Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Human cytomegalovirus wikipedia , lookup
Marburg virus disease wikipedia , lookup
Swine influenza wikipedia , lookup
Herpes simplex virus wikipedia , lookup
West Nile fever wikipedia , lookup
Hepatitis B wikipedia , lookup
Henipavirus wikipedia , lookup
Middle East respiratory syndrome wikipedia , lookup
Laboratory Diagnostics, Specimen Collection, and Biosafety Issues 1 Learning Objectives • Describe avian influenza-related laboratory • • • • • procedures Define laboratory safety List elements of specimen collection kit Explain how to collect & transport specimens Describe infectious waste disposal Explain how to manage laboratory data 2 Session Overview • Laboratory diagnosis of human cases of avian • • • • • influenza Laboratory safety Specimen collection and transportation Specimen storage, handling, packaging, and transportation Waste disposal and decontamination Managing and analyzing laboratory data 3 Laboratory Diagnosis of Human Cases of Avian Influenza 4 Laboratory Diagnostics for Avian Influenza Tests on respiratory samples: • Virus isolation • Immunofluorescence • Rapid antigen detection Tests on serum: • Measure specific antibodies • PCR-based techniques 5 H5N1 Virus Found in Other Human Specimens • H5N1 virus infection of cerebrospinal fluid documented in a fatal case with seizures and coma • H5N1 virus has also been found in: Rectal swab specimens and stool of fatal cases with diarrhea Serum and plasma of fatal cases • All respiratory secretions and bodily fluids of H5N1 patients should be considered potentially infected with H5N1 virus 6 PCR-based Techniques • Respiratory samples • Diagnostic assays and sequencing • Sensitivity depends on: Particular assay Influenza strain Type of the specimen Quality of the specimen 7 PCR: H5N1 Virus Testing Primary method: detection of H5N1 viral RNA by reverse-transcription polymerase chain reaction (RT-PCR) • Conventional RT-PCR • Real-time RT-PCR (rt-RT-PCR) • Highly sensitive and specific 8 Interpretation of Real-time RT-PCR Results Test reactions A H1 H3 H5 B RNP Results Sample 1 + - - + - + A/H5 Sample 2 - - - + - + Inconcl Sample 3 - - - - - + Neg Sample 4 - - - - - - Inconcl 9 Virus Isolation of H5N1 Viruses Gold Standard • Requires biosafety level 3 (BSL-3)- enhanced • • • • laboratory Requires select agent approval from USDA (United States) Allows antigenic & genetic characterization Allows drug susceptibilities tests Performed at WHO Influenza Centers Antigenic characterization of viral isolates Collect specimens for vaccine development 10 Immunofluorescence for H5N1 Testing • Requires H5 monoclonal antibody • Not part of WHO Reagent kit for seasonal • • influenza isolates Interpretation difficult Results: Presence of influenza virus Subtype identification 11 Indirect IF Staining of Cells From Tracheal Aspirate Anti-H5 Anti-H3 Anti-A/NP Anti-B 12 Taken from: World Bank Training by Alexander Klimov, CDC Rapid Antigen Tests • Quick results and commercially available • Low sensitivity • False negatives and false positives • Identifies viral type (type A or B) NOT subtype (H5, • H3, H1) Clinically useful to guide treatment, but must test for H5 with another assay RAPID TESTS NOT RECOMMENDED For Detection of human H5N1 virus infection 13 Using Serology Delay for anti-H5 levels to rise Requires acute and convalescent sera, 3 weeks apart Not useful for clinical management Can confirm epidemic cases, if respiratory specimens not available 14 Influenza Serology Tests • Micro-neutralization assays • Western Blot • Enzyme immunoassays Require acute and convalescent sera (serum obtained >21 days from onset) 15 Interpreting Serology Tests Acute Sample low low high Convalescent Sample Interpretation low No evidence of Infection high* Seroconversion (evidence of infection) high Inconclusive; past exposure to influenza or previous vaccination possible * four-fold or greater increase between acute and convalescent sera 16 Serological Specimens • Paired serum specimens most useful 4-fold rise in antibody titer • Single convalescent serum specimens: Useful in outbreak investigations of novel human influenza viruses If prevalence unknown, case serum specimen compared to age-matched control specimen 17 How to Avoid Misinterpretation of Diagnostic Tests • Use appropriate controls • Understand What is test identifying? What are limitations of assay? • Use more than one test to confirm results and use more than one sample from each patient 18 Review Question 1 Which of the following is the best technique to test respiratory samples for avian influenza? a. b. c. d. PCR-based techniques Virus isolation Immunofluorescence Rapid antigen detection Answers: a. PCR-based techniques 19 Review Question 2 Which of the following situations is most likely to lead to the misinterpretation of diagnostic tests? a. b. c. d. Use appropriate controls for each assay Understand what the test is identifying Understand the limitations of each assay Use only one test if you are sure that it is correct Answer: d. Instead, you should use multiple tests to confirm results. 