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147700.9892Multicenter 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
147810.9887Multicenter 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
140220.9884Detection 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
148530.9882Evaluation 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
148640.9881Multicenter 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
147450.9880Simple, 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
147960.9879BioFire 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
579570.9878Direct identification of Gram-positive bacteria and resistance determinants from blood cultures using a microarray-based nucleic acid assay: in-depth analysis of microarray data for undetermined results. BACKGROUND: The Verigene Gram-Positive Blood Culture (BC-GP) nucleic acid assay (Nanosphere, Inc., Northbrook, IL, USA) is a newly developed microarray-based test with which 12 Gram-positive bacterial genes and three resistance determinants can be detected using blood culture broths. We evaluated the performance of this assay and investigated the signal characteristics of the microarray images. METHODS: At the evaluation stage, we tested 80 blood cultures that were positive for various bacteria (68 bacteria covered and 12 not covered by the BC-GP panel) collected from the blood of 36 patients and 44 spiked samples. In instances where the automated system failed and errors were called, we manually inspected microarray images, measured the signal intensities of target spots, and reclassified the results. RESULTS: With the manual analysis of the microarray images of 14 samples for which error calls were reported, we could obtain correct identification results for 12 samples without the need for retesting, because strong signals in the target spots were clearly discriminable from background noise. With our interpretation strategy, we could obtain 97.1% sensitivity and 100% specificity for bacterial identification by using the BC-GP assay. The two unidentified bacteria were viridans group streptococci, which produced weaker target signals. During the application stage, among 25 consecutive samples positive for Gram-positive bacteria, we identified two specimens with error calls as Streptococcus spp. by using manual analysis. CONCLUSIONS: With help of the manual review of the microarray images, the BC-GP assay could successfully identify species and resistance markers for many clinically important Gram-positive bacteria.201525536666
147580.9877Evaluation of the FilmArray(®) Pneumonia Plus Panel for Rapid Diagnosis of Hospital-Acquired Pneumonia in Intensive Care Unit Patients. The FilmArray(®) Pneumonia plus Panel (FAPP) is a new multiplex molecular test for hospital-acquired pneumonia (HAP), which can rapidly detect 18 bacteria, 9 viruses, and 7 resistance genes. We aimed to compare the diagnosis performance of FAPP with conventional testing in 100 intensive care unit (ICU) patients who required mechanical ventilation, with clinically suspected HAP. A total of 237 samples [76 bronchoalveolar lavages (BAL(DS)) and 82 endotracheal aspirates (ETA(DS)) obtained at HAP diagnosis, and 79 ETA obtained during follow-up (ETA(TT))], were analyzed independently by routine microbiology testing and FAPP. 58 patients had paired BAL(DS) and ETA(DS). The positivity thresholds of semi-quantified bacteria were 10(3)-10(4) CFUs/mL or 10(4) copies/mL for BAL, and 10(5) CFUs/mL or copies/mL for ETA. Respiratory commensals (H. influenzae, S. aureus, E. coli, S. pneumoniae) were the most common pathogens. Discordant results for bacterial identification were observed in 33/76 (43.4%) BAL(DS) and 36/82 (43.9%) ETA(DS), and in most cases, FAPP identified one supplemental bacteria (23/33 BAL(DS) and 21/36 ETA(DS)). An absence of growth, or polybacterial cultures, explained almost equally the majority of the non-detections in culture. No linear relationship was observed between bin and CFUs/mL variables. Concordant results between paired BAL(DS) and ETA(DS) were obtained in 46/58 (79.3%) patients with FAPP. One of the 17 resistance genes detected with FAPP (mecA/C and MREJ) was not confirmed by conventional testing. Overall, FAPP enhanced the positivity rate of diagnostic testing, with increased recognition of coinfections. Implementing this strategy may allow clinicians to make more timely and informed decisions.202032983057
247790.9877Evaluation of targeted next-generation sequencing for microbiological diagnosis of acute lower respiratory infection. PURPOSE: To evaluate the performance of targeted next-generation sequencing (tNGS) in pathogen detection in acute lower respiratory infection. METHODS: The retrospective study was conducted between July 2023 and May 2024 at the Yantai Yuhuangding Hospital. Patients with acute lower respiratory infections were included. Qualified sputum or bronchoalveolar lavage fluid samples were collected for tNGS and conventional microbiological tests(CMTs), including culture, staining, polymerase chain reaction (PCR), and reverse transcription-PCR (RT-PCR). The time required and cost were counted. RESULTS: A total of 968 patients were enrolled. Study analysis discovered 1,019 strains of bacteria, 259 strains of fungi, 302 strains of viruses, 76 strains of Mycoplasma pneumoniae, and two strains of Chlamydia psittaci using tNGS. In addition, tNGS also identified 39 mecA, four KPC, 19 NDM, and two OXA-48 genes. The positive rates for bacteria, fungi, viruses, mycoplasma, and chlamydia obtained using tNGS were significantly higher than those determined using traditional methods. Among them, tNGS showed high consistence with mycobacterium DNA test, influenza A (H1N1) virus nucleic acid test and COVID-19 nucleic acid test. Poor consistency between drug resistance genes and bacterial resistance phenotypes was found. In addition, tNGS also had advantages over traditional methods in terms of detection time and cost. CONCLUSION: Compared to traditional methods, tNGS had higher sensitivity in detecting bacteria, fungi, viruses, and other pathogens in acute lower respiratory infection, and also had the advantages of timeliness and cost-effectiveness, making it a promising method for guiding clinical diagnosis.202540901079
