Genetic heterogeneity among Leishmania major isolates in Iran determined by restriction fragment length polymorphism [RFLP] and multilocus microsatellite typing [MLMT]

In recent years, molecular methods for characterizing genetic heterogeneity have found a major place in modern approaches. In this study, two different molecular techniques including Restriction Fragment Length Polymorphism [RFLP] and Multi Locus microsatellite typing [MLMT] were carried out in order to evaluate genetic heterogeneity among isolates of Leishmania major in Iran. In this experimental study, 24 Lmajor isolates from different endemic foci of cutaneous leishmaniasis were evaluated. All samples were amplified by specific primers for Internal Transcribed Spacer ribosomal RNA [ITS_rRNA] and miniexon genes [ME]. Ten different microsatellite markers were applied to 24 collected isolates as well. Restriction fragment length polymorphism of Polymerase chain reaction of ITS-rRNA and ME regions was identified in polyacrylamide gel electrophoresis. Size polymorphisms in PCR products of microsatellites markers were measured in the CEQ 8000 automated genetic analysis system. Population structure of the isolates was investigated by Structure Version 2.3.2 software. According to ITS- RFLP and ME-RFLP techniques, three and two different strains of Lmajor were determined, respectively, while microsattellites markers revealed 21 different genotypes, which were clustered in three genetic groups using structure software. Although genetic heterogeneity among studied L. major isolates was identified by molecular tools as used in this study, it seems that microsatellites markers are more useful in population structure and epidemiological studies. Our findings also showed correlation between different identified strains and their geographical regions

Comparison of the antimicrobial effect of garlic extract with two intracanal irrigants on enterococcus faecalis

It is very important to remove the microorganisms in the root canal before obturation. One of the causes of endodontic treatment failures is the existence of the bacteria responsible for resistant infections, including Enterococcus Faecalis. The aim of this study was to compare the antibacterial effect of the garlic extract with two intracanal irrigants on Enterococcus Faecalis. In this in-vitro study, the method of Well Agar Diffusion was used to compare the anti-bacterial effect of pure garlic extract [100%], garlic extract 80%, chlorhexidine 2%, sodium hypochlorite 5.25% and combined chlorhexidine 2% with pure garlic extract. Eighteen plates of Muller-Hinton agar were inoculated with E.faecalis. Each plate had 6 wells for test solutions and one of them was for sterile distilled water as the control. The prepared plates were distributed into aerobic [n=9] and anaerobic [n=9] groups, then incubated at 37°C for 24 hours. After that, the diameter of the zones of microbial inhibition around every well was measured and recorded. Our results demonstrated that Sodium hypochlorite 5.25% was more effective compared with the other antimicrobial materials in both aerobic and anaerobic groups. This difference was statistically significant [ANOVA, p<0.05]. The most effective antimicrobial agents in aerobic and anaerobic conditions were in this order sodium hypochlorite [5.25%], chlorhexidine 2%, pure garlic extract, combined chlorhexidine 2% with pure garlic extract and garlic extract 80% respectively [p<0.05]. However, the difference between pure garlic extract and combined chlorhexidine 2% with pure garlic extract in aerobic condition was not significant. The results showed that garlic extract is effective on Enterococcus Faecalis in both aerobic and anaerobic conditions; nevertheless it has less efficacy than chlorhexidine and sodium hypochlorite

[Detection of polymorphism within L.major isolates collected from Iranian patients suffering from cutaneous leishmaniasis by microsatellite markers]

Protozoan parasites of Leishmania major is one of the causative agents of cutaneous leishmaniasis in different parts of Iran. Currently different molecular biology tools based on different polymorphic microsatellite typing method [MLMT] is one of these interesting techniques that we applied to analyze leishmania major parasites collected from different endemic parts of zoonotic cutaneous Leishmaniasis [ZCL] in Iran. In this laboratory study, twenty-four leishmania major parasites collected from patients suffered from cutaneous leishmaniasis in different parts of Iran were investigated using MLMT method fro the first time in Iran. The isolates were amplified by seven different designed primers for microsatellite markers and then analysed after running on plyacrylamide gel electrophoresis for detection of polymorphism among these isolates. PCR products of 24 isolates using six microsatellite markers revealed polymorphic pattern, whereas monomorphic profile was observed with only one primer. Totally, seven different genotypes were demonstrated among studied isolates. Bases on our results different genotypes were detected within leishmania major isolates collected from endemic areas of ZCL in Esfahan, Semnan and Tehran provinces, although only one genotype was observed in the studied isolated obtained from Khuzestan and Ilam foci. Overall, seven different genotypes were identified based on MLMT in studied Leishmania major isolates in different endemic areas of ZCL in Iran. Based on our finding we suggest that MLMT can utilized as a reliable method for detection of polymorphism within Leishmania major species