Objective To explore the method of radiolabeling anti-Aspergillus monoclonal antibody (WF-AF-1)with 99mTc,and evaluate the in vitro and in vivo characteristics of 99mTc labeled WF-AF-1 (99mTc-WF-AF-1). Methods 99mTc-WF-AF-1 was prepared with indirect-labeling method.The labeled product was identified using thin layer chromatography.Suspensions of Aspergillus fumigatus,Staphylococcus aureus and Candida albicans were incubated with 99mTc-WF-AF-1 to evaluate the specificity of the labeled antibody.Mice were injected with 3.7MBq of labeled product.The biodistribution was measured at 40min,2h,4h and 7h after injection. Results The labeling efficiency of 99mTc-WF-AF-1 was over 95%,and the labeled product was stable in serum and phosphate buffer solution.In vitro binding of 99mTc-WF-AF-1 revealed that the labeled Mab-WF-AF-1 preferentially binds to Aspergillus fumigatus. Biodistrbution data showed that the labeled antibody was deposited mainly in liver,kidneys and spleen.The radioactivity uptake in blood at 40min and 7h was (2.51±0.23)%ID/g and (0.53±0.13)%ID/g,respectively. Conclusions The labeling efficiency and stability of 99mTc-WF-AF-1 are high.The labeled antibody is excreted mainly through the liver and kidneys with fast clearance in blood in normal mice.
Objective To explore the effects of prolonged inhalation of Aspergillus fumigatus ( Af)spores on epithelial cell injury and expression of epidermal growth factor receptor( EGFR) in airways of asthmatic rats. Methods 64 male Wistar rats were randomly divided into 8 groups, ie. chronic asthma group ( group A) , chronic asthma plus Af spores inhalation for 1 week ( group B) , 3 weeks ( group C) and 5 weeks ( group D) , chronic asthma plus saline inhalation for 5 weeks ( group E) , OVA-sensitized and salinechallenged group ( group F) , and OVA-sensitized and saline-challenged plus Af spores inhalation for 5 weeks ( group G) ( each n =8) . The airway resistance ( Raw) and change of Raw after acetylcholine provocation were detected using a computerized system. The concentrations of epidermal growth factor ( EGF) andtransforming growth factor alpha( TGF-α) in BALF were measured by ELISA. The extents of epithelial cell injury and goblet cell hyperplasia were evaluated on hematoxylin and eosin-stained( HE) and periodic acidschiff ( PAS) stained lung sections. The expression of EGFR in airway epithelia was demonstrated byimmunohistochemistry, and the level of EGFR protein in the rat lung tissues was measured by western blot.Results The concentration of EGF( pg/mL) ( 51. 72 ±8. 54, 68. 12 ±7. 85, 86. 24 ±9. 12, respectively)and TGF-α( pg/mL) ( 55. 26 ±9. 30, 75. 58 ±11. 56, 96. 75 ±14. 66, respectively) , detached/ inner perimeter of epithelium( % ) ( 11. 25 ±3. 12, 26. 45 ±5. 56, 28. 50 ±7. 50, respectively) , the ratio of goblet cell area to epithelial cell area ( % ) ( 16. 42 ±5. 24, 22. 64 ±6. 82, 36. 38 ±9. 21, respectively) , the integrated optical density ( IOD) of EGFR positive stain in airway epithelial cells ( 82 ±15,120 ±19, 165 ±21, respectively) , and the EGFR protein levels in lung tissues ( 0. 91 ±0. 26, 1. 61 ±0. 52, 2. 52 ±0. 78,respectively) in group B, C, and D were higher than those in group A, E, F and G( P lt; 0. 05 or P lt;0. 01) .The change rates of Raw( % ) ( 61. 91 ±5. 26, 84. 69 ±6. 38) in group C and D were higher than those in group A, E, F and G ( P lt; 0. 05 or P lt;0. 01) . The IOD of EGFR was positively correlated with detached/inner perimeter of epithelium( % ) and the ratio of goblet cell area to epithelial cell area( % ) ( r = 0. 692,P lt;0. 01; r = 0. 657, P lt; 0. 01, respectively) . Conclusion Prolonged inhalation of Aspergillus fumigatus spores can aggravate airway epithelial cell injury, up-regulate the expression of EGFR in airway epithelial cell and induce goblet cell hyperplasia, thus increase the airway responsiveness in rats with chronic asthma.
