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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.)

The prevalence of foodborne illnesses is a significant global concern, resulting in numerous illnesses, deaths, and substantial economic losses annually. Traditional detection methods for foodborne pathogens are often slow, limited, and impractical for field use, underscoring the need for rapid, sensitive, and portable assays. Microfluidic technology has emerged as a promising solution for sample preparation, reaction, and detection on a small scale. Our study introduces a novel microfluidic digital loop-mediated isothermal amplification (LAMP) assay platform, which employs digital microfluidic chips for absolute quantitative analysis of nucleic acids. This portable chip utilizes LAMP technology to achieve ultrasensitive detection of target nucleic acids within 30 min and reduces the detection limit to 1 fM without the need for complex instrumentation. By digitizing amplification signals directly from the target sample, our platform offers simplicity, affordability, portability, and quantitative molecular readouts. This innovation represents a crucial step toward the on-site detection of foodborne pathogens, thereby enhancing food safety and mitigating disease outbreaks.

Background: Valid diagnostic tests for human papillomavirus (HPV) detection are crucial to identify individuals at high risk of cervical cancer. We assessed and compared the validity of Mehrviru HPV genotyping and Sacace (HPV Genotypes 14 Real-TM Quant) for molecular detection of 14 high-risk human papillomaviruses., Methods: We used three HPV test results to identify HPV-positive individuals (14 high-risk genotypes) in a specialist gynecology clinic. The HPV test results were collected using Mehrviru ® , Sacace ® , and a third kit from the clinical diagnostic laboratory. We used Latent class analysis to determine the actual status of HPV infection in study participants. The sensitivity, specificity, positive and negative predictive values, positive and negative likelihood ratios, Youden, and area under the ROC curve indices of each diagnostic kit and their 95% confidence intervals were calculated. The agreement between the Mehrviru and Sacace kits was determined using the Kappa statistic., Results: We examined 117 women at high risk of HPV infection. The mean age (SD) was 37.2 (9.1). According to LCA, 28.6% of participants had an HPV infection. The sensitivity and specificity (95% CI) of the Mehrviru were 90.8% (73.7-97.2%) and 90.9% (82.1-95.6%), and corresponding figures for Sacace were 92.0% (72.3-98.1%) and 97.4% (90.2-99.3%). The kappa index between the Mehrviru and Sacace kits was 69.7% (55.6-83.9%). The area under the ROC curve for Mehrviru and Sacace test were 91.4% (85.7-97.1%) and 94.4% (89.3-99.5%)., Conclusions: There is an excellent agreement between Mehrviru and Sacace test results, and the diagnostic accuracy indices were similar., Competing Interests: Declarations Ethics approval and consent to participate This study was approved by Iran University of Medical Sciences’ Ethics Committee, reference: IR.IUMS.REC.1400.924, and written informed consent was obtained from all the participants. Consent for publication Not applicable. Competing interests MS is the technical manager and DH is the head of department of Pathology and Molecular Medicine at Mehr Clinical Diagnostic Laboratory. MSD advises MCDL on research project. HN and FRT declare no conflict of interest., (© 2024. The Author(s).)

Rickettsiosis is a disease caused by bacteria belonging to the genus Rickettsia, and is a potentially fatal zoonotic disease of great medical and veterinary importance. Given the urgent need to develop new diagnostic methods for detecting this disease, the present review aimed to evaluate the number of publications dedicated to the identification of Rickettsia spp. in human samples using molecular methods, such as polymerase chain reaction and its variations. To this end, a bibliographical survey covering articles published in the past ten years was conducted using the PudMed platform with the keywords "spotted fever" and "Rickettsia," both combined with "diagnosis." A growing number of publications in this area reflects an increasing interest in research, especially since 2015. From 2015 to February 2024, several promising results were tested and many studies were able to detect the genetic sequences of interest. Therefore, the absence of a standard diagnosis method highlights the critical need for developing an effective technique capable of accurately detecting the etiological agent and ensuring accurate diagnosis of the disease.

Developing instant detection systems with disease diagnostic capabilities holds immense importance for remote or resource-limited areas. However, the task of creating these systems-which are simultaneously easy to operate, rapid in detection, and cost-effective-remains a challenge. In this study, we present a compact highly sensitive photothermal reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP) chip (SPRC) designed for the detection of multiple diseases. The nucleic acid (NA) amplification on the chip is achieved through LAMP driven by either LED illumination or simple sunlight focusing. SPRC performs sample addition and amplification within a limited volume and autonomous enrichment of NA during the sample addition process, achieving a limit of detection (LOD) as low as 0.2 copies per microliter. Through 120 clinical samples, we achieved an accuracy of 95%, with a specificity exceeding 97.5%. Overall, SPRC has achieved promising progress in the application of point-of-care testing (POCT) by using light energy to simultaneously detect multiple diseases.

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.)

