| Aspergillosis |
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Sang J. Kim |
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Department of Bacteriology, Korean Institute of Tuberculosis Korean National Tuberculosis Association |
| Aspergillus 증 |
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김상재 |
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| Abstract |
INTRODUCTION
Aspergilli are ubiquitous in the nature and their airborne spores are frequently inhaled into the human respiratory tracts in which they they can cause the various forms of aspergillosis in susceptible individuals. A lthough aspergilli can produce otomycosis and rarely mycetoma in man, the most common and important forms of the disease, they cause, are pulmonary origined, allergic bronchopulmonar y aspergillosis, aspergilloma and invasive aspergillosis. Definitive diagnosis of as pergillosis can be made by the demonstration of the fungus in affected tissue by culture and histopathological examinations. However, in many cases, such examinations are not available, and show a very poor prodactivity. The various immanological procedares, therefore, are very useful adjuncts in establishing the diagnosis of asperg illosis.
ALLERGIC BRONCHOPULMONARY ASPERGILLOSIS (A BPA)
Eighty percent or more of extrinsic asthma wi t h pulmonary eosinophilia are related to aspergillus hypersensitivity in England. The spores of aspergilli can stimula te the production of IgE antibody particularly in atopic subjects, causing extrinsic, type I asthma upon subsequent introduction of aspergillus antigens. The production of precipitating antibody as well as IgE antibody leads to the development of allergic bronchopulmonary aspergillosis. Asthmatics are usually associated with fleeting nonsegmental lung shadow which is eosinophilic pneumonia, or with rather more persistant segmental shadows. Both types may be associated with febrile bouts -and plugs. More than one third of such patients tend to hav e chronic asthma, usu ally extrinsic. After a few episodes, some patients may go into a remission and may have no more episodes. Some go into a remission for a period of months or even years and then appear to relapse. Recurrences eventually tend to cause irreversible lung damage. Aspergilloma, apical or midzone fibrosis, or bronchiectasis occur in affected areas. Some of the patients develop progressive airway obstruction and ultimately go into respiratory failure. Non-atopic subjects may develop precipitins against aspergiIIus antigens upon heavy exposure, with the production of reactions in the bronchi demonstrable as extrinsic. late asthmatic reactions which appear after several hours. or reactions in the peripherallung tissuse. extrinsic allergic alveolitis.
Diagnosis of ABPA is usually made on the basis of the skin test. serological and mycological examinations with clinical and radiological informations. Positive sputum culture significantly correlate with ABPA but it is indistinguishable from contamination. Of the asthmatics. 7.8-8.3% produced positive sputm culture and this figure is significantly higher than 1.5-3.4% of the patients with other lung diseases. Of the patients with ABPA.
82.6% produce culture positive and 91.3% of culture positives produce positive precipitin.
The demonstration of antibody against aspergillus antigens in the sera has virtually diagnostic value. Levels of total IgE, specific IgE and precipitating antibody vary according to the disease activity. A rising level of IgE may portend a fIare, while a stable or declining value implies disease remission. By radioimmunoprecipitation (RIP) assay total IgE level of over 82% of the patients with ABPA exceeded those of normal or other lung diseases and 91% show higher IgE level specific to aspergillus antigens than those of normal persons on radioallergosorbent tests (RAST).
Positive IgE level will be nearly 100% if the serum specimens are collected during active disease state.
Ammonium sulfate tests (AST) with radiolabeled antigen showed 96% positive rate and the indirect fluorescent antibody tests produced 80% positives in ABPA. Fifty to 70% of ABPA produce positive precipitins against aspergillus antigens on double immunodiffusion tests (DID). However sera from the patients with asthma only react poorly to aspergillus antigens, showing 9-13% positive resusts. while sera from the patients with asthma plus pulmonary eosinophilia show 63-71% positive rate. If the serum specimens are concentrated this figure will be well over 90%. Counterimmunoelectrophoresis (CIE) can double or triple the positive rate obtained by DID in the subjects with asthma only.
