The Trypanosoma cruzi agent of Chagas disease produces long-lasting asymptomatic infections that abruptly develop into clinically recognized pathology. The following research protocol describes a short-run family-based epidemiological study to unravel the T. cruzi infection transmitted sexually from parent to progeny.
American trypanosomiasis is transmitted to humans by triatomine bugs through the ingestion of contaminated food, by blood transfusions or accidently in hospitals and research laboratories. In addition, the Trypanosoma cruzi infection is transmitted congenitally from a chagasic mother to her offspring, but the male partner's contribution to in utero contamination is unknown. The findings of nests and clumps of amastigotes and of trypomastigotes in the theca cells of the ovary, in the goniablasts and in the lumen of seminiferous tubules suggest that T. cruzi infections are sexually transmitted. The research protocol herein presents the results of a family study population showing parasite nuclear DNA in the diploid blood mononuclear cells and in the haploid gametes of human subjects. Thus, three independent biological samples collected one year apart confirmed that T. cruzi infections were sexually transmitted to progeny. Interestingly, the specific T. cruzi antibody was absent in the majority of family progeny that bore immune tolerance to the parasite antigen. Immune tolerance was demonstrated in chicken refractory to T. cruzi after the first week of embryonic growth, and chicks hatched from the flagellate-inoculated eggs were unable to produce the specific antibody. Moreover, the instillation of the human semen ejaculates intraperitoneally or into the vagina of naive mice yielded T. cruzi amastigotes in the epididymis, seminiferous tubule, vas deferens and uterine tube with an absence of inflammatory reactions in the immune privileged organs of reproduction. The breeding of T. cruzi-infected male and female mice with naive mates resulted in acquisition of the infections, which were later transmitted to the progeny. Therefore, a robust education, information and communication program that involves the population and social organizations is deemed necessary to prevent Chagas disease.
The protozoan parasite Trypanosoma cruzi belonging to the family Trypanosomatidae undergoes trypomastigote and amastigote life cycle stages in mammalian hosts and exists as epimastigotes in the insect-vector's (Reduviid: Triatominae) gut and in axenic culture. In recent decades, several studies have shown the presence of Chagas disease in countries on four continents considered triatomine bug free1,2,3,4,5,6,7,8,9,10,11,12,13; the dispersion of American trypanosomes was initially attributed to Latin American immigrants to the Northern Hemisphere, but the possibility that some are autochthonous cases of Chagas disease can no longer be denied3,4,5,6,7,8,9,10,11,12,13,14. The only recognizable endogenous source of T. cruzi transmission has been ascribed to the chagasic mother's transfer of the parasite to the offspring in approximately 10% of pregnancies15; the male partner's contribution to in utero infections through semen ejaculates has remained unrecognized.
Over one century ago, investigators16,17 observed intracellular T. cruzi amastigotes in the theca cells of the ovary and in the germ line cells of the testicles of acute cases of Chagas disease. The nests and clumps of T. cruzi trypomastigotes and amastigotes in theca cells of the ovary, in goniablasts and in the lumen of seminiferous tubules (Figure 1) of fatal acute Chagas disease cases develop immune privilege in the organs of reproduction in the absence of inflammatory infiltrates18,19. In recent decades, a few experimental studies have shown nests of the round amastigote forms of T. cruzi in the seminiferous tubule, epididymis, and vas deferens as well as in the uterus, tubes and ovary theca cells of acutely infected mice1,20,21,22. Furthermore, in the course of family studies to document the transfer of protozoan mitochondrial DNA from parental Chagas patients to their descendants, T. cruzi nuclear DNA (nDNA) was verified in human haploid germ line cells23, and parasite life cycle stages were observed in the ejaculates of chagasic mice24. These findings are in agreement with reports on the immune tolerance attained by the progeny of T. cruzi-infected hosts in the absence of the specific antibody1,25,26. Additionally, epidemiological reports that suggested the spread of endemic Chagas disease to the other continents3,4,5,6,7,8,9,10,11,12,13 are now supported by experimental studies showing that Chagas disease can be transmitted sexually1. The present investigation presents an epidemiologic family study protocol and shows that T. cruzi infection propagates by sexual intercourse.
