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

SIMPLE SUMMARY: Tunga penetrans is an invasive flea that causes tungiasis—a neglected skin parasitosis—in humans and animals. All juvenile T. penetrans stages (eggs, larvae; pupa) are found in sandy soil, while adults survive on their hosts, with females penetrating the skin to breed. Morphological identification of adult fleas is possible, but due to the absence of a morphological key for the juvenile stages, it is currently impossible to conclusively identify these stages of the flea that use morphological features. To understand the ecology of T. penetrans, it is important to identify where the off-host development of fleas occurs by sampling soil for monitoring and surveillance studies. For this purpose, a low-cost PCR-based tool for the identification of T. penetrans is desirable, since the flea is endemic, predominantly in low-income regions. Since flea larvae feed on organic material in the soil, which is known to be rich in PCR inhibitors, this is rather challenging. We tested six protocol combinations based on three DNA preparation methods and two PCR enzymes to determine the most efficient and economical protocol. The developed protocols can be used in future studies and reduce the costs by more than 80%, when compared with more conventional approaches. 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, C(q) 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.