Activity of methylene blue against the asexual ring stages of artemisinin-resistant Plasmodium falciparum

Activity of methylene blue against the asexual ring stages of artemisinin-resistant Plasmodium falciparum | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Activity of methylene blue against the asexual ring stages of artemisinin-resistant Plasmodium falciparum Olivia Verdier, Peter Christensen, Chalita Kaewkanya, Pachinee Kobphan, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8558391/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Artemisinin resistance in Plasmodium falciparum is now well established in three continents and challenges the efficacy of antimalarial drug regimens. This study examined whether methylene blue, an ancient antimalarial drug with a broad spectrum of activity against Plasmodium blood stages, retains its activity against the young rings of artemisinin-resistant parasites. Clinical isolates carrying kelch13 wild type (n = 3), R561H (n = 9), or P441L (n = 10) genotypes were tested with a modified ring survival assay whereby 0- to 3-hour post-invasion rings were exposed to a range of methylene blue concentrations. Ring survival was analysed with a Bayesian mixed effects E max model accounting for variability across isolates and experimental replicates. Methylene blue suppressed ring-stage survival at low nanomolar concentrations with no evidence of kelch13 -mediated cross-resistance: the mean 50% inhibitory concentration (IC 50 ) estimates were 23 nM (95% credible interval [CrI]: 15 to 37) for wild type, 27 nM (95%CrI: 21 to 36) for R561H and 14 nM (95%CrI: 10 to 20) for P441L. These findings indicate that methylene blue remains active in vitro against ring-stage artemisinin-resistant parasites. Methylene blue in vitro sensitivity Plasmodium falciparum artemisinin resistance kelch13 P441L R561H ring survival assay Thailand-Myanmar border Emax model. Figures Figure 1 Figure 2 Introduction Falciparum malaria remains a devastating infectious disease globally, with an estimated 245 million cases and 698,000 deaths in 2022, mostly among children under five and pregnant women in sub-Saharan Africa [ 1 ]. Antimalarial drugs play a crucial role in the treatment of malaria [ 2 ] and artemisinin derivatives have become the cornerstone of antimalarial drug regimens used worldwide [ 3 ]. This is because artemisinins are potent, fast-acting, well-tolerated drugs with a broad activity spectrum extended to circulating young rings (thereby quickly reducing parasitaemia and preventing development of more pathological cytoadhering mature stages) [ 4 ] and immature gametocytes (thereby reducing transmission) [ 5 ]. Artesunate is the treatment of choice for severe malaria [ 6 ]. In a pivotal randomized controlled trial of parenteral artesunate versus quinine in African children, artesunate reduced mortality and the development of coma and convulsions, and caused less post-treatment hypoglycaemia [ 7 ]. Artemisinin resistance, characterised by slower parasite clearance in vivo , has emerged and spread independently in Southeast Asia, East Africa and South America, driven by mutations in the kelch13 propeller domain [ 8 ]. Transcriptomic studies showed that resistance is associated with an altered gene expression profile during the early asexual blood stage cycle, linked with increased stress response and asexual life changes, conferring young ring stages the ability to survive artemisinin exposures and resume their development after drug clearance [ 9 ]. Hence, artemisinin resistance is not detected in standard in vitro drug susceptibility tests, whereby parasites are exposed to low drug concentrations for 48–72 hours [ 10 ]. An alternative phenotypic ring survival assay has been developed whereby synchronized ring stages are exposed to a single high dose of drug for a short time and ring survival is assessed after one cycle of re-invasion [ 11 ]. Methylene blue, the first synthetic antimalarial, is a phenothiazine dye discovered at the end of the 19th century [ 12 ]. Early observation that it can stain alive malaria parasites and concentrates in parasitized red blood cells motivated its assessment in the treatment of malaria [ 13 ]. Methylene blue was rapidly abandoned as synthetic quinolines (e.g. 4-aminoquinoline chloroquine and 8-aminoquinoline primaquine) were discovered and became widely available [ 14 ]. Interestingly, it remained frequently used only in some countries, in particular for rescuing treatment failures with quinine [ 15 ]. Methylene blue was systematically re-evaluated only 100 years later in the context of increasing drug resistance worldwide [ 16 ]. Since its mode of action is different than that of the artemisinins, it could be useful in areas where resistance to the later has emerged. Methylene blue is active against Plasmodium asexual blood stages (including young rings) with 50% inhibitory concentration (IC 50 ) in the nanomolar range against chloroquine and multi-drug resistant parasites, using schizont growth as an outcome [ 17 – 21 ]. Activity against artemisinin-resistant parasites is not well known: only the laboratory-adapted K1 clone and two field isolates were tested in vitro [ 22 , 23 ]. When used alone, parasite clearance is slow and long treatments are needed to cure the infection [ 24 ]. Evidences suggest that methylene blue potentiates the action of quinine, pyrimethamine and artemisinins but not chloroquine [ 19 , 25 ]. Methylene blue has strong and rapid transmission-blocking effects against the gametocytes [ 26 ] and is not active against the liver stages [ 27 ]. This study sought to assess the activity of methylene blue against the ring stage of artemisinin-resistant P. falciparum isolates from the Thai-Myanmar border with a modified ring survival assay capturing the dose-response relationship between drug exposures and parasite survival to test whether kelch13 mutations confer cross-resistance to methylene blue in vitro . Methods Participants and parasite samples A total of 28 P. falciparum isolates were sourced from the cryopreserved collection maintained by the Shoklo Malaria Research Unit, Mae Ramat, Thailand. These isolates originated from patients attending clinics along the Thai-Myanmar border between 2010 and 2014. They were selected based on kelch13 genotype including wild type (n = 3), R561H (n = 9) and P441L (n = 10) because these mutations are now predominant in this area [ 28 ]. The well-characterized culture-adapted NF54 strain was used as a reference to validate the assay. Artemisinin resistance was assessed by determining parasite clearance and kelch13 genotype as described previously [ 28 ]. Compound Proveblue®, a pharmaceutical-grade formulation of methylene blue that shows in vitro antiplasmodial activity against drug-resistant Plasmodium falciparum [ 21 ], was kindly provided by Provepharm (Marseille, France) and used as a 13,370 µM stock solution (methylene blue trihydrate 50 mg/10 mL). Single-use aliquots were made from unopened vials and stored at 4°C protected from light until use. Parasite culture and synchronisation Parasite culture and ring survival assay were performed following procedures described previously, with some modifications to allow for batch testing and assessment of the dose-response [ 11 ]. Incomplete culture medium was composed of RPMI-1640 (Sigma-Aldrich, catalog no. R6504), supplemented with 2 g/L of glucose (Sigma-Aldrich, catalog no. G7528), 2 g/L of NaHCO3 (Sigma-Aldrich, catalog no. S6014), 5.7 g/L of HEPES (Sigma-Aldrich, catalog no. H4034), 18 mg/L of hypoxanthine (Sigma-Aldrich, catalog no. H9636) and 40 mg/L of gentamicin (L.B.S. Laboratory Co. Ltd., registration no. 1A 396/30). Complete culture medium consisted of incomplete medium supplemented with 10% heat-inactivated serum matched to the sample’s blood group. Cultures were maintained at 2–3% haematocrit and incubated at 37°C in a 90% N₂, 5% CO₂, and 5% O₂ atmosphere. Parasite numbers were expanded and partially synchronized with D-sorbitol (Sigma-Aldrich, catalog no. S6021) until ring population predominated before cryopreservation in glycerolyte (Sigma-Aldrich, catalog no. G2025). Parasites were thawed, cultured to schizont stages, purified using a Percoll (Merck, catalog no. P1644) gradient and allowed to invade for 3 hours on fresh red blood cells (RBCs) before sorbitol and washing treatment. The resultant 0- to 3-hour post-invasion rings were used in the ring survival assay. Ring survival assay 0–3 h post-invasion