The Human and the Animal Research Committees of the Faculty of Medicine of the University of Brasilia approved all the procedures with human subjects and laboratory animals, respectively, in research protocols 2500.167567 and 10411/2011. The Ethics Committee of the Public Foundation Hospital Gaspar Vianna (protocol nº 054/2009 and CONEP 11163/2009) approved the free consent forms for the field study, with extension to the Ministry of Health National Commission on Human Research (CONEP 2585/04). The protocol was adjusted to assess T. cruzi DNA in diploid blood mononuclear cells and in haploid gametes of semen ejaculates. The laboratory animals received humane care; the mice, subjected to heart puncture before sacrifice, were under anesthesia.
1. Recruitment of human participants
2. Growth of parasites
3. DNA extraction and PCR analyses
4. Southern hybridization
NOTE: Southern hybridization was used to discard most of the false positive PCR amplicons in the agarose gel.
5. Immunological assays
NOTE: The sensitivity and specificity of the indirect immunofluorescence (IIF) and of the enzyme-linked immunosorbent assay (ELISA) were assessed in the serum from six Chagas patients with demonstrable parasitemia and from six Chagas-free, deidentified serum bank samples. The assays conducted with the double serum dilutions in PBS, pH 7.4, revealed that the IIF at 1:100 dilutions and the ELISA optical densities (ODs) at 0.150 and above separated the positive from the negative results.
6. Assessments of immune tolerance
NOTE: A chicken model system was used to test T. cruzi infections after the first week of embryo development.
7. Infection of mice with T. cruzi from Chagas patients' semen ejaculates
8. Transmission of the T. cruzi infection by intercourse
9. Assessment of immune privilege
10. Statistical analyses
This research, conducted according to the protocol, aimed to detect acute cases of Chagas disease by clinical and parasitological exams. Venous blood samples were subjected to direct microscopic examination and in vitro culture for parasite growth. Twenty-one acute cases of Chagas disease showed T. cruzi in blood. The research protocol secured the isolation of T. cruzi ECI1-to ECI21 from acute Chagas disease, and the DNA samples exhibited positive DNA footprints in the remainder of the study population: nDNA-PCR assays yielded the typical telomere repeat sequence with the 188-nt bands present as well as the T. cruzi Berenice archetype1. The Chagas cases and their family members who volunteered to participate in the study were grouped into four families1.
In this family study, the T. cruzi nDNA was PCR amplified with primer sets1,23 annealed to the specific telomere sequence from each of the 21 acute Chagas disease cases. These T. cruzi nDNA amplicons hybridized with the specific radiolabeled sequence probe (Figure 2); the cloning and sequencing revealed that the amplicons comprised the T. cruzi 188-nt telomere repeat motif. The specificity of these hybridization procedures was shown in the negative control performed with L. braziliensis promastigotes. The pathology analysis validated that the hemoflagellates in the acute Chagas disease patients were truly virulent T. cruzi. We conclude that the T. cruzi nDNA (188-bp) band found in the 21 acute Chagas cases (Figure 2) is a direct demonstration of persistent infections.
Sexual Transmission of Trypanosoma cruzi in Humans
To evaluate the ratios of the T. cruzi infections, we applied the nucleic acid test for high sensitivity detection of the parasite footprints in the family study population1. In these PCR assays, the amplification products that hybridized with the specific radiolabeled 188-nt probe formed nDNA bands in 76.1% (83/109) of the test samples; the results of Southern hybridization of the nDNA-PCR amplification products with the specific 188-nt radiolabeled probe are shown in Figure 3. Furthermore, the hybridizations showed the parasite DNA in the germ cell line of the volunteer family members (Figure 4).
IIF was employed to phenotype the ECI1 to ECI21 T. cruzi trypomastigotes with the human serum IgG from a Chagas disease serum bank sample with parasitological demonstration of the protozoan in the blood, and a fluorescein conjugated anti-human IgG was used. T. cruzi Berenice was a positive control for the Chagas antibody, and the negative control was the promastigote of its family relative L. braziliensis. Figure 5 shows that the positive apple-green silhouette of the Berenice archetype correlates with the wild-type T. cruzi envelope shown in the video.
