Fungi for Fellows

David N. Fredricks, MD

Fred Hutchinson Cancer Research Center

Infection Classification Schemes

Invasiveness

• Superficial mycoses– Trichophyton infxn hair– Diaper rash (Candida)– Thrush (Candida)– Onychomycosis

• Subcutaneous mycoses– Madurella (madura foot)– Sporotrichosis– Chromoblastomycosis

• Deep mycoses– Invasive aspergillosis– Disseminated Candidiasis– Histoplasmosis– Cryptococcosis

Source of fungi: Endogenous/Exogenous

• Endogenous human– Candida– Pneumocystis jiroveci

• Ubiquitous Environmental– Aspergillus– Zygomycetes

• Regional endemic mycoses– Histoplasma– Coccidioides– Blastomyces– Paracoccidioides

• Widespread endemic mycoses– Cryptococcus– Sporothrix

Morphology

Yeasts

Moulds

Dimorphic

Other (Cocci)

Diagnosis of Fungal Infection• Clinical: fever, pulmonary symptoms, skin lesions,

sinus tenderness, pain, etc.– Risk factors (neutropenia, steroids)

• Radiological– Chest x-ray– CT scans of chest, sinuses, abdomen

Microbiological• Histological: biopsy of suspect lesions• Serological: Antibody and antigen tests• Molecular: Nucleic acids (PCR)• Cultivation

– Blood cultures: low yield, especially for moulds– Culture of suspect lesions by biopsy– BAL fluid culture in patients with pneumonia

General

Specific

• Candida • Aspergillus• Rhizopus• Cryptococcus• Blastomyces• Histoplasma• Coccidioides• Paracoccidioides

Membrane FunctionPolyenes: Amphotericin B

Lipid Formulation AmB (Abelcet, AmBisome)Nystatin

Ergosterol SynthesisAzoles: Fluconazole

KetoconazoleItraconazoleClotrimazole

VoriconazolePosaconazole

Squalene epoxidase inhibitors:Terbinafine

Cell Wall SynthesisEchinocandins: glucan Caspofungin

MicafunginAnidulafungin

Nucleic Acid SynthesisPyrimidine analog: 5-Fluorocytosine (5-FC)

Antifungal Review

Microtubules

Griseofulvin

Spectrum of Antifungal Agents

Drug / Fungus Aspergillus spp.

Candida spp.

Endemic Zygos

Fluconazole - + + -Voriconazole + + + -Micafungin + + - -Itraconazole + + + -Amphotericin + + + +Posaconazole + + + +

Case 1

• A 54 year-old man with acute myelogenous leukemia develops persistent fever with neutropenia after consolidation chemotherapy with Ara-C and is currently receiving ceftazidime– Previous rounds of febrile neutropenia treated with

empiric antibacterial and antifungal agents

• Blood cultures grow a yeast.– Germ tube negative

• What are the likely pathogens?

Candida species

• Yeasts, pseudohyphae (elongated single cells with constricted ends), and true hyphae (septations)

• Candida species: C. albicans, C. dubliniensis, C. glabrata, C. krusei, C. parapsilosis, C. tropicalis, C. pseudotropicalis, C.lusitaniae, C. guilliermondii

• Part of the normal human flora: opportunists– Oral cavity, GI and genital tracts

• Disease Risk factors: AIDS, diabetes, surgery, catheters,

antibiotics, neutropenia, burns, dialysis

Candida Diseases

• Superficial– Thrush: white patches on oral mucosa

– Vaginal candidiasis: thick curdlike discharge, puritis & burning

– Dermatitis: diaper rash, intertriginous assoc w/ moisture• erythema, papules, fissures, itching, burning

– Onychomycosis and paronychia:nails– Chronic mucocutaneous candidiasis

• immune defect

Candida Diseases

• Deep and disseminated– Esophagitis: odynophagia, ulceration

– GI tract: hematolgical malignancy• hepatosplenic (Alk P) from portal circulation

– Urinary tract: bladder catheterization

– Blood and vascular catheter infections• Third to fourth most common Bld Cx isolate; 10-15% mortality

– Disseminated disease: fungemic, nephric, cutaneous

– Endophthalmitis: expanding white cotton ball (retina/vitreous)

