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Withdrawing antimalarial drugs: impact on parasite resistance and implications for malaria treatment policies
Drug Resistance Updates 7 (2004) 279–288
Withdrawing antimalarial drugs: impact on parasite resistance and implications for malaria treatment policies Miriam K. Laufer, Christopher V. Plowe∗ Malaria Section, Center for Vaccine Development, University of Maryland School of Medicine, 685 W. Baltimore Street, HSF-1 480, Baltimore, MD 21201, USA Accepted 25 August 2004
Abstract Malaria continues to be a leading cause of death in the tropics, taking the heaviest toll on children in Africa, where drug resistant Plasmodium falciparum has led to rising malaria mortality. High rates of chloroquine resistance prompted many countries in Africa to switch to alternative therapies to treat malaria. Parasites carrying mutations that render them chloroquine resistant may lose their survival advantage with the removal of chloroquine drug pressure. Alternatively, organisms may have undergone compensatory mutation that provides a survival advantage even in the absence of drug pressure. Decreasing drug resistant malaria has been reported following discontinuation of antimalarial drugs. However, most such reports are limited by the incomplete removal of chloroquine drug pressure, unreliable in vitro susceptibility assays and/or small, poorly described study populations. In Africa, Malawi was the first country to switch from chloroquine to sulfadoxine-pyrimethamine for the first line treatment of malaria. An effective campaign to end chloroquine use provided an excellent opportunity to study the natural history of drug resistance following the reduction of drug pressure. The finding that drug resistance decreases with the removal of drug pressure could provide a new paradigm for malaria treatment policies in Africa. © 2004 Published by Elsevier Ltd. Keywords: Malaria; Plasmodium falciparum; Chloroquine; PfCRT; Drug resistance; Sulfadoxine–pyrimethamine; Dihydrofolate reductase; Dihydropteroate synthase
1. Introduction Malaria continues to be a leading cause of death in the tropics, taking the heaviest toll on children in Africa. In areas of Southern and Eastern Africa with high levels of antimalarial drug resistance, malaria mortality has nearly doubled in the last decade (Korenromp et al., 2003). Chloroquine, an inexpensive, safe and initially highly effective drug, became the antimalarial of choice worldwide near the end of World War II and was the cornerstone of the effort to eradicate malaria in the 1950s and 1960s. Unfortunately, chloroquine-resistant Plasmodium falciparum arose and was first detected in Southeast Asia and South America in the late 1950s (Peters, 1969), reaching Africa two decades later (Campbell et al., 1979; Fogh et al., 1979, 1984; Jepsen et al., 1983). ∗
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Within 10 years, chloroquine resistance began to reach high levels in many parts of Southern and Eastern Africa. In a study of chloroquine efficacy in Kenya and Malawi in the early 1980s, 75 and 82% of infections were found to be resistant to chloroquine in vivo (Bloland et al., 1993). As chloroquine resistance rates increased in Africa, so did morbidity and mortality attributable to malaria (Zucker et al., 1996, 2003; Trape et al., 1998). In response to unacceptably high rates of chloroquine failure, in 1993 Malawi became the first sub-Saharan African country to discontinue the routine use of chloroquine and to elevate the antifolate combination sulfadoxine–pyrimethamine to the antimalarial of first choice nationwide (Bloland et al., 1993). Subsequently, Kenya, Botswana and Tanzania also adopted the policy change from chloroquine to sulfadoxine–pyrimethamine, and other countries have switched from chloroquine to a variety of other drugs and drug combinations. As chloroquine drug pressure is reduced or eliminated in
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these settings, what will become of chloroquine-resistant malaria?
2. Molecular basis for chloroquine resistance Chloroquine is thought to exert its antimalarial effect by interfering with hematin detoxification in the digestive vacuole of the parasite (Chou et al., 1980; Dorn et al., 1998). Chloroquine-resistant P. falciparum isolates exhibit decreased accumulation of the drug in the vacuole. This was originally thought to be due to an efflux mechanism (Verdier et al., 1985; Krogstad et al., 1987) but it is now clear that such a simple explanation does not fully explain all the experimental data. PfCRT is a predicted transporter located on the digestive vacuole membrane and may be responsible for modulating chloroquine accumulation within the vacuole. Mutations in PfCRT were found to be perfectly associated with in vitro chloroquine resistance in a geographically diverse set of P. falciparum isolates and among all progeny of a genetic cross between chloroquine sensitive and resistant parental clones. One mutation, the substitution of threonine for lysine at amino acid position 76 (K76T), was present in all in vitro resistant isolates and no sensitive isolates (Fidock et al., 2000; Wellems and Plowe, 2001). Genetic transformation experiments, in which replacement of wild-type pfcrt with mutant forms encoding 76T rendered chloroquinesusceptible clones resistant to chloroquine, demonstrating that mutation at PfCRT codon 76 is the essential determinant of chloroquine resistance (Sidhu et al., 2002). Polymorphisms in pfmdr1 encoding the P. falciparum Pglycoprotein homologue 1 (Pgh1), modulate the level of in vitro chloroquine resistance in parasites that already harbor PfCRT 76T (Reed et al., 2000). The relevance of the variations caused by pfmdr1 mutations in IC50 values within the chloroquine-resistant range cannot necessarily be extrapolated to parasite responses to treatment in vivo, however, and role of pfmdr1 in chloroquine treatment failure, if any, remains unclear. The mutations most often cited as potential contributors to chloroquine resistance are pfmdr1 N86Y and D1246Y. At the same time that its key role in conferring chloroquine resistance in vitro was being confirmed, the utility of the pfcrt 76T mutation as a molecular marker for clinical outcomes of chloroquine treatment was assessed. In vivo drug resistance is defined and measured differently from in vitro resistance. In the laboratory, in vitro resistance is defined according to the concentration of the drug required to inhibit asexual parasite replication, or schizont formation. In the case of chloroquine, in vitro resistance is partially reversible by the addition of verapamil to the assay (Martin et al., 1987). Classically, in vivo resistance is based on the presence or absence at specified time points of asexual parasites in the peripheral blood of symptomatic individuals treated with the drug. Parasites are resistant at the RIII level if asexual parasitemia continues to rise or is reduced by less than 75% during the first 48–72 h
after treatment; at the RII level if asexual parasitemia does not clear but is reduced to 25% or less of the pre-treatment parasitemia by 48–72 h after treatment; and at the RI level if asexual parasites disappear but return within the specified follow-up period, usually 14 or 28 days. Infections that clear by day 7 after treatment and do not recur within the follow-up period are considered sensitive (Rieckmann, 1990). In the mid-1990s, the World Health Organization released a new protocol for monitoring antimalarial drug efficacy that did not consider recurrent or persistent asexual parasitemia occurring 4 or more days after treatment to constitute treatment failure unless fever was present (WHO, 1996). This protocol is now being replaced with one that is more similar to the classic definitions of clinical resistance, with outcome categories including early treatment failure (corresponding to RIII and some cases of RII resistance); late treatment failure (corresponding to RII or RI resistance occurring 4 or more days after treatment and accompanied by fever); late parasitological failure (corresponding to RI resistance in the absence of fever); and adequate clinical and parasitological response (corresponding to sensitive outcomes) (WHO, 2002). In a human infections, the host immune response plays an important role in clearing malaria parasites, and persons who have acquired partial immunity to malaria through prolonged, intense exposure to infection are often able to clear infections after treatment with a drug to which the parasites are resistant (Hoffman et al., 1984). Thus, field studies assessing the role of pfcrt mutations in clinical outcomes to chloroquine treatment were predictably less straightforward than the absolute correlations found between pfcrt genotypes and in vitro phenotypes. Djimde and colleagues in Mali conducted a clinical efficacy study of chloroquine, assessing pfcrt genotypes of both pre-treatment and post-treatment infections. Eighty-six percent of participants had sensitive clinical outcomes, and as expected, the molecular marker for chloroquine resistance was more prevalent than clinical resistance. Nevertheless, 92% of infections carrying pfcrt 76T had resistant in vivo outcomes as compared with 37% of parasites carrying the wild-type pfcrt. The odds ratio of resistance to chloroquine treatment for parasites carrying the resistant genotype versus the wild-type pfcrt was 18.8 (P = 0.001). Among 60 samples from post-treatment infections, 100% carried the 76T mutation (Djimde et al., 2001). The investigators concluded that there was a strong association between the presence of the pfcrt 76T mutation at the time of chloroquine treatment and a resistant in vivo outcome. Subsequently, studies in other countries in Africa and Asia have confirmed the primary role of pfcrt 76T in conferring chloroquine resistance in vivo, as summarized in a recent review (Wellems and Plowe, 2001). Pfmdr1 mutations and other parasite genetic factors may play a secondary role in chloroquine treatment outcomes, but these appear to be neither necessary nor sufficient to cause chloroquine treatment failure, and multivariate analyses have failed to detect statistical interactions between mutations in pfcrt and pfmdr1 (Djimde et al., 2001; Jelinek et al., 2002), i.e.