The ELISA and IIF revealed the specific T. cruzi antibodies1,28 in 28.4% (31/109) of the test samples. The results of the ELISA and IIF, as well as those from the nDNA-PCR amplicon and Southern hybridizations, are plotted in Figure 6. The discrepancies among the results of the nDNA-PCR footprints and those from the specific antibody assays are depicted in the heredograms (Figure 7), Family A, four subjects had positive nDNA and the anti-T. cruzi antibody, and 11 had only the positive nDNA; five males had T. cruzi in the semen ejaculate. In family B with 44 people, 11 had the specific T. cruzi antibody, and 23 had both the specific antibody and the parasite nDNA; seven individuals had T. cruzi in the semen ejaculate. Family C with 29 members had the antibody and the T. cruzi nDNA in five individuals, and 17 had the parasite nDNA alone; four males had the nDNA-PCR positive in the semen ejaculate. In Family D, among 21 subjects, 11 had the specific anti-T. cruzi antibody and the nDNA footprint, and nine had positive nDNA-PCR alone. Figure 3, Figure 6 and Figure 7 depict the broad discrepancies among the results, consistently, in the biological samples obtained from family subjects in three independent experiments run one year apart.
Table 1 shows the quantitative differences between the IIF, the ELISA, and the nDNA-PCR assays in the samples from the human study families A-to-D. The discrepancies between the ratios of antibody assays (28.4%) and those of positive nucleic acid assays (76.1%) are statistically significant (p < 0.005). In these families, the differences among groups of T. cruzi-infected people (III and IV) accounted for 62.6% (52/83) of the population, showing a positive nucleic acid test alone. The broad discrepancies among the ratios of positive nDNA footprints and those of the specific T. cruzi antibody were explained by the experiments conducted in the chicken model system.
Immune Tolerance
The evaluation of the immune responses conducted in groups of chickens raised to the adult stage in individual cages in separate aisles containing naive control chickens (A); mock control chickens hatched from eggs inoculated with culture medium (B); and chickens hatched from the T. cruzi trypomastigotes-inoculated eggs (C)26. The adult chickens in groups B and C were challenged three times with the formalin-killed T. cruzi trypomastigote antigen, weekly, as shown in the video. The ELISA and the IIF assays were run with the serum collected from group A, B and C chickens one month after challenge. Figure 8 shows the absence of the specific antibody in group A and C chickens, which contrasted sharply with the specific antibody production in group B immunized with the T. cruzi antigen. The results clearly showed the immune tolerance in group C hatched from the T. cruzi-inoculated eggs.
Sexual Transmission of Trypanosoma cruzi in a Mouse Model System
Moreover, the infectivity of T. cruzi from a Chagas patient's ejaculate, which tested positive in the PCR and lacked the specific antibody, was demonstrated through instillations of 100 µL of semen into the peritoneal cavity of male mice and through an equal amount of semen instilled into the vagina. Five weeks later, the T. cruzi amastigote nests were detected in the heart and skeletal muscles, and clumps of differentiating parasites were present in the lumen of the vas deferens and uterine tube. Interestingly, the destructive inflammatory reactions did not surround the nests and clumps of the T. cruzi amastigotes (Figure 9).
The assessment of the sexual transmission of T. cruzi infections was further conducted in two groups of mice inoculated intraperitoneally with 1 x 105 T. cruzi Berenice trypomastigotes forms1,30,31. In experimental group I, 10 T. cruzi-infected males mated with 10 naive control female mice. In experimental group II, 10 T. cruzi-infected females mated with 10 naive control males. Figure 10 shows that the T. cruzi-infected male mice (A-to-E) and the T. cruzi-infected female mice (F-to G) yielded 188-bp nDNA bands (odd numbers). After breeding, the naive mates (even numbers) readily acquired T. cruzi following a unique sexual encounter with a chagasic mate. Similar repeat experiments performed under identical conditions confirmed that each naive female or male mouse that sexually mated with a T. cruzi-infected male or female acquired the flagellate infection. These nDNA-positive founders (F0) generated progeny that they raised until the age of six weeks. Then, the test and the control uninfected mice were bled via heart puncture to collect approximately 0.5 mL of blood. The nDNA-PCR assays showed that the founders' (F0) sexually acquired infections were transmitted to the F1 progeny, as shown by the 188-bp nDNA bands (Figure 11). The F1 progeny were nDNA-positive in 41 of the 70 (58.6%) samples examined. Of these mice with nDNA bands suggestive of vertically acquired infections, as few as 9 of 41 (22%) had T. cruzi antibodies.
