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.
Chagas disease is caused by the parasitic infection of Trypanosoma cruzi (T. cruzi). The STOP CHAGAS clinical trial was initiated in 2011 to evaluate posaconazole in treating Chagas disease, with treatment success defined as negative qualitative PCR results of detecting the parasites in blood specim...
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ftdoajarticles:oai:doaj.org/article:2c5528da53d44a28bdfff3094acf39c3 2023-05-15T15:08:19+02:00 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. Bo Wei Lei Chen Miho Kibukawa John Kang Hetty Waskin Matthew Marton 2016-12-01T00:00:00Z https://doi.org/10.1371/journal.pntd.0005146 https://doaj.org/article/2c5528da53d44a28bdfff3094acf39c3 EN eng Public Library of Science (PLoS) http://europepmc.org/articles/PMC5131911?pdf=render https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0005146 https://doaj.org/article/2c5528da53d44a28bdfff3094acf39c3 PLoS Neglected Tropical Diseases, Vol 10, Iss 12, p e0005146 (2016) Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 article 2016 ftdoajarticles https://doi.org/10.1371/journal.pntd.0005146 2022-12-31T14:37:43Z Chagas disease is caused by the parasitic infection of Trypanosoma cruzi (T. cruzi). The STOP CHAGAS clinical trial was initiated in 2011 to evaluate posaconazole in treating Chagas disease, with treatment success defined as negative qualitative PCR results of detecting the parasites in blood specimens collected post-treatment. PAXgene Blood DNA tubes were utilized as a simple procedure to collect and process blood specimens. However, the PAXgene blood specimens challenged published T. cruzi PCR methods, resulting in poor sensitivity and reproducibility. To accurately evaluate the treatment efficacy of the clinical study, we developed and validated a robust PCR assay for detecting low level T. cruzi in PAXgene blood specimens. The assay combines a new DNA extraction method with a custom designed qPCR assay, resulting in limit of detection of 0.005 and 0.01 fg/μl for K98 and CL Brener, two representative strains of two of T. cruzi's discrete typing units. Reliable qPCR standard curves were established for both strains to measure parasite loads, with amplification efficiency ≥ 90% and the lower limit of linearity ≥ 0.05 fg/μl. The assay successfully analyzed the samples collected from the STOP CHAGAS study and may prove useful for future global clinical trials evaluating new therapies for asymptomatic chronic Chagas disease. Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic PLOS Neglected Tropical Diseases 10 12 e0005146 |
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Directory of Open Access Journals: DOAJ Articles |
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ftdoajarticles |
language |
English |
topic |
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 Bo Wei Lei Chen Miho Kibukawa John Kang Hetty Waskin Matthew Marton 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. |
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Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
description |
Chagas disease is caused by the parasitic infection of Trypanosoma cruzi (T. cruzi). The STOP CHAGAS clinical trial was initiated in 2011 to evaluate posaconazole in treating Chagas disease, with treatment success defined as negative qualitative PCR results of detecting the parasites in blood specimens collected post-treatment. PAXgene Blood DNA tubes were utilized as a simple procedure to collect and process blood specimens. However, the PAXgene blood specimens challenged published T. cruzi PCR methods, resulting in poor sensitivity and reproducibility. To accurately evaluate the treatment efficacy of the clinical study, we developed and validated a robust PCR assay for detecting low level T. cruzi in PAXgene blood specimens. The assay combines a new DNA extraction method with a custom designed qPCR assay, resulting in limit of detection of 0.005 and 0.01 fg/μl for K98 and CL Brener, two representative strains of two of T. cruzi's discrete typing units. Reliable qPCR standard curves were established for both strains to measure parasite loads, with amplification efficiency ≥ 90% and the lower limit of linearity ≥ 0.05 fg/μl. The assay successfully analyzed the samples collected from the STOP CHAGAS study and may prove useful for future global clinical trials evaluating new therapies for asymptomatic chronic Chagas disease. |
format |
Article in Journal/Newspaper |
author |
Bo Wei Lei Chen Miho Kibukawa John Kang Hetty Waskin Matthew Marton |
author_facet |
Bo Wei Lei Chen Miho Kibukawa John Kang Hetty Waskin Matthew Marton |
author_sort |
Bo Wei |
title |
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. |
title_short |
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. |
title_full |
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. |
title_fullStr |
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. |
title_full_unstemmed |
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. |
title_sort |
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. |
publisher |
Public Library of Science (PLoS) |
publishDate |
2016 |
url |
https://doi.org/10.1371/journal.pntd.0005146 https://doaj.org/article/2c5528da53d44a28bdfff3094acf39c3 |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
PLoS Neglected Tropical Diseases, Vol 10, Iss 12, p e0005146 (2016) |
op_relation |
http://europepmc.org/articles/PMC5131911?pdf=render https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0005146 https://doaj.org/article/2c5528da53d44a28bdfff3094acf39c3 |
op_doi |
https://doi.org/10.1371/journal.pntd.0005146 |
container_title |
PLOS Neglected Tropical Diseases |
container_volume |
10 |
container_issue |
12 |
container_start_page |
e0005146 |
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1766339700130316288 |