Laboratory diagnosis of Laboratory diagnosis of influenzainfluenza

Dr Dr WilinaWilina LimLimGovernment Virus UnitGovernment Virus UnitDepartment of HealthDepartment of Health

Influenza VirusesInfluenza Viruses

OrthomyxovirusesOrthomyxoviruses can be identified as influenza can be identified as influenza A, B and C virusesA, B and C virusesInfluenza A infects a variety of different host Influenza A infects a variety of different host speciesspeciesThere are 15 subtypes of H and 9N. H1N1, There are 15 subtypes of H and 9N. H1N1, H2N2 and H3N2 have been recognized to cause H2N2 and H3N2 have been recognized to cause human infection for decadeshuman infection for decadesH1N1 H1N1 -- 1918 1918 -- 19571957

1977 1977 --H2N2 H2N2 -- 1957 1957 -- 19681968H3N2 H3N2 -- 1968 1968 ––H1N2 H1N2 –– 2002 2002 ----

Single stranded RNA virus with a core of 8 Single stranded RNA virus with a core of 8 segments. Each segment corresponds to a gene segments. Each segment corresponds to a gene

Influenza VirusesInfluenza VirusesThe envelop The envelop haemagglutininhaemagglutinin (HA) attaches to (HA) attaches to hosthost--cell receptors and stimulates the cell receptors and stimulates the production of neutralizing antibodyproduction of neutralizing antibodyThe surface spike neuraminidase (NA) permits The surface spike neuraminidase (NA) permits transport of virus through transport of virus through mucinmucin on cells of on cells of respiratory tractrespiratory tractThe nucleoprotein (NP) contains type specific The nucleoprotein (NP) contains type specific antigens that distinguish between influenza antigens that distinguish between influenza type A, B and Ctype A, B and CPB1 and PB2 are polymerases involved in PB1 and PB2 are polymerases involved in mRNA synthesis and PA in mRNA synthesis and PA in virionvirion RNA RNA synthesissynthesisM gene codes for membrane or matrix proteinM gene codes for membrane or matrix proteinNS genes code for two proteins in infected NS genes code for two proteins in infected cells involve in protein and RNA synthesiscells involve in protein and RNA synthesis

Source of specimensSource of specimens

Clinical specimens from hospitals for Clinical specimens from hospitals for virology studies.virology studies.Swabs from sentinel surveillance sitesSwabs from sentinel surveillance sitesSwabs from investigation of institutional Swabs from investigation of institutional outbreaksoutbreaksConfirmatory service for private Confirmatory service for private laboratories with positive screening laboratories with positive screening tests.tests.

Specimen collectionSpecimen collectionTake specimen early during illness. Best Take specimen early during illness. Best result in the first 3 days of illnessresult in the first 3 days of illnessThroat / nose swabsThroat / nose swabsNasopharyngeal aspirateNasopharyngeal aspirateTracheal aspirateTracheal aspirateBronchioBronchio--alveolar alveolar lavagelavageSputumSputumTransport to lab. as soon as possible. Place at Transport to lab. as soon as possible. Place at 44°°C if delay is inevitable (Isolation rate C if delay is inevitable (Isolation rate unchanged for up to 4 days at unchanged for up to 4 days at 44°°C)C)Place at Place at --7070°°C if not cultured C if not cultured withinwithin 4 days4 days

Laboratory MethodsLaboratory Methods

Cell cultureCell cultureImmunofluorescence testImmunofluorescence testHaemagglutination inhibition testHaemagglutination inhibition testPolymerase chain reactionPolymerase chain reactionNucleotide sequencingNucleotide sequencing

Rapid diagnosis of influenza A virusRapid diagnosis of influenza A virus

DirectigenDirectigen Flu A (Becton Dickinson)Flu A (Becton Dickinson)

NowNow®® Flu A (Flu A (BinaxBinax))

ImmunofluorescenceImmunofluorescence testtest

Reverse transcriptionReverse transcription--PCR (InPCR (In--house)house)

DirectigenDirectigen Flu A (Becton Dickinson)Flu A (Becton Dickinson)

•• An enzyme immunoassay (EIA test) membrane An enzyme immunoassay (EIA test) membrane test for influenza A viral antigen (nucleoprotein)test for influenza A viral antigen (nucleoprotein)

•• Results are available in 15 minutesResults are available in 15 minutes

Result of Result of DirectigenDirectigen Flu AFlu A

Comparison of Comparison of DirectigenDirectigen to Cultureto Culture

Specimen typeSpecimen typeSensitivitySensitivity SpecificitySpecificityNPANPA 92%92% 8888PSPS 67%67% 92%92%

NPSNPS 88%88% 97%97%

NowNow®® Flu A (Flu A (BinaxBinax))

