Species specific detection of Trypanosoma cruzi and Trypanosoma rangeli in vector and mammalian hosts by polymerase chain reaction amplification of kinetoplast minicircle DNA.

Several groups have recently developed molecular tests for the detection of Trypanosoma cruzi, the causative agent of Chagas' disease. Polymerase chain reaction (PCR) amplification of kinetoplast minicircle DNA sequences appears to be the most sensitive method. However, the specificity of PCR-based diagnostic methods was challenged when the complete sequence of Trypanosoma rangeli DNA minicircles was discovered. In the present study, we conducted. PCR experiments using the S35/S36 primers in Rhodnius prolixus and Balb/c mice with single and mixed infections of T. cruzi and/or T. rangeli. In single infections, the profile of each trypanosome was easily distinguishable in haemolymph, salivary gland and intestinal tissues and faeces of insect vectors. In mixed infections of anterior intestine (where T. rangeli is more predominant than T. cruzi), the DNA amplification profile of both parasites was observed simultaneously. Conversely, only the T. cruzi profile was observed in rectal ampulla (where T. cruzi is more abundant than T. rangeli). In mice with single infections of T. cruzi or T. rangeli, the profiles of amplified DNA were easily distinguishable in each case. The T. cruzi profile was dominant in most mixed infections, probably due to the fact that T. cruzi minicircles are more abundant and consequently compete more eagerly for annealing with the S35/S36 primers. In cases of mixed infections where T. rangeli was initially more abundant than T. cruzi, the specific T. rangeli 760 bp band was present for 7 days after infection and then this band and others ranging from 300 to 450 bp disappeared and only the typical T. cruzi 330 bp band remained. The S35/S36 primers used in polyacrylamide gel electrophoresis (PAGE) detected T. cruzi specifically, and prevented misdiagnosis due to the presence of T. rangeli. This technique can also be used to identify parasites in different stages of the infection (acute or chronic) in vertebrate hosts and to localize the parasites in the insect vector.

Vallejo GA ，Guhl F ，Chiari E ，Macedo AM ... -  《ACTA TROPICA》

Amplification of a specific repetitive DNA sequence for Trypanosoma rangeli identification and its potential application in epidemiological investigations.

Trypanosoma rangeli can infect humans as well as the same domestic and wild animals and triatomine vectors infected by Trypanosoma cruzi in Central and South America. This overlapping distribution complicates the epidemiology of American trypanosomiasis due to the cross-reactivity between T. rangeli and T. cruzi antigens and the presence of conserved DNA sequences in these parasites. We have isolated a T. rangeli-specific DNA repetitive element which is represented in approximately 103 copies per parasite genome and is distributed in several chromosomal bands. The 542-bp nucleotide sequence of this element, named P542, was determined and a PCR assay was standardized for its amplification. The sensitivity of the assay is high, allowing the detection of one tenth of the DNA content of a single parasite. The presence of the P542 element was confirmed in 11 T. rangeli isolates from mammalian hosts and insect vectors originating from several countries in Latin America. Negative amplification was observed with different T. cruzi strains and other trypanosomatids. The potential field application of the P542 PCR assay was investigated in simulated samples containing T. rangeli and/or T. cruzi and intestinal tract and feces of Rhodnius prolixus. Epidemiological studies were conducted in DNA preparations obtained from the digestive tracts of 12 Rhodnius colombiensis insects collected in a sylvatic area in Colombia. Positive amplification of the P542 element was obtained in 9/12 insects. We have also compared in the same samples the diagnostic performance of two PCR assays for the amplification of the variable domain of minicircle kinetoplast DNA (kDNA) and of the large subunit (LSU) of the ribosomal RNA gene of T. cruzi and T. rangeli. Data indicate that the kDNA PCR assay does not allow diagnosis of mixed infections in most insects. On the other hand, the PCR assay of the LSU RNA gene showed lower sensitivity in the detection of T. rangeli than the PCR assay of the P542 element. It is predicted that the use of sensitive detection techniques will indicate that the actual distribution of T. rangeli in America is wider than presumed.

