Phylogenetic Analysis of Trypanosoma cruzi from Pregnant Women and Newborns from Argentina, Honduras, and Mexico Suggests an Association of Parasite Haplotypes with Congenital Transmission of the Parasite

Abstract

Trypanosoma cruzi, the causative agent of Chagas disease, exhibits a high genetic variability and has been classified into six discrete typing units (DTUs) named TcI through TcVI. This genetic diversity is believed to be associated with clinical characteristics and outcomes, but evidence supporting such associations has been limited. Herein, we performed a phylogenetic analysis of T. cruzi sequences of the mini-exon intergenic region obtained from a large cohort of pregnant women and newborns from Argentina, Honduras, and Mexico, to assess parasite genetic diversity and possible associations with congenital transmission. Analysis of 105 samples (including five paired samples) from maternal and umbilical cord blood indicated that T. cruzi DTU distribution was similar among pregnant women and newborns from these three countries, with a high frequency of TcII-TcV-TcVI DTUs, including mixed infections with TcI. However, phylogenetic analysis revealed that although the same parasite haplotypes circulated in these three countries, they were present at different frequencies, leading to significant geographic differences. Of importance, a strong association was observed between parasite haplotypes and congenital infection of newborns. Thus, the identification of parasite haplotypes in pregnant women, but not of parasite DTUs, may help predict congenital transmission of T. cruzi.

Figure 1

Phylogenetic analysis of Trypanosoma cruzi TcI sequences. The phylogram depicting the phylogenetic relationships among 19 T. cruzi DNA sequences recovered from maternal and umbilical cord blood (MB and CB, respectively), from Argentina (ARG), Honduras (HON), and Mexico (MEX), compared with reference T. cruzi sequences from GenBank (https://www.ncbi.nlm.nih.gov/genbank; Materials and Methods), based on mini-exon intergenic gene sequencing. Bootstrap values appear on each clustering branch. Asterisks indicates cases of mixed infections with non-TcI; red circles, cases of congenital transmission; blue arrows, cases with negative PCR results; green arrows, cases with negative/inconclusive serology. n = 8 (ARG); n = 9 (HON); n = 2 (MEX). Int, Intibucá; Mer, Merida; SBa, Santa Barbara; Tuc, Tucuman.

Figure 2

Phylogenetic analysis of Trypanosoma cruzi non-TcI sequences. The phylogram depicting the phylogenetic relationships among selected T. cruzi DNA sequences representative from a total 98 sequences (https://www.ncbi.nlm.nih.gov/genbank; accession numbers MH629842 to MH629958) recovered from maternal and umbilical cord blood (MB and CB, respectively), with T. cruzi infection from Argentina (ARG), Honduras (HON), and Mexico (MEX), compared with reference T. cruzi sequences from GenBank (see Materials and Methods), based on mini-exon intergenic gene sequencing. Bootstrap values appear on each clustering branch. Text in blue indicates MB-CB paired samples. Asterisks indicate cases of mixed infections with TcI; red circles, cases of congenital transmission; blue arrows, cases with negative PCR results; green arrows, cases with negative/inconclusive serology. n = 28 (ARG); n = 24 (HON); n = 34 (MEX). Int, Intibucá; Mer, Merida; SBa, Santa Barbara; Tu, Tucuman; Val, Valladolid.

Figure 3

Distribution of Trypanosoma cruzi haplotypes among countries and populations. The proportions of T. cruzi haplotypes of the mini-exon gene partial sequence identified in Tables 3 and 4 are shown color coded as indicated. TcI haplotypes were grouped into a single category because of the low frequency of variant haplotypes. Similarly, non–TcI-H4 to non–TcI-H9 were grouped as other non-TcI haplotype because of their low frequency. A: Comparison of haplotype distribution among countries (P = 0.009 between all groups). B: Comparison of haplotypes in congenital cases and noncongenital cases (P = 0.021). C: Comparison of haplotype distribution between maternal and cord blood (MB and CB, respectively) samples (P = 0.0003). D: Comparison of haplotype distribution between confirmed seropositive and seronegative samples (P = 0.007). E: Comparison of haplotype distribution between PCR-positive and PCR-negative samples (P = 0.258).