Identification of tungiasis infection hotspots with a low-cost, high-throughput method for extracting Tunga penetrans (Siphonaptera) off-host stages from soil samples–An observational study

Background The sand flea, Tunga penetrans , is the cause of a severely neglected parasitic skin disease (tungiasis) in the tropics and has received little attention from entomologists to understand its transmission ecology. Like all fleas, T . penetrans has environmental off-host stages presenting a constant source of reinfection. We adapted the Berlese-Tullgren funnel method using heat from light bulbs to extract off-host stages from soil samples to identify the major development sites within rural households in Kenya and Uganda. Methods and findings Simple, low-cost units of multiple funnels were designed to allow the extraction of >60 soil samples in parallel. We calibrated the method by investigating the impact of different bulb wattage and extraction time on resulting abundance and quality of off-host stages. A cross-sectional field survey was conducted in 49 tungiasis affected households. A total of 238 soil samples from indoor and outdoor living spaces were collected and extracted. Associations between environmental factors, household member infection status and the presence and abundance of off-host stages in the soil samples were explored using generalized models. The impact of heat (bulb wattage) and time (hours) on the efficiency of extraction was demonstrated and, through a stepwise approach, standard operating conditions defined that consistently resulted in the recovery of 75% (95% CI 63–85%) of all present off-host stages from any given soil sample. To extract off-host stages alive, potentially for consecutive laboratory bioassays, a low wattage (15–25 W) and short extraction time (4 h) will be required. The odds of finding off-host stages in indoor samples were 3.7-fold higher than in outdoor samples (95% CI 1.8–7.7). For every one larva outdoors, four (95% CI 1.3–12.7) larvae were found indoors. We collected 67% of all off-host specimen from indoor sleeping locations and the presence of off-host stages in these locations was strongly associated with an infected person sleeping in the room (OR ...