• 1.

    Steverding D, 2014. The history of Chagas disease. Parasit Vectors 7: 18.

  • 2.

    WHO, 2012. Research priorities for Chagas disease, human African trypanosomiasis and leishmaniasis. World Health Organ Tech Rep Ser 975: 1100.

    • Search Google Scholar
    • Export Citation
  • 3.

    Bern C, Montgomery SP, 2009. An estimate of the burden of Chagas disease in the United States. Clin Infect Dis 49: e52e54.

  • 4.

    Rassi A, Marin-Neto JA, 2010. Chagas disease. Lancet 375: 13881402.

  • 5.

    Jackson Y, Gétaz L, Wolff H, Holst M, Mauris A, Tardin A, Sztajzel J, Besse V, Loutan L, Gaspoz JM, Jannin J, Albajar P, Luquetti A, Chappuis F, 2010. Prevalence, clinical staging and risk for blood-borne transmission of Chagas disease among Latin American migrants in Geneva, Switzerland. PLoS Negl Trop Dis 4: e592.

    • Search Google Scholar
    • Export Citation
  • 6.

    Bern C, Montgomery SP, Katz L, Caglioti S, Stramer SL, 2008. Chagas disease and the US blood supply. Curr Opin Infect Dis 21: 476482.

  • 7.

    Cancino-Faure B, Fisa R, Riera C, Bula I, Girona-Llobera E, Jimenez-Marco T, 2015. Evidence of meaningful levels of Trypanosoma cruzi in platelet concentrates from seropositive blood donors. Transfusion 55: 12491255.

    • Search Google Scholar
    • Export Citation
  • 8.

    Roca Saumell C, Soriano-Arandes A, Solsona Díaz L, Gascón Brustenga J, 2015. Documento de consenso sobre el abordaje de la enfermedad de Chagas en atención primaria de salud de áreas no endémicas. Aten Primaria 47: 308317.

    • Search Google Scholar
    • Export Citation
  • 9.

    Nóbrega AA, Garcia MH, Tatto E, Obara MT, Costa E, Sobel J, Araujo WN, 2009. Oral transmission of Chagas disease by consumption of Açaí palm fruit, Brazil. Emerg Infect Dis 15: 653655.

    • Search Google Scholar
    • Export Citation
  • 10.

    Coura JR, Junqueira ACV, 2015. Ecological diversity of Trypanosoma cruzi transmission in the Amazon basin. The main scenaries in the Brazilian Amazon. Acta Trop 151: 5157.

    • Search Google Scholar
    • Export Citation
  • 11.

    Toso MA, 2011. Oral transmission of Chagas disease. Rev Med Chil 139: 258266.

  • 12.

    Barreto-de-Albuquerque J, Silva-dos-Santos D, Pérez AR, Berbert LR, Santana-van-Vliet ED, Farias-de-Oliveira DA, Moreira OC, Roggero E, Carvalho-Pinto CE, Jurberg J, Cotta-de-Almeida V, Bottasso O, Savino W, Meis JD, 2015. Trypanosoma cruzi infection through the oral route promotes a severe infection in mice: new disease form from an old infection? PLoS Negl Trop Dis 9: e0003849.

    • Search Google Scholar
    • Export Citation
  • 13.

    Britto C, Cardoso MA, Monteiro Vanni CM, Hasslocher-Moreno A, Xavier SS, Oelemann W, Santoro A, Pirmez C, Morel CM, Wincker P, 1995. Polymerase chain reaction detection of Trypanosoma cruzi in human blood samples as a tool for diagnosis and treatment evaluation. Parasitology 110: 241247.

    • Search Google Scholar
    • Export Citation
  • 14.

    Virreira M, Torrico F, Truyens C, Alonso-Vega C, Solano M, Carlier Y, Svoboda M, 2003. Comparison of polymerase chain reaction methods for reliable and easy detection of congenital Trypanosoma cruzi infection. Am J Trop Med Hyg 68: 574582.

    • Search Google Scholar
    • Export Citation
  • 15.

    Schijman AG, Altcheh J, Burgos JM, Biancardi M, Bisio M, Levin MJ, Freilij H, 2003. Aetiological treatment of congenital Chagas' disease diagnosed and monitored by the polymerase chain reaction. J Antimicrob Chemother 52: 441449.

    • Search Google Scholar
    • Export Citation
  • 16.

    Sánchez G, Coronado X, Zulantay I, Apt W, Gajardo M, Solari S, Venegas J, 2005. Monitoring the efficacy of specific treatment in chronic Chagas disease by polymerase chain reaction and flow cytometry analysis. Parasite 12: 353357.

    • Search Google Scholar
    • Export Citation
  • 17.

