Sexual transmission of American trypanosomiasis in humans: a new potential pandemic route for Chagas parasites

Abstract

Background: The Trypanosoma cruzi infection endemic in Latin America has now spread to several countries across four continents; this endemic involves triatomine vector-free protists. We hypothesised that the sexual transmission of T. cruzi contributes to the ongoing spread of Chagas disease.

Objectives: A short-term longitudinal study was conducted to evaluate this hypothesis.

Methods: The study population comprised 109 subjects from four families, among whom 21 had been diagnosed with acute Chagas disease by direct parasitological analysis. Blood mononuclear cells and serum samples were obtained from each study subject once per year for three consecutive years. Enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence serological examinations were used to detect specific T. cruzi antibodies. Polymerase chain reaction of T. cruzi DNA revealed 188-nucleotide bands, which hybridised to a specific radiolabelled probe and were confirmed by cloning and sequencing.

Results: Three independent assessments at different time points revealed T. cruzi nuclear DNA footprints in 76% (83/109) of the study population with active infection. In contrast, the ELISA and indirect immunofluorescence assays detected the T. cruzi antibody in 28.4% (31/109) of the study samples. Moreover, the semen from 82.6% (19/23) of subjects people revealed harboured the 188- bp base pair T. cruzi footprint. Interestingly, the ejaculates of nuclear DNA-positive Chagas patient transmitted the T. cruzi upon peritoneal injection or infusion in the vagina of mice, and amastigotes were detected in the skeletal muscle, myocardium, vas deferens, and uterine tube.

Main conclusions: T. cruzi infections can be transmitted from females or males to naïve mates through intercourse, and progeny showed discrepancies between the ratios of nuclear DNA footprints and specific antibody that can be explained by the tolerance attained during early embryo growth. Additional studies are needed to develop drugs to eradicate the infections. Additionally, the importance of a vigorous education, information, and communication program to prevent sexually transmitted Chagas disease in humans cannot be underemphasised.

Fig. 1. : the phenotype of the Trypanosoma cruzi isolates from acutely infected humans. The Berenice protozoan flagellate and the ECI-4 wild-type flagellate shown by bright field (BF) imaging and DAPI (4’, 6-diamidino-2-phenylindole) used to stain parasite kinetoplast (kDNA) and nucleus (nDNA) in blue. These parasite isolates identified with the specific Chagas antiserum IgG that recognizes T. cruzi Berenice, ECI-4, but it does not recognize Leishmania braziliensis promastigotes after treatment with an FITC-labeled monoclonal antibody to human IgG. The insets show the negative controls. Bars, 20 µM.

Fig. 2. : the Trypanosoma cruzi genotype identified in the haemo flagellates from the acute Chagas disease cases. (A) The T. cruzi infections identified by the PCR nDNA footprints formed 188 nt bands and catamers after Southern blot with the specific radiolabeled probe; (B) the EC1-4 flagellate identified by the nDNA footprints. The wild-type T. cruzi shown by hybridisation with the digoxigenin-labeled 188 nt nDNA probe. From left to right: bright field image; the T. cruzi trypomastigote green DNA visible with a primary IgG to the digoxigenin-labeled 188 nt probe after interaction with a secondary fluorescent antibody; and, merge of previous images. The inserts show negative controls; (C) the ECI-10 trypomastigote shown by hybridisation with the Berenice T. cruzi 188 nt digoxigenin-labeled probe. The EC1-13 isolate shows a dark nucleus stained with an alkaline phosphatase-labeled monoclonal Ab to digoxigenin. Tc, T. cruzi; nc, negative control.

Fig. 3. : demonstration of the Trypanosoma cruzi nuclear DNA and of its mitochondrial kDNA by Southern blot with the specific radio labeled probes. Family A - T. cruzi infections in 15 people, showing positive nDNA and kDNA bands. Family B - In this family, 35 out of and 43 subjects formed the nDNA and kDNA bands. The individual 25 had the kDNA band alone. Family C - Among 29 members of the family C, 22 formed the nDNA and the kDNA bands and five had the kDNA alone. Family D - Eleven out of 21 subjects of the family D formed the nDNA and the kDNA bands; additionally, 10 subjects had the kDNA band alone.

Fig. 4. : the Trypanosoma cruzi infections demonstrated in the study families. (A) Profiles of T. cruzi infections shown by the specific antibody and by the nDNA footprints. Group I (n = 10) and group II (n = 20) were, respectively, the negative and the positive control antiserum to T. cruzi infections. Group III split subgroups: III-a (n = 31) families’ cases with the infections confirmed by nDNA-positive and specific antibodies to T. cruzi; III-b (n = 52) included families with the infections detected by the T. cruzi nDNA-positive in the absence of specific antibodies; III-c (n = 18) included families displaying kDNA-positive only in the absence of nDNA and of specific antibodies; III-d (n = 8) included the study population individuals with all tests negative. (B) Profiles of the nDNA and kDNA present in the germ cell line. Nineteen out of 23 semen samples contained the T. cruzi infections shown by the positive nDNA and kDNA assays. Tc, T. cruzi; nc, negative control. (C) The infectivity of the T. cruzi from human ejaculate inoculated in mice. Left, T. cruzi amastigotes nest in the heart (top) and in the lumen of the vas deferens (bottom). Right, T. cruzi amastigotes nest in the skeletal muscle (top) and in the uterine tube (bottom). The insert shows a dividing amastigote (circle). Bars, 25 µM. Arrows show amastigote parasites.

Fig. 5. : heredograms and personalised profiles of the Trypanosoma cruzi infections in four study families. Family A - of the 15 members tested, four had the T. cruzi IgG antibody, nDNA and kDNA; 11 had the parasite nDNA and kDNA, and five (yellow numbers) had T. cruzi in their ejaculates. Family B - of the 44 samples, 11 had the T. cruzi IgG antibody, nDNA and kDNA and 23 had the parasite nDNA and kDNA footprints in the absence of antibody. In this family, T. cruzi nDNA found in the ejaculates of seven subjects: three members had kDNA alone integrated into the genome, and seven had none of the above (numbered white circles and squares). Family C: - of the 29 members tested, five had the T. cruzi IgG antibody, nDNA and kDNA, 17 had the parasite nDNA and kDNA footprints, six had integrated kDNA only and one patient had none of the above. In this family four members had the T. cruzi active infections (yellow numbers) in the ejaculates; Family D: - of the 21 subjects tested, 11 had the T. cruzi IgG, nDNA and kDNA; one had the nDNA and kDNA, nine members had integrated kDNA only; two subjects (92 and 95) had the T. cruzi active infections in the ejaculates.