Optimal turnaround time for direct identification of microorganisms by mass spectrometry in blood culture

https://doi.org/10.1016/j.mimet.2016.08.019Get rights and content

Highlights

  • Identification of microorganisms directly performed from positive blood culture.

  • A short hands-on time and a turnaround time shorter than 15 min

  • 77.8% of detection at the species level with positive predictive value of 99.5%

  • The method can easily take place in the workflow of a laboratory with low staff.

Abstract

Introduction

During the past few years, several studies describing direct identification of bacteria from blood culture using mass spectrometry have been published.

These methods cannot, however, be easily integrated into a common laboratory workflow because of the high hands-on time they require. In this paper, we propose a new method of identification with a short hands-on time and a turnaround time shorter than 15 min.

Materials and methods

Positive blood bottles were homogenised and 600 μL of blood were transferred to an Eppendorf tube where 600 μL of lysis buffer were added. After homogenisation, a centrifugation step of 4 min at 10,500g was performed and the supernatant was discarded. The pellet was then washed and loaded in quadruplicate into wells of a Vitek® MS-DS plate. Each well was covered with a saturated matrix solution and a MALDI-TOF mass spectrometry analysis was performed. Species were identified using the software Myla 3.2.0-2.

Results

We analysed 266 positive blood culture bottles. A microorganism grew in 261 cultures, while five bottles remained sterile after 48 h of incubation in subculture. Our method reaches a probability of detection at the species level of 77.8% (203/261) with a positive predictive value of 99.5% (202/203).

Conclusion

We developed a new method for the identification of microorganisms using mass spectrometry, directly performed from a positive blood culture. This method has short hands-on time and turnaround time and can easily take place in the workflow of a laboratory, with comparable results in performance with other methods reported in the literature.

Introduction

Bloodstream infections are severe pathologies associated with high rates of morbidity and mortality in hospitals (Wisplinghoff et al., 2004). Blood culture is the gold standard for detecting and identifying microorganisms causing sepsis (Dellinger et al., 2013) and rapid identification of pathogenic organisms from blood cultures enables early initiation of antibiotic treatment in patients with bacteraemia (Seifert, 2009).

Introduction of Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) in microbiology laboratories allowed a significant time saving for identification of microorganisms after using solid phase media. This technology generates characteristic mass spectra that are unique signatures for each microorganism. The major limitation is the amount of bacteria present in the samples and the limit of detection of current MALDI-TOF protocols (Croxatto et al., 2012).

Conventionally, identification using MALDI-TOF MS is performed on colonies grown on agar plate after 18–48 h of incubation. During the past few years, several studies related to direct identification of bacteria from blood culture using mass spectrometry have been published (Bessède et al., 2014, Verroken et al., 2015, Machen et al., 2014, Jakovljev and Bergh, 2015, Frederic et al., 2015, Monteiro et al., 2015, Rodríguez-Sánchez et al., 2013, Lagacé-Wiens et al., 2012). The reported methods were based on identification after a short period of subculture from 3 to 5 h after the bottle became positive (Bessède et al., 2014, Verroken et al., 2015); or an aliquot directly taken from blood culture bottle when it becomes positive (Machen et al., 2014, Jakovljev and Bergh, 2015, Frederic et al., 2015, Monteiro et al., 2015, Rodríguez-Sánchez et al., 2013, Lagacé-Wiens et al., 2012). These methods cannot, however, be easily integrated into a common laboratory workflow because of the variety of the internal organization of microbiology laboratories (working 24 h/24 or not, dedicated staff, etc.) and the high hands-on time they may require.

In this paper, we propose a new method of identification, directly performed from blood culture, with a short hands-on time and a turnaround time smaller than 15 min once the bottle became positive and the moment we have identified the pathogen. The results were compared with the classical process used in our laboratory.

Section snippets

Blood culture collection and classical process

From August 2015 to December 2015, blood cultures bottles from adult patients were collected at the Laboratory of Clinical Biology of the Cliniques du Sud Luxembourg in Arlon, Belgium. They were systematically collected in BacT/Alert® FA Plus aerobic and BacT/Alert® SN anaerobic bottles (Biomérieux®, Marcy-l'Etoile, France). After the reception and encoding at the laboratory, the bottles were immediately incubated at 37 °C in the BacT/Alert 3D system (Biomérieux®, Marcy-l'Etoile, France) for a

Results

During the period of the study, we analysed 266 positive blood culture bottles: 157 from aerobic and 109 from anaerobic bottles. A total of 279 microorganisms grew in 261 cultures, while five bottles remained sterile after 48 h of incubation in subculture. Identification of a microorganism was possible in 214 of the 261 positive bottles (82.0%) through direct identification with a matching of 97.7% with the culture (209/214). These results correspond with a detection of 78.5% for the positive

Turnaround time

Rapid identification of pathogenic organisms from blood cultures enables early initiation and adaptation of antibiotic treatment in patients with bacteraemia. We developed a new method, which involves taking an aliquot from blood culture bottle as soon as it becomes positive, without the incubation step on agar plates. We therefore obtained a turnaround time for pathogen identification shorter than 15 min. Furthermore, if we do not take in consideration the identification time spent in the Vitek

Conclusion

We developed a new faster and easier method for the identification of microorganisms using mass spectrometry, directly performed from a positive blood culture. This method has short hands-on time and turnaround time and takes place easily in the workflow of the laboratory, with comparable results in performance with other methods reported in the literature.

Conflict of interest

The authors declare no conflicts of interest.

References (12)

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