ESCMID Online Lecture Library - ESCMID: ESCMID

Pan fungal PCR RA Barnes

ESCMID Online Lecture Library

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Fungal disease

• More than 2 million fungal species recognized • 600 reported to cause disease

• 99% due to 30 species

• Diseases • Skin, hair and nail

• Mucosal

• Allergic

• Chronic

• Acute invasive


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What are we looking for?


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At risk groups

• Haematological malignancy and SCT 3-15%

• Aspergillus 3-12%

• Pneumocystis 2%

• Candida 2%

• Mucorales, Fusarium, Scedosporium <1%

• Solid organ transplant • Candida 3%

• Pneumocystis 1%

• Critical care and special care baby units

• Candida 1%

• Cystic fibrosis • Aspergillus, Scedosporium,

Exophiala sp

• HIV • Cryptococcus

• Pneumocystis


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Incidence in haematological malignancy

• Aspergillus accounts for more than 90% of moulds in most centres

• But • Notable exceptions where

Scedosporium, Fusarium spp predominate

• Little known about epidemiology on many countries

Incidence IFD % Moulds incidence

% Acute myeloid leukaemia 12 7.9

Acute lymphoid leukaemia 6.5 4.3

Chronic myeloid leukaemia 2.5 2.3

Chronic lymphoid leukaemia 0.5 0.4

Lymphoma 1.6 0.9

Hodgkin disease 0.7 0.35

Multiple myeloma 0.5 0.3

adapted from Pagano L, et al. Haematologica 2006;91:1068-1075 ESCMID Online Lecture Library

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What do you want of your test?

• Screening test • Rule out a diagnosis

• To avoid empirical therapy immunocompromised patients

• Identify carriage Pick up cases before overt infection develops

• Regular testing required • Restricts use of invasive samples

• Prognostic test • Assess need for antifungals • Monitor response to therapy

• Diagnostic test • Rule in a diagnosis • Symptomatic Patients • Targeted (invasive) specimens


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Testing

Unlikely to be cost effective to “screen” for a disease with prevalence <5% negative predictive value most important Sensitivity needs to be high For diagnosis (once signs of clinical disease are present and fungal infection is suspected) Positive predictive value is important Specificity needs to be high


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Benefits of identifying fungal pathogens?

• Unknown organism/outbreaks

• Species may predict antifungal susceptibility

• molecular detection of resistance • multiplexing can detect TR34, L98H, T289A, and Y121F mutations in CYP51A

• Targeted therapy

• Improved epidemiology • Identify trends and geographical differences

• Inform local policy making


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Challenges for pan-fungal PCR • low amount of fungal DNA in clinical specimens

• Limits of PCR detection • Impacts on sensitivity

• Not ideal for screening where high NPV is important

• Broad-multiplexing can reduce analytical sensitivity

• Ubiquitous environmental contamination • Collection tubes

• Molecular reagents

• Human fungal biome largely unexplored

• Fungal disease has a broad range of clinical manifestations • Within specific diseases

• Across specific diseases

• Prohibits the use of a single specimen type

• Pan-fungal PCR requires performance robustness across this range ESCMID Online Lecture Library

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Hyphae/ Conidia

Serum/Plasma

Luminex/SERRS technology Broad range multiplex PCR Genus/species specific RT PCR

CSF

Blood Clot

Whole Blood Red cell lysis

White cell lysis

Fungal lysis – Bead beating

DNA purification/ppt/elution

PCR amplification

Real-time PCR

Pan-fungal RT PCR

Proteinase K/SDS digestion

Cerebral Disease

IPA, Invasive Sinusitis, Localised respiratory disease

Fungaemia, Vascular Invasion, Tissue Invasion

Cell associated DNA

Combination?

