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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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The standardization and optimization of the in vitro drug sensitivity assay systems for field isolates of Plasmodium falciparum are one of the prerequisites for validating molecular markers for drug resistance. In areas where high rates of therapeutic failure to chloroquine and sulfadoxine-pyrimethamine monotherapy are encountered, it has become difficult to justify, on ethical grounds, prospective clinical studies that aim to correlate clinical and parasitologic response and the corresponding markers: P. falciparum chloroquine resistance transporter for chloroquine, dihydropteroate synthase for sulfadoxine, and dihydrofolate reductase for pyrimethamine. The current strategy adopted by many malaria-endemic countries to combat drug-resistant malaria is based on combination therapies, in particular artemisinin-based combination therapies.3 In this context, the correlation between the in vivo response and molecular markers is difficult to establish. In vitro drug assay performed on field isolates is a useful research tool in these situations to determine the drug response independently of the treatment that the patient receives.
The cultivation method for P. falciparum that is routinely performed at present was developed by Trager and Jensen approximately 30 years ago.4 The mixture of RPMI 1640 medium, buffered with 25 mM HEPES buffer and 25 mM sodium bicarbonate, and 10% human serum has been the standard culture medium. Some minor modifications, such as hypoxanthine and glucose supplements and serum substitutes (fetal calf serum and Albumax®; Invitrogen Life Technologies, Cergy Pontoise, France) have been introduced and used as routine procedures for continuous cultivation in some laboratories. For field isolates, the initial phase of abrupt transition from in vivo to in vitro conditions is one of the key steps in the adaptation process that the parasites undergo before continuous cultivation becomes possible. Since some fresh isolates fail to grow, transform massively into gametocytes, or develop poorly in vitro, one of the possible reasons may be suboptimal culture conditions. Based on the assumption that the rings of all field isolates freshly obtained from patients without recent antimalarial medication are capable of developing into schizonts, the present study was designed to explore alternative media to propagate P. falciparum for at least a single erythrocytic cycle and compare chloroquine response of isolates cultivated in alternative media.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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12 years of age and adults visiting the Nlongkak Catholic Missionary Dispensary in 2003–2005 in Yaounde, Cameroon if the following criteria were met: the presence of P. falciparum, without other Plasmodium species, at a parasitemia 0.2%, signs and symptoms of acute uncomplicated malaria, and denial of recent self-medication with an antimalarial drug, confirmed by a negative Saker-Solomon’s urine test result.5 Blood samples (7 mL) were collected by venipuncture in tubes containing the anticoagulant EDTA. 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, with or without sulfadoxine-pyrimethamine, 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. Parasite growth was evaluated in the following media: the standard RPMI 1640 medium containing 1 mg/L of p-aminobenzoic acid and 1 mg/L of folic acid, Dulbecco’s modified Eagle’s medium (DME), Dulbecco’s modified Eagle’s medium–Ham’s F-12 nutrient medium mixture (DME/F-12, 1:1 [v/v]), Ham’s F-12 nutrient medium (F-12), Iscove’s modified Dulbecco’s medium (IMDM), L-15 medium Leibovitz (L-15), medium 199 with Earle’s salts (M199E), medium 199 with Hanks’ salts (M199H), minimum essential medium with Earle’s salts (MEM), McCoy’s 5A medium, Waymouth MB 752/1 medium, OptiMEM I® (Invitrogen Life Technologies) (derived from MEM), and GIT medium (1:1 mixture of IMDM and F-12 supplemented with ammonium sulfate fraction of adult bovine serum). All media were obtained from Invitrogen Life Technologies, except for GIT medium, which was obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan). All media were buffered with 25 mM HEPES and 25 mM NaHCO3. D-glucose (2 g/L) was added to L-15 medium, which contains D-galactose instead of D-glucose. Glucose concentration in other media (range = 1–5 g/L; RPMI 1640 medium contains 2 g/L) was not modified. Because of the unknown composition, OptiMEM I® and GIT medium were used without further modifications.
