Roche Diagnostics Symposium 2014 Current developm ents and - - PowerPoint PPT Presentation

Roche Diagnostics Symposium 2014

Friday, 26 September 2014 2

Current developm ents and challenges in the diagnosis and m anagem ent of congenital CMV infection Vincent Em ery Professor of Translational Virology

• Brief summary of congenital CMV infection
• Symptomatic
• Asymptomatic
• Advances in diagnosis
• Treatment options
• Looking to the future
• Benefits of universal screening
• vaccines

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Overview of presentation

• In resource rich settings CMV is:
• the commonest non-genetic cause of childhood

hearing loss

• Important cause of neurodevelopmental delay

Friday, 26 September 2014 4

Congenital CMV infection

Boppana et al (2001) NEJM 344, 1366-1371

Disability attributable to congenital CMV

Reviews in Medical Virology RMV-2013-055.R1, 24 APR 2014 DOI: 10.1002/rmv.1790 http://onlinelibrary.wiley.com/doi/10.1002/rmv.1790/full#rmv1790-fig-0004

Annual number of affected children in the USA

1000 2000 3000 4000 5000 6000

Disability attributable to congenital CMV

Reviews in Medical Virology RMV-2013-055.R1, 24 APR 2014 DOI: 10.1002/rmv.1790 http://onlinelibrary.wiley.com/doi/10.1002/rmv.1790/full#rmv1790-fig-0004

Annual number of affected children in the USA

1000 2000 3000 4000 5000 6000

Causes of Deafness at Birth and at Four Years

Morton C, NEJM 354, 2151, 2006.

• In resource rich settings CMV is:
• the commonest non-genetic cause of childhood

hearing loss

• Important cause of neurodevelopmental delay
• Primary infection most important risk but……
• Reinfection of seropositive women can lead to intrauterine

transmission and congenital disease1

• 8000 children with neurological disease per year (USA)
• Estimated cost in 1990s $1.86 billion per annum

(>$300,000 per child)

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Congenital CMV infection

1Boppana et al (2001) NEJM 344, 1366-1371

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Risk of intrauterine transm ission: prim ary infection

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Congenital infection rates ( )

CMV seroprevalence in wom en of reproductive age

Annual number of: Australia (populn ~ 22m) England and Wales (populn ~ 50m) United States (populn ~ 307m) Live births 296,000 709,000 4,248,000 Congenital CMV infections (0.6%) 1,780 4,254 25,488 Symptomatic at birth (12.8%) 228 544 3,262 Symptomatic who develop disability (50%) 114 272 1,631 Asymptomatic at birth (87.2%) 1,552 3,710 22,226 Asymptomatic who develop disability (13.5%) 210 501 3,001 Total 324 773 4,632

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Estim ates of congenital CMV – related disability

Cannon et al Rev Med Virology 2014 10.1002/ rmv.1790

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Congenital CMV in low and high seroprevalence settings

Parameter South Africa Thailand Annual birth rate 1,000,000 830,000 Antenatal HIV prevalence 30% 0.7% HIV perinatal transmission rate 3.5% 2.8% No of congenital CMV infections HIV unexposed (risk,1%) 700,000 (7,000) 824,190 (8,242) HIV exposed (risk, 3%) 265,000 (7,950) 5,647 (169) HIV infected (risk, 10%) 35,000 (3,500) 163 (16) Total no of congenital CMV infections 18,450 8,427

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Resource poor setting: estim ates of congenital CMV infection

Manicklal et al Clinical Micro Rev 2013, 26, 86-102

taken from, Lazzarotto et al 2011

Managem ent during pregnancy

Congenital CMV - outcom e

n=1000 CCMV cases

Congenital CMV - outcom e

n=1000 CCMV cases

2/3 of children with hearing loss are ‘asymptomatic’ at birth

Diagnosis of congenital CMV (CCMV)

Birth

+ + + + + + + + + + + + + + + + + + + + + + +

• - - - - - - - - - - - - - - - - - - - + + +

DAY 21 CCMV Perinatal infection

• Currently made by CMV positive PCR or culture before day 21 of life
• Urine and saliva most common sample received

Virus secretion

• Differentiating congenital CMV from perinatal is important
• perinatal infection much less likely to be associated with disease
• Could other samples be used?

