Trypanosoma cruzi has a multiclonal structure with wide biological diversity and high genetic polymorphism. This heterogeneity is associated with the presence of different parasite populations in the same host, a broad geographic distribution, infection stage, and susceptibility or resistance to specific treatments1.
Trypanosoma cruzi populations can be divided into six distinct discrete typing units (DTUs) named TcI-VI2, which show highly heterogeneity within and between types. TcII, TcV, and TcVI are associated with the domestic transmission cycle and are the main causes of Chagas disease in the southern and central regions of South America. TcI isolates are associated with wild and domestic cycles, and are frequently detected in the northern parts of the Amazon region and in endemic areas of Venezuela, Colombia, and Mexico. TcIV and TcIII circulate in the wild and are relatively poorly studied3.
The prevalence and geographic distributions of clinical forms of Chagas disease show differences among and within countries. These differences appear to be related to the genetic characteristics of hosts and strains of the parasite within a specific region. However, the data reported to date are inconclusive, making assessment of epidemiological studies difficult3.
Currently, the application of molecular techniques for epidemiological studies on Chagas disease has been proposed, which has stimulated research aiming to evaluate the relationship between the transmission mechanisms, DTUs of T. cruzi, and clinical forms of the disease3.
The low-stringency single-specific-primer-polymerase chain reaction (LSSP-PCR) technique has been successfully applied for T. cruzi characterization. This method has shown excellent potential for evaluating intraspecific differences or similarities of T. cruzipopulations and shows stability and reproducibility in experimental and human studies4.
In this study, the epidemiological application of LSSP-PCR in the characterization of TcII samples demonstrated similar genetic profiles circulating among individuals with very close kinship. Seventy-six chronic Chagas disease individuals from an endemic post-control vector program area in the State of Bahia (Brazil) were evaluated; the age of subjects ranged from 2 to 56 years (mean, 31.62 ± 12.25 years). Trypanosoma cruzi infection was detected by positive serology of anti-T. cruzi using indirect immunofluorescence and an enzyme-linked immunosorbent assay. This research project was approved by the Ethics Committee (n° 388) of the Universidade Federal do Triângulo Mineiro (UFTM) and all procedures were carried out with the informed consent of patients.
The clinical records of the patients were classified according to their symptoms and electrocardiographic and radiological abnormalities (esophagogram, opaque enema, and chest X-ray); 96.1% of patients presented the indeterminate form and 4% presented the cardiac form of Chagas disease.
Parasitemia and parasite isolation were evaluated by hemoculture5 and demonstrated the presence of T. cruzi in 46.1% (35/76) of the individuals, without significant differences with respect to gender (40% male and 60% female) and age group (range, 4-56 years; mean, 31.43 ± 11.95 years) (p> 0.05). The degree of kinship in the study population was also determined, and 13 pairs of the 35 individuals with positive hemoculture showed kinship.
Deoxyribonucleic acid (DNA) extraction of the positive hemoculture samples preserved in guanidine-ethylenediaminetetraacetic acid was performed in duplicate by the phenol-chloroform method6. Trypanosoma cruzi DNA controls represented by negative and positive samples were included in all DNA extractions and PCR procedures.
Genetic characterization of T. cruzi kinetoplast-DNA (kDNA) was performed by LSSP-PCR in two steps4. The first step consisted of specific amplification of a variable region of T. cruzi minicircles, using the primers 121 (5′-AAATAATGTACGGGGGAGATGCATGA-3′) and 122 (5′-GGTTCGATTGGGGTTGGTGTAATATA-3′)7. The PCR was carried out in a final volume of 20µL containing 10mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 3.5mM MgCl2, 75mM KCl, 0.2mM each of deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), and deoxythymidine triphosphate (dTTP), 1.0 unit of Taq DNA polymerase (Promega; Madison, WI, USA), 20pmol of each primer, 2µL (1ng/µL) of DNA, and 30µL of mineral oil. The amplification consisted of initial denaturing at 95°C (5min) and 35 cycles: 95°C (1min), 65°C (1min), and 72°C (1min), followed by final extension at 72°C (10min). The PCR-amplified products were submitted to electrophoresis on a 1.5% agarose gel (1% agarose, 0.5% low-melting-point agarose) and stained with ethidium bromide. The 330-bp fragments of individual amplifications, corresponding to approximately 150ng of DNA, were excised from the gel, melted, diluted 10-fold in double-distilled water, and used as the template for the second step of amplification. The second PCR was performed using a single S35 primer (5′-AAATAATGTACGGGGGAGATGCATGA-3′) in a final volume of 10µL, containing 10mM Tris-HCl (pH 8.5), 0.1% Triton X-100, 1.5mM MgCl2, 0.2mM each of dATP, dCTP, dGTP, and dTTP, 1.0 unit of Taq DNA polymerase (Promega; Madison, WI, USA), 45 pmol of the S35 primer, 3µL (1.5ng/µL) of DNA template, and 30µL of mineral oil. The amplification consisted of 40 cycles: 94°C (1min), 30°C (1min), and 72°C (1min), preceded by initial denaturing at 94°C (5min) and followed by final extension at 72°C (7min). In order to demonstrate the stability of the amplification, each DNA sample was analyzed in duplicate. The LSSP-PCR products were separated by electrophoresis on a 7.5% polyacrylamide gel and stained with 0.2% silver nitrate. The genetic profiles obtained by visual scan photography of the gel were compared using the Nei and Li coefficient in GelCompar II versão 5.0 software (Fingerprint and Gel Analysis Software-Applied Maths NV).
