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259900.9965Evaluation of whole-genome sequencing protocols for detection of antimicrobial resistance, virulence factors and mobile genetic elements in antimicrobial-resistant bacteria. Introduction. Antimicrobial resistance (AMR) poses a critical threat to global health, underscoring the need for rapid and accurate diagnostic tools. Methicillin-resistant Staphylococcus aureus (MRSA) and extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae (ESBL-Kp) are listed among the World Health Organization's priority pathogens.Hypothesis. A rapid nanopore-based protocol can accurately and efficiently detect AMR genes, virulence factors (VFs) and mobile genetic elements (MGEs) in MRSA and ESBL-Kp, offering performance comparable to or superior to traditional sequencing methods.Aim. Evaluate whole-genome sequencing (WGS) protocols for detecting AMR genes, VFs and MGEs in MRSA and ESBL-Kp, to identify the most accurate and efficient tool for pathogen profiling.Methodology. Five distinct WGS protocols, including a rapid nanopore-based protocol (ONT20h) and four slower sequencing methods, were evaluated for their effectiveness in detecting genetic markers. The protocols' performances were compared across AMR genes, VFs and MGEs. Additionally, phenotypic antimicrobial susceptibility testing was performed to assess concordance with the genomic findings.Results. Compared to four slower sequencing protocols, the rapid nanopore-based protocol (ONT20h) demonstrated comparable or superior performance in AMR gene detection and equivalent VF identification. Although MGE detection varied among protocols, ONT20h showed a high level of agreement with phenotypic antimicrobial susceptibility testing.Conclusion. The findings highlight the potential of rapid WGS as a valuable tool for clinical microbiology, enabling timely implementation of infection control measures and informed therapeutic decisions. However, further studies are required to optimize the clinical application of this technology, considering costs, availability of bioinformatics tools and quality of reference databases.202540105741
582810.9965Target-enriched sequencing enables accurate identification of bloodstream infections in whole blood. Bloodstream infections are within the top ten causes of death globally, with a mortality rate of up to 70%. Gold standard blood culture testing is time-consuming, resulting in delayed, but accurate, treatment. Molecular methods, such as RT-qPCR, have limited targets in one run. We present a new Ampliseq detection system (ADS) combining target amplification and next-generation sequencing for accurate identification of bacteria, fungi, and antimicrobial resistance determinants directly from blood samples. In this study, we included removal of human genomic DNA during nucleic acid extraction, optimized the target sequence set and drug resistance genes, performed antimicrobial resistance profiling of clinical isolates, and evaluated mock specimens and clinical samples by ADS. ADS successfully identified pathogens at the species-level in 36 h, from nucleic acid extraction to results. Besides pathogen identification, ADS can also present drug resistance profiles. ADS enabled detection of all bacteria and accurate identification of 47 pathogens. In 20 spiked samples and 8 clinical specimens, ADS detected at least 92.81% of reads mapped to pathogens. ADS also showed consistency with the three culture-negative samples, and correctly identified pathogens in four of five culture-positive clinical blood specimens. This Ampliseq-based technology promises broad coverage and accurate pathogen identification, helping clinicians to accurately diagnose and treat bloodstream infections.202234915067
506820.9965Ultrasensitive Label-Free Detection of Unamplified Multidrug-Resistance Bacteria Genes with a Bimodal Waveguide Interferometric Biosensor. Infections by multidrug-resistant bacteria are becoming a major healthcare emergence with millions of reported cases every year and an increasing incidence of deaths. An advanced diagnostic platform able to directly detect and identify antimicrobial resistance in a faster way than conventional techniques could help in the adoption of early and accurate therapeutic interventions, limiting the actual negative impact on patient outcomes. With this objective, we have developed a new biosensor methodology using an ultrasensitive nanophotonic bimodal waveguide interferometer (BiMW), which allows a rapid and direct detection, without amplification, of two prevalent and clinically relevant Gram-negative antimicrobial resistance encoding sequences: the extended-spectrum betalactamase-encoding gene blaCTX-M-15 and the carbapenemase-encoding gene blaNDM-5 We demonstrate the extreme sensitivity and specificity of our biosensor methodology for the detection of both gene sequences. Our results show that the BiMW biosensor can be employed as an ultrasensitive (attomolar level) and specific diagnostic tool for rapidly (less than 30 min) identifying drug resistance. The BiMW nanobiosensor holds great promise as a powerful tool for the control and management of healthcare-associated infections by multidrug-resistant bacteria.202033086716