2236100.9877Development 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
5037110.9876Development 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
2238120.9876Rapid detection of carbapenem resistance among gram-negative organisms directly from positive blood culture bottles. BACKGROUND: Carbapenemase producing gram-negative bacteria (GNB) has become a huge problem in majority of tertiary care centers worldwide. They are associated with very high morbidity and mortality rates, especially when they cause invasive infections. Therefore, rapid detection of these organisms is very important for prompt and adequate antibiotic therapy as well as infection control. The aim of this study was rapid detection of carbapenemase genes and thereby likely carbapenem resistance, 24-48 hours in advance, directly from the positive-flagged blood culture bottles using CHROMagar and Xpert® Carba-R. METHODS: Aspirate from positively flagged blood culture bottles was subjected to differential centrifuge. All gram-negative bacilli on gram stain from the deposit were processed in Xpert® Carba-R and inoculated on CHROMagar. The presence of genes and growth on CHROMagar was compared with carbapenem resistance on VITEK-2 Compact. RESULTS: A total of 119 GNB isolates were processed. One or more of the carbapenemase genes were detected in 80 isolates. On comparison with VITEK-2 result, 92 samples showed concordance for carbapenem resistance 48 hours in advance. There was discordance in 21 isolates with 12 major errors and 09 minor errors. The sensitivity of direct Xpert® Carba-R test for rapid detection of carbapenem resistance, 48 hours in advance, was 81.42%. The sensitivity of direct CHROMagar test for accurate detection of carbapenem resistance, 24 hours in advance, was 92.06%. CONCLUSION: The ability to detect carbapenem resistance with very high accuracy, 48 hours in advance, helps in appropriate antibiotic therapy and implementation of effective infection control practices.202337193528
2223130.9875Evaluation 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
5194140.9875Evaluation of the CosmosID Bioinformatics Platform for Prosthetic Joint-Associated Sonicate Fluid Shotgun Metagenomic Data Analysis. We previously demonstrated that shotgun metagenomic sequencing can detect bacteria in sonicate fluid, providing a diagnosis of prosthetic joint infection (PJI). A limitation of the approach that we used is that data analysis was time-consuming and specialized bioinformatics expertise was required, both of which are barriers to routine clinical use. Fortunately, automated commercial analytic platforms that can interpret shotgun metagenomic data are emerging. In this study, we evaluated the CosmosID bioinformatics platform using shotgun metagenomic sequencing data derived from 408 sonicate fluid samples from our prior study with the goal of evaluating the platform vis-à-vis bacterial detection and antibiotic resistance gene detection for predicting staphylococcal antibacterial susceptibility. Samples were divided into a derivation set and a validation set, each consisting of 204 samples; results from the derivation set were used to establish cutoffs, which were then tested in the validation set for identifying pathogens and predicting staphylococcal antibacterial resistance. Metagenomic analysis detected bacteria in 94.8% (109/115) of sonicate fluid culture-positive PJIs and 37.8% (37/98) of sonicate fluid culture-negative PJIs. Metagenomic analysis showed sensitivities ranging from 65.7 to 85.0% for predicting staphylococcal antibacterial resistance. In conclusion, the CosmosID platform has the potential to provide fast, reliable bacterial detection and identification from metagenomic shotgun sequencing data derived from sonicate fluid for the diagnosis of PJI. Strategies for metagenomic detection of antibiotic resistance genes for predicting staphylococcal antibacterial resistance need further development.201930429253
1480150.9874Prospective observational pilot study of the T2Resistance panel in the T2Dx system for detection of resistance genes in bacterial bloodstream infections. Early initiation of antimicrobial therapy targeting resistant bacterial pathogens causing sepsis and bloodstream infections (BSIs) is critical for a successful outcome. The T2Resistance Panel (T2R) detects the following resistance genes within organisms that commonly cause BSIs directly from patient blood samples: bla(KPC), bla(CTXM-14/15), bla(NDM)/bla(/IMP)/bla(VIM), bla(AmpC), bla(OXA), vanA, vanB, and mecA/mecC. We conducted a prospective study in two major medical centers for the detection of circulating resistance genes by T2R in patients with BSIs. T2R reports were compared to antimicrobial susceptibility testing (AST), phenotypic identification, and standard molecular detection assays. Among 59 enrolled patients, 25 resistance