Sample processing for Formalin-ethyl acetate sedimentation (FEASD)[18]

Briefly, fifty (50) grams of fresh intrarectally collected stool was sieved in tap water through a rough and then a fine mesh until the filtrate became clear. The fine mesh retentate was washed thrice in 50 ml saline by sedimentation, a 5 ml aliquot obtained for qPCR, and the remainder was washed twice in 10% formalin. Two 5 mL aliquots were treated with ethyl acetate, centrifuged, and the debris layers discarded. The pellets were then digested overnight in 10% KOH at 37°C, centrifuged, washed in water, and resuspended in 4 mL of 10% formalin. 100 uL aliquots were examined under the microscope, and S. japonicum eggs quantified by trained microscopists. A water buffalo is considered cumulated positive by FEASD if EPG>0 was identified by any of the stool exams after exposure. The cumulated EPG is calculated as the mean of all EPGs detected during 6 to 9 months’ exposure, missing data points are excluded.