Role of the laboratory in disease surveillance

2. 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

3. 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

4. Expected results Laboratory: Confirmation of clinical diagnostic: 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

5. 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

6. 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)

7. Outbreak detection Outbreak detection by the laboratory Outbreak detection with assistance from the laboratory

8. 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.

9. 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

10. 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

11. Laboratory role during outbreaks For new and emerging pathogens: Identify the pathogen Develop laboratory tests Patient treatment/management

12. 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

13. 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

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

15. Cases of malaria by species, Region A 1992-1996

16. 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

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

18. Predicting future AIDS trends for health service planning

19. 

20. V - Monitoring seroconversion/susceptibility Systematic control of immune status for specific diseases Tuberculin reaction Toxoplasmosis

21. 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

22. Define roles and responsibilities, identify referral system Ensure supplies, logistics, guidelines and forms Organize communication between lab and epi Prompt, regular reporting of results and feedback Plan quality assurance, biosafety and waste management Supervise and monitor Develop epidemic preparedness and response plans Establishing laboratory support for public health surveillance

23. 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

24. Genoserotyping or PCR Group

25. Pulsed-field gel electrophoresis (PFGE) Photo WHOPhoto WHO

26. Pulsed-field gel electrophoresis (PFGE) Photo WHO Photo WHO

27. Pulsed-Field Gel Electrophoresis

28. Universal PFGE standards

29. 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

30. Kirby-Bauer disc testing Antibiotic-impregnated discs placed on an agar plate at the interface between test organism and susceptible control organism Resulting zones of inhibition compared, use of controls Susceptibility is inferred (standard tables) Photos taken from Manual for the laboratory Identification and Antimicrobial Susceptibility Testing of Bacterial Pathogens of Public Health Importance in the Developing World WHO/CDS/CSR/RMD/2003.6 http://www.who.int/csr/resources/publications/drugresist/WHO_CDS_CSR_RMD_2003_6/en/Photos taken from Manual for the laboratory Identification and Antimicrobial Susceptibility Testing of Bacterial Pathogens of Public Health Importance in the Developing World WHO/CDS/CSR/RMD/2003.6 http://www.who.int/csr/resources/publications/drugresist/WHO_CDS_CSR_RMD_2003_6/en/

31. E-test Plastic strips with a predefined gradient of One antibiotic One antifungal Only one manufacturer One strip per antibiotic Wide range of antibiotics Easy to use Storage at -20�C Short shelf life, expensive Photos taken from Manual for the laboratory Identification and Antimicrobial Susceptibility Testing of Bacterial Pathogens of Public Health Importance in the Developing World WHO/CDS/CSR/RMD/2003.6 http://www.who.int/csr/resources/publications/drugresist/WHO_CDS_CSR_RMD_2003_6/en/ Photos taken from Manual for the laboratory Identification and Antimicrobial Susceptibility Testing of Bacterial Pathogens of Public Health Importance in the Developing World WHO/CDS/CSR/RMD/2003.6 http://www.who.int/csr/resources/publications/drugresist/WHO_CDS_CSR_RMD_2003_6/en/

32. Reading E-tests Photos taken from Manual for the laboratory Identification and Antimicrobial Susceptibility Testing of Bacterial Pathogens of Public Health Importance in the Developing World WHO/CDS/CSR/RMD/2003.6 http://www.who.int/csr/resources/publications/drugresist/WHO_CDS_CSR_RMD_2003_6/en/ Photos taken from Manual for the laboratory Identification and Antimicrobial Susceptibility Testing of Bacterial Pathogens of Public Health Importance in the Developing World WHO/CDS/CSR/RMD/2003.6 http://www.who.int/csr/resources/publications/drugresist/WHO_CDS_CSR_RMD_2003_6/en/

