Genotyping of Trypanosoma cruzi Sublineage in Human Samples from a North-East Argentina Area by Hybridization with DNA Probes and Specific Polymerase Chain Reaction (PCR)

Cristina Diez Laboratorio de Tecnología Inmunológica, Facultad de Bioquímica y Cs Biológicas, Universidad Nacional del Litoral, Argentina; Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Chile; Hospital Central de Reconquista. Santa Fe, Argentina

Search for other papers by Cristina Diez in
Current site
Google Scholar
PubMed
Close
,
Virginia Lorenz Laboratorio de Tecnología Inmunológica, Facultad de Bioquímica y Cs Biológicas, Universidad Nacional del Litoral, Argentina; Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Chile; Hospital Central de Reconquista. Santa Fe, Argentina

Search for other papers by Virginia Lorenz in
Current site
Google Scholar
PubMed
Close
,
Silvia Ortiz Laboratorio de Tecnología Inmunológica, Facultad de Bioquímica y Cs Biológicas, Universidad Nacional del Litoral, Argentina; Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Chile; Hospital Central de Reconquista. Santa Fe, Argentina

Search for other papers by Silvia Ortiz in
Current site
Google Scholar
PubMed
Close
,
Verónica Gonzalez Laboratorio de Tecnología Inmunológica, Facultad de Bioquímica y Cs Biológicas, Universidad Nacional del Litoral, Argentina; Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Chile; Hospital Central de Reconquista. Santa Fe, Argentina

Search for other papers by Verónica Gonzalez in
Current site
Google Scholar
PubMed
Close
,
Andrea Racca Laboratorio de Tecnología Inmunológica, Facultad de Bioquímica y Cs Biológicas, Universidad Nacional del Litoral, Argentina; Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Chile; Hospital Central de Reconquista. Santa Fe, Argentina

Search for other papers by Andrea Racca in
Current site
Google Scholar
PubMed
Close
,
Iván Bontempi Laboratorio de Tecnología Inmunológica, Facultad de Bioquímica y Cs Biológicas, Universidad Nacional del Litoral, Argentina; Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Chile; Hospital Central de Reconquista. Santa Fe, Argentina

Search for other papers by Iván Bontempi in
Current site
Google Scholar
PubMed
Close
,
Silvia Manattini Laboratorio de Tecnología Inmunológica, Facultad de Bioquímica y Cs Biológicas, Universidad Nacional del Litoral, Argentina; Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Chile; Hospital Central de Reconquista. Santa Fe, Argentina

Search for other papers by Silvia Manattini in
Current site
Google Scholar
PubMed
Close
,
Aldo Solari Laboratorio de Tecnología Inmunológica, Facultad de Bioquímica y Cs Biológicas, Universidad Nacional del Litoral, Argentina; Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Chile; Hospital Central de Reconquista. Santa Fe, Argentina

Search for other papers by Aldo Solari in
Current site
Google Scholar
PubMed
Close
, and
Iván Marcipar Laboratorio de Tecnología Inmunológica, Facultad de Bioquímica y Cs Biológicas, Universidad Nacional del Litoral, Argentina; Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Chile; Hospital Central de Reconquista. Santa Fe, Argentina

Search for other papers by Iván Marcipar in
Current site
Google Scholar
PubMed
Close
Restricted access

We have evaluated blood samples of chronic and congenital Trypanosoma cruzi-infected patients from the city of Reconquista located in the northeast of Argentina where no information was previously obtained about the genotype of infecting parasites. Fourteen samples of congenital and 19 chronical patients were analyzed by hybridization with DNA probes of minicircle hypervariable regions (mHVR). In congenital patients, 50% had single infections with TcIId, 7% single infections with TcIIe, 29% mixed infections with TcIId/e, and 7% had mixed infections with TcIId/b and 7% TcIId/b, respectively. In Chronical patients, 52% had single infections with TcIId, 11% single infections with TcIIe, 26% had mixed infections with TcIId/e, and 11% had non-identified genotypes. With these samples, we evaluated the minicircle lineage-specific polymerase chain reaction assay (MLS-PCR), which involves a nested PCR to HVR minicircle sequences and we found a correlation with hybridization probes of 96.4% for TcIId and 54.8% for TcIIe.

