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Broad range PCR tests for the detection of microorganisms:
opportunities and limitationsKatrien 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
GE Madico and PA Rice, Curr Infec Dis Rep 2008: 10 (4): 280-6
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.
16S ribosomal DNA
Morel AS et al, Eur J Clin Microbiol Infect Dis, 2014 Oct 28.
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
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
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)
Sepsis
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
(SIRS-Lab)
MagicplexSepsis Real-
Time test (Seegene)
BAC assay,IRIDICA
(Abbott)
Multiplex real-time PCR, species specificprobes
Broad range PCR + sequencing
Multiplex PCRplus micro-array hybridization
3 PCRs (1 conventional + 2 real time)
Broad-range PCR + ESI-MS
25 pathogens
(5 Candida, A. fumigatus)
> 345 bacteria and fungi
34 pathogens (6 Candida, A. fumigatus), mecA, vanA,vanB,blaSHV, blaCTX-M
21 bacterial species, 5 Candida species and A. fumigatus
> 780 bacteria Candida and mecA, vanA, vanBand 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
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 anti-
infective therapy Can supplement but not replace BC
Multiplex PCR: conclusions
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
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
Stenotrophomonasmaltophilia
Pathogens detectable using SeptiFast
0.68 (95% CI 0.63-0.73)
0.86(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
IRIDICA Workflow Steps and Instruments
Nucleic Acids Extraction & PCR Setup
PCR AmplificationDesalting & ESI-TOF MS Analysis
Sample Lysis
Bead Beater (BB) Sample Prep (SP) Thermal Cycler (TC) Desalter (DS) Mass Spectrometer (MS)
Organism Identification
Sample Prep
Isolated DNA
PP1 PP2 PP3 PP4 PP5 PP6 PP7 PP8
Organism Identified
Detection A Detection B
PP1 PP2 PP3 PP4 PP5 PP6 PP7 PP8
Organism Identified
Detection A Detection B
Assay Menu and Sample Types
Coverage Sample Type
780+ Bacteria, Candida and 4 Antibiotic Resistance Makers: mecA, vanA, vanB and kpc
5ml EDTA whole blood
Sterile fluid and tissues
ASSAY
BAC BSI (Blood Stream Infections)
BAC SFT (Sterile Fluids & Tissues)
BAC LRT (Lower Respiratory Tract)
Fungal
Viral IC (Immunocompromised)
BAL and ETA
200+ fungi and yeast BAL and Culture Isolates
13 distinct groups of viruses
130+ Viral speciesPlasma
BAC Assay Configuration
Bacterial identificationAntibiotic resistanceCandida detection and speciation
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
Gammaproteobacteria
Beta/Gammaproteobacteria
346 879A
1 2
34837674675B
361 3768C
349 3030D
3350 3031E
2249358
3766F
3346 3865G
3921 4437H
16S rDNABroad Bacterial
23S rDNA Broad Bacterial
Firmicutes
StaphylococcusEnterobactetriaceae
mecA
vanAKPC
vanB
Candida Identification & Speciation
Pumpkin DNA Extraction Control
Fungal Assay Configuration
Fungal identification
Sample Types:
- BAL
- Culture Isolates
Culture samples to be tested separate from BAL samples
Each sample is tested with 16 primer pairs in a 16 well setup
3030 5181
5185 4837
3766 5178
5186 5172
3865 5174
3867 4836
3862 5187
4145 4437
1 2
Ascomycetes(mtDNA SSU)
Fusarium(B-tubulin)
Broad Fungal Range(SSU rDNA)
(mtDNA cytB)
(SSU rDNA)
Miscellaneous Fungi(SSU rDNA)
Broad Fungal Range(LSU rDNA)
Mucorales
Broad Fungal Range(LSU rDNA)
Candida(mtDNA SSU)
Aspergillus(mtDNA SSU)
Cryptococcus(mtDNA SSU)
A
B
C
D
E
F
G
HPumpkin DNA Extraction Control
Viral IC Assay Configuration
Sample Type: Plasma
Mastermix for Reverse Transcription Reaction has to be added
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 pre-filled with water only
1 2
A
B
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
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
Endocarditis
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