Monitoring antimalarial drug resistance: making the most of the tools at hand
Malaria Section, Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, MD 21201, USA
(e-mail: cplowe{at}medicine.umaryland.edu)
Accepted 26 June 2003
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Summary |
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Key words: Plasmodium falciparum, drug resistance, PfCRT, dihydrofolate reductase, dihydropteroate synthase, chloroquine, sulfadoxine/pyrimethamine
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Introduction |
---|
A consensus has begun to emerge that the development of drug-resistant
malaria should be delayed through a strategy of routinely employing
combinations of drugs (White et al.,
1999; Nosten and Brasseur,
2002
), as is done with HIV and tuberculosis. Despite some
obstacles to implementing effective combination therapies in Africa
(Bloland et al., 2000
),
programs evaluating the efficacy and effectiveness of new combinations are now
underway in Africa (von Seidlein et al.,
2000
; Doherty et al.,
1999
; Adjuik et al.,
2002
). Among the challenges facing combination antimalarial
therapy in Africa are cost and safety issues.
Because of their rapid reduction of parasite biomass and the complete
absence of documented resistance despite over 2000 years of use, the
artemisinin derivatives are components of many candidate combinations.
Artemisinin derivatives may also reduce malaria transmission and spread of
resistance through their gametocytocidal properties
(Price et al., 1996;
Nosten et al., 2000
). However,
some safety concerns persist about this class of drugs, and their eventual
cost is uncertain, so it is prudent to consider other drugs suitable for
combination therapy.
Chlorproguanil-dapsone (also known as LapudrineTM-dapsone or LapDap)
is an antifolate combination similar to sulfadoxine/pyrimethamine (SP) but for
two important features: (1) it is rapidly eliminated and therefore exerts less
selective pressure for resistance-conferring parasite mutations than does SP
(Winstanley et al., 1997;
Nzila et al., 2000b
) and (2)
it is active against the SP-resistant forms of the parasite that are found in
Africa (Mutabingwa et al.,
2001a
,b
;
Kublin et al., 2002
).
Moreover, a pediatric course of treatment of LapDap is estimated to cost $0.15
(Mutabingwa et al., 2001b
),
making it orders of magnitude less expensive than any marketed antimalarial
drug other than chloroquine and SP.
However, the present reality is that many African countries with high rates of chloroquine-resistant malaria continue to use chloroquine as their first-line antimalarial, largely due to concerns that alternative drugs are either too expensive or would only provide interim solutions. This prolonged state of near-paralysis is also exacerbated by a paucity of current and comprehensive information on antimalarial drug efficacy in most endemic areas.
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In vivo and in vitro methods for measuring drug-resistant malaria |
---|
These standard methods for measuring antimalarial drug efficacy use a 14-day follow-up period and thus provide information only on short-term efficacy, leading some to advocate longitudinal studies of drug efficacy. In addition to measuring efficacy at 14 days, longitudinal studies measure sustained efficacy with repeated use of the same regimen over time and measure incidence of malaria episodes, reflecting how the treatment would hold up under `real world' conditions. The incidence of treatment episodes is an outcome that is highly relevant to public health policy makers, as it reflects not only the burden of disease but also the utilization of health resources. If a drug has a moderately impaired ability to eradicate parasites, it may still have high rates of therapeutic efficacy at 14 days but have more late recrudescenses associated with higher rates of recurrent episodes of symptomatic malaria requiring treatment. Longitudinal studies also permit assessment of how pharmacokinetic properties of drugs affect the incidence of treatment episodes.
The first such longitudinal trial of antimalarial drug efficacy was a
double-blind, placebo controlled trial of SP and LapDap in Kenya and Malawi
that compared the cumulative number of treatment episodes and rates of
treatment failure for SP and LapDap when each drug was used to treat
sequential episodes of malaria over the course of one year
(Sulo et al., 2002). It was
hypothesized that children receiving SP might have fewer malaria episodes than
those being treated with LapDap because the long half-life of SP provided at
least a month of prophylaxis against re-infection. However, this study found
that despite the rapid elimination of LapDap, children treated with this drug
did not have a higher incidence of malaria episodes than those treated with
SP. This is probably because LapDap had higher efficacy and thus fewer late
recrudescenses leading to retreatment. In effect, the potential disadvantage
of lacking a long prophylactic effect due to LapDap's short half-life was
offset by its better efficacy and possibly by less selection for
antifolate-resistant parasites. Another recent longitudinal study found fewer
treatment episodes in children treated sequentially for one year with
artesunate-SP and amodiaquine-SP than in those treated with SP alone
(Dorsey et al., 2002
).
