0. Diagnosis and importance of viral respiratory tract infections in children
M. Ieven
VAKB Leuven

1. 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?

2. 2001: hMPV Unidentified CPE on tMK cells
Sucrose gradient: EM paramyxovirus
RNA isolation
Random Arbitrarily Primed PCR – cloning sequencing

3. 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

4. Novel coronavirus
identified in SARS patients
2003: HCoV SARS

5. 2005: HCoV NL 63
2005: HCoV HKU1

6. 2005: Boca virus
2007: rhinovirus C

7. The main respiratory targets in molecular diagnostic tests
Those in our routine panel:
Influenza A (IFVA)
Influenza B (IFVB)
Parainfluenza (PIV) 1-4
Respiratory syncytial virus (RSV)
Adenoviruses (ADV)
Metapneumovirus (hMPV)
Those extra assays that many would consider important:
Rhinoviruses
Enteroviruses
Coronaviruses (OC43, 229E, NL63 and HKU1)
IFVA sub-typing
Bocavirus
Atypicals: M.pn., C. pn., Leg. pn., Bordetella pertussis

8. 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?

9. 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:

10. 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

11. Flocked swabs or conventional rayon swabs?
Mean respiratory epithelial cell yield among volunteers sampled by collecting NPS and NS using flocked or rayon
Mean of total and infected respiratory cells from NP samples by flocked and rayon swabs
Type of viral infection
Total no. of cells/hpf
No. of infected cells/hpf
(95% CI)
Flocked swab
Rayon swab
Flockes swab
Influenza A virus (20)
67.2
29.3
15.8 ( )
7.2 ( )
RSV (21)
51.7
19.6
32.6 ( )
11.0 ( )
DFA negative (20)
82.4
24.8
Daley P. et al J Clin Microbiol. 2006; 44:

12. 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=
NFS was non inferior to NW for detection of RSV by IF
NFS showed a 96.7% agreement with NPA or 93% sensitivity
Munywoki PK et al. J Clin Microbiol 2011; 49:
Faden H J Clin Microbiol 2010; 48:

13. 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?

14. Effect of Variables on Detection rates of the Flu A RT-PCR, Isolation, and ELISA
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:
Casiano-Colon et al. J Clin Virol. 2003; 28: 169

15. 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

16. 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
3M A+B: superior results compared to BinaxNOW; effective 1st line triage
BinaxNOW RSV is highly sensitive in children with bronchiolitis, but sens is low in non-bronchiolitis illness: 89% vs 38%
De Witte E et al. Eur J Clin Microbiol Infect Dis 2011: R1
Ginocchio C et al. J Clin Virol 2009; 45:
Miernyk K et al. J Clin Virol 2011; 50:

17. 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 < )
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

18. Conventional and Real-Time Mono- and Multiplex NAAT
Author targets Species detected
Fan, J et al RSVA, RSVB
Scheltinga et al hMPN, RHI
McDonough et al M. pn., C. pn., L. pn., B. pertussis
Gunson et al IFL A and B, PFL 1,2,3 RHI, hMPN
RSVA and B, COR E229, OC 43,
NL63 in 4 triplex reactions
Loens et al M. pn., C. pn., L. pn
Choi et al in 4 multiplex and one monoreaction
Tiveljung et al 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
Ieven M, J Clin Virol 2007; 40:

19. Commercially available
Mono- and Multiplex tests
kit targets Species detected
Xpert FluA, Cepheid Influenza A and subtyping
RSV,ASR, Cepheid RSVA, RSVB
ProPneumo-1, Prodesse M. pneumoniae, C. pneumoniae
RespiFinder plus, Pathofinder IFL A/B, PFL 1-4, RHI, hMPN,
RSV A/B, AV, 3 coronaviruses, M. pn., C.pn., L.pn., Bordetella pertussis
SeeplexRV, Seegene 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
xTAG RVP, Luminex IFL A ( H1, H3, H5, non-specific ) and B, PFL 1-4, RSV A/B, ADE, hMPV, RHI/ENT,SARS-COR, HCoV OC43, HCoV E, HCoV NL63 and HKU1

20. 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
Influenza, Legionella spp, Mycoplasma pn., B. pertussis
Outside normal lab working hrs
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
In immunocompromised
For virus discovery studies

21. 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:
Hamano- Hasegawa K et al, J Infect Chemother 2008; 14:
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:
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

22. 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

23. 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; )

24. 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

25. 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?

