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Immunodiagnosis of aspergillosis.

Aspergillus species are ubiquitous fungi and have been implicated as the causative agents of a variety of lung disorders in humans. These disorders include allergic, saprophytic, and systemic manifestations. The allergic disorders mainly affect atopic persons, and invasive or systemic diseases affect immunosuppressed individuals. Immunodiagnosis can help the practitioner diagnose these diseases. Demonstration of circulating antibodies is a useful criterion, but the lack of dependable and standardized antigens is a limiting factor in the diagnosis of most Aspergillus-induced diseases. Despite this limitation, however, immunodiffusion and enzyme-linked immunosorbent assays have been widely used for the detection of antibodies in the sera of patients with aspergillosis. Similarly, crude and semipurified antigens are being used to demonstrate skin hypersensitivity in patients, and several methods have been useful in the detection of antigenemia in patients with invasive aspergillosis. With a growing number of reports on the incidence of aspergillosis and an increase in the number of immunosuppressed individuals in the population, more rapid methods and more reliable reagents for immunodiagnosis are needed. With recent attempts at obtaining reliable reagents for through hybridoma technology and molecular biological techniques, substantial progress toward efficient immunodiagnosis may be achieved.

Animals↗

Lack of vessel wall elastolysis in human invasive pulmonary aspergillosis.

In experimental studies, the apparent ability of Aspergillus fumigatus isolates to produce elastase in agar plates correlates with their ability to cause invasive pulmonary aspergillosis in mice pretreated with cortisone. Thus, elastase production may govern the pathogenicity of particular isolates. If this is so, then disruption of the elastic layers within blood vessel walls in invasive aspergillosis would be expected. To test this hypothesis, tissue blocks were prepared from nine patients with invasive pulmonary aspergillosis. Separate but immediately adjacent histological sections were stained by the Grocott and periodic acid-Schiff methods for fungal hyphae and by the elastic van Gieson technique for elastic tissue. Comparison of those segments of vessel walls infiltrated by hyphae with those not infiltrated by hyphae showed no overall loss of elastic tissue. Material from five of the cases was also stained with an unconventional combination of histochemical stains, allowing accurate identification of both fungal hyphae and elastic laminae in the same histological sections. The results showed no more disruption of elastic laminae than would be expected from simple physical displacement of elastic laminae. We conclude that if elastolysis contributes at all to invasion of vessel walls by aspergilli, then it seems to be very localized and/or transient.

Adult↗

Development of a ligand-directed approach to study the pathogenesis of invasive aspergillosis.

Invasive aspergillosis is a leading cause of infectious death in immunosuppressed patients. Here, we adapted a phage display library-based selection to screen and identify binding peptides to the surface of Aspergillus fumigatus conidia and hyphae. We identified a peptide (sequence CGGRLGPFC) that reliably binds to the surface of Aspergillus fumigatus hyphae. Binding was not Aspergillus strain specific, as it was also observed in hyphae of other Aspergillus clinical isolates. Furthermore, CGGRLGPFC-displaying phage targets Aspergillus fumigatus hyphae on formalin-fixed paraffin-embedded histopathology sections of lung tissue recovered from mice with invasive pulmonary aspergillosis. This approach may yield reagents such as peptidomimetics for novel diagnostic and therapeutic interventions in invasive aspergillosis.

Amino Acid Motifs↗

Murine model of invasive pulmonary aspergillosis following an earlier stage, noninvasive Aspergillus infection.

Aspergillus spp. occasionally cause invasive pulmonary aspergillosis following noninvasive infection in patients with underlying bronchopulmonary disorders regardless of their systemic immunological conditions. We developed a murine model of invasive pulmonary aspergillosis following an earlier stage, noninvasive Aspergillus infection. BALB/c mice were inoculated intratracheally with agarose beads containing Aspergillus fumigatus conidia. Two weeks after inoculation, half of the mice were immunosuppressed with cortisone acetate. During a 4-week observation period, the survival rate of infected immunosuppressed mice was significantly lower (P < 0.01) than that of infected nonimmunosuppressed mice. The number of CFU in the lungs gradually decreased in the nonimmunosuppressed mice, whereas a time-related significant increase (P < 0.05) of CFU was demonstrated in the immunosuppressed mice. In the lungs of the nonimmunosuppressed mice, there was marked accumulation of neutrophils, lymphocytes, and macrophages (in this order) around the agarose beads in the bronchi. Aspergillus hyphae were surrounded by the inflammatory cells and did not invade the lung parenchyma. In contrast, in the immunosuppressed mice, Aspergillus hyphae proliferated markedly and invaded the lung parenchyma after immunosuppression. In this model, the two-dimensional extents of the lesions were also evaluated with an image-processing system. Time-related increase of the area of peribronchial necrotic lesions was significant (P < 0.05) after immunosuppression. This model should therefore be useful for investigating the pathophysiology of noninvasive Aspergillus infection and invasive pulmonary aspergillosis and also for clarifying the mechanism of conversion to the invasive disease from the noninvasive stage.

