Journal of Antimicrobial Chemotherapy (1992) 30, 571-585

Review Antimalarial drug resistance: the pace quickens N. J. White

Introduction

For an infection affecting approximately 5% of the World's population at any time, and killing between one and two million children each year, there are remarkably few drugs available for the treatment of faknparum malaria. Traditionally, antimalarial drug research has been stimulated by wars in tropical malarious areas involving economically-powerful temperate-climate powers. Armies fighting in the tropics lose more soldiers to malaria than bullets (Melville, 1911). The Second World War and the conflict in Vietnam brought us most of the drugs available today. The list is small, and the parasite has not been idle: Plasmodum falciparum has now developed resistance to all of our available drugs. The situation is particularly bad in South-East Asia. History Antimalarial drugs have never been effective in every patient (Bruce-Chwatt, 1981). In the last three centuries, quinine was the only specific remedy available, but a con, tinuous debate ranged as to the efficacy of cinchona bark or its extracts in the treatment of agues (febrile illnesses). Much of the controversy resulted from an inability to distinguish malaria from other causes of fever (Dawson, 1930). Although Binz had shown that quinine could kill Paramecium spp. in 1868, it was not until Laveran's discovery of the malaria parasite in 1880, and his subsequent demonstration that quinine killed these intraerythrocytic organisms, that the specific action of the alkoloid was characterized. Guttman and Ehrlich reported in 1891 that malaria could be treated with the dye methylene blue, but subsequent attempts to modify the molecule proved fruitless. During the First World War, stocks of quinine in Germany ran low, and considerable efforts were directed at developing synthetic antimalarials there in the 1920s. Through work on the dyestuffs, attention focused on the quinoline compounds and, in 1925, this led to an effective 8-aminoquinoline: plasmoquine (pamaquine), a predecessor of primaquine. Thefirstquinolines to be evaluated were fairly toxic, and development work switched to the acridines. In 1930, Mauss and Mietzch synthesized mepacrine (atebrin, quinacrine), and this was marketed in 1932. In 1934, Andersag discovered Resochin, later known as SN-7618 or chloroquine, while working in the 571 0305-7453/92/110571 + 15 $08.00/0

© 1992 The British Society for Antimicrobial Chemotherapy

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Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand, and Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Headington, Oxford 0X3 9DU, UK

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N. J. White

Mechanisms of antimalarial drug resistance

The mode of action of the quinoline and acridine antimalarials remains incompletely understood. These drugs are weak bases and are, therefore, concentrated in the acid food vacuole of the parasite (Krogstad & Schlesinger, 1987). Plasmodicidal action thereafter has not been explained fully. Chloroquine intercalates DNA, but at concentrations (1-2 mM) that are considerably higher than those required to kill susceptible parasites (10-20 nM). Chloroquine binds also to ferriprotoporphyrin-(FP)-IX, a product of haemoglobin degradation. It has been suggested that the FP-IX-chloroquine complex could be toxic to the parasite (Chou, Chevli & Fitch,

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Elberfield laboratories of Bayer IG Farbenindustrie AG. This was considered initially to be too toxic for human use on the basis of preliminary studies in bird malarias. Some clinical studies were, however, conducted in 1935 and 1936, but these again concluded that chloroquine was 'too toxic for practical use in humans'. Bayer abandoned further work on Resochin (chloroquine), and asked Andersag to synthesize a less toxic compound. His team then produced methylated chloroquine resorcinate, later known as Sontochin or Sontoquine. By the beginning of the Second World War over a thousand cases of malaria had been treated with Sontochin. The extraordinary wartime history of these compounds has been chronicled by Coatney (1963). Suffice to say that although the Allied Powers had detailed information on the 4-aminoquinolines through reciprocal arrangements with pre-war Germany, their potential value was not realized until the end of the war. However, by 1946 chloroquine had become the drug of choice for all malaria the world over. Thus research stimulated by two World Wars and, in particular, the threat and subsequent reality of Japanese occupation of the majority of the world's cinchona plantations (Taylor, 1945), ted to the introduction of mepacrine (quinacrine) and chloroquine, and the discovery of the antimalarial biguanides proguanil (chloroguanide), chlorproguanil and, somewhat later, amodiaquinc and pyrimethamine (Covell et al., 1955; Coatney, 1963). These drugs comprise the vast majority of antimalarials used in the world today. In the 1950s, interest in malaria and antimalarial drug research waned with the prospect of imminent global eradication. However, the achievements of the eradication programme in Europe, North America and northern Asia could not be reproduced in the tropics, and with the growing conflict in South-East Asia, interest in antimalarial drugs revived in the following decade. The US Army screened over a quarter of a million potential antimalarial compounds between 1963 and 1976. This led to the discovery of mefloquine and halofantrine. The reports in 1961 of chloroquine resistance in P. falciparum, both in South America and South-East Asia, were major stimuli to this research effort. Chloroquine had become the standard antimalarial for treatment and prophylaxis of all the human malarias and, as a consequence, one of the most widely-used drugs in the world. At first, resistance was low-grade and focal, but during the ensuing years, treatment failures increased in number and degree. By the beginning of the 1980s, chloroquine was no longer useful in many parts of South-East Asia and South America, and the ominous first reports of resistance to chloroquine were emerging from the east coast of Africa. Over the past ten years, resistance has marched steadily from the east to the western coasts of Africa (Bjorkman & Phillips-Howard, 1990). Few countries in the tropics are now unaffected (World Health Organization, 1990a).

