| Objective:Scopulariopsis brevicaulis is a saprophytic conditional pathomycte, usually it causes tinea unguium, seldom it produces the infection of deep tissues, but now there are many reports with regard to the infection of deep tissues. We isolated the strain from a patient of tinea unguium in the clinic service. It was identified as Scopulariopsis brevicaulis by China Institute of Microbiology in Acdemt Science.This study on the one hand by the Scopulariopsis brevicaulis in four different culture media on the situation observed in order to find out suited to their growth medium and temperature; Further more, to provide the guidance on the treatment of systemic infection of Geotricum sivicola, Fluconazol, Itraconazole and Terbinafine were emploied agaist the systemic infection of Scopulariopsis brevicaulis of immunodepressed mouse model. Histopath-ological can helped to know more about its tissue forms.Methods:1 Experimental morphologyPut Scopulariopsis brevicaulis solation at theordinary temperature and inoculate it respectively on the media of Sabouraud's Agar (SDA), Patato Glucose Agar (PDA), rice medium, CM A with Tw80 after three days, and culture it at 37℃and 27℃for two weeks, then observe it's development and measure the diameters of colonies every day, finally draw growth curves.2 Physiology Experiment2.1 Urease test:Inoculate the blood isolation on the Urea agar, and culture it at the ordinary temperature for 1 week, then observe the medium color.3 the sensitivity to antifungal agents in-vivo of Scopulariopsis brevicaulis3.1 Establition of animal model suffered from systemic infection of Scopulariopsis brevicaulis. The Scopulariopsis brevicaulis were subcultivated onto the media of SDA, cultured under 37℃for one week, employed to prepare the fungus suspension with stroke-physiological saline solution, then inoculate the immunodepressed Kunming mouse with the suspension through intraperitoneal injection.3.2 Survival ObservationMedication Fluconazol, Itraconazole and Terbinafine are administered to mouse model with the dose of 10mg/(kg.d) one day at a time via intraperitoneal injection two hours after inoculation. Continuous observation of mice within 21 days after vaccination and its general condition, died of natural causes, recording the date of death and death is only a few.3.3 Reverse cultivation of tissue:Execution of three mice in each fungus burden of kidney group in eighth day after inoculation. Levigate the internal organs with tissue grinder, then take a few sample from them and innoculate them onto the media of SDA and culture them under 27℃3.4 Determination of fungus burden of kidneyWere taking fluconazole group, itraconazole group, terbinafine group and the control group of 15 mice in each group of 15 mice from each of the side of the kidney, The kidney were levigated with tissue grinder, emploied to prepare the tissue suspension with 1ml stroke-physiological saline solution, distributed onto the plate media with 10μL suspension under 27 for 48 hours℃, observed for the number of clonal formation unit.3.5 Histopathological Examination:pathological tissues of the white patches were removed out, fixing, dehydrating, clearing, embedding, slicing, stainning with HE and PAS respectively, then observed under light microscope.Results:1. Experimental morphologyMorphology observation on the five kinds of media1.1 General colony observation1.1.1 Draw growth curves The fungus growed faster in the first week on the PDA and SDA than Rice medium and CMA with Tw80.Thegrowth was better at 27℃than 37℃.1.1.2 The colony diameterThe comparison under the two temperatures on the same medium showed that the colony diameters at 27℃was larger than 37℃in the two weeks(P<0.05).The comparison between random two media at 27℃showed that the colony diameters on PDA was the largest in all the media, and the diameters on SDA were smaller than that on PDA. No difference between Rice medium and CMA with Tw80 on the seventh and fourteenth day(P>0.05). No difference between PDA and SDA on the seventh and fourteenth day(P>0.05); The remaining medium were significantly difference(P<0.05).1.2 Morphology under naked eyes:When cultured for 3—4 days, they began to grow presented with whitish hymeno-type colony, growing fast, they became dust color.1.3 Morphology under light microscope:It showed abundant branched, separate mycelium and penicilli. conidiospore grew from annellophore with cluster, globular, thick wall, slick or rough hard sphere.2. Physiology Experiment2.1 Urease test was positive.3. Survival observed:observe the change of the survival in the group of Itraconazole,Fluconazol,Terbinafine and Control.21 days after the administration, the number survival were 17,16,15and 9.The first died was the sixth day in Itraconazole,Fluconazol and Terbinafine group, the first died day was the second day in Control group. Mortality peak was the seventh day in Fluconazol,Itraconazole and Control group, was eighth day in Terbinafine group.4.Results of reverse fungus culture The ratios of positive culture among heart, lung in the group of Itraconazole,Fluconazol and Terbinafine were significantly lower than that of Control. No difference in rate of positive culture among the same organs in others. 5. Results of fungus burden of kidney The fungus burden of kidney in the group of Itraconazole,Fluconazol and Terbinafine were much lower than that of and Control; No difference in fungus burden of kidney among the same organs in others.6 Histopathological Examination:pathological tissues of the white patches were removed out, fixing, dehydrating, clearing, embedding, slicing, stainning with HE and PAS respectively, then observed under light microscope.Conclusion:1 Sabouraud's agar medium (SDA), potato dextrose agar (PDA) are suitable for the growth of Scopulariopsis brevicaulis, rice and corn Tween 80 agar medium unsuitable for their growth; Compared with the temperature 37℃, Scopulariopsis brevicaulis is more suited for growing under 27℃; Urease test of Scopulariopsis brevicaulis was positive.2 Fluconazole, Itraconazole and Terbinafine show good activity against Geotrichum Silvicola in vivo because they prolong the median survial time of the immunodepressed mouse suffered withsystemic infection of Scopulariopsis brevicaulis, improve their survival rates, cut down their rates of positive fungus culture among heart and lung, decrease their fungus burden of kidney3 For the infection of Scopulariopsis brevicaulis in mice, No significant difference in the treatment of fluconazole,itraconazole and terbinafine. |
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