SYSTEMIC PHAEOHYPHOMYCOSIS DUE BY ALTERNARIA ALTERNATA: CASE REPORTS, IN-VITRO SENSITIVITY AND EXOANTIGEN STUDIES.

Alternaria alternata is the most frequently encountered species of Alternaria causing opportunistic mycoses. We report two cases of phaeohyphomycosis in which Alternaria alternata was isolated from peripheral blood samples of 35 year and 21 year old male TB and post operative patients respectively. The direct microscopic examination of the blood samples revealed dark dematiaceous, septate mycelium. A. alternata was repeatedly isolated from the clinical samples of both the patients. Ketoconazole with the MIC of 10 mgml-1 was found to be the best effective drug against both the Alternaria alternata strains tested in vitro. In the present study we tried to evaluate the role of exoantigens in the serological diagnosis of such infections. The exoantigen of A. alternate on SDS-PAGE analysis exhibited two bands of 15Kda and 67Kda. The antisera raised in rabbit against. A alternata exoantigen showed humoral response after one week of immunization of ODD method. No cross reactivity was seen with antisera raised in rabbit against Curvularia verreculosa and Cladosporum cladosporiodides. Formation of specific precipitin bands and no cross reactivity suggests the usefulness of Alternaria alternata exoantigen as diagnostic tool. The antisera analysis by SDS-PAGE revealed several additional bands particularly higher than 67 Kda molecular weight as compared to the normal (unchallenged) sera from the same rabbit. Whether any of these bands could be treated as specific marker for diagnosis needs further investigations. Keywords- opportunistic, phaeohypomycosis, Alternaria alternata, minimal inhibitory concentration (MIC

Systemic Phaeohyphomycosis Caused by Cladosporium Cladosporioides: In Vitro Sensitivity and its Serological Diagnosis

Phaeohyphomycosis is a clinical entity caused by dematiaceous fungi. Cladosporium cladosporioides is a phaeoid fungi commonly found in man’s environment and has been reported to cause infection in man.  We report a cause of pheohyphomycosis in a 60 year old male suffering from tuberculosis and gangrene. C. Cladoporioides was isolated from peripheral blood of the patient.  The direct microscopic examination of the blood sample revealed the presence of dark color dematiaceous septate & branched mycelium.                 In-vitro antifungal sensitivity of C. cladosporioides against Ketoconazole, Fluconazole, Itaconazole and Clotrimazole performed by Polak’s 1:3 dilution method.  Ketoconazole was the most effective drug against the isolated strain with MIC 10 ?g ml–1 after 96 hrs. of incubation. The exoantigen of C. cladosporioides was prepared and subjected to SDS PAGE (Polyacrylamide gel electrophoresis) analysis which revealed two bands of 15 and 67 K Dalton.  Antisera was raised against the antigen and subjected to ODD (Ouchterlony’s double diffusion) test which showed highly specific band of identity.  No cross reactivity was observed against the exoantigen of  C. cladosporioides with antisera of 2 strains of Curvularia verruculosa and one strain of Alternaria alternata. In this study it is concluded that cases of phaeohyphomycoses caused by C. cladosporioides can be rapidly diagnosed using exoantigenic method. Keywords; opportunistic, phaeohypomycosis, Cladosporium cladosporioides , minimal inhibitory concentration (MIC

Toxicity of Imidacloprid on Peripheral Blood Lymphocytes by MTT Assay and the Ameliorative Effect of Extract of Tinospora cordifolia (Gilloe) Extract

Imidacloprid (IMI) is a widely used insecticide which has a specific affinity for insect neonicotinoid acetylcholine receptors. Like all insecticides which are used in excess it tends to bioaccumulate in the environment. So it was thought worthwhile to study its cytotoxicity to human peripheral blood lymphocytes in concentrations ranging from 1.5mM to 4mM after 2 hours and 18 hours exposure by MTT method. Trypan blue test was also used to determine the percentage of living cells. The ameliorative effect of an extract of the stem in water and ethanolic extract of leaves of Tinospora cordifolia (Thunb.) Miers, was also studied. The viability of the lymphocytes showed a fall with increasing concentrations at an exposure of 2 hours. After 18 hours exposure to the IMI only, the viability showed a significant dose dependent drop. Trypan blue test for viability was also conducted. Addition of Tinospora extract raised the viability significantly at 2 hours of incubation. In fact this increase was greatest at 3.5mM and 4mM concentration of drug. The ameliorative effect was maximum at 2 hours. Addition of Tinospora leaf extract showed a significant increase in cell viability at 18 hours of incubation as compared to values obtained with only the drug. Thus a considerable loss of viability of lymphocytes was seen after exposure to the drug in the selected concentrations but herbal extracts seem to help to make the damage less marked. The cells showed a significant rise in viability when incubated with Tinospora leaf extract only, confirming its supportive action in cell proliferation. However, taking into account the evident fall in cell viability caused by exposure to the considerably dilute concentrations tested, caution is needed to prevent over exposure to the pesticide while spraying