[Surveillance of falciparum malaria susceptibility to antimalarial drugs and policy change in the Comoros]. / Surveillance de la chimiosensibilite du paludisme do à Plasmodium falciparum et changement de politique dans l'Union des Comores.

Between May and June 2001, efficacy of chloroquine was assessed in 5 sentinel sites in the 3 Comoro islands. Among the 183 children, age range between 6 and 59 months, followed up for 14 days, clinical failure rates ranged between 31.2 and 73.1% and the total failure rate (clinical and parasitological) between 50 and 88.5%. Failures were mainly early treatment failures. The Ministry of health, during a consensus meeting decided to change the first line drug and to gather baseline information on the efficacy and the tolerance of the combination artemether-lumefantrine. Between June and September 2004, among the 164 children, age range between 6 and 59 months included, the success rate of the combination was 99.4% in the 3 sites with a follow-up of 28 days. No serious drug related adverse event was reported.

Monitoring of the therapeutic efficacy of chloroquine for the treatment of uncomplicated, Plasmodium falciparum malaria in Iran.

Between 2002 and 2004, the standardized 28-day protocol recently developed by the World Health Organization was used to explore the efficacy of chloroquine, in the treatment of uncomplicated, Plasmodium falciparum malaria, in five sentinel sites in southern Iran. All but 14 of the 158 patients enrolled (128, 28 and two from the provinces of Sistan-Baluchestan, Hormozgan and Kerman, respectively) were successfully followed-up. The overall frequency of treatment failure by day 28 was 78.5%, with 17.4% of the patients being classed as early treatment failures, 34.7% as late clinical failures, and 26.4% as late parasitological failures. There appeared to be no significant change in the frequency of treatment failure between the 2002-2003 and 2003-2004 transmission seasons, nor any significant between-site variation in the efficacy of chloroquine. Given these observations, the replacement of chloroquine, as the first-line drug for the treatment of uncomplicated, P. falciparum malaria in Iran, was inevitable. Artesunate-sulfadoxine-pyrimethamine is now the recommended first-line treatment, with artemether-lumefantrine used for second-line treatment. The efficacies of these combination therapies are currently being evaluated and monitored.

Point mutations in the Plasmodium falciparum cg2 gene, polymorphism of the kappa repeat region, and their relationship with chloroquine resistance.

Based on the available DNA sequence data of the Plasmodium falciparum cg2 gene, we have hypothesized that 3 amino-acid substitutions, His275Gln, Gly281Ala, and His299Gln, may represent the key mutations that confer resistance to chloroquine. The presence of 14 tandemly repeated hexamer units in the kappa region has also been suggested to be indicative of chloroquine resistance. These 2 hypotheses were tested by determining the sequence of DNA fragments containing all 3 codons and kappa repetitive region (approximately 450-basepairs) for 53 randomly selected clinical isolates (obtained in Cameroon in 1994-97) with known response in vivo and/or in vitro to chloroquine. The cg2 genotypes based on the 3 codons and the response in vitro to chloroquine, as well as the number of kappa repeat units and responses in vivo and in vitro to chloroquine, were associated (P < 0.05). cg2 gene mutations were more common in parasites from patients with failure in vivo. However, this difference did not achieve statistical significance (P = 0.055). The sensitivity and specificity of the 3 codons and kappa repeat region to predict the response in vitro to chloroquine ranged between 75% and 85%. The sensitivity and specificity of these genetic markers to predict the response in vivo to chloroquine were of lower values. The kappa repeat region of the clinical isolates is polymorphic but characterized by several conserved features.

Analysis of the key pfcrt point mutation and in vitro and in vivo response to chloroquine in Yaoundé, Cameroon.

The putative key codon (Lys-76 in sensitive parasites and Thr-76 in resistant parasites) of the novel candidate gene for chloroquine resistance, Plasmodium falciparum chloroquine resistance transporter (pfcrt), was determined by polymerase chain reaction-restriction fragment length polymorphism from 111 Cameroonian isolates and was compared with in vivo and in vitro responses to chloroquine. The key codon was significantly associated (P< .001) with responses in vivo (92% sensitivity and 76% specificity) and in vitro (97% sensitivity and 81% specificity). Some discordant results were due to multiclonal infections. The high, but not perfect, correlation between the pfcrt polymorphism and the phenotype implies that a single point mutation in codon 76 of the pfcrt gene is the major, but possibly not the sole, determinant for chloroquine resistance.

