Pythiosis is a life-threatening infectious disease caused by the oomycete Pythium insidiosum. The disease has been increasingly reported worldwide. Most patients with pythiosis undergo surgical removal of an infected organ. Early diagnosis contributes to better prognosis of pythiosis patients. Here, we assessed the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for identification and biotyping of P. insidiosum. A comprehensive set of mass spectra of P. insidiosum was generated to evaluate MALDI-TOF MS for identification and biotyping of P. insidiosum. MALDI-TOF MS accurately identified all 13P. insidiosum strains tested, at the species level. Mass spectra of P. insidiosum did not match any other microorganisms, including fungi (i.e., Aspergillus species, Fusarium species, and fungal species of the class Zygomycetes), which have similar microscopic morphologies with this oomycete. MALDI-TOF MS- and rDNA sequence-based biotyping methods consistently classified P. insidiosum into three groups: Clade-I (American strains), II (Asian and Australian strains), and III (mostly Thai strains). MALDI-TOF MS has been successfully used for identification and biotyping of P. insidiosum. The obtained mass spectral database allows clinical microbiology laboratories, well-equipped with a MALDI-TOF mass spectrometer, to conveniently identify P. insidiosum, without requiring any pathogen-specific reagents (i.e., antigen, antibody or primers).

Pythium insidiosum causes a life-threatening condition called pythiosis. High morbidity and mortality of pythiosis are consequences of delayed diagnosis. We aimed to develop a loop-mediated isothermal amplification (LAMP) assay for the rapid detection of P. insidiosum for use in remote areas, where pythiosis is prevalent. We designed four LAMP primers to amplify the rDNA sequence. A side-by-side comparison evaluated performances of LAMP and the previously-established multiplex PCR (M-PCR), using gDNA samples extracted from colonies of P. insidiosum (n = 28) and other fungi (n = 54), and tissues of animals with (n = 16) or without (n = 13) pythiosis. LAMP demonstrated a 50% shorter assay duration (1.5 h) and a 10-fold lower limit of detection (10^-4 ng) than did M-PCR. Based on colony-extracted gDNAs, LAMP and M-PCR correctly reported P. insidiosum in all 28 samples, providing 100% sensitivity. While M-PCR did not amplify all fungal controls (100% specificity), LAMP falsely detected one organism (98% specificity). Based on the clinical samples, LAMP and M-PCR provided an equivalently-high specificity (100%). However, LAMP showed a markedly-higher sensitivity than that of M-PCR (88% vs. 56%). LAMP is a simple, useful, efficient assay for the detection of P. insidiosum in clinical specimens and pure cultures in resource-limited laboratories.

Antimicrobial peptides are promising molecules to address the global antibiotic resistance problem, however, optimization to achieve favorable potency and safety is required. Here, a peptide-template modification approach was employed to design physicochemical variants based on net charge, hydrophobicity, enantiomer, and terminal group. All variants of the scorpion venom peptide BmKn-2 with amphipathic α-helical cationic structure exhibited an increased antibacterial potency when evaluated against multidrug-resistant Salmonella isolates at a MIC range of 4–8 µM. They revealed antibiofilm activity in a dose-dependent manner. Sheep red blood cells were used to evaluate hemolytic and cell selectivity properties. Peptide Kn2-5R-NH₂, dKn2-5R-NH₂, and 2F-Kn2-5R-NH₂ (variants with +6 charges carrying amidated C-terminus) showed stronger antibacterial activity than Kn2-5R (a variant with +5 charges bearing free-carboxyl group at C-terminus). Peptide dKn2-5R-NH₂ (d-enantiomer) exhibited slightly weaker antibacterial activity with much less hemolytic activity (higher hemolytic concentration 50) than Kn2-5R-NH₂ (l-enantiomer). Furthermore, peptide Kn2-5R with the least hydrophobicity had the lowest hemolytic activity and showed the highest specificity to Salmonella (the highest selectivity index). This study also explained the relationship of peptide physicochemical properties and bioactivities that would fulfill and accelerate progress in peptide antibiotic research and development.

Outbreaks of trichinellosis caused by Trichinella papuae have been reported in South-East Asia. Mebendazole and thiabendazole are the treatments of choice for trichinellosis; however, both drugs result in significant side effects and are less effective for muscle-stage larvae (L1). An alternative therapeutic agent is needed to improve treatment. Information on lipid composition and metabolic pathways may bridge gaps in our knowledge and lead to new antiparasitics. The T. papuae L1 lipidome was analysed using a mass spectrometry-based approach, and 403 lipid components were identified. Eight lipid classes were found and glycerophospholipids were dominant, corresponding to 63% of total lipids, of which the glycerolipid DG (20:1[11Z]/22:4[7Z,10Z,13Z,16Z]/0:0) (iso2) was the most abundant. Overall, 57% of T. papuae lipids were absent in humans; therefore, lipid metabolism may be dissimilar in the two species. Proteins involved T. papuae lipid metabolism were explored using bioinformatics. We found that 4-hydroxybutyrate coenzyme A transferase, uncharacterized protein (A0A0V1MCB5) and ML-domain-containing protein are not present in humans. T. papuae glycerophospholipid metabolic and phosphatidylinositol dephosphorylation processes contain several proteins that are dissimilar to those in humans. These findings provide insights into T. papuae lipid composition and metabolism, which may facilitate the development of novel trichinellosis treatments.

Leptospirosis is a zoonosis with a worldwide distribution, caused by pathogenic spirochetes of the genus Leptospira. The classification and identification of leptospires can be conducted by both genotyping and serotyping which are time-consuming and established in few reference laboratories. This study used matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as rapid and accurate tool for the identification of leptospires. The whole cell protein spectra of 116 Leptospira isolates including 15 references Leptospira spp. (pathogenic, n = 8; intermediate, n = 2; non-pathogenic, n = 5) and 101 Leptospira spp. clinical isolates was created as an in-house MALDI-TOF MS database. Ninety-seven clinical isolates from Thailand and Laos was validated with these protein spectra and revealed 98.9% correct identification when compared with 16S rRNA gene sequences method. Moreover, MALDI-TOF MS could identify spiked leptospires whole cell in urine. Biomarkers for differentiation of leptospires phylogeny and specific protein spectra for most found Leptospira spp. in this area (L. interrogans, L. kirschneri, L. borgpetersenii) based on MALDI-MS algorithm were demonstrated. Leptospirosis is a world-wide zoonosis caused by pathogenic members of the genus Leptospira. Severity of infection ranges from mild features to multi-organ failure and death, although the reason for this is unclear. Clinical symptoms are similar to several other febrile illnesses such as dengue, scrub typhus, malaria, viral hepatitis. Identification of Leptospira spp. is generally based on genotyping and serotyping. This study used matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for rapid and accurate identification of Leptospira whole cell. The methods generated protein spectra of Leptospira isolates and created large protein database which can be used to compare and identify other Leptospira isolates. The machine learning based on the computational mathematic analysis of this database provide the classification of Leptospira phylogeny and biomarkers for species identification.