Pan fungal PCR RA Barnes
ESCMID Online Lecture Library
@ by author
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
ESCMID Online Lecture Library
@ by author
What are we looking for?
ESCMID Online Lecture Library
@ by author
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
ESCMID Online Lecture Library
@ by author
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
@ by author
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
ESCMID Online Lecture Library
@ by author
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
ESCMID Online Lecture Library
@ by author
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
ESCMID Online Lecture Library
@ by author
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
@ by author
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
ESCMID Online Lecture Library
@ by author
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
ESCMID Online Lecture Library
@ by author
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
ESCMID Online Lecture Library
@ by author
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
@ by author
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
ESCMID Online Lecture Library
@ by author
• 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
@ by author
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
@ by author
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
ESCMID Online Lecture Library
@ by author
xTAG liquid-phase microarray
Blake W. Buchan, and Nathan A. Ledeboer Clin. Microbiol. Rev. 2014;27:783-822
• 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
ESCMID Online Lecture Library
@ by author
Digital PCR
Blake W. Buchan, and Nathan A. Ledeboer Clin. Microbiol. Rev. 2014;27:783-822
• 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
ESCMID Online Lecture Library
@ by author
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…
ESCMID Online Lecture Library
@ by author
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
ESCMID Online Lecture Library
@ by author
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
@ by author
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
ESCMID Online Lecture Library
@ by author
PCR Electron spray Ionisation TOF /Plex ID
ESCMID Online Lecture Library
@ by author
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
ESCMID Online Lecture Library
@ by author
NGS
• Automatic sequence library construction
• No cloning, transformation , colony picking
• array based detection system
• Faster, cheaper better
• How do we interpret results?
ESCMID Online Lecture Library
@ by author
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
@ by author
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
@ by author
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
ESCMID Online Lecture Library
@ by author