Evaluation of a possible synergistic effect of meglumine antimoniate with paromomycin, miltefosine or allopurinol on in vitro susceptibility of leishmania tropica resistant isolate

Pentavalent antimonials are still the first choice treatment for leishmaniasis, but with low efficacy and resistance is emerging. In the present study, the effect of meglumine antimoniate [MA, Glucantime] combined with paromomycin, miltefosine or allopurinol on in vitro susceptibility of Leishmania tropica resistant isolate was evaluated. The drugs were obtained from commercial sources and diluents of each drug in medium were prepared on the day of experiment. J774 A.1 murine macro-phage cell lines were attached to the cultured on slide and incubated at 37 [degree sign]C with 5% CO[2] for 24 h. Then the stationary phase promastigotes were added to the cells and after 4 hrs of incubation different concentrations of MA, paromomycin, miltefosine or allopurinol were added and incubated for an additional of 72 h. Then the slides were dried and fixed with methanol, stained by Giemsa and studied under a light microscope. Drug activity was evaluated by assessing the macrophage infection rate and the number of amastigotes per infected macrophage was done by examining 100 macrophages. The experiment was done in triplicates. Various concentrations of MA along with paromomycin, miltefosine or allopurinol significantly inhibited [P<0.01] the proliferation of L. tropica amastigote stage in the macrophage cell line as compared with MA alone or positive control. Combination of Glucantime with paromomycin, miltefosine or allopurinol showed a synergistic effect on the clinical isolate of L. tropica in vitro. Use of combination therapy is a new hope and a logical basis for therapy of the patients with cutaneous leishmaniasis. Further investigations are needed to evaluate the therapeutic effects of these drugs on the CL patients

[Identification of Malassezia species associated with seborrheic dermatitis using PCR-RFLP]

Malassezia is a lipophilic and dimorphic fungus which has different species. Some of them can be found as natural flora on the skin and in some conditions may cause seborrheic dermatitis. The aim of this study was to identify Malassezia species associated with seborrheic dermatitis in Iranian patients, using PCR-RFLP. In this study out of 79 patients with seborrheic dermatitis, isolates of 70 patients were positive for Malassezia species using PCR-RFLP. The Internal Transcribed Spacer 2 [ITS2] region was amplified by PCR employing the ITS3 and ITS4 primers and the restriction endonucleases AluI, BanI and MspAI were selected for producing distinct RFLP patterns. M. globosa [48.6%], M. furfur [40.0%], M. slooffiae [8.6%] and M. sympodialis [2.8%], were the microorganisms responsible for the infection among participants. M. pachydermatis, M. japonica, M. dermatis, M. restricta, M. obtuse, M. nana and M. yamatoensis were not isolated from any samples. Our findings suggest that the most common Malassezia species associated with seborrheic dermatitis was M. globosa, followed by M. furfur

[Identification of Malassezia species associated with pityriasis versicolor using PCR-RFLP]

Malassezia is a lipophilic and dimorphic fungus which has different species. Some of them can be found as natural flora on skin and in some conditions may cause pityriasis versicolor. The aim of this study was to identify Malassezia species associated with pityriasis versicolor in Iranian patients, using PCR-RFLP. In this study out of 65 patients with pityriasis versicolor to have pityriasis versicolor,isolates of 60 patients were positive. Malassezia species. using by PCR-RFLP. The Internal Transcribed Spacer 2 [ITS2] region was amplified by PCR employing the ITS3 and ITS4 primers and the restriction endonucleases AluI, BanI and MspAI were selected for producing distinct RFLP patterns. M. furfur [36.7%], M. globosa [30.0%], M. sympodialis [20.0%], M. slooffiae [8.3%], M. restricta [3.3%] and M. obtusa [1.7%] were the microorganisms responsible for the infection among participants. The M. sympodialis infection was strongly correlated with the female gender [P=0.02]. Our findings suggest that, the most common Malassezia species associated with pityriasis versicolor was M. furfur, followed by M. globosa

[Phenotyping of peripheral memory T cell subsets in cutaneous leishmaniasis]

The heterogenous population of memory T lymphocytes is distinguished based on surface markers and effector functions such as cytokine secretion. Recently, two subsets of memory T cells are defined by expression of chemokine receptor CCR7 and CD45RA designating as "central memory" T cells [TCM] and "effector memory" T cells [TEM]. The objective of this study was to evaluate the phenotype and function of these lymphocytes in healed cases of cutaneous leishmaniasis. The phenotype of lymphocytes were determined in blood samples of 13 volunteers with history of self healing cutaneous leishmaniasis [HCL] and in 6 healthy controls. No significant difference was found in memory T cell subsets between HCL volunteers and healthy controls using flow cytometry. However, following sorting of different memory subsets, a significantly higher proliferation was seen in cells of HCL volunteers comparing to the control group. A significantly higher IFN-gamma response in TEM and a significantly higher IL-2 response in TCM were observed in cell culture of HCL volunteers comparing controls. The responses were elicited when the cells were stimulate with SLA in vitro, it is concluded Leishunania-specific TEM and Leishmania-specific TEM subsets exist in HCL volunteers and since the volunteers with history of CL presumed to be protected against reinfection, it seems that both TCM and TEM play role in the protection against Leishmania infection in these individuals

Evaluation of protectivity and immunogenicity of alum precipitated autoclaved Leishmania major vaccine mixed with Mycobacterium vaccae in the murine model of leishmaniasis

Leishmaniases represent a group of diseases caused by protozoan parasite of the genus Leishmania. Control strategies are not always effective, it seems that the sole control measure is to search for an effective vaccine. The objective of this study was evaluation of the rate of protection and immune response induction in Balb/c mice immunized with alum precipitated autoclaved Leishmania major [Alum-ALM] vaccine mixed with M. vaccae. Eleven groups of female 8-10 week old Balb/c mice were immunized subcutaneously [SC] three times, 21 days apart, with different doses of Alum-ALM mixed with different doses of either M. vaccae or BCG. The immunized animals and control group were challenged with 1 x 10 [6] L. major. Development and progression of leishmania infection were assessed by footpad swelling measurement at the site of challenge and parasite burden in lymph nodes. The immune responses of vaccinated animals were evaluated in vivo by leishmanin skin test [LST] and in vitro by measurement of cytokine [IFN-lamda and IL-4] levels in mononuclear cell culture] supernatants and titration of serum anti-leishmania antibodies [IgG and its sub-classes]. Footpad thickness measurment after challenge with live L. major showed no significant difference between immunized groups and control group. However, there were some prominent exceptional cases in the parasite burden titration in groups 1, 4, 6, and 8. Immunization with low dose of Alum-ALM mixed with M. vaccae or BCG induced IFN-lamda production, and diminished IL-4 level [in vitro], and caused a stronger LST response in a group that received BCG as an adjuvant. Mice that were immunized with high doses of Alum-ALM mixed with high doses of M. vaccae showed an increase in footpad thickness at the site of challenge and higher levels of IL-4 and IgGl. It seems that immunization of mice with a low dose of Alum-ALM mixed with M. vaccae as an adjuvant might induce a Thl type response. M.vaccae mixed with low dose of Alum-ALM has an inhibitory influence on the parasite burden in the infected tissues of mice. Also, BCG mixed with different doses of Alum-ALM induces a Thl immune response in Balb/c mice. Apparently, there is no significant difference in adjuvancity of BCG and M. vaccae. High dose of Alum-ALM mixed with high dose of M. vaccae induces a Th2 response