The extended recovery ring-stage survival assay provides a superior association with patient clearance half-life and increases throughput
Abstract Background Tracking and understanding artemisinin resistance is key for preventing global setbacks in malaria eradication efforts. The ring-stage survival assay (RSA) is the current gold standard for in vitro artemisinin resistance phenotyping. However, the RSA has several drawbacks: it is...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2020-01-01
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| Series: | Malaria Journal |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12936-020-3139-6 |
| _version_ | 1818930624093749248 |
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| author | Sage Z. Davis Puspendra P. Singh Katelyn M. Vendrely Douglas A. Shoue Lisa A. Checkley Marina McDew-White Katrina A. Button-Simons Zione Cassady Mackenzie A. C. Sievert Gabriel J. Foster François H. Nosten Timothy J. C. Anderson Michael T. Ferdig |
| author_facet | Sage Z. Davis Puspendra P. Singh Katelyn M. Vendrely Douglas A. Shoue Lisa A. Checkley Marina McDew-White Katrina A. Button-Simons Zione Cassady Mackenzie A. C. Sievert Gabriel J. Foster François H. Nosten Timothy J. C. Anderson Michael T. Ferdig |
| author_sort | Sage Z. Davis |
| collection | DOAJ |
| description | Abstract Background Tracking and understanding artemisinin resistance is key for preventing global setbacks in malaria eradication efforts. The ring-stage survival assay (RSA) is the current gold standard for in vitro artemisinin resistance phenotyping. However, the RSA has several drawbacks: it is relatively low throughput, has high variance due to microscopy readout, and correlates poorly with the current benchmark for in vivo resistance, patient clearance half-life post-artemisinin treatment. Here a modified RSA is presented, the extended Recovery Ring-stage Survival Assay (eRRSA), using 15 cloned patient isolates from Southeast Asia with a range of patient clearance half-lives, including parasite isolates with and without kelch13 mutations. Methods Plasmodium falciparum cultures were synchronized with single layer Percoll during the schizont stage of the intraerythrocytic development cycle. Cultures were left to reinvade to early ring-stage and parasitaemia was quantified using flow cytometry. Cultures were diluted to 2% haematocrit and 0.5% parasitaemia in a 96-well plate to start the assay, allowing for increased throughput and decreased variability between biological replicates. Parasites were treated with 700 nM of dihydroartemisinin or 0.02% dimethyl sulfoxide (DMSO) for 6 h, washed three times in drug-free media, and incubated for 66 or 114 h, when samples were collected and frozen for PCR amplification. A SYBR Green-based quantitative PCR method was used to quantify the fold-change between treated and untreated samples. Results 15 cloned patient isolates from Southeast Asia with a range of patient clearance half-lives were assayed using the eRRSA. Due to the large number of pyknotic and dying parasites at 66 h post-exposure (72 h sample), parasites were grown for an additional cell cycle (114 h post-exposure, 120 h sample), which drastically improved correlation with patient clearance half-life compared to the 66 h post-exposure sample. A Spearman correlation of − 0.8393 between fold change and patient clearance half-life was identified in these 15 isolates from Southeast Asia, which is the strongest correlation reported to date. Conclusions eRRSA drastically increases the efficiency and accuracy of in vitro artemisinin resistance phenotyping compared to the traditional RSA, which paves the way for extensive in vitro phenotyping of hundreds of artemisinin resistant parasites. |
| first_indexed | 2024-12-20T04:03:39Z |
| format | Article |
| id | doaj.art-958d81353f984036befdcac01447da43 |
| institution | Directory Open Access Journal |
| issn | 1475-2875 |
| language | English |
| last_indexed | 2024-12-20T04:03:39Z |
| publishDate | 2020-01-01 |
| publisher | BMC |
| record_format | Article |
| series | Malaria Journal |
| spelling | doaj.art-958d81353f984036befdcac01447da432022-12-21T19:54:06ZengBMCMalaria Journal1475-28752020-01-011911910.1186/s12936-020-3139-6The extended recovery ring-stage survival assay provides a superior association with patient clearance half-life and increases throughputSage Z. Davis0Puspendra P. Singh1Katelyn M. Vendrely2Douglas A. Shoue3Lisa A. Checkley4Marina McDew-White5Katrina A. Button-Simons6Zione Cassady7Mackenzie A. C. Sievert8Gabriel J. Foster9François H. Nosten10Timothy J. C. Anderson11Michael T. Ferdig12Eck Institute for Global Health, Dept. of Biological Sciences, University of Notre DameEck Institute for Global Health, Dept. of Biological Sciences, University of Notre DameEck Institute for Global Health, Dept. of Biological Sciences, University