Distribution of Trypanosoma cruzi discrete typing units in Bolivian migrants in Spain

Infection, Genetics and Evolution 21 (2014) 440–442

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Distribution of Trypanosoma cruzi discrete typing units in Bolivian migrants in Spain José A. Perez-Molina a,⇑, Cristina Poveda b, Angela Martinez-Perez a, Felipe Guhl b, Begoña Monge-Maillo a, Manuel Fresno c, Rogelio López-Velez a, Juan D. Ramírez b, Nuria Girones c a b c

Hospital Ramón y Cajal, Medicina Tropical y Parasitología Clínica, Servicio de Enfermedades Infecciosas, Madrid, Spain Centro de Investigaciones en Microbiología y Parasitología Tropical, CIMPAT, Universidad de los Andes, Cra 1 No. 18A-10, Bogotá, Colombia Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Cantoblanco, Madrid, Spain

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Article history: Received 13 September 2013 Received in revised form 14 December 2013 Accepted 17 December 2013 Available online 3 January 2014 Keywords: Trypanosoma cruzi Discrete typing units Chagas disease Migration

a b s t r a c t Chagas disease is caused by the protozoan Trypanosoma cruzi. This parasite is transmitted to humans mainly through the faeces of infected triatomine ‘‘kissing’’ bugs, by blood transfusions or organ donation from infected donors, and can be transmitted from mother to child. This disease is endemic in the Americas, where Bolivia has up to 28.8% prevalence in general population. Increased migration to Europe has made it emerge in countries where it was previously unknown, being Spain the second country in number of patients after the United States. T. cruzi is an organism with a rich genetic diversity, what has been grouped into six discrete typing units (DTUs). Some authors have linked these DTUs either to specific geographical distribution or to the different clinical presentations. Nevertheless little is known about its distribution in migrant populations. Our aim was to describe the T. cruzi strains isolated from a population of chronically infected Bolivian patients attending our clinic in Madrid. Thirty-three consecutive patients meeting this condition were selected for the study. Molecular characterization was performed by an algorithm that combines PCR of the intergenic region of the mini exon-gene, the 24Sa and 18S regions of rDNA and the variable region of the satellite DNA. A descriptive analysis was performed and associations between epidemiological/clinical data and the different DTUs were tested. Twenty-seven out of thirty-three patients had their DTU detected. Mean age was 36 years (IQR 31–43.3) and 23 were women (76.7%). The median time since arrival to Spain was 60 months (IQR 43–81). The most common DTU were TcV, TcIV and TcI. Four patients had cardiac involvement: 2 had TcV and 2 could not have their DTU determined. TcIII was not isolated from any patient. DTUs distribution in migrant population seems to be similar to that observed in the patients’ countries of origin. Ó 2013 Elsevier B.V. All rights reserved.

1. Introduction Chagas disease is caused by the protozoan parasite Trypanosoma cruzi. It has an acute initial form that is asymptomatic for most patients and a chronic form that manifests with cardiac, digestive, or cardiodigestive involvement after the acute infection. Patients can remain in a chronic indeterminate stage for decades, although every year 2–5% progress to a clinical stage. This disease is endemic in the Americas, and prevalence has reached 28.8% in the general population (Moncayo and Silveira, 2009). Increased migration to Europe means that Chagas disease has emerged in countries where it was previously unknown (Schmunis and Yadon, 2010). It is particularly frequent in Spain, which is the second country in number ⇑ Corresponding author. Address: Medicina Tropical, Servicio de Enfermedades Infecciosas, Hospital Universitario Ramón y Cajal, Carretera de Colmenar Km 9,1, 28034 Madrid, Spain. Tel.: +34 913368108; fax: +34 913368238. E-mail address: [email protected] (J.A. Perez-Molina). 1567-1348/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.meegid.2013.12.018

of cases after the US (Pérez-Molina et al., 2012). The increasing number of diagnoses made during the last decade have intensified the burden on the Spanish health system, which now has to streamline identification and treatment of a disease that was previously classed as tropical (Navarro et al., 2012). Many other European countries have faced a similar problem, and it is estimated that this disease goes undiagnosed in 92–96% of cases in its chronic indeterminate form (Basile et al., 2011). Different typing methods have made it possible to identify several ancestral parasite lineages, and six near-clades (TcI to TcVI) have been identified. These near-clades are known as discrete typing units (DTU), which have been defined as ‘‘sets 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’’ (Zingales et al., 2009). DTUs have been linked to specific geographical distributions and various clinical presentations, and data on the isolation of T. cruzi in vectors, reservoirs, and human samples from different origins have added to our knowledge