Pathogenic gram-negative bacteria frequently carry genes encoding extended-spectrum beta-lactamases (ESBL) and/or carbapenemases. Of great concern are carbapenem resistant Escherichia coli , Klebsiella pneumoniae , Pseudomonas aeruginosa, and Acinetobacter baumannii . Despite the need for rapid AMR diagnostics globally, current molecular detection methods often require expensive equipment and trained personnel. Here, we present a novel machine-learning-aided platform for the rapid detection of ESBLs and carbapenemases using Loop-mediated isothermal Amplification (LAMP). The platform consists of (i) an affordable device for sample lysis, LAMP amplification, and visual fluorometric detection; (ii) a LAMP screening panel to detect the most common ESBL and carbapenemase genes; and (iii) a smartphone application for automated interpretation of results. Validation studies on clinical isolates and urine samples demonstrated percent positive and negative agreements above 95% for all targets. Accuracy, precision, and recall values of the machine learning model deployed in the smartphone application were all above 92%. Providing a simplified workflow, minimal operation training, and results in less than an hour, this study demonstrated the platform's feasibility for near-patient testing in resource-limited settings.IMPORTANCEExtended-spectrum beta-lactamases (ESBL) and carbapenemases confer resistance to third-generation cephalosporins and carbapenems in pathogenic Gram-negative bacteria such as Escherichia coli , Klebsiella pneumoniae , Pseudomonas aeruginosa, and Acinetobacter baumannii . Conventional antimicrobial susceptibility testing is based on phenotypic methods, and results can take several days to be obtained. Current genotypic detection methods can be rapid but require expensive equipment and trained personnel. In this study, we present a novel machine learning-aided platform for the rapid detection of ESBLs and carbapenemases using Loop-mediated isothermal Amplification (LAMP). The validation of the platform demonstrated percent positive and negative agreements above 95% for all targets. The newly developed platform provided a simplified workflow, minimal technical training, and results in less than an hour. This study demonstrated the platform's feasibility for rapid testing of ESBL and carbapenemases in bacteria and urine specimens., Competing Interests: D.R.P. is scientific advisor and co-founder of Illucidx Inc., a local startup.

The aim was to develop an RT-qPCR targeting Aspergillus fumigatus and compare its performance to that of Aspergillus fumigatus qPCR for the diagnosis of invasive aspergillosis (IA). Samples from patients of the Lyon University hospitals for whom a suspicion of IA led to the realization of an Aspergillus fumigatus qPCR molecular diagnostic test over a 2-year period were included. The patients were classified according to the European Organization for Research and Treatment of Cancer/Mycoses Study Group (EORTC-MSGERC) criteria for suspected IA; RT-qPCR and qPCR assays were performed on all included samples. The sensitivities and specificities of RT-qPCR and qPCR were calculated and compared using the results of the EORTC-MSGERC classification as reference. The cycle threshold (Ct) results were compared according to IA classification and sample type. Among the 193 samples analyzed, 91 were classified as IA excluded, 46 as possible IA, 53 as probable IA, and 3 as proven IA. For all sample types, RT-qPCR was significantly more sensitive than qPCR for all IA classifications with an additional 17/102 samples detected ( P -value < 0.01). For plasma samples, sensitivity was significantly higher and specificity significantly lower using RT-qPCR for all IA classifications ( P -value < 0.001). The mean Ct obtained with RT-qPCR were significantly lower than those obtained with qPCR for all IA classifications and all sample types ( P -value < 0.001 and P -value < 0.0001, respectively). RT-qPCR presents a higher sensitivity than qPCR for the diagnosis of IA due to Aspergillus fumigatus , particularly in samples with an intrinsically low fungal load.IMPORTANCE Aspergillus fumigatus belongs to the critical priority group of the World Health Organization fungal priority pathogens list. Invasive aspergillosis (IA) is a life-threatening infection with poor prognosis and challenging diagnosis. PCR has been integrated into the 2020 European Organization for Research and Treatment of Cancer/Mycoses Study Group consensus definitions for IA diagnosis. However, due to frequent low fungal burdens, its sensitivity needs to be improved. This work presents an innovative method for detecting total nucleic acids, corresponding to both ribosomal RNA and DNA, that enables IA diagnosis with greater sensitivity than conventional techniques, especially in non-invasive samples such as blood, enhancing the monitoring of this infection in high-risk patients., Competing Interests: The authors declare no conflict of interest.

Diagnostic stewardship (DxS) for infectious disease testing requires a multi-disciplinary approach to optimize test selection, performance, interpretation and patient treatment. Nucleic acid amplification-based tests for the diagnosis of infectious diseases, or "molecular microbiology tests," have rapidly expanded over the past two decades. With the increased availability and complexity of these tests, there is also an increased need for collaborative approaches to optimize test use to promote positive impacts on patient care, while mitigating potential negative impact or resource waste. In this review, we provide recommendations on building collaborative DxS teams, including microbiologists and the diverse stakeholders that use and interpret molecular microbiology tests. We then detail approaches to identify high-priority molecular microbiology tests that may need utilization assessment, select appropriate diagnostic stewardship interventions, and monitor the impact of implemented interventions. This strategic process may be employed by laboratories to realize optimal testing for selected tests at their institution., Competing Interests: The authors declare no conflict of interest.

The BioFire Joint Infection (JI) Panel offers a significant advancement in the rapid diagnosis of joint infections by facilitating the simultaneous detection of multiple bacterial and fungal pathogens, as well as resistance markers, directly from synovial fluid samples. An article published in the Journal of Clinical Microbiology by Moran et al. (J Clin Microbiol 62:e00182-24, 2024, https://doi.org/10.1128/jcm.00182-24) presents both prospective and retrospective analyses of the panel's real-world clinical application. The study highlights the panel's benefits, such as its rapid turnaround time and ability to identify challenging pathogens, while also discussing its limitations, particularly in detecting certain off-panel organisms., Competing Interests: The author declares no conflict of interest.