genes were identified: bla(KPC) (n = 10), bla(NDM)/bla(/IMP)/bla(VIM) (n = 5), bla(CTXM-14/15) (n = 4), bla(AmpC) (n = 2), and mecA/mecC (n = 4). Median time-to-positive-T2R in both hospitals was 4.4 hours [interquartile range (IQR): 3.65-4.97 hours] in comparison to that for positive blood cultures with final reporting of AST of 58.34 h (IQR: 45.51-111.2 hours; P < 0.0001). The sensitivity of T2R to detect the following genes in comparison to AST was 100% for bla(CTXM-14/15), bla(NDM)/bla(/)(IMP)/bla(VIM), bla(AmpC), mecA/mecC and 87.5% for bla(KPC). When monitored for the impact of significant antimicrobial changes, there were 32 events of discontinuation of unnecessary antibiotics and 17 events of escalation of antibiotics, including initiation of ceftazidime/avibactam in six patients in response to positive T2R results for bla(KPC). In summary, T2R markers were highly sensitive for the detection of drug resistance genes in patients with bacterial BSIs, when compared with standard molecular resistance detection systems and phenotypic identification assays while significantly reducing by approximately 90% the time to detection of resistance compared to standard methodology and impacting clinical decisions for antimicrobial therapy. IMPORTANCE: This is the first reported study to our knowledge to identify key bacterial resistance genes directly from the bloodstream within 3 to 5 hours in patients with bloodstream infections and sepsis. The study further demonstrated a direct effect in modifying initial empirical antibacterial therapy in response to T2R signal to treat resistant bacteria causing bloodstream infections and sepsis.202438456690
5069160.9872MC-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
2237170.9872Evaluation of Sepsis Flow Chip for identification of Gram-negative bacilli and detection of antimicrobial resistance genes directly from positive blood cultures. Blood stream infections are serious conditions associated with high morbi-mortality. In this study, the new Sepsis Flow Chip (SFC) assay for identification of Gram-negative bacteria and their antimicrobial resistance genes was evaluated in positive blood cultures (BCs). SFC is a microarray with a broad panel comprising the most frequent causative agents of sepsis and antimicrobial resistance genes associated with them. A total of 100 prospective BCs, positive for Gram-negative bacilli, were assessed in the routine of the clinical microbiology laboratory and also applying the SFC assay. Moreover, 19 BCs spiked with well-characterized enterobacterial isolates, harboring antimicrobial resistance genes, were analyzed by the latter. Among the monomicrobial BCs (90), the concordance between SFC identification and the reference method was 94.4%; however, it achieved 100% when SFC was combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry after 4-h incubation. Regarding polymicrobial BCs (10), 15 out of the 22 bacteria present (68.2%) were correctly identified, including all contained in 50% of the cultures. With regard to antimicrobial resistance genes, 98.8%, 98.9%, and 99% concordance was obtained for bla(CTX-M), bla(OXA-48), and bla(VIM), respectively, in comparison with polymerase chain reaction amplification. SFC assay gives results in only 4 h and showed a high concordance rate with the reference method. Although further evaluation studies are necessary, SFC assay implementation, together with antimicrobial stewardship programs, could contribute to improve the therapeutic approaches and to reduce the morbi-mortality, length of hospital stay, and healthcare-associated costs in patients with sepsis.201829551362
2226180.9871Evaluation 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
1491190.9871Evaluation of an expanded antibiotic resistance gene panel on prediction of antimicrobial susceptibility results for Gram-negative bacteria in blood cultures. The QIAstat-Dx BCID Panels (RUO) ("QIAstat," QIAGEN, Hilden, Germany) for identification of 13 Gram-negative bacteria and 18 antimicrobial resistance (AMR) gene groups was evaluated. The study was conducted in two phases; in phase 1, analytical performance was evaluated against 154 challenge isolates against whole genome sequencing data. In this phase, sensitivity and specificity of organism identification calls were 153/154 (99.3%) and 1,748/1,749 (99.8%), respectively. For AMR genes, sensitivity was 434/435 (99.8%) and specificity was 2,334/2,337 (99.9%). One false-negative bla(IMP), one false-positive bla(CTX-M), and two false-positive aac-6'-lb detections were noted in this challenge set of organisms. In phase 2, 101 clinical blood culture isolates of Gram-negative rods were evaluated by the multiplexed PCR versus reference broth microdilution, for the ability of identification combined with AMR genes to predict final susceptibility results. Negative predictive values were 92.8% for ampicillin resistance (100% for Escherichia coli), 93.4% for ceftriaxone, 97.4% for ceftazidime, and 98.7% for cefepime. In constrast, negative predictive values for current standard of care (identification plus detection of bla(CTX-M)) ranged from 56.5% to 88.8%. This study demonstrated additive value of additional beta-lactamase genes for bacteria isolated from blood cultures. IMPORTANCE: Prediction of Gram-negative bacteria resistance through detection of resistance genes is complex. This study evaluated a novel, direct-from-blood or bacterial isolate multiplexed PCR for the detection of 17 resistance genes, and evaluated the prediction of antimicrobial susceptibility.202439297627