Molecular Detection Methods Research Articles

Identification of fine antigenic epitopes of Tamdy virus glycoprotein Gn fragment and establishment of ELISA detection method

BackgroundTamdy virus (TAMV) was first isolated in Uzbekistan and Turkmenistan. In 2018, it was found in China, marking its entry into the molecular research era. TAMV is linked to febrile diseases, but its epidemiology and spillover risks are poorly understood, necessitating urgent molecular research and detection method development.MethodsThe secondary structure of TAMV glycoprotein Gn was predicted, and the results showed that it had rich antigenic epitopes. According to the predicted results, glycoprotein Gn was divided into 46 truncated 16-peptides by modified synthetic peptide method, and the antigenicity of 46 truncated 16-peptides was verified by western blotting analysis.ResultsThe results showed that P8, P9, P24, P25, P28, P29, and P39 had antigenicity. Subsequently, the seven positive 16-peptide sequences with antigenicity were truncated to form 8-peptide sequences with an overlap of seven amino acids. After analysis with the same method, eight fine antigenic epitopes E1 (58VINSTLDH65), E2 (65HVGSWGMP72), E3 (68SWGMPVTT75), E4 (187IRNQPFKS194), E5 (195FNVEVQ200), E6 (226AVVEHH231), E7 (228VEHHGNKA235), and E8 (310RGGRR314) were identified, all of which were located on the three-dimensional surface of glycoprotein Gn and were highly conserved in different TAMV strains.ConclusionsEight precise epitopes were identified, and an indirect ELISA method based on fusion multiepitope peptide (r-Gn-MEPX2) was developed and implemented, featuring high sensitivity, accuracy, and specificity.Graphical

The development of rapid detection techniques for Alexandrium catenella.

Alexandrium catenella is an important toxic algal species in the Alexandrium genus, which can form toxic red tides in large numbers. The paralytic shellfish poisoning (PSP) produced by Alexandrium catenella can seriously endanger human health and threaten the production and development of the aquaculture and fishery industries. Therefore, it is important to explore and develop effective detection and early warning methods for toxic red tides. Traditional microscopy and molecular biology detection methods require specialized technicians and equipment, which are time-consuming and complicated to operate and cannot be adapted to the needs of rapid on-site detection. This study utilized the colloidal gold immunochromatography technique to prepare polyclonal and monoclonal antibodies against Alexandrium catenella, which were then assembled into colloidal gold test strips. These test strips enable rapid detection of Alexandrium catenella in aquatic environments and facilitate semi-quantitative analysis of its concentration. They allow for a quick assessment of whether the algal concentration in the environment has reached the baseline for red tide conditions, facilitating the timely implementation of preventive and control measures. This method holds significant practical importance and promising application prospects.

EVALUATION OF THE APPLICABILITY OF THE O.K.N.V.I. RESIST-5 AND THE KPC&MBL&0XA-48 DISK TESTS IN A ROUTINE MICROBIOLOGY LABORATORY

Background: The global spread of carbapenemase-producing Enterobacterales (CPE) and the increasing emergence of clinical Enterobacterales harboring multiple carbapenemases of different molecular classes have prioritized the use of rapid molecular detection methods in routine microbiology laboratories. The aim of this study was to evaluate the applicability of the immunochromatographic O. K.N.V.I. RESIST-5 and the KPC&MBL&0XA-48 disc tests in a clinical microbiology laboratory. Material and methods: The tests were performed with 50 CPE belonging to 8 species and producing 7 molecularly characterized carbapenemases. Six of these isolates carried two different carbapenemases. To assess the specificity of the assays, 18 non-carbapenemase-producing but carbapenem-resistant Enterobacterales (non-CP CRE) were also included. Both tests were performed from a common overnight culture on Mueller-Hinton agar with inoculum harvested around an ertapenem disk. Results: The O.K.N.V.I. RESIST-5 correctly detected all 56 carbapenemases, including KPC-2 in Klebsiella pneumoniae; OX-48 in Serratia marcescens, Citrobacter freundii, Enterobacter hormaechei and K. pneumoniae; NDM-1 in Escherichia coli, Morganella morganii, E. hormaechei, C. freundii, S. marcescens and K. pneumoniae; VIM-1 in Proteus mirabilis; VIM-4 in C. freundii and S. marcescens; VIM-86 with and without NDM-1 in Providencia stuartii, and NDM-5 with and without OXA-232 in K. pneumoniae. The KPC&MBL&0XA-48 disc tests correctly confirmed KPC, OX-48-like and most MBL except VIM in P. mirabilis. Furthermore, the combination disc tests failed to detect OXA-48-like in pairs with MBL in K. pneumoniae. Conclusions:The O.K.N.V.I. RESIST-5 multiplex lateral assay is an excellent test for rapid diagnostic of CPE in routine microbiology laboratories. It is easy to handle and provides results with 100% sensitivity and specificity when an inoculum around ertapenem disc from routine antibiogram was used.

