Working with the laboratory during outbreak investigations

1. Working with the laboratory during outbreak investigations Integrated Disease Surveillance Programme (IDSP) district surveillance officers (DSO) course

2. Preliminary question to the group • What is your experience in working with the laboratory on outbreak investigations? • If yes, what difficulties did you face? • What would you like to learn about working with a laboratory? 2

3. Outline of this session • Communicating with the laboratory • Specimen collection, storage and transportation • Biosafety • Quality assurance 3

4. Participation of laboratory specialists in field investigation • Presence in the field: The ideal option • Laboratory specialist provide real time input • Time consuming, expensive • Most useful in difficult situations • Remote participation: The common option • Involve the laboratory early • Exchange information • Most efficient in routine situations 4 Communication

5. Communicating with the laboratory • Share initial information at the earliest about the investigation • Epidemiological characteristics • Suspected pathogens (differential diagnosis) • Organize communication on an ongoing basis • Identify focal person, obtain contact information • Generate outbreak number • Provide updates • Send the final epidemiological report 5 Communication

6. General framework to decide what kind of specimens to take • What are the suspected pathogens? • What tests are used to diagnose the suspected pathogens? • What is the stage of the illness? • No virus isolation at a late stage of illness 6 Communication

7. Elements to consider when choosing a laboratory • Location • Referral protocols • Capacity • Biosafety level • Quality, accreditation or certification • (e.g., Polio) • Credibility, track record with your team • Costs 7 Communication

8. Whom to sample during a classical outbreak? • Typical cases • Should represent the majority of the specimens • Untreated patients • Without antibiotics • Cases likely to carry the pathogen • Children • Atypical cases • Few specimens • Healthy contacts • Few specimens 8 Collection

9. When to sample? • Isolation of agent, PCR or antigen: • At the earliest • Before anti-microbial administration • For antibody estimation: • Ideally two paired specimens • At earliest • After 7 - 10 days • Alternately, one specimen 4-5 weeks after onset 9 Collection

10. How many specimens to take? • Ensure sufficient number of specimens (At least 20) • Avoid sampling error • Obtain reliable results • Avoid overwhelming the laboratory with excessive specimens • Repeat sampling in some case-patients • Acute and convalescent sera • Exploration of chronic carriage • Intermittent shedding (e.g., Stool microscopy for parasites) • Unknown etiology 10 Collection

11. Rule of thumb regarding the number of specimens to take during a cholera outbreak • 10 specimens to confirm the outbreak • Five specimens per week during the outbreak • Specimens at the end to confirm that the outbreak is over WHO guide 11 Collection

12. Transport medium • Allows organisms to survive under adverse conditions • Does not allow organisms to proliferate • Available for bacteria • e.g., Cary Blair • Available for viruses • Virus transport media (VTM) 12 Collection

13. What is a Viral Transport Medium? • Sterile buffered solution (Pink coloured) containing antibiotics for preservation of viruses • Used in the collection of specimens for viral isolation and testing • Save the viruses from drying • Nutrient, glycerol • Prevents specimen from drying out • Prevents bacterial and fungus growth • Prepared in the lab or commercially obtained • Storage for short periods at 4 - 8 ºC 13 Collection

14. To avoiding hemolysis for blood specimens, avoid: • Fine needles • Forced suction of blood with syringe • Unclean tube (residual detergents) • Shaking tube vigorously • Forced expulsion of the blood through needle • Freezing / thawing of blood • High speed centrifugation before complete clotting 14 Collection

15. Vacutainers • Vacuum tube with rubber stopper mounted on a needle system • Tubes may be changed for collection of different tubes for different purposes • Smooth blood flow, lower risk of hemolysis • Reduces risk of spillage 15 Collection

16. Collecting and handling blood for smears • Collection • Take capillary blood from finger prick (Lancet) • Make smear on clean glass slide • Dry and fix with methanol or other fixative • Handling and transportation • Transport slides within 24 hours • Do not refrigerate • May alter the morphology of the cells 16 Collection

17. Collecting blood for cultures • Collect within 10-30 mn of fever • Aseptic technique • Quantity • 0.5 – 2 ml venous blood for infants • 2 – 5 ml venous blood for children • 5 – 10 ml venous blood for adults • Take three sets of blood culture when suspecting bacterial endocarditis 17 Collection

