Direct Analysis and Identification of Pathogenic Lichtheimia Species by Matrix-Assisted Laser Desorption Ionization–Time of Flight Analyzer-Mediated Mass Spectrometry

Abstract

Zygomycetes of the order Mucorales can cause life-threatening infections in humans. These mucormycoses are emerging and associated with a rapid tissue destruction and high mortality. The resistance of Mucorales to antimycotic substances varies between and within clinically important genera such as Mucor, Rhizopus, and Lichtheimia. Thus, an accurate diagnosis before onset of antimycotic therapy is recommended. Matrix-assisted laser desorption ionization (MALDI)-time of flight (TOF) mass spectrometry (MS) is a potentially powerful tool to rapidly identify infectious agents on the species level. We investigated the potential of MALDI-TOF MS to differentiate Lichtheimia species, one of the most important agents of mucormycoses. Using the Bruker Daltonics FlexAnalysis (version 3.0) software package, a spectral database library with m/z ratios of 2,000 to 20,000 Da was created for 19 type and reference strains of clinically relevant Zygomycetes of the order Mucorales (12 species in 7 genera). The database was tested for accuracy by use of 34 clinical and environmental isolates of Lichtheimia comprising a total of five species. Our data demonstrate that MALDI-TOF MS can be used to clearly discriminate Lichtheimia species from other pathogenic species of the Mucorales. Furthermore, the method is suitable to discriminate species within the genus. The reliability and robustness of the MALDI-TOF-based identification are evidenced by high score values (above 2.3) for the designation to a certain species and by moderate score values (below 2.0) for the discrimination between clinically relevant (Lichtheimia corymbifera, L. ramosa, and L. ornata) and irrelevant (L. hyalospora and L. sphaerocystis) species. In total, all 34 strains were unequivocally identified by MALDI-TOF MS with score values of >1.8 down to the generic level, 32 out of 34 of the Lichtheimia isolates (except CNM-CM 5399 and FSU 10566) were identified accurately with score values of >2 (probable species identification), and 25 of 34 isolates were identified to the species level with score values of >2.3 (highly probable species identification). The MALDI-TOF MS-based method reported here was found to be reproducible and accurate, with low consumable costs and minimal preparation time.

Matrix-Assisted Laser Desorption Ionization–Time of Flight (MALDI-TOF) Mass Spectrometry Using the Vitek MS System for Rapid and Accurate Identification of Dermatophytes on Solid Cultures

“…It is also most convenient in the routing laboratory, where solid culture media are mainly used; for instance, nails and hairs suspected to contain dermatophytes are generally incubated on agar plates. Identification by MALDI-TOF MS can, therefore, be carried out immediately from material derived from the original culture, with no additional time-consuming steps described for other protocols (28). The use of this protocol allows also the user to visually identify distinct types of molds growing on the same agar plate, thus avoiding the potential pitfall of liquid cultures that could mask the presence of several organisms.…”

Use of the Bruker MALDI Biotyper for Identification of Molds in the Clinical Mycology Laboratory

“…Preparation of mold isolates for MALDI-TOF MS measurement was done as previously described (14,17). Briefly, 1 ml of fungus-containing medium was transferred to a 1.5-ml Eppendorf tube and centrifuged at 16,000 ϫ g for 5 min.…”

“…Compared to that of bacteria, the cell wall of molds is more robust and rigid, making sample preparation more labor-and time-intensive (13,14). Several studies reported the generation of in-house databases for the Bruker MALDI Biotyper (Bruker Daltonik, Bremen, Germany) covering mainly single genera, such as Aspergillus (15), Fusarium (15,16), Lichtheimia (17), or the Pseudallescheria/Scedosporium complex (18); orders, such as the Mucorales (15); and individual groups of fungi, such as the dermatophytes (19,20) or clinically important molds (14,(21)(22)(23). However, the species coverage of most of these fungus databases is limited, and many are neither publicly nor commercially available.…”

Identification of Molds by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

“…In support of this, the results of antifungal susceptibility testing performed on the isolates from that study indicated that discriminating between Fusarium species complexes may be unnecessary for members of the same species complex (25). (26), which is the second (after Rhizopus) most common causative agent of mucormycosis in Europe. Thus, studies related to Rhizopus species have remained neglected so far, despite the fact that the identification of Rhizopus microsporus and Rhizopus arrhizus (previously known as Rhizopus oryzae), for which the antifungal drug susceptibility profiles are known, may be important for the treatment of mucormycosis.…”

“…It is worth noting that A. flavus, the second leading cause of human aspergillosis, is not separable from Aspergillus oryzae by means of molecular biology techniques, whereas the closely related species T. mentagrophytes and T. interdigitale are not separable by means of ITS sequence analysis. In contrast, MALDI-TOF MS seems to be more powerful for discrimination between these species (10,32) as well as to distinguish clinically relevant from irrelevant species of Lichtheimia (26). However, as MALDI-TOF MS-based identification should be consistent with the aspect of the organism in culture, relatively mature growth must be achieved before the identification is certain.…”