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the presence of mutations in both genes is not more strongly associated with treatment failure than the presence of the pfcrt T76 mutation alone.
3. Antibiotic pressure and fitness Drug resistance has developed in response to the use of most antimicrobials. Drug exposure is thought to select genetic mutations that allow the organism to survive and continue to replicate under drug pressure. Observational cohort studies in Australia and Iceland have shown that children who had recently received antibiotics were at higher risk of colonization with penicillin-resistant pneumococci (Arason et al., 1996; Nasrin et al., 2002). Quantity of antibiotic use within a community (as measured by doses of antibiotics per 1000 children per day) has also been also highly correlated with colonization with resistant organisms (Arason et al., 1996; Melander et al., 2000; Seppala et al., 1995). Mutations that confer drug resistance are commonly assumed to impose a cost to bacterial fitness. It would seem to follow that with the removal of drug pressure, the organism should revert back to the more fit, wild-type genotype, either through back-mutation or selection of residual wildtype organisms that have a survival advantage in the absence of drug pressure. Seppala et al. reported a dramatic increase in erythromycin resistance among group A streptococci in Finland and found a strong positive association between local erythromycin use and local erythromycin resistance rates (Seppala et al., 1995). In 1992, a nationwide effort was initiated to decrease erythromycin use in outpatients. Macrolide use decreased from 2.4 to 1.4 daily doses per 1000 inhabitants per day and erythromycin resistance fell from 16.5 to 8.6% among group A streptococci clinical isolates (Seppala et al., 1997). This observation supported the hypothesis that the removal of drug pressure permits the reemergence of susceptible organisms. Other clinical studies, however, have failed to find similar decreases in resistance following curtailment of drug use. Despite a 50-fold decrease in sulfonamide use in the United Kingdom, there was no decline in the prevalence of sulfonamide-resistant Escherichia coli (Enne et al., 2001). On an individual level, infection with HIV that develops protease inhibitor resistance has been shown to maintain that resistance after changing to non-protease inhibitor based treatment regimens (Boeri et al., 2003). The phenomenon of persistence of genetically mediated drug resistance despite removal of drug pressure has been investigated in the laboratory and through computer modeling. Among a wide variety of microorganisms that have been studied in experiments that apply and then remove drug pressure, many organisms develop additional, compensatory mutations that improve their ability to survive in the absence of drug pressure rather than reverting back to the wild-type drug sensitive form (Schrag et al., 1997; Levin et al., 2000; Sander et al., 2002; Menzo et al., 2003). Although the initial
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mutation leading to drug resistance may incur some cost to organism survival, through a process referred to as compensatory evolution, secondary mutations may restore the fitness of the drug-resistant organism and allow it to remain fixed in a population despite removal of drug pressure. Based on these studies, in areas where chloroquine use has been significantly decreased, the following possible outcomes can be entertained: (1) chloroquine resistance comes at a cost to parasite fitness that is overcome by compensatory evolution, allowing chloroquine-resistant parasites to remain fixed in the population; (2) chloroquine resistance comes at a cost to parasite fitness that is not overcome by compensatory evolution, and chloroquine sensitive parasites will reemerge in the absence of chloroquine drug pressure; or, (3) chloroquine resistance does not come at a cost to parasite fitness, and chloroquine-resistant parasites will remain prevalent after withdrawal of chloroquine from use.
4. Early studies The first suggestion that chloroquine susceptibile parasites might reemerge under reduced drug pressure came from French researchers who conducted a study in Vietnam. Over 5 years after a switch from chloroquine to sulfadoxine–pyrimethamine for treatment of P. falciparum in Vietnam, they enrolled 68 children with uncomplicated falciparum malaria in a chloroquine efficacy study in Ho Chi Minh City, and found that the rate of cure was 90%, with follow up from 1 to 12 weeks. The good clinical response to chloroquine was attributed to the decreased use of chloroquine since 1975 (Jacquier et al., 1985). Subsequently, however, representatives of the World Health Organization and the Institute of Malariology, Parasitology and Entomology in Vietnam presented conflicting data. They found no decrease in clinical resistance to chloroquine and documented a decrease, but not elimination of the use of chloroquine in the area. In 1985, there were still between 500,000 and 1 million chloroquine tablets dispensed per year in Ho Chi Minh City alone (Onori and Vu, 1986). The reemergence of chloroquine resistance was not addressed in the literature again until the early 1990s. On the island of Hainan, China, antimalarial treatment was changed from chloroquine to piperaquine in 1979, and community surveys documented that chloroquine use had largely ceased. Over a 5–8-year period, in vivo resistance reportedly decreased from 84 to 40%, and from 1981 to 1991 in vitro resistance decreased from 98 to 61% (Liu et al., 1995). The clinical studies were limited by small sample sizes (only 20 cases were assessed from 1986 to 1989) and details about the participants’ age and history of malaria exposure were not provided. The investigators used locally developed microtitration plates and followed a slightly altered protocol from the standard WHO recommendation. Nevertheless, the possibility of the return of chloroquine sensitivity was raised.
