• View in gallery

    Growth of 22 fresh clinical isolates of Plasmodium falciparum over a 42-hour incubation period with 10% fetal calf serum (FCS), different concentrations of Albumax II, and RPMI 1640 medium alone (RPMI), expressed as the mean percentage of parasite growth obtained with RPMI 1640 medium supplemented with 10% human serum. Bars show the mean ± SD.

  • View in gallery

    Growth of 17 fresh clinical isolates of Plasmodium falciparum over a 42-hour incubation period with serum-free supplements (PK, 3:1, 1:1, and 1:3 denote PFEK-1/RPMI 1640 [v/v] mixtures; OM = OptiMAb; UL = Ultroser) and a serum substitute (AM = Amniomax), expressed as the mean percentage of parasite growth obtained with RPMI 1640 medium supplemented with 10% human serum. Bars show the mean ± SD.

  • 1

    Reeder JC, Rieckmann KH, Genton B, Lorry K, Wines B, Cowman AF, 1996. Point mutations in the dihydrofolate reductase and dihydropteroate synthetase genes and in vitro susceptibility to pyrimethamine and cycloguanil of Plasmodium falciparum isolates from Papua New Guinea. Am J Trop Med Hyg 55 :209–213.

    • Search Google Scholar
    • Export Citation
  • 2

    Nzila-Mounda A, Mberu E, Sibley C, Plowe C, Winstanley P, Watkins W, 1998. Kenyan Plasmodium falciparum field isolates: Correlation between pyrimethamine and chlorcycloguanil activity in vitro and point mutations in the dihydrofolate reductase domain. Antimicrob Agents Chemother 42 :164–169.

    • Search Google Scholar
    • Export Citation
  • 3

    Basco LK, Tahar R, Keundjian A, Ringwald P, 2000. Sequence variations in the genes encoding dihydropteroate synthase and dihydrofolate reductase and clinical response to sulfadoxine-pyrimethamine in patients with acute uncomplicated falciparum malaria. J Infect Dis 182 :624–628.

    • Search Google Scholar
    • Export Citation
  • 4

    Babiker HA, Pringle SJ, Abdel-Muhsin A, Mackinnon M, Hunt P, Walliker D, 2001. High-level chloroquine resistance in Sudanese isolates of Plasmodium falciparum is associated with mutations in the chloroquine resistance transporter gene pfcrt and the multidrug resistance gene pfmdr1.J Infect Dis 183 :1535–1538.

    • Search Google Scholar
    • Export Citation
  • 5

    Basco LK, Ringwald P, 2001. Analysis of the key pfcrt point mutation and in vitro and in vivo response to chloroquine in Yaounde, Cameroon. J Infect Dis 183 :1828–1831.

    • Search Google Scholar
    • Export Citation
  • 6

    Price RN, Cassar C, Brockman A, Duraisingh M, van Vugt M, White NJ, Nosten F, Krishna S, 1999. The pfmdr1 gene is associated with a multidrug-resistant phenotype in Plasmodium falciparum from the western border of Thailand. Antimicrob Agents Chemother 43 :2943–2949.

    • Search Google Scholar
    • Export Citation
  • 7

    Duraisingh MT, Jones P, Sambou I, von Seidlein L, Pinder M, Warhurst DC, 2000. The tyrosine-86 allele of the pfmdr1 gene of Plasmodium falciparum is associated with increased sensitivity to the anti-malarials mefloquine and artemisinin. Mol Biochem Parasitol 108 :13–23.

    • Search Google Scholar
    • Export Citation
  • 8

    Basco LK, Ringwald P, 2002. Molecular epidemiology of malaria in Cameroon. X. Evaluation of pfmdr1 mutations as genetic marker for resistance to amino alcohols and artemisinin derivatives. Am J Trop Med Hyg 66 :667–671.

    • Search Google Scholar
    • Export Citation
  • 9

    Korsinczky M, Chen NH, Kotecka B, Saul A, Rieckmann K, Cheng Q, 2000. Mutations in Plasmodium falciparum cytochrome b that are associated with atovaquone resistance are located at a putative drug-binding site. Antimicrob Agents Chemother 44 :2100–2108.

