Identification of Demodex Hominis using Matrix-assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry

This research explored the utilization of Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for developmental stage differentiation in Demodex hominis and the creation of a reliable spectral reference database. VITEK® Mass Spectrometry (MS) was employed to capture the spectral profiles, with SARAMIS™ super spectra tool establishing the reference database. Validation experiments and blinded assessments demonstrated that species-level and developmental stage identification accuracy was confirmed through SARAMIS™ software evaluation. Consistent and reproducible MS profiles were obtained in 28 specimens. A spectrum database, including 4 super spectra, revealed 15 discriminating mass peaks. Peaks 4304.7 and 8609.8 were specific peaks for Demodex hominis. Peaks 3346.8 and 5236.6 are shown in adult Demodex brevis spectra. Similarly, peaks 3788.2 and 7673.3 emerged in the larva Demodex brevis. Peaks 4109.6, 5383.2, and 7518.3 presented in adult Demodex follicurolum spectra. Peak 4696.8 appeared only in larva Demodex follicurolum. Validation experiments revealed successful developmental stage determination in 44 out of 48 target Demodex samples during blinded testing. This research demonstrates the diagnostic potential of MALDI-TOF-MS-based proteomic analysis for precise differentiation of Demodex hominis subspecies and their maturation phases. A preliminary spectral reference database has been compiled, documenting protein profiles of two clinically significant Demodex hominis variants identified in China.