PCR-based screening and lineage identification of Trypanosoma cruzi directly from faecal samples of triatomine bugs from northwestern Argentina

Abstract

This study applied improved DNA extraction and polymerase chain reaction strategies for screening and identification of Trypanosoma cruzi lineages directly from faeces of triatomines collected in a well-defined rural area in northwestern Argentina. Amplification of the variable regions of the kinetoplastid minicircle genome (kDNA-PCR) was performed in faecal lysates from 33 microscope (MO)-positive and 93 MO-negative Triatoma infestans, 2 MO-positive and 38 MO-negative Triatoma guasayana and 2 MO-positive and 73 MO-negative Triatoma garciabesi. kDNA-PCR detected T. cruzi in 91% MO-positive and 7.5% MO-negative T. infestans, which were confirmed by amplification of the minicircle conserved region. In contrast, kDNA-PCR was negative in all faecal samples from the other triatomine species. A panel of PCR-based genomic markers (intergenic region of spliced-leader DNA, 24Salpha and 18S rRNA genes and A10 sequence) was implemented to identify the parasite lineages directly in DNA lysates from faeces and culture isolates from 28 infected specimens. Two were found to be infected with TCI, 24 with TCIIe, 1 with TCIId and 1 revealed a mixed TCI+TCII infection in the faecal sample whose corresponding culture only showed TCII, providing evidence of the advantages of direct typing of biological samples. This study provides an upgrade in the current diagnosis and lineage identification of T. cruzi in field-collected triatomines and shows T. cruziII strains as predominant in the region.

Fig. 1

PCR-based identification of Trypanosoma cruzi lineages in faeces and culture isolates of naturally infected T. infestans. (A) Identification of TCI and TCII by SL-DNA PCR. TCI is identified from a culture isolate of specimen IG-41 and TCII from faeces of specimen P-15. DG-100-F and DG-100-10 are faecal and culture samples from a same triatomine, which revealed a dual TCI+TCII infection in faeces (DG-100 F) but only TCII in culture (DG-100-10). (B) Distinction between TCIId and TCIIb or TCIIe by 24Sα rDNA-PCR. TCIId is identified in a culture isolate from specimen SI-2-6 (125 bp + 140 bp amplicons), whereas TCIIb or TCIIe are detected in the culture isolate of DG-100-10 (140 bp product alone). (C) Differential amplification of TCIIb and TCIIe by A10 and 18S rDNA-PCR. TCIIb control strain shows only amplification of 18S rDNA genes. In contrast, TCIIe control strain and culture isolate from specimen DG-100-10 show amplification of both markers. T. cruzi control strains: TCI, T. cruzi I (X-10); TCllb, T. cruzi IIb (Tu 18); TC IId, T. cruzi IId (Mn CL2); TCIIe, T. cruzi IIe (CL-Brener). M1: 100 bp ladder DNA molecular weight marker, M2, 50 bp ladder DNA molecular weight marker; 3% agarose gel electrophoresis stained with ethidium bromide.