582630.9964Rapid and accurate sepsis diagnostics via a novel probe-based multiplex real-time PCR system. Sepsis is a critical clinical emergency that requires prompt diagnosis and intervention. Its prevalence has increased due to the aging population and increased antibiotic resistance. Early identification and the use of innovative technologies are crucial for improving patient outcomes. Modern methodologies are needed to minimize the turnaround time for diagnosis and improve outcomes. Rapid diagnostic tests and multiplex PCR are effective but have limitations in identifying a range of pathogens and target genes. Our study evaluated two novel probe-based multiplex real-time PCR systems: the SEPSI ID and SEPSI DR panels. These systems can quickly identify bacterial and fungal pathogens, alongside antibiotic resistance genes. The assays cover 29 microorganisms (gram-negative bacteria, gram-positive bacteria, yeast, and mold species), alongside 23 resistance genes and four virulence factors. A streamlined workflow uses 2 µL of broth from positive blood cultures (BCs) without nucleic acid extraction and provides results in approximately 1 h. We present the results from an evaluation of 228 BCs and 22 isolates previously characterized by whole-genome sequencing. In comparison to the reference methods, the SEPSI ID panel demonstrated a sensitivity of 96.88%, a specificity of 100%, and a PPV of 100%, whereas the SEPSI DR panel showed a sensitivity of 97.8%, a PPV of 89.7%, and a specificity of 96.7%. Both panels also identified additional pathogens and resistance-related targets not detected by conventional methods. This assay shows promise for rapidly and accurately diagnosing sepsis. Future studies should validate its performance in various clinical settings to enhance sepsis management and improve patient outcomes.IMPORTANCEWe present a new diagnostic method that enables the quick and precise identification of pathogens and resistance genes from positive blood cultures, eliminating the need for nucleic acid extraction. This technique can also be used on fresh pathogen cultures. It has the potential to greatly improve treatment protocols, leading to better patient outcomes, more responsible antibiotic use, and more efficient management of healthcare resources.202541025980
582740.9963Duplex dPCR System for Rapid Identification of Gram-Negative Pathogens in the Blood of Patients with Bloodstream Infection: A Culture-Independent Approach. Early and accurate detection of pathogens is important to improve clinical outcomes of bloodstream infections (BSI), especially in the case of drug-resistant pathogens. In this study, we aimed to develop a culture-independent digital PCR (dPCR) system for multiplex detection of major sepsiscausing gram-negative pathogens and antimicrobial resistance genes using plasma DNA from BSI patients. Our duplex dPCR system successfully detected nine targets (five bacteria-specific targets and four antimicrobial resistance genes) through five reactions within 3 hours. The minimum detection limit was 50 ag of bacterial DNA, suggesting that 1 CFU/ml of bacteria in the blood can be detected. To validate the clinical applicability, cell-free DNA samples from febrile patients were tested with our system and confirmed high consistency with conventional blood culture. This system can support early identification of some drug-resistant gram-negative pathogens, which can help improving treatment outcomes of BSI.202134528911
249750.9963Rapid Simultaneous Detection of the Clinically Relevant Carbapenemase Resistance Genes blaKPC, blaOXA48, blaVIM and blaNDM with the Newly Developed Ready-to-Use qPCR CarbaScan LyoBead. Antibiotic resistance, in particular the dissemination of carbapenemase-producing organisms, poses a significant threat to global healthcare. This study introduces the qPCR CarbaScan LyoBead assay, a robust, accurate, and efficient tool for detecting key carbapenemase genes, including blaKPC, blaNDM, blaOXA-48, and blaVIM. The assay utilizes lyophilized beads, a technological advancement that enhances stability, simplifies handling, and eliminates the need for refrigeration. This feature renders it particularly well-suited for point-of-care diagnostics and resource-limited settings. The