Plasmodium falciparum: pfcrtandDHFRMutations Are Associated with Failure of Chloroquine plus Proguanil Prophylaxis in Travelers

To the Editor—French clinicians still support the combination of chloroquine and proguanil for antimalarial chemoprophylaxis in most of West Africa, where falciparum chloroquine resistance (FCR) remains moderate. Recently, a polymorphism in the pfcrt gene was reported as strongly associated with in vitro FCR in parasite lines and in natural isolates [1–3]. In vivo studies demonstrated absolute selection of the pfcrt K76T mutant allele in therapeutic failure [4]. However, in areas where FCR rates are high, the pfcrt mutant allele was ubiquitous and thus was not predictive of clinical outcomes in immune patients [5, 6]. As has been shown previously in many studies, point mutations in Plasmodium falciparum DHFR are linked to antifolate resistance. The aim of the present work was to determine the DHFR and pfcrt genotypes of isolates obtained from travelers who had used combination chloroquine-proguanil prophylaxis From 1997 to 2000, 62 isolates of P. falciparum were obtained, at the Bichat–Claude Bernard Hospital (Paris), from symptomatic malaria-infected travelers who had correctly taken chloroquine-proguanil prophylaxis. The countries visited were categorized into 2 groups, according to the classification adopted by French experts: group 2 countries are those where the recommended chemoprophylaxis regimen is chloroquine-proguanil, and group 3 countries are those where the recommended chemoprophylaxis regimen is mefloquine. Isolates came from 15 countries in sub-Saharan Africa, Southeast Asia, and South America; 1 isolate was from an undetermined African country. Twenty-eight isolates were from group 2 countries, and 33 were from group 3 countries. The presence of chloroquine, monodesethyl-chloroquine, proguanil, and cycloguanil in plasma was determined by high performance liquid chromatography [7]. Drugs or metabolites in plasma were detected for at least 3 of the 4 drugs or metabolites tested in 48 of the 62 cases. In 14 cases, only chloroquine and monodesethyl-chloroquine were detected DNA was extracted from clinical isolates, and DHFR 108, 51, and 59 and pfcrt 76 genotypes were determined by polymerase chain reaction followed by DNA sequencing (table 1).(see table 1, published only in the electronic edition of the Journal [http://www.journals.uchicago.edu/JID/home.html]). All isolates had the pfcrt K76T mutation. Sixty-one isolates (98%) presented the DHFR S108N mutation. Fifty isolates (81%) had the triple DHFR mutation S108N, N51I, C59R. Eleven isolates (18%) had double DHFR mutations, either S108N, N51I (2 isolates) or S108N, C59R (9 isolates). One isolate had a wild-type DHFR genotype. There was no significant association between the number of mutations in DHFR and the classification of the country. There also was no significant association between the number of mutations in DHFR and drug concentrations in plasma Amino acid residue 76 of the pfcrt gene and amino acid residues 108, 51, and 59 of the DHFR gene of Plasmodium falciparum isolates and drug concentrations in plasma from 62 travelers who used chloroquine-proguanil prophylaxis. The interpretation of the efficacy of a prophylactic regimen depends on drug compliance and may be controversial. In this regard, well-conducted interrogations were crucial to validate the prophylaxis failures included in this series. Measurements of proguanil and its metabolite cycloguanil were not always useful because their short half-life limited interpretation of drug ingestion. In particular, most patients who did not show a protective cycloguanil level in plasma developed malaria >4 weeks after returning to France and discontinuing prophylaxis. In addition, a large interindividual variability of drug or metabolite levels in plasma has often been reported. However, the only patient who presented an isolate having a DHFR wild-type genotype had no detectable cycloguanil in plasma The mutant allele pfcrt K76T was found in all isolates of this series. This could reflect a very high frequency of this allele in countries visited by the patients, but results of previous studies did not confirm this hypothesis [2–4, 8]. The more likely explanation is a selection of the mutant allele by the chloroquine included in the prophylactic regimen of patients. These results, obtained with prophylactic doses given mostly to nonimmune subjects, confirmed the association of the mutation pfcrt K76T with chloroquine resistance Mutations observed in the DHFR gene were dramatically frequent among isolates of the series. All isolates but 1 showed the S108N mutation, and most had a triple mutation. In a comparable series of isolates from travelers who had not taken antifolate prophylaxis, the proportion of wild-type (S108) isolates was reported as near 50% [9]. The rapid selection of DHFR point mutations in positions 108, 51, and 59 has already been described under antifolate pressure Among isolates with double mutations in the present series, a mutant allele was selected more frequently in codon 59 than in codon 51, as reported by Doumbo et al. [10] in a study using prophylaxis by pyrimethamine alone. More than one-half of isolates in the present series came from group 3 countries, where chloroquine-proguanil prophylaxis is not recommended by French clinicians. Authentic prophylaxis failures with this combination remain rare in group 2 countries. To continue to recommend combination chloroquine-proguanil prophylaxis to travelers, it is important to regularly monitor the susceptibility of parasites in areas where FCR is not already extensively present. It may be stressed that prophylaxis failures in travelers may detect resistance earlier and at weaker levels than treatment failures within local populations, because the doses used are lower and because travelers usually are nonimmune subjects