First report on natural Leishmania infection of Phlebotomus sergenti due Leishmania tropica by high resolution melting curve method in South-eastern Iran

OBJECTIVE@#To identify the Leishmania species in infected sand flies by Real-time PCR coupled with HRM analysis.@*METHODS@#Real-time PCR coupled with HRM analysis targeting the first internal transcribed spacer (ITS1) of nuclear ribosomal DNA as the genetic marker was used to identify and distinguish Leishmania species in sand flies specimens.@*RESULTS@#Three out of 115 females of Phlebotomus sergenti (P. sergenti) (2.6%) were positive to Leishmania tropica (L. tropica).@*CONCLUSIONS@#This is the first report on P. sergenti as the main and proven vector of anthroponitic cutaneous leishmaniasis in Dehbakri County using Real-time PCR coupled with HRM analysis. This method is rapid, sensitive and specific for diagnosing of parasites in infected Sand flies and ideal for large scale genotyping projects.

Molecular detection of leishmania infantum in naturally infected phlebotomus perfiliewi transcaucasicus in bilesavar district, Northwestern Iran

Visceral leishmaniasis is caused by Leishmania infantum, transmitted to humans by bites of phlebotomine sand flies and is one of the most important public health problems in Iran. To identify the vector[s], an investigation was carried out in Bilesavar District, one of the important foci of the disease in Ardebil Province in northwestern Iran, during July-September 2008. Using sticky papers, 2,110 sand flies were collected from indoors [bedroom, guestroom, toilet and stable] and outdoors [wall cracks, crevices and animal burrows] and identified morphologically. Species-specific amplification of promastigotes revealed specific PCR products of L. infantum DNA. Six sand fly species were found in the district, including: Phlebotomus perfiliewi transcaucasicus, P. papatasi, P. tobbi, P. sergenti, Sergentomyia dentata and S. sintoni. Phlebotomus perfiliewi transcaucasicus was the dominant species of the genus Phlebotomus [62.8%]. Of 270 female dissected P. perfiliewi transcuacasicus, 4 [1.5%] were found naturally infected with promastigotes. Based on natural infections of P. perfiliewi transcaucasicus with L. infantum and the fact that it was the only species found infected with L. infantum, it seems, this sand fly could be the principal vector of visceral leishmaniasis in the region

[Determination of parasite species of cutaneous leishmaniasis using nested PCR in Damghan-Iran, during 2008]

Cutaneous leishmaniases with two forms of rural and urban is the endemic diseases and as a health problem in our country. Identification of parasite species and type of disease is very important for treatment of disease as well as for planning of control program. The microscopic observations by Giemsa-stained smears is the most common laboratory test for the diagnosis of cutaneous leishmaniasis, but the determination of parasite species is impossible and utilization of other ways such as biochemical and molecular methods is required. This study was carried out to determine the parasite species caused cutaneous Leishmaniasis by Nested PCR in Damghan, Iran. This descriptive study was performed on 67 patients with dermal lesions that referred to Damghan health center laboratory in Iran during 2008. The patient's information were recorded in questionnaire. DNA of Giemsa-stained slides from patients was extracted and evaluated by specific primers of kinetoplast DNA using Nested PCR. Leishmania parasites were observed in 57 patients under light microscope. The 10 patients were infected by other dermal diseases. The PCR result showed the parasite presence in lesions of 57 patients is Leismania major. 54% of patients were male and 46% were female. 72% of the patients were lived in rural areas. 50.9% of disease was observed in over 25 years old patients. Hands were the most common region of ulcer [44.7%]. 48% of the patients had one ulcer and the other patients had two or more ulcers. High prevalence [31.6%] of disease was observed in October. This study showed that zoonotic cutaneous leishmaniasis to be prevalent in this area and Nested PCR method is a sensitive and accurate to leishmania species characterization

Identification of sand flies of the subgenus Larroussius based on molecular and morphological characters in North Western Iran

