RESUMEN
During the surveys on antimalarial drug efficacy carried out from 2003 to 2006, we systematically checked the presence of Plasmodium falciparum in patients consulting in two health centres located in the south of Brazzaville. The first centre is situated in the urban zone; the second, in the semi rural area. The objective of this survey was to determine the prevalence of malaria-infected patients among the consulting patients and the prevalence of symptomatic patients with acute malaria attacks based on the parasitic density. Patients with parasites were assigned to one of the 5 following classifications: <2000, > or =2000, <5000, > or =5000 and > or =10,000 asexual parasites/microl of blood. Based on the threshold of parasite density 10,000 asexual parasites/microl, 10% and 24% of febrile patients in Tenrikyo and Madibou health centres were diagnosed as cases of malaria, respectively; 13.6% and 26.8% of patients under 5 years old consulting in these two health centres had malaria attacks. If the threshold of parasite density is lowered to 2000 asexual parasites/microl for patients > or =15 years old, 8% and 14% of adults in Tenrikyo and Madibou had malaria attacks, respectively The malaria burden was higher in the periphery of the city of Brazzaville than in the urbanized central districts. The Madibou health centre located in semi rural zone receives twice as many malaria cases for consultation than Tenrikyo located in the urban zone.
Asunto(s)
Antimaláricos/uso terapéutico , Malaria/epidemiología , Adolescente , Adulto , Animales , Niño , Preescolar , Congo/epidemiología , Brotes de Enfermedades , Fiebre , Humanos , Incidencia , Malaria/tratamiento farmacológico , Plasmodium , Población Rural/estadística & datos numéricos , Población Urbana/estadística & datos numéricosRESUMEN
Primaquine, an 8-aminoquinoline, is a relatively unknown and underutilized drug in French-speaking African countries. It acts against the liver stage parasites of all human malaria species, asexual blood stages of Plasmodium vivax and, to a lesser degree, Plasmodium falciparum; P. falciparum mature gametocytes, and P. vivax and Plasmodium ovale hypnozoites. Gastrointestinal disturbances are its most common side effects. The clinical utility of primaquine is limited due to its hematological side effects in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency and other contraindications (pregnant woman, breastfeeding woman, infants less than 6 months old). In the light of the recent recommendations of the World Health Organization (WHO), we propose to examine how primaquine can be used in French-speaking Africa to improve malaria control and move towards malaria elimination. Two indications supported by the WHO are of relevance in Africa. First, artemisinin-based combination therapies and primaquine given as a single low dose (0.25 mg base/kg) are effective to kill asexual and sexual parasites of P. falciparum, are well-tolerated, and have very little risk even in mild to moderate G6PD-deficient patients. This strategy may be helpful to contain transmission in an area in Africa where P. falciparum malaria incidence has decreased considerably. There is an ethical concern in administering primaquine as a gametocytocide as it does not confer any direct benefit to the treated patient. However, the single low-dose primaquine is most likely associated with very low risk for adverse hematological effects, and WHO recommends its use even without prior G6PD testing. In our opinion, clinical studies including G6PD test should be conducted to assess the safety of low-dose primaquine in African patients. Second, primaquine is effective and necessary for radical treatment of P. vivax and P. ovale, but the standard 14-day treatment (0.25-0.5 mg base/kg/day) is not recommended in patients with G6PD deficiency. Prior G6PD testing is required before prescribing primaquine for radical treatment. The use of primaquine for radical treatment in patients without contraindications does not raise any major ethical problem since the probability of relapse in patients who do not receive anti-hypnozoite treatment can be relatively high and each relapse can cause or aggravate anemia, especially in children. In our opinion, patients with mild or moderate G6PD deficiency should not be treated with primaquine at present. Further clinical studies are necessary to define the role of this drug for radical treatment in G6PD-deficient African patients. Without primaquine, the eventual elimination of P. vivax and P. ovale malaria appears to be very difficult. Updated epidemiological data on G6PD, Duffy antigen, and the current distribution of and burden due to P. vivax and P. ovale are required for a rational use of primaquine in the African continent. Moreover, clinical studies on primaquine are required in Africa.
