ASPERGILLOSIS,ASPERGILLUS fumigatus,flavus,niger,mycotoxicosis

1. Dr.Praveen kumar.V. Doddamani

2. Aspergillosis
Aspergillosis is a spectrum of diseases of humans and animals caused by
members 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.

3. 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.

4.  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.

5.  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.

6.  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.

7. A.fumigatus A.versicolor A.niger

8. 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

9. Invasive aspergillosis 1. PULMONARY ASPERGILLOSIS
2. CNS ASPERGILLOSIS
3. SINO-NASAL ASPERGILLOSIS
4. OSTEOMYELITIS
5. ENDOPHTHALMITIS
 Sinuses & 6.
7.
ENDOCARDITIS
RENAL ASPERGILLOSIS
8. CUTANEOUS ASPERGILLOSIS
lungs
Pulmonary Aspergilloma Pre-exisiting lung cavity
colonization Sinuses & lungs
Allergic Bronchopulmonary Sinuses & lungs
aspergillosis
others CUTANEOUS ASPERGILLOSIS
Burns , Post surgical wound, I.V insertion
sites
Otomycosis, Exogenous endophthalmitis,
Allergic fungal sinusitis

10. 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

11.  1. Pulmonary Aspergillosis: including
allergic, aspergilloma and invasive aspergillosis.
The clinical manifestations of pulmonary aspergillosis
are many, ranging from harmless saprophytic
a)Allergic aspergillosis invasive disease. clinical entities
colonisation to acute : is a continuum of
ranging from extrinsic asthma to extrinsic allergic alveolitis to
allergic bronchopulmonary aspergillosis(ABPA)
(hypersensitivity pneumonitis) caused by the inhalation of
Aspergillus conidia. Features include asthma, intermittent or
persistent pulmonary infiltrates, peripheral
eosinophilia, positive skin test to Aspergillus antigenic
extracts, positive immunodiffusion precipitin tests for antibody
to Aspergillus, elevated total IgE, and elevated specific IgE

12.  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 IgE
Aspergilloma found at post-mortem in themany cases with leukaemia. Note
against Aspergillus. However, lung of a child are
fungus ball occupying and are usually found by routine chest
asymptomatic cavity.
roentenogram.

13. Cavity wall

14.  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

15.  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

16. d)Chronic narcotising aspergillosis is an
indolent, slowly progressive, "semi-invasive" form of
infection seen in mildly immunosuppressed
patients, especially those with a previous history of lung
disease. Diabetes mellitus, sarcoidosis and treatment
with low-dose glucocorticoids may be other predisposing
factors. Common symptoms include fever, cough and
4. Cutaneous Aspergillosis:
sputum production; positive serum antibody precipitins
 Cutaneous aspergillosis is a rare manifestation that is
may 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

17. 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.

18. LABORATORAY  Mycological Diagnosis:
DIAGNOSIS
1. Collection specimen
2. Direct microscopy
3. Culture & LPCB
4. Identification
 Histopathological Diagnosis,
Serological Diagnosis. & Skin
tests
 Molecular methods.

19.  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

20. For the diagnosis of bronchopulmonary infection
morning sputum or BAL (bronchioalveolar lavarge) should
be collected in a sterile container.
For systemic mycosis, pus swab from an ulcer or
aspiration from unruptured abscess, or biopsy during
surgical operation are collected by strict aseptic
technique.
For urinary tract infection, mid-stream urine samples
are collected into a wide mouth sterile container.

21. 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.

22. 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.

24. DICHOTOMOUS BRANCHING
Grocott’s methenamine silver (GMS) stained
tissue section of lung showing dichotomously
branched, septate hyphae of Aspergillus
fumigatus.

25. 033
Grocott’s methenamine silver Grocott’s methenamine silver (GMS)
(GMS) stained tissue section of stained tissue sections showing
lung showing fungal balls of Aspergillus fumigatus in lung tissue, note
hyphae of Aspergillus fumigatus. conidial heads forming in an alveolus.

26. 036
Grocott’s methenamine silver (GMS) stained tissue sections showing
Aspergillus fumigatus in lung tissue, note conidial heads forming in

27. 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)

28. Aspergillus fumigatus
SDA:Colonies are velvety/powdery at first, turning to smoky-green. Reverse is
white to tan.

29. 037
Aspergillus fumigatus on Czapek dox agar showing typical blue-green surface pigmentation
with a suede-like surface consisting of a dense felt of conidiophores.

30. 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 a
single row of phialides on the upper two thirds of the vesicle. Conidia are produced in basipetal
succession forming long chains (slide 1), however, during preparation of slides the conidial chains are
usually disrupted giving the more typical microscopic appearance seen in slide 2. Conidia are globose to
subglobose, green and rough-walled to echinulate.

