Diagnosis of Fungal Infections:

Challenges and Opportunities

Session #12

November 17, 2016

John W. Baddley, MD, MSPH

University of Alabama at Birmingham, USA

Birmingham VAMC

jbaddley@uab.edu

Disclosures

• Pfizer: trial adjudication committee

• R Pharma: DSMB

• Merck: consulting/honoraria (fungal)

Objectives

1. Understand the epidemiology of common

fungal infections

2. Describe the clinical specimens required for

diagnosis of fungal infections and current

limitations

3. Compare available and emerging diagnostic

tests for invasive fungal infections

Fungi

• There are approximately 90,000 fungal

species described

• It is estimated there are 1-5 million

species on the planet

Beneficial FungiUse Organism(s)

Antibiotics Penicillium chrysogenum, etc.

Antifungals Aspergillus nidulans, P. griseofulvin

Cholesterol Monascus purpureus

Wine Saccharomyces spp., others

Cheese Penicillium roqueforti

Beer Saccharomyces spp.

Chocolate C. krusei, Geotrichum, etc.

Bread Saccharomyces cerevisiae

Stone washed jeans Trichoderma reesii

Diagnosis of Fungal Infection

Case History

Physical Exam

Direct Microscopy

Histopathology

Culture and

Identification

Serodiagnostic

Testing

Laboratory Diagnosis Imaging Studies

Clinical Recognition

Yeast

Mould

Transbronchial Biopsy

Laboratory Specimens

• Tissue

• Blood

• Pulmonary (sputum,

BAL, pleural fluid)

• Cerebrospinal fluid

• Urine

• Bone marrow

• Skin and nail

scrapings

• Peritoneal fluid

• Synovial fluid

• Throat swabs

• Vitreous/corneal

scrapings

General Laboratory Diagnosis

• Direct microscopy and culture of all specimens• Microscopy:

Wet mounts (KOH, India ink, Calcofluor white)

Gram stain

H & E (hematoxylin and eosin)

PAS (periodic-acid shiff)

GMS (Gomori-methenamine silver)

Lactophenol blue

Specialty stains: mucicarmine

• Cultures should be maintained at least 4-6

weeks

Culture Media

• Blood agar

• Sabouraud dextrose Agar (SDA)

• Brain-heart-infusion (BHI) agar

• Potato dextrose

• Potato flake agar

• Mycobiotic agar

Diagnostic Value of Specimens

Organism CSF Sputum Biopsy Blood Urine

Tissue Marrow

Candida + + +++ ++ +

Cryptococcus +++ + +++ + +

Aspergillus - ++ +++ - -

Dimorphics + ++ +++ + +

- no value + limited value ++ frequently useful +++ very suitable specimen

CASE 1

• 26-year old man presents to his primary

care provider complaining of scratchy

throat x 1 week. Also c/o pain upon

swallowing

• No medical problems

• He is a bartender and sexually active with

men and women. Multiple partners in past

6 months.

• He smokes one pack per day

Candidiasis-OPC

• Oropharyngeal candidiasis (“thrush”)

Most frequent OI in patients with AIDS (75%)

Recurrent in 1/3 of patients

Risk factors: prior colonization, prior thrush, low CD4

Mostly due to C. albicans; C. glabrata and C. tropicalis

• Clinical manifestations: include oral discomfort, altered taste sensation, and the appearance of creamy white plaques on an erythematous base. Can also present as erythema alone.

