David N. Fredricks, MD Fred Hutchinson Cancer Research Center Fungi for Fellows David N. Fredricks, MD Fred Hutchinson Cancer Research Center

Infection Classification Schemes Source of fungi: Endogenous/Exogenous Endogenous human Candida spp. Pneumocystis jirovecii Ubiquitous Environmental Aspergillus Zygomycetes (Mucorales) Regional endemic mycoses Histoplasma Coccidioides Blastomyces Paracoccidioides *Widespread endemic mycoses Cryptococcus (haploid/diploid) Sporothrix Morphology Yeasts Moulds Dimorphic Invasiveness Superficial mycoses Trichophyton infxn hair Onychomycosis Subcutaneous mycoses Madurella (madura foot) Chromoblastomycosis Deep mycoses Invasive pulm. aspergillosis Candidemia Disseminated histoplasmosis Cryptococcal meningitis Haploid crypto is not dimorphic Diploid crypto is dimorphic * But not ubiquitous

Candida Aspergillus Rhizopus Cryptococcus Blastomyces Histoplasma Coccidioides Paracoccidioides Morphology: Yeasts, moulds Match picture to name

Diagnosis of Fungal Infection Clinical: fever, pulmonary symptoms, skin lesions, sinus tenderness, pain, etc. Risk factors (host) Neutropenia, steroids, iron overload, SOT Radiological Chest x-ray CT scans of chest, sinuses, abdomen Microbiological Histological: biopsy of suspect lesions Serological: Antibody (limited) and antigen tests (galactomannan and CRAG) Molecular: Nucleic acids (PCR) Cultivation Blood cultures: low yield for most moulds Culture of suspect lesions by biopsy BAL fluid culture in patients with pneumonia General Specific

Case 1 A 58 year old man s/p partial colectomy for colon cancer develops fever, tachycardia, and hypotension (85/60) and is transferred to the ICU. He is on broad spectrum antibiotics, has a central venous catheter (CVC), a foley catheter, and is receiving total parenteral nutrition. Creatinine is rising, now 2.5. The lab reports that a blood culture is growing yeasts.

Case 1: What is the best intervention? Remove the CVC and repeat blood culture Start an amphotericin B product IV (e.g. ambisome) Start fluconazole IV Start an echinocandin IV (e.g. micafungin), and switch to fluconazole if yeast is susceptible

Debates Should you remove the central venous catheter with candidemia? IDSA guidelines say YES: studies show faster clearance of fungemia and improved survival Recent study in Clinical Infectious Diseases showed that in a cohort of 842 patients with candidemia, line removal was associated with decreased mortality and improved treatment success, but not in multivariate analysis (CID 2010;51:295) This study has been criticized on grounds that authors over-adjusted in multivariate analysis. Should care teams obtain an ID consult? IDSA Abstract: Marwa Shoeb et al. Patients who received IDCs were 9 times more likely to survive at 30 days than patients without IDCs when controlling for Apache-II score (hazard 9.03, 95% CI 2.68-30.36, p=0.0004). Conclusion: Infectious diseases consultations for patients with candidemia were associated with improved adherence to standards of care and decreased mortality without increasing costs or hospital stays.

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 microbiota: 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 CMC: defect in TH17 signaling

Candida Diseases Deep and disseminated Esophagitis: odynophagia, ulceration Candidemia: 4th most common organism GI tract: hematologic malignancy Urinary tract: bladder catheterization Vascular catheter infections Disseminated disease: hepatosplenic (Alk P), nephric, fungemic Endophthalmitis: expanding white cotton ball (retina/vitreous) Endocarditis: usually requires surgery Clinical pearl: Candida pneumonia is rare

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.

