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Pan-stage real-time PCR for quantitation of Trypanosoma cruzi parasitic loads in blood samples

  • Juan David Ramírez
    Correspondence
    Corresponding author:
    Affiliations
    Molecular Microbiology Laboratory, Department of Pathology, Molecular and Cell-based Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

    Centro de Investigaciones en Microbiología y Biotecnología-UR (CIM BIUR), Facultad de Ciencias Naturales, Universidad del Rosario, Bogotá, Colombia.
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  • Liyong Cao
    Affiliations
    Molecular Microbiology Laboratory, Department of Pathology, Molecular and Cell-based Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
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  • Lissa Cruz-Saavedra
    Affiliations
    Centro de Investigaciones en Microbiología y Biotecnología-UR (CIM BIUR), Facultad de Ciencias Naturales, Universidad del Rosario, Bogotá, Colombia.
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  • Carolina Hernandez
    Affiliations
    Centro de Investigaciones en Microbiología y Biotecnología-UR (CIM BIUR), Facultad de Ciencias Naturales, Universidad del Rosario, Bogotá, Colombia.

    Centro de Tecnología en Salud (CETESA), Innovaseq SAS, Bogotá, Colombia.
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  • Sergio Castañeda
    Affiliations
    Centro de Investigaciones en Microbiología y Biotecnología-UR (CIM BIUR), Facultad de Ciencias Naturales, Universidad del Rosario, Bogotá, Colombia.
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  • Marina Muñoz
    Affiliations
    Centro de Investigaciones en Microbiología y Biotecnología-UR (CIM BIUR), Facultad de Ciencias Naturales, Universidad del Rosario, Bogotá, Colombia.
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  • Nathalia Ballesteros
    Affiliations
    Centro de Investigaciones en Microbiología y Biotecnología-UR (CIM BIUR), Facultad de Ciencias Naturales, Universidad del Rosario, Bogotá, Colombia.
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  • Radhika Banu
    Affiliations
    Molecular Microbiology Laboratory, Department of Pathology, Molecular and Cell-based Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
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  • Paras Shrestha
    Affiliations
    Molecular Microbiology Laboratory, Department of Pathology, Molecular and Cell-based Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
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  • Carlos Cordon-Cardo
    Affiliations
    Molecular Microbiology Laboratory, Department of Pathology, Molecular and Cell-based Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
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  • Emilia Mia Sordillo
    Affiliations
    Molecular Microbiology Laboratory, Department of Pathology, Molecular and Cell-based Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
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  • Alberto Paniz-Mondolfi
    Affiliations
    Molecular Microbiology Laboratory, Department of Pathology, Molecular and Cell-based Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

    Instituto de Investigaciones Biomédicas IDB/Incubadora Venezolana de la Ciencia, Barquisimeto, Venezuela.
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Open AccessPublished:June 08, 2022DOI:https://doi.org/10.1016/j.ijid.2022.06.006

      Highlights

      • The Trypanosoma cruzi parasitic load is not life-stage dependent.
      • Epimastigotes can be used to quantify T. cruzi parasitic loads.
      • Molecular diagnostics of T. cruzi must be improved.

      Abstract

      Chagas disease is a complex zoonosis caused by Trypanosoma cruzi. The diagnosis of this infection is complex and molecular tools are suggested to detect the parasite in blood samples. A long-standing question arises in Chagas disease molecular diagnostics and is related to the feasibility of using epimastigotes in standard curves to quantify parasitic loads. Herein, we conducted experiments running standard curves with all the known life stages of T. cruzi. Our results indicate that regardless of the life stage employed, there are no statistically significant differences when calculating parasitic loads in blood samples. Our results have practical implications from a laboratory perspective in terms of the usability of epimastigotes to build standard curves for T. cruzi pan-stage assessment. Future studies are needed to further improve T. cruzi molecular diagnostic methods and enhance their impact in clinical practice.

