Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for the Identification of Clinically Relevant Bacteria

Petrini O (2011) Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for the Identification of Clinically Relevant Bacteria. PLoS ONE 6(1): e16424. doi:10.1371/journal.pone.0016424 Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for the Identification of Clinically Relevant Bacteria Cinzia Benagli 0 Viviana Rossi 0 Marisa Dolina 0 Mauro Tonolla 0 Orlando Petrini 0 Niyaz Ahmed, University of Hyderabad, India 0 1 Cantonal Institute of Microbiology, Bellinzona, Switzerland, 2 Microbiology Unit, Plant Biology Department, University of Geneva , Switzerland Background: Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) allows rapid and reliable identification of microorganisms, particularly clinically important pathogens. Methodology/Principal Findings: We compared the identification efficiency of MALDI-TOF MS with that of PhoenixH, APIH and 16S ribosomal DNA sequence analysis on 1,019 strains obtained from routine diagnostics. Further, we determined the agreement of MALDI-TOF MS identifications as compared to 16S gene sequencing for additional 545 strains belonging to species of Enterococcus, Gardnerella, Staphylococcus, and Streptococcus. For 94.7% of the isolates MALDI-TOF MS results were identical with those obtained with conventional systems. 16S sequencing confirmed MALDI-TOF MS identification in 63% of the discordant results. Agreement of identification of Gardnerella, Enterococcus, Streptococcus and Staphylococcus species between MALDI-TOF MS and traditional method was high (Crohn's kappa values: 0.9 to 0.93). Conclusions/Significance: MALDI-TOF MS represents a rapid, reliable and cost-effective identification technique for clinically relevant bacteria. - Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is rapidly attracting the interest of microbiologists working in the routine labs, because of its powerful features that allow rapid and reliable identification of microorganisms. Standardized test systems such as APIH and VITEKH 2 (bioMerieux), or PHOENIXH (BD Diagnostics), complemented by traditional culture and microscopy methods, have so far been used in routine labs for the rapid identification of clinical microorganisms. With the introduction of these methods, the average time needed for a reliable and validated identification ranged from 6 h to 18 h and in the last few years, sequence analysis of small-subunit rRNAs or selected genes by PCR methods has complemented the biochemical methods, additionally decreasing throughput time and becoming in several cases the gold standard [1]. The recent developments of MALDI-TOF MS are rapidly changing the routine diagnostics scene. MALDI-TOF MS is a powerful method to detect and identify proteins by molecular weight determination of individual, specific fragments [2]. The method is accurate and easy to use, allowing quick determination of molecular weights of proteins with minimal sample requirements. MALDI-TOF MS is now widely used for the identification and characterization of clinically important microorganisms [3]. The currently available identification databases target the identification of human pathogens [4] and MALDI-TOF MS represents a valid and rapid alternative to conventional methods of identification and classification of human pathogens in microbiology. Traditionally, validation of a new identification system to be introduced in routine diagnostics consists of running parallel identifications of a large number of isolates using the new method concomitantly with set standards. In this study we compared the identification efficiency of MALDI-TOF MS with that of PhoenixH, APIH and 16S ribosomal DNA sequence analysis. In a first step we analyzed 1,019 strains obtained sequentially during three months from our routine diagnostic laboratory. In a second step, we studied in more detail 545 isolates of species belonging to the genera Enterococcus, Gardnerella, Staphylococcus, and Streptococcus and determined the agreement (and, when possible, efficiency, sensitivity and specificity) of the MALDI-TOF MS identifications as compared to 16S gene sequencing as the gold standard. In a first step we analysed 1,019 strains obtained from the routine diagnostic lab. The results are described in Fig. 1. For 965 isolates (94.7%) the results of MALDI-TOF MS were identical with those obtained with the BD PHOENIX system and the confidence level of the MALDI-TOF MS identification was almost 100% for approximately 75% of the isolates tested. API or 16S sequencing confirmed MALDI-TOF MS identification in 63% of the discordant results. Overall, therefore, MALDI-TOF was able to identify correctly more than 98% of the isolates tested. Table 2 reports the specificity, sensitivity, PPV, NPV and efficiency values of the MALDI-TOF MS identification, compared to the classical methods, for those bacterial species for which we could analyse at least 15 isolates. With the exception of Enterobacter cloacae and Klebsiella oxytoca, for which the sensitivity values for identifications at the confidence level of at least 90% are relatively low, MALDI-TOF MS has a quite high efficiency of identification. In most cases these values are still even if the limit of acceptance of the confidence level for correct identification by MALDI-TOF MS is set at 99.9% (Table 2). No. of isolates *includes Aerococcus (1), Aeromonas (3), Alcaligenes (2), Bacillus (1), Chryseobacterium (2), Corynebacterium (3), Delftia (1), Hafnia (3), Micrococcus (2), Pasteurella (3), Providencia (2), Raoultella (1), Shewanella (1), Vibrio (1). doi:10.1371/journal.pone.0016424.t001 We analysed separately 76 isolates of Gardnerella, 50 of Enterococcus, and 76 of Streptococcus (mainly S. agalactiae and S. pneumoniae) by MALDI-TOF MS, API/PHOENIX and 16S sequencing. The results are described in Table 3. For Gardnerella spp., the %AI was almost 100%. Very good concordance between 16S sequencing and MALDI-TOF MS was also observed for Enterococcus faecalis and E. faecium, for which the concordance was 100%. Results for E. gallinarum were not particularly good (44.4%), but the number of isolates investigated (9) was low. MALDI-TOF MS showed a fair performance also with Streptococcus spp., with quite high sensitivity and specificity values for S. agalactiae and S. pneumoniae (Table 4). In a separate experiment we evaluated the efficiency of MALDI-TOF MS in the identification of Staphylococcus spp. We considered a sample of 343 staphylococci belonging to 17 species (S. aureus, S. auricularis, S. capitis, S. carnosus, S. cohnii, S. epidermidis, S. equorum, S. haemolyticus, S. hominis, S. lugdunensis, S. pasteuri, S. saprophyticus, S. schleiferi, S. sciuri, S. simulans, S. warneri, S. xylosus) and we computed sensitivity, specificity, PPV and NPV values for the four most commonly isolated taxa in our routine laboratory (S. aureus, S. epidermidis (...truncated)