Molecular diagnosis of Molecular diagnosis of respiratory viruses and its respiratory viruses and its

impact on clinical impact on clinical managementmanagement

Prof G KudesiaProf G Kudesia

Sheffield Teaching Hospitals Sheffield Teaching Hospitals NHS TrustNHS Trust

Cell CultureCell Culture

Widely usedWidely used Result in 7-14 days Result in 7-14 days

or longeror longer

Adenovirus CPE in RMKAdenovirus CPE in RMK

Un-infected RMKUn-infected RMK

Live cells required Live cells required

Cytopathic effect Cytopathic effect needs to be needs to be confirmed by confirmed by specific tests .specific tests .

Technical expertiseTechnical expertise

Time delayTime delay

Catch ‘all’Catch ‘all’

Antigen detection by Antigen detection by ImmunofluorescenceImmunofluorescence

RapidRapid Relatively Relatively

insensitiveinsensitive Not suitable for all Not suitable for all

speciemn typesspeciemn types SubjectiveSubjective

SerologySerology

Technically Technically demandingdemanding

InsensitiveInsensitive

Acute and Acute and convalescent convalescent serum sampleserum sample

Respiratory Viruses: Diagnosis Pre 1990’s.Respiratory Viruses: Diagnosis Pre 1990’s.

AdvantagesAdvantages DisadvantageDisadvantagess

Tissue Tissue CultureCulture

““Open” TechniqueOpen” TechniqueSensitiveSensitiveFurther Further characterisationcharacterisationEvidence of active Evidence of active infectioninfection

Not applicable to Not applicable to allallTime consumingTime consumingviable virus onlyviable virus onlyContamination/Contamination/toxinstoxins

SerologySerology Detects current and Detects current and past infection past infection (immunity)(immunity)Important for Important for fastidious virusesfastidious viruses

Prolonged testing Prolonged testing timetime

Antigen Antigen detectiondetection

RapidRapidDetects non-viable Detects non-viable virusvirusCan test large Can test large numbers of samplesnumbers of samples

Not applicable to Not applicable to allallinterferenceinterference

Polymerase Chain Reaction Polymerase Chain Reaction (PCR)-Xeroxing DNA!(PCR)-Xeroxing DNA!

Kary MullisKary Mullis Won the NobelPrize Won the NobelPrize

in 1993 for in 1993 for describing the describing the methodology in methodology in 1985 to replicate 1985 to replicate DNA in a test tube.DNA in a test tube.

PCRPCR

Impact of PCR testing on Impact of PCR testing on respiratory virus investigationsrespiratory virus investigations

Comparison of cell culture with PCR for Influenza Comparison of cell culture with PCR for Influenza A and B and RSV-200 specimen tested winter A and B and RSV-200 specimen tested winter

2006/07-Sheffield2006/07-Sheffield

VirusVirus RSVRSV Flu AFlu A RhinoRhino HMPV TotalHMPV Total

PCRPCR

PositivePositive3838 2929 3232 11 11011 110

Cell Cell CultureCulture

PositivePositive

1212

(32%)(32%)1717

(59%)(59%)1 1 (3%)(3%)

0 300 30

(27%)(27%)

TotalTotal 3838 2929 3232 11 11011 110

Respiratory PCR from Children –winter Respiratory PCR from Children –winter 07/08(haematology/oncology)07/08(haematology/oncology)

0

5

10

15

20

25

30

35

40

45

50

%Positive

Rhinovirus

'Flu A

'Flu B

RSV

HMPV

Paraflu1,2,3

Adeno

Rhino+HMPV

Dual

Advantages of PCR over traditional methods-R Gunson, GlasgowAdvantages of PCR over traditional methods-R Gunson, Glasgow

PositivePositive Flu AFlu A RhinoRhino RSVRSV TotalTotal

CultureCulture 1616 22 22 2020

DIFDIF 1414 n/an/a 66 2020

Total Total IsolationIsolation

2020 22 88 3030

PCRPCR 4141 1414 1515 7070

Sensitivity Sensitivity

(Iso Vs PCR)(Iso Vs PCR)49%49% 14%14% 53%53% 43%43%

Improved detection rateImproved detection rate

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

1996-97 1997-98 1998-99 1999-2000 2000-01 2001-02 2002-03 2003-04 2004-05 2005-06

Respiratory season

Det

ectio

n r

ate

(%)

Flu A Flu B RSV PF1 PF2 PF3 PF4 hMPV Coronavirus Rhino Adenovirus

0

25

50

75

100

0-1 2-3 4-5 6-8 9-10 11-14 >14

Days

Acc

um

ula

tive

per

cen

tag

e 1996-97

1997-98

1998-99

1999-00

2000-01

2001-02

2002-03

2003-04

2004-05

2005-06

Improvements in TRTImprovements in TRT

Clinical impactClinical impact

InfluenzaInfluenza TreatmentTreatment ProphylaxisProphylaxis Outbreak ManagementOutbreak Management Control of infectionControl of infection

