Tanzania Journal of Health Research Health User's Trust Fund (HRUTF) ISSN: 1821-6404 Vol. 12, Num. 1, 2011

Tanzania Journal of Health Research, Vol. 12, No. 1, January, 2010

In vitro susceptibility of Plasmodium falciparum isolates from Abidjan (Côte d’Ivoire) to artemisinin, chloroquine, dihydroartemisinin and pyronaridine

B.K. BRICE¹, Y. WILLIAM^1,2, O. LACINA¹, Y. FÉLIX¹, A. HUGUES¹, B. LÉONARDO³, M. ANDRÉ⁴, D. JOSEPH^1,4*

¹Université de Cocody (22 BP 582 Abidjan 22), Institut Pasteur (IPCI) de Côte d’Ivoire, 01 BP 490 Abidjan
²Institut National de Santé Publique, BPV47 Abidjan, Côte d’Ivoire.
³Institut de recherche pour le développement (IRD)-Organisation de Coordination pour la lutte Contre les Endémies en Afrique Centrale, BP 288 Yaoundé, Cameroun.
⁴Université Paris-Sud XI, UMR 8080, CNRS, 91405, Orsay, France

* Correspondence: Dr. Djaman A. Joseph; E-mail: djamanj@yahoo.fr ; joseph.djaman@u-psud.fr

Received 28 August 2009
Revised 10 December 2009
Accepted 11 December 2009

Code Number: th10009

Abstract

Côte d’Ivoire is an endemic area for Plasmodium falciparum malaria, with perennial transmission in the southern forest and seasonal transmission in the northern savannah. Change of first-line treatment of uncomplicated malaria to artemisinin-combination therapy (ACT) is widespread in the country as elsewhere in Africa. The present study was conducted to assess the in vitro response of Plasmodium. falciparum to antimalarial drugs  currently used in the country (chloroquine, artemisinin and dihydroartemisinin) and new drugs that could be used in the near future (pyronaridine) and to analyse the pattern of cross-resistance between these drugs. The standard in vitro drug sensitivity microtechnique recommended by the World Health Organization was used to assess the sensitivity of Plasmodium falciparum isolates collected in Abidjan (Côte d’Ivoire) between April and December 2006. Of 128 in vitro tests performed, 112 (87.5%) were successful. Among them, 32, 27, 25, and 28 P. falciparum isolates grew satisfactorily and yield interpretable results for chloroquine, pyronaridine, artemisinin, and dihydroartemisinin respectively. The proportions of resistant isolates were 56.2% for chloroquine, 48% for pyronaridine, 36% for artemisinin and 3.6% for dihydroartemisinin. The most potent drug was dihydroartemisinin with a geometric mean IC₅₀ of 2.72 nM ranged from 1.45 to 3.99 nM. No multi-resistant isolates (showing resistance to more than three drugs) were found. A positive correlation was found between the IC₅₀ values for the following drugs: chloroquine and pyronaridine (r=0.45), pyronaridine and dihydroartemisinin (r=0.40), chloroquine and artemisinin (r=0.68), artemisinin and dihydroartemisinin (r=0.62). Data suggested cross-resistance between these drugs and warrant an improved surveillance programme for drug resistance to malaria in Côte d’Ivoire.

Keywords: malaria, drug resistance, in vitro assay, cross-resistance, Côte d’Ivoire

Introduction

Malaria continues to be a major cause of consultation (50.2% according the national programme of malaria control, 2008), morbidity and mortality in Côte d’Ivoire and, children of less than five years and pregnant women are more affected by this disease. Until 2003, the national program of malaria control in the country recommended chloroquine (CQ), sulfadoxine-pyrimethamine (SP) and quinine for the first-, second- and third-line treatment of uncomplicated P. falciparum malaria, respectively. In 2003, however, after it was realised that 25% of those given CQ were not showing an adequate response (Henry et al., 1998; Djaman et al., 2004), amodiaquine replaced chloroquine as the recommended first-line drug for a short time, before the use of artemisinin in monotherapy. SP remains recommended for second-line treatment and is also recommended for intermittent preventive treatment (IPT) for pregnant women. So, after a consensus for a change of strategy for malaria control in the country, in 2007 (12 January) officially antimalarial drugs used in monotherapy have been withdrawn in favour of drug combinations including artemisinin derivatives (artemisinin-based combination therapies) (Zongo et al., 2007). Amodiaquine-artesunate, artemether-lumefantrine and quinine as first, second and third-line drugs respectively.

