Walther H. Wernsdorfer

Epidemiology of drug-resistant malaria.

Since the first reports of chloroquine-resistant falciparum malaria in southeast Asia and South America almost half a century ago, drug-resistant malaria has posed a major problem in malaria control. By the late 1980s, resistance to sulfadoxine-pyrimethamine and to mefloquine was also prevalent on the Thai-Cambodian and Thai-Myanmar (Thai-Burmese) borders, rendering them established multidrug-resistant (MDR) areas. Chloroquine resistance spread across Africa during the 1980s, and severe resistance is especially found in east Africa. As a result, more than ten African countries have switched their first-line drug to sulfadoxine-pyrimethamine. Of great concern is the fact that the efficacy of this drug in Africa is progressively deteriorating, especially in foci in east Africa, which are classified as emerging MDR areas. Urgent efforts are needed to lengthen the lifespan of sulfadoxine-pyrimethamine and to identify effective, affordable, alternative antimalarial regimens. Molecular markers for antimalarial resistance have been identified, including pfcrt polymorphisms associated with chloroquine resistance and dhfr and dhps polymorphisms associated with sulfadoxine-pyrimethamine resistance. Polymorphisms in pfmdr1 may also be associated with resistance to chloroquine, mefloquine, quinine, and artemisinin. Use of such genetic information for the early detection of resistance foci and future monitoring of drug-resistant malaria is a potentially useful epidemiological tool, in conjunction with the conventional in-vivo and in-vitro drug-sensitivity assessments. This review describes the various features of drug resistance in Plasmodium falciparum, including its determinants, current status in diverse geographical areas, molecular markers, and their implications.

EPIDEMIOLOGY OF DRUG RESISTANCE IN MALARIA

In the late 1950s chloroquine resistance to Plasmodium falciparum occurred in South America and on the Indochina Subcontinent. Since then it has conquered most of the areas where the parasite species is endemic. This has necessitated the use of alternative drugs such as sulphonamide-pyrimethamine combinations, quinine/tetracyclines, mefloquine, halofantrine, and recently also artemisinin-based compounds. In wide areas of South-east Asia, western Oceania and South America sulphonamide-pyrimethamine combinations have lost adequate efficacy. The situation is most serious in the Thai/Cambodia and Thai/Myanmar border areas where multiresistance necessitated the shift to the last line drug, i.e., the artemisinin derivatives. Selection of resistant parasites due to drug pressure, and their subsequent propagation by local transmission and migration of reservoirs are key factors in the dynamics of drug resistance. Selection is the result of the interplay of parasite, drug and human host, and is largely influenced by immune factors and the pharmacokinetics and pharmacodynamics of the drug. Spread of resistance is determined by eco-epidemiological factors among which migration and vectorial parameters play a major role. Rational drug use, especially adequate, monitored, therapeutic administration according to strict criteria, should curb the onset and spread of resistance, but this concept may not be readily accepted by health services whose primary goal is clinical amelioration of the disease rather than the more stringent target of epidemiologically desirable results.

From malaria control to eradication: The WHO perspective

Efforts to control malaria have been boosted in the past few years with increased international funding and greater political commitment. Consequently, the reported malaria burden is being reduced in a number of countries throughout the world, including in some countries in tropical Africa where the burden of malaria is greatest. These achievements have raised new hopes of eradicating malaria. This paper summarizes the outcomes of a World Health Organization’s expert meeting on the feasibility of such a goal. Given the hindsight and experience of the Global Malaria Eradication Programme of the 1950s and 1960s, and current knowledge of the effectiveness of antimalarial tools and interventions, it would be feasible to effectively control malaria in all parts of the world and greatly reduce the enormous morbidity and mortality of malaria. It would also be entirely feasible to eliminate malaria from countries and regions where the intensity of transmission is low to moderate, and where health systems are strong. Elimination of malaria requires a re‐orientation of control activity, moving away from a population‐based coverage of interventions, to one based on a programme of effective surveillance and response. Sustained efforts will be required to prevent the resurgence of malaria from where it is eliminated. Eliminating malaria from countries where the intensity of transmission is high and stable such as in tropical Africa will require more potent tools and stronger health systems than are available today. When such countries have effectively reduced the burden of malaria, the achievements will need to be consolidated before a programme re‐orientation towards malaria elimination is contemplated. Malaria control and elimination are under the constant threat of the parasite and vector mosquito developing resistance to medicines and insecticides, which are the cornerstones of current antimalarial interventions. The prospects of malaria eradication, therefore, rest heavily on the outcomes of research and development for new and improved tools. Malaria control and elimination are complementary objectives in the global fight against malaria.