Listeria monocytogenes is a major foodborne pathogen affecting developing, and developed countries. The analysis of food contact surfaces in food industries is key for better controlling this pathogen. The current study focused on the development, optimization, and evaluation of a rapid and simple method for the detection of L. monocytogenes on stainless steel surfaces, suitable for decentralized setups, taking advantage of Loop-mediated isothermal amplification (LAMP). This was accomplished using a general pre-enrichment broth (TSB), with a simple DNA extraction based on a chelating resin, and final isothermal amplification. Two different detection strategies were tested, real-time fluorescence and naked-eye colorimetric, which were evaluated after 5, 7, and 24 h of pre-enrichment. Regardless the detection chemistry selected, after 5-7 h of pre-enrichment, 10 3 -10 4  CFU/cm 2 were needed to obtain a positive result, while after 24 h, it was possible to detect concentrations below 10 CFU/cm 2 . Within each given time, all the performance parameters calculated, relative sensitivity, specificity, and accuracy, reached values higher than 80-90%; likewise, a Cohen's k of concordance with a culture-based approach higher than 0.8. Overall, the most sensitive assay can be performed in roughly 25 h. This time-to-result outperforms commercial kits with the added value of specifically detecting L. monocytogenes instead of Listeria spp. KEY POINTS: • Real-time fluorescence and naked-eye colorimetric, were compared for the novel assay. • An LOD50 of 3.4 CFU/cm 2 and 4.2 CFU/cm 2 was calculated for the two assays. • Three pre-enrichment times were compared providing 24 h better results., Competing Interests: Declarations Ethics approval This article does not contain any studies with human participants or animals performed by any of the authors. Conflict of interest The authors declare no conflict of interest., (© 2024. The Author(s).)

Real-time PCR assays are valuable tools for the rapid and accurate diagnosis of infectious diseases by identifying the nucleic acid sequences of pathogens. Here, we aimed to develop a recombinant plasmid-based standard for validating the sensitivity of different molecular diagnostic methods adopting the Jonstrup assay (J assay). Chimeric plasmid DNA (cpDNA) harboring various pathogen genes and the target site of the J assay was constructed. Our findings revealed that the J assay could detect a single copy of the target gene similar to digital droplet PCR. The detection sensitivity of each established real-time PCR method for various disease diagnoses was evaluated using the cpDNA. Although most methods showed high sensitivity, similar to that of the J assay, the VHS Garver and SARS-CoV-2 diagnostic methods exhibited detection sensitivities tenfold lower than that of the J assay. To ensure accuracy of results and avoid genetic contamination from positive controls, we introduced an additional probe attachment site emitting distinct fluorescent signals within the cpDNA. Target genes and exogenous sequences within the plasmid DNA were simultaneously detected in a single assay using this unique method. Our approach will help improve the sensitivity of diagnostic methods and develop novel diagnostic methods based on molecular techniques., (© 2024. The Author(s).)

Objectives: Viral hepatitis caused by hepatitis B virus (HBV) and hepatitis C virus (HCV) infections remain a major global public health challenge, particularly in low- and middle-income countries. It is crucial to utilize a pointof-care (POC) testing platform that is sensitive, specific, rapid, and user-friendly for screening and diagnosis of the two infections. Here, a novel molecular diagnostic assay, integrating multiplex loop-mediated isothermal amplification with a gold nanoparticle-based lateral flow biosensor (mLAMP-AuNPs-LFB) was developed and applied for one-step, visual, rapid, sensitive, and specific identification of HBV and HCV., Methods: The AuNPs-based LFB was devised and constructed for the simultaneous detection of HBV and HCV. The HBV-LAMP and HCV-LAMP primers were designed against the S and 5'-untranslated region (5'-UTR) genes from the major HBV genotypes (B, C, D, B/C recombinant, and C/D recombinant) and HCV subtypes (1b, 2a, 3a, 3b, and 6a) in China, respectively. Our assay conditions, both multiplex-LAMP amplification temperature and time were optimized. The sensitivity and specificity of our assay were tested, and the feasibility of our assay was verified through clinical samples., Results: The AuNPs-based LFB used here was successfully manufactured according to our devise manual. The two unique independent primer pairs were successfully designed based on the S and 5'-UTR genes, respectively. The optimal mLAMP-AuNPs-LFB detection process, involving rapid nucleic acid isolation (10 min), mLAMP (63 °C for 35 min), and visual AuNPs-LFB interpretation (less than 2 min), could be completed within 50 min. The HBV&HCV-mLAMP-AuNPs-LFB assay can detect the target genes (HBV-S and HCV-5'-UTR) with as low as 20 copies of plasmid template per test, and the specificity was 100% for the experimental pathogens., Conclusions: The preliminary results manifested that our mLAMP-AuNPs-LFB assay is a valuable tool and has tremendous potential as a POC testing approach for HBV and HCV identification, especially in undeveloped regions., (© 2024. The Author(s).)

