Antimalarial Drug Susceptibility and Point Mutations Associated with Drug Resistance in 248 Plasmodium falciparum Isolates Imported from Comoros to Marseille, France in 2004–2006

Philippe Parola . 0 1 Bruno Pradines 0 1 Fabrice Simon 0 1 Marie-Paule Carlotti 0 1 Philippe Minodier 0 1 Marie-Pierre Ranjeva 0 1 Skn Badiaga 0 1 Lionel Bertaux 0 1 Jean Delmont 0 1 Marc Morillon 0 1 Ramatou Silai 0 1 Philippe Brouqui 0 1 Daniel Parzy 0 1 0 Infectieuses et Tropicales, Hopital Nord, Assistance Publique- Hopitaux de Marseille , 13015 Marseille , France 1 Service des Maladies Infectieuses et Tropicales, Hopital Nord, Assistance Publique-Hopitaux de Marseille , Marseille , France; Unite de Recherche en Physiologie et Pharmacocinetique Parasitaires, et Unite de Recherche en Biologie et Epidemiologie Parasitaires, Institut de Medecine Tropicale du Service de Sante des Arme es , Marseille , France; Service de Pathologies Infectieuses et Tropicales, et Laboratoire de Biologie, Hopital d'Instruction des Armees Laveran , Marseille , France; Service des Urgences, Hopital Nord , Marseille , France; Unite de Pharmacologie Clinique et d'Evaluation Therapeutique, Hopital Timone, Assistance Publique-Hopitaux de Marseille , Marseille , France; Centre de Formation et Recherche en Medecine et Sante Tropicales, Faculte de Medecine Secteur Nord , Marseille , France; Programme National de Lutte Contre le Paludisme , Moroni, Union des Comores A total of 248 Plasmodium falciparum isolates were sampled in travelers with malaria who came to Marseille, France from Comoros to investigate in vitro activities of antimalarial drugs and molecular markers of drug resistance. Of the 248 isolates, 126 were maintained in culture. Of these, 53% were resistant to chloroquine, and 3% had reduced susceptibility to quinine, mefloquine, and atovaquone; 1% had reduced susceptibility to halofantrine and dihydroartemisinin; 7% had reduced susceptibility to monodesethylamodiaquine; 37% had reduced susceptibility to cycloguanil; and none had reduced susceptibility to lumefantrine. Resistance-associated point mutations were screened in 207 isolates. No mutations in the cytochrome b gene were found. Of the 207 isolates, 119 (58%) had a mutation in the P. falciparum dihydrofolate reductase (Pfdhfr) gene at codon 108, 6 (5%) had mutations in both Pfdhfr codon 108 and the P. falciparum dihydropteroate synthase codon 437, and 115 (56%) had the chloroquine resistance-associated K76T mutation in the P. falciparum chloroquine resistance transporter gene. This study represents a unique opportunity to improve surveillance of P. falciparum drug resistance in Comoros with consequences for treatment and chemoprophylaxis guidelines. - Plasmodium falciparum drug resistance represents a major health problem in malaria-endemic countries. It is assessed by therapeutic response (World Health Organization in vivo tests), by in vitro assays measuring the intrinsic sensitivity of P. falciparum to inhibition of growth or schizont maturation in the presence of drugs, by the use of molecular markers associated with P. falciparum parasite drug resistance, and by pharmacokinetic analysis.13 However, monitoring in vitro P. falciparum drug resistance is often difficult in malariaendemic areas because of the lack of laboratory facilities, expertise, and funding. Immigrants returning to their country of origin to visit friends and relatives are a major risk group for imported malaria in Europe and the United States.4,5 In Marseille, France, there are approximately 50,00070,000 immigrants from the Comoros Islands. Persons originating from these Indian Ocean islands represent most patients in Marseille diagnosed with imported malaria.6,7 In 20002005, of 1,310 patients with malaria hospitalized at the University and Military Hospitals of Marseille, 67% of the adults and 90% of the children had been infected after travel to the Comoros Islands (Parola P, Simon F, unpublished data). The Comoros archipelago (4311 4519 E, 1120 1300 S) includes four islands located in the Indian Ocean. Three islands, Grande Comore (Njazidja), Moheli (Mwali), and Anjouan (Ndzouani), constitute Comoros (Union des Comores), which has been an independent country since 1975. The nearby island of Mayotte remains a French Territorial possession (Figure 1). These four islands have an area of approximately 2,300 km2 and a population of approximately 726,000 inhabitants. In Comoros, malaria is a major public health problem and causes 1520% of the registered deaths in the hospital pediatric services.8 Recently, artemisinin-based combination therapy (ACT) has been evaluated in Comoros and proposed as the national therapy for acute uncomplicated P. falciparum malaria. However, until 2005, chloroquine remained the first-line regimen with sulfadoxinepyrimethamine as a second-line drug. In Comoros, in vitro testing of P. falciparum susceptibility is difficult, with the closest reference center at the Institut Pasteur in Madagascar, where strains from Comoros have been studied.9 Thus, obtaining P. falciparum strains from travelers returning from Comoros to Marseille, where extensive laboratory facilities are available, is a unique opportunity to improve surveillance of P. falciparum drug resistance in the Comoros Islands. We present results of in vitro and molecular studies of P. falciparum isolates obtained from immigrants who returned from Comoros to Marseille with malaria in 20042006. MATERIALS AND METHODS Parasites. Between January 1, 2004, and October, 31, 2006, fresh P. falciparum isolates were obtained in Marseille from patients hospitalized with malaria after returning from the Comoros Islands. Approximately 1 mL of venous blood was collected before treatment, placed in Vacutainer ACD tubes (Becton Dickinson, Rutherford, NJ), and stored at 4C. Within 24 hours, the tubes were transported to the South National Reference Laboratory for Malaria Chemosusceptibility in Marseille. Thin blood smears were stained by using FIGURE 1. Maps showing Marseille, France and the Comoros archipelago. This archipelago includes the four islands of Grand Comore (Njazidja), Moheli (Mwali), and Anjouan (Ndzouani), which form Comoros (Union des Comores), which has been an independent country since 1975, and the nearby island of Mayotte, which is a French Territorial possession. an RAL kit (Ractifs RAL, Paris, France) and examined to determine the P. falciparum density. Informed written consent was obtained from patients before blood collection and institutional ethical guidelines of the Assistance Publique Hpitaux de Marseille were followed. Adults were treated according to French national guidelines and the protocols of each medical unit with either a standard three-day regimen of oral atovaquone-proguanil, four tablets/day for three days (Malarone ; GlaxoSmithKline, Research Triangle Park, NC), a 7-day regimen of oral quinine, or a three-day regimen of intravenous quinineclindamycin10,11 for those who vomited (initially or after having taken a first-line oral drug). Children less than 15 years of age were treated with standard regimens of mefloquine or halofantrine.12 Al (...truncated)