33. 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

34. Les puces � ADN ont suscit� beaucoup d�espoirs lors de leur apparition. Le principe est simplement bas� sur une amplification d�acides nucl�iques suivie d�une hybridation. Elle ne comporte rien de nouveau, si ce n�est une miniaturisation du syst�me, ce qui permet d�analyser non pas une s�quence, mais des milliers. Une sonde nucl�otidique sur laquelle peut venir s�hybrider la cible, est fix�e sur un support. Le signal est ensuite d�tect� puis analys�. Les puces � ADN ont suscit� beaucoup d�espoirs lors de leur apparition. Le principe est simplement bas� sur une amplification d�acides nucl�iques suivie d�une hybridation. Elle ne comporte rien de nouveau, si ce n�est une miniaturisation du syst�me, ce qui permet d�analyser non pas une s�quence, mais des milliers. Une sonde nucl�otidique sur laquelle peut venir s�hybrider la cible, est fix�e sur un support. Le signal est ensuite d�tect� puis analys�.

35. DNA chips characteristics

36. Pour une s�quence de r�f�rence, des amorces de 25 pb sont synth�tis�s et couvrent la totalit� de la s�quence. Le principe des puces � ADN de res�quen�age est assez simple du moment qu�une s�quence de r�f�rence dont on veut res�quencer la totalit� ou seulement une partie. A partir de cette s�quence, qui peut �tre un g�ne pour l�identification du pathog�ne ou celui d�un g�ne de r�f�rence, des sondes de 25 mers vont �tre d�sign�es. La premi�re sonde d�marre en position 1 de la s�quence jusqu�� la position 25. Les sondes suivantes sont d�cal�es d�une base � chaque fois, donc la deuxi�me sonde correspond � la s�quence de la base 2 � la base 26 et ainsi de suite jusqu�� la fin de la s�quence. Pour chaque position, il existe 4 oligos sens et 4 oligos antisens de 25 pb. La position centrale de ces oligo est variable : soit A; soit T, soit G, soit C En tout pour chaque position, il existe 8 oligonucl�otides de 25 pb. La cible c�est-� dire la s�quence que l�on souhaite hybrider sur la puce, va s�hybrider avec l�oligo pour lequel elle a le plus d�affinit�. La fluorescence �mise est directement proportionnelle � la quantit� de cibles marqu�es. Ona donc une image de fluorescence qui permet ensuite de res�quenc�r la s�quence hybrid�e sur la puce. Ex : en poisition 23, s�il y a un A sur la s�quence de r�f�rence et un T sur la s�quence cible, celle ci va s�hybrider pr�f�rentiellement avec l�oligo porteur d�un T en position 23 Pour une s�quence de r�f�rence, des amorces de 25 pb sont synth�tis�s et couvrent la totalit� de la s�quence. Le principe des puces � ADN de res�quen�age est assez simple du moment qu�une s�quence de r�f�rence dont on veut res�quencer la totalit� ou seulement une partie. A partir de cette s�quence, qui peut �tre un g�ne pour l�identification du pathog�ne ou celui d�un g�ne de r�f�rence, des sondes de 25 mers vont �tre d�sign�es. La premi�re sonde d�marre en position 1 de la s�quence jusqu�� la position 25. Les sondes suivantes sont d�cal�es d�une base � chaque fois, donc la deuxi�me sonde correspond � la s�quence de la base 2 � la base 26 et ainsi de suite jusqu�� la fin de la s�quence. Pour chaque position, il existe 4 oligos sens et 4 oligos antisens de 25 pb. La position centrale de ces oligo est variable : soit A; soit T, soit G, soit C En tout pour chaque position, il existe 8 oligonucl�otides de 25 pb. La cible c�est-� dire la s�quence que l�on souhaite hybrider sur la puce, va s�hybrider avec l�oligo pour lequel elle a le plus d�affinit�. La fluorescence �mise est directement proportionnelle � la quantit� de cibles marqu�es. Ona donc une image de fluorescence qui permet ensuite de res�quenc�r la s�quence hybrid�e sur la puce. Ex : en poisition 23, s�il y a un A sur la s�quence de r�f�rence et un T sur la s�quence cible, celle ci va s�hybrider pr�f�rentiellement avec l�oligo porteur d�un T en position 23

