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Background Therapeutic efficacy studies in uncomplicated Plasmodium falciparum malaria are confounded by new infections, which constitute competing risk events since they can potentially preclude/pre-empt the detection of subsequent recrudescence of persistent, sub-microscopic primary infections. Methods Antimalarial studies typically report the risk of recrudescence derived using the Kaplan–Meier (K–M) method, which considers new infections acquired during the follow-up period as censored. Cumulative Incidence Function (CIF) provides an alternative approach for handling new infections, which accounts for them as a competing risk event. The complement of the estimate derived using the K–M method (1 minus K–M), and the CIF were used to derive the risk of recrudescence at the end of the follow-up period using data from studies collated in the WorldWide Antimalarial Resistance Network data repository. Absolute differences in the failure estimates derived using these two methods were quantified. In comparative studies, the equality of two K–M curves was assessed using the log-rank test, and the equality of CIFs using Gray’s k-sample test (both at 5% level of significance). Two different regression modelling strategies for recrudescence were considered: cause-specific Cox model and Fine and Gray’s sub-distributional hazard model. Results Data were available from 92 studies (233 treatment arms, 31,379 patients) conducted between 1996 and 2014. At the end of follow-up, the median absolute overestimation in the estimated risk of cumulative recrudescence by using 1 minus K–M approach was 0.04% (interquartile range (IQR): 0.00–0.27%, Range: 0.00–3.60%). The overestimation was correlated positively with the proportion of patients with recrudescence [Pearson’s correlation coefficient (ρ): 0.38, 95% Confidence Interval (CI) 0.30–0.46] or new infection [ρ: 0.43; 95% CI 0.35–0.54]. In three study arms, the point estimates of failure were greater than 10% (the WHO threshold for withdrawing antimalarials) when the K–M method was used, but remained below 10% when using the CIF approach, but the 95% confidence interval included this threshold. Conclusions The 1 minus K–M method resulted in a marginal overestimation of recrudescence that became increasingly pronounced as antimalarial efficacy declined, particularly when the observed proportion of new infection was high. The CIF approach provides an alternative approach for derivation of failure estimates in antimalarial trials, particularly in high transmission settings. Electronic supplementary material The online version of this article (10.1186/s12936-019-2837-4) contains supplementary material, which is available to authorized users.

Background The screening of lead compounds against in vitro parasite cultures is an essential step in the development of novel anti-malarial drugs, but currently relies on laboratory parasite lines established in vitro during the last century. This study sought to establish in continuous culture a series of recent Plasmodium falciparum isolates to represent the current parasite populations in Africa, all of which are now exposed to artemisinin combination therapy. Methods Pre-treatment P. falciparum isolates were obtained in EDTA, and placed into continuous culture after sampling of DNA. One post-treatment blood sample was also collected for each donor to monitor parasite clonality during clearance in vivo. IC50 estimates were obtained for 11 anti-malarial compounds for each established parasite line, clonal multiplicity measured in vivo and in vitro, and polymorphic sites implicated in parasite sensitivity to drugs were investigated at the pfmdr1, pfcrt, pfdhfr, pfdhps and pfap2mu loci before and after treatment, and in the cultured lines. Results Plasmodium falciparum isolates from seven malaria patients with recent travel to three West African and two East African countries were successfully established in long-term culture. One of these, HL1211, was from a patient with recrudescent parasitaemia 14 days after a full course of artemether-lumefantrine. All established culture lines were shown to be polyclonal, reflecting the in vivo isolates from which they were derived, and at least two lines reliably produce gametocytes in vitro. Two lines displayed high chloroquine IC50 estimates, and carried the CVIET haplotype at codons 72–76, whereas the remaining five lines carried the CVMNK haplotype and were sensitive in vitro. All were sensitive to the endoperoxides dihydroartemisinin and OZ277, but IC50 estimates for lumefantrine varied, with the least sensitive parasites carrying pfmdr1 alleles encoding Asn at codon 86. Conclusions This study describes the establishment in continuous culture, in vitro drug sensitivity testing and molecular characterization of a series of multiclonal P. falciparum isolates taken directly from UK malaria patients following recent travel to various malaria-endemic countries in Africa. These “HL” isolates are available as an open resource for studies of drug response, antigenic diversity and other aspects of parasite biology.

Background Parasite resistance to the anti-malarial drug chloroquine is common in eastern Sudan. Dynamic within-host changes in the relative abundance of both sensitive and resistant Plasmodium falciparum parasites were examined in a cohort of chloroquine-treated patients presenting with uncomplicated falciparum malaria, using a novel allele-specific quantitative approach. Methods Treatment outcomes were determined for 93 patients of all ages in a per protocol cohort using a modified 14-day WHO protocol. Parasite DNA samples at days 0, 1, 2, 3, 7 and 14 following treatment were analysed using real-time quantitative PCR methods that distinguished resistant and sensitive genotypes at amino acids 72 - 76 of the pfcrt locus. Results Chloroquine treatment was not efficacious, and of 93 assessable patients, only 10 individuals (10.7%; 95% C.I. 4.34 - 17.2%) enjoyed an adequate clinical and parasitological response. Resistant parasites with the haplotype CVIET at codons 72-76 of the pfcrt locus were dominant in the starting population. Chloroquine sensitive parasites with the haplotype CVMNK were detected in 19 individuals prior to treatment (20.43%; 95% C.I. 5.14 - 18.5%). In these patients, CQ treatment rapidly selected CVIET parasites, and this haplotype overwhelmingly dominated the parasite population in each individual by day 2 after treatment. Conclusions Such rapid intra-host selection of particular genotypes after the introduction of drug will cause frequent misidentification of parasite genotypes present in the starting population. This will have a potentially serious confounding effect on clinical trials which employ PCR-corrected estimates of treatment failure, as resistant parasites below the detection threshold in the pre-treatment sample can be erroneously classified as "new" infections during follow-up, over-estimating drug efficacy.