    Piron M, Fisa R, Casamitjana N, López-Chejade P, Puig L, Vergés M, Gascón J, Gómez i Prat J, Portús M, Sauleda S, 2007. Development of a real-time PCR assay for Trypanosoma cruzi detection in blood samples. Acta Trop 103: 195200.

    • Search Google Scholar
    • Export Citation
  • 18.

    Cominetti MC, Csordas BG, Cunha RC, Andreotti R, 2014. Geographical distribution of Trypanosoma cruzi in triatomine vectors in the State of Mato Grosso do Sul, Brazil. Rev Soc Bras Med Trop 47: 747755.

    • Search Google Scholar
    • Export Citation
  • 19.

    Herrera CP, Licon MH, Nation CS, Jameson SB, Wesson DM, 2015. Genotype diversity of Trypanosoma cruzi in small rodents and Triatoma sanguisuga from a rural area in New Orleans, Louisiana. Parasit Vectors 8: 19.

    • Search Google Scholar
    • Export Citation
  • 20.

    Chiari E, Dias JC, Lana M, Chiari CA, 1989. Hemocultures for the parasitological diagnosis of human chronic Chagas' disease. Rev Soc Bras Med Trop 22: 1923.

    • Search Google Scholar
    • Export Citation
  • 21.

    Luz ZMP, 1999. Changes in the hemoculture methodology improve the test positivity. Mem Inst Oswaldo Cruz 94 (Suppl 1): 295298.

  • 22.

    Saavedra M, Zulantay I, Apt W, Martínez G, Rojas A, Rodríguez J, 2013. Chronic Chagas disease: PCR-xenodiagnosis without previous microscopic observation is a useful tool to detect viable Trypanosoma cruzi. Biol Res 46: 295298.

    • Search Google Scholar
    • Export Citation
  • 23.

    Nogva HK, Drømtorp SM, Nissen H, Rudi K, 2003. Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5′-nuclease PCR. Biotechniques 34: 804808, 810, 812–813.

    • Search Google Scholar
    • Export Citation
  • 24.

    Nocker A, Cheung C-Y, Camper AK, 2006. Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells. J Microbiol Methods 67: 310320.

    • Search Google Scholar
    • Export Citation
  • 25.

    Fittipaldi M, Nocker A, Codony F, 2012. Progress in understanding preferential detection of live cells using viability dyes in combination with DNA amplification. J Microbiol Methods 91: 276289.

    • Search Google Scholar
    • Export Citation
  • 26.

    Agustí G, Codony F, Fittipaldi M, Adrados B, Morató J, 2010. Viability determination of Helicobacter pylori using propidium monoazide quantitative PCR. Helicobacter 15: 4736.

    • Search Google Scholar
    • Export Citation
  • 27.

    Josefsen MH, Löfström C, Hansen TB, Christensen LS, Olsen JE, Hoorfar J, 2010. Rapid quantification of viable Campylobacter bacteria on chicken carcasses, using real-time PCR and propidium monoazide treatment, as a tool for quantitative risk assessment. Appl Environ Microbiol 76: 50975104.

    • Search Google Scholar
    • Export Citation
  • 28.

    Andorrà I, Esteve-Zarzoso B, Guillamón JM, Mas A, 2010. Determination of viable wine yeast using DNA binding dyes and quantitative PCR. Int J Food Microbiol 144: 257262.

    • Search Google Scholar
    • Export Citation
  • 29.

    Fittipaldi M, Pino Rodriguez NJ, Adrados B, Agustí G, Peñuela G, Morató J, Codony F, 2011. Discrimination of viable Acanthamoeba castellani trophozoites and cysts by propidium monoazide real-time polymerase chain reaction. J Eukaryot Microbiol 58: 359364.

    • Search Google Scholar
    • Export Citation
  • 30.

    Li B, Chen J-Q, 2013. Development of a sensitive and specific qPCR assay in conjunction with propidium monoazide for enhanced detection of live Salmonella spp. in food. BMC Microbiol 13: 273.

    • Search Google Scholar
    • Export Citation
  • 31.

    Alonso JL, Amorós I, Guy RA, 2014. Quantification of viable Giardia cysts and Cryptosporidium oocysts in wastewater using propidium monoazide quantitative real-time PCR. Parasitol Res 113: 26712678.

    • Search Google Scholar
    • Export Citation
  • 32.

    Rogers GB, Stressmann FA, Koller G, Daniels T, Carroll MP, Bruce KD, 2008. Assessing the diagnostic importance of nonviable bacterial cells in respiratory infections. Diagn Microbiol Infect Dis 62: 133141.

