Comparative diagnostic and analytical performance of PCR and LAMP-based trypanosome detection methods estimated using pooled whole tsetse flies and midguts.

Detection of trypanosomes that cause disease in human beings and livestock within their tsetse fly hosts is an essential component of vector and disease control programmes. Several molecular-based diagnostic tests have been developed for this purpose. Many of these tests, while sensitive, require analysis of trypanosome DNA extracted from single flies, or from pooled tsetse fly heads and amplified trypanosome DNA. In this study, we evaluated the relative analytical and diagnostic sensitivities of two PCR-based tests (ITS and TBR) and a Trypanozoon specific LAMP assay using pooled whole tsetse flies and midguts spiked with serially diluted procyclics of a laboratory strain of Trypanosoma brucei brucei (KETRI 3386). Test sensitivity was also evaluated using experimentally infected tsetse flies. The aim was to determine the most appropriate pooling strategy for whole tsetse and midguts. RIME-LAMP had the highest diagnostic sensitivity (100%) followed by TBR-PCR (95%) and ITS-PCR (50%) in detecting trypanosome DNA from pooled tsetse midguts. RIME-LAMP also had the best diagnostic specificity (75%) followed by ITS-PCR (68%) and TBR-PCR (50%). The relative detection limit determined by serial dilution of procyclics was below 10(-6) (equivalent to 1parasite/ml). Using TBR-PCR, ITS-PCR and RIME-LAMP, it was possible to detect trypanosome DNA in single flies or in pools of 2, 3, 4, 5, 10, or 15 flies/midguts. The proportion of positive pools declined by up to 60% when testing pools of 15 whole flies as opposed to testing pools of 5-10 flies. Additionally, it was possible to detect DNA in a single infected tsetse fly in the background of 4, 9, or 14 uninfected tsetse flies. Averaged across pool sizes and tsetse species, RIME-LAMP detected the highest proportion of positive pools in spiked whole tsetse and midguts (86.6% and 87.2%) followed by TBR-PCR (78. 6% and 79.2%) and ITS-PCR (34.3% and 40.2%). There were no significant differences between the proportions of positive pools detected in whole flies and midguts. We conclude that pooling of whole tsetse/midguts is an effective strategy to reduce hands-on-time and hence has potential application in large scale xenomonitoring to generate epidemiological data for decision making. RIME-LAMP offers the best diagnostic sensitivity and specificity on pooled tsetse midguts, thus demonstrating its superior diagnostic performance when compared with TBR-PCR and ITS-PCR. Using pools of whole tsetse or midguts as source of DNA does not have any significant effect on test results and is more representative of the field conditions where the proportion of flies with infected midguts tends to be higher than flies with infected salivary glands. Therefore to save time and minimize costs, pooling of whole tsetse flies is recommended.

Prevalence of SGHV among tsetse species of economic importance in Tanzania and their implication for SIT application.

Sterile Insect technique is an important component in area-wide integrated tsetse control. The presence of the salivary glands hypertrophy virus (SGHV) in the wild tsetse, which are the seeds for colony adaptations in the laboratory has become a stumbling block in establishing and maintaining colonies in the laboratory. The virus is transmitted both vertically (in the wild) and horizontally (in the laboratory). However, its prevalence is magnified in the laboratory as a result of the use of in vitro membrane feeding regimen. Fly species of Glossina fuscipes fuscipes, G. pallidipes, G. morsitans and G. swynnertoni were collected from the coastal and inland areas of Tanzania and virus infection rates were assessed microscopically and by PCR. The data showed that in a period of 4years, the virus was present in all species tested irrespective of their ages, sex, and season of the year. However, infection levels differed among species and from one location to another. Symptomatic infection determined by dissection was 1.2% (25/2164) from the coast as compared to 0.4% (6/1725) for inland collected flies. PCR analysis indicated a higher infection rate of 19.81% (104/525) of asymptomatic flies. From these observations, we conclude that care should be taken when planning to initiate tsetse laboratory colonies for use in SIT eradication program. All efforts should be made to select non-infected flies when initiating laboratory colonies and to try to minimize the infection with SGHV. Also management of SGHV infection in the established colony should be applied.

Multiple Trypanosoma infections are common amongst Glossina species in the new farming areas of Rufiji district, Tanzania.

BACKGROUND: Tsetse flies and trypanosomiasis are among several factors that constrain livestock development in Tanzania. Over the years Rufiji District was excluded from livestock production owing to tsetse fly infestation, however, a few years ago there was an influx of livestock following evictions aimed at conserving the Usangu wetlands. METHODS: A study was conducted to determine the efficiency of available traps for catching tsetse flies, Glossina species infesting the area, their infection rates and Trypanosoma species circulating in the area. Trapping was conducted during the semi dry season for a total of 30 days (ten days each month) during the onset of the dry season of May - July 2009. Harvested flies after every 24 hours were dissected and examined under a light microscope for trypanosome infections and whole fly DNA was extracted from 82 flies and analyzed for trypanosomes by polymerase chain reaction (PCR) using different sets of primers. RESULTS: The proportions of total tsetse catches per trap were in the following decreasing order S3 (33%), H-Trap (27%), Pyramidal (19%), sticky panel (11%) and biconical trap (10%). Of the 1200 trapped flies, 75.6% were identified as Glossina pallidipes, 11.7% as G. brevipalpis, 9.6% as G. austeni and 3.0% G. morsitans morsitans. Dissections revealed the overall infection rate of 6.6% (13/197). Whole DNA was extracted from 82 tsetse flies and the prevalence of trypanosomes circulating in the area in descending order was 92.7% (76/82) for T. simiae; 70.7% (58/82) for T. brucei types; 48.8% (40/82) for the T. vivax types and 32.9% (27/82) for the T. congolense types as determined by PCR. All trypanosome types were found in all tsetse species analysed except for the T. congolense types, which were absent in G. m. morsitans. None of the T. brucei positive samples contained human infective trypanosomes by SRA - PCR test CONCLUSION: All tsetse species found in Rufiji are biologically important in the transmission of animal trypanosomiasis and the absence of T. congolense in G. m. morsitans could be a matter of chance only. Therefore, plans for control should consider all tsetse species.