•• An An immunochromatographyimmunochromatography (ICT) technology(ICT) technology•• Requires only one stepRequires only one step•• Results are available in 15 minutesResults are available in 15 minutes

Result of NowResult of Now®® Flu A (Flu A (BinaxBinax))

Correlation between culture and Correlation between culture and immunofluorescenceimmunofluorescence

IF screen +IF screen +veve IF screen IF screen ––veve IF screen +IF screen +veveBoth IF Both IF andand

Culture +Culture +veve Culture +Culture +veve Culture Culture ––veve Culture Culture ––veve

RSVRSV 5454 55 1515Influenza AInfluenza A 115115 1313 22Influenza BInfluenza B 9494 2929 00ParainfluenzaParainfluenza 1 41 4 00 11ParainfluenzaParainfluenza 2 32 3 00 00ParainfluenzaParainfluenza 3 703 70 88 1010Adenovirus Adenovirus 9999 5858 22

TOTALTOTAL 439439 113113 3030 15661566

# some specimens had more than a single virus detected. There # some specimens had more than a single virus detected. There the total number of viruses detected was greater than the the total number of viruses detected was greater than the number of specimens examined.number of specimens examined.

Reverse transcriptionReverse transcription--PCRPCR

For subFor sub--typing and detection typing and detection PrinciplePrinciple

SubSub--type (H1 or H3) specific primertype (H1 or H3) specific primerTarget on viral Target on viral hemagglutininhemagglutinin genegeneViral RNA is extracted and transcribed into Viral RNA is extracted and transcribed into cDNAcDNAcDNAcDNA is then used as a target for polymerase chain is then used as a target for polymerase chain reactionreactionEach reaction is Each reaction is analysedanalysed by by agaroseagarose gel electrophoresis gel electrophoresis and and ethidiumethidium bromide stainingbromide staining

RTRT--PCR result (H3)PCR result (H3)

1 2 3 4 -/c Marker

Cell cultureCell culture

Inoculate into MDCK cells to isolate Inoculate into MDCK cells to isolate influenza virusinfluenza virus

Identify by IF and HI testsIdentify by IF and HI tests

Specimens are also inoculated into Specimens are also inoculated into LLCLLC--MK2, HEpMK2, HEp--2c, RD to isolate other 2c, RD to isolate other respiratory virusesrespiratory viruses

SerologySerology

Based upon a significant rise Based upon a significant rise (generally 4 fold) in antibody (generally 4 fold) in antibody titretitre to a to a given viral antigen over the course of the given viral antigen over the course of the patientspatients’’ illness.illness.--ComplexmentComplexment fixation test (CF)fixation test (CF)

type specifictype specific--HaemagglutinationHaemagglutination inhibition inhibition test(HItest(HI))

subtype specificsubtype specific--Neutralization testNeutralization test

measure protectionmeasure protectionmost sensitive and specificmost sensitive and specific

Typing of VirusesTyping of VirusesAll the influenza virus isolates All the influenza virus isolates would be typed with would be typed with antiseraantisera from from WHO.WHO.Atypical isolates would be sent to Atypical isolates would be sent to WHO Collaborating Reference WHO Collaborating Reference Laboratories (e.g. US, UK etc) for Laboratories (e.g. US, UK etc) for further typing.further typing.

WHO Influenza SurveillanceWHO Influenza Surveillance

WHO Influenza Collaborating Reference Laboratories

110 WHO National Influenza Centres

United States United Kingdom Australia Japan

World Health Organization

Specimens by Age GroupsSpecimens by Age Groups (1998(1998--2002)2002)

Age GroupAge Group Specimens numberSpecimens number No. (%) No. (%) positivepositive0 0 –– 4 53944 19259 (35.7)4 53944 19259 (35.7)5 5 –– 14 13108 5118 (39.0)14 13108 5118 (39.0)

15 15 –– 24 2516 820 (32.6)24 2516 820 (32.6)25 25 –– 29 7115 1908 (26.8)29 7115 1908 (26.8)≥≥60 4184 1092 (26.1)60 4184 1092 (26.1)

Respiratory Viruses by YearRespiratory Viruses by Year (1998(1998--2002)2002)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

1998 1999 2000 2001 2002

FluAdenoRSVPara

Lab influenza virus isolates 1991 - 2003 August

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003

Year

020040060080010001200140016001800

No. of isolates

Lab isolates

Recent Infections by Animal and Avian Recent Infections by Animal and Avian Influenza A VirusesInfluenza A Viruses

1997 1997 Influenza A H5N1 virus (chick)Influenza A H5N1 virus (chick)1999 Influenza A H9N2 virus (quail)1999 Influenza A H9N2 virus (quail)1999 Influenza A H3N2 virus (swine)1999 Influenza A H3N2 virus (swine)2001 Influenza A H1N1 virus (swine)2001 Influenza A H1N1 virus (swine)2003 Influenza A H5N1 virus (chick) 2003 Influenza A H5N1 virus (chick)

