Molecular and MALDI Identification Techniques

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The page below is a sample from the LabCE course Fungal Infections in Humans. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Molecular and MALDI Identification Techniques

At present, yeasts are not routinely identified using molecular methods, although methods have been developed for use in research. Specialized equipment, a high degree of expertise, and additional safety measures are required. In addition, DNA sequence databases must be in place and well-established. According to Wickes and Wiederhold62:
  • Polymerase chain reaction (PCR): PCR techniques amplify specific (predetermined) sections of DNA in patient samples. One limitation is the generally low amount of fungal DNA compared with viral or bacterial DNA in patient samples.
  • DNA sequencing: ribosomal RNA gene (rDNA) identification methods are quite useful. While public databases continue to grow, the method is still mostly restricted to research investigators.
  • Whole-genome sequencing (WGS): At this time, remains too expensive, too complex, and too slow. Applications may prove best in the field of public health (epidemiology).
  • DNA hybridization: One vendor offers a rapid DNA probe test (using DNA hybridization) to identify Histoplasma, Blastomyces, and Coccidioides. Culture isolates are used in this method.
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a rapidly growing platform, used to detect a laser to ionize molecules, then detect and measure their mass-to-charge ratio. This results in a "fingerprint" of the organism, which is compared to a known database. Similar to molecular techniques (sequencing), one limitation of MALDI is the limited database.
"MALDI-TOF MS has had good success identifying several major human fungal pathogens, including Aspergillus, Candida, Cryptococcus, and Fusarium species, the Mucorales, dimorphic fungi (Histoplasma capsulatum, Blastomyces dermatitidis, and Coccidioides species), and some dermatophytes."63 Some commercial assays for use on medical laboratory MALDI platforms have been approved for common Candida species, Cryptococcus, or Aspergillus.
Note: Safety considerations must be considered when performing any of these methods. Every mold must be considered a potential pathogen. The yeast forms of suspected dimorphic fungi are also considered dangerous.
The fields of molecular testing and MALDI techniques are rapidly changing. This content has been provided to give a basic idea of some of the methods currently available to researchers, while very few are available in the routine medical microbiology/mycology laboratory. Much research is currently underway.
63. Wickes BL, Wiederhold NP. Molecular diagnostics in medical mycology. Nat Commun. 2018;9(1):5135. Published 2018 Dec 3. doi:10.1038/s41467-018-07556-5
64. Robert MG, Cornet M, Hennebique A, et al. MALDI-TOF MS in a Medical Mycology Laboratory: On Stage and Backstage. Microorganisms. 2021;9(6):1283. Published 2021 Jun 12. doi:10.3390/microorganisms9061283