Author Notes

*Address correspondence to Iván Marcipar, Laboratorio de Tecnología Inmunológica, Facultad de Bioquímica y Cs Biológicas Universidad Nacional del Litoral, Santa Fe 3000, Argentina. E-mail: imarcipr@fbcb.unl.edu.ar

Financial support: This work received financial support from CAI+D 2009 PI 15-86, Universidad Nacional del Litoral and FONDECYT-Chile 1085154.

Disclosure: ISM and VG are researchers from the CONICET (Argentina).

Authors' addresses: Cristina Diez, Virginia Lorenz, Verónica Gonzalez, Andrea Racca, Iván Bontempi, and Iván Marcipar, Laboratorio de Tecnología Inmunológica, Facultad de Bioquímica y Cs Biológicas, Universidad Nacional del Litoral, Santa Fe, Argentina, E-mail: imarcipr@fbcb.unl.edu.ar. Silvia Ortiz and Aldo Solari, Program of Cellular and Molecular Biology, Biomedical Sciences Institute, Faculty of Medicine, University of Chile, Santiago, Chile, E-mail: asolari@med.uchile.cl. Silvia Manattini, Hospital Central de Reconquista, Santa Fe, Argentina, E-mail: equiroz@arnet.com.ar.

  • 1.

    World Health Organization, 2002. Control of Chagas disease. Report of a WHO expert committee. World Health Organ Tech Rep Ser 905: 1109.

  • 2.

    Moncayo A, 2003. Chagas disease: current epidemiological trends after the interruption of vectorial and transfusional transmission in the southern Cone countries. Mem Inst Oswaldo Cruz 98: 577591.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 3.

    Andrade SG, Magalhaes JB, Pontes AL, 1985. Evaluation of chemotherapy with benznidazole and nifurtimox in mice infected with Trypanosoma cruzi strains of different types. Bull World Health Organ 63: 721726.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 4.

    Filardi LS, Brener Z, 1987. Susceptibility and natural resistance to Trypanosoma cruzi strains to drugs used clinically in Chagas disease. Trans R Soc Trop Med Hyg 81: 755759.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 5.

    Risso MG, Garbarino GB, Mocetti E, Campetella O, Gonzalez Cappa SM, Buscaglia CA, Leguizamon MS, 2004. Differential expression of a virulence factor, the trans-sialidase, by the main Trypanosoma cruzi phylogenetic lineages. J Infect Dis 189: 22502259.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 6.

    Vago AR, Macedo AM, Adad SJ, Reis DD, Corrêa-Oliveira R, 1996. PCR detection of Trypanosoma cruzi DNA in oesophageal tissues of patients with chronic digestive Chagas' disease. Lancet 348: 891892.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 7.

    Vago AR, Macedo AM, Oliveira RP, Andrade LO, Chiari E, Galvão LM, Reis D, Pereira ME, Simpson AJ, Tostes S, Pena SD, 1996. Kinetoplast DNA signatures of Trypanosoma cruzi strains obtained directly from infected tissues. Am J Pathol 149: 21532159.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 8.

    Vago A, Andrade L, Leite A, Reis D, Macedo A, Adad S, Tostes S, Moreira M, Filo G, Pena S, 2000. Genetic characterization of Trypanosoma cruzi directly from tissues of patients with chronic Chagas disease: differential distribution of genetic types into diverse organs. Am J Pathol 156: 18051809.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 9.

    Brenière SF, Bosseno MF, Telleria J, Bastrenta B, Yacsik N, Noireau F, Alcazar JL, Barnabé C, Wincker P, Tibayrenc M, 1998. Different behavior of two Trypanosoma cruzi major clones: transmission and circulation in young Bolivian patients. Exp Parasitol 89: 285295.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 10.

    de Ornelas Toledo MJ, Bahia MT, Carneiro CM, Martins-Filho OA, Tibayrenc M, Barnabe C, Tafuri WL, de Lana M, 2003. Chemotherapy with benznidazole and itraconazole for mice infected with different Trypanosoma cruzi clonal genotypes. Antimicrob Agents Chemother 47: 223230.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 11.

    Coronado X, Zulantai I, Rozas M, Apt W, Sanchez G, Rodriguez J, Ortiz S, Solari A, 2006. Dissimilar distribution of Trypanosoma cruzi clones in humans after chemotherapy with allopurinol anad itraconazole. J Antimicrob Chemother 58: 216219.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 12.

