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Am. J. Trop. Med. Hyg., 73(2), 2005, pp. 239-243
Copyright © 2005 by The American Society of Tropical Medicine and Hygiene

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DRUG-RESISTANT MALARIA IN BANGUI, CENTRAL AFRICAN REPUBLIC: AN IN VITRO ASSESSMENT

DIDIER MENARD*, DJIBRINE DJALLE, ALEXANDRE MANIRAKIZA, FERDINAND YAPOU, VALERIE SIADOUA, SERGE SANA, MARCELLE DIANE MATSIKA-CLAQUIN, MADJI NESTOR, AND ANTOINE TALARMIN
Pasteur Institute of Bangui, Bangui, Central African Republic; National Malaria Control Program, Central African Republic Ministry of Health; Bangui, Central African Republic


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We used an in vitro isotopic drug sensitivity assay to assess the sensitivity of Plasmodium falciparum isolates collected in Bangui, Central African Republic between March and July 2004. We tested antimalarials that are currently in use in this country (chloroquine, amodiaquine, quinine, and pyrimethamine), antimalarials that will become available in this region in the future (artemisinin and halofantrine), and prophylactic antimalarials (mefloquine, doxycycline, and atovaquone). The proportions of resistant isolates were 37% for chloroquine, 15.9% for amodiaquine, 0% for quinine, 0% for dihydroartemisinin, 1.6% for mefloquine, 3.8% for halofantrine, 4.0% for atovaquone, and 38.3% for pyrimethamine. No multi-resistant isolates (showing resistance to more than three drugs) were found. A positive correlation was found between the 50% inhibitory concentrations values for the following drugs: chloroquine and amodiaquine; quinine and halofantrine; chloroquine and dihydroartemisinin; chloroquine and halofantrine; amodiaquine and dihydroartemisinin; dihydroartemisinin and mefloquine; chloroquine and quinine; and quinine and dihydroartemisinin. These findings suggest that the Ministry of Health should recommend a interim policy with the amodiaquine plus sulfadoxine-pyrimethamine combination as the first-line antimalarial drug in Bangui until better alternative treatments such as artemisinin-based combination therapies become available at low prices in the Central African Republic.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Malaria is a major infectious disease worldwide and is the leading cause of morbidity and mortality, particularly among children. According to the World Health Organization (WHO), 300–500 million people contract malaria each year. In Africa, more than 90% of the cases and between 1.5 and 3 million deaths occur in children less than five years of age.1

Chemotherapy is the principal method of treating malaria parasite infections in human hosts. Chloroquine has been the drug of choice for treating Plasmodium falciparum malaria for more than 50 years. However, the use of chloroquine as a prophylactic drug and as a malaria treatment is now limited because of the spread of chloroquine-resistant P. falciparum strains throughout most malaria-endemic areas. In the Central African Republic, P. falciparum resistance to chloroquine and sulfadoxine-pyrimethamine has been documented since 19832 and 1987,3 respectively. The most recent in vivo study based on the WHO standard protocol (WHO 2001) was conducted between 2002 and 2004 by the National Malaria Control Program and the Pasteur Institute of Bangui. This study showed that the overall rates of treatment failure with chloroquine, amodiaquine, sulfadoxine-pyrimethamine, the combination of chloroquine plus sulfadoxine-pyrimethamine, and the combination of amodiaquine plus sulfadoxine-pyrimethamine were 40.9%, 20.0%, 22.8%, 7.2%, and 0%, respectively.4 These findings led the Ministry of Health of the Central African Republic to replace chloroquine with amodiaquine plus sulfadoxine-pyrimethamine, which was to be used as an interim first-line antimalarial drug until better alternative treatments, such as artemisinin-based combination therapies, became available at low prices in this country.

In vivo resistance studies are useful for estimating the rate of clinical failure of antimalarials, whereas in vitro resistance studies are a useful tool for assessing the spread of P. falciparum resistance and have shown a decrease in the drug sensitivity of parasites to several antimalarial drugs. The aims of this study were 1) to determine the in vitro response of clinical isolates of P. falciparum to the following types of antimalarial drugs: a) those used routinely in the Central African Republic (chloroquine, amodiaquine, quinine, and pyrimethamine); b) those that will be available and appropriate for use in this country during the next few years (artemisinin and halofantrine); and c) those used in prophylaxis (mefloquine, amodiaquine, and doxycycline); and (2) to analyze the pattern of cross-resistance to these antimalarial drugs in vitro.


