Identification Of Positive Blood Cultures Research Articles

MALDI-TOF direct identification of positive blood cultures: A four-year analytical evaluation of A Triton based workflow

Rapid and reliable identification of the causal organism in bloodstream infections and sepsis is crucial for both individual patient care and public health. We have implemented a rapid in-house identification protocol (with 10 % Triton) using MALDI-TOF MS for identifying the causative organism in positive blood cultures without prior culture. Our objective was to retrospectively analyze data collected over a four-year period while implementing this rapid in-house identification protocol and to develop a guide for evaluating and reporting the obtained results. Overall, our method utilizing MALDI-TOF MS for rapid in-house identification, demonstrated comparable results to other commercially available and in-house methods reported in the literature. Over the past four years, direct identification has facilitated the distinction between clinically relevant positive blood cultures and irrelevant ones, guiding rapid focus control and appropriate antibiotic treatment. The established guide can serve as a valuable tool in reporting positive blood cultures and associated antibiotic treatments.

Direct inoculation method for identification and antimicrobial susceptibility testing using matrix-assisted laser desorption ionization-time of flight mass spectrometry and both the Vitek 2 and MicroScan Walkaway 96 Plus systems

The aim of our study was to evaluate a protocol utilizing serum separator tubes (SST) to facilitate a faster, cost-effective, direct method for rapid sensitivity testing and identification of positive blood cultures. Spiked cultures were inoculated into either Becton Dickinson (BD) BACTECTM Aerobic Plus or Anaerobic/F bottles containing sterile human blood. Bottles were immediately processed when positive. A parallel study using patient isolates was used in which bacteria were pelleted by SST from positive blood cultures. For identification, a portion of the pellet was tested by matrix-assisted laser desorption/ionization as described by the manufacturer. MicroScan panels and Vitek 2 results were compared. Categorical agreement was used as comparison to standard subculture and/or polymerase chain reaction methods. No discordant identifications were observed, and 86% generated a successful identification when compared to subculture methods. For the Vitek 2, we observed a 99% essential agreement when compared to the subculture method. For the MicroScan Walkaway, we observed 94.9%, 97.4%, and 100% categorical agreement for MIC panels 53, 38, and MICroSTREP Plus 2, respectively. Turnaround times were reduced from 4 hours for identification and 11 hours for antimicrobial sensitivity testing. We conclude that the SST method results in timelier, actionable results for antimicrobial stewardship initiatives.

Impact of Short-Incubation MALDI-TOF MS on Rapid Identification of Pathogens and Antibiotic Therapy

Background: It can be a critical point for reducing pathogen identification time and accurate antibiotic treatment for patients with blood circulation infection since it causes high mortality. Objective: The objectives of this study were to evaluate the time differences between conventional identification and MALDI-TOF conventional identification and short-incubation MALDI-TOF identification for positive blood cultures, and to explore the impact of short-incubation matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) identification on empirical antibiotic therapy. Methods: Positive blood cultures were collected in our hospital from 2017 to 2019, clinical data were collected from the medical records, which were analyzed retrospectively to determine the empirical antibiotic therapy. Results: Compared with the conventional identification method, the short-incubation MALDI-TOF identification time to initial identification of Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus, Enterococcus faecium, and E. faecalis decreased by 22.28 h, 22 h, 23.59 h, 23.63 h, 22.63 h, 23.92 h, and 21.59 h, respectively (P < 0.05). The time to final reporting was decreased by 48.85 h, 47.99 h, 55.40 h, 51.07 h, 49.60 h, 51.78h, and 51.73h, respectively (P < 0.05). However, the antimicrobial susceptibility test time of E. coli, A. baumannii, and S. aureus increased to 2.02 h, 2.19 h, and 3.86 h, respectively (P < 0.05). The coincidence rate of antimicrobial susceptibility was 98.48% between short-incubation MALDI-TOF identification and conventional identification method of all Gram-negative bacilli, and there were no extremely major errors or major errors. The coincidence rate of antimicrobial susceptibility of Gram-positive cocci was 99.53%, one strain of E. faecium and S. aureus had major errors. Patients received earlier correct empirical antibiotic 19.89 h earlier by short-incubation MALDI-TOF identification than the conventional identification method (P < 0.001). Conclusions: The short-incubation MALDI-TOF identification significantly shortens the pathogen identification time and the final report time, it is a reliable method for rapid identification of positive blood cultures; the results of antimicrobial susceptibility are highly consistent, which significantly lead to earlier appropriate empirical therapy of bacteremia.

