Methylene blue inhibits replication of SARS-CoV-2 in vitro

Int J Antimicrob Agents Jan 2020

Authors: Mathieu Gendrot, Julien Andreani, Isabelle Duflot, Manon Boxberger, Marion Le Bideau, Joel Mosnier, Priscilla Jardot, Isabelle Fonta, Clara Rolland, Hervé Bogreau, Sébastien Hutter, Bernard La Scola, Bruno Pradines


In December 2019, a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing coronavirus diseases 2019 (COVID-19) emerged in Wuhan, China. Currently there is no antiviral treatment recommended against SARS-CoV-2. Identifying effective antiviral drugs is urgently required. Methylene blue has already demonstrated in vitro antiviral activity in photodynamic therapy as well as antibacterial, antifungal and antiparasitic activities in non-photodynamic assays. In this study. non-photoactivated methylene blue showed in vitro activity at very low micromolar range with an EC50 (median effective concentration) of 0.30 ± 0.03 μM and an EC90 (90% effective concentration) of 0.75 ± 0.21 μM at a multiplicity of infection (MOI) of 0.25 against SARS-CoV-2 (strain IHUMI-3). The EC50 and EC90 values for methylene blue are lower than those obtained for hydroxychloroquine (1.5 μM and 3.0 μM) and azithromycin (20.1 μM and 41.9 μM). The ratios Cmax/EC50 and Cmax/EC90 in blood for methylene blue were estimated at 10.1 and 4.0, respectively, following oral administration and 33.3 and 13.3 following intravenous administration. Methylene blue EC50 and EC90 values are consistent with concentrations observed in human blood. We propose that methylene blue is a promising drug for treatment of COVID-19. In vivo evaluation in animal experimental models is now required to confirm its antiviral effects on SARS-CoV-2. The potential interest of methylene blue to treat COVID-19 needs to be confirmed by prospective comparative clinical studies.

Keywords: Antiviral; COVID-19; Coronavirus; In vitro; Methylene blue; SARS-CoV-2.


