Reduced sensitivity of Plasmodium falciparum to formerly recommended cheap and well-known antimalarial drugs places an increasing burden on malaria control programs and national health systems in endemic countries. The high costs of the new artemisinin-based combination treatments underline the use of rational and updated malaria treatment policies, but defining and updating such policies requires a sufficient volume of high-quality drug-resistance data collected at national and regional levels. Three main tools are used for drug resistance monitoring, including therapeutic efficacy tests, in vitro tests, and analyses of molecular markers. Data obtained with the therapeutic efficacy test conducted according to the standard protocol of the World Health Organization are most useful for updating national treatment policies, while the in vitro test and molecular markers can provide important additional information about changing patterns of resistance. However, some of the tests are technically demanding, and thus there is a need for more resources for training and capacity building in endemic countries to be able to adequately respond to the challenge of drug resistance.

Introduction

Global efforts toward controlling malaria are greatly challenged by the increasing spread of antimalarial drug resistance. Use of ineffective antimalarials is thus considered partly responsible for the difficulties in reducing malaria morbidity and mortality. This is a problem particularly in sub-Saharan Africa, where susceptibility of Plasmodium falciparum to previously used cheap and commonplace antimalarials, such as chloroquine and sulfadoxine–pyrimethamine (SP), has been declining rapidly in many high-transmission areas.¹ To ensure that malaria control strategies and malaria treatment policies rely on the deployment of effective anti-malarials, there is a need for systematic monitoring of anti-malarial drug efficacy and drug resistance. Artemisinin-based combination treatments (ACTs) are today the only consistently effective antimalarials available, and as ACTs now become more and more widely used, careful monitoring of their therapeutic efficacy and of any emerging resistance is needed. Preventing development of artemisinin resistance requires that the short-acting artemisinin component is “protected” by a longer-acting partner drug of known high efficacy, and therefore it is important also to keep an eye on the efficacy of the different partner drugs currently recommended for use in artemisinin-based combinations.² The aims of this overview are to briefly describe the current situation of drug-resistant malaria and to discuss the available tools for monitoring of drug efficacy and early detection of resistance, emphasizing the importance of a standardized and coordinated use of these tools to update national malaria treatment policies.

Malaria Treatment Failure and Drug Resistance

Before the spread and assessment of antimalarial drug resistance can be described, a few words on the definition of “resistance” are needed. Resistance of antimalarial drugs is defined as the “the ability of a parasite strain to survive and/ or multiply despite the administration and absorption of a drug given in doses equal to or higher than those usually recommended but within the tolerance of the subject.”³ This definition was originally drawn up before techniques allowing for in vitro drug-sensitivity testing and detection of molecular markers of resistance were available, and thus the definition is based on clinical observation. Today, this is still the case, although confirmation of true resistance to an antimalarial requires proof that the parasites are recrudescent (rather than arising from re-infection) in a patient who recently received treatment and a demonstration that an effective blood concentration of the drug or its metabolites has been maintained for at least 4 parasite cycles.⁴ The results of in vitro drug-sensitivity tests and the presence of gene mutations implicated in resistance are additional indicators of resistance (see later), but as these tests are only rarely conducted simultaneously with therapeutic efficacy tests, especially pharmacokinetic drug profiles, merely the failure to clear malarial parasitemia and/or resolve clinical symptoms is conventionally considered to be an indicator of drug resistance. In the event of a failing treatment, it is import to realize, however, that treatment failure may not necessarily be the same as true antimalarial drug resistance. Failure to clear an episode of malaria despite administration of an antimalarial may just be explained by insufficient blood concentrations of the drug, and thus in daily clinical practice, many factors may explain a treatment failure, such as incorrect dosing, problems of patient compliance, poor drug quality, interactions with other drugs, inter-individual variation in pharmacokinetics including poor absorption, rapid elimination (due to diarrhea or vomiting), or insufficient biotransformation of prodrugs because of human genetic characteristics. Furthermore, it should be considered whether a “failure” of malarial treatment may only be a matter of misdiagnosis of the patient.

Summary of the Epidemiology of Drug-Resistant Malaria

During the last 50 years, resistance to antimalarial drugs has been documented for P. falciparum, P. vivax, and recently also P. malariae (Figure 1). Resistance of P. falciparum has been observed to almost all currently used antimalarials except for artemisinin and its derivatives, although the geographical distributions and rates of spread have varied considerably between regions and countries. The spread of resistance to some of the common antimalarials is briefly summarized in the following, and a detailed account of the global levels of resistance can be found in a recent WHO report.⁵

Figure 1

Malaria transmission and reported treatment failure and drug resistance in 2005. (The designations employed and the presentation of material on this map do not imply the expression of any opinion whatsoever on the part of the World Health Organization (more...)