Animals↗

Quantitative galactomannan detection is superior to PCR in diagnosing and monitoring invasive pulmonary aspergillosis in an experimental rat model.

Two diagnostic tests, an Aspergillus-specific PCR and an enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of galactomannan, were compared for diagnosing and monitoring invasive pulmonary aspergillosis. Persistently neutropenic rats with left-sided invasive pulmonary aspergillosis were sacrificed at regular intervals after inoculation. Blood samples and bronchoalveolar lavage (BAL) fluid were cultured and tested by PCR as well as by ELISA. Disseminated fungal infection in extrapulmonary organs was determined. The sensitivity of the ELISA was higher than that of the PCR on all days of measurements, in both blood and BAL fluid. Positive PCR or ELISA results in blood were not significantly associated with disseminated fungal infection. Serial testing in a separate group of rats showed consistently increasing concentrations of circulating galactomannan during the course of disease, while a positive PCR could be followed by negative results. The concentration of galactomannan was highly predictive for the time of survival (P < 0.0001). It was concluded that, in this model, quantitative galactomannan detection is superior to PCR in diagnosing and monitoring invasive pulmonary aspergillosis.

Animals↗

Detection of cell wall galactomannoprotein Afmp1p in culture supernatants of Aspergillus fumigatus and in sera of aspergillosis patients.

Mannoproteins are important and abundant structural components of fungal cell walls. The AFMP1 gene encodes a cell wall galactomannoprotein of Aspergillus fumigatus. In the present study, we show that Afmp1p is secreted into the cell culture supernatant at a level that can be detected by Western blotting. A sensitive enzyme-linked immunosorbent assay (ELISA) developed with antibodies against Afmp1p was capable of detecting this protein from the cell culture supernatant of A. fumigatus. The anti-Afmp1p antibody is specific since it fails to react with any protein from lysates of Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Penicillium marneffei, Candida albicans, Cryptococcus neoformans, Blastomyces dermatitidis, and Histoplasma capsulatum by Western blotting. In addition, this Afmp1p antigen-based ELISA is also specific for A. fumigatus since the cell culture supernatants of the other eight fungi gave negative results. Finally, a clinical evaluation of sera from invasive aspergillosis patients indicates that 8 of 15 (53%) patients are Afmp1p antigen test positive. Furthermore, an Afmp1p antibody test was performed with these serum specimens. The combined antibody and antigen tests for invasive aspergillosis carry a sensitivity of 86.7% (13 of 15). The specificities of the tests are high since none of the 138 control sera, including 100 from normal blood donors, 20 from patients with penicilliosis marneffei, 6 from patients with candidemia, 8 from patients with typhoid fever, and 4 from patients with melioidosis, was positive by either test. In conclusion, the combined Afmp1p antibody and antigen tests are highly sensitive and specific for A. fumigatus invasive aspergillosis.

Antibodies, Fungal↗

Detection of antibodies specific to an antigenic cell wall galactomannoprotein for serodiagnosis of Aspergillus fumigatus aspergillosis.

Aspergilloma and invasive aspergillosis are important opportunistic infections caused by Aspergillus species, among which Aspergillus fumigatus is the most common species associated with human disease. We developed an enzyme-linked immunosorbent assay (ELISA)-based antibody assay with Afmp1p, a purified recombinant antigenic cell wall galactomannoprotein of A. fumigatus. Evaluation of the test with guinea pig sera against A. fumigatus and other pathogenic fungi indicated that this assay was specific for A. fumigatus. Clinical evaluation revealed that the assay was 100% sensitive for patients with aspergilloma and 33.3% sensitive for patients with invasive aspergillosis. No false-positive results were found for serum samples from 80 healthy blood donors, 6 patients with typhoid fever, 4 patients with melioidosis, 20 patients with penicilliosis marneffei, 5 patients with candidiasis, and 4 patients with cryptococcosis, indicating a high specificity of the test. Thus, this ELISA-based test for the detection of anti-Afmp1p antibody can be of significant value as a diagnostic for aspergillosis.