Cardiac effects of amodiaquine and sulfadoxine-pyrimethamine in malaria-infected African patients.

The cardiac effect of amodiaquine and sulfadoxine-pyrimethamine was studied in adult Cameroonian patients with acute uncomplicated Plasmodium falciparum malaria by electrocardiographic monitoring over the course of 7 days. Clinical and parasitological responses were monitored until Day 14. Bradycardia was observed in 16 of 20 amodiaquine-treated patients on Day 2, which corresponds to the time when maximal cumulative plasma concentration is reached, and in 12 of 20 patients on Day 7. A bradycardic effect lasting several days was not noted in patients treated with sulfadoxine-pyrimethamine. Significantly prolonged P, PQ, QRS, and QTc intervals were recorded on Day 2 after both 30 and 35 mg of amodiaquine base per kilogram of body weight had been administered, but these intervals were not correlated with the plasma monodesethylamodiaquine (main human active metabolite of amodiaquine) level. Electrocardiographic changes after therapy with sulfadoxine-pyrimethamine were minor and transient. All patients had fever and parasite clearance on or before Day 3 and remained free of fever and parasites until Day 14. None of the patients complained of cardiovascular adverse effects during the follow-up. These results suggest the absence of significant cardiac effects of amodiaquine and sulfadoxine-pyrimethamine at usual therapeutic doses, but they should draw the attention of clinicians treating malaria-infected patients who have taken other antimalarial drugs with cardiovascular side effects or those who are under treatment with cardiovascular drugs.

Molecular epidemiology of malaria in Yaounde, Cameroon. VIII. Multiple Plasmodium falciparum infections in symptomatic patients.

The extent of genetically distinct parasite populations coinfecting individual human hosts (i.e., multiplicity) was studied by polymerase chain reaction amplification of 3 polymorphic genetic markers, circumsporozoite protein and merozoite surface antigens (MSA) 1 and 2, in symptomatic children and adults and analyzed in relation with age and initial parasitemia. Of the total of 177 DNA samples analyzed (of which 115 were paired pre- and posttreatment samples), 101 (57%) were composed of multiclonal infections, with up to 7 distinguishable parasite populations. Among the 3 polymorphic markers, msa-2 yielded the highest proportion of clinical isolates with multiclonal populations. Patients with multiclonal infections before treatment had, on average, 2.9 genetically distinct parasite populations. The extent of multiplicity decreased significantly (P < 0.05) in recrudescent parasites, but not with reinfections, as compared with the pretreatment samples. Neither age (5-60 years) nor initial parasitemia was correlated with multiplicity. Further studies in different epidemiological settings are required to understand the role of multiclonal Plasmodium falciparum infections in influencing malaria transmission.

Chemoresistance of P. falciparum in urban areas of Yaounde, Cameroon. Part 1: Surveillance of in vitro and in vivo resistance of Plasmodium falciparum to chloroquine from 1994 to 1999 in Yaounde, Cameroon. / Chimiorésistance de P. falciparum en milieu urbain à Yaoundé, Cameroun. Part 1: S urveillance in vitro et in vivo de la résistance de Plasmodium falciparum à la chloroquine entre 1994 et 1999 à Yaoundé, Cameroun.

Chloroquine is indicated for the first-line treatment of uncomplicated malaria in most African countries. However, the spread of chloroquine-resistant Plasmodium falciparum requires periodic monitoring. Between 1994 and 1999, we studied the evolution of chloroquine resistance in adults (aged > 15 years) and children aged 5-15 years by using tests of therapeutic efficacy and in vitro assays. Responses to the 14-day in vivo test were classified according to the new criteria established by the World Health Organization. The results of the semi-microtest and the microtest were expressed as the 50% inhibitory concentration (IC50), and the threshold level of resistance was set at IC50 > 100 nM. The overall percentages of clinical and parasitological failures were 39.7% (31. 3% - 48.1%) and 48.8% (40.2% - 57.4%), respectively. Similarly, the percentage of isolates that were resistant in vitro was 52.5%. During the study, IC50 geometric mean varied between 84,6 nM and 149, 8 nM. The results of the in vitro assays agreed with those of tests of therapeutic efficacy (kappa coefficient = 0.69). The patients' chloroquine plasma levels were measured on day 0, day 3, day 7, and day 14. Drug measurement showed wide inter-individual variations and higher plasma levels in adults than in children. Some cases of therapeutic failure were associated with inadequate plasma levels of chloroquine. Our results confirm the high level of chloroquine resistance in Yaoundé and suggest that the use of an alternative antimalarial drug for the first-line treatment of uncomplicated malaria is warranted.

