Evaluation of formic acid sandwich (FA-sandwich): A pretreatment method for filamentous fungi, for the identification of clinically relevant filamentous fungi by two MALDI-TOF MS systems

Abstract

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been successfully applied to identify microorganisms. However, unlike bacteria and yeast where identification results can be obtained rapidly and accurately by using a simple direct-coating pretreatment method, the traditional pretreatment methods for filamentous fungi are more complex, involving ethanol, formic acid, acetonitrile, and a protein extraction process by centrifugation, i.e., the EtOH-FA full extraction. This cumbersome pretreatment for filamentous fungi is a major reason for the lack of widespread use of MALDI-TOF MS for the identification of filamentous fungi in clinical settings. The present study describes an alternative method, the FA-sandwich, and demonstrates that the approach is efficient and effective. 148 clinical filamentous fungal isolates collected from three large general hospitals in Hubei Province, China, were processed by the FA-sandwich method and identified by two MALDI-TOF MS platforms, Autof ms and Vitek MS. The FA-sandwich allowed a 93.9% species-level identification with Autof ms, and 97.3% species-level identification rates were found for Vitek MS when the IVD, the RUO and in-house databases are used in combination. Further comparison of the ease of FA-sandwich with the EtOH-FA full extraction showed that the FA-sandwich is a more convenient, time- and reagent-saving, and sensitive pretreatment method. These findings indicate that the FA-sandwich method is suitable for pretreating filamentous fungi followed by MALDI-TOF MS identification in clinical microbiology laboratories.

Lay summary: The FA-sandwich method improves the efficiency of identification of filamentous fungi using MALDI-TOF MS while ensuring identification accuracy. The method is easy to perform and very suitable for detecting mold in the microbiology laboratory with the goal of promoting timely and accurate therapies.

Keywords: Autof ms; FA-sandwich; Vitek MS; filamentous fungi; identification; pretreatment.