Specimen collection

1. Specimen collection Sample collection and shipping May 2007

2. Learning objectives At the end of the presentation, participants should understand the: • Procedures, preparation, processing and transport of specimens

3. Successful laboratory investigations • Advance planning • Collection of adequate and appropriate specimens • Sufficient documentation • Biosafety and decontamination • Correct packaging • Rapid transport • Choice of a laboratory that can accurately perform the tests • Timely communication of results

4. Specimen collection: key issues Consider differential diagnoses Decide on test(s) to be conducted Decide on clinical samples to be collected to conduct these tests • consultation between microbiologist, clinicians and epidemiologist

5. Transport medium Allows organisms (pathogens and contaminants) to survive Non-nutritive - does not allow organisms to proliferate For bacteria – i.e., Cary Blair For viruses - virus transport media (VTM)

6. Blood for smears Collection Capillary blood from finger prick • make smear • fix with methanol or other fixative Handling and transport Transport slides within 24 hours Do not refrigerate (can alter cell morphology)

7. Blood for cultures Collection Venous blood • infants: 0.5 – 2 ml • children: 2 – 5 ml • adults: 5 – 10 ml Requires aseptic technique Collect within 10 minutes of fever • if suspect bacterial endocarditis: 3 sets of blood culture

8. Blood for cultures Handling and Transport Collect into bottles with infusion broth • change needle to inoculate the broth Transport upright with cushion • prevents hemolysis Wrap tubes with absorbent cotton Travel at ambient temperature Store at 4oC if can’t reach laboratory in 24h

9. Serum Collection Venous blood in sterile test tube • let clot for 30 minutes at ambient temperature • glass better than plastic Handling Place at 4-8oC for clot retraction for at least 1-2 hours Centrifuge at 1 500 RPM for 5-10 min • separates serum from the clot

10. Serum Transport 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 Avoid repeated freeze-thaw cycles • destroys IgM To avoid hemolysis: do not freeze unseparated blood

11. Cerebrospinal fluid (CSF) Collection Lumbar puncture Sterile tubes Aseptic conditions Trained person

12. CSF Handling and transportation Bacteria • preferably in trans-isolate medium, pre-warmed to 25-37°C before inoculation OR • transport at ambient temperature (relevant pathogens do not survive at low temperatures) Viruses • transport at 4-8oC (if up to 48hrs or -70oC for longer duration)

13. Stool samples Collection: Freshly passed stool samples • avoid specimens from a bed pan Use sterile or clean container • do not clean with disinfectant During an outbreak - collect from 10-20 patients

14. Rectal swabs Advantage • convenient • adapted to small children, debilitated patients and other situations where voided stool sample not feasible Drawbacks • no macroscopic assessment possible • less material available • not recommended for viruses

15. Stool samples for viruses Timing • within 48 hours of onset Sample amount • 5-10 ml fresh stool from patients (and controls) Methods • fresh stool unmixed with urine in clean, dry and sterile container Storage • refrigerate at 4oC; do not freeze • store at -15oC - for Ag detection,polymerase chain reaction (PCR) Transport • 4oC (do not freeze); dry ice for (Ag detection and PCR)

16. Stool samples for bacteria Timing • during active phase Sample amount and size • fresh sample and two swabs from patients, controls and carriers (if indicated) Method • Cary-Blair medium • For Ag detection/PCR – no transport medium Storage • refrigerate at 4oC if testing within 48 hours, -70oC if longer; store at -15oC for Ag detection and PCR Transport • 4oC (do not freeze); dry ice for Ag, PCR detection

17. Stool samples for parasites Timing • as soon as possible after onset Sample amount and size • at least 3 x 5-10 ml fresh stool from patients and controls Method • mix with 10% formalin or polyvinyl chloride, 3 parts stool to 1 part preservative • unpreserved samples for Ag detection and PCR Storage • refrigerate at 4oC; store at -15oC for Ag detection and PCR Transport • 4oC (do not freeze); dry ice for antigen detection and PCR

18. Throat swab (posterior pharyngeal swab) WHO/CDS/EPR/ARO/2006.1 Hold tongue away with tongue depressor Locate areas of inflammation and exudate in posterior pharynx, tonsillar region of throat behind uvula Avoid swabbing soft palate; do not touch tongue Rub area back and forth with cotton or Dacron swab

19. Nasopharyngeal swab Tilt head backwards Insert flexible fine-shafted polyester swab into nostril and back to nasopharynx Leave in place a few seconds Withdraw slowly; rotating motion WHO/CDS/EPR/ARO/2006.1

20. Naso-pharyngeal aspirate Tilt head slightly backward Instill 1-1.5 ml of VTM /sterile normal saline into one nostril Use aspiration trap Insert silicon catheter in nostril and aspirate the secretion gently by suction in each nostril WHO/CDS/EPR/ARO/2006.1

