Diagnosis and importance of viral
respiratory tract infections in children
M. Ieven
VAKB Leuven
08. 02. 2012
Diagnosis and importance of viral
respiratory tract infections in children
• Which viruses can we detect?
• What is the appropriate specimen?
• Diagnosis of pediatric viral respiratory infections
- Do antigen tests still have a role in diagnosis of pediatric RTI ?
- Molecular based tests
- Does serology have an additional value?
• Epidemiology of pediatric viral respiratory infections
-
Prevalence of known and new respiratory viruses
Single versus co-infections
Role of respiratory viruses: pathogens or colonizers?
Quantitative testing to predict severity?
1
•
•
•
•
2001: hMPV
Unidentified CPE on tMK cells
Sucrose gradient: EM paramyxovirus
RNA isolation
Random Arbitrarily Primed PCR – cloning sequencing
2
Human coronaviruses:
common cold viruses
• (+) RNA genome
• Discovered early 60’s after inoculation of material of human common
cold on human embryonic trachea cultures
• Multiply very slowly and poorly in human kidney cultures and some
cell lines
• For years only OC43, 229E known: molecular techniques
discovered more
3
Novel coronavirus
identified in SARS patients
2003: HCoV SARS
4
2005: HCoV NL 63
2005: HCoV HKU1
5
2005: Boca virus
2007: rhinovirus C
6
The main respiratory targets in
molecular diagnostic tests
• Those in our routine
• Those extra assays that many
panel:
would consider important:
- Influenza A (IFVA)
- Rhinoviruses
- Influenza B (IFVB)
- Enteroviruses
- Parainfluenza
- Coronaviruses (OC43,
(PIV) 1-4
229E, NL63 and HKU1)
- Respiratory syncytial
- IFVA sub-typing
virus (RSV)
- Bocavirus
- Adenoviruses (ADV)
- Atypicals: M.pn., C. pn., Leg.
- Metapneumovirus
pn., Bordetella pertussis
(hMPV)
7
Diagnosis and importance of viral
respiratory tract infections in children
• Which viruses can we detect?
• What is the appropriate specimen?
• Diagnosis of pediatric viral respiratory infections
- Do antigen tests still have a role in diagnosis of pediatric RTI ?
- Molecular based tests
- Does serology have an additional value?
• Epidemiology of pediatric viral respiratory infections
-
Prevalence of known and new respiratory viruses
Single versus co-infections
Role of respiratory viruses: pathogens or colonizers?
Quantitative testing to predict severity?
8
What is most appropriate specimen?
Nasopharyngeal aspirates or washes
•
Specimens of choice for viral
detection
•
Advantage
- Enough epithelial cells to
detect respiratory viruses
•
Disadvantage
– Hard on the patients?
– Require suction device
mucus extractor
– Impractical to have in a
doctor office setting
RSV for NPA sample
Mucus Extractor
Hindiyeh M et al, 2007
Meerhoff TJ et al Eur J Clin Microbiol Infect Dis 2010; 29: 365-71
9
Sampling method: Flocked NPS
Pernasal Flocked Swab
The fibers have an hydrophilic action
•
•
Soft brush for improved
epithelial cells collection
Less traumatic on the
patient
10
Flocked swabs or conventional rayon
swabs?
70
50
Mean respiratory epithelial cell yield
among volunteers sampled by collecting
NPS and NS using flocked or rayon
40
Nasal (N=16)
30
Nasopharyngeal
(N=15)
60
20
10
Mean of total and infected respiratory cells
from NP samples by flocked and rayon swabs
0
Conventional Swab
Type of viral
infection
New Swab
Total no. of cells/hpf
No. of infected cells/hpf
(95% CI)
Flocked swab
Rayon swab
Flockes swab
Rayon swab
Influenza A virus
(20)
67.2
29.3
15.8 (9.7-21.9)
7.2 (3.6-10.8)
RSV (21)
51.7
19.6
32.6 (18.7-46.7)
11.0 (6.1-15.9)
DFA negative (20)
82.4
24.8
0
0
Daley P. et al J Clin Microbiol. 2006; 44: 2265-7.
11
Improved detection of respiratory
viruses in children using flocked swabs
• Nasopharyngeal flocked swabs (NFS) and nasal washing
(NW) compared for detection of respiratory viruses by Mx
PCR and RSV by IF
• NFS was superior to NW for detection of viruses by Mx-PCR
- Sens 89.6% vs 79.2% P= 0.0043
• NFS was non inferior to NW for detection of RSV by IF
Munywoki PK et al. J Clin Microbiol 2011; 49: 3365-3367
• NFS showed a 96.7% agreement with NPA or 93% sensitivity
Faden H J Clin Microbiol 2010; 48: 3742-3743
12
Diagnosis and importance of viral
respiratory tract infections in children
• Which viruses can we detect?
