Jogarao Vedula (Presenter)
Div. of Clinical Microbiology, Icahn School of Med
Bio: I first had exposure to Clinical MS, studying the N-Glycosylation changes in Immunoglobulin, and characterizing them by MALDI-TOF MS, after separation from sera using HPLC. Following graduation from St. George's Univ. School of Medicine (2015) with a MD, I wanted to further my experience, and exposure in clinical microbiology before applying for a Residency in Pathology. I find the application of MALDI-TOF MS a fascinating, and key innovation in the accurate and reliable identification of microorganisms. This in turn allows for appropriate targeting of antimicrobial therapy in a far shorter time than conventional methods.
Authorship: Jogarao Vedula (1), Rose Hanna (2), Michelle Lam (1), Camille Hamula (1,2), Tanis Dingle (1,2)
(1) Division of Clinical Microbiology, Department of Pathology, Icahn School of Medicine at Mount Sinai, Mount Sinai Health System, New York, NY, United States of America, (2) Division of Clinical Microbiology, Department of Pathology, Mount Sinai Beth Israel, Mount Sinai Health System, New York, NY
| Short Abstract In this study, the accuracy of identification of anaerobic microorganisms from two different anaerobic blood culture (BC) media using the Bruker MALDI Sepsityper kit followed by MALDI-TOF MS was evaluated. Comparison of the BACTEC and BacT/ALERT systems for detection of anaerobic bacteria showed that the BACTEC instrument was more rapidly able to detect anaerobic organisms spiked into the BACTEC BC bottles (median 43.13h) as compared to the BacT/ALERT BC bottles (median 65.12h). Conversely, anaerobic organisms present in the BacT/ALERT BC bottles were more accurately identified by MALDI-TOF MS after Sepsityper processing (15/26; 58%) then the BACTEC BC bottles (6/26; 23%). The MALDI Sepsityper method must be further refined for identification of anaerobes from positive blood culture bottles to enable more accurate identification of these organisms. |
Long Abstract
Introduction:
Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) has emerged as an accurate, rapid, and reliable method compared to conventional phenotypic methods to identify organisms in the microbiology laboratory. Two of the primary MALDI-TOF MS methods used currently in clinical microbiology laboratories include identification of isolates growing on solid culture media and direct identification of organisms from positive blood culture (BC) bottles. Anaerobic bacteria are commonly known to be fastidious, slow growing, and difficult to identify by traditional identification methods. As such, identification of anaerobes causing bacteremia can take several days after a blood culture becomes positive. The purpose of our study is to compare two automated blood culture instruments and anaerobic blood culture media in their ability to detect anaerobic microorganisms, and the subsequent identification of these anaerobic microorganisms by MALDI-TOF MS using the Bruker MALDI Sepsityper kit.
Methods:
Clinical anaerobic isolates previously identified by VITEK II (Biomerieux Diagnostics, France) or RapID ANA II (Lenexa, KS), from Mount Sinai Hospital and Mount Sinai Beth Israel Hospital, were collected and stored at -70°C. The isolates (n=33) in the study included, Propionibacterium acnes (4), Actinobaculum schalii (2), Bacteroides fragilis (2), Clostridium perfringens (2), Bacteroides ovatus (2), Bacteroides thetaiotomicron (2), Finegoldia magna (2), Peptostreptococcus anaerobius (2), Atopobium parvulum (2), Peptostreptococcus micros (2), Eubacterium limosum (2), Propionibacterium granulosum (2), and Actinomyces meyeri, Bacteroides caccae, Bacteroides vulgatus, Bacteroides uniformis, Clostridium sporogenes, Eggerthella lenta, Fusobacterium necrophorum, (1 each respectively).