The adult female sand flies [Diptera: Psychodidae] of the subgenus Larroussius are important vectors of Leishmania infantum [Kinetoplastida: Tripanosomatidae] in Meshkinshahr district, Northwest of Iran. Four Phlebotomus [Larroussius] species are present in this area, i.e. Phlebotomus [Larroussius] kandelakii, P. [La] major, P. [La] perfiliewi and P. [La] tobbi. The objective of the present study was to identify and distinguish the females of P. perfiliewi, P. major and P.tobbi, in this district. Adult sand flies were collected with sticky papers, CDC light traps, and aspirator in 2006. Individual sand flies of this four species from thirty different locations were characterized morphologically and by comparative DNA sequences analyses of a fragment of mitochondrial gene Cytochrome b [Cyt b] and nuclear gene Elongation Factor 1- alpha [EF -1 alpha]. PCR amplification was carried out for all three species P. major, P. perfiliewi, and P. tobbi in the sub-genus Larroussius. Phylogenetic analyses of P. major populations in this study displayed two different populations and genetic diversity. Spermathecal segment number, pharyngeal armature and other morphological characters of these three species were examined and found to present consistent interspecific differences. According to our findings, the phylogeny of Cyt b and EF-1 alpha haplotypes confirms the relationships between P. major, P. tobbi and P. perfiliewi as already defined by their morphological similarities

Molecular detection of leishmania major in the vectors and reservoir hosts of cutaneous leishmaniasis in Kakaleh district, Golestan province, Iran

An epidemiological study was carried out on the vector[s] and reservoir[s] of cutaneous leishmaniasis in rural areas of Kalaleh District, Golestan Province during 2006 - 2007. Totally 4900 sand flies were collected using sticky papers and were subjected to molecular methods for detection of leishmanial parasite. Phlebotomus papatasi was the common species in outdoor and indoor resting places. Employing PCR technique showed only 1 out of 372 P. papatasi [0.3%] was positive to parasite due Leishmania major. Sixteen rodent reservoir hosts were captured by Sherman traps and identified as Rhombomys opimus. Microscopic investigation on blood smear of the animals for amastigote parasites revealed 6[37.5%] infected rodents. Infection of these animals to L. major was then confirmed by PCR against rDNA loci of the parasite. This is the first molecular report of parasite infection of both vector [P. papatas] and reservoir [R. opimus] to L. major. The results indicated that P. papatas was the primary vector of the disease and circulating the parasite between human and reservoirs, and R. opimus was the most important host reservoir for maintenance of the parasite source in the area

[Phlebotomus [Paraphlebotomus] alexandri, the probable vector of visceral leishmaniasis [kalaazar] in southern Iran]

Visceral leishmaniasis [kala-azar], the most dangerous form of leishmaniasis, is endemic in some parts of Iran, e.g. Ardabil, Fars, East Azerbaijan, Bushehr and Qom provinces. In recent years, the incidence of VL has increased in the Nourabad-Mamassani district in Fars Province. This study was carried out to detect VL vectors and infection rates in this region over the 2003-2004 period. Sand flies were captured in the selected villages by means of sticky traps, aspirators and CDC miniature light traps. Heads and distal abdominal segments were used for species identification and other body parts were used for DNA extraction. We employed a semi-nested PCR technique to detect Leishmania, with specific kDNA primers [LIN R4 - LIN 17 - LIN19]. Some specimens were dissected for leptomonad infection. A total of 12688 sand flies were collected. Phlebotomus [Paraphlebotomus] alexandri was the second most prevalent species [17.34%]. The anthropophilic index of this species was 32.5%. Five cases [4.17%] of L. infantum infection were detected among the 120 P. alexandri examined by PCR method. We also observed two cases of leptomonad infection among the 112 dissected specimens. High prevalence rates and anthropophilic index of P. alexandri plus its natural infection with L. infantum provide enough evidence to implicate this species as the main vector species of VL in the region and the second proven kala-azar vector in Iran. Besides, the Mahoor-Milaty district of Noorabad-Mamassani was identified as a new endemic focus

seminested PCR based detection of Leishmania infantum infection in asymptomatic dogs in a new endemic focus of visceral leishmaniasis in Iran