    • Search Google Scholar
    • Export Citation
  • 10

    Triglia T, Menting JGT, Wilson C, Cowman AF, 1997. Mutations in dihydropteroate synthase are responsible for sulfone and sulfonamide resistance in Plasmodium falciparum.Proc Natl Acad Sci USA 94 :13944–13949.

    • Search Google Scholar
    • Export Citation
  • 11

    Wang P, Sims PFG, Hyde JE, 1997. A modified in vitro sulfadoxine susceptibility assay for Plasmodium falciparum suitable for investigating Fansidar resistance. Parasitology 115 :223–230.

    • Search Google Scholar
    • Export Citation
  • 12

    Ndounga M, Basco LK, Ringwald P, 2001. Evaluation of a new sulfadoxine sensitivity assay in vitro for field isolates of Plasmodium falciparum.Trans R Soc Trop Med Hyg 95 :55–57.

    • Search Google Scholar
    • Export Citation
  • 13

    Trager W, Jensen JB, 1976. Human malaria parasites in continuous culture. Science 193 :673–675.

  • 14

    Mount DL, Nahlen BL, Patchen LC, Churchill FC, 1989. Adaptations of the Saker-Solomons test: Simple, reliable colorimetric field assays for chloroquine and its metabolites in urine. Bull World Health Organ 67 :295–300.

    • Search Google Scholar
    • Export Citation
  • 15

    Desjardins RE, Canfield CJ, Haynes JD, Chulay JD, 1979. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob Agents Chemother 16 :710–718.

    • Search Google Scholar
    • Export Citation
  • 16

    Le Bras J, Deloron P, Ricour A, Andrieu B, Savel J, Coulaud JP, 1983. Plasmodium falciparum: drug sensitivity in vitro of isolates before and after adaptation to continuous culture. Exp Parasitol 56 :9–14.

    • Search Google Scholar
    • Export Citation
  • 17

    Pologe LG, Ravetch JV, 1986. A chromosome rearrangement in Plasmodium falciparum histidine-rich protein gene is associated with the knobless phenotype. Nature 322 :474–477.

    • Search Google Scholar
    • Export Citation
  • 18

    Siddiqui WA, Richmond-Crum SM, 1977. Fatty acid-free bovine albumin as plasma replacement for in vitro cultivation of Plasmodium falciparum.J Parasitol 63 :583–584.

    • Search Google Scholar
    • Export Citation
  • 19

    Ifediba T, Vanderberg JP, 1980. Peptones and calf serum as a replacement for human serum in the cultivation of Plasmodium falciparum.J Parasitol 66 :236–239.

    • Search Google Scholar
    • Export Citation
  • 20

    Divo AA, Jensen JB, 1982. Studies on serum requirements for the cultivation of Plasmodium falciparum. I. Animal sera. Bull World Health Organ 60 :565–569.

    • Search Google Scholar
    • Export Citation
  • 21

    Willet GP, Canfield CJ, 1984. Plasmodium falciparum: continuous cultivation of erythrocyte stages in plasma-free culture medium. Exp Parasitol 57 :76–80.

    • Search Google Scholar
    • Export Citation
  • 22

    Asahi H, Kanazawa T, 1994. Continuous cultivation of intraerythrocytic Plasmodium falciparum in a serum-free medium with the use of a growth-promoting factor. Parasitology 109 :397–401.

    • Search Google Scholar
    • Export Citation
  • 23

    Lingnau A, Margos G, Maier WA, Seitz HM, 1994. Serum-free cultivation of several Plasmodium falciparum strains. Parasitol Res 80 :84–86.

    • Search Google Scholar
    • Export Citation
  • 24

    Ofulla AVO, Okoye VCN, Khan B, Githure JI, Roberts CR, Johnson AJ, Martin SK, 1993. Cultivation of Plasmodium falciparum parasites in a serum-free medium. Am J Trop Med Hyg 49 :335–340.

    • Search Google Scholar
    • Export Citation
  • 25

    Binh VQ, Luty AJF, Kremsner PG, 1997. Differential effects of human serum and cells on the growth of Plasmodium falciparum adapted to serum-free in vitro culture conditions. Am J Trop Med Hyg 57 :594–600.

    • Search Google Scholar
    • Export Citation
  • 26

    Cranmer SL, Magowan C, Liang J, Coppel RL, Cooke BM, 1997. An alternative to serum for cultivation of Plasmodium falciparum in vitro.Trans R Soc Trop Med Hyg 91 :363–365.

