Abstract
Malaria remains an overwhelming problem in Africa, where control is threatened by drug resistance. Ex vivo assays have been used to evaluate the sensitivity of P. falciparum to antimalarials. Short-term refrigerated storage of field samples facilitates surveillance at sites distant from the laboratory, but the effects of parasite storage on ex vivo assay results are unknown. We assessed 130 P. falciparum samples collected from malaria patients in Tororo and Busia in eastern Uganda, using a standard 72-h SYBR Green assay, to compare ex vivo sensitivities of isolates assayed on the same day of sample collection (day 0) and after overnight storage at 4-8º C (day 1), against 8 antimalarials. We compared median 50% inhibitory concentrations (IC50s) on day 0 and on day 1 using the
Wilcoxon matched-pairs signed rank test. The median time from sample collection to initiation of ex vivo assays was 4.9 h on day 0 and 23 h on day 1. IC50s differed only slightly between day 0 and day 1 for all tested drugs, with results mostly slightly lower on day 1: chloroquine (24 vs 22 nM, p=0.02), MDAQ (7.3 vs 6.8 nM, p=0.008), piperaquine (8.1 vs 7.2 nM, p=0.08), pyronaridine (1.6 vs 1.1 nM, p<0.0001), mefloquine (12 vs 9.9 nM, p=0.10), lumefantrine (5.8 vs 4.9 nM, p=0.0002), DHA (1.9 vs 1.3 nM, p<0.0001), and pyrimethamine (41,645 vs 43,055 nM, p=0.19). We also observed that parasite growth over 72 h in drug-free wells was reduced by 20% in day 1 assays relative to day 0 assays (p<0.0001). We have found that sensitivities against laboratory and field strains can vary significantly for certain drugs based on methods of drug dilution and storage, which likely effects limits on solubility and stability for many compounds. We note concerns for lumefantrine and mefloquine, for which unusually high IC50s can be seen if compound solubilization is inadequate before serial dilution or resulting from multiple freeze-thaws of DMSO stocks. In summary, preparation of diluted drug stocks up to 24 h before assay initiation had minimal effects on ex vivo IC50 determinations, but care to assure solubilization and limiting freeze-thaws is essential to obtaining reliable measures of ex vivo drug sensitivity.
Wilcoxon matched-pairs signed rank test. The median time from sample collection to initiation of ex vivo assays was 4.9 h on day 0 and 23 h on day 1. IC50s differed only slightly between day 0 and day 1 for all tested drugs, with results mostly slightly lower on day 1: chloroquine (24 vs 22 nM, p=0.02), MDAQ (7.3 vs 6.8 nM, p=0.008), piperaquine (8.1 vs 7.2 nM, p=0.08), pyronaridine (1.6 vs 1.1 nM, p<0.0001), mefloquine (12 vs 9.9 nM, p=0.10), lumefantrine (5.8 vs 4.9 nM, p=0.0002), DHA (1.9 vs 1.3 nM, p<0.0001), and pyrimethamine (41,645 vs 43,055 nM, p=0.19). We also observed that parasite growth over 72 h in drug-free wells was reduced by 20% in day 1 assays relative to day 0 assays (p<0.0001). We have found that sensitivities against laboratory and field strains can vary significantly for certain drugs based on methods of drug dilution and storage, which likely effects limits on solubility and stability for many compounds. We note concerns for lumefantrine and mefloquine, for which unusually high IC50s can be seen if compound solubilization is inadequate before serial dilution or resulting from multiple freeze-thaws of DMSO stocks. In summary, preparation of diluted drug stocks up to 24 h before assay initiation had minimal effects on ex vivo IC50 determinations, but care to assure solubilization and limiting freeze-thaws is essential to obtaining reliable measures of ex vivo drug sensitivity.
| Original language | American English |
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| State | Published - 2020 |
| Event | American Society of Tropical Medicine and Hygiene Annual Conference - Online Duration: Nov 15 2020 → Nov 19 2020 |
Conference
| Conference | American Society of Tropical Medicine and Hygiene Annual Conference |
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| Abbreviated title | ASTMH2020 |
| Period | 11/15/20 → 11/19/20 |