Challenges in establishing a PCR assay forthe diagnosis of invasive aspergillosis

Stéphane BretagneProfessor of Medicine, Paris Diderot University; Chair of Parasitology and Mycology, Saint Louis University Hospital

Deputy Director, French National Reference Centre for Invasive Mycoses and Antifungals, Institut Pasteur, Paris, France

Conflicts of interest

• None

• Disclaimer– Research grant: MSD

– Bureau member: Gilead

– Travel grants: Pfizer, MSD.

Invasive aspergillosis

● Aspergillus fumigatus

● Environmental mould

● Deeply immunocompromised patients

(haematology, solid organ transplantation)

● Pneumopathy:● Many other possible diagnoses

● Mortality >40% in 3 months

Courtoisy Raoul HerbrechtOncologie et Hématologie, Strasbourg, France

Microbiological diagnosis of invasive aspergillosis

● Respiratory specimens● Bronchoalveolar lavage

● Sputum

● Direct examination● brightener

● Culture● 24-48h

● Phenotypic identification

(microscopy, MaldiTof)

● Molecular identification

Microbiological diagnosis of invasive aspergillosis

● Respiratory specimens● Bronchoalveolar lavage

● Sputum

● Direct examination● brightener

● Culture● 24-48h

● Phenotypic identification

(microscopy, MaldiTof)

Sensitivity ≈ 50%

Specificity: environmental mould

Biomarker

● Galactomannan● Antigen produced by growing

Aspergillus spp. and other

moulds

● ELISA test● False+

● Serum

Guigue N et al , Importance of Operational Factors in the Reproducibility of Aspergillus Galactomannan Enzyme Immune Assay, PLOS ONE | DOI:10.1371/journal.pone.0124044 April 10, 2015

Diagnosis of invasive aspergillosis

● Proven, Probable, Possible cases

● A combination of several criteria:● Host factors

● Imaging

● Microbiology (including antigens)

● PCR not included: no consensus

Ascioglu S. et al Clinical Infectious Diseases 2002; 34:7–14

The ISHAM Working Group“Towards a Standard for Aspergillus PCR”

2006

WHOLE BLOOD

- Most PCR amplification systems provided similar detectionthresholds, although positivity was a function of the fungalburden. When PCR amplification was combined with DNAextraction, 50% of the centers failed to achieve the same level ofdetection.

- The efficiency of the Aspergillus PCR is limited by the extractionprocedure and not by PCR amplification.

Natural history of invasive aspergillosis?

M

MΦ

Natural history of invasive aspergillosis?

M

MΦPositive blood culture? never

Natural history of invasive aspergillosis?

M

MΦ

Bretagne S et al, Comparison of Serum Galactomannan Antigen Detection and CompetitivePolymerase Chain Reaction for Diagnosing Invasive Aspergillosis Clin Infect Dis. 1998 Jun;26(6):1407-12.

Serum

Digital PCR

Digital PCR enzymatic digestion

No digestion After digestion

Digital PCR with enzymatic digestion

Alanio A. et al, J Microbiol Methods. 2016 Aug;127:160-3.

Real human

serum samples

(not spiked with

Aspergillus DNA)

Free DNA in

patient serum

- Most Aspergillus PCR protocols used to test serum generate satisfactory

analytical performance.

- Testing serum requires less standardization, and the specific

recommendations shown in this article will only improve performance.

- Plasma > serum (White PL, et al 2015 J Clin Microbiol 53:2832–2837.

doi:10.1128/JCM.00905-15.)

Adding an internal control

• Modified M13 phage fragment1

– Target specific

• Exogenous DNA

– Animal virus 2

– Plasmids3

– SPUD 4

– …

– Not human DNA!!!

(1) Bretagne S et al, J Clin Microbiol 1995

(2) Van Doornum et al JCM 2003

(3) White L, et al, Clin Inf Dis 2006

(4) Nolan et al, Anal Biochem 2006

Kinetics of invasive aspergillosis

antifungal

time

GM

aplasia

Kinetics of invasive aspergillosis

antifungal

time

GM

aplasia

qPCR

Results (PREVERT study)Performance of qPCR overall and according to sample selection

Definition of a patient tested positive for IA

No. patients tested

positive / No. patients

with IA

Sensitivity %

(95% CI)

No. patients tested

negative / No.

patients without IA

Specificity %

(95% CI)Youden indexa

Overall performance: all serum samples collected during study enrollment (n=1,534)

≥1 positive test with ß-glucan 9/11 82 (48-98) 79/174 45 (38-53) 0.27

≥1 positive test with qPCR 8/11 73 (39-94) 79/174 45 (38-53) 0.18

≥1 positive test with ß-glucan or qPCR 11/11 100 (72-100) 39/174 22 (16-29) 0.22

Early diagnosis of IA: selection of serum samples before early antifungal therapy (n=747)

