Occurrence of the p019 Gene in the blaKPC-Harboring Plasmids: Adverse Clinical Impact for Direct Tracking of KPC-Producing Klebsiella pneumoniae by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been used for the direct detection of KPC-producing isolates by analysis of the 11,109 Da mass peak representing the P019 protein. In this study, we evaluate the presence of the 11,109 Da mass peak in a collection of 435 unduplicated Klebsiella pneumoniae clinical isolates. The prevalence of the P019 peak in the blaKPC K. pneumoniae isolates was 49.2% (32/65). The 11,109 Da mass peak was not observed in any of the other carbapenemase (319) or noncarbapenemase producers (116). Computational analysis of the presence of the p019 gene was performed in the aforementioned carbapenemase-producing K. pneumoniae isolates fully characterized by whole-genome sequencing (WGS) and in a further collection of 1,649 K. pneumoniae genomes included in EuSCAPE. Herein, we have demonstrated that the p019 gene is not exclusively linked to the pKpQil plasmid but that it is present in the following plasmids: IncFIB(K)/IncFII(K)/ColRNAI, IncFIB(pQil), IncFIB(pQil)/ColRNAI, IncFIB(pQil)/IncFII(K), IncFIB(K)/IncFII(K), and IncX3. In addition, we have proven the independent movement of the Tn4401 and the ISKpn31, of which the p019 gene is a component. The absence of the p019 gene was obvious in Col440I, Col(pHAD28), IncFIB(K)/IncX3/IncFII(K), and IncFIB(K)/IncFII(K) plasmids. In addition, we also observed another plasmid in which neither Tn4401 nor ISKpn31 was found, IncP6. In the EuSCAPE, the occurrence of p019 varied from 0% to 100% among the different geographical locations. The adverse clinical impact of the diminished prevalence of the p019 gene within the plasmid encoding KPC-producing Klebsiella pneumoniae puts forward the need for reconsideration when applying this technique in a clinical setting.

An Improved Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Data Analysis Pipeline for the Identification of Carbapenemase-Producing Klebsiella pneumoniae.

The increasing emergence of carbapenemase-producing Klebsiella pneumoniae (CPK) isolates is a global health alarm. Rapid methods that require minimum sample preparation and rapid data analysis are urgently required. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been used by clinical laboratories for identification of antibiotic-resistant bacteria; however, discrepancies have arisen regarding biological and technical issues. The aim of this study was to standardize an operating procedure and data analysis for identification of CPK by MALDI-TOF MS. To evaluate this approach, a series of 162 K. pneumoniae isolates (112 CPK and 50 non-CPK) were processed in the MALDI BioTyper system (Bruker Daltonik, Germany) following a standard operating procedure. The study was conducted in two stages; the first is denominated the "reproducibility stage" and the second "CPK identification." The first stage was designed to evaluate the biological and technical variation associated with the entire analysis of CPK and the second stage to assess the final accuracy of MALDI-TOF MS for the identification of CPK. Therefore, we present an improved MALDI-TOF MS data analysis pipeline using neural network analysis implemented in Clover MS Data Analysis Software (Clover Biosoft, Spain) that is designed to reduce variability, guarantee interlaboratory reproducibility, and maximize the information selected from the bacterial proteome. Using the random forest (RF) algorithm, 100% of CPK isolates were correctly identified when all the peaks in the spectra were selected as input features and total ion current (TIC) normalization was applied. Thus, we have demonstrated that real-time direct tracking of CPK is possible using MALDI-TOF MS.

Citric/citrate buffer: an effective antiglycolytic agent.

