PCR-based identification of Trypanosoma lewisi and Trypanosoma musculi using maxicircle kinetoplast DNA.

Trypanosoma lewisi, transmitted by rat fleas, is a widespread pathogen specific to rats with records of human infection cases. Its closely related species with global distribution, Trypanosoma musculi, is transmitted between mice by ingestion of infected fleas. These trypanosomes are of similar morphology, making it difficult to distinguish them by microscopy. In this study, we have developed a rapid, sensitive and reliable PCR method for the diagnosis of T. lewisi and T. musculi. The T. lewisi-specific amplicons were not produced by other Trypanosoma, such as T. musculi, T. brucei complex or T. cruzi, neither by an outgroup of Leishmania amazonensis. The detection limits of the three pairs of T. lewisi-specific primers were 50ng, 1ng and 10ng of total DNA, respectively. The primers designed for T. musculi primers showed specifically that amplicon strictly in T. musculi and their detection limits were 10ng and 1ng of total DNA. To simplify the detection process, we managed to apply our method directly on tail blood samples without complicated DNA purification. In conclusion, PCR with our primers could be a highly sensitive, specific protocol to detect and distinguish T. lewisi and T. musculi from other trypanosomes.

Resistance to normal human serum reveals Trypanosoma lewisi as an underestimated human pathogen.

Human-infectious trypanosomes such as Trypanosoma cruzi, T. brucei rhodesiense, and T. b. gambiense can be discriminated from those only infecting animals by their resistance to normal human serum (NHS). These parasites are naturally resistant to trypanolysis induced by the human-specific pore-forming serum protein apolipoprotein L1 (ApoL-1). T. lewisi, a worldwide distributed parasite, has been considered as rat-specific and non-pathogenic to the natural hosts. Here we provide evidence that 19 tested T. lewisi isolates from Thailand and China share resistance to NHS. Further investigation on one selected isolate CPO02 showed that it could resist at least 90% NHS or 30 µg/ml recombinant human ApoL-1 (rhApoL-1) in vitro, in contrast to T. b. brucei which could not survive in 0.0001% NHS and 0.1 µg/ml rhApoL-1. In vivo tests in rats also demonstrated that this parasite is fully resistant to lysis by NHS. Together with recent reports of atypical human infection by T. lewisi, these data allow the conclusion that T. lewisi is potentially an underestimated and thus a neglected human pathogen.

Detection of Trypanosoma lewisi from wild rats in Southern China and its genetic diversity based on the ITS1 and ITS2 sequences.

Trypanosoma lewisi has widely been considered as a non-pathogenic rat trypanosome. However, more and more cases of humans infected with T. lewisi have been reported around the world, indicating that it can infect humans in some undetermined circumstances. Quick and sensitive diagnosis of infection by T. lewisi is important for both treatment of patients and epidemiological studies of this parasite. In this paper, three methods i.e. wet blood smear (diagnosis by microscopy), PCR and LAMP were used to detect T. lewisi from 238 wild rats (Rattus norvegicus) collected from the field in Huadu, Guangdong province, China. Infection rates of these samples detected by the 3 methods was 6.7% (16/238), 12.6% (30/238), and 18.9% (45/238), respectively. LAMP could detect all samples shown positive by microscopical observation of wet smear and by single PCR indicating good potential for application in the detection of T. lewisi. So far as we know, this is the first report of the LAMP method being used to detect T. lewisi in wild rats. The specific T. lewisi LAMP primers were able to amplify the target fragment from the genomic DNA of 19 T. lewisi strains isolated from Huadu, Guangdong province (n=16), Changchun, Jilin province of China (n=1) and from Thailand (n=2). Based on the analyses of ITS1 (internal transcribed spacer 1) and ITS2 sequences, these 19 strains show a very close genetic relationship with over 96-97% similarity to the other corresponding sequences of T. lewisi published in Genbank. Phylogenetic trees of the species in the subgenus Herpetosoma were constructed, based on the ITS1 and ITS2 sequences, and these results also indicate that they are closely related and in the same clade.