• What is the appropriate specimen?
• Diagnosis of pediatric viral respiratory infections
- Do antigen tests still have a role in diagnosis of pediatric RTI ?
- Molecular based tests
- Does serology have an additional value?
• Epidemiology of pediatric viral respiratory infections
-
Prevalence of known and new respiratory viruses
Single versus co-infections
Role of respiratory viruses: pathogens or colonizers?
Quantitative testing to predict severity?
13
Effect of Variables on Detection rates
of the Flu A RT-PCR, Isolation, and
ELISA
A
OIA
B
Virus isolation
ELISA
100
Percent of Positive specimens
RT-PCR
100
80
80
60
60
40
40
20
20
0
0
0-4
5 - 19
20 - 40
Patient age (years)
> 40
100
90
80
70
60
50
40
30
20
10
0
RT-PCR
0-2
Virus isolation
3-5
14 Day Culture
ELISA
>5
NA n=54
NPS n=65
TS n=56
Onset-Testing (days)
Specimen Type
SP n=41
 Rapid Ag tests useful in young children but in of
limited value in adults !
 NPA are superior to Nasal or Throat Swabs !
Steininger C et al. J Clin Microbiol. 2002; 40: 2051-56
Casiano-Colon et al. J Clin Virol. 2003; 28: 169
14
Antigen-based Rapid
Diagnostic Assays
• Usually less sensitive than
other methods
- Median sensitivity: e.g.
Zstat Flu 69%
- Directigen Flu A + B 87%,
Flu OIA 72%, RSV OIA
88%
- Quick Vue Flu 79%,
Testpack RSV 70%
• Sensitivity depends on
specimen type
• Individual or pooled
monoclonal antibodies: Flu
A/B, PU 1-3, RSV, adenovirus
• Sensitivity generally higher
than other rapid tests
- Flu A
: 40-90%
- Flu A+B : 60-90%
- RSV
: 94%
- PIV
: 70-80%
- Adenovirus: 22-67%
Henrickson K. Ped Infect Dis J 2004; 23:S6-10
15
New Antigen-based Rapid
Diagnostic Assays
• The ESPLINE Influenza A & B-N is a user friendly, rapid direct antigen
assay with a very good performance: sens 93% and spec 97%
• The test is less sensitive to detect H1N1 compared to seasonal flu.
• Due to its simplicity it facilitates urgent testing
De Witte E et al. Eur J Clin Microbiol Infect Dis 2011: 100212R1
• 3M A+B: superior results compared to BinaxNOW; effective 1st line triage
Ginocchio C et al. J Clin Virol 2009; 45: 146-149
• BinaxNOW RSV is highly sensitive in children with bronchiolitis, but sens
is low in non-bronchiolitis illness: 89% vs 38%
Miernyk K et al. J Clin Virol 2011; 50: 240-243
16
New Antigen-based
Rapid Diagnostic Assays
• Addition of assays for detection of picornaviruses and hMPV increased
the diagnostic yield by DFA from 35% to 58% (P < 0.0001)
• DFA, or EIA, is even in the “PCR era” a valid, rapid, flexible and cheap
method for detection of respiratory viruses in a pediatric population
Fuanzalida L et al. Clin Microbiol Infect 2010; 16: 1663
Sadeghi C et al. BMC Infect Dis 2011: 11: 41
17
Conventional and Real-Time
Mono- and Multiplex NAAT
Author
targets
Fan, J et al. 1998
Scheltinga et al. 2005
McDonough et al. 2005
Gunson et al. 2005
2
2
4
12
Loens et al. 2007
Choi et al. 2006
Tiveljung et al. 2009
3
12
16
Ieven M, J Clin Virol 2007; 40: 259-76
Species detected
RSVA, RSVB
hMPN, RHI
M. pn., C. pn., L. pn., B. pertussis
IFL A and B, PFL 1,2,3 RHI, hMPN
RSVA and B, COR E229, OC 43,
NL63 in 4 triplex reactions
M. pn., C. pn., L. pn
in 4 multiplex and one monoreaction
in 13 reactions: IFL A and B, RSV A+B,
PFL 1+3, PFL 2+ hCoV-229E, ADE, hMPV, RHI,
ENT, HCoV-OC43, HCoV-NL63 and HKU, HBoV
18
Commercially available
Mono- and Multiplex tests
kit
targets
Xpert FluA, Cepheid
2
RSV,ASR, Cepheid
2
ProPneumo-1, Prodesse
2
RespiFinder plus, Pathofinder 18
SeeplexRV, Seegene
19
xTAG RVP, Luminex
19
Species detected
Influenza A and subtyping
RSVA, RSVB
M. pneumoniae, C. pneumoniae
IFL A/B, PFL 1-4, RHI, hMPN,
RSV A/B, AV, 3 coronaviruses,
M. pn., C.pn., L.pn., Bordetella pertussis
S. pneumoniae, H. influenzae, M. pn., C.pn.,
L.pn., IFL A and B, RSV A/B, PFL 1-3,
RHI, 3 coronaviruses, AV, HBoV, EV
IFL A ( H1, H3, H5, non-specific ) and B,
PFL 1-4, RSV A/B, ADE, hMPV,
RHI/ENT,SARS-COR, HCoV OC43, HCoV
229E, HCoV NL63 and HKU1
19
PCR based tests: limited target
versus multiplex detection
Limited target detection
• Usually analytical sens.
