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.

Metabolomics-Based Study of Logarithmic and Stationary Phases of Promastigotes in Leishmania major by 1H NMR Spectroscopy.

BACKGROUND: Cutaneous leishmaniasis is one of the most important parasitic diseases in humans. In this disease, one of the responsible organisms is Leishmania major, which is transmitted by sandfly vector. There are specific differences in biochemical profiles and metabolite pathways in logarithmic and stationary phases of Leishmania parasites. In the present study, 1H NMR spectroscopy was used to examine the metabolites outliers in the logarithmic and stationary phases of promastigotes in L. major to enlighten more about the transmission mechanism in metacyclogenesis of L. major. METHODS: Promastigote was cultured, logarithmic and stationary phases were separated by the peanut agglutinin, and cell metabolites were extracted. 1H NMR spectroscopy was applied, and outliers were analyzed using principal component analysis. RESULTS: The most altered metabolites in stationary and logarithmic phases were limited to citraconic acid, isopropylmalic acid, L-leucine, ornithine, caprylic acid, capric acid, and acetic acid. CONCLUSION: 1H NMR spectroscopy could play an important role in the characterization of metabolites in biochemical pathways during a metacyclogenesis process. These metabolites and their pathways can help in exploiting a transmission mechanism in metacyclogenesis, and outcoming data might be used in the metabolic network reconstruction of L. major modeling.

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.

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. .

Comparative of three methods (ELIZA, MAIPA and flow cytometry) to determine anti-platelet antibody in children with ITP.

Immune (idiopathic) thrombocytopenic purpurea (ITP) is an autoimmune disease characterized by the increased anti-platelet antibodies in the patient's sera and decreased platelets in the blood circulation. This study has determined and characterized the antiplatelet glycoproteins in children with ITP. Thirty eight children, who were hospitalized with clinical signs of ITP in Mofid Children Hospital (Tehran, Iran) during 18 months, went under our clinical studies in a research project. ELISA, Flow cytometry and MAIPA (Monoclonal Antibody Immobilization of Platelet Antigens) methods were employed to determine serum anti-platelet antibodies level. The anti-platelet antibodies level above mean + 3SD of control group was assumed as positive. The platelet counts ranged between 2 × 10(9)/L and 100 × 10(9)/L. Among the patients 63.5% of them were anti-platelet antibodies positive with ELISA method. Results of platelet lysate method showed that 51.7% of patients had antibodies against platelet antigens. Antibody against platelet GPIIb/IIIa, GPIb/IX and GPIa/IIa using MAIPA method were 48%, 54% and 25% respectively. In flow cytometry 62% of patients showed anti-platelet antibodies. The comparison of three methods shows that since MAIPA is the specific method for the detection of very small amount of antibody against glycoprotein antigens, it has the advantage of differentiating between immune and non-immune thrombocytopenia.

The route of Leishmania tropica infection determines disease outcome and protection against Leishmania major in BALB/c mice.

Leishmania tropica is one of the causative agents of leishmaniasis in humans. Routes of infection have been reported to be an important variable for some species of Leishmania parasites. The role of this variable is not clear for L. tropica infection. The aim of this study was to explore the effects of route of L. tropica infection on the disease outcome and immunologic parameters in BALB/c mice. Two routes were used; subcutaneous in the footpad and intradermal in the ear. Mice were challenged by Leishmani major, after establishment of the L. tropica infection, to evaluate the level of protective immunity. Immune responses were assayed at week 1 and week 4 after challenge. The subcutaneous route in the footpad in comparison to the intradermal route in the ear induced significantly more protective immunity against L. major challenge, including higher delayed-type hypersensitivity responses, more rapid lesion resolution, lower parasite loads, and lower levels of IL-10. Our data showed that the route of infection in BALB/c model of L. tropica infection is an important variable and should be considered in developing an appropriate experimental model for L. tropica infections.

Histopathological changes induced by Hemiscorpius lepturus scorpion venom in mice.

Envenomation by Hemiscorpius lepturus (H. lepturus) is associated with local necrosis, followed by systemic manifestations. In this work the LD50 of H. lepturus venom were determined by subcutaneous (SC) injection in white Balb/c mice (5 mg/kg). Histopathological alterations in organs such as kidney, heart, liver, lungs, stomach and intestine were determined in 3, 6, 12 and 24 h following experimental (SC) envenoming injection of one LD 50 of the venom in Balb/c mice. Histological studies showed degenerative changes in the kidney with disorganized glomeruli and necrotic tubular in 3 h and reached to its climax in 6 h. Myocardium showed massive myocytolysis with interstitial necrosis in 3 h and reached to its peak after 6 h past envenoming. Bowels showed edema of lamina propria and slight villous necrosis. The enzymatic activities of creatine kinase (CK) and lactate dehydrogenase (LDH) were significantly increased in the serum in 9 h. No necrotic lesion observed in lungs and liver. The results indicate that the venom of H. lepturus is a highly cytotoxic, and induces massive tissue damages in specific organs, starting from the heart and kidney as the first target in 3 h and ends to the bowels in 6 h post envenomation.

