Rapid Microbial Detection: Current and future trends in healthcare
Surbhi MALHOTRA-KUMARSenior Research Associate
PhD (Med Microb ) MSc (Med Microb ) MSc (MolPhD (Med. Microb.), MSc (Med. Microb.), MSc (Mol. Biol. & Biotech.)
Department of Medical MicrobiologyVaccine & Infectious Disease InstituteVaccine & Infectious Disease Institute
Universiteit Antwerpen, Belgium
Rapid Tests in Infectious Diseases
• Current assays– Screening Assaysg y
• MRSA• MDR Enterobacteriaceae
Infection Detection Assays– Infection Detection Assays• Sepsis
• Future trendsutu e t e ds
Classical Methods in Bacterial Identification
• Gram’s stain
• Culture / antibiotic susceptibility
Christian GramRobert Koch
• Culture / antibiotic susceptibility
• Biochemical identification Christian Gram
Rapid Screening Tests for MRSA
BD GeneOhm MRSA and Cepheid GeneXpert MRSA assays targeting the SCCmec-orfX junctiony g g j
– Real-time PCR– Definitive identification of MRSA
Target both HA- and CA-MRSA– Target both HA- and CA-MRSA– Time to result is ≈ 1.30 hrs
SCCmec-orfX junction
Rapid Screening Tests for MRSA
BD GeneOhm MRSA and Cepheid GeneXpert are comparable and useful p
• GeneOhm: Batched and may need technical expertise, € 25/test
• Xpert: Modular/batched, user-friendly, € 35/test
Malhotra-Kumar et al., JCM, 2010
Rapid Screening Tests for MRSA
• High sensitivity • So specific that clinical
ADVANTAGES DISADVANTAGES
g y– Theoretically detects a
single organism
Hi h ifi it
pdata of aetiology needed before testing
• High specificity– Specific genotypes– Drug resistance
• New organisms missed unless molecular unknowns are sequenced
Culture remains the preferred method for MRSA detection
– Predict virulence
• Speed
q
• Expensive
– Quicker than culture
• SimplicityS– Some assays are now automated
Impact of Rapid MRSA Tests
• On MRSA acquisition rates/1000 patient-days– No effect on MRSA acquisition (vs. culture screening)q ( g)
• On incidence of MRSA bloodstream infections/1000 patient days– Significant decrease (46%) in MRSA bloodstream infections (vs
no screening)g)
• On MRSA surgical site infections/100 surgical procedures– Non-significant trend to reduction in surgical-site infections (vs
no screening)
Tacconnelli et al, LID, 2010
Decreasing MRSA Trends in Europe
Invasive MRSA trends, EARS-Net, 2009Courtesy D. Monnet
MDR Enterobacteriaceae: Emerging Superbugs
Invasive K. pneumoniae resistant to 3rd gen cephalosporins EARS-Net, 2009
– Optimum control strategy unclear
g p pCourtesy D. Monnet
– Screening for MDR Enterobacteriaceae limited
Screening for MDR Enterobacteriaceae
• Check-Points arrays– Check-ESBL: CTX-M, TEM, SHV– Check-KPC ESBL: KPC, CTX-M,
TEM, SHV– Check-MDR CT101: NDM-1, KPC,Check MDR CT101: NDM 1, KPC,
CTX-M, TEM, SHV, and several AmpCs
– Check-MDR CT102: NDM-1, VIM,Check MDR CT102: NDM 1, VIM, IMP, OXA-48, KPC, and CTX-M, TEM, SHV
S iti it S ifi it
PCR-microarray based commercial assays
Gene Sensitivity%
Specificity%
SHV 98.8 100TEM 100 96.4KPC 100 100
Endimiani et al., JCM, 2010
KPC 100 100
Screening for MDR Enterobacteriaceae
• Identibac AMR-ve genotyping array– vs. in-house PCR-sequencingq g
Amplification Hybridisation Detection Analysis
Gazin et al., ECCMID, 2011
Screening for MDR Enterobacteriaceae
• Direct detection from clinical samples is still culture-based– Diagnostic delay of at least 24 hoursg y
