RESUMO
We have developed a new fragment-analysis method to enumerate the clones and to quantify their proportions within Plasmodium falciparum isolates. We prospectively enrolled 20 adult patients with uncomplicated malaria who were returning to France from various sub-Saharan countries, from January 2000 through July 2001. The analysis of clonal populations was performed on blood samples obtained at 10 times: 1 before treatment with oral quinine and 9 during the first 96 h of the treatment. The resistance genotypes pfcrt and dhfr were determined for chloroquine and antifolinics. Multiple P. falciparum genotypes were detected in 19 (95%) of 20 patients: 2, 3, 4, and 5 genotypes were found in 4, 9, 4 and 2 patients, respectively. Disappearance and reappearance of some clones within a few hours was observed. Individual clones represented 0.4%-99.4% of total parasitemia. Surprisingly, in 10 of 15 subjects tested, resistance genotypes varied according to the time of blood collection. These findings may have important implications with regard to the interpretations of resistance studies.
Assuntos
Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Animais , Resistência a Medicamentos , Genótipo , Humanos , Malária Falciparum/parasitologia , Plasmodium falciparum/classificação , Plasmodium falciparum/genética , Dinâmica Populacional , Estudos Prospectivos , Quinina/uso terapêuticoRESUMO
Msp-1 and Msp-2 genes, each present as a unique copy in the genome of Plasmodium, contain polymorphic repeats in bloc 2. We studied allelic polymorphism of Msp-1 and Msp-2 by amplifying bloc 2 with a fluorescent primer, and analysing the fragment generated. We validated this method by mixing two cloned strains: chloroquine-susceptible HB3-Honduras and chloroquine-resistant FCM29-Cameroon. This method was then used to quantify the clones in natural isolates of 19 infected persons during quinine treatment. The fragment analysis method detects efficiently clone numbers and the proportions of each in isolates.
Assuntos
Antígenos de Protozoários/genética , Antimaláricos/farmacologia , Cloroquina/farmacologia , Malária Falciparum/parasitologia , Proteína 1 de Superfície de Merozoito/genética , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/genética , Quinina/farmacologia , África Ocidental/etnologia , Alelos , Animais , Células Cultivadas/efeitos dos fármacos , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , Resistência a Medicamentos/genética , Eletroforese em Gel de Poliacrilamida , Evolução Molecular , França/epidemiologia , Amplificação de Genes , Genótipo , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/epidemiologia , Repetições Minissatélites , Fenótipo , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificação , Reação em Cadeia da Polimerase , Polimorfismo GenéticoRESUMO
A PCR-based technique using molecular beacons was developed to detect the chloroquine resistance-associated pfcrt K76T point mutation in Plasmodium falciparum. One hundred thirty African clinical isolates were tested by the new method in comparison with the PCR-restriction fragment length polymorphism method. This rapid and inexpensive genomic assay could expand the possibilities for monitoring chloroquine resistance.
Assuntos
Antimaláricos/farmacologia , Cloroquina/farmacologia , Malária Falciparum/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Animais , Clonagem Molecular , DNA de Protozoário/genética , Resistência a Medicamentos , Corantes Fluorescentes , Marcadores Genéticos , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras , Mutação Puntual/genética , Proteínas de Protozoários , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVES: To assess the relationship between the presence of DHFR and DHPS mutations in Plasmodium falciparum, parasite in vitro resistance, and in vivo efficacy of sulfadoxine-pyrimethamine (SP) treatment. PATIENTS AND METHODS: Measurement of SP treatment efficacy in malaria-infected children in Gabon was combined with in vitro tests of susceptibility to pyrimethamine and cycloguanil, and molecular genotyping at several DHFR and DHPS loci of parasites isolated before treatment. DHFR was studied at codons 108, 51, and 59, whereas DHPS gene was typed at positions 436, 437, 540 and 581. RESULTS: SP treatment was effective in 86% of children by day 28. Seventy-five percent of isolates were in vitro resistant to pyrimethamine and 65.5% to cycloguanil. No mutation was detected at codons 540 and 581 of the DHPS gene. Most isolates (71.8%) presented with the triple mutant DHFR genotype, whereas 64.3% combined at least three DHFR and one DHPS mutations. The increase in the number of DHFR mutations was associated with an increase in in vitro resistance to pyrimethamine and cycloguanil; three DHFR mutations conferred pyrimethamine and to a lesser extent cycloguanil resistance. Treatment failures only occurred with isolates presenting at least two DHFR mutations (S108N and C59R) and one DHPS mutation (S436A or A437G), but SP treatment of infections with such parasites gave treatment success in 82.0% of children. CONCLUSIONS: DHFR mutations that lead to high-level in vitro resistance to pyrimethamine plus 1-2 DHPS mutations are not sufficient to induce in vivo failure of SP treatment in young children from Gabon.