Serum and serum substitutes. With the exception of OptiMEM I® (1–5% human serum) and GIT medium (free of human serum), media were supplemented with 10% (v/v) type AB+ pooled non-immune human serum (batch no. S02909S4190; Bio West, Nuaillé, France). Sera from several animal sources were used to supplement the standard RPMI 1640 medium (10% [v/v]). Mycoplasma-free fetal calf serum was obtained from three sources (batch 8R02; Seromed Bio-chrom KG, Berlin, Germany; batch no. 5–41201, Integro b. v., Amsterdam, The Netherlands; and batch no. 382245 from New Zealand, Invitrogen Life Technologies). Mycoplasma-tested sera from rabbit (batch no. 1132782), horse (batch no. 413335, from New Zealand), and goat (batch no. 394315, from New Zealand) were obtained from Invitrogen Life Technologies. Protein-free, cholesterol–lipid concentrate (Invitrogen Life Technologies) and Daigo’s GF21 (a chemically defined fraction of adult bovine serum that is also in the GIT medium; Wako Pure Chemical Industries Ltd.) were used as serum-free supplements of RPMI 1640 medium for the evaluation of parasite growth. Serum- and protein-free hybridoma medium (PFHM II; Invitrogen Life Technologies) was also assessed for its capacity to support parasite growth without serum supplement.
Parasite growth. The in vitro growth of freshly collected P. falciparum isolates in various culture media and sera or serum substitutes was compared with that of RPMI 1640 medium supplemented with 10% human serum. Fresh blood samples were washed with RPMI 1640 medium once (to provide D-glucose to the parasites during the washing procedures) and with phosphate-buffered saline twice by centrifugation. In all experiments, 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 test medium, using 1 µCi/well of 3H-hypoxanthine (Amersham International, Buckinghamshire, United Kingdom) added at the beginning of incubation as an index for parasite growth over a single life cycle. The initial parasitemia (0.2–2.0%) was not adjusted in these experiments. The parasites were incubated in 96-well microtiter plates in an atmosphere of 5% CO2 at 37°C for 42 hours. The incubation was terminated by freezing the test plates at –20°C. To assess parasite growth during the second erythrocytic cycle, the isolates were cultivated in test media without 3H-hypoxanthine for 42 hours in 35-mm petri dishes. Spent media were replaced with fresh complete media, and growth was assessed as described above, terminating the incubation at 72 hours. The second-cycle growth index was defined as the percentage of cpm during the second cycle divided by cpm during the first cycle.
In vitro assays. Chloroquine phosphate was obtained from Sigma Chemical Co (St. Louis, MO). Stock solutions and working solutions were prepared in sterile distilled water. Test microtiter plates were predosed with drug solutions and air dried. The final concentrations ranged from 3.12 nM to 3,200 nM (two-fold dilutions in duplicate).
Infected erythrocytes were resuspended in different test media to determine the in vitro activity of chloroquine. The technical procedures of the radioisotopic microtest were have been previously described.6,7 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 determined by non-linear regression analysis of logarithm of concentrations plotted against growth inhibition. A sigmoid curve was fitted to the plot using the PrismTM software (GraphPad Software, Inc., San Diego, CA). The threshold value for in vitro resistance to chloroquine was estimated to be 100 nM.8,9
Data interpretation. Parasite growth in various culture media was expressed as the relative growth index, defined as the percentage of cpm obtained with test media compared with cpm obtained with RPMI 1640 medium supplemented with 10% human serum. The quantity of 3H-hypoxanthine incorporated by the parasites cultivated in different test media and the geometric mean IC50 values determined using different culture media were compared by the repeated measures one-way analysis of variance and Dunnett’s multiple comparison post test. The significance level was set at 0.05 for all statistical tests.
