Between-laboratory quality control of automated analysis of IgG antibodies against Aspergillus fumigatus

Measurement of IgG antibodies against Aspergillus fumigatus is an important criterion for the diagnosis of aspergilloma, allergic bronchopulmonary aspergillosis, and extrinsic allergic alveolitis. In the present study, we compared IgG antibody analysis against A. fumigatus using 2 widely used automated immunochemistry systems. In a between-laboratory quality control program, good agreement was found between the results from laboratories that used the ImmunoCAP system; however, a laboratory that used the Immulite 2000 system found approx. 2-fold higher antibody levels in the quality control samples than did the ImmunoCAP system. Measurements of IgG against A. fumigatus in patient sera were significantly correlated (r_s = 0.77, P < 0.0001). These results demonstrate that analysis of IgG antibodies against A. fumigatus with these 2 systems has reached a level of standardization that allows for direct comparison of quantitative results from different laboratories. For longitudinal analysis of IgG against A. fumigatus, reagents from the same manufacturer should be used.     

血清半乳甘露聚糖抗原及曲霉抗体检测在慢性阻塞性肺疾病合并侵袭性肺曲霉病的早期诊断价值

目的 探讨血清半乳甘露聚糖(GM)抗原及烟曲霉特异性抗体检测在慢性阻塞性肺疾病(COPD)合并侵袭性肺曲霉病(IPA)的早期诊断价值。方法 对2008年1月至2010年1月在我院行血清GM抗原及烟曲霉特异性抗体IgG、IgM检测的51例COPD患者临床资料进行回顾性分析。结果 11例患者临床拟诊IPA,治愈7例,死亡4...     

Aspergillus Infection of Supramid Orbital Implant and Hyperostosis of Orbital Bone: Report of a Unique Case

Purpose: The authors report the clinical findings in a patient who developed proptosis fourteen years after an orbital floor fracture repair with a Supramid orbital implant due to hyperostosis of the orbital floor and lateral orbital wall bone secondary to aspergillus infection.

Methods: Clinical, radiological, microbiological and histological findings and the management of this patient are presented.

Results: A 25-year-old male was referred with proptosis and lower lid retraction, fourteen years after a traumatic orbital floor fracture repair with a Supramid implant. Orbital exploration revealed a thick irregular sheet of bone covering the orbital floor implant and extending laterally along the lateral orbital wall. Aspergillus fumigatus was grown from the Supramid implant as well as from the bone and histology showed chronic inflammatory process with reactive bone formation. Patient was treated with a course of oral Voriconazole and post-operatively the patient is asymptomatic with reduction in proptosis.

Conclusion: To the best of our knowledge, this is the first case of Apergillus fumigatus infection secondary to a Supramid orbital floor implant, associated with hyperostosis of orbital bone.     

应用锁定核酸水解探针实时荧光定量PCR检测烟曲霉

目的 建立检测烟曲霉的锁定核酸(LNA)水解探针实时荧光定量PCR(qPCR)方法.方法 实验菌株来自北京同仁医院临床分离曲霉菌株,均通过形态学和DNA测序方法鉴定到种.实验组:烟曲霉48株;对照组:黄曲霉55株、杂色曲霉16株、构巢曲霉10株、聚多曲霉5株、寄生曲霉1株;l临床标本组:已经纯培养出烟曲霉的冻存临床标本,鼻窦炎鼻窦组织标本20份、肺泡灌洗液1份.提取标本的DNA,LNA水解探针qPCR检测炯曲霉特异性β-微管蛋白基因,评价此方法的分析特异性、扩增效率、线性动态范围和检测限.结果 烟曲霉特异性β-微管蛋白基因LNA水解探针qPCR检测烟曲霉方法的分析特异性为100%,扩增效率为98.2%,线性动态范围6个数量级,检测限2.5pg.结论 烟曲霉特异性β-微管蛋白基因LNA水解探针qPCR法能快速、准确地检测烟曲霉,而且此方法易操作、经济实惠,应用前景广阔.

