Molecular epidemiology of Schistosoma mansoni: A robust, high-throughput method to assess multiple microsatellite markers from individual miracidia

Schistosomiasis is one of the major unconquered infectious diseases afflicting people of developing countries, particularly in Africa. A deeper understanding of the epidemiology of schistosomes is complicated by the intravascular location of adult worms which makes them routinely unavailable for study. Their progeny, miracidia, which are hatched from eggs that are passed in feces, are available and can provide valuable insights about human infections, but they are small in size, hindering robust molecular analyses. Here we present a new high-throughput technique to assess the genotypes at 21 previously published microsatellite loci for individual miracidia of S. mansoni. The 21 loci can be amplified in four multiplexed PCR reactions; however, enough template is produced for approximately six PCR reactions, which allows for additional PCR reactions for resampling or obtaining additional data. We validated this technique using a pedigree study employing laboratory crosses of S. mansoni from Kenya to obtain sets of parents and offspring. Of 23 loci examined, 21 loci were found to be reliable: false alleles were rare and missing alleles due to allelic dropout occurred at only two loci in approximately 5% of the offspring. The latter type of error can be further reduced by reamplification which is possible with our method. This technique is amenable to a 96-well format thus facilitating analysis of larger samples of miracidia, allowing more robust molecular epidemiological studies of S. mansoni to infer population size, population structure, gene flow, mating systems, speciation, and host race formation.