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1980), but there seems to be no differences between the FP-DC produced by resistant and sensitive parasites. Recently, it has been shown that the quinoline antimalarials selectively inhibit the plasmodial enzyme haem polymerase which polymerizes, and thus detoxifies FP-EX. This provides a plausible explanation for their antimalarial activity (Slater & Cerami, 1992). The mechanism of action of the sulphonamides, proguanil (or more correctly, its active metabolite cycloguanil), and pyrimethamine are known precisely. These drugs interfere with folic acid metabolism in the parasite. The sulphonamides inhibit dihydropteroate synthetase, while pyrimethamine and cycloguanil inhibit the bifunctional enzyme dihydrofolate reductase (DHFR)/thymidylate synthase. Resistance to quinine in P.falciparum was first reported in 1910 (Werner, 1910), but did not become a significant problem until the last IS years. However, within a few years of their introduction, the first reports of treatment failures with the DHFR inhibitors appeared (Gyde & Shute, 1954). Resistance in both P. faldparum and P. vivax soon began to limit the usefulness of pyrimethamine—a safe single-dose treatment and well-tolerated prophylactic drug (World Health Organization, 1975). Pyrimethamine resistance is associated with a point mutation in the DHFR gene (vide infra). Initially, it was assumed that resistance to pyrimethamine conferred resistance to cycloguanil automatically, but recent studies have shown that the two are not necessarily linked (Foote, Galatis & Cowman, 1990). Some mutations in the DHFR gene may result in reduced affinity of DHFR for either pyrimethamine or the antimalarial biguanides alone, while others result in reduced affinity for both drugs. Thus proguanil and chloroproguanil may be useful in some areas where pyrimethamine alone is not, and vice versa. P. vivax appears primarily to be relatively insensitive to pyrimethamine. As P. vivax cannot be grown in culture, the mechanism of resistance has not been characterized. Resistance to the DHFR inhibitors pyrimethamine and proguanil in P.falciparum results from reduced affinity of the DHFR-thymidylate synthase enzyme complex for the drug. Several distinct single base-pair mutations have been identified which result in affinities 100-1000 times less than that of the drug-sensitive DHFR complex. Analysis of a genetic cross between pyrimethamine-resistant and pyrimethamine-sensitive parasites showed that a single point mutation (Ser/108 -+ Asn/108) in the active site of the enzyme complex conferred pyrimethamine resistance (Peterson, WaUiker & Wellems, 1988). Other mutations were discovered subsequently. Interestingly, parasites with a paired mutation at positions 108 (Ser-»Thr) and 16 (Ala-»Val) were cycloguanilresistant, but showed only a small reduction in pyrimethamine sensitivity. None of these mutations appears to affect the function of the enzyme adversely. As only a single or double step mutation is required to move from 'sensitivity' to 'resistance', the DHFR type of resistance occurs readily. The mechanisms underlying resistance to the other antimalarials are less well-characterized. Chloroquine resistance has been studied extensively, but progress has been slow because the mode of action of quinoline antimalarials remains incompletely understood. Chloroquine resistance is associated with reduced concentrations of the drug in the acid food vacuole of the parasite. This is not associated with reduced uptake, but with increased efflux from the cell. Resistant parasites pump chloroquine out of the cell more rapidly (40-50-fold) than sensitive parasites. This process can be inhibited by a number of diverse drugs (calcium channel blockers, tricyclic antidepressants, phencthiazines, cyproheptadine etc.) and drug resistance reversed. Thus chloroquine-resistant malaria parasites are very similar to multi-drug-resistant (MDR) mammalian cancer

