Study On The Rapid Diagnosis Of Sporotrichosis By Sporotrichin Skin Test

Background:Sporotrichosis is a deep fungal disease that can cause sub-acute and/or chronic infections of the skin,subcutaneous tissues,and nearby lymphoid tissues.It can also spread to internal organs and bones.Severe cases can cause systemic disseminated infections.The disease is relatively frequent in the northeast region of China and poses a serious threat to health.At present,the diagnosis of the disease requires fungal culture and histopathological examination,which takes a long time.Some European and American regions use the method of sporotrichin skin test for the diagnosis and screening of sporotrichosis.However,our country has not carried out yet.Whether this method can be widely used requires further experimental research and clinical validation.Objectives:By establishing a mouse sporotrichosis model,extracting sporotrichin,and skin test which was used to observe the positive rate results to verify whether the sporotrichin skin test can provide a rapid diagnosis of sporotrichosis.Methods:1.Select clinically isolated strains of sporotrichosis preserved in department of Dermatology,Second Hospital of Jilin University and culture them on Sabouraud Dextrose Agar(SDA)plate culture medium at 25°C for 2 weeks to 4 weeks to prepare Sporothrix suspension.2.40 Kunming outbred male mice were selected and divided into model group and control group.Each group was divided into tail vein group and subcutaneous injection group.The model group was injected with Sporothrix suspension,and the control group was injected with normal saline.At the fourth week,2 mice were randomly selected from each group of the model group to be sacrificed for fungal culture and histopathological examination to verify the success of the mouse sporotrichosis model.3.The clinical isolates of sporotrichosis preserved in department of Dermatology,Second Hospital of Jilin University were selected and cultured in Sabouraud Dextrose Broth(SDB)medium at 25°C for 1 week to 2 weeks.The SDB forming the pellicle was centrifuged,homogenized,sterilized,filtered and dispensed to obtain a sporotrichin solution.The protein standard curve was measured using the BCA kit and the antigen concentration of sporotrichin was calculated.4.After the mouse sporotrichosis model was successfully established,the model group was injected intradermally with 0.1 ml sporotrichin solution,1:50 dilution solution,1:100 dilution solution,and the control group was injected intradermally with 0.1 ml physiological saline solution.The results were observed and recorded at 48 and 72 hours after injection,respectively.Criteria: erythema diameter <0.5 cm negative,erythema diameter 0.5-1.0 cm weak positive,erythema diameter 1.0-2.0 cm positive,erythema diameter> 2.0 cm strong positive.5.SPSS19.0 software was used for statistical analysis.Fisher’s exact test was used to statistically test the difference in the positive rate of each group.Results:1.All mice in the model group had skin lesion changes.Most of them had erythema,nodules,and scarring at the inoculation site from day 7 to day 14 and peaked at 28 days.The lesions in the subcutaneous injection group were mostly located at the inoculation site and adjacent skin.The lesions in the tail vein group were mostly confined to the tail.Tissue culture and pathological examinations were consistent with sporotrichosis.2.The protein content of the extracted sporotrichin solution was 106 ug/ml.3.The 48-hour and 72-hour positive results were consistent across the model group.Using SPSS19.0 software,Fisher’s exact probability algorithm was used to test the positive rate results of each group.There was no significant difference in the positive rate(12.5%)of the raw liquid skin test in the tail vein group and the positive rate(0.0%)of the skin test in the subcutaneous injection group(P=1.0,P>0.05).There was no significant difference in the skin test positive rate(37.5%)in the tail vein group 1:50 dilution and the skin test positive rate(50%)in the subcutaneous injection group 1:50 dilution(P=1.0,P>0.05).There was no significant difference in the skin test positive rate(25%)in the 1:100 dilution of the tail vein group and the skin test positive rate(37.5%)in the 1:100 dilution subcutaneous injection group P=1.0,P>0.05).The positive rate of skin test in the model group,1:50 dilution and 1:100 dilution was 6.3%,43.8%,and 31.3%,respectively.The 1:50 dilution was the effective concentration of 2.15 μg/ml skin test highest positive rate.Conclusion:1.The successful rate of the normal immunized mice sporotrichosis model was 100%.2.The mice sporotrichosis model has a low positive rate for sporotrichin skin test,and whether it can be used for rapid diagnosis of sporotrichosis needs further verification.