Rapid method for direct identification of positive blood cultures by MALDI-TOF MS.

Application of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using positive blood cultures (BCs) is a revolution in identification of microorganisms in clinical microbiology laboratories. Although there are several commercial pretreatment protocols they are expensive. Here, we evaluated the performance of a locally produced Bioyong pre-treatment kit for the direct identification of microorganisms in positive BCs by MALDI-TOF MS method. The mocked positive BCs were performed using 200 Thermo aerobic blood culture bottles and 200 aerobic Scenker blood culture bottles. A total of 200 organisms were invovled, including 91 strains of Gram-positive bacteria, 97 strains of Gram-negative bacteria and 12 strains of Candida. The positive BCs were subcultured and identified by classical biochemical Vitek II testing as the gold standard of identification. The Bioyong pre-treatment kit could successfully identify microorganisms in 189 (94.5%) Thermo positive BCs and 189 (94.5%) Scenker positive blood cultures, respectively. In total, 94 (96.9%) Gram-negative bacteria, 86 (94.5%) Gram-positive bacteria and 9 (75.0%) candida isolated from Thermo positive BCs were correctly identified to species level and 95 (97.9%) Gram-negative bacteria, 86 (94.5%) Gram-positive bacteria and 8 (66.7%) candida isolated from Scenker positive BCs were correctly identified to species level. This method provides a rapid, accurate identification of bacteria and Candida within one hour in positive blood cultures. Routine application of this technique will improve the antimicrobial treatment within 24 h among the patients with bacteremia and candidemia.

Clinical Impact of Rapid Identification of Positive Blood Cultures vs. Internal Laboratory Standard

Clinical Impact of Rapid Identification of Positive Blood Cultures vs. Internal Laboratory Standard

Candida Intestinal Domination Precedes Fungal Infections Bloodstream in Allogeneic Hematopoietic Cell Transplant Patients

Allogeneic hematopoietic cell transplantation (allo-HCT) recipients are at high risk of developing invasive bloodstream infections during the period of neutropenia prior to engraftment, even with prophylactic administration of antibacterial and antifungal drugs. Previous studies have demonstrated that disruption of the diversity and stability of commensal intestinal bacteria facilitates intestinal domination by pathobionts, a process that is associated with subsequent bacteremia. The relationship and dynamics of intestinal fungal communities (i.e., mycobiota) and the development of fungemia during allo-HCT remains unknown. In this study, we identified 8 allo-HCT patients that developed fungemia and participated in a prospective collection of fecal samples between 2014 and 2017 at MSKCC. All 8 patients developed Candida species fungemia (6 C. parasilopsis, 1 C. albicans, 1 C. lusitaniae). Using quantitative PCR to analyze fungal 18S rDNA, we found a temporal expansion of total intestinal fungi that coincided with the onset of fungemia (Figure A). We then analyzed the composition of the mycobiota by sequencing the internal transcribed spacer 1 (ITS1) region of the fungal rDNA, and found a significant loss of diversity of gut fungal flora (Figure B). Moreover, in seven of the eight cases, we observed intestinal dominance of the specific Candida species that was associated with fungemia, prior to the identification of positive blood cultures. Through whole genome sequencing of the fecal and the bloodstream fungal isolates, we further confirmed that the bloodstream fungal strain was the same clone as the one that had robustly colonized in the gut. Taken together, our results indicate that Candida intestinal domination precedes Candida bloodstream infection. Thus, monitoring the dynamics and composition of the mycobiota may facilitate identification of patients at-risk for this life-threatening complication and enable improved strategies for prophylactic or therapeutic gain.

Cheap and rapid in-house method for direct identification of positive blood cultures by MALDI-TOF MS technology

