Evaluation of transgenic Leishmania infantum expressing mLLO-BAX-SMAC in the apoptosis of the infected macrophages in vitro and in vivo.

BACKGROUND: Leishmaniasis is an important infectious disease that develops because of escaping parasite from the host immune system or preventing host macrophages apoptosis. Recently, the development of transgenic methods and the manipulation of the parasite genome has provided many advantages. So, in this study, the effect of the transgenic Leishmania infantum expressing mLLO-BAX-SMAC proteins was examined in accelerating host cell apoptosis. METHOD: The entire coding sequence of designed codon-optimized mLLO-Bax-Smac was cloned in the pLexyNeo2 vector and integrated downstream of the 18srRNA locus of L infantum genome by homologous recombination. Next, the expression of mLLO-BAX-SMAC fusion protein was evaluated by the Western blotting technique and the pathogenesis of transgenic parasite was surveyed in vitro and in vivo. RESULTS: The results of PCR and Western blot confirmed proper integration and expression of mLLO-Bax-Smac sequence into the 18srRNA locus of L infantum. Flow cytometry showed accelerating apoptosis of transgenic Leishmania-infected macrophages compared to wild-type parasite. Also, transgenic parasites were less virulent as a fewer parasitic burden was found in the spleen and liver of transgenic-infected mice compared to the control. CONCLUSION: The data suggested that the transgenic L infantum expressing BAX-SMAC can be used as an experimental model for developing vaccination against leishmaniasis.

Insights into the trypanothione system in antimony-resistant and sensitive Leishmania tropica clinical isolates.

Pentavalent antimonials are the mainstay treatment against different clinical forms of leishmaniasis. The emergence of resistant isolates in endemic areas has led to treatment failure. Unraveling the underlying resistance mechanism would assist in improving the treatment strategies against resistant isolates. This study aimed to investigate the RNA expression level of glutathione synthetase (GS), Spermidine synthetase (SpS), trypanothione synthetase (TryS) genes involved in trypanothione synthesis, and thiol-dependent reductase (TDR) implicated in drug reduction, in antimony-sensitive and -resistant Leishmania tropica isolates. We investigated 11 antimony-resistant and 11 antimony-sensitive L. tropica clinical isolates from ACL patients. Drug sensitivity of amastigotes was determined in mouse macrophage cell line J774A.1. The RNA expression level in the promastigote forms was analyzed by quantitative real-time PCR. The results revealed a significant increase in the average expression of GS, SpS, and TrpS genes by 2.19, 1.56, and 2.33-fold in resistant isolates compared to sensitive ones. The average expression of TDR was 1.24-fold higher in resistant isolates, which was insignificant. The highest correlation coefficient between inhibitory concentration (IC50) values and gene expression belonged to the TryS, GS, SpS, and TDR genes. Moreover, the intracellular thiol content was increased 2.17-fold in resistant isolates compared to sensitive ones and positively correlated with IC50 values. Our findings suggest that overexpression of trypanothione biosynthesis genes and increased thiol content might play a key role in the antimony resistance of L. tropica clinical isolates. In addition, the diversity of gene expression in the trypanothione system and thiol content among L. tropica clinical isolates highlighted the phenotypic heterogeneity of antimony resistance among the parasite population.

Data coverage of a cancer registry in southern Iran before and after implementation of a population-based reporting system: a 10-year trend study.

