Loop-mediated isothermal amplification (LAMP) method for rapid detection of Trypanosoma brucei rhodesiense

Loop-mediated isothermal amplification (LAMP) of DNA is a novel technique that rapidly amplifies target DNA under isothermal conditions. In the present study, a LAMP test was designed from the serum resistance-associated (SRA) gene of Trypanosoma brucei rhodesiense, the cause of the acute form of African sleeping sickness, and used to detect parasite DNA from processed and heat-treated infected blood samples. The SRA gene is specific to T. b. rhodesiense and has been shown to confer resistance to lysis by normal human serum. The assay was performed at 62°C for 1 h, using six primers that recognised eight targets. The template was varying concentrations of trypanosome DNA and supernatant from heat-treated infected blood samples. The resulting amplicons were detected using SYTO-9 fluorescence dye in a real-time thermocycler, visual observation after the addition of SYBR Green I, and gel electrophoresis. DNA amplification was detected within 35 min. The SRA LAMP test had an unequivocal detection limit of one pg of purified DNA (equivalent to 10 trypanosomes/ml) and 0.1 pg (1 trypanosome/ml) using heat-treated buffy coat, while the detection limit for conventional SRA PCR was ∼1,000 trypanosomes/ml. The expected LAMP amplicon was confirmed through restriction enzyme RsaI digestion, identical melt curves, and sequence analysis. The reproducibility of the SRA LAMP assay using water bath and heat-processed template, and the ease in results readout show great potential for the diagnosis of T. b. rhodesiense in endemic regions

Prevalence of trypanosomosis in camel calves : A pilot study in Laikipia District of Kenya

La trypanosomose est l'une des maladies les plus importantes du chamelon. Elle se présente sous une forme aiguë et est généralement fatale si aucun traitement n'est administré. Une étude a été effectuée au ranch de Mogwooni dans le district de Laikipia au Kenya pour déterminer la prévalence de la trypanosomose chez des chamelons de races diverses et pour évaluer la technique de centrifugation en tube pour microhématocrite (Tcmh), le test d'agglutination sur carte au latex sensibilisé avec des anticorps monoclonaux (Suratex®), l'examen à l'état frais et l'inoculation à la souris (IS) dans le diagnostic de la maladie chez le chamelon. Les tests ont été évalués pendant une période de 16 mois. Les prévalences moyennes de Trypanosoma evansi ont été de 4,5 p. 100 (test à l'état frais), 11,1 p. 100 (Tcmh), 14,6 p. 100 (IS) et 28,3 p. 100 (Suratex®). Le taux de mortalité des chamelons dû à la trypanosomose a été de 12,3 p. 100 pour un taux global de mortalité de 15 p. 100. Le coût des soins vétérinaires (anthelminthiques, acaricides et trypanocides) a été en moyenne de 4,6 dollars américains par chamelon et par an. Il est donc recommandé que le diagnostic soit systématiquement accompagné d'un traitement approprié pour assurer la survie des chamelons dans les zones où la trypanosomose est endémique. (Résumé d'auteur

Sleeping sickness: the wake-up of translational biomarker research

Comunicaciones a congreso

Extent and implications of incorrect offspring-sire relationships in pastoral production system in Kajiado District, Kenya

The aim of this study was to evaluate accuracy of farmer's paternity identification which determines success of future breed selection and hence genetic gain. Paternity of 269 Orma/zebu and Sahiwal/zebu calves was evaluated using genetic markers and the likelihood based method. Results indicate that only 6.7% farmer alleged paternities were confirmed, 88% parent-offspring relationships were rejected and 18% parent-offspring relationships were undetermined. However, 82% of offsprings were assigned at least 80% confident paternities to one of the sampled candidate males. These results suggest that there is need to institute proper breeding program in the pastoral area if farmers are to benefit from their current efforts of breed improvement

Some pharmacokinetic parameters of the trypanocidal drug homidium bromide in Friesian and Boran steers using an enzyme-linked immunosorbent assay (ELISA)

