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10700.9242Common ancestry of iron oxide- and iron-sulfide-based biomineralization in magnetotactic bacteria. Magnetosomes are prokaryotic organelles produced by magnetotactic bacteria that consist of nanometer-sized magnetite (Fe(3)O(4)) or/and greigite (Fe(3)S(4)) magnetic crystals enveloped by a lipid bilayer membrane. In magnetite-producing magnetotactic bacteria, proteins present in the magnetosome membrane modulate biomineralization of the magnetite crystal. In these microorganisms, genes that encode for magnetosome membrane proteins as well as genes involved in the construction of the magnetite magnetosome chain, the mam and mms genes, are organized within a genomic island. However, partially because there are presently no greigite-producing magnetotactic bacteria in pure culture, little is known regarding the greigite biomineralization process in these organisms including whether similar genes are involved in the process. Here using culture-independent techniques, we now show that mam genes involved in the production of magnetite magnetosomes are also present in greigite-producing magnetotactic bacteria. This finding suggest that the biomineralization of magnetite and greigite did not have evolve independently (that is, magnetotaxis is polyphyletic) as once suggested. Instead, results presented here are consistent with a model in which the ability to biomineralize magnetosomes and the possession of the mam genes was acquired by bacteria from a common ancestor, that is, the magnetotactic trait is monophyletic.201121509043
147410.9154Simple, rapid, and cost-effective modified Carba NP test for carbapenemase detection among Gram-negative bacteria. PURPOSE: Detection of carbapenemases among Gram-negative bacteria (GNB) is important for both clinicians and infection control practitioners. The Clinical and Laboratory Standards Institute recommends Carba NP (CNP) as confirmatory test for carbapenemase production. The reagents required for CNP test are costly and hence the test cannot be performed on a routine basis. The present study evaluates modifications of CNP test for rapid detection of carbapenemases among GNB. MATERIALS AND METHODS: The GNB were screened for carbapenemase production using CNP, CarbAcineto NP (CANP), and modified CNP (mCNP) test. A multiplex polymerase chain reaction (PCR) was performed on all the carbapenem-resistant bacteria for carbapenemase genes. The results of three phenotypic tests were compared with PCR. RESULTS: A total of 765 gram negative bacteria were screened for carbapenem resistance. Carbapenem resistance was found in 144 GNB. The metallo-β-lactamases were most common carbapenemases followed by OXA-48-like enzymes. The CANP test was most sensitive (80.6%) for carbapenemases detection. The mCNP test was 62.1% sensitive for detection of carbapenemases. The mCNP, CNP, and CANP tests were equally sensitive (95%) for detection of NDM enzymes among Enterobacteriaceae. The mCNP test had poor sensitivity for detection of OXA-48-like enzymes. CONCLUSION: The mCNP test was rapid, cost-effective, and easily adoptable on routine basis. The early detection of carbapenemases using mCNP test will help in preventing the spread of multidrug-resistant organisms in the hospital settings.201728966495
140220.9150Detection of β-lactam resistance genes in Gram-negative bacteria from positive blood cultures using a microchip-based molecular assay. BACKGROUND: Accurate detection of β-lactam resistance genes in bloodstream infections is critical for guiding antimicrobial therapy. This study evaluates the Alifax Gram-negative resistance (GNR) microchip assay for detecting β-lactam resistance genes directly from positive blood cultures (PBCs) for Gram-negative (GN) bacteria, including Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii. METHODS: Simulated (n=146) and clinical (n=106) GN-PBC samples were tested for bla (KPC), bla (VIM), bla (NDM), bla (IMP), bla (OXA-23)-like, bla (OXA-48)-like, bla (SHV)-ESBL, bla (CTX-M-1/9) group, and bla (CMY-2)-like genes using the GNR microchip assay. Whole-genome sequencing (WGS) served as the reference assay for simulated samples and, selectively, for clinical samples. The bioMérieux BioFire Blood Culture Identification 2 (BCID2) panel assay was used as a comparator for clinical samples. RESULTS: The GNR microchip assay correctly identified 203 (99.5%) of 204 β-lactam resistance genes in simulated samples. One sample tested false negative for a bla (SHV)-ESBL gene but true positive for a bla (KPC) gene. In clinical samples, GNR results were concordant with BCID2 for 113 (100%) of 113 genes included in both assays. Additionally, the GNR assay detected bla (CMY-2) -like (n=6), bla (OXA-23)-like (n=5), and bla (SHV)-ESBL (n=2), which are not targeted by BCID2, all confirmed by WGS. In two β-lactam-resistant P. aeruginosa samples but negative by the GNR assay, WGS confirmed the absence of acquired β-lactam resistance genes, suggesting alternative resistance mechanisms. CONCLUSION: The GNR microchip assay demonstrated high concordance and broader β-lactam resistance gene coverage compared to BCID2, supporting its potential role in routine diagnostics. Further validation in larger, prospective studies is warranted.202540529307
222630.9141Evaluation of the Microbiological Performance and Potential Clinical Impact of New Rapid Molecular Assays for the Diagnosis of Bloodstream Infections. Bloodstream infection (BSI) is a critical medical emergency associated with a high mortality rate. Rapid and accurate identification of the causative pathogen and the results of antimicrobial susceptibility testing are crucial for initiating appropriate antimicrobial therapy. The aim of this study was to evaluate the performance of a new rapid PCR Molecular Mouse System (MMS) for the identification of Gram-negative bacteria (GNB) and GNB resistance genes directly from a positive blood culture (BC). The validation of these rapid multiplex assays was carried out in a real hospital setting. A total of 80 BSI episodes were included in our study and the results were compared with culture-based methods. BC samples in which GNB had previously been detected microscopically and which originated from different hospital wards were analysed. The MMS GNB identification assay achieved a sensitivity of 98.7% and a specificity of 100% for the covered pathogens. In one BC sample, Klebsiella aerogenes was identified at the family level (Enterobacteriaceae) with MMS. However, in three polymicrobial samples, MMS identified bacteria that were not detected by culture-based methods (Klebsiella pneumoniae, K. aerogenes and Stenotrophomonas maltophilia). MMS also showed excellent overall performance in the detection of GNB resistance markers (100% sensitivity and 100% specificity). The type of extended-spectrum beta-lactamase (ESBL) resistance gene identified correctly with MMS was CTX-M-1/9 (n = 17/20), alone or in combination with SHV-type β-lactamase or with the different types of carbapenemase genes. MMS detected one carbapenemase gene of each type (KPC, NDM and OXA-23) and six OXA-48 genes. In addition, the colistin resistance gene mcr-1 was detected in one positive BC with Escherichia coli (E. coli). The time to result was significantly shorter for MMS than for routine culture methods. A retrospective analysis of the patients' medical records revealed that a change in empirical antimicrobial therapy would have been made in around half of the patients following the MMS results. These results support the use of MMS as a valuable complement to conventional culture methods for more rapid BSI diagnosis and adjustment of empirical therapy.202540142509
140440.9138Evaluation of a DNA microarray for rapid detection of the most prevalent extended-spectrum β-lactamases, plasmid-mediated cephalosporinases and carbapenemases in Enterobacteriaceae, Pseudomonas and Acinetobacter. The dissemination of Gram-negative bacteria (GNB) producing extended-spectrum β-lactamases (ESBLs), plasmid-encoded cephalosporinases (pAmpCs) and carbapenemases is a matter of great clinical concern. In this study, we evaluated a new low-density DNA array 'Check-MDR CT103 XL' (Check-Points, Wageningen, The Netherlands) that identifies the most clinically relevant β-lactamase genes of ESBLs (blaTEM, blaSHV, blaCTX-M, blaBEL, blaPER, blaGES and blaVEB), pAmpCs (blaCMY-2-like, blaDHA, blaFOX, blaACC-1, blaACT/MIR and blaCMY-1-like/MOX) and carbapenemases (blaKPC, blaOXA-48, blaVIM, blaIMP, blaNDM, blaGIM, blaSPM and blaOXA-23, -24 and -58) in cultured bacteria. In total, 223 GNB isolates with well-characterised resistance mechanisms to β-lactams were analysed. A specificity and sensitivity of 100% were recorded for most bla genes, with a slightly lower signal observed for blaIMP. The Check-MDR CT103 XL array proved highly accurate for the identification of epidemiologically relevant ESBL, pAmpC and carbapenemase genes harboured in Enterobacteriaceae, Pseudomonas and Acinetobacter spp. The Check-MDR CT103 XL assay is a significant improvement compared with Check-MDR CT103 and it highlights the ability of this array to evolve rapidly to adjust to the current needs for the detection of resistance mechanisms to β-lactam agents.201627374747
