CMV Congenital PDF

Roche Diagnostics Symposium 2014
Current developments and challenges in the diagnosis

and management of congenital CMV infection
Vincent Emery
Professor of Translational Virology
Friday, 26 September 2014
Overview of presentation
Brief summary of congenital CMV infection

Symptomatic
Asymptomatic
Advances in diagnosis
Treatment options
Looking to the future
Benefits of universal screening
vaccines
Congenital CMV infection
In resource rich settings CMV is:

the commonest non-genetic cause of childhood
hearing loss
Important cause of neurodevelopmental delay
Boppana et al (2001) NEJM 344, 1366-1371

Disability attributable to congenital CMV
6000
5000
Annual
number of
affected
children in
the USA
4000
3000
2000
1000
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
Disability attributable to congenital CMV
6000
5000
Annual
number of
affected
children in
the USA
4000
3000
2000
1000
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
Causes of Deafness at Birth and at Four Years
Morton C, NEJM 354, 2151, 2006.
Congenital CMV infection

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

8
Risk of intrauterine transmission:

primary infection
CMV seroprevalence in women of

reproductive age
Congenital infection rates ( )
10
Estimates of congenital CMV related disability

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
Cannon et al Rev Med Virology 2014 10.1002/rmv.1790
11
Congenital CMV in low and high

seroprevalence settings
12
Resource poor setting:

estimates of congenital CMV infection
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%
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
No of congenital CMV
infections
Manicklal et al Clinical Micro Rev 2013, 26, 86-102
13
Management during pregnancy
taken from, Lazzarotto et al 2011
Congenital CMV - outcome
n=1000 CCMV cases
Congenital CMV - outcome
n=1000 CCMV cases
2/3 of children with hearing loss are asymptomatic at birth
Diagnosis of congenital CMV (CCMV)
Currently made by CMV positive PCR or culture before day 21 of life

Urine and saliva most common sample received
DAY 21
Birth
Virus + + + + + + + + + + + + + + + + + + + + + + + CCMV
secretion
- - - - - - - - - - - - - - - - - - - - + + + Perinatal
infection
Differentiating congenital CMV from perinatal is important

perinatal infection much less likely to be associated with disease
Could other samples be used?
CMV titer (log10-TCID50/0.2 ml urine)
CMV load in congenital infection

Symptomatic congenital infection
106
Asymptomatic
Natal infection
105
Standard error of the mean
104
103
102
Birth
12
15
18
Age (months)
Stagno et al (1975) J Infect Dis, 132, 568
24
30
36
42
Viral load thresholds and disease
Griffiths PD. The Lancet Infectious

Diseases,
Volume 12, Issue 10, 2012, 790 - 798
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
CCMV: Diagnosis using DBS
DBS taken day 5-8
DAY 21
Birth
CCMV
Virus + + + + + + + + + + + + + + + + + + + + + + +
secretion
- - - - - - - - - - - - - - - - - - - - + + + Perinatal
infection
Dried blood spots (DBS) taken on every child born in the UK
Why diagnose congenital CMV?
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
Dried Blood Spot testing
1/2 Dried Blood

Spot cut from card
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 betaglobin gene
Screening algorithm- retrospective

diagnosis with DBS
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 algorihmretrospective diagnosis with DBS

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
>1 year old urine/Saliva

(+/ - HCMV IgG )
Urine, saliva
or IgG
positive
IgG
positive
Urine
/saliva
negative
IgG
negative
exclude
CCMV
Obtain parents 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
Is there potential for national

screening?
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)
To Screen or not?
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
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-Time Polymerase-Chain-Reaction (PCR) Assays of Liquid- and DriedSaliva Specimens, vs. Rapid Culture, in 5276 Newborns Who Underwent
All Three Assays Used to Screen for Congenital Cytomegalovirus Infection.
Performance of the saliva assay
Why DBS for CCMV?
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
of HCMV from dried blood spots developed1
1. Atkinson, Emery and Griffiths (2014) J Virol Methods 196:40-44
Evaluation a nested PCR approach
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
1. Williams, Kadambari et al., 2014
Sensitivity of a nested PCR approach
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
Atkinson, Emery and Griffiths (2014) J Virol Methods 196:40-44
Clinical Analysis
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:
one 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
Correlations between neonatal

presentation/outcome and DBS test results
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.
DBS viral load and SNHL
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
(CHIC study)
Correlation between CMV loads in whole blood

and DBS
log10 CMV DNA viral load in

DBS (copies/ml)
R2=0.904
P value = <0.0001
0
3.0
3.5
4.0
4.5
Log10 CMV DNA viral load in

w hole blood (copies/m l)
5.0
5.5
Benefits of a Nested PCR approach

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
Algorithm including DBS screening

Virus detection from urine and/saliva in first
2 weeks of life
Virus detection from DBS

(>2 weeks of life)
Negative
Positive
Uninfected
newborn
Congenitally
Infected newborn
Asymptomatic
Negative
does not exclude congenital CMV
(sensitivity 81% 95%CI 60-93%)
Symptomatic
Including SNHL
Therapy*
No further
testing
adapted from Lazzarotto et al; 2011
Long term follow-up

