Sero-diagnosis of invasive aspergillosis: Attempts to determine antigen and antibody relevance to infection

Attempt was made to define antigens and antisera which might prove useful in diagnosis of invasive aspergillosis in man. A convalescent antiserum (serum from rabbits after live infection withAspergillus fumigatus conidia) which might be more representative of immunological reaction to fungal growthin vivo, did not react in enzyme-linked immunosorbent assay with commercial antigens which are used at present in attempts to detect antibody response in systemic infections in man. However, this convalescent antiserum reacted with antigens from a range of fungal extracts. Antigens from young culture filtrates, in particular the 24h culture filtrate are advocated as the standard antigens for antibody detection using conventional immunoprecipitation techniques. For the detection of circulating antigens, the use of convalescent antiserum in enzyme-linked immunosorbent assay might be promising in the early diagnosis of invasive aspergillosis.     

Usefulness of galactomannan detection in the diagnosis and follow-up of hematological patients with invasive aspergillosis

The usefulness of galactomannan detection using the Platelia Aspergillus test for the diagnosis of invasive aspergillosis was studied in 849 sera from 54 hematological patients with prolonged neutropenia, which were classified according to the risk for invasive aspergillosis. Three patients developed a proven invasive aspergillosis, one a probable invasive aspergillosis and 17 patients a possible invasive aspergillosis. Thirty-three patients showed no evidence of invasive aspergillosis. All patients with proven invasive aspergillosis had a high risk for invasive aspergillosis, while the one having probable invasive aspergillosis had intermediate risk. Detection of galactomannan in this study showed a sensitivity of 66.7% for patients with proven invasive aspergillosis and 50% for patients with proven and probable invasive aspergillosis. The specificity was 98% or higher in all groups studied. The predictive positive and negative values for patients with proven invasive aspergillosis were 66.7% and 98%, respectively. A rise in the concentration of galactomannan was observed in patients who failed to respond to the antifungal treatment. Galactomannan antigenemia preceded post-mortem histological diagnosis of invasive aspergillosis in two patients by 17 and 81 days, respectively. In conclusion, detection of galactomannan by the Platelia Aspergillus test allows for a specific and relatively sensitive diagnosis of invasive aspergillosis in hematological patients with a high and intermediate risk for invasive aspergillosis.     

The contribution of galactomannan detection in the diagnosis of invasive aspergillosis in bone marrow transplant recipients

Until recently, accurate microbiological diagnosis of invasive aspergillosis (IA) was seldom established in HSCT recipients. Blood samples are rarely positive for Aspergillus species, the reliability of the cultures depends of the specimen (if taken from a normally sterile site or not) and biopsy samples require invasive procedures, rarely recommended in patients with severe thrombocytopenia. Implementing the international consensus defining the microbiological criteria for the diagnosis of Aspergillus infection, we retrospectively evaluated the role of serum galactomannan (GM) detection by EIA to diagnose IA among HSCT patients with proven invasive fungal infection (IFI) and the impact of serum storage in GM concentrations. The EIA assay allowed categorizing as “probable” 5 of the 10 cases of “possible” aspergillosis (50%). Considering a lower cut-off level for the reaction (1.0), 80% of the cases could be categorized as “probable” aspergillosis. Positive or undetermined results were detected one to 4 months before the diagnosis of IA in eight of the 11 patients (72.7%) with proven IFI. Retesting the stored samples after a second storage for four years, we could observe lower reactivity in 20% of the samples. The detection of galactomannan by the EIA test represents a major advance in the diagnosis of IA in HSCT recipients at high risk of IA. A better understanding of the kinetics of the GM in different clinical situations is necessary to maximize the benefit of the test in Aspergillus surveillance.     

Value of antigen and antibody detection,and blood evaluation parameters in diagnosis of avian invasive aspergillosis

The applicability of ELISA detection of circulating Aspergillus spp. antigen (Ag) and systemic antibody (Ab) of IgG class, and the blood parameter values were evaluated for diagnosis of invasive aspergillosis in Aspergillus spp.-challenged Peking ducks (Anas platyrhynchos). The protective role of Aspergillus spp. IgG was evaluated in Cape shelducks (Tadorna cana) immunized with Aspergillus spp. Ag. Challenged but non-immunized A. platyrhynchos developed invasive aspergillosis on day 21 as demonstrated histopathologically by the presence of fungal microgranuloma in air sacs and lung tissue, with serum antigenemia fluctuating from 65 to 270 ng of 55-kD basic protein Ag per ml. Immunized A. platyrhynchos did not demonstrate Aspergillus spp. serum antigenemia but did show rare histopathological changes in some air sacs associated with fungal inflammation. Although the differences between immunized and non-immunized T. cana in blood evaluation parameters did not differ significantly, immunized birds mounted high Aspergillus spp.-specific IgG titer. There was no correlation between the blood parameter values and post-immunization timepoints in T. cana and in A. platyrhynchos. Intramuscular immunization with Aspergillus spp. mycelial phase cultures Ag provided protection against the pathogens. The lack of relations between blood parameter values and increasing Aspergillus spp. IgG titers (in T. cana and A. platyrhynchos) indicate low applicability of these parameters in evaluation of a bird Aspergillus spp. status. Detection of circulating 55-kDa Aspergillus spp. Ag has high early predictive values for invasive aspergillosis in birds. This revised version was published online in August 2006 with corrections to the Cover Date.     

