Hyaline molds

• Filamentous fungi that do not have melanin present in the fungal cells (although the spores may be coloured).

Aspergillus species

Epidemiology

• Aspergillus is a ubiquitous fungus present worldwide, most commonly found in soil, air, water, and decaying vegetation. Risk factors for invasive disease include prolonged neutropenia (such as after induction chemotherapy), hematopoietic stem cell transplantation, graft-vs-host disease (especially with intensified therapy), solid-organ transplant (especially lung), anti-TNF-alpha immunosuppression, and CMV reactivation. Outbreaks have happened after construction.

Pathogenesis

• Conidia are inhaled into the lungs and alveoli, where, if not cleared, the conidia can germinate and start to invade. The normal host defense involves ciliary clearance of the conidia, destruction of the conidia by pulmonary macrophages, or destruction of the hyphal form by PMNs.
  • A. fumigatus has smaller conidia and may be more likely to reach the alveoli, which may explain the increased pathogenicity.
• Opsonization and complement may also be important.
• T-cell response is also important, with a Th-1 response being more favourable than Th-2.
• Hydrocortisone appears to be a growth factor.

Spectrum of Disease

• Colonization and superficial infections
  • Pulmonary aspergilloma
  • Otomycosis
  • Onychomycosis
  • Keratitis
• Allergic syndromes
  • Allergic bronchopulmonary aspergillosis (ABPA)
  • Allergic fungal rhinosinusitis
• Invasive aspergillosis, also a spectrum
  • Chronic cavitary pulmonary aspergillosis (CCPA)
  • Chronic necrotizing pulmonary aspergillosis
  • Invasive pulmonary aspergillosis
• Other rare syndromes
  • Ulcerative tracheobronchitis
  • Invasive fungal rhinosinusitis
  • Hematogenous dissemination to any organ
    • Cerebral aspergillosis
    • Osteomyelitis
    • SSTI

Non-culture diagnostic methods

• Serology for aspergillus antibodies exists, but is unhelpful, since it is a ubiquitous mold
• Galactomannan (GM): in serum or bronchoalveolar lavage (maybe CSF)
  • Release by the fungal cell wall during growth
  • Cutoff of 0.5 has 80% Sn and Sp (higher Sp and lower Sn at higher threshold)
    • Up to 90% in HSCT patients (serum)
    • Lower sensitivity in SOT
    • BAL fluid has higher sensitivity
  • Can do it in CSF as well
  • False-positives with piptazo and other beta-lactams (but may not still be the case)
  • No longer useful in surveillance now that we use mold-active antifungal prophylaxis (per IDSA)
• 1,3-beta-D-glucan (BDG): can detect Candida and Pneumocystis as well, so less specific. May be useful in combination with GM.
  • Utility in invasive fungal infections: from a systematic review in 2015, it is about 80% sensitive and 85% specific for IFI. Identified Candida and Aspergillus. In a retrospective review from 2014, it had similar specific and inferior sensitivity compared to GM.
  • Combination serologies: GM (BAL) Sn 43-56% and Sp 97%; BDG (blood) Sn 56-65% and Sp 97%; combination of either test positive Sn 78-92%% and Sp 93%, while PCR did not have any additional benefit (source).
• Fungal PCR possible, but not routinely done; may not be helpful since the fungus is ubiquitous and wouldn’t differentiate invasive disease vs. colonization.
• Microarray DNA:
• Microbiologic diagnostics are often combined with imaging to diagnose probable invasive fungal infection.

Identification of Aspergillus spp.

• Identification is done based on macroscopic appearance, microscopic features (especially of the fruiting body), and sometimes with special tests.
• Complexes cannot be differentiated phenotypically, but rather need molecular methods
  • May have cryptic species that are more resistant.
  • Fumigatus: fumigatus, lentulus, udagawae
  • Ustus: A. calidoustus (inherent resistance to ampho B)
  • Niger: tubingensis and niger
  • Versicolor: versicolor and sydowii

Species	Colonies	Head	Conidiophore	Phialides	Other
A. flavus	Yellow green, yellow, brownish	File:Media/image11.png	Rough colourless	Uniseriate and biseriate	Sclerotia sometimes present
A. fumigatus complex	Grey-green, blue green, yellowish	File:Media/image5.png	Smooth, colourless or greenish	Uniseriate	Good growth at 48ºC
A. glaucus	Green and yellow, yellowish, brown	File:Media/image2.png	Smooth, colourless	Uniseriate	Yellow to orange cleistothecia present
A. nidulans	Green buff, purplish red, olive	File:Media/image8.png	Smooth, brown	Biseriate	Round hülle cells and cleistothecia with purple ascospores usually present
A. niger	Black, white, yellowish	File:Media/image1.png	Smooth, colourless or brown	Biseriate
A. terreus	Brown cinnamon, yellowish brown	File:Media/image4.png	Smooth, colourless	Biseriate	Round, solitary aleurioconidia produced directly on hyphae
A. ustus	Light brown, grayish brown, yellowish brown		Smooth, brown	Biseriate	Long, brown-walled conidiophores, small vesicles, rough-walled conidia
A. versicolor	White, buff, yellow, pink, pale green, white, yellow, purplish red		Smooth, colourless	Biseriate	Round hülle cells sometimes present

Antifungal resistance

• Antifungal mechanisms
  • Polyenes (amphotericin): binds ergosterol to create pores within the cell membrane.
  • Triazoles (except fluconazole): inhibit sterol synthesis of ergosterol by disrupting 14-alpha demethylase. The mechanisms of resistance are myriad: modification of target enzymes, an increased expression of drug efflux mechanisms, an overexpression of target enzymes, an incorporation of exogenous cholesterol, an overexpression of HSP90 and of a sterole-regulatory element binding protein.
  • Echinocandins (the fungins): disrupt synthesis of beta-glucan in the cell wall by inhibiting 1,3-beta glucan synthase.
• Resistance patterns
  • All species are resistant to fluconazole. Historically, amphotericin has been the most reliable anti-Aspergillus antifungal; now, voriconazole is the standard.
  • Resistance to amphotericin is seen in A. terreus, A. flavus, and other less common species.
  • A. niger has variable susceptibility to azoles. There is increasing A. fumigatus resistance to azoles, with reports being most common from Europe. A. calidoustus (within A. ustus complex) is a growing cause, with late presentation, intrinsic antifungal resistance, and high mortality.

Organism	AmB	Fluc	Itra	Vori	Posa	Anidula	Caspo	Mica	Flucyt
Aspergillus spp.	+	–	+	+	+	+	+	+	–
* A. flavus*	±	–	+	+	+	+	+	+	–
* A. fumigatus*	+	–	+	+	+	+	+	+	–
* A. terreus*	–	–	+	+	+	+	+	+	–
* A. niger*	+	–	±	+	+	+	+	+	–

Susceptibility testing

• Broth microdilution is the main method for determining Aspergillus susceptibility recommended by CLSI and EUCAST. Microdilution results are affected by a number of factors (shape of the microdilution well, inoculum concentration, temperature and length of incubation time), so testing must be rigorously standardized.