503750.9138Development of an immunochromatographic assay for diagnosing the production of IMP-type metallo-β-lactamases that mediate carbapenem resistance in Pseudomonas. Rapid and reliable detection of carbapenem-resistant bacteria is an important infection-control measure and a crucial aspect of antimicrobial chemotherapy. IMP-type metallo-β-lactamase (MBL) is an emzyme that mediate carbapenem resistance in bacteria. Here, an immunochromatographic assay was newly developed using novel monoclonal antibodies (mAbs) recognizing IMP-type MBL. Epitope mapping of mAbs and mutational analysis of the epitope region in IMP antigen suggested that the mAbs could react to all known subtypes of IMP-type MBL. Evaluation of the assay using Pseudomonas aeruginosa strains (n=248) showed that the results of the immunochromatographic detection of the IMP-type MBLs were fully consistent with those of the PCR analysis for bla(IMP) genes, showing false positives and negatives. All positive strains were resistant to carbapenem (MIC ≥ 16 μg/ml). The assay also accurately distinguished the production of IMP-type MBLs in Pseudomonas putida, Acinetobacter baumannii, and Alcaligenes xylosoxidans. The detection limit of the assay was 5.7×10(4)cfu per test. Taken together, these data suggest that the developed assay can be used for rapid and reliable diagnosis of the production of IMP-type MBLs in Gram-negative bacteria.201121986031
147860.9138Multicenter Evaluation of the FilmArray Blood Culture Identification 2 Panel for Pathogen Detection in Bloodstream Infections. The FilmArray Blood Culture Identification 2 panel (BCID2; bioMérieux) is a fully automated PCR-based assay for identifying bacteria, fungi, and bacterial resistance markers in positive blood cultures (BC) in about 1 h. In this multicenter study, we evaluated the performance of the BCID2 panel for pathogen detection in positive BC. Conventional culture and BCID2 were performed in parallel at four tertiary-care hospitals. We included 152 positive BC-130 monomicrobial and 22 polymicrobial cultures-in this analysis. The BCID2 assay correctly identified 90% (88/98) of Gram-negative and 89% (70/79) of Gram-positive bacteria. Five bacterial isolates targeted by the BCID2 panel and recovered from five positive BC, including three polymicrobial cultures, were missed by the BCID2 assay. Fifteen isolates were off-panel organisms, accounting for 8% (15/182) of the isolates obtained from BC. The mean positive percent agreement between the BCID2 assay and standard culture was 97% (95% confidence interval, 95 to 99%), with agreement ranging from 67% for Candida albicans to 100% for 17 targets included in the BCID2 panel. BCID2 also identified the bla(CTX-M) gene in seven BC, including one for which no extended-spectrum β-lactamase (ESBL)-producing isolate was obtained in culture. However, it failed to detect ESBL-encoding genes in three BC. Two of the 18 mecA/C genes detected by the BCID2 were not confirmed. No carbapenemase, mecA/C, or MREJ targets were detected. The median turnaround time was significantly shorter for BCID2 than for culture. The BCID2 panel may facilitate faster pathogen identification in bloodstream infections. IMPORTANCE Rapid molecular diagnosis combining the identification of pathogens and the detection of antibiotic resistance genes from positive blood cultures (BC) can improve the outcome for patients with bloodstream infections. The FilmArray BCID2 panel, an updated version of the original BCID, can detect 11 Gram-positive bacteria, 15 Gram-negative bacteria, 7 fungal pathogens, and 10 antimicrobial resistance genes directly from a positive BC. Here, we evaluated the real-life microbiological performance of the BCID2 assay in comparison to the results of standard methods used in routine practice at four tertiary care hospitals.202336519852
