Detection of Trypanosoma cruzi and Trypanosoma rangeli Infection by Duplex PCR Assay Based on Telomeric Sequences

Abstract

We used the species specificity and repetitious nature of subtelomeric kinetoplastida sequences to generate a duplex PCR assay for the simultaneous detection of Trypanosoma cruzi and Trypanosoma rangeli in experimentally and naturally infected triatomine (Reduviid) bugs and in infected human subjects. The assay was species specific and was capable of detecting 1/20th of T. cruzi and 1/4th of T. rangeli cell equivalents without complementary hybridization. In addition, the PCR-based assay was robust enough for direct application to difficult biological samples such as Reduviid feces or guts and was capable of recognizing all T. cruzi and T. rangeli strains and lineages. Because the assay primers amplify entirely different target sequences, no reaction interference was observed, facilitating future adaptation of this assay to an automated format.

Predominance of Trypanosoma rangeli infection in children from a Chagas disease endemic area in the west-shore of the Panama canal

“…Hemoculture of samples was performed as described by Vasquez et al (1997). To corroborate and identify the species of trypanosomes isolated, a duplex PCR assay (Chiurillo et al 2003) followed by dot blot hybridization with radiolabeled oligonucleotide probes specific for T. cruzi and T. rangeli was performed on initially microscopically positive hemocultures.Sera samples were at first screened for the presence of anti-T. cruzi antibodies using an indirect immunofluorescence antibody test (IFAT) with a local T. cruzi isolate (Burunga strain) as antigen, according to the technique described by Guhl and Nicholls (2001). The sensitivity and specificity of this assay is of 95.2 and 96.3%, respectively.…”

“…Hemoculture of samples was performed as described by Vasquez et al (1997). To corroborate and identify the species of trypanosomes isolated, a duplex PCR assay (Chiurillo et al 2003) followed by dot blot hybridization with radiolabeled oligonucleotide probes specific for T. cruzi and T. rangeli was performed on initially microscopically positive hemocultures.…”

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

“…With this, we can conclude that those primers do not allow the detection of mixed infections. A duplex PCR assay based on telomeres sequences have been developed to determine the presence of both parasites, nevertheless, since the number of samples analyzed was reduced to a single triatomine bug 6 , more studies are needy in order to asses the lack of interference or competition between T. cruzi and T. rangeli DNA telomeric sequences in field samples.…”

“…Due to direct microscopic detection of trypanosomes, the traditional method for assessment of infection in vectors is not able to distinguish T. cruzi from T. rangeli infection, several polymerase chain reaction techniques have been developed 3,4,5,6,9,13,19,28,33,36,40,41 . However, current PCR assays used for mixed infection detection show some disadvantages such as the amplification of bands of similar size both in T. cruzi and T. rangeli 27,28 , the amplification of polymorphic fragments 11,19 , and bias to T. cruzi in the case of mixed T. cruzi and T. rangeli infection 9,33,36 .…”

Semisolid liver infusion tryptose supplemented with human urine allows growth and isolation of Trypanosoma cruzi and Trypanosoma rangeli clonal lineages

“…For determination of the species comprising each colony, we performed PCR analysis of telomeric and subtelomeric sequences (9) , which allows amplification of distinct fragments for T. cruzi (100bp) and T. rangeli (170bp). Primers used for T. cruzi detection were Tc189Fw2 (5′-CCAACGCTCCGGGAAAAC-3′) and Tc189Rv3 (5′-GCGTCTTCTCAGTATGGACTT-3′), and those used for T. rangeli were TrF3 (5′-CCCCATACAAAACACCCTT-3′) and TrR8 (5′-TGGAATGACGGTGCGGCGAC-3′).…”