BACKGROUND: Cancer registries help to decrease the burden of cancers by collecting accurate and complete data. We aimed to measure the completeness of coverage of information recorded between 2000 and 2009 in a cancer registry program in Fars province, southern Iran. METHODS: The cancer registry program run by Shiraz University of Medical Sciences was investigated in two periods: pathology-based data from 2000 to 2007 and population-based data from 2007 to 2009. Completeness of yearly coverage was measured as the number of reported cases of cancer in each year divided by estimated cases based on 107.3 new cases per 100 000 individuals. The percentage of complete data registration (patient's name, age, gender, address, phone number and father's name) and correct cancer encoding was calculated for each year and compared to the maximum acceptable error rate for each item. RESULTS: A total of 29 277 non-duplicate cancer records were studied. Completeness of coverage varied from 22.68% in 2000 to 118.7% in 2008. Deficiencies in patients' demographic data were highest for name in 2002 (0.09%), age in 2006 (2.36%), gender in 2001 (0.06%) and father's name in 2001 (52.5%). Incomplete address (99.7%) and missing phone number (100%) were most frequent in 2000, and deficiencies in encoding information were highest in 2008 (6.36%). CONCLUSIONS: The cancer registry program in Fars province (southern Iran) was considered satisfactory in terms of completeness of coverage and information about age. However, it was deficient in recording patients' phone number and address, and father's name. The error level for cancer encoding was unacceptably high. Enhancing hardware and software resources, education and motivation in all public and private sectors involved in the cancer registry program, and greater attention to epidemiological research are needed to increase the quality of the cancer registry program, including its completeness.

Evaluation of anti-leishmanial effects of killed Leishmania vaccine with BCG adjuvant in BALB/c mice infected with Leishmania major MRHO/IR/75/ER.

There are many strategies to control leishmaniasis, but majority of them are inadequate. Killed Leishmania vaccine (KLV) has been applied for its immunogenicity in human and mouse model. Bacillus Calmette-Guerin (BCG) as adjuvant is an immune-modulator inducing humoral and cellular immune responses during zoonotic cutaneous leishmaniasis (ZCL). Both KLV and BCG have been applied for their immune responses in hosts for controlling leishmaniasis. In this study, KLV and BCG were applied to inhibit replication and visceralization of Leishmania major in BALB/c mice. Mice were injected with KLV and BCG, followed by infection with promastigotes of L. major. Six weeks after infection, a small nodule appeared, which was followed by development of a large lesion and visceralization. Effects of KLV and BCG, physiopathological changes, lesion size, delay of lesion formation, proliferation of amastigotes inside macrophages and detection of amastigotes in target organs were studied. Results showed that the KLV had anti-leishmanial activity by reducing lesion size on late infection. In KLV and BCG group, the average number of amastigotes in macrophages was lower than in other groups. Significant reductions in number of amastigotes in both spleen and lymph node were observed, indicating lower visceralization of Leishmania parasites in these target organs. No significant changes were presented in body weights, survival rates and degrees of splenomegaly in test group. It can be concluded that application of KLV and BCG had acceptable efficacy in reduction of skin lesions size and proliferation of parasites, even though a few side-effects were observed. It is indicated that KLV/BSG may have ability to modulate host immune responses against Leishmania parasites and to reduce pathophysiology of the disease during infection.

Performance of a universal PCR assay to identify different Leishmania species causative of Old World cutaneous leishmaniasis.

BACKGROUND: The characterization of Leishmania species is important for clinical management of the diseases and the epidemiological control of the parasite distribution. Most of the published polymerase chain reaction (PCR) amplification methods lack the ability to identify all species complexes, have low performance and usually need post-PCR procedures. There is a need for improving the diagnosis of CL by development of an accurate affordable PCR method to identify all Leishmania species in clinical specimens. METHODS: We designed an optimized a PCR amplifying the internal transcribed spacer 2 sequence of the ribosomal RNA gene (ITS2) aligned from different strains of CL-causing Leishmania species in the Old World. The performance of the method was evaluated on lesion samples from several CL suspected patients and the limit of detection (LOD) was determined on DNA of promastigotes from reference strains. RESULTS: The universal PCR enabled simultaneous discrimination of L. major, L. tropica and L. infantum using one primer pair in one reaction. Stained smear microscopy and Novy-MacNeal-Nicolle (NNN) medium culture alone detected 77.27% (17/22) and 72.73% (16/22) of the positive CL samples, respectively. The PCR assay showed 100% sensitivity (22/22) (95% CI: 84.56-100%) and 100% specificity (3/3) (95% CI: 29.24-100%) for species identification on isolates from lesion scraping/exudate and 100% sensitivity (13/13) (95% CI: 75.29-100%) and 100% specificity (11/11) (95% CI: 71.51-100%) for species identification on biopsy samples of CL patients, while being capable to successfully amplify as little as 0.01-0.1 pg of Leishmania DNA from cultured promastigotes. CONCLUSIONS: We present a validated easy-to-use affordable universal PCR assay to identify the most common Old World Leishmania species causing CL. This PCR assay could be used as a sensitive/specific technique to diagnose CL-causing Leishmania species in clinical samples with high accuracy.