57*

N. J. White

Resistance to mefloquine and quinine is less well-characterized. In-vitro studies suggest that resistance to the two drugs is linked, as is chloroquine and quinine resistance, but interestingly, not chloroquine and mefloquine resistance (Brasseur et al., 1988; Warsame et al., 1991). In rodent models, mefloquine resistance can be induced relatively easily, whereas quinine resistance is difficult to induce. P.falciparum may primarily be mefloquine-resistant (i.e. before exposure to the drug), as in some isolates from West Africa, whereas primary quinine resistance is most unusual. However, the linkage of mefloquine and quinine resistance raises the possibility that resistance to one might drive the other. In Thailand, where mefloquine has been available widely for seven years, in-vitro quinine sensitivity has declined at a faster rate in recent years, coincident with a major reduction in mefloquine sensitivity. In-vitro sensitivity to halofantrine is also linked to mefloquine sensitivity, although the former is intrinsically more active. Whether resistance to these two new drugs is linked closely in clinical practice remains to be determined.

The development of anttmtlarial drug resistance How do parasites develop resistance to the antimalarial drugs? It should be noted that selection by drugs takes place during asexual multiplication in the human host, that the sexual forms (gametocytes) of P. falciparum are relatively resistant to the antimalarial

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cells, which also pump out drugs to which they are resistant (Newbold, 1990). In such neoplastic cell lines, the efflux of structurally-diverse drugs is mediated by P-glycoprotein, a transmembrane pump which hydrolyses ATP as an energy source. Genes encoding these proteins have been sequenced, and found to be over-expressed in drug-resistant cancer cell lines. P-glycoprotein is homologous to a family of ATP-driven transport system proteins which were identified originally in bacteria. Genes encoding similar proteins have been identified in P.falciparum (designated PFnutrl and PFntdrl), and have been found to be amplified in some chloroquineresistant lines (Foote et al., 1989; Wilson et al., 1989). However, the association of chloroquine resistance with amplification and over-expression of the MDR genes has been challenged by recent studies involving careful cloning and genetic cross-overs (during meiosis in the anopheline mosquito). Chloroquine resistance did not cosegregate with MDR genes in these experiments (Wellems et al., 1990) which localized the gene(s) associated with resistance to a 400 kb fragment on chromosome 7 of the parasites studied (Wellems, 1991, Wellems, Walker-Jonah & Panton, 1991). Furthermore, kinetic modelling of observed chloroquine uptake and efflux rates in sensitive and resistant isolates do not support the concept of an MDR pump in drugresistant parasites (Krugliak & Ginsburg, 1991; Ginsburg & Stein, 1991). From a genetic standpoint, genes in addition to, or other than, MDR must be involved in chloroquine resistance. From a therapeutic standpoint, there is no evidence yet that observations of the reversal of chloroquine resistance in vitro by an assortment of currently-available drugs can be translated into practically useful drug regimens. Although the dose of calcium channel antagonists required (Martin, Oduola & Milhous, 1987) would undoubtedly have proved toxic, the concentrations of desiprimine, cyproheptadine and the phenothiazines predicted to be effective from the in-vitro studies are within the therapeutic range (Basco & Le Bras, 1990). Nevertheless, there have been no convincing clinical studies demonstrating the efficacy of a chloroquinepump inhibitor combination in chloroquine-resistant falciparum malaria.

