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Assessing Parasite Load in Chagas Disease Patients by Quantitative Multiplex Real-Time PCR

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Book cover T. cruzi Infection

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1955))

Abstract

The development of accurate diagnostic tools and surrogate markers of parasitological response to treatment are priorities in Chagas disease (CD) research. For years, the detection of Trypanosoma cruzi DNA by PCR has proved to be useful in some clinical scenarios like acute CD, including cases of congenital transmission, CD reactivation in immunosuppressed patients, and posttreatment follow-up. In that sense, the implementation of quantitative real-time PCR (qPCR) assays was an important step in the development of more reliable tools for CD molecular diagnostics and treatment follow-up. In the last decade, two multicenter PCR studies allowed the harmonization and validation of standard operating procedures for PCR-based detection and quantification of T. cruzi DNA in blood samples. Herein we describe the two most used protocols to quantify parasitic load in human blood samples by multiplex qPCR assays and discuss some aspects to consider during planning and executing these procedures.

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References

  1. TDR/WHO (2012) Research priorities for Chagas disease, human African trypanosomiasis and leishmaniasis. World Health Organization, Geneva

    Google Scholar 

  2. Porras AI, Yadon ZE, Altcheh J et al (2015) Target product profile (TPP) for Chagas disease point-of-care diagnosis and assessment of response to treatment. PLoS Negl Trop Dis 9:e0003697. https://doi.org/10.1371/journal.pntd.0003697

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. WHO Expert Committee (2002) Control of Chagas disease. World Health Organ Tech Rep Ser 905:i–vi, 1–109

    Google Scholar 

  4. Antas PR, Medrano-Mercado N, Torrico F et al (1999) Early, intermediate, and late acute stages in Chagas’ disease: a study combining anti-galactose IgG, specific serodiagnosis, and polymerase chain reaction analysis. Am J Trop Med Hyg 61:308–314

    Article  CAS  Google Scholar 

  5. de NBA, Diaz-Bello Z, Colmenares C et al (2012) The performance of laboratory tests in the management of a large outbreak of orally transmitted Chagas disease. Mem Inst Oswaldo Cruz 107:893–898

    Article  Google Scholar 

  6. Virreira M, Torrico F, Truyens C et al (2003) Comparison of polymerase chain reaction methods for reliable and easy detection of congenital Trypanosoma cruzi infection. Am J Trop Med Hyg 68:574–582

    Article  CAS  Google Scholar 

  7. Velazquez EB, Rivero R, De Rissio AM et al (2014) Predictive role of polymerase chain reaction in the early diagnosis of congenital Trypanosoma cruzi infection. Acta Trop 137:195–200. https://doi.org/10.1016/j.actatropica.2014.05.016

    Article  CAS  PubMed  Google Scholar 

  8. Diez M, Favaloro L, Bertolotti A et al (2007) Usefulness of PCR strategies for early diagnosis of Chagas’ disease reactivation and treatment follow-up in heart transplantation. Am J Transplant 7:1633–1640. https://doi.org/10.1111/j.1600-6143.2007.01820.x

    Article  CAS  PubMed  Google Scholar 

  9. Britto C, Cardoso MA, Vanni CM et al (1995) Polymerase chain reaction detection of Trypanosoma cruzi in human blood samples as a tool for diagnosis and treatment evaluation. Parasitology 110(Pt 3):241–247

    Article  CAS  Google Scholar 

  10. Piron M, Fisa R, Casamitjana N et al (2007) Development of a real-time PCR assay for Trypanosoma cruzi detection in blood samples. Acta Trop 103:195–200. https://doi.org/10.1016/j.actatropica.2007.05.019

    Article  CAS  PubMed  Google Scholar 

  11. Duffy T, Bisio M, Altcheh J et al (2009) Accurate real-time PCR strategy for monitoring bloodstream parasitic loads in chagas disease patients. PLoS Negl Trop Dis 3:e419. https://doi.org/10.1371/journal.pntd.0000419

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Qvarnstrom Y, Schijman AG, Veron V et al (2012) Sensitive and specific detection of Trypanosoma cruzi DNA in clinical specimens using a multi-target real-time PCR approach. PLoS Negl Trop Dis 6:e1689. https://doi.org/10.1371/journal.pntd.0001689

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Schijman AGA, Bisio M, Orellana L et al (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:e931. https://doi.org/10.1371/journal.pntd.0000931

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Duffy T, Cura CI, Ramirez JC et al (2013) Analytical performance of a multiplex Real-Time PCR assay using TaqMan probes for quantification of Trypanosoma cruzi satellite DNA in blood samples. PLoS Negl Trop Dis 7:e2000. https://doi.org/10.1371/journal.pntd.0002000