Although the information gained from in vivo studies is exactly what is needed to make rational and evidence-based malaria treatment policies, standard in vivo studies remain expensive and time-consuming, and longitudinal clinical efficacy trials are even more so. Most countries in malaria-endemic areas therefore conduct in vivo surveys at only a few sites and at infrequent intervals.
In vitro methods for measuring drug resistance
(Nguyen-Dinh and Payne, 1980)
have proven to be even more limited in scope and suitability for surveillance.
They require that venous blood with a high parasite density be quickly frozen
or transported cold to a facility for parasite cultivation, the methods are
laborious, and failure to establish primary parasite growth is frequent.
Micro-test assays of fresh parasite isolates are subject to variation and
artifact, and their reliability for different drugs varies, with particularly
poor results for SP. While more-rigorous in vitro tests of
culture-adapted isolates are more reproducible, the processes of freezing,
thawing and adaptation to culture also introduce the possibility of selecting
sub-populations of parasites, so that the parasites ultimately assayed may be
genetically and phenotypically unrepresentative of the original parasite
population. Nevertheless, in vitro methods are indispensable for
confirming and characterizing resistance and for establishing and confirming
the molecular mechanisms of resistance.
The limitations of in vivo and in vitro methods for measuring drug-resistant malaria and the elucidation of molecular mechanisms of resistance to some antimalarial drugs have led to considerable research on molecular markers for resistance. While development of field-friendly molecular assays has been rapid, validation and implementation of these assays has moved at a slower pace.
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Molecular basis of chloroquine resistance |
---|
|
Polymorphisms in pfmdr1, which encodes the P. falciparum
P glycoprotein homologue 1, modulate chloroquine resistance in mutant
pfcrt-harboring parasites in vitro
(Reed et al., 2000), although
their role in vivo has yet to be substantiated
(Djimde et al., 2001a
). The
mutations most often cited as potential contributors to chloroquine resistance
are pfmdr1 N86Y and D1246Y.
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Molecular basis of antifolate resistance |
---|
The dhfr mutations alter the shape of the active site cavity where
the DHFR inhibitors bind the enzyme, resulting in differential binding
affinities for the different drugs. A single point mutation causing a Ser
Asn change at codon 108 causes pyrimethamine resistance with a only a
moderate loss of susceptibility to chlorcycloguanil. The addition of Asn
Ile51 and/or Cys
Arg59 mutations confers higher levels of
pyrimethamine resistance. Ile
Leu164, when combined with Asn108 and
Ile51 and/or Arg59, confers high-level resistance to both drugs.
Point mutations in the gene encoding DHPS have similarly been associated
with in vitro resistance to the sulfa drugs and sulfones. This gene
(dhps-pppk) is bi-functional, also encoding hydroxymethylpterin
pyrophosphokinase. Mutations associated with decreased susceptibility to
sulfas include Ser Ala436, Ala
Gly437, Ala
Gly581, and
Ser
Phe436 coupled with Ala
Thr/Ser613
(Brooks et al., 1994
;
Triglia and Cowman, 1994
;
Wang et al., 1997b
;
Robson et al., 1992
). A Lys
Glu540 DHPS mutation was discovered in Bolivia
(Plowe et al., 1997
) and also
appears to be linked with in vitro resistance to sulfa drugs
(Triglia et al., 1997
). Both
the DHFR and DHPS mutations occur in a progressive, step-wise fashion, with
higher levels of in vitro resistance occurring in the presence of
multiple mutations (Plowe et al.,
1997
; Wang et al.,
1997a
).
