Genotyping of Trypanosoma cruzi: systematic selection of assays allowing rapid and accurate discrimination of all known lineages

Abstract

Trypanosoma cruzi, the agent of Chagas disease, can be subdivided into six discrete typing units (DTUs), TcI, TcIIa, TcIIb, TcIIc, TcIId or TcIIe, each having distinct epidemiologically important features. Dozens of genetic markers are available to determine the DTU to which a T. cruzi isolate belongs, but there is no consensus on which should be used. We selected five assays: three polymerase chain reaction (PCR)-restriction fragment length polymorphisms based on single nucleotide polymorphisms (SNPs) in the HSP60, Histone H1, and GPI loci, and PCR product size polymorphism of the LSU rDNA and mini-exon loci. Each assay was tested for its capacity to differentiate between DTUs using a panel of 48 genetically diverse T. cruzi clones. Some markers allowed unequivocal identification of individual DTUs, however, only by using a combination of multiple markers could all six DTUs be resolved. Based upon the results we recommend a triple-assay comprising the LSU rDNA, HSP60 and GPI markers for reliable, rapid, low-cost DTU assignment.

Figure 1

Example of LSU rDNA and mini-exon PCR product size polymorphism genotyping assay profiles. A, LSU rDNA—Lanes: 1, Rita cl5 (TcIIb); 2, Chaco23 col4 (TcIIb); 3, ARMA18 cl3 (TcIIc); 4, Sc43 cl1 (TcIId); 5, 92-80 cl2 (TcIId); 6, Bug2148 cl1 (TcIId), note absence of 125-bp band; 7, Chaco2 cl3 (TcIId); 8, PAH179 cl5 (TcIId); 9, Para6 cl4 (TcIId); 10, Para4 cl3 (TcIId); 11, Vinch101 cl1 (TcIId); 12, EPV20-1 cl1 (TcIIe); 13, P251 cl7 (TcIIe); 14, VFRA1 cl1 (TcIIe). B, LSU rDNA—Lanes: 1, C8 cl1 (TcI); 2, SAXP18 cl1 (TcI); 3, JR cl4 (TcI); 4, B187 cl10 (TcI); 5, 10R26 (TcIIa); 6, 92122102R [TcIIa(NA)]; 7, CanIII cl1 (TcIIa); 8, StC10R cl1 [TcIIa(NA)]; 9, Saimiri3 cl1 (TcIIa); 10, ERA cl2 (TcIIa); 11, JA2 cl2 (TcIIc); 12, SABP19 cl1 (TcIIc); 13, Vinch101 cl1 (TcIId); 14, LHVA cl4 (TcIIe). Note that comparison of Lanes 8–11 shows the four distinct product sizes: 130, 125, 117, and 110 bp. C, Mini-exon—Lanes: 1, M5631 cl5 (TcIIc); 2, JA2 cl2 (TcIIc); 3, ARMA18 cl3 (TcIIc); 4, 85/847 cl2 (TcIIc); 5, SABP cl1 (TcIIc); 6, VFRA1 cl1 (TcIIe); 7, Chaco2 cl3 (TcIId); 8, Esm cl3 (TcIIb); 9, X10/1 (TcI); 10, B187 cl10 (TcI); 11, JR cl4 (TcI); 12, 92101601P cl1 (TcI); 13, CanIII cl1 (TcIIa); 14, 92122102R [TcIIa(NA)]; 15, Saimiri3 cl1 (TcIIa); 16, X10610 cl5 (TcIIa); 17, ERA cl2 (TcIIa); 18, StC10R cl1 [TcIIa(NA)].

Figure2

Examples of PCR-RFLP genotyping profiles. A, HSP60: digestion products only are shown. Lanes: 1, X10610 cl5 (TcIIa); 2, Saimiri3 cl1 (TcIIa); 3, ERA cl2 (TcIIa); 4, JR cl4 (TcI); 5, 10R26 (TcIIa); 6, StC10R cl1 [TcIIa(NA)]; 7, CanIII cl1 (TcIIa); 8, X10/1 (TcI); 9, 92122102R [TcIIa(NA)]; 10, CJ005/PII (TcI); 11, B187 cl10 (TcI); 12, Chile C22 cl1 (TcI); 13, 92-80 cl2 (TcIId); 14, Rita cl5 (TcIIb); 15, Pot7a cl1 (TcIIb); 16, ARMA18 cl3 (TcIIc); 17, PAH179 cl5 (TcIId); 18, VFRA1 cl1 (TcIIe); 19, SABP19 cl1 (TcIIc); 20, M6241 cl6 (TcIIc); 21, Vinch101 cl1 (TcIId). B, Histone H1: digestion products from unpurified PCR products are shown. Lanes: 1, X10610 cl5 (TcIIa); 2, Saimiri3 cl1 (TcIIa); 3, ERA cl2 (TcIIa); 4, JR cl4 (TcIIc); 5, 10R26 (TcIIa); 6, StC10R cl1 [TcIIa(NA)]; 7, CanIII cl1 (TcIIa); 8, X10/1 (TcI); 9, Pot7b cl5 (TcIIb); 10, Rita cl5 (TcIIb); 11, JA2 cl2 (TcIIc); 12, ARMA13 cl1 (TcIIc); 13, CJ007/PI (TcI); 14, B187 cl10 (TcI); 15, SAXP18 cl1 (TcI); 16, 92122102R [TcIIa(NA)]; 17, SABP19 cl1 (TcIIc); 18, Para4 cl3 (TcIId); 19, CM25 cl2 (TcIIc); 20, PAH179 cl5 (TcIId); 21, Sc43 cl1 (TcIId); 22, Chaco17 col1 (TcIIe); 23, Tu18 cl2 (TcIIb); 24, Chaco23 col4 (TcIIb). C, GPI: each pair of lanes shows undigested PCR product followed by restriction digest products. Lanes: 1, SAXP18 cl1 (TcI); 2, VFRA1 cl1 (TcIIe); 3, StC10R cl1 [TcIIa(NA)]; 4, 10R26 (TcIIa); 5, Pot7a cl1 (TcIIb); 6, Rita cl5 (TcIIb); 7, JA2 cl2 (TcIIc); 8, Para4 cl3 (TcIId); 9, Vinch101 cl1 (TcIId); 10, Chaco9 col15 (TcIIe); 11, CanIII cl1 (TcIIa); 12, ARMA18 cl3 (TcIIc).

Figure3

Recommended triple-assay for discriminating T. cruzi DTUs.