Aspergillus and Aspergillosis

Dr Summiya Nizamuddin

A filamentous or mould form of fungus which is a vegetative growth of filaments.

– reproduction is by spores or conidia– conidia are borne on specialized hyphae or

conidiophores– Many moulds can be identified by

the morphology of these spores and by their arrangement on the hyphae

Mould• Multicellular form of fungus

• Microscopic: Septate hyphae Spores

• Macroscopic: Surface texture: Cottony/wooly/ velvety/ granular

:Pigmentation: observed from the reverse

Mould-Definitions

• Hyphae• Mycelium: a. Vegetative

b. Aerial

Mould identification

• Macroscopic characteristics: colonial form, surface color, pigmentation

• Microscopic appearance: fruiting bodies, appendages, spore shape and arrangement

Hyaline Hyphomycetes

• Hyaline Hyphomycetes include those conidial fungi which are – not darkly pigmented, – colonies may be colorless or brightly colored.

• Include the agents of hyalohyphomycosis: Aspergillosis, dermatophytosis and the dimorphic pathogens, like Histoplasma capsulatum

Hyaline Hyphomycetes include:

• Acremonium• Aspergillus• Beauveria• Chrysosporium• Cylindrocarpon• Fusarium• Geotrichum• Gliocladium• Graphium• Madurella

•Malbranchea•Onychocola•Paecilomyces•Penicillium•Scedosporium•Scopulariopsis•Sepedonium•Trichoderma•Trichothecium•Verticillium

0 hr 2 hrs 6- 8hrs14- 22 hrs

Asexual life cycle of Aspergillus

Conidium Germinating conidia

Germling Branched,multinucleate mycelium

Foot cell and vesicle

Conidiophore with conidia

Aspergillus

Colony colors in Aspergillus

Colony colors in Aspergillus

Image Courtesy: Dr. Lindsley

Conidiophore Structure

Aspergillosis

Aspergillosis- a spectrum of diseases of humans and animals caused by members of the genus Aspergillus

Disease spectrumAspergillosis includes:

(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) colonisation without extension in preformed cavities

and debilitated tissues; (4) invasive, inflammatory, granulomatous, narcotising

disease of lungs, and other organs; and rarely (5) 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

The etiological agents are cosmopolitan and include Aspergillus fumigatus, A. flavus, A. niger, A. nidulans and A. terreus.

Ecology Found in cellars, potted plants, pepper and spices Found all over the world – Antarctica and Sahara Primary ecological niche – decaying vegetable matter

Sources of Infection? Soil Air, spores may be inhaled Water / storage tanks in hospitals etc Food Compost and decaying vegetation Fire proofing materials Bedding, pillows Ventilation and air conditioning systems Computer fans

Bennett, Medical Mycology, 2009

Aerosolized conidia

Aspergillus hyphae in the lung

Cavitary lesion A. fumigatus

culture in vitro

Aspergillus sp.–cellular states

Aspergillus Identification Criteria

Macro-morphology

Colony color

Micro-morphology

Laboratory diagnosis:1. Clinical material: Sputum, bronchial washings and tracheal aspirates from patients with pulmonary disease and tissue

biopsies from patients with disseminated disease.

2. Direct Microscopy: (a) Sputum, washings and aspirates make wet mounts in

either 10% KOH & Parker ink or Calcofluor and/or Gram stained smears;

(b) Tissue sections should be stained with H&E, GMS and PAS digest. Note Aspergillus hyphae may be missed in H&E stained sections. Examine specimens for dichotomously branched, septate hyphae.

• Interpretation:• The presence of hyaline, branching septate hyphae, consistent

with Aspergillus in any specimen, from a patient with supporting clinical symptoms should be considered significant.

• Biopsy and evidence of tissue invasion is of particular importance.

• Remember direct microscopy or histopathology does not offer a specific identification of the causative agent.

3. Culture: Clinical specimens should be inoculated onto primary isolation media, like Sabouraud's dextrose agar. Colonies are fast growing and may be white, yellow, yellow-brown, brown to black or green in colour.

• Interpretation: • Aspergillus species are well recognised as common

environmental airborne contaminants, therefore a positive culture from a non-sterile specimen, such as sputum, is not proof of infection.

• However, the detection of Aspergillus (especially A. fumigatus and A. flavus) in sputum cultures, from patients with appropriate predisposing conditions, is likely to be of diagnostic importance and empiric antifungal therapy should be considered.

• Unfortunately, patients with invasive pulmonary aspergillosis, often have negative sputum cultures making a lung biopsy a prerequisite for a definitive diagnosis.

4. Serology: Immunodiffusion tests for the detection of antibodies to Aspergillus species have proven to be of value in the diagnosis of allergic, aspergilloma, and invasive aspergillosis.

However, they should never be used alone, and must be correlated with other clinical and diagnostic data.

5. Identification: Aspergillus colonies are usually fast growing, white, yellow, yellow-brown, brown to black or shades of green, and they mostly consist of a dense felt of erect conidiophores.

• Conidiophores terminate in a vesicle covered with either a single palisade-like layer of phialides (uniseriate) or a layer of subtending cells (metulae) which bear small whorls of phialides (the so-called biseriate structure).

• The vesicle, phialides, metulae (if present) and conidia form the conidial head.

• Conidia are one-celled, smooth- or rough-walled, hyaline or pigmented, forming long dry chains which may be divergent (radiate) or aggregated in compact columns (columnar). Some species may produce Hülle cells or sclerotia.

AspergillumA perforated containerfor sprinkling holy water

Sprinkling the world with spores

Conidiophore Structure

columnar radiate

• Most species sporulate within 7 days. Descriptions are primarily based on colony pigmentation and morphology of the conidial head.

• • Microscopic mounts are best made using a

cellotape flag or slide culture preparation mounted in lactophenol cotton blue.

• Key Features: Hyaline hyphomycete showing distinctive conidial heads with flask-shaped phialides arranged in whorls on a vesicle.

• Aspergillus flavus• Aspergillus fumigatus• Aspergillus nidulans• Aspergillus niger• Aspergillus terreus

Aspergillus flavus

Deep green to olive green colonies

Dark brown to black sclerotia sometimes present

Conidial heads radiate to columnar

Stipe walls roughened

Vesicles spherical

Largely biseriate

Conidia globose to ellipsoidal

Aspergillus fumigatus

Aspergillus niger

Black to very dark brown

Rare sclerotia

Conidial heads radiate

Smooth walled stipes

Spherical vesicles

Biseriate

Globose, rough conidia

Aspergillus terreus

Brownish orange colonies

Conidial heads columnar

Smooth walled stipes

Spherical vesicles

Smooth walled globose conidia

Globose accessory conidia produced laterally on submerged hyphae

A. nidulans

Image Courtesy: Mycology online

Hülle cells Emericella nidulansCleistothecium of Emericella nidulans