Despite the advent of Xpert MTB/RIF, pleural tuberculosis (TB) diagnosis in pleural fluid is still difficult. Hence, we assessed the diagnostic efficacy of its advanced version, Xpert MTB/RIF Ultra, for pleural TB diagnosis using pleural fluid as a sample. Tuberculosis pleuritis (TBP) suspects (n = 261) were enrolled in the study, of which 29 were excluded. The remaining patients (n = 232) were categorized into definite TBP (n = 31), probable TBP (n = 28), and non-TB controls (n = 173) based on the composite reference standard consisting of smear, culture, histopathology, and Xpert MTB/RIF as well as follow-up/clinical response to anti-TB therapy. Among the TBP suspects, 59 were diagnosed as TBP patients. The sensitivity of Xpert MTB/RIF Ultra (52.5%) using pleural fluid for TBP diagnosis was higher than sensitivity obtained with smear (22.4%), culture (17.6%), and Xpert MTB/RIF (25%) alone, carried out using either pleural fluid or pleural biopsy or both the samples. In cases of probable TBP, where none of the laboratory tests were positive, Xpert MTB/RIF Ultra use led to an increased diagnostic percentage of definite TBP from 52.5% to 69.4%. Overall, Xpert MTB/RIF Ultra showed promising results for a definitive diagnosis of TBP in pleural fluid samples., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Canine parvovirus (CPV) can cause high morbidity and mortality rates in puppies, posing a significant threat to both pet dogs and the breeding industry. Rapid, accurate, and convenient detection methods are important for the early intervention and treatment of canine parvovirus. In this study, we propose a visual CPV detection system called nucleic acid mismatch enzyme digestion (NMED). This system combines loop-mediated isothermal amplification (LAMP), endonuclease for gene mismatch detection, and colloidal gold lateral chromatography. We demonstrated that NMED can induce the binding of the amplicon from the sample to the specific labeling probe, which in turn triggers digestion by the endonuclease. The sensitivity and visual visibility of LAMP were increased by combining endonuclease and colloidal gold lateral chromatography assisted by a simple temperature-controlled device. The sensitivity of the NMED assay was 1 copy/μL, which was consistent with quantitative PCR (qPCR). The method was validated with 20 clinical samples that potentially had CPV infection; 15 positive samples and 5 negative samples were evaluated; and the detection accuracy was consistent with that of qPCR. As a rapid, accurate, and convenient molecular diagnostic method, NMED has great potential for application in the field of pathogenic microorganism detection., Importance: The NMED method has been established in the laboratory and used for CPV detection. The method has several advantages, including simple sampling, high sensitivity, intuitive results, and no requirement for expensive equipment. The establishment of this method has commercial potential and offers a novel approach and concept for the future development of clinical detection of pathogenic microorganisms., Competing Interests: The authors declare no conflict of interest.

Cost-effective molecular diagnostic techniques for bacterial pneumonia are limited. We designed primers for 13 bacteria, performed multiplex nucleic acid detection through fragment analysis to obtain pathogen identification results, and established a multiplex PCR-capillary electrophoresis (MPCE) method, which can simultaneously detect 13 pathogens associated with bacterial pneumonia. The sensitivity, specificity, and reproducibility of the MPCE assay were tested, and 420 clinical samples were used to assess the clinical detection ability of MPCE, with the culture method used as a reference. Samples with inconsistent results detected by the two methods were sent for Sanger sequencing. The minimum detection limit of MPCE for 13 bacteria was 6.0 × 10 3 cfu/mL~2.0 × 10 6 cfu/mL. No cross-reactivity was observed with other pathogens. The percentage of agreement for reproducibility analysis reached 100%. For the 420 sputum samples, when the culture method was used as the reference, the sensitivity of MPCE to 13 bacteria ranged from 80% to 100%. The specificity for 13 bacteria ranged from 67.1% to 100%. The percentage of agreement between the MPCE and the culture method ranged from 69.7% to 100%. There was no statistically significant difference ( P > 0.05) in the detection of Escherichia coli , Enterobacter cloacae complex , Staphylococcus aureus , methicillin-resistant S. aureus, Streptococcus pyogenes , Moraxella catarrhalis , or Legionella pneumophila between the MPCE and the culture method. Clinical samples with negative cultures but positive MPCE results were validated by Sanger sequencing, and the results were consistent with those of MPCE. The MPCE method has high sensitivity and specificity for bacterial pneumonia, enabling the simultaneous and rapid detection of multiple pathogens. It is cost-effective and has potential for clinical application., Importance: This study successfully established a multiplex PCR-capillary electrophoresis detection system that can simultaneously detect 13 pathogens through a single detection method, significantly improving clinical efficiency. It is cost-effective and has potential for clinical application., Competing Interests: The authors declare no conflict of interest.

The coronavirus disease 2019 pandemic emphasised the importance of laboratory preparedness, including molecular diagnostic capacity, in the control of infectious disease outbreaks. This article reflects on diagnostic capacity-building opportunities presented by the pandemic, the challenges experienced along the way and the lessons learned from the perspective of a university teaching hospital in Southern Nigeria. We advocate for these lessons to inform strategic planning for laboratory preparedness at subnational, national and continental levels., (© The Author(s) 2024. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene.)

