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The aim of this study was to provide a comprehensive analysis of the worldwide genetic polymorphism of ms4760 alleles of the pfnhe-1 gene and to discuss their usefulness as molecular marker of quinine resistance (QNR). A new numbering of ms4760 allele, classification grouping ms4760 alleles according to the number of DNNND and DDNHNDNHNND repeat motifs in blocks II and V was also proposed. A total of 1508 ms4760 sequences from isolates, culture-adapted parasites or reference strains from various geographical regions were retrieved from GenBank (last update on 15th June 2012) or from publications and were used for genetic analyses. The association of different alleles of pfnhe-1 with resistance to quinoline antimalarial drugs showed marked geographic disparities. The validity and reliability of candidate polymorphisms in pfnhe-1 gene as molecular markers of QNR appeared restricted to endemic areas from South Asia or possibly East African countries and needs to be confirmed.

The purpose of this review was to provide up-to-date information on the resistance of Plasmodium falciparum to the main antimalarials used in Madagascar and to assist implementation of the malaria control and elimination program. In 2006, the failure rate for chloroquine treatment was 44% (n = 300) and was comparable to the rate observed in continental Africa. Most treatment failures occurred after the first week of follow-up. P. falciparum resistance to chloroquine appeared to be special in Madagascar with only 3.2% of isolates showing in vitro resistance (n = 372, 7 sentinel sites) and less than 1% harbouring mutant parasites within the Pfcrt gene. Conversely, the Pfmdr1 N86Y point mutation was found in 64.3% (n = 174) of isolates in 2006 and in 51.7% (n = 343) in 2007. Failure of combined sulfadoxine-pyrimethamine therapy, i.e., the recommended intermittent preventive treatment for malaria during pregnancy, and in vitro resistance to pyrimethamine were rare. However, the Pfdhfr 51I/59R/108N allele showed consistently high prevalence levels reaching 33.3% in 2008. Moreover, the single Pfdhfr 164L mutant allele, a haplotype unique to Madagascar, was discovered in 2006 and showed prevalence rates up to 30% in some locations (southeast) in 2008. Up to now, the quadruple mutant allele Pfdhfr 51I/59R/108N/164L has not been observed. Susceptibility to the other antimalarials tested appeared excellent but the number of isolates showing in vitro susceptibility to artemisinin derivatives has been fallen in recent years and this decline may herald a decrease in the efficacy of these drugs.

We report the results of clinical studies carried out at six sites in Madagascar, between January and October 2006. The aims were (i) to update our knowledge of the burden of Plasmodium malariae infection and (ii) to assess the therapeutic efficacy of chloroquine for uncomplicated quartan malaria. Our findings confirm that P. malariae is the third leading cause of malaria, accounting for 1.1% of all malarial infections. They also demonstrate that chloroquine-currently recommended for the home management of presumed malaria in children under the age of five years and commonly used by adults-remains highly effective in patients with uncomplicated P. malariae infection.

Intermittent preventive treatment (IPT) of pregnant women with sulfadoxine-pyrimethamine (SP) is being considered as a routine practice in Madagascar, mainly to decrease the risks of malaria-associated severe anaemia in the women, and of low birthweight in their babies. There is, however, relatively little information available on the efficacy of SP when used, in Madagascar, to treat cases of Plasmodium falciparum malaria. In a preliminary study, carried out in 2003 in the village of Saharevo, 36 uncomplicated cases were each treated with a standard dose of SP and with paracetamol and then followed up for 28 days. No case of therapeutic failure occurred and all the asexual parasitaemias cleared by day 3. It therefore appears that SP is effective against P. falciparum in Saharevo (and probably in the whole, rural district of Moramanga in which the village lies). This is an encouraging observation to make before IPT is initiated throughout the country.

