Fungal Infections

Peter Heseltine MB, MA, BCh, FACP, FIDSA, FAACC

Professor of Clinical Medicine UCI

peter.heseltine@uci.edu

ZOT

2

Conflict of Interest

None for this lecture

Goals & Objectives

Recognize risk factors and presentations of

serious fungal disease:

Candida, Aspergillus, Cryptococcus, Mucor

Histoplasmosis, Blastomycosis, Coccidioidomycosis

Apply fungal diagnostics in clinical practice

Galactomannan, beta-D-glucan

Understand treatment choices for fungemia,

pulmonary & CSF infections

echinocandins vs azoles vs polyenes

3

Current State of Mycology

Overall Mortality

Invasive Aspergillosis: 20-60%

Candidemia: 30-60%

Other Molds: ~80%

Delay in recipt of therapy is asociated with

mortality

Aspergillus (Cordonnier CID 2009)

Candida (Garey CID 2006; Morrell AACC 2005)

Mucormycosis (Chamilos CID 2008)

4

Cordonnnier C Clin Infect Dis 2009 48:1042-51. Garey KW Clin Infect Dis 2006 43:25-31

Morrell Antmicrob Agents Chemother 2005 49:3640-3645

Chamilos G Clin Infect Dis 2008 47:503-9

Populations At Risk / Risk Factors

Non-immunocompromised

Broad spectrum antibiotics

Hemodialysis

Central venous catheter

IV drug use

Total parenteral nutrition

GI perforation or surgery

Colonization

Diabetes

LOS in ICU

Pancreatitis

Sepsis

5

Immunocompromised

Neutropenia

Stem Cell Transplant

Mucositis

GVHD

Chemotherapy

Organ transplant

AIDS

Neonates

Gestational age

H2 blockers

Candidemia Management: Whom and When to Treat?

6

No disease

Cultures/

antigen

Signs and

symptoms

Cultures/

histopathology Sequelae

Prophylaxis Pre-emptive Empiric Treatment Morbidity/

mortality

Broad spectrum antibiotics

Catheters

Neutropenia, steroids

Surgery, ICU, etc

Crude mortality 40%

In hospital mortality

doubles if antifungal

therapy is not started

within 12 hours*

*Morrell. Antimicrob Agents Chemother 2005 49:3640. Slide courtesy of Dimitrios Kontoyiannis

High-risk

neutropenic

patient (chemo,

HSCT)

Candida Infection/Septic Shock Importance of Empiric Therapy and Source Control

7

P < .001 for the comparison of patients receiving both antifungal therapy and

adequate source control within 24 hours of the onset of shock to the other 3 groups.

Kolleff MH CID 2012;54:1739

Rex summary of updated IDSA Candidiasis guidelines.ppt 8

Catheter Exchange? Yes!

Lots of consistent data

Without catheter removal, 82% had persistent

infection

Lecciones, Clin Infect Dis 1992;14:875-883

Shortened duration of fungemia from 5.6 to 2.6 days

P < 0.001 (Rex, Clin Infect Dis 1995;21:994-996)

Reduced mortality: 41% to 21%

P < 0.001 (Nguyen, Arch Intern Med 1995;155:2429-2435)

Especially true for C. parapsilosis

Very strong link with catheters

Kojic, Clin Microbiol Rev 2004;17:255-267

Rex summary of updated IDSA Candidiasis guidelines.ppt 9

Catheter Exchange? Other Sources

~ 15% of candidemia patients have another

obvious source (urine, abscess)

• The gut may

be a cryptic

source

• What does

this imply?

10

Catheter Exchange? Definite Maybe

In non-cancer patients, suspect the catheter

Unless another source is apparent

After cytotoxic chemotherapy, think twice

Gut may be source, effect of removal is less?

C. parapsilosis is the clear exception

More data & better tools needed here

Differential quantitative and time-to-growth central/peripheral BC

Bouza, Clin Infect Dis 2007;44:820-6.