Visceral Leishmaniasis [Kala-azar] is a serious health problem in some northern and south western parts of Iran. The incidence of kala-azar caused by Leishmania infantum has recently increased in Nourabad-Mamassani district of Fars Province, in the south of the country. This study was designed to determine the role of asymptomatic dogs as host reservoir of L. infantum in this new formed focus and detection of prevalence of infection near them. A total of 20 asymptomatic stray and sheep dogs were randomly sampled. The Buffy coat layer of their peripheral blood was used for DNA extraction and PCR. A species specific seminested PCR was used for DNA amplification using LINR4, LIN17 and LIN19 primers. These primers amplified variable area of the minicircle kDNA of Leishmania parasites. Of the 20 sampled dogs checked for leishmanial kDNA, six [30%] were found naturally infected. It is concluded that, dogs [Canis familiaris] even if asymptomatic, is considered as the domestic host reservoir of kala-azar in this endemic focus

[Determination of reservoir[s] and vector[s] of cutaneous leishmaniasis by nested-PCR in Marvdasht District, Fars Province, Southern Iran]

Cutaneous leishmaniasis [CL] is an increasing public health problem in several parts of Iran. In southern parts, the incidence of CL has been doubled over the last decade. This epidemiological study was done for determination of reservoir[s] and vector[s] of cutaneous leishmaniasis in rural regions of Marvdasht, Fars province, southern Iran during 2003 and 2004. A total of 126 rodents were collected from three villages using live traps and their Giemsa-stained smears were studied for leishmania infection. After DNA extraction from positive smears, Nested-PCR was used for the identification of parasite species. In another procedure, 200 sand flies were collected by aspirator and after species identification DNA extraction and PCR was done. The collected samples included Meriones libycus [75.4%], Cricetulus migratorius [14.3%] and Microtus arualis [10.3%]. Eight out of 95 Meriones libycus [8.4%] were found to be infected with Leishmania major. None of the other species were positive. Among the collected female sandflies 75% were identified to be Phlebotomus papatasi and 2.7% of them were found with L.major infection. Only 2.7% of Phlebotomus papatasi were found naturally infected with Leishmania major. This is the first report of detection of L.major by Nested-PCR in P.papatasi as a proven principal vector of zoonotic cutaneous leishmaniasis in Fars province, south of Iran

Vectors and reservoirs of cutaneous leishmaniasis in Marvdasht district, southern Islamic Republic of Iran

An epidemiological study was made of vectors and reservoirs of cutaneous leishmaniasis in rural regions of Marvdasht, Fars province, southern Islamic Republic of Iran during 2003-04. Using live traps, 126 rodents were collected: 75.4% were Meriones libycus, 14.3% Cricetulus migratorius and 10.3% Microtus arvalis. Eight out of 95 Meriones libycus [8.4%] were found to be infected with Leishmania major, identified by nested-PCR; none of the other rodents were positive. Female sandflies were collected from indoor locations: 75% were Phlebotomus papatasi and only 2.7% were found naturally infected with L. major. This is the first report of P. papatasi as a proven vector of zoonotic cutaneous leishmaniasis in this area

Meriones libycus is the main reservoir of zoonotic cutaneous leishmaniasis in south Islamic Republic of Iran

A study was made in rural regions around Neiriz city, Fars province in the south of the Islamic Republic of Iran during 2002-03 to further investigate a new focus of zoonotic cutaneous leishmaniasis. Meriones libycus was the dominant rodent [100% of 65 rodents collected] and 4.6% were naturally infected with the amastigote form of Leishmania spp. Promasigotes were identified as L. major. This confirms M. libycus as the main reservoir host of zoonotic cutaneous leishmaniasis in southern parts of the country