INTRODUCTION
Chagas disease, caused by Trypanosoma cruzi, represents a health threat to an estimated 28 million people in Latin America (World Health Organization, 2007). Trypanosoma cruzi is transmitted mainly by triatomine insect vectors, blood transfusion or by infected women to offspring during pregnancy. After infection, a short acute phase is recognized only in a low proportion of individuals. Without treatment, the infection evolves to a chronic phase, which in most patients is asymptomatic with no electrocardiographic or radiological alterations in the heart, oesophagus or colon. However, a proportion of patients can develop lesions in the heart or gastrointestinal tract. Chronic cardiomyopathy is the most common and severe manifestation, and it affects approximately 30% of the infected patients (World Health Organization, 2007). Specific outcomes may be determined by a variety of non-exclusive factors including parasite genetics, host genetics, mixed infections, and cultural and geographical factors (Campbell et al. Reference Campbell, Westenberger and Sturm2004; Macedo et al. Reference Macedo, Machado, Oliveira and Pena2004). T. cruzi presents a basically clonal population structure, with a typical pattern of reticulate evolution and evidence of genetic exchange, as rare events leading to hybrid genomes (Gaunt et al. Reference Gaunt, Yeo, Frame, Stothard, Carrasco, Taylor, Mena, Veazey, Miles, Acosta, de Arias and Miles2003). The genetic diversity of T. cruzi strains has long been recognized and accordingly, a plethora of approaches were used to characterize its population structure aiming at elucidating the relationship between relevant parasite subgroups and eco-epidemiological and clinical aspects of the infection (Macedo and Pena, Reference Macedo and Pena1998; Miles et al. Reference Miles, Llewellyn, Lewis, Yeo, Baleela, Fitzpatrick, Gaunt and Mauricio2009). In a Satellite Meeting of Experts held in Buzios (Brazil) in 2009, a consensus was reached towards the classification of T. cruzi strains into 6 Discrete Typing Units (DTUs), defined as “set of stocks that are genetically more related to each other than to any other stock and that are identifiable by common genetic, molecular, or immunological markers”, designated as Tc I to Tc VI (Zingales et al. Reference Zingales, Andrade, Briones, Campbell, Chiari, Fernandes, Guhl, Lages-Silva, Macedo, Machado, Miles, Romanha, Sturm, Tibayrenc and Schijman2009). The DTUs constitute reliable units for analysis in molecular epidemiology and experimental studies of evolution. The association of strains belonging to different DTUs with the vast spectrum of clinical forms of Chagas disease prevailing in different regions of Latin America is the subject of current research (Macedo et al. Reference Macedo, Machado, Oliveira and Pena2004; Burgos et al. Reference Burgos, Begher, Freitas, Bisio, Duffy, Altcheh, Teijeiro, Lopez Alcoba, Deccarlini, Freilij, Levin, Levalle, Macedo and Schijman2005, Reference Burgos, Altcheh, Bisio, Duffy, Valadares, Seidenstein, Piccinali, Freitas, Levin, Macchi, Macedo, Freilij and Schijman2007, Reference Burgos, Diez, Vigliano, Bisio, Risso, Duffy, Cura, Brusses, Favaloro, Leguizamon, Lucero, Laguens, Levin, Favaloro and Schijman2010; Bisio et al. Reference Bisio, Cura, Duffy, Altcheh, Giganti, Begher, Scapellato, Burgos, Levin, Schreck, Freilij and Schijman2009; Miles et al. Reference Miles, Llewellyn, Lewis, Yeo, Baleela, Fitzpatrick, Gaunt and Mauricio2009). Most strategies to type DTUs require large amounts of parasite DNA, which is only available from cultured stocks. This type of analysis may underestimate the parasite diversity in natural infections (Macedo and Pena, Reference Macedo and Pena1998; Macedo et al. Reference Macedo, Machado, Oliveira and Pena2004). We have recently improved PCR-based strategies targeted to different genomic markers to identify DTUs directly in clinical specimens of T. cruzi-infected patients with different disease forms (Burgos et al. Reference Burgos, Begher, Freitas, Bisio, Duffy, Altcheh, Teijeiro, Lopez Alcoba, Deccarlini, Freilij, Levin, Levalle, Macedo and Schijman2005, Reference Burgos, Altcheh, Bisio, Duffy, Valadares, Seidenstein, Piccinali, Freitas, Levin, Macchi, Macedo, Freilij and Schijman2007, Reference Burgos, Diez, Vigliano, Bisio, Risso, Duffy, Cura, Brusses, Favaloro, Leguizamon, Lucero, Laguens, Levin, Favaloro and Schijman2010; Bisio et al. Reference Bisio, Cura, Duffy, Altcheh, Giganti, Begher, Scapellato, Burgos, Levin, Schreck, Freilij and Schijman2009). This report aims to expand the application of these tools to map the distribution of DTUs in Chagas disease patients from different regions of Argentina.