Histidine-Rich Protein II: a Novel Approach to Malaria Drug Sensitivity Testing

ABSTRACT The production of histidine-rich protein II (HRP2), a histidine- and alanine-rich protein produced by Plasmodium falciparum, is closely associated with the development and proliferation of the parasite and therefore is perfectly suited to reflect growth inhibition as a measure of drug susceptibility. It was the aim of the present study to develop a malaria drug sensitivity assay based on the measurement of HRP2 in a simple enzyme-linked immunosorbent assay (ELISA). The new test proved to be as reliable as traditional in vitro assays, while it was considerably easier to establish and perform. Parasites are incubated at an initial level of parasitemia of 0.01 to 0.1% on microculture plates predosed with ascending concentrations of antimalarial drugs. After incubation for 48 to 72 h, the samples are freeze-thawed and transferred to ELISA plates. The complete ELISA takes about 2.5 h to perform, may be carried out with commercially available test kits, and requires relatively little technical equipment. In correlation analysis, the results closely paralleled those obtained by the isotopic assay (R = 0.892; P < 0.0001) and World Health Organization schizont maturation tests (R = 0.959; P < 0.0001). The novel HRP2 drug susceptibility assay proved to be very sensitive, simple to establish, and highly reproducible. It can be used for a wide range of applications, from epidemiological studies to the screening of new drugs, and may have the potential to replace traditional in vitro techniques. Standard operating procedures, updated information, and analytical software are available from http://malaria.farch.net .

High level of resistance of Plasmodium falciparum to sulfadoxine-pyrimethamine in children in Tanzania

In many areas of tropical Africa affected by chloroquine-resistant Plasmodium falciparum, a combination of sulfadoxine and pyrimethamine (S-P) is used for alternative medication, especially in young children. In Magoda village in Muheza District, north-eastern Tanzania, 38 children 1-10 years of age were enrolled in a therapeutic study of S-P in July 1994. All had monoinfections of P. falciparum and an asexual parasite count of 1000-80,000/microL of blood. S-P was given as a single dose corresponding to 0.8-1.4 mg pyrimethamine/kg body weight. Of the 38 children followed up to day 7, 10 showed an S/RI response, 26 an RII response, and 2 an RIII response. Older children had lower pre-treatment parasitaemia and a better therapeutic response than younger children. Among the various contributory factors responsible for the poor therapeutic result, drug pressure from a prophylactic intervention with weekly dapsone-pyrimethamine between May 1993 and May 1994 seems to have been the most important.

Malaria drug-sensitivity testing: new assays, new perspectives

Over the past five decades, the drug resistance of Plasmodium falciparum has become an issue of utmost concern. At the same time, in vitro assays for assessing antimalarial drug sensitivity have become indispensable tools for the surveillance of drug resistance and the planning of therapeutic guidelines. Several new in vitro assays have been introduced, designed to be easier to handle than previous tests and allow a faster identification of drug-resistant parasites, as well as for simple evaluation of new drugs. This review examines the various new approaches to the in vitro assessment of malaria drug sensitivity and their limitations.

Effects of Miltefosine and Other Alkylphosphocholines on Human Intestinal Parasite Entamoeba histolytica

ABSTRACT The protozoan parasite Entamoeba histolytica is the cause of amoebic dysentery and liver abscess. It is therefore responsible for significant morbidity and mortality in a number of countries. Infections with E. histolytica are treated with nitroimidazoles, primarily with metronidazole. At this time, there is a lack of useful alternative classes of substances for the treatment of invasive amoebiasis. Alkylphosphocholines (alkyl-PCs) such as hexadecyl-PC (miltefosine) were originally developed as antitumor agents, but recently they have been successfully used for the treatment of visceral leishmaniasis in humans. We examined hexadecyl-PC and several other alkyl-PCs with longer alkyl chains, with and without double bond(s), for their activity against two strains of E. histolytica. The compounds with the highest activity were oleyl-PC, octadecyl-PC, and nonadecenyl-PC, with 50% effective concentrations for 48 h of treatment between 15 and 21 μM for strain SFL-3 and between 73 and 98 μM for strain HM-1:IMSS. We also tested liposomal formulations of these alkyl-PCs and miltefosine. The alkyl-PC liposomes showed slightly lower activity, but are expected to be well tolerated. Liposomal formulations of oleyl-PC or closely related alkyl-PCs could be promising candidates for testing as broad-spectrum antiprotozoal and antitumor agents in humans.

