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AspergillosisAspergillosis is a spectrum of diseases of humans and animals caused bymembers of the genus Aspergillus. These include(1) mycotoxicosis due to ingestion of contaminated foods;(2) Allergy and sequelae to the presence of conidia or transient growth of the organism in body orifices;(3) Colonization without extension in preformed cavities and debilitated tissues;(4) Invasive, inflammatory, granulomatous, necrotizing disease of lungs, and other organs;(5) And rarely, systemic and fatal disseminated disease. The type of disease and severity depends upon the physiologic state of the host and the species of Aspergillus involved.Distribution: World-wide.Aetiological Agents: Aspergillus fumigatus, A. flavus, A. niger, A. nidulans &A. terreus.
Aspergillum: Latin aspergere, "to scatter" HISTORY: 1729--Micheli noted pattern of conidial head of aspergillus with spore heads radiating from central structure resembling Aspergillum (perforated globe used to sprinkle holy water) 1809--Link named Aspergillus flavus 1842--John higes Bennett described Aspergillosis(in lung) 1965—Raper & Fennel: 151 spp, 18 groups.
MYCOLOGY: Aspergillus spp are saprophytic moulds ( decaying matter world wide) Out of 185 spp only 20 causes human disease 3 out of 20 are consistently & regularly encountered as etiological agent in 95% cases A.flavus, A.fumigatus, A.niger. Other common spp: A.terreus, A.glaucus,A.nidulans, A.oryzae & A.clavatus.
Aspergillus produces conidia in basipetal manner which results in chain of asexual conidia where youngest conidium is at base and oldest at tip of chain.(conidiogenous cell called phialide) Conidiophores: Hypha like structure that enlarges at its apex to form swollen vesicle Foot cell: base of conidiophores, where it originates from parent vegetative hyphae.
Phialides may arise directly from vesicle in Uniseriate/ from metulae as in biseriate In Some spp phialides cover entire surface of vesicle, in others it may cover ½ or ¾ of vesicle, may vary in colour in different spp.
PATHOGENESIS & PATHOLOGY The spores of opportunistic fungus may bypass upper respiratory tract defenses and reach distal bronchial airway and pulmonary alveoli because of their smaller size <5µm, and aerodynamic properties. Host relies primarily upon phagocytic cells to remove spores, if phagocytic cell are unable to clear spores quickly, they germinate & colonize alveoli. The principal phagocytic cells responsible for maintaining sterility of lower respiratory tract are Pulmonary alveolar macrophages . Although other cells including polymorphonuclear
Clinical Manifestations: Pulmonary Aspergillosis: Allergic, Aspergilloma & Invasive Aspergillosis Disseminated Aspergillosis: cerebral,renal,heart(endocarditis,myocarditis), bone(osteomyelitis) , gastrointestinal, ocular lesions(mycotic keratitis,endophthalmitis & orbital aspergilloma) either by dissemination/ following local trauma/surgery. Aspergillosis of PNS:1. Non-invasive aspergilloma: normal immune pts(chr sinusitis) 2. Invasive form: immunosuppressed pts. Cutaneous Aspergillosis: cutaneous Aspergillosis is rare manifestation that is usually seen in immunosuppressed pts. However cases of primary cutaneous aspergillosis also occurs, usually as a result of trauma or colonisation.lesions menifest as erythematous papules or macules with central progressive necrosis. Burns, Post surgical wound, I.V insertion sites, Otomycosis, Exogenous endophthalmitis, Allergic fungal sinusitis
1. Pulmonary Aspergillosis: including allergic, aspergilloma and invasive aspergillosis. The clinical manifestations of pulmonary aspergillosis are many, ranging from harmless saprophytica)Allergic aspergillosis invasive disease. clinical entities colonisation to acute : is a continuum ofranging from extrinsic asthma to extrinsic allergic alveolitis toallergic bronchopulmonary aspergillosis(ABPA)(hypersensitivity pneumonitis) caused by the inhalation ofAspergillus conidia. Features include asthma, intermittent orpersistent pulmonary infiltrates, peripheraleosinophilia, positive skin test to Aspergillus antigenicextracts, positive immunodiffusion precipitin tests for antibodyto Aspergillus, elevated total IgE, and elevated specific IgE
b)Non-invasive aspergillosis or aspergilloma (fungus ball), is caused by the saprophytic colonisation of pre- formed cavities, usually secondary to tuberculosis or sarcoidosis. Features often include hemoptysis with blood stained sputum, positive immunodiffusion precipitin tests for antibody to Aspergillus, and elevated specific IgEAspergilloma found at post-mortem in themany cases with leukaemia. Note against Aspergillus. However, lung of a child arefungus ball occupying and are usually found by routine chest asymptomatic cavity. roentenogram.
