Molecular identification of leishmania species using samples obtained from negative stained smears

Cutaneous Leishmaniasis [CL] is a parasitic skin disease. Diagnosis primarily is based on clinical signs and microscopic observation of parasite on direct stained smears or tissue sections. Sensitivity of direct smear is not as high as molecular methods. The aim of this study was to identify and characterize Leishmania species among the negative direct smears obtained from skin ulcers suspected to CL by PCR method. Among 81 patients with suspicious skin lesions to CL referred to the Parasitology lab, negative Giemsa stained smears were collected. DNA extraction performed by scraping stained smears, then PCR was performed. Among the DNA extracted from smears, L. tropica was isolated from 9 [11.1%] of the smears and L.major was not isolated from any samples. Direct microscopy on stained smears for diagnosis of leishmaniasis is not enough accurate. PCR is recommended for clinically suspected lesions with negative result of direct smear.

Whatman paper [FTA Cards] for storing and transferring Leishmania DNA for PCR examination

Diagnosis of cutaneous leishmaniasis [CL] is often made based on clinical manifestation. Correct diagnosis and identification of the parasite are crucial for choosing the effective treatment and for epidemiological studies. On the other hand, determination of Leishmania species is necessary for designing appropriate control programs. Diagnosis by PCR is becoming a [gold standard]. For PCR preparation, storage and shipments of specimens are necessary. In this study, Whatman filter paper [FTA Card] was used to store and transfer samples for Leishmania identification using PCR. Among the patients who had CL lesion and referred to Parasitology Laboratory of Emam Reza Hospital, Mashhad, Iran, 44 consented cases with positive results in their direct smear were selected. An informed consent form and a questionnaire were completed and three different types of samples [direct smear, NNN culture, and spot on FTA card] were collected. DNA extraction and PCR were carried out on three different samples from each patient. PCR results using Whatman paper samples revealed a significant difference [P<0.0001] compared to the culture method but no significant difference was seen between PCR results using samples stored on Whatman paper and direct smears. The use of FTA cards is simple, rapid, and cost-effective, and can be readily employed for large-scale population screening, especially for regions where the specimens are to be transported from distant places to the laboratory

[ case report of alveolar echinococcosis]

Echinococcus multilocularis is the cause of alveolar hydatic cysts in intermediate host, found in the liver and lungs. Alveolar hydatid diseas is the most lethal of helminthic diseases, with radical surgery still being the only curative therapy. The aim of this study was to report a splenic alveolar hydatic cyst. The patient was a 62 years old man, from a rural region of Ghochan, Khorasan Province, Iran, who was referred to surgeon, complaining left upper quadran abdominal pain. In CT scan 2 cysts were seen in the right lobe of liver and a septal cyst in spleen. After operation, evacuating and unroofing of the liver cysts, splenectomy was performed. Diagnosis was based on pathologic and histologic examinations. In macroscopic examination spleen was massive and cystic, composed of multiple irregular cysts with infiltrative appearance. The inner part of cysts contained brown necrotic and gelatinous material, at microscopic examination there was a thin acellular laminated layer but the germinal layer and protoscolex were not seen. After a week the patient recovered and left the hospital with good general condition. In conclusion, the presence of this case showed that life cycle of echinococcus multilocularis can be completed in this province. Medical managers and practitioners must notice this disease as a differential diagnosis for malignant of liver and spleen, as well as for prevention and control of the disease itself

[ study on the vectors of the cutaneous leishmaniasis in the northern part of Mashhad, Iran]

Phlebotomine sand flies are the vectors of different forms of leishmaniasis in Iran. Study on their ecology is essential for suggestion of leishmaniasis control programs. Following annually new case reports of cutaneous leishmaniasis, at Hemat Abad an investigation on the fauna and monthly activating phlebotomines was carried out during Mar 2003. Nov. 2003 in this region. Phlebotomine sand-flies were collected every 10 days from outdoors and indoors with sticky traps, during Mar. 2003-Nov. 2003. In this study 521 sand-flies were collected from outdoors and indoors [2 species of phlebotomus genus and 2 species of sergentomyia genus], as follows, P. sergenti [74.7%], P. papatasi [1Ll %], S. sintoni [13.3%], S. sombarica [0.9%]. The sand flies active season begins from April till October in this region. P. sergenti was the predominant phlebotomines species in our investigation. Based on the results, P. sergenti is the probable vector of Cutaneous leishmaniasis in this region and P. Papatasi can transmit disease from rodents to humans

Treatment of cutaneous leishmaniasis in murine model by alcoholic extract of Berberis vulgaris

In order to evaluate the effect of Berberis vulgaris extract on the experimental ulcers of cutaneous leishmaniasis [CL] on Balb/c mice, a study was undertaken over a 12 months period. Forty Balb/c mice were divided into 2 main groups A and B. Each main group in turn was divided into 5 sub groups of 4 mice and each sub group were inoculated subcutaneously by 0.1ml liquid phase culture containing promastigotes of Leishmania major. After 2-3 weeks, nodules and ulcers appeared on 37 of 40 inoculated mice. Ethanol extract of the stem and leaves as well as roots of Berberis vulgaris in different concentrations, were used topically on CL lesions of 4 sub groups A and B, respectively. Ethanol alone was used on the lesions of control mice. The surface area of lesions were measured before and 1-2 weeks after treatment. Direct Geimsa stained smear prepared 20 days after treatment. The results showed that after 2 weeks, a statistically significant decrease of ulcer size of treated mice observed, while in the control group the lesion growth continued. The examinations showed that using higher concentration of the extract caused more decrease in surface area of CL lesions on day 15 and negative direct smear on day 20. Alcoholic extract of B.vulgaris root was more effective than leaves and stem extract. Alcoholic extract of B vulgaris might be further used in animal model