Animals↗

Semiquantitative detection by real-time PCR of Aspergillus fumigatus in bronchoalveolar lavage fluids and tissue biopsy specimens from patients with invasive aspergillosis.

A real-time PCR method was developed and used to detect Aspergillus fumigatus mitochondrial DNA (mtDNA) in bronchoalveolar lavage (BAL) fluids and tissue biopsy specimens. The analytical sensitivity of the assay was one A. fumigatus conidium per reaction, and the assay was linear at least over 4 orders of magnitude above the detection limit. BAL fluids from 66 immunocompromised patients at risk of invasive pulmonary aspergillosis (IPA) and 33 immunocompetent controls and tissue biopsy specimens from 10 immunocompromised patients were analyzed. The results were related to the clinical diagnosis established according to recently published consensus criteria. A. fumigatus mtDNA positivity was encountered in 16 of 81 (20%) BAL fluid specimens from patients at risk and 1 of 33 (3%) specimens from immunocompetent controls. PCRs were positive in six of seven, two of four, and four of five of the patients with proven, probable, and possible IPA, respectively, as well as in four patients at risk but without any other evidence of IPA. With qualitative detection, the diagnostic sensitivity of PCR was 73%, specificity was 93%, and predictive values of positive (PPV) and negative (NPV) results were 73 and 95%, respectively. Using a threshold cycle of <35 as a limit for positive PCR, the specificity and PPV of PCR in the diagnosis of invasive aspergillosis were 100%, but its sensitivity was only 45% and NPV was 92%. PCR was positive in tissue biopsy specimens from all patients with invasive aspergillosis caused by A. fumigatus. Semiquantitative detection of A. fumigatus mtDNA in BAL fluid may be helpful in the diagnosis of IPA. PCR is well suited for the verification of the presence of A. fumigatus in tissue biopsy specimens.

Adolescent↗

Localised invasive sino-orbital aspergillosis: characteristic features.

BACKGROUND/AIM: To describe the characteristic constellation of historical, clinical, radiographic, and histopathological findings of localised invasive sino-orbital aspergillosis based on the authors' recent experience of four consecutive cases presenting over a 6 month period. Treatment and outcome are reviewed. METHODS: A case series of four patients with review of the English language literature. RESULTS: There have been 17 reported cases of invasive sino-orbital aspergillosis in healthy individuals over the past 33 years. The authors report four patients who presented during a 6 month period with persistent and significant pain followed by progressive ophthalmic signs-clinical histories reflecting the literature. Similar imaging findings were also noted: focal hypodense areas within apical infiltrates on contrasted computed tomography correspond to abscesses seen at surgery, and sinus obliteration or involvement of the adjacent sinus lining was noted on magnetic resonance imaging. Bone erosion (often focal) was also seen. There is frequently a delay in making the correct diagnosis, and often disease progression occurs despite treatment. CONCLUSIONS: The authors encountered four cases of invasive sino-orbital aspergillosis, three of which occurred in otherwise healthy individuals. The clinician must be aware of the characteristic presentation so that earlier diagnosis, management, and improved outcomes can be achieved.

Aged↗

CNS aspergillosis in organ transplantation: a clinicopathological study.

The clinical characteristics and neuropathological findings of 22 organ transplant recipients with CNS aspergillosis were reviewed. Thirteen patients had liver, six kidney, two heart and one had cluster transplants. The most frequent neurological symptoms were alteration of mental status (86%), seizures (41%) and focal neurological deficits (32%). Meningeal signs were less common (19%). Aspergillus spp invasion of the blood vessels with subsequent ischaemic or haemorrhagic infarcts, and solitary or multiple abscesses were the predominant neuropathological findings. The lungs were the probable portal of entry; however, isolated CNS aspergillosis was seen in two patients. Antemortem diagnosis of the infection was made in half of the patients. Concomitant diabetes mellitus was noted in 59% of the patients and bacterial or other severe infections in 86%. No specific clinical or pathological pattern could be identified among patients with different types of organ transplants. In addition CNS aspergillosis was preceded by organ rejection and the need for intense immunosuppression and retransplantation in the majority of the patients.

Adolescent↗

Invasive pulmonary aspergillosis: a rare presentation of non-Hodgkin's lymphoma.

We describe a patient with rapidly progressive pneumonia and a high level of serum lactate dehydrogenase, in whom postmortem study revealed the presence of a diffuse, small and large-cell multicentric non-Hodgkin's lymphoma, together with an invasive pulmonary aspergillosis. Aspergillosis is rare as a presenting feature of a lymphoproliferative disease; only one previous case has been reported to the best of our knowledge. Invasive aspergillosis and lymphoma should be considered in patients presenting with pneumonia and high level of lactate dehydrogenase.