• Lower cost
• Often lower TAT
• In outbreak situations
- Influenza, H1N1
- RSV, L. pn, M.pn
• As first approach
- in high prevalence periods
- if therapeutic implications
Multiplex detection
• In >90% similar results
• Expensive
• TAT usually > 4-6hours
• For epidemiological studies
- Prevalence of respiratory
etiologies
- Role of respiratory viruses
• As add-on diagnostic test
- In severely ill patients
• Influenza, Legionella spp,
- In immunocompromised
Mycoplasma pn., B. pertussis
• For virus discovery studies
• Outside normal lab working hrs
20
Impact of molecular diagnostics
compared to conventional diagnostics
• Increase in diagnostic yield from 37% to 57%, or even > 75%
- Main improvement: previously not detected viruses
Tiveljung-Lindell A et al. J Med Virol 2009; 81: 167-175
Hamano- Hasegawa K et al, J Infect Chemother 2008; 14: 427-432
•
in diagnostic yield from 24% to 43% or even > 66% in children,
from 3.5% to 36% in adults
Van de Pol et al. J Clin Microbiol 2007;45: 2360-6
Gharabaghi F et al Clin Microbiol Infect 2011;
• Acute RTI in elderly and children: up to 40%:
mostly rhino, RSV, hMPV, and influenza
Renwick et al 2007, Regamey et al 2008 Jartti et al 2008,
Caram et al 2009, Jin et al 2009
Significant increase in diagnostic yield
21
Serology for the Diagnosis of
Viral RTI ?
• Rarely helpful in rapid diagnosis of acute infection:
- IgG: only 4 fold rise between acute and late phase serum
specimens are informative:
- Single high IgG denotes past infection
- IgM may appear late or not at all: 10 to 50% of patients with
documented infections remain serologically negative
• Useful in epidemiologic studies
• Useful in vaccine studies
22
Serodiagnosis of Human
Bocavirus Infection
• Paired serum samples from children with wheezing, previously
tested for 16 resp viruses
• Immunoblot assays using 2 recombinant HBoV antigens
• Results:
- 24/49 (49%) of PCR + had IgM antibodies
- 36/49 (73%) of PCR + had IgG antibodies
- 29/49 (59%) of PCR + had IgM + in IgG antibody level:
• 91% of
in IgG antibody level: high load of HBoV DNA: acute infection
 Serology on acute phase sample: too insensitive
 Serologic testing correlates with high viral loads and viremia
 Max diagnostic accuracy, both qPCR and serological testing
Kantola K et al., Clin Infect Dis 2008; 46 540 - 46)
23
Importance of PCR in the diagnosis of
Mycoplasma pneumoniae infections
• PCR based detection: most sensitive: 87%
• Sensitivity of serology: 58%
• 7/32 patients only diagnosed by serology
• serology too insensitive for diagnosis of M. pneumoniae
during early phase
• Combination of PCR and serology detects most cases
Dekeyser S et al., Pathol Biol 2011; 83-87
24
Impact of molecular methods on the
diagnosis of respiratory tract infections
• Which viruses can we detect?
• What is the appropriate specimen?
• Diagnosis of pediatric viral respiratory infections
- Do antigen tests still have a role in diagnosis of pediatric RTI ?
- Molecular based tests
- Does serology have an additional value?
• Epidemiology of pediatric viral respiratory infections
-
Prevalence of known and new respiratory viruses
Single versus co-infections
Role of respiratory viruses: pathogens or colonizers?
Quantitative testing to predict severity?