Symptomatic congenital infection Asymptomatic Natal infection Standard error of the mean

Age (months)

102 106 103 104

Birth

CMV titer (log10-TCID50/0.2 ml urine)

105

3 6 9 12 15 18 24 30 36 42

Stagno et al (1975) J Infect Dis, 132, 568

CMV load in congenital infection

Griffiths PD. The Lancet Infectious Diseases, Volume 12, Issue 10, 2012, 790 - 798

Viral load thresholds and disease

Dried blood spots (DBS)

• DBS are routinely collected for neonatal screening for metabolic and

hereditary diseases

• Heel prick taken in the first few days of life is used
• Cards are stored for up to 18 years depending on the Health Authority

Virus secretion

• Dried blood spots (DBS) taken on every child born in the UK

Birth

+ + + + + + + + + + + + + + + + + + + + + + +

• - - - - - - - - - - - - - - - - - - - + + +

DAY 21 CCMV Perinatal infection DBS taken day 5-8

CCMV: Diagnosis using DBS

• To retrospectively diagnose CCMV in asymptomatic infants
• Allows follow up and early intervention
• Early intervention minimises the impact of SNHL on language

and social development

• Recruitment to cochlear implant programme
• Treatment: ganciclovir reduces hearing deterioration and

CNS involvement in symptomatic CCMV

Why diagnose congenital CMV?

Dried Blood Spot testing

Nucleic acid extracted

• Real Time PCR for CMV,

gB target.

• Positive result confirmed

with 2nd RT PCR for different region of CMV (UL69)

• Normalised against beta-

globin gene

1/2 Dried Blood Spot cut from card

• Screening algorithm for retrospective diagnosis of CCMV in children with SNHL

Child With SNHL (Consider testing mother for HCMV IgG if negative exclude CCMV ) <1 year old Urine/ Saliva x2 Urine / saliva negative exclude CCMV Urine or saliva positive Obtain parent ’ s written permission to test DBS DBS HCMV DNA detected: Consistent with congenital HCMV

Screening algorithm - retrospective diagnosis with DBS

Child With SNHL (Consider testing mother for HCMV if negative exclude CCMV ) <1 year old Urine/ Saliva x2 >1 year old urine/Saliva (+/

• HCMV IgG )

Urine / saliva negative exclude CCMV Urine or saliva positive Urine, saliva

• r IgG

positive Urine /saliva negative IgG negative exclude CCMV IgG positive Obtain parent’s written permission to test DBS DBS HCMV DNA detected: Consistent with congenital HCMV DBS HCMV DNA not detected: SNHL unlikely to be caused by HCMV

• Screening algorithm for retrospective diagnosis of CCMV in children with SNHL

IgG

Screening algorihm - retrospective diagnosis with DBS

• Sensitivity of assay found to be ~70%
• Not all CCMV babies are born viremic
• However viremic babies may be at higher risk of developing

disease

• Screening would identify these individuals
• Ganciclovir reduces hearing deterioration and CNS

involvement in infants with symptomatic CCMV

• Potential harms must be investigated – 80% of children with

CCMV do not develop disease

• BEST Study (stopped recruiting Nov 2012, 400 infants

tested, currently in data analysis)

Is there potential for national screening?

Paul D Griffiths Burden of disease associated with human cytomegalovirus and prospects for elimination by universal immunisation The Lancet Infectious Diseases, Volume 12, Issue 10, 2012, 790 - 798 http://dx.doi.org/10.1016/S1473-3099(12)70197-4

To Screen or not?