rings were exposed to various methylene blue dilutions (0, 5.14, 10.28, 51.4, 102.8, 257, 514, 1028 nM) in triplicate on 96-well plates (Thermo Scientific, catalog no. AB2800) for six hours. Working dilutions were prepared extemporaneously in culture medium and plates were kept shielded from light to limit photoreduction of methylene blue. Cultures were washed once to remove methylene blue and incubated in fresh medium for an additional sixty-six hours post-exposure. After seventy-two hours total incubation, thin smears were prepared on clean glass slides (Sail Brand, catalog no. 7105), fixed in methanol (VWR international, catalog no. 20847.307), and stained for ten minutes with 10% Giemsa (Merck, catalog no. 1.09204.0500). The proportion of infected red blood cells was assessed by counting the number of viable asexual parasites per 10,000 red blood cells. Pyknotic forms, vacuolated parasites, and gametocytes were excluded. In order to facilitate counting, the average number of red blood cells per field was estimated by counting the number of red blood cells in one representative field of observation, and the value was used to calculate the number of fields to reach at least 10,000 red blood cells. Each slide was read independently by two microscopists, and the average parasite count was used in the analysis. Data analysis In the classical assessment of single-concentration ring survival assay, ring survival (defined as the ratio between the proportion of infected red blood cells in treated wells and the mean proportion of infected red blood cells in the drug-free wells) was analysed under a multi-level logistic regression model including kelch13 genotype as a linear predictor and a random effect across test to account for correlation in ring survival between experimental replicates within the same test. The relative risk was then calculated using the odds ratio estimate of pairwise comparisons between groups and overall survival of wild type parasites. A concentration of 50 nM was used in this assessment because it was the concentration closest to IC 50 in dose-response analysis (see below). In the analysis of dose-response, ring survival was analysed under a Bayesian E max multi-level model taking into account variability across experiments and experimental replicates. Under the model, the likelihood function for the proportion of infected red blood cells (P) was a function of the drug concentration (C), proportion of infected red blood cells in the drug-free control wells (E 0 ), concentration that halves ring survival (IC 50 ) and Hill’s coefficient or shape parameter (H): P = E 0 - C H x E 0 / (C H + IC 50 H ) NF54 and isolate data were analysed separately to account for potential differences in the characteristics of the dose-response between NF54 and wild type isolates and repeated testing of NF54. Isolate data were analysed with a two-level random error structure accounting for variability across tests performed with different isolates and across experimental replicates within the same test, and a fixed-effect of kelch13 genotype parameterized as a proportional variation of population mean IC 50 and H. The model used for the analysis of NF54 data was parameterized similarly, except genotype effects on IC 50 and H were not included, and the 1st level random effects were for variability across independent repeats of the assay with the same parasite rather than variability across tests performed with different isolates. Thus, for each isolate (test) i and technical replicate k, the model parameters E 0i , IC 50i,k and H i,k were expressed as the product of population-level mean (µ E0_population, µ IC50_population, µ H_ population ), isolate dependent random effect (ʎ E0[i], ʎ IC50[i] and ʎ H[i] ) and replicate random effect (ʎ IC50[k] and ʎ H[k] ). The Student-t distribution with 7 degrees of freedom was chosen to accommodate the observed heterogeneity across isolates and replicates: ʎ E0[i] ~ Student-t(7, 1, σ E0_test ), ʎ IC50[i] ~ Student-t(7, 1, σ IC50_test ), ʎ H[i] ~ Student-t(7, 1, σ H_test ), ʎ IC50[k] ~ Student-t(7, 1, σ IC50_replicate ) and ʎ H[k] ~ Student-t(7, 1, σ H_replicate ). For a given isolate, the genotype effect β G[i] was parameterized as a proportional fold-variation in µ IC50_population and µ H_population (β_IC 50G[i] and β_H G[i] , respectively). Thus, the likelihood of the proportion of infected red blood cells P i,k,G[i] (isolate i, technical replicate k, genotype G[i]) given the parameters is: P i,k,G[i] ~ E 0i,k,G[i] – C^H i,k,G[i] * E 0i,k,G[i] / (C^H i,k,G[i] + IC 50i,k,G[i] ) + ε i,k With: E 0i = µ E0_population * ʎ E0[i] IC 50i,k,G[i] = µ IC50_population * ʎ IC50[i] * ʎ IC50 [k] * β_IC50 G[i] H i,k,G[i] = µ H_population * ʎ H[i] * ʎ H[k] * β_H G[i] and residual error term ε i,k ~ Normal(0,1). We used weakly informative priors to help computational convergence. The models were run with 4 independent chains each consisting of 10000 iterations. Convergence of the chains was assessed by examining the values of effective sample size and Rhat and the traceplots (Additional file 1 Figures S1 -S4). Results A total of twenty-five ring survival assay experiments were conducted with three “wild type”, nine R561H and ten P441L isolates together with three experiment repeats carried out with NF54 (Additional file 1 Table S1 ). Mean survival after 50 nM exposures was 9%, 13%, and 37% for wild type, R561H, and P441L isolates, respectively. Median survival was 24% (IQR 22–42; range 11–46) for R561H and 12% (IQR 5–17; range 0.4–25) for P441L mutants (Fig. 1). The logistic mixed-effects model explained most of the data variability (conditional R² = 0.97; marginal R² = 0.39). Post-hoc comparisons indicated that ring survival was significantly higher in R561H than in P441L mutants (relative risk [RR] 1.99, 95% CI 1.42–2.57), but not significantly different between “wild type” and either mutant group (wild type vs R561H RR 0.66, 95% CI 0.32–1.26; wild type vs P441L RR 1.49, 95% CI 0.81–2.32). For NF54, the observed mean survival across three independent experiments was 5% (SD 1.3%), and the model estimate was 4.7% (95% CI 3.7-6.0). Figure 1. Distribution of observed ring survival in the ring survival assay at a single methylene blue concentration of 50 nM across experimental groups (NF54 reference strain and clinical isolates with different kelch13 genotypes). Parameter estimates for the Bayesian multilevel E max models are presented in Table 1 . In the isolate dataset, the model estimated a population mean IC 50 of 23 nM (95% CrI 15–37) and a Hill coefficient of 2.2 (1.4–4.1). Methylene blue maintained low-nanomolar activity across all kelch13 genotypes (Table 2 ). R561H isolates were slightly less susceptible than wild type, whereas P441L mutants were marginally more sensitive. These differences were small, and only the contrast between R561H and P441L reached statistical credibility (Additional file 1 Table S2). The NF54 reference strain showed comparable parameters (IC 50 17 nM, 95% CrI 11–23; H 2.7 [1.8–4.1]). The model described the data well (Fig. 2), and assay variability was low (Additional file 1 Figure S5), with IC 50 and H values varying by less than one-fold across isolates and replicates. Table 1 Parameter estimates given by the output of Bayesian multilevel Emax models using in the analysis of clinical isolate and NF54 data. Parameter Clinical isolates NF54 Median of posterior draws 95%CrI Median of posterior draws 95%CrI µ E0_population 1.16 0.81 to 1.52 6.28 4.08 to 8.36 σ E0_test 0.65 0.42 to 1.14 0.16 0.05 to 0.98 µ IC50_population 23.2 14.8 to 37.2 16.6 10.8 to 22.7 σ IC50_test 0.33 0.19 to 0.57 0.12 0.01 to 0.98 σ IC50_replicate 0.14 0.08 to 0.23 0.12 0.04 to 0.28 µ H_population 2.19 1.41 to 4.06 2.75 1.78 to 4.09 σ H_test 0.2 0.06 to 0.4 0.13 0.01 to 1.06 σ H_replicate 0.15 0.08 to 0.25 0.09 0.01 to 0.33 β_IC 50 wild-type 1 reference - - β_IC 50 R561H 1.17 0.69 to 1.92 - - β_IC 50 P441L 0.6 0.34 to 1.06 - - β_H wild-type 1 reference - - β_H R561H 0.67 0.36 to 1.1 - - β_H P441L 0.76 0.39 to 1.31 - - σ residual 0.14 0.13 to 0.15 0.18 0.15 to 0.22 Figure 2. Concentration-response relationship between methylene blue concentration and ring survival in the ring survival assay. (A) NF54 reference strain; (B) wild type isolates, (C) R561H isolates and (D) P441L isolates. The grey dots show the observed values and the coloured-line and shaded area show the model-fitted relationship plotted using µ IC50_population , µ H_population , β_IC 50G[i] and β_H G[i] estimates