25
Epidemiology of viral respiratory tract
infections in children
• 237 patients with ARTI included from 10.2006 to 04.2007
• 52.3% positive for 1 or more viruses (12%), more in hospitalized
• Picornaviruses: 43.6%
• RSV: 24.3%, leading to hospitalisation in 85.3% of cases
• More co-infections with hMPV: 55.6% compared to RSV: 11.8% or PIC
• PIC: most frequently involved in co-infections; not related to severity
Fabbiani M et al. J Med Virol 2009; 81: 750-756
26
The changing face of pediatric
respiratory tract infections
Viral RTI in children <1yr
• RSV remains important cause
of LRTI
• hRV and hCoV: not only in
UTRI but also in LRTI
hMPV 10%
• hMPV and hBoV joined the list
if significant contributors
Hustedt J et al.Yale J Biol Med 2010; 83: 193-200
Louie JK et al Pediatr Infect Dis J 2009; 28: 337-339
27
- Most prevalent in (young) children
~ 10 % of children with RTI
- Immunocompromised individuals (fatal cases!)
- Elderly
- Normal individuals
> 2-3 % of RTI in community surveillance studies
Osterhaus and Fouchier, The Lancet 2003
v.d. Hoogen et al., JID 2003
28
Human metapneumovirus
Seroprevalence in The Netherlands
Age (Years)
Immunofluorescence assays
n tested n positive (%)
0.5 - 1
1-2
2-5
5 - 10
10 - 20
> 20
8 - 991
1Sero-archeological
20
20
20
20
20
20
72
5 (25)
11 (55)
14 (70)
20 (100)
20 (100)
20 (100)
72 (100)
Virus neutralization assays
n tested n positive (%) Titre range
12
13
8
4
4
4
11
3 (25)
4 (31)
3 (38)
4 (100)
3 (75)
3 (75)
11 (100)
16-32
16-32
16-512
32-256
32-128
32-128
16-128
analysis using sera collected in 1958
Van den Hoogen BG et al. Nat. Med. 2001; 7:719-724
29
Clinical picture of hMPV infections
•
•
•
•
•
•
The mean age of children infected - 11.6 mo
Up to 12% of all LRTI
Most children have a mild upper URTI
Resembling RSV, slightly milder
Preterm infants may be more susceptible.
Reports have described
- bronchiolitis 59%
- pneumonia 8%
8%
14%
- croup 18%
- asthma exacerbation 14%
18%
59%
• Associated diseases:
- conjunctivitis, otitis media
- febrile seizures
- diarrhoea, rash
McAdam AJ et al. J Infect Dis 2004; 190: 20-6
Esper F et al. J Infect
1388-96
30 Dis 2004; 189:30
hMPV resembling RSV:
Similar but Different?
•
•
•
RSV: more common than hMPV in infants <6 mo.
hMPV similar to RSV, majority of hMPV cases occur in young (<5yrs)
Seasonality with RSV: hMPV later
•
co-infection with RSV: More severe?
Contradictory
In 1 study, hMPV/RSV coinfection in 70%
Disease severity and hospitalization appears more common with RSV
Osterhaus A, et al. Lancet. 2003; 361:890-891
Boivin G, et al. Emerg Infect Dis. 2003;9:634-640
Greensill J, et al. Emerg Infect Dis. 2003; 9: 372-375.
McAdam AJ et al. J Infect Dis 2004; 190: 20-26
31 Dis 2004; 189: 1388-96
31
Esper F et al. J Infect
Epidemiology of viral respiratory tract
infections in infants with bronchiolitis
In hospitalized infants, RSV is the most frequent agent in
bronchiolitis in winter, but other viruses may play a significant
role wit RV, hBoV and MPV as most significant ones;
Clinical characteristics are similar
Calvo C et al. Acta pediatrica 2010; 99:883-887
32
Emerging respiratory viruses in
children with severe acute wheezing
• viruses detected in
71% of acute
wheezing episodes
• RSV most commonly
detected virus: 27%
• Rhinovirus in 24%
• Adenovirus 18%
• Rate of viral detection
in infants (77%) than in
older children (60%)
• RSV and rhino most
prevalent in wheezing;
emerging viruses hBoV
and hMPV also important
Garcia-Garcia ML et al. Pediatric pulmonology 2010; 45: 585-91
33
Respiratory viruses and atypicals in
children with asthma exacerbations
• Potential causative
agent detected in 78%
of patients
• More in young children
• RSV most commonly
detected : 40%
• Rhinovirus in 25%
• M.pneumoniae: 4.5%
• C.pneumoniae: 2%
high prevalence of resp viruses in asthma exacerbations
RSV and rhino most prevalent; hMPV also important
Maffey A et al. Pediatric Pulmonology 2010; 45: 619-625
34
The role of rhinovirus infections in
children
• Retrospective study of 580 children during 1987-2006
- Median age: 1.9 years, 27% underlying medical condition
- 16% of in patients treated in pediatric ICU
• Prospective study including all children > 1 month
- Rhinovirus detected in 28% of 163 hospital episodes
- Acute wheezing in 61% children with RV and in 31% with RSV
- 50% of RV strains belonged to newly identified group C
• Group C RV accounts for a large part of RV hospitalizations
• Acute wheezing: most frequent manifestation in hospital setting
Peltola V et al. J Med Virology 2009, 81: 1831-1838
• Hospitalization rates of HRV positive children with wheezing is
similar to that of RSV
Pietrowska Z et al. Ped Infect Dis J 2009, 28: 25-29
35
Epidemiology of viral respiratory tract
infections in children in daycare
Viral RTI in children <30
months in fulltime daycare
• At least 1 virus detected in
67% of RTI : 2x more than
previously reported
• hRV most important
• Co-infections common: 27%
• Severity of illness not worse
Rhinovirus, RSV and adenovirus have greatest impact
on young children in daycare
Fairchok MP et al. J Clin Virol 2010; 49: 16-20
36
Clinical relevance of infection with
multiple viruses?
