Pentalfa3 maart 2016 - KU LeuvenPentalfa3 maart 2016 7/03/2016 2 Conventional Tests Histopathology Direct Microscopy Culture Mass Spectrometry (MALDI‐TOF) Susceptibility testing

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Diagnostiek van schimmelinfecties

Katrien Lagrou, Laboratoriumgeneeskunde, UZ Leuven

Pentalfa 3 maart 2016

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Con

vent

iona

l Te

sts

Con

vent

iona

l Te

sts

Histopathology

Direct Microscopy

Culture Mass Spectrometry  

(MALDI‐TOF)

Susceptibility testing 

Ant

igen

/Ser

olog

ical

/DN

AA

ntig

en/S

erol

ogic

al/D

NA

Antigen detectionGalactomannan (GM)

1,3 β-D-glucan (BDG)

Lateral Flow Assays (LFA)

PCR

Which tests can the lab offer to you?

Antibody detection

Panfungal(not Pneumocystis)

Invasive aspergillosis‘Panfungal’

Panfungal

Cryptococcosis

Panfungal

Panfungal

Chronic pulmonary aspergillosis

Culture of blood

Useful only for the diagnosis of yeast infections (with few exceptions such as Fusarium/Scedosporium infections)

BUT CANDIDA DOES NOT CAUSE LUNG INFECTIONS

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Time to Candida blood culture positivity overall and by species

MC Arendrup et al, JCM 2011, 49: 3300-3308

DIAGNOSIS OFINVASIVE ASPERGILLOSIS

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Patients at risk for invasive aspergillosis

Degree of immunocompromise

Ris

k o

f ac

qu

isit

ion

5%

10%

15%

20%

25%

By courtesy of J. Maertens

• 286 BAL from 221patients with underlying respiratory diseases (without hematological malignancy or SOT)

• 14% proven/probable IPA

• 02/2012 – 05/2014, Graz, Austria

Prattes J et al. Am J Resp Crit Care Med 2014; 190: 922-929

Novel tests for diagnosis of IA in patients with underlying respiratory disease

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Some issues about galactomannan detection

• Sensitivity of serum GM detection is high in neutropenic patients only

• Sensitivity of BAL GM detection is high for the broad population at risk of IA but may also be positive due to colonization

• No false positivity due to TazocinTM administration (but cave generic formulations - compare serum with BAL values)

• Retesting the same sample may help to distinguish between true and false positive results

Mikulska M et al. J Antimicrob Chemother 2012,67:1746-8Metan, G. Infection 2013, 41: 293-294.

False positive galactomannan results: testing the sample twice improves specificity

Furfaro E et al., Transpl Infect Dis 2012, 14:E38-9

Big difference between initial and retest result

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ODI ≥ 3.O: rules in disease!

ODI ≤ 0.5: virtually rules out!

The ROC curve identified 0.8 as the most appropriate cut‐off value; the area under de curve was 0.94 (95% CI, 0.90‐0.98)

D’Haese J et al. J Clin Microbiol 2012; 50: 1258-1263

Galactomannan detection in BAL: the optimal cut-off value?

• Trend for mold-active antifungal use to decrease sensitivity (p=0.07): pooled sensitivity decreased from 0.91 to 0.76 (meta-analysis in adult hematology patients).

• Single center retrospective study in hematology patients (28 episodes proven/probable IA, 71% of patients mold-active antifungals at time of BAL):

o Time effect: sensitivity of GM detection decreased when antifungal treatment lasted ≥ 2 days at the time of bronchoscopy Detection of GM in the alveolar samples better sensitivity than in bronchial sample (first aliquot returned).

o No significant relationship between the amount of administered solution and the GM assay, trend towards a higher volume of aspirated fluid in GM-negative BAL (p=0.093)

• Pre-treatment of samples with Sputasol® or SL solution leads to false negative GM results

Prattes J et al. J Infect 2015, 70: 541-543.Gils S et al. JCM 2016, Epub ahead of print.

Galactomannan detection in BAL:pre-analytical variables that impact sensitivity

Heng SC et al. Crit Rev Microbiol. 2015, 41:124-34Racil Z et al. Int J Infect Dis. 2011; 15 :e874-881.