This technique certainly has advantages such as high yield of positives .and fast result over DID. Many other procedures are available but either not fully evaluated or have some disadvantages. Skin tests are virtually diagnostic and 100% of ABP A respond type 1 immediate reaction upon intradermal injection of aspergillus antigens. Many patients also show late reactions. Twenty five percent of apparently normal subjects also show positive immediate reaction thus skin test alone can not verify positive diagnosis of ABPA. Corticosteroid therapy may suppress late skin reaction and precipitin reaction.
therefore serum collection should be avoided during the therapy of this drug.
Lymphocytes from the patients with ABPA are highly reactive to aspergilius antigens.
while the cells from aspergilloma patients are poorly reactive. This procedure needs further evaluation.
ASPERGILLOMA (M)
Aspergilloma is a compact mass of mycelium and cell debris (1-5cm in diameter) formed in ectatic bronchus or in the cavity without invasion of the lung parenchyma.
About 15% of tuberculosis patients with open healed cavities had aspergillus fungus ball in England in 1970 survey. Fungus ball can be easily seen by radiological examination.
Although a definitive diagnosis can be made by isolating the fungus from the resected
fungus balls, the radiological, mycological and serological data can establish a satisfactory diagnosis before surgery. In many cases the ftingtis is continuously isolated from spitum specimen, btit we have to keep in mind that contamination is comrribn. Some fungus balls are apparently dead therefore cultivation of the ftingus is not possible. Demonstration of antibodies specific to aspergillus antigens is very useful to the diagnosis of this type of aspergillosis. If the sera were collected before the removal of fungus ball, nearly 100% of the cases produce antibodies against aspergillus antigens, demonstrable by any type of conventional serologic procedures. After removal of fungus ball antibody level wanes rapidly.
INVASIVE ASPERGILLOSIS
Invasive aspergillosis is a most important type of aspergillosis and usually occur in immunologically committed individuals who have reticulo-endothelial impairment or who are on immunosuppressive treatment, Early diagnosis is so difficult in many cases that the prognosis is bad. Primary granulomatous lesions are formed in the lung with invasion of the fungal hyphae into lung parench yma causing pyogenic pneumonitis. Some lesions may heal with calcification to form coin lesions, but in most cases necrosis progress and for macavitation. Acute necrotizing pyogenic abscess spread beyond cavity . Vascular involvement occur causing thrombotic angiitis and the disease eventually spread hematogenously to other organs. Laboratory diagnosis of invasive aspergillosis solely depends on microscopical, cultural and histopathological examinations. The sputum may contain mycelial elements when the chest radiograph show extensive shadowing. In some cases precipitins to the species of Asþergillus involved are detectable in the serum.
Serological diagnosis, however, is not possible in the cases of wide spread invasive aspergillosis because they do not produce a measurable antibody to aspergiIlus antigens.
Demonstration of fungal components instead of antibody in the sera from the patients with wide spread invasive aspergillosis is in developmental stage in several laboratories.
CHARACTERIZATION OF ANTIGENS
The figures obtained by every serological procedures are not consistent between the different laboratories, mainly due to the absence cf standardized antigen preparations.
The multiplicity of antigens of varying specificity and potency provide a difficulty for the production of standardized mater ials. Variation of composition of reactive components in the preparations result from the age of culture, the conditions for growth such as media, position of growth and temperature, the qualitative and quantitative antigenic differences of strains and species of aspergilli, and the extraction procedures. The commonly used crude extracts contain the diffcring amount of nonantigenic materials.
The different individual patterns of antibody response in exposed subjects also contribute difficulty to the preparation of standard materials. The suitable antigen preparations must be produced using a battery of appropriate strains of the fungus under reproducible procedures and must be devoid of nonantigenic materials and some interfering substances such as C-polysaccharides reactive with CRP and intragcneric cross reacting substances if any .
