Neutrophil extracellular traps formation: effect of Leishmania major promastigotes and salivary gland homogenates of Phlebotomus papatasi in human neutrophil culture.

BACKGROUND: Leishmaniasis as a neglected tropical disease (NTD) is caused by the inoculation of Leishmania parasites via the bite of phlebotomine sand flies. After an infected bite, a series of innate and adaptive immune responses occurs, among which neutrophils can be mentioned as the initiators. Among the multiple functions of these fighting cells, neutrophil extracellular traps (NETs) were studied in the presence of Leishmania major promastigotes and salivary gland homogenates (SGH) of Phlebotomus papatasi alone, and in combination to mimic natural conditions of transmission. MATERIAL & METHODS: The effect of L. major and SGH on NETs formation was studied in three different groups: neutrophils + SGH (NS), neutrophils + L. major (NL), neutrophils + L. major + SGH (NLS) along with negative and positive controls in 2, 4 and 6 h post-incubation. Different microscopic methods were used to visualize NETs comprising: fluorescence microscopy by Acridine Orange/ Ethidium Bromide staining, optical microscopy by Giemsa staining and scanning electron microscopy. In addition, the expression level of three different genes NE, MPO and MMP9 was evaluated by Real-Time PCR. RESULTS: All three microscopical methods revealed similar results, as in NS group, chromatin extrusion as a sign of NETosis, was not very evident in each three time points; but, in NL and especially NLS group, more NETosis was observed and the interaction between neutrophils and promastigotes in NL and also with saliva in NLS group, gradually increased over times. Real-time reveals that, the expression of MPO, NE and MMP9 genes increased during 2 and 4 h after exposure, and then decreased at 6 h in most groups. CONCLUSION: Hence, it was determined that the simultaneous presence of parasite and saliva in NLS group has a greater impact on the formation of NETs compared to NL and NS groups.

Detection and phylogenetic analysis of kinetoplast DNA of Leishmania infantum infected humans, domestic dogs and sandflies in Northwest Iran.

Leishmaniasis refers to a disease with a wide range of manifestations; and there are three main forms of disease, cutaneous, mucocutaneous, and visceral. Leishmaniasis is one of the diseases with a protozoan agent which is vector-borne. Visceral leishmaniasis (VL) is the most severe form that can be fiercely life-threatening if left untreated. VL can be caused by members of Leishmania donovani complex, in Iran, Leishmania infantum is considered the primary causative agent of VL, resulting in a zoonotic form of VL. The two main goals of our work, which followed our prior sero-epidemiological and entomological survey, were to characterize and conduct a phylogenetic analysis of the Leishmania species that infect people, dogs, and sandflies. The samples were collected throughout 2017, from January to December, so blood samples were collected from humans and dogs, while sandfly samples were collected with sticky traps. DNA extracted from all seropositive samples of humans and dogs, 10% of sero-negative human samples, and all collected sandflies were subjected to kDNA-nested-PCR for tracing parasites. A total of 30 samples, including 20 human samples, 8 dog samples, and 2 sandfly samples, were found positive for the kDNA gene of L. infantum. Sequences were evaluated to study the genetic diversity among the six discovered L. infantum. Based on kDNA, the phylogenetic study of L. infantum demonstrated a high level of genetic variety and a relationship between the host, the parasite's geographic origin, and its genetic diversity.

The Impact of COVID-19 Pandemic on Arthropod-Related Diseases.

Background: The Covid-19 pandemic that caused by the infection with the novel Coronavirus SARS-CoV-2 has revealed individual and global vulnerabilities all over the world. Many countries that had been struggled with arthropod-borne diseases (VBDs) are now embroiled in another challenge caused by COVID-19 pandemic. The situation that poses major obstacles 1) by misdiagnosis 2) delay in early and appropriate treatment of VBDs 3) difficulties in applying regular strategy for vector control and prevention methods and finally 4) irregularity in financing supports. Given the possible scenario of syndemics, it is important to plan integrated and combined measurement with the maximum participation of the people and health authorities. Here, the impact of COVID-19 pandemic on some major arthropod-related diseases will be discussed. Methods: To access the collective data all related databases such as Science direct, PubMed, Elsevier, Google scholar, as well WHO web page were searched with key words "arthropoda-related diseases, COVID-19 with the name of each individual disease". Results: The results showed that the management, control, and treatment of most important arthropod-related diseases could be delayed due to COVID-19 pandemic. Conclusion: Dealing with COVID-19, it is crucial to consider the other main killers such as malaria, dengue fever, etc. more especially in vulnerable populations by greater political, financial and global commitment. Continued surveillance will be essential to monitor for any possible changes.