assay's capacity to detect carbapenemase genes directly from bacterial colonies without the need for extensive sample preparation has been demonstrated to streamline workflows and enable rapid diagnostic results. The assay demonstrated 100% specificity and sensitivity across a diverse range of bacterial strains, including multiple allelic variants of target genes, facilitating precise identification of resistance mechanisms. Bacterial strains of the species Acinetobacter baumannii, Citrobacter freundii, Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae and Pseudomonas aeruginosa were utilized as reference material for assay development (n = 9) and validation (n = 28). It is notable that the assay's long shelf life and minimal operational complexity further enhance its utility for large-scale implementation in healthcare, food safety, and environmental monitoring. The findings emphasize the necessity of continuous surveillance and the implementation of rapid diagnostic methods for the effective detection of resistance genes. Furthermore, the assay's potential applications in other fields, such as toxin-antitoxin system research and monitoring of resistant bacteria in the community, highlight its versatility. In conclusion, the qPCR CarbaScan LyoBead assay is a valuable tool that can contribute to the urgent need to combat antibiotic resistance and improve global public health outcomes.202539940986
582460.9962Evaluation of a micro/nanofluidic chip platform for the high-throughput detection of bacteria and their antibiotic resistance genes in post-neurosurgical meningitis. BACKGROUND: Post-neurosurgical meningitis (PNM) is one of the most severe hospital-acquired infections worldwide, and a large number of pathogens, especially those possessing multi-resistance genes, are related to these infections. Existing methods for detecting bacteria and measuring their response to antibiotics lack sensitivity and stability, and laboratory-based detection methods are inconvenient, requiring at least 24h to complete. Rapid identification of bacteria and the determination of their susceptibility to antibiotics are urgently needed, in order to combat the emergence of multi-resistant bacterial strains. METHODS: This study evaluated a novel, fast, and easy-to-use micro/nanofluidic chip platform (MNCP), which overcomes the difficulties of diagnosing bacterial infections in neurosurgery. This platform can identify 10 genus or species targets and 13 genetic resistance determinants within 1h, and it is very simple to operate. A total of 108 bacterium-containing cerebrospinal fluid (CSF) cultures were tested using the MNCP for the identification of bacteria and determinants of genetic resistance. The results were compared to those obtained with conventional identification and antimicrobial susceptibility testing methods. RESULTS: For the 108 CSF cultures, the concordance rate between the MNCP and the conventional identification method was 94.44%; six species attained 100% consistency. For the production of carbapenemase- and extended-spectrum beta-lactamase (ESBL)-related antibiotic resistance genes, both the sensitivity and specificity of the MNCP tests were high (>90.0%) and could fully meet the requirements of clinical diagnosis. CONCLUSIONS: The MNCP is fast, accurate, and easy to use, and has great clinical potential in the treatment of post-neurosurgical meningitis.201829559366
507370.9962Parallel Detection of the Unamplified Carbapenem Resistance Genes bla(NDM-1) and bla(OXA-1) Using a Plasmonic Nano-Biosensor with a Field-Portable DNA Extraction Method. Antimicrobial resistance (AMR) is a rapidly growing global concern resulting from the overuse of antibiotics in agricultural and clinical settings. The challenge is exacerbated by the lack of rapid surveillance for resistant bacteria in clinical, environmental, and food supply settings. The increasing resistance to carbapenems, an important sub-class of beta-lactam antibiotics, is a major concern in the healthcare community. Carbapenem resistance (CR) has been found in the environment and food supply chain, where it has the potential to spread to pathogens, animals, and humans through direct or indirect contact. Rapid detection for preventative and control measures should be developed. This study utilized a gold nanoparticle-based plasmonic biosensor for the parallel detection of the CR genes bla(NDM-1) and bla(OXA-1). To explore the field portability, DNA was extracted using two methods: a commercial extraction kit and a boiling method. The results were compared between the two methods using a spectrophotometer and a cellphone application for RGB values to quantify the visual results. The results showed that the boiling method of extraction was more effective than extraction with a commercial kit for this analysis. The parallel detection of unamplified genes extracted via the boiling method is novel. When combined with other portable testing equipment, the approach has the potential to be an inexpensive, rapid, and simple on-site CR gene detection protocol.202539997014
