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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In vitro drug sensitivity assays are indispensable for establishing the correlation between genetic changes in candidate genes and drug response of laboratory strains of P. falciparum, as well as for validating these molecular markers for drug resistance in field isolates. At present, several markers have been identified but not all of them have been validated with in vitro assays in field isolates and/or in vivo response to drug treatment. Molecular markers that have been validated in field isolates by several research groups using in vitro drug sensitivity assays include the dihydrofolate reductase (dhfr) gene versus pyrimethamine and cycloguanil and the P. falciparum chloroquine resistance transporter (pfcrt) gene versus chloroquine.1–5 The available data on the relationship between P. falciparum multidrug resistance gene 1 (pfmdr1) and in vitro resistance of P. falciparum isolates to amino alcohols and artemisinin derivatives are not conclusive.6–8 The relationship between point mutations in the cytochrome bc1 complex gene and atovaquone resistance has not been firmly established using field isolates.9 As for dihydropteroate synthase (dhps) gene versus sulfonamides and sulfones, gene expression studies in heterologous system have provided a biochemical proof of the causal relation between dhps mutations and sulfadoxine resistance,10 but so far there has been no reproducible in vitro assay system to validate this relationship in field isolates.11,12 The present state of limited knowledge on the relevance and utility of these molecular markers for drug-resistant P. falciparum in the field calls for further studies, using both in vitro drug sensitivity assays and tests for therapeutic efficacy.
Some of the major disadvantages of in vitro drug sensitivity assays are the absence of a universally accepted standardized protocol and the requirement for expensive and relatively scarce biologic reagents, especially in endemic countries (for example, type AB non-immune human serum and a constant supply of fresh non-infected erythrocytes from non-immune donors). Other potential technical problems associated with the assessment of parasite growth (counting of schizonts under microscopy or quantification of tritium-labeled hypoxanthine incorporated into the parasite’s DNA) may be solved in the near future by alternative methods. The basic principles of in vitro drug sensitivity assay, which is an application of in vitro culture of malaria parasites, have not changed since Trager and Jensen discovered the suitable experimental conditions for continuous in vitro culture of P. falciparum.13 These include RPMI 1640 culture medium buffered with 25 mM N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid [HEPES]) and 25 mM NaHCO3 and supplemented with 10% human serum, a thin layer of infected erythrocytes, and incubation in an atmosphere of low oxygen at 37°C. Of these requirements, the need for human serum is a major limitation that hinders a wide application of standardized in vitro drug sensitivity assays in endemic countries. The present study was undertaken to assess the suitability of alternative sera, serum substitutes, and serum supplements for both parasite growth and determination of in vitro drug response of fresh clinical isolates without prior adaptation to in vitro culture conditions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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12 years old and adults visiting the Nlongkak Catholic Missionary Dispensary in Yaounde, Cameroon if the following criteria were met: the presence of P. falciparum, without other Plasmodium species, at a parasitemia 0.1%, signs and symptoms of acute uncomplicated malaria, and denial of recent self-medication with an antimalarial drug, confirmed by a negative Saker-Solomons urine test result.14 Pregnant women and patients with signs and symptoms of severe and complicated malaria were excluded from the study. The patients were treated with oral amodiaquine and followed-up by the dispensary staff. This study was reviewed and approved by the Cameroonian National Ethics Committee and the Cameroonian Ministry of Public Health. Culture media. The following media were evaluated in this study: the standard RPMI 1640 medium containing 1 mg/L of p-aminobenzoic acid (PABA) and 1 mg/L of folic acid (Sigma, St. Louis, MO), PABA- and folic acid–free RPMI 1640 medium (Sigma), and PFEK-1 medium (Biochrom KG, Berlin, Germany). Both types of RPMI 1640 media were buffered with 25 mM HEPES and 25 mM NaHCO3.;13 The PFEK-1 medium containing 2.25 g/L of NaHCO3 was either used alone or mixed at various proportions with RPMI 1640 medium (v/v, 3:1, 1:1, 1:3, and 10% PFEK-1).
