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

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.

[1]  P. Zhou,et al.  Moving towards an integrated approach to molecular detection and identification of Toxoplasma gondii , 2009, Parasitology.

[2]  M. Doenhoff,et al.  Determination of ED50 values for praziquantel in praziquantel-resistant and -susceptible Schistosoma mansoni isolates. , 2004, International journal for parasitology.

[3]  Conor R. Caffrey,et al.  Drug Discovery for Schistosomiasis: Hit and Lead Compounds Identified in a Library of Known Drugs by Medium-Throughput Phenotypic Screening , 2009, PLoS neglected tropical diseases.

[4]  N. el-Badawy,et al.  Experimental trials of an artemisinin derivative in treatment of Schistosoma mansoni infected mice. , 2000, Journal of the Egyptian Society of Parasitology.

[5]  N. Katz,et al.  Preliminary trials with praziquantel in human infections due to Schistosoma mansoni. , 1979, Bulletin of the World Health Organization.

[6]  S. D. de Vlas,et al.  Are poor responses to praziquantel for the treatment of Schistosoma mansoni infections in Senegal due to resistance? An overview of the evidence , 2001, Tropical medicine & international health : TM & IH.

[7]  R. Prichard,et al.  Differential effects of oltipraz and its oxy-analogue on the viability of Schistosoma mansoni and the activity of glutathione S-transferase. , 1991, Biochemical pharmacology.

[8]  F. Zicker,et al.  Preliminary clinical trials with oxamniquine, a new antischistosomal agent. , 1973, Revista do Instituto de Medicina Tropical de Sao Paulo.

[9]  D. Cioli.  Praziquantel: is there real resistance and are there alternatives? , 2000, Current opinion in infectious diseases.

[10]  L. du Toit,et al.  Preliminary results in human schistosomiasis with Ro 11-3128. , 1979, South African medical journal = Suid-Afrikaanse tydskrif vir geneeskunde.

[11]  M. Tanner,et al.  Oral artemether for prevention of Schistosoma mansoni infection: randomised controlled trial , 2000, The Lancet.

[12]  S. Nwaka,et al.  The Concept Paper on the Helminth Drug Initiative. Onchocerciasis/lymphatic filariasis and schistosomiasis: opportunities and challenges for the discovery of new drugs/diagnostics , 2007, Expert opinion on drug discovery.

[13]  J. Keiser.  In vitro and in vivo trematode models for chemotherapeutic studies , 2009, Parasitology.

[14]  M. Doenhoff,et al.  Parasitological evaluation of curative and subcurative doses of 9-acridanone-hydrazone drugs against Schistosoma mansoni in baboons, and observations on changes in serum levels of anti-egg antibodies detected by ELISA. , 1987, Transactions of the Royal Society of Tropical Medicine and Hygiene.

[15]  P. Basch.  Cultivation of Schistosoma mansoni in vitro. I. Establishment of cultures from cercariae and development until pairing. , 1981, The Journal of parasitology.

[16]  C. Jaffe,et al.  Rapid fluorescent assay for screening drugs on Leishmania amastigotes. , 2008, Journal of microbiological methods.

[17]  M. Taylor,et al.  Transformation of cercariae to schistosomula: A quantitative comparison of transformation techniques and of infectivity by different injection routes of the organisms produced , 1976, Journal of Helminthology.

[18]  R. Kaminsky,et al.  The Alamar Blue assay to determine drug sensitivity of African trypanosomes (T.b. rhodesiense and T.b. gambiense) in vitro. , 1997, Acta tropica.

[19]  M. Doenhoff,et al.  Factors affecting the acquisition of resistance against Schistosoma mansoni in the mouse. I. Demonstration of resistance to reinfection using a model system that involves perfusion of mice within three weeks of challenge , 1978, Journal of Helminthology.

[20]  M. Tanner,et al.  Schistosomiasis and neglected tropical diseases: towards integrated and sustainable control and a word of caution , 2009, Parasitology.

[21]  A. Sher,et al.  Schistosoma mansoni: rapid isolation and purification of schistosomula of different developmental stages by centrifugation on discontinuous density gradients of Percoll. , 1982, Experimental parasitology.

[22]  Solomon Nwaka,et al.  Schistosomes: challenges in compound screening , 2007, Expert opinion on drug discovery.

[23]  J. Bennett.  Characteristics of antischistosomal benzodiazepine binding sites in Schistosoma mansoni. , 1980, The Journal of parasitology.

[24]  Y. Zhang,et al.  The Schistosomiasis Control Initiative (SCI): rationale, development and implementation from 2002–2008 , 2009, Parasitology.

[25]  Marcel Tanner,et al.  Schistosomiasis and water resources development: systematic review, meta-analysis, and estimates of people at risk. , 2006, The Lancet. Infectious diseases.