Aspergillus

Microbiology

• Aspergillus is a mold with hyaline (lightly-pigmented) hyphae, septated, and usually branched at acute angle (45º)
• Named for the appearance of the sporulating head, which looks like an aspergillum used to sprinkle holy water (in 1729)
• Most species reproduce asexually, although A. fumigatus and a few others have teleomorphs (sexual form with fruiting body)
• Culture is important, but molecular methods are often required to identify the particular species
  • Pathogenic species grow quickly on common media
  • Can grow at 37º C, and A. fumigatus can grow up to 50º C
• Organized into complexes, which cannot be differentiated phenotypically, but rather need molecular methods
  • Fumigatus: fumigatus, lentulus, udagawae
  • Ustus: A. calidoustus (inherent resistance to ampho B)
  • Niger: A. tubingensis and A. niger
  • Versicolor: A. versicolor and A. 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

• Of note, A. ustus complex (A. caladustus) are resistant to azoles and echinocandins, and variable resistance to amphotericin (but susceptible to terbinafine)

Epidemiology

• Ubiquitous worldwide, found in soil, water, food, air, and decaying vegetation
• There is increasing antifungal resistance worldwide
• Outbreaks can occur with construction
• May also be possible to have activation of latent infecton or colonization, making infection control more difficult

High-Risk Populations

• The major risk factor is defective function or decreased number of neutrophils
• In order of risk: CGD, alloSCT with GVHD, AML with induction or (worse) reinduction, everyone else, etc.
• CGD is the highest risk disease; other at-risk groups include lung disease, AIDS, etc.
• Hematopoitic stem cell transplants are highest risk (7% allo, 1% auto)
  • Peaks <40 days and >100 days
  • With or without neutropenia, most likely related to steroid use
• Hematologic malignancies
  • Usually following induction chemotherapy, or refractory or recurrenct disease (50% mortality)
  • 3+7 AML induction usually 14-21 days of neutropenia
• Solid-organ transplants
  • Highest among lung transplant recipients (6%) due to ongoing environmental exposure, decreased ciliary clearance, and common concomitant Aspergillus colonization
  • Followed by liver (4%), heart (2%), and kidney (0.5%)
  • Usually diagnosed at 6 to 12 months
• Therapeutic immunosuppression, including prednisone and anti-TNF-alpha
• GVHD increases the risk, due to the additional immune suppression
  • Highest risk within GVHD is with gut involvement
• Solid maligancies are relatively low risk due to the short courses of neutropenia, but increasing risk with newer chemotherapies

Pathophysiology

• Initially acquired by inhalation of conidia into lungs or sinuses, or rarely from local tissue invasion
• The conidia grow and germinate, transforming into hyphae and invading the vasculature
  • Hydrocortisone appears to be a growth factor for Aspergillus
  • Vascular invasion is typical of invasive aspergillosis
  • May cause pulmonary infarction
• This can be followed by hematogenous dissemination
• The host immune response begins with ciliary clearance to prevent the conidia from reaching the alveoli
• Once in the alveoli, the response depends on pulmonary macrophages to phagocytose the conidia
• Following germination and growth of hyphae, PMNs act to kill hyphae and swollen conidia
  • This is helped by opsonization of conidia by complement
  • Antibodies are common, given the mold's ubiquity, but not protective
• A. fumigatus has small conidia, allowing it to reach the alveoli more easily, and also produces a complement inhibitor

Clinical Presentation

Colonization and superficial infections

Aspergilloma (fungal ball)

• Ball of hyphae growing in a preexisting cavity, often in bullous emphysema, sarcoidosis, tuberculosis, histoplasmosis, congenital cysts, bacterial lung abscesses, or Pneumocystis bleb
• Often asymptomatic, but the most common symptom is hemoptysis, which can be fatal
• Can also occur in the sinuses

Other supreficial infections

• Otomycosis: chronic otitis externa caused by A. niger or A. fumigatus
• Onychomycosis
• Keratitis