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COPD and invasive aspergillosis: clinical definitions

Bulpa P et al. ERJ 2007; 30: 782-800

• Murine MAb JF5: IgG3 immunoglobulin

• Recognizes an extracellular, constitutive, glycoprotein antigen

• Antigen is secreted during active growth of hyphae and is not produced by dead or quiescent spores

• Displays superior specificity to rat MAb EB-A2 (Bio-Rad Platelia GM-EIA)

• Used to develop a rapid, user-friendly, diagnostic test for detection of IA

Thornton CR. Clin Vaccine Immunol. 2008 Jul;15(7):1095‐105

1 Cryptococcus neoformans 2 Candida albicans3 Fusarium solani4 Rhizopus oryzae5 Aspergillus fumigatus

The Aspergillus lateral flow device: a point-of-care test

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Monoclonal antibody: Mab JF-5Antibody source: mouse

Step 1

Step 2

Positive reaction Negative reaction

Capillary flow

Step 3

BA

Control line Test line Control line

BA

Aspergillus lateral flow assay

Sample padCapture zone

Clinical specimen

Test line(mAb)

Control line(anti-mouse IgG)

Sample pad Sample pad

Ag No-Ag

Pretreatment of serum samples necessary, not of BAL samples

Current Fungal Infection Reports (2013) 7: 244‐251. 

Expert Review of Clinical Immunology (2014)

Localisation of JF5 antigen

When commercially available?

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Aspergillus PCR

• European Aspergillus PCR Initiative (2006)

• Commercial and in house assays lack of standardization… but we’re on our way!

• DNA load in many samples close to the detection limit of even highly sensitive PCR protocols

• Aspergillus PCR in blood mainly evaluated in hematology patients

• Not yet included in international guidelines…and not yet broadly introduced in clinical practice

• New assay on the market that combines detection of Aspergillus with triazole resistance markers (AsperGenius® assay)-limited validation

• Very useful for biopsy samples in which fungal hyphae were seen (microscopically) that remain culture negative (also panfungal PCR is very useful in this context)

White L. et al. JCM 2010, 48: 3753-3755Springer J et al. JCM 2013; 51: 1445-1450

Chong GL et al. JCM 2015, 53: 868-874.

Any need to perform Aspergillus susceptibility testing?

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Routes of resistance development in A. fumigatus

ENVIRONMENT

PATIENT Long-term triazole treatment for aspergillomaor cavitary lung disease

Triazole fungicides in agriculture

TR34/L98HTR46/Y121F/T289A

Variety of resistance mechanisms

• High genetic diversity between azole-resistant isolates from unrelated patients

• Lack of sporulation and reduced growth rate may occur

• Patients with IA and chronic Aspergillus diseases

• Low genetic diversity between azole-resistant isolates from unrelated patients

• No apparent fitness cost

PE Verweij et al, CID 2016, 62: 362-368.

Global presence of azole resistance in A. fumigatus

Countries that reported the TR34/L98H and TR46/Y121F/T289A resistance mechanism in clinical or environmental A. fumigatus

PE Verweij et al, CID 2016, 62: 362-368.

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High prevalence of azole resistance in patients on the hematology and ICU ward in Utrecht

• 105 positive cultures collected; proven IA (5), probable IA (48) and no infection (52)

• 21/105 (20%) isolates were resistant to at least one azole

• 16/105 (15.2) isolates showed pan-azole resistance

• 16/17 (94.1%) of voriconazole resistant isolates exhibit cyp51A gene mutation

Fuhren et al., JAC 2015, 70: 2894-2898.

BELGIAN DATA

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Belgium: nationwide surveillance

- April 2011 - April 2012

- 18 Belgian hospitals

- Only isolates from patients with documented Aspergillus disease were included:

Invasive aspergillosis:• EORTC proven/probable

• Putative IA in ICU

ABPA

Aspergillus bronchitis

Chronic cavitary/necrotisizing aspergillosis

Aspergilloma

E Vermeulen et al., AAC 2015, 59, 4569-76

0

20

40

60

80

100

120

IA chronicaspergillosis/

bronchitis

ABPA singleaspergilloma

A. fumigatus

A. flavus

A. niger

A.terreus

A. nidulans

A. niger > A. flavus

# isolates A. fumigatus A. flavus A. niger A.terreus A. nidulans Total

IA 115 (86%) 6 (4,5%) 9 (6,7%) 2 2 134

Chronic aspergillosis/ Aspergillus bronchitis

22 5 0 0 1 28

ABPA 51 1 1 0 0 53

single aspergilloma 4 1 0 0 0 5

Total 192 13 10 2 3 220

Belgium nationwide surveillance

E Vermeulen et al., AAC 2015, 59, 4569-76

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E Vermeulen et al., AAC 2015, 59, 4569-76

Susceptibility testing Aspergillus niger (n=10)

0

1

2

3

4

5

0,06 0,12 0,25 0,5 1 2 4 8 16 >16

itraconazole

A. foetidus A. tubingensis

# is

olat

es

MIC (mg/L)

E Vermeulen et al., AAC 2015, 59, 4569-76

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CMI, 2014 20:O333-5.

CMI, 2014 20:O333-5.