      Keywords

      Chagas disease is a complex zoonotic parasitic disease caused by the protozoan Trypanosoma cruzi (
      • Paniz Mondolfi AE
      • Madigan R
      • Perez-Garcia L
      • Sordillo EM
      Chagas disease endemism in the United States.
      ). The progressive nature of Chagas disease and its transition into different clinical stages poses a diagnostic challenge, requiring a variety of laboratory diagnostic approaches that can be tailored to the clinical phase of the disease (direct observation on Wright-Giemsa- stained smears and/or by nucleic acid amplification, usually by PCR, or serological testing by at least two different methods). The performance of two independent serologic assays, in conjunction with real-time PCR testing, is recommended to improve diagnostic accuracy (
      • Forsyth CJ
      • Manne-Goehler J
      • Bern C
      • Whitman J
      • Hochberg NS
      • et al.
      Recommendations for screening and diagnosis of Chagas disease in the United States.
      ;
      Panamerican Health Organization
      Synthesis of evidence: guidance for the diagnosis and treatment of Chagas disease.
      ).
      During the past two decades, different investigators across South America have developed and successfully deployed molecular detection tests for T. cruzi, reviewed in-depth (
      • Alonso-Padilla J
      • Gallego M
      • Schijman AG
      • Gascon J.
      Molecular Diagnostics for Chagas disease: up to date and novel methodologies.
      ;
      • Schijman AG.
      Molecular diagnosis of Trypanosoma cruzi.
      ). For example, the tandem repeat satellite DNA of T. cruzi PCR assay has shown excellent analytical and diagnostic performance. However, standard curves for parasite load quantitation generated for these studies have been constructed using epimastigotes from several T. cruzi strains, a vector developmental stage not found in human hosts (
      • Ramírez JC
      • Cura CI
      • Moreira C
      • Lages-Silva E
      • Juiz N
      • Velázquez E
      • et al.
      Analytical validation of quantitative real-time PCR methods for quantification of Trypanosoma cruzi DNA in blood samples from Chagas disease patients.
      ) and recently using synthetic satellite unit DNA sequence (
      • Muñoz-Calderon A
      • Silva-Gomes NL
      • Apodaca S
      • Alarcón de Noya B
      • Díaz-Bello Z
      • et al.
      Towards the establishment of a single standard curve for quantification of Trypanosoma cruzi natural populations using synthetic a satellite unit DNA sequence.
      ).
      T. cruzi develops in four biological stages: as epimastigotes (EP), which inhabit the vector's midgut; as metacyclic trypomastigotes (MT), the infective stage found in the ampulla of the triatomine vector; and as amastigotes (AM), the intracellular stage associated with tissue damage to the infected host that later differentiates into the cell-derived trypomastigotes (CDT) found in the bloodstream of infected individuals. In this study, we aimed to assemble standard curves for all known biological stages of T. cruzi and determine whether this could affect parasite load calculation, a long-standing question in the molecular diagnostics of Chagas disease.
      All experiments were conducted using the T. cruzi MG strain MHOM/CO/04/MG. This reference strain, cataloged as discrete typing unit (DTU) I, is the most widespread DTU throughout the Americas (

      Velasquez-Ortiz N, Herrera G, Hernandez C, Muñoz M, Ramírez JD. Discrete typing units (DTUs) of Trypanosoma cruzi: geographical and biological distribution in the Americas, in press.

