Comparison of Three Methods for Diagnosis of Cutaneous Leishmaniasis

Background: Leishmaniasis is one of the infectious parasitic diseases of highest incidence in the world. Cutaneous Leishmaniasis (CL) has long been reported in Shiraz, Southern Iran. There is a need to find a sensitive and specific method for treatment and control of the disease.Methods: We have compared the sensitivity of the conventional methods microscopy and cultiva­tion of lesion scrapes against PCR amplification of parasite kinetoplast DNA from these sam­ples. The samples (n=219) were obtained from the patients clinically suspected of CL. The smears were stained with Giemsa for microscopy and cultured in Novy-Nicolle-McNeal (NNN) blood agar for promastigote growth. For PCR, the dry smears were scraped off the slides and DNA was extracted.Results: The positive rates from 219 specimens were 76.71%, 50.68%, and 93.61% for micros­copy, cultivation, and PCR, respectively. The highest correlation was found between PCR and micros­copy method (P= 0.014). In PCR assay, 95.61%, 3.9%, and 0.49% of the samples were identi­fied as Leishmania major, L. tropica, and dermatropic L. infantum, respectively.Conclusion: The PCR method appears to be the most sensitive for the diagnosis of CL and is valu­able for identifying the other species of Leishmania with confusing dermatropic signs

In Vitro Infectivity of Leishmania major Isolated from Patients with Different Clinical Forms of Cutaneous Leishmaniasis and Its Association with Parasite Zymodems

"nBackground: The aim of this study was to characterize the Leishmania parasites isolated from cuta­neous leishmaniasis (CL) patients in Fars Province in Iran and to compare the potential infectivity of the isolates in macrophage cell line. Moreover, attempt was made to find out the association between parasite infectivity and their zymodems. "nMethods: Twenty samples were taken from the skin lesion of CL patients. The samples were cultured in biphasic media followed by mass cultivation in RPMI medium. Each isolate was tested for the ac­tivity of the 5 enzymes including glucose phosphate isomerase (GPI), malate dehydrogenase (MDH), nucleoside hydrolase 1& 2 (NH1 & NH2), and phosphoglucomutase (PGM).   The enzymatic profiles of the isolates were compared with WHO reference strains. Specific PCR (primers: LIN17 & LIN R4) and RAPD-PCR were used as complementary methods for characterization of the isolates. "nResults:  Isoenzyme electrophoresis showed that all of the isolates were L. major. PCR with LIN17 and LIN R4 and RAPD-PCR with AB-07 primers further determined the isolates as L. major. Results of macrophage infectivity experiment, using J774 cell line, showed that the most virulent isolates were related to Z1 with 63% macrophage infectivity rate. A well correlation was found between the infec­tivity rate of the isolates and type of ulcer. Those isolates with high infectivity rate were involved in more severe, ulcerative or erythmatose lesions in CL patients. "nConclusion: The most invasive isolates might be a good candidate for immunological studies and for vaccine development

Chrysomya bezziana as a Causative Agent of Human Myiasis in Fars Province, Southern Iran

Myiasis is the invasion of body tissues of humans or animals by the larvae of the Diptera or two-winged flies. The vari­ous forms of myiasis may be classified from clinical or entomological point. This study describes the existence of Chry­somya bezziana (Diptera: Calliphoridae) cases as a causative agent of myiasis in 18 and 87 year-old men in two differ­ent regions in Fars Province. To our knowledge, this is the first observation of mentioned species in this prov­ince.

Direct Agglutination Test and Enzyme Linked Immunosorbent Assay with Urine Samples for the Diagnosis of Visceral Leishma-niasis

Background: Visceral leishmaniasis (VL) or Kala azar is an infectious disease caused by various species of Leishmania parasites. The aim of this study was to detect and compare the presence of anti-Leishmania antibodies in the urine of vis-ceral leishmaniasis patients using ELISA and DAT methods."nMethods: A total of 30 urine samples were collected from VL patients referred to Shiraz (southeast of Iran) hospitals. Moreover 31 urine samples were collected from healthy individuals and patients with other diseases such as malaria, brucellosis, hydatidosis and cutaneous leishmaniasis. Collected samples were examined to detect anti-Leishmania antibod-ies in urine, using ELISA and DAT."nResults: Anti-Leishmania antibody was detected in urine of 18 out of 30 (60%) VL patients by DAT while ELISA detected anti-Leishmania antibodies in urine of 28 out of 30 (93.3%) of VL cases. Sensitivity and specificity of urine-based DAT was 60% and 83.9%, respectively while sensitivity and specificity of urine-based ELISA were 93.3% and 93.5%, corre-spondingly. "nConclusion: Urine-based DAT and ELISA have a reasonable specificity and sensitivity in diagnosis of VL. Accordingly, urine-based ELISA might be a suitable alternative for serum based assays for diagnosis of VL

The 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 as¬ymptomatic 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 asympto¬matic, is considered as the domestic host reservoir of kala-azar in this endemic focus

Characterization of in Vitro Cultivated Amastigote like of Leishmania major: A Substitution for in Vivo Studies

Background: Promastigotes of Leishmania spp. have been readily cultured, but the axenic culture of amastigotes has been successful in L. donovani, L .infantum L. mexicana and L. pifanoi. However, some species such as L. major, is much less amenable to axenic cultivation. In present study, we describe an in vitro culture system for the generation and propagation of axenic amastigotes form of L. major.Methods: Promastigotes of L. major were cultivated in a biphasic NNN medium. The liquid phase was Schneider's medium, pH 3.5, supplemented by 25% FCS (fetal calf serum). The cultures were maintained at 33-34°C for 120 hours. Results: Fine structure analysis of these in vitro-grown amastigotes by electron microscopy, demonstrated that they have a pear-shaped body with abortive short terminal flagellum. The in vitro-grown cells are agglutinated by peanut lectin. SDS-PAGE pattern of these axenic amastigotes showed a 66-kDa band, which was not present in promastigotes. The axenic grown amastigotes were able to infect peritoneum macrophages of BALB/c mice. In supernatant of culture, biochemical, analysis showed decreased protein and acid phosphate activity. Conclusion: These amastigotes like cells might serve as a suitable strain for the study of amastigote biochemistry, in vitro drug testing, and immunology of L. major