≥1 positive test with ß-glucan 5/11 45 (17-77) 113/172 66 (58-73) 0.11

≥1 positive test with qPCR 4/11 36 (11-69) 113/172 66 (58-73) 0.02

≥1 positive test with ß-glucan or qPCR 7/11 64 (31-89) 76/172 44 (37-52) 0.08

Cordonnier, C. et al. Clin Infect Dis; 48:1042-51 (2009)Swcharzinger M et al Plos One; 8(6): e65776 (2013)

Definition of a patient tested positive for IA

No. patients tested

positive / No. patients

with IA

Sensitivity %

(95% CI)

No. patients tested

negative / No.

patients without IA

Specificity %

(95% CI)Youden indexa

Overall performance: all serum samples collected during study enrollment (n=1,534)

≥1 positive test with ß-glucan 9/11 82 (48-98) 79/174 45 (38-53) 0.27

≥1 positive test with qPCR 8/11 73 (39-94) 79/174 45 (38-53) 0.18

≥1 positive test with ß-glucan or qPCR 11/11 100 (72-100) 39/174 22 (16-29) 0.22

Early diagnosis of IA: selection of serum samples before early antifungal therapy (n=747)

≥1 positive test with ß-glucan 5/11 45 (17-77) 113/172 66 (58-73) 0.11

≥1 positive test with qPCR 4/11 36 (11-69) 113/172 66 (58-73) 0.02

≥1 positive test with ß-glucan or qPCR 7/11 64 (31-89) 76/172 44 (37-52) 0.08

Cordonnier, C. et al. Clin Infect Dis; 48:1042-51 (2009)Swcharzinger M et al Plos One; 8(6): e65776 (2013)

Results (PREVERT study)Performance of qPCR overall and according to sample selection

Results (PREVERT study)

• Question: a qPCR assay may increase the sensitivity of bi-weekly

blood screening with galactomannan?

• Result: qPCR positivity was an independent predictor of baseline

invasive aspergillosis (P=.010), while (1,3)--D glucan was not (P=.53).

Table 3. Performance of Fungal Biomarker Assays for the Detection of Invasive Aspergillosis as Determined by the Relevant Published Meta-analyses

White PL et al Clinical Infectious Diseases 2015;61(8):1293–303

Galactomannan-EIA PCR

Aspergillus PCR on BAL

M

MΦ

Cell free fungal

elements

Summary of the 15 manuscripts included in meta-analysis of Aspergillus PCR testing BAL (Tuon, 2007)

Sensitivity (%)

Manuscript Extraction Protocol Appropriate disease definitions Tuon Original

Spreadbury 1993 Bead-beating in liquid N2 Phe/Chloro/Alc ppt Unknown 100 100

Melchers 1994 Bead-beating, Phe/Chloro/Alc ppt yes 100 100

Bretagne 1995 Hot detergent (10mM NaOH 100°C/10min) yes 100 100

Jones 1998 PK digestion Phe/Chloro/Alc ppt Proven yes prob/poss combined 100 100

Verweij 1995 Bead-beating, Phe/Chloro/IIAA ppt yes 71 71

Tang 1993 Bead-beating in liquid N2 Phe/Chloro/Alc ppt Unknown 100 100

Einsele 1998 Lyticase, then PK/SDS Phe/Chloro/Alc ppt yes 66 63 (100)

Skladny 1999 Lyticase, then PK/SDS Phe/Chloro/Alc ppt yes 36 100*

Buchheidt 2001 Lyticase, then PK/SDS Phe/Chloro/Alc ppt yes 76 100

Hayette 2001 Bead-beating in liquid N2 Phe/Chloro/Alc ppt Proven yes, probable needs CT 100 100

Raad 2002 SDS/PK/ Phe/Chloro/Alc ppt yes 69 69

Sanguinetti 2003 DNeasy Plant mini kit yes 90 90

Jalava 2003 Lyticase, bead-beating, Phe/Chloro/Alc ppt yes 73 73

Musher 2003 Master Pure yeast kit Proven yes, prob/poss combined 67 67

Spiess 2003 Lyticase, then PK/SDS Phe/Chloro/Alc ppt Yes 100 100

*Note: Paper does not state that all the patients with proven probable disease had BAL specimens tested. It only presents

PCR positive results. Sensitivity in the original manuscript was calculated using a combined whole blood/BAL approach

Lyticase ; sensitivity = 70%

Bead-beating ; sensitivity = 94%

Broncho-Alveolar Lavage Results

Study 1 Study 2

Study design prospective retrospective

Patients ((n) 191 32

inclusion Suspicion pneumopathy Subset of another study

BAL 3x50 ml Not specified (local procedures)

