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 language | English (US) |
|---|---|
| Pages (from-to) | 83-89 |
| Number of pages | 7 |
| Journal | The American journal of tropical medicine and hygiene |
| Volume | 100 |
| Issue number | 1 |
| DOIs | |
| State | Published - Jan 1 2019 |
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ASJC Scopus subject areas
- Parasitology
- Infectious Diseases
- Virology
Cite this
Development of a Novel Protocol Based on Blood Clot to Improve the Sensitivity of qPCR Detection of Toxoplasma gondii in Peripheral Blood Specimens. / Gutierrez-Loli, Renzo; Ferradas, Cusi; Diestra, Andrea; Traianou, Aliki; Bowman, Natalie; Bok, Jeroen; Reimer-McAtee, Melissa; Ramal, Cesar; Ticona, Eduardo; Steinberg, Hannah; Mayta, Holger; Calderon, Maritza; Calla-Choque, Jaeson S.; Sterling, Charles R; Gilman, Robert H.; For The Toxoplasmosis Working Group In Peru, The Toxoplasmosis Working Group In Peru.
In: The American journal of tropical medicine and hygiene, Vol. 100, No. 1, 01.01.2019, p. 83-89.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Development of a Novel Protocol Based on Blood Clot to Improve the Sensitivity of qPCR Detection of Toxoplasma gondii in Peripheral Blood Specimens
AU - Gutierrez-Loli, Renzo
AU - Ferradas, Cusi
AU - Diestra, Andrea
AU - Traianou, Aliki
AU - Bowman, Natalie
AU - Bok, Jeroen
AU - Reimer-McAtee, Melissa
AU - Ramal, Cesar
AU - Ticona, Eduardo
AU - Steinberg, Hannah
AU - Mayta, Holger
AU - Calderon, Maritza
AU - Calla-Choque, Jaeson S.
AU - Sterling, Charles R
AU - Gilman, Robert H.
AU - For The Toxoplasmosis Working Group In Peru, The Toxoplasmosis Working Group In Peru
PY - 2019/1/1
Y1 - 2019/1/1
N2 - 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.
AB - 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.
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U2 - 10.4269/ajtmh.17-0920
DO - 10.4269/ajtmh.17-0920
M3 - Article
VL - 100
SP - 83
EP - 89
JO - American Journal of Tropical Medicine and Hygiene
JF - American Journal of Tropical Medicine and Hygiene
SN - 0002-9637
IS - 1
ER -