Short report: high prevalence and imbalanced age distribution of the Plasmodium falciparum dihydrofolate reductase gene Asn108 mutation in an area of low pyrimethamine usage in Nigeria.

Abstract

Abstract. Resistance of Plasmodium falciparum to pyrimethamine is associated with a non-silent point mutation of the parasite dihydrofolate reductase (DHFR) gene (Ser 108 → Asn 108 ). Wide-scale use of antimalarials is thought to contribute to the emergence of drug resistance. In 131 P. falciparum-infected children in rural Nigeria, the frequency of the resistant Asn 108 genotype was assessed by enzymatic restriction digestion of polymerase chain reaction-amplified DHFR sequences and compared with residual pyrimethamine blood levels. The prevalence of the Asn 108 variant was 41.2%. In 18.3% of the isolates, both the Asn 108 and the wild-type alleles were present. In contrast to the high prevalence of resistant genotypes, residual pyrimethamine blood levels were detected in only 4%. Furthermore, age was found to be a determinant of the parasite genotype since the proportion of Asn 108 variants decreased with age (P Ͻ 0.05). These findings indicate that additional, unidentified factors, rather than selection by residual drug levels alone, might be responsible for the emergence of pyrimethamine-resistant parasite genotypes.In vitro resistance to pyrimethamine (PYR) is conferred by a non-silent point mutation at codon 108 of the parasite dihydrofolate-reductase (DHFR) gene (Ser 108 → Asn 108 ). 1,2 The DHFR Asn 108 genotype has been shown to be selected by treatment with PYR-containing drugs. 3 Likewise, high frequencies of resistant parasites in Mali have been attributed to increased consumption of PYR. 4 In Ibadan, Nigeria, a continuous decrease in the efficacy of PYR-sulfadoxine has also been observed. 5 The study was performed in Abanla, a rural community 20 km south of Ibadan. The purpose of the study was to assess the frequency of P. falciparum parasites exhibiting the DHFR gene Asn 108 mutation and to compare it with the prevalence of blood levels of PYR. Blood was collected from 146 randomly selected children (1-11 years old) at schools and vaccination programs after informed consent was obtained from their guardians. Ethical approval was obtained from the Ethical Committee of the University of Ibadan. Parasites were counted on Giemsa-stained thick films. In addition, P. falciparum infection was diagnosed by a polymerase chain reaction (PCR) assay 6 after extraction of DNA from blood. Samples positive by the parasite-specific PCR were tested by a second PCR assay to amplify a fragment of the P. falciparum DHFR gene and to identify the variants exhibiting Ser 108 and Asn 108 by digestion with the restriction enzymes Alu I and Bsr I, respectively. 3 Levels of PYR in the blood were measured by an ELSA. 7 The lower limit of detection was 5 ng/ml.The PCR results showed that 91% (133 of 146) of the children were infected with P. falciparum. The infection rate increased with age (Figure 1 (Figure 2). The proportion of Asn 108 -variants decreased significantly with age (Figure 1). Parasite densities (range ϭ 0.05-27.3 parasites/microscope field) and the proportion of submicroscopic parasitemia did not differ with re...

Genetic diversity of Plasmodium falciparum field samples from an isolated Colombian village.

“…Asn-108 frequency was 60% in the 0-2 age group and 18% among children age 6 and over. 26 However, our analysis of Colombian samples-in contrast to the Nigerian study-did not find a decline in the frequency of Asn-108 with an increase in the age of the group studied. Nor was it possible to establish a statistically significant relationship between children's ages and presence of the Ser-108 genotype or the presence of mixed infections ( 2 ‫ס‬ 8,91).…”

Increase in the prevalence of mutations associated with sulfadoxine–pyrimethamine resistance in Plasmodium falciparum isolates collected from early to late pregnancy in Nanoro, Burkina Faso

“…However, age distribution was also a noticeable difference; GP participants were mostly children <15 years of age, whereas pregnant women were aged 15–45 years. In children, age has been shown to be inversely related to dhfr S108 and quintuple mutation prevalence [42, 43]. Nevertheless, the difference in prevalence of dhfr N51 and triple dhfr mutations compared with ANC samples remained even if only GP participants aged 15–45 years were included in the model.…”

Monitoring antifolate resistance in intermittent preventive therapy for malaria

“…By contrast, young non-immune children suffer repeated symptomatic episodes, undergo consecutive drug treatment, and are more likely to carry drug-resistant parasites. Corroborating this hypothesis, studies in areas of high malaria transmission found that prevalence of SP resistance markers declines with age 64,65 and that quintuple mutants are more prevalent in children under five years of age than in older children 66 . Conversely, prevalence of resistance mutations may not vary by age in areas of low or unstable malaria transmission with less immunity, as was observed when comparing children under and over the age of 4 years in Northern Tanzania 59 .…”