Objectives: Ascites, often associated with critical pathologies such as liver cirrhosis or bowel perforation, can be complicated by fungal infection, increasing mortality especially in intensive care settings and demanding rapid diagnosis and adequate treatment. Traditional microbiological diagnostic methods have limited sensitivity in accurately identifying fungal pathogens in ascitic fluid. Alternative diagnostic methods may offer important insights to enable guiding of antifungal therapy and refining empirical treatment strategies. The objective of this study was to evaluate the potential of next-generation sequencing methods to identify specific fungal pathogens responsible for ascitic fluid infections., Methods: We prospectively collected 50 ascitic fluid samples from ICU patients with suspected ascites infection. In addition to standard culture-based microbiological testing, an ascitic fluid aliquot underwent fungal DNA isolation and was analyzed by next-generation sequencing (NGS) methods for identification of fungal species., Results: Of 50 ascitic samples collected, five samples showed growth of Candida spp. in culture. After DNA isolation and ITS2 PCR, detectable amplification was achieved in 10 samples. Sequencing of the 50 patients' samples identified facultative pathogenic fungi in 19 patients. In 15 cases, culture alone would not have permitted the identification of all facultative pathogenic fungi. The identification of fungal DNA by sequencing was significantly associated with poor patient outcome and a number of clinical parameters., Conclusions: Our results show a higher sensitivity for NGS-based diagnostic methods in the identification of ascitic fluid fungal infections compared to culture-based diagnostics. This may be beneficial especially for patients in a critical care setting, who have an increased prevalence of comorbidities and high mortality. The implementation of such methods in standard diagnosis will require increased standardization of the workflows and interpretation of the sequencing results with respect to patients' clinical picture., 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 Posadas-Cantera, Mehrbarzin, Wetzel, Goelz, Kousoulas, Utzolino, Häcker and Badr.)

Chlamydia pneumoniae (C. pneumoniae) is a specialized intracellular parasitic pathogen capable of causing pneumonia, sinusitis, bronchitis, and other respiratory diseases, which pose significant public health challenges. Therefore, rapid, accurate, and sensitive diagnosis is crucial for the prevention and treatment of respiratory diseases caused by C. pneumoniae . In this study, we combined enzymatic recombination amplification (ERA) with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 12a system (CRISPR/Cas12a) to develop a dual detection platform termed the Cpn-ERA-CRISPR/Cas12a dual system. This system integrates both the ERA-CRISPR/Cas12a fluorescence system and the ERA-CRISPR/Cas12a lateral flow system. Detection results can be measured using a fluorescence detector or observed with the naked eye on lateral flow strips. The fluorescence system and the lateral flow system detect C. pneumoniae in 30 minutes and 15 minutes, respectively. This dual system exhibits no cross-reactivity with the other seven pathogens, demonstrating high specificity, and achieves a sensitivity of 10 0 copies/µL. Additionally, the Cpn-ERA-CRISPR/Cas12a dual system was employed to analyze 39 clinical samples, comprising 19 positive and 20 negative samples. The detection rate for positive samples was 100%, with no positive results in the negative samples, indicating a high level of concordance with qPCR results. In summary, the Cpn-ERA-CRISPR/Cas12a dual system represents a novel tool for diagnosing C. pneumoniae and holds promising application potential in grassroots community hospitals., 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 Zhou, Yan, Zhou, Li, Yang, Chen, Deng, Zeng, Sun and Wu.)

This paper discusses the administrative aspects of molecular diagnostics in oncology, including US Food and Drug Administration (FDA) oversight, the regulatory approval process, clinical, and operational workflows, and payment models. Comprehensive molecular testing is important to deliver optimal oncology care and improve patient outcomes. Despite the potential benefits of testing, utilization remains low. The FDA regulatory approval process is reviewed for in vitro diagnostic products, which includes classification into three regulatory classes on the basis of risk. Companion diagnostic devices are used to guide treatment decisions. The clinical and operational challenges associated with molecular testing in oncology are also discussed, including the rapidly evolving landscape of precision oncology, the wide range of biomarker testing options, and complexities of test ordering, interpretation, and result delivery. There is a need for a multifaceted support approach involving education, technology enhancements, and workflow support to overcome these challenges. In terms of payment models, coverage policies between Medicare and commercial payers are compared with differences in coverage criteria, with Medicare focusing on FDA approval or clearance, whereas commercial payers consider additional factors such as National Comprehensive Cancer Network and ASCO guidelines. Commercial payers tend to cover smaller panels on the basis of guideline-recommended biomarkers, whereas coverage for broad tumor profiling is limited. Several strategies can increase the utilization of molecular testing, including integrating test results into electronic medical record platforms, standardizing billing practices, increasing clinical trials, and primary literature supporting the use of molecular testing, educating physicians, and using tumor boards for result interpretation and treatment discussions.

Enterovirus A71 (EVA71) is a leading causative agent of hand, foot, and mouth disease, posing a significant threat to the health of young children, particularly in the Asia-Pacific region. Currently, there is no specific antiviral drug for EVA71 infection; therefore, early and rapid diagnosis is critical for disease prevention and control. Here, we report the development of a simple, rapid, and sensitive detection method for EVA71 infection using reverse transcription-multiple cross displacement amplification (RT-MCDA) combined with nanoparticle-based lateral flow biosensors (LFB). In the RT-MCDA system, a set of 10 primers was designed to target the highly conserved region of the VP1 gene of EVA71 and amplify the genes in an isothermal amplification device. The RT-MCDA amplification reaction products could then be identified by visual detection reagent (VDR) and LFB without the need for specialized equipment. The results demonstrated that the optimal reaction condition for the EVA71-RT-MCDA assay was 65℃ for 40 min. The EVA71-RT-MCDA assay could detect as low as 40 copies of plasmid and 50 copies of pseudotyped virus in a reaction. No cross-reaction was found between EVA71 strains and non-EVA71 strains. For 125 clinical anal swab samples, with EVA71-RT-MCDA assay, 30 samples were positive, which was in consistent with the the conventional real-time quantitative reverse transcription polymerase chain reaction assays. The entire procedure, including a 15-min specimen processing step, a 40-min MCDA reaction, and result reporting within 2 min, was completed in less than 60 min. In conclusion, the EVA71-RT-MCDA-LFB assay targeting the VP1 gene is a rapid, highly sensitive, simple, and specific test that could be widely applied in point-of-care settings and basic medical facilities in rural areas., (© 2024 Wiley Periodicals LLC.)

