Role of the laboratory in surveillance Sources: WHO Laboratory Training for Field Epidemiologists ECDC -EUPHEM Robert Koch Institute National Reference.

E P I D E M I C A L E R T A N D R E S P O N S E

Role of the laboratory in surveillance

Sources: WHO Laboratory Training for Field EpidemiologistsECDC -EUPHEMRobert Koch InstituteNational Reference Center for Listeriosis, ParisLaboratory for Urgent Response to Biological Threats, Paris

Ph. Dubois, Laboratory for Urgent Response to Biological Threats, Institut Pasteur, Paris

E P I D E M I C A L E R T A N D R E S P O N S E

Learning objectives

At the end of the presentation, participants should:

• Understand how the laboratory contributes to epidemiological surveillance

• Understand the principles of laboratory-based surveillance

• Understand some concerns of public health microbiologists

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Laboratories and disease surveillance

Before the outbreak

• Early warning signals

• Outbreak detection

During the outbreak

• Outbreak response and management

In between outbreaks

• Trend monitoring

• Intervention evaluation

• Monitoring progress towards a control objective

E P I D E M I C A L E R T A N D R E S P O N S E

Expected results Laboratory:

• Confirmation of clinical diagnostic:

• Direct identification of the bug

• Serology detection

• Identification of the strain/isolate/subtype

• Identification of new pathogen

• Characterization of pathogen sensitivity to antimicrobials

• Identification of seroconvertants/carriers in populations

• Collection of data/information from patients with various / different geographic origins

• Collection of data/information from environmental or animal origin

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Expected results Surveillance:

• Early warning

• Outbreak detection

• Post-outbreak surveillance

• Environment and reservoir analyses

• Surveillance of eradication-elimination of a bug

• Surveillance of vaccination campaign

• Surveillance of notifiable diseases

• Surveillance of national drug treatment efficacy

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I - Early warning signals

Detection of pathogens that have potential to spread

Sentinel events requiring early control measures

• Isolation of a single epidemic prone isolate (e.g. non-typhoidal salmonella isolated from a neonate in a hospital neonatal intensive care unit)

• Emergence of resistant strains in the hospital or the community (e.g. multi-drug resistant tuberculosis)

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Outbreak detection by the lab

Identification of a cluster of:

• Infections with an unusual pathogen

• Specific subtype of a pathogen

– Outbreak of antibiotic-resistant strains

– Subtypes of a pathogen (e.g. Shigella dysenteriae type I)

Reference centres may capture outbreaks disseminatedover a large area, or correlate events (food control-

cluster of human cases).

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Listeria monocytogenesGenoserotyping or PCR Group

Profile PCR Group Serovar

1, 5 IIa 1/2a ou 3a

2, 6, 10 IIb 1/2b ou 3b ou 7

3, 7 IIc 1/2c ou 3c

4, 8, 9 IVb 4b ou 4d ou 4e

11-16 LListeria monocytogenes 4a, 4ab, 4c or

other species of Listeria

ORF2819

lmo0737 1000

800 700 600 500 400 300

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

lmo1118

ORF2110

prs

IIa IIcIIb IVb

Doumith et al. JCM, 2004Doumith et al., J Food Protect 2005

Multiplex PCR : simultaneous PCR on 5 different DNA fragments

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II - Outbreak confirmation

Epidemiologist captures an increased incidence

Laboratory:

• Confirms the diagnosis

• Allows for a more specific case definition

• Detects a new pathogen

• Provides additional details on the pathogen (e.g., phage type)

Examples : detection H5N1, detection H1N1

Effective participation of the laboratory in surveillancerequires good communication between theepidemiologists and the laboratories

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Laboratory role during outbreaks

Laboratory confirmation of early cases

• On a subset of cases

Identification of new pathogens

Typing of the pathogen

• Link clusters when the epidemiological data is not sufficient

Antimicrobial susceptibility testing to guide treatment

Post-outbreak surveillance

Environmental investigations

Detection of carriers

E P I D E M I C A L E R T A N D R E S P O N S E

Laboratory role during outbreaks

For new and emerging pathogens:

•Identify the pathogen

•Develop laboratory tests

•Patient treatment/managementSARS

Courtesy:The University of Hong Kong

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III - Monitoring endemic disease trends

Confirm diagnosis

• Case definitions that include laboratory criteria:

Monitor resistance patterns

Monitor subtypes of a pathogen

• Detection Flu viruses subtypes, such as H5N1, H1N1

E P I D E M I C A L E R T A N D R E S P O N S E

Monitoring endemic disease trends

Examples:

Circulating strains of bacterial meningitis

• Impact on treatment protocols

• Impact on immunization policies

Antibiotic resistance

• Methicilin resistant staphylococcus aureus

• Vancomycin resistant enterococcus

• Tuberculosis

• Monitoring of Flu viruses circulation, vaccination policies

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0

100

200

300

400

500

600

85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 2000

Year

Num

ber o

f cas

es

C

B

A

Unknown

Source : InVS and NRC for N. meningitis, Pasteur Institute, Paris

Invasive meningococcal infection serogroups by year, France, 1985-2000

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IV - Eradication/elimination monitoring

The elimination phase requires more specific tests aspositive predictive value decreases

Laboratory confirmed diagnosis

• Polio surveillance

• Measles

Typing helps identifying the origin

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0

50

100

150

200

250

80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96

Years

Nu

mb

er

of c

ase

s

National immunization day

National immunization day

Cases of polio where wild poliovirus was isolated in children, District X 1980-1996

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Tuberculosis: new cases, treatment completion and cures, District X, 1994-1997

0

500

1000

1500

2000

1994 1995 1996 1997

Years

Num

ber o

f pat

ient

s

Cases

Completion

Cure

Monitoring TB control program to ensure complete treatment and cure

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V - Monitoring seroconversion/susceptibility

Systematic control of immune status for specific diseases

Tuberculin reaction

Toxoplasmosis

E P I D E M I C A L E R T A N D R E S P O N S E

Establishing laboratory support for public health surveillance

Identify diseases of public health importance

List diseases that require laboratory confirmation

Determine tests to be performed

Map laboratory facilities and human resources,including reference laboratories

Establish laboratory networking

Identify a focal person to coordinate laboratory activities

Determine information flow

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How to identify a new subtype?

• Look for genetic material : broad range of genetic probes and methods

• Direct isolation :

• culture on selective media to obtain clonal populations

• Characterize new serotypes

• Characterize new chemical or biochemical activities

• Characterize new toxins

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Pulsed-field gel electrophoresis (PFGE)

RFLPs of VRE isolates as determined by PFGE; all appear identical

RFLPs of two strains (B & C) from a patient as determined by PFGE; both different implying mixed infection; lane A is marker

RFLPs of MRSA isolates with similar ABT ST profile as determined by PFGE; only isolates B & C are identical

E P I D E M I C A L E R T A N D R E S P O N S E

How to identify a new resistance to antibiotics

• Look for genetic material : broad range of genetic probes and methods for identification of resistance genes.

• Direct isolation :

• Culture on selective media to obtain clonal populations

• Grow isolates on different antibiotics, and determine dose-response curves

E P I D E M I C A L E R T A N D R E S P O N S E

How to identify a new pathogen ?

Good question !!!!!

What if totally unknown, no clue from clinicians, or classical lab techniques ?

• Look for genetic material : broad range of genetic probes and methods

• Direct examination : light microscopy, electronic microscopy

• Direct isolation :

• culture on a whole spectrum of bacteriology media and conditions

• Culture on a whole spectrum of cell lines permissive for most known viruses

E P I D E M I C A L E R T A N D R E S P O N S E

DNA CHIPSPathogenID® Microarray :

Categories of genes Number of Genes 16S ribosomal RNA 51 Toxins/Pathogen effects 229 Resistance to antibiotics 390 Conserved genes 72 MLST 7

