In China alone, an estimated 30 million people are at risk of schistosomiasis, caused by the Schistosoma japonicum parasite. Disease has re-emerged in several regions that had previously attained transmission control, reinforcing the need for active surveillance. The environmental stage of the parasite is known to exhibit high spatial and temporal variability, and current detection techniques rely on a sentinel mouse method which has serious limitations in obtaining data in both time and space. Here we describe a real-time PCR assay to quantitatively detect S. japonicum cercariae in laboratory samples and in natural water that has been spiked with known numbers of S. japonicum. Multiple primers were designed and assessed, and the best performing set, along with a TaqMan probe, was used to quantify S. japonicum. The resulting assay was selective, with no amplification detected for Schistosoma mansoni, Schistosoma haematobium, avian schistosomes nor organisms present in non-endemic surface water samples. Repeated samples containing various concentrations of S. japonicum cercariae showed that the real-time PCR method had a strong linear correlation (R2 = 0.921) with light microscopy counts, and the detection limit was below the DNA equivalent of half of one cercaria. Various cercarial concentrations spiked in 1 liter of natural water followed by a filtration process produced positive detection from 93% of samples analyzed. The real-time PCR method performed well quantifying the relative concentrations of various spiked samples, although the absolute concentration estimates exhibited high variance across replicated samples. Overall, the method has the potential to be applied to environmental water samples to produce a rapid, reliable assay for cercarial location in endemic areas.
Schistosomiasis ranks second only to malaria among parasitic diseases with regard to the number of people infected and those at risk. Schistosoma japonicum is the species that causes human and animal disease in China, the Philippines, and to a lesser extent, Indonesia. Recent evidence of schistosomiasis re-emergence in China has reinforced the need for active disease surveillance in these areas. Schistosomiasis infection occurs through contact with water contaminated with S. japonicum cercariae, the free-living stage of the parasite shed from intermediate host snails. Current practice of detecting cercariae in the environment uses sentinel mice, a method with serious limitations in which mice are exposed to environmental water and then maintained for 6 weeks before being dissected to count worms. The method is labor intensive and costly in terms of time and resources, making it logistically prohibitive to monitor water contact sites regularly or comprehensively. Here we develop a quantitative PCR assay to measure S. japonicum cercariae concentration in water, providing a potential method for rapid and reliable data collection in the field, potentially replacing the use of live animal models.