20 Laboratory Safety Dr. Terrence Tumpey examines reconstructed 1918 Pandemic Influenza Virus inside a specimen vial in a Biosafety Level 3enhanced laboratory setting. Courtesy CDC/James Gathany. 21 Biosafety Laboratory Levels (BSL) • Risk of acquiring infections exists in laboratory • Laboratory safety guidelines evolved • Safe work sites result from: Engineering controls Management policies Work practices and procedures Medical interventions (occasional) Increasing biosafety levels = increasing levels of personnel & environmental protection http://www.cdc.gov/OD/ohs/symp5/ 22 BSL-2 and BSL-3 • BSL-2 Good microbiological technique Protective clothing Biohazard sign • BSL-3 = BSL-2 plus: Controlled access Directional air flow • BSL-3 enhanced = BSL-3 plus: Respiratory protection and clothing change Shower out of facility HEPA filters 23 BSL Requirements for Potential H5N1 Influenza Specimens • BSL2 lab with BSL3 work practices needed for: Aliquoting human specimens Nucleic acid extractions All Diagnostic testing except viral culture • BSL3+ safety measures and guidelines necessary to culture H5N1 If laboratory does not meet BSL2 requirements, ship appropriately packaged specimen to reference laboratory 24 WHO H5 Reference Laboratories 1. Collect specimens / virus isolates of A(H5N1) and other 2. 3. 4. 5. 6. 7. 8. influenza subtypes Provide timely surveillance data Conduct antigenic and genetic analyses Assist in developing methods for global surveillance and diagnosis of A(H5N1) Participate in selecting, developing, and distributing candidate A(H5N1) vaccines Develop and distribute reagents Monitor susceptibility of emerging subtypes Provide expertise and laboratory support to Member States 25 Review Question 3 What Biosafety Level needs to be in place in order to culture H5N1? a. b. c. d. BSL-1 BSL-2 BSL-3 BSL-3 enhanced Answer: d. BSL-3enhanced safety measures and guidelines should be used for culturing potential H5N1 viruses 26 Specimen Collection 27 Specimen Collection Kit • Personal protective equipment (PPE) • Viral transport medium (VTM) collection vials • Swabs: throat, nasal, & nasopharyngeal • Tongue depressors • Nasal Wash Equipment • Transfer pipettes • Secondary container • Ice packs • Items for blood collection • Field collection forms • Labels and pen or marker Store kit in a dry, cool place Keep kit accessible for after hours 28 Viral Transport Medium (VTM) • Used to store & transport specimens • Isolates & maintains virus integrity • Prevents bacteria and fungi growth • Can be made in a lab or purchased • Different types of VTM: Animal specimen collection Viral isolation of human specimens 29 Storing VTM • Sterile collection vials containing 1-3 ml of VTM • VTM can be stored in a freezer at -20ºC to -40ºC until use • VTM can be stored for short periods of time at 4 - 8 ºC 30 Polyester Fiber-Tipped Applicator • Should ideally be dacron, rayon, or polyester-fiber swabs Remember! Use throat swabs for avian influenza and nasopharyngeal swabs for seasonal influenza 31 Personal Protective Equipment for Caring for H5N1 Patients • Masks (N-95 or N/P/R-100) • Gloves • Protective eye wear (goggles) • Hair covers • Boot or shoe covers • Protective clothing (gown or apron) 32 33 Clinical Specimen Sources Prepare to collect specimens before you leave for the field •Persons meeting trigger criteria Includes WHO suspected and probable cases •Symptomatic contacts Symptomatic people living/working with suspected cases 34 What to Collect From an Ambulatory patient • Throat swab (priority) and • Nasopharyngeal swab • If necessary, collect into same VTM From an Intubated patient • Tracheal aspirate 35 When to Collect Specimens When to Collect Respiratory Specimens • As soon as possible after symptoms begin • Ideally before antiviral medicine administered • For multiple days • Sample multiple types of specimens 37 When to collect Serum Specimens Acute specimen As soon as possible, ideally within 7-10 days after symptom onset Convalescent specimen 2-3 weeks after the acute sample (> 21 days after symptom onset) 38 Oropharyngeal (Throat) Swab 39 Nasopharyngeal Swab http://www.nlm.nih.gov/medlineplus/ency/imagepages/9687.htm 40 Nasopharyngeal Aspirate (Nasal Wash) Collection Process 1. 2. 3. 4. 5. 