ImmunocompromisedImmunocompromised Treatment Treatment Control of infection Control of infection

Treatment/prophylaxis for Treatment/prophylaxis for influenza-start within 48 hours influenza-start within 48 hours

Oseltamivir Oseltamivir • Treat- 75 mg twice a day x 5 daysTreat- 75 mg twice a day x 5 days• Prophylaxis- 75mg once a day x10 daysProphylaxis- 75mg once a day x10 days

Speed for laboratory confirmation of essenceSpeed for laboratory confirmation of essence

PCR testing was invaluable in the late PCR testing was invaluable in the late influenza B activity this winter- both for influenza B activity this winter- both for outbreak and individual patient managementoutbreak and individual patient management

FluFlu outbreak- SVC west Scotlandoutbreak- SVC west Scotland

Hospital X

Patient A – flu positive

Index case

Doctor A – flu positive

Doctor B – flu positive

Patient B – flupositive

Patient C – flu positive

Nurse – flu positive

Occurred out with flu season

Flu virus sequenced

Phylogenetic tree created

Shown to be H3 Wisconsin

Sequence flu strain originated from Patient A – the index case

FluFlu outbreakoutbreak

Hospital Y

Patient D – flu positive

Doctor C – flu positive

Patient E – flupositive

Flu virus sequenced

Phylogenetic tree created

Was there a connection with hospital X?

No connection betweenHospital X and Y

Tree may have looked likethis

Hospital Y

Hospital X

BUT THERE WAS A CONNECTION

Showed both flu outbreaks were connectedAll were H3 Wisconsin

What was the connection?

Hospital Y

Patient D – flu positive

Doctor C – flu positive

Patient E – flupositive

Patient C from hospital X was transferred to hospital Y

Hospital X

Patient A – flu positive

Index case

Doctor B – flu positive

Patient B – flupositive

Patient C – flu positive

Nurse – flu positive

Doctor A – flu positive

Molecular epidemiology for Molecular epidemiology for outbreak sequencingoutbreak sequencing

ImplicationsImplications• Shows connections between Shows connections between

patients/staffpatients/staff• Raises infection control issuesRaises infection control issues

Patient transferred while illPatient transferred while ill Why were staff infected Why were staff infected

• Re-evaluate hospital proceduresRe-evaluate hospital procedures E.g. masks, gowns, gloves, hand washingE.g. masks, gowns, gloves, hand washing

A case of Respiratory infection in A case of Respiratory infection in BMT-SheffieldBMT-Sheffield

37 year old male post BMT37 year old male post BMT Presented with GVHD in December 07Presented with GVHD in December 07 Third week of march 08- respiratory symptoms- ? Third week of march 08- respiratory symptoms- ?

Infection, ? Respiratory GVHDInfection, ? Respiratory GVHD Respiratory and PCP PCRs- HMPV PCR positive Respiratory and PCP PCRs- HMPV PCR positive

25/3, 7/425/3, 7/4 Not treated initially but subsequently treated with Not treated initially but subsequently treated with

I/V and nebulised Ribavirin due to deterioration in I/V and nebulised Ribavirin due to deterioration in respiratory symptoms.respiratory symptoms.

Died 14/4Died 14/4

Post-mortem histology of lungPost-mortem histology of lung

Sections from both the lungs show fibrin Sections from both the lungs show fibrin and macrophages in the alveolar spacesand macrophages in the alveolar spaces

along with focal squamous metaplasia. along with focal squamous metaplasia. There are scattered large bizzare cellsThere are scattered large bizzare cells

with basophilic inclusions in the with basophilic inclusions in the cytoplasm. The features are those of ancytoplasm. The features are those of an

organizing pneumonia with virocytopathic organizing pneumonia with virocytopathic effect suggesting of viral aetiology.effect suggesting of viral aetiology.

Human MetapneumovirusHuman Metapneumovirus

Discovered in 2000.Discovered in 2000. ParamyxoviridaeParamyxoviridae Negative sense, Single stranded RNANegative sense, Single stranded RNA Two genotypes A and BTwo genotypes A and B

Clinical ProblemsClinical Problems

Upper respiratory infectionUpper respiratory infection Lower respiratory infectionLower respiratory infection Non-specific symptomsNon-specific symptoms Fatalities reported in BMT patientsFatalities reported in BMT patients

ObjectiveObjective

To determine the incidenceTo determine the incidence 11stst September 2005 to 31 May 2006 September 2005 to 31 May 2006

MethodsMethods

Data collection-retrospectivelyData collection-retrospectively Descriptive methodsDescriptive methods