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Several studies of in vitro chloroquine susceptibility testing were subsequently published, all reporting trends of decreasing chloroquine resistance after a change to an alternative antimalarial drug. In one province in Gabon, in vitro chloroquine resistance reportedly decreased from 100 to 45% between 1992 and 1996, in a study of 30–40 clinical isolates on a biannual basis. Concomitantly, no changes in mefloquine or quinine resistance were detected (Schwenke et al., 2001). As a result of this dramatic finding, a small in vivo chloroquine efficacy trial was conducted in 2000 among children with uncomplicated malaria in this region. One hundred percent of the infections were found to be clinically resistant and all parasites carried the molecular marker for chloroquine resistance, pfcrt 76T (Borrmann et al., 2002). The striking inconsistency between the in vitro results and the in vivo outcomes and molecular results highlights the potential inaccuracies of in vitro drug susceptibility tests and emphasizes the need for well designed clinical and molecular studies to confirm new findings. By the late 1990s, two groups of investigators had found similar increases in in vitro chloroquine susceptibility in Vietnam. Huong et al. cited unpublished data that in vitro chloroquine resistance was found in 83% of isolate in 1988 in contrast to their 1999 findings of 16% resistance among clinical isolates (Huong et al., 2001). Thanh et al. reported results from 1995 and 1998 that show the prevalence of in vitro resistance falling from 92 to 34% (Thanh et al., 2001). In response to these findings, a clinical study was conducted to test the hypothesis that malaria susceptibility to chloroquine and sulfadoxine–pyrimethamine had returned, since these drugs had reportedly not been used in Vietnam for several years. The study took place in two locations with two distinct populations: semi-immune children in Dac Lac province and malaria naive adults in Binh Phuoc province. Both drugs were administered in combination with artesunate, so the efficacy rates of sulfadoxine–pyrimethamine and chloroquine were masked by the efficacy of the partner drug. Among semi-immune children, both combinations were highly efficacious. Among the non-immune adults, 49% of the participants who were treated with chloroquine plus artesunate had resistant outcomes. The chloroquine resistanceconferring mutation at pfcrt 76T was carried by 96 and 61% of infected patients in the semi-immune pediatric and malaria-naive adult population, respectively (Nguyen et al., 2003). Due to combination with artesunate, this study was unable to measure directly the in vivo efficacy of sulfadoxine–pyrimethamine and chloroquine. However the high prevalence of the chloroquine-resistant form of pfcrt and the poor outcomes among non-immune patients call into question the earlier in vitro results. As described above, parasite resistance to chloroquine is reliably associated with pfcrt 76T, while in vitro studies conducted under field conditions have not infrequently proven to be unreliable. The high rates of recrudescence among non-immune adults treated with chloroquine–artesunate and the high prevalence of pfcrt 76T
provide strong evidence that chloroquine sensitive P. falciparum malaria has not reemerged in Vietnam. Several factors distinguish malaria in Vietnam from malaria in Africa. The removal of chloroquine in Vietnam was incomplete due to its continued use for treatment of Plasmodium vivax, which is absent in nearly all of sub-Saharan Africa. Although P. vivax is much less prevalent that P. falciparum in Vietnam (Hung et al., 2002), patients who are suspected or documented to have P. vivax infection are still treated with chloroquine, and chloroquine may be prescribed for patients who are infected with P. falciparum, either through incorrect diagnosis or reverting to older treatment practices. Malaria transmission is generally much lower in Southeast Asia than in most sub-Saharan African sites, and populations in low transmission areas acquire less immune protection from malaria disease (Luxemburger et al., 1996). Thus, in low transmission areas the vast majority of the overall malaria parasite population is likely to be under drug pressure at any given time, since most persons who are infected become ill and are treated. Moreover, in low transmission areas polyclonal infections are less common than in Africa, where multiclonal infections are the rule (Babiker and Walliker, 1997), so there is less opportunity both for competition among drug sensitive and resistant clones within the host leading to predominance of sensitive clones, and for sexual recombination leading to disassociation of resistance-conferring polymorphisms and/or the compensatory mutations that help to maintain them (Conway et al., 1999). Both of these factors could contribute to persistence of resistant parasites after reduction of drug pressure.
5. Removal of drug pressure: Malawi Malawi offers a more clear-cut opportunity to observe the natural history of chloroquine resistance after the removal of drug pressure. In the face of unacceptably high chloroquine failure rates, Malawi was the first country in Africa to switch from chloroquine to sulfadoxine–pyrimethamine for first line treatment of malaria. Since 1993, sulfadoxine–pyrimethamine has been the treatment provided by all government health facilities and is dispensed without prescriptions. Due to successful national information campaigns and tight control of its distribution, chloroquine use has largely been eliminated. Unlike Southeast Asia and South America, non-falciparum Plasmodial species are rare. Five years after the initiation of the change from chloroquine to sulfadoxine–pyrimethamine, clinical isolates were tested in vitro for chloroquine susceptibility. Sixty-five percent were found to be sensitive to chloroquine, 31% were borderline resistant and only 3% resistant (Takechi et al., 2001). In the same region along the shore of Lake Malawi, children with asymptomatic malaria infection were recruited into a study of chloroquine efficacy in 2000. Fifty-four children were enrolled and only one child developed recurrent
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Fig. 1. Declining prevalence of chloroquine resistance conferring mutation PfCRT T76 in Blantyre, Malawi from 1992 to 2004. Arrow denotes change from chloroquine to sulfadoxine–pyrimethamine as first line antimalarial drug in Malawi in 1993. Reproduced with permission from Kublin et al. (2003) and S. Nkoma and D. Bell, personal communications.
parasitemia by day 7 and four more by day 14. Ninety-one percent of the infections were sensitive in vivo. Among the parasites that were detected on enrollment, none carried the pure pfcrt 76T mutation. Only 1/53 of the infections was found to contain parasites of mixed pfcrt K76 and 76T genotypes. The participant with the mixed infection had a sensitive in vivo response (Mita et al., 2003). In Blantyre, the largest city in Malawi, archived thick blood smears and blood samples collected on filter paper from children with symptomatic malaria were analyzed for the polymorphic codon at pfct 76. The earliest samples were from 1992, prior to the change from chloroquine to SP, and the sample set extended through the year 2000. Through nested PCR and allele-specific restriction digest assays, the investigators demonstrated a steady decrease in the prevalence of pfcrt 76T from 85% in 1992 to 13% in 2000 (Fig. 1). In 2001, no parasites with the chloroquine resistance conferring mutation were found (Kublin et al., 2003). In 2002–2004, other investigators in Malawi have continued to find only wild-type parasites with the genotype pfcrt K76 (S. Nkoma and D. Bell, personal communications). Mutations in pfmdr1, which play no role in antifolate resistance, exhibited a modest decrease in frequency over the decade. The prevalence of one of the implicated mutations, D1246Y, declined from one-half of the
infections in 1993 to one-fourth in 2000 (P = 0.023). Genetic analyses of parasites from the neighboring countries where chloroquine continued to be used have shown a very different pattern. Over 90% of parasites in the late 1990s from Zambia and Mozambique carried the mutation pfcrt 76T (Kublin et al., 2003; Mayor et al., 2001). The Malawi investigators also conducted an in vivo study of 72 adults with asymptomatic parasitemia. All infections demonstrated sensitive parasitologic outcomes. This study, along with the previous study of asymptomatic children, did not include control groups. This raises the possibility that semi-immune persons with asymptomatic parasitemia might have cleared their infections without any treatment or after administration of drugs with impaired efficacy. However, the uniformity of the clearance of parasites among both adults and children treated with chloroquine, along with the molecular finding of the return of wild-type pfcrt K76, provides strong evidence that chloroquine sensitive P. falciparum has reemerged and predominates in Malawi, at least in the regions studied. During the 10-year period of respite from chloroquine use, sulfadoxine–pyrimethamine has been used as first line therapy to treat uncomplicated malaria. During this time, there was a steady increase in the prevalence of mutations
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in parasite dihydrofolate reductase (DHFR) associated with sulfadoxine–pyrimethamine resistance. This is reflected in increasing parasitologic resistance and late treatment failures in clinical studies (Kublin et al., 2003; Plowe et al., 2004). Malawi, the African country that pioneered antimalarial policy change, now stands in a unique position to serve as a case study of whether chloroquine could be reintroduced to an area from which it has been withdrawn, through clinical trials of chloroquine alone and in combination with other drugs intended to prevent reemergence of chloroquine resistance.
