Aspergillus

Background

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: A. fumigatus, A. lentulus, A. udagawae
  • Ustus: A. calidoustus (often resistant to ampho B)
  • Niger: A. tubingensis and A. niger
  • Versicolor: A. versicolor and A. sydowii

Species	Colonies	Conidiophore	Phialides	Other
A. flavus	Yellow green, yellow, brownish	Rough colourless	Uniseriate and biseriate	Sclerotia sometimes present
A. fumigatus complex	Grey-green, blue green, yellowish	Smooth, colourless or greenish	Uniseriate	Good growth at 48ºC
A. glaucus	Green and yellow, yellowish, brown	Smooth, colourless	Uniseriate	Yellow to orange cleistothecia present
A. nidulans	Green buff, purplish red, olive	Smooth, brown	Biseriate	Round hülle cells and cleistothecia with purple ascospores usually present
A. niger	Black, white, yellowish	Smooth, colourless or brown	Biseriate
A. terreus	Brown cinnamon, yellowish brown	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
• Highest risk, in order, are: CGD, allogeneic hematopoietic stem cell transplantation with GVHD, AML with induction or (worse) reinduction, everyone else
• Hematopoitic stem cell transplantion is high 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 transplantation
  • Highest among lung transplantation recipients (6%) due to ongoing environmental exposure, decreased ciliary clearance, and common concomitant Aspergillus colonization
  • Followed by small bowel, liver (4%), heart (2%), and kidney (0.5%)
  • Usually diagnosed at 6 to 12 months (half within the first 3 months)
• Therapeutic immunosuppression, including prednisone and TNF-α inhibitors
• 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

Other Risk Factors

• HIV (2.2 per 10,000/year), associated with low CD4 counts, neutropenia, cirrhosis, liver transplantation, and glucocorticoid therapy
• Liver cirrhosis (0.3%)
• Immunocompetent patients in critical condition from ARDS, COPD, influenza, pneumonia, burns, severe bacterial sepsis, surgery, or malnutrition
  • Glucocorticoid therapy is most common risk factor in these patients

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 Manifestations

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 Superficial Infections

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

Allergic Syndromes

Allergic Bronchopulmonary Aspergillosis (ABPA)

• Allergic reaction to airway colonization, usually in patients with asthma or cystic fibrosis
• Characterized by poorly-controlled asthma or pneumonia, mucoid impaction, persistent eosinophilia
• See also Allergic bronchopulmonary aspergillosis

Allergic Fungal Sinusitis

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

Chronic Pulmonary Aspergillosis (CPA)

• Inclues chronic cavitary pulmonary aspergillosis (CCPA), chronic necrotising pulmonary aspergillosis (CNPA), and chronic fibrosing aspergillosis

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

Subacute Invasive Aspergillosis

• Previously called chronic necrotizing pulmonary aspergillosis
• Occurs in mildly immunocompromised or very debilitated patients
  • Risk factors include diabetes mellitus, malnutrition, alcoholism, advanced age, prolonged corticosteroids or other immunosuppressive agents, chronic obstructive lung disease, connective tissue disorders, radiation therapy, non-tuberculous mycobacterial infection, or HIV infection
• Similar clinical and radiological findings as chronic cavitary pulmonary aspergillosis, but progresses more rapidly into frank invasive pulmonary 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 neurological signs, altered mental status, and headache
• 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, AML induction, and SOT, it tends to be isolated pulmonary aspergillosis
• In HSCT 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 released 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
  • See review 1

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)

• Not always treated with antifungals
• See Allergic bronchopulmonary aspergillosis

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
  • An RCT of itraconazole found that the relapse rate was significantly lower with 12 months of therapy compared to 6 months2
• 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 (caspofungin, or other)
  • If hepatotoxicity with voriconazole, switch to posaconazole
  • Voriconazole is superior to amphotericin for mortality
  • Combination voriconazole plus anidulafungin is no better than voriconazole except in post-hoc analysis of possible early treatment
  • Isuvaconazole may be superior to voriconazole
• Duration 6-12 weeks depending on immunosuppression
• Follow-up CT after a minimum of 2 weeks, or earlier if deterioration
• Non-pharmacologic management for neutropenic patients includes:
  • Reducing or eliminating immunosuppression
  • Granulocyte colony-stimulating factor
  • Granulocyte transfusions

Breakthrough Infection

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

Therapeutic Drug Monitoring

Antifungal	When to Measure Trough Level	Target for Treatment	Target for Prophylaxis	Target for Safety
Itraconazole	day 5 of therapy	1-4 mg/L by HPLC 3-17 mg/L by bioassay	0.5-4 mg/L by HPLC 3-17 mg/L by bioassay	≤~4 mg/L by HPLC ≤17.1 mg/L by bioassay
Voriconazole	after 2 to 5 days of therapy, and 4 days after any change	1-5.5 mg/L	1-5.5 mg/L
Posaconazole	day 5 of therapy	>1 mg/L	>0.7 mg/L	≤3.75 mg/L
Isavuconazole	day 5 of therapy, but not clearly needed

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-α inhibitors)
• Lung transplant: voriconazole, itraconazole, or inhaled amphotericin B 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
• Chronic granulomatous disease: itraconazole and interferon-γ

Further Reading

• Practice Guidelines for the Diagnosis and Management of Aspergillosis: 2016 Update by the IDSA. Clin Infect Dis. 2016;63(4):e1-e60. doi: 10.1093/cid/ciw326
• Diagnosis and management of Aspergillus diseases: executive summary of the 2017 ESCMID-ECMM-ERS guideline. Clin Microbiol Infect. 2018. doi: 10.1016/j.cmi.2018.01.002