Objectives: To monitor the sensitivity of Plasmodium falciparum to the drugs used to treat severe malaria and to prevent malaria in Comoros and Madagascar., Design: We used the in vitro isotopic method to test the sensitivity of P. falciparum to quinine, mefloquine and cycloguanil., Results: We tested fresh isolates of P. falciparum, collected from patients living in urban, suburban and rural areas and suffering from uncomplicated malaria in 2001, against at least one of the antimalarials cited above. In both countries all of the successfully tested isolates were sensitive to quinine (N = 243) and to cycloguanil (N = 67). The mean IC50 ranged from 85.7 to 133.7 nM for quinine. For cycloguanil, the mean IC50 ranged from 1.4 to 20.2 nM and the highest IC50 value (102.5 nM) was recorded in Comoros. Only 0.9% (1/110) of the informative isolates from Madagascar were mefloquine-resistant (0/18 in Comoros). The mefloquine mean IC50s were 8.2 nM, 14.1 nM and 11.6 nM respectively in the rural, suburban and urban areas of Madagascar, and 5.9 nM in Comoros. A positive correlation was found between quinine and mefloquine IC50s (N = 127, r = 0.48, p < 10(-6)), but in vitro mefloquine was 6-16 times more potent than quinine. No correlation was noticed between the activities of quinine and cycloguanil or between the activities of mefloquine and cycloguanil., Conclusion: We therefore advocate the use of a full-course regimen of quinine, as recommended by the World Health Organisation (WHO), to treat above all severe malaria in Madagascar and Comoros. Our results also demonstrate that the use of mefloquine- and cycloguanil-based antimalarials is still justified to prevent malaria in both countries, mainly in the case of travellers.

To alleviate the insufficient number of experienced medical teams invited to and accepting to monitor the effectiveness of drugs prescribed to patients with a diagnosis of uncomplicated malaria and to insure the surveillance of the susceptibility of P. falciparum to current antimalarials used in Madagascar, there is a need to draw a feasible study protocol carefully discussed with them. We carried out a preliminary study in two rural areas and assessed the efficacy of sulfadoxine-pyrimethamine (SP) for curing uncomplicated P. falciparum malaria, with a simplified protocol based on the principle of observational study. A single dose of SP was given on day 0 with paracetamol. The persons to whom the drugs were administered accepted two other interventions of one member of the medical teams on day 14 and day 28. Nineteen patients, 3-63 years old, fulfilled the follow-up. The efficacy of this combination was noted for the 19 persons. Our results show that P. falciparum strains are susceptible to SP. Since SP will be used in intermittent preventive treatment in pregnant women in Madagascar, one way to delay the occurrence of SP resistant parasites will be (a) to avoid massive use of SP for the non pregnant persons and (b) to monitor susceptibility of P. falciparum to SP as part of pilot studies using standard WHO protocol (which is not really easy for most of the peripheral health facilities--with the follow-up procedures with clinical examination and parasitological control at Days 0, 1, 2, 3, 7, 14, 21 and 28), and routinely with simplified protocol such as the analytical observational study illustrated in this present study. Limit and advantage of observational study are discussed.

Objective: To determine how sensitive Plasmodium falciparum is to the major antimalarial drugs in Madagascar., Design: Assessment of Plasmodium falciparum isolates sensitivity to antimalarials, by use of the in-vitro radioisotope method., Setting: Ankazobe and Saharevo in the foothill areas; and Toamasina and Tolagnaro in the coastal areas (between January 1998 and November 1999)., Subjects: Primary Plasmodium falciparum isolates from patients with uncomplicated malaria attack., Results: Between January 1998 and November 1999, of the 293 in-vitro tests done with at least one antimalarial, 70% (205/293) were interpretable. As there was no significant difference between results from the four study sites, the data have been expressed as a whole. All of the successfully tested isolates were sensitive to halofantrine (n = 56) and to quinine (n = 199), 5.8% (12/205) of the isolates were resistant to chloroquine and 2% (4/199) to mefloquine. The geometric mean IC50 was 0.3 microg/L for halofantrine (95% CI = 0.1-0.4 microg/L); 9.4 microg/L for chloroquine (95% CI = 7.3-10.8 microg/L); 3.8 microg/L for mefloquine (95% CI = 3.3-4.3 microg/L); and 26.8 microg/L for quinine (95% CI = 24.3-29.4 microg/L). The low positive correlation found between halofantrine and chloroquine IC50s (n = 56; r = 0.41, P = 0.002) suggests a risk of cross-resistance between these two drugs., Conclusion: The degree and frequency of chloroquine resistance in-vitro is stationary in Madagascar compared to previous results during the last decade. The in-vitro sensitivity of P. falciparum to quinine, mefloquine and halofantrine encourages the use of these drugs as alternative in case of chloroquine treatment failure. Nevertheless, it is important to maintain and to extend malaria and drug sensitivity surveillance in Madagascar.