DBS vs saliva testing for CCMV

• 20,448 babies screened using both

methods

• CCMV confirmed in 92 infants

(0.45%)

• DBS sensitivity found to be poor

compared to saliva (34%)

• Other groups show much better

sensitivity in CCMV cohorts

• Barbi et al 100% (nested PCR)
• Could nested PCR be adapted for

screening to improve assay sensitivity? Boppana et al, JAMA, 2010 April 14 :(1375-82)

Saliva for screening newborns

Boppana SB et al. N Engl J Med 2011;364:2111-2118.

• Real-Tim e Polym erase-Chain-Reaction (PCR) Assays of Liquid- and Dried-

Saliva Specim ens, vs. Rapid Culture, in 5276 Newborns Who Underwent All Three Assays Used to Screen for Congenital Cytom egalovirus Infection.

Boppana SB et al. N Engl J Med 20 11;36 4:2111-2118 .

Boppana SB et al. N Engl J Med 2011;364:2111-2118.

Perform ance of the saliva assay

Boppana SB et al. N Engl J Med 2011;364:2111-2118.

• Established sample collection route on every child in

the UK

• Only sample taken at correct time point
• Cost and logistics of setting up a additional sample

collection

• Can we improve current methods….........
• A novel single tube nested PCR for enhanced detection
• f HCMV from dried blood spots developed1

Why DBS for CCMV?

• 1. Atkinson, Emery and Griffiths (2014) J Virol Methods 196:40-44

DBS Samples

• 3 clinical DBS cohorts were tested
• The Quality Control Molecular Diagnostic (QCMD) 2011 CMV

DBS panel

• 20 DBS samples from newborns with CCMV infection obtained

from an earlier published study (17th BPSU Annual Report 2002).

• DBS samples received as part of an ethically approved study

(Benefits of Extended Screening Testing (BEST Study) from 6 children who failed their newborn hearing screen and were proven to have CCMV1 Evaluation a nested PCR approach

• 1. Williams, Kadambari et al., 2014

• Overall sensitivity of the one step nested PCR assay in identifying CCMV was 21/26 (81%; 95% CI,

60.6% to 93.4%)

• Single step gB real time PCR had a sensitivity of 18/26 (69%;95% CI, 48.2%-85.6%)
• increased detection rate of 12% in children with laboratory diagnosed CCMV infection

Sensitivity of a nested PCR approach

Atkinson, Emery and Griffiths (2014) J Virol Methods 196:40-44

• BPSU: the two negative samples came from babies with asymptomatic presentation

with a normal clinical outcome (no problems reported, apparently normal development) at follow up (20.8 and 20.5 months after birth)

• CCMV failed NHSP : the additional positive DBS in the samples had a sample of

whole blood tested in the neonatal period (prior to the DBS being taken) with a viral load of 7,700 copies/ml. The child presented with unilateral SNHL, subependymal cysts on cranial imaging and received 6 weeks’ treatment with valganciclovir

• Overall the outcome was known in 25/26 CCMV children. The mean follow up period

was 19.9 months (± 4.6 months)

• The one step nested PCR detected CMV DNA in 20/25 samples compared to the

gB assay with 17/25 testing positive

• Of the 3 samples positive only with the nested PCR:
• ne DBS was from a symptomatic infant with mild SNHL at follow up
• the other two DBS samples were from symptomatic children with bi –

lateral hearing loss at follow-up

Clinical Analysis

Neonatal Presentation Outcome* Number of DBS testing positive with Single round PCR Number of DBS testing positive with nested one step PCR Asymptomatic Normal 3/7 3/7 Symptomatic Normal 2/2 2/2 Symptomatic Mild 5/6 6/6 Symptomatic Moderate 5/7 7/7 Symptomatic Severe 3/3 3/3

*Outcome: Normal- No reported problems Mild: Unilateral hearing loss, mild cerebral palsy,

mild language delay, Moderate: Bi-lateral profound deafness, deafness and other problem Severe: Multiple serious problems e.g. Severe global delay.

