Comparison of Three Commonly Used Genetic Markers for Detection of Leishmania Major: An Experimental Study

BACKGROUND፡ Leishmaniasis is a vector-borne disease caused by an intracellular protozoan parasite called Leishmania spp. Different species produce different clinical outcomes; the majority of cases are cutaneous forms. Leishmania major is one of the main causative agents of cutaneous leishmaniasis (CL). Various methods are being using to diagnose CL, including microscopic examination, culture, and molecular detection of the parasite genome.METHOD: In the current study, we tried to compare three common molecular markers, including Kinetoplast DNA (kDNA), Cytochrome b (Cyt b), and Internal transcribed space 1 (ITS1), for the detection of Leishmania major. After cultivation of standard strain of L. major MHOM/IR/75/ER in RPMI 1640, certain number of promastigotes was subjected to DNA extraction and different PCR reactions.RESULTS: The lowest number of the parasite (5 promastigotes) can be detected by kDNA-PCR, followed by Cyt b-PCR (10 promastigotes), and ITS1-PCR (50 promastigotes).CONCLUSION: In conclusion, kDNA-PCR was the most sensitive marker and may provide more reliable data in the initial screening, especially in false-negative results provided by parasitological methods due to the low number of parasites

MOLECULAR SURVEILLANCE OF Plasmodium vivax AND Plasmodium falciparum DHFR MUTATIONS IN ISOLATES FROM SOUTHERN IRAN

In Iran, both Plasmodium vivax and P. falciparum malaria have been detected, but P. vivax is the predominant species. Point mutations in dihydrofolate reductase (dhfr) gene in both Plasmodia are the major mechanisms of pyrimethamine resistance. From April 2007 to June 2009, a total of 134 blood samples in two endemic areas of southern Iran were collected from patients infected with P. vivax and P. falciparum. The isolates were analyzed for P. vivax dihydrofolate reductase (pvdhfr) and P. falciparum dihydrofolate reductase (pfdhfr) point mutations using various PCR-based methods. The majority of the isolates (72.9%) had wild type amino acids at five codons of pvdhfr. Amongst mutant isolates, the most common pvdhfr alleles were double mutant in 58 and 117 amino acids (58R-117N). Triple mutation in 57, 58, and 117 amino acids (57L/58R/117N) was identified for the first time in the pvdhfr gene of Iranian P. vivax isolates. All the P. falciparumsamples analyzed (n = 16) possessed a double mutant pfdhfrallele (59R/108N) and retained a wild-type mutation at position 51. This may be attributed to the fact that the falciparum malaria patients were treated using sulfadoxine-pyrimethamine (SP) in Iran. The presence of mutant haplotypes in P. vivax is worrying, but has not yet reached an alarming threshold regarding drugs such as SP. The results of this study reinforce the importance of performing a molecular surveillance by means of a continuous chemoresistance assessment

ITS2-rDNA fragments of Leishmania species isolated from the great gerbil in Iran, 2021

Background: The great gerbil (Rhombomys opimus), is widely distributed in Asia and is a natural reservoir for zoonotic cutaneous leishmaniasis in many endemic areas, as well as Iran. Materials and Methods: In this study, infection to Leishmania species was investigated by two methods, parasitological and molecular survey, in the small number of R. opimus collected from Jovain, a Zoonotic Cutaneous Leishmaniasis (ZCL) focus located in North East of Iran. Results: Parasitological observation showed infection in only one of five rodents. But, ITS2-Nested-PCR revealed Leishmania infection in three out of 5 gerbils, including the parasitological positive one. Based on the PCR amplified size, two cases of infections were Leishmania major and one Leishmania turanica, their sequences are accessible in GenBank. The results of sequence analysis were consistent with the results obtained based on the size of the PCR. Conclusion: These findings re-confirm the important role of R. opimus in the natural circulation of Leishmania spp and indicate the need to be concerned about the disease in the study area

Chitosan-based Scaffolds, Suitable Structures for Wound Healing Dressing: A Short Review

Introduction: Different kinds of substances has been used for wound dressing, however, some disadvantages such as unsatisfactory mechanical stability, poor flexibility, severe shrinkage, low porosity, hard separation from the wound site, and non-antibacterial activity, has been reported. Over the last two decades, much effort has been made to find suitable biopolymer materials for wound healing applications. Chitosan has revealed various biological properties like biodegradable, biocompatible, non-toxic and non-allergenic, antibacterial effects thus can be used for the production of biofilms and nano-scaffolds. The poor solubility and thermal properties of chitosan restrict its widespread uses, but this polysaccharide is highly compatible with other biopolymers, and researchers are using this property to improve the limitations of chitosan and produce various types of chitosan-based hybrids materials. The purpose of this study is to provide an overview of various chitosan-based nanoscaffolds as wound healing dressings. Materials and Methods: This narrative review was performed using ISI Web of Science, PubMed, SID, Scholar, Scopus, and Science Direct and articles published up to Jan 2020 were included. The keywords of chitosan, chitosan-based scaffolds, chitosan-based composite, and wound dressing were  used. Results: Many researches have been accomplished to obtain chitosan-based scaffolds, including the construction of chitosan based blends and composite scaffolds and etc. The results of most of these researches showed positive effects of chitosan, and its nanocomposite scaffolds/biofilms in blood clotting, activated platelet activity, facilitated tissue regeneration and wound healing process. Conclusion: The use of chitosan-based scaffolds is effective in biological dressings and wound healing. Futuristic and innovative approaches in chitosan derivatives and nanocomposites can lead to the preparation of suitable co-polymers and the production of wound dressings with the desired properties. the authors hope that this review will help  for researchers