  1. Wu F, Zhao S, Yu B, Chen YM, Wang W, Song ZG. A new coronavirus associated with human respiratory disease in China. Nature. 2020;579:365–369.
  2. Fryk JJ, Marks DC, Hobson-Peters J, Prow NA, Watterson D, Hall RA. Dengue and chikungunya viruses in plasma are effectively inactivated after treatment with methylene blue and visible light. Transfusion. 2016;56:2278–2285. – PubMed
  3. Faddy HM, Fryk JJ, Hal RA, Young PR, Reichenberg S, Tolksdorf F. Inactivation of yellow fever virus in plasma after treatment with methylene blue and visible light and in platelet concentrates following treatment with ultraviolet C light. Transfusion. 2019;59:2223–2227. – PubMed
  4. Wang Y, Ren K, Liao X, Luo G, Kumthip K, Leetrakool N. Inactivation of Zika virus in plasma and derivatives by four different methods. J Med Virol. 2019;91:2059–2065. – PubMed
  5. Eickmann M, Gravemann U, Handke W, Tolksdorf F, Reichenberg S, Müller TH. Inactivation of Ebola virus and Middle East respiratory syndrome coronavirus in platelet concentrates and plasma by ultraviolet C light and methylene blue plus visible light, respectively. Transfusion. 2018;58:2202–2207. – PMC – PubMed
  6. Gazel D, Tatman Otkun M, Akçali A. In vitro activity of methylene blue and eosin methylene blue agar on colistin-resistant A. baumannii: an experimental study. J Med Microbiol. 2019;68:1607–1613. – PubMed
  7. Tian RBD, Asmar S, Napez C, Lépidi H, Drancourt M. Effectiveness of purified methylene blue in an experimental model of Mycobacterium ulcerans infection. Int J Antimicrob Agents. 2017;49:290–295. – PubMed
  8. Pal R, Ansari MA, Saibabu V, Das S, Fatima Z, Hameed S. Nonphotodynamic roles of methylene blue: display of distinct antimycobacterial and anticandidal mode of actions. J Pathog. 2018;2018 – PMC – PubMed
  9. Helfritz FA, Bojkova D, Wanders V, Kuklinski N, Westhaus S, von Horn C. Methylene blue treatment of grafts during cold ischemia time reduces the risk of hepatitis C virus transmission. J Infect Dis. 2018;218:1711–1721. – PubMed
  10. Guttman P, Ehrlich P. Ueber die Wirkung des Methylenblau bei Malaria [About the effect of methylene blue in malaria] Berl Klin Wochenschr. 1891;28:953–956.
  11. Pascual A, Henry M, Briolant S, Charras S, Baret E, Amalvict R. In vitro activity of Proveblue (methylene blue) on Plasmodium falciparum strains resistant to standard antimalarial drugs. Antimicrob Agents Chemother. 2011;55:2472–2474. – PMC – PubMed
  12. Fall B, Camara C, Fall M, Nakoulima A, Dionne P, Diatta B. Plasmodium falciparum susceptibility to standard and potential anti-malarial drugs in Dakar, Senegal, during the 2013–2014 malaria season. Malar J. 2015;14:60. – PMC – PubMed
  13. Fall B, Madamet M, Diawara S, Briolant S, Wade KA, Lo G. Ex vivo activity of Proveblue, a methylene blue, against filed isolates of Plasmodium falciparum in Dakar, Senegal from 2013 to 2015. Int J Antimicrob Agents. 2017;50:155–158. – PubMed
  14. Gendrot M, Madamet M, Mosnier J, Fonta I, Amalvict R, Benoit N. Baseline and multinormal distribution of ex vivo susceptibilities of Plasmodium falciparum to methylene blue in Africa, 2013–18. J Antimicrob Chemother. 2020;75:2141–2148. – PubMed
  15. Dormoi J, Pradines B. Dose responses of Proveblue methylene blue in an experimental murine cerebral malaria model. Antimicrob Agents Chemother. 2013;57:4080–4081. – PMC – PubMed
  16. Dormoi J, Briolant S, Desgrouas C, Pradines B. Efficacy of Proveblue (methylene blue) in an experimental cerebral malaria murine model. Antimicrob Agents Chemother. 2013;57:3412–3414. – PMC – PubMed
  17. Dormoi J, Briolant S, Desgrouas C, Pradines B. Impact of methylene blue and atorvastatin combination therapy on the apparition of cerebral malaria in a murine model. Malar J. 2013;12:127. – PMC – PubMed
  18. Mendes Jorge M, Ouermi L, Meissner P, Compaoré G, Coulibaly B, Nebie E. Safety and efficacy of artesunate–amodiaquine combined with either methylene blue or primaquine in children with falciparum malaria in Burkina Faso: a randomized controlled trial. PLoS One. 2019;14 – PMC – PubMed