Sergentomyia species identification and their screening for possible infection to Leishmania spp. in Kaleybar, East-Azerbaijan province, Iran.

Leishmaniasis is a protozoal and vector-borne disease. World health organization has considered the disease as a neglected tropical disease. Phlebotomus and Lutzumyia species (order: Diptera, family: Psychodidae) are human leishmaniasis vectors in new and old worlds. Sergentomyia spp. (Diptera, Psychodidae) are proven vectors of lizard leishmaniasis. Although some studies have identified human Leishmania parasites in Sergentomyia, their role in parasite circulation is unknown yet. Hence, the parasitological and molecular methods were used to study the possible Leishmania infection of Sergentomyia spp., in the human and canine visceral leishmaniasis endemic area in North West of Iran. Even though Sergentomyia specimens were caught in a dominant number compared to Phlebotomus spp., no Leishmania promastigote or DNA was detected in live-caught or sticky trap-caught specimens, respectively. Sergentomyia spp. are proven vectors of sauroleishmaniasis, and despite several global reports of Leishmania infection in Sergentomyia spp., such findings should be carefully interpreted to avoid false vector incriminations.

Successful Isolation of Leishmania RNA Virus (LRV) from Leishmania major in a Cutaneous Leishmaniasis Focus in Central Iran: An Update on Cases.

PURPOSE: Cutaneous leishmaniasis (CL) is a major vector-borne disease that affects people globally, including Iran. Different factors are associated with leishmaniasis pathogenicity; recently, a link of the possible relationship between Leishmania RNA Virus (LRV) and disease severity was proposed, especially in the New World leishmaniasis (NWL). This study was aimed to investigate the presence of LRV2 in Leishmania isolates in Aran o Bidgol, Isfahan province. METHODS: Samples were collected from 110 CL-suspected patients referred to the health center. In this study, we aimed to investigate CL cases (parasitologically and clinically), identify Leishmania species (by ITS1-PCR-RFLP), and finally detection of LRV2 (by RdRp-semi-nested PCR). RESULTS: Parasitological methods showed 60 positive cases, based on the HaeIII enzyme restriction profile, 59 cases were caused by L. major and 1 case by L. tropica. Our project is the first study on LRV2 isolation in Aran o Bidgol city and the LRV was successfully detected from a single L. major isolated in a women's hand lesion. Using BLAST, 94.8-100% similarity was observed in the RdRp sequence of current LRV isolate with those available in GenBank from Iran or overseas. CONCLUSION: L. major was the main cause of CL in Aran o Bidgol, although L. tropica is also present in a much lower proportion in the area. This is the first report on the presence of LRV2 in Aran o Bidgol and the fifth in Iran.

Molecular Detection of Plasmodium Infection among Anophelinae Mosquitoes and Differentiation of Biological Forms of Anopheles Stephensi Collected from Malarious Areas of Afghanistan and Iran.

Background: Updated information on the vectorial capacity of vectors is required in each malarious areas as well in Iran and its neighboring countries such as Afghanistan. The aims of this study were to investigate the potential infection of about 800 specimens collected from malarious areas of Afghanistan and Iran, and to differentiate biological forms of Anopheles stephensi. Method: Two molecular markers, 18S RNA gene subunit and AsteObp1 intron I, were used respectively for investigation Plasmodium infection and identifying the biological forms of An. stephensi. Results: Plasmodium infection was detected in 4 pools of Afghanistan specimens, including An. stephensi, collected from Nangarhar. Individually examination showed infection in 5 An. stephensi (infection rate: 1.25), to P. falciparum (2), P. vivax (2) and a mix infection. Out of five infected specimens, three were intermediate forms and two were mysorensis. No infection was found in specimens collected from Iran (Chabahar County), probably due to the active malaria control program in south-east of Iran. Conclusion: The key role of An. stephensi, as a known Asian malaria vector, was re-emphasized in Afghanistan by the results achieved here. The fauna of vectors and the pattern of biological forms of An. stephensi are similar in both countries that urge regional investigations to provide evidence-based and applied data for decision-maker in malaria control.