503980.9962Analytical validation of a novel high multiplexing real-time PCR array for the identification of key pathogens causative of bacterial ventilator-associated pneumonia and their associated resistance genes. OBJECTIVES: Rapid diagnosis and appropriate empirical antimicrobial therapy before the availability of conventional microbiological results is of pivotal importance for the clinical outcome of ventilator-associated pneumonia (VAP). We evaluated the VAPChip, a novel, closed cartridge molecular tool aiming to identify directly from clinical samples and within a working day the principal bacteria causative of VAP as well as clinically relevant β-lactam resistance genes. METHODS: The Real-time Array PCR for Infectious Diseases (RAP-ID) is a novel technology that combines multiplex PCR with real-time microarray detection. The VAPChip is a closed cartridge kit adapted to the RAP-ID instrument that targets 13 key respiratory pathogens causative of VAP and 24 relevant antimicrobial resistance genes that mediate resistance to β-lactam agents, including extended-spectrum cephalosporins and carbapenems. Analytical validation of the VAPChip was carried out blindly on a collection of 292 genotypically characterized bacterial reference and clinical isolates, including 225 isolates selected on the basis of their species identification and antimicrobial resistance profiles and 67 bacterial isolates belonging to the oropharyngeal flora not targeted by the array. RESULTS: The limit of detection of the assay lies between 10 and 100 genome copies/PCR and the dynamic range is five orders of magnitude permitting at least semi-quantitative reporting of the results. Sensitivity, specificity and negative and positive predictive values ranged from 95.8% to 100% for species identification and detection of resistance genes. CONCLUSIONS: VAPChip is a novel diagnostic tool able to identify resistant bacterial isolates by RAP-ID technology. The results of this analytical validation have to be confirmed on clinical specimens.201323065698
506990.9962MC-PRPA-HLFIA Cascade Detection System for Point-of-Care Testing Pan-Drug-Resistant Genes in Urinary Tract Infection Samples. Recently, urinary tract infection (UTI) triggered by bacteria carrying pan-drug-resistant genes, including carbapenem resistance gene bla(NDM) and bla(KPC), colistin resistance gene mcr-1, and tet(X) for tigecycline resistance, have been reported, posing a serious challenge to the treatment of clinical UTI. Therefore, point-of-care (POC) detection of these genes in UTI samples without the need for pre-culturing is urgently needed. Based on PEG 200-enhanced recombinase polymerase amplification (RPA) and a refined Chelex-100 lysis method with HRP-catalyzed lateral flow immunoassay (LFIA), we developed an MCL-PRPA-HLFIA cascade assay system for detecting these genes in UTI samples. The refined Chelex-100 lysis method extracts target DNA from UTI samples in 20 min without high-speed centrifugation or pre-incubation of urine samples. Following optimization, the cascade detection system achieved an LOD of 10(2) CFU/mL with satisfactory specificity and could detect these genes in both simulated and actual UTI samples. It takes less than an hour to complete the process without the use of high-speed centrifuges or other specialized equipment, such as PCR amplifiers. The MCL-PRPA-HLFIA cascade assay system provides new ideas for the construction of rapid detection methods for pan-drug-resistant genes in clinical UTI samples and provides the necessary medication guidance for UTI treatment.202337047757
5038100.9961Simple and quick detection of extended-spectrum β-lactamase and carbapenemase-encoding genes using isothermal nucleic acid amplification techniques. The spread of plasmid-mediated antibiotic-resistant bacteria must be controlled; to this end, developing kits for simple and rapid detection in food and clinical settings is desirable. This review describes the detection of antibiotic resistance genes in extended-spectrum β-lactamase （ESBL）- and carbapenemase-producing bacteria. Loop-mediated isothermal amplification （LAMP）, a technique developed in Japan, is a useful diffusion amplification method that does not require equipment like thermal cyclers, and amplifies the target gene in 30 min at about 65℃. Although most reports targeting ESBL and carbapenemase genes are intended for clinical use, environmental and food samples have also been targeted. Recombinase polymerase amplification （RPA） has recently been developed; in RPA, the reaction proceeds under the human skin with reaction conditions of 30 min at 37℃. Detection of ESBL and carbapenemase-encoding genes in food and clinical samples using RPA has been reported in limited studies. However, research on RPA has just begun, and further development is expected.202338233166