Chloroquine, like other 4-aminoquinolines, is thought to bind to ferriprotoporphyrine IX (FPP-IX), thereby preventing its polymerisation into non-toxic haemozoin. FPP-IX is formed in large quantities during digestion of haemoglobin in the parasite and represents a toxic waste production for the parasite (Kumar et al., 2007; Fidock et al., 2008). Pyronaridine is a Mannich base schizonticide developed in China in the 1980s. The mechanism of action is based on the inhibition of β-haematin formation in vitro, formation of drug-haematin complex, and enhancement of haematin-induced lysis of red blood cells (Auparakkitanon et al., 2006; Schlitzer et al., 2008). Furthermore, it exerts in vitro antagonistic action when combined with other antimalarial drugs (chloroquine, mefloquine, and quinine) (Auparakkitanon et al., 2003). As for artemisinin (ART) or qinghaosu, it is a sesquiterpene lactone that carries a peroxide group and, unlike most other antimalarial drugs, lacks a nitrogen-containing heterocyclic ring system. The endoperoxide of ART is believed to be cleaved by iron-II sources to yield carbon-centered radicals in the parasite (Schlitzer et al., 2008; Lelievre et al., 2007). This compound has been used successfully for a long time to treat malaria patients in China, including those with both chloroquine-sensitive and chloroquine-resistant strains of P. falciparum (Gay et al., 1994). Derivatives of this drug, such as dihydroartemisinin, artemether, and the water-soluble sodium artesunate, appear to be more potent than ART itself. The active metabolite is thought to inhibit the sarcoplasmic/endoplasmic reticulum calcium ATPase encoded by Plasmodium falciparum pfatp6, and the iron-dependent decomposition of artemisinin generates reactive oxygen species that destroy the parasite (Stockwin et al., 2009; Tahar et al., 2009).

Because of the rapid development of drug resistance in P. falciparum, according to the WHO recommendations, it is not advisable to use these molecules in monotherapy, except for the emergency treatment of severe and complicated malaria (Giao et al., 2001; Rehwagen et al., 2006). Unfortunately, these recommendations are not always followed by the populations who continue to resort to self-medication with chloroquine and sulfadoxine/pyrimethamine (Henry et al., 1998).

Although in vivo surveys are useful for estimating the rate of clinical failure and remain the gold standard to guide the national antimalarial drug policy, in vitro studies are also useful tool for assessing the spread of P. falciparum resistance without the interference of host factors (acquired immunity, pharmacokinetic variations, poor compliance). Furthermore, in vitro assays may indicate a decrease of sensitivity of P. falciparum isolates to the tested antimalarial drugs and may serve as a warning to a decreasing drug efficacy. After changing the national malaria treatment policy based on the use of ACTs for the past 3 years in Côte d’Ivoire, it is important to determine whether the situation of drug resistance has changed. The main objectives of this study were to assess the in vitro response of P. falciparum to the following two types of antimalarial drugs: -a) those used routinely in the country by the population, essentially for self-medication (chloroquine, artemisinin and dihydroartemisinin) and -b) a new drug that could be used in the near future (pyronaridine); and to analyse the pattern of cross-resistance between these drugs.

Material and methods

Study area

This study was conduced from April to December, 2006 in the district of Abobo which lies 15 km to the north of Abidjan. Patients were recruited in two health centres (El Rapha and Anokoua Kouté) of Abobo. Malaria transmission in this town occurs throughout the year, with peaks at the beginning of (April) and the end of (October) of the rainy season. Thereby, malaria is hyperendemic in this area (Koudou et al. 2007) and P. falciparum is the dominant Plasmodium species.

