Alessandro C. Pasqualotto [email protected] Porto Alegre, Brazil What are we looking at?...

Alessandro C. PasqualottoAlessandro C. Pasqualotto

[email protected]@santacasa.tche.br

Porto Alegre, BrazilPorto Alegre, Brazil

What are we looking at?What are we looking at?Challenges in the diagnosis of Challenges in the diagnosis of

Invasive Mould DiseasesInvasive Mould Diseases

Potential conflicts of interest

• Research GrantsResearch Grants Myconostica, Pfizer, Merck, Sigma-Tau, CAPES, Myconostica, Pfizer, Merck, Sigma-Tau, CAPES,

CNPq,CNPq, Fungal Research TrustFungal Research Trust

• Travel GrantsTravel Grants Pfizer, United Medical, Schering (now Merck), Bagó, Pfizer, United Medical, Schering (now Merck), Bagó,

Merck Merck

• Speaker honorariaSpeaker honoraria Pfizer, United Medical, Merck, Schering (now Merck), Pfizer, United Medical, Merck, Schering (now Merck),

BiometrixBiometrix

First assumption:First assumption:IFD are highly lethal diseasesIFD are highly lethal diseases

p<0.001

Incidence 13.3% in lung transplant recipients

Xavier MO, Pasqualotto AC, et al. ECCMID 2009

4 days later

Rapidly evolving diseases

www.aspergillus.org.uk

Disseminated infectionDisseminated infection

www.aspergillus.org.uk

Second assumption:Second assumption:We need to intervene asapWe need to intervene asap

Von Eiff, et al. Respiration 1995; 62: 241-7

Early versus late interventionM

ort

alit

y ra

te (

%)

0

20

40

60

80

100

Within 10 days

Von Eiff, et al. Respiration 1995; 62: 241-7

0

20

40

60

80

100

Within 10 days > 11 days

Mo

rtal

ity

rate

(%

)Early versus late intervention

But how can we achieve such an But how can we achieve such an early diagnosis?early diagnosis?

A small black scar 2 days earlier

+ serum GM

Patient died 1 day after this picture was taken


+ serum GM



+ serum GM



+ serum GM


Zygo + A. flavus

Zygo + A. flavus

MD Anderson Cancer CentreNecropsy study over a 15-years period

• IFD detected in 31% IFD detected in 31% over 1,017 over 1,017

necropsiesnecropsies

• Antemortem diagnosis in only 25%Antemortem diagnosis in only 25%

Chamilos G, et al. Haematologica 2006; 91: 986-9

MD Anderson Cancer CentreNecropsy rate has reduced over time


0

20

40

60

80

100

1989-1993 1994-1998 1999-2003

%%

By the way, what is the necropsy rate in your institution?

1. >40%

2. 10-39%

3. 1-10%

4. <1%

5. Are you kidding?

MD Anderson Cancer CentreTrends in the prevalence of IFD


0

20

40

60

80

100

1989-1993 1994-1998 1999-2003

Invasivefungal disease

Invasivemould disease

Mixedinfections

%%

No need to worry!No need to worry!

CT scan and CT scan and galactomannan galactomannan

are there to help us out!are there to help us out!

Day 0: halo

Day 4:size, halo

Day 7:air crescent

Caillot, et al. J Clin Oncol 1997; 15: 139-47

‘‘Halo sign’ surrounding a noduleHalo sign’ surrounding a nodule

The sign is not specific for IAThe sign is not specific for IA

• VasculitisVasculitis

• MetastasisMetastasis

• PseudomonasPseudomonas infections infections

• Zygomycosis and other angio-invasive Zygomycosis and other angio-invasive

infectionsinfections

Greene RE, et al. Clin Infect Dis 2007; 44: 373-9

Absence of typical findings at Absence of typical findings at chest CT scan chest CT scan

• COPDCOPD

• SteroidsSteroids

• Other non-neutropenic patients / ICUOther non-neutropenic patients / ICU

• Lung transplant recipientsLung transplant recipients

• ? Monoclonal antibodies? Monoclonal antibodies

‘‘Reversed halo sign’Reversed halo sign’Organising cryptogenic pneumoniaOrganising cryptogenic pneumonia