21. Sputum Collection Instruct patient to take a deep breath and cough up sputum directly into a wide-mouth sterile container • avoid saliva or postnasal discharge • 1 ml minimum volume

22. Respiratory samples Handling and Transport All respiratory specimens except sputum are transported in appropriate media • bacteria: Amie’s or Stuart’s transport medium • viruses: viral transport medium (VTM) 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

23. Post-mortem samples Collection Biopsy relevant tissues • place in formalin for histopathology • place in transport medium for microbiological testing • place in sterile saline for isolation of viral pathogens

24. Post-mortem samples Handling and transportation Fixed specimens can be transported at ambient temperatures • transport specimens in transport media within 24h at ambient temperature • transport specimens in sterile saline at 4-8oC within 48h

25. Virologic Investigations

26. Water for bacteriology Preparation Chlorinated water - add sodium thiosulphate (0.5ml of 10% solution or a small crystal) Tap/ pump • remove attachments • wipe, clean and flame outlet • allow to flow (at least one minute) Water course or reservoir - collect from a depth of at least 20 cm Dug well - do not allow the bottle to touch the sides of the well

27. Water for bacteriology Collection At least 200 ml of water sample from the source In sterile glass bottles OR autoclavable plastic bottles • tight screw capped lid • securely fitting stoppers/caps • an overhanging rim

28. Water for bacteriology Handling and transportation Test the water sample within 3 hours of collection • keep at ambient temperature If delayed: • pack sample on ice • test refrigerated sample within 24 hours

29. Food samples Collect suspect food earliest Collect aseptically - sterile tools, containers Solid Food • cut 100-200 grams from centre with sterile knife • raw meat or poultry - refrigerate in a sterile plastic jar Liquids • shake to mix, use sterile tube • water used for cooking -- 1-5 litres Contact surfaces (utensils and/or equipment) for food processing • moisten swab with sterile 0.1% peptone water or buffered distilled water; put the swab in an enrichment broth

30. Food samples Handling and transportation As fast as possible Keep perishable food at 2-8oC Cool hot food rapidly - put containers under cold running water Pack samples to prevent spillage Contact the laboratory regarding method of transport and anticipated time of receipt Seek help from environmental/veterinary microbiologist

31. Labeling specimens Patient’s name Clinical specimen Unique ID number (Research/Outbreak) Specimen type Date, time and place of collection Name/ initials of collector

32. Glass slides for microscopy Label slides individually • use glass marking pencil • ensure markings don’t interfere with staining process Each slide should bear: • patient name • unique identification number • date of collection

33. Case investigation form 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 – if large number of patients

34. Case investigation form Receiving laboratory records: Date and time when specimen was received Name and initials of the person receiving specimen Record of specimen quality

36. Biosafety: protect the patient Use single use equipment Disinfect Work in a clean, dedicated area

37. Biosafety: protect yourself Use personal protective equipment • disposable gloves • laboratory coats / gown • mask • protective eyewear / face shields if procedure is likely to generate aerosols If no sharps container: collect sharps immediately to prevent needle-stick injury Have first aid kit readily accessible Do not reuse contaminated equipment

38. Biosafety: protect others, the environment Package samples 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/material in 1% household bleach for 5 minutes • wash in soapy water before re-use, sterilize if necessary Place waste in leak-proof biohazard bags - ensure safe final management of waste Protect cleaning/decontamination personnel with protective coat, thick rubber gloves

39. Infection control precautions Precautions Use Requirements Contact precautions Patients known or suspected to have serious illnesses easily transmitted by direct patient contact or by contact with items in the patient's environment • Gloves • Gown Droplet precautions Barrier to stop infections spread by large (>5 microns), moist droplets produced by people when they cough, sneeze or speak • Contact precautions • Well-fitting mask • Eye protection Airborne precautions Patients known or suspected to have serious illnesses transmitted by airborne droplet nuclei • Contact precautions • Droplet precautions • N95 mask • Isolation room (In hospital)

40. Criteria for rejecting samples Mismatch of information on the label and the request Inappropriate transport temperature Excessive delay in transportation Inappropriate transport medium • specimen received in a fixative • dry specimen • sample with questionable relevance Insufficient quantity Leakage

41. WHO reference materials Guidelines for the collection of clinical specimens during field investigation of outbreaks, WHO, 2000 The role of laboratories and blood banks in disaster situations, WHO publication, 2001 Sampling during avian influenza investigations (2006) IDSR guidelines for specimen collection (2003) Laboratory Needs for Emergency Situations (2003) Overview of Laboratory Structure and Operational Needs for the Iraqi Crisis (2003) Costing for sampling materials and diagnostic reagents for the Iraq crisis (2003)

42. Module 3: Sample collection and shipping 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