of Notre DameEck Institute for Global Health, Dept. of Biological Sciences, University of Notre DameEck Institute for Global Health, Dept. of Biological Sciences, University of Notre DameTexas Biomedical Research InstituteEck Institute for Global Health, Dept. of Biological Sciences, University of Notre DameEck Institute for Global Health, Dept. of Biological Sciences, University of Notre DameEck Institute for Global Health, Dept. of Biological Sciences, University of Notre DameEck Institute for Global Health, Dept. of Biological Sciences, University of Notre DameShoklo Malaria Research Unit, Mahidol-Oxford Tropical Medicine Research Unit, Mahidol UniversityTexas Biomedical Research InstituteEck Institute for Global Health, Dept. of Biological Sciences, University of Notre DameAbstract Background Tracking and understanding artemisinin resistance is key for preventing global setbacks in malaria eradication efforts. The ring-stage survival assay (RSA) is the current gold standard for in vitro artemisinin resistance phenotyping. However, the RSA has several drawbacks: it is relatively low throughput, has high variance due to microscopy readout, and correlates poorly with the current benchmark for in vivo resistance, patient clearance half-life post-artemisinin treatment. Here a modified RSA is presented, the extended Recovery Ring-stage Survival Assay (eRRSA), using 15 cloned patient isolates from Southeast Asia with a range of patient clearance half-lives, including parasite isolates with and without kelch13 mutations. Methods Plasmodium falciparum cultures were synchronized with single layer Percoll during the schizont stage of the intraerythrocytic development cycle. Cultures were left to reinvade to early ring-stage and parasitaemia was quantified using flow cytometry. Cultures were diluted to 2% haematocrit and 0.5% parasitaemia in a 96-well plate to start the assay, allowing for increased throughput and decreased variability between biological replicates. Parasites were treated with 700 nM of dihydroartemisinin or 0.02% dimethyl sulfoxide (DMSO) for 6 h, washed three times in drug-free media, and incubated for 66 or 114 h, when samples were collected and frozen for PCR amplification. A SYBR Green-based quantitative PCR method was used to quantify the fold-change between treated and untreated samples. Results 15 cloned patient isolates from Southeast Asia with a range of patient clearance half-lives were assayed using the eRRSA. Due to the large number of pyknotic and dying parasites at 66 h post-exposure (72 h sample), parasites were grown for an additional cell cycle (114 h post-exposure, 120 h sample), which drastically improved correlation with patient clearance half-life compared to the 66 h post-exposure sample. A Spearman correlation of − 0.8393 between fold change and patient clearance half-life was identified in these 15 isolates from Southeast Asia, which is the strongest correlation reported to date. Conclusions eRRSA drastically increases the efficiency and accuracy of in vitro artemisinin resistance phenotyping compared to the traditional RSA, which paves the way for extensive in vitro phenotyping of hundreds of artemisinin resistant parasites.https://doi.org/10.1186/s12936-020-3139-6Ring-stage survival assayArtemisinin resistancekelch13 |
| spellingShingle | Sage Z. Davis Puspendra P. Singh Katelyn M. Vendrely Douglas A. Shoue Lisa A. Checkley Marina McDew-White Katrina A. Button-Simons Zione Cassady Mackenzie A. C. Sievert Gabriel J. Foster François H. Nosten Timothy J. C. Anderson Michael T. Ferdig The extended recovery ring-stage survival assay provides a superior association with patient clearance half-life and increases throughput Malaria Journal Ring-stage survival assay Artemisinin resistance kelch13 |
| title | The extended recovery ring-stage survival assay provides a superior association with patient clearance half-life and increases throughput |
| title_full | The extended recovery ring-stage survival assay provides a superior association with patient clearance half-life and increases throughput |
| title_fullStr | The extended recovery ring-stage survival assay provides a superior association with patient clearance half-life and increases throughput |
| title_full_unstemmed | The extended recovery ring-stage survival assay provides a superior association with patient clearance half-life and increases throughput |
| title_short | The extended recovery ring-stage survival assay provides a superior association with patient clearance half-life and increases throughput |
| title_sort | extended recovery ring stage survival assay provides a superior association with patient clearance half life and increases throughput |
| topic | Ring-stage survival assay Artemisinin resistance kelch13 |
| url | https://doi.org/10.1186/s12936-020-3139-6 |
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