Pharmacokinetic studies on the trypanocidal drug homidium bromide using a competitive enzyme immunoassay (detection limit 0.1 ng/mL) are reported for non-infected Friesian and Boran steers following treatment with homidium bromide at a dose of 1.0 mg/kg b.w. Following intravenous (i.v.) treatment of Friesian steers (n = 5), the mean serum drug concentrations were 31.9 ± 2.1 and 3.9 ± 0.4 ng/ml at 1 and 24 h, respectively. The decline in serum drug concentration was tri-exponential with half-lives of 0.064 ± 0.037 h for t( 1/2 ?), 7.17 ± 1.87 h for T( 1/2 ?) and 106.3 ± 6.6 h for t( 1/2 ?) for distribution and elimination phases 1 and 2, respectively. Drug was detectable in serum for 17 days following treatment. The mean residence time (MRT) was 63.4 ± 7.5 h. Following intramuscular (i.m.) treatment of Friesian steers (n = 5), the drug concentration at 1 h after treatment was 72.5 ± 2.2 ng/mL. This declined to 9.8 ± 1.8 ng/mL at 24 h. Low concentrations of between 0.1 and 0.3 ng/mL remained in circulation for up to 90 days post-treatment. Following intramuscular treatment of Boran steers (n = 5), the mean serum drug concentration at 1 h after treatment was 112.1 ± 40.3 ng/mL. By 24 h after treatment, the concentration had fallen to 13.0 ± 3.3 ng/mL. Thereafter, the serum drug concentration-versus-time profile and the pharmacokinetic parameters obtained following non-compartmental analysis were similar to those obtained following intramuscular treatment of Friesian steers

Clinical and pathological characterization of blood stream forms and cerebrospinal fluid T. b. rhodesiense trypanosomes isolated from a patient using rabbits

Clinical and pathological characterisation of blood stream (BSF) and cerebrospinal fluid (CSF) forms of Trypanosoma brucei rhodesiense trypanosome isolated from a sleeping sickness patient were investigated in rabbits. The study aimed at investigating whether there is any significant difference in clinical and pathological presentation in rabbits infected by the two forms of trypanosomes. Each form of parasite was inoculated into five rabbits at 10,000 trypanosomes/ml while five rabbits were used as un-infected controls. Parasitaemia development, body temperature, packed cell volume (PCV), body weight, food and water intake, heartbeat and respiration were monitored daily for 30 days post infection when the experiment was terminated. Pathological changes were evaluated following euthanasia. All the infected rabbits became parasitaemic 6 days post infection (dpi) and the parasitaemia levels were significantly higher (p=0.01) for the BSF than the CSF infected rabbits. No significant difference was observed in heartbeat, respiration, food and water intake as well as PCV. However, CSF infected rabbits had a significantly (p=0.01) higher body temperature and weights than BSF infected rabbits. There was no major difference in the clinical manifestation of the disease caused by the two forms of parasite. However, temporary paralysis was observed around the left side of the neck in one rabbit infected with CSF trypanosomes whereas mucoid stool with the presence of amoeba cysts were observed in the rabbits infected with the BSF trypanosomes. The spleen weights of CSF infected rabbits was heavier (3.59 ± 1.13 grams) than the BSF infected rabbits (2.92 ± 0.78 grams). The proportions of monocytes were significantly higher (p<0.05) in the CSF infected rabbits while neutrophils proportions were significantly higher (p<0.05) in the BSF infected rabbits. The rest of the haematological changes were not significantly different. Results from this study demonstrate that BSF trypanosomes appeared relatively more virulent than the CSF trypanosomes. It would be important to carry out similar studies using a higher number of both BSF and CSF trypanosomes isolated from the same patient and different patients to authenticate this observation

The effects of drug-sensitive and drug-resistant Trypanosoma congolense infections on the pharmacokinetics of homidium in Boran cattle

