Antimalarial Drug Resistance in Mali

Characterization of Novel Molecular Tools for the Epidemiological Surveillance of Antimalarial Drug Resistance in Mali

Resistance of Plasmodium falciparum (malaria) to current antimalarial drugs and the continuing development of resistance to new antimalarial formulations is one of the major obstacles to effective malaria control and case management. Efficient, comprehensive and validated methods for monitoring drug resistance in advance of the development of resistance to the antimalarial drugs that are in use are urgently needed. Molecular markers of genetic polymorphisms that give rise to resistant P. falciparum parasites and methods in population genetics for evaluating the data can be valuable tools for monitoring drug resistance in the field. This study aims to: Prospectively measure the in vivo response of P. falciparum malaria in Mali to several different antimalarial drugs and drug combinations: chloroquine (CQ), sulfadoxine-pyrimethamine (SP), amodiaquine (AQ), sulfadoxine-pyrimethamine in combination with amodiaquine (SP/AQ), amodiaquine in combination with artesunate (AQ/AS), sulfadoxine-pyrimethamine in combination with artesunate (SP/AS), and artemether-lumefantrine (Co-artem). In one site with preliminary data showing a high rate of P. falciparum resistance to mefloquine (MQ), this drug will also be tested. Measure the frequencies of molecular markers for antimalarial drug resistance, and examine how those results relate to the efficacy of these drugs in treating clinical malaria Measure drug levels at 3 days and correlate with efficacy results. Examine early clinical, parasitologic, and clinical predictors of late treatment failure. Use the knowledge gained in Aims 1-3 to develop a molecular tool for a countrywide resistance surveillance system for antimalarial drugs.

Resistance of Plasmodium falciparum to current antimalarial drugs and the continuing development of resistance to new antimalarial formulations is one of the major obstacles to effective malaria control and case management. Parasite populations are highly resistant to chloroquine on an almost worldwide basis (Central America and Haiti being the exceptions) and resistance to the next line of treatment, SP, is widespread in Asia and large parts of East Africa and South America. SP is also now recommended for use as intermittent preventative treatment (IPT) in pregnancy, which adds to concerns about the development and spread of SP resistance. More expensive combination drug therapy using artesunate and other antimalarials in combination is increasingly being recommended in an effort to extend the useful life of drugs and to slow the spread of antimalarial drug resistance. In all likelihood, resistance will eventually emerge for any new single drug or combination formulation that we deploy in the field. Given the above, efficient, comprehensive and validated methods for monitoring drug resistance in advance of the development of resistance to the antimalarial drugs that are in use are urgently needed. Such methods would help malaria control and prevention programs in guiding national treatment recommendations and policies. Integrating laboratory expertise, analytic methods based on population genetics, and more traditional methods of surveillance for anti-malarial drug resistance (e.g. in vivo drug efficacy studies) and networking with national and international partners will result in a multidisciplinary, geographically diverse team approach to assessing and monitoring drug resistant malaria, as well as developing and validating molecular methods. This type of effort will greatly assist in maximizing the useful life span of antimalarial drugs and in providing evidence-based guidance for drug policy decisions. Specific Aims: Prospectively measure the in vivo response of P. falciparum malaria in Mali to CQ, SP, AQ, SP/AQ in combination, AQ/artesunate (AS) in combination, SP/AS, and artemether-lumefantrine (Co-artem). In one site with preliminary data showing a high rate of P. falciparum resistance to MQ, MQ will also be tested. Measure the frequencies of dihydrofolate reductase (dhfr), dihydropteroate synthetase (dhps), P. falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multi-drug resistant (pfmdr 1) genotypes and establish their relationship with in vivo resistance to SP (dhfr and dhps), CQ, AQ, SP/AQ, AQ/AS, SP/AS, and MQ. Measure drug levels at 3 days and correlate with in vivo efficacy results. Examine early clinical, parasitologic, and clinical predictors of late treatment failure. Use the knowledge gained in Aims 1-3 to develop a molecular tool for a countrywide resistance surveillance system for SP, AQ, and MQ. Study Design: The study will entail two consecutive years of prospective 28 day in vivo drug efficacy studies carried out during the rainy season in three different malaria transmission sites: Koro (rural town with 71% of resistance to MQ at a lower dose of 15 mg/kg), Pongono (rural town with little exposure to antimalarials) and Faladje (rural village with > 30% of chloroquine resistance). Children aged 6-59 months with clinical symptoms consistent with malaria will be enrolled in the study after screening for fever (axillary temperature >=37.5 C) and malaria asexual parasites identified by microscopic examination of thick blood films. Blood spotted onto filter papers will be collected prior to treatment and during follow up. These filter paper samples will be used for the molecular detection of drug resistance-conferring gene polymorphisms as well as the HPLC detection and quantification of the respective drugs and their relevant metabolites. In vivo data interpretation will be done using the WHO 28-day protocol (WHO, 2003) and molecular markers will be used for the determination of the genotype resistance index (GRI). Venous blood will be collected at enrollment and at the time of in vivo failure to measure in vitro drug efficacy and cryopreserve parasites to search for novel molecular markers to new antimalarial drugs.

