Interpretation on result of laboratory diagnosis of avian influenza - - PowerPoint PPT Presentation

Interpretation on result of laboratory diagnosis of avian influenza

Prof.Pilaipan Puthavathana, Ph.D.

Department of Microbiology Faculty of Medicine Siriraj Hospital Mahidol University

Case definition of AI case

Thai Bureau of Epidemiology, 1 June 2006 AI suspected case Patients who have fever > 38C plus any of the followings : myalgia, cough, abnormal breath (dyspnea or respiratory distress) or suspected of pneumonia and History of direct exposure to dead or sick poultry within 7 days before onset of symptom, or, closely providing health care to patients with pneumonia 10 days before and No clinical specimen available for lab investigation

AI probable case

Patients who meet the definition of suspected cases and developed signs and symptoms of, or died from acute respiratory failure

AI confirmed cases

Patient who meet the definition of suspected cases and has a final standard laboratory confirmation of fluA/H5 through at least one of the following methods

• A. Single RT-PCR using two primer/probe sets or using

specimens collected from at least two different location or using at least two specimens collected at different period

• B. Viral culture
• C. NT test ( four fold antibody increase in paired blood)

H and N classification for FluA H1, 2, 3........16 N1, 2, 3.........9

H and N classification

• All kinds of H and N are present in

aquatic birds

• Only H1,H2 and H3 ; and N1 and N2

are found in most human population, except reports of the emerging of H5N1, H7N7 and H9N2 viruses

Fields Virology. Third edition. Lippincott Williams and Wilkins, Philadelphia

Genome segments of influenza viruses

Influenza A

Encoded protein RNA segment Name Function 1 PB2 polymerase 2 PB1 polymerase 3 PA polymerase 4 HA,H hemagglutinin 5 NP nucleoprotein 6 NA, N neuraminidase 7 M1, M2 matrix (membrane) proteins 8 NS1,NS2 Nonstructural proteins

Clinical samples

NP wash/aspirate, NP swab, throat swab, sputum, endotraheal swab/aspirate, tissues

• Antigen detection
• Virus isolation
• Molecular diagnosis

Paired blood for antibody

Molecular diagnosis

• RT-PCR, nucleotide sequencing
• M gene is used to design universal

primers for all flu A

• H and N genes for subtyping
• House keeping gene to check for quantity
• f RNA in clinical samples

100 bp Marker Influenza A (H3) Influenza B Patient Blank 100 bp Marker Influenza A (H3) Influenza B Patient Blank

Multiplex PCR for H1&H3 PCR for H5

663 bp 591 bp

Siriraj Virology Lab

BLAST of H nucleotide sequence (606 nt) from a patient

H5N1 : AF216737 H5N1 : AF216721 H5N1 : AY075030 H5N1 : AY075027 H5N1 : AF098545 H5N1 : AY075033

Identities

96% 96% 96% 96% 96% 96%

Siriraj Virology Lab

Failure to get promising result

• Inadequate specimen
• Lab techniques involve many steps that

might prone to an error result

• Viral gene change

Virus isolation technique

• It is the gold standard method
• It requires BSL 3 facilities
• Cell culture : MDCK, LLC MK2
• Chick embryonated eggs

• Virus isolation needs molecular

techniques (RT-PCR, nucleotide sequencing) for type and subtype identification

• Monoclonal antibody can be used in the

identification

LLC-MK2 Siriraj Virology Lab

Siriraj Virology Lab Identification of flu A isolate in cell culture by IFA

Siriraj Virology Lab

+

Hemagglutination test

Siriraj Virology Lab

Antibody detection

• Paired blood (2-3 weeks apart) for a four-

folded increase in antibody titer

• If single blood is obtained, what is the

significant titer ?

• Micro-neutralization (Micro-NT) test is

recommended as hemagglutination- inhibition (HI) test is less specific

What is the significant antibody titer ? : I What is the meaning for possessing H5 NT antibody Vaccination with H5 antibody Natural infection Exposure to viral antigen (dead viruses) in the environment

What is the significant antibody titer ? : II

• Some patients did not develop H5

antibody when they died.

• Cross reactivity with other H subtype in
• ld age
• Mild case can develop a four folded rise

in Ab titer

Significant antibody titers

• U.S. CDC. for H5N1 Ab : > 80
• Japan for H5N1 Ab

: > 10

• Europe for H7 Ab

: > 20 Confirmatory test by Western Blot assay

Antigen testing

Rapid test

Immunofluorescence test

Ag detection

• Quick tests are less sensitive and less

specific

• Quick test require specific solution for

specimen collection

• Immunofluorescence(IF) test is

subjective, but it can be used to test for

• ther respiratory viruses

Respiratory viruses

Flu A Flu B RSV Adeno PF1 PF2 PF3

Indirect immunofluorescence test for respiratory viruses

PF3

PF3

RSV

Adeno FluB

• GOLDEN RULE : Not process avian,

swine and human samples in the same lab

• More than one standard techniques will

give the precise diagnosis

• Network for proficiency test may be

needed

Risk groups of microorganisms

Risk gr.1 : Low individual risk and low community risk e.g., B. subtilis, E.coli Risk gr.2 : Moderate individual risk and limited community risk e.g., S typhi, HBV, M.tuberculosis Risk gr.3 : High individual risk and low community risk e.g., Brucella, Lassa fever virus, SARSCoV, bird flu, HIV culture Risk gr.4 : High individual risk and high community risk e.g., Ebola-Marburg virus, foot and mouth disease virus, Nipah virus

Lab classification

• Biosafety Level 1 : teaching lab
• Biosafety Level 2 : clinical, diagnostic

lab working with moderate risk agents

• Biosafety Level 3 : for indigenous or

exotic agents with a potential for respiratory transmission

• Biosafety Level 4 : for dangerous and

exotic agents which pose a life- threatening disease, no vaccine and therapy available