Asunto(s)
Erradicación de la Enfermedad/métodos , Control de Infecciones/métodos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/prevención & control , Malaria Vivax/tratamiento farmacológico , Malaria Vivax/prevención & control , Primaquina/uso terapéutico , África/epidemiología , África del Norte/epidemiología , Humanos , Lenguaje , Malaria Falciparum/epidemiología , Malaria Vivax/epidemiología , Plasmodium falciparum/aislamiento & purificación , Plasmodium vivax/aislamiento & purificaciónRESUMEN
BACKGROUND: Until 2006, the Mauritanian Ministry of Health recommended chloroquine and sulfadoxine-pyrimethamine for first- and second-line treatment of uncomplicated malaria, respectively. This study assessed the clinical efficacy of sulfadoxine-pyrimethamine in Kobeni as first-line treatment. MATERIALS AND METHODS: This study included 55 patients with Plasmodium falciparum infections, who were treated with sulfadoxine-pyrimethamine and followed up for 28 days. Isolates were genotyped to distinguish between recrudescence and reinfection. Treatment success rates and survival were analysed per protocol to evaluate drug efficacy. RESULTS: After inclusion, 2 patients were excluded for protocol violations, and 3 patients were lost to follow-up. Of the remaining 50 patients (per protocol population), 43 (86%) had adequate clinical and parasitological responses. Of the 7 patients with treatment failure, 5 (10%) were early failures, while 2 (4%) had initially responded and had late clinical failure on day 7, associated with recrudescence. With the exception of one adult weighing 91 kg, all treatment failures occurred in children aged from 7 to 12 years. CONCLUSIONS: Sulfadoxine-pyrimethamine monotherapy was moderately effective but insufficiently reliable in view of the relatively high rate of early treatment failure. The high prevalence of chloroquine resistance found in earlier studies and the results of the present study on sulfadoxine-pyrimethamine justify the change in national policy and systematic use of artemisinin-based combination therapy for first-line treatment of P. falciparum malaria in Mauritania.
Asunto(s)
Antimaláricos/uso terapéutico , Malaria Falciparum/tratamiento farmacológico , Pirimetamina/uso terapéutico , Sulfadoxina/uso terapéutico , Adolescente , Adulto , Niño , Preescolar , Combinación de Medicamentos , Femenino , Humanos , Masculino , Mauritania , Persona de Mediana Edad , Insuficiencia del Tratamiento , Adulto JovenRESUMEN
A basis for the intrinsic resistance of some Plasmodium vivax isolates to pyrimethamine is suggested following the isolation of the bifunctional gene encoding dihydrofolate reductase-thymidylate synthase (DHFR-TS) of this human malaria parasite. Malaria parasites are dependent on this enzyme for folate biosynthesis. Specific inhibition of the DHFR domain of the enzyme by pyrimethamine blocks pyrimidine biosynthesis, leading to an inhibition of DNA replication. The gene was isolated by the polymerase chain reaction (PCR) from genomic DNA using degenerate oligonucleotides designed to hybridize on the highly conserved regions of the sequence. The nucleotide sequence was completed by screening P. vivax genomic bank. Sequence analysis revealed an open reading frame (ORF) of 1872 nucleotides encoding a deduced protein of 623 amino acids (aa). Alignment with other malarial DHFR-TS genes showed that a 237-residue DHFR domain and a 286-residue TS domain were separated by a 100-aa linker region. Comparison with other malarial species showed low and essentially no isology in the DHFR and junctional domains, respectively, whereas an extensive isology was observed in the TS domain. The characteristic features of the P. vivax DHFR-TS gene sequence include an insertion of a short repetitive tandem array within the DHFR domain that is absent in another human malaria parasite, P. falciparum, and a GC-biased aa composition, giving rise to highly GC-rich DHFR (50.8%), junctional (58.7%), and TS (40.5%) domains, as compared with other malaria parasites. Analysis of the 5' noncoding region revealed the presence of a putative TATA box at 116 nucleotides upstream of the ATG start codon as well as a putative GC box at -636. Comparison of the DHFR sequences from pyrimethamine-sensitive and pyrimethamine-resistant P. vivax isolates revealed two residue changes: Ser Arg-58 and Ser Asn-117. These aa residues correspond to codons 59 and 108 in the P. falciparum DHFR active site in which similar aa substitutions (Cys Arg-59 and Ser Asn-108) are associated with pyrimethamine resistance. These findings may explain the intrinsic resistance of some P. vivax isolates to pyrimethamine.
