Authors

Discussion

Background: Over 240 million people are infected globally with schistosomiasis, a neglected tropical disease. Specific and sensitive diagnostic techniques are important to monitor and guide our progress towards controlling the disease and measuring drug efficacy. However, all commonly used diagnostics for schistosomiasis, cannot detect adult worms directly and cannot differentiate new from surviving adult worms post treatment. Therefore, in a highly endemic area where reinfection is common, we use molecular markers on offspring, to estimate sibship and parentage to distinguish between old and new infections.

Methods: Schistosoma mansoni miracidia (hatched from eggs excreted in human stool) were collected pre-treatment and over 22 weekly time points post-treatment. DNA was extracted from 471 miracidia individually, collected from a single child, and amplified using two multiplex microsatellite PCRs (17 microsatellites in total) and allele sizing scored using Geneious Prime software Version 2020.2. COLONY software was used to identify half siblings, full-siblings and non-sibling miracidia within each timepoint to estimate sampling saturation, and between pre-treatment and post-treatment timepoints to identify offspring from surviving adult worms.

Results and discussion: Sibship analysis pre-treatment:A total of 151 miracidia were successfully analyzed pretreatment from a single child with a light infection of 56 eggs per gram of stool (EPG), among which 54.3% (82 miracidia) were full-siblings, 13.3% (20 miracidia) were half-siblings, totaling 67.6% sibling miracidia and 32.4 % (49) non-sibling miracidia. This indicates that we have likely not yet fully saturated pre-treatment sampling in this individual host. In high endemicity areas, worm burdens can be very heavy, producing thousands of eggs per gram of stool yet 151 miracidia do not fully saturate the adult worm burden in this child with only a light infection intensity. Sibship analysis post-treatment:We analyzed a total of 320 miracidia collected from 3 to 22 weeks post-treatment. Across post-treatment timepoints between 5.7% and 48.8% of miracidia were full siblings within that timepoint. Even though EPGs were lower at these post-treatment timepoints we also have not fully saturated sampling at each timepoint. Half siblings were observed at 11, 19 and 21 weeks post-treatment with percentage compositions of 10.8%, 27.9 % and 51.3% respectively, potentially as a result of mate swapping among the schistosomes or one male holding more than one female schistosome. Sibship analysis between pre- and post-treatment miracidia: revealed both full- and half-sibling miracidia showing individual and pairs of adult worms surviving praziquantel treatment and variations in praziquantel susceptibility. A large proportion (40.0% to 86.7%) of non-sibling miracidia were also identified at different timepoints post-treatment indicating new reproductive schistosome infections, although some of these may be due to not saturating the miracidia pre-treatment sampling.

Conclusion: This study presents preliminary evidence that sibship and parentage analysis using microsatellite markers has the potential to distinguish between new reproductive schistosomes and surviving adult worms following praziquantel treatment. Though there is need to increase the miracidia sample size at timepoints pre- and post-praziquantel treatment, to achieve sampling saturation and increase our power to detect siblings and new infections. Despite the non-saturation, we show that S. mansoni individual adult worms, and worm pairs, survived praziquantel treatment, and went on to produce full-sibling and half-sibling miracidia, representing old infections surviving praziquantel treatment.