Real-Time Identification of Bacteria and Candida Species in Positive Blood Culture Broths by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Abstract

Delays in the identification of microorganisms are a barrier to the establishment of adequate empirical antibiotic therapy of bacteremia. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) allows the identification of microorganisms directly from colonies within minutes. In this study, we have adapted and tested this technology for use with blood culture broths, thus allowing identification in less than 30 min once the blood culture is detected as positive. Our method is based on the selective recovery of bacteria by adding a detergent that solubilizes blood cells but not microbial membranes. Microorganisms are then extracted by centrifugation and analyzed by MALDI-TOF-MS. This strategy was first tested by inoculating various bacterial and fungal species into negative blood culture bottles. We then tested positive patient blood or fluid samples grown in blood culture bottles, and the results obtained by MALDI-TOF-MS were compared with those obtained using conventional strategies. Three hundred twelve spiked bottles and 434 positive cultures from patients were analyzed. Among monomicrobial fluids, MALDI-TOF-MS allowed a reliable identification at the species, group, and genus/ family level in 91%, 5%, and 2% of cases, respectively, in 20 min. In only 2% of these samples, MALDI-TOF MS did not yield any result. When blood cultures were multibacterial, identification was improved by using specific databases based on the Gram staining results. MALDI-TOF-MS is currently the fastest technique to accurately identify microorganisms grown in positive blood culture broths.Blood cultures in liquid medium are the gold standard for the diagnosis of bloodstream infections. Species identification of bacteria that have grown in this biological fluid first requires an overnight subculture on solid agar medium, thus delaying the precise identification of the bacteria by 24 to 48 h. For bacteremic patients, this requirement prevents the rapid prescription of an adequate empirical anti-infective therapy prior to obtaining the results of the antibiotic sensitivity testing. This empirical therapy may be roughly adjusted on the basis of the Gram staining. However, these microscopic results are not accurate enough to reduce the patient's exposure to ineffective antibiotic therapy. In order to reduce the time required for the identification of microorganisms in blood cultures, various methods have been proposed, including identification using automated systems into which fluids from positive blood cultures are directly inoculated, fluorescent in situ hybridization (FISH), and PCR followed by sequencing, hybridization, pyrosequencing, or single-stranded conformation polymorphism. All these methods are expensive and require several hours (2, 4, 7-9, 12-15, 17-24, 26, 28, 29).Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) allows rapid identification of bacteria grown on solid media by the identification of species-specific profiles obtained from isolated ...

Comparison of two matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry methods and API 20AN for identification of clinically relevant anaerobic bacteria

“…They are used to identify aerobic and anaerobic bacteria (Fedorko et al, 2012;Nagy et al, 2009Nagy et al, , 2012van Veen et al, 2010;Veloo et al, 2011b), mycobacteria (Saleeb et al, 2011), Nocardia (Verroken et al, 2010) and yeasts (van Veen et al, 2010) isolated on solid media from clinical specimens. MALDI-TOF MS has also been recently used for the identification of bacteria and yeasts directly from positive blood culture bottles (Ferroni et al, 2010;Stevenson et al, 2010). The MALDI-TOF MS system appears to be associated with rapid turnaround time, low sample volume requirements and modest reagent costs.…”

An Evaluation Of Direct Identification Of Pathogens From Blood Cultures By Maldi-Tof Mass Spectrometry

“…Identification of microorganisms from positive blood culture using of MALDI Sepsity per processing kit requires approximately 20 minutes and it does independent of species (6, 10,13,18,20,25,26,32,33). Batch processing added approximately 1 min per blood culture analyzed.Our estimated time to identify bacteria from the positive blood culture, of fixed automated system BD BACTEC Fx signal time to MALDI-TOF mass spectrometry identification time, is not an objective, it includes the blood smear preparation time by Gram staining, …”

“…With a special set of MALDI Sepsityper is possible to identify bacteria directly from the positive blood culture (17)(18)(19)(20)(21)(22)(23)(24)(25). Most reports describe the blood culture must be grown in automated BACTEC system vials (26,27). MALDI-TOF mass spectrometry has a high sensitivity and specificity (32).…”

Utilisation en routine du MALDI-TOF-MS pour l’identification des pathogènes en microbiologie médicale

“…Parmi les cocci à Gram positif, les streptocoques du groupe mitis sont particulièrement difficiles à identifier et à distinguer de Streptococcus pneumoniae [13][14][15]. Une autre application de cette technologie est l'identification des bactéries directement à partir des prélèvements, soit à partir de flacons d'hémocultures détectés comme positifs [19][20][21][22][23][24][25], soit à partir des urines [23]. Le gain de temps est alors très important pour la mise en route d'une antibiothérapie.…”