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Schistosomiasis is an intravascular parasitic infection estimated to affect over 206 million people, the majority of whom live in Africa where the trematode worms Schistosoma mansoni and Schistosoma haematobium are the major causative agents. While a number of drugs have been used to treat schistosomiasis, praziquantel (PZQ) is the only one that is widely available, relatively cheap, and easy to use. The reliance on a single drug for the treatment of such a prevalent disease is a cause for concern due to the potential for resistance to render PZQ ineffective. In this study, we examine the transcriptome of three generations of a laboratory strain of S. mansoni (PR1) whose susceptibility to PZQ has been diminished across 9 passages through exposure to increasing sub-lethal doses of the drug. Miracidial susceptibility was significantly reduced after exposure to 2 × 50 mg/Kg PZQ during the first passage. Susceptibility of worms in vivo was first assessed during passage 5 when mice infected with PZQ-selected schistosomes were dosed with a lethal dose of 3 × 300 mg/kg PZQ resulting in only a 10% reduction in worm number compared to control treatment. The emergence of reduced sensitivity was marked by a shift in sex ratio from a predominantly male to a female population, a reduction in the length of females and ultimately the loss of the PZQ-selected line after passage 9. Analysis of differentially regulated transcripts did not suggest that any particular gene product or pathway was associated with drug resistance suggesting either a loss of function mutation to a single gene or an epistatic interaction of multiple gene products as the underlying cause of reduced susceptibility.

Schistosomiasis is a chronic parasitic disease caused by sexually dimorphic blood flukes of the genus Schistosoma. Praziquantel (PZQ) is the only drug widely available to treat the disease but does not kill juvenile parasites. Here we report the use of next generation sequencing to study the transcriptional effect of PZQ on murine hepatic inflammatory, immune and fibrotic responses to Schistosoma mansoni worms and eggs. An initial T helper cell 1 (Th1) response is induced against schistosomes in mice treated with drug vehicle (Vh) around the time egg laying begins, followed by a T helper cell 2 (Th2) response and the induction of genes whose action leads to granuloma formation and fibrosis. When PZQ is administered at this time, there is a significant reduction in egg burden yet the hepatic Th1, Th2 and fibrotic responses are still observed in the absence of granuloma formation suggesting some degree of gene regulation may be induced by antigens released from the dying adult worms. Quantitative real-time PCR was used to examine the relative expression of 16 juvenile and adult S. mansoni genes during infection and their response to Vh and PZQ treatment in vivo. While the response of stress genes in adult parasites suggests the worms were alive immediately following exposure to PZQ, they were unable to induce transcription of any of the 9 genes encoding ATP-binding cassette (ABC) transporters tested. In contrast, juvenile schistosomes were able to significantly induce the activities of ABCB, C and G family members, underscoring the possibility that these efflux systems play a major role in drug resistance., Author summary Schistosomiasis is a disease caused by parasitic worms that significantly impacts the lives of approximately 260 million people. During infection, female parasites can release thousands of eggs, a significant number of which become trapped in the liver. Entrapment of eggs triggers immune and inflammatory responses that in turn causes many of the health problems associated with the disease. In this study, we analyzed gene expression profiles of livers from mice infected with the human parasite Schistosoma mansoni over multiple time points, beginning when the parasites start to lay eggs. In mice treated with a lethal dose of the anti-schistosomal drug praziquantel, we identified a number of different genes and pathways central to immune and inflammatory responses that are active even in the absence of egg deposition. Praziquantel is the only drug available to treat schistosomiasis, however, it displays bimodal activity in that it is ineffective against juvenile parasites during the early stages of mammalian infection. We also show that development of drug resistance may be due to the improved efficacy of the juvenile parasite to actively excrete the drug. These results provide insights into the effect of praziquantel on the host response to infection as well as the ability of juvenile parasites to overcome the drug’s lethal effect.

Highlights • We isolated S. mansoni miracidia from 72 Kenyan adults and children. • We found no evidence of S. mansoni with reduced praziquantel sensitivity. • An S. mansoni lab isolate with reduced praziquantel sensitivity was established. • The potential for the emergence of praziquantel resistance remains., Schistosomiasis is a neglected tropical disease caused by blood-dwelling flukes of the genus Schistosoma. While the disease may affect as many as 249 million people, treatment largely relies on a single drug, praziquantel. The near exclusive use of this drug for such a prevalent disease has led to concerns regarding the potential for drug resistance to arise and the effect this would have on affected populations. In this study, we use an in vitro assay of drug sensitivity to test the effect of praziquantel on miracidia hatched from eggs obtained from fecal samples of Kenyan adult car washers and sand harvesters as well as school children. Whereas in a previous study we found the car washers and sand harvesters to harbor Schistosoma mansoni with reduced praziquantel sensitivity, we found no evidence for the presence of such strains in any of the groups tested here. Using miracidia derived from seven car washers to infect snails, we used the shed cercariae to establish a strain of S. mansoni with significantly reduced praziquantel sensitivity in mice. This was achieved within 5 generations by administering increasing doses of praziquantel to the infected mice until the parasites could withstand a normally lethal dose. This result indicates that while the threat of praziquantel resistance may have diminished in the Kenyan populations tested here, there is a strong likelihood it could return if sufficient praziquantel pressure is applied.

Schistosomiasis is one of the foremost health problems in developing countries and has been estimated to account for the loss of up to 56 million annual disability-adjusted life years. Control of the disease relies almost exclusively on praziquantel (PZQ) but this drug does not kill juvenile worms during the early stages of infection or prevent post-treatment reinfection. As the use of PZQ continues to grow, there are fears that drug resistance may become problematic thus there is a need to develop a new generation of more broadly effective anti-schistosomal drugs, a task that will be made easier by having an understanding of why PZQ kills sexually mature worms but fails to kill juveniles. Here, we describe the exposure of mixed-sex juvenile and sexually mature male and female Schistosoma mansoni to 1 μg/mL PZQ in vitro and the use of microarrays to observe changes to the transcriptome associated with drug treatment. Although there was no significant difference in the total number of genes expressed by adult and juvenile schistosomes after treatment, juveniles differentially regulated a greater proportion of their genes. These included genes encoding multiple drug transporter as well as calcium regulatory, stress and apoptosis-related proteins. We propose that it is the greater transcriptomic flexibility of juvenile schistosomes that allows them to respond to and survive exposure to PZQ in vivo.