4937110.9960Fast and Accurate Large-Scale Detection of β-Lactamase Genes Conferring Antibiotic Resistance. Fast detection of β-lactamase (bla) genes allows improved surveillance studies and infection control measures, which can minimize the spread of antibiotic resistance. Although several molecular diagnostic methods have been developed to detect limited bla gene types, these methods have significant limitations, such as their failure to detect almost all clinically available bla genes. We developed a fast and accurate molecular method to overcome these limitations using 62 primer pairs, which were designed through elaborate optimization processes. To verify the ability of this large-scale bla detection method (large-scaleblaFinder), assays were performed on previously reported bacterial control isolates/strains. To confirm the applicability of the large-scaleblaFinder, the assays were performed on unreported clinical isolates. With perfect specificity and sensitivity in 189 control isolates/strains and 403 clinical isolates, the large-scaleblaFinder detected almost all clinically available bla genes. Notably, the large-scaleblaFinder detected 24 additional unreported bla genes in the isolates/strains that were previously studied, suggesting that previous methods detecting only limited types of bla genes can miss unexpected bla genes existing in pathogenic bacteria, and our method has the ability to detect almost all bla genes existing in a clinical isolate. The ability of large-scaleblaFinder to detect bla genes on a large scale enables prompt application to the detection of almost all bla genes present in bacterial pathogens. The widespread use of the large-scaleblaFinder in the future will provide an important aid for monitoring the emergence and dissemination of bla genes and minimizing the spread of resistant bacteria.201526169415
2249120.9960Tracking Multidrug Resistance in Gram-Negative Bacteria in Alexandria, Egypt (2020-2023): An Integrated Analysis of Patient Data and Diagnostic Tools. BACKGROUND: The rise in carbapenem-resistant Enterobacteriaceae (CRE) in Egypt, particularly in hospital settings, poses a significant public health challenge. This study aims to develop a combined epidemiological surveillance tool utilizing the Microreact online platform (version 269) and molecular microarray technology to track and analyze carbapenem-resistant Escherichia coli strains in Egypt. The objective is to integrate molecular diagnostics and real-time data visualization to better understand the spread and evolution of multidrug-resistant (MDR) bacteria. METHODS: The study analyzed 43 E. coli isolates collected from Egyptian hospitals between 2020 and 2023. Nanopore sequencing and microarray analysis were used to identify carbapenemase genes and other resistance markers, whereas the VITEK2 system was employed for phenotypic antibiotic susceptibility testing. Microreact was used to visualize epidemiological data, mapping the geographic and temporal distribution of resistant strains. RESULTS: We found that 72.09% of the isolates, predominantly from pediatric patients, carried the blaNDM-5 gene, while other carbapenemase genes, including blaOXA-48 and blaVIM, were also detected. The microarray method demonstrated 92.9% diagnostic sensitivity and 87.7% diagnostic specificity compared to whole-genome sequencing. Phenotypic resistance correlated strongly with next-generation sequencing (NGS) genotypic data, achieving 95.6% sensitivity and 95.2% specificity. CONCLUSIONS: This method establishes the utility of combining microarray technology, NGS and real-time data visualization for the surveillance of carbapenem-resistant Enterobacteriaceae, especially E. coli. The high concordance between genotypic and phenotypic data underscores the potential of DNA microarrays as a cost-effective alternative to whole-genome sequencing, especially in resource-limited settings. This integrated approach can enhance public health responses to MDR bacteria in Egypt.202439766575