OLAgen: A Software Tool for Reagent Design to Expand Access to Single-Nucleotide Variant Detection by the Oligonucleotide Ligation Assay.

Single-nucleotide variants (SNVs) and polymorphisms are characteristic biomarkers in various biological contexts, including pathogen drug resistances and human diseases. Tools that lower the implementation barrier of molecular SNV detection methods would provide greater leverage of the expanding single-nucleotide polymorphism/SNV database. The oligonucleotide ligation assay (OLA) is a highly specific means for detection of known SNVs and is especially powerful when coupled with PCR. Yet, the OLA design process remains intensive, and criteria for success are uncertain. To assist in the design process, this study describes OLAgen, an open-source tool to automate development of OLAs and their coupled PCR assays. The software facilitates alignment of sequences surrounding SNVs and generates ligation probes while screening for dimerization potential. OLAgen successfully produced ligation probes that closely matched previously validated designs for HIV-1, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and KRAS, confirming its reliability and potential for clinical applications. The tool was used to generate new assays targeting Mycobacterium tuberculosis drug resistance and variants in the human JAK2, BRAF, and factor V genes, all of which demonstrated 100% sensitivity and specificity in controlled laboratory experiments. The OLAgen predicted assay designs detected mutant frequencies as low as 1% to 5% in wild-type backgrounds in proof-of-concept laboratory studies. OLAgen represents a significant advancement in accessible assay design, promoting the broader application of OLA technology in clinical and research settings.

Importance of Molecular Method for Detection of Microorganisms in the Exploration of the Infectious Etiology of Male Infertility in Burkina Faso

Importance of Molecular Method for Detection of Microorganisms in the Exploration of the Infectious Etiology of Male Infertility in Burkina Faso

FMD RAPID DIAGNOSTIC TEST IN ACEH INDONESIA: A TECHNICAL NOTE

This study aims to offer a detailed technical note thorough examination of the pioneering and effectiveness of FMD immediate diagnostic tests in Aceh, Indonesia. We discuss the key components of these tests, including their sensitivity, specificity, and practicality in resource-limited settings. Furthermore, we explore the challenges and opportunities associated with adopting these tests locally and highlight their potential impact on FMD surveillance and control efforts. The findings presented in this technical note contribute to the ongoing efforts to combat FMD in Aceh, providing valuable insights into using rapid diagnostic tests as a vital tool in disease management strategies. The results showed that the FMD rapid diagnostic test kit has limited sensitivity in detecting early clinical signs of FMD in cattle in Aceh. Therefore, Further examination with laboratory molecular detection methods is needed.

A six-year epidemiological study of selected zoonotic abortifacient agents in ovine and caprine foetuses in Türkiye.

