Comparison of agar dilution, broth dilution, cylinder plate and disk diffusion methods for evaluation of anti-leishmanial drugs on Leishmania promastigotes

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. 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. 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. Cylinder plate and disk diffusion methods seem to be acceptable methods for susceptibility testing of anti-leishmanial compounds on Leishmania promastigotes

Comparison of three methods for diagnosis of cutaneous leishmaniasis

Leishmaniasis is one of the infectious parasitic diseases of highest incidence in the world. Cutaneous Leishmaniasis [CL] has long been reported in Shiraz, Southern Iran. There is a need to find a sensitive and specific method for treatment and control of the disease. We have compared the sensitivity of the conventional methods microscopy and cultivation of lesion scrapes against PCR amplification of parasite kinetoplast DNA from these samples. The samples [n=219] were obtained from the patients clinically suspected of CL. The smears were stained with Giemsa for microscopy and cultured in Novy-Nicolle-McNeal [NNN] blood agar for promastigote growth. For PCR, the dry smears were scraped off the slides and DNA was extracted. The positive rates from 219 specimens were 76.71%, 50.68%, and 93.61% for microscopy, cultivation, and PCR, respectively. The highest correlation was found between PCR and microscopy method [P= 0.014]. In PCR assay, 95.61%, 3.9%, and 0.49% of the samples were identified as Leishmania major, L.tropica, and dermatropic L.infantum, respectively. The PCR method appears to be the most sensitive for the diagnosis of CL and is valuable for identifying the other species of Leishmania with confusing dermatropic signs

[Evaluation of hydatid cyst fluid as a substitute for fetal bovine serum [FBS] in culture of leishmania major]

Isolation and characterization of Leishmania parasites is a necessary strategy for control of Leishmaniasis. Free-cell culture media, rich with biologic fluids such as Fetal Bovine Serum [FBS] are among the best media for culturing of the parasite. In our country, FBS is very expensive and is not available everywhere. In this study, we evaluated Ovine Hydatid Cyst Fluid [HCF] as a substitute for FBS in liquid culture media of Leishmania major. Leishmania parasites were isolated from Balb/C mouse ulcer and characterized by PCR. Of six tubes by triplicate [totally 18 tubes] add to each tube 800,000 promastigotes of Leishmania major and then add 1 cc of media including, once BHI [Brain Heart Infusion broth], BHI plus 5% FBS, BHI plus 10% FBS, once Hydatid Cyst Fluid [HCF], BHI plus 5% HCF and BHI plus 10%HCF, respectively. After 24h, 72h, 144h, 192 hours the number of parasites in each tube counted and the means of them compared together. The result of this study showed that BHI plus HCF 10% medium could be use as a suitable substitute till 72 hours for Fetal Bovine Serum [FBS] in liquid Culture of Leishmania major parasites. In this study, we have introduced the Ovine Hydatid Cyst Fluid [HCF] as replacement for FBS in liquid Culture media