• Locations: buccal mucosa, palate, tongue, oropharynx. Candida spp. also cause angular cheilitis

Candidiasis- OPC

• Diagnosis:Based on clinical appearance

KOH prep of scraped lesions may be helpful

Culture is helpful in refractory cases

• Treatment: topical agents or azolesFluconazole, itraconazole (70-100% response)

Clotrimazole (Mycelex troches) (65-95%)

Nystatin

KOH Preparation

Case 2

• 55 y/o man in SICU develops fever and

leukocytosis

• PMed Hx: Diabetes, hypertension

• Surg Hx: GI surgery for pancreatic fluid

collection 1 week ago

• Has foley, CVC in place; Cr 1.1

• BP 120/70, pulse 110, Temp 101.5º

• No recent antifungal use

• Blood cultures grow yeast

Candida albicans

Candida —A High Priority in the ICU:

Bloodstream Infection Pathogens

Pathogen

% BSI

(n=10,515)

Crude

Mortality, %

Coagulase-negative Staph

Staphylococcus aureus

Candida species

Enterococcus species

Pseudomonas aeruginosa

35.9 (1)a

16.8 (2)a

10.1 (3)

9.8 (4)

4.7 (5)

25.7

34.4

47.1

43.0

47.9

aP<.05 for patients in ICU vs non-ICU settings.

SCOPE data. Wisplinghoff et al. Clin Infect Dis. 2004;39:309-317.

Risk Factors for Candidemia

• Central venous catheters

• Systemic antibiotic exposure

• Length of stay in ICU >72 hours

• Major surgery, especially abdominal

• Pancreatitis

• Dialysis

• Neutropenia

• Immunosuppressive agents

• Colonization with Candida species

• Total parenteral nutrition, hyperalimentation

Distribution of Common

Candida Species

Various sources, predominantly IDSA Guidelines: Pappas et al. Clin Infect Dis. 2004;38:161-189.

Frequency

Lab Turnaround Times

Organism Culture Ident. Susceptibility Total

C. albicans 48 hrs 3 hrs 24-48 hrs 3-5 d

Other yeasts 48 hrs 72-96 hrs 24-72 hrs 4-8 d

Aspergillus 48 hrs 3 hrs-7 d 24-48 hrs 4-8 d

Other moulds 2-14 d 1-28 d 24-72 hrs 4-42 d

• Cultures

• Blood, urine, other bodily fluids

• Candida blood cultures

• ~60% positive with 2 or more organs involved at autopsy

• Newer diagnostic techniques

• β-glucan (Fungitell™)

• T2 Candida (rapidity of species diagnosis)