We keep wishing for good serodiagnostic tools

We have several (beta-glucan, PCR) but none have produced

high levels of confidence

Kedzierska, Eur J Clin Microbiol Infect Dis 2007;26:755-766

Nett, J Infec Dis 2007;195:1705-1712.

Candida diagnosis

Blood culture

50-60% sensitivity for IC

Median 1CFU/mL

Slow

2-5 days

1,3-β-D Glucan

SN 81% (95%CI; 77-

85%)

SP 81% (95% CI; 80-

83%

PCR

SN 95% (95% CI;88-98%

SP 92% (95CI; 88-95%

Deep-seated SN 88%

11

Candida Colonization

Deep-seated

Candidiasis Candidemia

Invasive Candidiasis

Pfeiffer J Clin Micro 2011;49:2879 Avni J Clin Microbio 2011;49:665 Nguyen Clin Infect Dis 2012;54:1240

Candida:T2 v β-D-glucan Importance of disease prevalence

Prevalence of

disease

T2 bGD

PPV NPV PPV

1% 32.6% 99.9% 1%

2% 49.4% 99.8% 8%

5% 71.6% 99.5% 10%

10% 84.2% 99.0% 30%

20% 92.3% 97.8% 42%

35% 96.3% 95.3% 61%

50% 98.0% 91.6% 80%

NPV, negative predictive value. PPV, positive predictive value

12

Mylonakis E Clin Infect Dis 2015;60:892

T2 Candida Assay Whole blood sample

(hands on 5 mins)

Results in 3-5 hours

Five species detected

2046 subjects

SN 91.1% & SP 99.4%

12% invalids*

LOD 1 cfu/mL

13

Wilson NM Poster Presentation IDWeek 2016.

Mylonakis E Clin Infect Dis 2015;60:892

14

Other settings

Thinking of non-neutropenia as the start point

The less you know or

The more the patient scares you

The more the guidelines point to

An echinocandin

A (lipid-associated) amphotericin B

But, for resource constrained settings…

The guidelines do note that classic AmB works

15

Empirical Therapy

In the febrile non-neutropenic patient?

Early treatment is theoretically attractive

IDSA Guidelines

“The specific basis for selecting non-neutropenic patients who

should receive empiric antifungal therapy is unclear, but should be

based on at least one of the following: clinical assessment of risk

factors, serologic markers for invasive candidiasis, and/or culture

data from non-sterile sites (BIII).”

My rules

Antibiotics, lines, no other source, and…

Colonized somewhere with Candida

I don’t distinguish sites: anywhere works for me

Prophylaxis? Even hazier

16

Candiduria

Asymptomatic candiduria

No treatment unless high-risk dissemination (AIII).

Focus on elimination of predisposing factors. (BIII).

High risk for dissemination

Urologic manipulations (BIII)

Use short course fluconazole or even amphotericin B

Neutropenic patients and low birth weight infants

Treat as for invasive candidiasis.

Consider imaging kidneys/collecting system (BIII)

17

Key organism principles

Helpful to know tiers of progressive difference

(most virulent, most susceptible): albicans, parapsilosis, tropicalis,

dubliniensis

(intermediate): glabrata - do susceptibility tests!

(least virulent, least susceptible): krusei

You often know quickly if C. albicans

It’s the one that is “germ tube-positive”

Just knowing species is very helpful

P = Plastic = parapsilosis. Look for the device!

Resistance patterns

C. glabrata & C. krusei: Azoles are dicey. Newer azoles are better

C. parapsilosis: Echinocandins sometimes a little weaker than fluconazole

C. lusitaniae: Amphotericin resistance is frequent

C. guilliermondii: higher azole and candin MICs

C. auris: special infection control precautions

18

Key drug principles Voriconazole

Fatty meal reduces absorption

IV form uses cyclodextrin: not well cleared by dialysis

Lots of drug-drug interaction

Echinocandins

Very few convincing differences

Usually cross-resistant – but not always! (emerging data)

