Comparison of Microscopy and Alamar Blue Reduction in a Larval Based Assay for Schistosome Drug Screening

Abstract

BackgroundIn 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 FindingsWe 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.ConclusionWe conclude that an HTS combining AB readout and image-based analysis would provide an efficient and stringent primary assay for schistosome drug discovery.

Cross-disciplinary approaches for measuring parasitic helminth viability and phenotype

“…Motility, together with other microscopic characteristics (e.g. shape alterations and granularity) (Butterworth et al 1982) currently comprise the most common indicators for assessing schistosome viability and represent the 'gold standard' for assessing drug screening protocols and determining RNAi phenotype within the schistosomiasis research community (Abdulla et al 2009, Mansour and Bickle 2010, Štefani et al 2010). However, despite its wide application, bright-field, light microscopic assessment of schistosome viability has several inherent problems.…”

“…The development of high content screening systems, as previously discussed, is an important step on the road to developing real time visualisation data for all schistosome lifecycle stages. However, while there have been calls for its development (Mansour and Bickle 2010), there is no published evidence that this has been achieved to date.…”

Lactate as a Novel Quantitative Measure of Viability in Schistosoma mansoni Drug Sensitivity Assays

“…Even though several new drug discovery techniques have been developed in the recent past, methodologies in pharmaceutical drug discovery for schistosomiasis lack elaboration, interlaboratory standardization, harmonization, and automation (9)(10)(11)(12)(13). This is in contrast to other povertyrelated diseases, such as malaria, where tremendous attention and support in the last 10 years by The Global Fund, a nongovernmental funding organization, have allowed the development of elaborate drug discovery programs (14).…”

“…Even though the handling of schistosomula allows automation, at least to a certain degree, the viability of the worms, which determines the drug's activity, is commonly assessed by microscopy. To overcome the subjectivity and complexity of microscopy, several novel techniques to determine schistosomulum viability have recently been presented, although microscopy is still the standard reference methodology (12,13,15,16). However, the phenotype and the degree of changes in the worms' morphology and motility heavily depend on the chemical nature of the drug, and in adult worms it has been shown that damaged tissues have the potential to regenerate (17,18).…”

Isothermal Microcalorimetry To Study Drugs against Schistosoma mansoni

“…Several studies have therefore been launched in the recent past. These have explored novel tools to facilitate the readout of in vitro drug screening on schistosomes, such as fluorescence-labeled albumin (14), fluorophores (propidium iodide and fluorescein diacetate) (29), and colorimetric assays with alamarBlue (25).…”