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

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

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

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

Model estimated mean proportions (including 95% confidence intervals) of larvae extracted (total versus alive) from spiked soil samples in relation to bulb wattage and extraction time. Mean soil temperature is shown on secondary axis.

Model estimated mean proportions (including 95% confidence intervals) of larvae extracted (total versus alive) from spiked soil samples in relation to bulb wattage and extraction time. Mean soil temperature is shown on secondary axis.</p

Database-field soil sampling.

BackgroundThe 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 findingsSimple, 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 10.5 95% CI 3.6–28.4).ConclusionThe indoor sleeping areas are the transmission hotspots for tungiasis in rural homes in Kenya and Uganda and can be targeted for disease control and prevention measures. The soil extraction methods can be used as a simple tool for monitoring direct impact of such interventions.</div

Outputs from generalised models exploring the association between the proportion of <i>Tunga</i> larvae recovered and bulb wattage and extraction time in experiments.

Outputs from generalised models exploring the association between the proportion of Tunga larvae recovered and bulb wattage and extraction time in experiments.</p

Examples of representative soil sampling areas from different households.

Loose sand floors in coastal Kenya (A); compacted clay floors in eastern Uganda (B); and outdoor sitting location (Kenya), animal tethering place (Uganda) and an outdoor kitchen (Kenya) as examples of outdoor sampling locations (C).</p

Morphological features of the larvae of <i>Tunga penetrans</i>, <i>Echidnophaga gallinacea</i>, and <i>Ctenocephalides felis</i>.

Morphological features of the larvae of Tunga penetrans, Echidnophaga gallinacea, and Ctenocephalides felis.</p

Berlese-Tullgren funnel apparatus for extraction of <i>Tunga penetrans</i> off-host stages from soil samples.

Schematic diagram showing components of individual funnel made from 2 L PET soda bottle, mesh screen and light bulb (A). Wooden frame with twelve units of Berlese-Tullgren funnels (B). High-throughput set up of 60 Berlese-Tullgren Funnels assembled in 5 units in the laboratory (C). Experimental set-up for the calibration of optimum conditions using 15, 25 and 40 W bulbs and 4 versus 8 hours of extraction (D).</p

Fig 3 -

Tunga penetrans (sand flea) adult (A) and larva (B); Echidnophaga gallinacea (stick tight (hen) flea) adult (C) and larva (D); Ctenocephalides felis (cat flea) adult (E) and larva (F); Pupa cocoons of Tunga penetrans (small on left) and Ctenophalides felis (G); Polypod insect larva [24] which can be distinguished from the flea larvae by the 3 pairs of legs and 5 pairs of prolegs (Order Lepidoptera) (H). Photos of adult fleas captured using Macropod Pro macrophotography system (Macroscopic Solutions.com), photos of larvae captured using Axiocam ERc 5s Rev, 2.0 mounted on the Zeiss Stemi 508 stereo microscope (Carl Zeiss Suzhou Co., Ltd.) and photos of pupa cocoons captured using a DinoCapture 2.0 (Dunwell Tech., Inc. Dino-Lite US).</p

Database-optimisation conditions for extraction of off-host stages.

Database-optimisation conditions for extraction of off-host stages.</p