6. Other antimalarial drugs Recent experience in Northwest Thailand with the combination of mefloquine and artesunate indicates that removal of drug pressure might not even be necessary for restoration of susceptibility among malaria parasites. In 1985, mefloquine was introduced in combination with sulfadoxine–pyrimethamine, and then in 1990 mefloquine was used at a higher dose as monotherapy. Within 4 years, clinical cure rates had fallen from 90 to 60%, leading to the deployment of an artesunate–mefloquine combination. With this combination, cure rates remained nearly 100% until 1998, when the camp for displaced persons that was under surveillance closed. Of particular interest in this discussion of the reemergence of drug susceptibility, is the pattern of in vitro mefloquine resistance. At the time when clinical resistance to mefloquine reached its peak, the geometric mean concentration of mefloquine required to inhibit 50% of schizont formation (IC50 ) was 47–56 ng/ml. By 1999, 5 years after the switch from mefloquine monotherapy to mefloquine–artesunate combination therapy, the IC50 decreased to 25 ng/ml (Nosten et al., 2000). These data from Thailand suggest that if parasites develop resistance to a drug, combining the drug with an appropriate partner drug may in some cases effectively reduce drug pressure and allow for the reemergence of the susceptible forms of the parasite. While these in vitro results should be interpreted with caution, given the limitations of in vitro testing described above, the assays were performed using standard radio-labelled hypoxanthine uptake-inhibition rather than field microtest methods and were performed at a highly experienced laboratory (Brockman et al., 2000). Sulfadoxine–pyrimethamine, an antifolate combination, has been used throughout the world in areas where chloroquine has failed. Sulfadoxine–pyrimethamine inhibits folate synthesis, a function that is essential to parasite survival. Pyrimethamine inhibits DHFR and sulfadoxine inhibits dihydropteroate synthase (DHPS). Mutations in DHFR (Cowman et al., 1988; Peterson et al., 1988, 1990) and DHPS (Brooks et al., 1994; Triglia et al., 1997; Wang et al., 1997a,b) have been associated with sulfadoxine–pyrimethamine resistance in vitro. Based on both laboratory assays and epidemiolog-
ical studies, mutations occur in a stepwise fashion, with increasing numbers of mutations conferring higher level resistance to Sulfadoxine–pyrimethamine (Plowe et al., 1997; Sirawaraporn et al., 1997; Wang et al., 1997a,b). In clinical studies in Africa, five mutations have been identified that are most closely associated with clinical sulfadoxine–pyrimethamine failure: DHFR S108N, C59R and N51I and DHPS A437G and K540E (Nzila et al., 2000; Kublin et al., 2002). Sulfadoxine–pyrimethamine resistance may not have the same natural history as chloroquine resistance when the drug is effectively withdrawn. Sulfadoxine–pyrimethamine has similar mechanisms of action to trimethoprim– sulfamethoxazole, an antibiotic that is widely used in the developing world both for treatment and increasingly for prophylaxis against opportunistic infections in persons living with HIV. Laboratory experiments with naturally occurring P. falciparum isolates as well as genetically engineered DHFR in a yeast expression system demonstrated cross resistance between pyrimethamine and trimethoprim (Iyer et al., 2001), and complete cross resistance has been demonstrated among the sulfas and sulfones in isolates with a variety of DHPS genotypes (Triglia et al., 1997). In regions of high transmission in Africa, individuals who receive trimethoprim–sulfamethoxazole either for treatment of bacterial infections or prophylaxis for opportunistic infections are also frequently infected with malaria parasites. Thus, antifolate drug pressure will not be eliminated even if sulfadoxine–pyrimethamine is not administered for malaria treatment. Frequent observations of rapid development of resistance to the antifolate drugs in direct response to drug pressure have led many to posit that DHFR and DHPS mutations arise spontaneously in response to drug pressure (Doumbo et al., 2000; Clyde and Shute, 1957). Biochemical analyses have shown that the DHFR mutations come at a cost to enzyme function and therefore presumably at a cost to parasite fitness (Sirawaraporn et al., 1997). If the mutations conferring resistance to antifolates arise under drug pressure and are harmful to the parasite, it would seem likely that withdrawal of antifolate drug pressure (if this could be accomplished) would lead to reemergence of wild-type sensitive parasites. However, recent genetic analyses of sulfadoxine– pyrimethamine-resistant parasites has called into question the notion that resistance-conferring DHFR and DHPS mutations arising spontaneously in direct response to drug pressure are primarily responsible for antifolate resistance at the regional level. Studies of microsatellite markers and other genetic sequences flanking the dhfr and dhps genes among drug-resistant parasites in South America and Africa have demonstrated that allelic haplotypes of the same ancestral origin were driven through large regions in genetic sweeps. Cortese et al. analyzed parasites from five regions of the South American Amazon. The haplotypes with mid to high-level mutations (DHFR 50R, DHFR 164L, DHPS
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540E and DHPS 581G) appeared to have a common origin. From the lower Amazon, these resistance markers spread in a North–Northwestward direction, perhaps in response to the permissive environment of drug pressure (Cortese et al., 2002). In Africa, microsatellite markers were used to determine the evolutionary origins of sulfadoxine–pyrimethamineresistant mutations in malaria. One mutant dhps allele and three mutant dhfr alleles, each of independent origin, have been driven through the parasite population in KwaZuluNatal, a region of low malaria transmission in South Africa. This same allelic haplotype was found to be the basis for sulfadoxine–pyrimethamine resistance in Tanzania, 4000 km away (Roper et al., 2003). These data suggest that gene flow following the pattern of sulfadoxine–pyrimethamine drug pressure, rather than spontaneous mutation, may be the driving force of sulfadoxine–pyrimethamine resistance. This is not to say that spontaneous mutation or that rapid, focal selection of rare background mutations does not occur, but that specific, limited haplotypes appear to account for the predominant forms of antifolate-resistant parasites found at these sites. The most likely explanation for this phenomenon is that the successful resistant haplotpyes include compensatory mutations, not in dhfr or dhps genes themselves, but elsewhere in the genome, providing these haplotypes with an advantage over spontaneously arising or pre-existing mutant genotypes when drug pressure is applied. In other words, even if the local parasite population contains some antifolateresistant parasites, when substantial drug pressure is applied and new arrivals appear on the scene that are equally resistant but more fit, the new haplotypes take over. If this is the case, reemergence of antifolate sensitive P. falciparum might be less likely in areas of lower transmission for one of the reasons considered above in the discussion of the situation in Vietnam, i.e. fewer opportunities for disassociation between resistance-conferring and compensatory mutations through sexual recombination. However, in areas of higher transmission, competition among sensitive and resistant clones as well as recombinatory disassociation might still lead to reduced prevalence of DHFR and DHPS mutations in response to reduction or elimination of antifolate drug pressure. In African countries where sulfadoxine–pyrimethamine is withdrawn from use, molecular epidemiological studies such as those done after chloroquine was withdrawn from Malawi (Kublin et al., 2003) will be of great interest. If removing sulfadoxine–pyrimethamine from use in high transmission areas does not result in reemergence of susceptible parasites as was observed with chloroquine, what could account for the different results of withdrawing the two drugs? Two possible explanations suggest themselves: (1) continued drug pressure from other antifolate drugs such as trimethoprim–sulfamethoxazole may apply sufficient pressure to maintain antifolate resistance; and/or (2) the survival disadvantage of DHFR and DHPS mutations is either less than that of PfCRT mutations, or overcome by compensatory mutations.
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7. Future directions It has been speculated that years of reliance on antimalarials other than chloroquine might lead to the reemergence of chloroquine-sensitive P. falciparum and permit the reintroduction of this safe and affordable drug. If this is confirmed in controlled clinical trials, several new opportunities for managing malaria drug resistance become possible. Countries that continue to use chloroquine despite high levels of resistance will be encouraged to switch now to more effective drugs, or drug combinations, with the knowledge that they maybe able to return to chloroquine use in the future. Countries that have already replaced chloroquine with other antimalarial drugs could attempt to eliminate chloroquine use in their population as completely as possible, as Malawi has, with the expectation that if the current drug regimen fails, chloroquine could be reintroduced. It is important to emphasize that reintroduction of chloroquine as monotherapy is highly inadvisable and would likely lead to a rapid reemergence of resistance chloroquineresistant parasites that remain lurking in the neighborhood, as they do in the countries surrounding Malawi. However, where chloroquine is highly efficacious, it could be an ideal component of combination drug therapies designed to deter the emergence of resistance to the component drugs. And, if withdrawal of both chloroquine and sulfadoxine–pyrimethamine from Africa were to result in a reemergence of falciparum malaria sensitive to these drugs, a combination therapy regimen including an artemisinin derivative combined with chloroquine and sulfadoxine–pyrimethamine would be very attractive based on cost and safety. In addition, this combination would offer the advantage of including of one component that rapidly reduces parasite biomass, reducing the probability of resistant parasites surviving, with two longacting drugs to protect against emergence of resistant parasites during the post-treatment prophylaxis phase. Given the limited number of safe and affordable antimalarial drugs and the compliance and prophylactic benefits of longeracting drugs, chloroquine and sulfadoxine–pyrimethamine may continue to be important tools in the antimalarial armamentarium.