Correlations between neonatal presentation/ outcom e and DBS test results

• Mean log10 CMV viral load in DBS

higher in children with SNHL (2.69 vs1.64 p=0.01)

• Provides important rationale for

antiviral treatment studies in CCMV

DBS viral load and SNHL

(CHIC study)

3.0 3.5 4.0 4.5 5.0 5.5 1 2 3 4 5 R2=0.904 Log10 CMV DNA viral load in whole blood (copies/ml) log1

0 CMV DNA viral load in

DBS (copies/ml)

P value = <0.0001

Correlation between CMV loads in whole blood and DBS

• Rapid Real Time assay allows simultaneous nested amplification and

detection of HCMV DNA in a single tube in 90 minutes

• Single tube protocol removes associated contamination risk of

traditional two step nested PCR

• Assay shown to be more sensitive than single round qPCR in mock

and diagnostic DBS, addressing some diagnostic concerns surrounding DBS

• One step optimised assay suitable for high throughput testing

Benefits of a Nested PCR approach

Virus detection from urine and/saliva in first 2 weeks of life Negative Uninfected newborn Congenitally Infected newborn No further testing Asymptomatic Symptomatic Including SNHL Long term follow-up (neurodevelopmental,ophthalmologic and audiologic investigations) Therapy* Virus detection from DBS (>2 weeks of life)

Negative

does not exclude congenital CMV (sensitivity 81% 95%CI 60-93%) Positive

*CASG 403; A Phase II randomised and controlled investigation of six weeks of oral valganciclovir therapy in infants and children (1 month- 3yrs old) with congenital cytomegalovirus infection and hearing loss) adapted from Lazzarotto et al; 2011

Algorithm including DBS screening

Friday, 26 September 2014 41

Therapeutic intervention

• Currently antiviral treatment with GCV or VGCV only recommended for

symptomatic newborns with severe symptomatic focal organ disease

• One phase III trial of GCV (IV 6mg/kg bd vs no treatment) for 6 weeks1
• GCV shown to:
• reduce hearing loss at 6 months and > 1year
• Short term improvements in weight gain and liver abnormalities
• Long term reduction in developmental delays at 6 and 12 months
• VGCV at 16mg/kg bd provides similar drug levels to IV GCV
• Usual to deliver GCV through central line but…
• Roche VGCV liquid formulation is being used off-license
• Currently a placebo controlled trial of 6 weeks and 6 months of VGCV (CASG

112) underway

• Primary outcomes are hearing loss, safety profiles and neurological
• utcomes

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Therapy for congenital CMV disease

1. Kimberlin et al (2003) J peadiatr 143:16-25

• 2. Kadambari et al (2011) early Human development 78:723-28

• Monitoring of neutropenia, thrombocytopenia and anemia

essential

• Weekly monitoring of neutropenia recommended
• If neutrophil count drops <0.5 x 109/L medication should cease
• If platelet count drops < 50X109/L medication should cease
• Creatinine clearance monitored weekly and dose adjustment

made

• Monitor CMV loads in blood
• Usually 1-2 log drop in first weeks of therapy
• Treatment with continuing high level replication may indicate

drug resistance

• Genomic screens for mutations UL97 and UL54

Friday, 26 September 2014 43

Monitoring during therapy

Kadambari et al (2011) early Human development 78:723-28

1

• Blood tests (FBC, U &E, LFTs); Diagnostic auditory assessment; ophthalmic , CrUSS±MRI

2

• Symptomatic focal organ disease
• Symptomatic CNS disease

3

• GCV 6mg/kg bd 6 weeks (VGCV 16mg/kg bd PO 6 weeks
• FBC, LFT and U & E weekly
• Viral load weekly
• Therapeutic drug monitoring

4

• Pediatric clinical evaluation at 6 months and 12 months
• Audiology assessment 3-6 months until 3 years then annually until 6 years of age
• Neurodevelopmental assessment at 1 year
• Ophthalmic assessment annually until 5 years old