Caspofungin versus Amphotericin B for Invasive Candidiasis 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 Neutropenic and non-neutropenic Mora-Duarte J et al. New Eng J Med 2002 Dec 19;347(25):2020-9

Caspofungin versus Amphotericin B for Invasive Candidiasis Analysis of all patients (non-stratified) Caspofungin Amphotericin B 100 80 81% 73% 60 Successful outcomes (%) 62% 65% 40 P<0.05 20 Modified ITT Evaluable patients Successful outcome = symptom resolution and microbiological clearance Mora-Duarte J et al. New Eng J Med 2002 Dec 19;347(25):2020-9

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, prior azole Rx 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 When in doubt, echinocandinize and step down to fluconazole!

Candidemia: Take Home Messages Don’t blow off a positive blood culture! Remove central venous catheters with + blood culture 30% mortality overall, but goes from 15% when Rx started on day 0 of culture, to 41% when started day 4 Don’t delay! Echinocandins just as effective as other agents, but less toxic and greater spectrum of Candida covered Fluconazole is good choice for stable patient when not exposed to previous azoles, and not neutropenic Treat for at least 14 days

Case 2 A 45-year-old woman from Boston with poorly controlled insulin-dependent diabetes had facial and periorbital swelling for four days. On the day of admission she was unable to open her right eye (Panel A). On admission she had a white-cell count of 22,000 per cubic millimeter, with 84% neutrophils and bands and a moderate degree of metabolic acidosis (blood pH, 7.22; plasma bicarbonate concentration, 8 mmol per liter). A CT scan of the head (Panel B) showed involvement of the paranasal sinuses (arrow) and periorbital soft tissues. Material from the periorbital tissue (Panel C), stained with periodic acid-Schiff stain (×560), demonstrated irregularly shaped broad hyphae with right-angle branching (arrow).

Case 2 Zygomycetes N Engl J Med 1995; 333:564

Case 2: The most likely diagnosis is: A. Invasive aspergillosis with sinusitis B. Sino-orbital mucormycosis C. Blastomycosis D. Invasive dermatophyte infection E. Cryptococcosis

Case 2: The best treatment is: Sharpened steel: call the surgeons Treat underlying DKA with insulin drip Start ambisome IV at 5 mg/kg per day Start posaconazole at 200 mg PO QID A, B, and C.

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 Unproven approaches Iron chelation therapy with deferasirox? Combination therapy? Ampho + Echinocandins? Ampho + Posaconazole?

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 mucormycosis

Mucormycosis (a.k.a. zygomycosis) 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 Why do patients with DKA develop mucormycosis?

The Iron-pH Connection Deferoxamine: used by fungi as siderophore and therefore increased risk Serum from DKA: acidic = 69 ug/dl free iron vs. basic = 13 ug/dl better growth of Rhizopus in acidic serum due to increase iron

THM: Reverse underlying predisposing factors, call the surgeon, start ambisome

Case 3 Neutrophils A 39 year-old woman had relapse of her acute myeloid 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

Case 3: Chest CT

Case 3: What is most likely diagnosis and best treatment? Candida pneumonia; micafungin Aspergillosis; amphotericin B Aspergillosis; voriconazole Pulmonary mucormycosis; ambisome Cryptococcosis: ampho B + 5FC How would you confirm the diagnosis?

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

Who gets invasive aspergillosis? 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

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 THM: Think aspergillosis in an appropriate host with radiographic abnormalities. Dx: galactomannan, culture, histology.

Aspergillosis Treatment options What is the single best agent? Azoles: voriconazole, posaconazole, itraconazole Echinocandins: caspofungin, micafungin, anidulafungin Polyenes: Ampho B, Ambisome (liposomal), Abelcet (lipid) CombinationRx, voriconazole + anidulafungin: RCT underway What is the single best agent? Herbrecht R. et al. Voriconazole vs. amphotericin B for primary therapy of invasive aspergillosis NEJM 2002:347;408-415. Unblinded RCT. At 12 weeks, success in 53% on vori, 32% on AmphoB Survival in 71% on vori, 58% on AmphoB Much less toxicity in vori arm Fluconazole: no activity

Hazard ratio, 0.59 95 % CI, 0.40 to 0.88

Case 3: 12 days later… Treatment: voriconazole Prior CT New CT

Day 0, 4, 10 CTs Caillot 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

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 Results of 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) Small, retrospective, non-randomized, uncontrolled trial Bottom line: we need a RCT! Combination salvage therapy was associated with reduced mortality relative to voriconazole alone (HR, 0.27; 95% CI, 0.09 0.78; P = .008).