Amphotericins

Liposomal amphotericin B = AmBisome

Amphotericin B lipid complex = ABLC = Abelcet

Amphotericin B colloidal dispersion = ABCD = Amphotec

(Classic ampho = Amphotercin B deoxycholate = Fungizone)

19

IDSA Candida guidelines Update 2016

Echinocandins (anidulafungin, caspofungin, micafungin)

preferred choices for proven/suspected invasive disease

There is little distinction made among the echinocandins

Lipid-formulation amphotericin B is alternative

Azole resistant C. glabrata, C. auris

Concept of step-down therapy is strongly encouraged

Fluconazole or Voriconazole (C. krusei) advised as step down

therapy for selected isolates

Fluconazole (oral) for immunocompetent patients

Fluconazole prophylaxis limited to high risk sites

All patients who have candidemia, should undergo an

ophthalmologic examination by an ophthalmologist to

look for evidence of endophthalmitis

Cryptococcus: Epi and mortality Burden of common life-threatening

fungal infections:

25 million in Sub-Saharan Africa with HIV

4-12% infected with Cryptococcus

Responsible for 50% of HIV-related mortality

0.5 – 1 million deaths annually (TB ~ 350,000)

20

Cryptococcus: Current Diagnostics

CSF India Ink preparation

~80% sensitive

Dependent on fungal burden

Cultures – standard media

CGB media

+ C. gattii v. -C. neoformans

Cryptococcal antigen testing

(CrAg) >90% sensitive

Clear differences between developing world and

elsewhere in type of test & utility

21

Klein K J Clin Micro 2009;47:3669

CrAg (GXM) testing: new method - LFA

Stored at room temperature

No specimen pretreatment

required - 40MuL required

Results in 10 minutes

Qualitative and semi-

quantitative

Serum or CSF

Improved sensitivity for

serotype C

22

Cryptococcus: asymptomatic screening

Antigenemia precedes

symptoms by median of 22

days (range, 5-234)

Those with CD4<100 reflex

CrAg testing

Using screening with 8% incidence

$21US/year of life gained

NNS to detect CrAg+ = 11

NNS to prevent death CM=16

Compare NNS: Colon CA: 1000

Breast CA: 543

23

Kozel TR Expert Opin Med Diagnn 2012 May 6(3) 245-51. Boulware DR Emerg Infect Dis 2014 Jan; 20(1) 45-53

Cryptococcal screening algorithm

24

Cryptococcal screening algorithm

Smith RM, Nguyen TA, Ha HTT, Thang PH, Thuy C, et al. (2013) Prevalence of Cryptococcal Antigenemia and Cost-Effectiveness

of a Cryptococcal Antigen Screening Program – Vietnam. PLOS ONE 8(4): e62213. doi:10.1371/journal.pone.0062213

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0062213

25

Biofire Film Array Meningitis/Encephalitis Panel

Tests CSF for 14 pathogens

Simple: 2 minute “hands-on”

time

Easy: no precise measuring or

pipetting required

Fast: TAT ~ 1 hour

Bacteria

E. coli K1

H influenzae

Listeria monocytogenes

N meningitidis

S agalactiae

S pneumonia

Viral

CMV

Enterovirus

HSV 1 & 2

HHV-6

Human parechovirus

VZV

Fungi

Cryptococcus neoformans/gattii

26

Hypersensitivity reaction to Aspergillus fumigatus mold that has

colonized bronchi of patients with asthma or cystic fibrosis (genetic

defects).

Exposure of atopic people to fungal spore elements results in the

formation of IgE (50%) and IgG antibodies. Galactomannan is

usually negative, and 1,3-betaD-glucan is often positive

Presents as asthma complicated by bronchial obstruction, fever,

malaise, expectoration of brownish mucus plugs, peripheral blood

eosinophilia, and hemoptysis.

Treatment involves environmental control measures, corticosteroids,

and itraconazole, voriconazole or posaconazole

Early detection and treatment can prevent the development of

bronchiectasis or pulmonary fibrosis that otherwise occurs in the

later stages of the disease.

