Proguanil Resistance in Plasmodium falciparum African Isolates: Assessment by Mutation-Specific Polymerase Chain Reaction and in Vitro Susceptibility Testing

Parzy, Daniel; Doerig, Christian; Pradines, Bruno; Rico, Alain; Fusaı̈, Thierry; Doury, J. C.

doi:10.4269/ajtmh.1997.57.646

Cited by 43 publications

(25 citation statements)

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“…19 None of the African isolates studied so far displayed the Leu-164 codon. 19,24,30,32,35,36 Thus, contrary to the molecular criteria set by Foote and others 10 and Peterson and others, 11 we failed to identify a differential in vitro response associated with a unique genotype for cycloguanil resistance.…”

Section: Discussioncontrasting

confidence: 67%

“…These parameters may explain the different levels of IC 50 values for antifolate drugs determined in the previous studies. 19,[22][23][24]27,30,31 A standardized isotopic in vitro assay is required so that the in vitro responses obtained in one study can be compared with those of other studies. The standardization of the in vitro assay needs to address the problem of serum or serum substitute to be used, the type of culture medium, tissue culture plates (including the volume of erythrocyte suspension per well), incubation period, hematocrit, parasitemia, and oxygen content of the incubator.…”

Section: Discussionmentioning

confidence: 99%

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Molecular epidemiology of malaria in Yaounde, Cameroon. VI. Sequence variations in the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene and in vitro resistance to pyrimethamine and cycloguanil.

Basco¹,

Ringwald²

2000

The American Journal of Tropical Medicine and Hygiene

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Abstract. Pyrimethamine and cycloguanil, the major human metabolite of proguanil, are inhibitors of dihydrofolate reductase that play a key role in the treatment and prevention of chloroquine-resistant Plasmodium falciparum infections in sub-Saharan Africa. Resistance to these antifolate drugs has emerged in some areas of Africa. Earlier molecular studies have demonstrated that point mutations at key positions of the dihydrofolate reductase-thymidylate synthase gene are strongly associated with antifolate resistance. However, whether the same or distinct mutations are involved in the development of resistance to both pyrimethamine and cycloguanil has not been well established in naturally occurring P. falciparum isolates. In this study, the in vitro responses to both antifolate drugs were measured in 42 Cameroonian isolates and compared with the complete sequence of the dihydrofolate reductase domain of the gene (from 34 of 42 isolates) to analyze the genotype that may distinguish between pyrimethamine and cycloguanil resistance. The wild-type profile (n ϭ 11 isolates) was associated with low 50% inhibitory concentrations (IC 50 s) ranging from 0.32 to 21.4 nanamole for pyrimethamine and 0.60-6.40 nM for cycloguanil. Mutant isolates had at least one amino acid substitution, Asn-108. Only three mutant codons were observed among the antifolate-resistant isolates: Ile-51, Arg-59, and Asn-108. The increasing number of point mutations was associated with an increasing level of pyrimethamine IC 50 and, to a much lesser extent, cycloguanil IC 50 . These results support a partial crossresistance between pyrimethamine and cycloguanil that is based on similar amino acid substitutions in dihydrofolate reductase and suggest that two or three mutations, including at least Asn-108, may be necessary for cycloguanil resistance, whereas a single Asn-108 mutation is sufficient for pyrimethamine resistance.Pyrimethamine and proguanil are dihydrofolate reductase (DHFR) inhibitors that continue to play a major role in the chemotherapy of chloroquine-resistant Plasmodium falciparum.

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Section: Discussioncontrasting

confidence: 67%

Section: Discussionmentioning

confidence: 99%

Molecular epidemiology of malaria in Yaounde, Cameroon. VI. Sequence variations in the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene and in vitro resistance to pyrimethamine and cycloguanil.

Basco¹,

Ringwald²

2000

The American Journal of Tropical Medicine and Hygiene

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“…They may appear only in combination with Asn-108, 7,27 but without necessarily causing higher resistance than single Asn-108 mutations. 21,25 A Leu-164 mutation was rare and Val-16 and Thr-108 mutations were not detected in our patients. This confirms that these mutations are generally rare.…”

Section: Discussionmentioning

confidence: 49%

“…In laboratory-cultured parasite lines, a single Asn-108 mutation has resulted in pyrimethamine resistance, whereas the accumulation of mutations (Asn-108, Ile-51, and Arg-59 and/or Leu-164 or Thr-108 and Val-16) was necessary for high levels of cycloguanil resistance. 20,21 In endemic areas, the in vivo effect of chlorproguanil 26 and the in vitro activity of cycloguanil [22][23][24][25][26] have been associated with Asn-108 but generally in combination with Ile-51 and/or Arg-59. However, the importance of these additional mutations is not fully clear.…”

Section: Discussionmentioning

confidence: 99%

Polyclonal Plasmodium falciparum malaria in travelers and selection of antifolate mutations after proguanil prophylaxis.

