Aspergillus fumigatus and Aspergillus flavus-Specific IgG Cut-Offs for the Diagnosis of Chronic Pulmonary Aspergillosis in Pakistan.
ABSTRACT: Despite a high burden of chronic pulmonary aspergillosis (CPA) in Pakistan, Aspergillus-specific IgG testing is currently not available. Establishing cut-offs for Aspergillus-specific IgG for CPA diagnosis is crucial due to geographical variation. In settings such as Pakistan, where non-Aspergillus fumigatus (mainly A. flavus) Aspergillus species account for the majority of CPA cases, there is a need to explore additional benefit of Aspergillus flavus-specific IgG detection along with A. fumigatus-specific IgG detection. This study was conducted at the Aga Khan University, Karachi, Pakistan after ethical approval. Serum for IgG detection were collected after informed consent from healthy controls (n = 21), diseased controls (patients with lung diseases, n = 18), and CPA patients (n = 21). A. fumigatus and A. flavus IgG were detected using Siemens immulite assay. The sensitivity and specificity of A. fumigatus-specific IgG were 80.95% and 82.05%, respectively at a cut-off of 20 mg/L. The sensitivity and specificity of A. flavus-specific IgG were 80.95% and 79.49% at a cut-off of 30 mg/L. We report, for the first time, performance of A. flavus-specific IgG for CPA diagnosis. Although there was no statistically significant difference between the performance of both antigens, it seems contextually relevant to include A. flavus IgG in the CPA diagnostic algorithm in regions with higher non-A. fumigatus CPA infections.
Project description:BACKGROUND:Chronic pulmonary aspergillosis (CPA) is an underdiagnosed and misdiagnosed disease and now increasingly recognised. However, the diagnosis of CPA remains challenging. In this study, we aimed to investigate the diagnostic values of serum Aspergillus-specific IgG, IgA and IgM antibodies in patients with CPA. METHODS:The prospective study was performed at Chinese People's Liberation Army General Hospital in Beijing, from January 2017 to December 2017. Adult patients with lung lesions presented as cavity, nodule, mass, bronchiectasis or severe fibrotic destruction with at least two lobes in CT imaging were enrolled. One hundred healthy persons were also enrolled as additional controls. The serum levels of Aspergillus-specific IgG, IgA and IgM antibodies and galactomannan (GM) levels were measured simultaneously by plate ELISA kit. RESULTS:A total of 202 patients were enrolled in this study, including 42 CPA patients, 60 non-CPA patients and 100 healthy persons. The most common underlying lung diseases in CPA patients were bronchiectasis (28.6%) and COPD (19.0%). The most common symptoms in the CPA patients were cough (76.2%), sputum (71.4%), and fever (45.2%); chest pain (4.8%) was infrequent. Receiver operating characteristic (ROC) curve analysis revealed that the optimal CPA diagnostic cut-off of Aspergillus-specific IgG, IgA and IgM assays and GM test were 89.3?AU/mL, 8.2?U/mL, 73.3?AU/mL and 0.5?g/L, respectively. The serum levels of Aspergillus-specific IgG and IgA in CPA patients were higher than these in non-CPA patients or healthy persons. The sensitivities and specificities of Aspergillus-specific IgG, IgA, IgM tests and GM test were 78.6 and 94.4%, 64.3 and 89.4%, 50.0 and 53.7% and 71.4 and 58.1%, respectively. CONCLUSIONS:The sensitivity and specificity of serum Aspergillus-specific IgG assay are satisfactory for diagnosing CPA, while the performance of Aspergillus-specific IgA assay is moderate. Aspergillus-specific IgM assay and serum GM test have limited value for CPA diagnosis. TRIAL REGISTRATION:NCT03027089 . Registered 20 January 2017.