Asunto(s)
Complejos Multienzimáticos/química , Plasmodium vivax/enzimología , Pirimetamina/farmacología , Tetrahidrofolato Deshidrogenasa/química , Timidilato Sintasa/química , Animales , Clonación Molecular , Resistencia a Medicamentos/genética , Ácido Fólico/biosíntesis , Genes Protozoarios/genética , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Sistemas de Lectura Abierta/genética , Proteínas Protozoarias/química , Secuencias Repetitivas de Ácidos Nucleicos/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de SecuenciaRESUMEN
Polymorphism of the circumsporozoite protein (CSP) of Plasmodium malariae was characterized by comparing gene sequences of twelve field isolates obtained in Yaoundé, Cameroon, Central Africa, and one clinical isolate originating from Côte d'Ivoire, West Africa. The length of the CSP gene ranged from 1266 to 1302 bp. The size polymorphism was due to variation in the number of tandem repeat units. All P. malariae isolates displayed a highly conserved 5' non-repeat region. Seven non-synonymous and two synonymous nucleotide variations were observed in the 3' non-repeat region. In the deduced amino acid sequence the repetitive sequences consisted of a varying number of major (Asn Ala Ala Gly (NAAG); range between 42 and 46 units) and minor (Asn Asp Ala Gly (NDAG) or Asn Asp Gln Gly (NDEG); n = six or seven units) tetrapeptide units. None of the isolates had an identical sequence at nucleotide level. These findings suggest that polymorphism in CSP is essentially limited to the tandem repeat domain.
Asunto(s)
Genes Protozoarios , Heterogeneidad Genética , Plasmodium malariae/genética , Proteínas Protozoarias/genética , Animales , Camerún , Clonación Molecular , Côte d'Ivoire , ADN Protozoario , Código Genético , Datos de Secuencia Molecular , Mutación , Reacción en Cadena de la Polimerasa , Secuencias Repetitivas de Ácidos Nucleicos , Análisis de Secuencia de ADNRESUMEN
Resistance of Plasmodium falciparum to many therapeutic agents is an increasing problem in most endemic areas. The role of the mdr-like gene products of P. falciparum in resistance to quinoline-containing compounds is not clear. The purpose of this study was to further examine the role of pfmdr1 in drug resistance in fresh clinical isolates originating from Africa. Drug susceptibility testing (chloroquine, mefloquine, halofantrine and quinine) and a molecular analysis of pfmdr1 was completed for 51 fresh clinical isolates. A statistical association between the chloroquine sensitivity phenotype and an intragenic allele of pfmdr1 was noted at a position, amino acid 86, which was previously associated with chloroquine resistance. There was little variation in the other intragenic alleles previously associated with chloroquine resistance. No correlation between pfmdr1 intragenic allelic variation and susceptibility to mefloquine, halofantrine or quinine was found. There was no association between gene copy number of pfmdr1 and any drug resistant phenotype in an analysis of selected isolates. This, along with other data, suggests that mefloquine resistance may have arisen by two different mechanisms in African and Southeast Asian isolates. Much more variability in the polyasparaginated region of the pfmdr1 gene was noted in this study than previously reported. In addition, fingerprint analysis using multiplex PCR revealed considerable genetic variability among these isolates.
Asunto(s)
Resistencia a Múltiples Medicamentos/genética , Genes Protozoarios , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , África del Sur del Sahara , Alelos , Animales , Antimaláricos/farmacología , Secuencia de Bases , Cloroquina/farmacología , Codón/genética , Dermatoglifia del ADN , ADN Protozoario/genética , ADN Protozoario/aislamiento & purificación , Variación Genética , Humanos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Mefloquina/farmacología , Fenotipo , Plasmodium falciparum/aislamiento & purificaciónRESUMEN
Antifolate drugs inhibit malarial dihydrofolate reductase (DHFR). In Plasmodium falciparum, antifolate resistance has been associated with point mutations in the gene encoding DHFR. Recently, mutations at homologous positions have been observed in the P. vivax gene. Since P. vivax cannot be propagated in a continuous in vitro culture for drug sensitivity assays, the kinetic properties of DHFR were studied by expression of the DHFR domain in Escherichia coli. Induced expression yielded a protein product that precipitated as an inclusion body in E. coli. The soluble, active DHFR recovered after denaturation and renaturation was purified to homogeneity by affinity chromatography. Kinetic properties of the recombinant P. vivax DHFR showed that the wild-type DHFR (Ser-58 and Ser-117) and double mutant DHFR (Arg-58 and Asn-117) have similar K(m) values for dihydrofolate and NADPH. Antifolate drugs (pyrimethamine, cycloguanil, trimethoprim, and methotrexate), but not proguanil (parent compound of cycloguanil) inhibit DHFR activity, as expected. The kinetics of enzyme inhibition indicated that point mutations (Ser58Arg and Ser117Asn) are associated with lower affinity between the mutant enzyme and pyrimethamine and cycloguanil, which may be the origin of antifolate resistance.
