The changing profile of cutaneous leishmaniasis agent in a central province of Iran

Cutaneous leishmaniasis in Iran is usually caused by Leishmania major or L. tropica. However, the direct examination or the cultures of biopsies for diagnosis are not very sensitive. The objective of this study was to identify the responsible species obtained from patients suspected of cutaneous leishmaniasis referred to the reference laboratory at Yazd in Iran during 2010-2011 using parasitological and molecular assays. After completing a clinical/epidemiologic data questionnaire for 145 patients with suspected skin lesions, scraping samples were collected. Each specimen was examined using both direct microscopy and molecular assay using polymerase chain reaction-restriction length polymorphism (PCR-RFLP). Location of the lesions included 47.7% on hands, 30.7% on face, 15.4% on feet, and the remainder on other regions. Out of 145 samples, Leishman body was observed in 52 by direct smear and 73 by PCR assay. Molecular assay indicated 36 cases as L. major, 36 cases as L. tropica and one case as unknown.  In conclusion, molecular characterization showed changing profile of Leishmania species in the study area which may have implications on treatment and/or control strategies

Molecular diagnosis of Old World leishmaniasis: Real-time PCR based on tryparedoxin peroxidase gene for the detection and identification of Leishmania spp

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

Molecular Analysis of Aquaglyceroporin 1 Gene in Non-Healing Clinical Isolates Obtained from Patients with Cutaneous Leishmaniasis from Central of Iran

Background: Regarding the antimonial-resistant of Leishmania spp., understanding of related mechanism is necessary. One of the most important involved molecules is aquaglyceropin1 (AQP1). The aim of this study was molecular analysis of AQP1 gene from antimonial-resistant clinical isolates and its expression. Methods: Overall, 150 patients with cutaneous leishmaniasis referring to the reference laboratories of Yazd and Varzaneh,, located 105km southeast of Isfahan and 240km away from Yazd, were assessed from Jun 2015 to Dec 2017. After sampling, staining was done and evaluated for Leishman by microscope. Samples were collected in RNAlater solution for gene expression analysis in non-healing isolates. DNA extraction was performed from each slide with Leishman body. All patients with L. major isolates detected by ITS1-PCR-RFLP were followed for finding the resistant isolates, consequence of molecular characterization of AQP1 using PCR-RFLP. Gene expression of AQP1 from all resistant isolates was assessed in comparison with the one in a sensitive isolate. Statistical analysis was done using SPSS. The significance level was considered ≤0.05. Results: Five isolates were detected as antimonial resistant. Molecular detection and identification were appeared that all were L. major. The molecular characterization of AQP1 showed G562A mutation. Gene expression of AQP1 in resistant isolates showed 1.67 fold higher than the sensitive isolate. Conclusion: We reported a new point mutation of G562A in AQP1 gene involved in molecular mechanism in resistant isolates

Development of a chamber system for rapid, high yield and cost-effective purification of deoxyribonucleic acid fragments from agarose gel

Background: There are several methods commonly practicing for deoxyribonucleic acid (DNA) purification from agarose gel. In most laboratories, especially in developing countries, present methods for recovering of DNA fragments from the gel are mostly involved organic solvents. However, manual purification using organic solvents are toxic, labor intensive, time consuming and prone to contamination owing to several handling steps. The above mentioned burdens as well as cost and long time to import them, especially in developing countries, prompted us to design and develop a chamber system for rapid, non-toxic, cost-effective and user friendly device for polymerase chain reaction (PCR) products purification from agarose gel. Materials and Methods: The device was made from plexiglass plates. After amplification of two fragments of 250 and 850 bp, PCR products were electrophoresed. Subsequently, the desired bands were excised and purified with three method: HiPer Mini chamber, phenol extraction method and spin column procedure. To assess the suitability of the purified DNAs, restriction digestion was applied. Results: Results showed that the yield of recovered DNA in our method was above 95%, whereas the yields obtained with conventional phenol extraction and spin column methods were around 60%. Conclusion: In conclusion, the current method for DNA elution is quick, inexpensive and robust and it does not require the use of toxic organic solvents. In addition, the purified DNA was well has suited for further manipulations such as restriction digestion, ligation, cloning, sequencing and hybridization

Molecular identification of Leishmania isolates obtained from patients suspected as having cutaneous leishmaniasis referred to reference laboratories from Yazd province in central Iran