Evaluation of anti-leishmanial activity by induction of nitric oxide and inhibition of prostaglandin in Balb/c mice infected with Leishmania major.

Cutaneous leishmaniasis is still one of the health problems in Iran and in the region. Nitric oxide (NO) has a key mechanism in the elimination of parasite from the body by its anti-leishmanial activity. Prostaglandin (PG) is a critical inhibitory factor of infected macrophage to decrease their anti-leishmanial activity. This study was designed to induce NO by L-arginine (L-Arg) precursor and inhibit PG production by anti-inflammatory Indomethacin (INDO) in Leishmania major infected Balb/c mice, in order to evaluate the effects of NO and PG on delay of lesion formation, size of lesion and proliferation of amastigotes inside macrophages. Liver, spleen and lymph nodes were also studied as target organs to detect amastigotes. Serum, liver and spleen suspensions were investigated for NO induction by using Griess microassay and serum PG was determined by ELISA. The results indicated that NO production was inhibited by Leishmania in infected Balb/c mice as compared with naive animals. Serum NO was inhibited by a combination therapy of L-Arg and INDO. Although NO was decreased in the liver by L-Arg, however it increased in the spleen after L-Arg and INDO application. A significant decline was observed in lesion size from Week 6 after infection by INDO. Both L-Arg and INDO had significant inhibitory effects on visceralization of leishmania in target organs. Only L-Arg decreased proliferation of promastigotes in macrophages. Pathophysiological signs including hepatomegaly, splenomegaly, survival rate and body weight all were affected in this experiment. Statistical analysis of data revealed an association between NO induction and PG inhibition in leishmaniasis. These data may indicate a possible candidatory for L-Arg and INDO as novel drugs for the treatment of leishmaniasis in mouse model.

Anti-leishmanial effects of trinitroglycerin in BALB/C mice infected with Leishmania major via nitric oxide pathway.

This study investigated whether trinitroglycerine (TNG) as nitric oxide (NO) releasing agent had anti-leishmanial effects and mediated pathology in BALB/c mice infected with Leishmania major. Cutaneous leishmaniasis (CL), a zoonotic infection caused by leishmania protozoa is still one of the health problems in the world and in Iran. NO is involved in host immune responses against intracellular L. major, and leishmania killing by macrophages is mediated by this substance. Moreover, application of CL treatment with NO-donors has been recently indicated. In our study, TNG was used for its ability to increase NO and to modify CL infection in mice, in order to evaluate NO effects on lesion size and formation, parasite proliferation inside macrophages, amastigote visceralization in target organs, and NO induction in plasma and organ suspensions. Data obtained in this study indicated that TNG increased plasma and liver-NO, reduced lesion sizes, removed amastigotes from lesions, livers, spleens, and lymph nodes, declined proliferation of amastigotes, hepatomegaly, and increased survival rate. However, TNG reduced spleen-NO and had no significant effects on spelenomegaly. The results show that TNG therapy reduced leishmaniasis and pathology in association with raised NO levels. TNG had some antiparasitic activity by reduction of positive smears from lesions, livers, spleens, and lymph nodes, which could emphasize the role of TNG to inhibit visceralization of L. major in target organs.

Assessment of Recombinant A2-Latex Agglutination Test (RA2-LAT) and RA2-ELISA for Detection of Canine Visceral Leishmaniasis: A Comparative Field Study with Direct Agglutination Test in Northwestern Iran.