Confirmed Cases of H5N1 Infection in 1997Confirmed Cases of H5N1 Infection in 1997

18 cases in total18 cases in totalM : F = 8 : 10M : F = 8 : 1011 children under 14, 7 adults11 children under 14, 7 adults3 from Hong Kong Island3 from Hong Kong Island6 from 6 from KowloonKowloon3 from New Territories East3 from New Territories East6 from New Territories West6 from New Territories WestHistory of possible exposure to poultry History of possible exposure to poultry in 7in 76 died 2 children <14, 4 adults6 died 2 children <14, 4 adults

VirologyVirology

All eight DNA gene segments of each of All eight DNA gene segments of each of these viruses are of avian originthese viruses are of avian originThe human and chick viruses showed a The human and chick viruses showed a high degree of homologyhigh degree of homologyThe avian influenza virus H5N1 crossed The avian influenza virus H5N1 crossed the avianthe avian--human species barrier human species barrier without prior adaptation in another without prior adaptation in another mammalian speciesmammalian species

Confirmed cases of H5N1 in 2003Confirmed cases of H5N1 in 2003–– Father 33 and son 9Father 33 and son 9–– Returned from Returned from FujianFujian after after

staying for 10staying for 10--14 days 14 days ----Daughter 8 yrs. died of Daughter 8 yrs. died of

pneumonia in pneumonia in FujianFujian–– Father died on 17.2.2003Father died on 17.2.2003–– Son recovered uneventfullySon recovered uneventfully

Confirmed cases of H5N1Confirmed cases of H5N1

NPANPA H5N1 virus isolated in MDCK cells H5N1 virus isolated in MDCK cells PCR PCR -- H5 specificH5 specific

A/HK/212/03 similar to A/HK/213/03A/HK/212/03 similar to A/HK/213/03No human influenza gene segments No human influenza gene segments presentpresentAll the internal genes are different from All the internal genes are different from A/HK/156/97A/HK/156/97AntigenicallyAntigenically different from different from A/HK/156/97A/HK/156/97

AvianAvian--toto--Human Transmission of Influenza Human Transmission of Influenza H9N2 VirusH9N2 Virus

H9N2 viruses were isolated from two H9N2 viruses were isolated from two children with mild influenza like symptomschildren with mild influenza like symptomsH9N2 viruses have been isolated from H9N2 viruses have been isolated from different species of birds and pigsdifferent species of birds and pigsH9N2 viruses are widespread and have H9N2 viruses are widespread and have been found in Germany, Iran, Pakistan, been found in Germany, Iran, Pakistan, Saudi ArabiaSaudi Arabia

SwineSwine--toto--Human Transmission of Influenza Human Transmission of Influenza VirusVirus

H3N2 virus isolated from 10 monthH3N2 virus isolated from 10 month--old baby with old baby with mild influenza symptomsmild influenza symptomsAntigenically distinct from circulating human Antigenically distinct from circulating human virusvirusRelated to early human and swine H3N2 virus Related to early human and swine H3N2 virus infectioninfectionClosely related to swine H3N2 virus prevalent in Closely related to swine H3N2 virus prevalent in the Europe since the 80the Europe since the 80’’ssResistant to antiResistant to anti--influenza drugs amantadine and influenza drugs amantadine and rimantadinerimantadine

SwineSwine--toto--Human Transmission of Influenza Human Transmission of Influenza VirusVirus

H1N1 virus isolated from 3 monthH1N1 virus isolated from 3 month--old baby with old baby with mild influenza symptomsmild influenza symptomsAntigenically distinct from circulating human Antigenically distinct from circulating human virusvirusRelated to early swine H1N1 virus infectionRelated to early swine H1N1 virus infectionAntigenicallyAntigenically related to swine H1N1 virus A/New related to swine H1N1 virus A/New Jersey/8/76Jersey/8/76None of the other family members were positive None of the other family members were positive for the virusfor the virus

ConclusionConclusionLaboratory diagnosis important forLaboratory diagnosis important for

• Treatment

•• Infection controlInfection control

•• Investigation and management of outbreakInvestigation and management of outbreak

•• Systematic collection and analysis of dataSystematic collection and analysis of dataprovide coherent information on trends of infectionprovide coherent information on trends of infection

•• Facilitate rapid isolation of new strains ofFacilitate rapid isolation of new strains ofinfluenza virusinfluenza virus

•• Detailed characterization of pathogens provideDetailed characterization of pathogens provideinformation for vaccine formulationinformation for vaccine formulation