    • Search Google Scholar
    • Export Citation
  • 27

    Flores MVC., Berger-Eiszele SM, Stewart TS, 1997. Long-term cultivation of Plasmodium falciparum in media with commercial non-serum supplements. Parasitol Res 83 :734–736.

    • Search Google Scholar
    • Export Citation
  • 28

    Ringwald P, Meche FS, Bickii J, Basco LK, 1999. In vitro culture and drug sensitivity assay of Plasmodium falciparum with non-serum substitute and acute phase sera. J Clin Microbiol 37 :700–705.

    • Search Google Scholar
    • Export Citation
  • 29

    Oduola AMJ, Ogundahunsi OAT, Salako LA, 1992. Continuous cultivation and drug susceptibility testing of Plasmodium falciparum in a malaria endemic area. J Protozool 39 :605–608.

    • Search Google Scholar
    • Export Citation
  • 30

    Binh VQ, Luty AJF, Kremsner PG, 1997. Differential effects of human serum and cells on the growth of Plasmodium falciparum adapted to serum-free in vitro culture conditions. Am J Trop Med Hyg 57 :594–600.

    • Search Google Scholar
    • Export Citation
 
 
 

 

 
 
 

 

 

 

 

 

 

MOLECULAR EPIDEMIOLOGY OF MALARIA IN CAMEROON. XV. EXPERIMENTAL STUDIES ON SERUM SUBSTITUTES AND SUPPLEMENTS AND ALTERNATIVE CULTURE MEDIA FOR IN VITRO DRUG SENSITIVITY ASSAYS USING FRESH ISOLATES OF PLASMODIUM FALCIPARUM

View More View Less
  • 1 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, Yaounde, Cameroon

In vitro drug sensitivity assay is an important tool for various on-going studies aiming to establish the correlation between candidate molecular markers for drug resistance and drug response in laboratory-adapted strains and field isolates of Plasmodium falciparum. A widespread use of this technique in the field would require a suitable substitute that can replace human serum. In this study, several alternative sources of serum substitutes and supplements were evaluated for their capacity to sustain parasite growth for a single life cycle and their compatibility with in vitro assays for clinical isolates that have not been adapted to in vitro culture. Albumax, a commercial preparation of lipid-enriched bovine albumin, did not support parasite growth as much as human serum and fetal calf serum in several isolates. Other serum supplements (AmnioMax and Ultroser) supported parasite growth relatively well. The 50% inhibitory concentrations (IC50s) of chloroquine and antifolates determined with 0.05% Albumax were generally two or three times higher than with human serum. With 10% fetal calf serum, IC50s for chloroquine and antifolates were approximately two times higher and three times lower than with human serum, respectively. The use of AmnioMax and OptiMAb resulted in a greater than two-fold increase in IC50s and several uninterpretable assays. Despite possible batch-to-batch differences, fetal calf serum may be a suitable substitute for in vitro drug assays while awaiting the results of further studies on other serum substitutes and supplements.

INTRODUCTION

In vitro drug sensitivity assay is one of the laboratory tools that are useful for the analysis of phenotypes of Plasmodium falciparum isolates. Unlike in vivo tests for drug resistance, in vitro assays have the advantage of yielding objective results of the parasites’ response to drugs, without any interference of host factors, including pharmacokinetic profiles (poor absorption, biotransformation, concentration in certain tissues, rapid elimination) and acquired immunity. In vitro assays can be performed simultaneously for several antimalarial drugs, including experimental drugs, against the same isolate. When parts or most of the technical procedures are automated, in vitro assays are compatible with high-output activities.

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.

MATERIALS AND METHODS

Parasites.

After informed consent was obtained, venous blood samples were obtained in 2001 from symptomatic children ≥ 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). Albumax™ II, lipid-enriched bovine albumin, and AmnioMax™-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 OptiMAb™, 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.

RESULTS

Parasite growth.