The anaerobic isolates were sub-cultured onto CDC anaerobic 5% sheep blood agar and incubated under anaerobic conditions at 37°C for 48 hours. Five mL of human packed red blood cells and 10-100 CFU of each isolate were inoculated into both a BACTEC Lytic Anaerobic (BD Diagnostics, Sparks MD) BC bottle, and a BacT/ALERT-FN Plus (BioMérieux) BC bottle. Bottles were loaded into either the BACTEC FX (BD Diagnostics, Sparks MD) or the BacT/ALERT ALERT 3D (BioMérieux) system. The bottles were kept in the automated systems for a total of seven days or until signaling positive. Bottles signaling positive were subsequently Gram stained, and processed by the Bruker MALDI Sepsityper kit and evaluated by MALDI-TOF MS (Bruker Daltonics, Billerica, MA; MALDI Biotyper Database v3.1) according to manufacturer’s recommendations. Due to increased background peaks from blood proteins and resins, a lower log (score) value of >1.7 was considered acceptable for both genus and species level classification.
Results:
A total of 33 anaerobic bacteria were spiked into 66 blood culture bottles (33 BACTEC and 33 BacT/ALERT). Over the course of the seven days of incubation, 26/33 (79%) of the BACTEC BC bottles, and 26/33 (79%) of BacT/ALERT BC bottles signaled positive. It was observed that both P. anaerobius isolates failed to be detected by either BACTEC or BacT/ALERT. BACTEC failed to detect E. lenta, C. sporogenes and both P.micros isolates. BacT/ALERT failed to detect A. meyeri, and one B.ovatus, which were both detected by BACTEC.
When the times to detection of the spiked blood cultures were analyzed, the BACTEC had a median detection time of 43.13 hr, and a mean detection time of 54.79 hr with a standard deviation of 34.88 hr. BacT/ALERT had a median detection time of 65.12 hr, and a mean detection time of 76.83 hr with a standard deviation of 44.86 hr.
Of the 52 BC bottles (26 BACTEC and 26 BacT/ALERT) that flagged positive by both systems, 21/26 (81%) of the BACTEC BC bottles and 21/26 (81%) of the BacT/ALERT BC bottles had measurable spectra and gave an organism ID by MALDI Sepsityper coupled with MALDI-TOF MS. However, for the BACTEC only 6/26 (23%) gave a correct genus level classification, and only 4/26 (15%) gave a correct genus and species level classification. In contrast, the BacT/ALERT 16/26 (62%) gave a correct genus level classification, and 15/26 (58%) gave a correct genus and species level identification.
Discussion:
Rapid identification of pathogens involved in sepsis can allow patients to be placed on appropriate therapy in a timely manner. Anaerobes have traditionally been difficult to identify accurately by biochemical methods. MALDI-TOF MS has proven to be an alternate method for identification of some, but not all anaerobes. Comparison of the BACTEC and BacT/ALERT systems for detection of anaerobic bacteria showed that the BACTEC instrument was more rapidly able to detect anaerobic organisms spiked into the BACTEC Lytic Anaerobic medium. Conversely, anaerobic organisms present in the BacT/ALERT Anaerobic Plus FN bottles were more accurately identified after processing with the MALDI Sepsityper Kit and MALDI-TOF MS than organisms in the BACTEC Lytic Anaerobic bottles. We hypothesize that the differences observed in the ability of MALDI-TOF MS to identify anaerobic organisms in the two different BC bottles is due to differences in media composition and also differences in the limits of detection of both instruments. Further evaluation of the MALDI Sepsityper protocol for anaerobes is necessary to increase the accuracy of anaerobic identification by this method.
References & Acknowledgements:
Almuhayawi, M. et al. “The Performance of the Four Anaerobic Blood Culture Bottles BacT/ALERT-FN, -FN Plus, BACTEC-Plus and -Lytic in Detection of Anaerobic Bacteria and Identification by Direct MALDI-TOF MS.” PLoS ONE. 2015; 10(11): e0142398.
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Morgenthaler, N.G., and Markus Kostrzewa. “Rapid Identification of Pathogens in Positive Blood Culture of Patients with Sepsis: Review and Meta-Analysis of the Performance of the Sepsityper Kit.” International Journal of Microbiology. 2015: 827416.
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