Molecular Characteristics and Epidemiology of Plasmodium falciparum Susceptibility to Sulfadoxine-Pyrimethamine and Chloroquine in Haj Yousif and Gedarif Town, Sudan

Abstract

Malaria is the world's most important tropical parasitic disease. The problem of malaria was aggravated due to the emergence of resistance to antimalarials and insecticides in the absence of a suitable malaria vaccine. The present study was carried out in two areas during two years (1998 to 2001), with the objectives of assessment of resistance to chloroquine and sulphadoxine-pyrimethamine as first-line antimalarials. Another purpose was the validation of molecular markers related to chloroquine and sulphadoxinepyrimethamine resistance for prediction of treatment failure to these antimalarials; and evaluation of the contribution of host and parasite factors in the development of resistance to antimalarial drugs. The study clearly demonstrated that chloroquine was no longer effective in the treatment of falciparum malaria. High levels of chloroquine resistance were detected in both areas; it was 77.1% and 56.3% in Gedarif and Haj Yousif respectively. High levels of sulphadoxine-pyrimethamine susceptibility were observed in Haj Yousif (95.6% and 98% in the first and second malaria seasons respectively) compared to 76.7% in Gedarif. Estimation of genuine levels of resistance to both antimalarials in Haj Yousif was carried out by genotyping of polymorphic genes msp1, msp2 and glurp. The mutant allele of pfcrt K76T was predominant in both study areas. In Haj Yousif, all pre-treatment specimens were found to be carriers of the mutant allele 76T regardless of their response to chloroquine. Also, in Gedarif all pre-treatment specimens were found to be carriers of the mutant allele 76T either as a pure mutant allele (96.2%) or a mixed allele (3.8%) regardless of their response to chloroquine. Lack of a significant association between mutant allele of Pfmdr1 N86Y and chloroquine treatment failure in both areas was detected. In Haj Yousif the frequency of mutant allele 86Y, wild allele N86 and mixed allele was 82.9%, 10% and 7.1% respectively. All post-treatment specimens were carrying mutant allele 86Yexcept one which had a wild one. In Gedarif the frequencies of mutant allele 86Y, wild allele N86 and mixed allele were 68.4%, 7.6% and 24.1% respectively. Similarly the mutations in dhfr and dhps genes had no significant role on sulphadoxine-pyrimethamine resistance in both areas. The association was observed in dhps K540E in Haj Yousif first cross-sectional study. The mutant allele dhfr S108N III and dhfr N51I were predominant in both areas. Less mutation was observed in dhps gene. The pattern of dhfr and dhps mutations in Haj Yousif second cross-sectional study was similar to that of the first study except in dhfr C59R and dhps K540E where the mutant allele has completely disappeared in the second study. Single, double and quadruple mutations in both genes were detected in the first and the second study while the triple mutation was detected only in the first study. In Gedarif study the frequency of mutations in dhfr gene was similar to the pattern in Haj Yousif study. Also, less mutation was observed in dhps gene compared to dhfr gene. Double, triple, quadruple and quintuple mutations in both genes were detected in the area. Although a similar frequency of dhfr mutations was observed in both study areas, the frequency of dhps mutations was significantly higher in Gedarif compared to Haj Yousif. A high frequency of mixed allele in Pfmdr1, dhfr and dhps was observed in Gedarif area indicating the prevalence of multiplicity of infections in the area which were reported from previous studies. . It was found that the assessment of antimalarial efficacy using 14 days followup protocol underestimated the resistance level and therefore it is recommended to use the 28 days follow-up protocol or longer. The contribution of gender, age and initial parasitaemia to the development of treatment failure was investigated. The significant effect of age as a factor for acquisition of immunity against treatment outcome was confirmed in the chloroquine study in both study areas (P< 0.01). There was a significant reverse relationship between age and parasite density. This validated the value of age as an immunological marker. Initial parasitaemia has a significant effect on development of chloroquine resistance in Haj Yousif. The contribution of gender as a factor in treatment failure was ruled out. There was no significant association between gender and treatment failure in both areas. It was concluded that, further clinical studies were needed to define the usefulness of Pfcrt, Pfmdr-1, dhps and dhfr as molecular markers for chloroquine and sulfadoxinepyrimethamine resistance and identify host factors that may influence the clinical outcome of drug treatment and explain the clearance of parasitaemia of parasites carrying mutant Pfcrt Pfmdr-1, dhps and dhfr allele by some patients. The quality of antimalarial products and patient adherence to dosage regimens are important determinants of drug effectiveness, and should be measured alongside the clinical efficacy.