Point mutations 167

ARNr 18S 4 Viruses 42

Total 962

Genre SpeciesAlphavirus Eastern Equine Encephalitits virusAlphavirus Venezuelan equine encephalitis virusAlphavirus Western equine encephalomyelitis virusArenavirus Guanarito virusArenavirus Junin virusArenavirus Lymphocytic choriomeningitis virusArenavirus Machupo virusCoronavirus SARS coronavirus Ebolavirus Reston ebolavirusEbolavirus Zaire virusEphemerovirus Bovine ephemeral fever virusFlavivirus Dengue virus type 2Flavivirus Japanese encephalitis virusFlavivirus Kyasanur forest disease virus strain

W371Flavivirus Tick-borne encephalitis virus

etc…………..

42 virus sequences :26 genders11 families

126 virus sequences :37 genders14 families

E P I D E M I C A L E R T A N D R E S P O N S E

Surveillance: Lab functions

Confirmation of etiology to resolve syndromic presentation

Data intelligence for:

• Antimicrobial resistance monitoring

• Emergence of unusual isolates

• Detection of new pathogens

• Sero-surveillance

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French surveillance system of listeriosis

Laboratory of medical

microbiology (Private & Public)

Patient

Clinician

Regional Agency of Ministry of

Health (ANRS)

Ministry of Economy (DGCCRF)

Ministry of Agriculture(DGAl)

NRC

Public Health Agency(InVS)

Ministry of Health(DGS)

Regional Agency of Ministry of Economy

(DDCCRF)

Regional Agency of Ministry of Agriculture

(DDPP)

Laboratory of foodmicrobiology

(Private & Public)

Legend :FI, Food IncidentM, Mandatory NotificationS, StrainFQ, Food Questionnaire

M

FQ

Cell Listeria

FI

FI

SFood Information

M

M

FQ

SPatient

information

NRL

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Samples from the field to the lab

What samples should you take? And how?

• Blood, stools, swabs, water, food items, etc…

How should you ship the samples?

• High risk material?

• Cooling necessary?

• “Box in a box in a box principle”

Ask the lab and inform that samples are arriving!

E P I D E M I C A L E R T A N D R E S P O N S E

The result of any laboratory test is only as good as the

sample received in the laboratory

E P I D E M I C A L E R T A N D R E S P O N S E

Packaging infectious substances for shipment

Triple packaging system

Infectious substance (BIOHAZARD) label

Container for the first suspected anthrax letter / Berlin

Prof. Matthias Niedrig, RKI

Packaging Specification Marking example: 4H’’/Class 6.2/94 GB/2470 The packaging marking consists of: The United Nations packaging symbol Type of packaging The text “Class 6.2” The last two digits of the year of

manufacture of the packaging State authority manufacturer’s code

u n

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Sampling module

E P I D E M I C A L E R T A N D R E S P O N S E

Biological Risk ManagementBiological Risk Management

• Laboratory biosafety: containment principles, technologies, and practices implemented to prevent unintentional exposure to pathogens and toxins, or their unintentional release

• Laboratory biosecurity: institutional and personal security measures designed to prevent the loss, theft, misuse, diversion, or intentional release of pathogens and toxins

• 1Laboratory biosafety manual, Third edition, World Health Organization, 2004)

E P I D E M I C A L E R T A N D R E S P O N S E

Risk GraphL

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Low RiskLow RiskL

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Risk assessmentRisk assessment

E P I D E M I C A L E R T A N D R E S P O N S E

Laboratory Biorisk Management Laboratory Biorisk Management StandardStandard

• System or process to control safety and security risks associated with the handling or storage and disposal of biological agents and toxins in laboratories and facilities

• CWA 15793:2008

E P I D E M I C A L E R T A N D R E S P O N S E

Developed by the Department of Epidemic and Pandemic Alert and Response of the World Health Organization with assistance from:

European Program for Intervention Epidemiology Training

Canadian Field Epidemiology Program

Thailand Ministry of Health

Institut Pasteur

Role of laboratory in surveillance

E P I D E M I C A L E R T A N D R E S P O N S E

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