Background: Cutaneous leishmaniasis (CL) continues to be an increasing public health problem in Iran. The dominant etiologic agents of CL in the Old World are Leishmania major and Leishmania tropica. One of the important endemic foci of CL in Iran is Yazd. Recently, previous studies showed the equal prevalence of L. major and L. tropica as the agents of cutaneous leishmaniasis in this area. This prompted us to identify the genotype of L. major isolates obtained from patients with cutaneous leishmaniasis. Materials and Methods: After completing a clinical/epidemiologic data questionnaire for 218 patients with suspected skin lesions, scraping samples were collected, and each specimen was examined using both direct microscopy and molecular assay of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Results : Results showed that of the 218 samples, Leishman body was observed in 77 by direct smear and 104 by PCR assay. Molecular assay indicated 50 cases as L. major, 52 cases as L. tropica, and two cases as unknown. Molecular characterization of L. major isolates showed four patterns, named LmA1, LmA2, LmA3, and LmA4. Conclusion: Our study is the first report for molecular characterization of L. major from one of the important central province of Iran that could affect the control strategies in this field

The association between single nucleotide polymorphism in interleukin-27 gene and recurrent pregnancy loss in Iranian women

Background: Recurrent pregnancy loss (RPL) has been defined as two or more miscarriages before 20th week of gestation. It seems that IL-27 may reduce inflammatory responses and affect the survival of the embryo during human pregnancy. IL-27 polymorphisms may influence RPL by altering the levels or the activity of gene product. Objective: We studied for the first time the association of IL-27 -964 A>G single nucleotide polymorphism (SNP) with RPL in Iranian women. Materials and Methods: A case-controlled study was performed on two groups consisting of 150 healthy women with at least one delivery (control group) and 150 women with two or more primary RPLs history (RPL group). The -964 A>G SNP in IL-27 gene was determined by PCR-RFLP technique. Genotype and allele frequencies were compared using 2 tests between two groups. Results: There was no difference between the two groups regarding age of women (29±4.4 [control] vs. 30.84±5.2 years [case]). In the RPL group, the genotype frequencies of -964 A>G polymorphism were AG (49.3%), AA (40%), and GG (10.7%), and in the control group, they were AG (43.3%), AA (48.7%), and GG (8%). There was no significant difference between the genotypes of AA, AG, and GG in two groups (p=0.23). As the frequency of allele A was 64.7% in the RPL group and 70.3% in the control group, the difference in frequency of allele A in -964 A>G between two groups was not significant (p=0.19). Conclusion: Our findings indicate that SNP of -964 A>G in IL-27 gene is not a risk factor for RPL in Iranian women

Molecular diagnosis of Old World leishmaniasis: Real-time PCR based on tryparedoxin peroxidase gene for the detection and identification of Leishmania spp

Background & objectives: Rapid and accurate diagnosis and identification of Leishmania sp causing cutaneousleishmaniasis is crucial in control and therapeutic programs. The problem of diagnosis with traditional methodsis that they have a low sensitivity or time consuming but molecular techniques would be an alternative methodfor rapid and accurate diagnosis. In this work, tryparedoxine peroxidase gene-based real-time PCR was used foraccurate 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 fordirect microscopy, culture in NNN or fixed in alcohol for identification of Leishmania spp using tryparedoxinperoxidase gene-based realtime PCR (qPCR).Results: Using direct microscopy and culture method, Leishmania parasites were isolated from 68 out of 100patient samples. However, 13 patients with negative finding on traditional tests, had positive results on RT-PCRtest. After melting curve analysis of PCR product, Leishmania major in 75 and L. tropica in 4 cases wereidentified. The sensitivity and specificity of RT-PCR for diagnosis of cutaneous leishmaniasis was 98.7 and59.8%, respectively.Conclusion: Results of this study showed that RT-PCR was the most sensitive diagnostic test for cutaneousleishmaniasis and represents a tool for rapid species identification

The changing profile of cutaneous leishmaniasis agent in a central province of Iran

Cutaneous leishmaniasis in Iran is usually caused by Leishmania major or L. tropica . However, the direct examination or the cultures of biopsies for diagnosis are not very sensitive. The objective of this study was to identify the responsible species obtained from patients suspected of cutaneous leishmaniasis referred to the reference laboratory at Yazd in Iran during 2010-2011 using parasitological and molecular assays. After completing a clinical/epidemiologic data questionnaire for 145 patients with suspected skin lesions, scraping samples were collected. Each specimen was examined using both direct microscopy and molecular assay using polymerase chain reaction-restriction length polymorphism (PCR-RFLP). Location of the lesions included 47.7% on hands, 30.7% on face, 15.4% on feet, and the remainder on other regions. Out of 145 samples, Leishman body was observed in 52 by direct smear and 73 by PCR assay. Molecular assay indicated 36 cases as L. major, 36 cases as L. tropica and one case as unknown. In conclusion, molecular characterization showed changing profile of Leishmania species in the study area which may have implications on treatment and/or control strategies