Development of a Novel Protocol Based on Blood Clot to Improve the Sensitivity of qPCR Detection of Toxoplasma gondii in Peripheral Blood Specimens

Renzo Gutierrez-Loli, Cusi Ferradas, Andrea Diestra, Aliki Traianou, Natalie Bowman, Jeroen Bok, Melissa Reimer-McAtee, Cesar Ramal, Eduardo Ticona, Hannah Steinberg, Holger Mayta, Maritza Calderon, Jaeson S. Calla-Choque, Charles Sterling, Robert H Gilman, The Toxoplasmosis Working Group In Peru For The Toxoplasmosis Working Group In Peru

Research output: Contribution to journalArticle

Abstract

Quantitative polymerase chain reaction (qPCR) for Toxoplasma gondii multicopy genes has emerged as a promising strategy for sensitive detection of parasite DNA. qPCR can be performed from blood samples, which are minimally invasive to collect. However, there is no consensus about what type of blood specimen yields the best sensitivity. The development of a novel protocol for qPCR detection of T. gondii using blood clot, involving an appropriate DNA extraction method and the use of an internal amplification control to monitor the reaction is presented in the current study. Assays directed to the B1 and REP529 genes were performed in spiked specimens of whole blood, guanidine-ethylenediaminetetraacetic acid blood, and clot. The clot-based qPCR was shown to be more sensitive when compared with other types of specimens, detecting five and 0.05 T. gondii genomes, using B1 and REP529 targets, respectively. Finally, a comparative analysis with samples from HIV patients with clinical suspicion of toxoplasmosis was performed, demonstrating the detection of four positive suspected cases with clots compared with only one using guanidine-ethylenediaminetetraacetic acid blood. The high analytical sensitivity and the cost-effective advantages offered by clot supports this methodology as a good laboratory tool to monitor parasite burden.

Original languageEnglish (US)
Pages (from-to)83-89
Number of pages7
JournalThe American journal of tropical medicine and hygiene
Volume100
Issue number1
DOIs
StatePublished - Jan 1 2019

ASJC Scopus subject areas

  • Parasitology
  • Infectious Diseases
  • Virology

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    Gutierrez-Loli, R., Ferradas, C., Diestra, A., Traianou, A., Bowman, N., Bok, J., Reimer-McAtee, M., Ramal, C., Ticona, E., Steinberg, H., Mayta, H., Calderon, M., Calla-Choque, J. S., Sterling, C., Gilman, R. H., & For The Toxoplasmosis Working Group In Peru, T. T. W. G. I. P. (2019). Development of a Novel Protocol Based on Blood Clot to Improve the Sensitivity of qPCR Detection of Toxoplasma gondii in Peripheral Blood Specimens. The American journal of tropical medicine and hygiene, 100(1), 83-89. https://doi.org/10.4269/ajtmh.17-0920