Cost-Effective PCR-Based Identification of Tunga penetrans (Siphonaptera) Larvae Extracted from Soil Samples Containing PCR Inhibitor-Rich Material

Abstract

Tungiasis is a neglected tropical disease caused by skin-penetrating female Tunga penetrans fleas. Although tungiasis causes severe health problems, its ecology is poorly understood and morphological descriptions of the larvae are unavailable. To identify T. penetrans immature stages and sites where they develop, diagnostic PCRs are required. However, flea larvae feed on soil organic matter rich in PCR inhibitors. Here, three DNA preparation methods, including a soil DNA kit that removes inhibitors, a simple ammonium acetate precipitation approach (AmAcet) and a crude lysate of larvae (CL), were combined with amplification by the highly processive FIREPol® Taq or the inhibitor-resistant Phusion® polymerase. Independent of the polymerase used, the frequency of successful amplification, Cq values and PCR efficacies for the low-cost CL and AmAcet methods were superior to the commercial kit for amplification of a 278 bp partial internal transcribed spacer-2 (ITS-2) and a 730 bp pan-Siphonaptera cytochrome oxidase II PCR. For the CL method combined with Phusion® polymerase, the costs were approximately 20-fold lower than for the methods based on the soil DNA kit, which is a considerable advantage in resource-poor settings. The ITS-2 PCR did not amplify Ctenocephalides felis genomic or Tunga trimammilata ITS-2 plasmid DNA, meaning it can be used to specifically identify T. penetrans.

Myth or truth: investigation of the jumping ability of Tunga penetrans (Siphonaptera: Tungidae)

“…Samples were collected from sleeping areas of tungiasis-infected members in the context of field sampling activities of larger risk factor surveys prepared for publication elsewhere. The soil samples were exposed to heat using a Berlese Tullgren funnel (Amugune et al 2022). Tunga larvae were identified morphologically under a microscope (Hicks 1930, Nagy et al 2007) and their identity was confirmed through PCR-based methods (Amugune et al 2022).…”

“…The soil samples were exposed to heat using a Berlese Tullgren funnel (Amugune et al 2022). Tunga larvae were identified morphologically under a microscope (Hicks 1930, Nagy et al 2007) and their identity was confirmed through PCR-based methods (Amugune et al 2022). Larvae were gently transferred to a larval medium, a mixture of a pinch of fish food (Tropical Flake Fish Food, Supa Aquatic Supplies Ltd., Sheffield, UK) and soil, and reared till pupation at ambient conditions at 24.2–29.0°C, natural light and at a relative humidity of 60%–99%.…”