The Experimental And Clinical Study Of Galactomman And 1,3-β-D-glucan In Diagnosis Of Invasive Aspergillosis

ObjectiveTo investigate the method of animal model building of pulmonary aspergillus infection, and evaluate the significance of GM and BG detecting in plasm,BALFand sputum in the diagnosis of invasive aspergillosis.Method1.Setting up the Aspergillus infection rat model with pulmonaryExperimental group:72 female SD rats were Randomly divided into experimental group A1, A3, A5, and the normal control group B,the immunosuppressed group C, the inoculated control group D respectively,each group consists of 12 rats.Establish immunesuppression and infection animal model:Experiments A1, A2, A3 group and the immunosuppressed group C were given immunosuppressive intramuscular dexamethasone 0.6mg/kg, cyclophosphamide 75mg/kg, for 3 days. On the third day,the experimental groups A1, A2, A3 and the inoculated group D were given 1×109 aspergillus fumigatus spores, each 300-400ul,one times a day,for two days.Group B and group C were given the same amount of sterile saline.Plasm,lavage and pulmonary samples collection and treatmentAnimals were killed on the first,third,fifth day,at the same time collect the plasm and BALF preserved at -80℃.Lungs were get out and make part of these tissue as homogenate, inoculated into sabouraud plate cultivate more than 3d.Put the remaining organs tissue in 10% formalin fixed for mare than eight hours,paraffin-embeded,and dyed with HE and PASM to detected the shape of aspergillus funmigatus,and Immunohistochemical processing to proving aspergillus funmigatus infection.The collection of clinical specimens:According the Diagnostic criteria based on the Chinese Medical Association, patients with invasive fungal infection (IPFI) were divided into diagnosis, clinical diagnosis and suspected diagnosed levels. The excluded IPFI patients For control group, collect the clinical blood and sputum samples of Hospitalized patients of Zhujiang Hospital, Southern Medical University during September 2008 to November 2009.The detection of GM and BG:Collect plasma and bronchoalveolar lavage fluid samples of rat models, pathology as the standard, Operating in accordance with the kits request, and detect GM and BG levels of two kinds of specimens by Platelia Aspergillus kit Produced the Bio-rad of France and the Glucatell kit produced ACC of United States. Also detect the levels of GM and BG in plasma and sputum samples of in-patients.Result1. All the 36 rats of experimental group were confirmed the presence of pulmonary aspergillosisby pathology.The GM test result:The GM test sensitivities of plasm samples are 58.30%,66.67%,83.33% after infected 1d,3d,5d respectively, the average sensitivity is 69.44%; The GM test sensitivities of lavage fluid were 66.67%, 91.67%,100.00% after infected 1d,3d,5d respectively, the average sensitivity is 86.11%. Both the GM detection specificity of the two kind samples were 100%.The GM tests of both BALF and blood samples were negative of All the three control groups. Comparing the GM consequence of plasma and BALF with the result of all the control groups, they have statistically significant devitation(P<0.005). Experimental infection the mean GM level lavage fluid tended to increase with the days of infection. The GM values of lavage fluid was significantly higher than that of blood GM in the same experimental group (paired t test, P<0.05).G assay results:The G test sensitivities of plasm samples are 58.33%,75%,75% after infected 1d,3d,5d respectively, the average sensitivity is 69.44%; The GM test sensitivities of lavage fluid were 75%,83.33%,83.33% after infected 1d,3d,5d respectively, the average sensitivity is 80.56%. the specificity of the two kind samples were 94.44%,100% respectively. Comparing the G test consequence of plasma and BALF with the result of all the control groups, they have statistically significant devitation(P<0.005). The average plasma BG concentration of experimental group is greater than 20 pg/ml; All the three control groups were less than the final plasma BG concentration 20pg/ml.The average BG concentration of the BALF samples of Experimental group ere greater than 20 pg/ml; All the three control groups were less than the final plasma BG concentration 20pg/ml. Both of the BG concentration of plasm and BALF of experimental group have significant difference with the control groups (ANOVA, p<0.001).2. Test results of Clinical patients:55 cases patients were collected, of which 2 diagnosed patients,27 clinical diagnosis,16 suspected diagnosed cases,10 cases of normal controls. GM test results:The mean GM value detected in plasma of confirmed cases, clinical diagnosis,and suspected cases were all more than 1.5, the mean GM value of the normal control cases is less than 1.5. Compared the GM value with the control group respectively, in addition to suspected diagnosed group was not statistically difference (ANOVA, p<0.05) other groups were statistically significantly different(ANOVA, p<0.05); Multiple comparisons between clinically diagnosed group, suspected group and confirmed group both have significant statistical difference (Dunnett T3, P<0.05); But compared clinically diagnosed group and suspected group, there is no significant difference (Dunnett T3, P> 0.05). The mean GM value of sputum of confirmed cases, clinical diagnosis, and suspected cases were more than 1.5, the mean GM of the normal control cases is less than 1.5. Campare with the GM value of the normal control group,all all case groups have statistically significantly different (ANOVA, p<0.05); All case groups betweens have statistically significantly different (ANOVA, p<0.05).. The plasma samples is 71.11% sensitivity and specificity of 70%, positive predictive value of 91.43%, negative predictive value of 35%. Sputum samples is sensitivity of 80%, specificity of 80%, positive predictive value of 94.74%, negative predictive value of 47.06%. BG test results of clinical patients:The mean BG levels of confirmed cases clinical diagnosis of cases, suspected cases detected in plasma were higher than 20pg/ml, the mean BG concentration of normal control patients is less than 20 pg/ml. Campare with the BG concentration of the normal control group,all case groups betweens have statistically significantly different (ANOVA, p<0.05). BG concentrations in all cases between groups have statistically significant different (multiple comparison, p<0.05). Sputum test of confirmed cases, clinical diagnosis,suspected diagnosed cases were higher than the mean BG concentration of 20pg/ml,the mean BG concentration of normal control cases is less than 20 pg/ml. Patients in each case group compared with the control patients have significant statistical difference (ANOVA, p<0.05); all case groups betweens have statistically significantly different (ANOVA, p<0.05).G test of serum samples is 71.11% sensitivity and specificity of 70%, positive predictive value of 91.43%, negative predictive value of 35%. G test detected in Sputum samples is sensitivity of 80%, specificity of 90%, positive predictive value of 97.30%, negative predictive value of 50%.Conclusions1. Aspergillus is a common pathogen of fungal infection, which are highly pathogenic.2. Both GM and the BG test for diagnosis of deep fungal infection are reliable methods for diagnosis of invasive aspergillus infections, with high sensitivity and specificity.3. Determination of GM and BG of BALF and sputum samples also has a high sensitivity and specificity, and moreover specimen collection is simple, time-saving, less pain and psychological burden bringing to the patients.They are apply to be used for deep fungal infection in early diagnosis.4. For suspected diagnosed patients lacking of diagnosis of deep fungal infections the increases of GM and BG level in based on plasma and sputum can be used as method of early diagnosis of deep fungal infection.5.For patients with high risk factors of deep fungal infection, when GM or BG in plasma and sputum levels were significantly higher, suggesting occurrence of deep fungal infection may be.