Identification of Blood Culture Isolates Directly from Positive Blood Cultures by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry and a Commercial Extraction System: Analysis of Performance, Cost, and Turnaround Time

Abstract

M atrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has been routinely used for the identification of bacteria and fungi from agar culture in many centers in Europe and is increasingly used in North America and elsewhere for primary identification of microorganisms (1, 2, 25). MALDI-TOF instruments use an ionizing laser to vaporize the abundant structural elements (primarily ribosomal proteins) of bacteria and yeasts and analyze the weight and relative abundance of each particle to generate a spectrum. Spectra are compared to a computer database of reference or user-defined organism spectra, and identification is obtained by matching the most similar spectrum in the database to the unknown organism. Performance of the Bruker MALDI BioTyper has been extensively studied in multiple centers and confirms that reliable identification can be obtained for Ͼ95% of the isolates grown on solid media routinely encountered in the clinical laboratory (1,4,5,8,19).More recently, protocols for the direct identification of pathogens from positive blood culture broths have been developed (3,12,16,21,22). Although blood culture broths are usually monobacterial (or monofungal) cultures, the presence of proteins from red cells, white blood cells, and serum interferes with the analysis by adding spectral peaks not found in the organism database. Furthermore, interfering substances such as charcoal (when present) and low organism numbers (as might be encountered with slow-growing or contaminating bacteria) present additional challenges in the use and interpretation of MALDI-TOF spectra for identification pathogens directly from positive blood cultures (24). As a result, many centers have developed in-house methods for preprocessing of blood cultures to optimize recovery of the bacterial proteome. More recently, a commercial kit (Bruker Sepsityper) has been released to simplify the processing steps required for the purification and extraction of the bacterial proteome from positive blood cultures. The system serves to facilitate preprocessing and minimize the impact of the interfering human proteome on the MALDI-TOF analysis. Here we report the performance of the Sepsityper system on the Bruker MALDI BioTyper for the direct identification of pathogens from blood cultures and a cost and turnaround time analysis of the results. MATERIALS AND METHODS Blood cultures.Blood was collected at the bedside and directly inoculated into BacT/Alert SA (aerobic culture) and/or SN (anaerobic) (bioMérieux, Marcy l'Etoile, France). Both bottle types are charcoal free. Bottles were loaded onto the a BacT/Alert instrument (bioMérieux, Marcy l'Etoile, France) and incubated. Bottles were incubated for up to 5 days, and when the operator was notified of a positive blood culture, a Gram stain was performed. All positive bottles were subjected to subculture and routine

Identification of Microorganisms Grown in Blood Culture Flasks using Liquid Chromatography–Tandem Mass Spectrometry

“…The result of one strain of E.coli, that was cultured four-times, and identified correctly in all cases, indicates the robustness of the method. The reported identification rates with MALDI-TOF MS directly from positive blood cultures ranged from 66 to 85.2% for monomicrobial blood cultures, which is lower compared with the 100% identification found using the LC-MS/MS method [9,[23][24][25][26].…”

A commentary on the role of molecular technology and automation in clinical diagnostics

“…22 Exemplifying this, previously technically-challenging identification of microorganisms from sputum of cystic fibrosis patients, 8 utilizing multiple selective media and prolonged incubation, may now be completed within 48 hours of sampling. 23 More specifically, a 2012 Canadian study 24 reported a mean reduction in turnaround time of 34.3 hours when definitive identification of isolates directly from blood samples was possible, albeit that this reduction was lower, but still clinically important, at 26.5 hours when incubation was necessary before MALDI-TOF MS could be utilized. These advances are especially relevant clinically as identification of Gram-negative and Gram-positive bacteria as well as yeasts can now be obtained directly from 7-8 ml of blood when using systems such as the MALDI Sepsityper ® Kit (BrukerDaltonics 2010), thereby eliminating incubation times associated with growth on solid media (Fig.…”

La espectrometría de masas MALDI-TOF en el laboratorio de microbiología clínica

“…El estudio de Lagacé-Wiens y colaboradores 41 , mediante el uso del kit de preparación de la muestra Sepsityper ® (Bruker Daltonik GmbH, Bremen, Alemania) y análisis por MALDI Biotyper ® de 61 hemocultivos monomicrobianos y dos hemocultivos polimicrobianos encontró una concordancia del 95,1% frente a los métodos convencionales; con una reducción del tiempo promedio del resultado de 34,3 horas en un escenario ideal y de 26,5 horas en una situación más práctica en la que los microorganismos requieren subcultivos y pruebas adicionales para su caracterización definitiva. Un meta-análisis publicado recientemente, que incluyó 21 artículos de investigación que evaluaron el desempeño del estuche Sepsityper ® en el sistema de espectrometría de masas Microflex Biotyper ® , encontró un porcentaje global de identificación correcta a nivel de especie en el 80% de 3320 botellas de hemocultivo positivo.…”