New viruses causing respiratory tract infections Eric C.J. Claas
(Re) emerging infectious diseases: what is new? Morens et al. Nature 2004
Virus discovery New molecular methods result in frequent detection of new viruses: Re-emerging viruses New introduction in the human population (e.g. zoonoses) Present but previously undetected
Influenza A/H5N1 infections, since 1997
H5N1 does not spread efficiently among humans:reassortment required? 1918 H1N1 1957 H2N2 1968 H3N2 PB1, HA, and NA PB1 and HA hemagglutinin neuraminidase
Highly pathogenic Influenza A\H5N1 Widespread in birds Sporadic human infections with high mortality Unable to efficiently spread among humans Diagnosis by real-time PCR Oseltamivir (Tamiflu)
Common causes of CAP in otherwise healthy children Streptococcus pneumoniae Haemophilus influenzae Mycoplasma pneumoniae Chlamydophila pneumoniae Viruses RSV Influenza A and B Parainfluenza viruses 1, 2 and 3 Adenoviruses Rhinoviruses McIntosh K. N Engl J Med 2002; 346: 429-37
Human metapneumovirus (2001)
Procedure Unidentified CPE on tmk cells Sucrose gradient: EM paramyxovirus RNA isolation Random Arbitrarily Primed PCR cloning sequencing APV-like: hmpv
Very common
Human metapneumovirus: clinical symptoms Upper and lower respiratory infections Resembling RSV Seasonality with RSV Frequently co-infection with RSV: More severe or not? Contradictory More prevalent in young children < 1-3 years
Seasonality of hmpv, RSV and influenza McAdam AJ et al. J Infect Dis 2004; 190: 20-6
Age distribution of hmpv positive children Esper F et al. J Infect Dis 2004; 189: 1388-96
hmpv is included in routine diagnostic virology: real-time PCR
Human coronaviruses: common cold viruses (+) RNA genome, 30 Kb Discovered early 60 s after inoculation of material of human common cold on human embryoric 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
Severe Acute Respiratory Syndrome SARS 2003: Infection with a coronavirus causing acute interstitial pneumonia with a 10% mortality
SARS Outbreak 2002 2003 2003 Nov. 16 Feb. 14 Feb. 28 Mar. 12 July 4 months Atypical pneumonia in China Hong Kong hotel WHO alerted Global alert Human chain of transmission broken
SARS: November 2002 - July 2003 8096 cases and 774 deaths
Human coronavirus NL63 (2004)
Vidisca: Virus Discovery cdna AFLP v.d.hoek et al., Nat. Med. 2004
HCOV-NL63: clinical relevance
Other new coronaviruses: HKU1
Others start looking as well
Association with clinical symptoms
Only detection of new RNA viruses?
Identification of human bocavirus (HBoV) Enrichment of viruses from pooled NPAs Ultracentrifugation (41.000 rpm) Host DNA depletion Microfiltration (0.22- μm spin filter) DNase treatment Extraction nucleic acids DNA (QIAamp Blood Mini Kit); RNA (TRIzol LS reagent) Random DNA & RNA (cdna) PCR amplification Generic primer sequence with a random 3 end
HBoV Pool 1: 28 supernatants 192 RNA clones 288 DNA clones Pool 2: 20 supernatans 192 RNA clones 192 DNA clones Uncharaterized viruses TT-virus-like sequences (not analyzed further; ubiquitous, heterogeneous) 1 coronavirus (later coronavirus HKU1) 1 parvo-like-virus
HBoV ~ 43% AA homology MVC & BPV Human Bocavirus (HBoV) Diagnostic PCR
Diagnostic PCR
HBoV clinical characteristics Common!
Implementation as diagnostic assay? Prevalence studies Establish the role of newly identified virus in etiology of disease Decide on implementation Interpretation of results
Respiratory Multiplex RNA virus assay as rotuine diagnostic assay VIRUS Influenza A virus PROBE LABEL Fam BHQ1 Influenza B virus Texas Red BHQ2 RSV HEX BHQ1 PIV1 HEX BHQ1 PIV2 Texas Red BHQ2 PIV3 Fam BHQ1 PIV4 Cy-5 BHQ2 (3) Rhinovirus FAM BHQ1 Human metapneumovirus HEX BHQ1 Equine Artheritis Virus Cy5 BHQ2 HCOV 229E FAM BHQ1 HCOV OC43 HEX BHQ1 HCOV NL63 Texas Red BHQ2
Positive results by patients: n=549 Rhinovirus Respiratory syncytial virus Influenza virus A Adenovirus Influenza virus B Metapneumovirus Coronavirus OC43 Parainfluenzavirus 4 Coronavirus NL63 Parainfluenzavirus 1 Coronavirus 229E Parainfluenzavirus 3 Parainfluenzavirus 2 0 20 40 60 80 100 120 140 160
HRV and HCOV positives v. Gageldonk-Lafeber et al. CID 2005:41; 493
Current diagnostic strategies
New (knowledge on) viruses: common cold or.
Importance of rhinoviruses in children Rhinoviruses are the most prevalent pathogens during URTI and rhinitis in 2 year old infants (+/-40%) Van Benten et al. Ped Allergy Immunol. 2003; 14: 363 In the first 9 months, picornaviruses (rhino) were most common pathogens detected: 51% of all episodes, 58% in bronchiolitis Legg JP et al. Ped Infect Dis J. 2005; 24: 611-13 Viral infections in 65% in school age children hospitalised with CAP: rhinovirus was detected in 45% of patients Tsolia MN et al Clin Infect Dis 2004;39:681 Common cause of bronchitis (25 %), acute attacks of asthma (6 %) and pneumonia in children (2->10 %) Guittet V et al. Arch Pediatr. 2003; 10: 417-23 Papadopoulos N. Ped Resp Rev. 2004; 5: S191-95
From conventional to molecular diagnostics Conventional Methods Real-time PCR No infection No infection Single Infection Single Infection Mixed infection Mixed infection In over 90% of CAP cases with PSI IV and V an etilogical agent was detected Templeton et al., CID 2005; 41: 345
Routine Diagnostics Increased number of diagnoses by molecular testing Viruses do matter in LRTI A significant numer of LRTI cases remain without a known etiology: undiscovered pathogens That is where WP5 of GRACE comes in
Thanks Greet Ieven Lia van der Hoek Willy Spaan You for your attention