First Report of Natural Infection of Phlebotomus mongolensis to Leishmania major and Leishmania turanica in the Endemic Foci of Zoonotic Cutaneous Leishmaniasis in Iran.

Background: The primary aim of this study is to determine infection to Leishmania parasites in the wild population of Phlebotomus caucasicus and Phlebotomus mongolensis using molecular methods in some important zoonotic cutaneous leishmaniasis foci in Iran. Methods: Sand flies were collected from active colonies of rodent burrows from 16 trapping sites using sticky trap paper. In order to detect and identify of Leishmania parasites in females Ph. caucasicus and Ph. mongolensis, the Nested-PCR amplification of ITS2-rDNA region was performed to generate amplicon with 245bp for Leishmania major, 206bp for L. gerbilli and 141bp for L. turanica. Results: In the current study we found DNA of different gerbil parasites such as L. major and L. turanica, and mixed infection of L. major/L. turanica in Ph. caucasicus and Ph. mongolensis. It should be noted that, in Iran, natural infection with Leishmania parasites is recorded for the first time in this study in Ph. mongolensis. Conclusion: Both species of Ph. caucasicus and Ph. mongolensis not only may participate in the ZCL transmission cycle between reservoir hosts, but also results of this study support the role of these species as secondary vectors in the transmission of leishmaniasis to humans.

Probability of false-negative results in microscopical detection of cutaneous leishmaniasis: more accurate screening by kDNA-PCR during epidemiological survey.

Leishmaniasis has an important impact on global public health, and the common form of the disease is cutaneous form as well in Iran. Different species of Leishmania parasite make variable clinical manifestations, so prompt diagnosis and recognition at the species level are important due to their impact on the treatment and outcome of the disease. We aimed to examine the potential existence of the Leishmania parasite genome in the exudate materials derived from lesions of the cutaneous leishmaniasis suspected patients referred to Varamin Health Center Laboratory, that were reported negative microscopically. Regarding the object of the study, kDNA-Nested-PCR was used. A 570 bp band equal to what expected for Leishmania major was amplified in 18 out of 29 tested samples (62%). Findings indicate the effectivness of kDNA as a high copy number gene to avoid false-negative results.

Ecology of sand flies (Diptera: Psychodidae, Phlebotominae) in a new focus of leishmaniasis in northern Iran.

Leishmaniasis is an important infectious disease in Iran. Rudbar County of Guilan Province is introduced as a new focus of cutaneous leishmaniasis. The present study is going to investigate the sand flies fauna in the province and their seasonality and ecology in Rudbar County. To study the sand flies fauna, the sampling was carried out in ten localities of Guilan Province using light traps, sticky traps and manual aspirators during 2016. For the determination of seasonality, the sampling was carried out every two weeks in the fixed site of Pareh Village of Rudbar County during April-October 2016. Sand Flies were removed from sticky traps, rinsed in acetone and then conserved in 80% ethanol as well as the samples of light traps and hand catch. In total, 4430 specimens of sand flies of the genera Phlebotomus and Sergentomyia were collected and identified including Ph. kandelakii, Ph. neglectus, Ph. perfiliewi, Ph. sergenti, Ph. tobbi and Se. dentata. Sergentomyia dentata was found for the first time in Guilan Province. The most prevalent species were Ph. tobbi (55.87%), Ph. perfiliewi (36.03%) and Ph. kandelakii (6. 97%). The seasonality of sand flies started in late May and ended in middle October in the fixed site. The peak of activity of Ph. kandelakii was in late June; however that was in late August for Ph. perfiliewi and Ph. tobbi. The monthly abundance of total sand flies was positively correlated with saturation deficit (P = 0.043). The abundance of Ph. perfiliewi was positively correlated with maximum temperature (P = 0.016), relative humidity (P = 0.006), rainfall (P = 0.007), saturation deficit (P = 0.003) and negatively with minimum temperature (P = 0.005). The abundance of Ph. tobbi was positively correlated with saturation deficit (P = 0.041) and negatively with minimum temperature (P = 0.043). The presence of suspected vectors of cutaneous and visceral leishmaniasis, Ph. kandelakii, Ph. neglectus, Ph. perfiliewi, Ph. sergenti and Ph. tobbi, is noteworthy. Finding the exact vectors of leishmaniasis using specific tests in the province is suggested.