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Parasites. This study was conducted in Bangui, the capital of the Central African Republic, between March and July 2004. Bangui is located on the Oubangui River in the heart of the central Africa (7°N, 21°E). The climate is tropical and rainfall is highest from April to November. The average temperature varies from 19°C to 32°C. Malaria transmission occurs throughout the year, with peaks at the beginning and the end of the rainy season. Malaria is hyperendemic in this region and P. falciparum is the major parasite. The prevalence of this parasite in children less than five years of age is 31.8%.5

Three hundred eighty-one P. falciparum clinical isolates were obtained from symptomatic Central African patients before they were treated. These patients attended several health centers (north of Bangui: Boy Rabe and Gobongo; south of Bangui: La Kouanga; and east of Bangui: Ouango). Venous blood samples (10 mL) were collected in tubes coated with EDTA (Vacutainer®; Becton Dickinson, Rutherford, NJ) from patients who provided informed consent. Giemsa-stained thin and thick blood smears were examined to check for mono-infection with P. falciparum and to determine parasite density. In vitro assays were performed on blood samples with a parasite density > 0.1%, within eight hours after blood was obtained. The patients were treated with amodiaquine plus sulfadoxine-pyrimethamine or quinine. There is no National Ethics Committee in the Central African Republic. Therefore, the study was reviewed and approved by the expert committee for antimalarial drug policy and the Ministry of Health of the Central African Republic.

Drugs. The antimalarial drugs used in this study were obtained as follows: chloroquine diphosphate, quinine hydrochloride, dihydroartemisinin, doxycycline hydrochloride and pyrimethamine base were obtained from the Sigma Chemical Company (St. Louis, MO); monodesethylamodiaquine hydrochloride (the biologically active human metabolite of amodiaquine) was kindly provided by Dr. Pascal Ringwald (Roll Back Malaria, WHO, Geneva, Switzerland); mefloquine hydrochloride was obtained from Hoffmann-La Roche (Basel, Switzerland); halofantrine hydrochloride was obtained from SmithKline Beecham (Hertfordshire, United Kingdom) and atovaquone hydrochloride was obtained from Glaxo-Wellcome (Hertfordshire, United Kingdom).

Stock solutions of CQ, monodesethylamodiaquine, dihydroartemisinin, quinine, mefloquine, halofantrine, doxycycline, and amodiaquine were prepared in methanol. The stock solution of pyrimethamine was prepared in ethanol. The final concentration of methanol and ethanol did not exceed 0.05%. Two-fold (four-fold for pyrimethamine) serial dilutions of the stock solutions were made in distilled water. The concentrations of the solutions tested ranged from 12.5 to 3,200 nM for chloroquine, 25 to 3,200 nM for quinine, 7.5 to 1,920 nM for monodesethylamodiaquine, 0.25 to 64 nM for dihydroartemisinin, 0.25 to 32 nM for halofantrine, 0.1 to 500 µM for doxycycline, 1.5 to 400 nM for mefloquine, and 50 to 40,000 nM for pyrimethamine. Aliquots (20 µL) of each solution (at all concentrations tested) were transferred to 96-well tissue culture plates in triplicate.

In vitro assay. The venous blood samples were washed three times in RPMI 1640 medium. The white blood cell interface was removed after each wash. The erythrocytes were then resuspended in a volume of complete RPMI 1640 medium (RPMI 1640 medium supplemented with 10% human serum, lot no. S02909S4190; AbCys, Paris, France), 25 mM HEPES buffer, and 25 mM sodium bicarbonate to give a hematocrit of 1.5% and an initial parasitemia of 0.1–0.5%. Folic acid– and p-aminobenzoic acid–free RPMI 1640 medium was used to assess the in vitro sensitivity to pyrimethamine. If the blood sample had a parasitemia > 0.5%, fresh uninfected erythrocytes were added to adjust the parasitemia to 0.3%. The sensitivity of isolates to antimalarial drugs was assessed using the isotopic microtest developed by Desjardins and others6 as follows. A suspension of infected erythrocytes (200 µL) was placed in each well of the 96-well tissue culture plates containing the antimalarial drug solutions. Parasite growth was assessed after adding 3H-hypoxanthine (1 µCi/well; Amersham Pharmacia Biotech, Orsay, France). Parasites were incubated at 37° C in an atmosphere of 5% CO2 for 42 hours. After incubation, the in vitro assay was terminated by freezing. Plates were then thawed and the contents of each well were collected on glass fiber filter papers (Printed Filtermat A®; Wallac, Turku, Finland) using a cell harvester (Skatron Instruments AS, Lier, Norway). The filter disks were transferred to scintillation tubes (4-mL Pico Pro Vial®; Packard Instruments SA, Rungis, France) and 2 mL of scintillation cocktail (Ultima Gold F®; Packard Instruments SA) was added. Incorporation of 3H-hypoxanthine was quantified using a liquid scintillation counter (Betamatic®; Kontron Instruments, Milan, Italy). A chloroquine-sensitive reference strain (3D7) and a chloroquine-resistant reference strain (W2) of P. falciparum obtained from the Malaria Research Reference Reagent Resource Center (American Type Culture Collection, Manassas, VA) and used as controls.

