Pcr Para Trypanosoma Cruzi

Rev. Inst. Med. trop. S. Paulo 49(1):23-30, January-February, 2007

DETECTION OF Trypanosoma cruzi AND Trypanosoma rangeli INFECTION IN TRIATOMINE VECTORS BY AMPLIFICATION OF THE HISTONE H2A/SIRE AND THE SNO-RNA-CL1 GENES

Paula Ximena PAVIA(1), Gustavo Adolfo VALLEJO(2), Marleny MONTILLA(3), Rubén Santiago NICHOLLS(3) & Concepción Judith PUERTA(1)

SUMMARY Trypanosoma rangeli is nonpathogenic 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 histoneH2A/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% withTrF/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 forepidemiological field studies of T. cruzi and T. rangeli vector infections. KEYWORDS: Trypanosoma cruzi; Trypanosoma rangeli; Rhodnius prolixus; Rhodnius colombiensis; PCR, Histone H2A; SIRE; sno-RNA -Cl1.

INTRODUCTION Chagas disease, caused by the flagellate parasite Trypanosoma cruzi, affects fifteen countries throughout Latin America. The number of new cases has been estimated at 200,000 per year andabout 21,000 chagasic patients die each year from the disease17,42. In Colombia, Chagas disease is a major public health problem. It is estimated that 5% of the population is at a high risk of being infected and that approximately 700,000 people are currently infected17. Chagas disease is transmitted to humans by bloodsucking triatomine bugs, from which 23 species have been reported in Colombia. Themost important vector species that circulate in the domestic cycle in this country are Rhodnius prolixus, Triatoma dimidiata, Triatoma maculata, and Triatoma venosa whereas in the sylvatic regions Rhodnius pallescens and Rhodnius colombiensis are the prevalent ones12,15. Based on phenotypic and genotypic characters, T. cruzi has been divided into two principal lineages: T. cruzi I and T. cruzi II1.In addition, T. cruzi II is divided into five subgroups, named IIa-e2. While T. cruzi I, associated with opossums and an arboreal ecology, predominates from the Amazon basin northwards, T. cruzi II is associated with armadillos and a terrestrial ecology and predominates in southern cone countries of South America43.

On the other hand, although Trypanosoma rangeli infection in humans isharmless, this parasite is a serious concern for the epidemiology and diagnosis of Chagas disease due to its morphological similarity and immunological cross-reactivity with T. cruzi14. Moreover, these trypanosomes are sympatric and share triatomine insects as well as vertebrate hosts, allowing the occurrence of mixed infections7. Colombia is one of the countries in which T. cruzi shares vectors andreservoirs with T. rangeli7,13,14. Recently, two important epidemiological groups of T. rangeli have been described: KP1(-) strains, associated with the adaptive line of Rhodnius, represented by R. colombiensis, R. pallescens, and R. ecuadoriensis, and KP1(+) strains, associated with R. prolixus30-32. These two groups have been defined on the basis of independent mitochondrial (minicircle profile...