Tools for Monitoring

The increasing spread of antimalarial drug resistance has emphasized the need for systematic monitoring to suggest where malaria treatment policies should be revised to secure rational use and effective case management. The available monitoring procedures include first of all the therapeutic efficacy test (also known as the in vivo test), which involves the repeated assessment of clinical and parasitological outcomes of treatment during a fixed period of follow-up to detect any reappearance of symptoms and signs of clinical malaria and/ or parasites in the blood. Other available methods include in vitro studies of parasite susceptibility to drugs in culture and studies with molecular methods of gene mutations or gene amplifications associated with parasite resistance (Table 1).

Table 1

Tools for monitoring antimalarial drug efficacy and drug resistance

Therapeutic Efficacy Testing

Therapeutic efficacy testing in P. Falciparum

The therapeutic efficacy test remains the gold standard for determining antimalarial drug efficacy for the management of P. falciparum infections. It provides policy makers and national malaria control programs a straightforward indicator of the efficacy of an antimalarial drug or a combination treatment in a given population at risk, to suggest whether a drug is still appropriate as first- or second-line treatment. Studies of therapeutic efficacy are relatively straightforward to set up, although they tend to be lengthy and may be costly, unless integrated into a national malaria control program; in particular, medical and technical personnel (especially microscopists) must be trained. To interpret the results uniformly to follow trends over time and to compare levels of resistance between different regions, studies must be carried out with the same standardized protocol, at the same sentinel sites, in the same age groups, and, if possible, at the same time of year in a given site.^10,11

The WHO standard protocol is meant for the evaluation of antimalarial drugs or drug combinations (chloroquine, amodiaquine, quinine, SP, ACTs, etc.) for treatment of uncomplicated P. falciparum malaria. The design is simple: a one-armed prospective study of clinical and parasitological responses after administration of antimalarial treatment to children of age 6–59 months with a degree of immunity that is unlikely to have much impact on the outcome of the test. In areas of low or moderate transmission in which it is difficult or time-consuming to enroll enough children in this age group, children > 5 years old and adults can be included, although it should be borne in mind that the results will be biased, as adults always respond better than children. To avoid the inclusion of asymptomatic carriers, only patients with 2000 parasites per μL or more (1000 parasites per μL in areas of low or moderate transmission) are included. Calculation of the sample size required by estimating prevalence is now preferred to the lot quality assurance sampling method. If the selected confidence interval is 95% with a precision of 10%, the sample size ranges between 50 and 100 patients. A minimum of 50 patients must be enrolled in order for the sample to be representative. This sample size was appropriate to detect failure cases during routine monitoring of the efficacy of currently recommended ACTs after their implementation. The recommended duration of follow-up is ≥ 28 days in areas of intense as well as low-to-moderate malaria transmission. For treatment with drugs such as amodiaquine, chloroquine, and SP, a 28-day follow-up is considered appropriate; for slowly eliminated antimalarials (lumefantrine, mefloquine, piperaquine, pyronaridine), recrudescences may occur after 28 days, and so 42- or even 63-day follow-up periods are recommended.

Several changes to the WHO protocol have been introduced based on the feedback from the field since 1996.^10,12 The main difference between the protocols for areas of high transmission and for areas of low-to-moderate transmission, apart from the inclusion criteria, was in the management of parasitological failures without clinical signs. The differences reflect regional program priorities (treatment of clinical cure or for radical cure). In 2005, the malaria treatment guidelines stressed that the objective of antimalarial treatment, even in areas of high transmission, should be radical cure of the disease.² From a clinical point of view, the persistence of parasites resulting from treatment failure can lead to anemia, gametocyte carriage, and risk of recurrent clinical signs and symptoms. These 3 harmful consequences of treatment failure are taken into consideration in the modified 2005 WHO protocol.

Patients included in the efficacy study and not lost to follow-up or excluded (e.g., because of self-medication, development of concomitant febrile infections, or refusal to continue participation) are classified in one of the following categories: early treatment failure, late clinical failure, late parasitological failure, or adequate clinical and parasitological response. These classifications rely on the presence or absence of fever or other signs of clinical malaria and/or presence of parasitemia during the course of follow-up, as listed in Table 2. The rates of total failure are used to define cut-off points for drug-policy change. Monitoring antimalarial drug efficacy is based on presence or absence of asexual parasites detected by microscopy at admission and during the follow-up, and the use of rapid diagnostic tests has been suggested for the screening of febrile patients. So far, however, rapid tests cannot be used during the follow-up because quantitative parasitemia is needed to classify patient outcome as early treatment failure, and some tests show false-positive results related to the persistence of circulating antigens or presence of gametocytes, despite complete parasite clearance.

