A fluorescence-based assay for Trichomonas vaginalis drug screening.

BACKGROUND: The emergence and spread of drug resistance in Trichomonas vaginalis parasites has become an important concern in trichomoniasis treatment. Fast and reliable growth assessment is critical for validating in vitro drug susceptibility and high-throughput screening of newly developed drugs. METHODS: Modified media without yeast extract were evaluated for their ability to support the growth of T. vaginalis parasites. The potential of the nucleic acid-binding dye SYBR Green I for detecting T. vaginalis drug resistance was characterized, and seeding parasite concentration and incubation time were optimized. The fluorescence assay based on SYBR Green I was further validated in four T. vaginalis isolates with different susceptibilities to the antibiotics metronidazole, tinidazole, ornidazole and secnidazole, and compared with the traditional method that detects minimum lethal concentrations (MLCs). RESULTS: A modified medium consisting of RPMI 1640 and Tryptone Plus as replacements for yeast extract and tryptone, respectively, in traditional trypticase-yeast extract-maltose (TYM) medium exhibited similar performance as TYM medium in maintaining T. vaginalis growth, while it showed much lower background fluorescent signals. The T. vaginalis SYBR Green I-based fluorescence (TSF) drug assay was found to have to satisfy one of two conditions to demonstrate the 50% inhibitory concentration of metronidazole for the sensitive isolate TV-334: (i) a seeding density of 3 × 104 parasites/ml and an incubation time of 48 h; or (ii) a seeding density of 1 × 104 parasites/ml and an incubation time of 72 h. Subsequent validation experiments revealed that the 48-h incubation/3 × 104 parasites/ml seeding density condition had a greater sensitivity to detect drug resistance than the 72-h condition. The TSF assay also exhibited high efficiency in identifying parasite drug resistance, as evidenced by its strong correlation with the standard MLC assay results (P = 0.003). CONCLUSIONS: This study presents a robust TSF assay that has the potential to facilitate high-throughput, automated in vitro anti-trichomoniasis susceptibility testing for drug resistance monitoring and drug development. In comparison to the standard MLC method, this assay offers the advantages of reduced labor and elimination of subjective examination.

New holostan-type triterpene glycosides from the sea cucumber Apostichopus japonicus.

Two new holostan-type glycosides, holotoxin D1 (1) and 25,26-dihydroxy-holotoxin A1 (2), together with two known analogues, stichlorosides C1 (3) and bivittoside D (4), were isolated from the sea cucumber Apostichopus japonicus Selenka. The structures of the new compounds were elucidated on the basis of detailed spectroscopic analysis. Glycosides 1 and 3 exhibited potent antifungal activity.

Antifungal triterpene glycosides from the sea cucumber Holothuria (Microthele) axiloga.

Three new holostan-type triterpene glycosides, arguside F (1), impatienside B (2), and pervicoside D (3), together with a known saponin, holothurin B ( 4) were isolated from the sea cucumber Holothuria (Microthele) axiloga H. L. Clark. On the basis of spectroscopic data and chemical reactions, the structures of the new compounds were elucidated. Compound 2 showed significant antifungal activities against six strains (1 < or = MIC(80) < or = 4 microg/mL). The stereochemistry of holothurin B (4) isolated from the title sea cucumber was also solved through X-ray diffraction analysis.

Antifungal triterpene glycosides from the sea cucumber Bohadschia marmorata.

Four new holostan-type triterpene glycosides, marmoratoside A ( 1), 17 alpha-hydroxy impatienside A ( 2), marmoratoside B ( 3), 25-acetoxy bivittoside D ( 4), together with two known triterpene glycosides, impatienside A ( 5) and bivittoside D ( 6), were isolated from the sea cucumber Bohadschia Marmorata Jaeger. On the basis of spectroscopic analyses, including two-dimensional NMR techniques, and chemical reactions, the structures of the new triterpene glycosides were elucidated. Compounds 1, 2, 5, and 6 exhibited significant antifungal activity against six strains (0.70 < or = MIC (80) < or = 2.81 microM).