AJTMH ASTMH Job Mart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am. J. Trop. Med. Hyg., 77(3), 2007, pp. 431-437
Copyright © 2007 by The American Society of Tropical Medicine and Hygiene

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Parola, P.
Right arrow Articles by Parzy, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Parola, P.
Right arrow Articles by Parzy, D.
Related Collections
Right arrow Drug-Resistance
Right arrow Malaria

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*, Bruno Pradines, Fabrice Simon, Marie-Paule Carlotti, Philippe Minodier, Marie-Pierre Ranjeva, Sékéné Badiaga, Lionel Bertaux, Jean Delmont, Marc Morillon, Ramatou Silai, Philippe Brouqui, AND Daniel Parzy
Service des Maladies Infectieuses et Tropicales, Hôpital Nord, Assistance Publique–Hôpitaux de Marseille, Marseille, France; Unité de Recherche en Physiologie et Pharmacocinétique Parasitaires, et Unité de Recherche en Biologie et Epidémiologie Parasitaires, Institut de Medecine Tropicale du Service de Sante des Armées, Marseille, France; Service de Pathologies Infectieuses et Tropicales, et Laboratoire de Biologie, Hôpital d’Instruction des Armées Laveran, Marseille, France; Service des Urgences, Hôpital Nord, Marseille, France; Unité de Pharmacologie Clinique et d’Evaluation Thérapeutique, Hôpital Timone, Assistance Publique–Hôpitaux de Marseille, Marseille, France; Centre de Formation et Recherche en Médecine et Santé Tropicales, Faculté de Médecine Secteur Nord, Marseille, France; Programme National de Lutte Contre le Paludisme, Moroni, Union des Comores


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.1–3 However, monitoring in vitro P. falciparum drug resistance is often difficult in malaria-endemic 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 50,000–70,000 immigrants from the Comoros Islands. Persons originating from these Indian Ocean islands represent most patients in Marseille diagnosed with imported malaria.6,7 In 2000–2005, 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 (43°11'–45°19'E, 11°20'–13°00'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 1Go). 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 15–20% 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 sulfadoxine-pyrimethamine as a second-line drug.


Figure 1
View larger version (46K):
[in this window]
[in a new window]

  		    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.

 
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 2004–2006.


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
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 4°C. 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 an RAL kit (Réactifs 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–Hôpitaux 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 quinine-clindamycin10,11 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 All severe cases were treated with a standard intravenous seven-day regimen of quinine (Quinimax®; Sanofi Winthrop, Gentilly, France).

In vitro assays. Samples with parasitemias ≥ 0.05% were used to test drug susceptibility. Parasitized erythrocytes were washed three times in RPMI 1640 medium (Invitrogen, Paisley, United Kingdom). When parasitemia exceeded 0.8%, infected erythrocytes were diluted to 0.5–0.8% with uninfected erythrocytes and resuspended in culture medium to a hematocrit of 1.5%. Susceptibilities to chloroquine, quinine, mon-odesethylamodiaquine, mefloquine, halofantrine, lumefantrine, doxycycline, atovaquone, and dihydroartemisinin were determined after these drugs were suspended in RPMI 1640 medium and to cycloguanil after this drug was suspended in RPMI 1640 medium SP823 with reduced concentrations of p-aminobenzoic acid (0.5 µg/L) and folate (10 µg/L) (Invitrogen). The two suspensions were supplemented with 10% human serum (AbCys SA, Paris, France) and buffered with 25 mM HEPES and 25 mM (Sigma-Aldrich, St. NaHCO3 Louis, MO).

For in vitro isotopic microtests, 200 µL/well of the suspension of parasitized erythrocytes was distributed in 96-well plates predosed with antimalarial agents. Parasite growth was assessed by adding 1 µCi of 3H-hypoxanthine with a specific activity of 14.1 Ci/mmol (Perkin Elmer, Meriden, CT) in each well. Plates were incubated for 42 hours at 37°C in an atmosphere of 10% O2, 5% CO2, and 85% N2, and a relative humidity of 95%. Immediately after incubation, the plates were frozen and thawed to lyse the erythrocytes. The contents of each well were collected on standard filter microplates (UnifilterTM GF/B; Perkin Elmer) and washed using a cell harvester (FilterMateTM Cell Harvester; Perkin Elmer). Filter microplates were dried and 25 µL of scintillation cocktail (MicroscintTM O; Perkin Elmer) was placed in each well. Radioactivity incorporated by the parasites was measured using a scintillation counter (Top CountTM; Perkin Elmer).

