In-vivo and Molecular characterization of drug resistant plasmodium falciparum isolates from Solwezi, Zambia

The study assessed the prevalence of drug resistant malarial parasites in patients at
Solwezi urban clinic in northwestern province of Zambia in February 2004, using the
in-vivo and molecular methods. In the in-vivo method each patient was monitored for
signs and symptoms of the disease following treatment with either
sulfadoxine/pyrimethamine (fansidar®), lumefantrine/artemether (coartem®) or
quinine. Molecular determination of mutations on the Plasmodium falciparum
chloroquine resistance transporter gene (pfcrt), associated with chloroquine
resistance, and the dihydropteroate synthase gene (dhps) and the dihydrofolate
reductase gene (dhfr), associated with sulfadoxine and pyrimethamine resistance,
respectively, was also done on parasites obtained from the same patients, before
treatment, on the day of recruitment to the study. The extracted Plasmodium DNA
was amplified using nested PCR and the resulting secondary PCR products were
digested with restriction enzymes. The digested DNA fragments were separated by
electrophoresis using 2 - 2.5% agarose gels and visualized by using UV
transillumination after staining with ethidium bromide.
In-vivo analysis revealed a 27% (n=74) fansidar® treatment failure rate. The
molecular analysis revealed 11% (n=108) prevalence of the combination mutation at
codons 437 and 108 of the dhps and dhfr, respectively, associated with fansidar®
resistance. There was a significant association between the presence of a
combination of mutations on both genes and the in-vivo S/P treatment response in
patients (x , P = 0.001). There was, however, no significant association between the

in-vivo response in patients that received sulfadoxine/pyrimethamine treatment and
the presence of a single or combined mutation(s) on either the dhps (codon 437: x2,
P = 0.143; codon 540: x2, P = 0.205; codon 437/540: x2, P = 0.454) or the dhfr
(codon 108: x2, P = 0.390; codon 59: x2, P = 0.246; codons 108/59: x2, P = 0.969)
alone. Molecular analysis showed a 97% (152) prevalence of the pre-requisite
mutation K76T, for chloroquine resistance while the supporting mutation at codon 75
had a prevalence of 99% (n=155). The higher in-vivo treatment failure rate in
comparison with the lower prevalence of mutation associated with resistance
recorded could be attributed to other causes other than the presence of drug resistant
parasites.
The study demonstrated that the collection of P. falciparum infected blood samples
on filter papers and the use of restriction fragment length polymorphism molecular
analysis is a viable method that can be employed in the identification of drug
resistant Plasmodium isolates.