Zoonoses and Public Health
SHORT COMMUNICATION
First Report of Human Trypanosoma cruzi Infection Attributed to TcBat Genotype ndez1, M. Montilla2, P. Zambrano3, A. C. Flo rez2, E. Parra4 and Z. M. Cucunuba 1 J. D. Ramırez1, C. Herna 1 2 3 4
Red Chagas Colombia, Instituto Nacional de Salud, Bogot a, Colombia Grupo de Parasitologıa, Instituto Nacional de Salud, Bogot a, Colombia n de Vigilancia y Salud Pu blica, Instituto Nacional de Salud, Bogota, Colombia Subdireccio Grupo de Patologıa, Instituto Nacional de Salud, Bogot a, Colombia
Impacts
• This is the first report of the Trypanosoma cruzi genotype associated with bats in humans.
• Surveillance of this genotype is mandatory in endemic countries. • We believe that TcBat detection is underestimated. Keywords: Chagas disease; discrete typing units; genotypes; bats; human infection Correspondence: J. D. Ramırez. Red Chagas Colombia, Instituto Nacional de Salud, Av. Calle 26 No. 51-28, Bogot a, Colombia. Tel./Fax: +571 2207700 ext. 1315; E-mail: [email protected]
Summary Chagas disease is an endemic disease of the American continent caused by Trypanosoma cruzi and divided into six discrete typing units (TcI – TcVI). Nearly 10 million people harbour the infection representing a serious issue in public health. Epidemiological surveillance allowed us to detect a bat-related T. cruzi genotype (henceforth named TcBat) in a 5-year-old female living in a forest area in northwestern Colombia. Molecular tools determined a mixed infection of T. cruzi I and TcBat genotypes. This represents the first report of TcBat infection in humans; the epidemiological consequences of this finding are discussed herein.
Received for publication June 24, 2013 doi: 10.1111/zph.12094
Chagas disease or American trypanosomiasis, caused by Trypanosoma cruzi, is a complex neglected tropical disease that affects over 8 million people in Latin America (Schmunis and Yadon, 2010). This parasite can be transmitted via contaminated faeces of triatomine bugs, oral route, blood transfusion, congenital route, laboratory accidents and transplantation (Rassi et al., 2012). The aetiological agent is characterized by exhibiting a remarkable genetic variability evinced in at least six discrete typing units (DTUs) from T. cruzi I to T. cruzi VI (TcI-TcVI) (Zingales et al., 2012). Recently, intensive sampling has reported a new genotype associated with bats from the Noctilio spp., Myotis spp. and Artibeus spp. species named as TcBat (Marcili et al., 2009; Pinto et al., 2012). The role of TcBat in human infections is unclear; previous studies have demonstrated its ability to infect murine models and invade He-La culture lines, but there have not yet to be any reports of this genotype associated with human cases (Maeda et al., 2011). In 2012, a screening for acute Chagas disease was carried out in Colombia as part of the National Health Institute
surveillance system. Twenty-six patients from the municipality of Sabana del Crespo of the department of Cesar in northeastern Colombia were evaluated. This community lives in the middle of the dry forest, in rudimentary houses where they interact with the fauna of the region (triatomine bugs, mammals, etc.). We conducted serological tests on the 26 patients using crude antigenic preparations of T. cruzi I by ELISA, IFAT and HAI commercial kit (Wiener, Argentina), obtaining a positivity of 19% with a fatal case of death due to acute cardiomyopathy. When entomological and epidemiological surveillance was conducted, we observed the presence of adults of Rhodnius prolixus and Triatoma dimidiata inside the houses and adults of Triatoma maculata and Panstrongylus geniculatus around the dwellings. Previous entomological surveillance in the municipality has reported an infestation index of 50%. Likewise, the presence of mammals (rodents, didelphids and dogs) within the houses and the dwellings was observed. Blood samples were taken to seed haemocultures and PCR directed to kDNA using primers previously reported