    Martins HR, Silva RM, Valadares HM, Toledo MJ, Veloso VM, Vitelli-Avelar DM, Carneiro CM, Machado-Coelho GL, Bahia MT, Martins-Filho OA, Macedo AM, Lana M, 2007. Impact of dual infections on chemotherapeutic efficacy in BALB/c mice infected with major genotypes of Trypanosoma cruzi. Antimicrob Agents Chemother 51: 32823289.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 13.

    Martins HR, Figueiredo LM, Valamiel-Silva JC, Carneiro CM, Machado-Coelho GL, Vitelli-Avelar DM, Bahia MT, Martins-Filho OA, Macedo AM, Lana M, 2008. Persistence of PCR-positive tissue in benznidazole-treated mice with negative blood parasitological and serological tests in dual infections with Trypanosoma cruzi stocks from different genotypes. J Antimicrob Chemother 61: 13191327.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 14.

    Brisse S, Dujardin JC, Tibayrenc M, 2000. Identification of six Trypanosoma cruzi lineages by sequence-characterised amplified region markers. Mol Biochem Parasitol 111: 95105.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 15.

    Brisse S, Verhoef J, Tibayrenc M, 2001. Characterisation of large and small subunit rRNA and mini-exon genes further supports the distinction of six Trypanosoma cruzi lineages. Int J Parasitol 31: 12181226.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 16.

    Devera R, Fernandes O, Coura JR, 2003. Should Trypanosoma cruzi be called “cruzi” complex? A review of the parasite diversity and the potential of selecting population after in vitro culturing and mice infection. Mem Inst Oswaldo Cruz 98: 112.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 17.

    Britto C, Cardoso MA, Ravel C, Santoro A, Pereira JB, Coura JR, Morel CM, Wincker P, 1995. Trypanosoma cruzi: parasite detection and strain discrimination in chronic chagasic patients from northeastern Brazil using PCR amplification of kinetoplast DNA and nonradioactive hybridization. Exp Parasitol 81: 462471.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 18.

    Britto CM, Lima MM, Sarquis O, Pires MQ, Coutinho CF, Duarte R, Pacheco RS, 2008. Genetic polymorphism in Trypanosoma cruzi I isolated from Brazilian northeast triatomines revealed by low-stringency single specific primer-polymerase chain reaction. Parasitol Res 103: 11111117.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 19.

    Morel C, Chiari E, Camargo EP, Mattei DM, Romanha AJ, Simpson L, 1980. Strains and clones of Trypanosoma cruzi can be characterized by pattern of restriction endonuclease products of kinetoplast DNA minicircles. Proc Natl Acad Sci USA 77: 68106814.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 20.

    Sturm N, Degrave W, Morel C, Simpson L, 1989. Sensitive detection and schizodeme classification of Trypanosoma cruzi cells by amplification of kinetoplast minicircle DNA sequences: use in diagnosis of Chagas' disease. Mol Biochem Parasitol 33: 205214.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 21.

    Marcet PL, Duffy T, Cardinal MV, Burgos JM, Lauricella MA, Levin MJ, Kitron U, Gürtler RE, Schijman AG, 2006. PCR-based screening and lineage identification of Trypanosoma cruzi directly from faecal samples of triatomine bugs from northwestern Argentina. Parasitology 132: 5765.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 22.

    Triana O, Ortiz S, Dujardin JC, Solari A, 2006. Trypanosoma cruzi: variability of stocks from Colombia determined by molecular karyotype and minicircle Southern blot analysis. Vet Parasitol Exp Parasitol 113: 6266.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 23.

    Telleria J, Lafay B, Virreira M, Barnabe C, Tibayrenc M, Svoboda M, 2006. Tr(i) panosoma cruzi: sequence analysis of the variable region of kinetoplast minicircles. Exp Parasitol 114: 279288.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 24.

    Velázquez M, Diez CN, Mora C, Diosque P, Marcipar IS, 2008. Trypanosoma cruzi: an analysis of the minicircle hypervariable regions diversity and its influence on strain typing. Exp Parasitol 120: 235241.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 25.