Asunto(s)
Escherichia coli/genética , Plasmodium vivax/enzimología , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismo , Animales , Clonación Molecular , Resistencia a Medicamentos , Escherichia coli/enzimología , Antagonistas del Ácido Fólico/farmacología , Cinética , Plasmodium vivax/genética , Pirimetamina/farmacología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Tetrahidrofolato Deshidrogenasa/aislamiento & purificaciónRESUMEN
The gene encoding dihydrofolate reductase-thymidylate synthase of the human malaria parasite, Plasmodium vivax, was isolated by polymerase chain reaction from genomic DNA and cloned. The sequences of the dihydrofolate reductase domain of 30 clinical isolates originating from various geographic areas were compared. Interstrain analysis revealed several genotypic variations, including short tandem repeat arrays which produced length polymorphism between different parasite isolates and point mutations in the putative dihydrofolate reductase active site cavity corresponding to those associated with pyrimethamine resistance in P. falciparum and rodent malaria parasites. Amino acid substitutions Ser-->Asn-117 and Ser-->Arg-58 were associated with decreased level of in vitro pyrimethamine sensitivity. These findings suggest that the P. vivax dihydrofolate reductase domain is characterized by polymorphism that has not been observed in P. falciparum and may explain the resistance of some P. vivax isolates to pyrimethamine. Nucleotide sequence data reported in this paper are available in the EMBL, GenBank and DDJB databases under the accession numbers X98123 (isolate ARI/Pakistan), AJ003050 (isolate CNC/Thailand), AJ003051 (isolate COU/unknown geographic origin), AJ003052 (isolate DUF/French Guiana), AJ003053 (isolate GRO/Madagascar), AJ003054 (isolate HRT/Comoros Islands), AJ003071 (isolate LFT/Cambodia), AJ003072 (isolate LGF/'India), AJ003073 (isolate MAN/Comoros Islands), AJ003074 (isolate MAT/Surinam), AJ003075 (isolate PHI/Djibouti), AJ003076 (isolate PIT/Madagascar), AJ003077 (isolate YTZ/Indonesia), AJ222630 (isolate Burma-1), AJ222631 (isolate Burma-151), AJ222632 (isolate Burma-5), AJ222633 (isolate Burma-6), AJ222634 (isolate Burma-98).
Asunto(s)
Antimaláricos/farmacología , Variación Genética , Plasmodium vivax/genética , Pirimetamina/farmacología , Tetrahidrofolato Deshidrogenasa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Protozoario , Resistencia a Medicamentos/genética , Antagonistas del Ácido Fólico/farmacología , Datos de Secuencia Molecular , Plasmodium vivax/efectos de los fármacos , Plasmodium vivax/enzimología , Mutación Puntual , Reacción en Cadena de la Polimerasa , Alineación de Secuencia , Tetrahidrofolato Deshidrogenasa/metabolismoRESUMEN
It has been postulated that chloroquine resistance may be associated with a single point mutation at codon 86 of the Plasmodium falciparum multidrug resistance 1 (pfmdr 1) gene. Using a simple and rapid molecular technique involving polymerase chain reaction and restriction fragment length polymorphism, the frequency of the Asn-to-Tyr mutation associated with chloroquine resistance was established among 129 clinical isolates obtained from indigenous patients in Yaoundé, Cameroon. The results showed that 110 of 129 isolates display a mutant codon. The other clinical isolates had either a pure wild-type Asn-86 codon (n = 12) or mixed Asn/Tyr alleles (n = 7). In vitro drug assays were performed to compare the genotype and phenotype in 102 clinical isolates. Of these isolates, 86 displayed pure Tyr-86 mutant codon; 48 (56%) mutant isolates were chloroquine-resistant (50% inhibitory concentration [IC50] > 100 nM), as expected, but 38 (44%) mutant isolates were chloroquine-sensitive (IC50 < 100 nM). Three chloroquine-resistant isolates and seven chloroquine-sensitive parasites carried a wild-type Asn-86 codon. Mixed alleles were found in six isolates (four chloroquine-sensitive and two chloroquine-resistant isolates). Our results did not confirm previous observations on the possible association between chloroquine resistance phenotype and genotype based on the pfmdr 1 gene.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Antimaláricos/farmacología , Cloroquina/farmacología , Genes Protozoarios , Plasmodium falciparum/efectos de los fármacos , Mutación Puntual , Proteínas Protozoarias/genética , Animales , Plasmodium falciparum/genéticaRESUMEN
The in vitro activities of chloroquine, quinine, mefloquine, halofantrine, artemisinin, arteether, artemether, and artelinate were evaluated against African clones and isolates of Plasmodium falciparum, using an isotopic, semimicro, drug susceptibility test. The chloroquine-resistant FCM 29 clone was 1.6 and 6.2 times more susceptible to artemisinin when compared with the chloroquine-susceptible, mefloquine-, and halofantrine-resistant L-3 and L-16 clones, respectively. Cross-resistance patterns between the standard antimalarial drugs and artemisinin were determined against 36 African isolates of P. falciparum obtained from imported cases of malaria in France. Chloroquine-resistant isolates (n = 21) were significantly more susceptible to artemisinin (50% inhibitory concentration [IC50] 7.67 nM), arteether (IC50 3.88 nM), artemether (IC50 3.71 nM), and artelinate (IC50 3.46 nM), as compared with the 15 chloroquine-susceptible isolates (IC50 11.4, 5.66, 5.14, and 5.04 nM, respectively). Arteether, artemether, and artelinate were equally effective and twice as potent as artemisinin. A significant positive correlation was found between artemisinin and mefloquine (r = 0.424, P = 0.022), artemisinin and halofantrine (r = 0.569, P < 0.001), chloroquine and quinine (r = 0.651, P < 0.001), and mefloquine and halofantrine (r = 0.863, P < 0.001), suggesting in vitro cross-resistance among these drugs. The present in vitro findings require confirmation in clinical studies.