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drugs (with the exception of primaquine), and that random mating between sexual forms of the parasite takes place in the gut of the female anopheline mosquito. After sporogony in the mosquito, the genetically-mixed variants are then redistributed to humans. Naturally-occurring populations of P. falciparum are genetically diverse, with heterogenous sensitivity to the antimalarial drugs (Thaithong, 1983). Human infections are usually polyclonal. Resistance is considered to result from spontaneous chromosomal point mutations (a process thought to be independent of drug pressure), followed by selection of the more resistant mutants under drug pressure. Although resistance to the DHFR inhibitors can arise from single point mutations, epidemiclogical and laboratory observations suggest that resistance to the quinoline compounds probably requires a series of unlinked additive mutations. Models of the development of drug resistance (Curtis & Otoo, 1986; Cross & Singer, 1991) have interesting and important practical implications. Such models predict that if maximally effective treatment is given to more than 25% of the population in areas of intense transmission, then resistance will develop rapidly. In theory, use of drug combinations delays the onset of resistance, but only if the resistance genes are rare and free recombination can occur between them, and that less than 20-25% of the population is treated (Curtis & Otoo, 1986). Resistant parasites are most likely to be selected if the heterogenous population is exposed to a sub-therapeutic level of drug. In order for resistance to spread, the resistant parasites must then survive to produce gametocytes, and these parasites must be transmitted. Anopheline mosquitoes differ in their receptivity to different parasite strains; in some circumstances the principal vectors are more receptive, and in others less receptive to resistant parasites. (Wernsdorfer, 1991). Antimalarial drug resistance is likely to occur in three sets of circumstances: (a) large scale antimalarial drug use (b) inadequate dosing; and (c) adequate dosing with drugs that are eliminated slowly from the body. Inadequate dosing occurs commonly because of poor compliance with treatment regimens, or unregulated antimalarial drug distribution and self-prescribing. The greatest pressure occurs in circumstances where the whole population is exposed to low antimalarial drug concentrations, as in the ill-fated . population experiments in which pyrimethamine or chloroquine was added to table salt (thought by some to account for the development of chloroquine resistance in South-East Asia). Slowly-eliminated drugs persist in the blood at sub-therapeutic concentrations, and act as a selective pressure when the patient is reinfected. Mefloquine (T^, 3 weeks) and chloroquine (T^, 1-2 months) are detectable in blood for months after antimalarial treatment, whereas quinine (TifJ 16 h in malaria; 11 h in health) is gone in days, and the artemisinin compounds are probably gone in hours (White, 1985, 1988). As the level of resistance rises, more parasites escape the initial therapeutic assault, and the chance of selecting resistant mutants from the primary infection increases. The pace of resistance quickens. There has been considerable interest in preserving the antimalarial efficacy of chloroquine. Perhaps drug sensitivity would return if chloroquine was no longer used, thereby removing the selective pressure. This has been suggested, but never proved conclusively. It is very difficult to restrict use of chloroquine, and what of the other three human malarias for which chloroquine is the drug of choice? Cross-resistance is another potential source of drug pressure, but one which has not been quantified adequately. For example, could amodiaquine drive chloroquine resistance? Does widespread use of co-trimoxazole for treating bacterial infections encouraging resistance to antimalarial antifolates? Does mefloquine drive quinine resistance? Clearly these are very important questions to which we do not have complete answers.

576

N. J. White Current situation

Chloroquine

Amodiaquine, sulphonamide-pyrimethamine, and quinine What are the alternatives? Amodiaquine is usually more active than chloroquine against moderately-resistant strains, but the difference is not great. The high incidence of agranulocytosis (1 in 2000) and hepatitis associated with amodiaquine have stopped prophylactic use of the drug (Hatton et al., 1986), and cast a shadow over its role in treatment. The combination of a long-acting sulphonamide with pyrimethamine is synergic and active against P. falciparum in some areas where pyrimethamine alone is not. This is a well-tolerated treatment which can be given in a single therapeutic dose. Unfortunately, resistance has developed rapidly in South-East Asia and South America, although both sulphonamide-pyrimethamine and sulphone-biguanide combinations are still effective in East Africa (Watkins et al., 19886). Quinine has stood up remarkably well in over 350 years of use. Quinine sensitivity in P. falciparum has