MATERIALS AND METHODS
Patients
DTU identification was carried out in clinical samples from different groups of patients born in Argentina, that were classified according to provenance and infection phase. The different study groups were (a) adult and pediatric patients with asymptomatic (ACD) or chronic Chagas heart disease (CCHD), (b) congenitally infected children without signs or symptoms of disease (CI) and (c) adult patients with clinical reactivation in the context of immunosuppression, either in solid organ transplant recipients or in HIV co-infected patients (RCD). Full characterization of most RCD patients was reported elsewhere (Burgos et al. Reference Burgos, Begher, Freitas, Bisio, Duffy, Altcheh, Teijeiro, Lopez Alcoba, Deccarlini, Freilij, Levin, Levalle, Macedo and Schijman2005, Reference Burgos, Diez, Vigliano, Bisio, Risso, Duffy, Cura, Brusses, Favaloro, Leguizamon, Lucero, Laguens, Levin, Favaloro and Schijman2010; Bisio et al. Reference Bisio, Cura, Duffy, Altcheh, Giganti, Begher, Scapellato, Burgos, Levin, Schreck, Freilij and Schijman2009). From several patients, more than 1 sample (blood, heart explant and endomyocardial, skin or brain biopsy samples) was available for analysis.
Geographical endemic regions where patients acquired the infection, as well as the type of clinical specimen tested are specified in Table 1, and their corresponding DTUs are included in Fig. 1A. DTUs from patients residing in non-endemic localities in the Province of Buenos Aires or in the city of Buenos Aires, whose sources and routes of transmission were unknown, were also genotyped; however, their corresponding DTUs are not included in Fig. 1A.
Fig. 1. Distribution of Trypanosoma cruzi DTUs in Chagas disease patients from Argentina. T. cruzi DTUs findings in (A) Endemic Argentinean regions. Samples from asymptomatic chronic Chagas disease patients (ACD) are indicated by circles; from chronic Chagas heart disease patients (CCHD), by squares; from congenitally infected patients (CI), by triangles; and from reactivated patients due to immunosupression (RCD), by rhombus. Numbers inside figures indicate quantity of typed samples. Northeastern region (
), Northwestern region (
), Cuyo (
) and Pampean region (
). (B) Clinical groups of Chagas disease patients: CCHD, ACD, CI and RCD. (C) Clinical samples from Chagas disease patients. PB, peripheral blood; HE, heart explants; EMB, endomyocardial biopsy samples, Sk, skin chagomas and Br, brain biopsy samples. T. cruzi DTUs are colour coded: Tc I (
), Tc II (
), Tc V (
), Tc VI (
), Tc II/V/VI (
), Tc V or V+II/VI (
), Tc II/VI (
) and mixed infections (□).
Table 1. Trypanosoma cruzi DTUs in Chagas disease patients from endemic and non-endemic regions of Argentina
a kDNA positive samples from children whose mothers’ procedences are unknown.
b Mixed infection by T. cruzi I+II/VI in one skin biopsy sample.
kDNA Pos, kDNA PCR positive samples; DTUs, Discrete Typing Units; NOA, Northwestern Argentinean region; NEA, Northeastern Argentinean region; CCHD, Chronic Chagas Heart Disease; ACD, Asymptomatic chronic Chagas Disease; RCD, Chagas Disease reactivation; CI, Congenital infection; Tissue, Heart explants, endomyocardial, brain or skin biopsy samples.
All procedures have complied with the criteria of the Ethical Committees of the participating Institutions, including written informed consents.
DTU genotyping
Preparation of DNA from clinical specimens was done according to the type of sample. Peripheral blood samples were processed using blood extraction kits (QIAamp DNA mini kit –Qiagen, USA– or High Pure PCR Template Preparation Kit–Roche, Germany) following the recommendations of the manufacturers. Each experiment included a kDNA-PCR positive blood sample from a Chagas disease patient as a positive control, and a blood sample from a healthy subject as a negative control.