Asunto(s)
Antimaláricos/farmacología , Artemisininas , Plasmodium falciparum/efectos de los fármacos , Sesquiterpenos/farmacología , Animales , Arteméter , Cloroquina/farmacología , Resistencia a Medicamentos , Mefloquina/farmacología , Fenantrenos/farmacología , Quinina/farmacologíaRESUMEN
The in vitro activity of halofantrine was studied in chloroquine-susceptible and chloroquine-resistant African clones of Plasmodium falciparum over a period of six months. The susceptibility level remained stable in both clones. The chloroquine-susceptible clone (50% inhibitory concentration [IC50] 6.88 nM) was less susceptible to halofantrine than the chloroquine-resistant clone (IC50 2.98 nM). Using an isotopic semimicro drug susceptibility test, the in vitro activity of halofantrine was compared with the activities of chloroquine, quinine, and mefloquine to study the cross-resistance patterns against 76 African isolates of P. falciparum isolated from cases of malaria imported into France. Chloroquine-resistant isolates (n = 47) were significantly less susceptible to quinine (IC50 234 nM), but were more susceptible to both mefloquine (IC50 3.20 nM) and halofantrine (IC50 1.14 nM) compared with the chloroquine-susceptible isolates (n = 29; IC50 147 nM for quinine, 7.16 nM for mefloquine, and 2.62 nM for halofantrine). A significant positive correlation was found between the activities of chloroquine and quinine and between those of mefloquine and halofantrine, indicating cross-resistance between these drugs, while a negative correlation was observed between chloroquine and mefloquine or halofantrine. The responses to quinine and mefloquine or halofantrine showed no correlation with each other. These results reinforce the importance of a cautious use of antimalarial drugs in Africa.
Asunto(s)
Antimaláricos/farmacología , Fenantrenos/farmacología , Plasmodium falciparum/efectos de los fármacos , África , Animales , Cloroquina/farmacología , Resistencia a Medicamentos , Humanos , Malaria Falciparum/parasitologíaRESUMEN
In an endemic area where malaria transmission is intense and continuous, reappearance of asexual parasites may be ascribed to either recrudescence or reinfection. To distinguish between recrudescence and reinfection after oral treatment with chloroquine, amodiaquine, pyronaridine, sulfadoxine-pyrimethamine, halofantrine, or artesunate, three polymorphic markers (circumsporozoite protein, merozoite surface antigens 1 and 2) from pre-treatment and post-treatment samples were amplified by the polymerase chain reaction, and the in vitro response to chloroquine was determined for comparison. Of 52 paired samples, 22 (42%) were reinfections. Recrudescence occurred more frequently on or before Day 14 (22 of 30 cases, 73%). Except for one case, all reinfections were observed beyond Day 14. The phenotype determination was not sufficiently precise to distinguish between recrudescence and reinfection. Our results suggest that beyond Day 14 (and until Day 42), recrudescence and reinfection cannot be distinguished at our study site unless molecular techniques are used and that some results derived from the polymerase chain reaction need to be compared with the microscopic examination of thick blood smear to exclude gametocyte carriers without asexual parasites after treatment.