肝移植术后曲霉的气道定植与侵袭性曲霉病的关系

目的评价肝移植术后曲霉气道的定植及其发生侵袭性感染的风险。方法回顾2003—2004年南京八一医院和上海长征医院56例肝移植患者进行的连续组织学检查和曲霉培养结果。结果24例患者被分离出曲霉,2例发生侵袭性烟曲霉感染。这2例患者在移植后6个月内都有烟曲霉的定植,并且都死亡,占所有移植后死亡的29％。无曲霉定植者都未发生侵袭性曲霉感染。结论肝移植术后的侵袭性曲霉感染较为少见,但常致死,曲霉定植常见但为一过性,由于气管侵袭性曲霉感染只发生在有烟曲霉定植的移植后6个月内的患者,所以在此期间进行预防性的治疗能否有效降低侵袭性曲霉感染值得研究。     

Substrate specifity profiling of the Aspergillus fumigatus proteolytic secretome reveals consensus motifs with predominance of Ile/Leu and Phe/Tyr

Background

The filamentous fungus Aspergillus fumigatus (AF) can cause devastating infections in immunocompromised individuals. Early diagnosis improves patient outcomes but remains challenging because of the limitations of current methods. To augment the clinician''s toolkit for rapid diagnosis of AF infections, we are investigating AF secreted proteases as novel diagnostic targets. The AF genome encodes up to 100 secreted proteases, but fewer than 15 of these enzymes have been characterized thus far. Given the large number of proteases in the genome, studies focused on individual enzymes may overlook potential diagnostic biomarkers.

Methodology and Principal Findings

As an alternative, we employed a combinatorial library of internally quenched fluorogenic probes (IQFPs) to profile the global proteolytic secretome of an AF clinical isolate in vitro. Comparative protease activity profiling revealed 212 substrate sequences that were cleaved by AF secreted proteases but not by normal human serum. A central finding was that isoleucine, leucine, phenylalanine, and tyrosine predominated at each of the three variable positions of the library (44.1%, 59.1%, and 57.0%, respectively) among substrate sequences cleaved by AF secreted proteases. In contrast, fewer than 10% of the residues at each position of cleaved sequences were cationic or anionic. Consensus substrate motifs were cleaved by thermostable serine proteases that retained activity up to 50°C. Precise proteolytic cleavage sites were reliably determined by a simple, rapid mass spectrometry-based method, revealing predominantly non-prime side specificity. A comparison of the secreted protease activities of three AF clinical isolates revealed consistent protease substrate specificity fingerprints. However, secreted proteases of A. flavus, A. nidulans, and A. terreus strains exhibited striking differences in their proteolytic signatures.

Conclusions

This report provides proof-of-principle for the use of protease substrate specificity profiling to define the proteolytic secretome of Aspergillus fumigatus. Expansion of this technique to protease secretion during infection could lead to development of novel approaches to fungal diagnosis.     

烟曲霉Asp f3和Asp f4线性B细胞抗原表位最小基序鉴定

烟曲霉(Aspergillus fumigatus)是一种广泛存在于自然界中的条件致病菌,其产生的分生孢子被易感人群吸入后定植于肺部,引起3种曲霉病:致咯血的曲霉肿、致肺纤维化的变应性支气管肺曲霉病、致较高死亡率的侵袭性曲霉病。目前临床上用于诊断烟曲霉感染的血清免疫学指标有半乳甘露聚糖和1,3-β-D-葡聚糖,但特异度和灵敏度方面存在一定的局限性。Asp f3和Asp f4为烟曲霉主要抗原,在感染者血清中存在相应的循环抗体。本研究分别用兔抗Asp f3和Asp f4抗血清对其进行抗原表位扫描,鉴定了8个Asp f3和6个Asp f4最小表位基序肽。用所鉴定的最小表位基序与烟曲霉其他抗原的最小表位基序构建嵌合肽,可能有助于提高烟曲霉感染诊断的特异度和灵敏度。     