c)Acute invasive pulmonary aspergillosis. Predisposing factors include prolonged neutropenia, especially in leukemia patients or in bone marrow transplant recipients, corticosteroid therapy, cytotoxic chemotherapy and to a lesser extent patients with AIDS or chronic granulomatous disease. Clinical symptoms may mimic acute bacterial pneumonia and include fever, cough, pleuritic pain, with hemorrhagic infarction or a nectrotising bronchopneumonia. The typical patient is granulocytopenic and receiving broad-spectrum antibiotics for unexplained fever. Radiological features may be non- specific and tests for serum antibody precipitins are also usually negative. Clinical recognition is essential as this is the
2. Disseminated Aspergillosis: Hematogenous dissemination to other visceral organs may occur, especially in patients with severe immunosuppression or intravenous drug addiction. Abscesses may occur in the brain (cerebral aspergillosis), kidney (renal aspergillosis), heart, (endocarditis, myocarditis), bone (osteomyelitis), and gastrointestinal tract. Ocular lesions (mycotic keratitis, endophthalmitis and orbital aspergilloma) may also occur, either as a result of dissemination or following local trauma or surgery. 3. Aspergillosis of the paranasal sinuses: Two types of paranasal sinus aspergillosis are generally recognised. (1) A non-invasive "aspergilloma" form, primarily seen in non- immunosuppressed individuals. Predisposing factors include a history of chronic sinusitis and poorly draining sinuses with
d)Chronic narcotising aspergillosis is anindolent, slowly progressive, "semi-invasive" form ofinfection seen in mildly immunosuppressedpatients, especially those with a previous history of lungdisease. Diabetes mellitus, sarcoidosis and treatmentwith low-dose glucocorticoids may be other predisposingfactors. Common symptoms include fever, cough and4. Cutaneous Aspergillosis:sputum production; positive serum antibody precipitins Cutaneous aspergillosis is a rare manifestation that ismay also be detected. usually a result of dissemination from primary pulmonary infection in the immunosuppressed patient. However, cases of primary cutaneous aspergillosis also occur, usually as a result of trauma or colonisation. Lesions manifest as erythematous papules or macules
Differential Diagnosis It has to be differentiated from Deep mycotic infection particularly with immunocompramised pts. Cutaneous aspergillosis from Ecthyma gangrenosum (due to Pseudomonas, candida, herpes simplex virus inf , zygomycosis, cryptococcosis, phaeohypomycosis) Aspergillus granuloma from granulomatous diseases as well as neoplasia.
Diagnosis of Aspergillosis has been primarily confirmed using conventional means of culturing causative fungal organism from clinical material on SDA. Direct Microscopy & culture both in combination increase the diagnostic yeild. In Pts with +ve fungal cultures, Aspergillus spp are 2nd most common isolate after Candida spp but +ve culture alone may not indicate pathogenic process as Aspergillus spp exsists
For the diagnosis of bronchopulmonary infectionmorning sputum or BAL (bronchioalveolar lavarge) shouldbe collected in a sterile container.For systemic mycosis, pus swab from an ulcer oraspiration from unruptured abscess, or biopsy duringsurgical operation are collected by strict aseptictechnique.For urinary tract infection, mid-stream urine samplesare collected into a wide mouth sterile container.
For cerebrospinal infections, a lumbar puncture should de performed to collect CSF into sterile test tubes.For Pleural and Peritoneal Effusions, a sample is collected by needle aspiration into sterile container.Eye-corneal scrapings from the base and margins of the ulcer. -aspiration.
