A novel cell-free method to culture Schistosoma mansoni from cercariae to juvenile worm stages for in vitro drug testing

Abstract

AbstractBackgroundThe arsenal in anthelminthic treatment against schistosomiasis is limited and relies almost exclusively on a single drug, praziquantel (PZQ). Thus, resistance to PZQ could constitute a major threat. Even though PZQ is potent in killing adult worms, it has been shown to be limited in its activity against earlier developmental stages. Current in vitro screening strategies for new drugs depend on newly transformed schistosomulae (NTS) for initial hit identification, thereby limiting sensitivity to new compounds predominantly active in later developmental stages. Therefore, the aim of this study was to establish a highly standardized, straightforward and reliable culture method to generate and maintain advanced larval stages in vitro. We present here how this method can be a valuable tool to test drug efficacy at each discrete intermediate larval stage, reducing the reliance on animal use (3Rs).Methodology/principal findingsCercariae were mechanically transformed into skin-stage (SkS) schistosomulae and successfully cultured under serum-free and cell-independent conditions for up to four weeks with no loss in viability. Under these conditions, larval development halted at the lung-stage (LuS). Addition of human serum (HSe) propelled further development into juvenile worms within eight weeks. Skin and lung stages, as well as juvenile worms, were submitted to 96-well format drug screening assays using known anti-schistosomal compounds such as PZQ, oxamniquine (OXM), mefloquine (MFQ) and artemether (ART). Our findings showed stage-dependent differences in larval susceptibility.ConclusionWith this robust and highly standardized in vitro assay, important developmental stages of S. mansoni up to juvenile worms can be generated and maintained over prolonged periods of time. The phenotype of juvenile worms when exposed to reference drugs was comparable to previously published works for ex vivo harvested adult worms. Therefore, this in vitro assay can help reduce reliance on animal experiments in the search for new anti-schistosomal drugs.Author SummarySchistosomiasis remains a major health threat, predominantly in developing countries. Even though there has been some progress in search of new drugs, praziquantel remains the only available drug. Probably the most important advance in the search for new drugs was in vitro transformation of cercariae and their subsequent culture. However, hit identification in compound screenings is exclusively tested in skin stage parasites and is only confirmed for more mature worms in a subsequent step. This is in part due to the lack of an easy culture system for advanced-stage parasites. We present here a reliable and highly standardized way to generate juvenile worms in vitro in a cell-free culture system. The inclusion of in vitro drug tests on advanced-stage parasites in initial hit identification will help to identify compounds that might otherwise be overlooked. Furthermore, the ability to continuously observe the parasite’s development in vitro will provide an important platform for a better understanding of its maturation in the human host. Taken together, this opens up new avenues to investigate the influence of specific cell types or host proteins on the development of Schistosoma mansoni and provides an additional tool to reduce animal use in future drug discovery efforts (3Rs).