© 2013 Blackwell Verlag GmbH Zoonoses and Public Health, 2014, 61, 477–479
477
J. D. Ramırez et al.
TcBat Genotype in Humans
TcV
(a)
(b)
TcIII
1
2
3
4
TcIV TcVI TcII
TcBat
TcI
biological clones typed as T.cruzi Bat
biological clones typed as T.cruzi I
Fig. 1. (a) Maximum composite likelihood (MCL) phylogenetic reconstruction of cytb gene sequences discriminating the biological clones as TcI and TcBat. (b) Pathology images of microtome tissues stained with haematoxylin and eosin. 1. cardiac tissue, 2. skeletal muscle, 3. small intestine, 4. liver.
(Schijman et al., 2011). We observed only one haemoculture turning positive after 37 days of examination. This result was observed in a 5-year-old female patient with positive ELISA, kDNA PCR and negative IFAT and IHA. Her mother presented positive serology, kDNA PCR and died due to acute cardiomyopathy attributed to T. cruzi. Likewise, her father presented positive serology with absence of cardiac failure. This couple had nine additional children as offspring that showed negative serology, PCR and haemoculture. The positive haemoculture from the patient (5-year-old female) was subjected to biological cloning as reported elsewhere (Ramırez et al., 2013b). Ten biological clones were genotyped using the intergenic region of mini-exon gene, 24S and 18S genes including direct sequencing of cytb and SSU rDNA gene fragments (Burgos et al., 2010; Ramırez et al., 2010). This typing scheme and the use of cytb barcoding (reliable maximum composite likelihood phylogenetic reconstruction) demonstrated the presence of four clones as T. cruzi I and six clones typed as TcBat using appropriate reference sequences from the cytb gene retrieved from GenBank (Fig. 1a). Lastly, to observe the biological behaviour of the isolate herein described, we injected a 200-uL aliquot of 106 epimastigotes in ICR line mice observing the absence of histopathological damage in heart, liver, brain and spleen. We analysed the heart from the mouse by kDNA PCR which turned out positive corroborating the incipient presence of the parasite (Fig. 1b). This is the first report to our concern of TcBat genotype in humans. Hence, these findings highlight the need to pursue future research related to the biological origins of this foreseen genotype and also the epidemiological implications of this report. The haemoculture of the mother did not grow and a direct link between the parasite from the mother and the child could not be established. However, this means that the most likely route of TcBat transmission could be vectorial because of the absence of infection in the other nine off-spring children. Another possible route could be the oral route that is the
most common cause of acute Chagas disease in Colombia and also involving death due to acute cardiomyopathy, but further epidemiological evidence is required to fulfil this hypothesis (Ramırez et al., 2013a). TcBat genotype has been reported in Colombia among Carollia perscipillata, Myotis oxyotus and Rhynchonycteris naso that are considered species that feed on insects and/or fruits in eastern Colombia. The occurrence of this genotype within human populations can be explained in the light of the close interactions between humans and these mammals, but again, further research is needed to unravel the epidemiological risk of this link/interaction. Herein, we described the first report of T. cruzi infection attributed to TcBat genotype in humans. The clinical symptomatology of the patient corroborated that TcBat is able to invade mammalian cells, but is not able to cause tissue damage or any pathological alteration (absence of ECG alterations). We encourage the scientific groups from endemic countries and in those where human populations cohabit within forest dwellings to evaluate the plausible presence of TcBat genotype apparently in those isolations from cases of asymptomatic Chagas disease. We certainly believe that human infection caused by this genotype is underestimated due to unbiased sampling and detection tools.