Parasites

Clinical isolates of P. falciparum were obtained from patients before they were treated. Venous blood samples (5mL) were collected in tubes coated EDTA (Terumo Europe N.V., Leuven, Belgium) from patients who gave their 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 six hours after blood was collected. Patients were treated by amodiaquine-artesunate or artemether-lumefantrine according to the recommendations of the malaria control program. The study was reviewed and approved by the Ivorian national Ethics Committee.

Drugs

The antimalarial drugs used in this study were obtained from the following sources: artemisinin (Aldrich, France), dihydroartemisinin and pyronaridine (TDR/WHO drug discovery Research, Geneva, Switzerland) and chloroquine (Sanofi-Aventis, Antony, France). Stock solutions of artemisinin and dihydroartemisinin were prepared in 70% methanol. Stock solutions of pyronaridine and chloroquine were prepared in sterile distilled water. Two-fold serial dilutions of the stock solutions were prepared in RPMI 1640 medium. The final concentrations of the drugs tested ranged from 12.5 to 1,600 nM for chloroquine, 1.25 to 160 nM for pyronaridine, 0.5 to 64 nM for artemisinin and dihydroartemisinin.

In vitro assay

Venous blood samples were washed three times in RPMI 1640 medium. The erythrocytes were resuspended in the complete RPMI 1640 medium (RPMI 1640 supplemented with 10% type O⁺ human serum), 25 mM HEPES buffer, and 25 mM sodium bicarbonate) at a haematocrit of 1.5% and initial parasitaemia of 0.1-0.5%. If the parasitaemia > 0.5%, fresh uninfected erythrocytes were added to adjust it (0.1-0.5%). The sensitivity of isolates to antimalarial drugs was assessed using the World Health Organization (WHO) microtest technique, and the inhibition of schizont maturation was measured microscopically. A suspension of infected erythrocytes (200 µL) was distributed in each well of the 96-well tissue culture plates containing the antimalarial drug solutions. After incubation, parasites were harvested and Giemsa-stained thick blood films were prepared. The number of mature schizonts (defined as parasites with > 3 nuclei) was counted per 200 asexual parasites. Isolates with less than 20% of mature schizonts in the control well were excluded.

Data analysis

The results were expressed as 50% as inhibitory concentration values (IC₅₀) determined by log-probit model (Excel^®; Microsoft, Redmond, WA). The IC₅₀ values for in vitro drug resistance were as follows: chloroquine, 100 nM; artemisinin and dihydroartemisinin, 10 nM; and pyronaridine, 15 nM (Pradines et al., 1998; Pradines et al, 1999). Data were expressed as the geometric mean IC₅₀ values with 95% confidence intervals. Correlation between the IC₅₀ values for different drugs was assessed by using Spearman’s rank order correlation coefficient (rho) and a coefficient of determination (r²). The significance level was fixed at 0.05. The Symyx Draw 3.1^® software was used to draw different molecules (chloroquine, pyronaridine, artemisinin and dihydroartemisinin).

Results

Of 41 clinical isolates of P. falciparum, only 32 (78%) had a parasite density > 0.1% and a total of 128 in vitro tests has been performed. A total of 112 assays (87.5%) were successful and the corresponding geometric mean IC₅₀ (GMIC₅₀) values determined from these assays are shown for each drug tested in table 1. Dihydroartemisinin (DHA) was the most active drugs tested against P. falciparum isolates. Only 3.6% (1/28) of the parasites were resistant to this drug against 97.4% (27/28) of sensitivity P. falciparum isolates The following proportions of resistance were observed for the other drugs: 56.25%, 48% and, 36% respectively for chloroquine, pyronaridine and arteminisin (Figure 1).

