Polymerase chain reaction identification of Trypanosoma brucei rhodesiense in wild tsetse flies from Nkhotakota Wildlife Reserve, Malawi

Abstract

Background Trypanosoma brucei rhodesiense is the causative agent of acute human African trypanosomiasis. Identification of T. b. rhodesiense in tsetse populations is essential for understanding transmission dynamics, assessng human disease risk, and monitoring spatiotemporal trends and impact of control interventions. Accurate detection and characterisation of trypanosomes in vectors relies on molecular techniques. For the first time in Malawi, a molecular technique has been used to detect trypanosomes in tsetse flies in Nkhotakota Wildlife Reserve. Methods A polymerase chain reaction (PCR) technique was used to identify the serum resistance associated (SRA) gene of T. b. rhodesiense in tsetse flies. Of 257 tsetse flies that were randomly caught, 42 flies were dissected for microscopic examination. The midguts of 206 flies were positive and were individually put in eppendorf tubes containing phosphate-buffered saline (PBS buffer) for DNA extraction. Internal transcribed spacer (ITS)-PCR was first used to isolate all trypanosome species from the flies. TBR PCR was then used to isolate the Trypanozoon group. T. brucei-positive samples were further evaluated by SRA PCR for the presence of the SRA gene. Results Of 257 flies caught, 185 (72%) were Glossina morsitans morsitans and 72 (28%) were Glossinapallidipes. Three were tenerals and 242 were mature live flies. Of the 242 flies dissected, 206 were positive, representing an 85.1% infection rate. From 206 infected flies, 106 (51.5%) were positive using ITS-PCR, 68 (33.0%) being mixed infections, 18 (8.7%) T. brucei, 9 (4.4%) Trypanosoma vivax, 4 (1.9%) Trypanosomagodfrey, 3 (1.5%) Trypanosoma congolense savanna, (1.5%) Trypanosoma simae, and 1 (0.4%) Trypanosoma simaetsavo. When subjected to TBR PCR, 107(51.9%) were positive for T. brucei. Of the 107 T. brucei-positive samples, 5 (4.7%) were found to have the SRA gene. Conclusions These results suggest that wild tsetse flies in Malawi are infected with human-infective trypanosomes that put communities around wildlife reserves at risk of human African trypanosomiasis outbreaks. Further studies need to be done to identify sources of blood meals for the flies and for surveillance of communities around wildlife reserves.