Abortion is one of the major threats to the livestock industry, and it also poses significant threats to public health since some of the abortifacient agents are considered zoonotic. Chlamydia abortus (C. abortus), Coxiella burnetii (C. burnetii), Listeria monocytogenes (L. monocytogenes), and Cache Valley virus (CVV) are recognized as important zoonotic and abortifacient agents of reproductive failure in small ruminants. This study determined the prevalence of these agents in ovine and caprine foetuses in Türkiye. A total of 1226 foetuses were collected from the sheep (n=1144) and goats (n=82) from different flocks between 2012 and 2017. Molecular detection methods were used to detect C. abortus, C. burnetii, and L. monocytogenes DNA and CVV RNA in aborted foetuses. In this study, C. abortus was the most prevalent abortifacient agent among the investigated ovine (264/1144) and caprine (12/82) foetuses, followed by C. burnetii with a frequency of 2.8% (32/1144) and 8.5% (7/82) in ovine and caprine foetuses, respectively. L. monocytogenes DNA was detected in 28 (2.4%) and 2 (2.4%) of the ovine and caprine foetuses, respectively. However, CVV RNA was not detected. Although the predominant mixed infection was C. abortus and C. burnetii, mixed infection of C. abortus and L. monocytogenes, and C. burnetii and L. monocytogenes were also found. The information presented in this study contributes to the understanding of the roles of C. abortus, C. burnetii, L. monocytogenes, and CVV in abortions in small ruminants, and could be beneficial for developing more effective control strategies.

Molecular pathogen profiling of COVID-19 coinfections

ObjectiveThis study aims to investigate the prevalence, pathogen spectrum, clinical characteristics, and prognosis-related factors of other respiratory pathogens in COVID-19-infected patients, and to explore the application of molecular detection methods in the epidemiological investigation of multiple pathogen infections.MethodsRespiratory samples and clinical data from 384 patients with outpatient and inpatient respiratory infections were collected and analyzed. Multiplex PCR and capillary electrophoresis were conducted to detect the distribution characteristics of 26 pathogen species, comprising 13 viruses, 13 bacteria. Statistical analysis explored the relationship between pathogen distribution with COVID-19 development.ResultsThere was no statistical difference in prognosis between the 230 COVID-19-positive patients and the 154 COVID-19-positive patients. COVID-19 cases co-infected with other pathogens do not correlate with patient’s age and gender. The main distribution of pathogens was mainly mecA (n = 62, 26.96%), followed by SPN(n = 61; 26.52%) and KP(n = 22; 9.57%). Compared with non-COVID-19 cases, COVID-19 infected patients showed a significantly higher mecA carrying rate (26.96% vs. 15.58%, P < 0.01), while there was no statistical difference for other pathogens. Regression analysis found that mecA and KP were independent risk factors for severe illnesses (mechanical ventilation, endotracheal intubation, ECOMO, etc.) or death in COVID-19 patients had 2.391 times and 3.722 times risk of severe disease or death compared with COVID-19 patients without mecA and KP.ConclusionCOVID-19 patients show a higher mecA carrier rate than non-COVID-19 patients, and mecA and KP may increase the risk of severe disease or death in COVID-19 patients, which requires close attention.

Parasitic Protozoa and Other Vector-Borne Pathogens in Captive Mammals from Brazil

In captive environments, mammals are frequently exposed to various parasitic protozoa and other vector-borne pathogens that can impact both animal health and public health. Monitoring these pathogens is essential for animal welfare and zoonotic disease control. This study aimed to investigate the prevalence of parasitic protozoa and other vector-borne pathogens in captive mammals through molecular detection methods at the Belo Horizonte Zoo, Brazil. Between November 2021 and March 2023, whole blood samples were collected from 40 mammals. Molecular analyses identified piroplasms, Leishmania spp., granulocytic/platelet Anaplasma/Ehrlichia spp., monocytic Ehrlichia spp., Bartonella spp. and hemotropic Mycoplasma spp. with a 72.5% positivity rate. Piroplasms were found in 22.5% (two Pantanal cats, two gorillas, one white rhinoceros, one spider monkey, one jaguar, one tufted capuchin and one hippo) and Leishmania spp. in 12.9% (four maned wolves). Granulocytic/platelet Anaplasma/Ehrlichia spp. were found in 12.5% of the samples (one gorilla and four maned wolves), Ehrlichia canis in 2.5% of the animals (one maned wolf), Bartonella spp. in 42.5% (six howler monkeys, two maned wolves, one gorilla, one white rhino, one southern tamandua, one common woolly monkey, one tufted capuchin, one brown brocket deer, one agouti, one cougar and one hippo), hemotropic Mycoplasma spp. in 17.5% (one gorilla, one maned wolf, one white rhino, one howler monkey, two common woolly monkeys and one European fallow deer). Five Artiodactyla members tested negative for A. marginale. Coinfections occurred in 34.5% of the positive samples. Sequencing revealed that Theileria spp. and Cytauxzoon spp. are closely related to Theileria bicornis and Cytauxzoon felis; Ehrlichia canis and Bartonella spp. are closely related to B. clarridgeiae and B. henselae; and hemotropic Mycoplasma spp. are closely related to Candidatus Mycoplasma haemominutum. Our results showed a high occurrence of vector-borne pathogens in captive animals, including zoonotic species, which may pose a risk to animal and human public health.