The 50% inhibitory concentration (IC50), i.e., the drug concentration corresponding to 50% of the uptake of 3H-hypoxanthine by the parasites in drug-free control wells, was determined by non-linear regression analysis of log-dose/ response curves (RiasmartTM; Packard, Meriden, CT). Data were analyzed after logarithmic transformation and expressed as the geometric mean and 95% confidence intervals IC50 were calculated (StataTM version 9; Stata Corporation, College Station, TX). The cut-off values, defined as > 2 SD above the mean and/or after correlation with clinical failures, for in vitro resistance or reduced susceptibility to chloroquine, quinine, mefloquine, halofantrine, monodesethylamodiaquine, lumefantrine, dihydroartemisinin, atovaquone and cyloguanil were 100 nM, 800 nM, 30 nM, 6 nM, 60 nM, 150 nM, 10.5 nM, 30 nM, and 500 nM, respectively.13

DNA extraction, polymerase chain reaction, nucleotide sequencing, and analysis of point mutations. For all samples, parasite DNA was extracted from 250 µL of blood by using the EZNA Blood DNA kit (Omega Bio Tek, Doraville, GA), as recommended by the manufacturer. Gene or gene fragments for cytochrome b (cytb), P. falciparum dihydrolate re-ductase (Pfdhfr), and P. falciparum dihydropteroate synthase (Pfdhps) were amplified and sequences obtained were analyzed.

To amplify the entire cytb gene, we used a reaction mixture that contained 0.3 µM of each primer (sense, 5'-ATGAACTTTTACTCTATTAATT-3'; antisense, 5'-TTATATGTTTGCTTGGGAGCT-3'),14 200 µM of each dNTP, 3 mM GoldStar DNA poly-MgCl2, buffer, 1 U of merase (Eurogentec, Seraing, Belgium), and 2.5 µL of DNA extract in a total volume of 25 µL. Samples were denatured at 94 °C for 5 minutes, subjected to 40 cycles (denaturation at 94°C for 20 seconds, hybridization at 50°C for 20 seconds, and denaturation at 72°C for 40 seconds), and a primer extension at 72°C for 5 minutes. An 1,131-basepair amplicon was purified with the High Pure PCR Product Purification Kit (Roche Diagnostics, Meylan, France) and sequenced with the ABI PRISM Big Dye Terminator version 1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol. Fluorescent PCR products were sequenced in an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). We screened mutations in the cytb gene at codon 268, particularly A803C (Tyr268Ser), a potential molecular marker of atovaquone/proguanil resistance.

A 562-bp fragment coding region of Pfdhfr was amplified using the PCR described above with primers sense 5'-ACGTTTTCGATATTTATGC-3' and antisense 5'-TCACATTCATATGTACTATTTATTC-3'. The fragment was amplified as described above (except for a hybridization at 52°C) and sequenced. We analyzed point mutations of Pfdhfr codons that have been associated with resistance to pyrimethamine and a proguanil metabolite. These mutations included a Ser->Asn change at codon 108, an Asn->Ile change at codon 51, a Cys->Arg change at codon 59, and an Ile->Leu change at codon 164.15

A 672-bp fragment coding region of Pfdhps was amplified using the PCR described above with primers sense 5'-GTTGAACCTAAACGTGCTGT-3' and antisense 5'-TTCATCATGTAATTTTTGTTGTG-3'. The fragment was amplified as described above (except for a hybridization at 53 ° C and a MgCl2 concentration of 2.5 mM) and sequenced. We analyzed point mutations of Pfdhps codons that have been associated with resistance to sulfadoxine. These mutations included Ser->Ala or Ser->Phe changes at codon 436, an Ala->Gly change at codon 437, a Lys->Glu change at codon 540, an Ala->Gly change at codon 581, and Ala->Thr or Ala->Ser changes at codon 613.16

The chloroquine resistance–associated K76T mutation of P. falciparum chloroquine resistance transporter (Pfcrt) was analyzed by nested allele–specific PCR, as described.17