Asunto(s)
Antimaláricos/uso terapéutico , ADN Protozoario/sangre , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/epidemiología , Plasmodium falciparum/aislamiento & purificación , Administración Oral , Adolescente , Adulto , Animales , Antimaláricos/administración & dosificación , Antimaláricos/farmacología , Camerún/epidemiología , Niño , Cartilla de ADN , Femenino , Humanos , Masculino , Proteína 1 de Superficie de Merozoito/sangre , Persona de Mediana Edad , Pruebas de Sensibilidad Parasitaria , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Reacción en Cadena de la Polimerasa , Proteínas Protozoarias/sangre , Ensayos Clínicos Controlados Aleatorios como Asunto , Recurrencia , Factores de TiempoRESUMEN
The extent of genetically distinct parasite populations coinfecting individual human hosts (i.e., multiplicity) was studied by polymerase chain reaction amplification of 3 polymorphic genetic markers, circumsporozoite protein and merozoite surface antigens (MSA) 1 and 2, in symptomatic children and adults and analyzed in relation with age and initial parasitemia. Of the total of 177 DNA samples analyzed (of which 115 were paired pre- and posttreatment samples), 101 (57%) were composed of multiclonal infections, with up to 7 distinguishable parasite populations. Among the 3 polymorphic markers, msa-2 yielded the highest proportion of clinical isolates with multiclonal populations. Patients with multiclonal infections before treatment had, on average, 2.9 genetically distinct parasite populations. The extent of multiplicity decreased significantly (P < 0.05) in recrudescent parasites, but not with reinfections, as compared with the pretreatment samples. Neither age (5-60 years) nor initial parasitemia was correlated with multiplicity. Further studies in different epidemiological settings are required to understand the role of multiclonal Plasmodium falciparum infections in influencing malaria transmission.
Asunto(s)
ADN Protozoario/sangre , Malaria Falciparum/epidemiología , Plasmodium falciparum/genética , Adolescente , Adulto , Animales , Antimaláricos/uso terapéutico , Camerún/epidemiología , Niño , Preescolar , Cartilla de ADN , Femenino , Genotipo , Humanos , Malaria Falciparum/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Plasmodium falciparum/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Ensayos Clínicos Controlados Aleatorios como Asunto , RecurrenciaRESUMEN
Resistance to antifolate antimalarial drugs (cycloguanil, a biologically active metabolite of proguanil, and pyrimethamine) is associated with a Ser- to Asn-108 point mutation in the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum. The frequency of this mutation was studied in 127 clinical isolates obtained in Yaounde, Cameroon using a simple and rapid molecular technique based on the polymerase chain reaction and restriction fragment length polymorphism. Of the 127 isolates, pure wild-type Ser-108 codon, pure mutant-type Asn-108 codon, and mixed codons were observed in 66, 55, and six parasites, respectively. The proportion of antifolate-resistant, pure mutant-type codon, with respect to pure wild-type or mixed alleles, was 43% (55 of 127). The results of the molecular assay were compared with those of semimicro isotopic in vitro assay in 34 isolates. All 17 pure Ser-108 isolates and two isolates with mixed alleles were sensitive to both pyrimethamine (50% inhibitory concentration [IC50] < 100 nM) and cycloguanil (IC50 < 50 nM). Fourteen of 15 isolates with the mutant-type Asn-108 codon were resistant to pyrimethamine and cycloguanil. One isolate with Asn-108 showed a slightly elevated pyrimethamine IC50 (78 nM), which was within the sensitive range. This study provides further evidence that antifolate-resistant P. falciparum isolates are already present in Yaounde, Cameroon.
Asunto(s)
Malaria Falciparum/epidemiología , Plasmodium falciparum/genética , Mutación Puntual , Tetrahidrofolato Deshidrogenasa/genética , Timidilato Sintasa/genética , Alelos , Animales , Camerún/epidemiología , Codón/genética , ADN Protozoario/análisis , ADN Protozoario/química , ADN Protozoario/genética , Resistencia a Medicamentos/genética , Genes Protozoarios/genética , Humanos , Malaria Falciparum/parasitología , Epidemiología Molecular , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
Sulfadoxine-pyrimethamine is one of the alternative antimalarial drugs used to treat chloroquine-resistant Plasmodium falciparum malaria. The molecular target of sulfadoxine, an analog of p-aminobenzoic acid that inhibits the folate biosynthetic pathway, is dihydropteroate synthase (DHPS). The nucleotide sequence of the DHPS gene was determined in 32 clinical isolates obtained in Yaounde, Cameroon, and compared with the sequence of reference clones and Cambodian strains of P. falciparum. Of the 32 Cameroonian isolates, 31 displayed one of the sulfadoxine-sensitive mutation patterns: Ala-436/Ala-437/Ala-581/Ala-613 (n = 20), Ser-436/Gly-437/Ala-581/Ala-613 (n = 6), Ser-436/Ala-437/Ala-581/Ala-613 (n = 4), and Ala-436/Gly-437/Ala-581/Ala-613 (n = 1). One isolate had a sulfadoxine-resistant profile characterized by a double mutation: Phe-436/Ala-437/Ala-581/Ser-613. Although the majority of the isolates had a sulfadoxine-sensitive genetic profile, further studies are needed to correlate the mutation patterns and in vitro and in vivo sulfadoxine sensitivity.
