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P. falciparum in vitro killing rates allow to discriminate between different antimalarial mode-of-action.

Sanz LM, Crespo B, De-Cózar C, Ding XC, Llergo JL, Burrows JN, García-Bustos JF, Gamo FJ - PLoS ONE (2012)

Bottom Line: Moreover, traditional techniques do not allow to measure the speed-of-action of compounds on parasite viability, which is an essential efficacy determinant.We present here a comprehensive methodology to measure in vitro the direct effect of antimalarial compounds over the parasite viability, which is based on limiting serial dilution of treated parasites and re-growth monitoring.This methodology allows to precisely determine the killing rate of antimalarial compounds, which can be quantified by the parasite reduction ratio and parasite clearance time, which are key mode-of-action parameters.

View Article: PubMed Central - PubMed

Affiliation: Tres Cantos Medicine Development Campus, Diseases of the Developing World, GlaxoSmithKline, Tres Cantos, Madrid, Spain.

ABSTRACT
Chemotherapy is still the cornerstone for malaria control. Developing drugs against Plasmodium parasites and monitoring their efficacy requires methods to accurately determine the parasite killing rate in response to treatment. Commonly used techniques essentially measure metabolic activity as a proxy for parasite viability. However, these approaches are susceptible to artefacts, as viability and metabolism are two parameters that are coupled during the parasite life cycle but can be differentially affected in response to drug actions. Moreover, traditional techniques do not allow to measure the speed-of-action of compounds on parasite viability, which is an essential efficacy determinant. We present here a comprehensive methodology to measure in vitro the direct effect of antimalarial compounds over the parasite viability, which is based on limiting serial dilution of treated parasites and re-growth monitoring. This methodology allows to precisely determine the killing rate of antimalarial compounds, which can be quantified by the parasite reduction ratio and parasite clearance time, which are key mode-of-action parameters. Importantly, we demonstrate that this technique readily permits to determine compound killing activities that might be otherwise missed by traditional, metabolism-based techniques. The analysis of a large set of antimalarial drugs reveals that this viability-based assay allows to discriminate compounds based on their antimalarial mode-of-action. This approach has been adapted to perform medium throughput screening, facilitating the identification of fast-acting antimalarial compounds, which are crucially needed for the control and possibly the eradication of malaria.

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Schematic representation of the in vitro PRR assay.A. Intraerythrocytic P. falciparum cultured at 0.5% parasitemia and 2% hematocrit is treated with drugs. The medium is exchanged and the drug replenished every 24 hours. Aliquots corresponding to 105 parasites are taken out at defined time points, washed, and free-drug parasites cultured with fresh erythrocytes under limiting serial dilution conditions (see Material and Methods). Parasite growth is subsequently monitored after 21 days and confirmed after 28 days, allowing to calculate the initial number of viable parasite in the aliquot. B. Parasite viability measurement allows in turn to determine the drug lag phase (i.e. time needed to reach the maximal rate of killing), PRR over one life cycle, and 99.9% PCT (i.e. the time needed to decrease the number of viable parasites by 3 –log units). The data presented in this panel are for illustration purpose only. Axe Y shows log (viable parasites +1) to allow representation of logarithms when counting of number of viable parasites is equal to zero.
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pone-0030949-g001: Schematic representation of the in vitro PRR assay.A. Intraerythrocytic P. falciparum cultured at 0.5% parasitemia and 2% hematocrit is treated with drugs. The medium is exchanged and the drug replenished every 24 hours. Aliquots corresponding to 105 parasites are taken out at defined time points, washed, and free-drug parasites cultured with fresh erythrocytes under limiting serial dilution conditions (see Material and Methods). Parasite growth is subsequently monitored after 21 days and confirmed after 28 days, allowing to calculate the initial number of viable parasite in the aliquot. B. Parasite viability measurement allows in turn to determine the drug lag phase (i.e. time needed to reach the maximal rate of killing), PRR over one life cycle, and 99.9% PCT (i.e. the time needed to decrease the number of viable parasites by 3 –log units). The data presented in this panel are for illustration purpose only. Axe Y shows log (viable parasites +1) to allow representation of logarithms when counting of number of viable parasites is equal to zero.

Mentions: A comprehensive methodology has been developed to directly measure the net effect of chemical compounds on the viability of intraerythrocytic asexual forms of P. falciparum parasites (Figure 1A). The principle of the method is the following. An initial inoculum of 106 intraerythrocytic parasites per milliliter, as determined by microscopy, is established at 0.5% parasitemia and 2% hematocrit. These conditions are exactly identical to the ones used for standard IC50 determination using a tritiated [3H]-hypoxanthine incorporation assay [9]. To avoid multiple infections, original cultures are incubated under shaking conditions. Resulting cultures present less than 1% erythrocytes with multiple infections. Once drug treatment is initiated, the effect of the inhibitor on parasite viability is monitored by taking out an aliquot corresponding to 105 parasites of the initial population every 24 hours, washing it thoroughly, adding fresh erythrocytes and performing serial dilutions in a microtiter plate. The number of parasites in the initial inoculum is calculated by performing the same actions with an aliquot of the culture before starting treatment. The limiting serial dilution culture is maintained for up to 28 days and the presence of growing parasites is terminally determined in each well by using any standard technique able to detect parasite growth, such as pLDH detection or [3H]-hypoxanthine incorporation. The number of viable parasites initially present in the aliquot, that is the number of parasites able to recrudesce upon removal of the drug, can then be back-calculated based on the most diluted well able to render growth. By repeating this procedure for multiple time points, here every 24 hours for up to 120 hours, it is possible to obtain a direct measurement of parasite viability over time in response to drug treatment. In vitro PRR is calculated as the decrease in viable parasites over 48 hours, that is one parasite life cycle, and is a direct measurement of the killing rate for the compound investigated (Figure 1B). Hence, a compound leaving 103 parasites alive out of 105 after 48 hours of treatment has a PRR of 102 (105/103) or log10(PRR) of 2, hereafter referred to as log(PRR). A “lag phase” is considered to occur for as long as drug treatment does not produced the maximal rate of killing, and this period of time is excluded for PRR calculation. In a practical way, 0–24 or 0–48 hours stretches are considered part of the lag phase when estimated reduction of parasite viability over 48 hours, (extrapolated from 0–24 hours in the first case), is more than one order of magnitude below the calculated PRR using the linear stretch of the profile. It is important to note that a lag phase is not observed in all profiles. 99.9% PCT, that is the time needed to clear 99.9% of the initial parasite population, is determined using a regression calculated on the log-linear phase of the parasite reduction and takes the lag phase into account (Figure 1B). In summary, measuring parasite viability over time in response to drug treatment allows to determine key in vitro parameters of the compound killing rate such as lag phase, PRR and 99.9% PCT values.


P. falciparum in vitro killing rates allow to discriminate between different antimalarial mode-of-action.

Sanz LM, Crespo B, De-Cózar C, Ding XC, Llergo JL, Burrows JN, García-Bustos JF, Gamo FJ - PLoS ONE (2012)

Schematic representation of the in vitro PRR assay.A. Intraerythrocytic P. falciparum cultured at 0.5% parasitemia and 2% hematocrit is treated with drugs. The medium is exchanged and the drug replenished every 24 hours. Aliquots corresponding to 105 parasites are taken out at defined time points, washed, and free-drug parasites cultured with fresh erythrocytes under limiting serial dilution conditions (see Material and Methods). Parasite growth is subsequently monitored after 21 days and confirmed after 28 days, allowing to calculate the initial number of viable parasite in the aliquot. B. Parasite viability measurement allows in turn to determine the drug lag phase (i.e. time needed to reach the maximal rate of killing), PRR over one life cycle, and 99.9% PCT (i.e. the time needed to decrease the number of viable parasites by 3 –log units). The data presented in this panel are for illustration purpose only. Axe Y shows log (viable parasites +1) to allow representation of logarithms when counting of number of viable parasites is equal to zero.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3285618&req=5