BACKGROUND: In order to minimize the influence of glycolysis on diabetes mellitus (DM) diagnostic tests, we have compared the behavior of citric/citrate, fluoride additives and gel-serum with plasma-heparin under careful preanalytical conditions. Subsequently, we compared the effectiveness of both fluoride and citric additives at different pre-centrifugation times. Finally, the influence of citric/citrate collection tube on diagnostic tests results was evaluated. METHODS: The first study of 80 voluntary patients assessed the glucose bias of citric/citrate, fluoride additive tubes and gel-serum tubes versus plasma-heparin tubes at several medical decision cut-offs (MDC). The second study performed with 72 volunteers evaluated additives, simulating transport times to the laboratory and centrifugation delay periods. Final evaluation compares the proportion of positive tests in total tests carried out in two different periods. RESULTS: When citric/citrate (n=79) and fluoride tubes (n=60) were compared with plasma-heparin under controlled preanalytical conditions, both met the bias specification for plasma glucose (±1.8%) at seven MDC. On the contrary, serum samples (n=15) did not meet it at five MDC. In the second study, differences in glucose values at distinct pre-centrifugation times were not statistically significant for citric/citrate tubes, but significant for fluoride tubes and also for comparison of fluoride and citric/citrate tubes. Hemolysis in fluoride tubes was higher. Citric/citrate tube implementation in our laboratory caused an increase in positive diagnostic tests that were only statistically significant for gestational diabetes mellitus (GDM) screening. CONCLUSIONS: Citric/citrate additive tube is equivalent to plasma-heparin avoiding glycolysis completely and immediately under careful preanalytical conditions even with a 3-h delay in plasma separation. According to used MDC we have not statistically significantly increased the diagnoses of DM cases.

Multicenter Performance Evaluation of MALDI-TOF MS for Rapid Detection of Carbapenemase Activity in Enterobacterales: The Future of Networking Data Analysis With Online Software.

In this study, we evaluate the performance of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for rapid detection of carbapenemase activity in Enterobacterales in clinical microbiology laboratories during a multicenter networking validation study. The study was divided into three different stages: "software design," "intercenter evaluation," and "clinical validation." First, a standardized procedure with an online software for data analysis was designed. Carbapenem resistance was detected by measuring imipenem hydrolysis and the results were automatically interpreted using the Clover MS data analysis software (Clover BioSoft, Spain). Second, a series of 74 genotypically characterized Enterobacterales (46 carbapenemase-producers and 28 non carbapenemase-producers) were analyzed in 8 international centers to ensure the reproducibility of the method. Finally, the methodology was evaluated independently in all centers during a 2-month period and results were compared with the reference standard for carbapenemase detection used in each center. The overall agreement rate relative to the reference method for carbapenemase resistance detection in clinical samples was 92.5%. The sensitivity was 93.9% and the specificity, 100%. Results were obtained within 60 min and accuracy ranged from 83.3 to 100% among the different centers. Further, our results demonstrate that MALDI-TOF MS is an outstanding tool for rapid detection of carbapenemase activity in Enterobacterales in clinical microbiology laboratories. The use of a simple in-house procedure with online software allows routine screening of carbapenemases in diagnostics, thereby facilitating early and appropriate antimicrobial therapy.

Troponin levels within the normal range and probability of inducible myocardial ischemia and coronary events in patients with acute chest pain.

BACKGROUND/OBJECTIVES: Patients with suspected acute coronary syndromes and negative cardiac troponin (cTn) levels are deemed at low risk. Our aim was to assess the effect of cTn levels on the frequency of inducible myocardial ischemia and subsequent coronary events in patients with acute chest pain and cTn levels within the normal range. METHODS: We evaluated 4474 patients with suspected acute coronary syndromes, nondiagnostic electrocardiograms and serial cTnI levels below the diagnostic threshold for myocardial necrosis using a conventional or a sensitive cTnI assay. The end points were the probability of inducible myocardial ischemia and coronary events (i.e., coronary death, myocardial infarction or coronary revascularization within 3 months). RESULTS: The probability of inducible myocardial ischemia was significantly higher in patients with detectable peak cTnI levels (25%) than in those with undetectable concentrations (14.6%, p<0.001). These results were consistent regardless of the type of cTnI assay, the type of stress testing modality, or the timing for cTnI measurement, and remained significant after multivariate adjustment (odds ratio [OR] 1.47, 95% confidence interval [CI] 1.21-1.79, p<0.001). The rate of coronary events at 3 months was also significantly higher in patients with detectable cTnI levels (adjusted OR 2.08, 95% CI 1.64-2.64, p<0.001). CONCLUSIONS: Higher cTnI levels within the normal range were associated with a significantly increased probability of inducible myocardial ischemia and coronary events in patients with suspected acute coronary syndromes and seemingly negative cTnI.