An Inventory of the Sand Flies (Diptera: Psychodidae) of Rudbar County, a New Focus of Leishmaniasis in Northern Iran, with a Taxonomic Note on the Subgenus Larroussius.

BACKGROUND: Different forms of leishmaniasis are significant infectious diseases in Iran. While, Rudbar County of Guilan Province has been introduced as a new cutaneous leishmaniasis focus, there are few published data about the phlebotomine sand flies (Diptera: Psychodidae) of the province. METHODS: To study the phlebotomine fauna of Rudbar County, the sampling was performed in 12 collection sites by light traps, sticky traps and manual aspirators throughout August-December 2015. Sand flies were removed from the sticky traps, rinsed in acetone and stored in 80% ethanol along with the collections of light traps and hand catches. RESULTS: In total, 2186 sand flies were collected and ten species representing two genera were morphologically identified: Phlebotomus (Adlerius) halepensis (0.27%), Ph. (Larroussius) kandeladii (0.10%), Ph. (Lar.) neglectus (0.91%), Ph. (Lar.) perfiliewi (53.88%), Ph. (Lar.) tobbi (43.45%), Ph. (Paraphlebotomus) sergenti (0.82%), Ph. (Phlebotomus) papatasi (0.10%), Sergentomyia (Parrotomyia) baghdadis (0.27%), Se. (Sintonius) clydei (0.05%) and Se. (Sin.) tiberiadis (0.10%). The species Ph. halepensis, Ph. neglectus, Ph. perfiliewi, Se. baghdadis, Se. clydei and Se. tiberiadis were reported for the first time in Guilan Province. This study also verified the presence of Ph. neglectus (Ph. major krimensis as a synonym and morphotype) in Iran. Moreover, the taxonomy of the subgenus Larroussius of the province was discussed. CONCLUSION: The prevalence of suspected or proven cutaneous and visceral leishmaniasis vectors is noteworthy. The study of ecology of sand flies and detecting the exact vectors of leishmaniasis and phlebotomine fever by molecular specific tests in Guilan Province are recommended.

A Molecular Screening of Mosquitoes (Diptera: Culicidae) for Flaviviruses in a Focus of West Nile Virus Infection in Northern Iran.

BACKGROUND: Mosquito-borne arboviruses such as West Nile (WN), dengue, Rift Valley fever, and Sindbis viruses are reported in Iran, but large-scale studies have not been performed on mosquitoes to find their vectors. A molecular study of the adult mosquitoes (Diptera: Culicidae) for flaviviruses was carried out in a focus of WN infection, Guilan Province, northern Iran. METHODS: Mosquito collections were carried out in five stations of two counties (Anzali and Rasht) using light traps, hand catch by manual aspirators and night landing catch during August-September 2013 and 2014. Molecular screening of WN virus and more widely for Flavivirus RNA was carried out using a specific PCR technique. RESULTS: In total, 1015 adult mosquitoes were collected including eight species representing four genera. The most prevalent species were Aedes vexans (33.2%), Culex tritaeniorhynchus (22%), Cx. pipiens (20.7%), and Anopheles maculipennis s.l. (15.6%). Molecular screening was carried out on the 1015 mosquitoes after they were organized as 38 pools according to sex, species and trapping location. None of the pools were positive. CONCLUSION: Surveillance should be continued while increasing the sampling campaigns due to the presence of wetlands in the region and abundant species which are considered as vectors, feeding on both birds and humans.

Genetic Diversity in C-terminal of SERA5 Gene in the Blood Stage of Human Isolates of Plasmodium vivax in Sistan and Baluchistan, Iran.