478
© 2013 Blackwell Verlag GmbH Zoonoses and Public Health, 2014, 61, 477–479
Acknowledgement This work is supported by Departamento Administrativo Nacional de Ciencia y Tecnologıa de Colombia ‘Francisco Jose de Caldas – COLCIENCIAS’, Grant Number 3802011, code 5014-537-30398. References Burgos, J. M., M. Diez, C. Vigliano, M. Bisio, M. Risso, T. Duffy, C. Cura, B. Brusses, L. Favaloro, M. S. Leguizamon, R. H. Lucero, R. Laguens, M. J. Levin, R. Favaloro, and A. G. Schijman, 2010: Molecular identification of Trypanosoma cruzi discrete typing units in end-stage chronic Chagas heart disease
J. D. Ramırez et al.
TcBat Genotype in Humans
and reactivation after heart transplantation. Clin. Infect. Dis. 51, 485–495. Maeda, F. Y., R. M. Alves, C. Cortez, F. M. Lima, and N. Yoshida, 2011: Characterization of the infective properties of a new genetic group of Trypanosoma cruzi associated with bats. Acta Trop. 120, 231–237. Marcili, A., L. Lima, M. J. Cavazzana, A. C. V. Junqueira, H. H. Veludo, F. Maia da Silva, M. Campaner, F. Paiva, V. L. B. Nunes, and M. M. G. Teixeira, 2009: A new genotype of Trypanosoma cruzi associated with bats evidenced by phylogenetic analyses using SSU rDNA, cytochrome b and Histone H2B genes and genotyping based on ITS1 rDNA. Parasitology 136, 641–655. Pinto, C. M., E. K. V. Kalko, I. Cottontail, N. Wellinghausen, and V. M. Cottontail, 2012: TcBat a bat-exclusive lineage of Trypanosoma cruzi in the Panama Canal Zone, with comments on its classification and the use of the 18S rRNA gene for lineage identification. Infect. Genet. Evol. 12, 1328–1332. Ramırez, J. D., F. Guhl, L. M. Rend on, F. Rosas, J. A. MarinNeto, and C. A. Morillo, 2010: Chagas cardiomyopathy manifestations and Trypanosoma cruzi genotypes circulating in chronic chagasic patients. PLoS Negl. Trop. Dis. 4, e899. Ramırez, J. D., M. Montilla, Z. M. Cucunuba, A. C. Florez, P. Zambrano, and F. Guhl, 2013a: Molecular epidemiology of human oral Chagas disease outbreaks in Colombia. PLoS Negl. Trop. Dis. 7, e2041.
Ramırez, J. D., C. Herrera, Y. Bogota, M. C. Duque, A. SuarezRivillas, and F. Guhl, 2013b: Validation of a Poisson-distributed limiting dilution assay [LDA] for a rapid and accurate resolution of multiclonal infections in natural Trypanosoma cruzi populations. J. Microbiol. Methods 92, 220–225. Rassi, J. Jr, A. Rassi, and J. Marcondes de Rezende, 2012: American trypanosomiasis, Chagas disease. Infect. Dis. Clin. North Am. 26, 275–291. Schijman, A., M. Bisio, L. Orellana, M. Sued, T. Duffy, A. M. Mejıa-Jaramillo, C. Cura, F. Auter, V. Veron, S. Deborgraeve, G. Hijar, I. Zulantay, R. Lucero, E. Velazquez, T. Tellez, Z. Sanchez, L. Galvao, D. Nolder, M. Monje-Rumi, J. Levi, J. D. Ramırez, O. Triana, F. Guhl, S. Sosa-Estani, and J. Lazdins, 2011: International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients. PLoS Negl. Trop. Dis. 5, 931–939. Schmunis, G. A., and Z. E. Yadon, 2010: Chagas disease: a Latin American health problem becoming a world health problem. Acta Trop. 115, 14–21. Zingales, B., M. A. Miles, D. A. Campbell, M. Tibayrenc, A. M. Macedo, M. M. G. Teixeira, A. G. Schijman, M. S. Llewellyn, E. Lages-Silva, C. R. Machado, S. G. Andrade, and N. R. Sturm, 2012: The revised Trypanosoma cruzi subspecific nomenclature: rationale, epidemiological relevance and research applications. Infect. Genet. Evol. 12, 240–253.
