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