1. health and fitness
2. disease

Broad range PCR tests for the detection
of microorganisms:
opportunities and limitations
Katrien Lagrou
University Hospitals Leuven and KU Leuven, BELGIUM
Detection of a broad range of pathogens
Multi-parameter screening
(test panels)
Broad-range PCR tests
(16S rDNA, 18S rDNA)
• Species-specific hybridization probes
• Species identification by gene sequencing or
electronspray mass spectrometry
16S ribosomal DNA
Conserved (≥ 95% homology) and variable regions in bacterial 16S ribosomal DNA
based on the alignment of DNA sequences of Staphylococcus, Streptococcus,
Abiotrophia, Listeria, Coxiella, Legionella, Bartonella, Brucella, and Francisella spp.
GE Madico and PA Rice, Curr Infec Dis Rep 2008: 10 (4): 280-6
• Specific real time PCRs have a higher sensitivity than broad-range PCRs
• Targeted real-time specific PCR test and conventional broad-range PCR
are complementary
Morel AS et al, Eur J Clin Microbiol Infect Dis, 2014 Oct 28.
Ideal diagnostic platform
• Should identify a broad spectrum of pathogens
(bacteria, fungi, viruses, and protozoa)
• Determine the susceptibility to a battery of antibiotics
• Allow the analysis of specimens in high or low
throughput
• Have a low cost per sample
• Have minimum hands-on time
• Be user friendly
• Generate the results in a timely manner (for septic
patients, 6 hours or less)
E. Jordana-Lluch et al., BioMed Research International, 2014.
SEPSIS
Sepsis
• Life threatening
• Blood culture (BC): current gold standard for the
detection of bloodstream infection
• Value of BC in the diagnosis of sepsis is impaired by
the delay in the time to results and the fact that
positive BC can be found in only ± 30% of patients
• Two categories of ‘rapid test’ that emerged for the
detection of bacteria and fungi in blood:
– Detection and identification of pathogens from positive BC
(MALDI-TOF MS, PNA-FISH)
– Assays directly on blood (no incubation)
Reinhart K et al. Clin. Microbiol. Rev. 2012;25:609-634
Sepsis
• Treatment delay is associated with substantial
increases in mortality
• Empirical broad-spectrum antimicrobial drugs
– Unnecessary broad-spectrum antimicrobial use
– Development of drug-resistant pathogens
– Clostridium difficile infections
– Adverse effects
– High costs
Commercially available Molecular Assays for the Diagnosis
of Bloodstream infections from whole blood
SeptiFast
(Roche)
SepsiTest
(Molzym)
VYOO
Magicplex
(SIRS-Lab)
Sepsis RealTime test
(Seegene)
Multiplex realtime PCR,
species specific
probes
Broad range
PCR +
sequencing
25 pathogens
> 345 bacteria 34 pathogens
and fungi
(6 Candida, A.
fumigatus),
mecA, vanA,
vanB,blaSHV,
blaCTX-M
(5 Candida,
A. fumigatus)
BAC assay,
IRIDICA
(Abbott)
Multiplex PCR
3 PCRs
Broad-range PCR
plus micro-array (1 conventional + 2 + ESI-MS
hybridization
real time)
21 bacterial
species, 5
Candida species
and A. fumigatus
> 780 bacteria
Candida and
mecA, vanA, vanB
and KPC
3 mL (manual)/
1.5 mL
1 mL
5 mL
1 mL
5 mL
4.5-6h
8h30
8h
6h
6h
Positivity rates and concordance of multiplex PCR and blood
culture (BC) results from 27 published studies
Reinhart K et al. Clin. Microbiol. Rev. 2012;25:609-634
Multiplex PCR: conclusions
• Consistent inability to identify approximately 20-30%
of culture-positive results by multiplex PCR, even if
the pathogen should be covered by a primer pair
• Clinical utility of PCR remains to be defined
• Whole blood as a template for PCR faces limitations
due to its very high human DNA background level
• Are not fast enough to postpone empirical antiinfective therapy
• Can supplement but not replace BC
Reinhart K et al. Clin. Microbiol. Rev. 2012;25:609-634
• 41 phase III diagnostic accuracy studies
• Compared to blood culture
• 10,493 SeptiFast test
Pathogens detectable using SeptiFast
Gram-negative
Gram-positive
Fungi
Escherichia coli
Staphylococcus aureus
Candida albicans
Klebsiella pn/ox
Coagulase-negative staphylococci
Candida tropicalis
Serratia marcescens
Streptococcus pneumoniae
Candida parapsilosis
Enterobacter cl/ae
Streptococcus spp.
Candida glabrata
Proteus mirabilis
Enterococcus faecium
Candida krusei
Acinetobacter baumannii
Enterococcus faecalis
Aspergillus fumigatus
Pseudomonas aeruginosa
Stenotrophomonas
maltophilia
0.68
0.86
(95% CI 0.63-0.73)
(95% CI 0.84-0.89)
SepsiTest™
• Each lot of reagents is subjected to stringent quality
control in respect to contamination with microbial
DNA and assay sensitivity.
• DNA contamination in all PCR reagents is <3% falsepositive rate guaranteed.
• Sensitivity is guaranteed for a detection limit of <4
CFU S. aureus per 16S rDNA PCR (25 µl) at 40 PCR
cycles.