Reassessment of the resistance of Plasmodium falciparum to chloroquine in Gabon: implications for the validity of tests in vitro vs. in vivo.

Increasing resistance of Plasmodium falciparum to antimalarial drugs presents a major risk factor for people living in endemic areas of tropical Africa. In Lambaréné, Gabon, regular surveillance of chloroquine sensitivity of P. falciparum in vitro has been carried out since 1992 using the WHO standard microtest. Results indicated that from 1994 onwards chloroquine resistance in vitro decreased significantly and that by 2000, about 70% of parasite isolates seemed to be sensitive to chloroquine in vitro. In 2001, we conducted a clinical study to reassess the efficacy of chloroquine in vivo for the treatment of uncomplicated P. falciparum malaria. Twenty-six patients aged 4-15 years were included in this study. Most unexpectedly, the study demonstrated high-grade resistance to chloroquine in vivo (failure rate on day 28 of 100%). As a consequence, tests of parasite susceptibility to chloroquine in vitro were repeated using the same protocol except for the replacement of previously used commercially available predosed WHO culture plates by independently dosed plates. All tested P. falciparum isolates were highly resistant to chloroquine, correlating well with our clinical findings. We concluded that high level resistance of P. falciparum to chloroquine persists in the study area. Neglect or absence of quality controls of essential test material can lead to invalid study results and wrong conclusions and should always be suspected in the case of major fluctuations in the sensitivity patterns of an antimalarial drug in vitro. In addition, our results highlight the supreme value of tests in vivo in providing reliable estimates of the efficacy of an antimalarial in a specific area.

In Vitro Interactions of Artemisinin with Atovaquone, Quinine, and Mefloquine against Plasmodium falciparum

ABSTRACT The interactions of artemisinin with atovaquone, quinine, and mefloquine were investigated in three Plasmodium falciparum strains (strains F-32, FCR-3, and K-1) by an in vitro culture assay. The parasites were cultured for 48 h in the presence of different concentrations and proportions of two drugs at a time in a checkerboard design. The response parameters were determined, and the sums of the fractional inhibitory concentrations (ΣFICs) of the drug combinations were calculated for different degrees of inhibition (50% effective concentration [EC50], EC90, and EC99). Within therapeutically relevant molar ratios (19 to 200), the combination of quinine and artemisinin showed mean ΣFICs of 1.71 at the EC50, 0.36 at the EC90, and 0.13 at the EC99, indicating increasing synergism. Within the range of molar ratios of 4.3 to 50, the combination of mefloquine and artemisinin yielded mean ΣFCIs of 0.93, 0.44, and 0.31 at the EC50, EC90, and EC99, respectively, indicating synergism. The atovaquone combination showed additive activity to synergism at atovaquone/artemisinin proportions considered relevant to the in vivo situation, i.e., between 4.3 and 200, with the mean ΣFICs decreasing from 1.34 at the EC50 to 0.85 and 0.23 at the EC90 and EC99, respectively. Interstrain differences in the degree of drug interaction were seen with the three strains for all combinations. Synergism was most consistent with quinine.

Sensitivity of Plasmodium vivax to chloroquine in Sa Kaeo Province, Thailand

Following a recent, abrupt local increase in the incidence of vivax malaria, a study was conducted in order to evaluate the efficacy of chloroquine for the treatment of 26 adult patients with acute vivax malaria in Sa Kaeo Province, Thailand. The chloroquine sensitivity of Plasmodium vivax has been assessed in parallel, using a growth inhibition method. Blood samples for the in vitro tests were taken prior to the administration of the standard treatment with chloroquine--in total 25 mg base/kg over 3 days--and primaquine 0.25 mg base/kg once daily for 14 days. The efficacy has been assessed according to the WHO standard in vivo test. The cure rate was 100%. No recrudescence was observed during the follow-up period of 28 days. The mean fever clearance time (FCT) was 40 h, the mean parasite clearance time (PCT) was 49 h. Mean IC(50) and IC(90) of the parasites were 28 and 171 nM, respectively. These results show that local P. vivax is still sensitive to chloroquine. The epidemic outbreak was therefore obviously not due to the presence of chloroquine-resistant P. vivax.