140570.9131The threat of carbapenem resistance in Eastern Europe in patients with decompensated cirrhosis admitted to intensive care unit. BACKGROUND: Multidrug-resistant organisms are an increasing concern in patients with decompensated cirrhosis. AIM: We aimed to evaluate the prevalence of infections with carbapenem-resistant Enterobacteriaceae in patients with decompensated cirrhosis. METHODS: Patients with decompensated cirrhosis admitted to ICU were included. The isolated Enterobacteriaceae strains were tested for carbapenemase-producing genes using the Roche LightMix® Modular VIM/IMP/NDM/GES/KPC/OXA48-carbapenemase detection kit. RESULTS: 48 culture-positive infections were registered in 75 patients with acutely decompensated cirrhosis. Thirty patients contracted a second infection. 46% of bacteria isolated at admission and 60% of bacteria responsible for infections identified during ICU-stay were multiresistant. ESBL+ Enterobacteriaceae were predominant at admission, while carbapenem-resistance was dominant in both Enterobacteriaceae and Non-Fermenting-Gram-Negative Bacteria responsible for infections diagnosed during hospitalisation. OXA 48 or KPC type carbapenemases were present in 30% of the analyzed Enterobacteriaceae and in 40% of the phenotypically carbapenem-resistant Klebsiella pneumoniae strains. The length of ICU stay was a risk-factor for a second infection (p=0.04). Previous carbapenem usage was associated with occurence of infections with carbapenem-resistant Gram-negative bacteria during hospitalization (p=0.03). CONCLUSION: The prevalence of infections with carbapenem-resistant Enterobacteriaceae is high in patients with decompensated cirrhosis admitted to ICU. Carbapenemase-producing genes in Enterobacteriaceae in our center are bla(OXA-48) and bla(KPC).202235732546
148580.9131Evaluation of Verigene Blood Culture Test Systems for Rapid Identification of Positive Blood Cultures. The performance of molecular tests using the Verigene Gram-Positive and Gram-Negative Blood Culture nucleic acid tests (BC-GP and BC-GN, resp.; Naosphere, Northbrook, IL, USA) was evaluated for the identification of microorganisms detected from blood cultures. Ninety-nine blood cultures containing Gram-positive bacteria and 150 containing Gram-negative bacteria were analyzed using the BC-GP and BC-GN assays, respectively. Blood cultures were performed using the Bactec blood culture system (BD Diagnostic Systems, Franklin Lakes, NJ, USA) and conventional identification and antibiotic-susceptibility tests were performed using a MicroScan system (Siemens, West Sacramento, CA, USA). When a single strain of bacteria was isolated from the blood culture, Verigene assays correctly identified 97.9% (94/96) of Gram-positive bacteria and 93.8% (137/146) of Gram-negative bacteria. Resistance genes mecA and vanA were correctly detected by the BC-GP assay, while the extended-spectrum β-lactamase CTX-M and the carbapenemase OXA resistance gene were detected from 30 cases cultures by the BC-GN assay. The BC-GP and BC-GN assays showed high agreement with conventional identification and susceptibility tests. These tests are useful for rapid identification of microorganisms and the detection of clinically important resistance genes from positive Bactec blood cultures.201626904669
147790.9130Multicenter Evaluation of the BIOFIRE Blood Culture Identification 2 Panel for Detection of Bacteria, Yeasts, and Antimicrobial Resistance Genes in Positive Blood Culture Samples. Diagnostic tools that can rapidly identify and characterize microbes growing in blood cultures are important components of clinical microbiology practice because they help to provide timely information that can be used to optimize patient management. This publication describes the bioMérieux BIOFIRE Blood Culture Identification 2 (BCID2) Panel clinical study that was submitted to the U.S. Food & Drug Administration. Results obtained with the BIOFIRE BCID2 Panel were compared to standard-of-care (SoC) results, sequencing results, PCR results, and reference laboratory antimicrobial susceptibility testing results to evaluate the accuracy of its performance. Results for 1,093 retrospectively and prospectively collected positive blood culture samples were initially enrolled, and 1,074 samples met the study criteria and were included in the final analyses. The BIOFIRE BCID2 Panel demonstrated an overall sensitivity of 98.9% (1,712/1,731) and an overall specificity of 99.6% (33,592/33,711) for Gram-positive bacteria, Gram-negative bacteria and yeast targets which the panel is designed to detect. One hundred eighteen off-panel organisms, which the BIOFIRE BCID2 Panel is not designed to detect, were identified by SoC in 10.6% (114/1,074) of samples. The BIOFIRE BCID2 Panel also demonstrated an overall positive percent agreement (PPA) of 97.9% (325/332) and an overall negative percent agreement (NPA) of 99.9% (2,465/2,767) for antimicrobial resistance determinants which the panel is designed to detect. The presence or absence of resistance markers in Enterobacterales correlated closely with phenotypic susceptibility and resistance. We conclude that the BIOFIRE BCID2 Panel produced accurate results in this clinical trial.202337227281