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In vitro assays. A total of 248 fresh P. falciparum isolates was obtained, of which 122 isolates with parasitemias < 0.01% were not tested in vitro by isotopic assays. However, 126 samples with parasitemias ranging from 0.05% to 1.7% were used to test drug susceptibility. Twenty-two isolates with ratios of growth (maximum counts per minute/minimum counts per minute) < 4 were considered failures of in vitro culture. The following proportions of isolates were successfully cultured for each drug tested: 104 (83%) of 126 for chloroquine and quinine, 102 (98%) of 104 for cycloguanil, 101 (97%) of 104 for dihydroartemisinin, 99 (95%) of 104 for mefloquine and doxycycline, 93 (97%) of 104 for atovaquone, 82 (79%) of 104 for monodesethylamodiaquine, 81 (79%) of 104 for lumefantrine, and 70 (77%) of 91 for halofantrine.

Average parameter estimates for the 10 antimalarial drugs against the P. falciparum isolates are shown in Table 1Go. Fifty-three percent of the isolates tested were resistant to chloroquine in vitro. Three percent of the isolates were resistant or had reduced susceptibility to quinine, mefloquine, and atovaquone. Only 1% of the parasites had reduced in vitro susceptibility to halofantrine and dihydroartemisinin. Resistance to lumefantrine was not observed. Seven percent of the isolates had reduced susceptibility to monodesethylamodiaquine. Fifty-seven percent of the parasites were resistant or had intermediate susceptibility to cycloguanil.


View this table:
[in this window]
[in a new window]

 
TABLE 1
In vitro susceptibilities and prevalence of resistance or reduced susceptibility to antimalarial drugs of P. falciparum isolates obtained from patients with malaria imported from Comoros Islands and hospitalized in Marseille, France, 2004–2006*
 
Resistance-associated point mutations. No mutations in the cytb gene at codon 268 were found in any of 203 isolates tested. The prevalences of the Pfdhfr and Pfdhps mutations were obtained in 203 and 120 isolates, respectively, and are shown in Table 2Go. No mutants were detected in Pfdhfr codon 164. A total of 51 (25%) isolates had wild-type Pfdhfr codons 108, 59, and 51, 119 (58%) isolates had a mutation at position 108, and 108 (53%) isolates had the triple mutant haplotype S108N, N51I, and C59R. For Pfdhps, 110 (92%) isolates had wild-type codons and 9 (7%) isolates had a single haplotype mutation A437G. Six isolates had mutations in Pfdhfr codon 108 and Pfdhps codon 437. Conversely, 64 (31%) of 207 isolates had Pfcrt wild-type codon 76, and 115 (56%) isolates had the K76T mutation on both alleles. When the presence of associated mutations on Pfcrt and Pfdhfr codons was examined, 22 (11%) isolates had wild-type codons and 70 (34%) isolates had Pfcrt K76T and Pfdhfr S108N mutations (Table 2Go).


View this table:
[in this window]
[in a new window]

 
TABLE 2
Frequency of mutations of Pfdhfr (codons 51, 59, and 108), Pfdhps (codons 436, 437, 540, 581, 613), and Pfcrt (codon 76) genes of Plasmodium falciparum isolates obtained from patients with malaria imported from Comoros Islands and hospitalized in Marseille, France, 2004–2006*
 

DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, immigrants hospitalized in France for imported malaria were the source for the study of drug resistance of P. falciparum parasites in their tropical country of origin. Marseille is the city with the largest Comorian population outside Comoros and in 2002–2004, approximately 20,000 people living in France traveled to Comoros every year. Our patients who visited friends and relatives18 enabled analysis of 248 P. falciparum isolates from Comoros.

We found that 53% of 104 isolates maintained in culture were resistant in vitro to chloroquine. A similar proportion was obtained when genetic studies were performed, with 56% of 207 P. falciparum parasites having a mutation at position 76 of the gene for Pfcrt. This gene is located on chromosome 7, and codes PfCRT, a vacuolar membrane transporter protein. Although many polymorphisms associated with chloroquine resistance have been identified, the K76T mutation has been found consistently in chloroquine-resistant parasites. It is currently considered as a reliable marker of chloroquine resistance and treatment failure, although it is also present to a lesser degree in chloroquine-sensitive strains, which suggests that other polymorphisms in pfcrt are necessary or that several genes are involved.1 These results are important for public health authorities in Comoros. Chloroquine has been used as a first-line drug to treat uncomplicated P. falciparum malaria for the past 50 years in Comoros.