BackgroundRapid and accurate pathogen identification in blood cultures is very important for septic patients and has major consequences on morbidity and mortality rates. In recent years, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS)-based technology has become useful for highly specific and sensitive identification of bacteria and yeasts from clinical samples including sterile body fluids. Additional in-house methods enabled direct identification from blood cultures following various preparation protocols.MethodsBlood culture (5 ml) was harvested from each positive bottle following growth identification by BACTEC™ FX system and transferred into a VACUETTE® Z Serum Sep Clot Activator tube containing an inert gel, which following centrifugation separates microorganisms from the blood cells. We used MALDI-TOF MS analysis for identification of microorganisms collected from the gel surface.ResultsPositive blood culture bottles (186) were collected. In comparison with the routine method, 99% (184/186) and 90% (168/186) of the isolates were correctly identified by the SepsiTyper kit and the in-house method, respectively. We found high concordance (Pearson coefficient = 0.7, p < 0.0001) between our in-house method and the SepsiTyper kit. Additionally, high correlation was found in sub-groups of identified bacteria, with Pearson coefficients of 0.77 (p < 0.0001), 0.67 (p < 0.0001), and 0.73 (p < 0.007) for Gram negative, Gram positive, and anaerobic bacteria, respectively.ConclusionsOur in-house method was found to be in good agreement with the SepsiTyper kit. Considering the low costs and the rapid and easy implementation of this procedure, we propose our in-house method for the direct identification of bacteria from blood cultures.

An Automated Sample Preparation Instrument to Accelerate Positive Blood Cultures Microbial Identification by MALDI-TOF Mass Spectrometry (Vitek®MS).

Sepsis is the leading cause of death among patients in intensive care units (ICUs) requiring an early diagnosis to introduce efficient therapeutic intervention. Rapid identification (ID) of a causative pathogen is key to guide directed antimicrobial selection and was recently shown to reduce hospitalization length in ICUs. Direct processing of positive blood cultures by MALDI-TOF MS technology is one of the several currently available tools used to generate rapid microbial ID. However, all recently published protocols are still manual and time consuming, requiring dedicated technician availability and specific strategies for batch processing. We present here a new prototype instrument for automated preparation of Vitek®MS slides directly from positive blood culture broth based on an “all-in-one” extraction strip. This bench top instrument was evaluated on 111 and 22 organisms processed using artificially inoculated blood culture bottles in the BacT/ALERT® 3D (SA/SN blood culture bottles) or the BacT/ALERT VirtuoTM system (FA/FN Plus bottles), respectively. Overall, this new preparation station provided reliable and accurate Vitek MS species-level identification of 87% (Gram-negative bacteria = 85%, Gram-positive bacteria = 88%, and yeast = 100%) when used with BacT/ALERT® 3D and of 84% (Gram-negative bacteria = 86%, Gram-positive bacteria = 86%, and yeast = 75%) with Virtuo® instruments, respectively. The prototype was then evaluated in a clinical microbiology laboratory on 102 clinical blood culture bottles and compared to routine laboratory ID procedures. Overall, the correlation of ID on monomicrobial bottles was 83% (Gram-negative bacteria = 89%, Gram-positive bacteria = 79%, and yeast = 78%), demonstrating roughly equivalent performance between manual and automatized extraction methods. This prototype instrument exhibited a high level of performance regardless of bottle type or BacT/ALERT system. Furthermore, blood culture workflow could potentially be improved by converting direct ID of positive blood cultures from a batch-based to real-time and “on-demand” process.

Impact of rapid identification of positive blood cultures using the Verigene system on antibiotic prescriptions: A prospective study of community-onset bacteremia in a tertiary hospital in Japan.

BackgroundRapid identification of positive blood cultures is important for initiation of optimal treatment in septic patients. Effects of automated, microarray-based rapid identification systems on antibiotic prescription against community-onset bacteremia (COB) remain unclear.MethodsWe prospectively enrolled 177 patients with 185 COB episodes (occurring within 72 h of admission) over 17 months. Bacteremia episodes due to gram-positive bacteria (GP) and gram-negative bacteria (GN) in the same patient were counted separately. For GP bacteremia, patients with ≥2 sets of positive blood cultures were included. The primary study objective was evaluating the rates of antibiotic prescription changes within 2 days of rapid identification using the Verigene system.ResultsBacteremia due to GN and GP included 144/185 (77.8%) and 41/185 (22.2%) episodes, respectively. Antibiotic prescription changes occurred in 51/185 cases (27.6% [95%CI:21.3–34.6%]) after Verigene analysis and 70/185 cases (37.8% [30.8–45.2%]) after conventional identification and susceptibility testing. Prescription changes after Verigene identification were more frequent in GP (17/41[41.5%]) than in GN (34/144[23.5%]). Among bacteremia due to single pathogen targeted by Verigene test, bacterial identification agreement between the two tests was high (GP: 38/39[97.4%], GN: 116/116[100%]). The Verigene test correctly predicted targeted antimicrobial resistance. The durations between the initiation of incubation and reporting of the results for the Verigene system and conventional test was 28.3 h (IQR: 25.8–43.4 h) and 90.6 h (68.3–118.4 h), respectively. In only four of the seven episodes of COB in which two isolates were identified by conventional tests, the Verigene test correctly identified both organisms.ConclusionWe observed a high rate of antibiotic prescription changes after the Verigene test in a population with COB especially in GP. The Verigene test would be a useful tool in antimicrobial stewardship programs among patients with COB.