Diagnostic potential of rhinovirus C using reverse-transcription loop-mediated isothermal amplification (RT-LAMP).

Rhinoviruses (RVs), particularly RV-C, frequently cause acute respiratory infections and asthma exacerbations. However, there is a lack of routine detection methods. Thus, this study aims to develop a rapid molecular and differential diagnostic detection method for RV-C using the reverse transcription (RT) loop-mediated isothermal amplification (LAMP) approach. The RT-LAMP assay targeting the 5'UTR region of RV-C genome was optimized by varying the reaction temperature, magnesium sulphate, betaine concentrations, and reaction time. Compared with conventional RT-PCR with a sensitivity of 106 copies of RNA, RT-LAMP demonstrated a significant increase in efficiency and sensitivity with a quantifiable viral load of at least 101 copies of RNA by gel electrophoresis and colour change, and 104 copies of RNA for end-point detection with a turbidimeter for 40min. The assay is also specific without amplifying RV-A16 and RV-B72 genomic RNA. In the proof-of-concept assay using 30 clinical respiratory samples with known etiological agents, it detected all RV-C isolates, of which its accuracy was confirmed by sequencing. The newly developed RT-LAMP assay demonstrated good analytical sensitivity and specificity toward RV-C. The assay provides an alternative for improved RV-C diagnosis.

A Sensitive, Specific and Fast Electrochemical‐based Nanobiosensor Diagnostic for Xanthomonas albilineans, the Cause of Sugarcane Leaf Scald Disease

AbstractLeaf scald (LS) caused by Xanthomonas albilineans (Xalb), is a major bacterial disease of sugarcane. The unreliable symptom expressions make traditional visual detection challenging. The molecular methods of detection require expensive equipment, labor‐intensive, and time‐consuming. This study proposes a novel electrochemical (EC)‐approach, that is relatively easy to use and less expensive to detect Xalb DNA in LS‐infected sugarcane leaves, meristematic tissue, and xylem sap samples. This method involves three key steps: i) DNA isolation from sugarcane samples via boiling lysis; ii) magnetic purification of target sequences from the lysate using magnetic bead‐bound capture probes; and iii) EC detection of the target DNA. The method shows excellent detection sensitivity (10 cells µL−1), reproducibility (Standard deviation, SD &lt;5%, for n = 3), and a wide linear dynamic range (1 nM–1 fM or 106–10° copies µL−1, r = 0.99). The EC assay has a strong negative correlation with quantitative polymerase chain reaction (qPCR) results (r = −0.95–0.97, n = 24, p &lt; 0.001), and weak or no correlation with the varietal resistance ratings. This EC‐based assay can be a commercially viable alternative, providing a DNA isolation/purification‐free solution, and can potentially be adapted into a handheld device for on‐farm detection and quantification of the LS‐causing pathogen.