Objective To investigate the correlation between persistent wheezing and positive result of sputum fungal culture in patients with chronic obstructive pulmonary disease ( COPD) . Methods The COPD patients who hospitalized in the respiratory department of Shanghai First People’s Hospital, Zhongshan Hospital and Huadong Hospital fromJanuary 2005 to December 2007 were analyzed retrospectively. Results Thirty-five cases were enrolled in the persistent wheezing group and 43 cases in the non-wheezing group. In the wheezing group, sputumfungal culture revealed positive yield in 32 cases while Aspergillus were isolated in 12 cases. In the non-wheezing group, sputum fungal culture revealed only 11 cases positive, and none of which were Aspergillus positive. Aspergillus distributions in the two groups were significantly different( P lt;0. 05) . There was also significant difference in the positive result of sputum fungal culture ( 91. 4% vs 25. 6%, P lt;0. 01) , while there was no significant difference in positive result of bacterial culture( 28. 6% vs 39. 5%, P gt; 0. 05) . In the wheezing group, the patients with antifungal treatment showed better prognosis than those without antifungal treatment( 81. 0% vs 36. 4% , P lt;0. 05) . Conclusion The persistent wheezing in the patients with COPD is correlated with the fungi, especially Aspergillus airway colonization.
In recent years, due to the extensive usage of immunosuppressant and the rise of patients with cancers and organ transplantation, the incidence rate of invasive fungal infection, especially invasive pulmonary fungal infection, has increased. Besides the clinical manifestations, medical history and imaging, the diagnosis of pulmonary mycosis mainly depends on pathogen detection methods in clinical microbiology laboratory. However, due to the difficulty in fungi culturing and the low sensitivity of smear microscopy, better molecular biology methods are needed. To date, the emergence of metagenomic next-generation sequencing (mNGS) has improved the identification rate of pulmonary fungal infections. mNGS is significantly superior to traditional detection methods in rapid, accurate, and comprehensive determination of fungi from various clinical specimens, especially atypical fungi. However, some problems in mNGS method have to be addressed including sample collection, report interpretation, and its combination with traditional microbiology methods. With the in-depth discussion and solution of the above problems, mNGS will be indispensable to the etiological diagnosis of pulmonary invasive fungal infection.
Objective To explore the effects of Aspergillus fumigatus（A. fumigatus） spores on airway inflammation and responsiveness in asthmatic rats.Methods Seventy male Wistar rats were randomly divided into Ⅰ and Ⅱ groups（n=35 in each group），then Group Ⅰ and Group Ⅱ were subdivided into a normal control group（n=5），an asthma group（n=10），a spores-treated control group（n=10），and a spores-treated asthma group（n=10）.The rats were sensitized to ovalbumin（OVA） and challenged with aerosol OVA to establish the asthma model.The effects of A. fumigatus spores on asthmatic rats before and after OVA aerosol challenging were investigated in Group Ⅰ and Group Ⅱ，respectively.The parameters associated with bronchial epithelial damage were observed by total protein concentration in BALF measured by BCA method.Total and differential cell counts in BALF were also counted.The airway resistance and airway responsiveness were calculated by transpulmonary pressure and gas flow rate.Results In Group Ⅰ，the total protein in BALF in the asthma group treated with A. fumigatus spores before OVA challenging（Group CA） was increased remarkably compared to the asthma group（Group A1）[（1.125±0.254）μg/mL vs（0.825±0.173）μg/mL，Plt;0.01].The nonspecific airway resistances induced by different concentration of acetylcholine in Group CA ［（0.997±0.196）cm H2O•mL-1•s-1，（1.123±0.142）cm H2O•mL-1•s-1，（1.130±0.197）cm H2O•mL-1•s-1］were increased significantly compared to Group A1 [（0.655±0.089）cm H2O•mL-1•s-1，（0.687±0.048）cm H2O•mL-1•s-1，（0.821±0.043）cm H2O•mL-1•s-1]（all Plt;0.05）.In Group Ⅱ，however，the above parameters in the asthma group treated with A. fumigatus spores after OVA challenging（Group AC） were not dramatically increased compared with the asthma group（Group A2）（all Pgt;0.05）.The differences in the total and differential cell counts in BALF in Group CA were not remarkable compared to other subgroups in Group Ⅰ（all Pgt;0.05）.But the BALF neutrophil count in Group AC was increased obviously compared to Group A2 ［（2.488±0.420）×106 vs （0.936±0.459）×106，Plt;0.05］.Conclusion These data indicate that exposure to A. fumigatus spores before challenging causes aggravated epithelial damage and increased airway resistance in an asthma rat model.