Allergic syndromes

Allergic bronchopulmonary aspergillosis (ABPA)

• Caused by a Th2 response to Aspergillus, usually in patients with asthma or cystic fibrosis
• Criteria include: asthma, central bronchiectasis on CT, positive skin test for Aspergillus, total IgE >417 IU/mL, IgE or IgG antibodies to A. fumigatus, transient CXR infiltrates, Aspergillus precipitans, and eosinophilia
• Supported by Aspergillus on sputum culture, brown mucous plugs with dead eosinophils, and CXR showing bronchiectasis
• The course is characterized by exacerbations and remissions, leading to eventual pulmonary fibrosis and chronic pulmonary aspergillosis

Allergic fungal sinusitis

• Can be Aspergillus or other molds
• Mangement is mostly surgical

Chronic cavitary pulmonary aspergillosis (CCPA)

• One or more cavities that can contain solid or liquid material or a fungal ball, usually following creation of multiple cavities from another process
• May present with pulmonary or constitutional symptoms, including hemoptysis, dyspnea, and productive cough
  • Weight loss and fatigue are common and profound, while fevers are less common
  • May mimic TB
• Diagnosis requires:
  • 3 months of symptoms or chronic illness or progressive radiological abnormalities with cavitation, pleural thickening, perivacitary infiltrates +/- fungal ball
  • Aspergillus IgG antibodies
  • No or minimal immunocompromise
• Must rule out other causes of symptoms, including other causes of weight loss

Invasive aspergillosis

• aka. angioinvasive, invading the vasculature

Chronic necrotizing pulmonary aspergillosis

• With mild or moderate immunosuppression, patients may develop chronic necrotizing pulmonary aspergillosis (CNPA), essentially a subacute form of invasive aspergillosis

Invasive pulmonary aspergillosis

• Usually after 10 to 12 days of severe neutropenia
• Non-productive cough, dyspnea, pleuritic chest pain, and fever with pulmonary infiltrates despite broad-spectrum antibiotics
  • Symptoms may be less prominent in patients with defective immunity
  • Fever dampened by high dose steroids
• Also hemoptysis, pleural effusion, and pneumothorax
  • Can mimic a pulmonary embolism
• Imaging may show multiple dense nodular pulmonary infiltrates without air bronchograms, suggesting extensive infection
  • Classic, though, is pleural-based wedge-shaped densities or cavitary lesions
  • Pleural effusions are common
  • A nodular lesion wth a halo is suggestive of early aspergillosis, followed by cavitation in later disease

Other sites of invasive respiratory aspergillosis

• Ulcerative tracheobronchitis, a high concern in lung transplant
  • May mimic graft rejection
• Invasive rhinosinusitis, with mortality of 10-20%
• Hematogenous dissemination to any organ, associated with 90% mortality

Other sites of invasive aspergillosis

• Cerebral aspergillosis, which may explain half of all CNS lesions in HSCT
  • Presents >100 days after transplant, usually with concomitant pulmonary disease
  • Presents with focal neuro signs, altered mental status, and headaches
• Osteomyelitis
  • Vertebral osteomyelitis may result from extension of empyema, but is also the most common site of hematogenous dissemination
• Skin and soft tissue infection
  • Either from hematogenous spread or local invasion
  • Often around IVs or adhesive dressings
  • Neutropenic patients as well as burns and surgical sites

Specific risk groups

• For CGD and AML induction and SOT, it tends to be isolated pulmonary aspergillosis
• In SCT with GVHD, you tend to see more CNS aspergillosis and disseminated aspergillosis