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Molecular markers for resistance |
---|
In the case of chloroquine, pfcrt 76T provides a single marker for
chloroquine resistance, but establishing markers for SP resistance has been
more difficult. In one study, pre-treatment DHFR and DHPS genotypes and
treatment outcomes from a longitudinal trial of SP and LapDap for
uncomplicated falciparum malaria were analyzed using a standardized system for
interpreting PCR results. The presence together of all five DHFR and DHPS
mutations that are found in Africa were strongly associated with SP failure
and there was a statistical interaction between DHFR and DHPS mutations
(Kublin et al., 2002). This
`quintuple mutant' associated with SP treatment failure was not associated
with LapDap failure. Just two mutations, DHFR Arg59 and DHPS Glu540, were over
90% sensitive and specific for the presence of all five DHFR and DHPS
mutations. It is likely, but not yet proven, that DHFR and DHPS mutations that
are widespread in Southeast Asia and South America would cause resistance to
LapDap, but the critical mutations are either rare (DHPS A581G) or absent
(DHFR I164L) in surveys of clinical samples in Africa.
Mutations in pfmdr1 (Price et
al., 1999) and pfcrt
(Cooper et al., 2002
) have been
found in laboratory studies to be associated with changes in susceptibility to
artemisinin derivatives, but these in vitro differences are not
associated with clinical failure, and clinical resistance has yet to be
documented for this class of drug. Markers for resistance to most other
antimalarial drugs are lacking because mechanisms of resistance are not yet
understood at the molecular level. Molecular markers are beginning to be
applied as tools for surveillance for SP and chloroquine resistance but are,
at present, still primarily used as research tools. Molecular markers can
provide direct and convincing evidence of selection for resistant parasites by
antimalarial drug treatment (Curtis et al.,
1998
; Diourte et al.,
1999
; Nzila et al.,
2000a
; Djimde et al.,
2001a
) or prophylaxis (Doumbo
et al., 2000
). Using the prevalence of molecular markers to
measure selective pressure provides a means of assessing the ability of
different combinations of antimalarial drugs to deter resistance to the drugs
for which markers are available.
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Genotype-resistance and genotype-failure indices |
---|
After controlling for age, both GRIs and GFIs ranged from 1.6 to 2.8 at all study sites over the three-year period. This study introduced the intriguing possibility that the ratios between the prevalence rates of resistant genotypes and in vivo outcomes may remain stable over time even as rates of both change, and that once these ratios, or indices, are established, molecular surveys could be used to predict in vivo treatment outcomes on a much broader scale than is possible using standard in vivo efficacy studies. Where GRIs and GFIs have been established and validated, it should be possible to collect finger-stick filter paper blood samples routinely at outlying health facilities and to transport these samples at ambient temperature to a central laboratory where the prevalence of the molecular marker can be quickly assessed for each site. Predictions of rates of resistance and failure will be somewhat imprecise but still useful. For example, in Mali it is now possible to measure the prevalence of pfcrt 76T and predict in vivo chloroquine failure and resistance rates of approximately half of the prevalence of the molecular marker. At the very least, molecular surveys can be used to target the scarce resources required to conduct in vivo studies: sites with very low rates of resistant genotypes can be safely monitored by periodic molecular surveys, while sites where the prevalence of resistance markers is high or rising might warrant confirmatory in vivo efficacy studies.
Further studies are needed to validate this model in other and more varied epidemiological settings and to refine the model to determine whether molecular surveys can reliably predict high-level (RII and RIII) resistance and early treatment failures. The stability over time of the indices in Mali could be attributable to relatively small differences among sites and years in the levels of malaria transmission and acquired immunity. Similar studies at sites with much higher vs lower malaria transmission intensities or highly seasonal vs year-round transmission might yield very different results. One can speculate that just as the indices increased with age in Mali, they would be higher in settings with higher levels of transmission intensity and immunity. Where transmission is very low, few persons might be expected to clear chloroquine-resistant parasites when treated with chloroquine, so the prevalence rate of in vivo resistance would approach that of pfcrt 76T, yielding very low GRIs and GFIs; in a setting of much higher transmission and immunity, most people may have enough immunity to clear these resistant infections, resulting in higher GRIs and GFIs even after adjusting for age. Alternatively, if GRIs and GFIs are found to be similar and stable in such different settings, the model would be far more useful in that indices would not have to be established in each setting before they could be applied there.
It will also be important to validate this model with other antimalarial drugs, most importantly SP. If ratios between the prevalence rate of the two mutations that predict the presence of quintuple DHFR and DHPS mutants and the prevalence of SP parasitological resistance and treatment failure are found to be stable after adjusting for age and at different sites with different epidemiological characteristics, the GRI and GFI model may provide a good tool for mapping SP resistance. This would be useful not only where SP is already being employed, to monitor for declining efficacy, but where SP is being considered as a replacement for chloroquine.