Molecular Genetic Methods for Detection and Genotyping of Human Sapoviruses (Review)

Molecular Genetic Methods for Detection and Genotyping of Human Sapoviruses (Review)

Concordance in molecular methods for detection of antimicrobial resistance: A cross sectional study of the influent to a wastewater plant

Methods that are used to characterise microbiomes and antimicrobial resistance genes (ARGs) in wastewater are not standardised. We used shotgun metagenomic sequencing (SM-Seq), RNA sequencing (RNA-seq) and targeted qPCR to compare microbial and ARG diversity in the influent to a municipal wastewater treatment plant in Australia. ARGs were annotated with CARD-RGI and MEGARes databases, and bacterial diversity was characterised by 16S rRNA gene sequencing and SM-Seq, with species annotation in SILVA/GreenGenes databases or Kraken2 and the NCBI nucleotide database respectively. CARD and MEGARes identified evenly distributed ARG profiles but MEGARes detected a richer array of ARGs (richness = 475 vs 320). Qualitatively, ARGs encoding for aminoglycoside, macrolide-lincosamide-streptogramin and multidrug resistance were the most abundant in all examined databases. RNA-seq detected only 32 % of ARGs identified by SM-Seq, but there was concordance in the qualitative identification of aminoglycoside, macrolide-lincosamide, phenicol, sulfonamide and multidrug resistance by SM-Seq and RNA-seq. qPCR confirmed the detection of some ARGs, including OXA, VEB and EREB, that were identified by SM-Seq and RNA-seq in the influent. For bacteria, SM-Seq or 16S rRNA gene sequencing were equally effective in population profiling at phyla or class level. However, SM-Seq identified a significantly higher species richness (richness = 15,000 vs 3750). These results demonstrate that SM-Seq with gene annotation in CARD and MEGARes are equally sufficient for surveillance of antimicrobial resistance in wastewater. For more precise ARG identification and quantification however, MEGARes presented a better resolution. The functionality of detected ARGs was not confirmed, but general agreement on the putative phenotypic resistance profile by antimicrobial class was observed between RNA-Seq and SM-Seq.

In vitro/In vivo oxygen electrochemical nanosensor for bioanalysis

Direct in vivo monitoring of O2 is critical in the study of numerous biological processes, and the development of novel highly sensitive techniques for O2in vivo detection is of great potential importance. Electrochemical amperometric sensors are some of the most promising, easy to operate, inexpensive, sensitive and have a high temporal resolution. As part of this work, we have demonstrated a direct rapid method for molecular oxygen detection inside multicellular spheroids in vitro and rat brain in vivo. External and internal oxygen profiles in human adenocarcinoma MCF-7 spheroids were studied using developed nanoelectrode. The size of spheroids was shown to affect the level of hypoxia inside them, and also affected the oxygen consumption near spheroids in solution. To demonstrate the in vivo relevance of the novel sensor we used it to measure the oxygen concentrations in the superficial layers of the rat brain. This study demonstrates a minimally invasive electrochemical method for real-time oxygen profiling in vivo.

Respiratory viruses associated with severe acute respiratory infection in children aged

With the increased availability of licensed vaccines for respiratory viruses such as severe acute respiratory syndrome coronavirus 2, respiratory syncytial virus (RSV), and influenza virus, a better understanding of the viral aetiology of severe acute respiratory infections (SARI) among children could help in optimising the use of these vaccines. We conducted a study among children aged <5 years hospitalised with SARI at a tertiary care children's hospital in north India and tested for common respiratory pathogens. We randomly enrolled eligible SARI cases aged <5 years from August 2013 to July 2015. SARI cases were defined as either <7-day history of fever with cough or in children aged eight days to three months, a physician diagnosis of acute lower respiratory infection requiring hospitalisation. We also enrolled an age-group matched control without any acute illness in a 2:1 ratio from the outpatient clinic within 24 hours of case enrolment. Nasopharyngeal and/or oropharyngeal swabs were collected and tested using TaqMan Array Cards, a real-time reverse transcription polymerase chain reaction-based multi-pathogen testing platform for selected respiratory viruses among the enrolled cases and controls. We compared the prevalence of each pathogen among cases and controls using the χ2 (χ2) or Fisher exact test (P < 0.05). We used logistic regression to estimate adjusted odds ratios (aORs) which were then used to calculate aetiologic fractions (EFs). We enrolled 840 cases and 419 outpatient controls. Almost half of the individuals in the whole sample were aged <6 months (n = 521, 41.4%). Females made up 33.7% of cases and 37.2% of controls. Viral detections were more common among cases (69%, 95% confidence interval (CI) = 66, 73) compared to controls (33%; 95% CI = 29, 38) (P < 0.01). RSV (n = 257, 31%; 95% CI = 28, 34%) was the most common virus detected among cases. Influenza A was detected among 24 (3%; 95% CI = 2, 4%), and influenza B among 5 (1%; 95% CI = 0, 1%) cases. The association between the virus and SARI was strongest for RSV (aOR = 23; 95% CI = 12, 47; EF = 96%). Antivirals were administered to 1% of SARI cases while 78% received antibiotics. Using a multi-pathogen molecular detection method, we detected respiratory viruses among more than two-thirds of children aged <5 years admitted with SARI in the Delhi tertiary care children's hospital. The guidelines for preventing and managing SARI cases among children could be optimised further with the improved availability of antivirals and vaccines.