  19. Liu J, Cao R, Xu M, Wang X, Zhang H, Hu H. Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro. Cell Discov. 2020;6:16. – PMC – PubMed
  20. Andreani J, Le Bideau M, Duflot I, Jardot P, Rolland C, Boxberger M. In vitro testing of hydroxychloroquine and azithromycin on SARS-CoV-2 shows synergistic effect. Microb Pathog. 2020;145 – PMC – PubMed
  21. Gautret P, Lagier JC, Parola P, Hoang VT, Meddeb L, Sevestre J. Clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in 80 COVID-19 patients with at least a six-day follow up: an observational study. Travel Med Infect Dis. 2020;34 – PMC – PubMed
  22. Million M, Lagier JC, Gautret P, Colson P, Fournier PE, Amrane S. Early treatment of 1061 COVID-19 patients with hydroxychloroquine and azithromycin, Marseille, France. Travel Med Dis. 2020;35 – PMC – PubMed
  23. Gautret P, Lagier JC, Parola P, Hoang VT, Meddeb L, Mailhe M. Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an open-label non-randomized clinical trial. Int J Antimicrob Agents. 2020;56 – PMC – PubMed
  24. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65:55–63. – PubMed
  25. Amrane S, Tissot-Dupont H, Doudier B, Eldin C, Hocquart M, Mailhe M. Rapid viral diagnosis and ambulatory management of suspected COVID-19 cases presenting at the infection diseases referral hospital in Marseille, France, January 31st to March 1st, 2020: a respiratory virus snapshot. Travel Med Infect Dis. 2020;36 – PMC – PubMed
  26. Anh CX, Chavchich M, Birrell GW, van Breda K, Travers T, Rowcliffe K. Pharmacokinetics and ex vivo antimalarial activity of artesunate–amodiaquine plus methylene blue in healthy volunteers. Antimicrob Agents Chemother. 2020;64 e01441-19. – PMC – PubMed
  27. Center for Drug Evaluation and Research . October 2020. Clinical pharmacology and biopharmaceutics review(s): application number 204630Orig1s000.… [accessed 16 October 2020]
  28. Link EM, Costa DC, Lui D, Ell PJ, Lower PJ, Spittle MF. Targeting disseminated melanoma with radiolabelled methylene blue. Acta Oncol. 1996;35:331–341. – PubMed
  29. Rainsford KD, Parke AL, Clifford-Rashotte M, Kean WF. Therapy and pharmacological properties of hydroxychloroquine and chloroquine in the treatment of systemic lupus erythematosus, rheumatoid arthritis and related diseases. Inflammatopharmacology. 2015;23:231–269. – PubMed
  30. Chhonker YS, Sleightholm RL, Li J, Oupicky D, Murry DJ. Simultaneous quantification of hydroxychloroquine and its metabolites in mouse blood and tissues using LC-ESI-MS/MS: an application for pharmacokinetic studies. J Chromatogr B Analyt Technol Biomed Life Sci. 2018;1072:320–327. – PMC – PubMed
  31. Danesi R, Lupetti A, Barbara C, Ghelardi E, Chella A, Malizia T. Comparative distribution of azithromycin in lung tissue of patient given oral daily doses of 500 and 1000 mg. J Antimicrob Chemother. 2003;51:939–945. – PubMed
  32. Lucchi M, Damle B, Fang A, de Caprariis PJ, Mussi A, Sanchez SP. Pharmacokinetics of azithromycin in serum, bronchial washings, alveolar macrophages and lung tissue following a single oral dose of extended or immediate release formulations of azithromycin. J Antimicrob Chemother. 2008;61:884–891. – PubMed
  33. Davidson RJ. In vitro activity and pharmacodynamic/pharmacokinetic parameters of clarithromycin and azithromycin: why they matter in the treatment of respiratory tract infections. Infect Drug Resist. 2019;12:585–596. – PMC – PubMed
  34. Madrid PB, Panchal RG, Warren TK, Shurleff AC, Endsley AN, Green CE. Evaluation of Ebola virus inhibitors for drug repurposing. ACS Infect Dis. 2015;1:317–326. – PubMed
  35. Takano T, Satoh K, Doki T, Tanabe T, Hohdatsu T. Antiviral effects of hydroxychloroquine and type I interferon on in vitro fatal feline coronavirus infection. Viruses. 2020;12:576. – PMC – PubMed
  36. Lu G, Nagbanshi M, Goldau N, Mendes Jorge M, Meissner P, Jahn A. Efficacy and safety of methylene blue in the treatment of malaria: a systemic review. BMC Med. 2018;16:59. – PMC – PubMed
  37. Choy KT, Wong AYL, Kaewpreedee P, Sia SF, Chen D, Hui KPY. Remdesivir, lopinavir, emetine, and homoharringtonine inhibits SARS-CoV-2 replication in vitro. Antiviral Res. 2020;178 – PMC – PubMed