Role of the laboratory in surveillance

Ph. Dubois, Laboratory for Urgent Response to Biological Threats, Institut Pasteur, Paris Sources: WHO Laboratory Training for Field Epidemiologists ECDC -EUPHEM Robert Koch Institute National Reference Center for Listeriosis, Paris Laboratory for Urgent Response to Biological Threats, Paris

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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

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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

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 disseminated over a large area, or correlate events (food control cluster of human cases).

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Listeria monocytogenes Genoserotyping or PCR Group

1000 800 700 600 500 400 300

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

Profile

1, 5 2, 6, 10 3, 7 4, 8, 9 11-16

PCR Group

IIa IIb IIc IVb L

Serovar 1/2a

ou 3a

1/2b

ou 3b ou 7

1/2c

ou 3c

4b

ou 4d ou 4e Listeria monocytogenes 4a, 4ab, 4c or other species of Listeria

lmo1118 lmo0737 ORF2110 ORF2819 prs

Multiplex PCR : simultaneous PCR on 5 different DNA fragments

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

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 surveillance requires good communication between the epidemiologists 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

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Laboratory role during outbreaks

For new and emerging pathogens: • Identify the pathogen • Develop laboratory tests • Patient treatment/management SARS 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

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

600 500 400 300 200 100 0 C B A Unknown 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 2000 Year

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

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IV - Eradication/elimination monitoring

The elimination phase requires more specific tests as positive predictive value decreases Laboratory confirmed diagnosis • Polio surveillance • Measles Typing helps identifying the origin

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

250

National immunization day

200 150

National immunization day

100 50 0 80 81 82 83 84 85 86 87 88 Years 89 90 91 92 93 94 95 96

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Monitoring TB control program to ensure complete treatment and cure

Tuberculosis: new cases, treatment completion and cures, District X, 1994-1997 2000 1500 1000 500 0 Cases Completion Cure 1994 1995 1996 1997 Years

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V - Monitoring seroconversion/susceptibility

Systematic control of immune status for specific diseases Tuberculin reaction Toxoplasmosis

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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

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

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

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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

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DNA CHIPS PathogenID® Microarray :

Categories of genes 16S ribosomal RNA Toxins/Pathogen effects Resistance to antibiotics Conserved genes MLST Point mutations ARNr 18S

Viruses

Number of Genes 51 229 390 72 7 167 4

42

Total 962 Genre

Alphavirus Alphavirus Alphavirus Arenavirus Arenavirus Arenavirus Arenavirus Coronavirus Ebolavirus Ebolavirus Ephemerovirus Flavivirus Flavivirus Flavivirus Flavivirus

42 virus sequences : 26 genders 11 families

Species Eastern Equine Encephalitits virus Venezuelan equine encephalitis virus Western equine encephalomyelitis virus Guanarito virus Junin virus Lymphocytic choriomeningitis virus Machupo virus SARS coronavirus Reston ebolavirus Zaire virus Bovine ephemeral fever virus Dengue virus type 2 Japanese encephalitis virus Kyasanur forest disease virus strain W371 Tick-borne encephalitis virus

etc…………..

126 virus sequences : 37 genders 14 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 M S

Patient information

Ministry of Economy

(

DGCCRF) FI Ministry of Agriculture (DGAl) FI NRC FQ M Regional Agency of Ministry of Health

(

ANRS) M FQ Public Health Agency (InVS) Regional Agency of Ministry of Economy (DDCCRF) Regional Agency of Ministry of Agriculture ( DDPP) S

Food Information

NRL Laboratory of food microbiology (Private & Public) Clinician Ministry of Health (DGS) Cell Listeria Legend :

FI, Food Incident M, Mandatory Notification S, Strain FQ, Food Questionnaire

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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!

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The result of any laboratory test is only as good as the sample received in the laboratory

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Packaging infectious substances for shipment Packaging Specification Marking

Triple packaging system

example:

u n 4H’’/Class

Infectious substance

GB/2470

(BIOHAZARD) label

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

E P I D E M I C A L E R T A N D R E S P O N S E Prof. Matthias Niedrig, RKI

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 Management

•

Laboratory biosafety

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

Laboratory biosecurity

toxins : institutional and personal security measures designed to prevent the loss, theft, misuse, diversion, or intentional release of pathogens and • 1

Laboratory biosafety manual, Third edition

, World Health Organization, 2004)

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Risk Graph

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High Risk

Very High Low Moderate High Very Low Consequences E P I D E M I C A L E R T A N D R E S P O N S E

Low risk ?

Very High Low Moderate High Very Low Consequences E P I D E M I C A L E R T A N D R E S P O N S E

Low Risk

Very High Low Moderate High Very Low Consequences 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 assessment

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Laboratory Biorisk Management Standard

• 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

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Role of laboratory in surveillance 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

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