Back to the Basics: Biochemical Testing for Pathogen Identification in the Era of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS)

LETTER

Inarguably, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has revolutionized the pathogen identification process in the clinical microbiology laboratory. However, identification of closely related species of organisms using MALDI-TOF MS remains challenging (1–3). Our clinical laboratory implemented MALDI-TOF MS (Bruker Daltonics, Billerica, MA) in 2013 with the RUO MALDI Biotyper Compass Library, and it expeditiously became the primary method of identification for ∼90% of cultures, replacing most biochemical tests. However, in the past 3 years, we have encountered several challenges that have tested our confidence in the system and have necessitated the reintroduction of key biochemical tests alongside strict protocols. Here, we briefly touch on some MALDI-TOF MS species identifications using the RUO MALDI Biotyper Compass Library, revision D, that currently trigger orthogonal testing algorithms in our laboratory.

A critical case of misidentification with detrimental consequences prompted the initiation of steps to change testing practices in our laboratory. A Neisseria species isolated from the throat culture of a cystic fibrosis patient was misidentified by MALDI-TOF MS as Neisseria gonorrhoeae, with a logarithmic score of 1.93. The organism was reported in the clinical record as N. gonorrhoeae, since the laboratory threshold for reporting organisms to the species level is set at ≥1.90 (4). Examination of MALDI-TOF MS results revealed that although N. gonorrhoeae had the top score of 1.93, Neisseria meningitidis had the second and third top scores, at 1.85 and 1.78, respectively. The RUO MALDI Biotyper Compass Library, revision D database currently includes 145 Neisseria species. We also ran the spectrum obtained against the MALDI Biotyper CA system and the CDC MicrobeNet database, both of which identified the isolate as Neisseria gonorrhoeae, with the same score as the RUO database. The isolate was subsequently identified as N. meningitidis/Neisseria cinerea using 16S rRNA gene sequencing. Misidentification of commensal Neisseria species as pathogenic Neisseria by MALDI-TOF MS has also previously been reported (2, 5). The incorrect identification triggered significant clinical, social, and public health repercussions for the patient and family.

This encounter prompted implementation of stringent measures when Neisseria spp. are identified by MALDI-TOF MS, including reinstating the RapID NH (Thermo Scientific, Canoga Park, CA) panel to complement MALDI-TOF MS identification when species level identification is warranted and increasing scoring criteria to ≥2.2 for all N. gonorrhoeae isolates, followed by consultation with the microbiology director. We selected the score of ≥2.2 for species level identification of N. gonorrhoeae based on the following factors: (i) past studies have reported misidentifications of Neisseria species even at a score of >2.0 (2), (ii) in a small verification conducted in our laboratory as well as in retrospective data analysis of past isolates tested by MALDI-TOF MS, we found that all N. gonorrhoeae isolates tested had scores of at least 2.2, and (iii) lastly, we opted to be more conservative with our score in response to the significant clinical and social impact that was encountered in the case described.

Likewise, frequent inability of MALDI-TOF MS to differentiate between Citrobacter species and Salmonella species (at scores of >2.0) has necessitated further identification using the Phoenix identification system (BD, Franklin Lakes, NJ). We have encountered discrepancies where the top five logarithmic scores from MALDI-TOF MS were between Salmonella versus Citrobacter species in all 11 blood cultures positive for Salmonella species in the past 3 years. In all cases, the isolates were confirmed to be Salmonella species by Phoenix identification system (BD). For laboratories without access to automated identification methods/instruments, urea agar slants combined with citrate agar slants can also aid in screening for commonly encountered Citrobacter species. If biochemical results are inconclusive for Salmonella species, isolates should be referred to public health laboratory for confirmation. Similarly, we isolated Klebsiella variicola from blood cultures in two patients, and in both cases the top five identifications varied between Klebsiella pneumoniae and Klebsiella variicola, with scores of >1.9. The inability of MALDI-TOF MS to distinguish between Klebsiella variicola and Klebsiella pneumoniae has been previously documented (3, 6). Discrepancies between Streptococcus pyogenes and Streptococcus dysgalactiae were also encountered for five patients where scores of >1.9 were reported for both species. Lancefield antigen typing or PCR-based methods were then performed to obtain definitive identification. Two isolates were identified as S. pyogenes and the remaining three as S. dysgalactiae. At least three instances of inconsistent species-level identification between Haemophilus influenzae and Haemophilus haemolyticus at a score of >1.9 were also encountered. Finally, differentiating Streptococcus pneumoniae from other species of the viridans group streptococci is a known issue (7). Hence, confirmation using latex agglutination testing in conjunction with testing for sensitivity to sodium deoxycholate is performed by our laboratory for definitive identification of Streptococcus pneumoniae. However, possible resistance to sodium deoxycholate should also be considered. All of the examples mentioned above were documented by our laboratory in the past 3 years. To provide a clear protocol that prevents routine misidentification, we now also require examination of the top ten scores generated by MALDI-TOF MS before reporting, with consistent scores of >1.9 to the species level for at least the top five. We find this approach particularly helpful in differentiating members of the Streptococcus mitis and Bacillus cereus groups, as well as species of Staphylococcus.

Misidentifications using MALDI TOF MS-based methods have been reported for both Bruker and Vitek MS (bioMérieux, St. Louis, MO) instruments. Past studies have independently reported misidentification of Klebsiella variicola as Klebsiella pneumoniae using the Vitek instrument (3, 6). Misidentifications of other organisms using the Vitek instrument have also been reported (1, 8). The correct identification in these instances could only be established using biochemical and/or genetic testing. Although MALDI-TOF MS is agnostic to culture medium in most cases, Lagacé-Wiens et al. recently showed that the use of chromogenic media can lead to misidentification of Mycoplasma alkalescens and Mycoplasma arginini (9).

Assessing the top 10 scores alongside colony morphology and specimen source, followed by consultation with the laboratory director by laboratory personnel can reduce the chances of incorrect identification and misreporting. MALDI-TOF MS continues to be essential in our laboratory, but there is heightened awareness of its limitations. Clinical laboratories must have protocols in place that maximizes the strengths of the technology while averting false identification of certain organisms. Despite their shortcomings, such as extended incubation periods and user-dependent judgments, conventional identification approaches continue to be advantageous in certain scenarios.