Project description:BACKGROUND:At present, serum Aspergillus IgG and IgM antibody detection is mainly used in the diagnosis of chronic pulmonary aspergillosis (CPA), but its value in the diagnosis of invasive pulmonary aspergillosis (IPA) in non-agranulocytic patients is still unclear. IgM can be used as a marker of acute infection to help diagnose acute infection-related diseases. IgG is a marker of long-term infection and is used to assist in the diagnosis of pre-existing or chronic infection-related diseases. The aim of this study was to investigate and compare the value of serum Aspergillus IgG and IgM antibody detection in the diagnosis of IPA and CPA in non-agranulocytic patients. METHODS:Fifty-eight cases of pulmonary aspergillosis (37 IPA and 21 CPA cases), 15 cases of community-acquired bacterial pneumonia and 50 cases in the healthy control group were collected. The serum (1,3)-?-D-glucan test (G test) was performed with a chromogenic method, and the galactomannan test (GM test) and Aspergillus IgG and IgM antibody detection were performed by commercial enzyme-linked immunosorbent assay (ELISA) in all patients. The sensitivity and specificity, cut-off value and area under the curve (AUC) of Aspergillus IgG and IgM antibodies were further obtained by receiver operating characteristic (ROC) curves. RESULTS:The positive rate of the G test, Aspergillus IgG antibody detection and the GM test also showed notable differences among the IPA, CPA, community-acquired bacterial pneumonia and healthy groups (P?=?0.006, P?<? 0.001 and P?=?0.217, respectively). Only the positive rate of the GM test showed a significant difference between the IPA and CPA groups (P?=?0.04). ROC curves indicated that Aspergillus IgG antibody detection had a higher specificity in the IPA group than in the CPA group (0.952). The detection of Aspergillus IgG antibody can preferably distinguish IPA from community-acquired bacterial pneumonia and healthy controls (sensitivity?=?0.923, specificity?=?0.459, cut-off value?=?134.46, AUC?=?0.727). It can also distinguish CPA from community-acquired bacterial pneumonia and healthy controls (sensitivity?=?0.952, specificity?=?0.692, cut-off value?=?75.46, AUC?=?0.873). CONCLUSIONS:Serum Aspergillus IgG antibody detection may have certain clinical value in the diagnosis of IPA and CPA in non-agranulocytic patients.
Project description:Although belong to the same genus, <i>Aspergillus fumigatus</i> is primarily involved in invasive pulmonary infection, whereas <i>Aspergillus flavus</i> is a common cause of superficial infection. In this study, we compared conidia (the infective propagules) of these two <i>Aspergillus</i> species. In immunocompetent mice, intranasal inoculation with conidia of <i>A. flavus</i> resulted in significantly higher inflammatory responses in the lungs compared to mice inoculated with <i>A. fumigatus</i> conidia. <i>In vitro</i> assays revealed that the dormant conidia of <i>A. flavus</i>, unlike <i>A. fumigatus</i> dormant conidia, are immunostimulatory. The conidial surface of <i>A. fumigatus</i> was covered by a rodlet-layer, while that of <i>A. flavus</i> were presented with exposed polysaccharides. <i>A. flavus</i> harbored significantly higher number of proteins in its conidial cell wall compared to <i>A. fumigatus</i> conidia. Notably, ?-1,3-glucan in the <i>A. flavus</i> conidial cell-wall showed significantly higher percentage of branching compared to that of <i>A. fumigatus</i>. The polysaccharides ensemble of <i>A. flavus</i> conidial cell wall stimulated the secretion of proinflammatory cytokines, and conidial cell wall associated proteins specifically stimulated IL-8 secretion from the host immune cells. Furthermore, the two species exhibited different sensitivities to antifungal drugs targeting cell wall polysaccharides, proposing the efficacy of species-specific treatment strategies. Overall, the species-specific organization of the conidial cell wall could be important in establishing infection by the two <i>Aspergillus</i> species.
Project description:Cyclopiazonic acid (CPA) is an indole-tetramic acid neurotoxin produced by some of the same strains of A. flavus that produce aflatoxins and by some Aspergillus oryzae strains. Despite its discovery 40 years ago, few reviews of its toxicity and biosynthesis have been reported. This review examines what is currently known about the toxicity of CPA to animals and humans, both by itself or in combination with other mycotoxins. The review also discusses CPA biosynthesis and the genetic diversity of CPA production in A. flavus/oryzae populations.