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RESULTS |
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90% growth in RPMI 1640) in DME (280 ± 132%, 91–598%; P < 0.05) and IMDM (271 ± 109%, 107–520%; P < 0.05). The mean parasite growth (± SD, range) was relatively higher in L-15 (182 ± 112%, 50–475%), M199/E (132 ± 66%, 24–280%), M199/H (121 ± 58%, 33–257%), MEM (188 ± 97%, 52–401%), and McCoy’s 5A (112 ± 53%, 40–266%), but some isolates (5–13 of 33 isolates, 15–39%) grew better in RPMI 1640 medium than in these five alternative media. The addition of F-12 medium to DME was associated with a lower incorporation of 3H-hypoxanthine because of non-radioactive hypoxanthine that interferes with data interpretation (4.77 mg/L of hypoxanthine in F-12; for comparison, 0.354 mg/L in M199 and none in RPMI 1640 medium, DME, and IMDM). The assessment of growth in Waymouth medium was terminated after the first 10 isolates showed non-interpretable results because of the relatively high concentration (25 mg/L) of non-radioactive hypoxanthine present in the medium. Of 33 isolates, 26 were also cultivated for 72 hours. Six isolates showed evidence for an increased parasitemia during the second erythrocytic cycle, as suggested by the higher incorporation of 3H-hypoxanthine than during the first erythrocytic cycle. The mean second-cycle growth index (range) of these six isolates in RPMI 1640 medium was 201% (137–282%). The corresponding mean index values in the other test media were lower (82–161%). In four of six isolates, growth was poorer during the second cycle than during the first cycle in several alternative media. For seven isolates that showed either an unchanged second-cycle growth index (i.e., 100%, n = 1) or an index between 20% and 60% (n = 6) in RPMI 1640 medium, growth was generally poorer in other test media, with the exception of DME, which yielded a comparable growth index in three isolates. Thirteen isolates that showed poor growth during the second cycle (i.e., second-cycle growth index < 20%) in RPMI 1640 medium also had generally poor growth in other media.
In the second series of experiments, 32 of 40 consecutive fresh isolates (four excluded because of high parasitemia > 2% and four consecutive samples because of technical errors) were cultivated for 42 hours in serum-free media (cholesterol–lipid concentrate, GIT, GF21, PFHM II), low-serum medium (OptiMEM I® supplemented with 1% human serum), and RPMI 1640 medium supplemented with 10% animal serum. The positive controls were new preparations of DME, IMDM, and RPMI media supplemented with 10% human serum (range of growth in RPMI–10% human serum = 1,290–18,600 cpm, n = 32). GIT medium (only one isolate with < 80% relative growth compared with RPMI–10% human serum) and RPMI supplemented with 10% goat serum (only 2 of 32 isolates with < 80% relative growth) yielded a consistently high growth rate (P < 0.05; Figure 1
). Parasite growth was less consistent with OptiMEM I® supplemented with 1% human serum (mean relative growth = 134%, 16 of 32 grew better [i.e. 90% relative growth] than in RPMI 1640 medium with 10% human serum) and horse serum (mean relative growth = 80%, 13 of 32 isolates grew better than in RPMI 1640 medium with 10% human serum) and poor with RPMI 1640 medium supplemented with GF21 (mean relative growth = 70%). The experiments with PFHM II (mean relative growth = 66%), serum-free RPMI 1640 medium supplemented with cholesterol–lipid concentrate (mean relative growth = 56%), and RPMI 1640 medium supplemented with 10% rabbit serum (mean growth = 32%) were terminated after the first 12 isolates because of consistently poor growth. One batch of fetal calf serum obtained from Invitrogen Life Technologies yielded inconsistent results (six isolates grew as much as or better than in RPMI 1640 medium with 10% human serum, with a relative growth of 90–130%; 11 grew poorly, with a relative growth of 32–80%, n = 17). Two batches of fetal calf serum from other commercial suppliers resulted in a consistently good to better growth (94–435% relative growth) in nine isolates tested with three batches of fetal calf serum.
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In vitro drug sensitivity assays.
In the first series of drug assays, the in vitro chloroquine response of 10 isolates was determined in five different media (RPMI 1640, DME, IMDM, L-15, and MEM) supplemented with 10% human serum (Table 1). The results of two isolates were excluded because of bacterial contamination or poor parasite growth in RPMI 1640 medium (< 1,000 cpm in control wells). Except for one isolate (Y01/04), the isolates cultivated in drug-free control wells generally incorporated higher quantities of 3H-hypoxanthine in the alternative media than in RPMI 1640 medium during the 42-hour incubation period. Chloroquine IC50 values obtained in the alternative media were generally higher (statistical test not done because of small sample size) than those determined with the standard RPMI 1640 medium.