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Allergic bronchopulmonary aspergillosis

Allergic bronchopulmonary aspergillosis

28

Aspergillus

Galactomannan Serum

Proven disease: Sens 68-74%; Spec 90-99%

With proven & probable Sens 59-63%; Spec 92-94%

Performs best in hematologic malignancy >> transplant, ICU

BAL – Sens 50-70%; Spec 73%

1,3-β-D-glucan Sens 55-95%; Spec 77-96%

Non-specific – negative in Cryptococcus and Mucormycosis

False positives in heavily filtered products (albumin, IVIG, etc)

gauze, etc

Direct Comparison Higher specificity of GM (97 v 82%) but lower sensitivity (81 v 49%)

29

Erwig LP Nature Rev Micro 2016 14:163. Pfeiffer CD Clin Infect Dis 2006; 42: 1417

Koo Clin Infect Dis 200; 49:1650 Sulahian A J Clin Micro 2014;52:2328

Pip/tazo false

positives greatly

reduced

Galactomannan and 1,3-β-d-Glucan Testing for the Diagnosis of Invasive Aspergillosis

High-risk patients with heme malignancies and

chemotherapy-induced neutropenia or allogeneic HSCT:

both tests have a similar performance with a limited sensitivity

(60%–80%) and a specificity ≥90%.

Two consecutive positive tests results gives high

specificity (95%–99%), with a slight loss of sensitivity.

Insufficient data:

Solid-organ transplant recipients and others at low or moderate

risk of IA. Sensitivity <40% due to limited angio-invasion?

βdG test specificity poor in lung transplant patients.

? GM in pediatric patients ~ adults

Aspergillus-Lateral-Flow Device – POC, BAL

30

Utility of GM kinetics Serial eval to monitor progression

& response to therapy

Clinical assessment can be

obscured

Neutropenia/compromised host

Deconditioned patient

Delayed radiographic improvement

If GMI >0.5, decline of 35%

correlates with clinical success

If GMI < 0.5, no significant change

over 4 weeks = good outcome

Every 0.1 unit increase = 21%

higher rate of failure

Promising…remains investigational

31

Chai LYA et al. J Clin Micro 2012;50:2330 P. Pini et al. Infection 2016;44:223

GM index decrease during the month after the cessation of

antibiotherapy. The solid line represents the population

average kinetics as estimated by use of a nonlinear mixed

regression model with a pointwise 95% confidence band

(shaded region). Circles represent individual GM index values

measured during this period, and the dashed line represents

the 0.5 threshold for positive indices.

Limitations of antigen assays in the diagnosis of invasive fungal disease

Galactomannan β-D-glucan

Reactivity with

fungal species

Aspergillus spp., Fusarium spp., Paecilomyces

spp., Acremonium, spp., Penicillium spp.,

Alternaria spp., Histoplasma capsulatum,

Blastomyces dermatitidis, Cryptococcus

neoformans, Emmonsia spp., Wangiella

dermatitidis, Prototheca, Myceliophthora,

Geotrichum capitatum, Chaetomium globosum

Pneumocystis jirovecii, Aspergillus spp., Fusarium

spp., Histoplasma capsulatum, Candida spp.,

Acremonium spp., Trichosporon sp., Sporothrix

schenkii, Saccharomyces cerevisiae, Coccidioides

immitis, Prototheca

False-positive

test

results

Semi-synthetic b-lactam antibiotics*

Multiple myeloma

Blood products collected using Fresenius Kabi

bags Flavoured ice-pops/frozen dessert

containing sodium gluconate

Semi-synthetic b-lactam antibiotics*

Human blood products, including

immunoglobulins, albumin,plasma, coagulation

factor infusions, filtered through cellulose

membranes

Cellulose hemodialysis/hemofiltration membranes

Gluconate-containing plasma expanders (e.g.

Plasmalyte)

Exposure to (surgical) gauze

Bacterial bloodstream infections (e.g.

