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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One such drug, introduced for the treatment of multidrug-resistant P. falciparum malaria, is artesunate, a derivative of artemisinin. Artesunate is a blood schizontocide that acts by increasing the oxidative stress on intra-erythrocytic parasite stages.8 The drug is rapidly absorbed and then immediately hydrolyzed into its active metabolite, dihydroartemisinin, which has a short plasma half-life (< 2 hours).
Plasmodium falciparum resistance to artemisinin compounds has not been reported.9 However, several studies have shown high rates of recrudescence when artemisinin derivatives were used in monotherapy for three10–12 or five days.13–15 Consequently, since 2000, the WHO and the International Artemisinin Study Group have recommended that uncomplicated P. falciparum malaria be treated with artesunate either in combination with another effective blood schizontocide or alone for seven days to prevent recrudescence and to delay the selection of resistant strains.16,17
The aims of our study were to 1) determine the efficacy and the safety of a seven-day course of artesunate (4 mg/kg loading dose on the first day, followed by 2 mg/kg once a day for six days) for the treatment of uncomplicated P. falciparum malaria in non-immune patients (European expatriates) living in Bangui, Central African Republic; and 2) to compare host- and parasite-related factors between cured patients and patients in whom treatment failed.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In vivo assay. Patients were included in the study if they met the following criteria: 1) uncomplicated P. falciparum infection with an asexual parasitemia between 2,000 and 200,000 parasites/µL, and 2) a temperature > 37.5°C or a history of fever in the previous 24 hours, and 3) provided written informed consent. Exclusion criteria were 1) prior adequate treatment with anti-malarial drugs for the current attack, 2) an inability to drink, sit, or stand, 3) vomiting more than twice within the previous 24 hours, 4) at least one convulsion within the previous 24 hours, 5) a hemoglobin concentration < 8 g/dL, 6) severe underlying disease (cardiac, renal, or hepatic disease, malnutrition, and concomitant disease), and 7) a history of allergy to artemisinins.
Venous blood samples collected on day 0 were used to prepare thick and thin blood films and to determine hematocrit and hemoglobin levels and white blood cells counts using an automated hematology analyzer (T540; Beckman Coulter, Fullerton, CA). The smears were stained with 4% Giemsa for 20 minutes. Parasite density was calculated by counting the number of asexual parasites per 200 white blood cells (WBCs) and adjusting for the total WBC count. Thin blood smears were also examined for other Plasmodium spp.
Patients received artesunate (50-mg tablets; Sanofi, Gentilly, France) at a dose of 4 mg/kg of body weight on day 0 and 2 mg/kg of body weight once a day for the next six days. If the patient vomited within one hour of the first administration, the full dose was readministered. Patients who vomited more than once were withdrawn from the study and treated appropriately. Follow-up appointments were scheduled for days 1, 2, 3, 7, 14, and 28, and consisted of a physical examination and completion of a standardized form. Patients were encouraged to return to the heath center at any time if they felt ill. Blood samples were collected by venous puncture on days 3, 7, 14, and 28 (used to prepare thick and thin blood smears) and whenever necessary based on the results of a clinical examination.
The clinical outcomes were classified into three categories. Early treatment failure (ETF) was defined as the development of danger signs or severe malaria on days 1, 2, or 3 with parasitemia; an axillary temperature 
37.5°C on day 2 with parasitemia higher than on day 0; parasitemia on day 3 with an axillary temperature 37.5°C; or parasitemia on day 3 25% of count on day 0. Late treatment failure (LTF) was defined as development of danger signs or severe malaria after day 3 with parasitemia without previously meeting any of the criteria for ETF; or parasitemia and an axillary temperature 37.5°C on any day between days 4 and 14 without previously meeting any of the criteria for ETF. Adequate clinical response (ACR) was defined as absence of parasitemia on day 14, irrespective of axillary temperature, without previously meeting any of the criteria for ETF or LTF; or an axillary temperature < 37.5°C on day 14, irrespective of parasite count, without previously meeting any of the criteria for ETF or LTF. We also assessed parasitologic failure by dividing the ACR group into two groups: ACR without parasitemia on day 14 (ACR and adequate parasitologic response) and ACR with parasitemia on day 14 but without fever (ACR and parasitologic failure). An adverse event was defined as an unexpected change in the baseline situation, whether or not it was associated with the study drug.