BACKGROUND: Vivax malaria is more prevalent in the malarious areas of Iran, which makes vaccine research a high priority. Serine Repeat Antigens (SERA) have essential role in the parasite life cycle and high expression profiles of PvSERA5 make it suitable vaccine candidates. This study aimed to evaluate the genetic diversity of C-terminal region of PvSERA5 in Iranian isolates of Plasmodium vivax in Sistan and Baluchistan. METHODS: Totally, 49 blood samples were taken from symptomatic malaria patients in Sistan and Baluchistan Province in 2016. Mono-infection to P. vivax was confirmed by 18srRNA-Nested-PCR. Genomic DNA was extracted and C-terminal region of PvSERA5 was amplified by specific primers. PCR-products have been sequenced and analysis was done by using bioinformatics software, mainly DnaSP & MEGA5. RESULTS: Genetic diversity was calculated 14.8% in C-terminal region of PvSERA5 in Iranian isolates, 19 different sequences and 4 haplotypes existed. The amount of Tajima's D (0.3805) and ratio of non-synonymous to synonymous mutation (1.82) showed that C-terminal region of PvSERA5 is under positive natural selection; also intragenic recombination could interfere. CONCLUSION: Results could be helpful in any research, regarding this antigen as vaccine candidate in Iran or worldwide.

Molecular-Based Detection of Leishmania infantum in Human Blood Samples in a New Focus of Visceral Leishmaniasis in Lorestan Province, Iran.

BACKGROUND: The fatal form of leishmaniasis is visceral form (VL), found in some of the countries in the world. Visceral leishmaniasis has been reported sporadically from all provinces in Iran, including Lorestan. This study aimed to characterize parasite species in DAT positive and some of the DAT negative human blood samples of Delfan district, Lorestan Province, central Iran. METHODS: Blood samples were collected from different geographical areas of Delfan. Serum was used for DAT test and remained part of molecular study. DNA was extracted by using DNG-plus extracted kit (Cinagen, Iran). Polymerase chain reaction amplification of Leishmania kDNA and PCR-RFLP of ITS1 was done to identify Leishmania species. Some amplicons were sequenced, submitted to GenBank and analyzed by BLASTn. RESULTS: Expected band of kDNA for L. infantum (720bp) was amplified in 16 out of 186 (8.6%) samples which showed previously anti-Leishmania antibody at different titers or were negative serologically. Using BLASTn, 93% similarity with L. infantum has been shown. The rDNA-ITS1 was amplified only in 9 samples (4.7%). RFLP pattern was similar to what expected for L. infantum. CONCLUSION: A new emerging hypo-endemic focus, caused by L. infantum, is going to be established in Delphan District, Lorestan Province. Further studies on vector and reservoirs are necessary for the region and other parts of Lorestan Province.

Identification of Leishmania species using N-acetylglucosamine-1-phosphate transferase gene in a zoonotic cutaneous leishmaniasis focus of Iran.

BACKGROUND & OBJECTIVES: Ilam province is one of the oldest known endemic foci of zoonotic cutaneous leishmani- asis (CL) in Iran; and the recent studies have shown an increasing trend in the number of cases from the region. This study was aimed to investigate the parasite species and genetic diversity of isolates obtained from CL patients based on the N-acetylglucosamine-1-phosphate transferase (nagt) gene. METHODS: Exudate materials were collected from the swollen margin of the skin lesions of the patients suspected with CL who were referred to health centers laboratory of Mehran, Dehloran, Ilam and Malekshahi cities in the Ilam province. Demographic data were collected through a questionnaire. Smears were stained and examined microscopically. In total, 62 parasitologically positive samples were subjected to PCR-RFLP of nagt gene for identification of Leishmania species, in addition to genetic diversity investigation. RESULTS: Nearly, half of the positive cases were referred from Mehran followed by Dehloran City (40.4%). These included people from different age groups (1 to 73 yr), with majority being male (66.1%). The common site of lesions was hand (48.4%). Half of the patients had multiple lesions; most of them were wet ulcerative type. A 1450-60 bp band of the nagt gene was amplified from all the samples. Digestion patterns of acetyl-coenzyme A carboxylase 1 (ACC1) enzyme were similar to what expected for Leishmania major. No difference was observed at the nucleotide acid level or resulting amino acid in nine sequenced samples on the basis of phylogenetic analyses. However, intra- species differences (0.0015) were observed amongst the L. major isolates of present study and the other parts of Iran. INTERPRETATION & CONCLUSION: The findings of this study demonstrated that the main causative agent of CL in Ilam Province is L. major, and there is no considerable heterogeneity among the L. major isolates. Moreover, nagt gene proved to be an efficient marker for differentiating Leishmania species. Further studies with more samples need to be carried out to achieve a more comprehensive result on the genetic variation of L. major isolates.