Diagnosis

• Culture positive for Aspergillus and histology with invasive hyphae
  • Only 10-30% of patients with IA have a positive BAL culture, improved with use of fungal media
• Serology
  • Antibodies is unhelpful, given that the mold is ubiquitous
  • Galactomannan by EIA
    • Best-studied and most sensitive in HSCT patients
    • It is a meleased from the fungal cell wall on growth
    • Cutoff of 0.5 is good, 80% Sn and Sp (up to 90% in HSCT patients' serum)
    • BAL is more sensitive, but prophylaxis decreases sensitivity
    • Can be done from CSF
    • False-positives may occur with pip/tazo and other beta-lactams (though mostly of historical interest now)
  • 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).
• Molecular testing
  • 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.
• Imaging can be helpful
  • Halo sign on CT is present for about the first 7 days of disease in neutropenic patients
  • Can also have nodules, pleural-based infarctions, or vacitation, as well as non-specific consolidation

Management

Antifungal resistance

• 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.
• 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*	+	–	±	+	+	+	+	+	–

Aspergilloma

• If asymptomatic and single aspergilloma, monitor
• If symptoms, especially hemoptysis, surgical resection (if possible)
• No role for antifungals

Allergic bronchopulmonary aspergillosis (ABPA)

• Indications for treatment
  • Diagnose with Aspergillus-IgE
  • If ongoing symptoms despite appropriate management of asthma (including oral steroids), treat with itraconazole
  • If CF patient has frequent exacerbations or falling FEV1, treat with itraconazole
• Itraconazole 200 mg/day for 16 weeks, which decreases steroid use and increases patient function

Allergic fungal rhinosinusitis

• Polypectomy and sinus washout
• Topical nasal steroids
• Oral antifungal therapy can be tried if above does not work, but rarely effective

Chronic cavitary pulmonary aspergillosis (CCPA)

• If asymptomatic, monitor every 3-6 months, with investigations every 3-12 months including
  • Low-dose CT chest or CXR
  • ESR/CRP
  • Aspergillus IgG titres
  • Annual PFTs
• If pulmonary symptoms, constitutional symptoms, or worsening lung function, treat with 6+ months of antifungal therapy
  • Itraconazole or voriconazole
  • If this fails, try IV micafungin, caspofungin, or amphotericin B
• If hemoptysis, treat with tranexamic acid, pulmonary artery embolization, or antifungal therapy
• May need surgical resection if localized disease refractory to medical management

Invasive aspergillosis

• Voriconazole 6 mg/kg IV q12h x2 then 4 mg/kg IV q12h, or 200 mg po q12h
  • Alternative: liposomal amphotericin B 3 mg/kg/day
  • Salvage: echinocandins (caspo, or other)
  • If hepatotoxicity with voriconazole, switch to posaconazole
  • Vori superior to amphofor mortality
  • Combo vori+anidula no better than vori except in post-hoc analysis of possible early treatment
  • In the future, watch out for isuvaconazole—may be superior to vori
• Duration 6-12 weeks depending on immunosuppression
• Follow-up CT after a minimum of 2 weeks, or earlier if deterioration

Breakthrough infection

• Base empiric treatment on local epidemiology
• Probably fewer breakthroughs in HSCT patients with posaconazole prophylaxis

Failure

• Technically should be assessed at 6 weeks (2 weeks at a minimum, based on pharmacokinetics)

Prevention

• For high-risk patients in hospital (e.g. HSCT), use air filters, frequent air exchanges, and positive-pressure ventilation

Antifungal prophylaxis

• Posaconazole prophylaxis is first to demonstrate survival benefit for AML/induction patients
• AML induction: posaconazole, voriconazole, or micafungin
  • Caspofungin probably also effective
  • Itraconazole also effective but poorly tolerated
• HSCT with moderate to severe GVHD: posaconazole (voriconazole is alternative)
  • Reduces invasive fungal infections, but no mortality benefit
• Immunosuppression for GVHD: prophylaxis for duration of immunosuppression (steroids >1mg/kg/d for >2 weeks, or lymphocyte-depleting agents, or TNF-alpha inhibition)
• Lung transplant: vori/itra/inhaled amphoB for 3 to 4 months after transplant, and when receiving thymoglobulin, alemtuzumab, or high-dose steroids
• Other solid-organ transplant: decision based on per-patient risk factors
• Prior IA requiring new immunosuppression: may also benefit from prophylaxis