BACKGROUND: This study aimed to set-up latex agglutination test (LAT) and ELISA based on recombinant A2 from Iranian strain of Leishmania (L.) infantum (rA2-Ag) and evaluated for detection of anti-Leishmania antibodies in dogs compared to standard direct agglutination test (DAT). METHODS: The rA2-Ag was synthesized under a part of the A2 gene sequences which contain immune dominant sequences and less number of repetitive sequences. Latex beads, 0.8 µm (Sigma, USA) were sensitized with rA2-Ag. The tests were carried out on sera collected from 350 ownership dogs including symptomatic (n=67), asymptomatic (n=230) canine visceral leishmaniasis (CVL), and (n=53) uninfected domestic dogs as control group. RESULTS: Anti-leishmanial antibodies were detected in 97 (27.7%), 96 (27.4%) and 29 (%9) of the serum samples by using DAT, rA2-ELISA, and rA2-latex, respectively with ≥1:320 as a cut-off titer when DAT-confirmed cases were compared with the control groups. A combined sensitivity of 52% and specificity of 82.40% for rA2-ELISA and 23.8% and specificity 95.38%, respectively were found with ≥1:320 as a cut-off titer when DAT-confirmed cases were compared with the control groups. The concordance between rA2-ELISA and rA2 latex compared with DAT as a gold standard serological test for VL were found 73.7% and 77.5%, respectively. CONCLUSION: A good degree of agreement was found between rA2-ELISA and DAT (73.7%). rA2-ELISA could detect more seropositive serum samples than rA2-LAT and it may be recommended as an alternative tool for the diagnosis of CVL.

Application of Recombinant Proteins for Serodiagnosis of Visceral Leishmaniasis in Humans and Dogs.

Visceral leishmaniasis (VL) is a zoonotic disease caused by leishmania species. Dogs are considered to be the main reservoir of VL. A number of methods and antigen-based assays are used for the diagnosis of leishmaniasis. However, currently available methods are mainly based on direct examination of tissues for the presence of parasites, which is highly invasive. A variety of serological tests are commonly applied for VL diagnosis, including indirect fluorescence antibody test, enzyme-linked immunosorbent assay (ELISA), dot-ELISA, direct agglutination test, Western-blotting, and immunochromatographic test. However, when soluble antigens are used, serological tests are less specific due to cross-reactivity with other parasitic diseases. Several studies have attempted to replace soluble antigens with recombinant proteins to improve the sensitivity and the specificity of the immunodiagnostic tests. Major technological advances in recombinant antigens as reagents for the serological diagnosis of VL have led to high sensitivity and specificity of these serological tests. A great number of recombinant proteins have been shown to be effective for the diagnosis of leishmania infection in dogs, the major reservoir of L. infantum. Although few recombinant proteins with high efficacy provide reasonable results for the diagnosis of human and canine VL, more optimization is still needed for the appropriate antigens to provide high-throughput performance. This review aims to explore the application of different recombinant proteins for the serodiagnosis of VL in humans and dogs.

Reactive nitrogen intermediate production and tolerance variability in different mouse strains after in vivo treatment with lipopolysaccharide from Salmonella abortus equi.

Lipopolysaccharide (LPS) stimulation in animal models generates a large number of immune factors including cytokines and mediators. It also acts as a potent inducer of macrophage reactive oxygen intermediates and reactive nitrogen intermediates (RNI). RNI as stable metabolites of nitric oxide (NO) are produced by cells stimulated with LPS and cytokines. In this study, LPS from Salmonella abortus equi was investigated as an inducer of RNI in untreated controls and test groups of white Naval Medical Research Institute (NMRI) mice. Animals were humanely killed at 30, 60, 120 and 180 min after LPS injection, and plasma RNI was measured by Griess microassay. In a further experiment, host tolerance against bacterial LPS was evaluated by sequential intravenous injection of LPS concentrations of 4, 1 and 0.5 mg/kg at 24 h intervals in NMRI and with the same schedule but via subcutaneous injection in Balb/c mice. Statistical analysis of RNI values using analysis of variance test indicated that in vivo LPS stimulation induced high levels of NO in murine hosts (p<0.001). Comparison of RNI levels at different times after administration revealed the largest amount of RNI at 180 min after inoculation. Analysis of the time course until maximum RNI induction indicated that NMRI mice had the longest delay, suggesting a difference in tolerance of NMRI and Balb/c mice to LPS stimulation dependent on LPS concentration, dose, and route of inoculation.

Comparison of recombinant A2-ELISA with rKE16 dipstick and direct agglutination tests for diagnosis of visceral leishmaniasis in dogs in Northwestern Iran.