Pseudomonas)

False-negative

test

results

Concomitant use of mold-active antifungal

agents

Mucolytic agents (bronchoalveolar lavage) such

as Sputasol or SL solution

Concomitant use of antifungal agents

32

Maertens JA J Antimicrob Chemother 2016; 71 Suppl 2: ii23–ii29

Aspergillus: Utility of PCR

Emergence of useful test

in diagnosis of IA

Odds ratio comparable to

GM and b-D-glucan

Potential Advanteges

Finds SOME resistance

mutations

Identify to species level

Disadvantages

Validation method in

process

NOT yet available in US

Performance characteristics (DX)

Test Source Sens % Spec %

GM Blood 79 80-86

BAL 83-85 89

b-D-

glucan

Blood 56-77 81-97

PCR Blood 84-88 75-76

BAL 76-80 93-94

Culture Tissue 25-50 100

33

White PL Clin Infect Dis 2015;8:1293. Chong J Antimicrob Chemo 2016 Aug 15

Aspergillus – Resistant Isolates

Triazole resistant isolates

Specific mutations in CYP51A

(azole target)

Global emergence of point

mutations with TR in promoter

region (TR34/L98H &

TR46/Y121F/T289A)

environmental

Specific hotspots: G54, L98,

G138, M220, G448

Overexpression of cyp51B

Efflux pumps: CDR1B, ATRF etc

Cholesterol import: SrbA – import

of cholesterol as ergosterol

substitute

GOF mutation in HapE (P88L)

PCR will catch “some” of these

isolates

New pathways for RES continually

discovered

Genotype v Phenotype misses

some RES isolates

34

Buled A J Anticrob Chemother 2010;65:2116 Fraczek MG J Antimicrob Chemother 2015;68:1486

Buled A J Anticrob Chemother 2013;68:512 Gregson L Antimicrob Agents Chemother 2013;57:5778

Surveillance for Azole-Resistant Aspergillus fumigatus,

United States, 2011–2013

Cryptic Aspergillus spp. Leading causes of IA

A. fumigatus (45-60%)

A. flavus (6-10%)

A. terreus (4-10%)

A. niger (2-9%)

Molecular tools led to description of new spp.

Cryptic or sibling spp. difficult to classify by phenotype/morphology

TRANSNET (US 2001-2006)

11% cryptic spp among 218 Aspergillus isolates

A. lentulus (1.8%) A. udagawae (1.4%) A. tubingensis (2.8%) A. calidoustous (2.8%)

FILOP (Spain 2010-2011 –

14.5% cryptic spp among 323 Aspergillus isolates

A. lentulus (1.1%) A. alliaceus (1.1%) A. tubingensis (7.9%) A. calidoustus (1.4%)

35

High wild-type MICs to azoles

Rivero-Menendez J Fungi 2016;21

Aspergillus Treatment strong recommendations

Primary treatment with voriconazole

? Combination with echinocandin (caspofungin)?

Initiate antifungal therapy when IPA is strongly

suspected, while a diagnostic evaluation is

conducted.

Primary therapy with an echinocandin is not

recommended

Treat IPA for a minimum of 6–12 weeks,

Secondary prophylaxis should be initiated to

prevent recurrence in the immunosuppressed.

36

IDSA Guideline 2016; ATS Guideline 2011

Necessity of TDM

Accurate and

cost effective;

available

Therapeutic

range

Inter/intra patient

variability

Voriconazole

? Posaconazole

Intraconazole

? Isavuconazole

37

Wiederhold NP Antimicrob. Agents Chemother. 2014;58:424-431

Bloodstream concentration distributions measured by UPLC/MS (A,B,C), and itraconazole/hydroxyitraconazole as measured by bioassay (D).

Nathan P. Wiederhold et al. Antimicrob. Agents Chemother.

2014;58:424-431

Distribution of voriconazole concentrations within the cerebral spinal fluid (CSF) (A) and voriconazole bloodstream

and CSF concentrations in matched clinical samples (B).