Aged↗

Studies in chronic allergic bronchopulmonary aspergillosis. 1. Clinical and physiological findings.

This report outlines the clinical and physiological features in 50 asthmatic patients with chronic allergic bronchopulmonary aspergillosis in whom the diagnosis was made from 2 to 25 years ago (mean duration 10-9 years). From a questionnaire and analysis of the peak expiratory flow rate measurements it was found that they were worse in the winter months, corresponding to the maximal concentrations of Aspergillus fumigatus in the atmosphere. Nineteen patients reported daily sputum production of up to an eggcupful or more, and 24 had noticed sputum 'plugs' in the previous year. Reduction of vital capacity (VC) was found in 20 patients, of forced expiratory volume in one second (FEV1) in 38 patients, and of maximal expiratory flow at 50% VC breathing air (V50air) in 47 patients. Nine patients had significantly reduced gas transfer factor (DLCO). Significant improvement (more than 15%) in FEV1 after inhaled bronchodilator was shown by only 17 patients. There were statistically significant correlations between the degree of reduction in the physiological measurements of VC, FEV1, and V50air with the age of the patient at the time of the study and the later in life the diagnosis of aspergillosis was made, whereas the reduction in DLCO was also significantly related to the duration of aspergillosis. Prospective studies are needed for a proper assessment of any protective effect of treatment on the pathophysiological changes due to the disease over many years.

Adult↗

Studies in chronic allergic bronchopulmonary aspergillosis. 2. Radiological findings.

The characteristics and the incidence of changes in plain chest radiographs were analysed in detail in 50 asthmatic patients with chronic allergic bronchopulmonary aspergillosis in whom the diagnosis had been made from 2 to 25 years previously (mean duration 10-9 years). One thousand two hundred and forty-two chest radiographs, an average of about two per year per patient, were reviewed. Two hundred and sixty-seven acute episodes of transient shadows (a mean of 5-3 per patient), mostly homogeneous consolidations and band-like shadows, were recorded throughout the period of follow-up. Features of overinflation were present in 21 patients in the first radiograph and in 17 in the last, the decrease being attributed to the effects of permanent lung damage due to the allergic aspergillosis. Permanent changes, such as tubular and ring shadows, loss of vascular shadows, and lobar shrinkage were recorded in 41 patients at the start and in 49 at the end of the follow-up. The tubular shadows were found in 28 patients in the first radiograph and in 45 in the last, whereas ring shadows were seen in 18 and 30 patients respectively. Half of the episodes of consolidation and atelectasis left permanent changes, mainly ring shadows, as seen in the last radiograph. Of the band-like shadows, 32% were found to be followed by the subsequent appearance of tubular shadows. Statistically significant correlations were found between the duration of aspergillosis, the number of transient shadows throughout the period of follow-up, the extent of the permanent shadows, and the reduction of gas transfer factor at the time of the final follow-up.

Aspergillosis↗

Controlled trial of natamycin in the treatment of allergic bronchopulmonary aspergillosis.

Allergic bronchopulmonary aspergillosis often requires treatment with oral corticosteroids to control the host response to Aspergillus fumigatus. In a double blind study 25 patients with allergic bronchopulmonary aspergillosis taking maintenance oral corticosteroids were randomly allocated to receive 5 mg natamycin or placebo by nebuliser twice daily for one year. The primary aim of the study was to assess the steroid sparing potential of natamycin. Standardised reductions in corticosteroid dosage were therefore undertaken every five weeks, unless clinically contraindicated. Five patients were withdrawn in the first four months: two (1 natamycin, 1 placebo) died, two (1 natamycin, 1 placebo) had suspected drug reactions, and one (natamycin) was non-compliant. The pretreatment characteristics of the 20 patients (10 in each group) who completed the study were similar, 17 (9 natamycin, 8 placebo) having evidence of recent disease activity. At the end of the study prednisolone dose had been reduced by a similar amount in each group (median natamycin 2.25 mg, placebo 2.5 mg). Evidence of disease activity during the study year (transient shadowing on the chest radiograph, blood eosinophilia, or increases in antibodies to A fumigatus, or any combination of these) was observed in similar numbers of patients in each group (5 natamycin, 7 placebo). There was no evidence that natamycin conferred benefit on these patients with allergic bronchopulmonary aspergillosis.