IRIDICA Workflow Steps and Instruments
Sample Lysis
Bead Beater (BB)
Nucleic Acids
Extraction &
PCR Setup
Sample Prep (SP)
PCR Amplification
Thermal Cycler (TC)
Desalting & ESI-TOF MS
Analysis
Desalter (DS)
Mass Spectrometer (MS)
Organism Identification
Sample
Prep
Isolated
DNA
PP1
PP2
PP3
PP4
PP5
PP6
PP7
PP8
Detection A
Detection B
Organism Identified
Assay Menu and Sample Types
ASSAY
BAC BSI (Blood Stream Infections)
BAC SFT (Sterile Fluids & Tissues)
Coverage
Sample Type
5ml EDTA whole blood
780+ Bacteria, Candida and
4 Antibiotic Resistance Makers:
mecA, vanA, vanB and kpc
BAC LRT (Lower Respiratory Tract)
Sterile fluid and tissues
BAL and ETA
Fungal
200+ fungi and yeast
BAL and Culture
Isolates
Viral IC (Immunocompromised)
13 distinct groups of viruses
130+ Viral species
Plasma
BAC Assay Configuration
Bacterial identification
Antibiotic resistance
Candida detection and speciation
1
2
A
346
879
mecA
B
348
3767
4675
vanA
KPC
C
361
3768
vanB
Broad
Bacterial
D
349
3030
Firmicutes
E
3350
3031
Staphylococcus
Enterobactetriaceae
F
2249
358
3766
Gammaproteobacteria
G
3346
3865
16S rDNA
Broad Bacterial
23S rDNA
• Different kit versions (BAC BSI,
BAC SFT, BAC LRT) for different
sample types
• Each sample is tested with 18
primer pairs in a 16 well setup
Beta/Gammaproteobacteria
H
3921
4437
Candida
Identification
& Speciation
Pumpkin DNA
Extraction
Control
Fungal Assay Configuration
• Fungal identification
• Sample Types:
1
2
A
3030
5181
Ascomycetes
(mtDNA SSU)
B
5185
4837
Fusarium
(B-tubulin)
C
3766
5178
Broad Fungal Range
(SSU rDNA)
D
5186
5172
(mtDNA cytB)
Broad Fungal Range
(LSU rDNA)
- BAL
- Culture Isolates
Mucorales
• Culture samples to be tested
separate from BAL samples
• Each sample is tested with 16
primer pairs in a 16 well setup
E
3865
5174
(SSU rDNA)
F
3867
4836
Miscellaneous Fungi
(SSU rDNA)
Aspergillus
(mtDNA SSU)
G
3862
5187
Broad Fungal Range
(LSU rDNA)
Cryptococcus
(mtDNA SSU)
H
4145
4437
Pumpkin DNA
Extraction
Control
Candida
(mtDNA SSU)
Viral IC Assay Configuration
1
2
• Sample Type: Plasma
A
•
Mastermix for Reverse Transcription
Reaction has to be added
B
•
Assay consumables configured to
run in a batch size of 6 samples.
•
•
Sample tested with 15 primers in a 8
well setup
The second column of the strip
(wells A2- H2) is not used and prefilled with water only
C
D
E
F
G
H
Future for sepsis tests…
Very difficult to speculate what the implications will be
for direct clinical care!!!
– Can not be used to stop treatment immediately in case of
negative results
– Restrict therapy to detected micro-organism (G+/G-)???
– To broaden therapy based on the results? But generally
broad-spectrum AB are already initiated and only a few
resistance markers are tested…
– No systematic interventional clinical trials on the overall
impact on clinical, laboratory and cost-effectiveness of
these tests
– Who will pay for these assays (+/- 250 euro)
ENDOCARDITIS
Endocarditis
• 2.5%-48% of all cases of infectious endocarditis are
culture-negative: prior or concurrent antibiotic
treatment and slow-growing or fastidious organisms
• Sensitivity of PCR in bloods samples disappointing
and below that in resected valves
• Major limitation: contamination of PCR reactions with
background bacterial DNA
• 174 patients who underwent surgery and with definite
endocarditis according to Duke criteria Jan 1, 2010-Jan 1,
2013
• Valves were sent for culture and universal bacterial PCR
(16S rRNA primers), universal fungal PCR (28S rRNA and
ITS primers) or mycobacterial PCR (hsp65 gene and probe
hybridization, rpoB gene) and sequencing
• Microbiological etiology was defined using comprehensive
clinical, pathologic and microbiological criteria
• Examination of blood culture, valve culture and valve
sequencing
Test
Sensitivity (%)
False positivity rate (%)
Blood culture
79
10
Valve culture
31
33
Valve sequencing
90
3
Blood cultures were negative in 46 patients (26%)
In these patients:
causative pathogen was identified in 37 (80%) by valve
sequencing versus 13 (28%) by valve culture (p< 0.001)
Mycobacterial and fungal sequencing offer little value
Patiënt 1, vrouw 64 jaar
• Endocarditis bevestigd
macroscopisch en door histologie
• Panbacteriële PCR: S. epidermidis
Patiënt 2, man 47 jaar
• Acute myocardinfarct met hartfalen en chordaruptuur
• Kreeg preoperatief moxifloxacine omwille van koorts,
gestegen CRP, infiltraten pulmonair (longoedeem met
vermoedelijk surinfectie, DD endocarditis)
• Klinisch peroperatief: degeneratief kleplijden, geen
teken van endocarditis
Patiënt 2
Panbacteriële PCR: negatief
Patiënt 3, vrouw 81j
• Week therapie met moxifloxacine voor afname
bloedkweken
• Hemoculturen: ¼ flessen positief met S. epidermidis
• Diagnose van endocarditis, transfer naar UZ Leuven
voor chirurgie
Patiënt 3
And the future…..?
Thank you!