Single nucleotide polymorphisms and copy-number variations in the Trypanosoma brucei repeat (TBR) sequence can be used to enhance amplification and genotyping of Trypanozoon strains.

The Trypanosoma brucei repeat (TBR) is a tandem repeat sequence present on the Trypanozoon minichromosomes. Here, we report that the TBR sequence is not as homogenous as previously believed. BLAST analysis of the available T. brucei genomes reveals various TBR sequences of 177 bp and 176 bp in length, which can be sorted into two TBR groups based on a few key single nucleotide polymorphisms. Conventional and quantitative PCR with primers matched to consensus sequences that target either TBR group show substantial copy-number variations in the TBR repertoire within a collection of 77 Trypanozoon strains. We developed the qTBR, a novel PCR consisting of three primers and two probes, to simultaneously amplify target sequences from each of the two TBR groups into one single qPCR reaction. This dual probe setup offers increased analytical sensitivity for the molecular detection of all Trypanozoon taxa, in particular for T.b. gambiense and T. evansi, when compared to existing TBR PCRs. By combining the qTBR with 18S rDNA amplification as an internal standard, the relative copy-number of each TBR target sequence can be calculated and plotted, allowing for further classification of strains into TBR genotypes associated with East, West or Central Africa. Thus, the qTBR takes advantage of the single-nucleotide polymorphisms and copy number variations in the TBR sequences to enhance amplification and genotyping of all Trypanozoon strains, making it a promising tool for prevalence studies of African trypanosomiasis in both humans and animals.

Assessment of Trypanosoma evansi prevalence and associated risk factors by immune trypanolysis test in camels from Ghardaïa district, southern Algeria.

Trypanosoma evansi (T. evansi) is a flagellated parasite with worldwide distribution, mainly affecting camels, horses, dogs, buffaloes and wild animals. Trypanosomosis caused by T. evansi, known as surra, is a vector borne disease that affects the health and productivity of camels. The aim of our study was to assess the prevalence of trypanosomosis due to T. evansi in camels by the immune trypanolosis test and to identify associated risk factors. Our cross-sectional study was performed on 161 camels from Ghardaïa district, southern Algeria. A structured questionnaire was used to collect data on individual characteristics (age, gender and breed) husbandry management (herd size and activity of animals) and health conditions (history of abortion and clinical symptoms). The immune trypanolysis test revealed an overall seroprevalence of 9.3% (CI 95%, 5.9-14.9). Possible factors associated with T. evansi infection were analysed by univariate and multivariate logistic regression. The results showed that risk factors for camels were history of symptoms (P = 0.002, OR = 21.91, CI95% = 3.48-169.80), racing activities (P = 0.003, OR = 0.01, CI95% = 0.001-0.18) and small herd size (P = 0.013, OR = 8.22, CI95% = 1.64-49.75). In conclusion, this study showed that T. evansi is endemic in camels of Ghardaïa district. To reduce dissemination of the disease to non-endemic areas, it is recommended to minimise risk factors associated with the infection.

Comparison of serological and molecular tests for detection of Trypanosoma evansi in domestic animals from Ghardaïa district, South Algeria.

Trypanosoma evansi (T. evansi) is a hemoflagellate parasite that affects a broad range of mammalian hosts and that causes a disease called surra. Diagnosis of surra based on clinical symptoms alone is inaccurate. Therefore, a variety of serological and molecular diagnostic tests are used to assist in the detection of T. evansi infections. The aim of this study was to compare the diagnostic performance of four serological tests (CATT/T.evansi, immune trypanolysis, ELISA with purified variant surface glycoprotein RoTat 1.2 and with whole cell lysate) and two molecular PCR tests targeting sequences within the ribosomal genes locus (ITS1 TD PCR and 18S qPCR). Tests were carried out on blood samples from 161 dromedary camels, 93 horses, 129 goats, 168 sheep, 127 bovines and 76 dogs. Latent class analysis was carried out to calculate the sensitivity and specificity of each diagnostic test. Cohen's Kappa test was used to assess the concordance between the different diagnostic tests. Overall positivity rates observed with the serological tests were as follows: 3.1 % with CATT/T.evansi, 4.9 % with ELISA/RoTat 1.2, 3.4 % with ELISA/whole lysate and 2.0 % with immune trypanolysis (TL). Among the 754 samples tested with the molecular tests, 1.7 % were positive with 18S qPCR and 1.3 % with ITS1 TD PCR. Cohen's Kappa test showed agreement ranging from fair to substantial (k = 0.2-0.8) between serological diagnostic tests. However, it showed a perfect agreement (k = 0.868) between molecular diagnostic tests. Latent class analysis showed that all serological tests were 100 % sensitive, in contrast to the molecular tests with 47 % sensitivity. All tests, though, were highly specific (≥ 97 %). Given the persistence of circulating antibodies after cure, detectable by serological tests, it is recommend combining a serological and a molecular diagnostic test for accurate diagnosis of infection with T. evansi in domestic animals.