Parasite DTUs were identified by amplification of the intergenic region of spliced leader genes, by amplification of the D7 domain of the 24Sα ribosomal ribonucleic acid genes using nested-hot-start PCR assays and by nested amplification of the A-10 fragment, as previously reported8. Trypanosoma rangelidetection was performed using a multiplex PCR with the primers D72, D75, and RG39. For identification of T. cruzi DTUs, the PCR amplification products were viewed on a 6% polyacrylamide gel and the products of multiplex PCR were viewed on a 7.5% polyacrylamide gel stained with silver nitrate.
For the statistical analysis, the chi-squared test was used to determine the association between positive hemoculture with the patients’ age group and gender. The Shapiro-Wilk test was used to investigate whether the age distributions of the groups was normal. If so, the Student’s t-test for independent samples was performed. Associations between patient age groups and the complexity level of the genetic profiles for T. cruzi obtained from LSSP-PCR were investigated using regression analysis. The significance level used was 5% (p <0.05). The analyses were performed using the Statistica for Windows program, version 8.0 (StatSoft, Inc.; USA).
TcII populations were predominant and were detected in 97.1% (34/35) of the hemoculture-positive individuals; these data are concordant with previous reports, which confirm the predominance of TcII in Brazil3. TcI was found in a single six-year-old patient who did not receive blood transfusion and whose mother did not present positive serology for T. cruzi. This finding suggests the existence of vector transmission in the studied region. Trypanosoma rangeli was not detected in any of the analyzed samples.
Intense kDNA polymorphism with only 46% of the bands shared among the TcII populations (mean number of bands, 9.26 ± 2.68) was demonstrated by LSSP-PCR analysis (Figure 1), which agrees with previous reports3,4,10. High genetic variability has previously been detected by LSSP-PCR among TcI stocks11,12.
It has been demonstrated that the percentage of polyclonal T. cruzi populations progressively decreases during the chronic phase of Chagas disease1. Therefore, for a single endemic area, the T. cruzi populations isolated from younger patients should present complex genetic profiles. In the present study, a weak but not significant (p> 0.05) inverse correlation (r = −0.3) was observed between patient age and the complexity level of the genetic profiles of kDNA obtained by LSSP-PCR. This result may be related to the small number of patients in the youngest age group, from zero to 20 years (n = 5).
Despite the high polymorphism of TcII isolates, very similar genetic profiles were observed between specific pairs of T. cruzi samples, particularly for eight pairs of patients with very close degrees of kinship. The isolate pairs 34/35, 3/4, and 14/15 corresponded to mother-child pairs and shared 92%, 100%, and 100% of their bands, respectively. The pairs 16/22, 1/2, and 10/11 corresponded to siblings and presented band similarities of 80%, 90%, and 96%, respectively. The pairs 8/9 and 16/17 were cousins and shared 92% and 94% of their bands, respectively (Figure 1). It is important to emphasize that the electrophoretic kDNA profiles were reproducible for all samples when a second LSSP-PCR was performed.
Most of the data reported in the literature have demonstrated unique and exclusive LSSP-PCR profiles for individual patients4,10,13,14, although no report has described the differential distribution of T. cruzipopulations in tissue and peripheral blood samples obtained from the same human host1. Here, the high similarity of T. cruzi kDNA minicircles associated with kinship suggests the possibility of congenital transmission and/or the presence of similar or identical T. cruzi blood populations circulating within the same home or family group. This hypothesis can be supported by evidence of nearly identical patterns of kDNA minicircles between each mother and infant in congenital transmission described in a study using other molecular techniques15.
Similarities among the LSSP-PCR T. cruzi genetic profiles were also observed among different individuals infected with TcI from the same geographical region11, although it was not possible to associate the kDNA genetic profile with the geographical or biological origin of the studied samples.
These data reinforce the potential of the LSSP-PCR technique for field studies of human Chagas disease and show the importance of using different genetic markers of T. cruzi to monitor the transmission of human Chagas disease, particularly for endemic areas that have been certified as free from vector transmission by Triatoma infestans. These findings also suggest the existence of vector transmission in the study area and may represent an important warning sign for Brazilian epidemiological surveillance programs.
The detection of T. cruzi populations that are genetically related and associated with a high degree of kinship, i.e., children and their mothers, is a new approach in the molecular epidemiology of Chagas disease and may provide new strategies for future studies of T. cruzi congenital transmission.