1823130.9960Finding the Missing IMP Gene: Overcoming the Imipenemase IMP Gene Drop-Out in Automated Molecular Testing for Carbapenem-Resistant Bacteria Circulating in Latin America. Carbapenem resistance is considered one of the greatest current threats to public health, particularly in the management of infections in clinical settings. Carbapenem resistance in bacteria is mainly due to mechanisms such as the production of carbapenemases (such as the imipenemase IMP, or other enzymes like VIM, NDM, and KPC), that can be detected by several laboratory tests, including immunochromatography and automated real-time PCR (qPCR). Methods: As part of local studies to monitor carbapenem-resistant bacteria in Costa Rica, two cases were initially identified with inconsistent IMP detection results. A possible gene drop-out in the automated qPCR test was suggested based on the negative result, contrasting with the positive result by immunochromatography and whole-genome sequencing. We hypothesized that molecular testing could be optimized through the development of tailored assays to improve the detection of IMP genes. Thus, using IMP gene sequences from the local isolates and regional sequences in databases, primers were redesigned to extend the detection of IMP alleles of regional relevance. Results: The tailored qPCR was applied to a local collection of 119 carbapenem-resistant isolates. The genomes of all 14 positive cases were sequenced, verifying the results of the custom qPCR, despite the negative results of the automated testing. Conclusions: Guided by whole-genome sequencing, it was possible to extend the molecular detection of IMP alleles circulating in Latin America using a tailored qPCR to overcome IMP gene drop-out and false-negative results in an automated qPCR.202540867967
5825140.9960Polymerase Chain Reaction (PCR) Profiling of Extensively Drug-Resistant (XDR) Pathogenic Bacteria in Pulmonary Tuberculosis Patients. Introduction Pulmonary tuberculosis (TB) remains a global health concern, exacerbated by the emergence of extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis. This study employs advanced molecular techniques, specifically polymerase chain reaction (PCR) profiling, to comprehensively characterize the genetic landscape of XDR pathogenic bacteria in patients diagnosed with pulmonary TB. The objective of the study is to elucidate the genes that are associated with drug resistance in pulmonary TB strains through the application of PCR and analyze specific genetic loci that contribute to the development of resistance against multiple drugs. Materials and methods A total of 116 clinical samples suspected of TB were collected from the tertiary healthcare setting of Saveetha Medical College and Hospitals for the identification of MTB, which includes sputum (n = 35), nasal swabs (n = 17), blood (n = 44), and bronchoalveolar lavage (BAL) (n = 20). The collected specimens were processed and subjected to DNA extraction. As per the protocol, reconstitution of the DNA pellet was carried out. The reconstituted DNA was stored at -20 °C for the PCR assay. From the obtained positive sample specimens, XDR pulmonary TB specimens were focused on the targeted genes, specifically the rpoB gene for rifampicin resistance, inhA, and katG gene for thepromoter region for isoniazid resistance. Results Out of a total of 116 samples obtained, 53 tested positive for pulmonary TB, indicative of a mycobacterial infection. Among these positive cases, 43 patients underwent treatment at a tertiary healthcare facility. Subsequently, a PCR assay was performed with the extracted DNA for the target genes rpoB, inhA, and katG. Specifically, 22 sputum samples exhibited gene expression for rpoB, inhA, and katG, while nine nasal swabs showed expression of the rpoB and inhA genes. Additionally, rpoB gene expression was detected in seven blood specimens, and both rpoB and inhA genes were expressed in five BAL samples. Conclusion The swift diagnosis and efficient treatment of XDR-TB can be facilitated by employing advanced and rapid molecular tests and oral medication regimens. Utilizing both newly developed and repurposed anti-TB drugs like pretomanid, bedaquiline, linezolid, and ethionamide. Adhering to these current recommendations holds promise for managing XDR-TB effectively. Nevertheless, it is significant to conduct well-designed clinical trials and studies to further evaluate the efficacy of new agents and shorter treatment regimens, thus ensuring continuous improvement in the management of this challenging condition.202438953074
