Comparison of microscopy and alamar blue reduction in a larval based assay for schistosome drug screening.


Mansour, NR; Bickle, QD; (2010) Comparison of microscopy and alamar blue reduction in a larval based assay for schistosome drug screening. PLoS neglected tropical diseases, 4 (8). e795. ISSN 1935-2727 DOI: https://doi.org/10.1371/journal.pntd.0000795

[img]
Preview
Text - Published Version
License:

Download (713kB) | Preview

Abstract

BACKGROUND: In view of the current widespread use of and reliance on a single schistosomicide, praziquantel, there is a pressing need to discover and develop alternative drugs for schistosomiasis. One approach to this is to develop High Throughput in vitro whole organism screens (HTS) to identify hits amongst large compound libraries. METHODOLOGY/PRINCIPAL FINDINGS: We have been carrying out low throughput (24-well plate) in vitro testing based on microscopic evaluation of killing of ex-vivo adult S. mansoni worms using selected compound collections mainly provided through the WHO-TDR Helminth Drug Initiative. To increase throughput, we introduced a similar but higher throughput 96-well primary in vitro assay using the schistosomula stage which can be readily produced in vitro in large quantities. In addition to morphological readout of viability we have investigated using fluorometric determination of the reduction of Alamar blue (AB), a redox indicator of enzyme activity widely used in whole organism screening. A panel of 7 known schistosome active compounds including praziquantel, produced diverse effects on larval morphology within 3 days of culture although only two induced marked larval death within 7 days. The AB assay was very effective in detecting these lethal compounds but proved more inconsistent in detecting compounds which damaged but did not kill. The utility of the AB assay in detecting compounds which cause severe morbidity and/or death of schistosomula was confirmed in testing a panel of compounds previously selected in library screening as having activity against the adult worms. Furthermore, in prospective library screening, the AB assay was able to detect all compounds which induced killing and also the majority of compounds designated as hits based on morphological changes. CONCLUSION: We conclude that an HTS combining AB readout and image-based analysis would provide an efficient and stringent primary assay for schistosome drug discovery.

Item Type: Article
Faculty and Department: Faculty of Infectious and Tropical Diseases > Dept of Immunology and Infection
PubMed ID: 20706580
Web of Science ID: 281443200024
URI: http://researchonline.lshtm.ac.uk/id/eprint/3153

Statistics


Download activity - last 12 months
Downloads since deposit
222Downloads
329Hits
Accesses by country - last 12 months
Accesses by referrer - last 12 months
Impact and interest
Additional statistics for this record are available via IRStats2

Actions (login required)

Edit Item Edit Item