Direct microscopy Direct examination of clinical specimen is done with 10% KOH for demonstration of hyline septate hyphae of Aspergillus spp, septate hyphae are 3-6µm in diameter with dichotomous branching. Calcofluor white stain, fluorescent-antibody techniques also demonstrate septate hyphae. Biopsy material is kept in tube KOH for overnight at 37c and slide is prepared to see for septate hyphae. HPE of biopsy material is stained with H&E, GMS,PAS, shows acute angle branching hyaline septate hyphae with neutrophilic to granulomatous response.hyphae exhibits characterstic dichotomous acute-angle branching,often giving finger like apperance to branching elements. Chr infections may exhibit atypical hyphal features such as swellings (12µm in dia) & or absence of septa as seen in fungal balls.
DICHOTOMOUS BRANCHING Grocott’s methenamine silver (GMS) stained tissue section of lung showing dichotomously branched, septate hyphae of Aspergillus fumigatus.
033 Grocott’s methenamine silver Grocott’s methenamine silver (GMS)(GMS) stained tissue section of stained tissue sections showinglung showing fungal balls of Aspergillus fumigatus in lung tissue, notehyphae of Aspergillus fumigatus. conidial heads forming in an alveolus.
036Grocott’s methenamine silver (GMS) stained tissue sections showingAspergillus fumigatus in lung tissue, note conidial heads forming in
FUNGAL CULTURE Pathogenic Aspergillus spp generally grows easily & relatively quickly on routine mycological &bacteriological media . Clinical material inoculated on to SDA with antidiotics (without Cyclohexamide) at 25c & 37c. Culture examined daily during 1st week, twice a week for further 4weeks before considering negative. Sub-culture of an isolate to Czapek Dox agar & 2% malt extractagar with incubation at 25c allows identification of most aspergilli using std monographs and taxonomic keys. Potato dextrose agar is particularly useful for induction of Sporulation. +ve urine culture implies metastatic abscesses in kidney and are diagnostic of invasive Aspergillosis. Aspergillus spp seldomly recovered - blood, csf, urine.(blood also +ve in endocarditis pts)
Aspergillus fumigatusSDA:Colonies are velvety/powdery at first, turning to smoky-green. Reverse iswhite to tan.
037Aspergillus fumigatus on Czapek dox agar showing typical blue-green surface pigmentationwith a suede-like surface consisting of a dense felt of conidiophores.
038. Microscopic morphology of Aspergillus fumigatus showing typical columnar, uniseriate conidial heads.Conidiophores are short, smooth-walled and have conical shaped terminal vesicles, which support asingle row of phialides on the upper two thirds of the vesicle. Conidia are produced in basipetalsuccession forming long chains (slide 1), however, during preparation of slides the conidial chains areusually disrupted giving the more typical microscopic appearance seen in slide 2. Conidia are globose tosubglobose, green and rough-walled to echinulate.
Microscopic morphology of Aspergillus fumigatus showing typical columnar, uniseriate conidial heads.Conidiophores are short, smooth-walled and have conical shaped terminal vesicles, which support a single row ofphialides on the upper two thirds of the vesicle. Conidia are produced in basipetal succession forming longchains, however, during preparation of slides the conidial chains are usually disrupted giving the more typicalmicroscopic appearance .
040Aspergillus niger on Czapek dox agar. Colonies consist of a compact white or yellowbasal felt covered by a dense layer of dark-brown to black conidial heads.
041Microscopic morphology of Aspergillus niger showing large, globose, dark brownconidial heads, which become radiate, tending to split into several loose columns withage. Conidiophores are smooth-walled, hyaline or turning dark towards the vesicle.Conidial heads are biseriate with the phialides borne on brown, often septate metulae.Conidia are globose to subglobose, dark brown to black and rough-walled.
Aspergillus flavus on Czapek dox042 agar. Colonies are granular, flat, often with radial grooves, yellow at first but quickly becoming bright to dark yellow-green with age. SDA: colonies are velvety, yellow to green or brown. Reverse is golden to red brown.