Paraffin-embedded tissue slices from cardiac explants or skin biopsy samples were processed as detailed elsewhere (Burgos et al. Reference Burgos, Diez, Vigliano, Bisio, Risso, Duffy, Cura, Brusses, Favaloro, Leguizamon, Lucero, Laguens, Levin, Favaloro and Schijman2010). Total DNA from fresh endomyocardial, brain or skin biopsy samples was purified using the QIAmp DNA tissue kit (Qiagen, USA) as recommended by the manufacturer. A paraffin-embedded sample from heart explants of patients with cardiac diseases of no Chagasic etiology (Burgos et al. Reference Burgos, Diez, Vigliano, Bisio, Risso, Duffy, Cura, Brusses, Favaloro, Leguizamon, Lucero, Laguens, Levin, Favaloro and Schijman2010) was included as a negative control in each experiment.
Identification of DTUs was assessed in positive kinetoplastid DNA-PCR (kDNA-PCR) samples (Burgos et al. Reference Burgos, Altcheh, Bisio, Duffy, Valadares, Seidenstein, Piccinali, Freitas, Levin, Macchi, Macedo, Freilij and Schijman2007). Five μl of DNA samples were subjected to a PCR algorithm for DTU genotyping, as reported in Burgos et al. (Reference Burgos, Diez, Vigliano, Bisio, Risso, Duffy, Cura, Brusses, Favaloro, Leguizamon, Lucero, Laguens, Levin, Favaloro and Schijman2010). Briefly, spliced leader intergenic region PCR (SL-IR-PCR) was used to distinguish Tc I (150 bp), Tc II, Tc V and Tc VI (157 bp) from Tc III and Tc IV (200 bp). Hemi-nested SL-IR-I was used to identify Tc I (350 bp), and hemi-nested SL-IR-II was used to identify Tc II, Tc V, and Tc VI (300 bp). Hemi-nested PCR of 24Sα-ribosomal DNA (24Sα-rDNA) was used to distinguish Tc V (125 or 125+140 bp) from Tc II and Tc VI (140 bp), and Tc III (125 bp) from Tc IV (140 or 145 bp); and hemi-nested PCR targeted to genomic fragment A10 was used to discriminate Tc II (580 bp) from Tc VI (525 bp). Samples that yielded positive SL-IR-II PCR results but negative 24Sα-rDNA PCR results were reported as Tc II/V/VI group. Those samples that amplified the 140 bp 24Sα-rDNA fragment but had negative results of PCR targeted to A10 were reported as Tc II/VI group. In those samples that amplified 125+140 bp 24Sα-rDNA fragments, mixed populations by Tc V+II/VI could not be discarded.
Each PCR run included an amplification reaction without DNA as a negative PCR mixture control and an amplification reaction with 50 pg of total DNA from those T. cruzi strains representing the DTUs to be genotyped.
RESULTS AND DISCUSSION
In total 347 kDNA-PCR positive clinical samples from 325 T. cruzi-infected patients were studied: (a) 237 samples from 207 adult patients (mean age 47·3 years, range 20–80 years old) and 30 pediatric patients (mean age 10·9 years, range 2–17 years old) with ACD or CCHD; (b) 63 samples from 63 children with CI (mean age 5·1 years, range 2 days-14 years) without signs or symptoms of disease and (c) 47 samples from 25 adult patients (mean age 51·4 years, range 29–70) with RCD. Cardiac explants from patients who underwent heart transplantation are indicated in Table 1 as ‘CCHD (Tissue)’.