• MALDI-TOFF

Diagnostic Strategies

Use of Beta-D-Glucan Assay

to Diagnose Candidemia

1. Obayashi et al. Lancet. 1995;345:17-20.

2. Odabasi et al. Clin Infect Dis. 2004;39:199-205.

3. Ostrosky-Zeichner et al. Clin Infect Dis. 2005;41:654-659.

4. Mohr et al. Presented at: 45th ICAAC; Washington, DC. 2005. Abstract M-168.

Author Population Sampling Sensitivity, % Specificity, % PPV,% NPV, %

Obayashi1 Febrile

patients

Single 90 100 59 97

Odabasi2 AML / MDS Multiple, 2+ 65 96 57 97

Ostrosky-

Zeichner3

Hospitalized

patients

Single 64 92 89 73

Mohr4 ICU patients,

surveillance

Multiple, 2+ 100 63 – –

Limits of Detection for Five Candida Species

C. albicans C. tropicalis C. krusei C. parapsilosis C. glabrata

0 CFU/ml

Mean T2 41.2 44.6 33.9 37.3 32.6

CV (%) 2.8 1.2 1.4 1.0 2.0

1 CFU/ml

Detected (%) 93.8 75.0 81.3 100 93.8

Mean T2 546.5 525.9 383.2 480.1 327.8

%DT2 1226 1079 1030 1187 906

CV (%) 6.1 22.4 21.9 24.7 12

2 CFU/ml

Detected (%) 93.8 87.5 100 100 100

Mean T2 570.4 479.7 438.8 574.0 386

%DT2 1284 976 1194 1439 1084

CV (%) 6.1 30.0 12.0 6.8 12

3 CFU/ml

Detected (%) 100 100 100 100 100

Mean T2 485.4 611.9 347.3 567.4 357

%DT2 1078 1272 924 1421 995

CV (%) 5.4 11.7 10.6 8.1 11.9

LOD (CFU/ml) 3 3 2 1 2

Agreement of T2MR with Blood Culture

Using Spiked Whole-blood Samples

Data are number of samples

Science Translational Media.org 24 April 2013 Vol 5 Issue 182 182ra54

Blood Culture Total

+ ‒

90 43 133

T2MR Candida* 88 0 88

Candida 2 43 45

Positive agreement = 88/90 = 97%

Negative agreement = 43/43 = 100%

T2Candida and T2Dx: Just Minutes of

Hands-on Time

Directly load the

vacutainer

Place the vacutainer

assembly and reagent

tray on the cartridge

body

Results reported to LIS system in approximately 3 hours

T2Candida and T2Dx

• Unparalleled limit of detection: ~1 CFU/mL

• Results in approximately 3 hours – Multiple days faster than the gold standard

• Direct whole blood detection

• Species data to aid in therapy choices

• Reduced costs – ICU– Length of stay

– Inappropriate antimicrobial

therapy

Candida Resistance to Azoles

• Candida glabrata is a common cause of candidemia

(incidence up to 30%)

• Fluconazole resistance (C. glabrata) in 10-20% of isolates

• Cross-resistance to other triazoles is common

• Resistance is associated with worse outcomes (mortality,

response)

• Mechanisms: cyp51A mutations; ATP-binding cassette

(ABC) transporter and major facilitator superfamily (MFS)

proteins (CDR, CaMDr); CgPDR1 mutations

• Risk factors: antifungal exposure, age, antibacterials

Castanheira et al., Int J Antimicrob Agents 2014

Ben-Ami et al., AAC 2012; Pham et al., AAC 2014

Perlin D et al., Curr Clin Micro Rpt 2015

Lee I et al., Arch Int Med 2009

Candida Resistance to Echinocandins

• Resistance is uncommon (<3%)

• C. glabrata echinocandin resistance is increasing (up to

15%) and is increased in azole-resistant C. glabrata isolates

• Resistance among specific echinocandins similar (3.1%-

3.6%), but not always the same

• Mechanisms: FKS1 or FKS2 “hot spot” mutations; drug

efflux mediated by Cdr2p (ATP-binding cassette transporter)

• Resistance with FKS mutants is associated with treatment

failure

• Risk Factors: previous echinocandin exposure

Pham et al., AAC 2014; Beyda et al., CID 2014

Garcia-Effron et al, AAC 2009; Alexander B et al., Clin Infect Dis 2013

Perlin D et al., Curr Clin Micro Rpt 2015

CASE 3

• 50 y/o WM that came to clinic complaining of

low-grade fever and cough productive of

yellow sputum for 5 days

• PMHx: CML requiring allogeneic HSCT (90

days prior to symptoms)

GVHD of skin (HSCT +25)

• Meds: Cyclosporine, corticosteroids

History

• The mould Aspergillus was named in 1729 by the Italian priest and biologist Pietro Antonio Micheli. Under the microscope it resembled an aspergillum

• Aspergillus candidus identified from

the air sac of a Bullfinch in 1842

(Rayer and Montagne)

• First human infection in 1856 (Virchow)

• Aspergillus fumigatus described in 1863 in the bronchi of a great bustard (Fresenius)

• Over 200 Aspergillus species have been described, 40 of which are human pathogens

• We inhale 20-2000 Aspergillus spores (conidia) daily

Disease

1) Allergic response

-Allergic bronchopulmonary aspergillosis (ABPA)

-Allergic sinusitis

2) Colonization/Infection

-Aspergilloma

-Chronic cavitary pulmonary aspergillosis

-Chronic fibrosing pulmonary aspergillosis

-Semi-invasive with progression to IA

3) Other

-Tracheobronchitis

4) Tissue invasion

-Invasive aspergillosis

Diagnostics for IA

• Chest imaging: “halo sign”

• Cultures:

-Blood cultures: positive in <3%

-Respiratory tract: positive in 50-60%

-Transbronchial/open lung: positive in 55%

• Aspergillus galactomannan: Sensitivity 30-

95%; specificity 72-95%

• β-glucan: Sensitivity/specificity 70-90%

• PCR: Sensitivity 43-100%; specificity 93-100%

A. terreus

A. niger

A. flavus

A. fumigatus

A. versicolor

• Sandwich ELISA technique (using rat monclonal antibody EB-A2, which recognizes (15)--D-galactofuranose residues of the GM molecule)

• Sensitivity: 50-98%; specificity 85-99%

• Indications for testing:-Diagnosis of IA

-Monitoring during immunosuppression

-Monitoring response to treatment

• *Test is positive a median of:- 14 days before definitive diagnosis of IA

- 9 days before a positive culture for Aspergillus

- 6 days before initiation of antifungal therapy

- 6 days before CXR/CT abnormality

Platelia® Aspergillus Antigen (Galactomannan)

Courtesy of Larry Wheat*Maertens et al., JID 2002

Serum Galactomannan

• Meta-analyses of heterogeneous studies:

Pfeiffer CD, et al. Clin Infect Dis 2005;42:1417-27.

Leeflang MM, et al. Cochrane Database Syst Rev 2008;4:CD007394

Study Group Sensitivity Specificity LR+ LR-

Pfeiffer 2006 Overall

0.61

(0.59, 0.63)

0.93

(0.92, 0.94) 8.7 0.42

Pfeiffer 2006 Heme Malig

0.58

(0.52, 0.64)

0.95

(0.94, 0.96) 11.6 0.44

Pfeiffer 2006 SOT

0.41

(0.21, 0.64)

0.65

(0.44, 0.83) 1.2 0.91

Leeflang 2008 Overall

0.78

(0.61, 0.89)

0.81

(0.72, 0.88) 4.1 0.27

BAL Galactomannan

Guo YL, et al. Chest 2010;138:817-24. Zou M, et al. PLoS One 2012;7:e43347

Avni T, et al. J Clin Microbiol 2012;50:3652-8. Heng SC, et al. Crit Rev Microbiol 2015;41:124-34.

Meta-Analysis Cutoff Sensitivity Specificity LR+ LR- Context

Guo 2010 0.5-2 0.90 (0.79, 0.96) 0.94 (0.90, 0.96) 14.9 0.1

Zou 2012 0.5 0.87 (0.79, 0.92) 0.89 (0.85, 0.92) 8 0.15

Zou 2012 1 0.86 (0.76, 0.92) 0.95 (0.91, 0.97) 17 0.15

Avni 2012 0.5 0.82 (0.53, 0.95) 0.97 (0.92, 0.99) 27 0.19

Avni 2012 1 0.85 (0.63, 0.95) 0.99 (0.97, 1.0) 85 0.15

Heng 2015 0.5 0.82 (0.70, 0.91) 0.92 (0.85, 0.96) 10.9 0.19

Hematologic Malignancy

Heng 2015 1 0.75 (0.55, 0.88) 0.95 (0.87, 0.98) 16.1 0.26

Hematologic Malignancy

Heng 2015

1.5 (4

studies) 0.92 (0.48, 0.99) 0.98 (0.78, 1.00) 54 0.08

Hematologic Malignancy

Aspergillus PCR

Meta-Analysis Consecutive + Sensitivity Specificity LR+ LR-

Special

Population

Mengoli 2009 1 0.88 (0.75, 0.94) 0.75 (0.63, 0.84) 3.5 0.15

Arvanitis 2014 1 0.84 (0.75, 0.91) 0.76 (0.65, 0.84) 3.5 0.21

Heme

Malignancy

Cruciani 2015 1 0.81 (0.73, 0.86) 0.79 (0.68, 0.86) 3.9 0.24

Mengoli 2009 2 0.75 (0.54, 0.88) 0.87 (0.78, 0.93) 6 0.28

Arvanitis 2014 2 0.64 (0.38, 0.84) 0.95 (0.88, 0.98) 12.8 0.38

Heme

Malignancy

Cruciani 2015 2 0.58 (0.37, 0.77) 0.96 (0.90, 0.99) 14.5 0.44

Mengoli C, et al. Lancet Infect Dis 2009;9:89-96.

