Detection of drug resistant plasmodium Falciparum by polymerase chain reaction Using mutation-specific oligonucleotide Primers
Abstract
Due to increased chloroquine resistance, the antifolate-sulfa
combinations are becoming increasingly important in the
chemotherapy of falciparum malaria. However, resistance to the
antifolates exists and it is important to study it because they are
still effective in the above combinations. Point mutations in the
dihydrofolate reductase (DHFR) gene lead to resistance to the
antifolate drugs. It was considered important to establish the
prevalence of the six reported point mutations among Kenyan field
isolates of P. falciparum, and look for a correlation between these
mutations and resistance of these isolates to antimalarials.
Mutation specific polymerase chain reaction, (MSPCR), was
carried out on 21 Kenyan P. falciparum isolates to detect point
mutations. Optimal concentrations of each peR component were
established for each MSPCR reaction. Direct sequencing of the DHFR
gene was also carried out to firstly confirm the above point
mutations and secondly to look for any other base pair changes. IDso
values were calculated from drug sensitivity tests carried out by
measuring 3H Hypoxanthine uptake in the presence of increasing
concentrations of the appropriate drug.
Out of the 21 Kenyan isolates five were found to be
pyrimethamine sensitive and 16 were pyrimethamine resistant. Drug
sensitivity results have shown that compared to reference strains,
the Kenyan isolates examined are not resistant to the biguanides
with respect to their IDsovalues. At the same time none of the
base pair changes associated with biguanide resistance (at
positions 16 and 108 in the DHFR gene) were found and neither was
the change at 164 which confers cross resistance to both
pyrimethamine and biguanides. Of the reported six mutations, we
have so far only found three in Kenyan isolates. These are Ser-108
to Asn-108, Asn51 to lIe-51 and Cys-59 to Arg-59; all associated
with pyrimethamine resistance. The results obtained were confirmed
by direct sequencing. No other differences in the DHFR sequence
were detected.
Thus the point mutations correlated well with absence or
presence of pyrimethamine resistance and absence of cycloguanil
resistance. MSPCR has the advantage over drug sensitivity tests of
being specific, quicker and more reproducible. It should therefore
be feasible to screen large numbers of patients to monitor the
appearance, persistence and spread of anti-folate (e.g .,
pyrimethamine or biguanides) resistance of P.falciparum in a
population.
Citation
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University of NairobiPublisher
University of Nairobi Faculty of Science