Early Treatment Failure (ETF, defined as: Development of danger signs or severe malaria on Day 1, 2, or 3, in the presence of parasitemia

Late Clinical Failure (LCF), defined as: Development of danger signs or severe malaria from Day 4 to Day 28 in the presence of parasitemia, without previously meeting any of the criteria of ETF

Presence of parasitemia and axillary temperature ≥37.5° C on any day from Day 4 to Day 28, without previously meeting any of the criteria of ETF

Late parasitological failure (LPF), defined as: Presence of parasitemia on Day 14 to Day 28 and axillary temperature <37.5°C without previously meeting any of the criteria of ETF or LCF

Adequate Clinical and Parasitological Response (ACPR), defined as: Absence of parasitemia on Day 28 irrespective of axillary temperature, without previously meeting any of the criteria of ETF, LCF or LPF

Frequencies of dhfr, dhps, pfcrt and pfmdr1 P. falciparum genotypes and relationship with in vivo resistance to SP (dhfr and dhps), CQ, AQ, SP/AQ, AQ/AS, SP/AS, and MQ

Inclusion Criteria: Aged 6-59 months Absence of severe malnutrition (defined as a child whose weight-for-height is below 3 standard deviations of less than 70% of the median of World Health Organization (WHO) reference values, or who has symmetrical edema involving at least the feet) A slide-confirmed infection with P. falciparum only (i.e. no mixed infections) Initial parasite density between 2,000 and 200,000 asexual parasites per microliter. Absence of general danger signs among children < 5 years (inability to drink or breastfeed; vomiting everything; recent history of convulsions; lethargy or unconsciousness; inability to sit or stand up) or other signs of severe and complicated falciparum malaria according to WHO definitions Measured axillary temperature ≥ 37.5 °C Ability to attend stipulated follow-up visits Informed consent provided by parent/guardian Absence of history of hypersensitivity reactions to any of the drugs being evaluated Exclusion Criteria: Aged < 6 or >59 months Severe malnutrition (defined as a child whose weight-for-height is below 3 standard deviations of less than 70% of the median of WHO reference values, or who has symmetrical edema involving at least the feet) No slide confirmed infection with P. falciparum or a mixed infection that includes a non P. falciparum species Initial parasite density < 2,000 or > 200,000 asexual parasites per microliter. Presence of general danger signs among children < 5 years (inability to drink or breastfeed; vomiting everything; recent history of convulsions; lethargy or unconsciousness; inability to sit or stand up) or other signs of severe and complicated falciparum malaria according to WHO definitions Measured axillary temperature <37.5 °C Inability to attend stipulated follow-up visits Unwilling to provide informed consent provided by parent/guardian History of hypersensitivity reactions to any of the drugs being evaluated

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