Arvanitis M, et al. J Clin Microbiol 2014;52:3731-42.Cruciani M, et al. Cochrane Database Syst Rev. 2015 Sep 7;9:CD009551.

Lateral Flow Device

• Serum: sensitivity and specificity comparable to serum GM

• BAL: comparable to BAL GM in solid organ transplant recipients

(relatively high sensitivity), ICU patients, hematologic malignancy (lower

sensitivity in the context of antifungal therapy), lung disease

Pan Z, et al. J Med Microbiol 2015;64:702-7.

White PL, et al. J Clin Microbiol 2013;51:1510–6.

Hoenigl M, et al. J Clin Microbiol 2014;52:2039-45.

Dufresne SF, et al. PLoS One 2015; 7:e42736.

Matrix Sens Spec LR+ LR-

BAL 0.86 (0.76, 0.93) 0.93 (0.89, 0.96) 12.3 0.15

Serum 0.68 (0.52, 0.81) 0.87 (0.80, 0.92) 22.7 0.33

Willinger B, et al. Transplantation 2014;98:898-902.

Eigl S, et al. Crit Care 2015; 19:178.

Prattes J, et al. Mycoses 2015;58:461-69.

Prattes J, Am J Resp Crit Care Med 2014;190:922-9

Breath sampling in patients

with suspected fungal pneumonia

Metabolite overlap between breath and A.

fumigatus cultures

Patient Breath (VOCP001)In-vitro Culture

A: a-pinene

B: camphene

C: limonene

D: a-trans-

bergamotene

E: -trans-

bergamotene

A

A

B

B

C

C

D

DE

E

Aspergillus metabolites in breath

Koo S, et al. Clin Infect Dis 2014; 59:1733.

Sens: 0.94 (0.81, 0.95)

Spec: 0.93 (0.79, 0.98)

LR+ 13.4, LR-: 0.06

Resistance in Aspergillus

Itraconazole R - first described in 1997

Specific mutations in CYP51A (azole

target)

– Global emergence of point mutations

with TR in promoter region

(TR34/L98H &

TR46/Y121F/T289A)—environmental

– Specific “hot-spots”:

G54, L98, G138, M220, G448

Over expression of CYP51B

Efflux pump: cdr1B, atrF

“Pan” azole resistant isolates

now described – cavitary disease,

also in azole naïve patientsBuied A, et al. J Antimicrob Chemother. 2010 Oct;65(10):2116-8.

Fraczek MG, et al. J Antimicrob Chemother. 2013 Jul;68(7):1486-96. Buied A, et al. J Antimicrob Chemother. 2013 Mar;68(3):512-4

Verweij PE, et al. NEJM 2007;356:1481-3.

Slide adapted from GR Thompson. Verweij et al., Lancet Infectious Diseases 2009

CASE 4

• 52-year-old renal transplant patient with fever,

chills, cough, malaise for 1-2 months

• Presented to OSH and diagnosed with

pneumonia, UTI, AKI, CHF exacerbation

• Received multiple antibiotics

• Transferred to UAB for continued care

• Renal tx 12/2007; HTN, DM, CHF

• Soc: lives in Selma, AL; no tobacco/drugs, or

sick contacts. Had influenza vaccine

• No CMV, PJP prophylaxis

CASE 4, con’t

• BAL: forms c/w Histoplasma capsulatum

• Blasto antigen positive

• Histo antigen positive (12.1)

• BAL grew H. capsulatum

Histoplasmosis

• Caused by the dimorphic fungus Histoplasma

capsulatum

• Discovered by Darling in 1904. Other names:

Darling’s disease, Ohio Valley Disease

• Endemic to Ohio and Mississippi River Valleys,

also in parts of Central and South America. Seen

in South Africa

• Found in the soil; associated with bird roosts,

caves, excavation, and rivers

• Acquired by inhalation of spores

• Person-to-person transmission by organs only

Suitability model included three geographic layers: the 2006 National Land Cover Database, soil pH and Euclidean distance fro m the nearest open water. Layers were weighted 70% for land utilization, 20% for distance from water and 10% for soil pH. Our suitability score model predicted states

with high and mid-to-high histoplasmosis incidence with an AUC of 0.72 and 0.74, respectively, when compared to state -based CMS data.