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Withdrawing antimalarial drugs: impact on parasite resistance and implications for malaria treatment policies Miriam K. Laufer, Christopher V. Plowe∗ Malaria Section, Center for Vaccine Development, University of Maryland School of Medicine, 685 W. Baltimore Street, HSF-1 480, Baltimore, MD 21201, USA Accepted 25 August 2004
Abstract Malaria continues to be a leading cause of death in the tropics, taking the heaviest toll on children in Africa, where drug resistant Plasmodium falciparum has led to rising malaria mortality. High rates of chloroquine resistance prompted many countries in Africa to switch to alternative therapies to treat malaria. Parasites carrying mutations that render them chloroquine resistant may lose their survival advantage with the removal of chloroquine drug pressure. Alternatively, organisms may have undergone compensatory mutation that provides a survival advantage even in the absence of drug pressure. Decreasing drug resistant malaria has been reported following discontinuation of antimalarial drugs. However, most such reports are limited by the incomplete removal of chloroquine drug pressure, unreliable in vitro susceptibility assays and/or small, poorly described study populations. In Africa, Malawi was the first country to switch from chloroquine to sulfadoxine-pyrimethamine for the first line treatment of malaria. An effective campaign to end chloroquine use provided an excellent opportunity to study the natural history of drug resistance following the reduction of drug pressure. The finding that drug resistance decreases with the removal of drug pressure could provide a new paradigm for malaria treatment policies in Africa. © 2004 Published by Elsevier Ltd. Keywords: Malaria; Plasmodium falciparum; Chloroquine; PfCRT; Drug resistance; Sulfadoxine–pyrimethamine; Dihydrofolate reductase; Dihydropteroate synthase
1. Introduction Malaria continues to be a leading cause of death in the tropics, taking the heaviest toll on children in Africa. In areas of Southern and Eastern Africa with high levels of antimalarial drug resistance, malaria mortality has nearly doubled in the last decade (Korenromp et al., 2003). Chloroquine, an inexpensive, safe and initially highly effective drug, became the antimalarial of choice worldwide near the end of World War II and was the cornerstone of the effort to eradicate malaria in the 1950s and 1960s. Unfortunately, chloroquine-resistant Plasmodium falciparum arose and was first detected in Southeast Asia and South America in the late 1950s (Peters, 1969), reaching Africa two decades later (Campbell et al., 1979; Fogh et al., 1979, 1984; Jepsen et al., 1983). ∗
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Within 10 years, chloroquine resistance began to reach high levels in many parts of Southern and Eastern Africa. In a study of chloroquine efficacy in Kenya and Malawi in the early 1980s, 75 and 82% of infections were found to be resistant to chloroquine in vivo (Bloland et al., 1993). As chloroquine resistance rates increased in Africa, so did morbidity and mortality attributable to malaria (Zucker et al., 1996, 2003; Trape et al., 1998). In response to unacceptably high rates of chloroquine failure, in 1993 Malawi became the first sub-Saharan African country to discontinue the routine use of chloroquine and to elevate the antifolate combination sulfadoxine–pyrimethamine to the antimalarial of first choice nationwide (Bloland et al., 1993). Subsequently, Kenya, Botswana and Tanzania also adopted the policy change from chloroquine to sulfadoxine–pyrimethamine, and other countries have switched from chloroquine to a variety of other drugs and drug combinations. As chloroquine drug pressure is reduced or eliminated in
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these settings, what will become of chloroquine-resistant malaria?
2. Molecular basis for chloroquine resistance Chloroquine is thought to exert its antimalarial effect by interfering with hematin detoxification in the digestive vacuole of the parasite (Chou et al., 1980; Dorn et al., 1998). Chloroquine-resistant P. falciparum isolates exhibit decreased accumulation of the drug in the vacuole. This was originally thought to be due to an efflux mechanism (Verdier et al., 1985; Krogstad et al., 1987) but it is now clear that such a simple explanation does not fully explain all the experimental data. PfCRT is a predicted transporter located on the digestive vacuole membrane and may be responsible for modulating chloroquine accumulation within the vacuole. Mutations in PfCRT were found to be perfectly associated with in vitro chloroquine resistance in a geographically diverse set of P. falciparum isolates and among all progeny of a genetic cross between chloroquine sensitive and resistant parental clones. One mutation, the substitution of threonine for lysine at amino acid position 76 (K76T), was present in all in vitro resistant isolates and no sensitive isolates (Fidock et al., 2000; Wellems and Plowe, 2001). Genetic transformation experiments, in which replacement of wild-type pfcrt with mutant forms encoding 76T rendered chloroquinesusceptible clones resistant to chloroquine, demonstrating that mutation at PfCRT codon 76 is the essential determinant of chloroquine resistance (Sidhu et al., 2002). Polymorphisms in pfmdr1 encoding the P. falciparum Pglycoprotein homologue 1 (Pgh1), modulate the level of in vitro chloroquine resistance in parasites that already harbor PfCRT 76T (Reed et al., 2000). The relevance of the variations caused by pfmdr1 mutations in IC50 values within the chloroquine-resistant range cannot necessarily be extrapolated to parasite responses to treatment in vivo, however, and role of pfmdr1 in chloroquine treatment failure, if any, remains unclear. The mutations most often cited as potential contributors to chloroquine resistance are pfmdr1 N86Y and D1246Y. At the same time that its key role in conferring chloroquine resistance in vitro was being confirmed, the utility of the pfcrt 76T mutation as a molecular marker for clinical outcomes of chloroquine treatment was assessed. In vivo drug resistance is defined and measured differently from in vitro resistance. In the laboratory, in vitro resistance is defined according to the concentration of the drug required to inhibit asexual parasite replication, or schizont formation. In the case of chloroquine, in vitro resistance is partially reversible by the addition of verapamil to the assay (Martin et al., 1987). Classically, in vivo resistance is based on the presence or absence at specified time points of asexual parasites in the peripheral blood of symptomatic individuals treated with the drug. Parasites are resistant at the RIII level if asexual parasitemia continues to rise or is reduced by less than 75% during the first 48–72 h
after treatment; at the RII level if asexual parasitemia does not clear but is reduced to 25% or less of the pre-treatment parasitemia by 48–72 h after treatment; and at the RI level if asexual parasites disappear but return within the specified follow-up period, usually 14 or 28 days. Infections that clear by day 7 after treatment and do not recur within the follow-up period are considered sensitive (Rieckmann, 1990). In the mid-1990s, the World Health Organization released a new protocol for monitoring antimalarial drug efficacy that did not consider recurrent or persistent asexual parasitemia occurring 4 or more days after treatment to constitute treatment failure unless fever was present (WHO, 1996). This protocol is now being replaced with one that is more similar to the classic definitions of clinical resistance, with outcome categories including early treatment failure (corresponding to RIII and some cases of RII resistance); late treatment failure (corresponding to RII or RI resistance occurring 4 or more days after treatment and accompanied by fever); late parasitological failure (corresponding to RI resistance in the absence of fever); and adequate clinical and parasitological response (corresponding to sensitive outcomes) (WHO, 2002). In a human infections, the host immune response plays an important role in clearing malaria parasites, and persons who have acquired partial immunity to malaria through prolonged, intense exposure to infection are often able to clear infections after treatment with a drug to which the parasites are resistant (Hoffman et al., 1984). Thus, field studies assessing the role of pfcrt mutations in clinical outcomes to chloroquine treatment were predictably less straightforward than the absolute correlations found between pfcrt genotypes and in vitro phenotypes. Djimde and colleagues in Mali conducted a clinical efficacy study of chloroquine, assessing pfcrt genotypes of both pre-treatment and post-treatment infections. Eighty-six percent of participants had sensitive clinical outcomes, and as expected, the molecular marker for chloroquine resistance was more prevalent than clinical resistance. Nevertheless, 92% of infections carrying pfcrt 76T had resistant in vivo outcomes as compared with 37% of parasites carrying the wild-type pfcrt. The odds ratio of resistance to chloroquine treatment for parasites carrying the resistant genotype versus the wild-type pfcrt was 18.8 (P = 0.001). Among 60 samples from post-treatment infections, 100% carried the 76T mutation (Djimde et al., 2001). The investigators concluded that there was a strong association between the presence of the pfcrt 76T mutation at the time of chloroquine treatment and a resistant in vivo outcome. Subsequently, studies in other countries in Africa and Asia have confirmed the primary role of pfcrt 76T in conferring chloroquine resistance in vivo, as summarized in a recent review (Wellems and Plowe, 2001). Pfmdr1 mutations and other parasite genetic factors may play a secondary role in chloroquine treatment outcomes, but these appear to be neither necessary nor sufficient to cause chloroquine treatment failure, and multivariate analyses have failed to detect statistical interactions between mutations in pfcrt and pfmdr1 (Djimde et al., 2001; Jelinek et al., 2002), i.e.