    • Search Google Scholar
    • Export Citation
  • 33.

    Miotto P, Bigoni S, Migliori GB, Matteelli A, Cirillo DM, 2012. Early tuberculosis treatment monitoring by Xpert(R) MTB/RIF. Eur Respir J 39: 12691271.

    • Search Google Scholar
    • Export Citation
  • 34.

    De Oliveira-Silva JCV, Machado-de-Assis GF, Oliveira MT, Noguieira Paiva NC, Silva Araujo MS, Martins Carneiro C, Martins OA, Rodrigues Martins H, de Lana M, 2015. Experimental benznidazole treatment of Trypanosoma cruzi II strains isolated from children of the Jequitinhonha Valley, Minas Gerais, Brazil, with Chagas disease. Mem Inst Oswaldo Cruz 110: 8694.

    • Search Google Scholar
    • Export Citation
  • 35.

    Caldas S, Caldas IS, Cecílio AB, Diniz LF, Talvani A, Ribeiro I, Bahia MT, 2014. Therapeutic responses to different anti-Trypanosoma cruzi drugs in experimental infection by benznidazole-resistant parasite stock. Parasitology 141: 110.

    • Search Google Scholar
    • Export Citation
  • 36.

    Brescia CC, Griffin SM, Ware MW, Varughese EA, Egorov AI, Villegas EN, 2009. Cryptosporidium propidium monoazide-PCR, a molecular biology-based technique for genotyping of viable Cryptosporidium oocysts. Appl Environ Microbiol 75: 68566863.

    • Search Google Scholar
    • Export Citation
  • 37.

    Chang CW, Lu LW, Kuo CL, Hung NT, 2013. Density of environmental Acanthamoeba and their responses to superheating disinfection. Parasitol Res 112: 36873696.

    • Search Google Scholar
    • Export Citation
  • 38.

    De Assunção TM, Batista EL, Deves C, Villela AD, Pagnussatti VE, De Oliveira Dias AC, Kritski A, Rodrigues-Junior V, Basso LA, Santos DS, 2014. Real time PCR quantification of viable Mycobacterium tuberculosis from sputum samples treated with propidium monoazide. Tuberculosis (Edinb) 94: 421427.

    • Search Google Scholar
    • Export Citation
  • 39.

    Kralik P, Nocker A, Pavlik I, 2010. Mycobacterium avium subsp. paratuberculosis viability determination using F57 quantitative PCR in combination with propidium monoazide treatment. Int J Food Microbiol 141: S80S86.

    • Search Google Scholar
    • Export Citation
  • 40.

    Løvdal T, Hovda MB, Björkblom B, Møller SG, 2011. Propidium monoazide combined with real-time quantitative PCR underestimates heat-killed Listeria innocua. J Microbiol Methods 85: 164169.

    • Search Google Scholar
    • Export Citation
  • 41.

    Barbau-Piednoir E, Mahillon J, Pillyser J, Coucke W, Roosens NH, Botteldoorn N, 2014. Evaluation of viability-qPCR detection system on viable and dead Salmonella serovar Enteritidis. J Microbiol Methods 103: 131137.

    • Search Google Scholar
    • Export Citation
  • 42.

    Cawthorn DM, Witthuhn RC, 2008. Selective PCR detection of viable Enterobacter sakazakii cells utilizing propidium monoazide or ethidium bromide monoazide. J Appl Microbiol 105: 11781185.

    • Search Google Scholar
    • Export Citation
  • 43.

    Ditommaso S, Giacomuzzi M, Ricciardi E, Zotti CM, 2015. Viability-qPCR for detecting Legionella: comparison of two assays based on different amplicon lengths. Mol Cell Probes 29: 17.

    • Search Google Scholar
    • Export Citation
  • 44.

    Soejima T, Schlitt-Dittrich F, Yoshida S, 2011. Polymerase chain reaction amplification length-dependent ethidium monoazide suppression power for heat-killed cells of Enterobacteriaceae. Anal Biochem 418: 3743.

    • Search Google Scholar
    • Export Citation
  • 45.

    Contreras PJ, Urrutia H, Sossa K, Nocker A, 2011. Effect of PCR amplicon length on suppressing signals from membrane-compromised cells by propidium monoazide treatment. J Microbiol Methods 87: 8995.

    • Search Google Scholar
    • Export Citation
  • 46.

    Opel KL, Chung D, McCord BR, 2010. A study of PCR inhibition mechanisms using real time PCR. J Forensic Sci 55: 2533.

  • 47.

    Chang B, Taguri T, Sugiyama K, Amemura-Maekawa J, Kura F, Watanabe H, 2010. Comparison of ethidium monoazide and propidium monoazide for the selective detection of viable Legionella cells. Jpn J Infect Dis 63: 119123.