Wahba H, et al. Clin Infect Dis 2008; 46: 1733-7

‘‘Reversed halo sign’Reversed halo sign’


• Review of 189 cases Review of 189 cases of invasive mould of invasive mould

diseasedisease

Overall frequency 4%Overall frequency 4%

‘‘Reversed halo sign’Reversed halo sign’


• Review of 189 cases Review of 189 cases of invasive mould of invasive mould

diseasedisease

Overall frequency 4%Overall frequency 4%

Zygomycosis 19%Zygomycosis 19%

Aspergillosis <1%Aspergillosis <1%

Fusariosis 0% Fusariosis 0% (p<0.01)(p<0.01)

• >10 nodules>10 nodules

• Pleural effusionPleural effusion

• Concomitant sinusitisConcomitant sinusitis

• Treatment with voriconazoleTreatment with voriconazole

Other predictors of zygomycosisOther predictors of zygomycosis

Chamilos G, et al. Clin Infect Dis 2005; 41: 60-6

Pfeiffer CD, et al. Clin Infect Dis 2006; 42: 1417-27

Low PPV High NPVLow PPV High NPV

Meta-analysis of GM testing

Caution with low

+ve indexes!

Upton A, et al. J Clin Microbiol 2005; 43: 4796-800

Reproducibility

Aquino VR, Goldani LZ, Pasqualotto AC. Mycopathologia 2007; 163: 191-202

• Penicillium marneffei

• Geotricum capitatum

• Acremonium species

• Alternaria alternata

• Rhodotorula rubra

• Trichophyton species

• Paecilomyces variotii

• Botrytis tulipae

• Cladosporium species

• Exophiala dermatitidis

GM release by non-Aspergillus fungi

Cross-reaction with GM testing

Xavier MO, Pasqualotto AC, Severo LC. Clin Vaccin Immunol 2009; 16: 132-3

0

10

20

30

40

50

60

70

80

90

100

Paracocci Histo Cryptoneoformans

C gattii

%

Clinical case

Maertens J, et al. Clin Infect Dis 2004; 39: 289-90

• 19 year-old man, refractory leukaemia

• Febrile neutropenia

• Amox-clav for E. Coli bacteremia

Clinical case





• Daily GM determination

– D1 after antibiotic: GM index of >1.5

– 5 +ve tests afterwards

Clinical case





• Daily GM determination

– D1 after antibiotic: GM index of >1.5

– 5 +ve tests afterwards

• Fluoroquinolone: gradual reduction in GM index

Clinical case


• 1 wk later

– Pipe-tazo for appendicitis

– GM >2.5; bilateral nodular infiltrate

Clinical case


• 1 wk later



• Probable IA (EORTC / MSG)

– Antifungal therapy + meropenem

– Gradual ↓ in GM index

Clinical case


• 1 wk later



• Probable IA (EORTC / MSG)

– Antifungal therapy + meropenem

– Gradual ↓ in GM index

• Necropsy: leukaemia infiltrate

– Absence of IA

Pfeiffer CD, et al. Clin Infect Dis 2006; 42: 1417-27

Proven or probable IA

Sensitivity Specificity

Haematological malignancies

0.58 (52-64) 0.95 (94-96)

Solid organ transplantation

0.41 (21-64) 0.85 (80-89)

Meta-analysis of GM testing

Tuon FF. Rev Iberoam Micol 2007; 24: 89-94

Specificity 94%

Sensitivity 79%

Marked heterogeneity(particularly for sensitivity)

BAL PCR testing

PCR Critical points

• Variable sensitivity / specificity

• Lack of standardised targets / reagents

• Extraction method

• Platform (conventional PCR vs Real time)

• Poor understanding of DNA kinetics

• Not yet part of the EORTC/MSG criteriaDe Pauw B, et al. Clin Infect Dis 2008; 46: 1813-21