37. L�utilisation de puces � ADN n�cessite une quantit� suffisante d�ADN. L�aDN double brin est d�abord d�natur�. Puis des amorces al�atoires vont venir s�hybrider sur l�ADN cible simple brin. Celles-ci serviront d�amorces de polym�risation pour une polym�rase particuli�re issue d�un bact�riophage, la phi29. Elle poss�de une activit� de polym�risation tr�s rapide, une grande fid�lit� et une activit� de d�placement de brin. Elle ne sera pas d�croch�e quand elle rencontrera de l�ADN double brin devant elle. Sa fonction h�licase va lui permettre de d�placer et d�ouvrir l�ADN double brin et de continuer sa polym�risation. Sur ce nouveau brin, ainsi synth�tis�, de nouvelles amorces vont venir se fixer. On aboutit ainsi � des structures de type hyperbranch�es.L�utilisation de puces � ADN n�cessite une quantit� suffisante d�ADN. L�aDN double brin est d�abord d�natur�. Puis des amorces al�atoires vont venir s�hybrider sur l�ADN cible simple brin. Celles-ci serviront d�amorces de polym�risation pour une polym�rase particuli�re issue d�un bact�riophage, la phi29. Elle poss�de une activit� de polym�risation tr�s rapide, une grande fid�lit� et une activit� de d�placement de brin. Elle ne sera pas d�croch�e quand elle rencontrera de l�ADN double brin devant elle. Sa fonction h�licase va lui permettre de d�placer et d�ouvrir l�ADN double brin et de continuer sa polym�risation. Sur ce nouveau brin, ainsi synth�tis�, de nouvelles amorces vont venir se fixer. On aboutit ainsi � des structures de type hyperbranch�es.

38. Cette diapositive r�sume la m�thodologie utilis�e dans cette �tude.Cette diapositive r�sume la m�thodologie utilis�e dans cette �tude.

42. Peripheral level objectives Diagnosis and early warning signals Routine lab surveillance with intensification before epidemic season Environmental monitoring Epidemic prone disease monitoring Proper collection, transport and storage of samples Reporting of results Establishing laboratory support for public health surveillance

43. Intermediate level objectives Diagnosis and early warning signal Epidemic preparedness, response and capacity building In addition to activities at peripheral level, strengthen surveillance through: Supplies and logistics support Networking of laboratories, feedback and feed forward Monitoring, supervision Outbreak investigation, epi-lab coordination Establishing laboratory support for public health surveillance

44. Referral level objectives Confirmation and capacity building Key activities Referral investigations Outbreak investigation Development of guidelines Quality assurance program, bio-safety and waste management Training, monitoring, supervision and feedback Establishing laboratory support for public health surveillance

45. 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

47. Food Incident to Human cases Weekly comparison Microbiological characteristics (PCR Group and combined PFGE AscI/ApaI profiles) of each strain of food incident take as reference Search of all human strains with same (One band difference allowed) microbiological characteristics on three months before Report to the member of Listeria Cell Example of link between Human-Food Incident for smoked fish in 2009

48. Biochemical identification Purification on Columbia Determination of PIPLC, Gram, Catalase, Mobility Determination of biochemical reactions Arylamidase Esculin hydrolysis Alpha mannosidase D-arabitol acidification D-xylose acidification Rhamnose acidification Alpha-M�thyl-D-glucoside acidification Ribose acidification Glucose-I-phosphate acidification D-Tagatose acidification Mannitol Haemolysis on Horse Blood Camp Test

50. Flow chart of analyses

51. Flow chart of analyses

52. Outbreak detection within the laboratory Tracing spread through typing and characterization Detection of carriers and natural foci of infection Determine the end of an outbreak Determine elimination or eradication of disease Surveillance: Lab functions

53. Surveillance: Lab & epi functions Outbreak detection and investigation Develop case definition; determine case management Environmental monitoring Understand the natural history of disease Evaluate interventions Monitor progress towards control Develop immunization strategies Prevalence studies

54. Public health and clinical labs Public health laboratories Belong to the public sector Are involved in public health Participate in surveillance Clinical laboratories May be public or private Involved in management of patients May participate in public health surveillance (e.g. laboratory reporting)