DTUs were identified in 256 samples from 239 patients (Table 1). The rate of DTU detection was higher in younger pediatric CI and in RCD patients, than among ACD and CCHD patients (85·2% versus 69·2%, respectively, χ 2: 7·27206618, P<0·05). This reflects the typical higher parasitic loads in recent CI and in RCD (Duffy et al. Reference Duffy, Bisio, Altcheh, Burgos, Diez, Levin, Favaloro, Freilij and Schijman2009). Of the blood samples, 27% could not be typed due to lack of amplification in some or all tested PCR strategies, probably because of parasitic loads under the detection limits of the procedures (0·1–10 pg parasite DNA in the reaction tube; Burgos et al. Reference Burgos, Altcheh, Bisio, Duffy, Valadares, Seidenstein, Piccinali, Freitas, Levin, Macchi, Macedo, Freilij and Schijman2007). The identification of DTUs was possible in 66·7% of heart explants and endomyocardial biopsies (10/15) and reached 100% (10/10) in chagoma samples, in agreement with their high parasitic burdens, corroborated in some cases by the microscopical observation of amastigote nests (Burgos et al. Reference Burgos, Diez, Vigliano, Bisio, Risso, Duffy, Cura, Brusses, Favaloro, Leguizamon, Lucero, Laguens, Levin, Favaloro and Schijman2010).
Geographical and clinical distribution of DTUs
Tc I was detected in 4·2% (10/239) of patients and, exclusively, in heart explants of CCHD patients who underwent transplantation or in blood, endomyocardial biopsy samples and chagomas from RCD cases, mainly from the NEA (Northeastern) and NOA (Northwestern) regions (Table 1). Tc II was detected in only 2 RCD patients who acquired T. cruzi infection in the Pampean region, including 3 samples (1 blood sample, 1 skin and 1 brain biopsy samples) (Table 1). Tc III and Tc IV were not detected in the studied population (Table 1).
The Trypanosoma cruzi V or the Tc II/V/VI group were the most prevalent in the tested population and were detected in 89·1% (228/256) of tested samples corresponding to 94·6% (226/239) of the patients (Table 1). In 39 out of these 226 patients (17·3%), mixed populations composed by Tc V and Tc II, Tc V and Tc VI, or Tc V and Tc II plus Tc VI, could not be excluded because their tested samples amplified both 24Sα-rDNA fragments (see Materials and Methods section). Tc V DTUs belonging to the Tc II/V/VI group were identified in 100% (51/51) of CI samples, 97·9% (93/95) of ACD samples, 90·9% (70/77) of CCHD samples and 42·4% (14/33) of RCD samples (χ 2: 87·83, P<0·0001). Tc VI and Tc II/VI were found in 2·9% of cases (7/239), 57·1% of whom were RCD, mostly from the Pampean Region (Table 1).
The geographical distribution of DTUs in patients' groups whose routes of transmission were known is shown in Fig. 1. Our findings are in agreement with previous studies of serum samples (Di Noia et al. Reference Di Noia, Buscaglia, De Marchi, Almeida and Frasch2002), culture isolates recovered from peripheral blood (Diosque et al. Reference Diosque, Barnabe, Padilla, Marco, Cardozo, Cimino, Nasserd, Tibayrenc and Basombrıo2003; Carranza et al. Reference Carranza, Valadares, D'Avila, Baptista, Moreno, Galvão, Chiari, Sturm, Gontijo, Macedo and Zingales2009) and tissue samples (Freitas et al. Reference Freitas, Lages-Silva, Crema, Pena and Macedo2005) that revealed prevailing Tc II, V, and VI in human infections at the southern cone of America, whereas Tc I is less frequent and associated with cases from Chaco and Santiago del Estero, mainly detectable in target organs such as cardiac explants or blood samples in reactivation after immunosuppression due to organ transplantation (Fig. 1B and C). This has been discussed in more depth in a recent paper on heart transplant recipients (Burgos et al. Reference Burgos, Diez, Vigliano, Bisio, Risso, Duffy, Cura, Brusses, Favaloro, Leguizamon, Lucero, Laguens, Levin, Favaloro and Schijman2010) where it was shown that this DTU is not implicated only in human disease in the Amazonia, Andean region, Central America and Mexico (Higo et al. Reference Higo, Miura, Horio, Mimori, Hamano, Agatsuma, Yanagi, Cruz-Reyes, Uyema, Rojas de Arias, Matta, Akahane, Hirayama, Takeuchi, Tada and Himeno2004). The lack of detection of Tc III and Tc IV is in agreement with their association with the sylvatic cycle in other endemic countries, and their paucity in human infections (Miles et al. Reference Miles, Llewellyn, Lewis, Yeo, Baleela, Fitzpatrick, Gaunt and Mauricio2009).