Färnert¹,

Tengstam²,

Palme³

et al. 2002

The American Journal of Tropical Medicine and Hygiene

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Abstract. The polymorphism of malaria parasites will greatly influence the efficiency of antimalarial drugs and vaccines. This study determined the genetic diversity of Plasmodium falciparum infections in 107 travelers and estimated the importance of mutations in the parasite dihydrofolate reductase (dhfr) gene for clinical breakthrough during proguanil prophylaxis. Genotyping with regards to the three highly polymorphic antigen-coding regions (merozoite surface protein-1 [msp-1], msp-2, and the glutamate-rich protein [glurp]) revealed multiple genotypes (up to five) in 64% of the patients. Single genotype infections were mainly associated with prior intake of antimalarial drugs, but also with a shorter stay in a malaria-endemic area and low parasite density. Malaria breakthrough despite proguanil prophylaxis was always associated with mutations in the dhfr gene; always the Asn-108 mutation and often the Ile-51 and Arg-59 mutations. The Leu-164 mutation was found in four travelers from Africa. Travelers with limited time in an endemic area were often infected with polyclonal P. falciparum infections, which suggests that single mosquito inoculations are often composed of several genetically diverse parasites. Chemoprophylaxis reduces the number of infecting clones and selects for resistant parasites as shown for proguanil through mutations in the dhfr gene.

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“…Previous studies have established the high correlation between the in vitro response to chloroquine and cycloguanil (biologically active metabolite of proguanil) and P. falciparum chloroquine-resistance transporter (pfcrt) and dihydrofolate reductase (dhfr) gene mutations, respectively, both in reference clones and field isolates. [8][9][10][11][12][13] Likewise, the key pfcrt mutation at codon 76 has been associated with clinical resistance to chloroquine.…”

Section: Introductionmentioning

confidence: 99%

Molecular epidemiology of malaria in Cameroon. XII. In vitro drug assays and molecular surveillance of chloroquine and proguanil resistance.

Basco¹

2002

The American Journal of Tropical Medicine and Hygiene

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Abstract. Chloroquine-proguanil combination is one of the options for chemoprophylaxis. The rapid evolution of drug resistance status requires a constant upgrade of epidemiologic data. Due to various difficulties in conducting prospective clinical studies on the prophylactic efficacy of the drug combination, especially in highly chloroquineresistant zones, in vitro drug sensitivity assays and specific molecular markers for chloroquine (Plasmodium falciparum chloroquine-resistance transporter, pfcrt) and cycloguanil (a biologically active metabolite of proguanil; dihydrofolate reductase, dhfr) resistance were evaluated as an alternative approach in this study. Of 116 isolates, 62 (53.4%) were doubly resistant in vitro to chloroquine (IC 50 Ն 100 nM) and cycloguanil (IC 50 Ն 15 nM). Likewise, 62 of 118 isolates (52.5%) carried both the mutant Thr-76 pfcrt allele and at least one dhfr mutant allele (1 with a single Asn-108 allele, 8 with double Arg-59 and Asn-108 mutations, and 53 with triple Ile-51, Arg-59, and Asn-108 mutations). The in vitro drug response corresponded with the presence or absence of key mutation(s) in the pfcrt and dhfr genes. These results suggest the high proportion of P. falciparum isolates in southern Cameroon that may not respond to chloroquineproguanil combination.

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Proguanil Resistance in Plasmodium falciparum African Isolates: Assessment by Mutation-Specific Polymerase Chain Reaction and in Vitro Susceptibility Testing

Cited by 43 publications

References 0 publications

Molecular epidemiology of malaria in Yaounde, Cameroon. VI. Sequence variations in the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene and in vitro resistance to pyrimethamine and cycloguanil.

Molecular epidemiology of malaria in Yaounde, Cameroon. VI. Sequence variations in the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene and in vitro resistance to pyrimethamine and cycloguanil.

Polyclonal Plasmodium falciparum malaria in travelers and selection of antifolate mutations after proguanil prophylaxis.

Molecular epidemiology of malaria in Cameroon. XII. In vitro drug assays and molecular surveillance of chloroquine and proguanil resistance.

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