Project description:The emergence of resistant <i>Aspergillus</i> spp. is increasing worldwide. Long-term susceptibility surveillance for clinically isolated <i>Aspergillus</i> spp. strains is warranted for understanding the dynamic change in susceptibility and monitoring the emergence of resistance. Additionally, neither clinical breakpoints (CBPs) nor epidemiological cutoff values (ECVs) for <i>Aspergillus</i> spp. in China have been established. In this study, we performed a 20-year antifungal susceptibility surveillance for 706 isolates of <i>Aspergillus</i> spp. in a clinical laboratory at Peking University First Hospital from 1999 to 2019; and <i>in vitro</i> antifungal susceptibility to triazoles, caspofungin, and amphotericin B was determined by the Clinical and Laboratory Standards Institute (CLSI) broth microdilution method. It was observed that <i>Aspergillus fumigatus</i> was the most common species, followed by <i>Aspergillus flavus</i> and <i>Aspergillus terreus</i>. Forty isolates (5.7%), including <i>A. fumigatus</i>, <i>A. flavus</i>, <i>A. terreus</i>, <i>Aspergillus niger</i>, and <i>Aspergillus nidulans</i>, were classified as non-wild type (non-WT). Importantly, multidrug resistance was observed among <i>A. flavus</i>, <i>A. terreus</i>, and <i>A. niger</i> isolates. <i>Cyp51A</i> mutations were characterized for 19 non-WT <i>A. fumigatus</i> isolates, and TR<sub>34</sub>/L98H/S297T/F495I was the most prevalent mutation during the 20-year surveillance period. The overall resistance trend of <i>A. fumigatus</i> increased over 20 years in China. Furthermore, based on ECV establishment principles, proposed ECVs for <i>A. fumigatus</i> and <i>A. flavus</i> were established using gathered minimum inhibitory concentration (MIC)/minimum effective concentration (MEC) data. Consequently, all the proposed ECVs were identical to the CLSI ECVs, with the exception of itraconazole against <i>A. flavus</i>, resulting in a decrease in the non-WT rate from 6.0 to 0.6%.
Project description:A systematic review of literature data on the antifungal potential of extracted lichen compounds and individual secondary metabolites against mold species of the genus Aspergillus is provided. Crude extracts from 49 epiphytic, 16 epigeic and 22 epilithic species of lichens and 44 secondary metabolites against 10 species, Aspergillus candidus, A. flavus, A. fumigatus, A. nidulans, A. niger, A. ochraceus, A. parasiticus, A. restrictus, A. stellatus and A. ustus, were analysed. Several measuring techniques were employed for such analyses. Lichen substances were extracted with alcoholic and other organic solvents mainly using the Soxhlet apparatus. Among the three most-studied mold species, the results showed that the crude extracts from the thalli of the lichens Cladonia foliacea, Hypotrachyna cirrhata, Leucodermia leucomelos, Platismatia glauca and Pseudevernia furfuracea against Aspergillus flavus, from C. foliacea, Nephroma arcticum and Parmelia sulcata against A. fumigatus and from Evernia prunastri, Hypogymnia physodes, Umbilicaria cylindrica and Variospora dolomiticola against A. niger have the greatest antifungal potential. The lichen secondary metabolites showed a higher inhibitory potential, e.g. protolichesterinic acid against A. flavus, lecanoric acid against A. fumigatus and orsellinic acid against A. niger; the other seven species of Aspergillus have been poorly studied and require further investigation. A comparison of the inhibitory potential of the tested mixtures of lichen substances and their secondary metabolites shows that they can compete with commonly used antifungal substances, such as ketoconazole and clotrimazole against A. flavus, A. nidulans, A. niger and A. parasiticus and fluconazole in the case of A. fumigatus.
Project description:Cyclopiazonic acid (?-cyclopiazonic acid, ?-CPA) is an indole-hydrindane-tetramic acid neurotoxin produced by various fungal species, including the notorious food and feed contaminant <i>Aspergillus flavus</i>. Despite its discovery in <i>A. flavus</i> cultures approximately 40 years ago, its contribution to the <i>A. flavus</i> mycotoxin burden is consistently minimized by our focus on the more potent carcinogenic aflatoxins also produced by this fungus. Here, we report the screening and identification of several CPA-type alkaloids not previously found in <i>A. flavus</i> cultures. Our identifications of these CPA-type alkaloids are based on a dereplication strategy involving accurate mass high resolution mass spectrometry data and a careful study of the ?-CPA fragmentation pattern. In total, 22 CPA-type alkaloids were identified in extracts from the <i>A. flavus</i> strains examined. Of these metabolites, 13 have been previously reported in other fungi, though this is the first report of their existence in <i>A. flavus</i>. Two of our metabolite discoveries, 11,12-dehydro ?-CPA and 3-hydroxy-2-oxo CPA, have never been reported for any organism. The conspicuous presence of CPA and its numerous derivatives in <i>A. flavus</i> cultures raises concerns about the long-term and cumulative toxicological effects of these fungal secondary metabolites and their contributions to the entire <i>A. flavus</i> mycotoxin problem.
