Detection of Trypanosoma cruzi and Trypanosoma rangeli infection in triatomine vectors by amplification of the histone H2A/SIRE and the sno-RNA-C11 genes

Abstract

SUMMARYTrypanosoma rangeli is non pathogenic for humans but of important medical and epidemiological interest because it shares vertebrate hosts, insect vectors, reservoirs and geographic areas with T. cruzi, the etiological agent of Chagas disease. Therefore, in this work, we set up two PCR reactions, TcH2AF/R and TrFR2, to distinguish T. cruzi from T. rangeli in mixed infections of vectors based on amplification of the histone H2A/SIRE and the small nucleolar RNA Cl1 genes, respectively. Both PCRs were able to appropriately detect all T. cruzi or T. rangeli experimentally infected-triatomines, as well as the S35/S36 PCR which amplifies the variable region of minicircle kDNA of T. cruzi. In mixed infections, whereas T. cruzi DNA was amplified in 100% of samples with TcH2AF/R and S35/S36 PCRs, T. rangeli was detected in 71% with TrF/R2 and in 6% with S35/S36. In a group of Rhodnius colombiensis collected from Coyaima (Colombia), T. cruzi was identified in 100% with both PCRs and T. rangeli in 14% with TrF/R2 and 10% with S35/S36 PCR. These results show that TcH2AF/R and TrF/R2 PCRs which are capable of recognizing all T. cruzi and T. rangeli strains and lineages could be useful for diagnosis as well as for epidemiological field studies of T. cruzi and T. rangeli vector infections.

Cl gene cluster encoding several small nucleolar RNAs: a comparison amongst trypanosomatids

“…On the other hand, species-specific differences in the nontranscribed regions of Cl-snoRNAs could be useful for developing PCR-based diagnostic tools. Indeed, Morales et al (2002) have developed a PCR test specific for T. rangeli detection, which does not amplify the DNA of any T. cruzi groups (Pavia et al 2007). …”

Chagasic Cardiomyopathy

“…Their performance varies depending on aspects like type and number of the target amplification, lack of polymorphisms among the parasite DTU-annealing primer target, sample volume, treatment and conservation, DNA extraction method, type of DNA polymerase used, and thermo-cycling program, among variables (Schijman et al, 2011). Some PCR tests show disadvantages like the amplification of polymorphic fragments or of similar-size bands in both T. cruzi and T. rangeli infections, the deviation of the test towards T. cruzi in mixed infections with T. rangeli, and the possible integration of the parasite's kDNA in the human genome (Gil et al, 2007;Pavía et al, 2003Pavía et al, , 2007. Bearing all this in mind, the Molecular Parasitology Laboratory at Pontificia Universidad Javeriana designed and standardized the TcH2AF-R PCR, specific for T. cruzi (Pavia et al, 2003).…”

Evaluación de las pruebas de PCR TcH2AF-R y S35-S36 para la detección de Trypanosoma cruzi en tejido cardiaco de ratón

“…Se utilizaron los iniciadores TcH2AF (5'-GAGAGTGATCGTGGGAGAGC-3') y TcH2AR (5'-AGTGGCAGACTTTGGGGTC-3'), los cuales permiten la amplificación de una banda de 230 pb de la región 3' no codificante de la unidad de 1,2 kb perteneciente al gen que codifica para la histona H2A y a los nucleótidos 16 a 246 del elemento SIRE (10)(11)(12) (11). También se utilizaron los iniciadores denominados S35 (5'-AAATAATGTACGGG(T/G)GAGATGCATGA-3') y S36 (5'-GGGTTCGATTGGGGTTGGTGT-3'), los cuales amplifican un fragmento de 330 pb derivado de la región variable de los minicirculos de T. cruzi (13,20).…”