given by the model output and the corresponding 95% credible interval, respectively. Table 2 Characteristics of the dose-response relationship between exposure to methylene blue and ring survival in the ring survival assay for clinical isolates and NF54. Experimental group IC 50 (nM) a H a ED90 (nM) a ED99 (nM) a Wild type isolates 23 (15 to 37) 2.2 (1.4 to 4.1) 64 (34 to 124) 190 (68 to 625) R561H isolates 27 (21 to 36) 1.5 (1.2 to 1.8) 121 (82 to 179) 610 (320 to 1230) P441L isolates 14 (10 to 20) 1.7 (1.2 to 2.3) 52 (31 to 89) 218 (94 to 565) NF54 17 (11 to 23) 2.7 (1.8 to 4.1) 37 (22 to 63) 88 (44 to 221) a Values are median of posterior draws and 95% credible interval. Discussion This study demonstrates that methylene blue exhibits potent in vitro activity against both artemisinin-sensitive and resistant P. falciparum isolates from the Thai-Myanmar border, with IC 50 values in the nanomolar range using ring survival as the outcome. Although minor differences in susceptibility were detected between kelch13 genotypes (R561H mutants showing slightly higher IC 50 and ED 90 values than P441L mutants), the magnitude of these differences was small and unlikely to be biologically significant. These findings support the hypothesis that methylene blue retains efficacy against ring-stage artemisinin-resistant parasites and could be considered as a complementary or partner component in artemisinin therapies. Although methylene blue should be used with caution in individuals with glucose-6-phosphate dehydrogenase deficiency and during pregnancy because of the risk of haemolytic anaemia, it is already licensed for the treatment of methaemoglobinemia and used in other medical contexts (surgical staining, oncology), facilitating potential repurposing as an antimalarial [ 16 ]. Use of methylene blue for the chemotherapy of malaria requires careful consideration of its pharmacodynamic and pharmacokinetic properties. Methylene blue has a t max of ~ 2 hours after oral administration and a half-life of ~ 6 hours, comparable to that of artemisinins. Similar dosing gives higher C max with parenteral than oral formulation [ 29 ]. It is slow-acting in vivo and seven-day treatments (4 asexual cycles) are needed to kill all parasites [ 24 ]. Therefore, it is probably not valuable as an adjuvant to artemisinin-combination therapies used for the oral treatment of uncomplicated malaria. This statement is further supported by the similar parasite clearance rate and cure rate when comparing artesunate-amodiaquine versus artesunate-amodiaquine- methylene blue [ 30 ]. However, parenteral methylene blue combined with artesunate may be useful in the treatment of severe malaria in area where artemisinin-resistance is prevalent. Methylene blue activity against artemisinin-resistant ring stages could restore the benefit of artesunate over quinine in artemisinin-resistant infections but whether this holds in vivo warrants further investigation. Methylene blue effects on cytoadherence and rosetting have not been assessed but observations from recent primate models suggest this mechanism could also contribute [ 31 ]. The most plausible explanation for the consistent methylene blue activity across kelch13 genotypes lies in its redox-targeting effects distinct from artemisinin’s mechanisms of action, which likely explains the absence of cross-resistance. The IC 50 values estimated in this modified ring survival assay are higher than reported in previous studies using standard in vitro and ex vivo assays. The higher IC 50 estimates observed here are most likely explained by assay design rather than by reduced intrinsic susceptibility. Importantly, there is no clear methylene blue resistance in the field to date and the potential for selecting de novo resistance mutations under conditions of sublethal exposures is deemed low [ 32 ]. A strength of this study is the use of a modified ring survival assay protocol combined with Bayesian multilevel modelling, which allowed assessment of the dose-response while accounting for experimental variability thereby making the results more robust to outliers and increasing statistical power for detecting significant effects of genotype on parasite susceptibility [ 33 ]. This approach strengthens confidence in the inference of genotype effects on parasite susceptibility to methylene blue. The inclusion of reference strain NF54 adds external validity and supports the reproducibility of the results. Certain limitations should be acknowledged. The sample size was small and inter-experiment variability arising from random errors across repeated testing was not taken into account. Only two kelch13 mutations were assessed (R561H and P441L). These alleles were chosen because they are now predominant in Karen state (Eastern Myanmar) [ 28 ] but further evaluation against other resistant alleles, including C580Y and F446I, would provide a more comprehensive picture. In addition, in vitro effects on parasite growth do not necessarily correlate with parasite clearance in vivo , especially considering the central role of the spleen in removing damaged parasites and red blood cells from the circulation after drug exposures [ 34 ]. Future randomised trials should evaluate whether short course parenteral methylene blue added to standard intravenous artesunate can safely enhance parasite clearance and reduce mortality in severe falciparum malaria. Such trials would be particularly relevant in areas of East Africa where malaria incidence and mortality are high and artemisinin resistance is firmly established. Conclusions Methylene blue retains potent activity against artemisinin-resistant P. falciparum isolates and shows no evidence of kelch13- mediated cross-resistance. While its pharmacodynamic profile makes it unsuitable for the oral treatment of uncomplicated malaria, the compound may have value as an intravenous adjunct in the treatment of severe infection in settings where artemisinin resistance reduces the efficacy of standard therapies. Abbreviations CI confidence interval CrI Credible interval IC inhibitory concentration IQR interquartile range RR relative risk SD standard deviation Declarations Ethics approval and consent to participate The study was approved by the Oxford Tropical Research Ethics Committee (OxTREC, number 28 − 09). All participants provided their written informed consent to participate in the study. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Funding This research was funded in full by a Wellcome grant (#220211). Author Contribution OV performed ring survival assay experiments and wrote the initial draft of the manuscript. CK, PK performed ring survival assay experiments. PC developed assay methodology, performed ring survival assay experiments, supervised the laboratory staff and reviewed and edited the manuscript. APP and CB collected the samples. VC designed the study, administered the project, developed assay methodology, supervised the laboratory staff, analysed the data and wrote the initial draft of the manuscript. MI genotyped the parasites. KC designed the study, provided the NF54 parasite and reviewed and edited the manuscript. NJW designed the study and acquired funding. FN designed the study, acquired funding, administered the project and reviewed and edited the manuscript. All authors read and approved the final manuscript. Acknowledgement We are very grateful to the volunteers who participated in this study. We thank the staff of the Laboratory, Medical and Data Management Departments of the Shoklo Malaria Research Unit, NHP study investigators, and the staff of the MORU Malaria Molecular and Malaria In Vitro labs for their help with samples and data collection, processing and management. We thank Provepharm (Marseille, France) for providing Proveblue®. The Shoklo Malaria Research Unit is part of the Mahidol-Oxford Research Unit, supported by Wellcome (U.K.). A CC BY or equivalent licence is applied to the author accepted manuscript arising from this submission, in accordance with the grant’s open access conditions. Data Availability All analysis code and data are available via an accompanying github repository: [https://github.com/victorSMRU/methylene-blue-rsa](https:/github.com/victorSMRU/methylene-blue-rsa) References Weiss DJ, Dzianach PA, Saddler A, Lubinda J, Browne A, McPhail M, et al. Mapping the global prevalence, incidence, and mortality of Plasmodium falciparum and Plasmodium vivax malaria, 2000-22: a spatial and temporal modelling study. Lancet. 