Almost 30 years ago, a case of chloroquine treatment failure was reported in Germany in a patient who had visited Comoros.19 In the 1980s and early 1990s, the prevalence of chloroquine resistance was considered low in Comoros, although cases with RIII resistance had been recorded.20 Since the early part of the current decade, the frequency of chloroquine treatment failures in Comoros has become unacceptably high. The last published clinical study conducted in Moroni, Grande Comore, reported that chloroquine treatment failed in 23 (77%) of 30 children, and 83% of the isolates had the K76T mutation.21 Also, on the neighboring French island of Mayotte, a high prevalence of P. falciparum with in vitro resistance to chloroquine was observed (88% of 132 tested isolates). This finding is consistent with clinical failures reported by rural dispensaries and the increase in the number of cases referred to hospitals.22 Our series of isolates obtained over three years also indicate a high prevalence of chloroquine resistance in P. falciparum in the Comoros. These results are consistent with the results of in vivo studies conducted on several islands in Comoros in 2003, include a proportion of clinical failures after chloroquine therapy greater than 50%,23 and show the need to introduce new drugs as first-line treatments for acute uncomplicated P. falciparum malaria.

Using in vitro isotopic microtests, we showed that 57% of the parasites tested were resistant to cycloguanil or had intermediate susceptibility to this drug. A good correlation was observed with our molecular results because a similar proportion (58%) of point mutations at position 108 in Pfdhfr was found, and 108 isolates (53%) had the triple mutant haplotype S108N, N51I, and C59R. The S108N mutation is essential for pyrimethamine and cycloguanil resistance of P. falciparum, and additional point mutations in three other codons (Ile51, Arg59, and Leu164) are associated with higher degrees of resistance.1,3

In a smaller number of isolates obtained in 2001 from Grande Comore, 15 (50%) of 30 isolates also had a mutation at position 108 in Pfdhfr. These results have implications for chemoprophylaxis for travelers to Comoros. The current recommended drugs (mefloquine, doxycycline, and atovaquone-proguanil)24 may be contraindicated or too expensive for some of those traveling to Comoros, and chloroquine plus proguanil is sometimes the only chemoprophylaxis that can be used by travelers. However, 34% of our isolates had a K76T mutation in Pfcrt (associated with chloroquine resistance) and an S108N mutation in Pfdhfr (associated with antifolate resistance) on both alleles, and 49% had a mutation in at least one allele of the genes. Therefore, although chemoprophylaxis with chloroquine plus proguanil is sometimes prescribed as better than nothing for a trip to the Comoros, this regimen is probably poorly efficacious and inferior to other recommendations.

Another clinical implication of our results on in vitro susceptibility to cycloguanil and Pfdhfr mutations is linked to mutations in Pfdhps. The combination of pyrimethamine plus sulfadoxine has been the second-line drug treatment for uncomplicated P. falciparum malaria in Comoros islands for many years. Sulfadoxine and pyrimethamine act synergistically. Sulfadoxine inhibits DHPS and pyrimethamine inhibits DHFR; both of these enzymes are involved in folate synthesis. The dhps point mutations screened in our study have been implicated in conferring resistance by decreasing the binding affinity of the enzyme.1 Among these mutations, Gly-437 and Glu-540 are strongly associated with sulfadoxine-pyrimethamine treatment failure in Africa. In our study, the only mutation found in Pfdhps was 437 (Ala->Gly) in 7% of 120 tested isolates. No isolate had the quintuple mutant haplotype Pfdhfr S108N, N51I, and C59R and Pfdhps K540E and A437G or the Pfdhfr C59R and Pfdhps K540E combination, which have been associated with sulfadoxine-pyrimethamine clinical failure.25 Finally, 5% of 120 isolates had mutations in Pfdhfr codon 108 and Pfdhps codon 437. Although the precise relationship between mutations in the dhfr and dhps genes in clinical sulfadoxine-pyrimethamine resistance is unclear, most data show that a sensitive dhfr allele is predictive of sulfadoxine-pyrimethamine treatment success irrespective of the dhps allele.1 Therefore, our results suggest a problem with sulfadoxine-pyrimethamine efficacy in the future in Comoros.