Microscopic morphology of Aspergillus flavus.043 Conidial heads are typically radiate, later splitting to form loose columns, biseriate but having some heads with phialides borne directly on the vesicle. Conidiophores are hyaline and coarsely roughened, often more noticeable near the vesicle. Conidia are globose to subglobose, pale green and conspicuously echinulate. Some strains produce brownish sclerotia.
Aspergillus nidulansAspergillus nidulans on Czapek dox agar Microscopic morphology of Aspergillus nidulans.showing typical plain green colony with dark Conidial heads are short columnar and biseriate.red-brown cleistothecia developing within and Conidiophores are usually short, brownish andupon the conidial layer. Reverse may be olive to smooth-walled. Conidia are globose and rough-drab-grey or purple-brown. walled
Aspergillus terreus Conidial head of Aspergillus terreus.Conidial heads areAspergillus terreus on Czapek dox agar compact, columnar and biseriate. Conidiophores areshowing typical suede-like cinnamon- hyaline and smooth-walled. Conidia are globose tobuff to sand brown colonies. Reverse ellipsoidal, hyaline to slightly yellow and smooth-walled.yellow to deep dirty brown.
Summary of identificationSpecies morphology Phialides Color of conidiaA.Fumigat Velvety/powdery turning Uniseriate cover Grey,green, blue-us to smoky green,reverse upper ½ vesicle green white-tanA.Flavus Yellow to green/ brown Uniseriate/Biseriat Yellow to green velvety colonies,reverse ecovers entire golden to red brown vesiclesA.Niger Wooly white to yellow ten Biseriate covers Black turns to dark brown to entire vesicles blackA.Terreus Velvety cinnomon brown Biseriate, Orange to brown compact columnarA.nidulans Biseriate Dark green
Immunodiagnosis Immunological tests have been used as important tools in diagnosis of various clinical forms of aspergillosis. In Aspergillomma pts demonstrate precipitating IgG antibodies. In Allergic bronchopulomonary aspergillosis for diagnostic criteria includes +ve skin test reactions to Aspergillus Ags & elevated levels IgE & IgG precipitating Abs to Aspergillus Serological tests Immuno diffusion spp in serum. Indirect immunofluroscence immunoelectrophoresis ELISA Enzyme linked immunofiltration assay Immunobloting
Immunodiffusion test showing 048precipitins against Aspergillus.
Detection of Antibody = Immunodiffusion, BALISA(biotin-avidin amplification sys)Detection of Antigen = Latexagglutination, RIA,ELISA, BALISAMolecular techniques = DNAsequencing, PCR(realtimePCR),DNA probeMolecular typing = analysis of genomicDNA(mtDNA,rDNA),RFLPDetection of fungal metabolites =G-test, D-ELISA: Aspergillus galactomannan (content of cell wall) is used as indicatormannitol as marker diagnosis.of invasive aspergillosis for earlySkin tests = 0.1ml Ag(1000PNU/ml aspergillin) results: Type I -Hypersensitivity- erythema &
DETECTION OF METABOLITES:G-Test:Recently developed G-test by Japanese workers detects circulatingâ-(1,3)-D-glucan with use of modification of litmulus assay for endotoxins & has sensitivity of ~20pg/mlG-factor is horse-shoe crab coagulation factorThis test can confirm invasive mycosis(aspergillosis) but does not distinguish between Spp of Candida and aspergillus or other fungus.High concentration of D-mannitol, fungal metabolite recently found in serum of rats with experimentally
Antifungal susceptibility disk test showing the in vitro activity of voriconazole againstAspergillus fumigatus with Candida krusei as a control.
Medical Mycology is a Iceberg Thank youReferences:1. Practical Laboratory Mycology – E.Koneman(2nd edn)2. Color Atlas of Diagnostic Microbiology- luis,marie,ellen3. Text book of Diagnostic Microbiology- Murry(ASM)4. Gabrino J Aspergillosis (orphanet Encyclopedia 2004).5. Textbook of Medical Mycology- J.Chandra(3rd edn)