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of the evolution and distribution of the species in the Americas (Burgos et al., 2010; Ramírez et al., 2010; Zingales et al., 2012). Nevertheless, little is known about the exact distribution this species in the migrant population. 2. Objective Our hypothesis was that the presence of the most common DTUs in a specific endemic area would be uniform in migrants from that area; however, we cannot predict whether biological characteristics such as degree of persistence in blood, tissue tropism, or absence of reinfection could modify the distribution of DTUs in non-endemic areas. Our aim was to describe the T. cruzi strains identified from a population of chronically infected Bolivian patients attending our clinic in Madrid. 3. Methods We conducted a cross-sectional observational study. Thirtythree consecutive Bolivian patients who were chronically infected with T. cruzi were selected from those attended at our clinic during the period 2010–2011. They all had positive results in two serological tests: in-house IFAT and ELISA (ARCHITECT Chagas ELISA test, BiosChile, Abbott Laboratories, Wiesbaden, Germany). Our in-house IFAT was prepared with a combination of cultured epimastigote antigens (Mc, T, and Dm28), and human IgG (bioMérieuxÒ) was used as a marker. Thirteen patients had a positive PCR result in blood. Molecular characterization was performed using an algorithm combining the PCR of the intergenic region of the mini-exon gene, the 24Sa and 18S regions of rDNA, and the variable region of the satellite DNA, as described elsewhere (Ramírez et al., 2013). The primers used were kinetoplast DNA (primers 121 [50 -AAATAATGTA CGGGKGAGATGCATGA-30 ] and 122 [50 -GGTTCGATTGGGGTTGGT GTAATATA-30 ]) and the tandem repeat satellite region from T. cruzi (primers cruzi1 [50 -ASTCGGCTGATCGTTTTCGA-30 ] and cruzi2 [50 -AATTCCTCCAAGCAGCGGATA-30 ] (Liarte et al., 2009). The molecular markers used were the intergenic region of the nontranscribed mini-exon gene (primers TCC [50 -CCC CCC TCC CAG GCC ACA CTG-30 ], TC1 [50 -GTG TCC GCC ACC TCC TTCGGG CC-30 ], and TC2 [50 -CCT GCA GGC ACA CGT GTG TGT G-30 ]) (Souto et al., 1996). All mini-exon gene PCR assays were performed using two primers instead of a multiplex PCR assay to determine the presence of mixed infections. Thus, PCR was applied using the primers TCCTC1 and TCC-TC2. Segments containing the D7 domain of LSU rDNA were amplified from genomic DNA by PCR using the trypanosomatid conserved primers D75 (50 -GCAGATCTTGGTTGGCGTAG-30 ) and D76 (50 -GGTTCTCTGTTGCCCCTTTT-3) (Briones et al., 1999), as well as A10 with primers pr1 (5-CCGCTAAGCAGTTCTGTCCATA-3) and pr3 (5-GCTTTATTACCCCATGCCACAG-3) (Burgos et al., 2010). We performed a descriptive analysis and tested associations between epidemiological and clinical data and the different DTUs. We specifically analyzed age, sex, cardiac or digestive involvement, and time since arrival in Spain. Qualitative data were analyzed using the v2 and Fisher exact tests; quantitative data were analyzed using the t test.

presented a combination of two different DTUs: two patients (6.1%) presented TcI in combination with TcIV, two (6.1%) had TcIV with TcV, one (3%) had TcI and TcV, and one (3%) had TcII and TcVI. TcIII was not identified in any patient (Fig. 1). Cardiac involvement was detected in four patients. One patient had completed treatment during 2008 and presented right bundle branch block, which had been stable for five years of follow-up, although his DTU was not determined. Another patient also presented right bundle branch block; he received treatment in 2010, and his DTU was characterized as TcV. This TcV was also detected in a patient presenting ventricular extrasystoles, left anterior fascicular block, and incomplete right bundle branch block that had never been treated. Finally, DTUs were not characterized in a patient with a pacemaker for severe bradycardia. Findings regarding cardiac involvement were insufficient to test associations. Neither age nor sex nor time elapsed from arrival to Spain was significantly associated with any specific DTU. Similarly, no significant association was found with these three parameters when DTUs were grouped into TcV (whether alone or in combination with other DTUs) and non-TcV.

