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

2. 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

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

5. 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

6. 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.

7. 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

8. 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 , 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.

9. 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

10. 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

11. 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

12. Fluconazole vs. AmB for Candidemia
Study population: 237 non-neutropenic, immunocompetant patients with candidemia
206 evaluable subjects ( )
AmB 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:

13. 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

14. 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

15. Guidelines for Therapy of Candidemia
Not neutropenic, no prior azoles, germ-tube positive (C. albicans)
Fluconazole at mg/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!

16. 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

17. 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).

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

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

20. 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.

21. 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?

22. 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 = )
Limitations: retrospective, selection bias
Tedder M. et al. Ann Thorac Surg 1994;57:

23. 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

24. 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?

25. 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

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

27. 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

28. Case 3: Chest CT

29. 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?

30. 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

31. Who gets invasive aspergillosis?
Underlying lung disease
Hematological malignancy, chemotherapy
Immunosuppression
Steroids
Neutropenia
Chronic granulomatous disease, AIDS
Transplantation
Solid organ
Hematopoietic stem cells

32. 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

33. 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 % 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.

34. 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; 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

35. Hazard ratio, 0.59
95 % CI, 0.40 to 0.88

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

39. 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:

40. 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:
MacCallum DM et al. AAC :
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: )
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, ; P = .008).

41. 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

42. Post-Op

43. 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.

44. Frozen Section

45. Wedge resection - Mucicarmine Stain

46. 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

49. 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

50. 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

51. 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.

52. 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

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

54. References Doctor fungus: http://www.doctorfungus.org/
IDSA guidelines:
Infections by Organism: Fungi 
Aspergillus
Blastomycosis
Coccidioidomycosis
Cryptococcal Disease 
Candidiasis
Histoplasmosis
Sporotrichosis 

55. 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

56. 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?

57. 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

58. 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
banq-im/Images/cyto46.jpg

59. 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)

60. 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