The correlations between the drugs are given in table 2. A positive correlation (p<0.05) was observed between the IC₅₀ values for chloroquine and artemisinin (r=0.68), chloroquine and pyronaridine (r=0.45), artemisinin and dihydroartemisinin (r=0.62), pyronaridine and dihydroartemisinin (r=0.40). However, there was no significant correlation (p>0.05) between chloroquine and dihydroartemisinin (r=-0.29), and pyronaridine and artemisinin (r=0.25).

Table 1: In vitro drug susceptibility of Plasmodium falciparum isolates collected from Abobo

^§Threshold IC₅₀ value (nM) for resistance: chloroquine=100nM, pyronaridine=15nM, artemisinin=10nM, dihydroartemisinin=10 nM

Table 2: Correlation between the in vitro responses of Plasmodium falciparum to antimalarial drugs

^§ r=Spearman’s rank order correlation coefficient; NS= Not Significant

Discussion

Since 2003 the high rate of chloroquine treatment failure and the epidemiological data obtained in Côte d’Ivoire (Djaman et al., 2002; Yavo et al., 2002; Assi et al., 2004) have led to a consensus for a change of strategy for malaria control based on the use of amodiaquine-artesunate combination for first-line treatment. However, ACT is not affordable to the majority of population without national or international grant or subsidy and the prevalence of chloroquine resistance has not changed. Indeed, the outcome of our study showed more than 50% of chloroquine-resistant P. falciparum isolates. This result is probably due to the fact that, until now, chloroquine is widely used by individuals for self-treatment and is often prescribed by health personnel despite its withdrawal (Bergeri et al., 2003; Djaman et al., 2007). Moreover, poor compliance often leads to incomplete intake of the full treatment dose.

This is the first report on the evaluation of P. falciparum in vitro susceptibility to dihydroartemisinin in Côte d’Ivoire. Our results showed that DHA had a very high effect on P. falciparum isolates, compared with the other antimalarial drugs. Because all artemisinin derivatives are rapidly converted to DHA in humans (Gay et al., 1994; Lee & Hufford, 1990), this active metabolite is more suitable for in vitro assays. The geometric mean IC₅₀ value for this drug was similar to that obtained for isolates from other African countries, but was lower than that observed in Asian isolates (Thanh et al., 2001; Noedl et al., 2003).

There was a significant correlation between the in vitro responses to chloroquine and pyronaridine or artemisinin and between the response to artemisinin and dihydroartemisinin. Cross-resistance between these drugs may be explained, at least in part, by close similarities in chemical structures (ART and DHA). A positive correlation was also obtained with other artemisinin derivatives and amino alcohols in several in vitro studies (Bustos et al., 1994; Pardines et al., 1998) and between chloroquine and pyronaridine (Pradines et al., 1999). The clinical and epidemiological significance of in vitro cross-resistance between these two classes of drugs is still unknown. However, it was suggested that resistance to artemisinin derivatives and amino alcohols may be associated with multiple mutations in pfmdr1 gene, genetic code for protein transportation (Reed et al, 2000). Pyronaridine results showed that the parasite can develop resistance mechanism to an antimalarial drug before its using. One of the reasons for such high resistance could be their structural instability. Artemisinin is chemically unstable and poorly soluble in water. The carbonyl group at C-10 of the parent compound was reduced to obtain dihydroartemisinin. Several derivatives have been developed by adding ether, ester or other substituent to the hydroxyl group of dihydroartemisinin (Gay et al., 1994).

The present study demonstrates the high in vitro activity of dihydroartemisinin against clinical isolates of P. falciparum in Abidjan. Dihydroartemisinin was more active in vitro than chloroquine, pyronaridine, and artemisinin. It is thus important to follow the current recommendations of WHO in regards to the use of the ACTs. This will allow protecting artemisinin derivatives from the rapid emergence of resistant P. falciparum malaria isolates. However, for the pyronaridine and artemisinin, the high rates of resistant Plasmodium falciparum isolates found in this study suggest other investigations on a wider sampling.