26. Epidemiology of viral respiratory tract infections in children
237 patients with ARTI included from to
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:

27. 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 and hBoV joined the list if significant contributors
hMPV 10%
Hustedt J et al.Yale J Biol Med 2010; 83: Louie JK et al Pediatr Infect Dis J 2009; 28:

28. - 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

29. Human metapneumovirus
Seroprevalence in The Netherlands
n tested 20 72
n positive (%)
5 (25)
11 (55)
14 (70)
20 (100)
72 (100)
Immunofluorescence assays
Virus neutralization assays
n tested 12 13 8 4 11
n positive (%)
3 (25)
4 (31)
3 (38)
4 (100)
3 (75)
11 (100)
Titre range
16-32
16-512
32-256
32-128
16-128
Age (Years)
1 - 2
2 - 5
5 - 10
> 20
1Sero-archeological analysis using sera collected in 1958
Van den Hoogen BG et al. Nat. Med. 2001; 7:

30. Clinical picture of hMPV infections
The mean age of children infected 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%
croup 18%
asthma exacerbation 14%
Associated diseases:
conjunctivitis, otitis media
febrile seizures
diarrhoea, rash
59%
18%
14% 8%
McAdam AJ et al. J Infect Dis 2004; 190: 20-6
Esper F et al. J Infect Dis 2004; 189:

31. 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
Although it appears that hMPV is the cause of respiratory tract infections less often than RSV, it most often affects both young and older populations, as well as those who are immunocompromised.
Pelletier et al. found that over a single winter season ( ), hMPV was isolated from respiratory specimens of all age groups combined about half as often as RSV.
They also observed that children less than 5 years of age and elderly persons (>65 yrs) represented 35.1% and 45.9% of the hMPV-infected cases, respectively.
Boivin et al. noted that infections due to hMPV tended to peak later than RSV (March and January, respectively).
Falsey et al. examined the clinical spectrum of infection due to hMPV in adults. Young adults tended to have asymptomatic infection or mild illness limited to nasal congestion, sore throat, hoarsenss, and cough. In contrast, elderly patients experienced more serious illness similar to RSV, such as wheezing and dypsnea, sometimes requiring hospitalization.
Similar to RSV, hMPV can provoke severe infections in both children and the elderly [Osterhaus].
At least one study suggests that in young children, hMPV is less likely than RSV to cause severe infection [Boivin]. No hMPV-infected children were admitted to an ICU compared with 15% of those with RSV.
Pelletier G, et al 42nd ICAAC Abstract V-476 Falsey AR, et al need citation pdf Osterhaus A, et al. Lancet 2003;361:890-1 Boivin G, et al. Emerg Infect Dis 2003;9:634-40
Osterhaus A, et al. Lancet. 2003; 361:
Boivin G, et al. Emerg Infect Dis. 2003;9:
Greensill J, et al. Emerg Infect Dis. 2003; 9:
McAdam AJ et al. J Infect Dis 2004; 190: 20-26
Esper F et al. J Infect Dis 2004; 189:

32. Calvo C et al. Acta pediatrica 2010; 99:883-887
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:

33. Garcia-Garcia ML et al. Pediatric pulmonology 2010; 45: 585-91
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:

34. Maffey A et al. Pediatric Pulmonology 2010; 45: 619-625
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:

35. The role of rhinovirus infections in children
Retrospective study of 580 children during
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
Hospitalization rates of HRV positive children with wheezing is similar to that of RSV
Peltola V et al. J Med Virology 2009, 81:
Pietrowska Z et al. Ped Infect Dis J 2009, 28: 25-29

36. Fairchok MP et al. J Clin Virol 2010; 49: 16-20
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

37. 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

38. 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

39. 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%
Infection with 2 or more viruses: more frequent in children with mild or moderate disease than in those with severe disease
Children < 3m
Children >3m
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:
Brand HK et al. Pediatric Pulmonology 2011, 162: 88-90

40. De Vos N et al. Eur J Clin Microbiol Infect Dis 2009; 28: 1305-1310
The role of bocavirus infections in a belgian pediatric population
404 patients with ARTI included during winter
61% positive for 1 or more viruses
bocaviruses: 11%
Adenovirus: 13%
More co-infections with AdV: 62% compared to hBoV: 49%
Causal role for hBoV in RTI is still a topic for debate: Q-PCR?
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
De Vos N et al. Eur J Clin Microbiol Infect Dis 2009; 28:

41. 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
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
Utokaparch S et al. Pediatr Infect Dis J 2011; 30: e18-e23
At high viral loads (> 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?
Gerna G et al. J Med Virol 2009; 81:
Jansen R et al. J Clin Microbiol 2011; 49:

42. 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.