Data analysis. The IC50 values (defined as the drug concentration that resulted in a level of 3H-hypoxanthine uptake that was 50% lower than that measured in the drug-free control wells) were determined by nonlinear regression analysis of the plot of logarithm of concentration against growth inhibition. Data were adapted to fit the log-probit model (Excel®; Microsoft, Redmond, WA). The threshold IC50 values for in vitro resistance to chloroquine, monodesethylamodiaquine, quinine, mefloquine, halofantrine, atovaquone, and pyrimethamine have previously been estimated to be > 100 nM,7 > 60 nM,8 > 800 nM,9 > 30 nM,10 > 6 nM,11 > 7 nM,12 and > 100 nM,13 respectively. The values for dihydroartemisinin and doxycycline have not been clearly determined. Data are expressed as the geometric mean IC50 values with 95% confidence intervals. The mean logarithmic IC50 values of the various drugs with chloroquine-sensitive and chloroquine-resistant parasites were compared using the Student’s unpaired t-test. Correlations between the IC50 values for different drugs were assessed by using Spearman’s rank order correlation t-test. The significance level was fixed at 0.05. Data were analyzed using EpiInfo 2000 (Centers for Disease Control and Prevention, Atlanta, GA) and MedCalc® 7.4.3.0 software (MedCalc, Mariakerke, Belgium).


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Of the 381 clinical isolates, only 189 had a parasite density > 0.1%. These were assessed for resistance to chloroquine, monodesethylamodiaquine, and quinine. The in vitro activities of dihydroartemisinin, mefloquine, halofantrine, atovaquone, pyrimethamine, and doxycycline were assessed using 98 clinical isolates. The number successful assays performed and the IC50 values determined from these assays are shown for each drug tested in Table 1Go. The IC50 values for reference strains used as controls are shown in the footnotes in Table 1Go.


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TABLE 1
In vitro drug susceptibility of Plasmodium falciparum isolates collected from Bangui, Central African Republic*
 
The in vitro activities of the antimalarial drugs and the number of chloroquine-sensitive and chloroquine-resistant isolates are shown in Table 2Go. Monodesethylamodiaquine and quinine were more active against the chloroquine-sensitive isolates than against the chloroquine-resistant parasites (P < 0.05). The other drugs tested were equally active against the chloroquine-sensitive and the chloroquine-resistant isolates (P > 0.05, not significant).


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TABLE 2
In vitro response of chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum isolates from the Central African Republic*
 
The chloroquine IC50 value was unusually high for the halofantrine-resistant isolate (IC50 = 347 nM). In contrast, the IC50 values of several of the drugs tested (chloroquine, monodesethylamodiaquine, quinine, dihydroartemisinin, and doxycycline) were unusually low for the mefloquine-resistant isolate. The two atovaquone-resistant isolates were sensitive to all drugs tested. The IC50 value for dihydroartemisinin was < 3.7 nM, except for two isolates in which the values were 5.6 and 5.9 nM. A wide range of IC50 values were observed for pyrimethamine (1–16,206 nM). None of the isolates were resistant to three drugs.

A positive correlation (P < 0.05) was observed between the IC50 values for chloroquine and monodesethylamodiaquine (r = 0.61), quinine and halofantrine (r = 0.61), chloroquine and dihydroartemisinin (r = 0.41), chloroquine and halofantrine (r = 0.41), monodesethylamodiaquine and dihydroartemisinin (r = 0.39), dihydroartemisinin and mefloquine (r = 0.34), chloroquine and quinine (r = 0.33), quinine and dihydroartemisinin (r = 0.31) (Table 3Go).