18. Handling and transporting blood for cultures • Collect into blood culture bottles with infusion broth • Change the needle to inoculate the broth • Travel at ambient temperature 18 Collection

19. Collecting serum • Collect venous blood in a sterile test tube • Let specimen clot for 30 minutes at ambient temperature • Place at 4-8oC for clot retraction for at least 1-2 hours • Centrifuge at 1500 RPM for 5-10 min • Separate the serum from the clot with pipette / micro-pipette 19 Collection

20. Handling and transporting serum • Transport at 4-8oC if transport lasts less than 10 days • Freeze at -20oC if storage for weeks or months before processing and shipment to reference laboratory • Ship frozen • Avoid repeated freeze-thaw cycles • Destroy IgM (e.g., Measles diagnosis) 20 Collection

21. Collecting and handling cerebrospinal fluid • Collection • Lumbar puncture • Aseptic conditions • Trained person • Sterile tubes • Handling and transportation • For bacteria, transport at ambient temperature or preferably in trans-isolate medium (pre-warmed to 25-37°C before inoculation) • For viruses, transport at 4-8oC for up to 48 hours or at -70oC for longer duration Trans-isolate biphasic medium 21 Collection

22. Collecting and handling stool specimens • Take freshly passed stool specimen • Avoid collecting specimen from a bed pan • Collect specimen in a sterile container (if available) or clean container (not cleaned with a disinfectant) 22 Collection

23. Rectal swabs • Advantage • Convenient • Adapted to small children, debilitated patients and other situation where voided stool specimen collection is not feasible • Drawbacks • No macroscopic assessment possible • Less material available • Not recommended for viruses 23 Collection

24. Collecting stool specimens for viruses • Timing • Within 48 hours of onset • Specimen amount and size • At least 5-10 ml fresh stool from patients (and controls) • Method • Fresh stool unmixed with urines in clean, dry and sterile container • Storage • Refrigerate at 4oC. Do not freeze • Store at -15oC for antigen detection and protein chain reaction (PCR) • Transport • 4oC (Do not freeze) • Dry ice for antigen detection and PCR 24 Collection

25. Collecting stool specimens for bacteria • Timing • During active phase • Specimen amount and size • Fresh specimens and two swabs from patients, controls and carriers (if indicated) • Method • In Cary-Blair medium (+ specimen without transport medium for antigen detection / PCR) • Storage • Refrigerate at 4oC if testing within 48 hours, -70oC if longer 25 Collection

26. Collecting stool specimens for parasites • Timing • As soon as possible after onset • Specimen amount and size • At least 3 x 5-10 ml fresh stool from patients (and controls) • Method • Mixed with 10% formalin or polyvinyl chloride, 3 parts stool to 1 part preservative • Unpreserved specimens for antigen detection and PCR • Storage • Refrigerate at 4oC • Store at -15oC for antigen detection and PCR • Transport • 4oC (Do not freeze) • Dry ice for antigen detection and PCR 26 Collection

27. Collecting a sputum • Instruct patient to take a deep breath and cough up sputum directly into a wide-mouth sterile container • Avoid saliva or postnasal discharge • Minimum volume should be about 1 ml 27 Collection

28. Handling and transportation of respiratory specimens • All respiratory specimens except sputum are transported in appropriate media • Amie’s or Stuart’s transport medium for bacteria • Viral transport medium for viruses • Transport as quickly as possible to the laboratory to reduce overgrowth by oral flora • For transit periods up to 24 hours • Ambient temperature for bacteria • 4-8°C for viruses 28 Collection

29. The label of the specimen • Name: _________ • Age: ______ • ID number: _____ • Specimen type: ______ • Date, time of collection:___________ • Place of collection:___________ 29 Collection

30. Labeling glass slides for microscopy • Label slides individually • Use glass marking pencil • Make sure procedure will not interfere with the staining process • Each slide should bear: • Patient' name • Unique identification number • Date of collection 30 Collection