Serum and serum substitutes. Type AB+ human serum free of virus and other pathogens was obtained from non-immune European donors (Blood Transfusion Center, Strasbourg, France) and pooled. Mycoplasma-free fetal calf serum was obtained from two sources (Seromed®, batch 8R02; Biochrom KG and batch no. 5-41201, Integro b. v., Amsterdam, The Netherlands). AlbumaxTM II, lipid-enriched bovine albumin, and AmnioMaxTM-C100 (probably containing serum from animal sources) were obtained from Invitrogen Life Technologies (Cergy-Pontoise, France). Various concentrations of the following serum-free supplements supplied by Invitrogen Life Technologies were added to RPMI 1640 medium for the evaluation of parasite growth: protein-free OptiMAbTM, chemically-defined emulsified lipid concentrates, Ultroser® HY (constituents including albumin, transferrin, lipids, insulin, vitamins C and B12, selenium, and phosphate-buffered saline), insulin-transferrin-selenium X mixture, and insulin.
Parasite growth. Stock solution of 10% Albumax was prepared in sterile distilled water and filtered through a 0.2-µm filter. Before each experiment, the stock solution was diluted to 2% in RPMI 1640 medium in a volume of 200 µL. Eight additional two-fold serial dilutions, ranging from 0.0078% to 1%, were prepared in RPMI 1640 medium (100 µL). A red blood cell suspension (3% hematocrit) containing 3H-hypoxanthine was prepared in serum-free RPMI 1640 medium and distributed in duplicate (100 µL per well). Parasite growth in various concentrations of Albumax was compared with that of RPMI 1640 medium containing 10% human serum or 10% fetal calf serum, as well as in RPMI 1640 medium alone without serum supplement. The final concentrations of Albumax ranged from 0.0039% to 1% in a final volume of 200 µL (1.5% hematocrit).
In all experiments testing PFEK-1 medium and serum-free media with various concentrations of serum substitutes and supplements, the hematocrit was fixed at 1.5% in a final volume of 200 µL of culture medium. Parasite growth was assessed in duplicate for each concentration of test media, using 0.5 µCi of 3H-hypoxanthine added at the beginning of incubation as an index for parasite growth over a single life cycle. The initial parasitemia was not adjusted in these experiments. The parasites were incubated in an atmosphere of 5% CO2 at 37°C for 42 hours. The incubation was terminated by freezing the test plates at -20°C.
In vitro assays. Chloroquine sulfate, monodesethylamodiaquine dihydrochloride, and cycloguanil base were kindly provided by Aventis (Antony, France), Parke-Davis (Dakar, Senegal), and AstraZeneca (Rueil-Malmaison, France), respectively. Pyrimethamine base was obtained from Sigma. Stock solutions and working solutions of chloroquine, monodesethylamodiaquine, and cycloguanil were prepared in sterile distilled water. Pyrimethamine was dissolved in absolute ethanol. The final concentrations ranged from 25-1, 600 nM to 1,600 nM for chloroquine (two-fold dilutions in triplicate), 5–320 nM for monodesethylamodiaquine (two-fold dilutions in triplicate), and 0.0488–51,200 nM (four-fold dilutions in duplicate) for cycloguanil and pyrimethamine.
Blood samples were washed with PABA- and folic acid–free RPMI 1640 medium three times by centrifugation within two hours after blood extraction. Infected erythrocytes were resuspended in different test media to determine the in vitro activity of the test compounds. For 4-aminoquinolines, the standard RPMI 1640 medium containing 1 mg/L of PABA and 1 mg/L of folic acid was used. For antifolate drugs, PABA- and folic acid–free RPMI 1640 medium was used. The technical procedures of the isotopic microtest were described by Desjardins and others.15 The calculation of the 50% inhibitory concentration (IC50), defined as the drug concentration at which 50% of the parasite growth is inhibited, compared with drug-free control wells, was described in previous reports.3,5,8
Data interpretation. Parasite growth in drug-free wells was expressed as the relative growth index, defined as the percentage of counts per minute (cpm) obtained with test media compared with cpm obtained with RPMI 1640 medium supplemented with 10% human serum. The mean relative growth index for different concentrations of test media obtained from different isolates was compared by the unpaired t-test. The level of significance was fixed at 0.05. The IC50s obtained from the same isolate cultivated in different test media were expressed as ratios.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Overall, parasite growth in 22 isolates did not differ significantly (P > 0.05) with the concentration of Albumax II ranging from 0.004% to 1% (Figure 1
). Albumax II was indispensable to ameliorate parasite growth, as seen when parasite growth was compared with that observed in serum-free RPMI 1640 medium. However, because of the wide inter-strain variation in in vitro growth and a tendency towards decreased growth with decreasing concentration of Albumax II in a few isolates, 0.05% Albumax was determined to be the minimal concentration yielding parasite growth comparable to or equivalent to 70% of growth in culture medium containing 10% human serum in a majority of isolates tested in this study.