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TABLE 3
Correlations between the in vitro responses of Plasmodium falciparum isolates from the Central African Republic to antimalarial drug*
 

DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Since 2003, the Central African Republic Ministry of Health has been recommending the use of the amodiaquine plus sulfadoxine-pyrimethamine combination for the first-line treatment of uncomplicated P. falciparum malaria, and the use of the artesunate plus lumefantrine combination, followed by quinine for second-line and third-line treatments, respectively. These recommendations were based on data obtained from the in vivo study carried out in Bangui from 2002 to 2004.4 As part of this study, we conducted an investigation of antimalarial drug susceptibility in P. falciparum isolates collected in the Central African Republic.

In the first part of this study, we assessed the sensitivity of P. falciparum isolates to affordable and available antimalarial drugs currently being used in the Central African Republic (chloroquine, amodiaquine, quinine, and pyrimethamine). The percentage of isolates resistant to these drugs was strongly correlated with the overall number of treatment failures reported in our most recent in vivo study 4 and with data obtained from other studies conducted in central Africa.14,15

Two factors may explain the high levels of resistance to antimalarial drugs detected in the P. falciparum isolates: 1) selective pressure exerted by extensive use of chloroquine and 2) cross-resistance. Chloroquine is widely used by physicians in health centers and by individuals undergoing self treatment.4 Unfortunately, this drug is often prescribed in insufficient doses and treatment is frequently stopped too early.16 In contrast, amodiaquine and quinine are rarely used as antimalarial treatments. We found that the IC50 values for monodesethylamodiaquine and quinine differed significantly between chloroquine-sensitive and chloroquine-resistant isolates, and that there was a significant correlation between the in vitro responses to chloroquine and monodesethylamodiaquine (r = 0.61) and between those to chloroquine and quinine (r = 0.33). Cross-resistance between these drugs may be explained, at least in part, by similarities in chemical structure. Our results are similar to those obtained in a study of an area with high levels of chloroquine resistance.17–20 However, as in other studies of central African countries,15 none of our P. falciparum isolates showed resistance to quinine. These data show that quinine is a very efficient antimalarial treatment and thus should be used to treat severe malaria in the Central African Republic. However, several studies have reported borderline-resistant P. falciparum isolates collected from central Africa, in particular, from Gabon 20,21 and west Africa.17,18 Over next few years, as chloroquine is withdrawn from use as a first-line treatment, it will be important to monitor the spread of P. falciparum resistance to chloroquine, amodiaquine, and quinine. In vitro assays will provide an invaluable tool for these studies. Studies conducted under similar conditions in Malawi suggest that that P. falciparum resistance to chloroquine will decrease.22

The rapid spread of pyrimethamine resistance may also be explained by pressures exerted by drug use. The sulfadoxine-pyrimethamine and sulfamethoxazole-trimethoprim combinations are extensively used in the Central African Republic as prophylactic treatments for opportunistic diseases occurring in immunocompromised patients or as treatments for bacterial disease. Only one or two mutations in the dihydrofolate reductase (dhfr) domain, mainly at codons 108 (S108N), 59 (C59R), and 51 (N51I) are necessary to cause high levels of resistance.23

In the second part of the study, we assessed the level of resistance to antimalarial drugs that are planned for use in the Central African Republic over the next few years (artemisinin and halofantrine). These drugs are currently available only at private pharmacies, are expensive, and are only used by expatriates and wealthy individuals.

We tested resistance to artemisinin using its derivative dihydroartemisinin because all artemisinin derivatives are rapidly converted to dihydroartemisinin in humans,15 and because this metabolite is relatively stable and thus more suitable for use in in vitro assays. Compared with the other antimalarial drugs tested, we found that dihydroartemisinin had very high activity against P. falciparum isolates. The geometric mean IC50 value we obtained for this drug was similar to that obtained for isolates from other African countries,15 but was lower than that observed for Asian isolates.24,25 Our data showed that dihydroartemisinin was as active against the chloroquine-sensitive isolates as it was against the chloroquine-resistant isolates. Unfortunately, lumefantrine was not test in our study because we did not obtain the drug. Thus, it was impossible to access the efficacy of the artemisininlumefantrine combination, a treatment that Malaria Control Program in the Central African Republic expects to used as a first-line treatment of malaria in 2005.

