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404 DRUG RESISTANCE
An alternative laboratory strategy aimed at
identifying the molecular basis of chloroquine
resistance employed a genetic cross between
a chloroquine-susceptible and a chloroquineresistant
parasite clone. The resulting drug
sensitivity of the progeny was characteristic of
one of the parent clones with no intermediates,
suggesting simple monogenic inheritance of
chloroquine resistance from these two parent
lines. Linkage analysis mapped the chloroquine
resistance locus to a 36 kb region of chromosome
7 (pfmdr1 is located on chromosome 5).
Detailed dissection of this chromosomal locus
initially provided a list of ten candidate genes,
and eventually uncovered the chloroquineresistance
gene pfCRT hiding in an assumed
non-coding region between two candidate
genes. The gene is contained within 13 exons,
and difficulties in establishing the intron–exon
boundaries are blamed for the delays experienced
in tracking down the gene. It is proposed
that the translated product of this gene, PfCRT,
is a membrane transporter containing ten
predicted transmembrane domains. In keeping
with chloroquine’s accepted mechanism of
action, PfCRT has been immunolocalized to
the boundaries of the digestive food vacuole
(Figure 16.3).
Sequence analysis of PfCRT from the genetic
cross have revealed eight point mutations
(M741I, N75E, K76T, A220S, Q271E, N326S,
I356T and R371I) capable of distinguishing
chloroquine-susceptible from resistant parasites.
Analysis of parasite isolates from different
origins around the world has consistently
identified the 76T and 220S mutations in all
chloroquine-resistant isolates (IC 50 100 nM).
With the exception of the I356T mutation, the
remaining six mutations were regularly found
in association with resistant isolates from Africa
and SE Asia. Based on the pattern of additional
mutations that accompany the K76T mutation,
it has been suggested that resistance must have
developed from four independent foci.
Interestingly, analysis of chloroquine-sensitive
parasites, as defined by the 100 nM cut-off, indicated
that with the exception of one isolate all
isolates shared the wild type PfCRT sequence.
The rogue isolate, which actually displays intermediate
chloroquine sensitivity without the
verapamil effect, contained all the PfCRT mutations
associated with resistance with the exception
of the critical K76T. Furthermore, under
chloroquine pressure it was possible to select
high-level chloroquine resistance with a verapamil
effect from this parasite clone. The
resultant parasites had acquired an additional
mutation, K76I. This experiment highlights
the importance of position 76 in PfCRT,
and indicates that against the correct genetic
backdrop selection of high-level chloroquine
resistance is relatively easy. Episomal transfection
studies claiming to provide further laboratory-based
evidence for the role of PfCRT
in resistance are compromised by the use of
chloroquine as the selection agent and the
acquisition of the K76I mutation in the resultant
parasite line rather than the 76T encoded in the
plasmid.
There is increasing field-based evidence
from a number of distinct geographical settings
including Mali, Cameroon, Sudan, Mozambique,
Brazil, Laos, Thailand and Papua New
Guinea, confirming that the PfCRT 76T mutation
is universally present in patients failing
standard chloroquine treatment. Equally of
note are the observations of apparent treatment
success against parasites containing the
76T mutation in semi-immune individuals,
emphasizing the important contribution of host
immunity to drug response. A number of studies
have also suggested that the use of both
pfCRT and pfmdr1 as markers of chloroquine
resistance improves predictive outcome. However,
a recent multivariate analysis has found
no improvement in predicting chloroquine
MEDICAL APPLICATIONS