DNA extraction Pellet of 2.5 ml 200 µl

Method EZ1 DNA Tissue Kit (QIAGEN) EZ1 DNA Tissue Kit (QIAGEN)

Elution Not provided 50 µl

PCR format Nested PCR qPCR

Primer design “pan fungal” “pan fungal”

IC* no Yes

EORTC/MSG IA criteria 3 pv/8 pr/42 po 8 pv./3 pr/6 po vs. no IA=15

sensitivity 26% 100%

specificity 70% 87%

ppv 26% 80%

npv 71% 100%

Conclusion Limited usefulness Complete agreement with antigen marker (LAF)

(1) Buess M et al BMC Infectious Diseases 2012 :237; (2) Johnson GL et al JCM 2015, 53, 2103-8

Challenges using BAL

• Procedure: different according to country and wards (ICU, haematology)

– Volume (2x20 ml; 5x50 ml)

– Lung segment washed (localized disease)

• DNA extraction

– Pellet: very stringent (spores, hyphae)

– Supernatant: no bead-beating (free DNA)

– Note: no satisfactory control of DNA extraction if spores or hyphae are the

source of the DNA

• Human cell recovery (human DNA quantification)1, 2

• Meaning of a PCR positive result? Same as culture

• Purpose: only invasive aspergillosis? or concomitant diagnosis of pneumocystosis?

1- Fréalle E et al Eur J Clin Microbiol Infect Dis (2009) 28:223–2322- Khot PD et al BMC Infectious Diseases 2008, 8:73

High variability of mould species recovered in

respiratory specimens

Garcia–Hermoso et al Mycoses 2015 DOI: 10.1111/myc.12356

Primer design

Use of species specific primers:Scherer et al JCM 2018, Baldin et al JCM 2018, Wehrle-Wieland et al TID 2018

Table 3. Performance of Fungal Biomarker Assays for the Detection of Invasive Aspergillosis as Determined by the Relevant Published Meta-analyses

White PL et al Clinical Infectious Diseases 2015;61(8):1293–303

Alanio et al Frontiers in Microbiology | www.frontiersin.org 1 October 2017 | Volume 8 | Article 2040

Correlation between galactomannan index (GM-ODI) and quantification cycle (Cq) of Aspergillus fumigatus PCR assay in GM-positive serum samples

before (A) or after (B) initiation of antifungal therapy.

Combining several biomarkers

Combined surveillance/detection of antigens and Aspergillus DNA

• GM and Aspergillus DNA

– decrease the incidence of IA in high-risk haematological patients 1

– reduce empirical anti-fungal treatment in haematology patients at

high risk of IA 2

• Use of other biomarkers (LFD Lateral Flow Device) and qPCR 3

1- Aguado JM, et al. Clin Infect Dis. 2015; 60(3): 405–14.2- Morrissey CO et al Lancet Infect Dis. 2013; 13(6): 519–28.3- White PL, et al. J Clin Microbiol. 2013; 51(5): 1510–6.

Commercial Aspergillus qPCR kits & LBA

• MycAssay®*

• A. fumigatus® Bio-Evolution

• MycoGENIE ® Ademtech

• AsperGenius®

• A. fumigatus Genesig®

• Fungiplex Renishaw®

• Aspergillus spp. ELITe MGB Kit

• AspID PCR assay (OLM Diagnostics, Newcastle, UK)

• …

*not commercialized anymore

Commercial PCR kits and BAL

N Invasive aspergillosis

(EORTC/MSG criteria)

PCR assay Sensitivity

(%)

Specificity

(%)

Reference

22 2 proven, 9 probable Commercial kit 88.9-80 89.3-93.3 Chong et al 2015

63 31 (11 possible) 2 commercial kits 71-81 100 Denis et al 2018

387 61 (23 possible)2 in-house;

2 commercial71.1-73.7 92.8-95.4 Guegan et al 2018

13321 (probable-possible

gathered)Commercial kit 90 97 Grancini et al 2018

36 18 Commercial kit 94.1 76.5 Prattes et al 2018

Conclusion-Perspectives

• PCR itself is not the main issue

– providing qPCR format, internal control, MIQE validation …

– huge difficulties in designing primers for multiplex PCRs for use in complex biological

specimens (respiratory specimens)

• Knowledge of the physiopathology/epidemiology of the disease

– development of the fungus in the organism (steroids ≠ neutropenia)

– decreasing prevalence (use of antifungal prophylaxis) = expected difficulties in assessing

performance of the tests

• Nucleic acids extraction/choice of the clinical specimen

– expected pitfalls of the “syndromic panels” because extraction cannot be optimal for every

microorganism whatever the clinical specimen.

Many thanks to EAPCRI laboratory group

Thank you for your attention

(1607 AD)