Molecular xenomonitoring of trypanosomes in tsetse flies

Abstract

Monitoring trypanosomes infections in wild-caught tsetse flies in a given area, is important in prediction of epidemic outbreaks and spread of disease, and could help focus control programs for areas requiring immediate attention in order to limit disease transmission and spread. The main objective of this study is to evaluate the recently developed RIME LAMP and PanTryp LAMP for screening large numbers of tsetse flies for trypanosomes and to assess their sensitivities and specificities for trypanosomes in endemic areas. Wild-caught tsetse flies were dissected and the mid-guts examined by microscopy. The mid-guts were pooled in fives (including one infected gut where applicable), homogenised and DNA extracted by Quiagen kits. TBR- and ITS-PCRs were carried out and examined under ethidium bromide-stained agarose gels while RIMELAMP and PanTryp LAMP were carried out and stained with SYBR green and also observed under ethidium bromide stained agarose gels. A total of 14912 tsetse flies identified as Glossina fuscipes fuscipes, Glossina. pallidipes, Glossina morsitans, Glossina. swynnertoni, Glossina fuscipes quazensis were trapped from the six different countries. Of these, 8789 were dissected. Both males and female tsetse flies had equal infection rates (12.2%) although overall infection rates varied with country. The highest number of infected tsetse flies was obtained by PanTryp LAMP followed by RIME LAMP, ITS-PCR, TBR-PCR and microscopy respectively. PanTryp LAMP was the most sensitive method followed by ITS-PCR, RIME LAMP and TBR-PCR respectively. However, ITS-PCR was the most specific followed by TBR-PCR, RIME LAMP and PanTryp LAMP respectively. Carrying out LAMP tests in the field provides the simplest and quickest means to estimate trypanosome infection rates in the vector tsetse flies. Key words: Xenomonitoring, trypanosome, tsetse fly, LAMP.