2025. https://doi.org/10.1016/S0140-6736(25)00038-8 . S0140-6736(25)00038 – 8. White NJ. The role of anti-malarial drugs in eliminating malaria. Malar J Engl. 2008;7(Suppl 1):S8. https://doi.org/10.1186/1475-2875-7-S1-S8 . White NJ. Qinghaosu (artemisinin): the price of success. 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Nat Commun Nat Publishing Group. 2025;16:10534. https://doi.org/10.1038/s41467-025-65552-y . Thurston JP. The chemotherapy of Plasmodium berghei . I. Resistance to drugs. Parasitology. 1953;43:246–52. https://doi.org/10.1017/s0031182000018618 . Simpson JA, Jamsen KM, Anderson TJC, Zaloumis S, Nair S, Woodrow C, et al. Nonlinear mixed-effects modelling of in vitro drug susceptibility and molecular correlates of multidrug resistant Plasmodium falciparum . PLoS One United States. 2013;8:e69505. https://doi.org/10.1371/journal.pone.0069505 . White NJ. Malaria parasite clearance. Malar J Engl. 2017;16:88. https://doi.org/10.1186/s12936-017-1731-1 . Additional Declarations No competing interests reported. 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07:44:37","extension":"html","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":115952,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8558391/v1/499ce4b8b3bd28a8af6593e8.html"},{"id":101397620,"identity":"239c70cf-25d5-4570-be45-dee2ca5d27bb","added_by":"auto","created_at":"2026-01-29 09:32:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":80351,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of observed ring survival in the ring survival assay at a single methylene blue concentration of 50 nM across experimental groups (NF54 reference strain and clinical isolates with different \u003cem\u003ekelch13\u003c/em\u003egenotypes).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8558391/v1/50ca3f8d5477638fc1aa9488.png"},{"id":100957036,"identity":"886f8d1a-2614-4f6b-9829-61a56e18c519","added_by":"auto","created_at":"2026-01-23 07:44:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":380493,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConcentration-response relationship between methylene blue concentration and ring survival in the ring survival assay. \u003c/strong\u003e(A) NF54 reference strain; (B) wild type isolates, (C) R561H isolates and (D) P441L isolates. The grey dots show the observed values and the coloured-line and shaded area show the model-fitted relationship plotted using μ\u003csub\u003eIC50_population\u003c/sub\u003e, μ\u003csub\u003eH_population\u003c/sub\u003e, β_IC\u003csub\u003e50G[i] \u003c/sub\u003eand β_H\u003csub\u003eG[i] \u003c/sub\u003eestimates given by the model output and the corresponding 95% credible interval, respectively.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8558391/v1/fe9a3f2572cf4e53fd3eae9e.png"},{"id":101398840,"identity":"efafb300-d358-42b4-a29d-135ddead9301","added_by":"auto","created_at":"2026-01-29 09:49:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1352371,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8558391/v1/aa730376-9579-43a5-a1d2-e6bb0b85997d.pdf"},{"id":101297239,"identity":"aaaa57df-e640-4340-be13-9910218015e5","added_by":"auto","created_at":"2026-01-28 09:26:06","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":396001,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8558391/v1/6520968d268945dd005778a8.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Activity of methylene blue against the asexual ring stages of artemisinin-resistant Plasmodium falciparum","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFalciparum malaria remains a devastating infectious disease globally, with an estimated 245\u0026nbsp;million cases and 698,000 deaths in 2022, mostly among children under five and pregnant women in sub-Saharan Africa [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAntimalarial drugs play a crucial role in the treatment of malaria [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] and artemisinin derivatives have become the cornerstone of antimalarial drug regimens used worldwide [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This is because artemisinins are potent, fast-acting, well-tolerated drugs with a broad activity spectrum extended to circulating young rings (thereby quickly reducing parasitaemia and preventing development of more pathological cytoadhering mature stages) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and immature gametocytes (thereby reducing transmission) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Artesunate is the treatment of choice for severe malaria [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In a pivotal randomized controlled trial of parenteral artesunate \u003cem\u003eversus\u003c/em\u003e quinine in African children, artesunate reduced mortality and the development of coma and convulsions, and caused less post-treatment hypoglycaemia [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eArtemisinin resistance, characterised by slower parasite clearance \u003cem\u003ein vivo\u003c/em\u003e, has emerged and spread independently in Southeast Asia, East Africa and South America, driven by mutations in the \u003cem\u003ekelch13\u003c/em\u003e propeller domain [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Transcriptomic studies showed that resistance is associated with an altered gene expression profile during the early asexual blood stage cycle, linked with increased stress response and asexual life changes, conferring young ring stages the ability to survive artemisinin exposures and resume their development after drug clearance [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Hence, artemisinin resistance is not detected in standard \u003cem\u003ein vitro\u003c/em\u003e drug susceptibility tests, whereby parasites are exposed to low drug concentrations for 48\u0026ndash;72 hours [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. An alternative phenotypic ring survival assay has been developed whereby synchronized ring stages are exposed to a single high dose of drug for a short time and ring survival is assessed after one cycle of re-invasion [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMethylene blue, the first synthetic antimalarial, is a phenothiazine dye discovered at the end of the 19th century [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Early observation that it can stain alive malaria parasites and concentrates in parasitized red blood cells motivated its assessment in the treatment of malaria [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Methylene blue was rapidly abandoned as synthetic quinolines (e.g. 4-aminoquinoline chloroquine and 8-aminoquinoline primaquine) were discovered and became widely available [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Interestingly, it remained frequently used only in some countries, in particular for rescuing treatment failures with quinine [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Methylene blue was systematically re-evaluated only 100 years later in the context of increasing drug resistance worldwide [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Since its mode of action is different than that of the artemisinins, it could be useful in areas where resistance to the later has emerged.