No mutations in the cytb gene (changes in codon 268), a potential molecular marker of atovaquone/proguanil resistance, were found in any of the Comorian isolates tested. These data are important because the fixed-dose atovaquone-proguanil combination (Malarone®) is now commonly used for the treatment and prophylaxis of P. falciparum malaria. Using in vitro isotopic assays, we found that 3% of the isolates were resistant in vitro or had reduced susceptibility to atovaquone, but consequences for the clinical efficacy have yet to be evaluated. Although we demonstrated a high rate of in vitro resistance or intermediate susceptibility to pyrimethamine and a high prevalence of Pfdhfr mutations, which suggests poor efficacy of antifolates including proguanil, this drug is synergistic with atovaquone. When combined with atovaquone, unmetabolized proguanil lowers the effective concentration at which atovaquone collapses the mitochondrial membrane potential. However, the molecular basis of this enhancement is unclear. However, only resistance to atovaquone is predictive of Malarone® clinical failures. There are no data that suggest a decreased efficacy of the atovaquone/proguanil combination for the treatment and prophylaxis of P. falciparum malaria from Comoros. To our knowledge, there is no report of any documented clinical failures of patients with P. falciparum malaria imported from Comoros and who received atovaquone/proguanil as a treatment or prophylaxis (except those in whom vomiting led to changing to an intravenous medication).

A total of 16% of 104 isolates showed decreased in vitro susceptibility to quinine (> 500 nM) with the highest IC50 of 1,253 nM. The same substantial prevalence of decreased in vitro susceptibility to quinine was found in the neighboring French territory of Mayotte.22 Quinine has been used as a third-line treatment in Comoros highlands for a long time and as a first-line drug in severe cases of malaria. However, when used to treat uncomplicated malaria, quinine is often used for 3–5 days and infrequently for 7 days, the recommended duration (Silai R, unpublished data). The consequences of our in vitro results for clinical efficacy of quinine in Comoros have yet to be evaluated, but if confirmed by clinical failure, this would leave few treatment alternatives other than ACTs.

Few isolates were classified as resistant to halofantrine and mefloquine, which are both used in the treatment of imported malaria in France (children are currently treated only with halofantrine and not mefloquine), and mefloquine is used for prophylaxis. These results are supported by good clinical efficacy of both drugs in treating P. falciparum malaria imported in children from Comoros.12 The correlation between halofantrine, mefloquine, and lumefantrine can be partly explained by their similar chemical structure and their similar mode of action or mechanism of resistance. Only 7% of the isolates were resistant to the active metabolite of amodiaquine, monodesethylamodiaquine, which suggested that amodiaquine may still be effective in Comoros. However, the relationship between the in vitro cut-off value and the in vivo outcome is uncertain.13 We did not observe resistance to lumefantrine, and one strain had reduced in vitro susceptibility to dihydroartemisinin. Because the artemisinin derivatives recommended are usually combined with lumefantrine, mefloquine, or amodiaquine, our results strongly support the use of ACT in Comoros.

The absence of resistance to monodesethylamodiaquine, mefloquine, halofantrine, lumefantrine, dihydroartemisinin, and atovaquone is consistent with other observations in Africa, including the Republic of Congo, Gabon, Senegal, and Djibouti.13,26 The level of chloroquine resistance we observed is comparable with that in Senegal, and the levels of resistance to quinine and cycloguanil are comparable with those of Gabon, Senegal, or the Republic of Congo. The current antimalarial drug situation in Comoros seems to be similar to that in many African countries.

In the absence of in vitro sensitivity assays in Comoros, obtaining Plasmodium strains from Comoros among patients becoming ill in Marseille, where all laboratory facilities are available, is a unique opportunity to establish surveillance of P. falciparum drug resistance.6 This opportunity provides data of public health importance that is essential for planning of appropriate antimalarial treatment policy in Comoros, as well as for prophylaxis and treatment guidelines for travelers to Comoros. Our results suggest that chloroquine should not be used as a first-line drug to treat P. falciparum malaria in the local population. The use of pyrimethamine-sulfadoxine has to be further evaluated. Data also suggest that clinically important decreased susceptibility of some isolates to quinine may occur in Comoros, and suggests that ACTs should be used. Although the cut-off value for in vitro reduced susceptibility to doxycycline has yet to be determined, data suggest that mefloquine and atovaquone-proguanil remain appropriate chemoprophylaxis for travelers to Comoros.

