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Objective: To investigate whether the severity of Plasmodium falciparum attack in endemic areas was associated with the multiplicity of infection (MOI) and/or with a particular genotype(s)., Method: In two areas of different malaria transmission pattern in Madagascar (Sainte-Marie - mesoendemic and Tsiroanomandidy - hypoendemic) the number and the proportions of msp-2 genotypes within isolates were determined for each patient using a capillary electrophoresis genotyping method. DNA sequencing was performed to identify the msp-2 allelic family of dominant clones., Results: Eighty six uncomplicated and 33 severe cases were included in Sainte-Marie and 48 uncomplicated and 69 severe cases were included in Tsiroanomandidy. We found no association between the MOI and severity of malaria as the same mean number of msp-2 genotypes was found in isolates from uncomplicated and from severe malaria cases (3.72 and 3.73, respectively, P>0.05). The study of the association of dominant clones with clinical status showed no particular genotype or allelic family associated with malaria severity., Conclusions: Severity of malaria was not associated with higher MOI in our study. Severity did not appear restricted to some particular genotypes either. On the contrary, severe malaria appeared to be caused by very common genotypes in the studied areas. More comprehensive explorations including immunity and genetic factors of the host are needed to acquire new information about this complex condition.

Background: The study of urban malaria is an area undergoing rapid expansion, after many years of neglect. The problem of over-diagnosis of malaria, especially in low transmission settings including urban areas, is also receiving deserved attention. The primary objective of the present study was to assess the frequency of malaria among febrile outpatients seen in private and public primary care facilities of Antananarivo. The second aim was to determine, among the diagnosed malaria cases, the contribution of autochthonous urban malaria., Methods: Two cross-sectional surveys in 43 health centres in Antananarivo in February 2003 (rainy season) and in July 2003 (dry season) were conducted. Consenting clinically suspected malaria patients with fever or history of fever in the past 48 hours were included. Malaria rapid diagnostic tests and microscopy were used to diagnose malaria. Basic information was collected from patients to try to identify the origin of the infection: autochthonous or introduced., Results: In February, among 771 patients, 15 (1.9%) positive cases were detected. Three malaria parasites were implicated: Plasmodium. falciparum (n = 12), Plasmodium vivax (n = 2) and Plasmodium. ovale (n = 1). Only two cases, both P. falciparum, were likely to have been autochthonous (0.26%). In July, among 739 blood smears examined, 11 (1.5%) were positive: P. falciparum (n = 9) and P. vivax (n = 2). Three cases of P. falciparum malaria were considered to be of local origin (0.4%)., Conclusion: This study demonstrates that malaria cases among febrile episodes are low in Antananarivo and autochthonous malaria cases exist but are rare.

The in vivo and in vitro response of Plasmodium falciparum to chloroquine was conducted in Ankazobe, a village located in the high plateau area. These studies confirmed the low level of chloroquine-resistance. The in vivo data indicate the absence of increase resistance during the 2 years study. Chloroquine is still the drug of choice for the treatment of malaria attack in this area.

One thousand and twenty six P. falciparum strains isolated from cases imported in France and field surveys in four regions of Africa and Madagascar were studied in vitro against chloroquine, monodesethylamodiaquine, quinine and mefloquine, 917 in vivo tests were performed during field studies with chloroquine (10 and 25 mg/kg) and amodiaquine (10, 25, 35 mg/kg). In Madagascar, the chemoresistance remained low and stable during the study period, concerning mostly chloroquine (11% in vitro and in vivo) without obvious geographical variation. 25 mg/kg chloroquine or amodiaquine were satisfactory as respectively first and second line therapeutic regimen. In Central Africa, chemoresistance emerged with an epidemic profile and increased dramatically in disseminated urban focus. High level and prevalence of chloroquine resistance and multiresistance were observed few months after the index cases in these foci. In South West Cameroon, amodiaquine remained efficient as curative treatment but only at a dose of 35 mg/kg/5 days. Decrease of in vitro sensitivity and in vivo efficacy of quinine is a matter of concern. Given the heterogeneous and evolutive situation of drug resistance, the need for epidemiological surveillance and monitoring of P. falciparum drug sensitivity in Africa is obvious to adjust therapeutic regimen.

34 case-reports of type RI resistance of P. falciparum to chloroquine published between 1975 and 1982 are reviewed. The patients were non-immune travellers infected in East Africa and in the neighbouring islands, especially Tanzania, Kenya and Madagascar. The increase in number and level of resistance might be a problem in Africa. A monitoring system of the patient and in the field is proposed and the possibilities of prevention and control are discussed.

563 cases of Falciparum malaria were detected in 1984 and 1985 in 7 malarial zones covering 3 climatic regions in Madagascar. All subjects underwent a therapeutic test; 175 strains of Plasmodium falciparum were isolated for in vitro drug sensitivity studies. 28 strains which were moderately chloroquine resistant in vitro were identified in 1983 in these various zones. However, 16% strains studied in 1984 in Alatsinainy (plateaux area), were chloroquine-resistant in vitro. The in vitro sensitivity to the other amino-4-quinolines seemed to be retained. In vivo, 7% of resistance type RI or RII were noted with 25 mg/kg of chloroquine but none with 25 mg/kg of amodiaquine. The usual therapeutic schedule for partially immune subjects (10 mg/kg in one dose) was ineffective on day 7 in 34% of the cases with chloroquine and 5% of the cases with amodiaquine. In conclusion to this study, we recommend that chemoprophylaxis should be stopped in schools in Madagascar and that presumed malarial attacks should be treated with a minimum dose of 25 mg/kg of chloroquine in 3 days. We suggest that amodiaquine should be used in cases of therapeutic failure with chloroquine.