Abstract：

Objective To evaluate the performance of locked nucleic acid(LNA)probe Real-time polymerase chain reaction(PCR)in the detection of Aspergillus fumigatus(A.fumigatus).Methods All clinically cultured isolates of Aspergillus at our hospital were identified by morphology and DNA sequencing assay.The experimental group consists of A.fumigatus(n=48)while the control group Was made up of A.flavus(n=55),A.versicolor(n=16),A.nidulans(n=10),A.sydowii(n=5)and A.parasiticus (n=1).The clinical samples consisted of A.fumigatus sinusitis tissue(n=20)and bronchoalveolar lavage fluid(n=1).DNA was extracted from all samples.A.fumigatus β-tubulin gene Was targeted with LNA probe Real-time PCR assay.LNA probe Real-time PCR was evaluated with regards to specificity,efficiency,linear dynamic range in PCR amplification and limits of detection.Results All clinical samples were positively amplified.The specificity was 100%and the PCR efficiency 98.2%.Linear dynamic range Was at least six orders of magnitude and the limit of detection 2.5 pg.Conclusion LNA probe Real-time PCR is a promisingly accurate assay of rapidly detecting A.fumigatus practically and cost-efficiently.     

盐胁迫对胶霉毒素生物合成基因gliT表达的影响

从烟曲霉菌株F3中克隆得到gliT基因（胶霉毒素生物合成基因簇成员之一）,其编码产物GliT是一个假定的硫氧还蛋白还原酶,含有保守的Pyr_redox和TrxB结构域,与来自于烟曲酶菌株Af293的GliT在氨基酸水平上的同源性高达97%。不同盐浓度培养条件下,烟曲霉菌株F3中gliT基因的表达变化与发酵液中胶霉毒素的含量变化之间存在着明显的相关性,均在盐浓度70g/L培养条件下达到峰值,提示gliT基因在胶霉毒素生物合成过程中的作用可能与盐胁迫有关。     

侵袭性肺曲霉菌病11例临床分析

目的探讨侵袭性肺曲霉菌病（IPA）的危险因素、临床表现、诊断和治疗特点。方法回顾性分析2006年1月至2010年10月收治的11例IPA的基础疾病、危险因素、临床表现、实验室及影像学特征、治疗和预后情况。结果 11例IPA患者确诊2例,临床诊断9例。3例患者无特殊病史,其余8例均有基础疾病。主要症状为发热、咳嗽、咯痰、咯血、胸闷和气喘。痰涂片3例、痰培养1例、肺泡灌洗液1例、胸水1例均查见曲霉菌。1,3-β-D-葡聚糖试验（G试验）及半乳甘露聚糖试验（GM试验）阳性6例。1例支气管镜活检、1例经皮肺活检病理形态学及特殊染色结果符合曲霉菌感染。影像学表现呈多样性。11例患者均接受抗真菌药物治疗,目前仍在治疗中2例,7例随诊,失访2例。6例选用伏立康唑、3例选用伊曲康唑、2例选用卡泊芬净作为初始治疗,均有疗效。结论 IPA与基础疾病及长期使用大剂量广谱抗生素、大剂量糖皮质激素等因素密切相关,主要临床表现不典型,病情进展迅速,病死率极高。诊断有赖于病理学及病原微生学结果,但随着高分辨率CT及G试验与GM实验在临床中的开展,使早期拟诊成为可能,早期予以经验性抗真菌治疗,可明显提高IPA的治愈率。     

Dectin-1 expression at early period of Aspergillus fumigatus infection in rat’s corneal epithelium