In vitro assay. We used dihydroartemisinin (Sigma, St. Louis, MO) for the in vitro assay because this derivative is relatively stable and because all currently used artemisinin derivatives and those that are at advanced phases of development are rapidly transformed into dihydroartemisinin in humans.18,19 A stock solution of dihydroartemisinin was prepared in methanol. Two-fold serial dilutions of the stock solution were made in distilled water, giving final concentrations between 0.25 and 64 nM. All concentrations, as well as drug-free controls, were distributed in 20-µL aliquots in triplicate in 96-well tissue culture plates.
Venous blood samples were washed three times in RPMI 1640 medium. Each time, the white blood cell interface was removed. The erythrocytes were resuspended in complete RPMI 1640 medium consisting of 10% human serum (batch no. S02909S4190; Serum AbCys, Paris, France), 25 mM HEPES buffer, and 25 mM sodium bicarbonate at a hematocrit of 1.5% and an initial parasitemia of 0.1–0.5%. If the blood sample had a parasitemia > 0.5%, fresh uninfected erythrocytes were added to adjust the parasitemia to 0.3%. The isotopic microtest developed by Desjardins and others20 was used in this study. A suspension of infected erythrocytes (200 µL) was added to each well of the 96-well tissue culture plates. To assess parasite growth, 3H-hypoxanthine (1 µCi/ well; Amersham Pharmacia Biotech, Orsay, France) was added. The parasites were incubated at 37°C in an atmosphere of 5% CO2 for 42 hours. After incubation, the plates were frozen to terminate the in vitro assay. The plates were then thawed, and the contents of each well were collected on fiberglass filter papers (Printed Filtermat A®; Wallac, Turku, Finland) using a cell harvester (Skatron Instruments AS, Tranby, Norway). The filter disks were transferred into scintillation tubes (4 mL of Pico Pro Vial®; Packard Instrument SA) and 2 mL of scintillation cocktail (Ultima Gold F®; Packard Instrument SA, Perkin Elmer Life and Analytical Sciences, Inc., Boston, MA) was added. The incorporation of 3H-hypoxanthine was quantitated using a liquid scintillation counter (Betamatic®; Kontron, Milan, Italy). A CQ-sensitive reference strain (3D7) and a CQ-resistant reference strain (W2) obtained from the Malaria Research Reference Reagent Resource Center/American Type Culture Collection (Manassas, VA) were used as controls in the in vitro assay.
The 50% and 90% inhibitory concentrations (IC50 and IC90) were defined as the drug concentrations corresponding to 50% and 90% of the uptake of 3H-hypoxanthine measured in the drug-free control wells. They were determined by nonlinear regression analysis of log-transformed concentrations plotted against growth inhibition. For graphic display, the data were adapted to a log-probit model (Excel®; Microsoft, Redmond, WA). Data are expressed as geometric mean IC50 or IC90 values and 95% confidence intervals (CIs).
Isolation of DNA. Parasite DNA was obtained from 100 µL of thawed red blood cell pellets by incubation at 100°C for three minutes with 0.1M NaOH. The supernatant was incubated at 37°C for one hour with 250 µL of lysing solution (15 mM Tris-HCl, pH 7.5, 1% [v/v] Triton 100X, 150 mM NaCl, 1% sodium dodecyl sulfate, 1 mM EDTA) and 20 mg/mL of proteinase K, followed by two extractions with phenol/ chloroform (1:1). The DNA was then precipitated with ethanol and resuspended in 100 µL of distilled water.
Genotyping of merozoite surface protein 1 (msp-1) and merozoite surface protein 2 (msp-2). The msp-1 and msp-2 genes were genotyped to distinguish cases of recrudescence from new infections by comparing matched pairs of parasite isolates obtained at time of inclusion and on the day of reappearing parasitemia. The polymorphic regions of msp-1 (block 2) and msp-2 (block 3) were amplified by a nested polymerase chain reaction (PCR). First-round PCR primers corresponded to conserved sequences flanking these regions.21 Second-round PCR primers were used to amplify the K1, MAD20 and RO33 allelic families of msp-1,22 and the 3D7 and FC27 allelic families of msp-2.23 Nested PCR products were analyzed for size polymorphism by electrophoresis using 3% agarose gels. The DNA was stained with ethidium bromide, visualized under ultraviolet light, and photographed. Samples from an individual patient were run in adjacent lanes.
Genotyping results were used to calculate PCR-corrected cure rates. We classified parasitemia as re-infection if all bands seen on gels on the day of reappearing parasitemia were distinct from those seen on the day of inclusion (the samples were tested in parallel). We also calculated multiplicity of infection (MOI) or the number of genotypes per isolate, i.e., the highest number of genotypes at either of the two msp loci.