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. Compared with the serum-free RPMI 1640 control medium, parasite growth was significantly inhibited (P < 0.05) with pure PFEK-1, a PFEK-1/RPMI 1640 mixture at a 3:1 ratio, and high lipid concentrations. There was a significant improvement (P < 0.05) in parasite growth compared with the serum-free RPMI 1640 control medium with 1–4% Ultroser, but not at higher concentrations (10% and 20%) and 3–20% AmnioMax. Other serum supplements tested in this study did not significantly improve parasite growth as compared with RPMI 1640 medium alone.
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). A similar trend was observed with 0.05% Albumax versus 10% human serum added to PABA- and folic acid–free RPMI 1640 medium to determine antifolate sensitivity (a 2.3-fold increase for pyrimethamine and 2.8-fold for cycloguanil). In contrast, an opposite trend was consistently observed with 10% fetal calf serum, which resulted in an average of a three-fold decrease in the IC50s for pyrimethamine and cycloguanil compared with 10% human serum.
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). Similarly, 3% AmnioMax in RPMI 1640 medium yielded IC50s for chloroquine and monodesethylamodiaquine that were 2.2-fold and 1.6-fold higher than the corresponding values obtained with RPMI 1640 medium supplemented with 10% human serum. In one isolate tested with 10% PFEK-1, the chloroquine IC50 was 3.2-fold higher than with RPMI 1640 medium supplemented with 10% human serum. The pyrimethamine IC50 was determined in seven isolates (IC50 range with 10% human serum = 0.038–3,850 nM). Three isolates yielded interpretable results with OptiMAb, but the pyrimethamine IC50s were, on the average, 12 times higher than those obtained with human serum. Three isolates also yielded IC50s with AmnioMax, which were 2.0- fold higher than those determined with human serum. Four isolates were not sufficiently inhibited at the highest drug concentration used or the experimental points were dispersed so that curve-fitting failed in these cases. There was no interpretable result with PFEK-1 in any of the isolates due to the failure of sigmoidal curve-fitting (n = 5) or failure to grow (n = 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because of the difficulties in obtaining type AB human sera from non-immune donors for malaria parasite cultivation, a number of studies have evaluated alternative sources of animal sera and non-serum substitutes.18–23 Although some of these alternatives support parasite growth, their use has generally been confined to a few laboratories and have not replaced human serum for routine use. An important breakthrough was attained when Ofulla and others have identified serum albumin and lipids as the key serum components necessary for optimal parasite growth.24 Subsequent studies have shown that Albumax, a commercial preparation of lipid-enriched bovine serum albumin, can replace human serum to maintain laboratory-adapted P. falciparum strains in vitro, leading to its increasing routine use in laboratories around the world.11,25–27
Albumax is one of the ideal candidates because it costs less than human serum, is compatible with any blood type, and is not expected to display a wide batch-to-batch difference as in human or animal sera. Based on the success of Albumax II for maintaining laboratory-adapted parasites, its suitability for fresh isolates was assessed. In a previous study on a limited number of fresh isolates, six of nine isolates grew with 0.5% Albumax at least as much as, or better than, with human serum.28 However, when applied to in vitro drug sensitivity assay, the use of 0.5% Albumax resulted in a 1.6-fold increase in IC50s (up to a 13-fold increase for pyrimethamine), compared with those determined with 10% human serum, for most antimalarial drugs, with the exception of halofantrine. The underlying explanation that we proposed includes the difference in drug-protein affinity between human and bovine albumin. In the present study, we initially determined the minimal concentration of Albumax required for parasite growth so that the effect of protein binding can be reduced by decreasing Albumax concentration. The use of 0.05% Albumax did not result in lower IC50s to the level comparable with that for 10% human serum. Even though the comparison was based on a limited number of isolates, further tests were not performed due to the relatively consistent tendency, as well as some uninterpretable results obtained with Albumax and serum supplement-free RPMI 1640 medium. Moreover, in this series of experiments, Albumax did not sustain parasite growth as much as human serum in all fresh isolates.