Molds that cause Mucormycosis Produce Large Ribbon-Like, Irregular Aseptate Hyphae

58 Cx-proven mold

infections – 4 tertiary

centers; blinded

assessment

Aspergillus infections

were misidentified as

MCR in 11%

Concordance between

histopath and culture for

non-mucormycosis

cases: only 68%

39

Haurany N 4th Advances Against Aspergillosis 2010 Rome,

Italy Poster #78

Taxonomic organization of the zygomycetes

40

Ribes JA Clin Micro Rev 2000;13:236

Mucormycosis Risk Factors

Uncontrolled diabetes

IED blast injuries

Deferoxamine

Severe burns

Lymphoma

Organ transplantation

Cancer chemotherapy

Corticosteroids

41

Mucormycosis: problems in diagnostic testing

Lung Disease

Very similar to Aspergillosis radiographically

(exceptions such as no of nodules, reverse halo sign)

Antigen tests not well validated,

Diverse genera cause disease

PCR not well validated

Serum 1,3-β-D-glucan not helpful

25% of sputum or BAL positive pre-mortem

Species identification

Specialized reference labs

MALDI-TOF MS not yet validated

Susceptibility testing

Reproducible? Breakpoints? Improve outcomes?

42

Mucormycosis: Prognostic Factors

Series of 391 patients with

heme malignancy, most die

with 12 weeks

The primary sites of infection

lungs (85%), nose and paranasal

sinus (10%), and other sites (5%).

The diagnosis was made only

at autopsy in 81 patients (21%).

Infection with Cunninghamella

spp – 2.78 fold increase in risk

of death (95% CI, 1.11-6.96;

p=0.029)

High MICs more common in

Rhizopus and Cunninghamella

43

Pagano Haematologica 2001;86:862

Kontoyiannis et al. BMC Infect Diseas 2016;16:730

Roden Clin Infect Dis 2005;41:634

44%

22%

5%

9%

4% 3%

3% 2% 8%

Mucorales infections

Rhizopus oryzae

Rhizopus microsporus

Lichthelmia corymbifera

Mucor circinelloides

Rhizopus pusillus

Cunninghamella bertholletiae

Mucor indicus

Apophycomyces

Other

Coccidioidomycosis

Affects 150,000 / yr - 1/2 to 1/3 subclinical

Cause of CAP in 17-29% of patients in endemic areas

CMS data: 10% of cases diagnosed outside endemic region

No definitive IDSA or IDSA/ATS CAP guideline for testing

44

https://www.cdc.gov/fungal/diseases/coccidioidomycosis/statistics.html 2017

Baddley JW Emerg Infect Dis. 2011; 17:1664

Coccy diagnostics Culture/Histology

Culture: definitive, laboratory hazard

Histopath dx: characteristic forms in tissue

Serologic diagnosis

ID/CF: used to establish diagnosis

Dissem infections ID/CF titers >1:16

+CSF ab: meningeal infection

EIA: sensitivity, false+; x-react w/other

endemic fungi – good for rapid screen

IgM has good PPV for acute infection

Skin Test

Spherulin replaces coccidioidin ST

Spherulin reactive in ~1/3 more individuals

than coccidioidin

Prior therapy with an azole antifungal did not

appear to alter the sensitivity of the test

45

Thompson GR J Clin Micro 2012;50:3060 Thompson GR Chest 2012;143:776

Kozell TR Cold Spring Harb Perspect Med 2014;4:a019299

Wack EE, Clin Infect Dis. 2015;61:787-91.

Emerging Diagnostics

Lateral Flow Assay

Developed specifically to

improve TAT

Simple to use

Yes/No answer in Urgent

Care/Clinic/ER

IgM and IgG

Semi-quantitative

MVista® Coccidioides

Antigen Quant EIA

CSF, Serum

Lab developed test

Also Blastomyces,

Histoplasma AG

46

Histoplamosis: cases/100,000 person-years

Blastomycosis: cases/100,000 person-years

MVista® Antigen Quant EIA

Histoplasmosis

Consider for “culture-negative” TB syndromes

Consider in differential for sarcoid

Galactomannan: test both blood and urine

Treatment: itraconazole or amphotericin

Measure itraconazole levels (1-3 mcg/mL)

Posaconazole for salvage therapy

Fluconazole NOT recommended

Echincandins NOT effective in animal models

47

2007 update by the Infectious Diseases Society of America.