      ). All T. cruzi-developmental stages were cultured as reported elsewhere (
      • Cruz-Saavedra L
      • Vallejo G
      • Guhl F
      • Ramírez JD.
      Transcriptomic changes across the life cycle of Trypanosoma cruzi II.
      ) and then washed twice with phosphate-buffered saline (PBS) 1X after achieving the logarithmic growth phase, followed later by quantification in the Neubauer chamber to reach a concentration of 1.000.000 equivalent parasites/mL. A concentrated stock from each stage was then subjected to DNA extraction using the High Pure PCR Template Preparation Kit (Roche) as per the manufacturer's instructions.
      A standard calibration curve for each biological stage (EP, MT, CDT, AM) was performed to quantify parasite DNA. The calibration curve was performed in triplicate using a six-point serial dilution starting from 1,00,000 parasite equivalents/mL to 0.01 p-eq/mL. To determine the limit of detection (LOD), a Probit regression analysis was conducted. The parasites’ DNAs were diluted using as matrix human DNA from donors who tested negative for T. cruzi by PCR/serology and living in non-endemic areas for Chagas disease. All DNAs from point serial dilutions were tested under conditions described previously (
      • Hernández C
      • Cucunubá Z
      • Flórez C
      • Olivera M
      • Valencia C
      • Zambrano P
      • León C
      • Ramírez JD.
      Molecular diagnosis of Chagas disease in Colombia: parasitic loads and discrete typing units in patients from acute and chronic phases.
      ). The resulting cycle threshold (Ct) values of each calibration curve were plotted by linear regression to determine the dynamic range of the test. The Probit analysis showed that the LOD was 0.1 p-eq/mL for all stages. There were no statistically significant differences (Kruskal-Wallis chi-squared = 1.0274, df = 3, P-value = 0.7946) among stages (Figure 1 and Table S1). In addition, the R-squared was over 0.980 in EP, AM, and CDT, the R-squared of AM was 0.940; this could be attributed to the lower content of DNA in the AM.
      Figure 1
      Figure 1Dynamic range of the calibration curves using different lifecycle stages of T. cruzi a. MT b. EP c. AM d. CDT CDT = cell-derived trypomastigotes; EP = epimastigotes; MT = metacyclic trypomastigotes.
      DNA from blood samples that tested positive by real-time PCR and serology for T. cruzi in a previous study (
      • Ramírez JD
      • Herrera G
      • Hernández C
      • Cruz-Saavedra L
      • Muñoz M
      • et al.
      Evaluation of the analytical and diagnostic performance of a digital droplet polymerase chain reaction (ddPCR) assay to detect Trypanosoma cruzi DNA in blood samples.
      ) was used to test diagnostic performance. A set of 43 positive samples were analyzed, including 10 samples from donors that were negative by serology living in non-endemic areas. DNAs were subjected blind to two independent assay runs. There were no statistically significant differences between parasitic loads when calculated with each stage calibration curve (Kruskal-Wallis chi-squared = 7.5645, df = 3, P-value = 0.06592), revealing that molecular detection and quantitation of T. cruzi are not affected by the biological stage of the parasite (Table S2).
      Our results provide valuable insights regarding a long-lasting question in the molecular diagnostics of Chagas disease and provide positive evidence regarding the suitability of using EP to build standard curves necessary for parasitic load assessment in clinical settings. This is advantageous as its use would decrease the odds of laboratory staff acquiring Chagas disease because of laboratory accidents. A limitation of this study is that only DTU I was evaluated for this estimation; it is well known that TcI strains have 30 to 50 times less satellite DNA than other DTUs (
      • Muñoz-Calderon A
      • Silva-Gomes NL
      • Apodaca S
      • Alarcón de Noya B
      • Díaz-Bello Z
      • et al.
      Towards the establishment of a single standard curve for quantification of Trypanosoma cruzi natural populations using synthetic a satellite unit DNA sequence.
      ). Therefore, subsequent studies must include a wider range of DTUs to assess T. cruzi's broader range of pheno/genotypic topographies, including the deployment of the use of synthetic satellite DNA as recently reported (
      • Muñoz-Calderon A
      • Silva-Gomes NL
      • Apodaca S
      • Alarcón de Noya B
      • Díaz-Bello Z
      • et al.
      Towards the establishment of a single standard curve for quantification of Trypanosoma cruzi natural populations using synthetic a satellite unit DNA sequence.
      ). Although T. cruzi PCR has proven successful for the diagnosis of acute-phase disease, early diagnosis of oral and congenital transmission, early detection in transplant receptors from infected donors, reactivation, and as a tool for monitoring trypanocidal chemotherapy, its role in the diagnosis of chronic phase remains challenging. Future studies are needed to further improve T. cruzi molecular diagnostic methods and enhance their impact in clinical practice.

      Funding source

      This publication is based on work supported by The Pershing Square Foundation for the Mount Sinai Health System COVID-19 Saliva Testing Project.

      Ethical statement

      The protocol was approved by the London School of Hygiene & Tropical Medicine Observational Ethics Committee (protocol number 15515).

      Conflict of interest

      The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

      Appendix. Supplementary materials

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