Recently we have made an effort to produce the preparations which could meet such requirements, to the utmost, using a strain of A , fumigats NIH 5233. The fungus was found to grow well in completely synthetic asparagine glycerol medium. Four-day oid mycelia were totally disrupted by vigorous mechanical shaking with 0. 45mm glass beads.
Such mechanicall y produced extract contained at lea st 52 precipita t ing antigens. The number of protein and carbohydrate components was maximal in 4-day old organisms and decreased in older cultures. Conversely, the number of components in culture filtrates increased with longer incubation periods, but was only approximately one-half that produced by the 4-day-old mycelial extracts (ME). Mycelial extracts were then fractionated by precipitation with ammonium sulfate, by ion-exchange chromatography, and by gel filtration. The fractions were characterized chemically, by analytic polyacrylamide gel electrophoresis, and by test for serologic and biologic activity. The distribution of the antigens in the fractions was determined by double diffusion in geI and fused-rocket immunoelectrophoresis (FRIEP).
Activity greater than that of the parent materials serologically, in guinea pig skin -tests, and in vitro lymphocyte transformation was observed with one fraction (APIFB) -that was found in the 75% ammonium sulfate precipitable portion and was eluted early by gel filtration. Another ammonium sulfate-precipitable components (APIFA) and one that was not precipitable at 75% saturation of ammonium sulfate, but was firmly bound by ion exchange columns, were found to have activity equal or more to unfractionated ME. These fractions were also active by RAST for IgE in ABPA patients. A carbohydrate componts (ASII) comprising almost 40% of ME was found to be devoid of any immunologic activity. CommerciaIly available preparations, ME and the fractions of A.
fumigatus and extracts of 5 other aspergilli were evaluated in a blind manner for their abilities to detect antibody in 66 serum specimens from patients with aspergiIloma, a Ilergic bronchopulmonary and invasive aspergillosis, and unconfirmed aspergiIlosis Although qualitative differences in antigen composition could be'd etected, commercial preparations compared favorably with the best experimental extracts in detecting positive specimens. Extracts from young, actively-growing myceliums were most effective and produced the largest number of precipitin bands. In contrast to aIl unfractionated preparations, several fractions that do not react with CRP of sera, yet were as effective as the best preparations in detecting sera positive for Aspergillus.
Seventy-three to 89% of the numbers of antigens detected between strains of A. fumigatus, A. fumigatus var. ellipticus and A. phialiseptus were shared. The degree of sharing between antigens of A. flavus, A. fumigatus series, and A. niger was much lower and ranged from 19 to 35%.
Enzyme-linked immunoassay (ELISA) offer a number of advantanges includig exquisite sensitivity approaching that seen with procedures using radiolabeled reagents, simplicity, and quantitation. In several serum specimens in which no precipitins could be detected titers of up to 1 : 810 were recorded. The procedure should be a more useful adjunct in the diagnosis of aspergillosis than currently available routine tests. Poorly reactive polysaccharide components (ASII) have been detected in the sera from the rabbits with established invasive aspergillosis by radioimmunoassay or by a reverse ELISA inhibition technique. Development of sensitive technique in detecting polysaccharides liberated from the fungus in the sera seems to be promising in the diagnosis of invasive form of aspergillosis.
CONCLUSION
Aspergilli are ubiquitous in the nature, causing the various forms of aspergillosis. The diagnosis of aspergillosis largely depends on the mycological and immunological tests with appropriate specimens and reagents.
Different values were apparent in the mycological and serological tests in various forms of aspergillosis. Precipitation tests are most valuable in the diagnosis of aspergilloma.
Counterimmunoelectrophoresis is a most sensitive technique in detecting precipitating antibodies to aspergillus antigens and this procedure can be suited for the diagnosis of allergic form of aspergillosis in addition to skin test. Sensitivity of serological procedures largely depend on the nature of the antigen preparations used. Most reactive components distributed maximally in the extracts from young actively growing mycelium. Proteinic substances separated from the mycelial extract can exclude poorly antigenic and Cpolysaccharides and interspecific and intergeneric cross reactive substances. |
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