Received March 15, 2007. Accepted for publication June 13, 2007.

Acknowledgments: We thank E. Baret, N. Benoit, P. Bigot, J. Cren, and J. Mosnier for technical support, and Dr. P. Newton for editing the paper and helpful comments.

Financial support: This study was supported by the Programme Hospitalier de Recherche Clinique Régional 2003, Assistance Publique–Hôpitaux de Marseille; the French Ministry of Health (grant to the National Reference Centre of Malaria, Institut de Veille Sanitaire) and the French Armed Forces Health Service.

Disclosure: None of the authors has any conflicts of interest.

* Address correspondence to Philippe Parola, Service des Maladies Infectieuses et Tropicales, Hôpital Nord, Assistance Publique–Hôpitaux de Marseille, 13015 Marseille, France. E-mail: philippe.parola{at}medecine.univ-mrs.fr Back

Authors’ addresses: Philippe Parola, Jean Delmont, and Philippe Brouqui, Service des Maladies Infectieuses et Tropicales, Hôpital Nord, Assistance Publique–Hôpitaux de Marseille, 13015 Marseille, France, Telephone: 33-4-91-96-89-35, Fax: 33-4-91-96-89-38. Bruno Pradines, Unité de Recherche en Biologie et Epidémiologie Parasi-taires, Institut de Medecine Tropicale du Service de Sante des Armees, Boulevard Charles Livon, 13007 Marseille, France, Telephone: 33-4-91-15-01-10, Fax: 33-4-91-15-01-64. Fabrice Simon, Service de Pathologies Infectieuses et Tropicales, Hôpital d’Instruction des Armées Laveran, 13013 Marseille, France, Telephone: 33-4-91-61-72-48, Fax: 33-4-91-61-75-04. Marie-Paule Carlotti and Daniel Parzy, Unité de Recherche en Physiologie et Pharmacocinétique Parasitaires, Institut de Medecine Tropicale du Service de Sante des Armees, Boulevard Charles Livon, 13007 Marseille, France, Telephone: 33-4-91-15-01-14, Fax: 33-4-91-15-01-64. Philippe Minodier and Sékéné Badiaga, Service des Urgences, Hôpital Nord, Assistance Publique–Hôpitaux de Marseille, 13015 Marseille, France, Telephone: 33-4-91-96-83-84, Fax: 33-4-91-96-87-65. Marie-Pierre Ran-jeva, Unité de Pharmacologie Clinique et d’Evaluation Thérapeu-tique, Hôpital Timone, Assistance Publique–Hôpitaux de Marseille, Boulevard Jean Moulin, 13005 Marseille, Telephone: 33-4-91-38-80-01, Fax: 33-4-91-47-21-40. Marc Morillon, Laboratoire de Biologie, Hôpital d’Instruction des Armées Laveran, 13013 Marseille, France, Telephone: 33-4-91-61-71-10, Fax: 33-4-91-61-71-12. Ramatou Silai, Programme National de Lutte Contre le Paludisme, Moroni, Coulée, B 2108, Union des Comores, Telephone: 269-752-110.


REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Wongsrichanalai C, Pickard AL, Wernsdorfer WH, Meshnick SR, 2002. Epidemiology of drug-resistant malaria. Lancet Infect Dis 2: 209–218.[Web of Science][Medline]
  2. Plowe CV, 2005. Antimalarial drug resistance in Africa: strategies for monitoring and deterrence. Curr Top Microbiol Immunol 295: 55–79.[Web of Science][Medline]
  3. Hyde JE, 2005. Drug-resistant malaria. Trends Parasitol 21: 494–498.[Web of Science][Medline]
  4. Schlagenhauf P, Steffen R, Loutan L, 2003. Migrants as a major risk group for imported malaria in European countries. J Travel Med 10: 106–107.[Web of Science][Medline]
  5. Eliades MJ, Shah S, Nguyen-Dinh P, Newman RD, Barber AM, Roberts JM, Mali S, Parisse ME, Steketee R, 2005. Malaria surveillance–United States 2003. MMWR Surveill Summ 54: 25–40.[Medline]
  6. Parola P, Gazin P, Pradines B, Parzy D, Delmont J, Brouqui P, 2004. Marseille: a surveillance site for malaria from Comoros Islands. J Travel Med 11: 184–186.[Web of Science][Medline]
  7. Parola P, Soula G, Gazin P, Foucault C, Delmont J, Brouqui P, 2006. Fever in travelers returning from tropical areas: prospective observational study of 613 cases hospitalized in Marseille, France, 1999–2003. Travel Med Infect Dis 4: 61–70.[Medline]
  8. Ouledi A, 1995. Epidemiology and control of malaria in the Islamic Federal Republic of Comoros. Sante 5: 368–371.[Medline]
  9. Randrianarivelojosia M, Sahondra-Harisoa JL, Raharimalala LA, Raveloson A, Mauclere P, Ariey F, 2003. A network RER rooted on in vitro readout assays of Plasmodium falciparum sensitivity to chloroquine in the Indian Ocean Region. Sante 13: 95–100.[Medline]
  10. Parola P, Ranque S, Badiaga S, Niang M, Blin O, Charbit JJ, Delmont J, Brouqui P, 2001. Controlled trial of 3-day quinine-clindamycin treatment versus 7-day quinine treatment for adult travelers with uncomplicated falciparum malaria imported from the tropics. Antimicrob Agents Chemother 45: 932–935.[Abstract/Free Full Text]
  11. Adehossi E, Parola P, Foucault C, Delmont J, Brouqui P, Badiaga S, Ranque S, 2003. Three-day quinine-clindamycin treatment of uncomplicated falciparum malaria imported from the tropics. Antimicrob Agents Chemother 47: 1173.[Free Full Text]
  12. Minodier P, Noel G, Salles MK, Retornaz K, Walters H, Combes JC, Garnier JM, 2005. Mefloquine versus halofantrine in children suffering from acute uncomplicated falciparum malaria. Arch Pediatr 12 (Suppl 1): S67–S71.[Web of Science][Medline]
  13. Pradines B, Hovette P, Fusai TH, Atanda L, Baret E, Cheval P, Mosnier J, Callec A, Cren J, Amalvict R, Gardair JP, Rogier C, 2006. Prevalence of in vitro resistance to eleven standard or new antimalarial drugs among Plasmodium falciparum isolates from Pointe-Noire, Republic of the Congo. J Clin Microbiol 44: 2404–2408.[Abstract/Free Full Text]
  14. Musset L, Pradines B, Parzy D, Durand R, Bigot P, Le Bras J, 2006. Apparent absence of atovaquone/proguanil resistance in 477 Plasmodium falciparum isolates from untreated French travellers. J Antimicrob Chemother 57: 110–115.[Abstract/Free Full Text]
  15. Parzy D, Doerig C, Pradines B, Rico A, Fusai T, Doury JC, 2007. Proguanil resistance in Plasmodium falciparum African isolates: assessment by mutation-specific polymerase chain reaction and in vitro susceptibility testing. Am J Trop Med Hyg 57: 646–650.
  16. Reeder JC, Rieckmann KH, Genton B, Lorry K, Wines B, Cowman AF, 1996. Point mutations in the dihydrofolate reductase and dihydropteroate synthetase genes and in vitro susceptibility to pyrimethamine and cycloguanil of Plasmodium falciparum isolates from Papua New Guinea. Am J Trop Med Hyg 55: 209–213.[Abstract/Free Full Text]
  17. Djimde A, Doumbo OK, Cortese JF, Kayentao K, Doumbo S, Diourte Y, Dicko A, Su XZ, Nomura T, Fidock DA, Wellems TE, Plowe CV, Coulibaly D, 2001. A molecular marker for chloroquine-resistant falciparum malaria. N Engl J Med 344: 257–263.[Abstract/Free Full Text]
  18. Leder K, Tong S, Weld L, Kain KC, Wilder-Smith A, von Sonnenburg F, Black J, Brown GV, Torresi J, GeoSentinel Surveillance Network, 2006. Illness in travelers visiting friends and relatives: a review of the GeoSentinel Surveillance Network. Clin Infect Dis 43: 1185–1193.[Web of Science][Medline]
  19. Eichenlaub D, Pohle HD, 1980. A case of chloroquine-resistant (R1) falciparum malaria from the East African Comoros Island. Infection 8: 90–92.[Web of Science][Medline]
  20. Feuillet NP, Agnamey P, Brasseur P, Druilhe P, 1993. In vivo resistance of Plasmodium falciparum to chloroquine in Anjouan (Comoros). Bull Soc Pathol Exot 86: 48–51.[Web of Science][Medline]
  21. Randrianarivelojosia M, Raherinjafy RH, Migliani R, Mercereau-Puijalon O, Ariey F, Bedja SA, 2004. Plasmodium falciparum resistant to chloroquine and to pyrimethamine in Comoros. Parasite 11: 419–423.[Web of Science][Medline]
  22. Pettinelli F, Pettinelli ME, Eldin de Pecoulas P, Millet J, Michel D, Brasseur P, Druilhe P, 2004. High prevalence of multidrug-resistant Plasmodium falciparum malaria in the French territory of Mayotte. Am J Trop Med Hyg 70: 635–637.[Abstract/Free Full Text]
  23. Silai R, Moussa M, Abdalli Mari M, Astafieva-Djaza M, Hafidhou M, Oumadi A, Randrianarivelojosia M, Said Ankili A, Said Ahmed B, Gayibor AH, Ariey F, Ringwald P, 2007. Surveillance of falciparum malaria susceptibility to antimalarial drugs and policy change in the Comoros. Bull Soc Pathol Exot 100: 6–9.[Medline]
  24. Anonymous, 2006. Travelers’ health and advices for travelers. Bull Epidemiol Hebd 23–24: 154–163.
  25. Kublin JG, Dzinjalamala FK, Kamwendo DD, Malkin EM, Cortese JF, Martino LM, Mukadam RA, Rogerson SL, Lescano AG, Molyneux ME, Winstanley PA, Chimpeni P, Taylor TE, Plowe CV, 2002. Molecular markers for failure of sulfadoxine-pyrimethamine and chlorproguanil-dapsone treatment of Plasmodium falciparum malaria. J Infect Dis 185: 380–388.[Web of Science][Medline]
  26. Pradines B, Mabika Mamfoumbi M, Tall A, Sokhna C, Hoeck JL, Fusai T, Mosnier J, Czarnecki E, Spiegel A, Trape JF, Kombila M, Rogier C, 2006. In vitro activity of tafenoquine against the asexual blood stages of Plasmodium falciparum isolates from Gabon, Senegal, and Djibouti. Antimicrob Agents Chemother 50: 3225–3226.[Free Full Text]