– Endocarditis: usually requires surgery

Case 1: Treatment

A. Fluconazole 400 mg daily

B. Amphotericin B 0.7 mg/kg IV daily

C. Ambisome 3.0 mg/kg IV daily

D. Voriconazole 200 mg bid

E. Micafungin 100 mg IV qd

F. Amphotericin + Fluconazole

G. Amphotericin + 5FC

Fluconazole vs. AmB for Candidemia

• Study population: 237 non-neutropenic, immunocompetant patients with candidemia– 206 evaluable subjects (103 + 103)

• AmB 0.5-0.6 mg/kg/day vs. fluconazole 400 mg/day for 14 days after last + blood culture

• Success: 79% AmB vs. 70% Fluconazole (95% CI –5%, 23%, not significant)

• 41 deaths with AmB, 34 with Flu (p = 0.2)• More toxicity with AmB

Rex JH et al. N Engl J Med. 1994;331:1325-1330.

Fluconazole + Amphotericin B for Candidemia

• Study Population: 219 adults; no neutropenia– Flu+Placebo: Flu 800 mg/day plus MVI (placebo)– Flu+AmB: Flu 800 mg/day+0.7 mg/kg AmB for first 5-6 days– Lower APACHE II scores in combination arm

• Results: – Success at day 30 in 57% Flu vs. 69% combo (p=.08)– Overall success in 56% Flu vs. 69% combo (p=.043)– Persistent +BC in 7% combo vs. 17% Flu– Higher toxicity in combination arm; renal dysfunction led to

reduction in the study drug dosage in 3% and 23% (P < .001)– Death within 90 days of starting study therapy occurred for

42 (39%) of 107 FP-treated subjects and 45 (40%) of 112 FA-treated subjects

• Conclusion: No evidence of antagonism and trend toward better outcomes in combination arm but with increased toxicity; no benefit in patients with very high or very low APACHE scores

Rex JH et al. Clin Infect Dis 2003;36:1221-1228

• Study population: 224 subjects with clinical evidence of infection and a culture positive for Candida from blood or other sterile site were stratified by APACHE II score and presence of neutropenia

• Randomized, double-blind, multi-center study:– IV caspofungin OR IV amphotericin B– Minimum of 10 days of IV therapy required;

antifungal therapy continued for 14 days after last positive Candida culture

• Lower toxicity in the caspofungin arm

Caspofungin versus Amphotericin B for Invasive Candidiasis

Mora-Duarte J et al. New Eng J Med 2002 Dec 19;347(25):2020-9

Caspofungin versus Amphotericin B for Invasive Candidiasis

0

20

40

60

80

100 Caspofungin

Amphotericin B

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ssf

ul o

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73%62%

81%

65%

Analysis of all patients (non-stratified)

Successful outcome = symptom resolution and microbiological clearance

Modified ITT Evaluable patients

Mora-Duarte J et al. New Eng J Med 2002 Dec 19;347(25):2020-9

P<0.05

Mora-Duarte J et al. N Engl J Med 2002;347:2020-2029

Favorable Responses to Treatment

Guidelines for Therapy of Candidemia

• Not neutropenic, no prior azoles, germ-tube positive (C. albicans) – Fluconazole at 400-800mg/d (clinically stable)– Echinocandins: Caspofungin, Micafungin, Anidulafungin– AmB (0.7 mg/kg/d): increased toxicity (cost vs. toxicity)– Lipid formulations of AmB 3-5 mg/kg (LFAB) in setting of renal

toxicities• Non-albicans Candida; neutropenic patients

– Echinocandins– Amphotericin B 0.7 mg/kg/d; LFAB– Sequential AmB Flu therapy

• Susceptible organism & clinical response– Consider susceptibility testing

• Voriconazole approved for candidemia; C. glabrata still a problem• Catheter should be pulled when feasible

– Several studies suggest mortality benefit, but sicker patients tend to have catheter retained

Case 2

• A 45 year old diabetic man presents to the ER with fever and left facial pain. There is new diplopia.

• ABG reveals pH of 7.22; serum glucose is 675.

• What is your diagnosis?