© 2013 Blackwell Verlag GmbH Zoonoses and Public Health, 2014, 61, 477–479
479
SHORT COMMUNICATION
First Report of Human Trypanosoma cruzi Infection Attributed to TcBat Genotype ndez1, M. Montilla2, P. Zambrano3, A. C. Flo rez2, E. Parra4 and Z. M. Cucunuba 1 J. D. Ramırez1, C. Herna 1 2 3 4
Red Chagas Colombia, Instituto Nacional de Salud, Bogot a, Colombia Grupo de Parasitologıa, Instituto Nacional de Salud, Bogot a, Colombia n de Vigilancia y Salud Pu blica, Instituto Nacional de Salud, Bogota, Colombia Subdireccio Grupo de Patologıa, Instituto Nacional de Salud, Bogot a, Colombia
Impacts
• This is the first report of the Trypanosoma cruzi genotype associated with bats in humans.
• Surveillance of this genotype is mandatory in endemic countries. • We believe that TcBat detection is underestimated. Keywords: Chagas disease; discrete typing units; genotypes; bats; human infection Correspondence: J. D. Ramırez. Red Chagas Colombia, Instituto Nacional de Salud, Av. Calle 26 No. 51-28, Bogot a, Colombia. Tel./Fax: +571 2207700 ext. 1315; E-mail: [email protected]
Summary Chagas disease is an endemic disease of the American continent caused by Trypanosoma cruzi and divided into six discrete typing units (TcI – TcVI). Nearly 10 million people harbour the infection representing a serious issue in public health. Epidemiological surveillance allowed us to detect a bat-related T. cruzi genotype (henceforth named TcBat) in a 5-year-old female living in a forest area in northwestern Colombia. Molecular tools determined a mixed infection of T. cruzi I and TcBat genotypes. This represents the first report of TcBat infection in humans; the epidemiological consequences of this finding are discussed herein.
Received for publication June 24, 2013 doi: 10.1111/zph.12094
Chagas disease or American trypanosomiasis, caused by Trypanosoma cruzi, is a complex neglected tropical disease that affects over 8 million people in Latin America (Schmunis and Yadon, 2010). This parasite can be transmitted via contaminated faeces of triatomine bugs, oral route, blood transfusion, congenital route, laboratory accidents and transplantation (Rassi et al., 2012). The aetiological agent is characterized by exhibiting a remarkable genetic variability evinced in at least six discrete typing units (DTUs) from T. cruzi I to T. cruzi VI (TcI-TcVI) (Zingales et al., 2012). Recently, intensive sampling has reported a new genotype associated with bats from the Noctilio spp., Myotis spp. and Artibeus spp. species named as TcBat (Marcili et al., 2009; Pinto et al., 2012). The role of TcBat in human infections is unclear; previous studies have demonstrated its ability to infect murine models and invade He-La culture lines, but there have not yet to be any reports of this genotype associated with human cases (Maeda et al., 2011). In 2012, a screening for acute Chagas disease was carried out in Colombia as part of the National Health Institute
surveillance system. Twenty-six patients from the municipality of Sabana del Crespo of the department of Cesar in northeastern Colombia were evaluated. This community lives in the middle of the dry forest, in rudimentary houses where they interact with the fauna of the region (triatomine bugs, mammals, etc.). We conducted serological tests on the 26 patients using crude antigenic preparations of T. cruzi I by ELISA, IFAT and HAI commercial kit (Wiener, Argentina), obtaining a positivity of 19% with a fatal case of death due to acute cardiomyopathy. When entomological and epidemiological surveillance was conducted, we observed the presence of adults of Rhodnius prolixus and Triatoma dimidiata inside the houses and adults of Triatoma maculata and Panstrongylus geniculatus around the dwellings. Previous entomological surveillance in the municipality has reported an infestation index of 50%. Likewise, the presence of mammals (rodents, didelphids and dogs) within the houses and the dwellings was observed. Blood samples were taken to seed haemocultures and PCR directed to kDNA using primers previously reported