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Ramírez JC, Cura CII, Moreira C et al (2015) Analytical validation of quantitative real-time PCR methods for quantification of Trypanosoma cruzi DNA in blood samples from Chagas disease patients. J Mol Diagn 17:605–615. https://doi.org/10.1016/j.jmoldx.2015.04.010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Moreira OC, Ramirez JD, Velazquez E et al (2013) Towards the establishment of a consensus real-time qPCR to monitor Trypanosoma cruzi parasitemia in patients with chronic Chagas disease cardiomyopathy: a substudy from the BENEFIT trial. Acta Trop 125:23–31. https://doi.org/10.1016/j.actatropica.2012.08.020

    Article  CAS  PubMed  Google Scholar 

  17. Molina I, Gomez i Prat J, Salvador F et al (2014) Randomized trial of posaconazole and benznidazole for chronic Chagas’ disease. N Engl J Med 370:1899–1908. https://doi.org/10.1056/NEJMoa1313122

    Article  CAS  PubMed  Google Scholar 

  18. Wei B, Chen L, Kibukawa M et al (2016) Development of a PCR assay to detect low level Trypanosoma cruzi in blood specimens collected with PAXgene blood DNA tubes for clinical trials treating chagas disease. PLoS Negl Trop Dis 10:e0005146. https://doi.org/10.1371/journal.pntd.0005146

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Torrico F, Gascon J, Ortiz L et al (2018) Treatment of adult chronic indeterminate Chagas disease with benznidazole and three E1224 dosing regimens: a proof-of-concept, randomised, placebo-controlled trial. Lancet Infect Dis 18:419–430. https://doi.org/10.1016/S1473-3099(17)30538-8

    Article  CAS  PubMed  Google Scholar 

  20. Cura CI, Ramirez JC, Rodriguez M et al (2017) Comparative study and analytical verification of PCR methods for the diagnosis of congenital Chagas disease. J Mol Diagn 19:673–681. https://doi.org/10.1016/j.jmoldx.2017.05.010

    Article  CAS  PubMed  Google Scholar 

  21. Guhl F, Jaramillo C, Carranza JC, Vallejo GA (2002) Molecular characterization and diagnosis of Trypanosoma cruzi and T. rangeli. Arch Med Res 33:362–370

    Article  CAS  Google Scholar 

  22. Ferreira KAM, Fajardo EF, Baptista RP et al (2014) Species-specific markers for the differential diagnosis of Trypanosoma cruzi and Trypanosoma rangeli and polymorphisms detection in Trypanosoma rangeli. Parasitol Res 113:2199–2207. https://doi.org/10.1007/s00436-014-3872-2

    Article  PubMed  Google Scholar 

  23. Burns MJ, Nixon GJ, Foy CA, Harris N (2005) Standardisation of data from real-time quantitative PCR methods – evaluation of outliers and comparison of calibration curves. BMC Biotechnol 5:31. https://doi.org/10.1186/1472-6750-5-31

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Demeke T, Jenkins GR (2010) Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits. Anal Bioanal Chem 396:1977–1990. https://doi.org/10.1007/s00216-009-3150-9

    Article  CAS  PubMed  Google Scholar 

  25. Burd EM (2010) Validation of laboratory-developed molecular assays for infectious diseases. Clin Microbiol Rev 23:550–576. https://doi.org/10.1128/CMR.00074-09

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Authors and Affiliations

  1. Laboratorio de Biología Molecular de la Enfermedad de Chagas, Instituto de Investigaciones en Ingeniería Genética y Biología Molecular “Dr. Héctor N. Torres” (INGEBI), CONICET, Buenos Aires, Argentina

    Juan Carlos Ramírez

  2. Laboratório de Biología Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz/Fiocruz, Rio de Janeiro, Brazil

    Otacilio da Cruz Moreira

Authors
  1. Juan Carlos Ramírez
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  2. Otacilio da Cruz Moreira
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Corresponding author

Correspondence to Juan Carlos Ramírez .

Editor information

Editors and Affiliations

  1. INGEBI-CONICET, Buenos Aires, Argentina

    Karina Andrea Gómez

  2. UNSAM-CONICET, IIB-INTECH, San Martin, Buenos Aires, Argentina

    Carlos Andrés Buscaglia

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Ramírez, J.C., da Cruz Moreira, O. (2019). Assessing Parasite Load in Chagas Disease Patients by Quantitative Multiplex Real-Time PCR. In: Gómez, K., Buscaglia, C. (eds) T. cruzi Infection. Methods in Molecular Biology, vol 1955. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9148-8_16

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  • DOI: https://doi.org/10.1007/978-1-4939-9148-8_16

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9147-1

  • Online ISBN: 978-1-4939-9148-8

  • eBook Packages: Springer Protocols

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