Objective To investigate diagnosis and treatment strategies of patients with pulmonary tuberculosis （TB） complicated by Aspergillus infection. Methods Clinical data of 38 patients with pulmonary TB complicated by Aspergillus infection who underwent surgical treatment from January 2008 to December 2010 in Chengdu Infectious Disease Hospital were retrospectively analyzed. There were 23 male patients and 15 female patients with their average age of 37.8 （23-59） years. Preoperatively，all the patients received regular anti-TB treatment for more than 2 weeks，and patients with definite Aspergillus infection received anti-Aspergillus therapy for more than 3 days with consultation of infectious disease physicians. After above treatment，26 patients underwent lobectomy，1 patient underwent right pneumonectomy，and 11 patients underwent left pneumonectomy. All the patients were followed up at the outpatient department after discharge. They were evaluated every 2 weeks in the first 3 months，every 1 month after 3 months，and every 6 months after 1 year. During follow-up，they received acid-fast bacillus smear and sputum culture to check Aspergillus，as well as CT chest scan. Results All the patients successfully received surgical resection of the pulmonary lesion without perioperative death or severe complication. Postoperative pathology examination confirmed pulmonary TB with Aspergillosis infection in all the 38 patients，whose basic diseases included TB cavity in 17 patients，TB-destroyed lung in 12 patients，and post-TB bronchiectasis in 9 patients. All the patients were followed up after discharge for 1.5-4.5 years. During follow-up，they received regular anti-TB therapy for adequate duration in addition to antifungal medications such as voriconazole. None of the 38 patients had recurrence of Aspergillus infection or pulmonary TB. One patient had hemoptysis which was controlled after proper treatment during follow-up. Conclusion Missed diagnosis rate of pulmonary TB complicated by Aspergillus infection is high. Surgical resection of the pulmonary lesion and postoperative medication treatment are the most effective treatment strategies for patients with pulmonary TB complicated by Aspergillus infection.
ObjectiveTo investigate antifungal activity in vitro of single or combination of triazole and echinocandin against Aspergillus species. MethodsBased on EUCAST protocol,the susceptibilities of 62 isolates of Aspergillus spp. were determined for voriconazole (VRC),itraconazole (ICZ),caspofungin (CAS) and micafungin (MICA). For VRC and ICZ,MIC-0 and MIC-2 were determined. For CAS and MICA,minimum effective concentration (MEC) and MIC-2 were determined. The fractional inhibitory concentration (FIC) was used to evaluate the effect of combination of triazole and echinocandin. ResultsIndifference was found in 2 isolates of Aspergillus fumigatus in combination of ICZ and CAS or MICA by using MIC-0 endpoint. Synergy was found in all other isolates of Aspergillus spp.With MIC-2 and MEC endpoints,synergy for VRC and CAS,VRC and MICA,ICZ and CAS and ICZ and MICA was found in 16,21,11 and 14 isolates of Aspergillus fumigatus,9,13,9 and 11 isolates of Aspergillus flavus,0,2,1 and 1 isolates of Aspergillus niger,respectively. ConclusionThe in vitro sensitivity results of combination of triazole and echinocandin are different with different endpoints. Thus,the efficacy of combination of triazole and echinocandin can not predicted by in vitro sensitivity and should be further confirmed in invasive aspergillosis animal experiments.
