Chlorproguanil/Dapsone An Alternative Antimalarial Combination:In-vitro Chemosensitivity, Molecular basis of resistance and Crossresistance.

Abstract

Resistance to available antimalarial drugs by Plasmodium talciparum has become more prevalent, prompting renewed interest in the mechanisms involved and a search for alternative antimalarial compounds. One promising group of -compounds is the short half-life antifolates in combination with sulphonamides or sui phone. The aims of this study were to assess in-vitro chemosensitivities, cross-resistance and to determine the prevalence of genetic makers for resistance to an alternative antifolate drug combination (chlorproguanilldapsone) in parasite isolates from Kilifi on the Kenyan coast. 108 field isolates were tested in-vitro against pyrimethamine (PYR), chlorcycloguanil (CCG), sulfadoxine (SO) and dapsone (DDS) in minimal folate medium, and their dihydrofolate reductase (DHFR) genotype determined. The proportion of PYR-resistant isolates of P. talciparum was shown to be about 90%, in sharp contrast to 20% in late 1980's in the same area. This finding supports the suggestion that the replacement of chloroquine with pyrimetha.mine/sulfadoxine, (PYRlSO), commonly referred to as Fansidar" in an area of continuous malaria transmission rapidly selects for pyrimethamine resistance, making the combination less effective. In addition, 95% of these isolates' ·were sensitive to CCG, the active metabolite of chlorproguanil (CPG), indicating that isolates resistant to CCG and possibly cycloguanil (CG)' are rare in the study area. The chemosensitivity of SO and DDS showed that the range of in-vitro response (ICso values) between sensitive and resistant isolates was narrow in comparison to PYR However, DDS was more potent than SO. Polymerase chain reaction (PCR) analysis of the OHFR domain revealed the presence of Asn-108, a mutation known to confer resistance to pyrimethamine, in 92% of the isolates. However, 56% of these isolates had both the mutated (Asn-108) and wild type (Ser-108) alleles. This indicates that about 44% of malaria infections in this area were due to isolates with pure mutated alleles (Asn-108) in respect to the 108 codon.peR analysis for Thr-108 (a mutation that confers resistance to cycloguanil and possibly the other triazines) in these isolates showed that none possessed this mutation. This finding was in agreement with similar studies undertaken elsewhere. The observed in-vitro CCG chemosensitivity not only confirms the low prevalence of Thr-108 but also suggests that this mutation may not be an important one in field isolates with respect to their response to CCG. This implies that Asn-108 in combination with I/e-51 and Arg-59 is the primary mutation that not only governs resistanoe against PYR but may also lead to a general reduction in antifolate chemosensitivity. DHFR genotype was highly correlated with PYR and CCG drug response and the isolates could be classified into three distinct groups in respect to the two variables. Based on 43 isolates (44%) that yielded a PCR product with only one set of primers (pure OHFR alleles), the first group comprises wild type isolates with mean PYR and CCG IC50 values of 3.71 and 0.24 nM respectively. The second group includes isolates which have mutations at codon 108 alone or paired with mutations at codon 51 or 59. This group represents a homogeneous population with IC50 values of 116.21 and 1.37 nM for PYR and CCG respectively. Isolates with mutations at codons 108, 51 and 59 (triple mutants) form the third group with IC50 of 785.5 and 10.80 nM for PYR and CCG respectively. The simultaneous appearance of Asn-108 lie-51 and Arg-59 mutations may also be responsible for reduced responses to CCG, SO and DDS by these isolates which were highly PYR- resistant as well. These findings suggest that the useful therapeutic life of PYR-containing antimalarial drugs in the management of nonsevere falciparum malaria in the study area is approaching its endpoint This is a matter of great concern because there are no affordable, safe, and effective alternative antimalarial drug(s) to replace Fansidar