Two groups of five Boran (Bos indicus) cattle were infected with one of two populations of Trypanosoma congolense; one drug-sensitive (IL1180), and one drug-resistant (IL3330). The animals were then treated intramuscularly with homidium bromide at a dose rate of 1.0 mg kg - bodyweight 7 days after trypanosomes were detected in the peripheral blood of all the five animals in each grouFollowing treatment of cattle infected with drug-sensitive trypanosomes. parasites could no longer be detected in the bloodstream of four out of five cattle after 24 h, and after 48 h for the fifth animal. The animals remained aparasitaemic up to the end of the observation period of 90 days and serum drug concentrations determined by enzyme-linked immunosorbent assay (ELISA) remained above the detection limit of 0.1 ng ml for the entire period. Following treatment of cattle infected with drug-resistant trypanosomes. parasites did not disappear from the bloodstream in any of the five animals. The rate of drug elimination was greater in cattle infected with drug-resistant trypanosomes and the drug was no longer detectable approximately 3 weeks after treatment. Non-compartmental pharmacokinetic analysis showed that the values for t1 beta, of 75.5 ± 16.9 h, the area under the curve (AUC a-x ) of 1.33 ± 0.156 mce g h ml - and the MRT a-x of 32.8 ± 4.45 h obtained in cattle infected with the drug-resistant trypanosome population were significantly lower than the values of 424 ± 146 h for tJl, 1.67 ± 0.233 mug h ml -1 for AUC a-x and 297 ± 159 h for MRT a-x obtained in cattle infected with the drug sensitive population. The persistence of drug-resistant infections in cattle following homidium treatment was associated with more rapid drug elimination than in those in which infections with drug-sensitive parasites were cleared by the drug

Investigation into the effects of Trypanosoma congolense infections on the pharmacokinetics of Homidium in Boran cattle

Control of trypanosomes in cattle, sheep and goats in endemic areas has depended largely on the use of chemotherapeutic or chemoprophylactic agents. One such agent is homidium. The normal use of homidium has been in the treatment of infections due to both Trypanosoma congolense and T.vivax in cattle, sheep and goats at the recommended does rate of 1.0 mg kg-1b.w. This paper describes the results of an investigation into the chemotherapeutic activity of homidium against T.congolense infections in cattle. Two groups of five Boran cattle were infected with two populations of T.Congolense; one drug-sensitive IL 1180 and one drug-resistant (IL 3330). Parasitaemia was estimated using the methods of Murray et al. (1977); the serum drug levels by the method described by Murilla (1996) and the pharmacokinetic parameters using the formulae described by Baggot (1977). After infection, there was a rapid drop in packed cell volume (PCV) values from a mean pre-infection value of approximately 40% to approximately 25% within 14 days of infection in cattle infected with IL 1180. However, in cattle infected with IL 3330, the drop in PCV was more gradual from approximately 40% to approximately 30% within the same period of time. The animals were treated with homidium bromide at a dose rate of 1.0 mg kg-1 body weight (b.w.) seven days after the last animal in each group was detected positive. Following intermuscular (i.m.) treatment of cattle infected with drug-sensitive trypanosomes, no parasites were detected in the bloodstream of four out of five cattle within 24 hours; the fifth within 48 hours. During this period and for the next 10 days after treatment, an accelaration in the rate of drug elimination was observed. Thereafter, the rate of elimination reverted back to that observed in Non-infected cattle (Murilla, 1996). This was accompanied by an elevation in PCV to pre-infection values. The animals remained aparasitaemic up to the end of the 90 days observation period with low serum drug concentrations of between 0.1 and 0.3 ng ml-1 in circulation

Development and evaluation of an enzyme-linked immunosorbent assay (ELISA) for the determination of the trypanocidal drug homidium in serum of treated cattle

Two enzyme-linked immunosorbent assays (ELISA) for the determination of homidium in serum of treated cattle have been developed and evaluated. One is a direct competition (Assay 1) and the other an indirect competition assay (Assay 2). Both assays are highly sensitive with a limit of detection of 0.1 ng homidium per mL serum. Homidium levels were measurable in serum of cattle for over 2 months following administration of a single intramuscular (i.m.) dose at 1 mg/kg bodyweight. The level of sensitivity afforded by these assays makes them potentially useful tools in the pharmacokinetic evaluation of homidium and for invetigating drug resistance or causes of drug failure. Assay 2 was chosen as being most suitable for further studies