Project description:BACKGROUND: Aspergillus flavus is intensively studied for its role in infecting crop plants and contaminating produce with aflatoxin, but its role as a human pathogen is less well understood. In parts of the Middle East and India, A. flavus surpasses A. fumigatus as a cause of invasive aspergillosis and is a significant cause of cutaneous, sinus, nasal and nail infections. METHODS: A collection of 45 clinical and 10 environmental A. flavus isolates from Iran were analysed using Variable-Number Tandem-Repeat (VNTR) markers with MICROSAT and goeBURST to determine their genetic diversity and their relatedness to clinical and environmental A. flavus isolates from Australia. Phylogeny was assessed using partial ?-tubulin and calmodulin gene sequencing, and mating type was determined by PCR. Antifungal susceptibility testing was performed on selected isolates using a reference microbroth dilution method. RESULTS: There was considerable diversity in the A. flavus collection, with no segregation on goeBURST networks according to source or geographic location. Three Iranian isolates, two from sinus infections and one from a paranasal infection grouped with Aspergillus minisclerotigenes, and all produced B and G aflatoxin. Phylogenic analysis using partial ?-tubulin and calmodulin sequencing confirmed two of these as A. minisclerotigenes, while the third could not be differentiated from A. flavus and related species within Aspergillus section flavi. Based on epidemiological cut-off values, the A. minisclerotigens and A. flavus isolates tested were susceptible to commonly used antifungal drugs. CONCLUSIONS: This is the first report of human infection due to A. minisclerotigenes, and it raises the possiblity that other species within Aspergillus section flavi may also cause clinical disease. Clinical isolates of A. flavus from Iran are not distinct from Australian isolates, indicating local environmental, climatic or host features, rather than fungal features, govern the high incidence of A. flavus infection in this region. The results of this study have important implications for biological control strategies that aim to reduce aflatoxin by the introduction of non-toxigenic strains, as potentially any strain of A. flavus, and closely related species like A. minisclerotigenes, might be capable of human infection.
Project description:Understanding the nature of species" boundaries is a fundamental question in evolutionary biology. The availability of genomes from several species of the genus Aspergillus allows us for the first time to examine the demarcation of fungal species at the whole-genome level. Here, we examine four case studies, two of which involve intraspecific comparisons, whereas the other two deal with interspecific genomic comparisons between closely related species. These four comparisons reveal significant variation in the nature of species boundaries across Aspergillus. For example, comparisons between A. fumigatus and Neosartorya fischeri (the teleomorph of A. fischerianus) and between A. oryzae and A. flavus suggest that measures of sequence similarity and species-specific genes are significantly higher for the A. fumigatus - N. fischeri pair. Importantly, the values obtained from the comparison between A. oryzae and A. flavus are remarkably similar to those obtained from an intra-specific comparison of A. fumigatus strains, giving support to the proposal that A. oryzae represents a distinct ecotype of A. flavus and not a distinct species. We argue that genomic data can aid Aspergillus taxonomy by serving as a source of novel and unprecedented amounts of comparative data, as a resource for the development of additional diagnostic tools, and finally as a knowledge database about the biological differences between strains and species.
Project description:Aspergillus fumigatus is the most common species that causes invasive aspergillosis. In order to identify A. fumigatus, partial ribosomal DNA (rDNA) from two to six strains of five different Aspergillus species was sequenced. By comparing sequence data from GenBank, we designed specific primer pairs targeting rDNA internal transcribed spacer (ITS) regions of A. fumigatus. A nested PCR method for identification of other A. fumigatus-related species was established by using the primers. To evaluate the specificities and sensitivities of those primers, 24 isolates of A. fumigatus and variants, 8 isolates of Aspergillus nidulans, 7 isolates of Aspergillus flavus and variants, 8 isolates of Aspergillus terreus, 9 isolates of Aspergillus niger, 1 isolate each of Aspergillus parasiticus, Aspergillus penicilloides, Aspergillus versicolor, Aspergillus wangduanlii, Aspergillus qizutongii, Aspergillus beijingensis, and Exophiala dermatitidis, 4 isolates of Candida, 4 isolates of bacteria, and human DNA were used. The nested PCR method specifically identified the A. fumigatus isolates and closely related species and showed a high degree of sensitivity. Additionally, four A. fumigatus strains that were recently isolated from our clinic were correctly identified by this method. Our results demonstrate that these primers are useful for the identification of A. fumigatus and closely related species in culture and suggest further studies for the identification of Aspergillus fumigatus species in clinical specimens.