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Management algorithm

Web based registry for treated infants at www.ecci.ac.uk

Kadambari et al (2011) early Human development 78:723-28

Friday, 26 September 2014 45

Looking to the future

• What impact would screening have?
• Impact of vaccination

Screening neonates – hearing loss

Cannon, 2014

4,248,000 Live births 4,222,512 Children born without congenital CMV infection 25,488 Children born with congenital CMV infection 22,226 Children who are asymptomatic at birth 3,262 Children who are symptomatic at birth

99.4% 87.2% 0.6% 12.8%

815 Symptomatic children diagnosed clinically with congenital CMV

1,067 Delayed hearing loss 24–72 months 178 Delayed hearing loss 9–24 months

222 Delayed hearing loss <9 months 1,245 Hearing loss at birth

5.6% 1% 1% 5.3% 25% 75%

2,447 Symptomatic children not diagnosed clinically with congenital CMV 1,504 No hearing loss 670 Hearing loss at birth 78 Delayed hearing loss <9 months

78 Delayed hearing loss 9–24 months 117 Delayed hearing loss 24–72 months

61.4% 27.4% 3.2% 3.2% 4.8%

Quality of evidence Good evidence Fair evidence Poor evidence No benefit

Screening neonates – cognitive deficit

Cannon, 2014

21,181 Children with no cognitive deficit 1,045 Children with cognitive deficit 1,337 Children with cognitive deficit 763 Children with cognitive deficit who are diagnosed clinically with congenital CMV 574 Children with cognitive deficit who are not diagnosed clinically with congenital CMV

95.3% 4.7%

4,248,000 Live births 4,222,512 Children born without congenital CMV infection 25,488 Children born with congenital CMV infection 22,226 Children who are asymptomatic at birth

99.4% 87.2% 0.6% 41% 57.1% 42.9%

3,262 Children who are symptomatic at birth

12.8%

Quality of evidence Fair evidence No benefit

Modelling the effects of vaccination on congenital infection

Green = congenital infection (seropositive women) Red = congenital infection (seropositive women reinfected) Blue = congenital infection (seronegative women)

Griffiths PD. The Lancet Infectious Diseases, Volume 12, Issue 10, 2012, 790 - 798

• Phase 2, placebo controlled double blind trials of

recombinant gB + MF59 adjuvant1

• Target population:
• HCMV seronegative women within 1 year after

giving birth

• 3 doses of vaccine/placebo given (0,1,6 months)
• HCMV infection assessed by quarterly IgG serology for

HCMV proteins other than gB

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Preventing congenital infection: vaccination

• 1. Pass et al (2009) N Engl J Med 360:1191-99

gB vaccine reduced infection by 50 %

• 1. Pass et al (2009) N Engl J Med 360:1191-99

• Congenital CMV infection remains an major cause of morbidity in the

neonate

• Current diagnosis of congenital infection relies on PCR/culture in the

first 3 weeks of life

• CMV load at birth indicative of symtomatology
• Asymptomatic neonates are at risk of developing SNHL
• Saliva and DBS can be used for screening and DBS useful for

retrospective diagnosis of congential infection

• Therapy warranted for symptomatic infection and can reduce SNHL

but drug toxicity must be considered

• New drugs such as neutralising monoclonal antibodies and

letermovir (terminase inhibitor) undergoing trials but not in neonates

• CMV vaccination would significantly impact on infection/disease but

at present no licensed vaccine available

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Conclusions

Friday, 26 September 2014 52

Thank You

University of Washington Dr Jenn Slyker Grace John-Stewart Michael Boeckh University of Nairobi, Kenya Dorothy Mbori-Ngacha Jam es Kiarie UCL Claire Atkinson Paul Griffiths University of Cape Town Sheetal Manicklal University of Bologna Tiziana Lazzarotto

National Institutes of Health

Roche Diagnostics Symposium 2014