Case 2: Treatment

A. Amphotericin B 1 mg/kg

B. Abelcet 5 mg/kg

C. Ambisome 10 mg/kg

D. Micafungin 200 mg

E. Posaconazole 400 mg bid

F. Voriconazole 200 mg bid

G. None of the above

• Broad, non-septate hyaline hyphae• Irregular diameter, non-parallel walls with 90 degree angle branching possible

Treatment of Mucormycois

• Surgery: early consultation and intervention• Treat underlying host factors; reverse

immunosuppression, acidosis, hyperglycemia

• Antifungal therapy:– Lipid-AmB: high doses, empiric and directed– Posaconazole: oral salvage, follow on Rx

Surgery for zygomycosis

• 255 cases of pulmonary zygomycosis– 30 Duke, 225 literature

• Overall mortality = 80%• Retrospective analysis of mortality by treatment

group (in subset of treated pts)– Surgical (n= 36): 11%– Medical (n= 56): 68%– Difference highly significant (p = 0.0004)– Limitations: retrospective, selection bias

Tedder M. et al. Ann Thorac Surg 1994;57:1044-50.

Posaconazole for zygomycosis

• van Burik JA CID 2006;42:e61-65– Salvage therapy in 91 patients who were refractory

(81) or intolerant (10) of initial therapy; 800 mg/day– Complete and partial responses in 60%, with stable

disease in another 21% at 12 weeks• Greenberg RN et al AAC 2006;50:126-33

– Salvage therapy in 24 patients with zygomycosis who failed or were intolerant of conventional antifungal therapy (11 rhinocerebral, 4 disseminated) 800 mg /day orally divided

– Survival in 19/24 = 79% compared with historical survival rates of 50-70% using first line therapy

These were both salvage studies; there are no large published studies examining the efficacy of posaconazole for initial treatment of zygomycosis

Zygomycosis(a.k.a. mucormycosis)

• Rhinocerebral and sino-orbital disease– Risks: Diabetes (DKA), iron chelation with deferoxamine– Invasion of orbit and brain from sinuses– Rx: surgery and high dose lipid Ampho

• Pulmonary– Risks: Stem cell tx, leukemia, lymphoma, Solid organ tx– May disseminate to brain– Behaves like Aspergillus infection

• Halo, crescent signs; angioinvasion

• Other: GI, cutaneous, disseminated, isolated cerebral

Most commonly affected organ systems are host specific

Diabetes: rhinocerebral/sino-orbital

No underlying dz: cutaneous

Deferoxamine: None dominant

IVDU: Cerebral

Malignancy /BMT: pulmonary

SOT: Pulmonary and sinus

The Iron-pH Connection

Deferoxamine: used by fungi as siderophore

Serum from DKA: acidic = 69 ug/dl free iron vs. basic = 13 ug/dl better growth of Rhizopus in acidic serum

Deferasirox: chelates iron but not transported by fungi potential Rx

Case 3

• 30 year-old HIV+ man admitted to the VA hospital complaining of fever, weight loss, and anorexia for 2 months

• SH: Born in Louisiana, resident of California• Exam: 39.7oC, Pulse 105, BP 90/50 General:

cachectic with umbilicated papules on skin. Lungs: clear. CV: tachycardic without murmur. Abd: No hepatosplenomegaly. Neuro: non-focal

• Labs: Pancytopenia Meds: TMP/SMX• Admission diagnosis: dehydration, fever

Case 3

• Chest radiograph: No acute disease• Treatment: IVF, Ceftriaxone + metronidazole

– Persistent fever despite antibiotics

• Blood, urine, sputum cultures: no growth • New skin lesions apparent in hospital• CD4 count 50• Serum ferritin level 21, 240• Serum cryptococcal antigen: negative• Differential diagnosis?

What organism is this?What is the best treatment?

Giemsa blood smear

Skin biopsy: Silver stain

Histoplasmosis

• Agent: Histoplasma capsulatum– Dimorphic, endemic mycosis– Has no capsule

• Diagnosis• Treatment• Epidemiology: sporadic worldwide with

hyperendemic region in U.S.– Mississippi and Ohio river valleys– Associated with exposure to

• bird (not infected) and bat (infected) guano• Caves and spelunking• Building demolition

www.medvet.umontreal.ca/clinpath/ banq-im/Images/cyto46.jpg

Other Regional Endemic Mycoses in the United States

CoccidioidomycosisCoccidioides immitis & posadasii

• Desert Southwest: CA, AZ and Mexican border

• Pneumonia, Meningitis• Diagnosis: cx, histology,

serology (good)• Rx: Flucon, AmphoB, Itra

BlastomycosisBlastomyces dermatitidis

• Distribution: shadows histo in mid-western and SE US

• Pneumonia, skin, bone dz• Diagnosis: cx, histology

(BBBBY)• Rx: Itra, AmphoB, Flucon

US Endemic Mycoses: Common Themes• Fungus grows in the environment as mould

– Release spores into air inhaled, form yeasts in tissue

• Primary pulmonary infection– No person to person transmission– Frequently asymptomatic