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Monitoring resistance after cessation of drug use |
---|
In 1993, Malawi became the first sub-Saharan African country to switch from
chloroquine to the antifolate combination SP as the first-line antimalarial
nationwide (Bloland et al.,
1993). Since 1993, SP has been the only treatment for
uncomplicated malaria available in government health facilities, where it is
dispensed without prescription. Chloroquine is legally available only by
prescription, and a national information campaign was largely successful in
convincing health practitioners and the public to accept SP as the standard
treatment for malaria. Some other eastern and southern African countries,
including Kenya in 1999 (Shretta et al.,
2000
), have attempted to institute similar changes in national
drug policy in the face of rising chloroquine resistance, with more mixed
results.
Recent studies have found that the cessation of chloroquine use in Malawi
was followed by reemergence and predominance of chloroquine-sensitive P.
falciparum. The prevalence of the chloroquine-resistant pfcrt
76T genotype decreased steadily from 85% in 1992 to 13% in 2000 in Blantyre, a
large city in central Malawi. In 2001, chloroquine cleared 100% of 63
asymptomatic P. falciparum infections, no isolates were resistant to
chloroquine in vitro, and no infections with the
chloroquine-resistant pfcrt genotype were detected
(Kublin et al., 2003). This
same study found that in 1999, 92% of P. falciparum infections
carried this mutation in neighboring Zambia, where chloroquine remains the
first-line drug. Another study found a 17% prevalence of pfcrt 76T in
1998 and only 2% in 2000 in a district further north in Malawi
(Mita et al., 2003
),
confirming that the withdrawal of chloroquine from Malawi has resulted in the
return of chloroquine-sensitive falciparum malaria there. This is most likely
explained by a cost to the fitness of the parasite of the chloroquine
resistance-conferring pfcrt mutations and selection for parasites
with wild-type pfcrt in the absence of chloroquine drug pressure,
although this remains to be demonstrated.
These studies provide a rationale for conducting controlled trials of chloroquine efficacy in areas where chloroquine use has been substantially reduced for a period of years. If such trials confirm a return of chloroquine's clinical efficacy, governments can consider withdrawing chloroquine and switching to other drugs on an interim basis, knowing that they may be able to later reintroduce chloroquine, which is unparalleled in its safety and low cost. As such antimalarial drug policy changes are considered and implemented, careful and integrated use of in vivo, in vitro and molecular assays for resistance have the potential to provide timely and practical information to help guide these policies. Already, molecular surveys have been integrated into the National Malaria Control Program in Mali and used to provide timely advice in a malaria epidemic in northern Mali (A. A. Djimde, A. Dolo, S. Diakite, A. Ouattara, C. V. Plowe and O. K. Doumbo, unpublished). Laboratories capable of performing basic molecular assays are well established in many African countries. The challenge now for scientists and policy makers is to work together to exploit advances in our understanding of the molecular mechanisms of drug-resistant malaria in order to make rational and informed decisions about malaria chemotherapy and prophylaxis policies.
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Appendix. Definitions of in vivo therapeutic efficacy and parasitological resistance |
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axillary temperature of >37.5°C on day 2 with parasitemia greater than the day 0 level;
axillary temperature of >37.5°C on day 3 in the presence of parasitemia; or
parasitemia on day 3 of >25% of day 0 level.
Late treatment failure:
failure to meet any of the criteria for early treatment failure;
development of danger signs or severe malaria in the presence of parasitemia during days 4-14; or
axillary temperature of >37.5°C in the presence of parasitemia during days 4-14.
Adequate clinical response:
failure to meet any of the criteria of early or late treatment failure;
absence of parasitemia on day 14 irrespective of temperature; or
axillary temperature of <37.5°C irrespective of the presence of parasitemia.
Parasitological resistance outcomes
RIII: no reduction in parasitemia, or reduction to >25% of day 0 level,
by day 3.
RII: reduction in parasitemia to <25% of day 0 level without clearance leading to re-treatment or followed by persistent parasitemia.
RI: initial clearance of parasites indicated by negative thick smear after day 0, with subsequent positive thick smear by day 14.
Sensitive: clearance of parasites by day 14 with no recurrence of parasitemia.
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Acknowledgments |
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