A molecular detection method for HBV pgRNA without RNA extraction based on nucleic acid hybridization

BackgroundEarly diagnosis is critical for patients with chronic hepatitis B. Here, we utilized a sandwich RNA hybridization assay to directly detect HBV pgRNA, thereby avoiding the need for RNA extraction and purification. MethodsWe designed a hybridization cascade reaction on a solid surface using a set of oligonucleotide probes that target several highly conserved regions in pgRNA. The detection performance was validated by concurrently testing serum samples from CHB patients and healthy individuals. ResultsThe optimal detection conditions were: a universal probe coating concentration of 0.003 µg/µl with a coating duration of 2 h; capture probe and detection probe concentrations of 0.1 nM; a hybridization capture duration of 4 h; and an antibody incubation duration of 60 minutes. The sensitivity and specificity were calculated to be 91.47 % and 90.63 %, respectively. ConclusionThis novel detection method is both simple and high-throughput, making it particularly suitable for active CHB infection screening

Prevalence, Molecular Characterization and Antibiogram Pattern of Livestock Associated Methicillin Resistant Staphylococcus aureus in Livebird Markets Gusau, Zamfara State, Nigeria

Study’s Excerpt The prevalence and antibiotic resistance patterns of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) in poultry markets within the Gusau metropolis was examined. Phenotypic and molecular (PCR) detection methods were combined to identify the LA-MRSA isolates. Multidrug resistance (MDR) phenotypes were observed in 72.2% of the isolates. Full Abstract Poultry farms have been identified as hotspots for the emergence and spread of antibiotic-resistant bacteria, especially livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA). In this study, a total of 320 swab samples were randomly collected from fecal litter, Hands of poultry handlers, Soil, Feed, and Water from five different poultry markets in the Gusau metropolis. The LA-MRSA isolates involved were detected culturally and identified phenotypically by the use of PCR assay. The pooled prevalence of the LAMRSA isolates in the samples was 22(100%). The chi-square test was used to compare the prevalence of LAMRSA among the samples collected at 95% confidence interval and 0.05 level of significance. There is no statistically significant difference in the prevalence of LAMRSA among the samples. Findings from the antibiogram study conducted revealed that all the 22(100%) LAMRSA isolates were resistant to Penicillin antibiotics, with resistance to other antibiotics following the order: Nitrofurantoin, 17(77.3%), Sulphamethoxazole, 13(59.1%), Chloramphenicol 6(27.28%), Ciprofloxacin 5(22.7%) and Erythromycin 4(18.2%). A total of 14 multidrug resistant phenotypes were identified from the 22 LAMRSA isolates with 16(72.2%) demonstrating cross resistant to ≥3 of the six different classes of antibiotics used. The MDR rate in soil samples was the highest (27.3%) (p&lt;0.05) compared to other MDR isolates from the other sources. Of all the isolates, 22(100%) demonstrated a MAR Index ranging from 0.2-0.8. These are disturbing trends with significant risks to humans and animals. Suggesting a potential for inoculation of self and transmission to others, particularly through those who sell and make daily contact with live birds. Therefore, the government should awaken the consciousness of poultry farmers/handlers by way of sensitization on the need to improve water quality and practice routine personal and environmental hygiene as it relates to poultry management, with a focus on antibiotic stewardship within the one health framework so as to lower endemicity and equally break the chain of transmission of LA- MRSA across the farm to fork continuum.

