Rapid and sensitive detection of mycobacterium ulcerans by use of a loop-mediated isothermal amplification test

This work reports the design and evaluation of a rapid loop-mediated isothermal amplification test for detecting Mycobacterium ulcerans DNA based on the multicopy insertion sequence IS2404. The test is robust and specific with a detection limit equivalent to 20 copies of the target sequence (0.01 to 0.1 genome). The test has potential for the diagnosis of Buruli ulcer under field conditions

Multiple evolutionary origins of Trypanosoma evansi in Kenya

Trypanosoma evansi is the parasite causing surra, a form of trypanosomiasis in camels and other livestock, and a serious economic burden in Kenya and many other parts of the world. Trypanosoma evansi transmission can be sustained mechanically by tabanid and Stomoxys biting flies, whereas the closely related African trypanosomes T. brucei brucei and T. b. rhodesiense require cyclical development in tsetse flies (genus Glossina) for transmission. In this study, we investigated the evolutionary origins of T. evansi. We used 15 polymorphic microsatellites to quantify levels and patterns of genetic diversity among 41 T. evansi isolates and 66 isolates of T. b. brucei (n = 51) and T. b. rhodesiense (n = 15), including many from Kenya, a region where T. evansi may have evolved from T. brucei. We found that T. evansi strains belong to at least two distinct T. brucei genetic units and contain genetic diversity that is similar to that in T. brucei strains. Results indicated that the 41 T. evansi isolates originated from multiple T. brucei strains from different genetic backgrounds, implying independent origins of T. evansi from T. brucei strains. This surprising finding further suggested that the acquisition of the ability of T. evansi to be transmitted mechanically, and thus the ability to escape the obligate link with the African tsetse fly vector, has occurred repeatedly. These findings, if confirmed, have epidemiological implications, as T. brucei strains from different genetic backgrounds can become either causative agents of a dangerous, cosmopolitan livestock disease or of a lethal human disease, like for T. b. rhodesiense

Population Genetics of Trypanosoma evansi from Camel in the Sudan

Genetic variation of microsatellite loci is a widely used method for the analysis of population genetic structure of microorganisms. We have investigated genetic variation at 15 microsatellite loci of T. evansi isolated from camels in Sudan and Kenya to evaluate the genetic information partitioned within and between individuals and between sites. We detected a strong signal of isolation by distance across the area sampled. The results also indicate that either, and as expected, T. evansi is purely clonal and structured in small units at very local scales and that there are numerous allelic dropouts in the data, or that this species often sexually recombines without the need of the “normal” definitive host, the tsetse fly or as the recurrent immigration from sexually recombined T. brucei brucei. Though the first hypothesis is the most likely, discriminating between these two incompatible hypotheses will require further studies at much localized scales

Improvements on Restricted Insecticide Application Protocol for Control of Human and Animal African Trypanosomiasis in Eastern Uganda

African trypanosomes constrain livestock and human health in Sub-Saharan Africa, and aggravate poverty and hunger of these otherwise largely livestock-keeping communities. To solve this, there is need to develop and use effective and cheap tsetse control methods. To this end, we aimed at determining the smallest proportion of a cattle herd that needs to be sprayed on the legs, bellies and ears (RAP) for effective Human and Animal African Trypanosomiasis (HAT/AAT) control.; Cattle in 20 villages were ear-tagged and injected with two doses of diminazene diaceturate (DA) forty days apart, and randomly allocated to one of five treatment regimens namely; no treatment, 25%, 50%, 75% monthly RAP and every 3 month Albendazole drench. Cattle trypanosome re-infection rate was determined by molecular techniques. ArcMap V10.3 was used to map apparent tsetse density (FTD) from trap catches. The effect of graded RAP on incidence risk ratios and trypanosome prevalence was determined using Poisson and logistic random effect models in R and STATA V12.1 respectively. Incidence was estimated at 9.8/100 years in RAP regimens, significantly lower compared to 25.7/100 years in the non-RAP regimens (incidence rate ratio: 0.37; 95% CI: 0.22-0.65; P>0.001). Likewise, trypanosome prevalence after one year of follow up was significantly lower in RAP animals than in non-RAP animals (4% vs 15%, OR: 0.20, 95% CI: 0.08-0.44; P>0.001). Contrary to our expectation, level of protection did not increase with increasing proportion of animals treated.; Reduction in RAP coverage did not significantly affect efficacy of treatment. This is envisaged to improve RAP adaptability to low income livestock keepers but needs further evaluation in different tsetse challenge, HAT/AAT transmission rates and management systems before adopting it for routine tsetse control programs

The typing of Trypanosoma evansi isolates using mobile genetic element (MGE) PCR

The mobile genetic element PCR (MGE-PCR) is a simple and sensitive technique that can be used to detect genetic variability in Trypanosoma brucei ssp. To investigate the reliability of MGE-PCR in genotyping Trypanosoma evansi, stocks that were isolated directly from camels and after their respective passage in mice were analyzed. Construction of a dendrogram using the MGE-PCR banding profiles revealed a clear distinction between T. evansi and T. brucei, as well as discriminating the T. evansi strains (T. evansi with minicircle types B and A). A minor host-dependent clustering shows a genetic difference o

Detection of Group 1 Trypanosoma brucei gambiense by Loop-Mediated Isothermal Amplification▿

Trypanosoma brucei gambiense group 1 is the major causative agent of the Gambian human African trypanosomiasis (HAT). Accurate diagnosis of Gambian HAT is still challenged by lack of precise diagnostic methods, low and fluctuating parasitemia, and generally poor services in the areas of endemicity. In this study, we designed a rapid loop-mediated isothermal amplification (LAMP) test for T. b. gambiense based on the 3′ end of the T. b. gambiense-specific glycoprotein (TgsGP) gene. The test is specific and amplifies DNA from T. b. gambiense isolates and clinical samples at 62°C within 40 min using a normal water bath. The analytical sensitivity of the TgsGP LAMP was equivalent to 10 trypanosomes/ml using purified DNA and ∼1 trypanosome/ml using supernatant prepared from boiled blood, while those of classical PCR tests ranged from 10 to 103 trypanosomes/ml. There was 100% agreement in the detection of the LAMP product by real-time gel electrophoresis and the DNA-intercalating dye SYBR green I. The LAMP amplicons were unequivocally confirmed through sequencing and analysis of melting curves. The assay was able to amplify parasite DNA from native cerebrospinal fluid (CSF) and double-centrifuged supernatant prepared from boiled buffy coat and bone marrow aspirate. The robustness, superior sensitivity, and ability to inspect results visually through color change indicate the potential of TgsGP LAMP as a future point-of-care test

Loop-mediated isothermal amplification test for Trypanosoma vivax based on satellite repeat DNA

Trypanosoma vivax is major cause of animal trypanosomiasis and responsible for enormous economic burden in Africa and South America animal industry. T. vivax infections mostly run low parasitaemia with no apparent clinical symptoms, making diagnosis a challenge. This work reports the design and evaluation of a loop-mediated isothermal amplification (LAMP) test for detecting T. vivax DNA based on the nuclear satellite repeat sequence. The assay is rapid with results obtained within 35min. The analytical sensitivity is ∼1trypanosome/ml while that of the classical PCR tests ranged from 10 to 10 trypanosomes/ml. The T. vivax LAMP test reported here is simple, robust and has future potential in diagnosis of animal trypanosomiasis in the field