\u003c/p\u003e \u003cp\u003eMethylene blue is active against \u003cem\u003ePlasmodium\u003c/em\u003e asexual blood stages (including young rings) with 50% inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) in the nanomolar range against chloroquine and multi-drug resistant parasites, using schizont growth as an outcome [\u003cspan additionalcitationids=\"CR18 CR19 CR20\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Activity against artemisinin-resistant parasites is not well known: only the laboratory-adapted K1 clone and two field isolates were tested \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. When used alone, parasite clearance is slow and long treatments are needed to cure the infection [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Evidences suggest that methylene blue potentiates the action of quinine, pyrimethamine and artemisinins but not chloroquine [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Methylene blue has strong and rapid transmission-blocking effects against the gametocytes [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and is not active against the liver stages [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study sought to assess the activity of methylene blue against the ring stage of artemisinin-resistant \u003cem\u003eP. falciparum\u003c/em\u003e isolates from the Thai-Myanmar border with a modified ring survival assay capturing the dose-response relationship between drug exposures and parasite survival to test whether \u003cem\u003ekelch13\u003c/em\u003e mutations confer cross-resistance to methylene blue \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eParticipants and parasite samples\u003c/h2\u003e \u003cp\u003eA total of 28 \u003cem\u003eP. falciparum\u003c/em\u003e isolates were sourced from the cryopreserved collection maintained by the Shoklo Malaria Research Unit, Mae Ramat, Thailand. These isolates originated from patients attending clinics along the Thai-Myanmar border between 2010 and 2014. They were selected based on \u003cem\u003ekelch13\u003c/em\u003e genotype including wild type (n\u0026thinsp;=\u0026thinsp;3), R561H (n\u0026thinsp;=\u0026thinsp;9) and P441L (n\u0026thinsp;=\u0026thinsp;10) because these mutations are now predominant in this area [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The well-characterized culture-adapted NF54 strain was used as a reference to validate the assay. Artemisinin resistance was assessed by determining parasite clearance and \u003cem\u003ekelch13\u003c/em\u003e genotype as described previously [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCompound\u003c/h3\u003e\n\u003cp\u003eProveblue\u0026reg;, a pharmaceutical-grade formulation of methylene blue that shows \u003cem\u003ein vitro\u003c/em\u003e antiplasmodial activity against drug-resistant \u003cem\u003ePlasmodium falciparum\u003c/em\u003e [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], was kindly provided by Provepharm (Marseille, France) and used as a 13,370 \u0026micro;M stock solution (methylene blue trihydrate 50 mg/10 mL). Single-use aliquots were made from unopened vials and stored at 4\u0026deg;C protected from light until use.\u003c/p\u003e\n\u003ch3\u003eParasite culture and synchronisation\u003c/h3\u003e\n\u003cp\u003eParasite culture and ring survival assay were performed following procedures described previously, with some modifications to allow for batch testing and assessment of the dose-response [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Incomplete culture medium was composed of RPMI-1640 (Sigma-Aldrich, catalog no. R6504), supplemented with 2 g/L of glucose (Sigma-Aldrich, catalog no. G7528), 2 g/L of NaHCO3 (Sigma-Aldrich, catalog no. S6014), 5.7 g/L of HEPES (Sigma-Aldrich, catalog no. H4034), 18 mg/L of hypoxanthine (Sigma-Aldrich, catalog no. H9636) and 40 mg/L of gentamicin (L.B.S. Laboratory Co. Ltd., registration no. 1A 396/30). Complete culture medium consisted of incomplete medium supplemented with 10% heat-inactivated serum matched to the sample\u0026rsquo;s blood group. Cultures were maintained at 2\u0026ndash;3% haematocrit and incubated at 37\u0026deg;C in a 90% N₂, 5% CO₂, and 5% O₂ atmosphere. Parasite numbers were expanded and partially synchronized with D-sorbitol (Sigma-Aldrich, catalog no. S6021) until ring population predominated before cryopreservation in glycerolyte (Sigma-Aldrich, catalog no. G2025). Parasites were thawed, cultured to schizont stages, purified using a Percoll (Merck, catalog no. P1644) gradient and allowed to invade for 3 hours on fresh red blood cells (RBCs) before sorbitol and washing treatment. The resultant 0- to 3-hour post-invasion rings were used in the ring survival assay.\u003c/p\u003e\n\u003ch3\u003eRing survival assay\u003c/h3\u003e\n\u003cp\u003e0\u0026ndash;3 h post-invasion rings were exposed to various methylene blue dilutions (0, 5.14, 10.28, 51.4, 102.8, 257, 514, 1028 nM) in triplicate on 96-well plates (Thermo Scientific, catalog no. AB2800) for six hours. Working dilutions were prepared extemporaneously in culture medium and plates were kept shielded from light to limit photoreduction of methylene blue. Cultures were washed once to remove methylene blue and incubated in fresh medium for an additional sixty-six hours post-exposure. After seventy-two hours total incubation, thin smears were prepared on clean glass slides (Sail Brand, catalog no. 7105), fixed in methanol (VWR international, catalog no. 20847.307), and stained for ten minutes with 10% Giemsa (Merck, catalog no. 1.09204.0500). The proportion of infected red blood cells was assessed by counting the number of viable asexual parasites per 10,000 red blood cells. Pyknotic forms, vacuolated parasites, and gametocytes were excluded. In order to facilitate counting, the average number of red blood cells per field was estimated by counting the number of red blood cells in one representative field of observation, and the value was used to calculate the number of fields to reach at least 10,000 red blood cells. Each slide was read independently by two microscopists, and the average parasite count was used in the analysis.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eIn the classical assessment of single-concentration ring survival assay, ring survival (defined as the ratio between the proportion of infected red blood cells in treated wells and the mean proportion of infected red blood cells in the drug-free wells) was analysed under a multi-level logistic regression model including \u003cem\u003ekelch13\u003c/em\u003e genotype as a linear predictor and a random effect across test to account for correlation in ring survival between experimental replicates within the same test. The relative risk was then calculated using the odds ratio estimate of pairwise comparisons between groups and overall survival of wild type parasites. A concentration of 50 nM was used in this assessment because it was the concentration closest to IC\u003csub\u003e50\u003c/sub\u003e in dose-response analysis (see below).\u003c/p\u003e \u003cp\u003eIn the analysis of dose-response, ring survival was analysed under a Bayesian E\u003csub\u003emax\u003c/sub\u003e multi-level model taking into account variability across experiments and experimental replicates. Under the model, the likelihood function for the proportion of infected red blood cells (P) was a function of the drug concentration (C), proportion of infected red blood cells in the drug-free control wells (E\u003csub\u003e0\u003c/sub\u003e), concentration that halves ring survival (IC\u003csub\u003e50\u003c/sub\u003e) and Hill\u0026rsquo;s coefficient or shape parameter (H):\u003c/p\u003e \u003cp\u003eP\u0026thinsp;=\u0026thinsp;E\u003csub\u003e0\u003c/sub\u003e - C\u003csup\u003eH\u003c/sup\u003e x E\u003csub\u003e0\u003c/sub\u003e / (C\u003csup\u003eH\u003c/sup\u003e + IC\u003csub\u003e50\u003c/sub\u003e\u003csup\u003eH\u003c/sup\u003e)\u003c/p\u003e \u003cp\u003eNF54 and isolate data were analysed separately to account for potential differences in the characteristics of the dose-response between NF54 and wild type isolates and repeated testing of NF54. Isolate data were analysed with a two-level random error structure accounting for variability across tests performed with different isolates and across experimental replicates within the same test, and a fixed-effect of \u003cem\u003ekelch13\u003c/em\u003e genotype parameterized as a proportional variation of population mean IC\u003csub\u003e50\u003c/sub\u003e and H. The model used for the analysis of NF54 data was parameterized similarly, except genotype effects on IC\u003csub\u003e50\u003c/sub\u003e and H were not included, and the 1st level random effects were for variability across independent repeats of the assay with the same parasite rather than variability across tests performed with different isolates.