Laboratory Diagnosis for avian flu (H5N1) in human

Laboratory Diagnosis for avian flu (H5N1) in human Wattana Auwanit MT, PhD Biohazard Laboratory and Immunology Section National Institute of Health Department of Medical Sciences Ministry of Public Health Thailand

Influenza Virus • Influenza A - variety of different host species (avian, human, swine, etc.) - HA undergoes minor and occasional major changes - Cause severe disease and associated with epidemic and pandemics • Influenza B - Infects only in human but not severe as type A virus - Relative slow change in HA • Influenza C - Uncommon strain, infects in man only

Influenza type A Human influenza : H1N1, H3N2, H1N2 Avian influenza : H5N1

H5N1 infection laboratory diagnosis

Questions • When to collect specimen? • What specimens to collect ? • How to store / transport specimens ? • How to test ? • How to interpret results?

specimens • Quality of specimen is a most importance. • Best test cannot give a accurate result on poor specimen.

Type of specimens • Respiratory specimens in VTM – viral RNA detection • Nasopharyngeal aspirate • Nasopharyngeal swab • Throat swab • Nose swab (not recommend) • Feces – low virus level • Blood for serology – viral antibody detection • Autopsy tissues – viral RNA detection

Timing • 2-3 days after illness onset • End the peak at 7-8 days • Nasopharyngeal wash/aspirate > Nasopharyngeal swab ~ throat swab

Storage of specimens • At hospital and during transportation • wet ice, refrigerator (4 oC) • At the laboratory • Store at – 70 oC not at -20 oC • Avoid freeze-thaw

Laboratory diagnosis • Detection of viral antigen • Immunofluorescence assay (IFA) • Rapid test (Bed-side rapid assay) • Detection of viral genome • Reverese transcription and polymerase chain reaction assay (RT-PCR) • Real-time RT-PCR • Virus culture in cell lines • Serology test • Micro-neutralization test for seroconversion (blood)

Rapid test

Rapid tests • Simple (1 hr), differentiate only A/B not H3,H1,H5 • Commercially available - Directigen Flu A - Now Flu A+B - etc. • Less sensitivity (17/35 : 48%)

Sensitivity of rapid antigen tests for H5N1 disease Pos (%) Test Hong Kong 1997 9/11 (82%) DirectigenFluA Yuen et al Lancet 1998 Thailand 2004 17/35 (48%) Directigen, Binax NOW Thai MOPH/US CDC- unpublished Vietnam 2004 3/15 (20%) Directigen and QuickVue de Jong M & Farrar J - Unpublished Data from : JSM Peiris, The University of Hong Kong, and Queen Mary Hospital

Immunofluorescence assay (IFA) • Sensitive method to identify influenza A and B, parainfluenza, adenovirus, etc. • Using type specific monoclonal antibody to viral antigen (A/H5, A/H1, A/H3, etc.) • 2-3 hours of testing • Need enough cells

Molecular assay Reverse Transcription-PCR (RT-PCR) • For detection of viral gene and sub-typing (influenza A, B, A/H1, A/H3, A/H5) using specific primers and probe • Target on viral haemagglutinin gene • 24 hours testing • High specificity

Molecular assay Real-time RT-PCR • Increase sensitivity • Increase speed (4-5 hr) • Quantitative • Multiple genetic target (H and N gene)

Virus culture • Virus culture in MDCK cell, LLC-MK2, HEp-2c and RD cells • Results time 7-14 days • The virus identification by IFA or haemagglutination inhibition assays using reference antisera is required • Need Biosafety laboratory level 2/3

Serology test : Detect antibody response Infection Incubation Disease IgG antibody IgM antibody

Serology • For viral antibody detection • Micro-neutralization test (BSL-3) • Detection of 4-fold rising of specific antibody between acute (3days) and convalescence (14 days) sera. • For confirmation of suspected case, not for routine diagnosis. • 14 days or more.

Gold standard for diagnosis of H5N1 infection • Culture • RT-PCR • Sero-conversion by micro-neutralization tests (BSL-3)

ขั้นตอนการตรวจวิเคราะห์ขั้นตอนการตรวจวิเคราะห์ Sample  viral RNA  RT-PCR (FluA/B, H1, H5) การแปลผล : FluA/B -, H1-, H5 -  ไม่พบสารพันธุกรรมของเชื้อไวรัสไข้หวัดใหญ่และไข้หวัดนก FluB +, H1-, H5 -  พบสารพันธุกรรมของเชื้อไวรัสไข้หวัดใหญ่ชนิด B แต่ไม่พบสารพันธุกรรมของเชื้อไข้หวัดนก FluA + H1-, H5 -  พบสารพันธุกรรมของเชื้อไวรัสไข้หวัดใหญ่ชนิด A แต่ไม่พบสารพันธุกรรมของเชื้อไข้หวัดนก

การรายงานผล FluA +, H1+, H5 -  พบสารพันธุกรรมของเชื้อไวรัสไข้หวัดใหญ่ชนิด A สายพันธุ์ H1 แต่ไม่พบสารพันธุกรรมของเชื้อไวรัสไข้หวัดนก FluA +, H1-, H5 +  พบสารพันธุกรรมของเชื้อไวรัสไข้หวัดนก FluA - H1-, H5 -  ไม่พบสารพันธุกรรมของเชื้อไวรัสไข้หวัดใหญ่และใวรัสไข้หวัดนก

การรายงานผล FluA -, H1-, H5 - และ internal control -  ตัวอย่างไม่ได้คุณภาพ

Control • Internal control เป็นการตรวจหาสารพันธุกรรมของ human cell ในตัวอย่าง หากให้ผลบวกแสดงว่ามีเซลล์ที่เพียงพอในการตรวจวิเคราะห์ หากให้ผลลบควรเก็บตัวอย่างตรวจอีกครั้ง • Positive and negative control (A/B, H1, H5) เป็นการควบคุมความถูกต้องของการตรวจ ซึ่งจะทำตัวอย่างที่บวกจริงและลบจริงควบคู่กันไปด้วย

M s1 s2 s3 s4 s5 s6 A B Neg M s1 s2 s3 A B - Human cell

Interpretation Interpretation of H5N1 infection Positive by RT-PCR or real-time PCR with at least 2 sets of specific primers to H5 If only 1 set of primer is positive  report inconclusive  then follow-up samples will be requested Seroconversion of H5N1 antibody (1day, >14 days sera) by microneutralization test is a confirmation of H5N1 infection DNA sequencing will be used only for risk assessment and molecular epidemiology not forconfirmation of H5N1 infection

DNA Sequencing • Genetic analysis of influenza viruses • Risk assessment • Molecular epidemiology

Influenza A Viruses • Influenza virus contains 8 segments of viral genome which increase the potential to form the recombinants by interchange of gene segments from two different viruses, both among human strains and avian and human strains. This may cause virulent human strains to evolve. • A major change in HA and sometime a new NA as well (antigenic shift) result in pandemic strain appears in man (10-15 years interval) • A minor change in HA and NA(antigenic drift) result in epidemic (2-3 years interval)

Thank you

Laboratory Diagnosis for avian flu (H5N1) in human