Table 2

Classification of treatment outcome according to the WHO Protocol 2005 for all endemic areas

Therapeutic efficacy testing in P. Vivax, P. Ovale, and P. Malariae

Relapse, re-infection, and recrudescence cannot be distinguished reliably in the infections with P. vivax and P. ovale. Nevertheless, in vivo assessments of chloroquine susceptibility can be performed using the same protocol format as for P. falciparum, with a follow-up period of 28 days and preferably accompanied by measurement of whole-blood chloroquine and desethychloroquine levels at the day of failure. Recurrent infections within this period presenting with whole-blood chloroquine + desethychloroquine concentrations exceeding 100 ng/mL are currently considered as resistant whether they are a relapse, a recrudescence, or even a new infection, as this concentration should be suppressive.¹³ This definition will most probably need to be reconsidered according to the on-going trials.

WHO Database on Therapeutic Efficacy Studies

To facilitate the use of data from such standardized assessments, a comprehensive database has been established by WHO, where reported standardized drug-efficacy results are compiled and summarized to better inform endemic countries and their national malaria control programs about the current situation and of changing patterns of resistance. The data in the database originate from 3 sources: 1) published studies; 2) unpublished studies (available in reports by ministries of health, national control programs, or nongovernmental organizations, consultant reports, theses, and papers, or posters presented at national and international conferences); and 3) regular data from surveillance studies conducted according to the WHO standard protocol and sent from countries to WHO for validation. On the basis of this database, detailed estimates of global levels of drug efficacy in the period from 1996 to 2004 were recently reported as mentioned above.⁵

Coordinated Monitoring at National and Regional Level

To allow for the collection of accurate and comparable data on drug resistance, WHO recommends a systematic and uniform organization of drug-efficacy monitoring, coordinated by ministries of health and national malaria control programs. Many countries are already collecting drug-efficacy data in a systematic and timely manner, and monitoring systems in other countries can be improved, even if only limited resources are available. National monitoring of drug efficacy mainly requires a well-planned and coordinated system for resistance-data collection, including established sentinel sites with the necessary trained staff, some minimum laboratory facilities, and agreed standards for data collection, analysis, and reporting of results according to the WHO standard efficacy protocol. Monitoring may involve teams working centrally, which then conduct studies across the country, or teams working at the district level with support from teams at the central level. In both cases, the initial and regular training of central teams is crucial to avoid fundamental errors such as sampling errors, incorrect patient follow-up, or incorrect classification of therapeutic responses. Only the WHO standard efficacy testing protocol should be used for monitoring, from which no deviations should be made that would disturb the direct comparison of results between studies and therefore complicate guidance of national treatment policy. Additional research questions, in particular during new antimalarial drug phase III studies, may be addressed as part of the studies, but the collection of additional information to serve this purpose should not affect the collection of recommended routine data, which serves the purpose of national monitoring. For the comparability of results from year to year and from one site to another, similar study populations must be enrolled, and the studies should be conducted during the same malaria transmission season. Only standard drug administration and regimens should be tested, using drugs of normal standard formulation and quality. In all areas of transmission, 28 days of follow-up should also be the standard. The selection of testing sites should be done carefully to cover different geographical areas and different levels of transmission intensity and may also need to consider operational feasibility to allow a high quality of patient follow-up. Although no definitive scientific advice can be given regarding the number of sites needed, experience suggests that a balance between representativeness and practicality can be achieved with 4–8 sites in a country collecting data for the purpose of policy change. For routine monitoring after the implementation of an ACT, a more limited number of sites is necessary. But a new site can be created in case of an epidemic outbreak or when the distribution of confirmed malaria treatment failures is reported to be higher in some areas of the country through simplified routine surveillance or health information systems. Based on accumulated experience rather than definitive science, it is recommended that assessments be conducted at least once every 2 years, unless the data show a trend of increasing failure rate. Some programs conducting sentinel site surveillance prefer to alternate test sites, for example, testing 4 sites per year with each site being assessed every other year. Other programs find it more easy and sustainable to monitor a first-line drug in all sites the first year and the second-line drug the next year. Both first- and second-line drugs should be monitored because the second-line drug is not always an ACT and because most of the current ACTs and their partner drugs as monotherapies are available in the private sector.