1403100.9127Evaluation of the AusDiagnostics MT CRE EU assay for the detection of carbapenemase genes and transferable colistin resistance determinants mcr-1/-2 in MDR Gram-negative bacteria. OBJECTIVES: To evaluate the AusDiagnostics MT CRE EU assay for the detection of carbapenemase and acquired colistin resistance genes in Gram-negative bacteria. METHODS: The assay allows the detection of blaKPC, blaOXA-48-like, blaNDM, blaVIM, blaIMP, blaSIM, blaGIM, blaSPM, blaFRI, blaIMI, blaGES (differentiating ESBL and carbapenemase variants), blaSME and mcr-1/-2. It was evaluated against a panel of isolates including Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp. retrospectively (n = 210) and prospectively (n = 182). RESULTS: The CRE EU assay was able to detect 268/268 carbapenemase genes, with 239 belonging to the 'big five' families (KPC, OXA-48-like, NDM, VIM and IMP) and 29 carbapenemase genes of the SIM, GIM, SPM, FRI, IMI, SME and GES families. It could distinguish between ESBL and carbapenemase variants of GES. It also allowed detection of mcr-1/-2 colistin resistance genes on their own or in isolates co-producing a carbapenemase. CONCLUSIONS: The AusDiagnostics MT CRE EU assay offered wide coverage for detection of acquired carbapenemase genes. It required minimal hands-on time and delivered results in less than 4 h from bacterial culture.201830189011
1479110.9124BioFire FilmArray BCID2 versus VITEK-2 System in Determining Microbial Etiology and Antibiotic-Resistant Genes of Pathogens Recovered from Central Line-Associated Bloodstream Infections. Central line-associated bloodstream infection (CLABSI) is among the most serious hospital acquired infections. Therefore, the rapid detection of the causative microorganism is of crucial importance to allow for the appropriate antimicrobial therapy. In the present study, we analyzed the clinical performance of the BioFire FilmArray Blood Culture Identification 2 (BCID2) panel in the identification of 33 microbial species and 10 antibiotic resistance genes in comparison to the VITEK-2 system. A total of 104 blood specimens were included. The FilmArray BCID2 results were concordant with the VITEK-2 system in 69/97 specimens (71.1%). Non-concordance was either due to the detection of more pathogens by the FilmArray BCID2 23/28 (82%) or microbial species were misidentified 5/28 (18%). Hence, in comparison to the VITEK-2 system, the FilmArray BCID2 panel showed an overall sensitivity of 75.8% (95% CI, 66-83%) and an overall specificity of 98% (95% CI, 97-98.8%) in detecting microbial species. For the resistance genes, the FilmArray BCID was able to detect the presence of blaCTX-M gene in 23 Gram-negative isolates, blaNDM and blaOXA-48- like genes in 14 and 13 isolates, respectively. The mecA and mecC genes were found in 23 Staphylococcus species, while mecA, mecC and MREJ genes were found in 4 Staphylococcus aureus isolates. The sensitivity and specificity for detecting resistance genes by the FilmArray BCID2 was 90% (95% CI, 81.4-95%) and 99.6% (95% CI, 99-100%), respectively. As concluded, the present study emphasizes the high sensitivity and specificity of the FilmArray BCID2 in the rapid and reliable detection of different bacteria and fungi from positive blood culture bottles, as well as the accurate detection of various antibiotic resistance markers.202236358274
5050120.9120Genomic Insights into Drug Resistance Determinants in Cedecea neteri, A Rare Opportunistic Pathogen. Cedecea, a genus in the Enterobacteriaceae family, includes several opportunistic pathogens reported to cause an array of sporadic acute infections, most notably of the lung and bloodstream. One species, Cedecea neteri, is associated with cases of bacteremia in immunocompromised hosts and has documented resistance to different antibiotics, including β-lactams and colistin. Despite the potential to inflict serious infections, knowledge about drug resistance determinants in Cedecea is limited. In this study, we utilized whole-genome sequence data available for three environmental strains (SSMD04, M006, ND14a) of C. neteri and various bioinformatics tools to analyze drug resistance genes in this bacterium. All three genomes harbor multiple chromosome-encoded