• Cell mediated immunity contains infection• Exposure based on geography• Immunocompetent and immunocompromised hosts are

both at risk– Most disease is in the immunocompetent host: self limited– Severe, disseminated and reactivation disease more common in

compromised hosts

Case 4

• A 39 year-old woman had relapse of her acute myelogenous leukemia and was treated with cytarabine and mylotarg (Anti-CD33

Ab) resulting in prolonged neutropenia• She developed unexplained fevers (“febrile

neutropenia”) despite treatment with ceftazidime and fluconazole

• Remote tobacco use. WA resident, no travel• Exam: fever without localizing signs• Labs: ANC = 0, UA negative, Blood cx neg• CXR: Bibasilar opacities

Neutrophils

Case 4: Chest CT

Case 4

• Bronchoscopy with bronchoalveolar lavage– BAL galactomannan + (2.8 and 5.2)– Aspergillus PCR + (A. fumigatus)

• Serum galactomannan +: 1.4

What is the diagnosis?

How would you treat this patient?

Case 4: 12 days later…

Treatment: voriconazole + caspofungin

New CT

New CT

Prior CT

Day 0, 4, 10 CTsCaillot D et al. Increasing Volume and Changing Characteristics of Invasive Pulmonary Aspergillosis on Sequential Thoracic Computed Tomography Scans in Patients With Neutropenia Journal of Clinical Oncology, Vol 19, Issue 1 (January), 2001: 253-259

Invasive Aspergillosis

• Aspergillus species: ubiquitous moulds– A. fumigatus, A. flavus, A. terreus, A. niger, A. ustus– Ubiquity means no geographic predisposition– Opportunistic pathogens: it’s the host!

• Conidia are infectious unit inhaled, form hyphae

• Primary respiratory infection– Angioinvasion leads to necrosis of tissue– Pneumonia, sinusitis– Dissemination associated with high mortality

• Skin, brain, GI tract, pericardium, myocardium

www.med.unipg.it/ imagelab/microbio.html

Invasive Aspergillosis: Risks

• Underlying lung disease• Hematological malignancy, chemotherapy• Immunosuppression

– Steroids– Neutropenia– Chronic granulomatous disease, AIDS

• Transplantation– Solid organ– Hematopoietic stem cells

Invasive Aspergillosis

• Labs: routine labs not helpful; neutropenia is risk• Diagnosis

– Suspect when risk factors and radiological findings are present; may be clinically silent

• CT chest: nodules, halo, crescent, infiltrate, effusion

– Histology of tissue: hyphae proven fungal infection• Septate hyphae with 45 0 angle dichotomous branching

– Culture: sputum, BAL fluid, tissue• Blood cultures negative even with dissemination

– Galactomannan antigen, PCR

Aspergillus Histology

Narow septate hyphae with 45 degree angle branching and parallel walls, dichotomously branching = split into two equal branches.

hill.biology.rhodes.edu/ hill/Aspergillus.jpg

Aspergillosis• Treatment options

– Azoles: voriconazole, posaconazole, itraconazole– Echinocandins: caspofungin, micafungin,

anidulafungin – Polyenes: Ampho B, Ambisome (liposomal), Abelcet

(lipid)– Combination: voriconazole + caspofungin?

Combination Therapy: Aspergillosis• Animal models of infection show combination therapy

with an azole and echinocandin improves survival, reduces pathology, and reduces organism burden– Petraitis V et al. J Infect Dis. 2003:187:1834-43.– MacCallum DM et al. AAC. 2005 49:3697-701

• No randomized controlled trials of combo therapy• Non-randomized, uncontrolled study of 47 subjects who

failed initial therapy with an AmphoB regimen, salvage– 31 vori, 16 vori + caspo (Marr K et al. CID 2004;39:797-802)

Combination salvage therapy was associated with reduced mortality relative to voriconazole alone (HR, 0.27; 95% CI, 0.09 0.78; P = .008).

Marr K et al.

Clinical Infectious Diseases 2005;40:1075-76

1 year follow up data showed no long term survival benefit to combination therapy, though death due to aspergillosis was reduced

• Retrospective review of >400 subjects with IA fails to confirm benefit of combo Rx

What is the single best agent?Paucity of randomized controlled trials to inform agent selection• Herbrecht R. et al. Voriconazole vs. amphotericin B for primary therapy of invasive aspergillosis NEJM 2002:347;408-415. Unblinded RCT.

