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