1. SEARO – CSR Training on Outbreak Investigation Laboratory specimen: collection, safe transport and biosafety Specimen Collection

2. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation Learning objectives At the end of the presentation participants should be able to: –Explain the procedures, preparation, processing and transport of specimens

3. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation Successful laboratory investigations advance planning collection of appropriate and adequate specimens labeling and documentation of laboratory specimen storage, packaging and transport to appropriate laboratory the ability of the laboratory to accurately perform the diagnostic tests biosafety and decontamination procedures to reduce the risk of further spread of the disease timely communication of results

4. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation 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 microbiologists, clinicians and epidemiologists

5. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation 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. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation Some tips Laboratory investigation should start as early as possible Specimens obtained early, preferably prior to antimicrobial treatment likely to yield the infective pathogen Before doing anything, explain the procedure to patient and relatives When collecting the specimen, avoid contamination Take a sufficient quantity of material Follow the appropriate precautions for safety

7. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation 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)

8. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation 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

9. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation 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 4 o C if can’t reach laboratory in 24 hours

10. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation Serum Collection Venous blood in sterile tube let clot for 30 minutes at ambient temperature glass better than plastic Handling Place at 4-8°C for clot retraction for at least 1-2 hours Centrifuge at 1 500 RPM for 5-10 min separates serum from the clot

11. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation Serum Transport 4-8 o C if transport lasts less than 10 days Freeze at -20°C 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

12. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation Collection –Lumbar puncture –Sterile tubes –A septic conditions –Trained person Cerebrospinal fluid (CSF)

13. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation 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 4-8 ° C (if up to 48hrs or -70 ° C for longer duration)

14. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation 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. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation 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

16. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation 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 4°C; do not freeze –store at -15°C - for Ag detection, polymerase chain reaction (PCR) Transport –4°C (do not freeze); dry ice for (Ag detection and PCR)

17. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation 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 4°C if testing within 48 hours, -70°C if longer; store at -15°C for Ag detection and PCR Transport –4°C (do not freeze); dry ice for Ag, PCR detection

18. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation 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 4°C; store at -15°C for Ag detection and PCR Transport –4°C (do not freeze); dry ice for antigen detection and PCR

19. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation Throat swab (posterior pharyngeal swab) 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 WHO/CDS/EPR/ARO/2006.1

20. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation 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

21. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation Nasopharyngeal aspirate Tilt head slightly backward Instill 1-1.5 ml of VTM /sterile normal saline into one nostril Use aspiration mucus trap Insert silicon catheter in nostril and aspirate the secretion gently by suction in each nostril WHO/CDS/EPR/ARO/2006. 1

22. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation 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

23. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation 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

24. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation 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 Post-mortem samples

25. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation 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- 8 o C within 48h

26. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation Specimen Transport media Storage condition Purpose/ Lab investigation TransportPending test Throat swabVTM2-8 0 C-20 0 CIsolation NPA/ swabVTM2-8 0 C-20 0 CIsolation CSFNo2-8 0 C-20 0 CIsolation, serology StoolNo2-8 0 C-20 0 CIsolation UrineNo2-8 0 C-20 0 CIsolation Serum/ Clotted blood No2-8 0 C-20 0 C 2-8 0 C Isolation, serology Whole bloodNo2-8 0 C Isolation, serology Virologic Investigations

27. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation Specimen Transport media Storage condition Purpose/ Lab investigation TransportPending test Throat swabAmie’s or Stuart’s TM RT2-8 0 CIsolation SputumNo2-8 0 C Isolation CSFTrans-isolate media RT37 0 CIsolation StoolCary-Blair2-8 0 C Isolation UrineNoRT2-8 0 CIsolation Serum/ Clotted blood No2-8 0 C Isolation Whole bloodInfusion brothRT or 4 0 C if > 24 hours 37 0 CIsolation Visualization Bacteriologic Investigations

28. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation 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

29. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation 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

30. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation 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

31. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation 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 liters 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

32. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation Food samples Handling and transportation –As fast as possible –Keep perishable food at 2-8 °C –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

33. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation Labeling specimens Patient’s name (or Patient Identifier) Unique ID number (Research/Outbreak) Specimen type Date, time and place of collection Name/ initials of collector Patient’s Name/Identifier Unique ID Number RRR-0023 001712643003 Date, Time, Place of Collection Sept 22, 2006, 1515 hr, Maka Community Hospital, Barton, Capiz, Philippines Specimen Type Serum Collected by: MDR

34. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation 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

35. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation Some Tips Pre-print labels Permanently affix label to the specimen container. Glass slides for microscopy labeled individually One specimen – one lab request Each slide should bear the patient’s name, unique identifier, and date of collection Use line listing for multiple patients Original documents kept by investigation team

36. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation 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

37. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation 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

38. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation

39. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation 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

40. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation Reference Most of the slides used in this presentation are 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

41. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation References Communicable Disease Toolkit: Iraq Crisis. Guidelines for the collection of specimens for laboratory testing. WHO, 2003 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. Laboratory specimen: Collection SEARO – CSR Training on Outbreak Investigation