Validation of novel fluorescence assays for the routine screening of drug susceptibilities of Trichomonas vaginalis
Bibliographic record
Abstract
OBJECTIVES: To evaluate existing protocols, based on Alamar blue (resazurin), for the routine determination of drug susceptibility in trichomonads, develop new ones and validate these by screening small antiprotozoal libraries. METHODS: The resazurin-based assay was evaluated by determining fluorescence development in Trichomonas media with various cell densities after various intervals and in the presence of metronidazole. Similar investigations were performed with the alternative fluorophores propidium iodide (PI) and resorufin. The optimized protocols were used to screen for new antitrichomonal compounds. RESULTS: Anaerobic cultures of Trichomonas vaginalis rapidly reduced blue resazurin to red, fluorescent resorufin. However, the ascorbic acid in the culture medium produced similar effects, even in the absence of cells, causing high background fluorescence and variability. Moreover, T. vaginalis rapidly metabolized resorufin to the non-fluorescent and colourless metabolite dihydroresorufin, making the fluorescent signal transient. In contrast, resorufin proved to be an excellent viability probe for Trichomonas due to its chemical stability in media and rapid metabolism by the parasite. We also show that staining with PI after cell permeabilization similarly constitutes a reliable measurement of trophozoite numbers. Using the PI and resorufin assays we determined reproducible EC(50) values and identified potent antitrichomonal compounds from a limited screen of phosphodiesterase inhibitors and phosphonium salts. CONCLUSIONS: The resorufin- and PI-based assays are suitable for routine and high-throughput drug screening, whereas resazurin-based assays are not. These assays constitute a major advance in the current protocols as demonstrated by a successful screen for new antitrichomonal lead compounds.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.003 | 0.004 |
| Meta-epidemiology (narrow) | 0.002 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.000 | 0.001 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".