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Drug resistance has to be placed in context. There has been a tendency in the past to declare a country's falciparum malaria 'chloroquine-resistant' on the basis of in-vivo observations in returned (non-immune) travellers or in-vitro sensitivity tests. These 'sentinel reports' do not assess the usefulness of the drug in the indigenous population. Unfortunately, maps have been drawn and, on the basis of such information, drug regulatory authorities have been led to believe that chloroquine can no longer be used. Despite the fact that nearly all countries in sub-Saharan Africa now have evidence of chloroquine-resistant P. falciparum, chloroquine remains the most widely-used antimalarial on the continent, and is still a most valuable drug. Transmission is usually intense in Africa and, as a consequence, malaria is largely a problem of childhood. The scale of chloroquine use in Africa is phenomenal. In 1988, 91 metric tons of chloroquine were consumed (World Health Organization, 1990), corresponding to approximately five hundred million child-treatment doses. Chloroquine can be purchased readily without prescription, and is used as a cure-all for a variety of minor ailments. It has an extremely long (c. 1-2 months) terminal elimination half-life (White, 1985). Thus, in many places, the majority of the population has detectable blood concentrations of chloroquine at any time. This is part of the 'drug-pressure' that drives resistance. Where transmission of malaria is intense, reinfection occurs rapidly after treatment If there is a low-grade resistance and some background immunity, there will be a symptomatic response to chloroquine (Brandling-Bennett et a!., 1988). Most importantly, severe infections will respond to treatment, i.e. the drug is still very useful. However, as resistance rises in areas of high transmission, the therapeutic response (times to fever clearance, to return to school or work, or less importantly, to parasite clearance) slows, and increasing numbers of infants with severe anaemia are seen. Eventually the number of infections with an unsatisfactory early response to treatment rises to the point where the drug is no longer useful, and alternative drugs are recommended. It should be noted that the decision at this point on whether to change treatment recommendations depends on knowledge of the local therapeutic response in vivo, and the cost and availability of alternative drugs. Chloroquine is still useful, and used, in much of Africa, but as a treatment of falciparum malaria, its days are numbered.

Anttmalarial ding resistance

577

Combinations Cure rates with quinine can be increased by the addition of a sulphonamide/ pyrimethamine combination if there is still sensitivity to these drugs. Tetracycline is a more reliably-effective addition, but it cannot be given to the two most vulnerable patient groups: young children and pregnant women. Combinations with macrolide antibiotics have given disappointing results in South-East Asia, but clindamycin has proved useful in antimalarial combinations in South America (Kremsner et al., 1988). Preliminary studies with the fluoroquinolone antibiotics indicated clinical antimalarial efficacy (Sarma, 1989), but more rigorous investigations failed to confirm the earlier report (Deloron et al., 1991; Watt et al., 1991; McClean, Hitchman & Shafran, 1992). Mefloquine Faced with increasing problems of drug resistance in P. falciparum, Thailand introduced the US Army-developed quinoline-methanol compound, mefloquine, in November 1984 (UNDP/World Bank/WHO, 1985). Mefloquine is active against most multi-drug-resistant strains of P. falciparum, although some West African strains appear to be intrinsically-resistant (Simon et al., 1988). Until 1991, mefloquine was available in Thailand only in combination with sulphadoxine and pyrimethamine (MSP). The MSP combination was an attempt to delay the onset of mefloquine resistance in P. falciparum. Unfortunately, because of pharmacokinetic dissimilarities between the three compounds (White, 1987)—mefloquine is eliminated much more slowly than the other two compounds—and the fact that P. falciparum was already highly-resistant to sulphadoxine and pyrimethamine when the combined drug was introduced in 1984, the strategy to protect mefloquine has not worked. Despite strict regulation of drug use (mefloquine is not commercially available in Thailand, and its prescription is confined to microscopically confirmed falciparum malaria) resistance has developed at an alarming rate over the past three years (Figure). Failure rates are now over 50% in some parts of the country (Nosten et al., 1991), and an increasing proportion of patients do not clear their peripheral parasitaemia (i.e. high-grade resistance). Furthermore, there is the aforementioned concern that mefloquine resist-

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decreased in some areas, but fortunately there are still no well-documented clinical reports of high-grade resistance, i.e. complete failure to respond in the presence of adequate blood levels (Looareesuwan et al., 1990). In the treatment of severe fakiparum malaria in Thailand, where multi-drug resistance is a particular problem, the therapeutic response assessed from the times to recover consciousness in cerebral malaria, and the times for parasite and fever clearance have not changed since 1980 (S. Pukrittayakamee and N. J. White, unpublished observations). Thus quinine (or quinidine) can still be relied upon in severe malaria (World Health Organization 19906), but for how much longer? In-vitro tests in Thailand are now indicating a worrying increase in the rate at which quinine sensitivity is declining (H. K. Webster, personal communication). Although quinine is well tolerated in severe malaria, common sideeffects ('cinchonism'; tinnitus, nausea, dysphoria) and an exceedingly bitter taste conspire to reduce compliance with the seven-day treatment regimens required for the cure of uncomplicated malaria in drug-resistant areas (White, 1988). In general, patients do not like to take antimalarial drugs for longer than they feel ill (i.e. three to four days).