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). The results of two isolates with low parasitemia (0.2%) were excluded because of poor growth in RPMI 1640 medium. Although preliminary assays suggested possible differences in IC50 values (Table 1
), the geometric mean chloroquine IC50 (95% confidence intervals) determined with RPMI 1640–10% human serum (112 nM, 73.3–171 nM) did not differ significantly (P > 0.05) from the corresponding values obtained with DME–10% human serum (124 nM, 75.9–204 nM) and IMDM–10% human serum (89.3 nM, 57.7–138 nM). In six isolates, the classification of in vitro chloroquine response, based on the arbitrary cut-off value of 100 nM, was discordant in these three media, but in two of these isolates, the IC50 values were within borderline values (80–120 nM). In contrast, the geometric mean chloroquine IC50 values (95% confidence intervals) were significantly higher (P < 0.05) in RPMI 1640–10% goat serum (235 nM, 153–362 nM) and GIT (179 nM, 109–295 nM) and significantly lower (P < 0.05) in OptiMEM I®–5% human serum (27.8 nM, 17.6–43.9 nM).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For in vitro drug sensitivity assays using field isolates, an optimal growth during the first 48 hours is the minimal requirement for a successful cultivation and interpretable test. In this study, microscopic examination of thin blood smears at 18 and 42 hours suggested that the underlying explanations for an enhanced parasite growth observed with DME, IMDM, and GIT media, compared with that in RPMI 1640 medium, are an earlier onset of schizogony and possibly higher numbers of parasites that appeared to develop into mature forms. In an earlier study by Tan-ariya and Brockelman,12 the onset of schizogony of laboratory-adapted P. falciparum strains cultivated in Waymouth medium was approximately four hours earlier than in RPMI 1640 medium during the 48-hour incubation. This observation is in agreement with their findings that although the rate of glucose use in both media was similar during the first 24 hours of incubation (ring stage), parasites cultivated in Waymouth (glucose concentration = 5 g/L) consumed twice as much glucose as those in RPMI 1640 medium (glucose concentration = 2 g/L) during the second 24-hour incubation (trophozoites and schizonts). Although precise kinetics of schizogony in different test media were not analyzed in our study, an earlier onset of schizogony would explain the 2–4-fold higher incorporation of 3H-hypoxanthine, which indirectly reflects the geometric, and not arithmetic, progression of nuclei formation in schizonts.
The results of the present study on serum-free culture media suggest that in addition to RPMI 1640–0.5% Albumax® mixture,15,16 GIT is a suitable alternative medium for the initiation of cultures of fresh isolates. Other serum-free media tested in the present study, as well as in previous studies, did not support parasite growth as much as or better than RPMI 1640–10% human serum mixture.17 In this study, the ability of GIT to sustain the growth of fresh field isolates for at least three complete erythrocytic cycles was assessed. Cultures were initiated at 1.5% hematocrit, and the medium was changed on day 2 and day 4, then daily from day 5. Uninfected erythrocytes were added to a final hematocrit of 2.5–3.0% on day 4. Trophozoites and/or schizonts were observed on day 4 in 25 of 27 isolates and on day 5 or 6 in 17 of 27 isolates. Four isolates were successfully maintained in culture for 10–15 days, as shown by schizont formation. Other investigators have shown that GIT supports the growth of laboratory-adapted P. falciparum strains maintained in a continuous culture and that there is no batch-to-batch variation in parasite growth rate.13,18 Some of the current disadvantages of GIT include the lack of powder formulation (for ease of transport to malaria-endemic countries) and lack of commercial distributors outside Japan. Furthermore, a major drawback of GIT for in vitro drug sensitivity assays is that it contains partially purified serum factors from bovine serum, including bovine serum albumin. As with RPMI 1640 medium supplemented with Albumax® (lipid-enriched bovine albumin), previous studies have shown that the use of bovine albumin, instead of human albumin in non-immune donor serum, considerably modifies IC50 values of most antimalarial drugs.17,19