(neurodevelopmental,ophthalmologic
and audiologic investigations)
*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)
Therapeutic intervention
41
Therapy for congenital CMV disease

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

2. Kadambari et al (2011) early Human development 78:723-28
42
Monitoring during therapy
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
Kadambari et al (2011) early Human development 78:723-28
43
Management algorithm
1
2
Blood tests (FBC, U &E, LFTs); Diagnostic auditory assessment; ophthalmic , CrUSSMRI
Symptomatic focal organ disease

Symptomatic CNS disease
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
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
Web based registry for treated infants at www.ecci.ac.uk
Kadambari et al (2011) early Human development 78:723-28

44
Looking to the future

What impact would screening have?
Impact of vaccination
45
Screening neonates hearing loss

Cannon, 2014
4,248,000
Live births
99.4%
4,222,512
Children born without
congenital CMV infection
0.6%
25,488
Children born with
87.2%
12.8%
25%
815
Symptomatic children
diagnosed clinically with
congenital CMV
670
Hearing loss
at birth
1%
1,245
Hearing loss
at birth
75%
61.4%
1,504
No hearing
loss
5.6%
3,262
Children who are
symptomatic at birth
2,447
Symptomatic children not
congenital CMV
27.4%
3.2%
78
Delayed hearing
loss <9 months
3.2%
22,226
Children who are
asymptomatic at birth
1,067
Delayed hearing
loss 2472 months
1%
222
Delayed hearing
loss <9 months
5.3%
178
Delayed hearing
loss 924 months
4.8%
117
Delayed hearing
loss 2472 months
78
Delayed hearing
loss 924 months
Quality of evidence
Good evidence
Fair evidence
Poor evidence
No benefit
Screening neonates cognitive deficit

Cannon, 2014
4,248,000
Live births
99.4%
4,222,512
Children born without
0.6%
25,488
Children born with
12.8%
3,262
Children who are
symptomatic at birth
87.2%
22,226
Children who are
asymptomatic at birth
95.3%
21,181
Children with no
cognitive deficit
41%
4.7%
1,045
Children with
cognitive deficit
1,337
Children with cognitive
deficit
57.1%
42.9%
Quality of evidence
763
deficit who are
congenital CMV
574
deficit who are not
congenital CMV
Fair evidence
No benefit
Modelling the effects of vaccination on congenital infection
Green
Red
Blue
= congenital infection (seropositive women)

= congenital infection (seropositive women reinfected)
= congenital infection (seronegative women)
Griffiths PD. The Lancet Infectious Diseases, Volume 12, Issue 10, 2012, 790 - 798
Preventing congenital infection:

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

49
gB vaccine reduced infection by 50%
1. Pass et al (2009) N Engl J Med 360:1191-99
Conclusions
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
51
Thank You
University of Washington
Dr Jenn Slyker
Grace John-Stewart
Michael Boeckh
University of Nairobi, Kenya
Dorothy Mbori-Ngacha
James Kiarie
UCL
Claire Atkinson
Paul Griffiths
University of Cape Town

Sheetal Manicklal
University of Bologna
Tiziana Lazzarotto
National
Institutes of
Health
52
Roche Diagnostics Symposium 2014

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Roche Diagnostics Symposium 2014

Current developments and challenges in the diagnosis

Brief summary of congenital CMV infection

Friday, 26 September 2014

Congenital CMV infection

In resource rich settings CMV is:

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

Disability attributable to congenital CMV

Reviews in Medical Virology

Disability attributable to congenital CMV

Reviews in Medical Virology

Causes of Deafness at Birth and at Four Years

Morton C, NEJM 354, 2151, 2006.

Congenital CMV infection

Friday, 26 September 2014

et al (2001) NEJM 344, 1366-1371

Risk of intrauterine transmission:

Friday, 26 September 2014

CMV seroprevalence in women of

Friday, 26 September 2014

Estimates of congenital CMV related disability

England and Wales

Friday, 26 September 2014

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

Congenital CMV in low and high

Friday, 26 September 2014

Resource poor setting:

Annual birth rate

Antenatal HIV prevalence

HIV perinatal transmission

HIV unexposed (risk,1%)

HIV exposed (risk, 3%)

HIV infected (risk, 10%)

Total no of congenital CMV

Friday, 26 September 2014

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

Management during pregnancy

taken from, Lazzarotto et al 2011

Congenital CMV - outcome

n=1000 CCMV cases

Congenital CMV - outcome

n=1000 CCMV cases

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

Diagnosis of congenital CMV (CCMV)

Currently made by CMV positive PCR or culture before day 21 of life

Differentiating congenital CMV from perinatal is important

Could other samples be used?

CMV titer (log10-TCID50/0.2 ml urine)

CMV load in congenital infection

Standard error of the mean

Viral load thresholds and disease

Griffiths PD. The Lancet Infectious

Dried blood spots (DBS)

CCMV: Diagnosis using DBS

DBS taken day 5-8

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

Why diagnose congenital CMV?

To retrospectively diagnose CCMV in asymptomatic infants

Dried Blood Spot testing

1/2 Dried Blood

Nucleic acid extracted

Screening algorithm- retrospective

Screening algorithm for retrospective diagnosis of CCMV in children with SNHL