INTRODUCTION: Various methods are used for the diagnosis of visceral leishmaniasis (VL), such as microscopic examination, culture and inoculation of laboratory animals; however, serological assays are commonly used for the detection of antibodies in serum samples with a wide range of specificity and sensitivity. METHODS: The purpose of this study was to compare three serological methods, including rA2-ELISA, the recombinant KE16 (rKE16) dipstick test and the direct agglutination test (DAT), for the detection of antibodies against VL antigens. The assays utilized 350 statistically based random serum samples from domestic dogs with clinical symptoms as well as samples from asymptomatic and healthy dogs from rural and urban areas of the Meshkinshahr district, northwestern Iran. RESULTS: Samples were assessed, and the following positive rates were obtained: 11.5% by rKE16, 26.9% by DAT and 49.8% by ELISA. The sensitivity among symptomatic dogs was 32.4% with rKE16, 100% with DAT and 52.9% with ELISA. Conversely, rA2-ELISA was less specific for asymptomatic dogs, at 46.5%, compared with DAT, at 88.9%. CONCLUSIONS: This study recommends rA2-ELISA as a parallel assay combined with DAT to detect VL infection among dogs. Further evaluations should be performed to develop an inexpensive and reliable serologic test for the detection of Leishmania infantum among infected dogs.

Different Morphologies of Leishmania major Amastigotes with No Molecular Diversity in a Neglected Endemic Area of Zoonotic Cutaneous Leishmaniasis in Iran.

BACKGROUND: Molecular diversity of Leishmania major and its morphological changes have become a controversial issue among researchers. Some aspects of polymorphic shapes of amastigotes in clinical manifestations along with molecular variation were evaluated among suspected patients of some exceptional zoonotic cutaneous leishmaniasis locations in Northern Khuzestan, Southwestern Iran. METHODS: Suspected patients (n = 165) were sampled in zoonotic cutaneous leishmaniasis foci over two consecutive years during 2012-2014. Prepared smears were stained, scaled and measured by ocular micrometer. DNA was extracted from smears; ITS-rDNA and Cytochrome b (Cyt b) markers were amplified, and PCR products were digested by BsuR1 restriction enzyme. Then the RFLP and sequencing were employed. RESULTS: Only L. major was identified in patients containing regular amastigotes' shapes (oval or round) with a size of 2-4 µm in each of classical wet, dry, mixed lesions. Meanwhile, irregular shapes (spindle, pear, or cigarette) were observed separately in non-classical wet lesions with more than 4 µm. Interestingly, a few amastigotes with an external flagellum were observed in some lesions. All sequenced ITS-rDNA and Cyt b genes of L. major did not show any molecular variation (χ 2 P > 0.05), including only one common haplotype (GenBank access no. EF413075). CONCLUSION: Findings proved that unlike other endemic foci, there is not a meaningful correlation between phenotypic and genotypic features of L. major isolates. This study is considered as the first comprehensive report to incriminate morphometric shapes of L. major amastigotes, which enhances our knowledge concerning their relevance with various clinical appearances and genotypic traits.

Molecular epidemiology of malaria in endemic areas of Iran.

In this study, 333 blood samples of malaria cases positive by microscopic test (70.6% male and 29.4% female, p<0.05) were investigated. The group included 55 cases (16.52%) from Minab (Hormozgan Province), 116 cases (34.82%) from Iranshahr (Sistan-Baluchesta Province) and 162 cases (48.65%) from Kahnouj (Kerman Province). The results showed 244 cases (73.27%) were diagnosed as P. vivax, 87 cases (26.13%) P. falciparum and 2 cases (0.6%) showed a mixed infection of both Plasmodia. In a molecular study of the same samples using nested-polymerase chain reaction (nested-PCR), 185 cases (55.6%) were P. vivax, 50 cases (15%) P. falciparum and 95 cases (29%) both Plasmodia. Comparing the two methods used in this study, the highest rate of infection was found to be P. vivax. However, the rate of mixed infections (0.6% microscopy, 29% nested-PCR) varied and depended on the assay used. This indicated that the sensitivity of nested-PCR was greater than microscopic examination, especially for the detection of mixed-infections (p<0.05) in the current malaria epidemiology study.