Free DNA

Tissue biopsy Tissue Invasion

Respiratory specimens

Cellular material

Antifungal Therapy

Hyphae

Free DNA

Cell associated DNA

Conventional PCR Pan-fungal PCR

Sequencing/Micro Array

Options for Fungal PCR


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Targets

• Multicopy genes required for sensitivity

• 18S/28S rRNA gene • Truly panfungal • Assays allow for genus/species specific probes

• But • Lacks sensitivity if multiplexing • Lacks specificity if using a single pan-fungal probe • Can amplifiy human DNA (if poorly designed) • Prone to contamination

• ITS regions of rDNA complex • Improve species differentiation

• Ability to multiplex both PCR and detection

• New detection methods • Isothermic PCR • microarrays, xMAP technology, sequencing, Mas spec ToF

Human vs Fungi


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Oligonucleotide design

• rRNA operon • 18S rRNA gene • Pan-fungal primers

• Genus sp. probe(s) • Pan-fungal probe

Block-based/Sybr Green/Pan-fungal probes

– False positives – Cross-reactivity with Human DNA – Contamination - environmental

fungi – Careful assay design paramount

Genus sp probe(s) with pan-fungal primers – False negatives – Competitive PCR (Human DNA)

Human vs Fungi Human vs Bacteria


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New developments in PCR

• Isothermal amplification • PCR without the need for thermocycling

• Loop-mediated isothermal amplification

• Helicase-dependent amplification

• Cheaper, potential for POC • maximum amplifiable length of the products too small for most

fungal pathogens

Sakai K, Trabasso P, Moretti M, Mikami Y, Kamei K, Gonoi T. Identification of Fungal Pathogens by Visible Microarray System inCombination with Isothermal Gene Amplification. Mycopathologia. 2014;178(1-2):11-26 ESCMID Online Lectu

re Library

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Isothermal PCR

• Can use turbidity of biotin peroxisase systems to read with naked eye

• Can capture PCR products with additional probe to create multiplex microarray

Loop-mediated isothermal amplification

Helicase-dependent amplification

Uses 4 different primers to recognize 6 distinct regions on single target gene to generate a strand displacement reaction

Helicase enzyme uncoils and denatures DS DNA


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• Sakai et al devised microarray system to 42 species from 24 genera fungal pathogens

• Used recombinase polymerase amplification cycle for labelling and amplification

• Used biotin-peroxidase spot detection system • Read with naked eye

• Targeted ITS region of rRNA

• Used panfungal, species specific, genus specific probes, fixed to glass slide

Sakai K. et al Identification of Fungal Pathogens by Visible Microarray System in Combination with Isothermal Gene Amplification. Mycopathologia. 2014;178(1-2):11-26 ESCMID Online Lectu

re Library

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Isothermal PCR

• Tested sensitivity and specificity • Against 355 target and non-target fungal species

• In a mouse mode l(aspergillosis)

• Clinical BCs one positive one negative

• Sensitivity • Spiked whole blood 103 cfu/ml

• Serum 5 pg/ml • Nested 50 fg/ml

• Mouse model required nested PCR

• BC correct ID Sakai K. et al Identification of Fungal Pathogens by Visible Microarray System in Combination with Isothermal Gene Amplification. Mycopathologia. 2014;178(1-2):11-26 ESCMID Online Lectu

re Library

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Verigene solid-phase microarray

Blake W. Buchan, and Nathan A. Ledeboer Clin. Microbiol. Rev. 2014;27:783-822

• Denatured nucleic acid anneals to complementary capture probe

• Gold microspheres coated with single-stranded nucleic acid target sequence anneal to the capture probe

• colloidal silver increases the size • Target-specific capture probes

added • bound silver microspheres diffract

the light, which is then detected by an optical camera


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xTAG liquid-phase microarray


• Target sequences amplified

• set of target-specific primers containing “universal tags "used for primer extension reaction

• biotin label is also incorporated

• Labelled amplicons incubated with up to 100 coloured polystyrene microbeads

• Each colour bead coated with a single-strand nucleic acid probe

• streptavidin-fluorophore conjugate added

• beads are analyzed using a cell sorter equipped with two lasers


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Digital PCR


• A nucleic acid template diluted into individual microwells

• each well or droplet contains one or zero copies

• PCR performed

• amplicon is detected using fluorescent dyes or probes


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Limitations of multiplexing and microarrays

• Requires stringent probe detection systems

• Implies some pre- knowledge of what you are looking

• Will miss new and emerging pathogens

• For example…


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skin and soft tissue

• Serious infection following penetrating injuries contaminated with soil, water

• Troops returning from Afghanistan

• Natural disasters • Tornados Tsunamis, floods

Hiransuthikul N et al. Clin Infect Dis. 2005;41:e93-e96


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Disaster-associated infections