5819150.9959Application of mNGS in the Etiological Analysis of Lower Respiratory Tract Infections and the Prediction of Drug Resistance. Lower respiratory tract infections (LRTIs) have high morbidity and mortality rates. However, traditional etiological detection methods have not been able to meet the needs for the clinical diagnosis and prognosis of LRTIs. The rapid development of metagenomic next-generation sequencing (mNGS) provides new insights for the diagnosis and treatment of LRTIs; however, little is known about how to interpret the application of mNGS results in LRTIs. In this study, lower respiratory tract specimens from 46 patients with suspected LRTIs were tested simultaneously using conventional microbiological detection methods and mNGS. Receiver operating characteristic (ROC) curves were used to evaluate the performance of the logarithm of reads per kilobase per million mapped reads [lg(RPKM)], genomic coverage, and relative abundance of the organism in predicting the true-positive pathogenic bacteria. True-positive viruses were identified according to the lg(RPKM) threshold of bacteria. We also evaluated the ability to predict drug resistance genes using mNGS. Compared to that using conventional detection methods, the false-positive detection rate of pathogenic bacteria was significantly higher using mNGS. It was concluded from the ROC curves that the lg(RPKM) and genomic coverage contributed to the identification of pathogenic bacteria, with the performance of lg(RPKM) being the best (area under the curve [AUC] = 0.99). The corresponding lg(RPKM) threshold for identifying the pathogenic bacteria was -1.35. Thirty-five strains of true-positive virus were identified based on the lg(RPKM) threshold of bacteria, with the detection of human gammaherpesvirus 4 being the highest and prone to coinfection with Pseudomonas aeruginosa, Acinetobacter baumannii, and Stenotrophomonas maltophilia. Antimicrobial susceptibility tests (AST) revealed the resistance of bacteria containing drug resistance genes (detected by mNGS). However, the drug resistance genes of some multidrug-resistant bacteria were not detected. As an emerging technology, mNGS has shown many advantages for the unbiased etiological detection and the prediction of antibiotic resistance. However, a correct understanding of mNGS results is a prerequisite for its clinical application, especially for LRTIs. IMPORTANCE LRTIs are caused by hundreds of pathogens, and they have become a great threat to human health due to the limitations of traditional etiological detection methods. As an unbiased approach to detect pathogens, mNGS overcomes such etiological diagnostic challenges. However, there is no unified standard on how to use mNGS indicators (the sequencing reads, genomic coverage, and relative abundance of each organism) to distinguish between pathogens and colonizing microorganisms or contaminant microorganisms. Here, we selected the mNGS indicator with the best identification performance and established a cutoff value for the identification of pathogens in LRTIs using ROC curves. In addition, we also evaluated the accuracy of antibiotic resistance prediction using mNGS.202235171007
2236160.9959Development of a Multiplex PCR Platform for the Rapid Detection of Bacteria, Antibiotic Resistance, and Candida in Human Blood Samples. The diagnosis of bloodstream infections (BSIs) still relies on blood culture (BC), but low turnaround times may hinder the early initiation of an appropriate antimicrobial therapy, thus increasing the risk of infection-related death. We describe a direct and rapid multiplex PCR-based assay capable of detecting and identifying 16 bacterial and four Candida species, as well as three antibiotic-resistance determinants, in uncultured samples. Using whole-blood samples spiked with microorganisms at low densities, we found that the MicrobScan assay had a mean limit of detection of 15.1 ± 3.3 CFU of bacteria/Candida per ml of blood. When applied to positive BC samples, the assay allowed the sensitive and specific detection of BSI pathogens, including bla(KPC)-, mecA-, or vanA/vanB-positive bacteria. We evaluated the assay using prospectively collected blood samples from patients with suspected BSI. The sensitivity and specificity were 86.4 and 97.0%, respectively, among patients with positive BCs for the microorganisms targeted by the assay or patients fulfilling the criteria for infection. The mean times to positive or negative assay results were 5.3 ± 0.2 and 5.1 ± 0.1 h, respectively. Fifteen of 20 patients with MicrobScan assay-positive/BC-negative samples were receiving antimicrobial therapy. In conclusion, the MicrobScan assay is well suited to complement current diagnostic methods for BSIs.201931799215
