Morphological Identification of Sandflies and Their Species’ Diversity

LIntrodaction:Leishmaniasis is a zoonosis disease, presented in three forms including cutaneous, mucocutaneous, and visceral (kala-azar). The parasite, Leishmania spp, is transferred by the causative agent of the disease, namely the sand-fly. Accurate identification of sand-flies helps to prevent and treat the disease. Materials and Methods:This study was conducted to identify sand-fly species distributed in Birjand, east of Iran. Sand-flies were collected from different parts of Birjand city, Iran through using sticky traps in summer 2015. Captured female specimens were mounted and identified by specific keys. Result:In this study, a total of 1480 samples of sand-flies were collected, out of which 945 sand-flies in areas were related to animal and 535 sand-flies in areas related to human. The most prevalent species, among 654 collected female specimens were Phlebotomus sergenti (22.17%) and Sergentomyia dentata (49.54%). Conclusion:According to the current results, the dominant species among disease vectors of cutaneous leishmaniasis is the urban type. Because of the presence of Leishmania tropica, the city's health system must take necessary care to control cutaneous leishmaniasis

Molecular Identification and Phylogenetic Classification of Leishmania spp. Isolated from Human Cutaneous Leishmaniasis in Iran: A Cross-sectional Study

Background: In Iran, both forms of cutaneous (CL) and visceral leishmaniasis (VL) have been reported; so the accurate species identification of the parasite(s) and the analysis of genetic diversity are necessary. Methods: The smears were collected from lesions samples of 654 patients with CL, who attended local health centers in 12 provinces of Iran during 2013-2015. The smears were checked for the presence of amastigotes by light microscopy. DNA of 648 Leishmania isolates, amplified by targeting a partial sequence of ITS (18S rRNA–ITS1–5.8S rRNA–ITS2) gene. Twenty-five of all the amplicons were sequenced and analyzed with restriction fragment length polymorphism (RFLP) using the Taq1 enzyme. Results: All the smears were positive microscopically. The PCR-RFLP analysis revealed that 176 (27%) CL patients were infected with L. tropica and, 478 (73%) with L. major. The dominant species in all over Iran is L. major. The sequencing results of all CL patients and RFLP analysis confirmed each other. Based on our phylogenetic tree, 25 ITS DNA sequences were grouped into two clusters representing L. major and L. tropica species. Phylogenetic tree derived from the ITS sequences supports a clear divergence between L. major from the other species. Conclusion: Discrimination of Iranian Leishmania isolates using ITS gene gives us this opportunity to detect, identify, and construct the phylogenetic relationship of Iranian isolates

Investigation of Species Diversity of Leishmaniasis Vectors (Phlebotomine Sand Flies) in Khusf County, South Khorasan Province (Iran)

Background and Objectives: Leishmaniasis as a zoonosis disease, occurs in three forms of cutaneous, mucocutaneous, and visceral (kala-azar). The causative agent of this disease is a protozoa of the Leishmania genus, which is commonly transmitted by species of sand flies. Given that explanation of an effective control strategy for vector-borne diseases requires identification of the fauna of their vectors, the present study was carried out to detect the fauna of Leishmaniasis vectors in Khusf county.   Methods: In this cross-sectional study, sand flies were captured using sticky traps in different parts of Khusf county from May 2015 to October 2015, Then, the species of female samples were mounted and identified using valid species identification key.   Results: In this study, from total 370 collected sand flies samples, 238 sand flies were related to animal areas and 132 sand flies were from human areas. Out of 115 female sand flies, Phlebotomus sergenti with frequency of 27.82% and Sergentomyia dentata with frequency of 34.78%, were reported as the dominant species in the region.   Conclusion: According to the findings of this study, the dominant species in the region was among the vectors of urban cutaneous leishmaniasis, and considering the presence of Leishmania tropica in the region, the health system of this county must consider required care for the control of cutaneous leishmaniasis disease. &nbsp

Morphological and Molecular Identification of Acanthamoeba Spp from Surface Waters in Birjand, Iran, During 2014-2015

Background & Aims of the Study: Free-living amoebae (FLA) are opportunistic and ubiquitous protozoa that are widely found in various environmental sources. They are known to cause serious human infections including a fatal encephalitis, a blinding keratitis, and pneumonia. So, due to their medical importance, the identification of free living amoeba in water resources, as a source of human infection, is necessary. The objective of this study was to isolate the Acanthamoebaspp from the surface waters of Birjand, Iran, during 2014-2015 by Morphological and molecular method. Materials and Methods:  In a cross-sectional study, 50 samples were collected from different localities of Birjand city including the surface waters, pools and fountains in parks,squares and water stations from the October 2014 to the January 2015.Each sample was filtered through a nitrocellulose membrane filters and cultured on non-nutrient agar (NNA) with Escherichia coli suspension and incubated for 1 week to 2 months at room temperature.The plates were examined by the microscopy to morphologically identify Acanthamoeba species. Following DNA extraction, PCR specific primers was used to confirm the identification morphologically. Results:  Out of 50 water samples, 19 (38%) were positive for Acanthamoebatrophozoites and cysts according to the morphological criteria. In addition, Acanthamoebaspp was identified by PCR method, using genus specific primers pairs in 15 (78.9%) cases of positive cultures, showing anearly 500bp band. Conclusion: According to the prevalent of Acanthamoebaspp in the surface stagnant waters of Birjand, more attention to the potential role of such waters in transmission of infection by the regional clinicians and health practitioners is necessary

Microscopic isolation and characterization of free living amoebae (FLA) from surface water sources.in Birjand, the capital city of the South Khorasan

Background and Aim: Free living amoebae (FLA) are amphizoic protozoa that are widely found in various environmental sources. They are known to cause serious infections in human and animal nervous systems. . The aim of the current study was to determine the presence of Acanthamoeba spp in surface water sources in Birjand city employing microscopic culture analysis based on morphology features of the amoebae. Materials and Methods: In this cross-sectional study, 50 samples of surface water sources in Birjand city. including . parks pools, public squares, waterfronts, and water stations were collected and .transferred to the laboratory and were passed through nitrocellulose filter paper. The remained elements in the filters were cultured on non-nutrient agar (NNA) with 100μl Escherichia coli suspension. After a few weeks of using morphological features,the amoeba grown were identified. Results: Out of the total of 50 samples cultured on non-nutrient agar (NNA), 19 (38%) samples.were morphologically polluted with Acanthamoeba spp,. In 2 samples (4%) a colony of Vahlkampfiidae were observed. Conclusion: The results indicated that a significant percentage of surface water sources in Birjand city was contaminated with Acanthamoeba spp. It is necessary for physicians, therefore, to take into account the diseases caused by these infectious agents. Besides, local regional health professionals should take into consideration the potential role of surface stagnant water sources in transferring these infectious agents. Placing warning signs in areas contaminated with these infectious agents seems a useful measure

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.