ABSTRACT
Background & objectives: The interaction of Leishmania spp. with microbiota inside the midgut vector has significant output in pathogenesis. This study aimed to identify the profile of Leishmania major gene expression of LACK, gp63, and hsp70 after exposure to Staphylococcus aureus and group A beta-hemolytic Streptococci (GABHS). Methods: Leishmania major (MRHO/IR/75/ER) promastigotes were exposed with S. aureus, with GABHS, and with both GABHS and S. aureus at 25°C for 72 h. The gene expression analysis of Lmgp63, Lmhsp70, and LmLACK was assessed using SYBR Green real-time PCR by ??Ct. All experiments were repeated in triplicate. Statistical analysis was done using two-way ANOVA. A P-value less than 0.05 was considered significant. Results: Lmgp63 was expressed in the group exposed to GABHS with 1.75-fold lower than the control group (p=0.000). The LmLACK had expression in both groups exposed with GABHS and GABHS with S. aureus with 2.8 and 1.33-fold more than the control group, respectively (p=0.000). The Lmhsp70 gene expression was reported in the group exposed with GABHS with relative quantification of 5.7-fold more than the control group. Interpretation & conclusion: This study showed that the important genes encoding LACK, gp63, and hsp70 changed their expression after exposure to the S. aureus and GABHS.
ABSTRACT
Background & objectives: Leishmaniasis is a geographically widespread severe disease which includes visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). There are 350 million people at risk in over 80 countries. In the Old World, CL is usually caused by Leishmania major, L. tropica, and L. aetiopica complex of which 90% of cases occur in Afghanistan, Algeria, Iran, Iraq, Saudi Arabia, Syria, Brazil and Peru. Recently, Eslami et al (2011) reported a novel TRYP6 gene encoding tryparedoxin peroxidase from an Iranian L. major strain exhibiting homology with the related gene in a divergent genus of Kinetoplastida, the Crithidia. This prompted us to analyze the mentioned gene in 100 isolates obtained from patients with suspected CL. Consequently, we analyzed internal transcribed spacer 1 (ITS1) region, RNA polymerase II largest subunit (RPOIILS) and the mitochondrial DNA polymerase beta (DPOLB). Methods: After obtaining samples from 100 patients, DNA extraction was performed and TRYP6 was analyzed using conventional PCR. All samples harbouring TRYP6 with smaller size (555 bp) were analysed based on three other regions: ITS1, RPOIILS and DPOLB genes. Results: Results showed that 10% of the isolates have the same character as observed in our previous study. The ITS1-RFLP-PCR of this 10% isolates showed their similarity to the one from Crithidia fasciculata. RNA polymerase II largest subunit (RPOIILS) showed genetic diversity but the mitochondrial DNA polymerase beta (DPOLB) did not show any genetic diversity. Conclusion: This study might also help in solving the problems concerning Leishmaniasis outbreaks currently reported in Iran and some other endemic regions of the world.
ABSTRACT
Background & objectives: Rapid and accurate diagnosis and identification of Leishmania sp causing cutaneous leishmaniasis is crucial in control and therapeutic programs. The problem of diagnosis with traditional methods is that they have a low sensitivity or time consuming but molecular techniques would be an alternative method for rapid and accurate diagnosis. In this work, tryparedoxine peroxidase gene-based real-time PCR was used for accurate identification of Leishmania spp causing Old-World cutaneous leishmaniasis. Methods: In this study, biopsies of specimens were taken from the ulcerative sites in 100 patients and used for direct microscopy, culture in NNN or fixed in alcohol for identification of Leishmania spp using tryparedoxin peroxidase gene-based realtime PCR (qPCR). Results: Using direct microscopy and culture method, Leishmania parasites were isolated from 68 out of 100 patient samples. However, 13 patients with negative finding on traditional tests, had positive results on RT-PCR test. After melting curve analysis of PCR product, Leishmania major in 75 and L. tropica in 4 cases were identified. The sensitivity and specificity of RT-PCR for diagnosis of cutaneous leishmaniasis was 98.7 and 59.8%, respectively. Conclusion: Results of this study showed that RT-PCR was the most sensitive diagnostic test for cutaneous leishmaniasis and represents a tool for rapid species identification.