Case 4 58 y.o. woman s/p renal transplant in 12/2000 for polycystic kidney disease, on MMF, tacrolimus, and prednisone, was admitted for elective surgery Post-op CXR 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 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

Cryptococcus neoformans Yeast with worldwide distribution High concentrations in pigeon droppings, soil Thick polysaccharide capsule Sexual mould form detected: Filobasidiella neoformans Varieties of C. neoformans Serotype A : var. grubii Serotypes B and C: C. 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 Self limited pneumonia: well circumscribed lesions with surprisingly meager inflammatory response; commonly untreated in normal host 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!!) Rare lesions of bone, prostate

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 False + CRAG: Rheumatoid factor [216] Cancer. Very low-titer false-positive reactions have been reported [1043]. Infection due to Trichosporon spp. This organism produces the same polysaccharide in its capsule as is produced by the cryptococcus [1375, 1478]. Infection due to Stomatococcus mucilaginosis As with Trichosporon infections, this organism produces a polysaccharide that cross-reacts with that of the cryptococcus [410]. Infection due to Capnocytophaga canimorsus (formerly known as DF-2) Reported only once [2377], the cause of this cross-reaction is not known. Contamination during pipetting in the laboratory [999] Soaps and disinfectants use for slide washing [254] Hydroxyethyly starch (HES) for intravascular volume replacement (fluid resucitation) CSF shunting Interferon THM: Cryptococcus gattii outbreak in Pacific Northwest! Same Dx and Rx.

Antifungal Review Membrane Function Polyenes: Amphotericin B Lipid Formulation AmB (Abelcet, AmBisome) Nystatin Cell Wall Synthesis Echinocandins: glucan Caspofungin Micafungin Anidulafungin Microtubules Griseofulvin Ergosterol Synthesis Azoles: Fluconazole Ketoconazole Itraconazole Clotrimazole Voriconazole Posaconazole Squalene epoxidase inhibitors: Terbinafine Nucleic Acid Synthesis Pyrimidine analog: 5-Fluorocytosine (5-FC) Antifungal Review

Spectrum of Antifungal Agents Drug / Fungus Aspergillus spp. Candida spp. Endemic Zygos Fluconazole - + Voriconazole Micafungin Itraconazole Amphotericin Posaconazole Fluconazole has activity against Basidiobolus

References Doctor fungus: http://www.doctorfungus.org/ IDSA guidelines: http://www.idsociety.org/content.aspx?id=9088 Infections by Organism: Fungi  Aspergillus Blastomycosis Coccidioidomycosis Cryptococcal Disease  Candidiasis Histoplasmosis Sporotrichosis 

Case 5 30 year-old HIV+ man admitted 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 No hx OI

Case 5 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? Skin biopsy: Silver stain Blood cultures grew Histo at 4 weeks. He was treated with IV ampho B but expired. Giemsa blood smear

Histoplasmosis Agent: Histoplasma capsulatum Diagnosis Treatment 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 Coccidioidomycosis Coccidioides immitis & posadasii Desert Southwest: CA, AZ and Mexican border Pneumonia, Meningitis Diagnosis: cx, histology, serology (good) Rx: Flucon, AmphoB, Itra Blastomycosis Blastomyces dermatitidis Distribution: shadows histo in mid-western and SE US Pneumonia, skin, bone dz Diagnosis: cx, histology (BBBBY) Rx: Itra, AmphoB, Flucon Vori, caspo Immitis (CA) Posadasii (non-CA)

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 THM: Take a travel history, match travel to mental map of endemic fungi, consider disease presentation, order appropriate diagnostic tests to exclude