    • Search Google Scholar
    • Export Citation
  • 48.

    Dannelley JM, Boyce L, Gaubatz JW, 1986. Efficiency of photoaffinity labeling DNA homopolymers and copolymers with ethidium monoazide. Photochem Photobiol 43: 711.

    • Search Google Scholar
    • Export Citation
  • 49.

    Pan Y, Breidt F, 2007. Enumeration of viable Listeria monocytogenes cells by real-time PCR with propidium monoazide and ethidium monoazide in the presence of dead cells. Appl Environ Microbiol 73: 80288031.

    • Search Google Scholar
    • Export Citation
 
 
 

 

 
 
 

 

 

 

 

 

 

Detection and Quantification of Viable and Nonviable Trypanosoma cruzi Parasites by a Propidium Monoazide Real-Time Polymerase Chain Reaction Assay

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  • Laboratori de Parasitologia, Departament de Microbiologia i Parasitologia Sanitàries, Facultat de Farmàcia, Universitat de Barcelona, Barcelona, Spain; Fundació Banc de Sang i Teixits de les Illes Balears, Mallorca, Balearic Islands, Spain; IUNICS Institut Universitari d'Investigació en Ciències de la Salut, Universitat de les Illes Balears, Spain

Molecular techniques based on real-time polymerase chain reaction (qPCR) allow the detection and quantification of DNA but are unable to distinguish between signals from dead or live cells. Because of the lack of simple techniques to differentiate between viable and nonviable cells, the aim of this study was to optimize and evaluate a straightforward test based on propidium monoazide (PMA) dye action combined with a qPCR assay (PMA-qPCR) for the selective quantification of viable/nonviable epimastigotes of Trypanosoma cruzi. PMA has the ability to penetrate the plasma membrane of dead cells and covalently cross-link to the DNA during exposure to bright visible light, thereby inhibiting PCR amplification. Different concentrations of PMA (50–200 μM) and epimastigotes of the Maracay strain of T. cruzi (1 × 105–10 parasites/mL) were assayed; viable and nonviable parasites were tested and quantified by qPCR with a TaqMan probe specific for T. cruzi. In the PMA-qPCR assay optimized at 100 μM PMA, a significant qPCR signal reduction was observed in the nonviable versus viable epimastigotes treated with PMA, with a mean signal reduction of 2.5 logarithm units and a percentage of signal reduction > 98%, in all concentrations of parasites assayed. This signal reduction was also observed when PMA-qPCR was applied to a mixture of live/dead parasites, which allowed the detection of live cells, except when the concentration of live parasites was low (10 parasites/mL). The PMA-qPCR developed allows differentiation between viable and nonviable epimastigotes of T. cruzi and could thus be a potential method of parasite viability assessment and quantification.

Author Notes

* Address correspondence to Roser Fisa, Laboratori de Parasitologia, Departament de Microbiologia i Parasitologia Sanitàries, Facultat de Farmàcia, Universitat de Barcelona, Avinguda Joan XXIII s/n. E-08028, Barcelona, Spain. E-mail: rfisa@ub.edu

Financial support: This work is part of a research study supported by the National R&D+i Plan 2008–2011 and ISC III -Subdirección General de Evaluación y Fomento de la Investigación (PI 10/00533), was in part funded by CONICYT Becas Chile (72130155) and is part of the project 2014 SGR 1241 de la Generalitat de Catalunya.

Authors' addresses: Beatriz Cancino-Faure, Roser Fisa, M. Magdalena Alcover, and Cristina Riera, Laboratori de Parasitologia, Departament de Microbiologia i Parasitologia Sanitàries, Facultat de Farmacia, Universitat de Barcelona, Barcelona, Spain, E-mails: mbcancino@gmail.com, rfisa@ub.edu, mmagdalenaalcoveramengual@ub.edu, and mcriera@ub.edu. Teresa Jimenez-Marco, Fundació Banc de Sang i Teixits de les Illes Balears, Palma de Mallorca, Balearic Islands, Spain, and IUNICS Institut Universitari d'Investigació en Ciències de la Salut, Universitat de les Illes Balears, Cra. de Valldemossa, Balearic Islands, Spain, E-mail: tjimenez@fbstib.org.

Reprint requests: Roser Fisa, Laboratori de Parasitologia, Departament de Microbiologia i Parasitologia Sanitàries, Facultat de Farmàcia, Universitat de Barcelona, Avinguda Joan XXIII s/n. E-08028, Barcelona, Spain, E-mail: rfisa@ub.edu; Tel: +34 934024502; Fax: +34 934024504.

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