Several animal sera obtained from commercial suppliers were evaluated in this study. Because abattoirs exist in many malaria-endemic countries, animal sera, if suitable, are a potential alternative source of medium supplement that may replace the more expensive non-immune type AB+ human serum. Fetal calf serum is available from many commercial sources at lower cost because of its multiple uses in biomedical research, including mammalian cell cultures. Although there is a batch-to-batch variation in supporting parasite growth, once a suitable batch is identified, fetal calf serum is suitable for routine malaria cultivation. However, for in vitro drug assays, the IC50 values of antimalarial drugs determined in RPMI 1640–fetal calf serum mixture differ considerably from those determined in RPMI 1640 medium supplemented with human serum, probably because of the difference in drug-protein binding properties.17,20
A short-term cultivation was successful with RPMI 1640 medium supplemented with 10% goat serum. However, chloroquine IC50 values determined in RPMI 1640–10% goat serum were higher than those determined in RPMI 1640–10% human serum. Most isolates incorporated higher quantities of 3H-hypoxanthine during the 42-hour incubation period in RPMI 1640–10% goat serum mixture than in RPMI 1640–10% human serum. A long-term cultivation has also been successfully performed with goat serum.21 In their study, Oduola and others showed that chloroquine IC50 values did not differ significantly between RPMI 1640 medium supplemented with human plasma or goat plasma.21 Unlike in this study, once adapted to goat serum, laboratory strains of P. falciparum generally grew slightly less in the medium supplemented with goat plasma. The lower growth rate with goat plasma, adaptation to goat serum during the long-term cultivation, and batch-to-batch difference of goat sera may explain in part the discordance of results between the studies of Oduola and others and the present study. Although some investigators have reported a successful cultivation of laboratory-adapted P. falciparum strains in RPMI 1640 supplemented with 10% rabbit or horse serum,21,22 parasite growth was poor in these media in this study. The likely explanation is the batch-to-batch variation of the nutritional quality of animal sera. Sera from some other animals (e.g., pigs and sheep) do not support P. falciparum growth in vitro and were not tested in this study.21,23,24
In summary, the capacity of several commercially available media, animal sera, and serum-free media to support the in vitro growth of fresh isolates during the first erythrocytic cycle was assessed with the aim to optimize drug assays, both in this study and in previous studies.17 The results suggest that DME plus 10% human serum, IMDM plus 10% human serum, RPMI 1640 medium plus 10% goat serum, RPMI 1640 medium plus 10% fetal calf serum, RPMI 1640 medium plus 0.5% Albumax®, and serum-free GIT are of potential interest. Media supplemented with human or animal serum require testing to exclude batches that do not support parasite growth. Other media containing high concentrations of hypoxanthine, such as Waymouth MB 752/1 medium, should be further evaluated by means other than the 3H-hypoxanthine incorporation method. Further studies on other commercially prepared media and combinations of media are required to attain the ultimate objective of developing a reproducible, optimized, and standardized serum-free in vitro drug sensitivity assay system that yields results that are comparable to those determined in RPMI 1640–10% human serum mixture.
Received May 1, 2005. Accepted for publication August 2, 2006.
Acknowledgment: I thank the personnel of the Nlongkak Catholic missionary dispensary for their aid in recruiting patients.
Financial support: This study was supported by the French Ministry of Research (Programme PAL+).
* Address correspondence to Leonardo K. Basco, Laboratoire de Recherche sur le Paludisme, Organisation de Coordination pour la lutte contre les Endémies en Afrique Centrale, BP 288, Yaounde, Cameroon. E-mail: lkbasco{at}yahoo.fr 
Author’s address: Leonardo K. Basco, Unité de Recherche Paludologie Afro-Tropicale, Institut de Recherche pour le Développement and Laboratoire de Recherche sur le Paludisme, Organisation de Coordination pour la lutte contre les Endémies en Afrique Centrale, BP 288, Yaounde, Cameroon.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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