Evaluation of the anti-Leishmania major activity of Satureja bakhtiarica essential oil in vitro.

Leishmaniasis is a painless chronic skin disease that is caused by the protozoan parasite Leishmania. Due to the importance of this disease and the side effects of chemical drugs, use of drugs of plant origin to treat Leishmaniasis is very important. In the present study, the chemical composition and the anti-Leishmania major activity of the essential oils obtained from Satureja bakhtiarica were evaluated in vitro. The oils were extracted using a Clevenger apparatus and then the chemical composition was analyzed by GC-MS. Promastigotes of L. major were cultured in both N.N.N and RPMI1640 media. GC-MS analysis showed 13 compounds, in which the major components were the phenolic (37.4%) compounds, thymol (22.6%) and p-cymene (19.3%). The essential oil of S. bakhtiarica showed higher activity against L. major than the standard anti-Leishmania drug, glucantime,. Perhaps because of the high concentration of phenolic compounds in the essential oil, all the parasites were killed after 24 hours. The essential oil from S. bakhtiarica is a potential plant drug against leishmaniasis. Further studies are necessary to evaluate this oil in animal models (in vivo) for future drug applications.

Incidence of breast cancer in fars province, southern iran: a hospital-based study.

BACKGROUND: Breast cancer is still considered as one of the most common female cancers worldwide regardless of the countries' level of development. This study determines the incidence of breast cancer in Fars Province, Southern Iran. METHODS: This study used patients' records from Shiraz University of Medical Sciences Cancer Registry Centre, which is a Hospital-Based Registry of Nemazee Hospital. Data were recorded based on International Classification of Diseases for Oncology (ICD-O) and compromised all invasive cancers in ICD-10 categories of C-00 to C-80. The findings were shown as the number of cases by site (ICD-10) and gender, with crude incidence (CRs), age-specific incidence and age-standardized incidence rates (ASRs) per 100,000 persons per year, performed by direct method using the world standard population. RESULTS: The age group of 40-49 years had the highest rate of breast cancer and naturally most cases were post-menopause ones. Most cases were diagnosed in moderate differentiated state with an increasing trend. Early diagnosis of in situ neoplasms has not increased over time in comprised with malignant cases. The number of diagnosed cases has sharply increased after year 2004 especially during post-menopause period. CONCLUSION: As the number of diagnosed cases has increased during post-menopausal period, screening and health programs seem necessary for menopause women.

Molecular Analysis of A2-genes Encoding Stage-specific S Antigen-like Proteins among Isolates from Iranian Cutaneous and Visceral Leishmaniasis.

OBJECTIVES: Leishmania can lead to a broad spectrum of diseases, collectively known as leishmaniasis. The A2 gene/ protein family could be one of the most eligible candidate factors of virulence in visceral leishmaniasis (VL). The previous results confirmed that in Leishmania infantum, several A2 proteins are abundantly expressed by the amastigote, but not the promastigote stage. As there are no data available on the pattern of A2 gene / protein in Iranian Leishmania isolates of either cutaneous leishmaniasis (CL) or VL; the current study aimed to investigate molecular analysis of A2 gene in leishmania species among field isolates of . MATERIALS AND METHODS: An A2 gene was identified by sequencing of crude PCR products resulting from 20 samples of CL and 10 samples of VL isolates from Iranian patients. RESULTS: The results indicated the A2 gene in CL is only a single copy of 153 bp encoding a protein of 51 amino acids, as opposed to A2 of VL species with multi-copy genes of varying length. A2 sequences in Iranian L. major strains represented a homology with stage-specific S antigen-like protein (A2) of L. major and L. infantum. Moreover, A2 sequences in Iranian L. tropica strains have homology with A2 protein of L. major and L. tropica. CONCLUSION: It is concluded that A2 is an antigen candidate for vaccine development and diagnosis purposes and that A2 sequences are conserved among field isolates.

An overview of a diagnostic and epidemiologic reappraisal of cutaneous leishmaniasis in Iran.