The growth of 22 consecutive isolates in various serum-supplemented RPMI 1640 medium was compared in preliminary experiments. An adequate in vitro growth over a 42-hour period was observed in all isolates, with an incorporation of 3H-hypoxanthine ranging from 1,150 cpm to 39,600 cpm in drug-free, 10% human serum–supplemented RPMI 1640 medium. In negative controls containing uninfected erythrocytes, the mean background incorporation of 3H-hypoxanthine was 82.5 cpm (range = 40–119 cpm) and did not differ significantly (P > 0.05) with any of the serum or serum substitutes or serum-free medium. Compared with the parasite growth in 10% human serum–supplemented RPMI 1640 medium, 0.5% Albumax-supplemented medium yielded a superior growth (approximately two-fold) in two isolates, 50–100% growth (100% being an equivalent growth) in 10 isolates, and < 50% growth in 10 additional isolates. The mean ± SD relative growth (range) with 0.5% Albumax compared with 10% human serum was 64 ± 53% (10–217%). An improved growth was observed with 10% fetal calf serum, with a mean ± SD relative growth of 134 ± 56% (48–245%) compared with 10% human serum. When 10% fetal calf serum was used, 15 isolates had better growth (> 100%) than human serum, while seven had lower growth (< 100%).

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.

In the absence of a consistent parasite growth of fresh clinical isolates with Albumax, experiments were conducted with 17 additional isolates using serum-free media and AmnioMax. The results are summarized in Figure 2. 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.

In vitro assay.

The in vitro response to chloroquine, monodesethylamodiaquine, pyrimethamine, and cycloguanil was determined with different sera, serum substitute, or serum supplement. The use of 0.05% Albumax and 10% fetal calf serum resulted in an average increase of 2.8- and 2.3-fold in the chloroquine IC50s compared with those determined by using 10% human serum, respectively (Table 1). 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.

Additional experiments were carried out to assess the suitability of alternative media for the determination of IC50s. The use of RPMI 1640 medium supplemented with OptiMAb resulted in a 2.3-fold increase in chloroquine IC50s for five isolates (Table 2). 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).

DISCUSSION

Laboratory-adapted strains of P. falciparum are generally easier to cultivate than isolates freshly obtained from patients. Even if we exclude blood samples from patients with a recent history of antimalarial drug intake, some isolates either fail to initiate the erythrocytic schizogony or transform massively into gametocytes during the first in vitro life cycle. The presence of trace amounts of antimalarial drugs or intake of traditional medicine undetected by urine test for commonly used antimalarial drugs may partially explain why some fresh isolates do not grow in vitro. The biologic reasons underlying this difference between laboratory strains and fresh clinical isolates are not well known, although it has been observed that parasites adapted to in vitro conditions undergo selection and genetic changes.16,17 It is within this context that, to attain the objective of the present study, a candidate for serum replacement needs to fulfill the double requirement: the ability to sustain the in vitro growth of fresh clinical isolates that have not been previously adapted to in vitro culture and compatibility with in vitro drug sensitivity assays.

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.

Table 1

In vitro response of Plasmodium falciparum isolates to chloroquine and antifolates determined in RPMI 1640 medium containing different sera or serum substitute*

IC50, nM (mean parasite growth in drug-free control wells, cpm)
Isolate10% human serum10% fetal calf serum0.05% AlbumaxSerum-free RPMI†
* IC50 = 50% inhibitory concentration; ND = not done; PYR = pyrimethamine; CYC = cycloguanil; NI = not interpretable due to either inadequate parasite growth (<1,000 cpm in drug-free control wells) or insufficient growth inhibition at the highest drug concentration used, which precludes graph plotting and sigmoid curve fitting.
† Serum-free RPMI denotes the use of RPMI without any serum (or serum-substitute) supplement. The standard RPMI 1640 medium was used for chloroquine. p-aminobenzoic acid– and folic acid–free RPMI 1640 were used to determine the in vitro response to antifolates.
Chloroquine
    23/01211 (12,153)ND388 (3,308)ND
    24/01138 (5,795)ND380 (1,357)ND
    27/01204 (4,522)NDNI (389)ND
    30/01130 (13,316)390 (11,029)369 (5,720)412 (5,708)
    33/01143 (2,424)398 (2,881)NDND
    34/0127.6 (4,640)38.4 (11,352)NDND
    35/0193.1 (1,480)331 (4,847)NDND
    36/01270 (1,361)530 (1,744)NDND
    38/0136.0 (3,016)35.2 (8,963)NDND
    39/01214 (11,586)397 (16,918)375 (6,995)NI (3,927)
    41/0122.2 (4,009)36.1 (11,912)44.2 (1,610)47.5 (1,336)
    42/0137.0 (5,781)158 (7,736)199 (2,200)381 (1,403)
    43/01177 (2,600)253 (5,669)NI (518)NI (473)
Antifolates
    23/011,770 PYRND4,870 PYRND
74.4 CYC (11,931)344 CYC (9,693)
    24/010.040 PYRNDNIND
0.701 CYC (3,810)
(3,310)
    25/01594 PYRND1,100 PYRND
33.4 CYC (3,084)31.1 CYC (4,798)
    27/012,700 PYRNDNIND
98.5 CYC (9,534)
(8,848)
    28/014,120 PYRNDNDNI
205 CYC (19,783)
(3,371)
    29/013,980 PYRNDNIND
166 CYC (3,410)
(3,385)
    30/01933 PYR630 PYRNDND
46.4 CYC (16,625)31.9 CYC (9,988)
    31/013,330 PYR2,120 PYRNDND
135 CYC (14,621)99.1 CYC (13,469)
32/01731 PYR521 PYRNDND
45.1 CYC (2,412)30.8 CYC (3,185)
    33/01551 PYR105 PYRNDND
28.2 CYC (2,759)8.13 CYC (2,826)
    34/014,180 PYR634 PYRNDND
317 CYC (10,605)40.9 CYC (12,447)
    35/012,091 PYR1,315 PYRNDND
ND CYC (3,461)ND CYC (5,145)
Table 2

