Accurate real-time PCR strategy for monitoring bloodstream parasitic loads in chagas disease patients.
<h4>Background</h4>This report describes a real-time PCR (Q-PCR) strategy to quantify Trypanosoma cruzi (T. cruzi) DNA in peripheral blood samples from Chagas disease patients targeted to conserved motifs within the repetitive satellite sequence.<h4>Methodology/principal findings&l...
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2009
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oai:doaj.org-article:5f7dacb14bf04d5099766327b89ba8492021-11-25T06:32:54ZAccurate real-time PCR strategy for monitoring bloodstream parasitic loads in chagas disease patients.1935-27271935-273510.1371/journal.pntd.0000419https://doaj.org/article/5f7dacb14bf04d5099766327b89ba8492009-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/19381287/pdf/?tool=EBIhttps://doaj.org/toc/1935-2727https://doaj.org/toc/1935-2735<h4>Background</h4>This report describes a real-time PCR (Q-PCR) strategy to quantify Trypanosoma cruzi (T. cruzi) DNA in peripheral blood samples from Chagas disease patients targeted to conserved motifs within the repetitive satellite sequence.<h4>Methodology/principal findings</h4>The Q-PCR has a detection limit of 0.1 and 0.01 parasites/mL, with a dynamic range of 10(6) and 10(7) for Silvio X10 cl1 (T. cruzi I) and Cl Brener stocks (T. cruzi IIe), respectively, an efficiency of 99%, and a coefficient of determination (R(2)) of 0.998. In order to express accurately the parasitic loads: (1) we adapted a commercial kit based on silica-membrane technology to enable efficient processing of Guanidine Hydrochloride-EDTA treated blood samples and minimize PCR inhibition; (2) results were normalized incorporating a linearized plasmid as an internal standard of the whole procedure; and (3) a correction factor according to the representativity of satellite sequences in each parasite lineage group was determined using a modified real-time PCR protocol (Lg-PCR). The Q-PCR strategy was applied (1) to estimate basal parasite loads in 43 pediatric Chagas disease patients, (2) to follow-up 38 of them receiving treatment with benznidazole, and (3) to monitor three chronic Chagas heart disease patients who underwent heart-transplantation and displayed events of clinical reactivation due to immunosupression.<h4>Conclusion/significance</h4>All together, the high analytical sensitivity of the Q-PCR strategy, the low levels of intra- and inter-assay variations, as well as the accuracy provided by the Lg-PCR based correction factor support this methodology as a key laboratory tool for monitoring clinical reactivation and etiological treatment outcome in Chagas disease patients.Tomas DuffyMargarita BisioJaime AltchehJuan Miguel BurgosMirta DiezMariano Jorge LevinRoberto Rene FavaloroHector FreilijAlejandro Gabriel SchijmanPublic Library of Science (PLoS)articleArctic medicine. Tropical medicineRC955-962Public aspects of medicineRA1-1270ENPLoS Neglected Tropical Diseases, Vol 3, Iss 4, p e419 (2009) |
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Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
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Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 Tomas Duffy Margarita Bisio Jaime Altcheh Juan Miguel Burgos Mirta Diez Mariano Jorge Levin Roberto Rene Favaloro Hector Freilij Alejandro Gabriel Schijman Accurate real-time PCR strategy for monitoring bloodstream parasitic loads in chagas disease patients. |
| description |
<h4>Background</h4>This report describes a real-time PCR (Q-PCR) strategy to quantify Trypanosoma cruzi (T. cruzi) DNA in peripheral blood samples from Chagas disease patients targeted to conserved motifs within the repetitive satellite sequence.<h4>Methodology/principal findings</h4>The Q-PCR has a detection limit of 0.1 and 0.01 parasites/mL, with a dynamic range of 10(6) and 10(7) for Silvio X10 cl1 (T. cruzi I) and Cl Brener stocks (T. cruzi IIe), respectively, an efficiency of 99%, and a coefficient of determination (R(2)) of 0.998. In order to express accurately the parasitic loads: (1) we adapted a commercial kit based on silica-membrane technology to enable efficient processing of Guanidine Hydrochloride-EDTA treated blood samples and minimize PCR inhibition; (2) results were normalized incorporating a linearized plasmid as an internal standard of the whole procedure; and (3) a correction factor according to the representativity of satellite sequences in each parasite lineage group was determined using a modified real-time PCR protocol (Lg-PCR). The Q-PCR strategy was applied (1) to estimate basal parasite loads in 43 pediatric Chagas disease patients, (2) to follow-up 38 of them receiving treatment with benznidazole, and (3) to monitor three chronic Chagas heart disease patients who underwent heart-transplantation and displayed events of clinical reactivation due to immunosupression.<h4>Conclusion/significance</h4>All together, the high analytical sensitivity of the Q-PCR strategy, the low levels of intra- and inter-assay variations, as well as the accuracy provided by the Lg-PCR based correction factor support this methodology as a key laboratory tool for monitoring clinical reactivation and etiological treatment outcome in Chagas disease patients. |
| format |
article |
| author |
Tomas Duffy Margarita Bisio Jaime Altcheh Juan Miguel Burgos Mirta Diez Mariano Jorge Levin Roberto Rene Favaloro Hector Freilij Alejandro Gabriel Schijman |
| author_facet |
Tomas Duffy Margarita Bisio Jaime Altcheh Juan Miguel Burgos Mirta Diez Mariano Jorge Levin Roberto Rene Favaloro Hector Freilij Alejandro Gabriel Schijman |
| author_sort |
Tomas Duffy |
| title |
Accurate real-time PCR strategy for monitoring bloodstream parasitic loads in chagas disease patients. |
| title_short |
Accurate real-time PCR strategy for monitoring bloodstream parasitic loads in chagas disease patients. |
| title_full |
Accurate real-time PCR strategy for monitoring bloodstream parasitic loads in chagas disease patients. |
| title_fullStr |
Accurate real-time PCR strategy for monitoring bloodstream parasitic loads in chagas disease patients. |
| title_full_unstemmed |
Accurate real-time PCR strategy for monitoring bloodstream parasitic loads in chagas disease patients. |
| title_sort |
accurate real-time pcr strategy for monitoring bloodstream parasitic loads in chagas disease patients. |
| publisher |
Public Library of Science (PLoS) |
| publishDate |
2009 |
| url |
https://doaj.org/article/5f7dacb14bf04d5099766327b89ba849 |
| work_keys_str_mv |
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