Immunotherapy with Imiquimod increases the efficacy of Glucantime therapy of Leishmania major infection.

BACKGROUND: Leishmaniasis is a complex disease which presents as visceral, cutaneous and mucocutaneous forms. The current treatment options for this infection are very limited and the immunological state of the host appears to play an important role in the efficacy of the treatment. Immunostimulation through immune response activating agents such as Imiquimod is another rational approach for this purpose. OBJECTIVE: We assessed the efficacy of immunotherapy with Imiquimod alone or combined with Glucantime for treatment of Leishmania major infection in BALB/c mice. METHODS: Treatment efficacy was monitored by determination of thickness and parasite load of infected footpad of mice. RESULTS: The footpad thickness revealed that treatment with Imiquimod plus Glucantime has the highest efficacy. The results were confirmed by parasite load of infected footpad. Our data shows that treatment of Leishmania major infection in BALB/c mice by the combined Imiquimod and Glucantime is more efficient than each drug alone. CONCLUSION: The combination of Imiquimod with chemotherapy may offer a way for more efficient treatment of leishmaniasis.

Expression and Single-step Purification of GRA8 Antigen of Toxoplasma gondii in Escherichia coli.

Diagnosis of Toxoplasma gondii (T.gondii) infection is of great medical importance especially for pregnant women and immunosuppressed patients. Numerous studies have shown that the recombinant production of several toxoplasma antigens, including dense granule antigens (GRAs) has a great potential as diagnostic reagents. Previous studies reported expression of amino terminal GRA8 protein in fusion with large tags. In the present study, we produced truncated GRA8 (GRA8), excluded from the signal peptide and C-terminal transmembrane domain, with a short fusion tag in Escherichia coli (E.coli). GRA8 was purified using an optimized single-step Immobilized Metal ion Affinity Chromatography (IMAC). The purity and yield of GRA8 was highest at pH = 9.25. At this pH, 13.6 mg of GRA8 was obtained with the purity of 97.97%. Immunogenicity of the protein was evaluated in Western blot analysis showing the serum sample from a rabbit immunized with GRA8 recognized a single antigen of T.gondii tachyzoite at the expected molecular weight of native GRA8. To diagnosis acute toxoplasma infection in pregnant women, an indirect immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) was developed using GRA8 resulting in a test specificity and sensitivity of 97.1% and 60.6%, respectively. These results demonstrated that immunogenic GRA8 can be produced in fusion with a short tag and purified near to homogeneity using an optimized IMAC. GRA8-IgM-ELISA was useful for detection of acute toxoplasma infection.

Serodiagnosis of recently acquired Toxoplasma gondii infection in pregnant women using enzyme-linked immunosorbent assays with a recombinant dense granule GRA6 protein.

Indirect immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays (ELISAs) with a recombinant GRA6 protein of Toxoplasma gondii were developed and evaluated for accurate diagnosis of recently acquired infection in pregnant women. According to the results from Toxoplasma serodiagnostic tests, women were classified into 3 groups representing acute (group I), chronic (group II), or no Toxoplasma infection (group III). To discriminate group I from group II sera, the GRA6-IgG-ELISA reached sensitivity and specificity of 87.5% and 94.1%, respectively. Although 22 (91.7%) of 24 group I sera were positive by the GRA6-IgM-ELISA, only 1 (2.9%) of 34 group II sera scored positive. The GRA6-IgM-ELISA displayed a meaningful correlation with Vidas Toxo IgM and exhibited higher specificity (97.1%) than Euroimmun IgM ELISA (88.2%) (Euroimmun, Lübeck, Germany) for detection of recent infection. These results demonstrate that IgG and IgM ELISA with rGRA6 are useful to identify and discriminate recent from past Toxoplasma infection in pregnant women.

Allelic distribution of human leukocyte antigen in Iranian patients with pulmonary tuberculosis.

There is convincing evidence for a genetic component in susceptibility to tuberculosis (TB) in humans. Attempts to link susceptibility to TB to human leukocyte antigen (HLA) phenotype have produced conflicting data. The purpose of this study was to determine whether HLA phenotype is associated with clinical TB. We compared the frequencies of HLA alleles in 44 Iranian sputum smear positive pulmonary TB patients with allele frequencies of 108 healthy adults. HLA typing was performed by lymphocytotoxicity assay. The frequencies of HLA-B17 and -DR14 were higher in TB patients and the frequencies of HLA-A26 and -B27 were higher in healthy controls (p value < 0.05, corrected p value [pc] >0.05). Our findings suggest that these alleles are associated (either positively or negatively) with pulmonary TB in an Iranian population. However, considering corrected p value, our data are not conclusive and should be considered preliminary.