• More complex than MRSA detection– Fecal samples, numerous targets
• Molecular diagnostic solutions with off-board sample preparation
(Resistance in Gram-Negative Organisms: Studying Intervention Strategies)
Rapid Tests in Infectious Diseases
• Screening Assays– MRSAMRSA– Multi-resistant Enterobacteriaceae
• Infection Detection Assaysy– Sepsis
• Future trends
Diagnostic Tests for Sepsis
• Hybridization– Oligonucleotide–FISH, PNA-FISHg
• Prove-itTM Sepsis (Mobidiag)– Broad-range PCR + mecA detection
Highly conserved regions;
– 86% pathogen coverage– Sens 95% Spec 99%– Fast method
Highly conserved regions;Broad-range primers:
gyrB and parE
Target geneFast method
• LightCycler SeptiFast® PCR (Roche)
Variable regions;Capture oligos Tissari et al., Lancet, 2010
– TAT ≈ 6hrs for 8 samples– Sens 76–90% Spec 85–98% (vs. culture)– 82% pathogen coverage– 82% pathogen coverage– Limited sens/spec vs. culture Jaton-Ogay et al., ECCMID 2008
Regueiro BJ et al, ECCMID 2008
Diagnostic Tests for Sepsis
• Total TAT should still be ADVANTAGES DISADVANTAGES
• Relatively rapidshorter to directly impact therapeutic management
S l t li i ll l t
• Increase detection sensitivity especially useful i ti l li i l tti • Select clinically relevant
pathogens not in the panel
• Sample preparation laborious
in particular clinical settings– In patients who have been
treated with antibiotics• Sample preparation laborious
• Careful validation in real life is necessary
– Difficult to grow bacteria e.g. Bartonella spp,
– Neonatal sepsis, … is necessary
• Cost
Rapid Tests in Infectious Diseases
• Current assays– Screening Assaysg y
• MRSA• MDR Enterobacteriaceae
Infection Detection Assays– Infection Detection Assays• Sepsis
• Future trendsutu e t e ds
Paradigm Shift in Diagnostics
Integrated point of care test/personalized medicine
µPCR Photonic sensorsensor
Why Require Better, Rapid Point-of-CareAssays?
• There has been under-investment in rapid diagnostics for improving the quality of care for patients withfor improving the quality of care for patients with suspected infections– Diagnostics influence 60-70% of health care decision making but
account for less than 5% of hospital costs (Lewin report 2006)account for less than 5% of hospital costs (Lewin report 2006)
POCT i tibi ti t ti t l th h• POCTs improve antibiotic targeting to only those who will benefit, thus reducing overuse– The commonest reason for prescribing antibiotics in the p g
community is acute cough, and these prescriptions virtually never benefit patients (Butler et al, BMJ 2009)
Why Require Better, Rapid Point-of-CareAssays?
• POCTs enhance surveillance of pathogens and infectious diseasesinfectious diseases– e.g. H1N1 flu pandemic
• POCTs support rapid initiation and cessation of treatment
Sepsis is associated with 7% increased mortality for every hour– Sepsis is associated with 7% increased mortality for every hour delay in the administration of appropriate antibiotics (Kumar et al, CCM 2006)
• POCTs decrease the size and cost of antibacterial clinical trialsclinical trials– We URGENTLY need new antibiotics (ECDC/EMA report 2009)
Why Has It Not Happened Till Now?
Can you imagine the challenges of shrinkingchallenges of shrinking a huge laboratory filled with people and equipment onto a singleequipment onto a single chip the size of a matchbox?