31. The case investigation form:What the epidemiologist sends • Patient information • Age (or date of birth), sex, complete address • Clinical information • Date of onset of symptoms, clinical and immunization history, risk factors or contact history where relevant, anti-microbial drugs taken prior to specimen collection • Laboratory information • Acute or convalescent specimen • Other specimens from the same patient • Line listing of patients 31 Collection

32. The case investigation form:What the receiving laboratory records • Date and time when specimen was received • Name and initials of the person receiving specimen • Record of specimen quality 32 Collection

33. Biosafety 1/3:Protect the patient • Use single use equipment • Disinfection • Work in a clean, dedicated area 33 Biosafety

34. Biosafety 2/3:Protect yourself • Use personal protective equipment • Disposable gloves • Laboratory coats / gown • Mask • Protective eyewear / face shields if procedure is likely to generate aerosols • Collect sharps immediately, in the absence of recapping in sharps container to prevent needle-stick injury • Have first aid kit readily accessible • Do not reuse contaminated equipment / supplies such as gloves • Do not leave the specimen on the request form 34 Collection

35. Biosafety 3/3:Protect others and the environment • Package specimens appropriately for transport • Decontaminate spills • 10% bleach after wiping the surface clean • Disinfect working areas for future use • 1% household bleach daily • Soak contaminated non-disposable equipment or materials in 1% household bleach for 5 minutes. Wash in soapy water before use and sterilize if necessary • Place waste in leak-proof biohazard bags • Ensure safe final management of waste • Protect personnel in charge of cleaning or decontamination with protective coat and thick rubber gloves 35 Collection

36. Triple-packaging of specimens: Two goals • Protect the environment and the carrier • Protect the specimen 36 Collection

37. The basic triple packaging system:1/3: The primary receptacle • Sealed specimen container to be placed in a suitably sized plastic bag/ ziploc bag • Packaged with sufficient absorbent material to absorb the entire content of the primary receptacle in case of breakage • Specimens from different patients should never be sealed in the same bag • Two or more sealed specimens of the same patient may be put in a larger plastic bag and sealed 37 Collection

38. The basic triple packaging system:2/3: The secondary receptacle • Leak-proof secondary plastic container with screw capped lids • Enclose and protect the primary receptacle(s) • Place the sealed bags containing the specimens inside secondary plastic containers • Specimens from several patients may be packed inside the same secondary plastic container • Sufficient additional absorbent material used to absorb all fluid in case of breakage 38 Collection

39. The basic triple packaging system:3/3: The outer packaging • Secondary packaging(s) are placed in outer shipping packaging with suitable cushioning material • Outer packaging protect their contents from outside influences, such as physical damage, while in transit • Resistant, high density external cover (metal, wood, fiberboard) • Smallest overall external dimension • 10x10 cm 39 Collection

40. Refrigeration methods to obtain different temperatures • 2-8 °C/4 °C • Wet ice/ice packs/domestic refrigerator • -8/ -10 °C • Freezer of domestic refrigerator • -20 °C • Freezer cabinet • -70 °C • Deep freezer/dry ice • -170/ -196 °C • Liquid Nitrogen Vaccine carriers that have been used for specimen transport must never be reused for carrying vaccines! 40 Collection

41. Quality assurance Quality assurance Internal quality control(Continuous, concurrent control of laboratory work) External quality assessment(Retrospective and periodic assessment) + = 41 Quality

42. Internal quality control • Test request and specimen collection • Test processing • Temperature • Reagent • Maintenance of equipment • Reporting and using test results 42 Collection

43. External quality assessment • Within the state IDSP system • L1 by L2 • L2 by L3 • Through external agency • External quality assurance scheme for selected tests 43 Collection

44. Criteria for rejection of specimens • Mismatch of information on the label and the request • Inappropriate transport temperature • Inappropriate transport medium • Insufficient quantity • Leakage • Excessive delay in transportation • Specimen received in a fixative • Dry specimen • Specimen with questionable relevance 44 Collection

45. Take home messages • Develop rapport with the laboratory • Collect specimen according to the guidelines and access on-line resources if needed • Protect the patient, yourself and others • You can contribute to quality assurance! 45

46. Additional reading • WHO Guidelines for the collection of clinical specimens during field investigation of outbreaks • IDSP bio safety manual • Section 6 of operations manual • Module 6 of training manual 46 Collection