Blastomycosis

All patients with disseminated

blastomycosis need a bone scan

to detect occult osteoarticular dx

Genitourinary infection may be

asymptomatic or associated with

symptoms of prostatism – urine culture!

Extrapulmonary blastomycosis can occur in the absence of

lung disease.

Most patients require therapy: Itraconazole

“Current serologic assays serve no role” (MKSAP)

48

2008 update by the Infectious Diseases Society of America.

Sporotrichosis

49

Exposure

Roses etc.

Cats!

Diagnosis

Culture, histopath

Treatment

Itraconazole

SSKI

Exserohilum rostratum

Exposure

Contaminated lots of methylprednisolone acetate

from a single compounding pharmacy

Treament:

Voriconazole + liposomal Amphoterecin B

50

CBS/AP October 25, 2012, 5:02 PM

323 infected in meningitis outbreak as experts study fungal culprit

Barry J. Cadden, the onetime president of a booming drug

compounding company, is on trial on charges of racketeering

and 25 counts of second-degree murder. Cadden's now defunct

company, the New England Compounding Center, produced the

fungus-riddled drugs that sickened ..more than 700. It killed 76.

USA Today Feb. 2, 2017

Pan-fungal PCR Broad range amplification/detection of nucleic acid

Useful when:

Culture negative or not sent

Slow growing organisms

Variable sensitivity (SN)

Fresh tissue v. FFPE

Culture proven 86-94%

Histologically proven 64-89%

Specificity (SP) is a concern

Fungal DNA contamination

Database issues

52

• Rhizopus oryzae, Rhizopus microsporus

and the genus Mucor spp. in both culture

and clinical samples.

• Molecular beacon species-specific probes

with an internal control.

• This multiplex real-time PCR (MRT-PCR)

was tested in 22 cultured strains and 12

clinical samples from patients suffering

from a proven mucormycosis.

• Results showed 100% specificity and a

detection limit of 1fg of DNA per microlitre

of sample.

Bernal-Martınez L Clin Microbiol Infect. 2013;19:E1

Alternative methods Not hypothesis driven

Proteomics

Profiling/Fingerprinting

MALDI/TOF

Patterns of antibodies

Pan-Fungal Detection of b-

Glucan

favorable NPV

Able to detect multiple pathogens

PCR fungal ribosomal targets

53

The variable regions can provide information that discriminate to the

species level, depending on genus, include ITS1, ITS2, and D1/D2.

Diagnosis: Can imaging help?

A fumigatus labeled

monoclonal antibody

Allowed specific

localization of lung

infection when combined

with PET (in vivo

imaging)

Able to distinguish from

pneumococcus and

other bacterial

pathogens

54

Rolle AM PNAS 2016;113:E1026-33

Current Diagnostics Infection

Culture

/histopath

Biomarker for Diagnosis Response

To Therapy

Antibody

Antigen/marker

Aspergillosis Yes –

invasive

No-invasive

disease

Galatomannan/β

-D-glucan/PCR

Increasing

evidence

Candidiasis Routine Investigational

(anti-mannan)

PCR mannan/β-

D-glucan/PCR

No

Cryptococcus Routine No Yes/PCR Yes (CSF

antigen)

Histoplasmosis Culture-

delay

Limited Yes Yes

(Antigen)

Coccidioidomycosis Culture-

delay

Yes Antigen in CSF Yes

(Antibody)

Paracoccidioidomycosis Culture-

delay

Yes Yes Unknown

Mucormycosis Yes-

invasive

No Investigational No

Other molds Yes-

invasive

No Investigational No

55

56

What’s Learned?

Diagnosis: Targeted populations

New monitoring tests

Prognosis: Variability

Treatment: New agents