Plasmodium falciparum multidrug resistance 1 (pfmdr-1) PCR–restriction fragment length polymorphism. The pfmdr-1 gene was analyzed as described by Duraisingh and others.24 Parasite DNA was amplified with specific primers flanking codon 86 in two rounds of outer and nested PCRs. The pfmdr-1 alleles were identified for codon 86 by using Afl III, which cuts the coding sequence at allele Tyr86 (mutant) but not at Asn86 (wild type).
Statistical analysis. We calculated that 50 patients were required to obtain an estimate of the cure rate (estimated to be 90%) within a 95% binomial CI. Qualitative variables were compared by using either the chi-square test or Fisher’s exact test. Quantitative variables were compared by analysis of variance (normal data with Bartlett’s test) or the Kruskal-Wallis test (non-normal data with Bartlett’s test). The 95% CIs of percentages were calculated using the exact binomial test. The level of significance P was fixed at 0.05 for all statistical tests. The data were analyzed using the Epi-Info 2000 program (Centers for Disease Control and Prevention, Atlanta, GA) and MedCalc® version 7.4.3.0 software (Franck Schoojans, Department of Endocrinology, University Hospital of Ghent, Ghent, Belgium, available from http://www.medcale.be).
Ethical approval. Since there is no national ethical committee in the Central African Republic, this study was reviewed and approved by the expert committee for the anti-malarial drug policy and the Ministry of Health in this country. Written informed consent was obtained from all patients.
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RESULTS |
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Parasites were eliminated within 72 hours in all cases. The geometric mean time to parasite clearance was 32 hours (95% CI = 28–36 hours). Fever was also rapidly cleared (temperature < 37.5°C), with a geometric mean clearance time of 24 hours (95% CI = 22–26 hours). Artesunate was well tolerated and no serious adverse events were recorded.
The treatment and parasitologic cure rate was 100% on day 14, 93% on day 28 (51 of 55 patients, 95% CI = 83–98%), and 80% on day 42 (44 of 55 patients, 95% CI = 68–89%). The PCR showed that in 3 of the 11 cases of apparent recrudescence, the parasites recovered at baseline subsequently had different msp-1 and msp-2 genotypes. These patients were therefore considered re-infected and were classified as cured. These re-infections occurred from days 21 to 28 in one case and from days 28 to 42 in two cases. The PCR-corrected parasitologic cure rate was therefore 95% on day 28 (52 of 55 patients, 95% CI = 86–98%) and 85% on day 42 (47 of 55 patients, 95% CI = 74–93%). All recrudescent patients were symptomatic.
The demographic, clinical, and parasitologic characteristics of baseline isolates from recrudescent and non-recrudescent patients are summarized in Table 1
. There were no significant differences in parasitemia, IC50 for dihydroartemisinin (on available blood samples with parasitemias 0.1%). and frequency of mutant pfmdr-1 codon 86 between the two groups of patients. However, the IC90 for dihydroartemisinin was five-fold higher and the IC50:IC90 ratio for dihydroartemisinin was eight-fold lower, in isolates from recrudescent patients. The IC50 and IC90 for dihydroartemisinin of recrudescent and non-recrudescent patient isolates are showed in Figure 1. We also found that the multiplicity of infection was significantly 2-fold higher in isolates from recrudescent patients. The parasitologic characteristics of isolates at the baseline and at the failure treatment from recrudescent patients are shown in Table 2.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We confirmed that artesunate was well tolerated and rapidly cleared parasites and fever. We also demonstrated that a seven-day course of artesunate showed a high cure rate by day 14 (100%). However, the PCR-adjusted cure rate was 92% on day 28 and 85% on day 42.
Previous studies have shown high rates of recrudescence when artemisinin derivatives were used as monotherapy for three days (46–80% in non-immune patients on day 28 in Thailand and China11,12 and 72% in children on day 28 in Gabon10) and five days (80% in patients in Tanzania and Nigeria13–15). Few studies have investigated the effects of extending the duration of artemisinin monotherapy. In a Vietnamese study that compiled results of different non-comparative studies, the recrudescence rate was highest in patients treated for P. falciparum malaria with artemisinin for 5 days (50%), but ranged from 10% to 23% for patients receiving the drug for 5–10 days.26 Our study confirmed the results of another Vietnamese study,27 which showed that extending the duration of artesunate treatment from five to seven days is not useful, and that a seven-day course is no better than a five-day course. We conclude that a seven-day course of artesunate is not sufficiently effective for the treatment of uncomplicated P. falciparum malaria in non-immune patients in Bangui (European expatriates and central African citizens less than five years of age).