- Survival in 71% on vori, 58% on AmphoB- Less toxicity in vori arm: 80% on AmB switched to salvage vs. 36% on vori- Success of salvage therapy with OLAT (mostly lipid ampho) only 30% in Ampho B arm (Patterson TF et al. CID 2005: 41;1448-52)

Hazard ratio, 0.59

95 % CI, 0.40 to 0.88

Galactomannan antigen testing for diagnosis of invasive aspergillosis

• Sandwich ELISA detection limit = 0.5 ng/ml– Tissue: serum, BAL fluid, CSF– Approved in the United States and Europe as aid to

diagnosis of invasive aspergillosis in adults (0.5 OD cutoff)– False negatives: antifungal therapy, limited disease, SOT

• Sensitivities of 29-100% reported

– False positive rate is quite variable, depending on patient population (kids), underlying disease (mucositis), and cutoff

• False + or True +? Definition: biological vs. clinical?• Absorbed GM from food, cereal grains, Bifidobacterial antigen• Antibiotics: Zosyn, Augmentin

27 studies

May 15, 2006 Clinical Infectious Diseases

Case 5 (Courtesy of Linda Gorgos, MD)

• 58 y.o. woman s/p renal transplant in 12/2000 for polycystic kidney disease, on MMF, tacrolimus, and prednisone, was admitted for elective partial hepatectomy and cyst removal

• Post-op CXR on 2/9/07 showed a LLL lung mass that was new since 2001 when she had an episode of pneumonia

– Denied any fevers or pulmonary complaints

Post-Op

Exposures and Social History

• Social History: Microbiologist.

• Remote exposure to parakeets and limited exposure to dogs (all healthy)

• Most recent travel to Orcas and San Juan Islands in 9/06. Lopez Island in 7/05. Traveled to Italy 1 ½ yrs ago.

Frozen Section

Wedge resection - Mucicarmine StainWedge resection - Mucicarmine Stain

Further Evaluation

• CT head with contrast - no masses or abnormalities other than old aneurysm clip

• LP performed with normal OP – normal protein and glucose– acellular – CSF CrAg negative – fungal stain and culture negative

• Serum CrAg negative• Blood cultures negative

What is the best treatment for this patient?

Vancouver Island Outbreak

Cryptococcus neoformans

• Yeast with worldwide distribution– High concentrations in pigeon droppings, soil– Thick polysaccharide capsule– Sexual mould form detected in nature:

Filobasidiella neoformans

• Varieties of C. neoformans– Serotype A : var. grubii– Serotypes B and C: var. gattii

• Outbreak on Vancouver Is. = “B”

– Serotypes D: var. neoformans

Cryptococcosis

• Opportunistic infection: AIDS, cancer, organ transplantation, steroids, diabetes

• Primary pathogen in selected cases (10-40%)• Pathophysiology: Pneumonia dissemination

via blood to meninges, skin 1. Self limited pneumonia: well circumscribed lesions

with surprisingly meager inflammatory response; usually untreated in normal host

2. Meningitis: (or disseminated disease) / immunocompromised host: treat with antifungals

– AIDS: crypto meningitis common opportunistic infection– Complication: Elevated intracranial pressure (measure opening

pressure at time of LP!!)

Cryptococcosis

• Labs: Routine labs not generally helpful• Diagnosis

– India ink prep of CSF ( historical interest)– Cryptococcal antigen: serum , CSF

• Sensitivity >95% with high specificity

– Cultivation– Histology: narrow based budding yeast, capsule

• Mucicarmine stain for capsule: specific for crypto

• Treatment– Ampho B + 5-FC Fluconazole: meningitis– Fluconazole alone, Lipid-Ampho alone– Itraconazole for intolerance or refractory disease

Taxonomic Groups of Fungi-Based on sexual spore

• Basidiomycetes: Mushrooms, Cryptococcus neoformans

– Basidiospores

• Zygomycetes: Mucor, Rhizopus, Absidia

– Aseptate hyphae, hyaline, zygospores

• Ascomycetes: Aspergillus, Scedosporium, H. capsulatum, C. immitis

– Septate hyphae, hyaline or dematiaceous, ascospores

• Deuteromycetes: Fungi imperfecti = no known sexual state

– Yeasts or moulds with septate hyphae/hyaline or dematiaceous