β-lactamase genes. A deeper analysis of β-lactamase genes in SSMD04 revealed four metallo-β-lactamases, a novel variant, and a CMY/ACT-type AmpC putatively regulated by a divergently transcribed AmpR. Homologs of known resistance-nodulation-cell division (RND)-type multidrug efflux pumps such as OqxB, AcrB, AcrD, and MdtBC were also identified. Genomic island prediction for SSMD04 indicated that tolC, involved in drug and toxin export across the outer membrane of Gram-negative bacteria, was acquired by a transposase-mediated genetic transfer mechanism. Our study provides new insights into drug resistance mechanisms of an environmental microorganism capable of behaving as a clinically relevant opportunistic pathogen.202134442820
5044130.9118Detection of Colistin Resistance in Salmonella enterica Using MALDIxin Test on the Routine MALDI Biotyper Sirius Mass Spectrometer. Resistance to polymyxins in most Gram-negative bacteria arises from chemical modifications to the lipid A portion of their lipopolysaccharide (LPS) mediated by chromosomally encoded mutations or the recently discovered plasmid-encoded mcr genes that have further complicated the landscape of colistin resistance. Currently, minimal inhibitory concentration (MIC) determination by broth microdilution, the gold standard for the detection of polymyxin resistance, is time consuming (24 h) and challenging to perform in clinical and veterinary laboratories. Here we present the use of the MALDIxin to detect colistin resistant Salmonella enterica using the MALDxin test on the routine matrix-assisted laser desorption ionization (MALDI) Biotyper Sirius system.202032582090
1481140.9116Molecular versus conventional assay for diagnosis of hospital-acquired pneumonia in critically ill patients: a single center experience. PURPOSE: Lower respiratory tract infections are reported as one of top five causes of mortality and morbidity in the world. A bacterial etiology is often involved in HAP, most frequently from multidrug resistant gram-negative bacteria, and fast accurate diagnosis of etiologic agent(s) of LRTI is essential for an appropriate management. The aim of this retrospective study was to evaluate the analytical performance of Biofire Filmarray Pneumonia Plus for bacteria detection in bronchoalveolar lavage samples and the concordance of bacterial loads between BFPP and cultural gold standard methods. METHODS: A total of 111 BAL samples were obtained from 111 consecutive patients admitted to Intensive Care Unit of "Renato Dulbecco" Teaching Hospital of Catanzaro, from March 2023 to March 2024. RESULTS: Compared to conventional methods, BFPP showed a sensitivity of 99 % and a specificity of 64 %. The agreement between the two methods was assessed by calculating PPA and NPA, being 89 % and 95 %, respectively. The most common bacterial species identified at BFPP was Klebsiella pneumoniae, followed by Acinetobacter calcaceuticus-baumanii complex, Staphylococcus aureus and Pseudomonas aeruginosa. Bacterial load (CFU/ml) in relation to copy number detected by molecular analysis showed the best performance for value ≥10(6) copie/mL. About molecular mechanisms of resistance in comparison to phenotypic profiles, the highest level of performance was observed for presence of KPC genes, all isolates showing resistance to carbapenems, followed by OXA-48 like and NDM. CONCLUSION: The high concordance reported in this study between the identification of resistance genes and phenotypic indication can lead to an appropriate, fast and tailored antibiotic therapy.202540513663
5069150.9113MC-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
2214160.9112Development of multiplex recombinase polymerase amplification for the rapid detection of five carbapenemase (bla(KPC), bla(NDM), bla(OXA-48)-like, bla(IMP), and bla(VIM)) and 10 mcr (mcr-1 to mcr-10) genes in blood cultures. The emergence of plasmid-encoded carbapenemase and mobile colistin resistance (mcr) genes poses a significant challenge in controlling the spread of multidrug-resistant Gram-negative bacteria. Addressing this issue requires the development of rapid, accurate, and cost-effective tools for gene detection. For the first time, this study reports three multiplex recombinase polymerase amplification (RPA) assays, each designed to detect five resistance genes: carbapenemase (bla(KPC), bla(NDM), bla(OXA-48)-like, bla(IMP), and bla(VIM)), mcr-1 to mcr-5, and mcr-6 to mcr-10. Using agarose gel electrophoresis, all 15 target genes were successfully amplified by the