Event Place No Fungus Infection Deaths

Tornado 2011 Joplin, USA 13 Apophysomyces trapeziformis Soft tissue 38%

Great East Japan

Earthquake and

Tsunami, 2011

Japan 3 Aspergillus fumigatus

Scedosporium apiospermum

Pulmonary disseminated

Lung and brain abscesses

Sinusitis and meningitis

100%

Hurricane Ike 2008 USA 3 Unspecified agent of

chromoblastomycosis

Soft tissue 0

Hurricane

Katrina,2005

USA 1 Cladosporium sp. Pulmonary 0

Indian Ocean

Tsunami, 2004

Thailand, Sri

Lanka

14 Cladophialophora bantiana

Scedosporium apiospermum

A. fumigatus

Apophysomyces elegans

A. elegans

Fusarium sp.

Mucor sp.

Soft tissue

Spondylodiscitis,

brain abscess,

Meningitis

50%

Earthquake 1994 USA 203 Coccidioides immitis Pulmonary

disseminated

1.5%

Volcano 1985 Colombia 8 Rhizopus arrhizus Soft tissue 80%

Dust storm 1977 USA 133 C. immitis Pulmonary;

disseminated

7%

Emerg Infect Dis.2014; 20: 349–355.doi: 10.3201/eid2003.131230 ESCMID Online Lecture Library

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Man-made outbreaks

• Exserohilum rostratum • 2012 Multistate US outbreak of

meningitis, spinal and joint infection

• >750 cases • 61 deaths

• Contaminated corticosteroid injections

• Pan fungal PCR facilitated rapid and efficient management of the investigation


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PCR Electron spray Ionisation TOF /Plex ID


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PLEX-ID

• 101 BAL fluid specimens from 93 patients • Pneumonia or lung transplant work up

• Compared PLEX ID to standard microbiology

• Focus on concordance and impact on management

• 91% positivity • High rate of polymicrobial isolation

• only relatively small number of fungal isolates

• Concordance only 45% overall • 5/7 cases of Pneumocytis jirovecii missed

by PLEX ID

• Limited impact on decision making

Huttner A et al J. Polymerase-chain reaction/electrospray ionization-mass spectrometry for the detection of bacteria and fungi in bronchoalveolar lavage fluids: a prospective observational study. Clin Microbiol Infect. 2014;20(12):O1059-O66.

Organism Total SM PLEX- ID

Aspergillus fumigatus

1 1 1

Candida albicans

7 2 7

Candida glabrata

1 1 1

Candida tropicalis

2 2 1

Penicillium spp. 1 0 1

Pneumocystis jirovecii

5 5 2

Rhizomucor pusillus

3 3 3


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NGS

• Automatic sequence library construction

• No cloning, transformation , colony picking

• array based detection system

• Faster, cheaper better

• How do we interpret results?


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Microbiota of the Respiratory tract

• 185 BAL specimens from ICU patients

• Amplified genes in 16 S and ITS 18S rDNA

• cloned and sequenced

• Up to 16 different organisms per specimen

• Up to 5 fungal species per patient

Bousbia S et al. Repertoire of Intensive Care Unit Pneumonia Microbiota. PLoS One. 2012;7(2):14. ESCMID Online Lectu

re Library

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Pros Cons Broad-range detection.

Ideal for screening as reduces the chance of

“missed” fungal infection

Range of potential pathogens implicated limits

commercial interest

Can detect emerging infections and outbreaks Susceptible to false positives due to contamination

from environmental fungi

More suitable for multiplex syndromic diagnosis Compromised sensitivity at limit of current PCR

detection

Improved turnaround times Species specific probes needed as melt curve analysis

lacks specificity across the range of potential

pathogens

Reduced cost compared to multiple monoplex

reactions

Sensitivity compromised by commensal flora

More suited to emerging genomic and

proteomic technologies eg PCR Electron spray

ionization/mass spectrometry

Low prevalence of non-candida non-aspergillus

infections limits clinical utility

Increased chance of hybridizing with human DNA

Limited validation in different specimen types ESCMID Online Lecture Library

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Conclusions

• Fungal PCR has come a long way

• Species specific PCR advantageous where Aspergillus is the prominent pathogen

• Pan fungal has a role in certain geographical areas and patient groups

• New technologies are overcoming limitations with sensitivity

• Still need to know more about the fungal biome before we can interpret utility of results


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