Asunto(s)
Dihidropteroato Sintasa/genética , Malaria Falciparum/epidemiología , Plasmodium falciparum/genética , Mutación Puntual , Animales , Camerún/epidemiología , Clonación Molecular , Codón/genética , ADN Protozoario/análisis , ADN Protozoario/química , Humanos , Malaria Falciparum/parasitología , Epidemiología Molecular , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
The in vitro activity of monodesethylamodiaquine and amopyroquine was evaluated against chloroquine-susceptible and chloroquine-resistant African clones of Plasmodium falciparum. The chloroquine-resistant clone (50% inhibitory concentration [IC50] = 1,630 nM) was 26 and 3.7 times less susceptible to monodesethylamodiaquine and amopyroquine, respectively, than the chloroquine-susceptible clone (IC50 = 36.5 nM). Cross-resistance patterns of chloroquine, monodesethylamodiaquine, and amopyroquine were determined against 62 African isolates of P. falciparum obtained from imported cases of malaria in France, using an isotopic semimicro drug susceptibility test. Chloroquine-resistant isolates (n = 38) were significantly less susceptible to both monodesethylamodiaquine (IC50 = 74.8 nM) and amopyroquine (IC50 = 18.9 nM), as compared with the 24 chloroquine-susceptible isolates (IC50 = 28.2 nM and 16.1 nM, respectively). A significant positive correlation was found between chloroquine and monodesethylamodiaquine (r = 0.903) and between chloroquine and amopyroquine (r = 0.371), indicating in vitro cross-resistance among these drugs. These in vitro results suggest that the clinical response to amodiaquine and amopyroquine needs to be closely monitored in Africa.
Asunto(s)
Aminoquinolinas/farmacología , Amodiaquina/análogos & derivados , Antimaláricos/farmacología , Plasmodium falciparum/efectos de los fármacos , Amodiaquina/farmacología , Animales , Cloroquina/farmacología , Resistencia a Medicamentos , Humanos , Análisis de RegresiónRESUMEN
Pyrimethamine, in combination with sulfadoxine, is currently one of the major alternative drugs used for the treatment of chloroquine-resistant Plasmodium falciparum malaria infections in Africa. The mechanism of pyrimethamine resistance has been strongly associated with a single, key point mutation in the dihydrofolate reductase-thymidylate synthase gene, resulting in the substitution of the wild-type allele Ser-108 by either Asn-108 or Thr-108. The pyrimethamine-resistant phenotype and/or genotype were determined in 273 Cameroonian clinical isolates obtained in Yaounde by in vitro assays and polymerase chain reaction-restriction fragment length polymorphism over a 5-year period. The in vitro assays showed that 42% (18 of 43) and 63% (69 of 110) of the isolates obtained in 1994-1995 and 1997-1998, respectively, were resistant to pyrimethamine (50% inhibitory concentration [IC50] > 100 nM). The polymerase chain reaction showed that 43% (55 of 127) and 59% (50 of 85) of the isolates in 1994-1995 and 1997-1998, respectively, had the mutant Asn-108 allele. The pyrimethamine-resistant genotype (Asn-108) corresponded with the pyrimethamine-resistant phenotype (IC50 > or = 100 nM) in a large majority (> 95%) of the isolates. The results of our study suggest an increasing prevalence of pyrimethamine resistance in Yaounde. Our study further suggests that pyrimethamine resistance can be monitored by a technique that can be adopted by malaria research centers in Africa.