Spatial Distribution of Phlebotomine Sand Flies (Diptera: Psychodidae) as Phlebovirus Vectors in Different Areas of Iran.

Sand fly fever is caused by Naples (SFNV) and Sicilian viruses (SFSV) and the closely related Toscana virus of the Phlebovirus genus in the family Bunyaviridae, and transmitted by Phlebotomine sand flies. Phlebotomus papatasi Scopoli, 1786 is known as the main vector of the disease. This study aimed to investigate the distribution of Phlebotomine sand flies as vector of sand fly fever viruses, and the effects of some environmental variables on their potential dispersion to new areas in some provinces of Iran. Sand flies were collected during their active season in the region using CDC light traps. Ecological parameters were recorded for each collection site. Arc GIS 10.3 software was used for data analysis and mapping the distribution of sand flies. Sampling in the study areas was carried out in six different climatic zones. Seventeen sand fly species were collected including eight species of genus Phlebotomus and nine species of genus Sergentomyia. The Medium Semi-Arid climatic zone had the highest species diversity. Fourteen species of sand flies were collected at altitude between 2 and 325 m, and seven species were collected between 326 and 1380 m above sea level. There was significant correlation between sand fly density and all tested environmental variables. Phlebotominae sand flies have wide distribution in Iran and have a major public health concern in the country. P. papatasi and Sergentomyia sintoni Pringle, 1933 prefers hot summers and rainfall. This poses a threat of transmission of sand fly fever caused by SFSV and SFNV across the country.

Isolation and sequencing of Dashli virus, a novel Sicilian-like virus in sandflies from Iran; genetic and phylogenetic evidence for the creation of one novel species within the Phlebovirus genus in the Phenuiviridae family.

Phlebotomine sandflies are vectors of phleboviruses that cause sandfly fever or meningitis with significant implications for public health. Although several strains of these viruses had been isolated in Iran in the late 1970's, there was no recent data about the present situation at the outset of this study. Entomological investigations performed in 2009 and 2011 in Iran collected 4,770 sandflies from 10 different regions. Based on morphological identification, they were sorted into 315 pools according to species, sex, trapping station and date of capture. A phlebovirus, provisionally named Dashli virus (DASHV), was isolated from one pool of Sergentomyia spp, and subsequently DASHV RNA was detected in a second pool of Phlebotomus papatasi. Genetic and phylogenetic analyses based on complete coding genomic sequences indicated that (i) DASHV is most closely related to the Iranian isolates of Sandfly fever Sicilian virus [SFSV], (ii) there is a common ancestor to DASHV, Sandfly fever Sicilian- (SFS) and SFS-like viruses isolated in Italy, India, Turkey, and Cyprus (lineage I), (iii) DASHV is more distantly related with Corfou and Toros viruses (lineage II) although common ancestry is supported with 100% bootstrap, (iii) lineage I can be subdivided into sublineage Ia including all SFSV, SFCV and SFTV except those isolated in Iran which forms sublineage Ib (DASHV). Accordingly, we suggest to approve Sandfly fever Sicilian virus species consisting of the all aforementioned viruses. Owing that most of these viruses have been identified in human patients with febrile illness, DASHV should be considered as a potential human pathogen in Iran.

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.

Composition of Anopheles Species Collected from Selected Malarious Areas of Afghanistan and Iran.