The F1 progeny mice were sacrificed under anesthesia, and the body tissues were subjected to pathological and immune peroxidase-staining analyses. The results of these experiments are shown in Figure 12. The results for the T. cruzi amastigotes were documented in the interstitial cells of the epididymis and in goniablasts; amastigotes differentiating into trypomastigotes were present in the lumen of seminiferous tubules in the absence of inflammatory reactions.
Figure 1. Trypanosoma cruzi infection in the seminiferous tubule of a boy who died of acute Chagas disease. Microphotograph from Doctor Teixeira's file, 197018. The T. cruzi forms are in goniablasts, and clumps of amastigotes and free trypomastigotes (arrows) are present in the lumen of the seminiferous tubule in the absence of inflammatory infiltrates1. Hematoxylin-eosin stains. Bar, 20 µm. Reprinted with permission from the publisher and the authors1,19.
Figure 2. The footprint of Trypanosoma cruzi from acute Chagas disease. The T. cruzi nDNA-PCR amplification products formed 188-nt bands with a specific radiolabeled 188-nt probe. Tc, T. cruzi; nc, negative control. Reprinted with permission from the publisher and the author1. Please click here to view a larger version of this figure.
Figure 3. Southern blotting analysis of Trypanosoma cruzi infections in subjects of human study families. Family A – All 15 subjects showed the specific nDNA-PCR 188-nt bands. In Family B, a total of 35 of 43 subjects (81.4%) formed the specific nDNA bands. In Family C, among 29 members, 22 (75.8%) formed the nDNA bands. In Family D, 11 of 21 subjects (52.4%) had the nDNA bands. The T. cruzi-specific nDNA bands were confirmed by cloning and sequencing. Reprinted with permission from the publisher and the author1. Please click here to view a larger version of this figure.
Figure 4. The active Trypanosoma cruzi infections in the semen ejaculate from study family volunteers. The infections in Chagas patients' ejaculates identified by the nDNA-PCR 188-bp bands. Tc, T. cruzi positive control. Nc, L. braziliensis negative control. Reprinted with permission from the publisher and the author1. Please click here to view a larger version of this figure.
Figure 5. The phenotype of Trypanosoma cruzi with the Chagas disease patients' serum antibody. T. cruzi identified with the Chagas serum IgG antibody that recognizes the parasite trypomastigote treated with an FITC-labeled monoclonal Ab anti-human IgG. The anti-T. cruzi Ab does not recognize Leishmania braziliensis promastigotes. The insets show the negative controls. Bars, 20 µm. Reprinted with permission from the publisher and the author1. Please click here to view a larger version of this figure.
Figure 6. Graphic representation of the ELISAs and nDNA-PCR assays in the family study population. Group I (n=10) and group II (n=20) were the negative control and the positive control sera, respectively, from T. cruzi infections with parasitological demonstration. Group III (n=31) included samples from family subjects with the 188-bp nDNA bands and specific antibodies to T. cruzi. Group IV (n=52) comprised sample from subjects with T. cruzi infections detected by the nDNA-PCR 188-nt amplicons in the absence of the specific antibody. Group V (n=26) were negative test samples comprising the infection-free people in the family study. Reprinted with permission from the publisher and the author1. Please click here to view a larger version of this figure.
Figure 7. The heredograms and mapping of the Trypanosoma cruzi-infected family population. The figure shows the discrepancies among the ratios of the anti-T. cruzi antibody and those of the nDNA-PCR assays. Open square and circle, negative male and female. Red squares and circles, positive anti-T. cruzi antibody and nDNA-PCR. Black squares and circles, positive nDNA-PCR alone. Please click here to view a larger version of this figure.
Figure 8. The immune tolerance in chickens hatched from Trypanosoma cruzi-inoculated eggs. A) Preimmune antibody profile in the mock control chickens (n = 10). B) The specific antibody response in the naive control chickens challenged with the T. cruzi antigen (n = 20). C) The absence of a specific immune response in chickens hatched from the T. cruzi-inoculated eggs after challenge with the T. cruzi antigen (n = 20). The optical density difference between A and C (024 ± 0.17) towards B (0.85 ± 0.6) is statistically significant (p<0.05). This figure has been modified from reference26 and is reprinted with permission from the publisher and the author.