In vitro response of clinical isolates of Plasmodium falciparum determined with RPMI 1640 medium containing serum-free substitutes*

IC50 (nM)
IsolateRPMI + 10% human serum†RPMI + OptiMAbRPMI + AmnioMax
* IC50 = 50% inhibitory concentration; ND = not done; NI = not interpretable.
† Standard RPMI 1640 medium containing 1 mg/L of p-aminobenzoic acid (PABA) and 1 mg/L of folic acid for chloroquine and monodesethylamodiaquine and PABA- and folate-free RPMI for pyrimethamine. Of 6 experiments with chloroquine and 7 experiments with pyrimethamine, there was only one interpretable result with 10% PFEK1 medium (chloroquine IC50 = 714 nM vs 221 nM with 10% human serum).
Chloroquine
    114/0188.6314ND
    117/0128.647.3ND
    120/0139.572.8ND
    128/01282355ND
    130/01119400ND
    105/01118ND254
    106/01234ND442
    107/01176ND453
    108/01155ND334
    109/01140ND367
    132/01221ND433
Monodesethylamodiaquine
    105/0118.4ND35.5
    106/0147.1ND61.8
    107/0131.0ND48.7
Pyrimethamine
    43/020.0381.17NI
    49/021,950NI2,900
    56/02553NINI
    57/022,4106,100NI
    64/023,850NINI
    66/02486NI965
    78/029662,6902,330
Figure 1.
Figure 1.

Growth of 22 fresh clinical isolates of Plasmodium falciparum over a 42-hour incubation period with 10% fetal calf serum (FCS), different concentrations of Albumax II, and RPMI 1640 medium alone (RPMI), expressed as the mean percentage of parasite growth obtained with RPMI 1640 medium supplemented with 10% human serum. Bars show the mean ± SD.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 2; 10.4269/ajtmh.2003.69.168

Figure 2.
Figure 2.

Growth of 17 fresh clinical isolates of Plasmodium falciparum over a 42-hour incubation period with serum-free supplements (PK, 3:1, 1:1, and 1:3 denote PFEK-1/RPMI 1640 [v/v] mixtures; OM = OptiMAb; UL = Ultroser) and a serum substitute (AM = Amniomax), expressed as the mean percentage of parasite growth obtained with RPMI 1640 medium supplemented with 10% human serum. Bars show the mean ± SD.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 2; 10.4269/ajtmh.2003.69.168

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@ibaic.u-psud.fr or lkbasco@yahoo.fr.

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).

REFERENCES

  • 1

    Reeder JC, Rieckmann KH, Genton B, Lorry K, Wines B, Cowman AF, 1996. Point mutations in the dihydrofolate reductase and dihydropteroate synthetase genes and in vitro susceptibility to pyrimethamine and cycloguanil of Plasmodium falciparum isolates from Papua New Guinea. Am J Trop Med Hyg 55 :209–213.