Impact of Rapid Identification of Positive Blood Cultures by Verigene System on Antibiotic Prescription: Prospective Study of Community-Onset Bacteremia in a Tertiary Hospital in Japan

Impact of Rapid Identification of Positive Blood Cultures by Verigene System on Antibiotic Prescription: Prospective Study of Community-Onset Bacteremia in a Tertiary Hospital in Japan

Impact of a rapid microarray-based assay for identification of positive blood cultures for treatment optimization for patients with streptococcal and enterococcal bacteremia.

Implementation of the Verigene Gram-positive blood culture test led to reductions in time to acceptable antibiotic overall (1.9 versus 13.2 h, respectively; P=0.04) and time to appropriate antibiotic for patients with vancomycin-resistant Enterococcus (4.2 versus 43.7 h; P=0.006) and viridans group Streptococcus (0.2 versus 7.1 h; P=0.02).

Impact of a Rapid Microarray-Based Assay for Identification of Positive Blood Cultures for Treatment Optimization for Patients with Streptococcal and Enterococcal Bacteremia

Impact of a Rapid Microarray-Based Assay for Identification of Positive Blood Cultures for Treatment Optimization for Patients with Streptococcal and Enterococcal Bacteremia

Reducing time to identification of positive blood cultures with MALDI-TOF MS analysis after a 5-h subculture

Speeding up the turn-around time of positive blood culture identifications is essential in order to optimize the treatment of septic patients. Several sample preparation techniques have been developed allowing direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) identification of positive blood cultures. Yet, the hands-on time restrains their routine workflow. In this study, we evaluated an approach whereby MALDI-TOF MS identification without any additional steps was carried out on short subcultured colonies from positive blood bottles with the objective of allowing results reporting on the day of positivity detection. Over a 7-month period in 2012, positive blood cultures detected by 9 am with an automated system were inoculated onto a Columbia blood agar and processed after a 5-h incubation on a MALDI-TOF MicroFlex platform (Bruker Daltonik GmbH). Single-spotted colonies were covered with 1 μl formic acid and 1 μl matrix solution. The results were compared to the validated identification techniques. A total of 925 positive blood culture bottles (representing 470 bacteremic episodes) were included. Concordant identification was obtained in 727 (81.1 %) of the 896 monomicrobial blood cultures, with failure being mostly observed with anaerobes and yeasts. In 17 episodes of polymicrobic bacteremia, the identification of one of the two isolates was achieved in 24/29 (82.7 %) positive cultures. Routine implementation of MALDI-TOF MS identification on young positive blood subcultures provides correct results to the clinician in more than 80 % of the bacteremic episodes and allows access to identification results on the day of blood culture positivity detection, potentially accelerating the implementation of targeted clinical treatments.

Evaluation of an Automated Rapid Diagnostic Assay for Detection of Gram-Negative Bacteria and Their Drug-Resistance Genes in Positive Blood Cultures

We evaluated the performance of the Verigene Gram-Negative Blood Culture Nucleic Acid Test (BC-GN; Nanosphere, Northbrook, IL, USA), an automated multiplex assay for rapid identification of positive blood cultures caused by 9 Gram-negative bacteria (GNB) and for detection of 9 genes associated with β-lactam resistance. The BC-GN assay can be performed directly from positive blood cultures with 5 minutes of hands-on and 2 hours of run time per sample. A total of 397 GNB positive blood cultures were analyzed using the BC-GN assay. Of the 397 samples, 295 were simulated samples prepared by inoculating GNB into blood culture bottles, and the remaining were clinical samples from 102 patients with positive blood cultures. Aliquots of the positive blood cultures were tested by the BC-GN assay. The results of bacterial identification between the BC-GN assay and standard laboratory methods were as follows: Acinetobacter spp. (39 isolates for the BC-GN assay/39 for the standard methods), Citrobacter spp. (7/7), Escherichia coli (87/87), Klebsiella oxytoca (13/13), and Proteus spp. (11/11); Enterobacter spp. (29/30); Klebsiella pneumoniae (62/72); Pseudomonas aeruginosa (124/125); and Serratia marcescens (18/21); respectively. From the 102 clinical samples, 104 bacterial species were identified with the BC-GN assay, whereas 110 were identified with the standard methods. The BC-GN assay also detected all β-lactam resistance genes tested (233 genes), including 54 bla CTX-M, 119 bla IMP, 8 bla KPC, 16 bla NDM, 24 bla OXA-23, 1 bla OXA-24/40, 1 bla OXA-48, 4 bla OXA-58, and 6 bla VIM. The data shows that the BC-GN assay provides rapid detection of GNB and β-lactam resistance genes in positive blood cultures and has the potential to contributing to optimal patient management by earlier detection of major antimicrobial resistance genes.