Nucleic acid extraction (NAE) is crucial for molecular diagnostics but presents challenges in point-of-care testing (POCT) and decentralized settings. We developed a streamlined, paper-based NAE method for hepatitis C virus (HCV) RNA amplification, suitable for integration into POCT and lab-on-a-chip systems. This method uses Fusion 5 paper discs, completing extraction in under 30 min without centrifugation. The nucleic acids on the disc can be directly used or eluted for amplification. We validated this method's compatibility with reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative PCR (RQ-PCR), and loop-mediated isothermal amplification (LAMP), demonstrating versatility across amplification platforms. Clinical evaluation (n = 60) showed 100% sensitivity and specificity with a low detection limit of ~10 1  IU/mL. Results matched those from standard HCV RQ-PCR, confirming accuracy. Additionally, incorporating polyethylene glycol (PEG) improves extraction efficiency, eliminating the need for ethanol treatment and washing/drying steps. This modification enhances performance and suitability for field applications. Our paper-based HCV amplification is affordable and user-friendly, making it valuable for decentralized HCV detection and supporting global health initiatives., (© 2024 Wiley Periodicals LLC.)

Background: Songling virus (SGLV) within the genus Orthonairovirus, family Nairoviridae, is an emerging tick-borne virus associated with human febrile illness. However, no rapid detection method for SGLV has been established., Methods: In this study, four primer sets targeting the nucleocapsid protein gene of SGLV were designed for use in the LAMP assay and evaluated to identify the optimal primer set. Recombinant plasmids were constructed and utilized for assessing the sensitivity of the assay. Tacheng tick virus 1 (TcTV-1)-, Beiji nairovirus (BJNV)-, Yezo virus (YEZV)-, severe fever with thrombocytopenia syndrome virus (SFTSV)-, and tick-borne encephalitis virus (TBEV)-positive tick samples were utilized to assess the specificity. Field-collected ticks were also evaluated as biological specimens to validate the assay., Results: A SGLV-specific LAMP assay was established with a detection limit of 1 × 10 -2  copies/μl and could be visually confirmed by a color change from purple to blue in SGLV-positive samples. No cross-reactivity was observed in the detection of TcTV-1, BJNV, YEZV, SFTSV, and TBEV using the LAMP assay. In addition to the detection of the same seven high-copy numbers of SGLV as the SYBR Green quantitative RT-PCR assay within a reduced timeframe, the developed LAMP method also effectively identified an additional sample with a low copy number in the field-collected tick samples., Conclusions: We successfully developed a sensitive, specific, and cost-effective visual method for the rapid detection of SGLV using the LAMP assay, which can be applied in pathogenesis and epidemiological surveillance studies of SGLV., (© 2024. The Author(s).)

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.)

Purpose: Loop-mediated isothermal amplification (LAMP) is a simple and rapid nucleic acid method for DNA amplification at a constant temperature. The "gold standard" culture method for yeast detection, has low sensitivity with severe consequences, increasing morbidity and mortality rates. Here, we report the development of a LAMP method for the specific detection of C. glabrata., Methodology: The specific LAMP primers for C. glabrata detection were designed and evaluated., Results: The LAMP assay accurately detected C. glabrata with no cross-reactivity with other Candida species., Conclusion: The developed molecular method would be a promising tool in the management of invasive candidiasis., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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.)

Concepts in thyroid diagnostics are evolving. As cytopathologists, we benefit from understanding the changes taking place in cytopathology practice as well as intersecting areas that may have implications for us. In this review, we discuss recent changes to 1. Classification systems, 2. Ancillary molecular testing modalities, and 3. Key metrics that affect thyroid cytopathology. The recent World Health Organization, Bethesda Thyroid Cytopathology, and American Joint Committee on Cancer classification systems have aspects that are designed to harmonize the clinical, cytopathologic, histomorphologic, and molecular findings for improved communication and patient management. New terminologies such as thyroid follicular nodular disease and low-risk follicular cell-derived thyroid neoplasms are introduced to reflect the subtle biologic nuances involving benign non-neoplastic and low-grade neoplastic conditions. The Bethesda Thyroid Cytopathology System has simplified its terminology, updated risk of malignancy estimates, and expanded the discussions on molecular testing, clinical and imaging assessments, and pediatric cytopathology. There is now a single term for each of the 6 diagnostic categories. The American Joint Committee on Cancer has refined the staging criteria to provide improved stratification of patient prognostication. Molecular testing using next-generation technology now offers large panels of markers that are sensitive for detecting the wide range of thyroid neoplasms. These panels were developed in North America and whether other regions of the world will choose similar tests remain to be seen. Finally, metrics such as molecular-derived risk of malignancy and molecular risk group may be viewed as surrogates of resection information and used to complement diagnostics, management, and quality assurance., (Copyright © 2024 American Society of Cytopathology. Published by Elsevier Inc. All rights reserved.)