6. Attach mucus trap to vacuum source Place catheter into nostril parallel to palate Apply vacuum Slowly remove catheter while slightly rotating it Repeat with other nostril using same catheter After collection, flush catheter with 3 ml VTM and return VTM to a plastic vial 41 Labeling Specimens Use pre-printed barcode labels: On specimen container On field data collection form In log book Label each specimen with: Subject’s unique identification number 42 Field Data Collection Forms 43 Review Question 4 True or False: Are nasopharyngeal swabs best for seasonal or avian influenza? Answer: Seasonal 44 Specimen Storage, Handling, Packaging and Transportation 45 How to Store Specimens For specimens in VTM: •Transport to laboratory as soon as possible •Within 48 hours store at 4 °C to 8 °C •Beyond 48 hours store at -70 °C to -80 °C •Do not use standard freezer; keep on ice or in refrigerator Avoid freeze-thaw cycles 46 How to Store Specimens For sera: •Store specimen at 4 °C to 8 °C for short periods of time -20 °C to - 40 °C for long term storage •Avoid freeze-thaw cycles •Centrifuge blood and aliquot serum to another container before shipping and before freezing 47 Packing Specimens for Transportation Goal: protect specimens during transportation •Use three packaging layers •Use water tight first layer •Use absorbent material in all layers •<500mL of liquid in specimen collection container •If transporting specimens a long distance, send on dry ice. If transporting a short distance, ice is acceptable 48 Transporting Specimens • WHO guidelines for the safe transport http://www.who.int/csr/emc97_3.pdf • Follow local regulations • Coordinate with the laboratory 49 Review Question 5 Which of the following is true about storing clinical specimens (sera or specimens in VTM)? a.They can be stored at 4°C up to a week b.They can be stored in a standard freezer Answer: a. Both sera and specimens in VTM can be stored for certain periods at 4°C for up to a week. Neither of these samples should be stored in a standard freezer, because the freeze-thaw cycle will destroy the virus 50 Packaging a Specimen for Shipment Demonstration 51 Transporting Specimens from Field to Lab 52 Waste Disposal and Decontamination 53 Items Requiring Disposal • Infectious blood, body fluids • Disposable or nonreusable gloves • Disposable needles and • Used laboratory syringes* • Disposable or nonreusable protective clothing* supplies* • Used disinfectants * Incineration recommended 54 Managing Contamination or Accidents • • • Contaminated work surface: Use 5% bleach solution for at least 5 minutes Make bleach solution fresh daily 70% ethanol, 5% Lysol is also adequate • • • Exposed laboratory worker: Remove infected clothing Wash any exposed areas Give post-exposure prophylaxis according to the established emergency procedure 55 Managing and Analyzing Laboratory Data 56 Specimen Tracking System Maintain a database to track: • Identification number same ID # as on epidemiologic data collection forms • Subject information Age, sex, exposures • Specimen collection date • Specimen collection location • Diagnostic test results 57 Data Management Rules • Double check data entry accuracy • Include unique identification numbers • Keep subject names confidential • Track testing dates and results • Back up the database 58 Summary • Maintain stocked specimen collection kits and • • • store them properly Throat swabs are the most important specimens to collect for human H5N1 detection Nasopharyngeal swabs are best for detecting seasonal influenza viruses Collect multiple specimens on multiple days 59 Summary • Proper specimen storage, handling, and • • • shipping is vital for laboratory test success Track specimen data in database or logbook Use safety precautions when handling infectious materials in the laboratory or in the field Properly dispose of any infectious material 60 Summary • If needed, ship specimens to national or regional laboratory • Share findings with local health officials and the WHO Global Influenza Program 61 Glossary Aliquot A portion of a total amount of a solution Centrifuge A machine that uses high-speed rotation to separate materials with different densities. Culture Growing of microorganisms in a nutrient-rich medium. Nucleic acid Component genetic material such as DNA or RNA found in all cells in humans, animals, bacteria, and viruses. Every species 62 and organism has a unique pattern. Glossary Pipette A glass or plastic tube used to measure or transfer small amounts of liquid. Saline A liquid solution made of salt and water. Viral Transport Medium (VTM) The preservative liquid in which specimens are stored until they are tested. 63 References and Resources • • • • Recommended laboratory tests to identify avian influenza A virus in specimens from humans. World Health Organization, June, 2005. http://www.who.int/csr/disease/avian_influenza/guidelines/avi an_labtests2.pdf WHO guidelines for the collection of human specimens for laboratory diagnosis of avian influenza infection, 12 Jan 2005. http://www.who.int/csr/disease/avian_influenza/guidelines/hu manspecimens/en/index.html WHO laboratory biosafety guidelines for handling specimens suspected of containing avian influenza A virus. 12 Jan 2005. http://www.who.int/csr/disease/avian_influenza/guidelines/han dlingspecimens/en/ Infection control for viral haemorrhagic fevers in the African health care setting. WHO/EMC/ESR/98.2 Section 6: Dispose of Waste Safely http://www.who.int/csr/resources/publications/ebola/WHO_E MC_ESR_98_2_EN/en/index.html 64