\u003c/p\u003e \u003cp\u003eThus, for each isolate (test) i and technical replicate k, the model parameters E\u003csub\u003e0i\u003c/sub\u003e, IC\u003csub\u003e50i,k\u003c/sub\u003e and H\u003csub\u003ei,k\u003c/sub\u003e were expressed as the product of population-level mean (\u0026micro;\u003csub\u003eE0_population,\u003c/sub\u003e \u0026micro;\u003csub\u003eIC50_population,\u003c/sub\u003e \u0026micro;\u003csub\u003eH_ population\u003c/sub\u003e), isolate dependent random effect (ʎ\u003csub\u003eE0[i],\u003c/sub\u003e ʎ\u003csub\u003eIC50[i]\u003c/sub\u003e and ʎ\u003csub\u003eH[i]\u003c/sub\u003e) and replicate random effect (ʎ\u003csub\u003eIC50[k]\u003c/sub\u003e and ʎ\u003csub\u003eH[k]\u003c/sub\u003e). The Student-t distribution with 7 degrees of freedom was chosen to accommodate the observed heterogeneity across isolates and replicates: ʎ\u003csub\u003eE0[i]\u003c/sub\u003e\u0026thinsp;~\u0026thinsp;Student-t(7, 1, σ\u003csub\u003eE0_test\u003c/sub\u003e), ʎ\u003csub\u003eIC50[i]\u003c/sub\u003e\u0026thinsp;~\u0026thinsp;Student-t(7, 1, σ\u003csub\u003eIC50_test\u003c/sub\u003e), ʎ\u003csub\u003eH[i]\u003c/sub\u003e\u0026thinsp;~\u0026thinsp;Student-t(7, 1, σ\u003csub\u003eH_test\u003c/sub\u003e), ʎ\u003csub\u003eIC50[k]\u003c/sub\u003e\u0026thinsp;~\u0026thinsp;Student-t(7, 1, σ\u003csub\u003eIC50_replicate\u003c/sub\u003e) and ʎ\u003csub\u003eH[k]\u003c/sub\u003e\u0026thinsp;~\u0026thinsp;Student-t(7, 1, σ\u003csub\u003eH_replicate\u003c/sub\u003e). For a given isolate, the genotype effect β\u003csub\u003eG[i]\u003c/sub\u003e was parameterized as a proportional fold-variation in \u0026micro;\u003csub\u003eIC50_population\u003c/sub\u003e and \u0026micro;\u003csub\u003eH_population\u003c/sub\u003e (β_IC\u003csub\u003e50G[i]\u003c/sub\u003e and β_H\u003csub\u003eG[i]\u003c/sub\u003e, respectively). Thus, the likelihood of the proportion of infected red blood cells P\u003csub\u003ei,k,G[i]\u003c/sub\u003e (isolate i, technical replicate k, genotype G[i]) given the parameters is:\u003c/p\u003e \u003cp\u003eP\u003csub\u003ei,k,G[i]\u003c/sub\u003e\u0026thinsp;~\u0026thinsp;E\u003csub\u003e0i,k,G[i]\u003c/sub\u003e \u0026ndash; C^H\u003csub\u003ei,k,G[i]\u003c/sub\u003e * E\u003csub\u003e0i,k,G[i]\u003c/sub\u003e / (C^H\u003csub\u003ei,k,G[i]\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;IC\u003csub\u003e50i,k,G[i]\u003c/sub\u003e) + ε\u003csub\u003ei,k\u003c/sub\u003e\u003c/p\u003e \u003cp\u003eWith:\u003c/p\u003e \u003cp\u003eE\u003csub\u003e0i\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;\u0026micro;\u003csub\u003eE0_population\u003c/sub\u003e * ʎ\u003csub\u003eE0[i]\u003c/sub\u003e\u003c/p\u003e \u003cp\u003eIC\u003csub\u003e50i,k,G[i]\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;\u0026micro;\u003csub\u003eIC50_population\u003c/sub\u003e * ʎ\u003csub\u003eIC50[i]\u003c/sub\u003e * ʎ \u003csub\u003eIC50 [k]\u003c/sub\u003e * β_IC50\u003csub\u003eG[i]\u003c/sub\u003e\u003c/p\u003e \u003cp\u003eH\u003csub\u003ei,k,G[i]\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;\u0026micro;\u003csub\u003eH_population\u003c/sub\u003e * ʎ\u003csub\u003eH[i]\u003c/sub\u003e * ʎ\u003csub\u003eH[k]\u003c/sub\u003e * β_H\u003csub\u003eG[i]\u003c/sub\u003e\u003c/p\u003e \u003cp\u003eand residual error term ε\u003csub\u003ei,k\u003c/sub\u003e\u0026thinsp;~\u0026thinsp;Normal(0,1).\u003c/p\u003e \u003cp\u003eWe used weakly informative priors to help computational convergence. The models were run with 4 independent chains each consisting of 10000 iterations. Convergence of the chains was assessed by examining the values of effective sample size and Rhat and the traceplots (Additional file 1 Figures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-S4).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eA total of twenty-five ring survival assay experiments were conducted with three \u0026ldquo;wild type\u0026rdquo;, nine R561H and ten P441L isolates together with three experiment repeats carried out with NF54 (Additional file 1 Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Mean survival after 50 nM exposures was 9%, 13%, and 37% for wild type, R561H, and P441L isolates, respectively. Median survival was 24% (IQR 22\u0026ndash;42; range 11\u0026ndash;46) for R561H and 12% (IQR 5\u0026ndash;17; range 0.4\u0026ndash;25) for P441L mutants (Fig.\u0026nbsp;1). The logistic mixed-effects model explained most of the data variability (conditional R\u0026sup2; = 0.97; marginal R\u0026sup2; = 0.39). Post-hoc comparisons indicated that ring survival was significantly higher in R561H than in P441L mutants (relative risk [RR] 1.99, 95% CI 1.42\u0026ndash;2.57), but not significantly different between \u0026ldquo;wild type\u0026rdquo; and either mutant group (wild type vs R561H RR 0.66, 95% CI 0.32\u0026ndash;1.26; wild type vs P441L RR 1.49, 95% CI 0.81\u0026ndash;2.32). For NF54, the observed mean survival across three independent experiments was 5% (SD 1.3%), and the model estimate was 4.7% (95% CI 3.7-6.0).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 1. Distribution of observed ring survival in the ring survival assay at a single methylene blue concentration of 50 nM across experimental groups (NF54 reference strain and clinical isolates with different\u003c/b\u003e \u003cb\u003ekelch13\u003c/b\u003e \u003cb\u003egenotypes).\u003c/b\u003e\u003c/p\u003e \u003cp\u003eParameter estimates for the Bayesian multilevel E\u003csub\u003emax\u003c/sub\u003e models are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. In the isolate dataset, the model estimated a population mean IC\u003csub\u003e50\u003c/sub\u003e of 23 nM (95% CrI 15\u0026ndash;37) and a Hill coefficient of 2.2 (1.4\u0026ndash;4.1). Methylene blue maintained low-nanomolar activity across all \u003cem\u003ekelch13\u003c/em\u003e genotypes (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). R561H isolates were slightly less susceptible than wild type, whereas P441L mutants were marginally more sensitive. These differences were small, and only the contrast between R561H and P441L reached statistical credibility (Additional file 1 Table S2). The NF54 reference strain showed comparable parameters (IC\u003csub\u003e50\u003c/sub\u003e 17 nM, 95% CrI 11\u0026ndash;23; H 2.7 [1.8\u0026ndash;4.1]). The model described the data well (Fig.\u0026nbsp;2), and assay variability was low (Additional file 1 Figure S5), with IC\u003csub\u003e50\u003c/sub\u003e and H values varying by less than one-fold across isolates and replicates.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eParameter estimates given by the output of Bayesian multilevel Emax models using in the analysis of clinical isolate and NF54 data.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eClinical isolates\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eNF54\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedian of posterior draws\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e95%CrI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMedian of posterior draws\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e95%CrI\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026micro;\u003csub\u003eE0_population\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.81 to 1.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.08 to 8.36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eσ\u003csub\u003eE0_test\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.42 to 1.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.05 to 0.98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026micro;\u003csub\u003eIC50_population\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.8 to 37.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.8 to 22.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eσ\u003csub\u003eIC50_test\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.19 to 0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.01 to 0.98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eσ\u003csub\u003eIC50_replicate\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.08 to 0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.04 to 0.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026micro;\u003csub\u003eH_population\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.41 to 4.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.78 to 4.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eσ\u003csub\u003eH_test\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.06 to 0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.01 to 1.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eσ\u003csub\u003eH_replicate\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.08 to 0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.01 to 0.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ_IC\u003csub\u003e50 wild-type\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ereference\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ_IC\u003csub\u003e50 R561H\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.69 to 1.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ_IC\u003csub\u003e50 P441L\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.34 to 1.