31. 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 of
phialides on the upper two thirds of the vesicle. Conidia are produced in basipetal succession forming long
chains, however, during preparation of slides the conidial chains are usually disrupted giving the more typical
microscopic appearance .

33. Aspergillus niger

34. 040
Aspergillus niger on Czapek dox agar. Colonies consist of a compact white or yellow
basal felt covered by a dense layer of dark-brown to black conidial heads.

35. 041
Microscopic morphology of Aspergillus niger showing large, globose, dark brown
conidial heads, which become radiate, tending to split into several loose columns with
age. 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.

36. Aspergillus flavus

37. Aspergillus flavus on Czapek dox
042 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.

38. 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.

40. Aspergillus nidulans
Aspergillus 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 and
upon the conidial layer. Reverse may be olive to smooth-walled. Conidia are globose and rough-
drab-grey or purple-brown. walled

41. Aspergillus terreus
Conidial head of Aspergillus terreus.Conidial heads are
Aspergillus terreus on Czapek dox agar
compact, columnar and biseriate. Conidiophores are
showing typical suede-like cinnamon- hyaline and smooth-walled. Conidia are globose to
buff to sand brown colonies. Reverse ellipsoidal, hyaline to slightly yellow and smooth-walled.
yellow to deep dirty brown.

44. Summary of identification
Species morphology Phialides Color of conidia
A.Fumigat Velvety/powdery turning Uniseriate cover Grey,green, blue-
us to smoky green,reverse upper ½ vesicle green
white-tan
A.Flavus Yellow to green/ brown Uniseriate/Biseriat Yellow to green
velvety colonies,reverse ecovers entire
golden to red brown vesicles
A.Niger Wooly white to yellow ten Biseriate covers Black
turns to dark brown to entire vesicles
black
A.Terreus Velvety cinnomon brown Biseriate, Orange to brown
compact columnar
A.nidulans Biseriate Dark green

45. 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

46. Immunodiffusion test showing
048
precipitins against Aspergillus.

47. Detection of Antibody = Immunodiffusion
, BALISA(biotin-avidin amplification sys)
Detection of Antigen = Latex
agglutination, RIA,ELISA, BALISA
Molecular techniques = DNA
sequencing, PCR(realtimePCR),DNA probe
Molecular typing = analysis of genomic
DNA(mtDNA,rDNA),RFLP
Detection of fungal metabolites =G-test, D-
ELISA: Aspergillus galactomannan (content of cell wall) is used as indicator
mannitol as marker diagnosis.
of invasive aspergillosis for early
Skin tests = 0.1ml Ag(1000PNU/ml aspergillin)
results: Type I -Hypersensitivity- erythema &

48. DETECTION OF METABOLITES:
G-Test:
Recently developed G-test by Japanese workers detects
circulating
â-(1,3)-D-glucan with use of modification of litmulus
assay for endotoxins & has sensitivity of ~20pg/ml
G-factor is horse-shoe crab coagulation factor
This 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

49. Antifungal susceptibility disk test showing the in vitro activity of voriconazole against
Aspergillus fumigatus with Candida krusei as a control.

50. MYCOTOXINS Producing fungal Source of exposure Clinical conditions
species
Aflatoxin A.flavus, A.nomius. Groundnut, Aflatoxicosis,
A.parasiticus maize Reye’s syndrome,
P.puberculum hepatoma
Fuminosins Fusarium moniliforme Maize ELEM,PPE,
esophageal ca
Trichothecenes F.graminearium.F.sporo Maize,sorghum Human toxicosis, ATA,
tricoides biological warfare
Ocratoxins A.ocraceus, A.niger Cereals,coffee Nephropathies
P.verrulosum beans,bread
Cyclopiazonic acid A.flavus, Groundnut,corn Cocontaminant,
A.versicolor kodua poisoning
Zearalenones F.graminearium Wheat,maize, Cervical Ca, precocious
sorghum puberty

51. Medical Mycology is a
Iceberg
Thank
you
References:
1. Practical Laboratory Mycology – E.Koneman(2nd edn)
2. Color Atlas of Diagnostic Microbiology- luis,marie,ellen
3. Text book of Diagnostic Microbiology- Murry(ASM)
4. Gabrino J Aspergillosis (orphanet Encyclopedia 2004).
5. Textbook of Medical Mycology- J.Chandra(3rd edn)

52. 1.

53. Aspergilloma found at post-mortem in the lung of a child with leukaemia
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54. Aspergillosis in air sacs of a hen during an epidemic of aspergillosis in poultry.
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