Acknowledgements

We thank the patients and their parents or guardians for participating in this study, and the managers of El Rapha and Anonkoua Kouté Health centres. We are also grateful to Dr. Leonardo Basco and TDR/WHO drug discovery Research for providing the antimalarial dugs.

References

• Assi S.B., Henry M.C., Nzeyimana, I. & Kone, M. (2004) Efficacité thérapeutique de la chloroquine en région de savane du nord de la Côte d’Ivoire en 1997. Bulletin de la Societe de Pathologie Exotique 97, 177-179.
• Auparakkitanon, S., Noonpakdee, W., Ralph, R.K., Denny, W.A. & Wilairat, P. (2003) Antimalarial 9-anilinoacridine compounds directed at hematin. Antimicrobial  Agents and Chemotherapy 47, 3708-3712.
• Auparakkitanon, S., Chapoomram, S., Kuaha, K., Chirachariyavej, T. & Wilairat, P. (2006) Targeting of hematin by the antimalarial pyronaridine. Antimicrobial Agents and Chemotherapy 50, 2197-2200.
• Bergeri I., Zoguereh, D.D., Madji, N., Barrau, K., Delmont, J., Namsenmo, A. & Ndoyo J. (2003) In vivo evaluation of chloroquine therapeutic efficacy in uncomplicated Plasmodium falciparum malaria in Central African Republic in 1997 and 1998. Bulletin de la Societe de Pathologie Exotique 96, 29–34.
• Bustos, M.D.G., Gay, F. & Diquet, B. (1994) In vitro tests on Philippine isolates of Plasmodium falciparum against four standard antimalarials and four qinghaosu derivatives. Bulletin of the World Health Organization 72, 729–735.
• Djaman, A.J., Basco, L.K. & Mazabraud, A. (2002) Monitoring the chemoresistance of Plasmodium falciparum malaria in Yopougon (Abidjan): in vivo study of chloroquine sensitivity and evaluation of pyrimethamine resistance following the analysis of point mutation in the dihydrofolate reductase gene. Sante 12, 363-367.
• Djaman, J., Abouanou, S., Basco, L. & Kone, M. (2004) Limits of the efficacy of chloroquine and sulfadoxine-pyrimethamine in Northern Abidjan (Cote d'Ivoire): Combined in vivo and in vitro studies]. Sante 14, 205-209.
• Djaman, J.A., Bla, B.K., Yavo, W., Yapi, H.F., Mazabraud, A. & Basco L.K. (2007) Polymorphism of PFCRT and PFMDR-1 Genes of Plasmodium falciparum and chloroquine susceptibility in Côte d’Ivoire. Acta Protozoologica 46, 361–365.
• Fidock, D.A., Eastman, R.T., Ward, S.A. & Meshnick, S.R. (2008) Recent highlights in antimalarial drug resistance and chemotherapy research. Trends in Parasitology 24, 537-544.
• Gay, F., Ciceron, L., Litaudon, M., Bustos, M.D., Astagneau, P., Diquet, B., Danis, M. & Gentilini, M. (1994) In-vitro resistance of Plasmodium falciparum to qinghaosu derivatives in West Africa. Lancet 343, 850-851.
• Giao, P.T., Binh, T.Q., Kager, P.A., Long, H.P., Van Thang, N., Van Nam, N. & de Vries, P.J. (2001) Artemisinin for treatment of uncomplicated falciparum malaria: is there a place for monotherapy? American Journal of Tropical Medicine and Hygiene 65, 690-695.
• Henry M.C., Koné M., Guillet P. & Mouchet J. (1998) Chloroquine resistance and malaria control in Ivory Coast. Sante 8, 287-291.
• Koudou B.G., Adja A.M., Matthys B., Cissé G., Koné M., Tanner M. & Utzinger G. (2007) Pratique agricole et transmission du paludisme dans deux zones eco-épidémiologique au centre de la Côte d’Ivoire. Bulletin de la Societe de Pathologie Exotique 100,119–123