Figure 9. The infective Trypanosoma cruzi in human ejaculates translates into an active murine infection. Aliquots of Chagas patient ejaculates were instilled into the peritoneal cavity or into the vagina of mice. The mice were sacrificed three weeks after instillation. Top lane, T. cruzi amastigotes nests in the heart (left) and in the skeletal muscle (right). Bottom lane, T. cruzi amastigote nests in the vas deferens (left) and in the uterine tube (right). The insert shows a dividing amastigote (circle). Notice the absence of inflammatory infiltrates in the tissue sections. Hematoxylin-eosin stains. Bars: top and bottom left, 20 µm; bottom right, 10 µm. Reprinted with permission from the publisher and the author1. Please click here to view a larger version of this figure.
Figure 10. The sexual transmission of Trypanosoma cruzi infections in the mouse model system by intercourse. The transmission of the T. cruzi infections from chagasic to naive mates demonstrated by the specific nDNA 188-bp bands revealed in the Southern hybridizations. Top lane) Prebreeding profiles of the PCR amplification products of the T. cruzi-infected mice and of the naive mice. The odd numbers indicate the T. cruzi-infected male A-to-E and female F-to-I mouse samples. The even numbers are naive female (2-to-10) and male (12-to-20) mice. Bottom lane, after breeding, the profiles show that even mice 2-to-20 acquired T. cruzi infections. Reprinted with permission from the publisher and the author1,30. Please click here to view a larger version of this figure.
Figure 11. The Trypanosoma cruzi infection is vertically transferred from the F0 chagasic parental to the F1 progeny mice. The chagasic parent transmitted the T. cruzi infections by a single breeding encounter. The T. cruzi-infected females were mated to naive males A-E, and the naive females were mated to the T. cruzi-infected males F-J. After breeding, all the founders (F0) showed the positive protozoan nDNA-PCR 188-bp band. Southern blotting revealed the specific nDNA band after hybridization with the radiolabeled 188-nt probe in a majority of the F1 litters. Nc, L. braziliensis negative control; Tc, T. cruzi positive control. Reprinted with permission from the publisher and the author1,31. Please click here to view a larger version of this figure.
Figure 12. The histopathology documented Trypanosoma cruzi sexually transmitted from F0 to F1 progeny and immune tolerance in the absence of an inflammatory reaction. The sections show growth of the T. cruzi forms in the epididymis, goniablasts and the seminiferous tubules of the F1 mice. The mice were sacrificed under anesthesia, and the immune peroxidase-stained sections were examined under a microscope. The photomicrographs show brownish immune peroxidase-stained T. cruzi amastigotes in the interstitial of the epididymis (A) and clumps of amastigotes differentiating into trypomastigotes shed into the lumen of the seminiferous tubule (B, C, and F). The amastigote nest seen in a goniablast (D). The positive control mouse's seminiferous tubule normal histology (E). Notice the absence of inflammatory infiltrates in the testes of the F1 mice showing loads of the Chagas parasites. Giemsa's stain. Bars: A, B, C and E, 20 µm; D and F, 10 µm. Please click here to view a larger version of this figure.
Table 1. The discrepancies between the ratios of positive IIF and ELISA exams and those of nDNA-PCR assays in the samples collected from the human study families A-to-D*
Groups** | ELISA: serum anti-T. cruzi Ab (%) | T. cruzi nDNA- PCR (%) | ||
I- Control Ab- PCR- (n=10) | 10/10 | 100 | 10/10 | 100 |
II- Control Ab+ PCR+ | 20/20 | 100 | 20/20 | 100 |
(n = 20) | ||||
III- Chagas Ab+ PCR+ ᵟ | 31/109 | 28.4 | 31/109 | 28.4 |
(n = 31) | ||||
IV- Chagas Ab- PCR+ ᵟ | – | – | 83/109 | 76.1 |
(n= 52) | ||||
V- Chagas-free Ab- PCR- | 26/109 | 23.9 | 26/109 | 23.9 |
(n = 26) | ||||
* Results of three independent ELISA and nDNA-PCR assays run in samples collected in three different occasions at years 1, 2, and 3. [1]. The amplification of the 188-nt T. cruzi DNA repeat confirmed by cloning and sequencing. **The differences among negative (groups I and V) and the positive subjects (groups III and IV) are statistically significant (p < 0.05). ᵟ The discrepancies between groups III and IV explained by the immune tolerance in the absence of the T. cruzi antibody attained in 62.6% (52/83) of the PCR positive subjects. |
Herein, we discuss a family-based research protocol that answered the question of whether human Chagas disease stems from sexually transmitted intraspecies T. cruzi infections. Early studies could not provide evidence of the sexual transmission of T. cruzi infections, probably because the available data and information on Chagas disease were obtained separately from the individual3,4,5,6,7,8,9,10,11,12,13. The finding of T. cruzi in the seminiferous tubule of a boy (Figure 1) was the spark that spurred clinical and epidemiological investigation. After several decades, conceivably when family study approaches and the technologies described in this research protocol were available, T. cruzi life cycle stages appeared in the human ejaculate1,19.