Vargas N ，Souto RP ，Carranza JC ，Vallejo GA ，Zingales B ... -  《EXPERIMENTAL PARASITOLOGY》

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

Trypanosoma 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.

Pavia PX ，Vallejo GA ，Montilla M ，Nicholls RS ，Puerta CJ ... -  《REVISTA DO INSTITUTO DE MEDICINA TROPICAL DE SAO PAULO》

High prevalence of Trypanosoma rangeli and Trypanosoma cruzi in opossums and triatomids in a formerly-endemic area of Chagas disease in Southeast Brazil.

In Brazil Trypanosoma rangeli has been detected in humans, sylvatic mammals and vectors in the Amazon Basin and in wild rodents in a Southern State. Here we report for the first time a high prevalence of T. rangeli in opossums and triatomids captured in peridomestic environments in a formerly-endemic area of Chagas disease in Southeast Brazil. Five molecular typing tools clearly indicate the presence of T. rangeli and Trypanosoma cruzi in mammalian reservoirs and triatomids. Twenty-one opossums (Didelphis albiventris) were captured and flagellates were detected in the blood of 57.1% (12/21) of the animals. Single infections with T. rangeli or T. cruzi were diagnosed, respectively, in 58.4 and 8.3% of the opossums. Mixed infections were observed in 33.3%. Forty-four triatomids (38 Rhodnius neglectus and 6 Panstrongylus megistus) were collected in palm trees within 50 m from human dwellings. Flagellates were observed in the digestive tract and feces of 50% of the insects. PCR assays performed in DNA samples obtained from 16 cultures of the intestinal tract revealed single infection with T. cruzi (68.7%) or T. rangeli (6.3%), as well as mixed infections (25%). T. rangeli was also detected in the hemolymph of two specimens. Genotyping revealed predominance of T. cruzi I. The data suggest that R. neglectus in conjunction with D. albiventris may be significant factors in the maintenance of the sylvatic and peridomestic cycles of T. rangeli in the region. The finding of T. cruzi and T. rangeli in triatomine species capable of domiciliation and therefore considered as alternative vectors for the parasite transmission opens up the possibility of re-establishment of Chagas disease following reinfestation of houses.

Ramirez LE ，Lages-Silva E ，Alvarenga-Franco F ，Matos A ，Vargas N ，Fernandes O ，Zingales B ... -  《ACTA TROPICA》

Characterization of a candidate Trypanosoma rangeli small nucleolar RNA gene and its application in a PCR-based parasite detection.

In this study, we report the isolation and characterization of a candidate Trypanosoma rangeli small nucleolar RNA (snoRNA) gene, and the development of a PCR assay for detection of the parasite based on its nucleotide sequence. This gene, isolated from a T. rangeli genomic sub-library, was named snoRNA-cl1 and is encoded by a multi-copy gene of 801bp in length. Computer sequence analysis of snoRNA-cl1 showed the presence of two sequence motifs, box C and box D, as well as of two long stretches that perfectly complement the universal core region of the mature rRNA 28S, suggesting that cl1 encodes for a Box C/D snoRNA from the parasite. Hybridization analysis using cl1 as probe, showed a weak hybridization signal with Trypanosoma cruzi DNA, demonstrating the existence of differences in this locus between these two species. Two oligonucleotide primers from this gene, which specifically amplified a 620-bp fragment in KP1 (+) and KP1 (-) strains of T. rangeli, were used in a PCR assay. The amplification allowed the detection of 1pg of DNA in the presence of heterologous DNA and no amplification was observed with different T. cruzi strains (groups I and II). In addition, the PCR assay reported here is able to detect T. rangeli in the presence of T. cruzi DNA, and is useful for detection of the parasite in samples from infected vectors.

Morales L ，Romero I ，Diez H ，Del Portillo P ，Montilla M ，Nicholls S ，Puerta C ... -  《EXPERIMENTAL PARASITOLOGY》