5125170.9959Do we still need Illumina sequencing data? Evaluating Oxford Nanopore Technologies R10.4.1 flow cells and the Rapid v14 library prep kit for Gram negative bacteria whole genome assemblies. The best whole genome assemblies are currently built from a combination of highly accurate short-read sequencing data and long-read sequencing data that can bridge repetitive and problematic regions. Oxford Nanopore Technologies (ONT) produce long-read sequencing platforms and they are continually improving their technology to obtain higher quality read data that is approaching the quality obtained from short-read platforms such as Illumina. As these innovations continue, we evaluated how much ONT read coverage produced by the Rapid Barcoding Kit v14 (SQK-RBK114) is necessary to generate high-quality hybrid and long-read-only genome assemblies for a panel of carbapenemase-producing Enterobacterales bacterial isolates. We found that 30× long-read coverage is sufficient if Illumina data are available, and that more (at least 100× long-read coverage is recommended for long-read-only assemblies. Illumina polishing is still improving single nucleotide variants (SNVs) and INDELs in long-read-only assemblies. We also examined if antimicrobial resistance genes could be accurately identified in long-read-only data, and found that Flye assemblies regardless of ONT coverage detected >96% of resistance genes at 100% identity and length. Overall, the Rapid Barcoding Kit v14 and long-read-only assemblies can be an optimal sequencing strategy (i.e., plasmid characterization and AMR detection) but finer-scale analyses (i.e., SNV) still benefit from short-read data.202438354391
5042180.9959Multiplex loop-mediated isothermal amplification (multi-LAMP) assay for rapid detection of mcr-1 to mcr-5 in colistin-resistant bacteria. Purpose: The discovery of the plasmid-mediated colistin resistance genes, mcr, revealed a mechanism of transmission of colistin resistance, which is a major, global public health concern especially among individuals infected with carbapenem-resistant Gram-negative bacteria. To monitor the spread and epidemiology of mcr genes, a convenient and reliable method to detect mcr genes in clinical isolates is needed, especially in the primary care institutions. This study aimed to establish a restriction endonuclease-based multiplex loop-mediated isothermal amplification (multi-LAMP) assay to detect mcr genes (mcr-1 to mcr-5) harbored by colistin-resistant bacteria. Methods: A triple-LAMP assay for mcr-1, mcr-3, and mcr-4 and a double-LAMP assay for mcr-2 and mcr-5 were established. The sensitivity and specificity of the LAMP reactions were determined via electrophoresis and visual detection. Results: The sensitivity of the LAMP assay was 10-fold greater than that of PCR, with high specificity among the screened primers. Specific mcr genes were distinguished in accordance with band numbers and the fragment length of the digested LAMP amplification products. Furthermore, the LAMP assay was confirmed as a rapid and reliable diagnostic technique upon application for clinical samples, and the results were consistent with those of conventional PCR assay. Conclusion: The multi-LAMP assay is a potentially promising method to detect mcr genes and will, if implemented, help prevent infections by drug-resistant bacteria in primary-care hospitals due to rapid and reliable surveillance. To our knowledge, this is the first study to report the application of LAMP to detect mcr-2 to mcr-5 genes and the first time that multi-LAMP has been applied to detect mcr genes.201931308708
4939190.9959Identification of bacterial pathogens and antimicrobial resistance directly from clinical urines by nanopore-based metagenomic sequencing. OBJECTIVES: The introduction of metagenomic sequencing to diagnostic microbiology has been hampered by slowness, cost and complexity. We explored whether MinION nanopore sequencing could accelerate diagnosis and resistance profiling, using complicated urinary tract infections as an exemplar. METHODS: Bacterial DNA was enriched from clinical urines (n = 10) and from healthy urines 'spiked' with multiresistant Escherichia coli (n = 5), then sequenced by MinION. Sequences were analysed using external databases and bioinformatic pipelines or, ultimately, using integrated real-time analysis applications. Results were compared with Illumina data and resistance phenotypes. RESULTS: MinION correctly identified pathogens without culture and, among 55 acquired resistance genes detected in the cultivated bacteria by Illumina sequencing, 51 were found by MinION sequencing directly from the urines; with three of the four failures in an early run with low genome coverage. Resistance-conferring mutations and allelic variants were not reliably identified. CONCLUSIONS: MinION sequencing comprehensively identified pathogens and acquired resistance genes from urine in a timeframe similar to PCR (4 h from sample to result). Bioinformatic pipeline optimization is needed to better detect resistances conferred by point mutations. Metagenomic-sequencing-based diagnosis will enable clinicians to adjust antimicrobial therapy before the second dose of a typical (i.e. every 8 h) antibiotic.201727667325