Comparison of traditional methods and pcr for diagnosis of cutaneous leishmaniasis in South-West of Iran

The usual method for laboratory diagnosis of cutaneous leishmaniasis was the direct observation of parasites under a light microscope. Although this method has high specificity, it has low sensitivity. The purpose of this study is to compare three methods of direct observation, culture and Mini-exon-PCR to diagnose cutaneous leishmaniasis in Khuzestan province. This study intends to compare sensitivity of PCR approach with sensitivity of the existing traditional methods to diagnose cutaneous leishmaniasis using Mini-exon gene. A total 216 skin biopsies prepared from patients with cutaneous leishmaniasis were studied though direct method, culture in NNN, culture in RPMI 1640 and Mini-exon-PCR and the sensitivity of these methods were compared with each other. In this study Mini-exon-PCR was considered as the gold standard method. Results showed that 46.7% with direct method, 35.1% with culture method in RPMI 1640,57.8% with culture methodin NNNand 70.3% withPCRwere positive.Sensitivity wasobtained 66.4% for microscopic observation, 50% for culture in RPMI1640, and 82.2% for culturein NNNand 100% forPCR. This study showed that PCR on samples stored in normal saline has higher sensitivity and specificity than other traditional methods [p<0.05]. Thus, Mini-exon-PCR on samples in normal saline is a reliable method to diagnose cutaneous leishmaniasis, especially in cases where the diagnosis is negative with the other methods.

Miltefosine-Induced Apoptotic Cell Death on Leishmania major and L. tropica Strains

The aim of this study was to assess the cytotoxic effects of various concentrations of miltefosine on Leishmania major (MRHO/IR/75/ER) and L. tropica (MHOM/IR/02/Mash10) promastigotes and to observe the programmed cell death features. The colorimetric MTT assay was used to find L. major and L. tropica viability and the obtained results were expressed as 50% inhibitory concentration (IC50). Also, 50% effective doses (ED50) for L. major and L. tropica amastigotes were also determined. Annexin-V FLUOS staining was performed to study the cell death properties of miltefosine using FACS analysis. Qualitative analysis of the total genomic DNA fragmentation was performed by agarose gel electrophoresis. Furthermore, to observe changes in cell morphology, promastigotes were examined using light microscopy. In both strains of L. major and L. tropica, miltefosine induced dose-dependent death with features of apoptosis, including cell shrinkage, DNA laddering, and externalization of phosphatidylserine. The IC50 was achieved at 22 microM and 11 microM for L. major and L. tropica after 48 hr of incubation, respectively. ED50 of L. major and L. tropica amastigotes were 5.7 microM and 4.2 microM, respectively. Our results indicate that miltefosine induces apoptosis of the causative agent of cutaneous leishmaniasis in a dose-dependent manner. Interestingly, L. major did not display any apoptotic changes when it was exposed to miltefosine in concentrations sufficient to kill L. tropica.