Early diagnosis and treatment of uncomplicated malaria with anti-malarial drugs remains the mainstay of disease control in endemic areas. The emergence and spread of Plasmodium falciparum resistance to chloroquine (CQ) and sulphadoxine/pyrimethamine (SP) has rendered these two inexpensive, first-line anti-malarials ineffective in most malarious areas of the world, and compromised malaria control programmes 12. To rationalize alternative anti-malarial drug policy, it is crucial to be able to predict and monitor parasite resistance and yet the challenges are immense 3. The in vivo test is widely used, but requires substantial logistical and financial support and its interpretation is confounded by factors such as reinfection, immunity, and pharmacokinetics 4. In vitro tests quantify the anti-malarial activity against parasites isolated from infected individuals, but the correlation between such assays and clinical outcome is mostly unsubstantiated 5. Identification of the molecular basis of anti-malarial drug resistance and its relationship to therapeutic failure represents a major advance in our ability to monitor anti-malarial drug resistance 6.

Linkage studies with parasite isolates from malaria patients have demonstrated a close association between the pfcrt K76T mutation and the in vitro chloroquine resistant phenotype 7. Sequence analyses of the multi-drug resistance (Pfmdr) genes, initially thought to confer resistance through gene and P-glycoprotein over-expression, have revealed a series of point mutations that were associated with resistance 8. More recently, gene copy number has been associated with decreased susceptibility to quinine, mefloquine, artemisinins, lumefantrine and halofantrine 9. Resistance to antifolates and sulphonamides is conferred by point mutations at specific codons in the genes coding for the dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) enzymes, respectively, resulting in decreased affinity of the enzyme for the drug 10. The molecular basis of artemisinin susceptibility has not been established yet, although an association with SERCA/ATPase6 has been proposed 11.

Understanding the relationship between putative molecular markers, parasite resistance and treatment failure has become a priority now that artemisin-based combination therapy (ACT) has replaced monotherapies as the first-line treatment of uncomplicated falciparum malaria 12. In combination, the contribution of the individual components of drug regimen cannot be disentangled from a clinical study. Furthermore, recent studies have highlighted that withdrawal of chloroquine drug pressure may lead to a reversion to chloroquine-susceptible phenotypes 13, and these might have gone undetected if molecular prevalence surveys had not been conducted 14. Amodiaquine is one of the most widely used artemisinin partner drugs in ACT, but the underlying mechanism of parasite resistance is poorly characterized 15. Sulphadoxine/pyrimethamine (SP) is the only drug currently studied in detail for intermittent preventive therapy (IPT) in pregnant women and infants, but neither the influence of antifolate resistance on IPT efficacy nor the impact of IPT on the selection of drug resistant parasites has been comprehensively addressed.

Although it is not customary to change treatment policies based on molecular studies alone, molecular studies from Mali and Tanzania have demonstrated that a high prevalence of resistance makers can inform policy change 1617. Hence, it is hoped that the identification of early markers of resistance will facilitate more widespread deployment of rational treatment policies that will retard the emergence of antimalarial drug resistant 18.

A key step in the process of validating experimental findings is to verify the correlation of parasite genetics with clinical response of the host. Collating this information is crucial to our ability to apply specific genetic markers to predict treatment failure. The aim of the current study was to conduct a systematic review and a meta-analysis of clinical trials reporting on putative genetic markers of P. falciparum resistance.

Despite the large number of studies published on anti-malarial drug efficacy, as reviewed by Myint et al 86, approximately 10% have specifically addressed the in vivo-molecular correlates of resistance with criteria proposed here. In total, 92 met the inclusion criteria, enrolling more than 1,000 patients for each of the major molecular markers of drug resistance. For the drugs presented in this analysis, resistance occurs via two fundamentally different mechanisms. Quinoline resistance is multigenic and epistatic and, at least for chloroquine, affects drug accumulation in the parasite food vacuole 8788. In contrast the underlying molecular mechanism of antifolate resistance involves accumulation of single mutations of the gene encoding for the respective target enzymes 89.

Both pfcrt and pfmdr1 polymorphisms have been associated with chloroquine resistance. The Odds Ratio (OR) of the pfcrt K76T mutation for therapeutic failure after chloroquine exceeded 7.0 at 28 days and 2.0 at day 14. The robustness of this association is confirmed by the high number of null studies (77) required to negate it.

The association between CRT polymorphism and amodiaquine failure has not been adequately addressed. In the analysis presented the pfmdr1 N86Y polymorphism was the most frequently studied mutation and predicted failure to both chloroquine (1.9 (95%CI: 1.3–2.7, p < 0.001)) and amodiaquine (5.4 (95%CI: 2.6 – 11.2, p < 0.001)). However the association of this mutation and clinical response to chloroquine was weak since few null studies would challenge this observation. The predictivity of the combined pfmdr1 + pfcrt was comparable (OR = 3.9 (95%CI: 2.6–5.8)) compared to pfcrt alone and the number needed to nullify this association decreased to 40. However few studies combined both markers.

