something old, something new: beyond lactophenol cotton blue · something old, something new:...
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Something Old, Something New: Beyond Lactophenol Cotton Blue
Donald C. Vinh, MD Assistant Professor, Clinician-Scientist
Division of Infectious Diseases Division of Allergy & Clinical Immunology
Dept of Medicine; Dept of Medical Microbiology; Dept of Human Genetics
McGill University Health Centre
IFI DIAGNOSTICS:
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Disclosures Advisory Board Member/Consultant: • CSL Behring Canada, Pfizer Canada Speaker: • CSL Behring Canada & Sunovion Grant & Honorarium: • CSL Behring Canada, Astellas Canada
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Objectives: • Overview of diagnostic strategies for IFI in
haematological patients • Discuss advantages and disadvantages of
different diagnostic modalities • State emerging diagnostic tools
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The Problem: At-Risk Patient
Signs/Symptoms compatible with IFI
Diagnostic Evaluation
Modifications in Therapy
• Antifungal Susceptibility Testing • Therapeutic Drug Monitoring
Survive
Treatment Delay in Therapy
↑Mortality
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The Problem: At-Risk Patient
Signs/Symptoms compatible with IFI
Diagnostic Evaluation
Modifications in Therapy
• Antifungal Susceptibility Testing • Therapeutic Drug Monitoring
Survive
Treatment Delay in Therapy
↑Mortality
EARLIER DIAGNOSIS
CLINICAL LABORATORY
• Clinical suspicion
• Clinical sampling
• Standardized methods
• Faster TAT • ↑Analytical Sensitivity
• ↑Test performance (sens; spec)
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FUNGI
YEAST MOLD
Clinical Mycology:
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Blood vessel
IFI DIAGNOSTICS:
IMAGING
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IMAGING: Chest CT: GOLD STANDARD • 1896: CXR… ? Time to move
forward ? Mod-High sensitivity: 80-85% (cf. autopsy)* Earlier detection: 5 d (median) “Characteristic signs”: • EARLY: Halo sign; Fluffy nodules;
Pleural-based wedges • LATE: Air-crescent sign; Cavitation • Non-specific findings… adjunctive
Dx tests
Typical radiological evolution: • On appropriate antifungal Tx, ↑ volume of infiltrates (4x) during 1st week (d7)
• Stable (x few days) • ↓ thereafter • F/U scan no sooner than 2
weeks (if clinically stable)
*Kami et al., Mycoses 2002
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CT IMAGING:
ADVANTAGES:
• Non-invasive • Availability • Speed of the test • Systematic use: associated
with improved outcomes
DISADVANTAGES:
• You have to know where to look: Lungs, Sinuses… elsewhere (?GI, GU?)
• Non-specific signs • incl. Non-specific radiology
report: “… could be compatible with fungal or mycobacterial disease, or parasites or alien plant life”
• Limited distinction capability • Fungus vs. Non-fungus • Which fungus?
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Emerging Imaging Modalities:
FDG PET/CT • FDG uptake in infl cells ; glc metabolism (“respiratory burst”) • Rationale: non-specific findings on CT
Kim et al.*: • IA (imm compr): hypermetabolic nodule, NO halo • NIA (imm compt): isometabolic noule, (+) halo
More studies needed Work done on
fungal labeling…
*J Comput Assist Tomogr 2013
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Blood vessel
IFI DIAGNOSTICS:
CULTURE
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CULTURE:
(Some) Different types of fungal media: • BHI (Brain-Heart-Infusion) agar • Czapek’s agar • Inhibitory Mould Agar • Mycosel agar • Potato Dextrose Agar • Sabouraud-BHI • Sabouraud’s Dextrose Agar • Potato flake agar
Septum
RIGHT Clinical sample
RIGHT Culture media
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CULTURE:
ADVANTAGES:
• HIGH specificity for IFI (70-95% cf. autopsy)*
• Provides an isolate for further testing, including
• Antifungal Susceptibility testing
DISADVANTAGES:
• Usually requires invasive procedure for sampling
• LOW sensitivity: 0 – 88% (cf. histology)*
• Precarious sampling / sample processing • Pan-Micro lab testing + Path
• Requires expertise • May be slow
• weeks to complete
*Guarner et al. CMR 2011
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Blood vessel
IFI DIAGNOSTICS:
HISTOPATHOLOGY
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Histopathology:
Pathology Stains: • H&E • PAS • GMS
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Histopathology:
ADVANTAGES:
• Pretty • Distinguishes:
contamination vs. colonization vs. infection • Micro stains: H&E, PAS, GMS,
Fontana-Masson, Calcofluor • Host response: Inflammation,
necrosis, hemorrhage
• Provides presumptive Dx pending cultures (if they do grow)
DISADVANTAGES: • Usually requires invasive
procedure for sampling • Overall accuracy: 20 – 80%
(cf. culture) • Morphological diagnosis (i.e. “they all look the same”)
• polymorphic fungal elements • No speciation (or even
genusiation) • Dual infection • Requires interaction with a
Pathologist
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Blood vessel
IFI DIAGNOSTICS:
ANTIGEN DETECTION
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ANTIGEN DETECTION:
GALACTOMANNAN (GM)
• Polysaccharide found in cell wall of most Aspergillus sp.