578

N.J. White 1

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Flfare. Antim«i«riai cure rates in faJctpanun malaria in Eastern Thailand since 1976. The arrow marks the introduction of mefloquine to malaria clinics for the treatment of slide-positive P.faldparum infections.

ance may 'drive' resistance to quinine—the only available drug for severe malaria. Fortunately, mefloquine is still effective in most countries of the world, and those infections which recrudesce after mefloquine treatment will respond to the quininetetracycHne combination. What next? The question of what to do next is not answered easily. The cupboard of reserve drugs is looking increasingly bare. Halofantrine is a valuable addition to the small armoury of antimalarial drugs. It is intrinsically more active as an antimalarial and, apart from occasional diarrhoea, is generally very well tolerated in comparison with other antimalarials (Watkins et al., 1988a). However, it has variable oral bioavailability and it is structurally similar to mefloquine. Cross-resistance is seen in vitro and may be a problem in clinical use (Webster et al., 1985). The one-day, three-dose halofantrine regimen is effective in semi-immunes, or where parasites are highly-sensitive, but longer courses are required for the treatment of multi-drug-resistant malaria (F. ter Kiule unpublished observations). Nevertheless, because it lacks adverse central nervous system effects, halofantrine is often preferred by patients to mefloquine. The hydroxynaphthoquinone atovaquone (566C80) is active against Toxoplasma gondii, Pneumocystis carinii and Plasmodium spp. (Hammond, Burchell & Pudney, 1985). It is very well tolerated, but shares with halofantrine the problem of variable oral bioavailability. Clinical trials are in progress. It is too early to speculate on the role of this compound in prophylaxis or treatment The Chinese drugs related to qinghaosu (artemisinin) are exciting new compounds, structurally unrelated to the other known antimalarials. Several different preparations and formulations are available for parenteral, rectal and oral administration (artemether, artesunate and the parent compound artemisinin), but all have a common

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20

Aotimalarial drag robtancc

579

How to chose? Prophylaxis This is a most difficult area. In many situations there are no right answers, and compromise recommendations must be under constant scrutiny and review. Where P. vivax or sensitive strains of P. faldparum only are seen, chloroquine alone is an appropriate prophylactic. As resistance begins, an increase in the usual weekly chloroquine dose from 5 to 10 mg base/kg (or daily administration of 1-5 mg/kg) will prove more effective, i.e. buy some time. Thereafter opinions diverge. The European practice is to recommend chloroquine and proguanil for most places in the knowledge that this will not be completely effective everywhere, but at least will prevent severe disease. The combination is well-tolerated and remains effective over much of Africa, southern Asia, Oceania and some parts of the Americas. Where multi-drug resistance is more prevalent in South-East Asia, there are three options: (a) weekly mefloquine; (b) daily doxycycline; and (c) presumptive treatment with either mefloquine, halofantrine or quinine plus tetracych'ne. None of these is entirely satisfactory. In all cases, local knowledge of transmission areas and antimalarial drug sensitivity is of paramount importance in giving the appropriate advice. Antimalarial prophylaxis is often not needed at all—for example, for most tourists visiting South-East Asia. Even if prophylaxis is incompletely effective, it will probably attenuate the resistant infection, and thereby reduce the risk of a fatal outcome (provided the patient and the physicians consider the possibility of malaria in the differential diagnosis of subsequent fever). Poor compliance is a major confusing factor, and an important contributor to 'apparent drug resistance' and malaria fatalities in returned travellers.