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the presence of mutations in both genes is not more strongly associated with treatment failure than the presence of the pfcrt T76 mutation alone.
3. Antibiotic pressure and fitness Drug resistance has developed in response to the use of most antimicrobials. Drug exposure is thought to select genetic mutations that allow the organism to survive and continue to replicate under drug pressure. Observational cohort studies in Australia and Iceland have shown that children who had recently received antibiotics were at higher risk of colonization with penicillin-resistant pneumococci (Arason et al., 1996; Nasrin et al., 2002). Quantity of antibiotic use within a community (as measured by doses of antibiotics per 1000 children per day) has also been also highly correlated with colonization with resistant organisms (Arason et al., 1996; Melander et al., 2000; Seppala et al., 1995). Mutations that confer drug resistance are commonly assumed to impose a cost to bacterial fitness. It would seem to follow that with the removal of drug pressure, the organism should revert back to the more fit, wild-type genotype, either through back-mutation or selection of residual wildtype organisms that have a survival advantage in the absence of drug pressure. Seppala et al. reported a dramatic increase in erythromycin resistance among group A streptococci in Finland and found a strong positive association between local erythromycin use and local erythromycin resistance rates (Seppala et al., 1995). In 1992, a nationwide effort was initiated to decrease erythromycin use in outpatients. Macrolide use decreased from 2.4 to 1.4 daily doses per 1000 inhabitants per day and erythromycin resistance fell from 16.5 to 8.6% among group A streptococci clinical isolates (Seppala et al., 1997). This observation supported the hypothesis that the removal of drug pressure permits the reemergence of susceptible organisms. Other clinical studies, however, have failed to find similar decreases in resistance following curtailment of drug use. Despite a 50-fold decrease in sulfonamide use in the United Kingdom, there was no decline in the prevalence of sulfonamide-resistant Escherichia coli (Enne et al., 2001). On an individual level, infection with HIV that develops protease inhibitor resistance has been shown to maintain that resistance after changing to non-protease inhibitor based treatment regimens (Boeri et al., 2003). The phenomenon of persistence of genetically mediated drug resistance despite removal of drug pressure has been investigated in the laboratory and through computer modeling. Among a wide variety of microorganisms that have been studied in experiments that apply and then remove drug pressure, many organisms develop additional, compensatory mutations that improve their ability to survive in the absence of drug pressure rather than reverting back to the wild-type drug sensitive form (Schrag et al., 1997; Levin et al., 2000; Sander et al., 2002; Menzo et al., 2003). Although the initial
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mutation leading to drug resistance may incur some cost to organism survival, through a process referred to as compensatory evolution, secondary mutations may restore the fitness of the drug-resistant organism and allow it to remain fixed in a population despite removal of drug pressure. Based on these studies, in areas where chloroquine use has been significantly decreased, the following possible outcomes can be entertained: (1) chloroquine resistance comes at a cost to parasite fitness that is overcome by compensatory evolution, allowing chloroquine-resistant parasites to remain fixed in the population; (2) chloroquine resistance comes at a cost to parasite fitness that is not overcome by compensatory evolution, and chloroquine sensitive parasites will reemerge in the absence of chloroquine drug pressure; or, (3) chloroquine resistance does not come at a cost to parasite fitness, and chloroquine-resistant parasites will remain prevalent after withdrawal of chloroquine from use.
4. Early studies The first suggestion that chloroquine susceptibile parasites might reemerge under reduced drug pressure came from French researchers who conducted a study in Vietnam. Over 5 years after a switch from chloroquine to sulfadoxine–pyrimethamine for treatment of P. falciparum in Vietnam, they enrolled 68 children with uncomplicated falciparum malaria in a chloroquine efficacy study in Ho Chi Minh City, and found that the rate of cure was 90%, with follow up from 1 to 12 weeks. The good clinical response to chloroquine was attributed to the decreased use of chloroquine since 1975 (Jacquier et al., 1985). Subsequently, however, representatives of the World Health Organization and the Institute of Malariology, Parasitology and Entomology in Vietnam presented conflicting data. They found no decrease in clinical resistance to chloroquine and documented a decrease, but not elimination of the use of chloroquine in the area. In 1985, there were still between 500,000 and 1 million chloroquine tablets dispensed per year in Ho Chi Minh City alone (Onori and Vu, 1986). The reemergence of chloroquine resistance was not addressed in the literature again until the early 1990s. On the island of Hainan, China, antimalarial treatment was changed from chloroquine to piperaquine in 1979, and community surveys documented that chloroquine use had largely ceased. Over a 5–8-year period, in vivo resistance reportedly decreased from 84 to 40%, and from 1981 to 1991 in vitro resistance decreased from 98 to 61% (Liu et al., 1995). The clinical studies were limited by small sample sizes (only 20 cases were assessed from 1986 to 1989) and details about the participants’ age and history of malaria exposure were not provided. The investigators used locally developed microtitration plates and followed a slightly altered protocol from the standard WHO recommendation. Nevertheless, the possibility of the return of chloroquine sensitivity was raised.