    Avila HA, Pereira JB, Thiemann O, De Paiva E, DeGrave W, Morel CM, Simpson L, 1993. Detection of Trypanosoma cruzi in blood specimens of chronic chagasic patients by polymerase chain reaction amplification of kinetoplast minicircle DNA: comparison with serology and xenodiagnosis. J Clin Microbiol 31: 24212426.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 26.

    Wincker P, Brito C, Borges Pereira J, Cardoso MA, Oelemann W, Morel CM, 1994. Use of a simplified polymerase chain reaction procedure to detect Trypanosoma cruzi in blood samples from chronic chagasic patients in a rural endemic area. Am J Trop Med Hyg 51: 133139.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 27.

    Rozas M, Botto-Mahan C, Coronado X, Ortiz S, Cattan PE, Solari A, 2007. Coexistence of Trypanosoma cruzi genotypes in wild and periodomestic mammals in Chile. Am J Trop Med Hyg 77: 647653.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 28.

    Diez M, Favaloro L, Bertolotti A, Burgos JM, Vigliano C, Lastra MP, Levin MJ, Arnedo A, Nagel C, Schijman AG, Favaloro RR, 2007. Usefuness of PCR strategies for early diagnosis of Chagas disease reactivation and treatment follow-up in heart transplantation. Am J Transplant 7: 16331640.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 29.

    Diez CN, Manattini S, Zanuttini JC, Bottasso O, Marcipar I, 2008. The value of molecular studies for the diagnosis of congenital Chagas disease in northeastern Argentina. Am J Trop Med Hyg 78: 624627.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 30.

    Solari A, Campillay R, Ortíz S, Wallace A, 2001. Identification of Trypanosoma cruzi genotypes circulating in Chilean chagasic patients. Exp Parasitol 97: 226233.

  • 31.

    Salazar A, Schijman AG, Triana-Chávez O, 2006. High variability of Colombian Trypanosoma cruzi lineage I stocks as revealed by low-stringency single primer-PCR minicircle signatures. Acta Trop 100: 110118.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 32.

    Lisboa CV, Pinho AP, Herrera HM, Gerhardt M, Cupolillo E, Jansen AM, 2008. Trypanosoma cruzi (Kinetoplastida, Trypanosomatidae) genotypes in neotropical bats in Brazil. Vet Parasitol 156: 314318.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 33.

    Burgos JM, Altcheh J, Bisio M, Duffy T, Valadares HM, Seidenstein ME, Piccinali R, Freitas JM, Levin MJ, Macchi L, Macedo AM, Freilij H, Schijman AG, 2007. Direct molecular profiling of minicircle signatures and lineages of Trypanosoma cruzi bloodstream populations causing congenital Chagas disease. Int J Parasitol 37: 13191327.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 34.

    Diosque P, Barnabé C, Padilla AM, Marco JD, Cardozo RM, Cimino RO, Nasser JR, Tibayrenc M, Basombrío MA, 2003. Multilocus enzyme electrophoresis analysis of Trypanosoma cruzi isolates from a geographically restricted endemic area for Chagas disease in Argentina. Int J Parasitol 33: 9971003.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 35.

    Cardinal MV, Lauricella MA, Ceballos LA, Lanati L, Marcet PL, Levin MJ, Kitron U, Gürtler RE, Schijman AG, 2008. Molecular epidemiology of domestic and sylvatic Trypanosoma cruzi infection in rural northwestern Argentina. Int J Parasitol 38: 15331543.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 36.

    Corrales RM, Mora MC, Negrette OS, Diosque P, Lacunza D, Virreira M, Brenière SF, Basombrio MA, 2009. Congenital Chagas disease involves Trypanosoma cruzi sub-lineage IId in the northwestern province of Salta, Argentina. Infect Genet Evol 9: 278282.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 37.

    Veas F, Breniere SF, Cuny G, Brengues C, Solari A, Tibayrenc M, 1991. General procedure to construct highly specific kDNA probes for clones of Trypanosoma cruzi for sensitive detection by polymerase chain reaction. Cell Mol Biol 37: 7384.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 38.

    Torres JP, Ortiz S, Muñoz S, Solari A, 2004. Trypanosoma cruzi isolates from Chile are heterogeneous and composed of mixed populations when characterized by schizodeme and Southern analyses. Parasitology 128: 161168.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 39.