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).)

Cotton root rot caused by Macrophomina phaseolina pose a significant threat to cotton production, leading to substantial yield and quality losses. Early and accurate diagnosis of this pathogen in soil is crucial for effective disease management. This study presents a pioneering investigation into the utilization of the nit gene encoding nitrilase for the development of a molecular diagnostic assay aimed at the rapid detection of M. phaseolina in field soils. The methodology involved the design and validation of primers targeting the Nit gene sequence, followed by the optimization of PCR conditions for efficient amplification. Leveraging state-of-the-art molecular techniques, the assay offers a novel protocol to accurately identify the presence of M. phaseolina in soil with high sensitivity and specificity. The specificity of the designed primers was confirmed through PCR amplification using DNA from M. phaseolina and other related fungi. Sensitivity tests demonstrated that the PCR assay reliably detected M. phaseolina DNA at concentrations as low as 1 ng. Furthermore, the performance of the diagnostic assay was rigorously evaluated using field soil samples with a known status of M. phaseolina infection, demonstrating its reliability and efficacy in real-world scenarios. This study introduces a novel molecular marker for the detection of M. phaseolina and offers a rapid and efficient means for screening M. phaseolina in large soil samples with minimal time and manpower., (© 2024 Wiley‐VCH GmbH.)

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.)

Biochemical phenotyping has been the milestone for diagnosing and managing patients affected by inborn errors of intermediary metabolism (IEiM); however, identifying the genotype responsible for these monogenic disorders greatly contributes to achieving these goals. Herein, whole-exome sequencing (WES) was used to determine the genotypes of 95 unrelated Mexican pediatric patients suspected of having IEiM. They were classified into those bearing specific biochemical abnormalities (Group 1), and those presenting unspecific biochemical profiles (Group 2). The overall concordance between the initial biochemical diagnosis and final genotypic diagnoses was 72.6% ( N = 69/95 patients), with the highest concordance achieved in Group 1 (91.3%, N = 63/69), whereas the concordance was limited in Group 2 (23.07%). This finding suggests that previous biochemical phenotyping correlated with the high WES diagnostic success. Concordance was high for urea cycle disorders (94.1%) and organic acid disorders (77.4%). The identified mutational spectrum comprised 83 IEiM-relevant variants (pathogenic, likely pathogenic, and variants of uncertain significance or VUS), including three novel ones, distributed among 29 different genes responsible for amino acid, organic acid, urea cycle, carbohydrate, and lipid disorders. Inconclusive WES results (7.3%, N = 7/95) relied on monoallelic pathogenic genotypes or those involving two VUS for autosomal-recessive IEiMs. A second monogenic disease was observed in 10.5% ( N = 10/95) of the patients. According to the WES results, modifications in treatment had to be made in 33.6% ( N = 32/95) of patients, mainly attributed to the presence of a second monogenic disease, or to an actionable trait. This study includes the largest cohort of Mexican patients to date with biochemically suspected IEiM who were genetically diagnosed through WES, underscoring its importance in medical management.

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.

Citrus canker, a highly transmissible bacterial disease, has three major types, with Asiatic canker (Canker A), caused by Xanthomonas citri pv. citri ( Xcc A), being the most widespread and severe, affecting most citrus varieties. Xcc A has two mild variants, Xcc A* and A w with a limited host range, reported in Southwest Asia and Florida, respectively. Since 2015, the canker caused by Xcc A w has been being reported in the Rio Grande Valley of South Texas where the Texas commercial citrus industry is located. In 2016, a more severe Canker A was reported in the upper Texas gulf coast region, north of the Rio Grande Valley, posing a potential threat to the Texas citrus industry. Given that existing diagnostic methods cannot reliably distinguish Xcc A w from Xcc A, we developed a loop-mediated isothermal amplification (LAMP) assay specific to Xcc A w (LAMP-A w ) for rapid, field-based identification of this bacterial variant. The detection limit of LAMP-A w was ~4.52 Log 10 copies of the target molecule. This study also evaluated the field applicability of the LAMP-A w assay by coupling the LAMP-A w assay with a lateral flow immunoassay system.