ResultsResults

Specimen typeSpecimen type NO of specimensNO of specimens

NPANPA 205205

BALBAL 7373

ETSETS 2222

OthersOthers 4848

TotalTotal 348348

The incidence of Respiratory The incidence of Respiratory PathogensPathogens

TotalTotal

AdenAden 13 13

MPVMPV 1111

FluAFluA 1111

FluBFluB 1717

ParaPara 88

RSVRSV 2525

MeasMeas 11

TotalTotal 86 86 (25%)(25%)

ageage wardward CondConditionition

ClinicClinicalal

SpecSpec OutcomeOutcome

11 <1<1 PICUPICU BronBron NPANPA

22 <1<1 PICUPICU BronBron NPANPA

33 22 PICUPICU BronBron NPANPA

44 33 M3M3 ALLALL URTIURTI NPANPA

55 55 M3M3 ALLALL NasalNasal NPANPA

66 55 M3M3 OncoOnco CoryzaCoryza NPANPA DischargedDischarged

77 1010 M3M3 ALLALL NPANPA DischargedDischarged

88 3838 E2E2 HIVHIV DyspDysp NPANPA

99 3939 E2E2 AtypiAtypi NPANPA DischargedDischarged

1010 5454 P3P3 BMTBMT CoryzaCoryza NPANPA

1111 6868 ITUITU NPANPA

FindingFinding

HMPV -4HMPV -4th th commonest respiratory commonest respiratory pathogenpathogen

Affected all age groupsAffected all age groups Detected in patients with both upper Detected in patients with both upper

and lower respiratory tract infectionsand lower respiratory tract infections Some patients discharged before Some patients discharged before

results were availableresults were available Further studies for clinical Further studies for clinical

significancesignificance

New viruses- human BocavirusNew viruses- human Bocavirus(HBoV)(HBoV)

Identified in 2005Identified in 2005 DNA virus belonging to family DNA virus belonging to family

ParvoviridaeParvoviridae Found in respiratory secretions from Found in respiratory secretions from

children with and with out respiratory children with and with out respiratory symptomssymptoms

Exact role in respiratory infections to Exact role in respiratory infections to be still worked outbe still worked out

How feasible is it to introduce PCR in How feasible is it to introduce PCR in routine diagnosisroutine diagnosis

Multiplex Real Time PCRMultiplex Real Time PCR

Multiplex 1Multiplex 1 Influenza AInfluenza A

Influenza BInfluenza B

Influenza CInfluenza C

MatrixMatrix

NSNS

MatrixMatrix

Multiplex 2Multiplex 2 HMPV AHMPV A

HMPV BHMPV B

Parainfluenza 1Parainfluenza 1

FusionFusion

FusionFusion

HNHN

Multiplex 3Multiplex 3 Parainfluenza 2Parainfluenza 2

Parainfluenza 3Parainfluenza 3

Parainfluenza 4Parainfluenza 4

HNHN

HNHN

FusionFusion

Multiplex 4Multiplex 4 HuCoV 229EHuCoV 229E

HuCoVOC43HuCoVOC43

HuCoV NL63HuCoV NL63

NucleocapsidNucleocapsid

NucleocapsidNucleocapsid

1a gene1a gene

Multiplex 5Multiplex 5 RSV ARSV A

RSV BRSV B

RhinoRhino

NPNP

NPNP

5-UTR5-UTR

Small numbers of tests/More pathogens detectedSmall numbers of tests/More pathogens detected

YearYear FormatFormat NNoo of tests of tests Tests performed (cumulative)Tests performed (cumulative)

2000-012000-01

Gel based nested Gel based nested 44

influenza A, B; RSV; adenovirus; picornavirusinfluenza A, B; RSV; adenovirus; picornavirus

2001-022001-02 influenza A, B; RSV; adenovirus; picornavirusinfluenza A, B; RSV; adenovirus; picornavirus

2002-032002-03 influenza A, B; RSV; adenovirus; picornavirus; influenza A, B; RSV; adenovirus; picornavirus;

2003-042003-04

Real time PCRReal time PCR

55influenza A, B; RSV; adenovirus; influenza A, B; RSV; adenovirus; Rhinovirus; PF1, 2, Rhinovirus; PF1, 2,

33

2004-052004-05 55influenza A, B; RSV A + B; adenovirus; rhinovirus; influenza A, B; RSV A + B; adenovirus; rhinovirus;

PF1, 2, 3; PF1, 2, 3; coronavirus NL63, 229e, OC43; HuMPVcoronavirus NL63, 229e, OC43; HuMPV

2005-062005-06 66influenza A, B, influenza A, B, CC; RSV A + B; adenovirus; rhinovirus; ; RSV A + B; adenovirus; rhinovirus;

