Diagnosis and importance of viral respiratory tract infections in children

1. Diagnosisand importance of viral respiratory tractinfections in children M. Ieven VAKBLeuven 08. 02. 2012

2. 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? Diagnosis and importance of viral respiratory tract infections in children

3. 2001: hMPV • Unidentified CPE on tMK cells • Sucrose gradient: EM paramyxovirus • RNA isolation • Random Arbitrarily Primed PCR – cloning sequencing

4. 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

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

6. 2005: HCoV NL 63 2005: HCoV HKU1

7. 2005: Boca virus 2007: rhinovirus C

8. The main respiratory targets in molecular diagnostic tests • 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 • Those in our routine panel: • Influenza A (IFVA) • Influenza B (IFVB) • Parainfluenza (PIV) 1-4 • Respiratory syncytial virus (RSV) • Adenoviruses (ADV) • Metapneumovirus (hMPV)

9. 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? Diagnosis and importance of viral respiratory tract infections in children

10. 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

11. 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

12. Mean respiratory epithelial cell yield among volunteers sampled by collecting NPS and NS using flocked or rayon Flocked swabs or conventional rayon swabs? Mean of total and infected respiratory cells from NP samples by flocked and rayon swabs Daley P. et al J Clin Microbiol. 2006; 44: 2265-7.

13. 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 • NFS showed a 96.7% agreement with NPA or 93% sensitivity Munywoki PK et al. J Clin Microbiol 2011; 49: 3365-3367 Faden H J Clin Microbiol 2010; 48: 3742-3743

14. 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? Diagnosis and importance of viral respiratory tract infections in children

15. 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: 2051-56 Casiano-Colon et al. J Clin Virol. 2003; 28: 169

16. 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% Antigen-based Rapid Diagnostic Assays Henrickson K. Ped Infect Dis J 2004; 23:S6-10

17. 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: 100212R1 Ginocchio C et al. J Clin Virol 2009; 45: 146-149 Miernyk K et al. J Clin Virol 2011; 50: 240-243

18. 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

19. Conventional andReal-Time Mono- and Multiplex NAAT Author targets Species detected Fan, J et al. 1998 2 RSVA, RSVB Scheltinga et al. 2005 2 hMPN, RHI McDonough et al. 2005 4 M. pn., C. pn., L. pn., B. pertussis Gunson et al. 2005 12 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. 2007 3 M. pn., C. pn., L. pn Choi et al. 2006 12 in 4 multiplex and one monoreaction Tiveljung et al. 2009 16 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: 259-76

20. Commercially available Mono- and Multiplex tests kit targets Species detected Xpert FluA, Cepheid 2 Influenza A and subtyping RSV,ASR, Cepheid 2 RSVA, RSVB ProPneumo-1, Prodesse 2 M. pneumoniae, C. pneumoniae RespiFinder plus, Pathofinder 18 IFL A/B, PFL 1-4, RHI, hMPN, RSV A/B, AV, 3 coronaviruses, M. pn., C.pn., L.pn., Bordetella pertussis SeeplexRV, Seegene 19 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 19 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

21. 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 PCR based tests: limited target versus multiplex detection

22. 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

23. 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

24. 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)

25. Importance of PCR in the diagnosis of Mycoplasma pneumoniae infections • PCR baseddetection: most sensitive: 87% • Sensitivity of serology: 58% • 7/32 patientsonlydiagnosedbyserology • serologytooinsensitivefor diagnosis of M. pneumoniaeduringearlyphase • Combination of PCR and serologydetects most cases Dekeyser S et al., Pathol Biol 2011; 83-87

26. 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? Impact of molecular methods on the diagnosis of respiratory tract infections

27. Epidemiology of viralrespiratorytractinfections 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

28. The changing face of pediatricrespiratorytractinfections • 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 Hustedt J et al.Yale J Biol Med 2010; 83: 193-200Louie JK et al Pediatr Infect Dis J 2009; 28: 337-339 hMPV 10%

29. - 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

30. Immunofluorescence assays Virus neutralization assays n tested 20 20 20 20 20 20 72 n positive (%) 5 (25) 11 (55) 14 (70) 20 (100) 20 (100) 20 (100) 72 (100) n tested 12 13 8 4 4 4 11 n positive (%) 3 (25) 4 (31) 3 (38) 4 (100) 3 (75) 3 (75) 11 (100) Titre range 16-32 16-32 16-512 32-256 32-128 32-128 16-128 Human metapneumovirus Seroprevalence in The Netherlands Age (Years) 0.5 - 1 1 - 2 2 - 5 5 - 10 10 - 20 > 20 8 - 991 1Sero-archeological analysis using sera collected in 1958 Van den Hoogen BG et al. Nat. Med. 2001; 7:719-724

31. 8% 14% 18% 59% 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% • croup 18% • asthma exacerbation 14% • 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 Dis 2004; 189: 1388-96

32. 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 • Esper F et al. J Infect Dis 2004; 189: 1388-96

33. Epidemiology of viralrespiratorytractinfections in infantswithbronchiolitis Calvo C et al. Acta pediatrica 2010; 99:883-887 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

34. Emergingrespiratoryviruses in childrenwithsevere 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

35. Respiratoryviruses and atypicals in childrenwithasthmaexacerbations • 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% Maffey A et al. Pediatric Pulmonology 2010; 45: 619-625 high prevalence of resp viruses in asthma exacerbations RSV and rhino most prevalent; hMPV also important

36. 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 • Hospitalization rates of HRV positive children with wheezing is similar to that of RSV Peltola V et al. J Med Virology 2009, 81: 1831-1838 Pietrowska Z et al. Ped Infect Dis J 2009, 28: 25-29

37. Epidemiology of viralrespiratorytractinfections 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 Fairchok MP et al. J Clin Virol 2010; 49: 16-20 Rhinovirus, RSV and adenovirus have greatest impact on young children in daycare

38. Clinicalrelevance of infectionwith 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

39. Clinicalrelevance of infectionwith 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

40. Importance of infectionwith 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 febrileRTI Suryadevara M et al. Clinical Pediatrics 2011, 50: 513-51 Brand HK et al. Pediatric Pulmonology 2011, 162: 88-90

41. The role of bocavirusinfections in a belgianpediatricpopulation • 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% • 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

42. 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 Individual patient care: Is viral quantification useful? Utokaparch S et al. Pediatr Infect Dis J 2011; 30:e18-e23 • At high viralloads (> 104.5 RNA cps/ml): HRVslikely to be the cause of presenting LRTI • At medium-lowviralloads (<104.5 RNA copies/ml): mayrepresentonlybystander • Q PCR: maybe the nextnecessary step? Gerna G et al. J Med Virol 2009; 81:1498-1507 JansenR et al. J Clin Microbiol 2011; 49: 2631-36

43. Molecularmethods have contributedsignificantly to anincreasedyield of etiologicagentsdetected in RTI. • NPA ornasopharyngealflockedswabs are the most appropriate specimens. • There is still a rolefor antigen basedmethodsespeciallyfordetection of RSV and hMPV in children. • Serology is of limitedvalue in the acute phase of RTI. • The role of hRV and hMPVbecome more clear in LRTI. Diagnosis and importance of viral respiratory tract infections in children