Chemoresistance of Plasmodium falciparum in the urban region of Yaounde, Cameroon. Part 2: Evaluation of the efficacy of amodiaquine and sulfadoxine-pyrimethamine combination in the treatment of uncomplicated Plasmodium falciparum malaria in Yaounde, Cameroon. / Chimiorésistance de P. falciparum En milieu urbain à Yaoundé, Cameroun. Part 2: evaluation de l'efficacité de l'amodiaquine et de l'association sulfadoxine-pyriméthamine pour le traitement de l'accès palustre simple à Plasmodium falciparum à Yaoundé, Cameroun.

The spread of chloroquine resistance or its stabilization at a high level calls for a change in the therapeutic strategy, including a possible replacement of chloroquine. We assessed and compared the efficacy of amodiaquine and sulfadoxine-pyrimethamine in Yaoundé. Of 140 adults and children > 5 years enrolled in the study, 59 in the amodiaquine and 58 in the sulfadoxine-pyrimethamine treatment group were followed until day 14. The efficacy of amodiaquine was 100%, whereas 12.1% of the patients treated with sulfadoxine-pyrimethamine responded with an early treatment failure. Side effects in both treatment groups were mild and did not require any specific treatment. We did in vitro drug assays for monodesethylamodiaquine (active metabolite of amodiaquine) and pyrimethamine and measured plasma levels of monodesethylamodiaquine, sulfadoxine, and pyrimethamine. Unlike amodiaquine, the results of the in vitro drug sensitivity test for pyrimethamine were not concordant with the clinical response. A wide inter-individual variation in the plasma drug levels was observed. Unlike chloroquine, the mean plasma concentrations did not vary with age. There was no significant difference in the plasma concentrations of sulfadoxine and pyrimethamine between patients responding with an adequate clinical response and those responding with treatment failure. Amodiaquine has several advantages over sulfadoxine-pyrimethamine combination and may be considered to be an effective drug in an endemic zone with a moderate level of chloroquine resistance.

Sequence variations in the genes encoding dihydropteroate synthase and dihydrofolate reductase and clinical response to sulfadoxine-pyrimethamine in patients with acute uncomplicated falciparum malaria.

Mutations in dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) are associated with in vitro resistance to sulfadoxine and pyrimethamine, respectively. The response of 75 patients to sulfadoxine-pyrimethamine was determined, and the genes of the corresponding Plasmodium falciparum isolates were sequenced. Of 12 different unmixed allelic combinations, the triple dhfr mutation Asn-108/Arg-59/Ile-51 was observed in all patients responding with early treatment failure. Some, but not all, patients with an adequate clinical response also harbored isolates with the triple dhfr mutation. Higher initial parasitemia and fever distinguished these 2 patient groups. The dhps genotype apparently had no influence on the clinical outcome. The other dhfr alleles with 1 or 2 mutations and the wild-type allele were found in patients with an adequate clinical response. The triple dhfr mutation is one of the genetic determinants associated with in vivo resistance to sulfadoxine-pyrimethamine.

[Drug-resistant malaria: problems with its definition and technical approaches]. / Chimiorésistance du paludism: problèmes de la définition et de l'approche technique.

In antimalarial chemotherapy, drug resistance is defined as "the ability of a parasite strain to survive and/or multiply despite the administration and absorption of a drug in doses equal to or higher than those usually recommended but within the limits of tolerance of the subject". This official World Health Organization definition, based on clinical and parasitological observations, was established in 1973, when genetics, pharmacology and in vitro culture techniques were still in the early stages of development. Several techniques are currently used to detect drug-resistant Plasmodium falciparum. Several in vivo tests, the traditional gold standard for the detection of drug resistance, have been developed. Classical tests include the 28-day extended test and the 7-day test, interpreted using the S-RI-RII-RIII classification system (S for susceptible and R for resistant, with three degrees of resistance, I to III, depending on parasitological response). These tests cannot be applied in practice, in field situations, and the results do not take into account the clinical condition of the patient, largely because they were designed for use with asymptomatic carriers. These limitations led to the development in 1994 (modified in 1996) of the more practical and simplified 14-day test of therapeutic efficacy. This test classifies the patient's clinical and parasitological response as "adequate clinical response", "late treatment failure" or "early treatment failure". This in vivo test of therapeutic efficacy can be applied in the field with a minimum of health facilities, personnel and other resources. However, true cases of drug resistance may not always be detected by in vivo tests due to pharmacokinetic variations, reinfection, multiple infections, noncompliance or interference with the acquired immune response. The most commonly used reliable in vitro assay, the isotopic microtest, determines the drug concentration at which 50% of parasite growth is inhibited (50% inhibitory concentration IC50). The in vitro assay not only yields quantitative results, it also determines the phenotype of the parasite independently of the immune and physiopathological conditions of the host. However, this in vitro assay requires highly skilled personnel and laboratory equipment. In addition, parasites isolated from patients who have taken medication on their own initiative a few days before consultation usually do not grow in vitro and the interpretation of assay results for patients with multiple infections may be equivocal. One of the major problems with in vitro tests is the determination of the threshold IC50 values that distinguish susceptible from resistant parasites. There are currently no fully validated cut-off points for assessing in vitro resistance. Despite these shortcomings, in vitro tests are of value, particularly if performed in parallel with the in vivo test. Molecular biology has made a major contribution to our understanding of the mechanisms of drug resistance. Discrete point mutations in the genes encoding dihydrofolate reductase and dihydropteroate synthase are strongly associated with resistance in vitro to pyrimethamine and sulfadoxine, respectively. Preliminary results have also suggested that these mutations are responsible for the failure of sulfadoxine-pyrimethamine combination treatment. No causal relationship between discrete polymorphisms in the candidate genes and in vitro chloroquine resistance has yet been established. High-performance liquid chromatography is being increasingly used to determine the plasma concentrations of antimalarial drugs in patients with prophylactic or therapeutic failure, to check that the failure of the treatment is not due to inadequate levels of the drug in the patient. Taking into account all these aspects of resistance to antimalarial drugs we think that the WHO definition of drug resistance is now inadequate. (ABSTRACT TRUNCATED)

Molecular epidemiology of malaria in Yaounde, Cameroon. VI. Sequence variations in the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene and in vitro resistance to pyrimethamine and cycloguanil.

Pyrimethamine and cycloguanil, the major human metabolite of proguanil, are inhibitors of dihydrofolate reductase that play a key role in the treatment and prevention of chloroquine-resistant Plasmodium falciparum infections in sub-Saharan Africa. Resistance to these antifolate drugs has emerged in some areas of Africa. Earlier molecular studies have demonstrated that point mutations at key positions of the dihydrofolate reductase-thymidylate synthase gene are strongly associated with antifolate resistance. However, whether the same or distinct mutations are involved in the development of resistance to both pyrimethamine and cycloguanil has not been well established in naturally occurring P. falciparum isolates. In this study, the in vitro responses to both antifolate drugs were measured in 42 Cameroonian isolates and compared with the complete sequence of the dihydrofolate reductase domain of the gene (from 34 of 42 isolates) to analyze the genotype that may distinguish between pyrimethamine and cycloguanil resistance. The wild-type profile (n = 11 isolates) was associated with low 50% inhibitory concentrations (IC50s) ranging from 0.32 to 21.4 nanamole for pyrimethamine and 0.60-6.40 nM for cycloguanil. Mutant isolates had at least one amino acid substitution, Asn-108. Only three mutant codons were observed among the antifolate-resistant isolates: Ile-51, Arg-59, and Asn-108. The increasing number of point mutations was associated with an increasing level of pyrimethamine IC50 and, to a much lesser extent, cycloguanil IC50. These results support a partial cross-resistance between pyrimethamine and cycloguanil that is based on similar amino acid substitutions in dihydrofolate reductase and suggest that two or three mutations, including at least Asn-108, may be necessary for cycloguanil resistance, whereas a single Asn-108 mutation is sufficient for pyrimethamine resistance.

In vitro antimalarial activity of limonoids from Khaya grandifoliola C.D.C. (Meliaceae).