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ_H\u003csub\u003ewild-type\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ereference\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ_H\u003csub\u003eR561H\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.36 to 1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eβ_H\u003csub\u003eP441L\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.39 to 1.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eσ\u003csub\u003eresidual\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.13 to 0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.15 to 0.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 2. Concentration-response relationship between methylene blue concentration and ring survival in the ring survival assay.\u003c/b\u003e (A) NF54 reference strain; (B) wild type isolates, (C) R561H isolates and (D) P441L isolates. The grey dots show the observed values and the coloured-line and shaded area show the model-fitted relationship plotted using \u0026micro;\u003csub\u003eIC50_population\u003c/sub\u003e, \u0026micro;\u003csub\u003eH_population\u003c/sub\u003e, β_IC\u003csub\u003e50G[i]\u003c/sub\u003e and β_H\u003csub\u003eG[i]\u003c/sub\u003e estimates given by the model output and the corresponding 95% credible interval, respectively.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacteristics of the dose-response relationship between exposure to methylene blue and ring survival in the ring survival assay for clinical isolates and NF54.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExperimental group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e (nM) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eH \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eED90 (nM) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eED99 (nM) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWild type isolates\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23 (15 to 37)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.2 (1.4 to 4.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e64 (34 to 124)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e190 (68 to 625)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eR561H isolates\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27 (21 to 36)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.5 (1.2 to 1.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e121 (82 to 179)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e610 (320 to 1230)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP441L isolates\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14 (10 to 20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.7 (1.2 to 2.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52 (31 to 89)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e218 (94 to 565)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNF54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17 (11 to 23)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.7 (1.8 to 4.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e37 (22 to 63)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e88 (44 to 221)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003ea\u003c/sup\u003e Values are median of posterior draws and 95% credible interval.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study demonstrates that methylene blue exhibits potent \u003cem\u003ein vitro\u003c/em\u003e activity against both artemisinin-sensitive and resistant \u003cem\u003eP. falciparum\u003c/em\u003e isolates from the Thai-Myanmar border, with IC\u003csub\u003e50\u003c/sub\u003e values in the nanomolar range using ring survival as the outcome. Although minor differences in susceptibility were detected between \u003cem\u003ekelch13\u003c/em\u003e genotypes (R561H mutants showing slightly higher IC\u003csub\u003e50\u003c/sub\u003e and ED\u003csub\u003e90\u003c/sub\u003e values than P441L mutants), the magnitude of these differences was small and unlikely to be biologically significant. These findings support the hypothesis that methylene blue retains efficacy against ring-stage artemisinin-resistant parasites and could be considered as a complementary or partner component in artemisinin therapies. Although methylene blue should be used with caution in individuals with glucose-6-phosphate dehydrogenase deficiency and during pregnancy because of the risk of haemolytic anaemia, it is already licensed for the treatment of methaemoglobinemia and used in other medical contexts (surgical staining, oncology), facilitating potential repurposing as an antimalarial [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUse of methylene blue for the chemotherapy of malaria requires careful consideration of its pharmacodynamic and pharmacokinetic properties. Methylene blue has a t\u003csub\u003emax\u003c/sub\u003e of ~\u0026thinsp;2 hours after oral administration and a half-life of ~\u0026thinsp;6 hours, comparable to that of artemisinins. Similar dosing gives higher C\u003csub\u003emax\u003c/sub\u003e with parenteral than oral formulation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. It is slow-acting \u003cem\u003ein vivo\u003c/em\u003e and seven-day treatments (4 asexual cycles) are needed to kill all parasites [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Therefore, it is probably not valuable as an adjuvant to artemisinin-combination therapies used for the oral treatment of uncomplicated malaria. This statement is further supported by the similar parasite clearance rate and cure rate when comparing artesunate-amodiaquine \u003cem\u003eversus\u003c/em\u003e artesunate-amodiaquine- methylene blue [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. However, parenteral methylene blue combined with artesunate may be useful in the treatment of severe malaria in area where artemisinin-resistance is prevalent. Methylene blue activity against artemisinin-resistant ring stages could restore the benefit of artesunate over quinine in artemisinin-resistant infections but whether this holds \u003cem\u003ein vivo\u003c/em\u003e warrants further investigation. Methylene blue effects on cytoadherence and rosetting have not been assessed but observations from recent primate models suggest this mechanism could also contribute [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe most plausible explanation for the consistent methylene blue activity across \u003cem\u003ekelch13\u003c/em\u003e genotypes lies in its redox-targeting effects distinct from artemisinin\u0026rsquo;s mechanisms of action, which likely explains the absence of cross-resistance. The IC\u003csub\u003e50\u003c/sub\u003e values estimated in this modified ring survival assay are higher than reported in previous studies using standard \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003eex vivo\u003c/em\u003e assays. The higher IC\u003csub\u003e50\u003c/sub\u003e estimates observed here are most likely explained by assay design rather than by reduced intrinsic susceptibility. Importantly, there is no clear methylene blue resistance in the field to date and the potential for selecting \u003cem\u003ede novo\u003c/em\u003e resistance mutations under conditions of sublethal exposures is deemed low [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA strength of this study is the use of a modified ring survival assay protocol combined with Bayesian multilevel modelling, which allowed assessment of the dose-response while accounting for experimental variability thereby making the results more robust to outliers and increasing statistical power for detecting significant effects of genotype on parasite susceptibility [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. This approach strengthens confidence in the inference of genotype effects on parasite susceptibility to methylene blue. The inclusion of reference strain NF54 adds external validity and supports the reproducibility of the results.\u003c/p\u003e \u003cp\u003eCertain limitations should be acknowledged. The sample size was small and inter-experiment variability arising from random errors across repeated testing was not taken into account. Only two \u003cem\u003ekelch13\u003c/em\u003e mutations were assessed (R561H and P441L). These alleles were chosen because they are now predominant in Karen state (Eastern Myanmar) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] but further evaluation against other resistant alleles, including C580Y and F446I, would provide a more comprehensive picture. In addition, \u003cem\u003ein vitro\u003c/em\u003e effects on parasite growth do not necessarily correlate with parasite clearance \u003cem\u003ein vivo\u003c/em\u003e, especially considering the central role of the spleen in removing damaged parasites and red blood cells from the circulation after drug exposures [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFuture randomised trials should evaluate whether short course parenteral methylene blue added to standard intravenous artesunate can safely enhance parasite clearance and reduce mortality in severe falciparum malaria. Such trials would be particularly relevant in areas of East Africa where malaria incidence and mortality are high and artemisinin resistance is firmly established.