A novel machine-learning aided platform for rapid detection of urine ESBLs and carbapenemases: URECA-LAMP.

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.

Development and Evaluation of Real-Time Quantitative PCR Assays for Detection of Phellinus noxius Causing Brown Root Rot Disease.

Brown root rot disease (BRRD) is a highly destructive tree disease. Early diagnosis of BRRD has been challenging because the first symptoms and signs are often observed after extensive tissue colonization. Existing molecular detection methods, all based on the internal transcribed spacer (ITS) region, were developed without testing against global Phellinus noxius isolates, other wood-decay fungi, or host plant tissues. This study aimed to develop SYBR Green real-time quantitative PCR (qPCR) assays for P. noxius. The primer pair Pn_ITS_F/Pn_ITS_R targets the ITS, and the primer pair Pn_NLR_F/Pn_NLR_R targets a P. noxius-unique group of homologous genes identified through a comparative genomics analysis. The homologous genes belong to the nucleotide-binding-oligomerization-domain-like receptor (NLR) superfamily. The new primer pairs and a previous primer pair G1F/G1R were optimized for qPCR conditions and tested for specificity using 61 global P. noxius isolates, 5 other Phellinus species, and 22 non-Phellinus wood-decay fungal species. Although all three primer pairs could detect as little as 100 fg (approximately 2.99 copies) of P. noxius genomic DNA, G1F/G1R had the highest specificity and Pn_NLR_F/Pn_NLR_R had the highest efficiency. To avoid false positives, the cutoff quantification cycle values were determined as 34 for G1F/G1R, 29 for Pn_ITS_F/Pn_ITS_R, and 32 for Pn_NLR_F/Pn_NLR_R. We further validated these qPCR assays using Ficus benjamina seedlings artificially inoculated with P. noxius, six tree species naturally infected by P. noxius, rhizosphere soil, and bulk soil. The newly developed qPCR assays provide sensitive detection and quantification of P. noxius, which is useful for long-term monitoring of BRRD status.

A comparative study of traditional and molecular diagnostic methods for detection of gastrointestinal parasites in Nepalese migrants to the UK

We evaluated the results of examining a single faecal sample for gastrointestinal parasites (GIP) using a combination of traditional methods with multiplex qPCR for helminths and protozoa, compared to a reference standard of examining three faecal samples from each person using traditional diagnostic methods alone. Three faecal samples were collected at weekly intervals from 596 healthy Nepalese men. Each sample underwent formalin-ethyl acetate (FEA) concentration and light microscopy, and charcoal culture. The combined results of these investigations for all three stool samples were designated the reference standard. The first sample was also analysed using a multiplex TaqMan™ qPCR assay, screening for five helminths and three protozoa. We compared sensitivity and specificity of analysing the first faecal sample with qPCR alone, or a hybrid approach combining qPCR with traditional methods, to the reference standard. Additionally, a serum sample was taken from each participant for Strongyloides stercoralis IgG ELISA. The reference standard identified 139 GIP infections in 133 (22.3%) participants. Use of qPCR alone in one stool identified 176 infections in 147 (24.8%) participants, rising to 187 infections in 156 (26.3%) when combined with FEA microscopy and charcoal culture. The sensitivity of this latter hybrid approach was 100% for Strongyloides spp., 90.9% for Trichuris trichiura, 86.8% for hookworm species and 75% for Giardia duodenalis compared to the reference standard. The hybrid approach increased the detected prevalence of G. duodenalis by 4.5% (27 cases) overall, T. trichiura by 2.9% (17 cases), Strongyloides spp. by 1% (6 cases), and hookworm by 0.5% (3 cases), compared to the reference standard. Examination of a single faecal sample using qPCR alone showed superior or equivalent sensitivity to traditional methods for most GIP infections when both were compared to the reference standard. Combining molecular and traditional methods to analyse a single stool improved the detection rate for most studied parasites. This approach has value in settings where repeated sampling and/or faecal culture for helminths is impractical, but molecular diagnostics are available.