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Diagnosis and importance of viral respiratory tract infections in children M. Ieven VAKB Leuven 08. 02. 2012.

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Presentation on theme: "Diagnosis and importance of viral respiratory tract infections in children M. Ieven VAKB Leuven 08. 02. 2012."— Presentation transcript:

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

2 1 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 2 Unidentified CPE on tMK cells Sucrose gradient: EM paramyxovirus RNA isolation Random Arbitrarily Primed PCR – cloning sequencing 2001: hMPV

4 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

5 4 2003: HCoV SARS Novel coronavirus identified in SARS patients

6 5 2005: HCoV NL : HCoV HKU1

7 6 2005: Boca virus 2007: rhinovirus C

8 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

9 8 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 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:

11 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

12 11 Flocked swabs or conventional rayon swabs? Daley P. et al J Clin Microbiol. 2006; 44: Mean respiratory epithelial cell yield among volunteers sampled by collecting NPS and NS using flocked or rayon Type of viral infection Total no. of cells/hpfNo. of infected cells/hpf (95% CI) Flocked swabRayon swabFlockes swabRayon swab Influenza A virus (20) ( )7.2 ( ) RSV (21) ( )11.0 ( ) DFA negative (20) Mean of total and infected respiratory cells from NP samples by flocked and rayon swabs

13 12 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 Improved detection of respiratory viruses in children using flocked swabs Munywoki PK et al. J Clin Microbiol 2011; 49: Faden H J Clin Microbiol 2010; 48:

14 13 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 14 Effect of Variables on Detection rates of the Flu A RT-PCR, Isolation, and ELISA Steininger C et al. J Clin Microbiol. 2002; 40: Casiano-Colon et al. J Clin Virol. 2003; 28: 169  Rapid Ag tests useful in young children but in of limited value in adults !  NPA are superior to Nasal or Throat Swabs !

16 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

17 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 1 st 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:

18 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 Sadeghi C et al. BMC Infect Dis 2011: 11: 41 Fuanzalida L et al. Clin Microbiol Infect 2010; 16: 1663

19 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:

20 19 kit targets Species detected Xpert FluA, Cepheid 2Influenza A and subtyping RSV,ASR, Cepheid 2RSVA, RSVB ProPneumo-1, Prodesse 2M. pneumoniae, C. pneumoniae RespiFinder plus, Pathofinder 18IFL 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 19IFL 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 Commercially available Mono- and Multiplex tests

21 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

22 21 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 Impact of molecular diagnostics compared to conventional diagnostics

23 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

24 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; )

25 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

26 25 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 26 Fabbiani M et al. J Med Virol 2009; 81: Epidemiology of viral respiratory tract infections in children 237 patients with ARTI included from to % 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

28 27 Hustedt J et al.Yale J Biol Med 2010; 83: Louie JK et al Pediatr Infect Dis J 2009; 28: 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%

29 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

30 29 Van den Hoogen BG et al. Nat. Med. 2001; 7: Age (Years) > n tested n positive (%) 5 (25) 11 (55) 14 (70) 20 (100) 72 (100) Immunofluorescence assaysVirus neutralization assays n tested n positive (%) 3 (25) 4 (31) 3 (38) 4 (100) 3 (75) 11 (100) Titre range Sero-archeological analysis using sera collected in 1958 Human metapneumovirus Seroprevalence in The Netherlands

31 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:

32 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 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: Esper F et al. J Infect Dis 2004; 189:

33 32 Calvo C et al. Acta pediatrica 2010; 99: 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

34 33 Garcia-Garcia ML et al. Pediatric pulmonology 2010; 45: 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

35 34 Maffey A et al. Pediatric Pulmonology 2010; 45: 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

36 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

37 36 Fairchok MP et al. J Clin Virol 2010; 49: 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

38 37 Brand HK et al. Pediatric Pulmonology 2011, 162: 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

39 38 Brand HK et al. Pediatric Pulmonology 2011, 162: 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

40 39 Importance of infection with multiple viruses? Children < 3mChildren >3m 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 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 Brand HK et al. Pediatric Pulmonology 2011, 162: Suryadevara M et al. Clinical Pediatrics 2011, 50:

41 40 De Vos N et al. Eur J Clin Microbiol Infect Dis 2009; 28: The role of bocavirus infections in a belgian pediatric population N(%) Total mono and co-infections272 (61%) Co-infections53 (19%) AdV/RSV18 hBoV/RSV10 hBoV/AdV7 AdV/hMPV4 All other combinations<=3 404 patients with ARTI included during winter % 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?

42 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 (> 10 6 RNA copies/ml): HRVs may cause severe LRTI At medium-low viral loads (<10 5 RNA copies/ml): may represent only bystander » Utokaparch S et al. Pediatr Infect Dis J 2011; 30: e18-e23 Gerna G et al. J Med Virol 2009; 81: At high viral loads (> RNA cps/ml): HRVs likely to be the cause of presenting LRTI At medium-low viral loads (< RNA copies/ml): may represent only bystander Q PCR: maybe the next necessary step? » Jansen R et al. J Clin Microbiol 2011; 49:

43 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.


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