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Several studies of in vitro chloroquine susceptibility testing were subsequently published, all reporting trends of decreasing chloroquine resistance after a change to an alternative antimalarial drug. In one province in Gabon, in vitro chloroquine resistance reportedly decreased from 100 to 45% between 1992 and 1996, in a study of 30–40 clinical isolates on a biannual basis. Concomitantly, no changes in mefloquine or quinine resistance were detected (Schwenke et al., 2001). As a result of this dramatic finding, a small in vivo chloroquine efficacy trial was conducted in 2000 among children with uncomplicated malaria in this region. One hundred percent of the infections were found to be clinically resistant and all parasites carried the molecular marker for chloroquine resistance, pfcrt 76T (Borrmann et al., 2002). The striking inconsistency between the in vitro results and the in vivo outcomes and molecular results highlights the potential inaccuracies of in vitro drug susceptibility tests and emphasizes the need for well designed clinical and molecular studies to confirm new findings. By the late 1990s, two groups of investigators had found similar increases in in vitro chloroquine susceptibility in Vietnam. Huong et al. cited unpublished data that in vitro chloroquine resistance was found in 83% of isolate in 1988 in contrast to their 1999 findings of 16% resistance among clinical isolates (Huong et al., 2001). Thanh et al. reported results from 1995 and 1998 that show the prevalence of in vitro resistance falling from 92 to 34% (Thanh et al., 2001). In response to these findings, a clinical study was conducted to test the hypothesis that malaria susceptibility to chloroquine and sulfadoxine–pyrimethamine had returned, since these drugs had reportedly not been used in Vietnam for several years. The study took place in two locations with two distinct populations: semi-immune children in Dac Lac province and malaria naive adults in Binh Phuoc province. Both drugs were administered in combination with artesunate, so the efficacy rates of sulfadoxine–pyrimethamine and chloroquine were masked by the efficacy of the partner drug. Among semi-immune children, both combinations were highly efficacious. Among the non-immune adults, 49% of the participants who were treated with chloroquine plus artesunate had resistant outcomes. The chloroquine resistanceconferring mutation at pfcrt 76T was carried by 96 and 61% of infected patients in the semi-immune pediatric and malaria-naive adult population, respectively (Nguyen et al., 2003). Due to combination with artesunate, this study was unable to measure directly the in vivo efficacy of sulfadoxine–pyrimethamine and chloroquine. However the high prevalence of the chloroquine-resistant form of pfcrt and the poor outcomes among non-immune patients call into question the earlier in vitro results. As described above, parasite resistance to chloroquine is reliably associated with pfcrt 76T, while in vitro studies conducted under field conditions have not infrequently proven to be unreliable. The high rates of recrudescence among non-immune adults treated with chloroquine–artesunate and the high prevalence of pfcrt 76T
provide strong evidence that chloroquine sensitive P. falciparum malaria has not reemerged in Vietnam. Several factors distinguish malaria in Vietnam from malaria in Africa. The removal of chloroquine in Vietnam was incomplete due to its continued use for treatment of Plasmodium vivax, which is absent in nearly all of sub-Saharan Africa. Although P. vivax is much less prevalent that P. falciparum in Vietnam (Hung et al., 2002), patients who are suspected or documented to have P. vivax infection are still treated with chloroquine, and chloroquine may be prescribed for patients who are infected with P. falciparum, either through incorrect diagnosis or reverting to older treatment practices. Malaria transmission is generally much lower in Southeast Asia than in most sub-Saharan African sites, and populations in low transmission areas acquire less immune protection from malaria disease (Luxemburger et al., 1996). Thus, in low transmission areas the vast majority of the overall malaria parasite population is likely to be under drug pressure at any given time, since most persons who are infected become ill and are treated. Moreover, in low transmission areas polyclonal infections are less common than in Africa, where multiclonal infections are the rule (Babiker and Walliker, 1997), so there is less opportunity both for competition among drug sensitive and resistant clones within the host leading to predominance of sensitive clones, and for sexual recombination leading to disassociation of resistance-conferring polymorphisms and/or the compensatory mutations that help to maintain them (Conway et al., 1999). Both of these factors could contribute to persistence of resistant parasites after reduction of drug pressure.
5. Removal of drug pressure: Malawi Malawi offers a more clear-cut opportunity to observe the natural history of chloroquine resistance after the removal of drug pressure. In the face of unacceptably high chloroquine failure rates, Malawi was the first country in Africa to switch from chloroquine to sulfadoxine–pyrimethamine for first line treatment of malaria. Since 1993, sulfadoxine–pyrimethamine has been the treatment provided by all government health facilities and is dispensed without prescriptions. Due to successful national information campaigns and tight control of its distribution, chloroquine use has largely been eliminated. Unlike Southeast Asia and South America, non-falciparum Plasmodial species are rare. Five years after the initiation of the change from chloroquine to sulfadoxine–pyrimethamine, clinical isolates were tested in vitro for chloroquine susceptibility. Sixty-five percent were found to be sensitive to chloroquine, 31% were borderline resistant and only 3% resistant (Takechi et al., 2001). In the same region along the shore of Lake Malawi, children with asymptomatic malaria infection were recruited into a study of chloroquine efficacy in 2000. Fifty-four children were enrolled and only one child developed recurrent
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Fig. 1. Declining prevalence of chloroquine resistance conferring mutation PfCRT T76 in Blantyre, Malawi from 1992 to 2004. Arrow denotes change from chloroquine to sulfadoxine–pyrimethamine as first line antimalarial drug in Malawi in 1993. Reproduced with permission from Kublin et al. (2003) and S. Nkoma and D. Bell, personal communications.
parasitemia by day 7 and four more by day 14. Ninety-one percent of the infections were sensitive in vivo. Among the parasites that were detected on enrollment, none carried the pure pfcrt 76T mutation. Only 1/53 of the infections was found to contain parasites of mixed pfcrt K76 and 76T genotypes. The participant with the mixed infection had a sensitive in vivo response (Mita et al., 2003). In Blantyre, the largest city in Malawi, archived thick blood smears and blood samples collected on filter paper from children with symptomatic malaria were analyzed for the polymorphic codon at pfct 76. The earliest samples were from 1992, prior to the change from chloroquine to SP, and the sample set extended through the year 2000. Through nested PCR and allele-specific restriction digest assays, the investigators demonstrated a steady decrease in the prevalence of pfcrt 76T from 85% in 1992 to 13% in 2000 (Fig. 1). In 2001, no parasites with the chloroquine resistance conferring mutation were found (Kublin et al., 2003). In 2002–2004, other investigators in Malawi have continued to find only wild-type parasites with the genotype pfcrt K76 (S. Nkoma and D. Bell, personal communications). Mutations in pfmdr1, which play no role in antifolate resistance, exhibited a modest decrease in frequency over the decade. The prevalence of one of the implicated mutations, D1246Y, declined from one-half of the
infections in 1993 to one-fourth in 2000 (P = 0.023). Genetic analyses of parasites from the neighboring countries where chloroquine continued to be used have shown a very different pattern. Over 90% of parasites in the late 1990s from Zambia and Mozambique carried the mutation pfcrt 76T (Kublin et al., 2003; Mayor et al., 2001). The Malawi investigators also conducted an in vivo study of 72 adults with asymptomatic parasitemia. All infections demonstrated sensitive parasitologic outcomes. This study, along with the previous study of asymptomatic children, did not include control groups. This raises the possibility that semi-immune persons with asymptomatic parasitemia might have cleared their infections without any treatment or after administration of drugs with impaired efficacy. However, the uniformity of the clearance of parasites among both adults and children treated with chloroquine, along with the molecular finding of the return of wild-type pfcrt K76, provides strong evidence that chloroquine sensitive P. falciparum has reemerged and predominates in Malawi, at least in the regions studied. During the 10-year period of respite from chloroquine use, sulfadoxine–pyrimethamine has been used as first line therapy to treat uncomplicated malaria. During this time, there was a steady increase in the prevalence of mutations
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in parasite dihydrofolate reductase (DHFR) associated with sulfadoxine–pyrimethamine resistance. This is reflected in increasing parasitologic resistance and late treatment failures in clinical studies (Kublin et al., 2003; Plowe et al., 2004). Malawi, the African country that pioneered antimalarial policy change, now stands in a unique position to serve as a case study of whether chloroquine could be reintroduced to an area from which it has been withdrawn, through clinical trials of chloroquine alone and in combination with other drugs intended to prevent reemergence of chloroquine resistance.
