Diagnosis and importance of viral respiratory tract infections in children

Diagnosis and importance of viral

respiratory tract infections in children

M. Ieven

VAKB Leuven

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

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

2001: hMPV

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

2003: HCoV SARS

Novel coronavirus identified in SARS patients

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 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

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10

What is most appropriate specimen?

Nasopharyngeal aspirates or washes• Specimens of choice for viral

detection• Advantage

- Enough epithelial cells todetect 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, 2007Meerhoff 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

Flocked swabs or conventional rayon swabs?

Daley P. et al J Clin Microbiol. 2006; 44: 2265-7.

0

10

20

30

40

50

60

70

Conventional Swab New Swab

Nasal (N=16)

Nasopharyngeal(N=15)

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/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

Mean of total and infected respiratory cells from NP

samples by flocked and rayon swabs

13

• 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

Improved detection of respiratory viruses in children using flocked swabs

Munywoki PK et al. J Clin Microbiol 2011; 49: 3365-3367

Faden H J Clin Microbiol 2010; 48: 3742-3743

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Effect of Variables on Detection rates of the Flu A RT-PCR, Isolation, and ELISA

0

20

40

60

80

100

0 - 4 5 - 19 20 - 40 > 40

RT-PCR Virus isolation ELISA

Patient age (years)

A

0

20

40

60

80

100

0 - 2 3 - 5 > 5

RT-PCR Virus isolation ELISA

B

Onset-Testing (days)

01020304050

60708090

100

NA n=54 NPS n=65 TS n=56 SP n=41

OIA 14 Day Culture

Per

cen

t o

f P

osi

tive

sp

ecim

ens

Specimen Type

Steininger C et al. J Clin Microbiol. 2002; 40: 2051-56 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

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

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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

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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

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

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Conventional and Real-Time Mono- and Multiplex NAAT

Author targets Species detected

Fan, J et al. 1998 2 RSVA, RSVBScheltinga et al. 2005 2 hMPN, RHIMcDonough et al. 2005 4 M. pn., C. pn., L. pn., B. pertussisGunson 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. pnChoi et al. 2006 12 in 4 multiplex and one monoreactionTiveljung 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

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kit targets Species detected

Xpert FluA, Cepheid 2 Influenza A and subtypingRSV,ASR, Cepheid 2 RSVA, RSVBProPneumo-1, Prodesse 2 M. pneumoniae, C. pneumoniaeRespiFinder 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

Commercially available Mono- and Multiplex tests

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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

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• 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: 167-175Hamano- 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

Impact of molecular diagnostics compared to conventional diagnostics

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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

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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)

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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

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Impact of molecular methods on the diagnosis of

respiratory tract infections

27Fabbiani M et al. J Med Virol 2009; 81: 750-756

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

28

Hustedt J et al.Yale J Biol Med 2010; 83: 193-200Louie JK et al Pediatr Infect Dis J 2009; 28: 337-339

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%

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- 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 2003v.d. Hoogen et al., JID 2003

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Van den Hoogen BG et al. Nat. Med. 2001; 7:719-724

Age (Years)

0.5 - 11 - 22 - 55 - 10

10 - 20> 20

8 - 991

n tested

20202020202072

n positive (%)

5 (25)11 (55)14 (70)20 (100)20 (100)20 (100)72 (100)

Immunofluorescence assays Virus neutralization assays

n tested

12138444

11

n positive (%)

3 (25)4 (31)3 (38)

4 (100)3 (75)3 (75)

11 (100)

Titre range

16-3216-32

16-51232-25632-12832-12816-128

1Sero-archeological analysis using sera collected in 1958

Human metapneumovirus

Seroprevalence in The Netherlands

3131

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

59%18%

14%8%

McAdam AJ et al. J Infect Dis 2004; 190: 20-6

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

3232

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

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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

34

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

35

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

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

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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

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Brand HK et al. Pediatric Pulmonology 2011, 162: 88-90

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

39Brand HK et al. Pediatric Pulmonology 2011, 162: 88-90

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

Importance of infection with multiple viruses?

Children < 3m Children >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 RTIBrand HK et al. Pediatric Pulmonology 2011, 162: 88-90

Suryadevara M et al. Clinical Pediatrics 2011, 50: 513-51

41

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

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

• 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?

42

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

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

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• 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.

Diagnosis and importance of viral respiratory tract infections in children

Documents

infected respiratory

new respiratory virusessingle

main respiratory targets

diagnosis of pediatric

viral detection advantage

flocked swabs

appropriate specimen

flocked npspernasal