BACKGROUND: Malarious areas in Iran are close to Afghanistan and Pakistan that urge the researchers to extend their knowledge on malaria epidemiology to the neighboring countries as well. Vectorial capacity differs at species or even at population level, the first essential step is accurate identification of vectors. This study aimed to identify Anopheles species composition in selected malarious areas of Afghanistan and Iran, providing further applied data for other research in two countries. METHODS: Adults Anopheles spp. were collected from four provinces in Afghanistan (Badakhshan, Herat, Kunduz, Nangarhar) by pyrethrum spray catch, hand collection methods through WHO/EMRO coordination and from Chabahar County in Iran by pyrethrum spray catch method. Identification was performed using reliable identification key. RESULTS: Totally, 800 female Anopheles mosquitos, 400 from each country were identified at species level. Anopheles composition in Afghanistan was An. superpictus, An. stephensi and An. hyrcanus. Most prevalent species in Badakhshan and Kunduz were An. superpictus, whereas An. stephensi and An. hyrcanus were respectively found in Nangarhar and Heart. Anopheles species in Chabahar County of Iran were An. stephensi, An. fluviatilis, An. culicifacies and An. sergentii. The most prevalent species was An. stephensi. CONCLUSION: Current study provides a basis for future research such as detection of Plasmodium infection in collected samples which is on process by the authors, also for effective implementation of evidence-based malaria vector intervention strategies.

Frequency of Cutaneous Leishmaniasis and Species Identification in Suspected Individuals from Golestan Province, Northern Iran in 2014.

BACKGROUND: Leishmaniasis is a zoonotic disease caused by species of protozoa of the genus Leishmania. In recent years, incidence of cutaneous leishmaniasis has increasing trend in Golestan Province, North of Iran. The aim of the present study was to identify the frequency of cutaneous leishmaniasis using PCR-RFLP in patients referred to Kalaleh Health Center, during 2013-14. METHODS: This descriptive cross-sectional study was conducted on 70 individuals with suspected cutaneous leishmaniasis that referred to health center of Kalaleh County, Golestan Province, Northern Iran, from Sep 2013 to Nov 2014. Samples of cutaneous lesions were examined microscopically. DNA was extracted from all of the positive smears and PCR was done on ITS-1 gene. RFLP was performed using HaeIII enzyme for species identification. RESULTS: Totally, 38 out of the 70 (54.3%) suspected individuals including 22 males (57.9%) were found positive by microscopic examination. All of microscopically positive samples were confirmed to be positive for Leishmania DNA (approximately 340 bp bands were detected). RFLP revealed 140 bp and 200 bp bands (approximate size), indicative of L. major. CONCLUSION: The detected species of studied region was L. major. Cutaneous leishmaniasis has high prevalence in Kalaleh County, thus more studies on leishmaniasis in the animal reservoirs, comparison of homology of animal and human isolates and a survey regarding natural infection of vectors in this region is highly recommended.

ITS2-rDNA Sequence Variation of Phlebotomus sergenti s.l. (Dip: Psychodidae) Populations in Iran.

BACKGROUND: Phlebotomus sergenti s.l. is considered the most likely vector of Leishmania tropica in Iran. Although two morphotypes- P. sergenti sergenti (A) and P. sergenti similis (B)-have been formally described, further morphological and a molecular analysis of mitochondrial cytochrome oxidase I (mtDNA-COI) gene revealed inconsistencies and suggests that the variation between the morphotypes is intraspecific and the morphotypes might be identical species. METHODS: We examined the sequence of the ITS2-rDNA of Iranian specimens of P. sergenti s.l., comprising P. cf sergenti, P. cf similis, and intermediate morphotypes, together with available data in Genbank. RESULTS: Sequence analysis showed 5.2% variation among P. sergenti s.l. morphotypes. Almost half of the variation was due to the number of an AT microsatellite repeats in the center of the spacer. Nine haplotypes were found in the species constructing three main lineages corresponding to the origin of the colonies located in southwest (SW), northeast (NE), and northwest-center-southeast (NCS). Lineages NCS and NE included both typical P. cf sergenti and P. cf similis and intermediate morphotypes. CONCLUSION: Phylogenetic sequence analysis revealed that, except for one Iranian sample, which was close to the European samples, other Iranian haplotypes were associated with the northeastern Mediterranean populations including Turkey, Cyprus, Syria, and Pakistan. Similar to the sequences of mtDNA COI gene, ITS2 sequences could not resolve P. sergenti from P. similis and did not support the possible existence of sibling species or subspecies within P. sergenti s.l..