5. Discussion In order of frequency, the most common DTUs identified in our sample were TcV, TcIV, and TcI. Consistent with published data on Bolivian patients (del Puerto et al., 2010), we also found a high number of mixed infections. TcII and TcVI coinfected only one patient; this finding seems reasonable, as both these DTUs are rarely reported in Bolivia. No TcIII was detected, maybe because it is associated with sylvatic cycles and is rarely present in human infection (Zingales et al., 2012). The largest series characterizing DTUs in Bolivians to date found TcV and TcI to be the most common near-clades. However, this study excluded specific probes to detect TcIV (del Puerto et al., 2010). Using these specific probes, we found that TcIV was the second most frequent DTU. A study performed in Venezuela also found TcIV to be the second most common cause of human infection (Carrasco et al., 2012). TcIV has been linked to sylvatic cycles and has been reported in the context of acute oral outbreaks in tropical wet areas of Colombia and Brazil (Monteiro et al., 2012; Ramirez et al., 2013). A recent report also related this DTU to an oral outbreak in Bolivia (Santalla Vargas, 2012). Finally, TcIV has

4. Results DTUs were identified in 27 of the 33 patients. Mean age was 36 years (IQR, 31-43.3), and 23 patients were women (76.7%). The median time since arrival in Spain was 60 months (IQR, 4381). These characteristics were similar to those of the population commonly attended at our clinic. DTU was not characterized in six patients. The most prevalent DTU was TcV (n = 15, 45.5%), followed by TcIV (n = 5, 15.2%) and TcI (n = 1, 3%). Six patients

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Fig. 1. DTUs distribution in a Bolivian population.

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recently been detected in dogs, suggesting a potential role in domestic transmission (Ramírez et al., 2013). Our study is limited by its small sample size and the fact that we did not record the exact geographical origin of our patients at the beginning of the study. Ours is the first study to characterize the T. cruzi genome in an adult migrant population in Europe. Given that the distribution of DTUs in non-endemic countries could be comparable to that of endemic countries, the molecular genetics of the DTUs of T. cruzi and its potential associations with clinical outcomes warrant further study. Acknowledgements This work was carried out with the support provided by the programme on I+D+I 2008–2011, ISCIII-Subdirección General de Redes y Centros de Investigación Cooperativa (RICET), file no. RD12/0018/0019. References Basile, L., Jansa, J.M., Carlier, Y., Salamanca, D.D., Angheben, A, Seixas, J., Bartoloni, A., Van Gool, T., Cañavate, C., Flores-Chávez, M., Jackson, Y., Chiodini, P.L., AlbajarViñas, P., 2011. Working group on chagas disease, chagas disease in European countries: the challenge of a surveillance system. Euro. Surveill. 16, pii=19968. Briones, M.R., Souto, R.P., Stolf, B.S., Zingales, B., 1999. The evolution of two Trypanosoma cruzi subgroups inferred from rRNA genes can be correlated with the interchange of American mammalian faunas in the Cenozoic and has implications to pathogenicity and host specificity. Mol. Biochem. Parasitol. 104, 219–232. Burgos, J.M., Diez, M., Vigliano, C., Bisio, M., Risso, M., Duffy, T., Cura, C., Brusses, B., Favaloro, L., Leguizamon, M.S., Lucero, R.H., Laguens, R., Levin, M.J., Favaloro, R., Schijman, A.G., 2010. Molecular identification of Trypanosoma cruzi discrete typing units in end-stage chronic chagas heart disease and reactivation after heart transplantation. Clin. Infect. Dis. 51, 485–495. Carrasco, H.J., Segovia, M., Llewellyn, M.S., Morocoima, A., Urdaneta-Morales, S., Martínez, C., Martínez, C.E., Garcia, C., Rodríguez, M., Espinosa, R., de Noya, B.A., Díaz-Bello, Z., Herrera, L., Fitzpatrick, S., Yeo, M., Miles, M.A., Feliciangeli, M.D., 2012. Geographical distribution of Trypanosoma cruzi genotypes in Venezuela. PLoS Negl. Trop. Dis. 6, e1707. del Puerto, R., Nishizawa, J.E., Kikuchi, M., Iihoshi, N., Roca, Y., Avilas, C., Gianella, A., Lora, J., Velarde, F.U., Renjel, L.A., Miura, S., Higo, H., Komiya, N., Maemura, K.,

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