Aspergillus conidia activate the complement by the mannan-binding lectin C2 bypass mechanism

Innate immunity is the major host defense against invasive aspergillosis. To determine whether the collectin mannan-binding lectin (MBL) is involved in the initial protective immunity through complement activation against opportunistic fungal infections caused by Aspergillus, we performed in vitro studies on 29 different strains of Aspergillus conidia from five different species. Incubation of Aspergillus conidia in human normal serum leads to activation of the alternative pathway, whereas neither the classical nor the lectin pathways through C4 and C2 cleavage are activated. Complement response to conidia was investigated using a MBL-deficient serum and reconstitution experiments were conducted with MBL/MASPs complexes. We found that MBL can directly support C3 activation by a C2 bypass mechanism. Finally, a stronger activation of the alternative pathway was observed for the clinical strains isolated from patients with invasive aspergillosis, compared with the environmental strains.     

SELDI-TOF-MS of saliva: methodology and pre-treatment effects

Interest in saliva as a diagnostic fluid for monitoring general health and for early diagnosis of disease has increased in the last few years. In particular, efforts have focused on the generation of protein maps of saliva using advanced proteomics technology. Surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) is a novel high throughput and extremely sensitive proteomic approach that allows protein expression profiling of large sets of complex biological specimens. In this study, large scale profiling of salivary proteins and peptides, ranging from 2 to 100kDa was demonstrated using SELDI-TOF-MS. Various methodological aspects and pre-analytical variables were analysed with respect to their effects on saliva SELDI-TOF-MS profiling. Results show that chip surface type and sample type (unstimulated versus stimulated) critically affect the amount and composition of detected salivary proteins. Factors that influenced normal saliva protein profiling were matrix composition, sample dilution and binding buffer properties. Delayed processing time experiments show certain new peptides evolving 3h post-saliva donation, and quantitative analyses indicate relative intensity of other proteins and peptides changing with time. The addition of protease inhibitors partly counteracted the destabilization of certain protein/peptide mass spectra over time suggesting that some proteins in saliva are subject to digestion by intrinsic salivary proteases. SELDI-TOF-MS profiles also changed by varying storage time and storage temperature whereas centrifugation speed and freeze-thaw cycles had minimal impact. In conclusion, SELDI-TOF-MS offers a high throughput platform for saliva protein and peptide profiling, however, (pre-)analytical conditions must be taken into account for valid interpretation of the acquired data.     

Multicenter evaluation of an enzyme immunoassay (Platelia® Aspergillus) for the detection of Aspergillus antigen in serum

Invasive aspergillosis is a serious problem for immunocompromised patients, especially if neutropenic. The diagnosis of this infection is complicated, since clinical symptoms are often similar to those of other fungal diseases. The chance of detecting the presence of a specific antigen in the serum could confirm the suspected clinical diagnosis and. perhaps, be useful for the follow-up of the patient. The Medical Mycology Committee of the Associazione Microbiologi Clinici Italiani (AMCLI) decided to evaluate in a multicenter prospective study (from I November 1998 to 28 February 1999) the performance of the Platelia Aspergillus Kit (Bio-Rad) for the detection of Aspergillus galactomannan in human serum. The enrolled patients included various groups of immunosuppressed patients (mostly neutropenic). Blood samples were drawn at the time of enrollment. This decision was based upon a clinical diagnosis of probable aspergillosis (antibiotic non-responsive fever for at least 96 hours, cough, hemophthosis and positive chest X-ray). Additional blood samples were drawn on days 3, 6, 9, 12, 15 and 21. Culture and histopathologic examinations were performed according to the individual laboratory workflow. For each patient the laboratory filled a form with all the available clinical information, to create a database on which to evaluate the results of the test. During the study, 187 patients with various kinds of immunosuppression were enrolled. A total of 256 sera were tested: for 117 patients (62.6%) only the basal sample was tested, whereas for the 70 symptomatic patients (37.4%) multiple specimens (range: 1-6) were tested. The results allowed the laboratories to exclude (68.6%) or confirm (31.5%: confirmed and/or probable) the clinical diagnosis of invasive aspergillosis; 4 cases remained undetermined. Based on the results of this study, it seems that the use of this test should be limited to those patients with clinical symptoms of aspergillosis.     

Invasive aspergillosis

Invasive aspergillosis is one of the most frequent fungal infections in neutropenic patients, in whom it is associated with a high mortality. Its diagnosis is difficult by the traditionally used laboratory tests. In the last years, an ELISA (Platelia Aspergillus, Bio-Rad, France) to detect galactomannan in neutropenic and cancer patients with high risk of suffering invasive aspergillosis has been developed. The experience accumulated in Spain in the diagnosis of invasive aspergillosis by Platelia Aspergillus is presented in this monograph.     