three assays, demonstrating the potential of these assays for integration with rapid reporting platforms. To increase their applicability, the assays were combined with SYBR(Ⓡ) Green I for visual identification of all 15 target genes and with lateral flow immunoassays (LFIAs) for detection of two carbapenemase (bla(NDM) and bla(OXA-48)-like) and two mcr genes (mcr-1 and mcr-3) genes. Specificity testing showed that RPA-SYBR(Ⓡ) Green I and RPA-LFIAs produced no cross-reactivity among the target genes. The limit of detection for RPA-SYBR(Ⓡ) Green I, for all genes, ranged from 2 × 10(0) to 2 × 10(2) CFU/reaction, and for RPA-LFIAs from 2 × 10(0) to 2 × 10(3) CFU/reaction. The developed RPA-SYBR(Ⓡ) Green I and RPA-LFIAs successfully detected 15 and four target genes, from positive haemoculture bottles. These assays offer a promising approach for point-of-care testing. Providing a valuable tool for antimicrobial resistance surveillance and timely guidance for effective antibiotic intervention.202540618792
1486170.9112Multicenter evaluation of the Verigene Gram-negative blood culture nucleic acid test for rapid detection of bacteria and resistance determinants in positive blood cultures. The Verigene Gram-Negative Blood Culture Nucleic Acid Test (BC-GN) is a microarray-based assay that enables rapid detection of 9 common Gram-negative bacteria and 6 resistance determinants directly from positive blood cultures. We compared the performance of BC-GN with currently used automated systems, testing 141 clinical blood cultures and 205 spiked blood cultures. For identification of BC-GN target organisms in clinical and spiked blood cultures, the BC-GN assay showed 98.5% (130/132) and 98.9% (182/184) concordance, respectively. Of 140 resistance genes positively detected in clinical and spiked blood cultures with the BC-GN test, 139 (99.3%) were confirmed by PCR, and the detection results were consistent with the resistance phenotypes observed. The BC-GN assay, thus, can potentially improve care for sepsis patients by enabling timely detection and targeted antimicrobial therapy.201526361710
2225180.9111Evaluation of the DNA microarray "AMR Direct Flow Chip Kit" for detection of antimicrobial resistance genes from Gram-positive and Gram-negative bacterial isolated colonies. INTRODUCTION: The AMR Direct Flow Chip assay allows the simultaneous detection of a large variety of antibiotic resistance genetic markers. To assess this kit's performance, we use isolated colonies as starting material. The assay has been approved by the European Economic Area as a suitable device for in vitro diagnosis (CE IVD) using clinical specimens. METHODS: A total of 210 bacterial isolates harbouring either one or more antimicrobial resistance genes including plasmid-encoded extended-spectrum β-lactamases (SHV, CTX-M) and carbapenemases (GES, SME, KPC, NMC/IMI, SIM, GIM, SPM, NDM, VIM, IMP, and OXA), mecA, vanA and vanB, and 30 controls were included. RESULTS: The assay displayed a sensitivity and specificity of 100% for all target genes included in the array. CONCLUSION: The AMR Direct Flow Chip Kit is an accurate assay for detecting genes which commonly confer resistance to β-lactams and vancomycin from isolated colonies in culture of Gram-positive and Gram-negative bacteria.201930857832
2223190.9111Evaluation of a new real-time PCR assay (Check-Direct CPE) for rapid detection of KPC, OXA-48, VIM, and NDM carbapenemases using spiked rectal swabs. To prevent the spread of carbapenemase-producing bacteria, a fast and accurate detection of patients carrying these bacteria is extremely important. The Check-Direct CPE assay (Check-Points, Wageningen, The Netherlands) is a new multiplex real-time PCR assay, which has been developed to detect and differentiate between the most prevalent carbapenemase genes encountered in Enterobacteriaceae (blaKPC, blaOXA-48, blaVIM, and blaNDM) directly from rectal swabs. Evaluation of this assay using 83 non-duplicate isolates demonstrated 100% sensitivity and specificity and the correct identification of the carbapenemase gene(s) present in all carbapenemase-producing isolates. Moreover, the limit of detection (LoD) of the real-time PCR assay in spiked rectal swabs was determined and showed comparable LoDs with the ChromID CARBA agar. With an excellent performance on clinical isolates and spiked rectal swabs, this assay appeared to be an accurate and rapid method to detect blaKPC, blaOXA-48, blaVIM, and blaNDM genes directly from a rectal screening swab.201324135412