Prevalence and Genetic Characterization of Cryptosporidium Spp. In Diarrheic Children from Gonbad Kavoos City, Iran

Background: Cryptosporidium is an intestinal protozean parasite causing water­borne and foodborne outbreaks of diarrheal diseases. The present study was per­formed in order to find prevalence and subtypes of Cryptosporidium among children with diarrhea in Gonbad Kavoos City, Northern Iran. Methods: Diarrheic samples were collected from 547 children. The initial parasitologi­cal diagnosis was made based on detection of oocysts using the modi­fied Ziehl-Neelsen acid-fast staining method. The positive microscopically samples were selected for sequence analysis of partial 60 kDa glycoprotein (gp60) gene. Results: Out of 547 collected samples, 27 (4.94%) were positive for Cryptosporid­ium oocysts. Fifteen from 27 positive samples successfully amplified in PCR. Se­quences analysis of gp60 gene in 15 Cryptosporidium isolates revealed that all of them (100%) were C. parvum. The results showed three subtypes of IIa subtype family (7 cases) including IIaA16G2R1, IIaA17G1R1, IIaA22G3R1 and one subtype of IId subtype family (8 cases). The most common allele was IId A17G1d (53.3%). Conclusion: The predominance of zoonotic subtype families of C. parvum species (IIa, IId) in the present study is in concordance with previous studies in Iran and emphasizes the significance of zoonotic transmission of cryptosporidiosis in the country

Cloning and Sequence Analysis of Recombinant Plasmodium vivax Merozoite Surface Protein 1 (PvMSP-142 kDa) In pTZ57R/T Vector

Background:Haemonchosis has a negative effect on the farming industry throughout the world, especially in the tropic and sub-tropic countries. The present study was carried out to differentiate Haemonchus species from its main hosts in Iran, including sheep, goat and camel.  Methods:The identification took place based on the morphometrics of the spic-ules and molecular characters. Two hundred seventy adult male nematodes were collected from the abomasums of different ruminants (90 samples from each ani-mal) at the slaughterhouses from different localities in Iran. Samples were morpho-logically identified according to the spicules’ morphometric measurements. In the section on molecular study, 10 samples of each Haemonchus isolates were genetically examined. A simple PCR-restriction fragment length polymorphism (PCR-RFLP) assay of the second internal transcribed spacer of ribosomal DNA (ITS2-rDNA) were described to confirm the PCR results. Results:PCR-RFLP profile obtained from the restriction enzyme HPa1 in H. con-tortus and H. longistipes indicated 1 (278 bp) and 2 (113 and 135 bp) different frag-ments, respectively. The morphological parameters clearly distinguish H. contortus from H. longistipes. Moreover, regarding the ITS2-rDNA, sequences of 295 bp and 314 bp were obtained from H. contortus and H. longistipes, respectively. Conclusion:High similarity of cloned PvMSP-142 kDa gene in comparison to reference sequence and other sequences could be beneficial as a remarkable mo-lecular marker for serological diagnostic

Molecular Identification of Leishmania Species in a Re-Emerged Focus of Cutaneous Leishmaniasis in Varamin District, Iran

Background: Cutaneous leishmaniasis (CL) is one of the most important neglected tropical diseases and a major public health challenge in Iran caused by Leishmania spp and transmitted by phlebotomine sand flies. The number of CL cases has shown an increasing pattern all over the country, including the district of Varamin, southeast of Tehran, Iran. This study aimed to identify the Leishmania spp isolated from CL patients using molecular methods in Varamin during 2012–2013. Methods: Exudate materials collected from the swollen edge of the skin lesions of 44 parasitological positive CL patients by disposable lancet. They were referred to Varamin Health Center by physician. The samples were sub­jected to molecular method for Leishmania species identification. Results: The digestion pattern of restriction enzyme revealed that 37 (84.1%) CL patients were infected with L. ma­jor and 7 (15.9%) were infected with L. tropica. They were mostly male than female. More than half of the patients (58%) had multiple lesions, and they were mostly observed on extremities, 34.1% on legs and 29.5% on hands. Le­sions were mostly of wet ulcerative type. Conclusion: Dominancy of L. major provides more evidence that Varamin District probably could be considered as Zoonotic Cutaneous Leishmaniasis (ZCL) areas. More investigation on other epidemiological aspects of disease is needed.