Use of the proteomic tool MALDI-TOF MS in termite identification

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) has proved effective for the identification of many arthropods. A total of 432 termite specimens were collected in Mali, Cote d’Ivoire, Togo, Senegal, Switzerland and France. Morphologically, 22 species were identified, including Ancistrotermes cavithorax, Amitermes evuncifer, Cryptotermes brevis, Cubitermes orthognathus, Kalotermes flavicollis, Macrotermes bellicosus, Macrotermes herus, Macrotermes ivorensis, Macrotermes subhyalinus, Microcerotermes parvus, Microtermes sp., Odontotermes latericius, Procubitermes sjostedti, Promirotermes holmgreni, Reticulitermes grassei, Reticulitermes lucifugus, Reticulitermes santonensis, Trinervitermes geminatus, Trinervitermes occidentalis, Trinervitermes togoensis, Trinervitermes sp., Trinervitermes trinervoides and Trinervitermes trinervius. Analysis of MALDI-TOF MS spectra profiles from termites revealed that all were of high quality, with intra-species reproducibility and inter-species specificity. Blind testing of the spectra of 389 termites against our updated database with the spectra of 43 specimens of different termite species revealed that all were correctly identified with log score values (LSVs) ranging from 1.65 to 2.851, mean 2.290 ± 0.225, median 2.299, and 98.4% (383) had LSVs > 1.8. This study is the first on the use of MALDI-TOF for termite identification and shows its importance as a tool for arthropod taxonomy and reinforces the idea that MALDI-TOF MS is a promising tool in the field of entomology.

MALDI-TOF MS Based Bacterial Antibiotics Resistance Finger Print for Diabetic Pedopathy

Diabetes mellitus has become a major global health issue. Currently, the use of antibiotics remains the best foundational strategy in the control of diabetic foot infections. However, the lack of accurate identification of pathogens and the empirical use of antibiotics at early stages of infection represents a non-targeted treatment approach with a poor curative effect that may increase the of bacterial drug resistance. Therefore, the timely identification of drug resistant bacteria is the key to increasing the efficacy of treatments for diabetic foot infections. The traditional identification method is based on bacterial morphology, cell physiology, and biochemistry. Despite the simplicity and low costs associated with this method, it is time-consuming and has limited clinical value, which delays early diagnosis and treatment. In the recent years, MALDI-TOF MS has emerged as a promising new technology in the field of clinical microbial identification. In this study, we developed a strategy for the identification of drug resistance in the diagnosis of diabetic foot infections using a combination of macro-proteomics and MALDI MS analysis. The macro-proteomics result was utilized to determine the differential proteins in the resistance group and the corresponding peptide fragments were used as the finger print in a MALDI MS analysis. This strategy was successfully used in the research of drug resistance in patients with diabetic foot infections and achieved several biomarkers that could be used as a finger print for 4 different drugs, including ceftazidime, piperacillin, levofloxacin, and tetracycline. This method can quickly confirm the drug resistance of clinical diabetic foot infections, which can help aid in the early treatment of patients.

Quantitative analysis of vitamin D using m/MALDI-TOF mass spectrometry based on a parylene matrix chip

Vitamin D deficiency is associated with various disorders and is diagnosed based on the concentration of 25-hydroxy vitamin D3 (25(OH)D3) in serum. The parylene matrix chip was fabricated to reduce the matrix background noise, and the homogenous distribution of the matrix was retained for the quantitative analysis of 25(OH)D3. The Amplex Red assay was performed to confirm that the sample-matrix mixing zone of the parylene matrix chip was formed below the surface of the parylene-N film. The homogeneous distribution of the matrix was verified from the fluorescence image. For effective analysis using a parylene matrix chip, 25(OH)D3 was modified through the nucleophilic addition of betaine aldehyde (BA) to form a hemiacetal salt. Such modified 25(OH)D3 with a positive charge from BA could be effectively analyzed using MALDI-TOF mass spectrometry. Serum 25(OH)D3 was extracted by liquid–liquid extraction (LLE) and quantified using MALDI-TOF mass spectrometry based on the parylene matrix chip. The intensity of the mass peak of 25(OH)D3 was linearly correlated (r2 = 0.992) with the concentration of 25(OH)D3 spiked in serum, and the LOD was 0.0056 pmol/μL. Energy drinks and vitamin D3 tablets were also employed for the real sample analysis. Finally, the results of the chemiluminescence binding assay and MALDI-TOF mass spectrometry were statistically analyzed to determine the applicability of the method using the Bland–Altman test and Passing–Bablok regression.

A New Filter Based Cultivation Approach for Improving Aspergillus Identification using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS)

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) is widely used in clinical laboratories for routine identification of bacteria and yeasts. However, methodological difficulties are still apparent when applied to filamentous fungi. The liquid cultivation method recommended by Bruker Daltonics GmbH for identification of filamentous fungi by MALDI-TOF MS is labour intensive and time-consuming. In this study, growth of Aspergillus species on different (porous) surfaces was investigated with the aim to develop a more reliable, quicker and less laborious identification method using MALDI-TOF MS. Mycelial growth without sporulation mimicking liquid cultivation and reliable MALDI-TOF MS spectra were obtained when A. fumigatus strains were grown on and in between a polycarbonate membrane filter on Sabouraud dextrose agar. A database of in-house reference spectra was created by growing Aspergillus reference strains (mainly focusing on sections Fumigati and Flavi) under these selected conditions. A test set of 50 molecularly identified strains grown under different conditions was used to select the best growth condition for identification and to perform an initial validation of the in-house database. Based on these results, the cultivation method on top of a polycarbonate filter proved to be most successful for species identification. This method was therefore selected for the identification of two sets of clinical isolates that mainly consisted of Aspergilli (100 strains originating from Indonesia, 70 isolates from Qatar). The results showed that this cultivation method is reliable for identification of clinically relevant Aspergillus species, with 67% and 76% correct identification of strains from Indonesia and Qatar, respectively. In conclusion, cultivation of Aspergilli on top of a polycarbonate filter showed improved results compared to the liquid cultivation protocol recommended by Bruker in terms of percentage of correct identification, ease of MSP creation, time consumption, cost and labour intensity. This method can be reliably applied for identification of clinically important Aspergilli and has potential for identification of other filamentous fungi.