PF1, 2, 3, PF1, 2, 3, 44; coronavirus ; coronavirus NL63, 229e, OC43NL63, 229e, OC43; ; HuMPV A + BHuMPV A + B

2007-082007-08 55

influenza A, B, C; RSV A + B; adenovirus; rhinovirus; influenza A, B, C; RSV A + B; adenovirus; rhinovirus; PF1, 2, 3, 4; coronavirus PF1, 2, 3, 4; coronavirus NL63, 229e, OC43NL63, 229e, OC43; ; HuMPV A + B; HuMPV A + B; M pneumoniaeM pneumoniae

But……………..:– Post amplification processing

– Contamination

– Prolonged testing time

– Non-automated

– Expensive to implement/expertise needed

– Qualitative (difficult to quantify)

Advantages of PCR

The utilisation of PCR conferred many advantages:– Highly sensitive/specific• Applicable to RNA or DNA virusesApplicable to RNA or DNA viruses• Rapid (turn around time of 24-48 hours)Rapid (turn around time of 24-48 hours)• Can detect multiple virusesCan detect multiple viruses• Products can be sequenced for epidemiological/resistance Products can be sequenced for epidemiological/resistance

study.study.• Improved patient management and disease surveillanceImproved patient management and disease surveillance

Submitted by R GunsonJune 2003Submitted by R GunsonJune 2003

– Closed systemClosed systemNo post-amplification processingNo post-amplification processing

RapidRapid

Reduced contaminationReduced contamination

Automation/high throughput/Cost effectiveAutomation/high throughput/Cost effective

Real time PCR: Real time PCR: Unlike conventional PCR:Unlike conventional PCR:

– Amplicon is visualised as the amplification progresses.Amplicon is visualised as the amplification progresses.– Exponential rather than endpoint analysisExponential rather than endpoint analysis

– More tests /less reagents/standardised cycling conditionsMore tests /less reagents/standardised cycling conditions

– Increased sensitivity/specificityIncreased sensitivity/specificity

Disadvantages of real time PCR:Disadvantages of real time PCR:– Risk of false negative reactions (due to miss-matches).Risk of false negative reactions (due to miss-matches).

– Number of amplicons detected is limited by the number of fluorophores.Number of amplicons detected is limited by the number of fluorophores.

– Expensive to implementExpensive to implement

Submitted by R GunsonJune 2003Submitted by R GunsonJune 2003

Examples of the benefits of real time PCR assays Examples of the benefits of real time PCR assays in viral respiratory infectionin viral respiratory infection

Gueudin Gueudin etet al: al:• Developed a real time PCR to detect, subgroup, and quantitate RSV A and BDeveloped a real time PCR to detect, subgroup, and quantitate RSV A and B

RSV A and B to be responsible to differing disease severitiesRSV A and B to be responsible to differing disease severities Found higher viral loads in more severe infectionsFound higher viral loads in more severe infections

Elden Elden et et al:al:• Developed a real time PCR for simultaneous detection of influenza A and B.Developed a real time PCR for simultaneous detection of influenza A and B.

Rapid diagnosis allowed timely therapeutic and infection control Rapid diagnosis allowed timely therapeutic and infection control interventionintervention

Quantitation could be used to examine the effects of antiviral therapyQuantitation could be used to examine the effects of antiviral therapy

Mackay Mackay etet al: al:• Developed a sensitive real time PCR for Human metapneumovirusDeveloped a sensitive real time PCR for Human metapneumovirus

Most sensitive assay currently availableMost sensitive assay currently available

Puhakka Puhakka et et al:al:• Examined the effect of zanamivir on the viral load of influenza Examined the effect of zanamivir on the viral load of influenza

Viral loads were reduced significantlyViral loads were reduced significantly

Submitted by R GunsonJune 2003Submitted by R GunsonJune 2003

SummarySummary

PCR for respiratory viruses are PCR for respiratory viruses are sensitive and specificsensitive and specific

Positivity rate of 50% or greater Positivity rate of 50% or greater Cell culture sensitivity 30-50% Cell culture sensitivity 30-50%

compared to PCR (for viruses that compared to PCR (for viruses that can be cultured)can be cultured)

Detection rate of PCR improved Detection rate of PCR improved further as many viruses not further as many viruses not culturable . culturable .

Summary- continuedSummary- continued

Several viruses can be tested for at Several viruses can be tested for at the same time by multiplex PCRthe same time by multiplex PCR

In-house PCR cost effective In-house PCR cost effective compared to cell culture compared to cell culture

PCR effective epidemiological tool in PCR effective epidemiological tool in investigation of outbreaksinvestigation of outbreaks

Rapid and sensitive assay aids in Rapid and sensitive assay aids in clinical management of respiratory clinical management of respiratory infections.infections.