Direct Identification Of Microorganisms Research Articles

653. Direct Identification of Microorganisms in Positive Blood Cultures by the BioFire® FilmArray® Blood Culture Identification Panel Leads to Faster Optimal Antibiotic Therapy: A Before–After Study

BackgroundRapid pathogen identification from positive blood cultures may help optimize empiric antibiotic therapy quickly by reducing unnecessary broad spectrum antibiotic use and may improve patient outcomes. The BioFire® FilmArray® Blood Culture Identification Panel 1 (BF-FA-BCIP) identifies 24 pathogens directly from positive blood cultures without subculture. 3 resistance genes are included. We aimed to compare the time to optimal antibiotic therapy between BF-FA-BCIP and conventional identification.MethodsWe performed a single-center retrospective case-control before-after study of 386 cases (November 2018 to October 2019) with BF-FA-BCIP compared to 414 controls (August 2017 to July 2018) with conventional identification. The primary study endpoint was the time from blood sampling to implementation of optimal antimicrobial therapy. Secondary endpoints were time to effective therapy, length of hospital stay, and in-hospital and 30-day mortality. Outcomes were assessed using cause-specific Cox Proportional Hazard models and logistic regressions.ResultsWe included 800 patients with comparable baseline characteristics. Main sources of blood stream infection (BSI) were urinary tract infection and intra-abdominal infection (19.2% vs. 22.0% and 16.8% vs. 15.7% for case and control groups, respectively). Overall, 212 positive blood cultures were considered as contaminations. Identification results were available after a median of 21.9 hours by the BF-FA-BCIP and 44.3 hours by the conventional method. Patients with BF-FA-BCIP received the optimal therapy after a median of 25.5 hours (95%CI 21.0 - 31.2) as compared to 45.7 hours (95%CI 37.7 - 51.2) in the control group (Figure 1). We found no effect of the identification method on secondary outcomes.Kaplan-Meier curve representing the probability of implementing the optimal therapy at any given time according to the identification method (Standard vs. BF-FA-BCIP). Shaded ribbons represent the 95 % confidence interval (CI). The vertical dashes represent censored data. The vertical dotted lines represent the median time, i.e. the time at which 50 % of the patients obtained the optimal therapy, for the two methods. Median (95 % CI) time to optimal therapy is 45.7 (37.7 - 51.4) hours with the Standard method and 25.5 (21.0- 31.2) hours with Biofire. The tables below the curves present the numbers expecting optimal therapy according to the bacteria identification method, as well as the number of censored data in parenthesis. Panel A shows data from 0 to 900 hours. Panel B shows the data from 0 to 90 hours to better visualize how the probability to implement optimal therapy varies in the first 72 hours.ConclusionIn conclusion, rapid pathogen identification by BF-FA-BCIP was associated with an almost 24h earlier initiation of the optimal antibiotic therapy in BSI. However, the overall benefit for individual patients seems to be limited. Future studies should assess the cost-effectiveness and impact on the prevention of antibiotic resistance using this diagnostic approach.Disclosures All Authors: No reported disclosures

Current Scenario and Challenges in the Direct Identification of Microorganisms Using MALDI TOF MS.

MALDI TOF MS-based microbial identification significantly lowers the operational costs because of minimal requirements of substrates and reagents for extraction. Therefore, it has been widely used in varied applications such as clinical, food, military, and ecological research. However, the MALDI TOF MS method is laced with many challenges including its limitation of the reference spectrum. This review briefly introduces the background of MALDI TOF MS technology, including sample preparation and workflow. We have primarily discussed the application of MALDI TOF MS in the identification of microorganisms. Furthermore, we have discussed the current trends for bioaerosol detection using MALDI TOF MS and the limitations and challenges involved, and finally the approaches to overcome these challenges.