© 2013 Blackwell Verlag GmbH Zoonoses and Public Health, 2014, 61, 477–479
477
J. D. Ramırez et al.
TcBat Genotype in Humans
TcV
(a)
(b)
TcIII
1
2
3
4
TcIV TcVI TcII
TcBat
TcI
biological clones typed as T.cruzi Bat
biological clones typed as T.cruzi I
Fig. 1. (a) Maximum composite likelihood (MCL) phylogenetic reconstruction of cytb gene sequences discriminating the biological clones as TcI and TcBat. (b) Pathology images of microtome tissues stained with haematoxylin and eosin. 1. cardiac tissue, 2. skeletal muscle, 3. small intestine, 4. liver.
(Schijman et al., 2011). We observed only one haemoculture turning positive after 37 days of examination. This result was observed in a 5-year-old female patient with positive ELISA, kDNA PCR and negative IFAT and IHA. Her mother presented positive serology, kDNA PCR and died due to acute cardiomyopathy attributed to T. cruzi. Likewise, her father presented positive serology with absence of cardiac failure. This couple had nine additional children as offspring that showed negative serology, PCR and haemoculture. The positive haemoculture from the patient (5-year-old female) was subjected to biological cloning as reported elsewhere (Ramırez et al., 2013b). Ten biological clones were genotyped using the intergenic region of mini-exon gene, 24S and 18S genes including direct sequencing of cytb and SSU rDNA gene fragments (Burgos et al., 2010; Ramırez et al., 2010). This typing scheme and the use of cytb barcoding (reliable maximum composite likelihood phylogenetic reconstruction) demonstrated the presence of four clones as T. cruzi I and six clones typed as TcBat using appropriate reference sequences from the cytb gene retrieved from GenBank (Fig. 1a). Lastly, to observe the biological behaviour of the isolate herein described, we injected a 200-uL aliquot of 106 epimastigotes in ICR line mice observing the absence of histopathological damage in heart, liver, brain and spleen. We analysed the heart from the mouse by kDNA PCR which turned out positive corroborating the incipient presence of the parasite (Fig. 1b). This is the first report to our concern of TcBat genotype in humans. Hence, these findings highlight the need to pursue future research related to the biological origins of this foreseen genotype and also the epidemiological implications of this report. The haemoculture of the mother did not grow and a direct link between the parasite from the mother and the child could not be established. However, this means that the most likely route of TcBat transmission could be vectorial because of the absence of infection in the other nine off-spring children. Another possible route could be the oral route that is the
most common cause of acute Chagas disease in Colombia and also involving death due to acute cardiomyopathy, but further epidemiological evidence is required to fulfil this hypothesis (Ramırez et al., 2013a). TcBat genotype has been reported in Colombia among Carollia perscipillata, Myotis oxyotus and Rhynchonycteris naso that are considered species that feed on insects and/or fruits in eastern Colombia. The occurrence of this genotype within human populations can be explained in the light of the close interactions between humans and these mammals, but again, further research is needed to unravel the epidemiological risk of this link/interaction. Herein, we described the first report of T. cruzi infection attributed to TcBat genotype in humans. The clinical symptomatology of the patient corroborated that TcBat is able to invade mammalian cells, but is not able to cause tissue damage or any pathological alteration (absence of ECG alterations). We encourage the scientific groups from endemic countries and in those where human populations cohabit within forest dwellings to evaluate the plausible presence of TcBat genotype apparently in those isolations from cases of asymptomatic Chagas disease. We certainly believe that human infection caused by this genotype is underestimated due to unbiased sampling and detection tools.