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biologically-active metabolite, dihydroartemisinin. They have proved safe and effective in China (Qinghaosu Antimalaria Coordinating Group, 1979), and over the past IS years have gained a reputation for rapidity of action in severe malaria (World Health Organization, 1990ft). Over 1000 patients have been included in 18 comparative trials with these compounds. Parasite and fever clearance times have been significantly faster than with other antimalarial drugs, and there have been no significant adverse effects (T. T. Hien & N. J. White, unpublished observation). Resistance can be induced in animal models and has been reported already in clinical use, but it is not dear how important this is in practice, nor whether the cases represented inadequate treatment or true resistance. Reproducible methods for measurement of the drugs in blood are not available generally, and thus dose regimens are largely empirical. Unfortunately, the artemisinin-related drugs are not yet licensed outside China, Brazil, Ivory Coast, Gabon, Burma, Thailand and Vietnam. Furthermore, neither artemisinin nor halofantrinc are recommended in pregnancy. The Chinese scientists have developed other antimalarial drugs, notably pyronaridine and nitroquine, but these have not been used outside China, and their potential role in treatment remains to be determined (Ding, 1988). Unfortunately, despite the enormous efforts of the US Army antimalarial drug development programme in the 1960s and 1970s, and the extensive research in China over the past 20 years, drug development has not kept pace with resistance. Hopefully, improvement in existing drug regimens and, if necessary, a return to quinine, will buy some time, but if no new drugs are forthcoming (which seems likely), then there is a real prospect of completely untreatable malaria within the next ten years.

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Treatment

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How are decisions made on what antimalarial drugs to recommend for treatment? Travellers returning with malaria are likely to have little or no immunity, and are unlikely to know the drug sensitivity patterns in the area where they contracted malaria. Treatment should err on the side of caution, and falciparum malaria should be considered resistant if there is any doubt. In most countries in the tropics, cost is probably the major factor determining therapeutic practice locally. The other factor is information on antimalarial drug sensitivity. In-vitro testing of antimalarial drug sensitivity has been refined and simplified over the past ten years, and is now available to most countries with malaria problems. The information it provides is interesting, but not very useful. It has never led to a change in treatment recommendations. It does not predict reliably the response to antimalarial drug treatment in an individual because it cannot account for the variability in antimalarial drug disposition, background immunity and other treatment given. What health authorities and workers need, and what they act on, is the in-vivo response to antimalarial treatment The source of this information varies from detailed prospective research assessments of drug efficacy in a particular community, to the ad hoc impressions of health workers that more and more patients seem to be coming back for re-treatment within a few weeks. The latter can be very insensitive; a high incidence of low-grade drug resistance(RI) can easily go unnoticed. As there are so few drugs available, their effectiveness needs to be monitored carefully and repeatedly, and the tolerance and compliance of different drug regimens evaluated. Failure of a malaria infection to respond to treatment results either from intrinsic resistance of the parasite to the drug treatment, or host factors (phannacokinetics, compliance) which result in an insufficient concentration of the drug in blood. Pharmacological studies during the Second World War (Shannon et al., 1944) showed that the slow clinical and parasitological response to mepacrine (quinacrine) resulted from inadequate blood concentrations early in the course of treatment. Use of a loading or 'priming' dose circumvented the problem. However, 40 years' ago, the measurement of antimalarial drug levels was in its infancy, and it was usually impossible to distinguish pharmacokinetic factors from intrinsic drug resistance in causing treatment failure. These assays are now available more widely, and antimalarial blood levels should be measured in studies investigating drug resistance. The classification of in-vivo resistance that is used most widely today was derived originally for evaluation of aminoquinoline resistance (Table). It is useful for other drugs as well but has certain limitations (White & Krishna, 1989). For example, following mefloquine treatment, infections may recrudesce up to ten weeks (and probably more) after treatment. These infections would be classified incorrectly as sensitive. In some fully drug-sensitive infections treated at a time when the majority of the infecting parasites are sequestered, the parasite count will rise after starting treatment, and will not have fallen to a value less than 23% of the admission parasite count within 48 h. An erroneous assessment of high-grade resistance can be made (R m classification). As resistance develops, increasing numbers of R, recrudescences are seen. The initial therapeutic response is satisfactory, but the infection returns later. Patients whose parasitaemias decline slowly may be more likely to have a subsequent recrudescence; in the case of mefloquine, persistence of parasitaemia beyond day 4 following treatment in Thailand predicts subsequent failure (ter Kiule et al., 1992). As resistance increases,

Anmnabuial drug r—to»nof

581

Table. The World Health Organization grading of resistance of asexual parasites (P. falciparum) to 'schizontotidaT drugs (4-aminoquinolines) Response