Invasive pulmonary aspergillosis due to a mixed infection caused by Aspergillus flavus and Aspergillus fumigatus

Invasive pulmonary aspergillosis is typically caused by a single Aspergillus species, most frequently Aspergillus fumigatus. Here we report that a lung transplant recipient developed invasive aspergillosis due to a mixed infection caused by Aspergillus flavus and A. fumigatus. The implications for this unusual finding are discussed.     

Use of the Immunodiffusion Test in the Serodiagnosis of Aspergillosis

The diagnostic value of an immunodiffusion (ID) test with standardized precipitinogens derived from five Aspergillus species was determined with sera from 60 proven and 12 suspected cases of aspergillosis. The data demonstrated that the greatest number of aspergillosis cases were detected by the concurrent use of A. fumigatus and A. niger precipitinogens. With these precipitinogens, the ID test permitted the serodiagnosis of aspergillosis in 82% of the 60 proven cases and in 83% of the 12 suspected cases. The presence of one or more precipitins was indicative of aspergilloma, of allergic bronchopulmonary aspergillosis, or of invasive aspergillosis. Precipitins were detected in 93% of the sera from patients with aspergilloma, in 50% of the sera from patients with allergic bronchopulmonary aspergillosis, and in 88% of the sera from patients with invasive aspergillosis. Although the presence of one or two precipitin bands could indicate any form of aspergillosis, the presence of three or four was strong evidence of either aspergilloma or invasive aspergillosis. The ID test was found to be 100% specific in an evaluation of its effectiveness with 65 sera from individuals with other systemic mycotic infections, bacterial or neoplastic diseases, and from apparently normal humans. In diagnosed cases of aspergillosis, the examination of serial serum specimens provided information about the clinical course of the disease. A reduction in the number of precipitin bands and significant titer changes were noted as the patients responded to therapy.     

Invasive aspergillosis promotes tumor growth and severity in a tumor-bearing mouse model

Invasive aspergillosis increases in chronic immunosuppressive diseases such as cancer. There is little information about the mechanisms by which Aspergillus infection affects the immune regulation and microenvironment of cancer cells. Hence, this study was aimed at investigating the effect of invasive aspergillosis on immunosurveillance, metastasis, and prognosis of cancer in tumor-bearing mice. After implantation of mouse mammary tumor in BALB/c mice, they were infected with Aspergillus conidia intravenously. For comparison, groups of mice were experimentally infected with Aspergillus conidia or implanted with tumor cells separately. Seven days after Aspergillus infection, the serum levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) were measured by ELISA, and subsequently regulatory T lymphocytes were analyzed by flow cytometry. The survival of animals and mean tumor size were then determined. Our results indicated that tumor sizes in mice increased significantly after infection with Aspergillus conidia. Moreover, invasive aspergillosis enhanced the population of regulatory lymphocytes and level of TIMP-1. This study supports the idea that massive Aspergillus infection could stimulate tumor growth and increases the possibility of a bad prognosis. As a result, treatment of Aspergillus infection could be considered an important issue for efficient cancer therapy.     

Immunoproteomics based identification of thioredoxin reductase GliT and novel <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> antigens for serologic diagnosis of invasive aspergillosis

Background  

There has been a rising incidence of invasive aspergillosis (IA) in critically ill patients, even in the absence of an apparent predisposing immunodeficiency. The diagnosis of IA is difficult because clinical signs are not sensitive and specific, and serum galactomannan has relatively low sensitivity in this group of patients. Therefore, more prompt and accurate disease markers for early diagnosis are needed. To establish disease markers demands a thorough knowledge of fungal antigens which may be detected in the serum or other body fluids of patients. Herein we report novel immunodominant antigens identified from extracellular proteins of Aspergillus fumigatus.     

Robust substrate profiling method reveals striking differences in specificities of serum and lung fluid proteases

Proteases are candidate biomarkers and therapeutic targets for many diseases. Sensitive and robust techniques are needed to quantify proteolytic activities within the complex biological milieu. We hypothesized that a combinatorial protease substrate library could be used effectively to identify similarities and differences between serum and bronchoalveolar lavage fluid (BALF), two body fluids that are clinically important for developing targeted therapies and diagnostics. We used a concise library of fluorogenic probes to map the protease substrate specificities of serum and BALF from guinea pigs. Differences in the proteolytic fingerprints of the two fluids were striking: serum proteases cleaved substrates containing cationic residues and proline, whereas BALF proteases cleaved substrates containing aliphatic and aromatic residues. Notably, cleavage of proline-containing substrates dominated all other protease activities in both human and guinea pig serum. This substrate profiling approach provides a foundation for quantitative comparisons of protease specificities between complex biological samples.     