478
© 2013 Blackwell Verlag GmbH Zoonoses and Public Health, 2014, 61, 477–479
Acknowledgement This work is supported by Departamento Administrativo Nacional de Ciencia y Tecnologıa de Colombia ‘Francisco Jose de Caldas – COLCIENCIAS’, Grant Number 3802011, code 5014-537-30398. References Burgos, J. M., M. Diez, C. Vigliano, M. Bisio, M. Risso, T. Duffy, C. Cura, B. Brusses, L. Favaloro, M. S. Leguizamon, R. H. Lucero, R. Laguens, M. J. Levin, R. Favaloro, and A. G. Schijman, 2010: Molecular identification of Trypanosoma cruzi discrete typing units in end-stage chronic Chagas heart disease
J. D. Ramırez et al.
TcBat Genotype in Humans
and reactivation after heart transplantation. Clin. Infect. Dis. 51, 485–495. Maeda, F. Y., R. M. Alves, C. Cortez, F. M. Lima, and N. Yoshida, 2011: Characterization of the infective properties of a new genetic group of Trypanosoma cruzi associated with bats. Acta Trop. 120, 231–237. Marcili, A., L. Lima, M. J. Cavazzana, A. C. V. Junqueira, H. H. Veludo, F. Maia da Silva, M. Campaner, F. Paiva, V. L. B. Nunes, and M. M. G. Teixeira, 2009: A new genotype of Trypanosoma cruzi associated with bats evidenced by phylogenetic analyses using SSU rDNA, cytochrome b and Histone H2B genes and genotyping based on ITS1 rDNA. Parasitology 136, 641–655. Pinto, C. M., E. K. V. Kalko, I. Cottontail, N. Wellinghausen, and V. M. Cottontail, 2012: TcBat a bat-exclusive lineage of Trypanosoma cruzi in the Panama Canal Zone, with comments on its classification and the use of the 18S rRNA gene for lineage identification. Infect. Genet. Evol. 12, 1328–1332. Ramırez, J. D., F. Guhl, L. M. Rend on, F. Rosas, J. A. MarinNeto, and C. A. Morillo, 2010: Chagas cardiomyopathy manifestations and Trypanosoma cruzi genotypes circulating in chronic chagasic patients. PLoS Negl. Trop. Dis. 4, e899. Ramırez, J. D., M. Montilla, Z. M. Cucunuba, A. C. Florez, P. Zambrano, and F. Guhl, 2013a: Molecular epidemiology of human oral Chagas disease outbreaks in Colombia. PLoS Negl. Trop. Dis. 7, e2041.
Ramırez, J. D., C. Herrera, Y. Bogota, M. C. Duque, A. SuarezRivillas, and F. Guhl, 2013b: Validation of a Poisson-distributed limiting dilution assay [LDA] for a rapid and accurate resolution of multiclonal infections in natural Trypanosoma cruzi populations. J. Microbiol. Methods 92, 220–225. Rassi, J. Jr, A. Rassi, and J. Marcondes de Rezende, 2012: American trypanosomiasis, Chagas disease. Infect. Dis. Clin. North Am. 26, 275–291. Schijman, A., M. Bisio, L. Orellana, M. Sued, T. Duffy, A. M. Mejıa-Jaramillo, C. Cura, F. Auter, V. Veron, S. Deborgraeve, G. Hijar, I. Zulantay, R. Lucero, E. Velazquez, T. Tellez, Z. Sanchez, L. Galvao, D. Nolder, M. Monje-Rumi, J. Levi, J. D. Ramırez, O. Triana, F. Guhl, S. Sosa-Estani, and J. Lazdins, 2011: International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients. PLoS Negl. Trop. Dis. 5, 931–939. Schmunis, G. A., and Z. E. Yadon, 2010: Chagas disease: a Latin American health problem becoming a world health problem. Acta Trop. 115, 14–21. Zingales, B., M. A. Miles, D. A. Campbell, M. Tibayrenc, A. M. Macedo, M. M. G. Teixeira, A. G. Schijman, M. S. Llewellyn, E. Lages-Silva, C. R. Machado, S. G. Andrade, and N. R. Sturm, 2012: The revised Trypanosoma cruzi subspecific nomenclature: rationale, epidemiological relevance and research applications. Infect. Genet. Evol. 12, 240–253.
© 2013 Blackwell Verlag GmbH Zoonoses and Public Health, 2014, 61, 477–479
479