We also tested resistance to halofantrine, an amino-alcohol and a congener of lumefantrine. The success rate of the in vitro assay with this drug was very low due to its rapid degradation in precoated plates. Despite this technical difficulty, the prevalence of in vitro resistance to halofantrine observed in our study was similar to that found for isolates from Cameroon,15 but was much lower than that found for isolates from Gabon14 or Burkina-Faso.18 As previously described for mefloquine,15 this prevalence rate is probably related to the cross-resistance with other antimalarials (chloroquine and quinine) that have been used in the Central African Republic.

We studied resistance to prophylactic antimalarial drugs (mefloquine, atovaquone, and doxycycline), and observed that the prevalence of primary resistance to mefloquine and atovaquone in vitro was low. The WHO recommends that mefloquine be used as a prophylactic antimalarial treatment for individuals visiting the Central African Republic for a period of less than three months. Doxycycline seems to be an effective treatment for malaria in the Central African Republic and is used by French soldiers living in Bangui for periods of four months. In addition, no multi-resistant P. falciparum isolates (showing resistance to more than three antimalarials) were found in our study.

The results of this in vitro study indicate that cross-resistance can occur between drugs that do not share any similar chemical features, such as dihydroartemisinin and 4-aminoquinolines (chloroquine, monodesethylamodiaquine), dihydroartemisinin and quinine and dihydroartemisinin, and amino-alcohols (mefloquine). Positive correlations between other artemisinin derivatives and amino-alcohols have been observed in several in vitro studies.26–28 The clinical and epidemiologic significance of this in vitro cross-resistance is still unknown. Eckstein and others recently reported that artemisinin derivatives act differently from other antimalarials, such 4-aminoquinolines and amino-alcohols, by inhibiting P. falciparum calcium-dependent ATPase outside the food vacuole after activation by iron.29

In conclusion, we used an in vitro assay to assess the levels of resistance of P. falciparum strains to antimalarials that are currently being used in the Central African Republic, to antimalarials that will be available for use in the future in this region, and to prophylactic antimalarials. These findings suggest that the Ministry of Health in this country should recommend an interim policy of the amodiaquine plus sulfadoxine-pyrimethamine combination as the first-line antimalarial drug until better alternative treatments such as artemisinin-based combination therapies become available at low prices. Regular monitoring of the in vitro activity of antimalarials (especially lumefantrine) and screening for resistance markers for antifolates (such as the dhfr and dihydropteroate synthase mutations) and for chloroquine (such as P. falciparum multidrug resistance 1 gene and P. falciparum chloroquine resistance transporter mutations) should be carried out both in Bangui and the rest of the Central African Republic so that the National Malaria Control Program can recommend the best available treatment for malaria.

Received October 4, 2004. Accepted for publication January 19, 2005.

Acknowledgments: We thank the patients and their parents or guardians for participating in this study, and the managers of the urban health centers in Bangui (Foyer de Charité; Dr. G. Service, Boy Rabe, and Dr. C. Begoua), and Dr. D. Senekian for their assistance. We are grateful to the members of the Groupe de Travail Malaria, Réseau International des Instituts Pasteur, especially Milijoana Randrianarivelojosia (Institut Pasteur, Antananarivo, Madagascar), Ronan Jambou (Institut Pasteur, Dakar, Senegal), Thierry Fandeur (Institut Pasteur du Cambodge, Phnom Penh, Cambodia), and Eric Legrand (Institut Pasteur de Guyanne, Cayenne, French Guiana) for their valuable assistance. We also thank Leonardo K. Basco, Pascal Ringwald, Hoffmann-La Roche, and SmithKline Beecham for providing the antimalarial drugs.

Financial support: This study was supported by the Pasteur Institute of Bangui and PAL+ (French Ministry of Foreign Affairs).

* Address correspondence to Didier Menard, Institut Pasteur de Madagascar, BP 1274, Antananarivo 101, Madagascar. E-mail: dmenard{at}pasteur.mg and didier.menard{at}laposte.net Back

Authors’ addresses: Didier Menard, Institut Pasteur de Madagascar, BP 1274, Antananarivo 101, Madagascar, Telephone: 261-20-22-412-72, Fax: 261-20-22-415-34, E-mails: dmenard{at}pasteur.mg and didier.menard{at}laposte.net. Djibrine Djalle, Alexandre Manirakiza, Ferdinand Yapou, Valerie Siadoua, Serge Sana, Marcelle Diane Matsika-Claquin, and Antoine Talarmin, Pasteur Institute of Bangui, BP 923, Bangui, Central African Republic. Madji Nestor, National Malaria Control Program, Central African Republic Ministry of Health, Bangui, Central African Republic.


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TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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