Direct Identification of Microorganisms from Positive Blood Cultures by MALDI-TOF MS Using an in-House Tween® 80 Method: Experimental and Clinical Study

It has been reported that direct identification from blood culture bottles with positive signals and reporting the results to the clinics earlier has positive effects on mortality and morbidity. Extraction methods especially using detergents are used for the direct identification from the bottles which give positive signal. For this purpose, in-house methods developed based on the usage of saponin are widely available in the literature. In this study, it was aimed to develop a simple, easy-to-apply and reliable protocol for identifying the agent directly from the blood culture bottle that gives positive signal with the use of detergent Tween® 80, and to study the obtained protocol in clinical samples in a routine microbiology laboratory and to evaluate the results. The study was carried out in two stages, the experimental stage where the method was developed and the clinical stage where the method was applied. In the experimental stage, blood culture bottles were created with standard strains and isolates previously diagnosed with the 16S rRNA method. 10% solution of Tween® 80 was prepared with distilled water. 1 ml sample was transferred from the bottle that gave positive signal to the microcentrifuge tube, 100 µl of 10% solution of Tween® 80 was added, vortexed for 10 seconds and then incubated for 5 minutes at room temperature. The tubes were centrifuged for 5 min at 14.000 rpm, the supernatant was discarded and the pellet was washed with 1 ml of distilled water and centrifuged at 14.000 rpm for 5 minutes in three times. Samples taken from the pellets were rubbed on the slide and dried on air. Firstly, 1 µl of 70% formic acid, then 1 µl, of matrix solution was added and it was used after drying. In the second stage of the study, the method was applied to the 502 vials giving positive signal in the Microbiology Laboratory of Ankara University Faculty of Medicine Ibni Sina Hospital between 17 April 2018-31 August 2018 and the results were compared with the subculture results. The results obtained at the end of extraction in the experimental stage were compared with the subculture results and no statistical difference was found. In 383 (82.9%) bottles among 462 (92.1%) bottles with monomicrobial positive cultures, compatible results with the subculture results were obtained. Of the microorganisms correctly identified, 350 (91.3%) were bacteria and 33 (8.7%) were fungi. On the other hand, 216 (56.4%) of the bacteria were gram positive and 134 (34.9%) of them were gram negative bacteria. At least one microorganism was correctly identified in 19 (47.5%) of 40 (7.9%) bottles with polymicrobial blood cultures. Their distribution was gram negative (n= 10) and gram positive (n= 8) and yeast (n= 1). No microorganisms were identified in six bottles with polymicrobial cultures. According to the results, we believe that this in-house method developed using Tween® 80 will be a routinely applicable method for blood culture bottles that give positive signal in microbiology laboratories and it will contribute to the early diagnosis.

Clinical evaluation of microorganisms identification from midstream urine by MALDI-TOF MS with different sample enrichment methods

Objective To evaluate the clinical effect of direct identification of microorganisms from the midstream urine by MALDI-TOF MS combined with separation gel tube and differential centrifugation. Methods A total of 2 150 samples of midstream urine were collected from the outpatient and inpatients in Huadong Hospital affiliated to Fudan University from May 2017 to April 2018, including 934 males and 1 216 females with an age of (72.0±17.5) years. After preliminary quantification by microscopic examination, samples with a bacterial acount of ≥ 105cfu/ml were treated with two kinds of pretreatment methods to enrich the bacteria for directly identification by MS. Based on the results of quantitative urine culture and MS identification,the coincidence rates of two kinds of pretreatment methods combined with MS were analyzed. All statistical analyses were performed using Stata/SE 14.0, differences between the groups were compared by Pearson Chi-square tests. Results 464 out of 2 150 midstream urine samples (21.6%) had a bacterial count ≥ 105cfu/ml after microscopic examination. In traditional culture combined with MALDI-TOF MS, 436 (94.0%) cases were single species, 28 (6.0%) cases were double species. Among single species of bacteria samples infection, the gram-negative bacteria accounted for 78.9% (344/436) , the Gram-positive bacteria accounted for 15.8% (69/436) , and the fungus accounted for 5.3% (23/436) . In two methods that the separation gel tube combined MALDI-TOF MS and the differential centrifugation combined MALDI-TOF MS, the gram-negative bacteria coincidence rates were 92.4% (318/344) and 89.0% (306/344) respectively, the Gram-positive bacteria coincidence rates were 68.1% (47/69) and 62.3% (43/69) respectively, and the fungi coincidence rates were 56.5% (13/23) and 34.8% (8/23) respectively. Meanwhile,the double bacteria infection coincidence rates were 64.3% (18/28) and 60.7% (17/28) . Conclusions The separation gel tube combined MALDI-TOF MS and the differential centrifugation combined MALDI-TOF MS identify directly the gram-negative bacteria in the midstream urine samples with high detection rate and accuracy, and they are rapid and simple pretreatment method, suitable for rapid screening of midstream urine. Key words: Urinary tract infections; Urinalysis; Spectrometry, mass, matrix-assisted laser desorption-ionization; Centrifugation