Huge Challenges and Synergies
BiotechnologiesIntegrated sample prep solutions Bi l i l
Clinical practiceSelection of relevantIntegrated sample prep solutions
Targeting NA + host/pathogen biomarkers
N l f h i t i
Biological Sciences
Selection of relevant targets/applications
Validation of analytical, clinical performanceNovel surface chemistries clinical performance
Physical Sciences
Clinical Practice
(Micro)technologiesLab-on-a-chip/microfluidics
PhotonicsPhotonics
Biosensors
A Big Bottleneck in Developing POCTs: Sample Preparation
• Off-chip (macroscale) sample prep– LaboriousLaborious– Refrigerated/frozen reagents– Large sample volumes
R i t if b d b t– Requires centrifuges, bead beaters, several machines
– Few hours
• On-chip sample prep• Room temperature stable reagents
(di bl hi ith hi(disposable chips with on-chip storage)
• Microliter volumes
• Few minutes!!
On-chip Bacterial Lysis and DNA Purification
Development of a proprietary bacterial lysis and DNApurification protocol and its successful application on apurification protocol and its successful application on aprototypal microfluidic chip for a CA-LRTI assay
In collaboration with Institut für Mikrotechnik, Mainz, Germany
Van Heirstraeten et al., ECCMID, 2011
On-chip Bacterial Lysis, DNA Purification/Amplification
Development of a sample prep solution and on-chip micro-PCR for a rapid patient bed-side sepsis assayPCR for a rapid patient bed side sepsis assay
In collaboration with KTH Royal Institute for Technology, Stockholm, Sweden
Developing an efficient, rapid and accurate POCT
• The joint efforts of academia and industry can bring this to realityy
• IMI supports collaborative research projects and builds networks of industrial and academic experts in order to boost pharmaceutical innovation in Europe
Microfluidic ChipShop GmbH, Germany
Primary care Hospital care
RAPP-ID Project Phases
EFPIA MEMBER COMPANIES
- GlaxoSmithKline
UNIVERSITIES, RESEARCH ORGANISATIONS, PUBLIC BODIES &
NON-PROFIT
SMEs
LIONEX GermanyGlaxoSmithKline- Virco-Janssen- Merck- Novartis
S f
NON-PROFIT- Cardiff University, UK- Catholic University of Leuven, Belgium- IMEC, Belgium
U i it f C b id UK
- LIONEX, Germany- Microfluidic ChipShop, Germany- Mobidiag, Finland
- Sanofi-Aventis - University of Cambridge, UK- Geneva University, Switzerland- Ghent University, Belgium- Royal Institute of Technology, Sweden
- Q-linea, Sweden
oya s u e o ec o ogy, S ede- University of Antwerp, Belgium- University of Twente, Netherlands- Uppsala University, Sweden
C ll bCollaborators
Overall Objective of RAPP-ID
RAPP-ID will develop a Point-of-Care Test (POCT) for rapid (hospital <2h primary care(hospital <2h, primary care <30min) detection of bacteria, mycobacteria, fungi, as well as viruses and host biomarkers by combining novel specific probes, novel methods of sample preparation, and demonstrated ultra-high sensitive detectionultra high sensitive detection methods. The platforms will also determine resistance to antimicrobial drugs
RAPP-ID Meets Clearly Defined Clinician Needs
• Sepsis
• Lower Respiratory Tract Infections (LRTI)– Community-Acquired Pneumonia (CAP)– Community-Acquired Pneumonia (CAP)– Ventilator-Associated Pneumonia (VAP)
• Tuberculosis (TB)
Why Should RAPP-ID Succeed?
• IMI is a unique instrument of joint academia-industry initiatives, and this is the ONLY way to successfullyinitiatives, and this is the ONLY way to successfully develop POCT for Infectious Diseases
• RAPP-ID partners have built up experience in other EU p p pfunded projects (GRACE, InTopSens, TheraEDGE, ...)
• RAPP-ID provides a unique combination of a whole range of novel assays and technologies
• POCT development will not only be “pathogen or t h l i ll d i ’ b t l d i d t t l ltechnologically driven’ but also designed to meet clearly defined clinical needs with optimal integration and implementation into diagnostic/clinical algorithms and p g ghealthcare programs
Visit Our Website: www.rapp-id.eu