Fetal calf serum seemed to be a good candidate because one of the two batches tested resulted in consistently satisfactory parasite growth. Chloroquine IC50s with fetal calf serum are 2.3 times higher, while antifolate IC50s are 3 times lower, than with human serum. Since the stock of fetal calf serum from commercial sources is abundant and relatively cheap, it may be an alternative serum that is useful for in vitro drug sensitivity assays. However, batch-to-batch differences in nutritional quality for malaria parasites exist, and the eventual presence of microorganisms that also incorporate 3H-hypoxanthine, such as mycoplasma, needs to be screened. In addition to these factors, more data will be required to define the conversion factor between IC50s determined by using human serum and fetal calf serum before the latter can be used interchangeably with the former.
The earlier-mentioned disadvantages of fetal calf serum for in vitro drug sensitivity assays led us to conduct other experiments with serum-free media. Among these supplements, AmnioMax yielded satisfactory parasite growth and IC50s for chloroquine and monodesethylamodiaquine. This serum supplement was prepared for the primary culture of human cells in amniotic fluid and chorionic villi. However, its components are unknown due to industrial secrecy, although it may be surmised that serum from an unknown animal source at an unknown concentration is present. The other supplements tested in this study are completely devoid of serum. The PFEK-1 medium was developed specifically for serum-free Vero cell culture. In addition to amino acids, vitamins, and various inorganic salts, this culture medium contains fatty acids and components necessary for phospholipid metabolism (choline precursors, cholesterol) as well as 0.02 mg/L of PABA and 1.2 mg/L of folic acid. This medium, when mixed with RPMI 1640 medium, did not yield satisfactory results. Ultroser HY is a serum substitute designed to replace fetal calf serum for in vitro culture of cells. The use of Ultroser supported parasite growth to some extent. However, further studies were not pursued to assess its suitability for in vitro drug sensitivity assays because it contains bovine albumin, as in Albumax, which seems to strongly bind to drugs and influence IC50s. Instead, serum-free RPMI 1640 medium containing other constituents of Ultroser, such as transferrin, lipids, insulin, and selenium, was evaluated but did not yield comparable results as Ultroser.
The results of this study reveal technical difficulties in improving the basic principles formulated by Trager and Jensen to initiate in vitro culture and maintain fresh clinical isolates of P. falciparum, even for a single life cycle, for the purpose of determining drug response. Several serum substitutes from animal sources and serum-free artificial supplements have been identified for continuous in vitro culture of laboratory-adapted parasite strains, but there is still no alternative that yields similar IC50s as human serum. An alternative choice would be to use animal sera or artificial supplements that consistently support parasite growth of fresh isolates and define a conversion factor that would allow expression of the expected IC50s in terms of human serum-supplemented RPMI 1640 medium. Another approach would be to use heat-inactivated human plasma or serum from local donors with no recent history of malarial infection and intake of antimalarial drugs, including some antibiotics.29,30 However, this latter approach would require control experiments to ensure that the batch of plasma or sera from semi-immune donors supports parasite growth. Other commercial serum substitutes or serum supplements need to be assessed for their suitability for culturing P. falciparum and determining in vitro response.
Received September 7, 2002. Accepted for publication April 26, 2003.
Acknowledgment: I thank Sister Marie-Solange Oko, the personnel of the Nlongkak Catholic Missionary Dispensary, and Delphine Ngo Ndombol for their aid in recruiting patients.
Financial support: This study was supported by the French Ministry of Research (Program VIHPAL/PAL+, Actions 2000 and 2001).
Author’s address: Leonardo K. Basco, Unité de Recherche Paludologie Afro-Tropicale, Institut de Recherche pour le Développement, Laboratoire de Recherche sur le Paludisme, Organisation de Coordination pour la Lutte contre les Endémies en Afrique Centrale, BP 288, Yaounde, Cameroon, Telephone: 237-223-22-32, Fax: 237-223-00-61, E-mail: Leonardo.Basco{at}ibaic.u-psud.fr or lkbasco{at}yahoo.fr.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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