Introduction: Early and accurate diagnosis of tuberculosis (TB) is crucial for initiating timely treatment and preventing new infections. In this study, we introduced the iFIND TBR assay, an automated all-in-one tuberculosis detection approach that simultaneously detect Mycobacterium tuberculosis (MTB) and rifampicin (RIF) resistance., Methods: The limits of detection (LOD), sensitivity, specificity, and RIF-R rpoB mutation detection of the iFIND TBR were tested on Mycobacterium tuberculosis DNA or sputum samples spiked with known numbers of M.tuberculosis H37Rv. Frozen clinical samples from patients suspected of having TB were also tested., Results: The LOD of the iFIND TBR for MTB detection were 13.34 CFU/ml (95% CI, 11.71-16.47), and for RIF resistance was 109.79CFU/mL (95% CI, 95-138.19). The iFIND TBR assay accurately distinguish MTB strains from non-tuberculous mycobacteria (NTM) without any cross reactivity. Testing on 157 clinical sputum samples, compared with the bacteriologically TB standard, the overall sensitivity and specificity of the iFIND TBR was 100% (95%CI, 94.64, 100) and 85.29% (95% CI, 74.61, 92.72), respectively. When assessing RIF susceptibility, the iFIND TBR achieved a sensitivity of 98.15% (95% CI, 90.11-99.95) and a specificity of 85.71% (95% CI, 67.33-95.97), compared with phenotypic drug susceptibility testing. Discordant RIF susceptibility results were more frequently observed in samples exhibiting heteroresistance., Discussion: These findings demonstrate that iFIND TBR assay performs well in detecting TB and RIF resistance, and shows promise as a point-of-care tool in resource-limited areas., 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 Ou, Song, Xing, Zhao, Pei, Teng, Zhang, Sun, Liu, Xia, Zhou, Zheng, Song, Zhang, Wang, Anthony and Zhao.)

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).)

Loop-mediated isothermal amplification (LAMP) is a highly effective molecular diagnostic technique, particularly advantageous for point-of-care (POC) settings. In recent years, LAMP has expanded to include various adaptations such as DARQ-LAMP, QUASR, FLOS-LAMP, displacement probes and molecular beacons. These methods enable multiplex detection of multiple targets in a single reaction, enhancing cost-effectiveness and diagnostic efficiency. Consequently, LAMP has gained significant traction in diagnosing diverse viruses, notably during the COVID-19 pandemic. However, its application for detecting Herpesviridae remains relatively unexplored. This group of viruses is of particular interest due to their latency and potential reactivation, crucial for immunocompromised patients, including organ and hematopoietic stem cell transplant recipients. This review highlights recent advancements in LAMP for virus diagnosis and explores current research trends and future prospects, emphasizing the detection challenges posed by Herpesviridae.

Isothermal amplification (IA) techniques like rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP) have gained significant attention in recent years due to their ability to rapidly amplify DNA or RNA targets at a constant temperature without the need for complex thermal cycling equipment. Such technologies, combined with colorimetric systems rendering visual confirmation of the amplification event, are ideal for the development of point-of-need detection methods suitable for field settings where access to specialized laboratory equipment is limited. The utility of these technologies, thus far limited to DNA and RNA targets, could be broadened to a wide range of targets by using aptamers. Composed of DNA or RNA themselves, aptamers can bind to substances, including proteins, metabolites, and inorganic substances. Their nucleic acid nature can potentially allow them to serve as a bridge, extending the reach of DNA/RNA-centric technologies to the broader molecular world. Indeed, the change in aptamer conformation occurring during ligand interaction can be used to elaborate ligand-responding RCA or LAMP templates. By using an existing aptamer targeting SARS-CoV-2 Spike protein as a model, we explored the possibility of establishing ligand-responsive IA systems. Our study used aptamers with simple sequence modifications as templates in LAMP assays and hyperbranched RCA (HRCA) by exploiting the dynamic nature of the model aptamer to trigger these IA systems. Importantly, our work uniquely demonstrates that this aptamer's dynamic response to ligand binding can regulate both RCA and LAMP processes. This novel approach of using aptamer conformational changes to trigger LAMP paves the way for new aptamer-based detection assays. Our system detects 50 nM of Spike protein, with LAMP occurring within 30 min in the presence of Spike. The colorimetric readout showed clear results, allowing for the detection of Spike protein presence.

Background: Senecavirus A (SVA) is a newly pathogenic virus correlated with the acute death of piglets and vesicular lesions in pigs. The further prevalence of SVA will cause considerable economic damage to the global pig farming industry. Therefore, rapid and accurate diagnostic tools for SVA are crucial for preventing and controlling the disease., Methods: We designed multiple primer pairs targeting the most conserved region of the SVA 3D gene and selected those with the highest specificity. Nfo-probes were subsequently developed based on the highest specificity primer pairs. Subsequently, the recombinase-assisted amplification (RAA) reaction was completed, and the reaction temperature and duration were optimized. The RAA amplicons were detected using a lateral flow device (LFD). Finally, a rapid and intuitive RAA-LFD assay was established against SVA., Results: The SVA RAA-LFD assay can be performed under reaction conditions of 35°C within 17 minutes, with results observable to the naked eye. We then evaluated the performance of this method. It exhibited high specificity and no cross-reaction with the other common swine pathogens. The lowest detectable limits of this method for the plasmid of pMD18-SVA-3D, DNA amplification product, and viral were 3.86×10 1 copies/µL, 8.76×10 -7 ng/µL, and 1×10 0.25 TCID 50 /mL, respectively. A total of 44 clinical samples were then tested using the RAA-LFD, PCR, and RT-qPCR methods. The results demonstrated a consistent detection rate between the RAA-LFD and RT-qPCR assays., Conclusion: The SVA RAA-LFD assay developed in our study exhibits excellent specificity, sensitivity, and time-saving attributes, making it ideally suited for utilization in lack-instrumented laboratory and field settings., Competing Interests: Authors ZL and LW were employed by the company Wen’s Foodstuffs Group Co., Ltd. The remaining 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 Song, Fang, Zhu, Wang, Wang, Chen, He, Yi, Ding, Zhao, Fan, Li and Chen.)

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.