Objective To investigate the clinical manifestation and histopathologic changes of the fungal necrotizing retinochoroiditis. Methods Collecting 7 cases of fungal retinochoroiditis with severe immunodepression and loss of visual acuity.Seven removed eyeballs were stained with HE,PAS and silver methenamine,and observed by light microscopy,and in addition,2 of them examined by electron microscopy.Also fungal cultures of blood and affected tissues were performed. Results The chief clinical macnifestation included ciliary injection of conjunctiva,opaque aqueous fluid and vitreous and diffuse hemorrhage and greyt white opacity with retinal detachment in severe cases.Pathologic changes included hemorrhage in the retina,chorioretinal tissue necrosis,hyphae in the blood vessels,affected tissue and vitreous.Fungal culture of blood was positive in three cases.Culture of affected tissues was positive in all cases. Conclusions Eedogenous fungal infection of choroid and retina may be due to the severe immunodepression of the sufferers and usually causes chorioretinal tissue destruction and blind. (Chin J Ocul Fundus Dis, 1999, 15: 235-237)
ObjectiveTo explore the effect of Aspergillus fumigatus on airway eosinophilia and hyperresponsiveness in rat model of chronic asthma. MethodsWistar rats were sensitized by intraperitoneal injections with ovalbumin (OVA) followed by chronic inhalation of nebulized OVA or physiologic saline. Rats were administered via the airways with placebo or aerosolized Aspergillus fumigatus spores suspension mimicking chronic Aspergillus fumigatus exposure. The Penh after acetylcholine provocation was detected using WBP system. The concentrations of IL-5 and eotaxin in BALF were measured by ELISA. The extents of eosinophil infiltration were evaluated on hematoxylin and eosin-stained(HE) and Wright-Giemsa stained BALF cells smear. ResultsAspergillus fumigatus worsened allergic airway inflammation in OVA-challenged rats,as evidenced by enhanced bronchial responsiveness to inhaled acetylcholine and increased bronchial eosinophilia. Elevated airway eosinophilia corresponded with higher levels of IL-5 and eotaxin in the Aspergillus Fumigatus exposure group. Aspergillus fumigatus,however,did not affect bronchial responses,numbers of eosinophils,IL-5 and eotaxin levels in saline challenged mice. ConclusionThe Results show that chronic Aspergillus fumigatus exposure aggravates eosinophilic airway inflammation in asthma rats by enhancing IL-5 and eotaxin production. Aspergillus fumigatus also increases bronchial hyperresponsiveness in asthma rats.
ObjectiveTo investigate the role of Aspergillus in the severe refractory exacerbations of chronic obstructive pulmonary disease (COPD).MethodsThe clinical data of two COPD patients suffering from refractory acute exacerbations were analyzed and the relevant literature were reviewed.ResultsTwo patients were male, aging 72 and 64 years respectively. Both of them had a history of frequent acute exacerbations with severe COPD recently. Meanwhile, they received intravenous use of antibiotics repeatedly, one of them took oral corticosteroids to control wheezing, but failed. Their serum Aspergillus-specific IgG antibody was weakly positive. Besides traditional treatment, they received additional antifungal therapy, and the symptoms alleviated. There was no acute exacerbation in the half a year follow-up period after appropriate therapy.ConclusionsAspergillus colonization, sensitization, infection should be considered in patients with severe COPD. When Aspergillus-associated evidence are acquired, antifungal therapy will be unexpected helpful.