This article has been cited by other articles:


Home page
 Antimicrob. Agents Chemother.Home page
V. Andriantsoanirina, A. Ratsimbasoa, C. Bouchier, M. Jahevitra, S. Rabearimanana, R. Radrianjafy, V. Andrianaranjaka, T. Randriantsoa, M. A. Rason, M. Tichit, et al.
Plasmodium falciparum Drug Resistance in Madagascar: Facing the Spread of Unusual pfdhfr and pfmdr-1 Haplotypes and the Decrease of Dihydroartemisinin Susceptibility
Antimicrob. Agents Chemother., November 1, 2009; 53(11): 4588 - 4597.
[Abstract] [Full Text] [PDF]

Home page
 Antimicrob. Agents Chemother.Home page
S. M. Kiara, J. Okombo, V. Masseno, L. Mwai, I. Ochola, S. Borrmann, and A. Nzila
In Vitro Activity of Antifolate and Polymorphism in Dihydrofolate Reductase of Plasmodium falciparum Isolates from the Kenyan Coast: Emergence of Parasites with Ile-164-Leu Mutation
Antimicrob. Agents Chemother., September 1, 2009; 53(9): 3793 - 3798.
[Abstract] [Full Text] [PDF]

Home page
 Antimicrob. Agents Chemother.Home page
H. Savini, H. Bogreau, L. Bertaux, H. Bouchiba, P. Kraemer, D. Parzy, E. Garnotel, C. Rogier, F. Simon, and B. Pradines
First Case of Emergence of Atovaquone-Proguanil Resistance in Plasmodium falciparum during Treatment in a Traveler in Comoros
Antimicrob. Agents Chemother., June 1, 2008; 52(6): 2283 - 2284.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Parola, P.
Right arrow Articles by Parzy, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Parola, P.
Right arrow Articles by Parzy, D.
Related Collections
Right arrow Drug-Resistance
Right arrow Malaria

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS