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Journal of Clinical Microbiology, August 2007, p. 2734-2736, Vol. 45, No. 8
0095-1137/07/$08.00+0     doi:10.1128/JCM.00479-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Shelf Life of Predosed Plates Containing Mefloquine, Artemisinin, Dihydroartemisinin, and Artesunate as Used for In Vitro Plasmodium falciparum Susceptibility Assessment{triangledown}

Sandrine Houzé,1* Aline Munier,1,{dagger} Xavier Paoletti,2 Halima Kaddouri,1 Pascal Ringwald,3 and Jacques Le Bras1

Laboratoire de Parasitologie, Centre National de Référence du Paludisme, Assistance Publique-Hôpitaux de Paris, Hôpital Bichat-Claude Bernard, and Université Paris Descartes, EA 209, Paris, France,1 Département d'Epidémiologie, de Biostatistique et de Recherche Clinique, Assistance Publique-Hôpitaux de Paris, Hôpital Bichat-Claude Bernard, Paris, France,2 World Health Organization, 1211 Geneva 27, Switzerland3

Received 3 March 2007/ Returned for modification 17 April 2007/ Accepted 30 May 2007


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ABSTRACT
 
The shelf lives of preserved antimalarial agent-predosed plates according to the type of wrapping and the temperature of storage were studied by measuring the 50% inhibitory concentrations of drug for Plasmodium falciparum 3D7. The shelf life of mefloquine was 8 weeks at 25°C; and those of artesunate, artemisinin, and dihydroartemisinin were a minimum of 24, 12, and 8 weeks, respectively, at 4°C.


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TEXT
 
Plasmodium falciparum antimalarial drug resistance contributes to morbidity and requires accurate surveillance. The results of in vitro drug susceptibility testing of P. falciparum have been reported to be disparate (2, 5). Drug susceptibility was mainly assessed by determination of the in vitro drug inhibition of parasite growth (8). Ready-to-use predosed plates are produced under WHO control (17), but the conservation of antimalarial activity over time is a concern.

The present study explored the influences of the storage temperature and the type of wrapping of preserved predosed plates on the 50% inhibitory concentrations (IC50s), which were compared to the IC50s obtained for predosed plates dried for 24 h as a reference, and determined the shelf life of each antimalarial drug.

Mefloquine hydrochloride was obtained from Hoffmann-La Roche (Basel, Switzerland), artesunate was from Aventis (Gentilly, France), and artemisinin and dihydroartemisinin (DHA) were from Sigma-Aldrich Company (St. Louis, MO). The final dilutions of the drugs in water (artemisinins) or methanol (mefloquine) from methanol stock solutions (which were identical throughout the study) were distributed with a MicroLab device (Hamilton, Reno, NV) in 2.5- to 25 µl volumes into 96-well culture plates and dried. The final concentrations ranged from 2.5 to 400 nM for mefloquine and from 0.25 to 40 nM for artemisinin and its derivatives (artemisinins).

One predosed plate dried for 24 h was tested, and the time for that plate was considered week 0. To test the storage conditions, the preserved predosed plates were divided: they were unwrapped, wrapped in aluminum foil and then in a plastic bag closed with a piece of self-adhesive tape (standard WHO packaging), or sealed in an aluminum bag with a desiccant. Each group was divided into three parts; and each part was stored at 4°C (except for the plates in an aluminum bag), 25°C, and 37°C, respectively.

Plasmodium falciparum strain 3D7 was cryopreserved and thawed before each measurement. It was cultivated in erythrocytes resuspended in RPMI 1640 medium supplemented with 10% human serum (Biowest, Nuaillé, France) at 37°C in a 5% CO2- 5% O2-85% N2 atmosphere until a minimum parasite density of 1% was achieved (16). The susceptibilities to the antimalarial drugs were determined after suspension in the same medium and addition of uninfected erythrocytes to obtain 0.3% parasitemia and 1.5% hematocrit. The culture obtained within three asexual cycles after thawing contained a minimum of 90% ring forms and did not require synchronization (14).

The isotopic microtest described previously (12) was used for the in vitro assay. IC50s were determined by nonlinear regression analysis with an inhibitory sigmoid maximum-effect model. The estimated parameters were the IC50 and its confidence interval (CI) (12).

Each condition except for the plates in an aluminum bag was tested at 1, 2, 3, 4, 6, 8, 12, 16, and 24 weeks; the plates in an aluminum bag were tested every two attempts (i.e., at 1, 3, 6, 12, and 24 weeks). As reference, a plate on which drugs had been distributed 24 h earlier and dried was tested concomitantly.

The repeatability analysis relied on a type III analysis of variance for investigation of the parasite suspension, drug, and experimental factors. We express the results as the coefficients of variation (CVs). CVs defined the limits of the plates’ acceptabilities. We then investigated the evolution of the IC50 over time compared to these limits.

The repeatability was assessed by the use of 16 independent IC50 measurements. The overall variation was between 14% (mefloquine) and 25% (artemisinin and derivatives), which were further considered the limits of acceptance. The plate was then considered not valid if the IC50 ± 95% CI was off from the limits of acceptance (IC50 ± CV for the reference plate) for two successive measurements for a given drug and the condition was not tested again.

At 37°C, whatever the conditions of packaging or the drug used, the plates were no longer valid after 1 week. The validity was longer at 4°C than at 25°C (P = 0.002) for the artemisinins (Fig. 1) but not for mefloquine (Table 1). The packaging had no influence on the shelf life (Table 1). The results for the predosed plates altered with time (P < 0.0001) more rapidly for the artemisinins (P < 0.0001) than for mefloquine (P = 0.04). In addition, the effect of time on the degradation of the plates was higher at 25°C (P < 0.0001) than at 4°C (P = 0.04) (Fig. 1). The limits of the shelf life at 4°C were 6 to 8 weeks for mefloquine, 8 weeks for DHA, 12 weeks for artemisinin, and 24 weeks for artesunate (Table 1). In the case of storage at 25°C, the limits were 6 to 8 weeks for mefloquine, 4 weeks for DHA, 8 weeks for artemisinin, and 12 weeks for artesunate, according to the packaging (Table 1).


Figure 1
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  		FIG. 1. Variations in IC50s of four antimalarials for P. falciparum clone 3D7 over time according to two shelf conditions until the limit of the shelf life: artemisinin and artesunate (A) and mefloquine and dihydroartemisinin (B). To make the IC50 evolution comparable for each condition, IC50s were standardized to the mean of the reference IC50. The same parasite suspension was used each week for all conditions. Gray bars, limits of acceptance for each weekly measure (IC50 ± CV); for artemisinin, open squares, reference; solid squares, +4°C without wrapping; gray squares,+25°C in a plastic bag; for artesunate, open circles, reference; solid circles, +4°C without wrapping; gray circles, +25°C in a plastic bag; for mefloquine, open triangles, reference; solid triangles, +4°C without wrapping; gray triangles, +25°C in a plastic bag; for DHA, open diamonds, reference; solid diamonds, +4°C without wrapping; gray diamonds, +25°C in a plastic bag.


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  		TABLE 1. Shelf lives of predosed plates according to drug, storage temperature, and packaging

One study on the conservation of predosed plates has been published (11). The exploration of drugs by measurement by high-pressure liquid chromatography is not possible due to the level of detection by that method. The only possible control is a biological assay. We observed that the shelf life is often better at 4°C without packaging.

The IC50 obtained by a microtest is the result of many interactions (3, 10). In fact, if practical procedures that have been suggested previously (18) are used, numerous variations in the details of these protocols make it impossible to compare these IC50 values between laboratories worldwide.

We consider the IC50 of a fresh dried plate (24 h) to be reference of the plate's stability. We had to define the limits of acceptability in the variation of the IC50s in different experiments. In previous studies, the CVs were low (less than 10%) (1) or high (10 to 40%), according to the test, the strain, and the drug used (7). CVs that depended on the drugs used (9) were chosen as limits of acceptability for the molecule.

The packaging in an aluminum bag with desiccant, which could allow degradation as a result of exposure to light and humidity to be avoided, was not better than the packaging recommended by WHO. The exact cause of the quick degradation (in terms of a decrease in bioavailability or chemical degradation) observed at 37°C and the slower degradation at 25°C has not been determined (13). The quality of the plates supplied or their incorrect use could be responsible for the false drug susceptibility or resistance results (4). Even with Plasmodium lactate dehydrogenase or histidine-rich protein 2 measurements (6, 14), the culture of parasites with drugs still remains a factor limiting the collection of sound data. The first step for the retrieval of reproducible and reliable results would be the availability of a ready-to-use method (15). As very few studies on the validation of plates for the study of antimalarial drug susceptibility are available, no comparison may help to clearly identify the weakness of using either predosed plates or plates prepared at the time of use.

In conclusion, we recommend drugs with very different shelf lives not be included in the same plate and that the drug with shortest shelf live be the plate life. When a drug with a very short shelf life is to be tested, it is better to prepare the plates at the time of use.


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ACKNOWLEDGMENTS
 
This work was supported by WHO, Geneva, Switzerland.


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratoire de Parasitologie, CNR Paludisme, Hôpital Bichat-Claude Bernard, 46, Rue Henri Huchard, 75877 Paris Cedex 18, France. Phone: (331) 40257881. Fax: (331) 40256763. E-mail: sandrine.houze{at}bch.aphp.fr Back

{triangledown} Published ahead of print on 6 June 2007. Back

{dagger} Present address: IRD UR 009, Dakar, Senegal. Back


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Journal of Clinical Microbiology, August 2007, p. 2734-2736, Vol. 45, No. 8
0095-1137/07/$08.00+0     doi:10.1128/JCM.00479-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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