity assays against host DNA and other turfgrass pathogens were negative. Successful LAMP amplification was also observed for dollar spot-infected turfgrass field samples. Further, a DNA extraction technique via rapid heat-chill cycles and visualization of LAMP results via a florescent flashlight was developed and adapted for fast, simple, and reliable detection in 1.25 h. This assimilating probe-based LAMP assay has proved successful as a rapid, sensitive, and specific method for the detection of C. monteithiana and C. jacksonii in pure cultures and from symptomatic turfgrass leaves blades. The assay represents a promising technology to be used in the field for on-site, point-of-care pathogen detection., Competing Interests: The author(s) declare no conflict of interest.