• Detected in IFDs caused by

– Candida and Aspergillus

Yoshida M, et al. J Med Veter Mycol 1997; 35: 371-4

Beta-Glucan

Phospholipid bilayerof the fungal cell

membrane

Fungalcell wall

-(1,3)-glucan-(1,3)-glucan synthase Ergosterol

Beta-Glucan

• Detected in IFDs caused by

– Candida and Aspergillus

– Trichosporon

– Fusarium

– Acremonium

– Saccharomyces

– Pneumocystis

Yoshida M, et al. J Med Veter Mycol 1997; 35: 371-4

There he is again,There he is again,

speaking about a test that nobody speaking about a test that nobody

uses in Brazil …uses in Brazil …

The reality in BrazilThe reality in Brazil

• A survey performed in collaboration A survey performed in collaboration

with ANVISAwith ANVISA

140 hospitals140 hospitals

>42,000 beds>42,000 beds

65% teaching hospitals65% teaching hospitals

90% belonging to the 90% belonging to the Sentinel NetworkSentinel Network

Complexity in hospital careComplexity in hospital care

0

20

40

60

80

100

%%

Is IFD a problem in your centre?

Yes

No38.7%38.7%

n=140n=140

Do you know your local epidemiology?

Yes

No

40.1%40.1%

n=140n=140

Specialised media for fungi

Yes

No19.7%19.7%

n=140n=140

Yes, always

Yes, occasionally

Never58.5%58.5%

Aspergillus identificationat the species level

n=140n=140

Fungal staining - biopsies

Yes, always

Yes, occasionally

No

51.1%51.1%

n=140n=140

Biopsy specimens are sent in formalin only?

Yes, always!

Occasionally

Never

26.0%26.0%

n=140n=140

Access to high resolution CT

Yes

No

34.3%34.3%

n=140n=140

Galactomannan

Sim

Não83.6%83.6%

n=140n=140

The appropriateness or

inappropriateness of feelings is

relative to the ground and to the

circumstances of those feelings

Aristotle The Doctrine of the Mean, 384-322 BC

Fight fire with fire

Metallica

Ride the Lightning, 1984 AD

Invasive diagnostic interventions

• CT-guided percutaneous lung biopsy:

yield of 70-100%

Nosari A, et al. Haematologica 2003; 88: 1405-9

Crawford SW, et al. Transplantation 1989; 48: 266-71

Hoffer FA, et al. Pediatr Radiol 2001; 31: 144-52

Lass-Florl C, et al. Clin Infect Dis 2007; 45: e1001-4






• CT-guided percutaneous lung biopsy:

yield of 70-100%

Platelets >60,000/ml are required

Pneumothorax 18%

Haemoptysis 3%Nosari A, et al. Haematologica 2003; 88: 1405-9









• Transbronchial biopsies:

False-negative results are frequently seen

Lass-Florl C, Freund MC. In: Aspergillosis: from diagnosis to prevention.

Pasqulaotto AC, ed. Springer, 2009




• Open lung biopsies:

Provide larger samples of tissue with

improved accuracy and specificity






• Open lung biopsies:

Provide larger samples of tissue with

improved accuracy and specificity

Contradictory results

Complication rate of 10-15%










Peripheral lesions Needle biopsy /Surgical resectionNeedle biopsy /Surgical resection

Focal lesions near thehilum / great vessels

Focal lesions near thehilum / great vessels

Urgent thoracotomy and resectionUrgent thoracotomy and resection

Bilateral /multifocal disease

Bilateral /multifocal disease

BALBAL

Conclusion

• It is mostly but not all about IA

Invasive mould diseases have to be

differentiated from each other

Conclusion




• Diagnosis is the most challenging step in

infectious diseases

Conclusion




• Diagnosis is the most challenging step in

infectious diseases

• We need a better understanding on the

performance of the available tests

Acknowledgments

• Mycology team Luiz Carlos Severo

Valerio R Aquino

Cecilia B Severo

Luciana Guazelli

Melissa Xavier

• Infection Control Dept Teresa Sukiennik

Alessandro C. Pasqualotto [email protected] Porto Alegre, Brazil What are we looking at?...

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