Review of the impact of MALDI-TOF MS in public health and hospital hygiene, 2018.

IntroductionMALDI-TOF MS represents a new technological era for microbiology laboratories. Improved sample processing and expanded databases have facilitated rapid and direct identification of microorganisms from some clinical samples. Automated analysis of protein spectra from different microbial populations is emerging as a potential tool for epidemiological studies and is expected to impact public health.AimTo demonstrate how implementation of MALDI-TOF MS has changed the way microorganisms are identified, how its applications keep increasing and its impact on public health and hospital hygiene.MethodsA review of the available literature in PubMED, published between 2009 and 2018, was carried out.ResultsOf 9,709 articles retrieved, 108 were included in the review. They show that rapid identification of a growing number of microorganisms using MALDI-TOF MS has allowed for optimisation of patient management through prompt initiation of directed antimicrobial treatment. The diagnosis of Gram-negative bacteraemia directly from blood culture pellets has positively impacted antibiotic streamlining, length of hospital stay and costs per patient. The flexibility of MALDI-TOF MS has encouraged new forms of use, such as detecting antibiotic resistance mechanisms (e.g. carbapenemases), which provides valuable information in a reduced turnaround time. MALDI-TOF MS has also been successfully applied to bacterial typing.ConclusionsMALDI-TOF MS is a powerful method for protein analysis. The increase in speed of pathogen detection enables improvement of antimicrobial therapy, infection prevention and control measures leading to positive impact on public health. For antibiotic susceptibility testing and bacterial typing, it represents a rapid alternative to time-consuming conventional techniques.

Facilitating effective treatment of septicaemia by direct identification of microorganisms in positive BacT/ALERT mds blood cultures using MALDI-TOF MS

Facilitating effective treatment of septicaemia by direct identification of microorganisms in positive BacT/ALERT mds blood cultures using MALDI-TOF MS

Direct identification of microorganisms from thioglycolate broth by MALDI-TOF MS.

We developed an easy MALDI-TOF MS-based assay to identify microorganisms directly from thioglycolate broth. A total of 101 positive thioglycolate broths inoculated with 15 different kinds of samples were evaluated. In 91 samples (90.1%), direct MALDI-TOF MS identifications were the same as those obtained after conventional laboratory procedures including subcultures. In 10 samples misidentified by direct processing, yeasts or mixed cultures grew in the thioglycolate subcultures, or high cellular debris hampered a correct analysis. This rapid method can provide a fast, clinically- relevant species-level identification without disturbing the daily workflow in clinical microbiology laboratories.

Direct identification of microorganisms from positive blood cultures by MALDI-TOF MS using an in-house saponin method

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a fast and reliable method for the identification of bacteria. A MALDI Sepsityper kit is generally used to prepare samples obtained directly from culture bottles. However, the relatively high cost of this kit is a major obstacle to introducing this method into routine clinical use. In this study, the accuracies of three different preparation methods for rapid direct identification of bacteria from positive blood culture bottles by MALDI-TOF MS analysis were compared. In total, 195 positive bottles were included in this study. Overall, 78.5%, 68.7%, and 76.4% of bacteria were correctly identified to the genus level (score ≥1.7) directly from positive blood cultures using the Sepsityper, centrifugation, and saponin methods, respectively. The identification rates using the Sepsityper and saponin methods were significantly higher than that using the centrifugation method (Sepsityper vs. centrifugation, p<0.001; saponin vs. centrifugation, p=0.003). These results suggest that the saponin method is superior to the centrifugation method and comparable to the Sepsityper method in the accuracy of rapid bacterial identification directly from blood culture bottles, and could be a less expensive alternative to the Sepsityper method.

