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

A New Endemic Focus of Visceral Leishmaniosis in Southeren IRAN

Introduction & Objective: Visceral leishmaniosis (VL) is endemic in some parts of Iran. Mediterranean type of disease is present in Iran where its causative agent is Leishmania infantum and dogs are the main reservoirs. Since many cases of the disease were reported from Noor-abad, in Fars provine, we aimed to carry out an epidemiological survey on VL in human and animal reservoirs (dogs) in Mahoor-Milaty district of Noor-Abad city at WestNorth of Fars province. Materials & Methods: In this cross-sectional descriptive survey, blood samples were randomly collected from all children 10 years old, 10% of the adult population and 20% of the dogs kept by owners in Mahoor-Milaty villages drawn by systematic sampling. The specimens were subjected to direct agglutination test (DAT) on serum and PCR on whole blood. The data were analyzed by standard statistical tests using SPSS software. Results: Of the 321 human samples, 234 samples belonged to children 10 years old and 87 samples were from adults. 182 (56.7%) out of 321 samples were prepared from males and 139 (43.3%) from females. Totally, 6 cases (1.86%) of human samples showed specific Leishmania antibodies with titers 1:3200 or higher by DAT. Of the 19 dog samples, 5 cases (26.3%) showed specific Leishmania antibodies with titers 1:160 or higher. Likewise, kinetoplast DNA (kDNA) of Leishmania infantum was identified in 6 cases (31.5%) of all dogs by PCR with specific primers on whole blood. None of these cases had seropositive titer. Conclusion: Results of this study show that dogs are the main sources of infection for human visceral leishmaniasis in this region and VL is endemic in Mahoor-Milaty district where the incidence rate of human VL cases has recently been increased. Besides it seems that PCR method is a good tool for primary screening of dogs' blood samples with overt or cryptic VL infectio

The Prevalence of Toxoplasma Infection among Free-Ranging Chickens in Southern Iran Using IFA and Nested-PCR

"nBackground: As consumption of chicken meat may be as one of the sources of human infection, this study was undertaken to determine the prevalence of T. gondii in farm chickens (Gallus gallus domesti­cus) in Shiraz, southern Iran. "nMethods: Two hundred and thirty one blood samples were collected from farm chickens by a cluster ran­dom sampling method and tested for toxoplasmosis by indirect fluorescent antibody technique (IFAT). The samples of the brain, heart, and liver of the chickens were tested by a Nested PCR method. The re­sults were analyzed by SPSS software using Chi-Square test and a P value <0.05 was considered statically sig­nificant. "nResults: Out of 58 seropositive chickens, 29 (1:16 in eight, 1:32 in 14, 1:64 in five and 1:128 in two birds) and out of seronegative chickens, three were enrolled in the study. The most infected tissue was liver (27 out of 29) and the lowest was the heart (16 out of 29) (α=0.05, P=0.002). None of the seronegative chick­ens was positive in PCR method. Only 2 out of 8 cases with a titer of 1:16 (as cut off point) were negative in PCR method whereas the remained were positive. "nConclusion: Based on cultural and food habits in our area, the meat and viscera of chicken may be impor­tant sources of infection in human when consuming semi-cooked meats. Considering the high prevalence of toxoplasmosis in chickens, standards in chicken breeding, education of environmental health personnel and standardization for preparation and handling techniques are required by Health and Veterinary organizations

"Investigation on Zoonotic Cutaneous Leishm- aniasis, Southern Iran "

Since occurrence of an epidemic of cutaneous leishmaniasis with high incidence of disease (8.9/1000) in Arsanjan county, Fars province, southern Iran, an attempt was made to investigate an epidemiological study during 2001-2002. Symptomatic investigation among 270 schoolchildren over one year of study revealed that 11.9% of population had active lesion with 28.1% scar rate. Parallel study showed that among 7968 people which comprise 1572 households, 6.18% were positive for scars and 1.2% for ulcer. In our study three species of rodents, including Meriones libycus (71%), Cricetulus migratorius (22%) and Microtus arvalis (6%) were captured. By employing Nested-PCR technique, 6.8% (4 of 56) of M.libycus were found infected with Leishmania major. About 83% of indoor collected sandflies comprised, Phlebotomus papatasi. From its density it can be concluded that this species plays an important role in leishmania transmission in the area. The seasonal activity of the sandflies starts at early April and ended in early November

First Report of Kala-azar from Qeshm Island in Persian Gulf

Visceral Leishmaniasis (VL) is a sever disease that is prevalent in Iran. We report a case of VL in a 3.5 year-old boy. Prolonged fever, chill, abdominal distention, and weight loss were important symptoms. Blood count showed pancytopenia and hypohemoglobinemia. Specific anti-leishmanial antibodies were detected by serological test (IFAT, DAT) but no Leishman body was observed in bone marrow. However, a 145 bp band of KDNA belong to L. infantum was detected by PCR method. Glucantime was administered and treatment was well tolerated. This is the first report of VL from Qeshm Island in Persian Gulf

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