The direct parasitological demonstration of the protozoan in 21 acute Chagas disease cases was crucial to validate the nDNA-PCR amplification products, which formed specific bands in samples from all the subjects acutely infected with T. cruzi. This point-of-care laboratory marker evaluated the results of the immunological and nucleic acid assays. The fundamental long-run Chagas disease family study, therefore, combines the findings in humans with those obtained in groups of laboratory animals. The research conducted according to the protocol revealed for the first time that T. cruzi infections are sexually transmitted in humans1.
The broad difference between the ratios from the parasite-specific antibody assays and those from the nucleic acid tests indicates that the majority of the nDNA footprints resulted from sexually transmitted cases in the absence of specific anti T. cruzi antibodies. Thus, the sexual transmission of T. cruzi in the family members exhibiting positive nDNA in the absence of the specific IgG antibody was due to immune tolerance.
Immune tolerance was demonstrated in a chicken model system refractory to T. cruzi infections after the first week of embryo growth1,25,26,32,33,34. Thereafter, the immature immune system's inability to recognize the parasite as a foreign component of the body indicated the chicken's late mature immune system tolerance towards T cruzi. In view of these results, tolerance is a natural phenomenon1 resulting from the immune system's self-recognition and maintenance of its own body components under physiological conditions25,26,32,33,34. The shift from the state of immune tolerance to autoimmune Chagas heart disease can therefore be associated with effector cell modifications resulting from T. cruzi kinetoplast (kDNA) mutations in the host's genome1,2,14,23,25,26.
The critical steps in the research protocol describe the main technique modification and troubleshooting in order to disclose the sexually transmitted Chagas parasites1,14,23,25,26: i) selecting study families with cases of acute Chagas disease35,36; ii) isolating of wild-type T. cruzi from the blood of the acute cases; iii) obtaining DNA samples from the families' participants blood flagellates, from the Berenice T. cruzi archetype, from positive deidentified bank DNA samples, and from the negative control L. braziliensis; iv) performing tidy technical procedures to demonstrate that the participants' flagellates nDNA footprint is identical to that of the Berenice T. cruzi archetype and of those positive bank DNA samples; v) running independent triplicate nDNA footprinting to demonstrate the T. cruzi infections in the family members on three occasions one year apart; vi) ensuring that the nDNA-PCR technique conducted at the point of care yields results confirmed by cloning and sequencing all the amplicons annealed to the specific primer sets, thus consistently showing the T. cruzi 188-nt sequence1,25,28,29,30; vii) using high-quality trademark reagents to reproduce the antibody titers still in serum samples collected at three different time points; viii) the family study protocol revealed the existing live infection in the germ line cells upon the demonstration of the T. cruzi nDNA in the absence of specific serum antibodies in semen ejaculates collected from Chagas parasite-infected individuals1; ix) the perspective is that the research protocol designed to unravel the sexually transmitted T. cruzi infections should disclose the autochthonous Chagas disease on five continents; x) the nDNA and the kDNA footprints secure the diagnosis of chronic asymptomatic Chagas disease in humans1,2; xi) in the absence of the nDNA, the mutationof the T. cruzi kDNA sequence1,2,23,25,26 alone is a laboratory marker for achieving the differential diagnosis from the idiopathic dilated cardiomyopathies23,25,37,38,39.
Additionally, the virulent T. cruzi documented in Chagas patient ejaculates were capable of initiating widespread infections upon instillation into the mouse vagina and into its peritoneal cavity. The pathology study showed T. cruzi amastigote nests in the heart and skeletal muscles as well as in the vas deferens and uterine tube. Interestingly, the parasite nests did not provoke inflammatory reactions that would hamper vital reproductive functions. The absence of inflammatory reactions renders the immune privilege in vital functional body structures40,41,42,43,44,45 and therefore explains the uncurbed growth of T. cruzi in the reproductive organs.