Recommended grading

Sensitivity

S

Low-grade resistance

R,

Clearance of asexual parasitaemia, as in sensitivity, followed by recrudescence*

High-grade resistance

Ru

Marked reduction ( > 75%) of asexual parasitaemia, but no clearance, i.e. the parasitaemia remains patent for seven days

Rm

No marked reduction in parasitaemia, i.e. the parasite count does not fall by more than 75% within 48 h

Evidence Clearance of asexual parasitaemia within seven days of initiation of treatment, without subsequent recrudescence

progressively more patients are seen whose infections do not resolve (R,, and R,,,; high-grade resistance). Antimalarial drug regimens have changed in recent years with a fuller understanding of the pharmacokinetic properties of these compounds (Winstanley & Watkins, 1992; White, 1992). There is probably some room for further improvement of current regimens, but the fundamental problem is that there are not enough new drugs, and not enough research is being conducted on antimalarial drug development Assessment tod practice Guidelines for the conduct of in-vivo evaluations of chloroquine sensitivity are available (Bruce-Chwatt, 1981). A recent World Health Organization Scientific Group (World Health Organization, 1990a) has advised that 'simple and sustainable systems for the identification and reporting of antimalarial side-effects should be developed' -and that the 'frequency of treatment failures should be carefully monitored and reported to health authorities', but it does not say how these recommendations could be effected. In general, antimalarial drug studies are too small for confident conclusions to be drawn on the differences between treatment regimens. Evaluations tend to concentrate on parasitological responses which is reasonable in low transmission areas, but in high transmission areas, clinical assessment is more important. There are often discrepancies between the two in semi-immune patient populations. For example, in The Gambia, antimalarial prophylaxis prevented disease and death better than it prevented parasitaemia (Greenwood el al., 1988). Symptomatic parasitaemia in Gambian children is approximately six-fold more common in the rainy season months, but the prevalence of asymptomatic parasitaemia (c. 20%) remains constant through the year (Lindsay et al., 1991). Thus symptomatic and asymptomatic recrudescences of parasitaemia after treatment should be distinguished, and the number of children who remain clinically well during the follow-up period should be compared. The haematocrit or haemoglobin concentration is a useful indicator of the therapeutic response in malaria. Ineffective treatment is associated with a delayed recovery from anaemia.

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The anessment is usually made 28 days after the last treatment dose, although it ii known that recrudescences can occur up to ten weeks following drug administration. Reinfection cannot be excluded if the patient re-enten an area of malaria transmission.

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In many places, the majority of patients have received antimalarial drugs before they attend the malaria clinic. Such patients tend to be excluded from drug studies, but if this is a genuine reflection of the patient population then they should be included. If necessary, the patient groups can be stratified for previous treatment. In endemic areas, reinfection cannot be excluded. The more intense the malaria transmission, the greater the likelihood of reinfection before the end of the follow-up period. Obviously, if the age-stratified infection rate in the community is known, then the treatment failure rate can be adjusted accordingly, but this figure is notoriously difficult to obtain. The agespecific prevalence of parasitaemia at the time of the study is a useful measure of transmission and immunity, but as it is not known for how long each individual has a patent parasitaemia, it cannot be substituted directly in the failure rate calculations. Nevertheless, in comparing two drug regimens in endemic areas, the number of patients with positive blood smears two to six weeks after treatment is still useful, as it will be higher in the less-effective drug regimen group (i.e. recrudescences plus reinfections). The sample sizes chosen will depend on the anticipated level of drug resistance, and the variance within different patient sub-groups (infants, pregnant women, variance in background immunity, etc.). It has been appreciated for many years that background immunity is an important factor determining treatment response (York & Macfie, 1924), and this is directly proportional to age and transmission intensity. The antimalarial drug reduces the parasite burden, and host defences eliminate it. It is important to stratify therapeutic assessments for age and, when interpreting the results of studies, to consider the likely level of background immunity in the population. Antimalarial drugs always do better in semi-immune populations. Treatment courses can be shorter than those required in non-immune subjects. If in-vivo testing stations could be set up in malarious areas, the results provided would give health authorities, and the medical profession, the information they need to decide upon appropriate antimalarial treatment More detailed assessments of drug toxicity, blood concentration measurements, and determination of other maliariametric indices could be added where necessary. The true costs and true benefits of newer, and nearly always more expensive, drug regimens could be gauged with certainty, and the march of drug resistance defined.

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