Our study indicates that the parasitologic cure rates on day 42 are potentially a more sensitive marker of the in vivo efficacy of an anti-malarial drug than the cure rates on days 14 or 28. This is particularly true for drugs with long plasma half-lives, but is also applicable, although to a lesser extent, to drugs with very short plasma half-lives such as artemisinin compounds. Use of validated PCR protocols28 to distinguish recrudescent infections from new infections increases the specificity of the in vivo test, as is recommended in the latest WHO in vivo protocol.29 The fact that the PCR-corrected cure rates decreased over time (from 100% to 85%) in our study clearly shows that the in vivo test was most sensitive on day 42. This confirms that 28 days should be the minimum follow-up period when assessing the efficacy of drugs against P. falciparum malaria.30
The second objective was to identify host- and parasite-related factors that differed between cured patients and patients in whom treatment failed. We observed no significant differences in host-related factors (age, sex, weight, temperature, hemoglobin concentration, hematocrit, or leukocyte count) between these two groups (Table 1
).
We also determined the IC50 and IC90 of dihydroartemisinin for baseline isolates from the two patient groups (Figure 1). The range of in vitro IC50 values observed (0.15–1.79 nM) was similar to those previously reported for Africans isolates,31 but lower than those reported for Asian isolates.32,33 We observed no significant differences in IC50 for dihydroartemisinin between the two groups. However, our study provides the first evidence that recrudescence is linked to diminished sensitivity of isolates to dihydroartemisinin. Indeed, the IC90 and the IC50:IC90 ratio for dihydroartemisinin of isolates from recrudescent patients were five-fold higher and eightfold lower, respectively, than that of isolates from non-recrudescent patients. Unlike previous studies,34 we did not find that the increase in the IC90 and the decrease in the IC50:IC90 ratio of dihydroartemisinin were associated with a mutation at codon 86 of the pfmdr-1 gene (N86Y).
No significant differences in parasite density were found between the two groups of patients, which is consistent with the findings of Ittarat and others35 or previous studies on quinine36 and mefloquine,37 which showed that recrudescent patients tended to have higher parasitemias before treatment than non-recrudescent patients. This difference could be explained by the limited size of our population and a resulting missed power in the statistical analysis.
Conversely, we found that the number of genotypes (MOI) was two-fold higher in the baseline isolates from recrudescent patients than in those from cured patients, and that only one survival genotype was found in the treatment failure isolates in all cases of recrudescence. This observation, combined with the higher IC90% observed in recrudescent patients, suggests that isolates from recrudescent patients contain three or four clones of which one is slightly diminished sensitivity to dihydroartemisinin. The presence of this slightly diminished sensitivity to dihydroartemisinin clone in recrudescent isolates did not modify IC50% values for dihydroartemisinin but increase CI90% values. We suspect that this clone was not completely eliminated with the seven-day courses of artesunate treatment and was responsible for the recrudescent cases. Unfortunately, the parasite density was too low in the treatment failure sample to measure the sensitivity of the isolate, so we cannot confirm this hypothesis.
In conclusion, our study demonstrates that a seven-day course of artesunate does not prevent recrudescence. Furthermore, the association between recrudescence and decreased sensitivity suggests that the use of artemisinin compounds alone will select resistant strains. Therefore, artesunate should not be used alone, even in seven-day courses as is recommended by the WHO, but only in combination with other anti-malarials to prevent the emergence of resistant P. falciparum.
Received February 4, 2005. Accepted for publication April 19, 2005.
Acknowledgments: We thank the patients for participating in the study.
Financial support: This work was supported by the French Government, via the PAL+ project (French Ministry of Research) and the Fonds de Solidarité Prioritaire, Résistance aux Anti Infectieux project (French Ministry of Foreign Affairs).
Disclaimer: None of the authors has any conflicts of interest.
* Address correspondence to Didier Menard, Unité du Paludisme, Institut Pasteur de Madagascar, BP 1274 Antananarivo 101, Madagascar. E-mail: dmenard{at}pasteur.mg 
Authors’ addresses: Didier Menard, Unité du Paludisme, Institut Pasteur de Madagascar, BP 1274, Antananarivo 101, Madagascar, Telephone: 261-20-22-412-72, Fax: 261-20-22-415-34, E-mail: dmenard{at}pasteur.mg. Marcelle Diane Matsika-Claquin, Djibrine Djalle, Ferdinand Yapou, Alexandre Manirakiza, Virginie Dolmazon, and Antoine Talarmin, Institut Pasteur de Bangui, BP 923, Bangui, Central African Republic. Jacques Sarda, Centre Médico-Social de l’Ambassade de France à Bangui, Bangui, Central African Republic.
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REFERENCES |
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