• Released from growing hyphae
• Commercial kit
β-D-GLUCAN (BDG)
• Polysaccharide found in cell wall of MANY FUNGI
• Commercial kit • Manufacturer recommendations
made on 2 studies (mainly, non-neutropenic patients)
• Meta-analysis: (+)=2++ samples
• LOW Sens: 50% • HIGH Specificity: 99%
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GALACTOMANNAN: • Standard test: EIA • Extensively studied in Heme population
(Allo-SCT, AML/chemoTx) • Serum*: Sens: 58–65%; Spec:65–95%
• Heterogeneity in : design/patients/OD cut-off • May precede radiologic/microbiologic Dx by 5-8 d (median) • Useful for PRE-EMPTIVE strategy in at-risk (non-prophylaxed
patients) • False (+):
• Pip/Tazo (previously… ?generic) • Other fungi (e.g. Fusarium) • Prophylaxed patients (low pre-test prob; ∴Dx-driven)
*Pfeiffer et al., CID 2006
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GALACTOMANNAN: • BAL:
• Diagnostic-driven, NOT screening • In meta-analysis restricted to hematology patients*,
using GAL-GM cut-off of 1.5:
• Combining BAL-GM to S-GM or BAL-PCR: ↑Sens by 5-9%
*Heng et al., Crit Rev Microbiol. 2015
SENS: 82% SPEC: 92%
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GALACTOMANNAN:
• Not technically demanding • Costs can be reduced by
sample batching • Fast TAT: 4 h • Serial serum GM: monitor
response to therapy
• (+) results need confirmation (repeat testing)
• Limited microbiological spectrum:
• Aspergillus vs. NON-Aspergillus • WHICH Aspergillus ??
ADVANTAGES: DISADVANTAGES:
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Will the real A. fumigatus please stand up? PHENOTYPE
(MORPHOLOGY) GENOTYPE
Aspergillus species complex (section Fumigati)
ANTI-FUNGAL SUSCEPTIBILITY
PROFILE
A.fumi (ss)
pheno-copies
AmB 0.25-1 >1
Vori 0.25 – 1 >1
Terb 2 – 8 < 1.66
(+)GM ≠ Vori(S)
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GM by Lateral Flow Assay:
*Wiederhold et al., Clin Vaccine Immunol 2009
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Blood vessel
IFI DIAGNOSTICS:
NUCLEIC ACID-based Testing
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NUCLEIC ACID-based TESTING (NAT):
• PCR: Polymerase Chain Reaction • “Why not just PCR it out ?”
Specimen type
Specimen processing
DNA extraction
PCR assay design:
• Target? • Primers? • Reagents?
Downstream analysis
• (+)/(-) ? 2++? • Sequencing • Database ?
QUALITY (QC / QA)
Clinical Dx
Micro Dx
§ Screen vs. Diagnose § Sample? Blood (which component)? BAL?
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PCR: • Aspergillus-specific PCR
• Serum: SENS: 72%; SPEC: 96.5%
• Lack of standardization of methods
• Commercial kit (Candida 7-plex; Mould 11-plex)
• European Aspergillus PCR Initiative (EAPCRI)
• Real-time PCR for detection of Candida
• Pan-Fungal PCR: detect unknown fungi in clinical specimens • Path(+), Culture(-) • Blood
• May be esp. useful for
Mucormycosis
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Emerging NAT: T2Candida assay
• Detection of Candida from whole blood Whole blood (2mL)
Lyse blood cells
Lyse Candida
PCR lysate Amplified DNA detecting using
magnetic resonance technology
• Time to detection: 3 hours • Detects: C. albicans, C. tropicalis, C. glabrata, C. krusei, C. parapsilosis • Analytical senstivity: 1 CFU / mL
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Blood vessel
IFI DIAGNOSTICS:
PROTEOMIC Analysis
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Proteomic Analysis: MALDI-TOF MS:
• Identifies protein fingerprints of unknown fungi based on spectral patterns
• >90-95% concordance with conventional identification
Advantages Restrictions
↓ Time to identification Standardize fungal growth
Identify FF Standardize protein extraction
Has been applied to clinical specimens (e.g. Blood/Candida)
Database curation
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SUMMARY: • EARLIER DIAGNOSIS: Key to Improved
outcomes • CLASSICAL micro / MOLECULAR micro • Ongoing performance optimization &
validation • Incorporation into clinical trials
→ Integration into clinical practice
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Summary & Closing Remarks
Dr. Shariq Haider
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Thank you!