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Module 5 Supplemental Information Laboratory Diagnostics, Specimen Collection, and Biosafety Issues 1 Laboratory Diagnosis of Avian Influenza 2 Laboratory Diagnosis for Avian Influenza • Only test samples that were packed during transport • Only test samples with identification information • Use positive and negative controls for all tests 3 Laboratory Diagnosis for Avian Influenza Tests for respiratory samples: • • • • Virus isolation PCR-based techniques Immunofluorescence Rapid antigen detection Blood sera used for: • Measurement of specific antibodies • Viral isolation • PCR-based techniques 4 Virus Isolation • Gold standard for detection of avian influenza • Allows identification of virus and test for drug susceptibilities • Technically difficult and requires BSL-3 laboratory 5 PCR-based Techniques • Examples: nested reverse transcriptase polymerase chain reaction (RT-PCR) and real-time RT-PCR • Sensitivity depends on the particular test, the influenza strain, and the type of specimen used – Sensitivity: 80 - 100% – Specificity: 90 - 100% 6 Immunofluorescence • Works best on clinical specimens collected immediately after symptoms begin • Results will tell you whether a particular influenza virus is present • Sensitivity and specificity not established 7 Indirect IF Staining of Cells From Tracheal Aspirate Anti-H5 Anti-H3 Anti-A/NP Anti-B 8 Taken from: World Bank Training by Alexander Klimov, CDC Rapid Antigen Tests • Detects influenza A and B virus • Detects human strains of influenza • Limitations: – Not yet used to test for avian influenza – Specificity and sensitivity not yet established 9 Serology Tests • Used to confirm infection because it takes several weeks to get results • Useful when the sample is taken too late for virus isolation • Sensitivity and specificity of 80 - 100% 10 Serology Tests Types: • Haemagglutination inhibition test • Enzyme immunoassay • Virus neutralization test • Western Blot 11 Serological Tests Paired serum samples are most useful Acute sample Within 7 days after symptom onset Convalescent sample More than 12 days after symptom onset 12 When Single Samples are Appropriate 1. Convalescent samples collected in outbreak investigations of novel viruses (such as H7 or H9) 2. Single samples collected in convalescent phase are compared to age-matched controls 13 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 avian influenza possible 14 How to Avoid Misinterpretations • Use positive and negative controls • Make sure you understand what the test is identifying • Use multiple tests to confirm results 15 Laboratory Practice Exercise 16 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/avian _labtests2.pdf • WHO guidelines for the collection of human specimens for laboratory diagnosis of avian influenza infection, 12 January 2005. http://www.who.int/csr/disease/avian_influenza/guidelines/hum anspecimens/en/index.html • 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_EMC _ESR_98_2_EN/en/index.html 17