国产血清半乳甘露聚糖检测试剂对侵袭性肺曲霉菌病的诊断价值评估

目的：评估国产血清半乳甘露聚糖（Galactomannan,GM）检测试剂对侵袭性肺曲霉菌病的诊断价值。方法根据血液病/恶性肿瘤患者侵袭性真菌病的诊断标准与治疗原则（第四次修订版）[1]收集临床确诊侵袭性肺曲霉菌病（inva-sive pulmonary aspergillosis,IPA）、临床诊断IPA、拟诊IPA、排除IPA四组病例。采用天津贻诺琦公司酶联免疫吸附法（ELISA）试剂检测纳入的86例患者血清标本的GM浓度,分析其敏感性、特异性、阳性预测值（PPV）、阴性预测值（NPV）。结果86例病例中,临床诊断27例、拟诊12例、排除47例。在3种不同的阳性判断标准下,敏感性：9444%、9630%、6296%;特异性：5625%、4576%、6441%;PPV：4474%、4483%、4474%;NPV：9643%、9643%、7917%。统计学分析证实标准1（即血清GM值〉095μg/L为阳性,〈075μg/L为阴性,075~095μg/L为灰区,未将灰区加入计算）在3种判断标准中最优,故选择其为最终判断标准。结论该血清GM检测试剂盒诊断性能较好,可以用于侵袭性肺曲霉菌病的辅助诊断。     

Successful Antifungal Combination Therapy and Surgical Approach for A<Emphasis Type="Italic">spergillus fumigatus</Emphasis> Suppurative Thyroiditis Associated with Thyrotoxicosis and Review of Published Reports

In immunocompromised patients, Aspergillus infections are important causes of morbidity and mortality. We describe a patient with cryoglobulinemic vasculitis who developed disseminated invasive aspergillosis with thyrotoxicosis caused by Aspergillus fumigatus. The diagnosis was based upon radiological, microbiological and pathological findings. The patient was treated successfully with voriconazole and caspofungin treatment followed by total thyroidectomy. We provide an overview of published reports on Aspergillus thyroiditis with an emphasis on therapeutic approaches.     

Value of Aspergillus galactomannan antigen detection in the diagnosis and follow-up of invasive aspergillosis in hematological patients

Serum galactomannan detection is considered to be a useful test for early diagnosis and follow-up of invasive aspergillosis. From February to September 2002, adult patients hospitalized in our Hematology Unit for receiving intensive chemotherapy and/or hematopoietic stem cell transplant were prospectively studied. We analyzed a total of 760 samples obtained from 100 patients. Eleven patients (11%) having a positive result (OD index >1.5 ng/ml) in two consecutive Platelia Aspergillus tests were considered galactomannan-positive cases. On the other hand, 12 patients (12%) were diagnosed of proven or probable invasive aspergillosis. Sensitivity (66.6%), specificity (95.5%), positive predictive value (72.7%) and negative predictive value (96.7%) were comparable to those of larger series. Galactomannan positivity allowed also to anticipate invasive aspergillosis diagnosis (from two to 17 days before radiographic findings and from two to 15 days before mycological culture). Moreover, kinetics of antigenemia could be useful for assessing therapeutic response. Once accepted galactomannan test as a diagnostic criterium for invasive aspergillosis knowing potential causes of false positive results is of paramount importance.     

X-ray Structure Analysis and Characterization of AFUEI,an Elastase Inhibitor from Aspergillus fumigatus

Elastase from Aspergillus sp. is an important factor for aspergillosis. AFUEI is an inhibitor of the elastase derived from Aspergillus fumigatus. AFUEI is a member of the I78 inhibitor family and has a high inhibitory activity against elastases of Aspergillus fumigatus and Aspergillus flavus, human neutrophil elastase and bovine chymotrypsin, but does not inhibit bovine trypsin. Here we report the crystal structure of AFUEI in two crystal forms. AFUEI is a wedge-shaped protein composed of an extended loop and a scaffold protein core. The structure of AFUEI shows remarkable similarity to serine protease inhibitors of the potato inhibitor I family, although they are classified into different inhibitor families. A structural comparison with the potato I family inhibitors suggests that the extended loop of AFUEI corresponds to the binding loop of the potato inhibitor I family, and AFUEI inhibits its cognate proteases through the same mechanism as the potato I family inhibitors.