Point mutations in dihydrofolate reductase and dihydropteroate synthase genes of Plasmodium falciparum isolates from Venezuela.

Abstract

Abstract. The present study was designed to characterize mutations in dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genes of Plasmodium falciparum in the Bolivar region of Venezuela, where high levels of clinical resistance to sulfadoxine-pyrimethamine (SP, Fansidar; F. Hoffman-La Roche, Basel, Switzerland) has been documented. We used a nested mutation-specific polymerase chain reaction and restriction digestion methods to measure 1) the prevalence of DHFR mutations at 16, 50, 51, 59, 108, and 164 codon positions, and 2) the prevalence of mutations in the 436, 437, 581, and 613 codon sites in DHPS gene. In the case of the DHFR gene, of the 54 parasite isolates analyzed, we detected the presence of Asn-108 and Ile-51 in 96% of the isolates and Arg-50 mutation in 64% of the isolates. Each of these mutations has been associated with high level of resistance to pyrimethamine. Only 2 samples (4%) showed the wild type Ser-108 mutation and none showed Thr-108 and Val-16 mutations that are specific for resistance to cycloguanil. In the case of DHPS gene, we found a mutation at position 437 (Gly) in 100% of the isolates and Gly-581 in 96% of the isolates. The simultaneous presence of mutations Asn-108 and Ile-51 in the DHFR gene and Gly-437 and Gly-581 in the DHPS gene in 96% of the samples tested suggested that a cumulative effect of mutations could be the major mechanism conferring high SP resistance in this area.

Common Origin and Fixation of Plasmodium falciparum dhfr and dhps Mutations Associated with Sulfadoxine-Pyrimethamine Resistance in a Low-Transmission Area in South America

“…One study noted that 4% of 54 samples from 1995 had wild-type codons 51 and 108; it is unclear whether all of the dhfr codons were wild type on a given chromosome. That same study did not find any wild-type dhps alleles (30). Another study using samples from 1998 to 2000 did not have any wild-type dhfr or dhps alleles (2).…”

Molecular Analysis of Chloroquine and Sulfadoxine-Pyrimethamine Resistance-Associated Alleles in Plasmodium falciparum Isolates from Nicaragua

“…[11][12][13][14][15][16] Alleles consisting of multiple polymorphisms, such as the C50R, N51I, S108N type and the C50R, N51I, S108N, I164L type, have been identified in Brazil, Peru, and Venezuela. 17,18 In pfdhps, polymorphisms, including S436A/F, A437G, K540E, A581G, and A613S/T, have been shown to affect parasite susceptibility to sulfadoxine. 19 -21 All sulfadoxine resistance pfdhps alleles that have been identified in the Americas have the A437G polymorphism alone or in combination with K540E and/or A581G.…”

Mutaciones puntuales en los genes dhfr y dhps de Plasmodium falciparum de tres regiones endémicas

“…El producto obtenido en esta reacción (711 pb) fue analizado por restricción enzimática utilizando las enzimas MSPA1 I que detectan el alelo mutante 436A, Ava II que detecta el alelo mutante 437G y Fok I que detecta el alelo mutante 540E (10,21). Como controles se utilizaron las cepas T9-96, 3D7, HB3, T9-94 e IEC513/86.…”

“…500 pb C el estado de Bolívar, zona que informó altos niveles de falla terapéutica a sulfadoxina-pirimetamina. Los resultados mostraron la presencia de las mutaciones 108N, 50R e 51I en el gen dhfr con frecuencias de 96%, 64% y 96%, respectivamente, y las mutaciones 437G y 581G en el gen dhps con frecuencias del 100% y 96%, respectivamente (21). Por otra parte, Contreras et al (23) encontraron la misma tendencia de mutaciones, lo cual sugiere que los parásitos mantienen en su genoma estas mutaciones desde antes del 2000.…”

“…En este estudio no se detectó el genotipo mutante en el codón 59 del gen dhfr en ninguno de los pacientes de las tres regiones analizadas, resultado que está de acuerdo con el trabajo de Méndez (24), realizado con pacientes procedentes de Buenaventura (Costa Pacífica), y con varios reportes de diferentes países suramericanos (8,(21)(22)(23)(25)(26)(27). Lo anterior sugiere que el codón 59 no es afectado por la presión ejercida con la sulfadoxinapirimetamina y que el mecanismo de selección de mutaciones puede ser distinto en parásitos de Suramérica comparado con el de parásitos de África (28,29), pero esto debe ser corroborado con estudios de parásitos provenientes de diferentes regiones del país y de otros países, así como con muestras tomadas en diferentes periodos.…”