• children < 2yrs old with bronchiolitis:
• Mild: no supportive treatment
• Moderate: supplemental oxygen and/or nasogastric feeding
• Severe: mechanical ventilation
• Mx PCR for 15 viruses on NPA
• Results: overall 211 viruses detected in 142 NPA
• RSV most commonly detected virus: 73%
• Rhinovirus in 30%
• Other respiratory viruses in < 10% of samples
Brand HK et al. Pediatric Pulmonology 2011, 162: 88-90
37
Clinical relevance of infection with
multiple viruses?
• RSV detected as a single virus infection in 59% of positives followed
by hMPV as single infection in 56% of hMPV positives
• Other viruses less frequently detected as single virus infections
• hBoV, PeV and AdV: only detected in combination with other viruses
Brand HK et al. Pediatric Pulmonology 2011, 162: 88-90
38
Importance of infection with multiple
viruses?
• Children younger than 3
months: less often infected
by multiple viruses
compared to children older
than 3 months: 25% vs 65%
Children < 3m
Children >3m
• Infection with 2 or more
viruses: more frequent in
children with mild or
moderate disease than in
those with severe disease
The detection of more than one virus is not associated with increased
disease severity in children with bronchiolitis
Co- infections not associated with illness severity in acute febrile RTI
Suryadevara M et al. Clinical Pediatrics 2011, 50: 513-51
Brand HK et al. Pediatric Pulmonology 2011, 162: 88-90
39
The role of bocavirus infections in a
belgian pediatric population
• 404 patients with ARTI included during winter 2004 - 2005
• 61% positive for 1 or more viruses
• bocaviruses: 11%
• Adenovirus: 13%
• More co-infections with AdV: 62% compared to hBoV: 49%
N(%)
Total mono and co-infections
272 (61%)
Co-infections
53 (19%)
AdV/RSV
18
hBoV/RSV
10
hBoV/AdV
7
AdV/hMPV
4
All other combinations
<=3
• Causal role for hBoV in RTI is still a topic for debate: Q-PCR?
De Vos N et al. Eur J Clin Microbiol Infect Dis 2009; 28: 1305-1310
40
Individual patient care: Is viral
quantification useful?
In case of PIV, rhinovirus
• Total viral load is related to clinical diagnosis in children presenting at emergency
room
Utokaparch S et al. Pediatr Infect Dis J 2011; 30: e18-e23
In case of rhinovirus
• At high viral loads (> 106 RNA copies/ml): HRVs may cause severe LRTI
• At medium-low viral loads (<105 RNA copies/ml): may represent only bystander
Gerna G et al. J Med Virol 2009; 81:1498-1507
•
•
»
At high viral loads (> 104.5 RNA cps/ml):
HRVs likely to be the cause of presenting
LRTI
At medium-low viral loads (<104.5 RNA
copies/ml): may represent only bystander
• Q PCR: maybe the next necessary
step?
Jansen R et al. J Clin Microbiol 2011; 49: 2631-36
41
Diagnosis and importance of viral
respiratory tract infections in children
• Molecular methods have contributed significantly to an
increased yield of etiologic agents detected in RTI.
• NPA or nasopharyngeal flocked swabs are the most
appropriate specimens.
• There is still a role for antigen based methods especially
for detection of RSV and hMPV in children.
• Serology is of limited value in the acute phase of RTI.
• The role of hRV and hMPV become more clear in LRTI.
42