The crude extract from the bark and seeds of Khaya grandifoliola was active in vitro against Plasmodium falciparum with an IC50 value of 13.23 microg/ml. The extract was purified to obtain seven limonoids--methylangolensate (1), 6-methylhydroxyangolensate (2), gedunin (3), 7-deacetylkhivorin (5), 1-deacetylkhivorin (6), swietenolide (7), 6-acetylswietenolide (8)--and one flavonoid, catechin (4). Five limonoids (1, 3, 5, 6, 8) were active with IC50 values between 1.25 and 9.63 microg/ml. Catechin was practically devoid of activity. The most active limonoid, gedunin, exhibited an additive effect when combined with chloroquine.

Immune response to Plasmodium falciparum liver stage antigen-1: geographical variations within Central Africa and their relationship with protection from clinical malaria.

Two populations of schoolchildren from Gabon and Cameroon were tested in 1995 for their immunological reactivity to synthetic peptides (LSA-Rep, LSA-J and LSA-CTL) from Plasmodium falciparum liver stage antigen-1 (LSA-1). The prevalence and levels of both cellular (lymphocyte proliferation, tumour necrosis factor alpha (TNF alpha), interferon gamma (IFN gamma), and interleukin-10 (IL-10)) and humoral (immunoglobulin G) responses were determined. Protection from clinical malaria, determined after a prospective 1 year study in both sites, was associated with elevated proliferative responses to LSA-Rep and LSA-CTL in the Gabonese children, as well as with higher antibody levels to both schizont extract and LSA-Rep. The prevalence of peptide-stimulated TNF-alpha secretion was higher in the Cameroonian group, but higher levels of antibodies to LSA-Rep and LSA-J were found in the Gabonese children. The immunological differences observed between children in the 2 study sites are discussed in the context of both epidemiological and individual host factors.

Site-based study on polymorphism of Plasmodium falciparum MSP-1 and MSP-2 genes in isolates from two villages in Central Africa.

We investigated Plasmodium falciparum genetic diversity in isolates collected from school-going residents aged from 5 to 15 years in the village of Pouma (Cameroon, Central Africa). Seventy-six children were grouped according to the clinical status. Asymptomatic status was defined as parasite carriage in the absence of any clinical symptom and malaria symptomatic status with patent parasitemia over 5000 parasites/microliter of blood and an axillary temperature > 37.5 degrees C. Parasite DNA was analysed prior to malaria treatment. Genotyping of the P. falciparum merozoite surface proteins (MSP) 1 and 2 was performed by polymerase chain reaction using allele-specific primers. K1, MAD20, Ro33 and 3D7/CAMP, FC27 allelic families were attributed to MSP-1 and MSP-2 genes, respectively. No association was found between P. falciparum MSP-1 and MSP-2 genotypes and the clinical status of children. Mixed P. falciparum infections were detected in 78% of overall samples and all isolates from symptomatic children contained more than 1 clone. The results obtained in the village of Pouma were compared to those of the village of Dienga in Gabon where a similar study, using the same genotyping methods, had been carried out in the same age group of schoolchildren. Data are interpreted in the context of malaria epidemiology in both settings.

Molecular epidemiology of malaria in Yaounde, Cameroon. VII. Analysis of recrudescence and reinfection in patients with uncomplicated falciparum malaria.

In an endemic area where malaria transmission is intense and continuous, reappearance of asexual parasites may be ascribed to either recrudescence or reinfection. To distinguish between recrudescence and reinfection after oral treatment with chloroquine, amodiaquine, pyronaridine, sulfadoxine-pyrimethamine, halofantrine, or artesunate, three polymorphic markers (circumsporozoite protein, merozoite surface antigens 1 and 2) from pre-treatment and post-treatment samples were amplified by the polymerase chain reaction, and the in vitro response to chloroquine was determined for comparison. Of 52 paired samples, 22 (42%) were reinfections. Recrudescence occurred more frequently on or before Day 14 (22 of 30 cases, 73%). Except for one case, all reinfections were observed beyond Day 14. The phenotype determination was not sufficiently precise to distinguish between recrudescence and reinfection. Our results suggest that beyond Day 14 (and until Day 42), recrudescence and reinfection cannot be distinguished at our study site unless molecular techniques are used and that some results derived from the polymerase chain reaction need to be compared with the microscopic examination of thick blood smear to exclude gametocyte carriers without asexual parasites after treatment.