    • Search Google Scholar
    • Export Citation
  • 2

    Nzila-Mounda A, Mberu E, Sibley C, Plowe C, Winstanley P, Watkins W, 1998. Kenyan Plasmodium falciparum field isolates: Correlation between pyrimethamine and chlorcycloguanil activity in vitro and point mutations in the dihydrofolate reductase domain. Antimicrob Agents Chemother 42 :164–169.

    • Search Google Scholar
    • Export Citation
  • 3

    Basco LK, Tahar R, Keundjian A, Ringwald P, 2000. Sequence variations in the genes encoding dihydropteroate synthase and dihydrofolate reductase and clinical response to sulfadoxine-pyrimethamine in patients with acute uncomplicated falciparum malaria. J Infect Dis 182 :624–628.

    • Search Google Scholar
    • Export Citation
  • 4

    Babiker HA, Pringle SJ, Abdel-Muhsin A, Mackinnon M, Hunt P, Walliker D, 2001. High-level chloroquine resistance in Sudanese isolates of Plasmodium falciparum is associated with mutations in the chloroquine resistance transporter gene pfcrt and the multidrug resistance gene pfmdr1.J Infect Dis 183 :1535–1538.

    • Search Google Scholar
    • Export Citation
  • 5

    Basco LK, Ringwald P, 2001. Analysis of the key pfcrt point mutation and in vitro and in vivo response to chloroquine in Yaounde, Cameroon. J Infect Dis 183 :1828–1831.

    • Search Google Scholar
    • Export Citation
  • 6

    Price RN, Cassar C, Brockman A, Duraisingh M, van Vugt M, White NJ, Nosten F, Krishna S, 1999. The pfmdr1 gene is associated with a multidrug-resistant phenotype in Plasmodium falciparum from the western border of Thailand. Antimicrob Agents Chemother 43 :2943–2949.

    • Search Google Scholar
    • Export Citation
  • 7

    Duraisingh MT, Jones P, Sambou I, von Seidlein L, Pinder M, Warhurst DC, 2000. The tyrosine-86 allele of the pfmdr1 gene of Plasmodium falciparum is associated with increased sensitivity to the anti-malarials mefloquine and artemisinin. Mol Biochem Parasitol 108 :13–23.

    • Search Google Scholar
    • Export Citation
  • 8

    Basco LK, Ringwald P, 2002. Molecular epidemiology of malaria in Cameroon. X. Evaluation of pfmdr1 mutations as genetic marker for resistance to amino alcohols and artemisinin derivatives. Am J Trop Med Hyg 66 :667–671.

    • Search Google Scholar
    • Export Citation
  • 9

    Korsinczky M, Chen NH, Kotecka B, Saul A, Rieckmann K, Cheng Q, 2000. Mutations in Plasmodium falciparum cytochrome b that are associated with atovaquone resistance are located at a putative drug-binding site. Antimicrob Agents Chemother 44 :2100–2108.

    • Search Google Scholar
    • Export Citation
  • 10

    Triglia T, Menting JGT, Wilson C, Cowman AF, 1997. Mutations in dihydropteroate synthase are responsible for sulfone and sulfonamide resistance in Plasmodium falciparum.Proc Natl Acad Sci USA 94 :13944–13949.

    • Search Google Scholar
    • Export Citation
  • 11

    Wang P, Sims PFG, Hyde JE, 1997. A modified in vitro sulfadoxine susceptibility assay for Plasmodium falciparum suitable for investigating Fansidar resistance. Parasitology 115 :223–230.

    • Search Google Scholar
    • Export Citation
  • 12

    Ndounga M, Basco LK, Ringwald P, 2001. Evaluation of a new sulfadoxine sensitivity assay in vitro for field isolates of Plasmodium falciparum.Trans R Soc Trop Med Hyg 95 :55–57.

    • Search Google Scholar
    • Export Citation
  • 13

    Trager W, Jensen JB, 1976. Human malaria parasites in continuous culture. Science 193 :673–675.

  • 14

    Mount DL, Nahlen BL, Patchen LC, Churchill FC, 1989. Adaptations of the Saker-Solomons test: Simple, reliable colorimetric field assays for chloroquine and its metabolites in urine. Bull World Health Organ 67 :295–300.

