Laboratory advanced technologies in Laboratory advanced technologies in the support of Public Health the support of Public Health

interventions in infectious disease interventions in infectious disease

LA PREPARAZIONE E LA RISPOSTA ALLE EMERGENZE INFETT IVELA PREPARAZIONE E LA RISPOSTA ALLE EMERGENZE INFETT IVE

Padova, 20 settembre 2012Padova, 20 settembre 2012

Prof. Giorgio Palù, M.D.Prof. Giorgio Palù, M.D.

Director, Department of Molecular Medicine,Director, Department of Molecular Medicine,University of PadovaUniversity of Padova

Head, Clinical Microbiology and Virology, Head, Clinical Microbiology and Virology, Padova University HospitalPadova University Hospital

interventions in infectious disease interventions in infectious disease emergenciesemergencies

THE ROLE OF A THIRD LEVEL REFERENCE CENTER THE ROLE OF A THIRD LEVEL REFERENCE CENTER (REGIONAL AND MACROREGIONAL DISTRICT)(REGIONAL AND MACROREGIONAL DISTRICT)

•• DiagnosisDiagnosis of infectious diseases and confirmationconfirmation of diagnosis

• Diagnosis requiring special expertise and containmentspecial expertise and containment (BL3 or plus)

•• Molecular epidemiologyMolecular epidemiology of emerging/re-emerging pathogens

• Genotypic-phenotypic dissection of microbial resistant traitsmicrobial resistant traits• Genotypic-phenotypic dissection of microbial resistant traitsmicrobial resistant traits

• Introduction and validation of innovative technologiesinnovative technologies

• Probing new guidelinesguidelines in diagnostic procedures

• Role as a proponent of clinical protocolsclinical protocols of therapy and prevention

OUTCOMES OF INFECTIOUS DISEASE SURVEILLANCE OUTCOMES OF INFECTIOUS DISEASE SURVEILLANCE BY A THIRD LEVEL REFERENCE CENTERBY A THIRD LEVEL REFERENCE CENTER

•• Best practice & appropriatenessBest practice & appropriateness in the management of major issues

in clinical and laboratory medicine

• Impact on costs costs of medicines, reagents, personnel and procedures

• Impact on public health controlpublic health control• Impact on public health controlpublic health control

•• Scientific contributionsScientific contributions

•• InnovationInnovation, R&D, technology transfer, Intellectual property

•• CollaborationCollaboration with international, national and regional authorities

Clinical microbiology in the genomics eraClinical microbiology in the genomics era

• Technology evolution since the late 19th century has lead to a great change

in our understanding of microbiology today.

– Recognition of new microbial and viral speciesnew microbial and viral species

–– Whole sequencingWhole sequencing of microbial genomes

–– MetagenomicsMetagenomics

–– New diagnostic toolsNew diagnostic tools (MS, NGS, automation)–– New diagnostic toolsNew diagnostic tools (MS, NGS, automation)

– Easier identificationidentification of microorganisms (e.g., MALDI-TOF)

–– MultiplexingMultiplexing (e.g., multiplex PCR, microarray, 454 pyrosequencing)

–– RealReal--timetime microbial genome sequencingsequencing (e.g., EHEC, WNV)

– Fast and sensitive antibiotic resistance testingantibiotic resistance testing (e.g., MALDI-TOF, NGS)

Clinical Microbiology Clinical Microbiology

Padova Clinical Microbiology LabPadova Clinical Microbiology Lab

Department of Molecular Medicine

Department of Molecular Medicine

& Virology& Virology

Microbiology and Virology Unit, Padova University HospitalMicrobiology and Virology Unit, Padova University Hospital

Facilities and research activitiesFacilities and research activities

•• BSL3 lab for virus isolation and cultureBSL3 lab for virus isolation and culture

•• MS and HPLC equipments for TDM MS and HPLC equipments for TDM

•• StateState--ofof--thethe--art genomics and art genomics and proteomics facilities. proteomics facilities.

•• Bioinformatics expertiseBioinformatics expertise

•• Tools for immunity testingTools for immunity testing

•• Research activityResearch activity•• Research activityResearch activity– molecular epidemiology and

pathogenesis of infectious diseases, – genomics of microorganisms and

host, – proteomics,– antimicrobial drug resistance, – antimicrobial drug-design, – infection and immunity,– new vaccine development, – cell and gene therapy of infectious

diseases and cancer.