To redefine strategy and policy to cure or to prevent malaria, there is a need to get relevant and updated data on Plasmodium sp sensitivity level to antimalarial drugs. Thus, in September 1999, the Madagascan Ministry of Health and the Institut Pasteur de Madagascar (IPM) formed a network named RER for malaria resistance surveillance. To alleviate the lack of experienced medical teams within the health centres, and due to technical and logistic matters, as part of the network activities, it was decided to give a start with the in vitro studies which are carried out at IPM. In vitro sensitivity testing is done by use of the isotopic method. Results from the study done in 2001 demonstrate that the Madagascan P. falciparum isolates are susceptible to amodiaquine (n = 215), to cycloguanil (n = 56), to pyrimethamine (n = 98) and to quinine (n = 214). One isolate (1/110 i.e. 0.9%) of mefloquine-resistant phenotype is detected from the Eastern region. P. falciparum susceptibility to chloroquine is satisfactory with 95.4% (206/216) of in vitro sensitive isolates. RER arises from the partnership and collaboration between the Madagascan Ministry of Health and the IPM. The network set-up is presented. The usefulness of the in vivo approach, and the in vitro investigations (chemosusceptibility test and screening of mutations accounting for resistance to chloroquine) to monitor the emergence and the dissemination of drug-resistant parasites in Madagascar as well as in the subregion of the Indian Ocean is discussed.

In Madagascar, although chloroquine (CQ) remains the first-line treatment of choice for malaria, the gradual spread of resistance to this antimalarial drug is of increasing concern. As part of a larger investigation of the effectiveness of the second- and third-line drugs used to treat malaria, the in-vitro susceptibilities of Plasmodium falciparum collected in Madagascar to CQ, mefloquine (MQ) and artemether (ART) were therefore investigated. Median inhibitory concentrations (IC(50)) were determined for isolates collected from residents of two villages in the foothills of the central highlands. The IC(50) for ART ranged from 0.23-17.50 nM [N = 51; geometric mean = 4.02 nM; 95% confidence interval (CI) = 2.99-5.05 nM], four isolates exhibiting IC(50) (> 12 nM) indicative of resistance to this drug. The artemether IC(50) were found to be correlated with those of CQ (N = 46; Spearman's r = 0.51; P = 0.0002), which varied widely (0.4-254.3 nM; mean = 23.4 nM; CI = 7.1-39.7 nM; N = 46). Five (11%) of the 46 isolates exposed to CQ in vitro were considered resistant to this drug (i.e. to have IC(50) > 100 nM), with IC(50) ranging from 109-245.3 nM (mean = 171.6 nM; CI = 110.4-232.8 nM). However, all the CQ-resistant isolates were considered sensitive to ART and vice versa. All the isolates tested also appeared sensitive to MQ (IC(50) = 2.21-43.1 nM; mean = 10.5 nM; CI = 7.95-13.07 nM; N = 46), the IC(50) for MQ being correlated with those for CQ (N = 46; Spearman's r =0.46; P = 0.001). There was no significant correlation between ART and MQ activities. Although the sample was fairly small, the present results indicate that P. falciparum in Madagascar is generally becoming less sensitive to CQ and ART. The observation of a correlation between the IC(50) for these two drugs perhaps indicates that artemisinin derivatives would be better used in combination with antimalarial drugs other than 4-aminoquinolines.