    • Search Google Scholar
    • Export Citation
  • 15

    Desjardins RE, Canfield CJ, Haynes JD, Chulay JD, 1979. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob Agents Chemother 16 :710–718.

    • Search Google Scholar
    • Export Citation
  • 16

    Le Bras J, Deloron P, Ricour A, Andrieu B, Savel J, Coulaud JP, 1983. Plasmodium falciparum: drug sensitivity in vitro of isolates before and after adaptation to continuous culture. Exp Parasitol 56 :9–14.

    • Search Google Scholar
    • Export Citation
  • 17

    Pologe LG, Ravetch JV, 1986. A chromosome rearrangement in Plasmodium falciparum histidine-rich protein gene is associated with the knobless phenotype. Nature 322 :474–477.

    • Search Google Scholar
    • Export Citation
  • 18

    Siddiqui WA, Richmond-Crum SM, 1977. Fatty acid-free bovine albumin as plasma replacement for in vitro cultivation of Plasmodium falciparum.J Parasitol 63 :583–584.

    • Search Google Scholar
    • Export Citation
  • 19

    Ifediba T, Vanderberg JP, 1980. Peptones and calf serum as a replacement for human serum in the cultivation of Plasmodium falciparum.J Parasitol 66 :236–239.

    • Search Google Scholar
    • Export Citation
  • 20

    Divo AA, Jensen JB, 1982. Studies on serum requirements for the cultivation of Plasmodium falciparum. I. Animal sera. Bull World Health Organ 60 :565–569.

    • Search Google Scholar
    • Export Citation
  • 21

    Willet GP, Canfield CJ, 1984. Plasmodium falciparum: continuous cultivation of erythrocyte stages in plasma-free culture medium. Exp Parasitol 57 :76–80.

    • Search Google Scholar
    • Export Citation
  • 22

    Asahi H, Kanazawa T, 1994. Continuous cultivation of intraerythrocytic Plasmodium falciparum in a serum-free medium with the use of a growth-promoting factor. Parasitology 109 :397–401.

    • Search Google Scholar
    • Export Citation
  • 23

    Lingnau A, Margos G, Maier WA, Seitz HM, 1994. Serum-free cultivation of several Plasmodium falciparum strains. Parasitol Res 80 :84–86.

    • Search Google Scholar
    • Export Citation
  • 24

    Ofulla AVO, Okoye VCN, Khan B, Githure JI, Roberts CR, Johnson AJ, Martin SK, 1993. Cultivation of Plasmodium falciparum parasites in a serum-free medium. Am J Trop Med Hyg 49 :335–340.

    • Search Google Scholar
    • Export Citation
  • 25

    Binh VQ, Luty AJF, Kremsner PG, 1997. Differential effects of human serum and cells on the growth of Plasmodium falciparum adapted to serum-free in vitro culture conditions. Am J Trop Med Hyg 57 :594–600.

    • Search Google Scholar
    • Export Citation
  • 26

    Cranmer SL, Magowan C, Liang J, Coppel RL, Cooke BM, 1997. An alternative to serum for cultivation of Plasmodium falciparum in vitro.Trans R Soc Trop Med Hyg 91 :363–365.

    • Search Google Scholar
    • Export Citation
  • 27

    Flores MVC., Berger-Eiszele SM, Stewart TS, 1997. Long-term cultivation of Plasmodium falciparum in media with commercial non-serum supplements. Parasitol Res 83 :734–736.

    • Search Google Scholar
    • Export Citation
  • 28

    Ringwald P, Meche FS, Bickii J, Basco LK, 1999. In vitro culture and drug sensitivity assay of Plasmodium falciparum with non-serum substitute and acute phase sera. J Clin Microbiol 37 :700–705.

    • Search Google Scholar
    • Export Citation
  • 29

    Oduola AMJ, Ogundahunsi OAT, Salako LA, 1992. Continuous cultivation and drug susceptibility testing of Plasmodium falciparum in a malaria endemic area. J Protozool 39 :605–608.

    • Search Google Scholar
    • Export Citation
  • 30

    Binh VQ, Luty AJF, Kremsner PG, 1997. Differential effects of human serum and cells on the growth of Plasmodium falciparum adapted to serum-free in vitro culture conditions. Am J Trop Med Hyg 57 :594–600.

    • Search Google Scholar
    • Export Citation
Save