To better allow for the detection of changing patterns of resistance at national, subregional, and regional levels, increased collaboration between national institutions has proven useful. The East Africa Network for Monitoring of Antimalarial Treatment (EANMAT)²⁴ serves as a good example of such coordinated efforts

Updating National Malaria Treatment Policies

The cut-off points for changing a treatment policy, which are sometimes based on parasitological or economic parameters, are often determined arbitrarily. Such suggested cut-off points are indicative, and decision makers at the national level should feel free to initiate change at any time. In the past, many countries were too slow or too cautious in deciding on policy change, despite a high failure rate with chloroquine reported in the field. In 1999, a scale of dynamic change was established on the basis of clinical failure rates on day 14, but it should be noted that this system was drawn up for regions of high transmission.²⁵ The WHO malaria treatment guidelines from 2005 suggest that, with the introduction of more effective combination therapies, efficacy of an antimalarial treatment should reach 95% and that a policy change should be seriously considered if the efficacy is < 90% on day 28.²

Between 2001 and 2006, 68 countries (nearly 40 in Africa) have changed their policies. Although it might be clear from surveillance at sentinel sites that a first-line treatment is ineffective, the choice of a replacement drug or drug combination at consensus meetings has sometimes proved difficult. According to the 2001 WHO recommendations, an endemic country in which drug resistance to monotherapy is observed should change to a combination therapy based on artemisinin. The currently recommended options are artemether–lumefantrine, artesunate + amodiaquine, artesunate + SP, amodiaquine + SP, and artesunate + mefloquine.²⁶

Results of tests for therapeutic efficacy are the most important information for determining whether an antimalarial drug is still effective. Surveillance of therapeutic efficacy over time is an essential component of malaria control. With a few exceptions, all policy changes have been based on the results of in vivo tests. In South Africa, data on in vitro drug sensitivity were also taken into consideration when chloroquine was abandoned as a first-line drug. In Mali, the results of use of molecular markers were determinant in deciding to use sulfadoxine–pyrimethamine instead of chloroquine during an epidemic outbreak.²⁷ In vitro tests and detection of genetic markers of resistance conducted over time can provide early warning of impending resistance before it becomes clinically apparent and can help guide therapeutic efficacy studies.²⁸ These tests are also useful for monitoring changes over time in susceptibility to a drug that has been withdrawn.²⁹ The usefulness of these tests has become evident with the ever-increasing use of combination therapy. It is often impossible to conduct therapeutic efficacy tests for each component, owing to ethical problems, nonavailability of the drug as a single therapy, and the need to study a large number of patients. In vitro tests can be used to monitor susceptibility to each drug in a combination.

Future Challenges

The need for high-quality monitoring of antimalarial drug resistance has increased in recent years. It is increasingly important to know if the required high levels of efficacy of the new and costly combination treatments are maintained, and a more proactive approach to malaria treatment policy change is considered a key to effective malaria control. Technical advances in recent years have made it possible to provide more accurate efficacy results than previously, but the technical requirements in routine monitoring place an additional burden on malaria control efforts, such as the increase in the recommended duration of follow-up and the need of PCR analysis, which will inevitably increase the cost of routine monitoring. Sustained funding for routine monitoring activities is becoming a major issue. In the last decade, countries had to carry out a limited numbers of studies to demonstrate that the monotherapies used as first- and second-line drugs were failing and to compile the baseline data on ACT efficacy. Monitoring ACTs is becoming crucial because the long-acting partner drug of some of the recommended ACTs are marketed as monotherapies, and their efficacy is compromised by high resistance rates, exposing the artemisinin derivatives to the development of resistance. Funding agencies should request the countries to include systematically in their proposals a budget for monitoring activities, particularly when ACTs are going to be implemented at a large scale.

National malaria control programs should be collecting information on the cost of testing to better support their requests for funding. Despite support from the Global Fund, the World Bank or the President’s Malaria Initiative, many countries are still facing either a funding gap for monitoring drug efficacy or an absence of sustainability in funding for this specific activity. When constraints caused by limited budgets or limited numbers of qualified personnel are difficult to overcome, the available resources should be channeled into more complete and regular studies at fewer sites to obtain better information.

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Reprint requests: P. Ringwald, Global Malaria Programme, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzer-land, Telephone: +41-22-7913469, Fax: +41-22-7914824, E-mail: tni.ohw@pdlawgnir.

Authors’ addresses: Lasse S. Vestergaard, Department of Epidemiology, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark, Telephone: +45-32-683695, Fax: +45-32-683874, E-mail: kd.iss@val. Pascal Ringwald, Global Malaria Programme, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland, Telephone: +41-22-7913469, Fax: +41-22-7914824, E-mail: tni.ohw@pdlawgnir.