Antibodies directed against antigens of the asexual blood stages of Plasmodium falciparum were studied in the plasma of 29 individuals infected with P. falciparum and living in two areas of Madagascar. These plasma samples were investigated by four immunological methods: indirect fluorescence, immunoprecipitation of radiolabeled P. falciparum polypeptides, inhibition of the in vitro growth of P. falciparum, and double diffusion in a gelose plate. A multifactorial correspondence analysis of the results obtained for each sample revealed that the nature of several of the antibodies varied according to the age and place of residence of the subjects. In comparison with plasma samples from older individuals, specimens from young children had a higher immunofluorescence titer, immunoprecipitated several additional peptides (90, 110, and 118 kilodaltons), revealed more precipitation lines in the Ouchterlony plate technique, and did not inhibit the in vitro growth of P. falciparum to the same extent. Furthermore, as opposed to plasma samples from individuals living in the high central plateau, plasma samples from individuals living on the east coast of the island inhibited the penetration of erythrocytes by merozoites of one of the two studied P. falciparum strains and preferentially immunoprecipitated low-, rather than high-, molecular-weight peptides.

Two three weeks field studies were performed in Morondava (West Coast) and Nosy Be (North West Coast) respectively in January and April 1983. Among 110 malarial patients recruited through passive detection, 70 in vivo tests and 37 in vitro tests were performed. In vivo, within 7 days 10 mg/kg chloroquine failed in 8 of the 21 treated patients, 25 mg/kg chloroquine failed in 1/43 and none of these patients treated with 10 mg/kg amodiaquine failed. In vitro, 5 among 37 isolates had chloroquine IC50 more than 250 nM/1 which is predictive for resistance with the semi-microtest. All isolates were sensitive in vitro to low doses of amodiaquine, piperaquine and dichlorquinazine.

Among 273 malarial patients recruited through passive detection, 177 in vivo tests were performed in Andekaleka and Ankazobe and 23 in vitro tests in Ifanadiana, and Ankazobe (inland of Madagascar). In vivo, chloroquine failed within 7 days at 10 mg/kg in 28/86 patients, at 25 mg/kg in 7/34 and amodiaquine failed in 5/55 with 10 mg/kg and in 0/14 with 25 mg/kg. In vitro, the semi-microtest revealed resistance to chloroquine of 1/23 isolates (IC50 = 750 nM/1) but high sensitivity to amodiaquine, dichlorquinazine and piperaquine of all studied isolates (max IC50 = 92 nM/1).

The sensitivity level of Plasmodium falciparum isolates to chloroquine and the activity of six other antimalarials were studied in the different climatic zones of Madagascar in 1983. In vivo tests were done with 10 and 25 mg kg-1 of chloroquine and amodiaquine. Early recrudescence or RII resistance was observed after treatment with 10 mg kg-1 of these drugs in 34% of the cases for chloroquine and 6.5% for amodiaquine, and after the 25 mg kg-1 dose in 7% and 0% of the cases respectively. In vitro sensitivity of 84 P. falciparum isolates to seven drugs were studied with a semi-microtest. For chloroquine, 9% of the isolates had an IC50 above 250 nM, indicating resistance. In vitro activity of piperaquine was high for all isolates except two. In vitro activity of amodiaquine, dichlorquinazine, quinine, mefloquine and halofantrine was good against all isolates (maximum IC50 was 76, 92, 560, less than or equal to 20 and less than or equal to 12 nM, respectively). Correlation between the WHO standard field test and the in vitro semi-microtest was good. Resistance of P. falciparum to chloroquine was observed in the six survey areas, but the other tested drugs showed good activity. Since no cross-resistance to 4-aminoquinolines seems to exist in Madagascar, amodiaquine (the only one available at present) should be studied as an alternative to chloroquine in the prevention and treatment of falciparum malaria in this area.

Plasma samples from 50 subjects living in three distinct regions in Madagascar (Ankazobe, Manakara, and Foulpointe) were studied by sodium dodecyl sulphate polyacrylamide gel electrophoresis of immunoprecipitated proteins from a Plasmodium falciparum strain (FCM22/Madagascar) which had been biosynthetically labelled with 35S-methionine and maintained in long-term in-vitro culture. Four major proteins with molecular weights of 96, 100, 110, and 118 kD were precipitated by plasma from Ankazobe, whereas low molecular weight proteins (27, 40, and 47 kD) were recognised more often by plasma from Foulpointe. Plasma from Manakara immunoprecipitated proteins of intermediate molecular weight. Variations in the immunological response according to the area, or antigenic diversity among the parasitic isolates might account for these results.