The clinical microbiology labThe clinical microbiology lab

• Subspecialty Labs• Conventional culture, identification, and

isolation methods• Antimicrobial susceptibility testing • Molecular methods• Automation• Proteomics• Genomics• Emergency examinations

Quality management in microbiologyQuality management in microbiology

• Accreditation (ISO standard 15189)

• Quality management system

• Continual improvement• Continual improvement

• Process approach

Process map for the clinical microbiology labProcess map for the clinical microbiology lab

Strategic and management processStrategic and management process

Operational processesOperational processes

• Organization management• Collaboration, contracts• Customer feedback, complaints• Nonconformity management,corrective actions

• Evaluations (audits, indicators,mangement review)

• Technology review• Sustainable development• Continual improvement

Pre-analytical phase Analytical phase• method validation and

Post-analytical phase• biological validationPatient Results

Guidelines, plans of action

Information

Support processesSupport processes

• sampling• transport• reception – allocation• dispatch to other labs• storage

• method validation and performace verification

• technical validation• internal and external QC• in-vitro medical device vigilance

• biological validation• reporting• invoicing• storage of samples

• Human resource management• Documentation management• Equipment and reagent control• IT resource management• Hygiene, safety, premises and environment

• Record control, traceability, archiving

• Procurement

Patient

Prescription

Results

Resources allocated

Resource requirements

ImplementationImplementation of NGS of NGS technologytechnology in the in the clinicalclinicalmicrobiologymicrobiology and and virologyvirology laboratorylaboratory

Diagnostic needs

Research needs

NGS workflow Skills in NGS data

assembly and analysis

Skills in NGS protocols

set up (wet lab team)(wet lab team)

NGS platform

NGS workflowassembly and analysis

(bioinformatic team)(bioinformatic team)

QA/QCAnalytical validation

Clinical validation

Routine testing

NGS PlatformsNGS Platforms

• Roche/454 FLX: 2004• Illumina Solexa Genome Analyzer: 2006• Applied Biosystems SOLiDTM System: 2007• Helicos HeliscopeTM : 2008• Pacific Biosciencies SMRT: 2010• Ion TorrentTM PGS: 2011• Oxford Nanopore Sequencing: 2012• Oxford Nanopore Sequencing: 2012

Applications of advanced technologies Applications of advanced technologies by the Regional Reference Lab to by the Regional Reference Lab to support Public Health in infectious support Public Health in infectious

disease emergencies: disease emergencies: disease emergencies: disease emergencies: Some examples….Some examples….

Phenotypic and molecular characterization Phenotypic and molecular characterization of MDR pathogensof MDR pathogens

• Hospital infections• Antimicrobial susceptibility testing• K. pneumoniae MDR • MLST• Antiviral drug resistance• Antiviral drug resistance• Whole genome sequencing• New antimicrobial drug

development

UltraUltra--rapid detection of blarapid detection of blaKPC1/2KPC1/2--1212 by realby real--time PCR from perirectal and time PCR from perirectal and nasal swabsnasal swabs

New method: less than 2 hrs to detect KPC presence in easily available specimens high sensitivity (100%) and specificity (98%)

Application: KPC surveillance and containment in ICU, surgical and medical

Preventing and monitoring carbapenemase spreadingPreventing and monitoring carbapenemase spreading

Application: KPC surveillance and containment in ICU, surgical and medical wards

60 bp amplicon

detection of bladetection of blaKPC1/2KPC1/2--1212

Richter et al. JCM 2012Richter et al. JCM 2012

pKPN101pKPN101--IT, a novel fully sequenced blaIT, a novel fully sequenced blaKPCKPC--22 plasmid, from an plasmid, from an extremely drugextremely drug--resistant resistant Klebsiella pneumoniaeKlebsiella pneumoniae ST101 isolateST101 isolate

Frasson et al. J Clin Microbiol 2012Frasson et al. J Clin Microbiol 2012

AntiAnti--Influenza Compounds Influenza Compounds

that Act by a Novel that Act by a Novel that Act by a Novel that Act by a Novel MechanismMechanism

Our Target:

The Interaction between the PA and PB1 subunits of Flu RNA Polymerase

� Flu polymerase is composed of three subunits: PA, PB1, and PB2; their interactions are essential for virus replication

� In 2008, two crystal structures of the PA-PB1 binding interface have been published:

both structures showed that the C-terminal domain of PA forms a highly hydrophobic groove into which the amino-terminal residues of PB1 can fit by

forming an α-helix

He et al., Nature 2008, 454:1123-6 Obayashi et al., Nature 2008, 454:1127-31

The new compound

New Compounds Acting on Flu Virus Polymerase

PB2

PB1PA

1 12

N

O Cl

NH2O

O

O

O

N

N

N

F

FO

NH

S

OH2N

N+O

O-

O

NN

N

N+H

5

Surveillance for emerging Surveillance for emerging infections and outbreaksinfections and outbreaks

• N. meningitidis infection• Influenza • Measles• West Nile virus disease• West Nile virus disease• Dengue and chikungunya• Etc.

Clinical microbiologists have the expertise Clinical microbiologists have the expertise necessary to handle the unexpected !necessary to handle the unexpected !