pone-0030949-g001: Schematic representation of the in vitro PRR assay.A. Intraerythrocytic P. falciparum cultured at 0.5% parasitemia and 2% hematocrit is treated with drugs. The medium is exchanged and the drug replenished every 24 hours. Aliquots corresponding to 105 parasites are taken out at defined time points, washed, and free-drug parasites cultured with fresh erythrocytes under limiting serial dilution conditions (see Material and Methods). Parasite growth is subsequently monitored after 21 days and confirmed after 28 days, allowing to calculate the initial number of viable parasite in the aliquot. B. Parasite viability measurement allows in turn to determine the drug lag phase (i.e. time needed to reach the maximal rate of killing), PRR over one life cycle, and 99.9% PCT (i.e. the time needed to decrease the number of viable parasites by 3 –log units). The data presented in this panel are for illustration purpose only. Axe Y shows log (viable parasites +1) to allow representation of logarithms when counting of number of viable parasites is equal to zero.
Mentions: A comprehensive methodology has been developed to directly measure the net effect of chemical compounds on the viability of intraerythrocytic asexual forms of P. falciparum parasites (Figure 1A). The principle of the method is the following. An initial inoculum of 106 intraerythrocytic parasites per milliliter, as determined by microscopy, is established at 0.5% parasitemia and 2% hematocrit. These conditions are exactly identical to the ones used for standard IC50 determination using a tritiated [3H]-hypoxanthine incorporation assay [9]. To avoid multiple infections, original cultures are incubated under shaking conditions. Resulting cultures present less than 1% erythrocytes with multiple infections. Once drug treatment is initiated, the effect of the inhibitor on parasite viability is monitored by taking out an aliquot corresponding to 105 parasites of the initial population every 24 hours, washing it thoroughly, adding fresh erythrocytes and performing serial dilutions in a microtiter plate. The number of parasites in the initial inoculum is calculated by performing the same actions with an aliquot of the culture before starting treatment. The limiting serial dilution culture is maintained for up to 28 days and the presence of growing parasites is terminally determined in each well by using any standard technique able to detect parasite growth, such as pLDH detection or [3H]-hypoxanthine incorporation. The number of viable parasites initially present in the aliquot, that is the number of parasites able to recrudesce upon removal of the drug, can then be back-calculated based on the most diluted well able to render growth. By repeating this procedure for multiple time points, here every 24 hours for up to 120 hours, it is possible to obtain a direct measurement of parasite viability over time in response to drug treatment. In vitro PRR is calculated as the decrease in viable parasites over 48 hours, that is one parasite life cycle, and is a direct measurement of the killing rate for the compound investigated (Figure 1B). Hence, a compound leaving 103 parasites alive out of 105 after 48 hours of treatment has a PRR of 102 (105/103) or log10(PRR) of 2, hereafter referred to as log(PRR). A “lag phase” is considered to occur for as long as drug treatment does not produced the maximal rate of killing, and this period of time is excluded for PRR calculation. In a practical way, 0–24 or 0–48 hours stretches are considered part of the lag phase when estimated reduction of parasite viability over 48 hours, (extrapolated from 0–24 hours in the first case), is more than one order of magnitude below the calculated PRR using the linear stretch of the profile. It is important to note that a lag phase is not observed in all profiles. 99.9% PCT, that is the time needed to clear 99.9% of the initial parasite population, is determined using a regression calculated on the log-linear phase of the parasite reduction and takes the lag phase into account (Figure 1B). In summary, measuring parasite viability over time in response to drug treatment allows to determine key in vitro parameters of the compound killing rate such as lag phase, PRR and 99.9% PCT values.

Bottom Line: Moreover, traditional techniques do not allow to measure the speed-of-action of compounds on parasite viability, which is an essential efficacy determinant.We present here a comprehensive methodology to measure in vitro the direct effect of antimalarial compounds over the parasite viability, which is based on limiting serial dilution of treated parasites and re-growth monitoring.This methodology allows to precisely determine the killing rate of antimalarial compounds, which can be quantified by the parasite reduction ratio and parasite clearance time, which are key mode-of-action parameters.

View Article: PubMed Central - PubMed

Affiliation: Tres Cantos Medicine Development Campus, Diseases of the Developing World, GlaxoSmithKline, Tres Cantos, Madrid, Spain.

ABSTRACT
Chemotherapy is still the cornerstone for malaria control. Developing drugs against Plasmodium parasites and monitoring their efficacy requires methods to accurately determine the parasite killing rate in response to treatment. Commonly used techniques essentially measure metabolic activity as a proxy for parasite viability. However, these approaches are susceptible to artefacts, as viability and metabolism are two parameters that are coupled during the parasite life cycle but can be differentially affected in response to drug actions. Moreover, traditional techniques do not allow to measure the speed-of-action of compounds on parasite viability, which is an essential efficacy determinant. We present here a comprehensive methodology to measure in vitro the direct effect of antimalarial compounds over the parasite viability, which is based on limiting serial dilution of treated parasites and re-growth monitoring. This methodology allows to precisely determine the killing rate of antimalarial compounds, which can be quantified by the parasite reduction ratio and parasite clearance time, which are key mode-of-action parameters. Importantly, we demonstrate that this technique readily permits to determine compound killing activities that might be otherwise missed by traditional, metabolism-based techniques. The analysis of a large set of antimalarial drugs reveals that this viability-based assay allows to discriminate compounds based on their antimalarial mode-of-action. This approach has been adapted to perform medium throughput screening, facilitating the identification of fast-acting antimalarial compounds, which are crucially needed for the control and possibly the eradication of malaria.

Show MeSH
Related in: MedlinePlus