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DIAGNOSIS OFPNEUMOCYSTIS JIROVECII PNEUMONIA

ECIL 5, 2013. J. Maertens et al, www.kobe.fr/ecil/

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Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases, 6th Edition

Analysis of rRNA gene in the 1980s suggested that the organism is more closely related to fungi than to protozoa

P. carinii ratsP. jirovecii humans (in recognition of Otto Jirovec)

• BAL is het preferred sample type, highest sensitivity, induced sputum is an alternative

• Fungal load gradient BAL>induced sputum> sputum> upper respiratory specimens (nasopharyngeal aspirate, oral wash, nasal swab)• Sensitivity to detect Pneumocystis in sputum and upper respiratory tract

samples is significantly lower. A negative result (also with PCR) does not exclude PCP!

Diagnosis of PCP: sample

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• Culture based methods have no role

• Microscopic demonstration of P. jirovecii

• Different stains are possible but immunofluorescence is the most sensitive method and thus recommended

• In non-HIV+ immunocompromised pts, the fungal load is lower, making microscopical diagnosis generally strenuous

• PCR

• -D-glucan detection

Diagnostic methods

• P. jirovecii DNA detection by PCR in lower respiratory specimen is highly sensitive and has a high negative predictive value, resulting in the exclusion of PJP when negative

• A positive PCR does not necessarily correspond to infection! Quantitative PCR (qPCR) is required to determine the fungal load. Clinical validation of the PCR is essential to determine interpretation thresholds especially when no microscopy is performed anymore.

• > High threshold: always correspond to PCP

• < Lower threshold: always correspond to colonization

• Grey zone in between: interpretation may be different according to the underlying disease/ immune suppression

PCR

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1,3 β-D-Glucan detection

• ‘Panfungal’:

• Aspergillus, Candida, many other fungi

• NO detection of Mucorales and Cryptococcus

• Several commercial kits available (different cut-off values): Fungitell™,Glucatell™ in US and Europe

• Approved for serum testing only. Not recommended on BAL samples (frequent Candida colonization of respiratory tract)

• Test not really user-friendly

• Many cause for false positivity:

o Hemodialysis/hemofiltrationo -lactam antibiotics o Blood components: immunoglobulin preparationso Surgical gauzeso Bacterial infections

Endotoxin (LPS)

Endotoxin (LPS)

Pro-clotting enzyme

Pro-clotting enzyme Clotting

enzymeClotting enzyme

Factor BFactor BFactor B

(activated)Factor B

(activated)

Factor CFactor CFactor C

(activated)Factor C

(activated)

Factor GFactor G

Factor G(activated)Factor G

(activated)

(1-3) β-D glucan

Syntetic peptidechromogenic

substrate

Syntetic peptidechromogenic

substrateYellow

artificial substrate

Absorbance405 nm

Lysate source: horseshoe crab

1,3 β-D-Glucan Assay

A B C

Clinical specimen

Reaction Assay

D

Clinical specimenClinical

specimen

Limus Amebocyte Lysate (LAL) Pathway

Limus Amebocyte Lysate (LAL) Pathway

Synetic peptidechromogenic

substrate

Synetic peptidechromogenic

substrate

E

Microplate

Well

Miceli MH, Maertens. J.Semin Respir Crit Care Med. 2015;36:650-61

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-D-glucan detection in serum• BG assay can be used as a screening tool for PCP

• Serum BDG shows excellent sensitivity and very good specificity in the diagnosis of PCP. Still, in clinical practice the test results should be interpreted in the context of the underlying clinical characteristics of the individual patient

o A negative serum BG can be used to rule out PCP in patients with a low or moderate pretest probability for the disease

o If the BG assay is positive, other invasive fungal infections than PCP should be considered for every individual patient

• It cannot be excluded that the accuracy of BDG testing for the diagnosis of PCP may differ between different patient groups.

• BG kinetics can not be used for the assessment of treatment response

• Role of BG in patients receiving PCP prophylaxis has not been adequately evaluated

Strong suspicion of invasive fungal infection while all (other) tests remain negative or inconclusive

Inability to perform bronchoalveolar lavage in a patient with suspicion of Pneumocystis jirovecii pneumonia

1-3 -D-glucan performed by the National Reference Center for Mycosis

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DIAGNOSIS OFCRYPTOCOCCOSIS

• Antigen: the major capsular polysaccharide glucuronoxylomannan (shed during infection)

• Detection in serum/CSF

• Latex-agglutination/Lateral flow assay

• Excellent sensitivity and specificity

• “One of the most useful serologic tests in mycology”

Cryptococcus antigen detection

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THINK FUNGAL

CONTACT YOUR MICROBIOLOGIST WHEN FACING DIFFICULT CASES