Furthermore, the experimental studies in chagasic mice that bred with naive mates further explained the sexual transmission of T. cruzi infections in humans. The infected females and males transmitted the T. cruzi infections to the uninfected naive mates during intercourse, and the majority of their litters acquired the T. cruzi vertically transferred from parent to progeny. In these experiments, the initial phase referred to the growth of T. cruzi in the tube and in the uterus, as well as in the seminiferous tubule and vas deferens, where the immune privilege took place. Then, sexual transmission occurred through the parasitic stages in the semen or in the uterine secretions into the vagina. Immune privilege40,41,42,43,44,45 is a phenomenon that allows some organs (reproductive system, eyes, and brain) to downregulate inflammatory reactions and avoid damage to important, sensitive and specific functions40. Hormones41 and several immune factors downregulate macrophages41,42,43, natural killer cells41, T-lymphocytes, and T-regulatory (Treg) cells, thus orchestrating the inhibition of a number of proinflammatory cytokines and immune-privilege triggers40,41,42,43,44,45.
The sexual transmission of T. cruzi infections from males and females to naive partners indicates that the control of Chagas disease requires international solidarity. The results discussed herein suggest that more creative research is needed. The following immediate goals are achievable: i) to develop high-throughput platforms for specific and highly sensitive nucleic acid testing to reach an accurate diagnosis, aiming for the prevention of infections transmitted by sexual intercourse, blood transfusions and organ transplantation as well as facilitating clinical and epidemiologic enquiries to determine the diagnosis and the prevalence of Chagas disease; ii) to promote a multicenter drug development program to obtain new drugs for the eradication of T. cruzi infections; and iii) to implement a suitable education, information and communication program that includes the participation of schools, churches, social organizations, and health institutions to prevent the spread of Chagas disease.
The authors have nothing to disclose.
We acknowledge the laboratory facilities and the critical comments of Izabela Dourado, Carla Araujo, and Clever Gomes and the technical assistance of Bruno Dalago and Rafael Andrade. We are indebted to the Foundation for the Advancement of Science (FAPDF), The National Research Council, Ministry of Science and Technology (CNPq/MCT), and The Agency for Training Human Resources, Ministry of Education (CAPES/ME), Brazil, for supporting these investigations.
BCIP and NBT redox system | Sigma-Aldrich | 681 451 001 | |
Blood DNA Purification columns | Amersham Biosciences | 27-9603-01 | |
d-ATP, [α-32P], 250 µCi. | Perkin Elmer | BLU012H | |
DNA, Solution Salt Fish Sperm | AMRESCO | 064-10G | |
dNTP Set, 100 mM Solutions | GE Healthcare | 28-4065-51 | |
Eco RI | Invitrogen | 15202-021 | |
Goat anti-human IgG- alkaline phosphatase conjugated | Southern Biotech | 2040-04 | |
Goat anti-human IgG- FITC conjugated | Biocompare | MB5198020 | |
Hybond – N+ nylon membrane | GE Healthcare | RPN303B | |
Hybridization oven | Thomas Scientific | 95-0031-02 | |
Micro imaging software cell Sens software | Olympus, Japan | ||
Molecular probes labeling System | Invitrogen | 700-0030 | |
Nsi I | Sigma-Aldrich | R5584 1KU | |
Plasmid Prep Mini Spin Kit | GE Healthcare | 28-9042-70 | |
Plate reader | Bio-Tek GmBH | 2015 | |
Rabbit anti-chicken IgG-alkaline phosphatase conjugated | Sigma-Aldrich | A9171 | |
Rabbit anti-chicken IgG-FITC conjugated | Sigma-Aldrich | F8888 | |
Rabbit anti-mouse IgG- alkaline phosphatase conjugated | Sigma Aldrich | A2418 | |
Rabbit anti-mouse IgG-FITC conjugated | Biorad | MCA5787 | |
Spin Columns for radio labeled DNA purification, Sephadex G-25, fine | Sigma-Aldrich | G25DNA-RO | |
Taq DNA Polymerase Recombinant | Invitrogen | 11615-010 | |
Thermal cycler system | Biorad, USA | 1709703 | |
Vector Systems | Promega | A1380 |