Cutaneous leishmaniasis (CL) is a widespread tropical infection which has a high incidence rate in Iran. Leishmania tropica, the causative agent of anthroponotic cutaneous leishmaniasis (ACL), and Leishmania major, which causes zoonotic cutaneous leishmaniasis (ZCL), are endemic in various parts of Iran with a high incidence rate. The aim of this study was to evaluate the reappraisal of the diagnosis and epidemiology of CL in Iran, by different clinical, parasitological and molecular assays among patients suspected of CL referred to the Department of Parasitology, at the Pasteur Institute of Iran during 2006-2009. Two hundred samples from patients with ulcerative skin lesions were collected, clinical analyses were applied, data questionnaire was completed and samples were examined for CL by using both direct microscopic and culture methods. Moreover, PCR assay was applied for detection of Leishmania species in CL isolates resulting from parasitological assay. Clinical observation revealed that the majority (58%) of lesions was single; double lesions were observed in 22% of patients, and only 20% of CL had multiple lesions. Out of 200 patients, Leishman body was observed in 77 samples (38.5%) by direct smear and 40% by cultivation assay. Most patients (21.3%) had a travel history to the Isfahan province, one of the most important endemic areas of CL located in center of Iran. PCR assay by kDNA indicated 32 and 18 out of 50 isolates respectively had similar patterns with standard L. major and L. tropica. In conclusion, clinical manifestations and an appropriate diagnostic assay with a parallel molecular characterization of CL may lead to a screening evaluation of disease, prognosis, treatment and control strategies.

Comparison of recombinant A2-ELISA with rKE16 dipstick and direct agglutination tests for diagnosis of visceral leishmaniasis in dogs in Northwestern Iran

INTRODUCTION: Various methods are used for the diagnosis of visceral leishmaniasis (VL), such as microscopic examination, culture and inoculation of laboratory animals; however, serological assays are commonly used for the detection of antibodies in serum samples with a wide range of specificity and sensitivity. METHODS: The purpose of this study was to compare three serological methods, including rA2-ELISA, the recombinant KE16 (rKE16) dipstick test and the direct agglutination test (DAT), for the detection of antibodies against VL antigens. The assays utilized 350 statistically based random serum samples from domestic dogs with clinical symptoms as well as samples from asymptomatic and healthy dogs from rural and urban areas of the Meshkinshahr district, northwestern Iran. RESULTS: Samples were assessed, and the following positive rates were obtained: 11.5% by rKE16, 26.9% by DAT and 49.8% by ELISA. The sensitivity among symptomatic dogs was 32.4% with rKE16, 100% with DAT and 52.9% with ELISA. Conversely, rA2-ELISA was less specific for asymptomatic dogs, at 46.5%, compared with DAT, at 88.9%. CONCLUSIONS : This study recommends rA2-ELISA as a parallel assay combined with DAT to detect VL infection among dogs. Further evaluations should be performed to develop an inexpensive and reliable serologic test for the detection of Leishmania infantum among infected dogs. .

Biochemical association between essential trace elements and susceptibility to Leishmania major in BALB/c and C57BL/6 mice.

Several enzymes that contribute to immune system responses require zinc and copper as trace elements for their activity. We examined zinc and copper levels in two susceptible Balb/c mouse lines and resistant C57bl/6 mice infected with Leishmania major MRHO/IR/75/ER, a prevalent strain that causes cutaneous leishmaniasis in Iran. Serum Zn and Cu were determined by flame atomic absorption spectrophotometry. Higher Cu levels were found in infected C57bl/6 mice and higher Zn levels were found in infected Balb/c mice. Also, Cu/Zn ratios were increased in both the Balb/c and the C57bl/6 mice. We conclude that concentrations of essential trace elements vary during cutaneous leishmaniasis infection and that this variation is associated with susceptibility/resistance to Leishmania major in Balb/c and C57bl/6 mice. We detected Zn deficiency in the plasma of infected Balb/c mice; possibly, therapeutic administration of Zn would be useful for treating this form of leishmaniasis. Increases in Cu level might increase resistance to leishmaniasis. Based on our findings, the Cu/Zn ratio could be a useful marker for the pathophysiology of leishmaniasis.