Comparison of Agar Dilution, Broth Dilution, Cylinder Plate and Disk Diffusion Methods for Evaluation of Anti-leishmanial Drugs on Leishmania promastigotes
Abstract
Background: Leishmaniases are a group of diseases caused by Leishmania parasites. Growing of drug unresponsiveness in leishmaniasis patients necessitates the development of new drugs and accordingly a suitable assay is needed for evaluation of any modalities. The aim of this study was to compare four drug assays methods, agar dilution, broth dilution, cylinder plate and disk diffusion, for evaluation of anti-leishmanial drugs on Leishmania promastigotes, using glucantime as a currently available drug for treatment of leishmaniasis.
Methods: For broth dilution method, different concentration of glucantime was added to the parasite culture (promastigotes of Leishmania), while in cylinder plate method wells were punched in agar gel and filled with different concentration of drug and zone of inhibition was measured in each well. In disk diffusion method, the parasites were cultivated on the surface of agar; filter paper disks were enriched with various concentration of glucantime and were placed on the surface of agar. In agar dilution method, various concentrations of drug were incorporated onto blood agar and the parasites were cultivated on the surface of the agar.
Results: A direct correlation was found between the drug concentration and size of inhibitory zones in cylinder plate and disk diffusion methods. These two drug assays methods provided much better performance in comparison with broth and agar dilution methods.
Conclusion: Cylinder plate and disk diffusion methods seem to be acceptable methods for susceptibility testing of anti-leishmanial compounds on Leishmania promastigotes.
Herwaldt BL. Leishmaniasis. Lancet. 1999; 354:1191-9.
Berman JD. Human leishmaniasis: clinical, diagnostic and chemotherapeutic developments in the last 10 years. Clin Infect Dis.1997; 24:684-703.
Vouldoukis I, Rougier S, Dugas B, Pino P, Mazier D, Woehrlé F. Canine visceral leishmaniasis: comparison of in vitro leishmanicidal activity of marbofloxacin, meglumine antimoniate and sodium stibogluconate. Vet Parasitol. 2006; 135:137-46.
Fatima F, Khalid A, Nazar N, Abdalla M, Mohomed H, Toum AM, Magzoub M, Ali MS. In vitro assessment of anti-cutaneous leishmaniasis activity of some Sudanese plants. Turkiye Parazitol Dreg. 2005; 29:3-6.
Mittal MK, Rai S, Ashutosh, Ravinder, Gupta S, Sundar S, Goyal N. Characterization of natural antimony resistance in Leishmania donovani isolates. Am J Trop Med Hyg. 2007; 76:681-8.
Wright CW, Philipson JD. Natural products and the development of selective antiprotozoal drugs. Phytother Res. 1990; 4:127-39.
Pourmohammadi B, Motazedian MH, Handjani F, Hatam GH, Habibi S, Sarkari B. Glucantime efficacy in the treatment of zoonotic cutaneous leishmaniasis. Southeast Asian J Trop Med Public Health. 2011; 42:502-8.
Hauser KJ, Johnston JA, Zabransky RJ. Economical agar dilution technique for susceptibility testing of anaerobes. Antimicrob Agents Chemother. 1975; 7:712-4.
Buron EJ, Finegold M. Methods for testing antimicrobial effectiveness in: Diagnostic Microbology.1990; 8th ed. Pp. 181-188.
Hammer KA, Carson CF, Riley TV. In-vitro activity of essential oils, in particular Melaleuca alternifolia (tea tree) oil and tea tree oil products, against Candida spp. J Antimicrob Chemother. 1998; 42:591-5.
Matar MJ, Ostrosky-Zeichner L, Paetznick VL, Rodriguez JR, Chen E, Rex JH. Correlation between E-Test, disk diffusion, and microdilution methods for antifungal susceptibility testing of fluconazole and voriconazole. Antimicrob Agents Chemother. 2003; 47:1647-51.
Pfaller MA, Diekema DJ, Gibbs DL, Newell VA, Meis JF, Gould IM, Fu W, Colombo AL, Rodriguez-Noriega E. Global antifungal surveillance study. Results from the ARTEMIS DISK global antifungal surveillance study, 1997 to 2005: an 8.5-year analysis of susceptibilities of Candida species and other yeast species to fluconazole and voriconazole determined by CLSI standardized disk diffusion testing. J Clin Microbiol. 2007; 45:1735-45.
Inouye S, Uchida K, Maruyama N, Yamaguchi H, Abe S. A novel method to estimate the contribution of the vapor activity of essential oils in agar diffusion assay. Jpn J Med Mycol. 2006; 47:91-98.
Lino A, Deogracious O. The in-vitro antibacterial activity of Annona senegalensis, Securidacca longipendiculata and Steganotaenia araliacea Ugandan medicinal plants. Afr Health Sci. 2006; 6:31-5.
St George S, Bishop JV, Titus RG, Selitrennikoff CP. Novel compounds active against Leishmania major. Antimicrob Agents Chemother. 2006; 50:474-9.
Bhattacharyya A, Mukherjee M, Duttagupta S. Studies on stibanate unresponsive isolates of Leishmania donovani. J Biosci. 2002; 27:503-8.
Habibi P, Motazdian MH, Esfandiari F, Fakhar M. Introduction a simple and rapid modified blood agar media for mass cultivation of Leishmania Spp. Armaghane-Danesh, 2005; 10 :47-54. [In Persian].
Habibi P, Sadjjadi SM, Owji M, Moattari A, Sarkari B, Naghibalhosseini F, Hatam GR, Kazemian S. Characterization of in vitro cultivated amastigote like of Leishmania major: a substitution for in vivo studies. Iranian J Parasitol. 2008; 3: 6-15.
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| Issue | Vol 7 No 3 (2012) | |
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| Leishmaniasis Drug Assay Agar Dilution Cylinder Plate Disk Diffusion | ||
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