A similar DTU distribution was found between congenitally infected children and chronic patients infected in areas of endemicity, in agreement with other studies showing that prevalence of DTUs in congenitally infected cases reflects DTU prevalence in the general infected population (Brenière et al. Reference Brenière, Bosseno, Telleria, Bastrenta, Yacsik, Noireau, Alcazar, Barnabé, Wincker and Tibayrenc1998; Virreira et al. Reference Virreira, Alonso-Vega, Solano, Jijena, Brutus, Bustamante, Truyens, Schneider, Torrico, Carlier and Svoboda2006; Burgos et al. Reference Burgos, Altcheh, Bisio, Duffy, Valadares, Seidenstein, Piccinali, Freitas, Levin, Macchi, Macedo, Freilij and Schijman2007).
Differential tissue distribution of DTUs in RCD patients
In 15 RCD patients, clinical samples were withdrawn from different body sites. Tc V was detected in blood and Tc II in a brain biopsy sample from a RCD patient with AIDS (Burgos et al. Reference Burgos, Begher, Freitas, Bisio, Duffy, Altcheh, Teijeiro, Lopez Alcoba, Deccarlini, Freilij, Levin, Levalle, Macedo and Schijman2005). Tc I and Tc II/VI were detected in blood and in the heart explant and skin chagoma, respectively, from a CCHD patient with skin reactivation. Tc I was found in the heart explant and Tc V, in blood and skin chagoma from another CCHD patient with skin reactivation (Burgos et al. Reference Burgos, Diez, Vigliano, Bisio, Risso, Duffy, Cura, Brusses, Favaloro, Leguizamon, Lucero, Laguens, Levin, Favaloro and Schijman2010). These data suggest the occurrence of mixed DTU polyclonal parasitic populations in the human chronic infection (Vago et al. Reference Vago, Andrade, Leite, d'Avila Reis, Macedo, Adad, Tostes, Moreira, Filho and Pena2000; Burgos et al. Reference Burgos, Begher, Freitas, Bisio, Duffy, Altcheh, Teijeiro, Lopez Alcoba, Deccarlini, Freilij, Levin, Levalle, Macedo and Schijman2005, Reference Burgos, Diez, Vigliano, Bisio, Risso, Duffy, Cura, Brusses, Favaloro, Leguizamon, Lucero, Laguens, Levin, Favaloro and Schijman2010) in higher proportions than previously assumed from the analysis of culture isolates of peripheral blood samples (Macedo et al. Reference Macedo and Pena1998, Reference Macedo, Machado, Oliveira and Pena2004). Furthermore, infection by more than 1 DTU was found in a skin lesion of lower limbs from a kidney transplanted patient residing in Buenos Aires city (Tc I plus Tc II/VI) (Table 1, Fig. 1B). T. cruzi has the potential for genetic exchange (Gaunt et al. Reference Gaunt, Yeo, Frame, Stothard, Carrasco, Taylor, Mena, Veazey, Miles, Acosta, de Arias and Miles2003). Some localized transmission cycles suggest that genetic recombination does occur within DTUs (Carranza et al. Reference Carranza, Valadares, D'Avila, Baptista, Moreno, Galvão, Chiari, Sturm, Gontijo, Macedo and Zingales2009; Ocaña-Mayorga et al. Reference Ocaña-Mayorga, Llewellyn, Costales, Miles and Grijalva2010). The above-mentioned case illustrates a natural scenario where this type of event could occur.
Finally, our findings provide evidence of the complexity of the dynamics of T. cruzi diversity in the natural history of human Chagas disease and allege the pathogenic role of DTUs I, II, V and VI in our population.
ACKNOWLEDGEMENTS
We thank Dr Laura A. Barcan (Sección Infectología, Servicio de Clínica Médica, Hospital Italiano, Buenos Aires, Argentina) for providing blood samples from an RCD case, and Dr Salvador O. Giganti (Servicios de Neurocirugía y Clínica Médica, HIGA Eva Perón, San Martín, Provincia de Buenos Aires, Argentina) for providing a brain biopsy sample from another RCD case.
FINANCIAL SUPPORT
This work was supported by the Ministry of Science and Technology (FONCyT) (A.G.S., PICT 33955); CONICET (A.G.S., PIP 112-2008-01-02913); Secretaría General de Ciencia y Tecnología de la UNNE (D.E.M., Res. ANPCyT N° 163/08 – PICTO 00167); and the Ministry of Health (E.O., Beca Ramón Carrillo-Arturo Oñativia).