Diagnosis

• Definitive test is culture from clinical samples

• Grows in up to 6 weeks

• Histopathology shows 2-4 m oval, budding

yeasts, often intracellular in macrophages. Seen

best on GMS staining

• Antigen testing is most useful in patients with

disseminated disease

• Antibody testing (comp. fix, EIA) also sensitive in

acute and chronic pulmonary disease, but may

take up to 6 weeks to develop antibody response

FINAL CASE!

• 42-year-old lady with a history of renal

transplantation

• She was driving her car home and ran into a

house. At the scene, she did not remember that

this happened

• Taken to UAB and admitted for evaluation

• Bad headache x 2 weeks

• Fever on admission

• CT of brain was negative

• CXR showed multiple small nodules

Cryptococcosis

• Caused by the encapsulated, budding yeasts Cryptococcus neoformans or Cryptococcus gattii

• These fungi are particularly abundant in soil contaminated by pigeon droppings

• C. gattii infection has been described throughout the world but endemnicity has primarily been limited to tropical and subtropical regions. A notable exception is the emergence of C. gattii in Vancouver, Canada and the pacific northwest United States since 1999

• Pulmonary infection results from inhalation of the organism from an environmental source, and the organism has a propensity to metastasize to the central nervous system

• Rare cause of pneumonia, with incidence of <0.1%

Cryptococcosis: Pulmonary Disease

Clinical features:

-Symptoms: Cough, fever, malaise, chest pain, weight loss, dyspnea, night sweats, and hemoptysis

-CXR with focal or diffuse infiltrates, nodules, or cavitary lesions

A significantly lower prevalence of symptoms has been described in immunocompetent versus immunocompromised patients, with as many as 25% of HIV-negative patients with pulmonary involvement lacking respiratory symptoms

Cryptococcal Meningitis

-Headache, fever, malaise, lethargy, altered mentation

-Symptoms are usually present 2-4 weeks before diagnosis

-May have cranial nerve palsies, hyperreflexia

-Much more common in HIV-infected patients (when compared to HIV-negative)

Diagnosis• Definitive diagnosis is made by culture. Best yields will occur

from bronchoscopy with BAL, transbronchial biopsy or open lung

biopsy.

• C. neoformans can grow on most bacterial and fungal culture

media and can be detected after 2-7 days of growth

• On histopathology, Cryptococcus is recognized by its oval shape

and narrow-based budding. With use of the mucicarmine stain,

the cryptococcal capsule will stain rose to burgundy in color and

help to differentiate it from Blastomyces dermatiditis, and

Histoplasma capsulatum

• Cryptococcal antigen testing: in HIV-negative patients with

pulmonary cryptococcosis, serum cryptococcal antigen will be

positive in 25-56%

• Patients with extrapulmonary and pulmonary cryptococcosis are

more likely to have higher antigen titers when compared to

patients with only cryptococcal lung disease

Diagnosis (2)

-CSF shows lymphocyte predominance, elevated

protein, low glucose

-Lumbar puncture opening pressure is elevated in

>50%

-India ink shows thick capsule (positive in 50-80% of

patients with AIDS and cryptococcal meningitis)

-CSF cryptococcal antigen (positive in 99%)

-CSF, blood cultures often positive

Mucicarmine staining

Cryptococcal Antigen Lateral Flow Assay

CrAg LFA

• Qualitative

• Semi-Quantitative (titer)

20ul ; incubation

at 5, 15 min

THANKS!