Illuminating the Prevalence of Trypanosoma brucei s.l. in Glossina Using LAMP as a Tool for Xenomonitoring

Journal article

Background As the reality of eliminating human African trypanosomiasis (HAT) by 2020 draws closer, the need to detect and identify the remaining areas of transmission increases. Here, we have explored the feasibility of using commercially available LAMP kits, designed to detect the Trypanozoon group of trypanosomes, as a xenomonitoring tool to screen tsetse flies for trypanosomes to be used in future epidemiological surveys. Methods and Findings The DNA extraction method was simplified and worked with the LAMP kits to detect a single positive fly when pooled with 19 negative flies, and the absolute lowest limit of detection that the kits were able to work at was the equivalent of 0.1 trypanosome per ml. The DNA from Trypanosoma brucei brucei could be detected six days after the fly had taken a blood meal containing dead trypanosomes, and when confronted with a range of non-target species, from both laboratory-reared flies and wild-caught flies, the kits showed no evidence of cross-reacting. Conclusion We have shown that it is possible to use a simplified DNA extraction method in conjunction with the pooling of tsetse flies to decrease the time it would take to screen large numbers of flies for the presence of Trypanozoon trypanosomes. The use of commercially-available LAMP kits provides a reliable and highly sensitive tool for xenomonitoring and identifying potential sleeping sickness transmission sites. Author Summary Recent control efforts have reduced the global incidence of Gambiense human African trypanosomiasis (HAT) to <5,000 cases per year, strengthening the prospect of eliminating the disease as a public health problem by 2020. To meet this goal, new methods for identifying transmission must be explored to provide a cost-effective way of identifying hotspots and areas of re-emergence; commercial loop-mediated isothermal amplification (LAMP) kits that detect the trypanosome subgenus, responsible for the two forms of sleeping sickness, have been developed. The LAMP kits were tested to assess their sensitivity, specificity and suitability as a method of screening the vector of the disease, Glossina, for Trypanozoon infection, in xenomonitoring campaigns. A simplified DNA extraction process that worked in conjunction with the LAMP kits on pooled samples demonstrated a faster method of processing large numbers of flies compared to other molecular tools. The kits performed well in our experiments and demonstrated the ability of detecting low levels of target DNA, equivalent to 0.1 trypanosome per ml. The lack of cross reaction with non-target species of trypanosomes makes the kits reliable in so far as they will only react with the Trypanozoon group of parasites of which the two human forms of the disease belong, however, further species-specific tests would need to be undertaken to identify HAT areas on selected samples

Authors: Cunningham, LJ; Lingley, JK; Haines, LR; Ndung'u, JM; Torr, SJ

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: Public Library of Science
• Journal: PLoS Neglected Tropical Diseases
• Volume: 10
• Issue: 2
• Pages: e0004441-e0004441
• Publication date: 2016-02-18
• DOI: 10.1371/journal.pntd.0004441
• EISSN: 1935-2735
• ISSN: 1935-2727
• Language: English
• Keywords: Trypanosomiasis; Veterinary medicine; DNA extraction; Virology; Trypanosoma; Tsetse fly; Blood meal; Polymerase chain reaction; Medicine; Ecology; Transmission (telecommunications); African trypanosomiasis; Genetics; Biology; Trypanosoma brucei; Zoology