• Kumar, S., Guha, M., Choubey, V., Maity, P. & Bandyopadhyay, U. (2007) Antimalarial drugs inhibiting hemozoin (beta-hematin) formation: a mechanistic update. Life Sciences 80, 813-828.
• Lee, I.S. & Hufford, C.D. (1990) Metabolism of antimalarial sesquiterpene lactones. Pharmacology and  Therapeutics 48, 345–355.
• Lelievre, J., Berry, A. & Benoit-Vical, F. (2007) Artemisinin and chloroquine: do mode of action and mechanism of resistance involve the same protagonists? Current Opinions in the Investigational  Drugs 8, 117-124.
• Noedl H., Faiz M.A., Yunus E.B., Rahman M.R., Hossain M.A., Samad R., Miller R.S., Pang L.W., Wongsrichanalai C., Drug-resistant malaria in Bangladesh: an in vitro assessment. American Journal of Tropical Medicine and Hygiene   68, 140–142.
• Pradines, B.C., Tall, A, Parzy, D., Spiegel, A., Fusai, A., Hienne, R. & Doury, J.C. (1998) In vitro activity of pyronaridine and amodiaquine against African isolates (Senegal) of Plasmodium falciparum in comparison with standard antimalarial agents. Journal of Antimicrobial Agents and Chemotherapy  42, 333-339.
• Pradines B.C., Rogier T., Fusai A., Tall A., Trape J.F. & Doury J.C. (1999) In vitro activity of artemether against African isolates (Senegal) of Plasmodium falciparum in comparison with standard antimalarial drugs. American Journal of Tropical Medicine and Hygiene 58, 354-357.
• Rehwagen, C. (2006) WHO ultimatum on artemisinin monotherapy is showing results. BMJ 332: 1176.
• Schlitzer, M. (2008) Antimalarial drugs - what is in use and what is in the pipeline. Archiv der Pharmazie  (Weinheim) 341, 149-163.
• Stockwin, L.H., Han, B., Yu, S.X., Hollingshead, M.G., ElSohly, M.A., Gul, W., Slade, D., Galal, A.M. & Newton, D.L. (2009) Artemisinin dimer anticancer activity correlates with heme-catalyzed reactive oxygen species generation and endoplasmic reticulum stress induction. International Journal of  Cancer 125, 1266-1275.
• Tahar, R., Ringwald, P. & Basco, L.K. (2009) Molecular epidemiology of malaria in Cameroon. XXVIII. In vitro activity of dihydroartemisinin against clinical isolates of Plasmodium falciparum and sequence analysis of the P. falciparum ATPase 6 gene. American Journal of Tropical Medicine and Hygiene 81, 13-18.
• Thanh, N.V., Cowman, A.F., Hipgrave, D., Kim, T.B., Phuc, B.Q., Cong, L.D. & Biggs, B.A. (2001) Assessment of susceptibility of Plasmodium falciparum to chloroquine, quinine, mefloquine, sulfadoxine-pyrimethamine and artemisinin in southern Vietnam. Transactions of the Royal Society of Tropical Medicine and Hygiene 95, 513–517.
• Yavo, W., Menan, E.I., Adjetey, T.A., Barro-Kiki, P.C., Nigue, L., Konan, Y.J., Nebavi, N.G. & Kone, M. (2002) In vivo sensitivity of Plasmodium falciparum to amino-4-quinolines and sulfadoxine/pyrimethamine in Agou (Ivory Coast). Pathologie Biologie (Paris) 50, 184-188.
• Zongo, I., Dorsey, G., Rouamba, N., Tinto, H., Dokomajilar, C., Guiguemde, R.T., Rosenthal, P.J. & Ouedraogo, J.B. (2007) Artemether-lumefantrine versus amodiaquine plus sulfadoxine-pyrimethamine for uncomplicated falciparum malaria in Burkina Faso: a randomised non-inferiority trial. Lancet 369, 491-498.

Copyright 2010 - Tanzania Journal of Health Research