Chemical analysis and medicine

The main directions in using chemical analysis in medicine are considered, such as diagnosis of diseases, sanitation and hygiene control, doping control, direct identification of microorganisms, DNA-analysis, etc.

Integration of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in blood culture diagnostics: a fast and effective approach

Sepsis is a major cause of mortality in hospitalized patients worldwide, with lethality rates ranging from 30 to 70 %. Sepsis is caused by a variety of different pathogens, and rapid diagnosis is of outstanding importance, as early and adequate antimicrobial therapy correlates with positive clinical outcome. In recent years, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) fingerprinting has become a powerful tool in microbiological diagnostics. The direct identification of micro-organisms in a positive blood culture by MALDI-TOF MS can shorten the diagnostic procedure significantly. Therefore, the aim of the present study was to evaluate whether identification rates could be improved by using the new Sepsityper kit from Bruker Daltonics for direct isolation and identification of bacteria from positive blood cultures by MALDI-TOF MS compared with the use of conventional separator gel columns, and to integrate the MALDI-TOF MS-based identification method into the routine course of blood culture diagnostics in the setting of a microbiological laboratory at a university hospital in Germany. The identification of Gram-negative bacteria by MALDI-TOF MS was significantly better using the Sepsityper kit compared with a separator gel tube-based method (99 and 68 % correct identification, respectively). For Gram-positive bacteria, only 73 % were correctly identified by MALDI-TOF with the Sepsityper kit and 59 % with the separator gel tube assay. A major problem of both methods was the poor identification of Gram-positive grape-like clustered cocci. As differentiation of Staphylococcus aureus from coagulase-negative staphylococci is of clinical importance, a PCR was additionally established that was capable of identifying S. aureus directly from positive blood cultures, thus closing this diagnostic gap. Another benefit of the PCR approach is the possibility of directly detecting the genes responsible for meticillin resistance in staphylococci and for vancomycin resistance in enterococci, which is of high importance for early adequate treatment. Both of the described methods were finally integrated into a protocol for fast and effective identification of bacteria from positive blood cultures.

Linking microbial community structure with function: fluorescence in situ hybridization-microautoradiography and isotope arrays

The ecophysiology of microorganisms has been at the heart of microbial ecology since its early days, but only during the past decade have methods become available for cultivation-independent, direct identification of microorganisms in complex communities and for the simultaneous investigation of their activity and substrate uptake patterns. The combination of fluorescence in situ hybridization (FISH) and microautoradiography (MAR) is currently the most widely applied tool for revealing physiological properties of microorganisms in their natural environment with single-cell resolution. For example, this technique has been used in wastewater treatment and marine systems to describe the functional properties of newly discovered species, and to identify microorganisms responsible for key physiological processes. Recently, the scope of FISH-MAR was extended by rendering it quantitative and by combining it with microelectrode measurements or stable isotope probing. Isotope arrays have also been developed that exploit the parallel detection offered by DNA microarrays to measure incorporation of labelled substrate into the rRNA of many community members in a single experiment.

Immunofluorescence in Diagnostic Bacteriology

Fluorescent antibody techniques have shown promise in recent years of becoming a valuable addition to the bacteriologic and serologic procedures of the diagnostic laboratory. They are particularly applicable to the diagnosis of those infections in which rapid identification of the etiologic agent is essential. The feasibility of using fluorescent antibodies to identify micro-organisms directly in smears or in sections of infected tissues has already been demonstrated for a variety of infectious agents. 1-12 These methods seemed especially suitable for a rapid and direct identification of micro-organisms causing bacterial meningitis, where prompt initiation of specific antibiotic therapy depends on the immediate recognition of the etiologic agent. Of the variety of bacterial species capable of causing meningitis in infancy and childhood, Hemophilus influenzae is the most frequent offender. 13,14 Conventional bacteriologic methods rely on presumptive iden tification of the organisms in Gram-stained spinal fluid sediments and confirmation by cultural procedures. Gram-stained smears