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. .

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 percent) of lesions was single; double lesions were observed in 22 percent of patients, and only 20 percent of CL had multiple lesions. Out of 200 patients, Leishman body was observed in 77 samples (38.5 percent) by direct smear and 40 percent by cultivation assay. Most patients (21.3 percent) 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.

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.

Patterns of co-association of C-reactive protein and nitric oxide in malaria in endemic areas of Iran

In addition to numerous immune factors, C-reactive protein (CRP) and nitric oxide (NO) are believed to be molecules of malaria immunopathology. The objective of this study was to detect CRP and NO inductions by agglutination latex test and Griess microassay respectively in both control and malaria groups from endemic areas of Iran, including Southeastern (SE) (Sistan & Balouchestan, Hormozgan, Kerman) and Northwestern (NW) provinces (Ardabil). The results indicated that CRP and NO are produced in all malaria endemic areas of Iran. In addition, more CRP and NO positive cases were observed amongst malaria patients in comparison with those in control group. A variable co-association of CRP/NO production were detected between control and malaria groups, which depended upon the malaria endemic areas and the type of plasmodia infection. The percentage of CRP/NO positive cases was observed to be lower in NW compare to SE region, which may be due to the different type of plasmodium in the NW (Plasmodium vivax) with SE area (P. vivax, Plasmodium falciparum, mixed infection). The fluctuations in CRP/NO induction may be consistent with genetic background of patients. Although, CRP/NO may play important role in malaria, their actual function and interaction in clinical forms of disease remains unclear.

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.