Adult↗

Corticosteroid treatment as a risk factor for invasive aspergillosis in patients with lung disease.

Invasive pulmonary aspergillosis usually occurs in severely immunocompromised or neutropenic patients. Six patients with invasive aspergillosis are described whose only defence impairment was underlying lung disease and corticosteroid treatment. Cough, fever, and sputum production were the usual reasons for presentation and four patients developed the sepsis syndrome. Radiographic findings included de novo cavitation in three patients and rapid radiographic progression in four. Aspergillus species were isolated from respiratory secretions of all patients early in the course of the disease. Treatment was effective in only two patients and the subsequent progress of the others was consistent with a chronic necrotising process. Invasive pulmonary aspergillosis is uncommon in patients with respiratory diseases receiving corticosteroids, but should be considered when pneumonia and cavitary infiltrates occur.

Adult↗

Invasive aspergillosis of the airways: radiographic, CT, and pathologic findings.

PURPOSE: To assess the radiographic, computed tomographic (CT), and pathologic findings in invasive aspergillosis of the airways. MATERIALS AND METHODS: The study included nine consecutive patients (aged 17-65 years [median, 49 years]) with pathologically proved invasive aspergillosis of the airways. All nine underwent chest radiography and seven underwent CT within 3 days of diagnosis. RESULTS: The radiographic findings include normal parenchyma (n = 1), unilateral consolidation (n = 1), bilateral consolidation (n = 5), and ill-defined nodules (n = 2). The main findings at CT included lobar consolidation (n = 1), bilateral predominantly peribronchial consolidation (n = 3), ground-glass attenuation (n = 1), and centrilobular nodules less than 5 mm in diameter (n = 2). At pathologic examination, the peribronchial infiltrates represented bronchopneumonia and the nodules represented Aspergillus bronchiolitis with a variable degree of peribronchiolar organizing pneumonia and hemorrhage. CONCLUSION: Radiographic findings of invasive aspergillosis of the airways consist of consolidation or ill-defined nodules. At CT, the consolidation can be seen to be peribronchial and the nodules centrilobular.

Adolescent↗

Invasive pulmonary aspergillosis in AIDS: radiographic, CT, and pathologic findings.

PURPOSE: To review the radiographic and computed tomographic (CT) manifestations of invasive pulmonary aspergillosis and to correlate the imaging and pathologic findings in patients with acquired immunodeficiency syndrome (AIDS). MATERIALS AND METHODS: Chest radiographs, CT scans, and pathologic specimens were reviewed retrospectively in 10 AIDS patients with proved invasive pulmonary aspergillosis. RESULTS: The most common radiographic finding was the presence of thick-walled cavitary lesions. Less common findings included nodules, consolidation, and pleural effusion. CT depicted more nodules and cavities than did radiography. The predominant pathologic abnormalities consisted of tissue invasion and abscess formation and angioinvasion with or without infarction. All patients had infection with Aspergillus fumigatus as well as other pathogens, the most common being cytomegalovirus and Pseudomonas aeruginosa. CONCLUSION: Thick-walled cavitary lesions are the most common radiologic manifestation of invasive pulmonary aspergillosis in AIDS. The findings are more numerous and better defined on CT scans. The radiologic findings reflect a spectrum of pathologic abnormalities.

AIDS-Related Opportunistic Infections↗

Invasive sphenoidal aspergillosis: successful treatment with sphenoidotomy and voriconazole.

Treatment of invasive sphenoidal aspergillosis is surgical, followed by antifungal therapy, mostly amphotericin B. To optimize the adjuvant antifungal treatment, which is often limited by severe side effects, the new triazole antifungal agent voriconazole with broad coverage of fungal pathogens including Aspergillus was investigated in a study of 4 patients with clinical, radiological and histological signs of invasive sphenoidal aspergillosis. They first underwent endoscopic sphenoidotomy with drainage and extraction of the fungal mass. Postoperatively, 2 patients were immediately treated with voriconazole. Two patients initially received amphotericin B; but this treatment had to be stopped because of acute renal toxicity. Finally, all patients were treated orally with 200 mg voriconazole twice a day for 12-14 weeks. After this combined treatment all patients were asymptomatic and there were no endoscopic or radiological signs of residual fungal disease. The only side effects were nausea in one and transient visual disturbances in 2 other patients. In the 4 patients presented and treated, voriconazole was shown to be effective and less toxic than amphotericin B in adjuvant treatment of invasive sphenoidal aspergillosis.

Adult↗