    Arimone Y, Miremont-Salamé G, Haramburu F, Molimard M, Moore N, Fourrier-Réglat A, Bégaud B, 2007. Inter-expert agreement of seven criteria in causality assessment of adverse drug reactions. Br J Clin Pharmacol 64: 482488.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 40.

    Virreira M, Alonso-Vega C, Solano M, Jijena J, Brutus L, Bustamante Z, Truyens C, Schneider D, Torrico F, Carlier Y, Svoboda M, 2006. Congenital Chagas disease in Bolivia is not associated with DNA polymorphism of Trypanosoma cruzi. Am J Trop Med Hyg 75: 871879.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 41.

    Burgos JM, Begher S, Silva HM, Bisio M, Duffy T, Levin MJ, Macedo AM, Schijman AG, 2008. Molecular identification of Trypanosoma cruzi I tropism for central nervous system in Chagas reactivation due to AIDS. Am J Trop Med Hyg 78: 294297.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 42.

    Di Noia J, Buscaglia C, De Marchi C, Almeida I, Frasch A, 2002. A Trypanosoma cruzi small surface molecule provides the first immunological evidence that Chagas' disease is due to a single parasite lineage. J Exp Med 195: 401413.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 43.

    Montamat EE, Arauzo S, Cazzulo JJ, Subias E, 1987. Characterization by electrophoretic zymograms of 19 Trypanosoma cruzi clones derived from two chronic chagasic patients. Comp Biochem Physiol 87: 417422.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 44.

    Montamat EE, De Luca d'Oro GM, Perret B, Rivas C, 1992. Characterization of Trypanosoma cruzi from Argentina by electrophoretic zymograms. Acta Trop 50: 125133.

  • 45.

    Montamat EE, De Luca d'Oro GM, Gallerano RH, Sosa R, Blanco A, 1996. Characterization of Trypanosoma cruzi populations by zymodemes: correlation with clinical picture. Am J Trop Med Hyg 55: 625628.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 46.

    De Luca d'Oro GM, Gardenal CN, Perret B, Crisci JV, Montamat EE, 1993. Genetic structure of Trypanosoma cruzi populations from Argentina estimated from enzyme polymorphism. Parasitology 107: 405410.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 47.

    Fabbro De Suasnábar D, Arias E, Streiger M, Piacenza M, Ingaramo M, Del Barco M, Amicone N, 2000. Evolutive behavior towards cardiomyopathy of treated (nifurtimox or benznidazole) and untreated chronic chagasic patients. Rev Inst Med Trop Sao Paulo 42: 99109.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 48.

    Dubner S, Schapachnik E, Riera AR, Valero E, 2008. Chagas disease: state-of-the-art of diagnosis and management. Cardiol J 15: 493504.

  • 49.

    Abello M, González-Zuelgaray J, López C, Labadet C, 2008. Initiation modes of spontaneous monomorphic ventricular tachycardia in patients with Chagas heart disease. Rev Esp Cardiol 61: 487493.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 50.

    Storino R, Auger S, Caravello O, Urrutia MI, Sanmartino M, Jörg M, 2002. Chagasic cardiopathy in endemic area versus sporadically infected patients. Rev Saude Publica 36: 755758.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 51.

    Venegas J, Coñoepan W, Pichuantes S, Miranda S, Apt W, Arribada A, Zulantay I, Coronado X, Rodriguez J, Reyes E, Solari A, Sanchez G, 2009. Differential distribution of Trypanosoma cruzi clones in human chronic chagasic cardiopathic and non-cardiopathic individuals. Acta Trop 109: 187193.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 52.

    Carranza JC, Valadares HM, D'Avila DA, Baptista RP, Moreno M, Galvão LM, Chiari E, Sturm NR, Gontijo ED, Macedo AM, Zingales B, 2009. Trypanosoma cruzi maxicircle heterogeneity in Chagas disease patients from Brazil. Int J Parasitol 39: 963973.

    • PubMed
    • Search Google Scholar
    • Export Citation
Past two years Past Year Past 30 Days
Abstract Views 459 356 127
Full Text Views 274 8 4
PDF Downloads 52 9 4
 

 

 

 
 
Affiliate Membership Banner
 
 
Research for Health Information Banner
 
 
CLOCKSS
 
 
 
Society Publishers Coalition Banner
Save