Procalcitonin and interleukin 6 for predicting blood culture positivity in sepsis

BackgroundProcalcitonin and interleukin 6 (IL-6) are well-known predictors of blood culture positivity in patients with sepsis. However, the association of procalcitonin and IL-6 with blood culture positivity was assessed separately in previous studies. This study aims to examine and compare the performance of procalcitonin and IL-6, measured concomitantly, in predicting blood culture positivity in patients with sepsis. MethodsForty adult patients with sepsis were enrolled in the study. Blood cultures were drawn before the institution of antibiotic therapy. The area under the curve (AUC) of the receiver operating characteristic curve was estimated to assess the performance of procalcitonin and IL-6 in predicting blood culture positivity. ResultsPositive blood cultures were detected in 10 patients (25%). The AUC of procalcitonin and IL-6 was 0.85 and 0.61, respectively. The combined performance of procalcitonin and IL-6 was similar to that of procalcitonin alone, AUC of 0.85. On univariate analysis, only procalcitonin and IL-6 were associated with blood culture positivity. Multivariate logistic regression analysis showed that only procalcitonin was associated with blood culture positivity (odds ratio, 12.15 [1.29-114.0] for levels above the median compared with levels below the median). Using procalcitonin cut points of 1.35 and 2.14 (nanogram per milliliter) enabled 100% and 90% identification of positive blood cultures and reduced the need of blood cultures by 47.5% and 57.5%, respectively. ConclusionsCompared with IL-6, procalcitonin better predicts blood culture positivity in patients with sepsis. Using a predefined procalcitonin cut points will predict most positive blood cultures and reduce the need of blood cultures in almost half of patients with sepsis.

Utility of direct genetic probes for the rapid presumptive identification of positive blood cultures

The changes in morphology and proliferation kinetics produced by colchicine in the intestinal mucosa are known. Contrarily, the functional changes have yet not been investigated systematically. The aim of this study is to investigate the effects of colchicine treatment on rat intestinal disaccharidase activities.Material and methods: For the experiments male SPF-Wistar rats, body weight 180-200 g-(supplied by Mus-Rattus AG, 8011 Brunnthal, FRG) were used. The animals were kept under constant conditions (6 rats per cage) and fed Altromin® (Altromin GmbH, Lage/Lippe), tap water ad libitum.Per day 6 animals of each test group were scarificed by cervical dislocation at defined time. About 3 cm of the intestine were removed 8-10 cm distally of the pylorus and cut longitudinally. After cautious cleaning the mucosa was detached using a scalpel. The thus obtained specimens of the mucosa were frozen at –20 °C in an air-tight humidity chamber and preserved there until further handling. For histological examination the correspondent parts of the intestine were fixed in 10 % formol. From each specimen 4 different stainings were made (haematoxylin-eosin, Giemsa, PAS and Feulgen stain).Per test series 60–66 rats were used. The following doses of colchicine were applied:1 × 1.0 mg/kg body weight,1 × 0.5 mg/kg body weight,5 × 0.1 mg/kg body weight within 24 hours.The single doses were dissolved in about 0.5 physiological saline and applied intraperitoneally. In each 6 rats per group the activities of the following diasccharidases were measured at 24 hours intervals from the 1st to 10th or 14th day: sucrase, maltase, lactase, and cellobiase.The enzyme activities were determined after the two-step method developed by Dahlqvist (5, 6) which can be used as micromethod. The ascertained enzyme activities were always related to the protein content. Protein determination was done after the method of Folin-Ciocalteu as modified by Lowry et al. (14). The enzyme activities were expressed in international units per g protein (U/g protein).The results obtained were statistically ascertained by the non-parametric test after Kruskal-Wallis, variance analysis with linear contrasts after Scheffe as well as by double variance analyses (26).