Asunto(s)
Antimaláricos/farmacología , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparum/efectos de los fármacos , Pirimetamina/farmacología , Sulfadoxina/farmacología , Tetrahidrofolato Deshidrogenasa/genética , Adolescente , Adulto , Animales , Camerún/epidemiología , Niño , Cartilla de ADN/química , ADN Protozoario/química , Combinación de Medicamentos , Resistencia a Medicamentos/genética , Electroforesis en Gel de Agar , Humanos , Concentración 50 Inhibidora , Malaria Falciparum/epidemiología , Malaria Falciparum/parasitología , Fenotipo , Plasmodium falciparum/enzimología , Plasmodium falciparum/genética , Mutación Puntual , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Conteo por Cintilación , Tetrahidrofolato Deshidrogenasa/químicaRESUMEN
A novel Plasmodium falciparum gene, denoted cg2 gene, has been recently discovered, and a distinct genotype, characterized by 12 point mutations and 3 size polymorphisms, has been shown to be associated with chloroquine resistance in laboratory-adapted parasite strains. One of the polymorphic regions, denoted the omega region, consists of 16 tandem repeat units in chloroquine-resistant strains, while the chloroquine-sensitive strains have either < or = 15 or > or = 17 repeat units. In this study, the in vivo and in vitro responses were compared with the number of repeat units in the omega region of the cg2 gene for 75 Cameroonian isolates determined either by DNA sequencing or agarose gel electrophoresis. The 16-repeat units that characterize the resistant strains were found in 10 chloroquine-sensitive isolates (50% inhibitory concentration [IC50] < 100 nM) and 30 chloroquine-resistant isolates (IC50 > or = 100 nM). Thirty-five isolates (28 chloroquine-sensitive isolates and 7 chloroquine-resistant isolates) displayed < or = 15 or > or = 17 repeat units. Of the 18 patients responding with treatment failure, 15 were infected with parasites carrying 16 repeat units. Twenty-eight patients (11 with isolates carrying 16 repeat units and 17 with isolates carrying < or = 15 or > or = 17 repeat units) showed an adequate clinical response. The sensitivity, specificity, and predictive value were 81% (83%), 74% (61%), and 75% (58%), respectively compared with in vitro (or in vivo) responses. Neither the level of IC50 nor the key P. falciparum multidrug resistance gene 1 (pfmdr 1) allele at position 86 was associated with the number of omega repeat units. Although in vitro and in vivo resistance to chloroquine was statistically associated with the presence of 16 repeat units in the omega region (P < 0.05), the number of omega repeat units did not adequately discriminate patients infected with chloroquine-resistant parasites from those infected with chloroquine-sensitive parasites. Further studies on the cg2 gene are needed to determine whether cg2 gene is a reliable genetic marker for chloroquine resistance.
Asunto(s)
Antimaláricos/farmacología , Cloroquina/farmacología , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparum/genética , Adolescente , Adulto , Animales , Antimaláricos/normas , Antimaláricos/uso terapéutico , Camerún/epidemiología , Niño , Cloroquina/uso terapéutico , Cartilla de ADN/química , ADN Protozoario/química , Resistencia a Medicamentos/genética , Electroforesis en Gel de Agar , Humanos , Concentración 50 Inhibidora , Malaria Falciparum/epidemiología , Naftiridinas/uso terapéutico , Parasitemia , Fenantrenos/uso terapéutico , Plasmodium falciparum/química , Plasmodium falciparum/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Análisis de Regresión , Secuencias Repetitivas de Ácidos Nucleicos/genética , Conteo por Cintilación , Análisis de Secuencia de ADNRESUMEN
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 = 11 isolates) was associated with low 50% inhibitory concentrations (IC50s) 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 IC50 and, to a much lesser extent, cycloguanil IC50. These results support a partial cross-resistance 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.
Asunto(s)
Antimaláricos/farmacología , Antagonistas del Ácido Fólico/farmacología , Malaria Falciparum/parasitología , Plasmodium falciparum/genética , Pirimetamina/farmacología , Tetrahidrofolato Deshidrogenasa/genética , Triazinas/farmacología , Animales , Antimaláricos/uso terapéutico , Camerún/epidemiología , Cartilla de ADN/química , ADN Protozoario/química , ADN Protozoario/aislamiento & purificación , Resistencia a Medicamentos/genética , Antagonistas del Ácido Fólico/uso terapéutico , Humanos , Concentración 50 Inhibidora , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/epidemiología , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Mutación Puntual , Reacción en Cadena de la Polimerasa , Proguanil , Pirimetamina/uso terapéutico , Análisis de Secuencia de ADN , Tetrahidrofolato Deshidrogenasa/química , Triazinas/uso terapéuticoRESUMEN
The in vitro activity of two dihydrofolate reductase (DHFR) inhibitors, pyrimethamine and cycloguanil, was evaluated against African clones and isolates of Plasmodium falciparum using an isotopic, semimicro drug susceptibility test. Three susceptibility levels (susceptible, intermediate, and resistant) were observed in the response of culture-adapted clones and strains to pyrimethamine (50% inhibitory concentration [IC50]) < 100, 100-2,000, and > 2,000 nM) and cycloguanil (IC50 < 50, 50-500, and > 500 nM). Based on these susceptibility levels, 73 and 68 of 96 fresh clinical isolates were susceptible to pyrimethamine (mean IC50 15.4 nM) and cycloguanil (mean IC50 11.1 nM), respectively. Thirteen and 18 isolates were resistant to pyrimethamine (mean IC50 9,440 nM) and cycloguanil (mean IC50 2,030 nM), respectively. A highly significant positive correlation was found between pyrimethamine and cycloguanil (r = 0.786), indicating in vitro cross-resistance between these antifolates. The spread of resistance to one of the DHFR inhibitors in Africa may compromise the utility of the other DHFR inhibitor.