AIM:To investigate the expression of dendritic cell-associated C-type lectin-1(dectin-1) at the early period of Aspergillus fumigatus infection in rat’s corneal epithelium.METHODS:A total of 72 Wistar rats were randomly divided into three groups:A, B and C. The right eyes were chosen as experimental eyes. Group A was control group. Rats in group B were not inoculated with Aspergillus fumigatus. Group C was taken as Aspergillus fumigatus keratitis model. Rats in group B and C (six from each group) were executed randomly at 4, 8, 16 and 24 hours after experimental model being established to assess the expression of dectin-1 mRNA through real-time PCR. Another six rats in group B and C were executed randomly at 24 hours to assess the expression of dectin-1 protein through immunohistochemistry.RESULTS:The results of real-time PCR indicated that dectin-1 mRNA expression was low in corneal epithelium of normal rats’. There was no significantly difference of dectin-1 mRNA expression in group A and B (P>0.05). The expression of Aspergillus fumigatus infected corneal epithelium increased gradually after 8 hours in group C. The synchronous expression of group A and C had significant difference (P<0.01). Immunohistochemisty discovered that dectin-1 receptor existed in normal rat’s corneal epithelium . Dectin-1 protein increased after 24 hours in group C. There was a significant difference of synchronous expression in group B and C(P<0.01).CONCLUSION:Dectin-1 exists in rat’s corneal epithelium and its expression significantly increases at the early period of Aspergillus fumigatus infection. Dectin-1 is a pattern recognition receptor that expresses in corneal epithelium and involves in immune response to Aspergillus fungal keratitis.     

固有免疫在脂多糖、烟曲霉菌诱导的角膜细胞耐受中的作用

研究烟曲霉菌(AF)、脂多糖(LPS)能否引起角膜细胞的耐受反应,增强角膜对病原体刺激的防御功能。小剂量AF、LPS分别预处理永生化人角膜上皮细胞(THCE)和永生化人角膜基质细胞(THSF),20 h后给予大剂量AF或LPS再次刺激。于再次刺激后4 h收集细胞上清,酶联免疫吸附试验(ELISA)检测细胞因子IL-8的分泌,趋化实验检测LPS预处理引起THSF对PMN趋化的改变。于再次刺激后1 h收集细胞,实时RT-PCR检测细胞因子IL-8 mRNA和抗菌肽CCL20和LL-37 mRNA的表达。结果显示AF、LPS分别预处理THCE和THSF均可显著抑制IL-8的分泌。LPS预处理THSF抑制IL-8 mRNA的表达,增加CCL20和LL-37 mRNA的表达,并且抑制中性粒细胞(PMN)向角膜细胞的趋化。因此角膜细胞的耐受反应可避免过度的炎症反应,减轻角膜感染,在角膜炎症中起保护性作用。     

Species assignment and antifungal susceptibilities of black aspergilli recovered from otomycosis cases in Iran

Black aspergilli are among the main causative agents of otomycosis worldwide. In this study, the species assignment of black aspergilli isolated from otomycosis cases in Iran was carried out using sequence analysis of part of the calmodulin gene. The results indicate that Aspergillus niger is not the only black Aspergillus species involved in otomycosis cases in Iran: Aspergillus awamori and Aspergillus tubingensis are also able to cause ear infections. Antifungal susceptibility tests were carried out against five antifungal drugs including amphotericin B, fluconazole, itraconazole, ketoconazole and terbinafine. All isolates were highly susceptible to terbinafine, while they exhibited moderate susceptibilities against amphotericin B, fluconazole and ketoconazole. Aspergillus niger and A. awamori were found to have higher minimal inhibitory concentrations for azoles than A. tubingensis, in accordance with previous findings.     

Caspofungin: when and how? The microbiologist’s view

The echinocandins are antifungal agents, which act by inhibiting the synthesis of β-(1,3)-D-glucan, an integral component of fungal cell walls. Caspofungin, the first approved echinocandin, demonstrates good in vitro and in vivo activity against a range of Candida species and is an alternative therapy for Aspergillus infections. Caspofungin provides an excellent safety profile and is therefore favoured in patients with moderately severe to severe illness, recent azole exposure and in those who are at high risk of infections due to Candida glabrata or Candida krusei. In vivo/in vitro resistance to caspofungin and breakthrough infections in patients receiving this agent have been reported for Candida and Aspergillus species. The types of pathogens and the frequency causing breakthrough mycoses are not well delineated. Caspofungin resistance resulting in clinical failure has been linked to mutations in the Fksp subunit of glucan synthase complex. European Committee for Antimicrobial Susceptibility Testing and Clinical and Laboratory Standards Institute need to improve the in vitro susceptibility testing methods to detect fks hot spot mutants. Caspofungin represents a significant advance in the care of patients with serious fungal infections.