6. Other antimalarial drugs Recent experience in Northwest Thailand with the combination of mefloquine and artesunate indicates that removal of drug pressure might not even be necessary for restoration of susceptibility among malaria parasites. In 1985, mefloquine was introduced in combination with sulfadoxine–pyrimethamine, and then in 1990 mefloquine was used at a higher dose as monotherapy. Within 4 years, clinical cure rates had fallen from 90 to 60%, leading to the deployment of an artesunate–mefloquine combination. With this combination, cure rates remained nearly 100% until 1998, when the camp for displaced persons that was under surveillance closed. Of particular interest in this discussion of the reemergence of drug susceptibility, is the pattern of in vitro mefloquine resistance. At the time when clinical resistance to mefloquine reached its peak, the geometric mean concentration of mefloquine required to inhibit 50% of schizont formation (IC50 ) was 47–56 ng/ml. By 1999, 5 years after the switch from mefloquine monotherapy to mefloquine–artesunate combination therapy, the IC50 decreased to 25 ng/ml (Nosten et al., 2000). These data from Thailand suggest that if parasites develop resistance to a drug, combining the drug with an appropriate partner drug may in some cases effectively reduce drug pressure and allow for the reemergence of the susceptible forms of the parasite. While these in vitro results should be interpreted with caution, given the limitations of in vitro testing described above, the assays were performed using standard radio-labelled hypoxanthine uptake-inhibition rather than field microtest methods and were performed at a highly experienced laboratory (Brockman et al., 2000). Sulfadoxine–pyrimethamine, an antifolate combination, has been used throughout the world in areas where chloroquine has failed. Sulfadoxine–pyrimethamine inhibits folate synthesis, a function that is essential to parasite survival. Pyrimethamine inhibits DHFR and sulfadoxine inhibits dihydropteroate synthase (DHPS). Mutations in DHFR (Cowman et al., 1988; Peterson et al., 1988, 1990) and DHPS (Brooks et al., 1994; Triglia et al., 1997; Wang et al., 1997a,b) have been associated with sulfadoxine–pyrimethamine resistance in vitro. Based on both laboratory assays and epidemiolog-
ical studies, mutations occur in a stepwise fashion, with increasing numbers of mutations conferring higher level resistance to Sulfadoxine–pyrimethamine (Plowe et al., 1997; Sirawaraporn et al., 1997; Wang et al., 1997a,b). In clinical studies in Africa, five mutations have been identified that are most closely associated with clinical sulfadoxine–pyrimethamine failure: DHFR S108N, C59R and N51I and DHPS A437G and K540E (Nzila et al., 2000; Kublin et al., 2002). Sulfadoxine–pyrimethamine resistance may not have the same natural history as chloroquine resistance when the drug is effectively withdrawn. Sulfadoxine–pyrimethamine has similar mechanisms of action to trimethoprim– sulfamethoxazole, an antibiotic that is widely used in the developing world both for treatment and increasingly for prophylaxis against opportunistic infections in persons living with HIV. Laboratory experiments with naturally occurring P. falciparum isolates as well as genetically engineered DHFR in a yeast expression system demonstrated cross resistance between pyrimethamine and trimethoprim (Iyer et al., 2001), and complete cross resistance has been demonstrated among the sulfas and sulfones in isolates with a variety of DHPS genotypes (Triglia et al., 1997). In regions of high transmission in Africa, individuals who receive trimethoprim–sulfamethoxazole either for treatment of bacterial infections or prophylaxis for opportunistic infections are also frequently infected with malaria parasites. Thus, antifolate drug pressure will not be eliminated even if sulfadoxine–pyrimethamine is not administered for malaria treatment. Frequent observations of rapid development of resistance to the antifolate drugs in direct response to drug pressure have led many to posit that DHFR and DHPS mutations arise spontaneously in response to drug pressure (Doumbo et al., 2000; Clyde and Shute, 1957). Biochemical analyses have shown that the DHFR mutations come at a cost to enzyme function and therefore presumably at a cost to parasite fitness (Sirawaraporn et al., 1997). If the mutations conferring resistance to antifolates arise under drug pressure and are harmful to the parasite, it would seem likely that withdrawal of antifolate drug pressure (if this could be accomplished) would lead to reemergence of wild-type sensitive parasites. However, recent genetic analyses of sulfadoxine– pyrimethamine-resistant parasites has called into question the notion that resistance-conferring DHFR and DHPS mutations arising spontaneously in direct response to drug pressure are primarily responsible for antifolate resistance at the regional level. Studies of microsatellite markers and other genetic sequences flanking the dhfr and dhps genes among drug-resistant parasites in South America and Africa have demonstrated that allelic haplotypes of the same ancestral origin were driven through large regions in genetic sweeps. Cortese et al. analyzed parasites from five regions of the South American Amazon. The haplotypes with mid to high-level mutations (DHFR 50R, DHFR 164L, DHPS
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540E and DHPS 581G) appeared to have a common origin. From the lower Amazon, these resistance markers spread in a North–Northwestward direction, perhaps in response to the permissive environment of drug pressure (Cortese et al., 2002). In Africa, microsatellite markers were used to determine the evolutionary origins of sulfadoxine–pyrimethamineresistant mutations in malaria. One mutant dhps allele and three mutant dhfr alleles, each of independent origin, have been driven through the parasite population in KwaZuluNatal, a region of low malaria transmission in South Africa. This same allelic haplotype was found to be the basis for sulfadoxine–pyrimethamine resistance in Tanzania, 4000 km away (Roper et al., 2003). These data suggest that gene flow following the pattern of sulfadoxine–pyrimethamine drug pressure, rather than spontaneous mutation, may be the driving force of sulfadoxine–pyrimethamine resistance. This is not to say that spontaneous mutation or that rapid, focal selection of rare background mutations does not occur, but that specific, limited haplotypes appear to account for the predominant forms of antifolate-resistant parasites found at these sites. The most likely explanation for this phenomenon is that the successful resistant haplotpyes include compensatory mutations, not in dhfr or dhps genes themselves, but elsewhere in the genome, providing these haplotypes with an advantage over spontaneously arising or pre-existing mutant genotypes when drug pressure is applied. In other words, even if the local parasite population contains some antifolateresistant parasites, when substantial drug pressure is applied and new arrivals appear on the scene that are equally resistant but more fit, the new haplotypes take over. If this is the case, reemergence of antifolate sensitive P. falciparum might be less likely in areas of lower transmission for one of the reasons considered above in the discussion of the situation in Vietnam, i.e. fewer opportunities for disassociation between resistance-conferring and compensatory mutations through sexual recombination. However, in areas of higher transmission, competition among sensitive and resistant clones as well as recombinatory disassociation might still lead to reduced prevalence of DHFR and DHPS mutations in response to reduction or elimination of antifolate drug pressure. In African countries where sulfadoxine–pyrimethamine is withdrawn from use, molecular epidemiological studies such as those done after chloroquine was withdrawn from Malawi (Kublin et al., 2003) will be of great interest. If removing sulfadoxine–pyrimethamine from use in high transmission areas does not result in reemergence of susceptible parasites as was observed with chloroquine, what could account for the different results of withdrawing the two drugs? Two possible explanations suggest themselves: (1) continued drug pressure from other antifolate drugs such as trimethoprim–sulfamethoxazole may apply sufficient pressure to maintain antifolate resistance; and/or (2) the survival disadvantage of DHFR and DHPS mutations is either less than that of PfCRT mutations, or overcome by compensatory mutations.
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7. Future directions It has been speculated that years of reliance on antimalarials other than chloroquine might lead to the reemergence of chloroquine-sensitive P. falciparum and permit the reintroduction of this safe and affordable drug. If this is confirmed in controlled clinical trials, several new opportunities for managing malaria drug resistance become possible. Countries that continue to use chloroquine despite high levels of resistance will be encouraged to switch now to more effective drugs, or drug combinations, with the knowledge that they maybe able to return to chloroquine use in the future. Countries that have already replaced chloroquine with other antimalarial drugs could attempt to eliminate chloroquine use in their population as completely as possible, as Malawi has, with the expectation that if the current drug regimen fails, chloroquine could be reintroduced. It is important to emphasize that reintroduction of chloroquine as monotherapy is highly inadvisable and would likely lead to a rapid reemergence of resistance chloroquineresistant parasites that remain lurking in the neighborhood, as they do in the countries surrounding Malawi. However, where chloroquine is highly efficacious, it could be an ideal component of combination drug therapies designed to deter the emergence of resistance to the component drugs. And, if withdrawal of both chloroquine and sulfadoxine–pyrimethamine from Africa were to result in a reemergence of falciparum malaria sensitive to these drugs, a combination therapy regimen including an artemisinin derivative combined with chloroquine and sulfadoxine–pyrimethamine would be very attractive based on cost and safety. In addition, this combination would offer the advantage of including of one component that rapidly reduces parasite biomass, reducing the probability of resistant parasites surviving, with two longacting drugs to protect against emergence of resistant parasites during the post-treatment prophylaxis phase. Given the limited number of safe and affordable antimalarial drugs and the compliance and prophylactic benefits of longeracting drugs, chloroquine and sulfadoxine–pyrimethamine may continue to be important tools in the antimalarial armamentarium.
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