While the relationship between mutations in the Pfdhfr and Pfdhps genes and parasite resistance to antifolates is well described 90, the relative role of different mutations in either gene in determining treatment outcome is less clear. Although the degree of in vitro resistance and treatment failures to antifolates in this meta-analysis was expected to be proportional to accumulating mutations of Pfdhfr, there was no clear difference in the predictive values of single and triple mutants: OR = 3.5 (95%CI: 1.9–6.3, p < 0.001) and OR = 4.3 (95%CI: 3.0–6.3, p < 0.001) respectively. Most studies failed to analyse the link between mutations at codons 51, 59 and 108. When data were provided on the therapeutic failure rates associated with each of these codons, it was not always possible to carry out a cumulative analysis. The low difference for OR between single and triple mutants suggest that single mutants maybe markers for presence of other point mutations. Due to these limitations, the only predictive value that should be taken into account was the OR for triple mutants. Several other mutant patterns or drug combinations have been studied, but none provided sufficient data to be included in the meta-analysis.

Overall polymorphisms in Pfdhps at positions 437 and 540 were predictive of therapeutic failure (OR = 3.9 (95%CI: 2.6–5.8, p < 0.001), but these data should be considered with caution because of methodological issues with the studies included (different duration of follow-up and different use of genotyping). Pfdhfr + Pfdhps quintuple mutants were analysed from three different studies, providing an OR = 5.2 (95%CI: 3.2–8.8, p < 0.001), with 38 null studies required -suggesting the high predictive value of this composite genotype. It was impossible however to clearly address the question of the predictive role of the increase number of Pfdhfr + Pfdhps mutations since cumulative data from the same patient were rare.

The meta-analysis confirmed and quantified the association of the four genes studied and their underlying associated with the risk of therapeutic failure (Table 1). However there are several caveats. Firstly the resistance of the infecting parasite is only one determinant of treatment outcome. Multiple studies have highlighted the importance of host immunity to the underlying therapeutic efficacy in clinical studies. Such immunity is acquired over time with multiple exposures and thus related to the age of the patient and the transmission intensity 91. Other contributing factors include the biomass of parasites at the start of treatment, the patient's adherence to treatment, the dose of drug used and its adequate absorption 92. There were no enough studies in the present analysis for a subgroup or multivariate analysis incorporating age and other confounding factors, which reduces the power of the analysis to detect independent parasite factors associated with treatment failure.

Second, most of the studies included were conducted in Africa over the past 10 years, limiting the relevance of the conclusions in space and time 93. For instance, during the study period chloroquine resistance was well established, and failure and prevalence of mutations rates were often at saturation, decreasing the power to detect a significant association. In view of the low number of studies meeting inclusion criteria, it was not possible to compare the OR between areas or periods with low mutation rates to areas or periods with mutations close to fixation.

Third, only published studies indexed in PubMed were considered for this meta-analysis, and one cannot exclude a publication bias towards positive studies. However, considering the number of null studies needed to change the data obtained, the effect of unpublished studies is likely to be limited.

Fourth, study methodology varied with respect to inclusion criteria, age of subjects, treatment schedules, PCR methods and reporting, level of transmission at trial site. A frequent reason for excluding a study was insufficient details in the paper to allow coherent data extraction. Moreover, approximately half of the studies included followed patients for only 14 days (Table 1), and as such will not identify late treatment failures, often the earliest manifestation of resistance 94. Genotyping of recurrent infections to distinguish between re-infection and recrudescence was only available in 53% of studies assessed. When the analyses were restricted to studies where true failures could be determined, the ORs varied significantly and power was lost.

Lastly, the proportion of patients studied for molecular markers represents a fraction of those enrolled or analysed at the end of follow-up. As no explanation is given for patient attrition, a selection bias cannot be excluded.

Recent initiatives, such as the consensus meeting on use of genotyping in clinical trials 95 and the World-Wide Antimalarial Resistance Network 96, will hopefully provide guidelines on how to analyse and report field data on clinical, in vitro, molecular and pharmacokinetic determinants of resistance. As a result of these methodological issues, when inclusion criteria were applied, very few studies were eligible for the meta-analysis compared to the number of studies identified. Despite the limitations listed above, the results of this meta-analysis were reassuringly homogeneous (funnel plots were highly symmetrical) for all markers except pfmdr1 + pfcrt for chloroquine.

While the trials considered studied mostly monotherapies with variable degrees of parasite resistance, these results are still relevant now that combinations have become standard treatment of uncomplicated malaria. Amodiaquine and SP are used combined with artesunate and with each other. SP is currently the drug of choice for intermittent preventive treatment (IPT) in pregnancy and infancy. However, data on amodiaquine are limited and the relevance of the genetic mechanisms of resistance of chloroquine to other quinolines (pyronaridine, piperaquine) used in these combinations remains to be established.

With the extended use of combination therapies including old and newer drugs, genetic markers can discriminate the individual role of each component. Obviously more research is needed into the molecular basis of resistance, which are largely unknown especially for artemisin compounds. Early mapping of known and new resistance genes might be achieved by genome-wide scanning of polymorphisms 97.

Whatever the drug to be tested and the mutation to be surveyed, it is of utmost importance to reach a consensus on the methodology of futures studies, especially if comparison between areas and time is the objective of the network of team involved in molecular surveillance of drug resistance. A checklist is proposed here (Table 2), including a series items which need to be fulfilled before designing a study and before preparing data report. This template could be used by colleagues to increase the portability of molecular studies. It could be amended with the experience of experts in the field.

The authors declare no conflict of interest. PO and PR are staff members of the World Health Organization. The authors alone are responsible for the views expressed in this publication and they do not necessarily represent the decisions, policy or views of the World Health Organization.

SP wrote the protocol, selected the studies, performed the meta-analysis, wrote the first draft, and edited the manuscript. FdM reviewed abstracts. ALB reviewed abstracts and full text and controlled the statistics. All authors contributed to the interpretation of the analysis, read and approved the final manuscript.