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eMethylene blue retains potent activity against artemisinin-resistant \u003cem\u003eP. falciparum\u003c/em\u003e isolates and shows no evidence of \u003cem\u003ekelch13-\u003c/em\u003emediated cross-resistance. While its pharmacodynamic profile makes it unsuitable for the oral treatment of uncomplicated malaria, the compound may have value as an intravenous adjunct in the treatment of severe infection in settings where artemisinin resistance reduces the efficacy of standard therapies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003econfidence interval\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCrI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCredible interval\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003einhibitory concentration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIQR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003einterquartile range\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003erelative risk\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003estandard deviation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eThe study was approved by the Oxford Tropical Research Ethics Committee (OxTREC, number 28\u0026thinsp;\u0026minus;\u0026thinsp;09). All participants provided their written informed consent to participate in the study.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was funded in full by a Wellcome grant (#220211).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eOV performed ring survival assay experiments and wrote the initial draft of the manuscript. CK, PK performed ring survival assay experiments. PC developed assay methodology, performed ring survival assay experiments, supervised the laboratory staff and reviewed and edited the manuscript. APP and CB collected the samples. VC designed the study, administered the project, developed assay methodology, supervised the laboratory staff, analysed the data and wrote the initial draft of the manuscript. MI genotyped the parasites. KC designed the study, provided the NF54 parasite and reviewed and edited the manuscript. NJW designed the study and acquired funding. FN designed the study, acquired funding, administered the project and reviewed and edited the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe are very grateful to the volunteers who participated in this study. We thank the staff of the Laboratory, Medical and Data Management Departments of the Shoklo Malaria Research Unit, NHP study investigators, and the staff of the MORU Malaria Molecular and Malaria In Vitro labs for their help with samples and data collection, processing and management. We thank Provepharm (Marseille, France) for providing Proveblue\u0026reg;. The Shoklo Malaria Research Unit is part of the Mahidol-Oxford Research Unit, supported by Wellcome (U.K.). A CC BY or equivalent licence is applied to the author accepted manuscript arising from this submission, in accordance with the grant\u0026rsquo;s open access conditions.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll analysis code and data are available via an accompanying github repository: [https://github.com/victorSMRU/methylene-blue-rsa](https:/github.com/victorSMRU/methylene-blue-rsa)\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWeiss DJ, Dzianach PA, Saddler A, Lubinda J, Browne A, McPhail M, et al. Mapping the global prevalence, incidence, and mortality of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e and \u003cem\u003ePlasmodium vivax\u003c/em\u003e malaria, 2000-22: a spatial and temporal modelling study. 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Malar J Engl. 2017;16:88. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12936-017-1731-1\u003c/span\u003e\u003cspan address=\"10.1186/s12936-017-1731-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"malaria-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"malj","sideBox":"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)","snPcode":"12936","submissionUrl":"https://submission.nature.com/new-submission/12936/3","title":"Malaria Journal","twitterHandle":"@malariajournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Methylene blue, in vitro sensitivity, Plasmodium falciparum, artemisinin resistance, kelch13, P441L, R561H, ring survival assay, Thailand-Myanmar border, Emax model.","lastPublishedDoi":"10.21203/rs.3.rs-8558391/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8558391/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eArtemisinin resistance in \u003cem\u003ePlasmodium falciparum\u003c/em\u003e is now well established in three continents and challenges the efficacy of antimalarial drug regimens. This study examined whether methylene blue, an ancient antimalarial drug with a broad spectrum of activity against \u003cem\u003ePlasmodium\u003c/em\u003e blood stages, retains its activity against the young rings of artemisinin-resistant parasites. Clinical isolates carrying \u003cem\u003ekelch13\u003c/em\u003e wild type (n\u0026thinsp;=\u0026thinsp;3), R561H (n\u0026thinsp;=\u0026thinsp;9), or P441L (n\u0026thinsp;=\u0026thinsp;10) genotypes were tested with a modified ring survival assay whereby 0- to 3-hour post-invasion rings were exposed to a range of methylene blue concentrations. Ring survival was analysed with a Bayesian mixed effects E\u003csub\u003emax\u003c/sub\u003e model accounting for variability across isolates and experimental replicates. Methylene blue suppressed ring-stage survival at low nanomolar concentrations with no evidence of \u003cem\u003ekelch13\u003c/em\u003e-mediated cross-resistance: the mean 50% inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) estimates were 23 nM (95% credible interval [CrI]: 15 to 37) for wild type, 27 nM (95%CrI: 21 to 36) for R561H and 14 nM (95%CrI: 10 to 20) for P441L. These findings indicate that methylene blue remains active \u003cem\u003ein vitro\u003c/em\u003e against ring-stage artemisinin-resistant parasites.\u003c/p\u003e","manuscriptTitle":"Activity of methylene blue against the asexual ring stages of artemisinin-resistant Plasmodium falciparum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-23 07:44:26","doi":"10.21203/rs.3.rs-8558391/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-06T16:43:15+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-05T13:49:59+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-16T23:21:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"302683325645692852657252746579055526128","date":"2026-01-21T23:40:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"199280518370608175587830709879460764832","date":"2026-01-21T23:15:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"73929589047818772852543853882799731257","date":"2026-01-21T17:02:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-21T16:32:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-21T15:19:56+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-18T23:48:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-09T18:11:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Malaria Journal","date":"2026-01-09T07:49:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"malaria-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"malj","sideBox":"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)","snPcode":"12936","submissionUrl":"https://submission.nature.com/new-submission/12936/3","title":"Malaria Journal","twitterHandle":"@malariajournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b4e2ae07-7a6a-4b28-b7c1-357717446896","owner":[],"postedDate":"January 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-29T12:53:50+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-23 07:44:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8558391","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8558391","identity":"rs-8558391","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}