Detection of Leishmania metacyclogenesis within the sand fly vector employing a real-time PCR for sherp gene expression: A tool for Leishmania surveillance and transmission potential
Bibliographic record
Abstract
Surveillance of infected insect vectors of vector-transmitted diseases has been recognized for its ability to estimate pathogen prevalence and transmission potential. Classically restricted to microscopic dissection and examination of individual insects, the potential of entomological monitoring has grown due to the advent of rapid molecular DNA detection methods with high specificity and sensitivity. Despite such advancement, a recurring question concerning DNA detection of parasitic pathogens is related to the fact that DNA amplification, by itself, does not differentiate between insects carrying infectious versus dead, non- or poorly-infectious life-cycle stages, thereby limiting it's programmatic usefulness for accurately measuring the transmission potential of infected insects in endemic areas or within experimentally infected populations. Herein, we developed a quantitative real-time PCR with Reverse Transcription (RT-qPCR) based sherp (small hydrophilic endoplasmic reticulum-associated protein) detection assay employing a novel set of sherp-RT-qPCR primers to detect and quantify infectious Leishmania parasites in infected vector sand flies. The sherp RT-qPCR showed significantly increased expression of sherp transcripts in infectious Leishmania metacyclic versus non-metacyclic promastigotes or mammalian-derived amastigotes. The assay displayed detection performance ranging from 106 to 1 parasite and could reliably quantify parasites within infected sand flies without the need for dissection. Sherp transcripts were also successfully amplified from flies stored in ethanol at room temperature, a practical and economical method of sample preservation in resource-limited field settings. Lastly, in conjunction with an established RT-qPCR assay for Leishmania kinetoplast DNA minicircles, we were able to calculate a score for the degree of metacyclogenesis within infected sand flies, a known predictor of transmission potential. These results highlight the potential of the sherp-RT-qPCR assay to identify hotspots of potential transmission, areas of re-emergence, vector competence, and the transmission potential of infected sand fly populations.
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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 | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.001 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
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".