Identifying Respiratory Viral Pathogens

Transcription

1 Identifying Respiratory Viral Pathogens Theo Sloots Unit Director Queensland Paediatric Infectious Diseases Laboratory Children s Health Service, Brisbane Queensland Children s Medical Research Institute Sakzewski Virus Research Centre Pathology Queensland Central The University of Queensland Helping Kids Get Better Sooner

2 QPID laboratory core business Activities are focussed on 3 areas: Clinical investigations and clinical trials Development of diagnostic assays Research in paediatric infectious disease. Together with the Director of Infectious Diseases, the QPID laboratory performes clinical investigations to diagnose unresolved infectious disease in children. Quantitative PCR for BKV and CMV in immunosuppressed kids. Fungal infections in BMT The QPID laboratory also includes a Clinical Trial Unit Over the last 5 years the CTU has completed approximately 42 clinical trials, sponsored by industry. Viruses in May, 2011

3 QPID laboratory core business Develop and validate new (molecular) diagnostic procedures for infectious diseases. Diagnostic PCR assays Quantitative real-time PCR Typing assays (rep PCR, MLST, SNP, Sequenom technologies) Drug resistance detection and characterisation Validate and transfer assays for routine use at Pathology Queensland More than 70 assays developed so far Most notable; meningococcus, gonococcus enteric viruses respiratory viruses, including swine flu Funded by Qld Health, PQ, RCH Foundation Viruses in May, 2011

4 QPID laboratory core business Conduct research in (paediatric) infectious diseases Pseudomonas aeruginosa in Cystic Fibrosis Typing using rep-pcr, MLST, Sequenom Antibiotic resistance markers and mechanisms Virulence factors Characterisation of viral enteric disease Epidemiology of known and new enteric viruses Characterisation of paediatric respiratory disease Epidemiology of known and new respiratory viruses Birth Cohort - acquisition of respiratory viruses during the first 2 years of life New virus discovery Funded by RCH Foundation, NHMRC, ARC, NIH. Viruses in May, 2011

5 Detection of Respiratory Viruses Until 1999 the Pathology Queensland Diagnostic Virology laboratory employed traditional methods for the detection of respiratory viruses Virus isolation in cell culture using roller tubes Direct Fluorescent staining (DFA) The algorithm was modified to a more rapid protocol. Direct Fluorescent staining (DFA) Culture amplified DFA (48 hr culture followed by DFA staining) This method showed 99.7% sensitivity and 99.3% specificity compared to the traditional method. It reduced the turn around time for 85% of the results from 7 days to 3 days

6 Diagnosis of Respiratory Viruses Then in 2003 we developed the first molecular test for respiratory viruses and applied it to routine diagnostic use. This method employed conventional multiplex PCR for 7 respiratory viruses. Amplification products were detected by individual virus-specific biotinylated probes in a 96-well microtitration plate using digoxigenin (Roche) and colour detection (ELAHA). This assay was validated by analysing 1500 respiratory samples (at least 20 positives) and comparing the results against DFA /CA-DFA Syrmis et al. Journal of Molecular Diagnostics (2004), 6:

7 Diagnosis of Respiratory Viruses The PCR assay showed a significant increase in sensitivity (approximately 23% overall) with 99.8% specificity compared to the DFA/CA-DFA algorithm DFA/CA-DFA (+) PCR (+) % Increase RSV PIV PIV PIV INF A INF B ADV Positive Negative During 2003 approximately 3000 respiratory samples were tested using the PCR assay. Viruses in May, 2011

8 Real-time PCR Assays Tests were performed on a LightCycler I as monoplex assays. RSV and ADV PIV 1,2,3 INF A and B HMPV and EHV (internal control) Applied Biosystems 7500 QIAGEN RotorGene LightCycler 480 Viruses in May, 2011

9 Number of Samples Real-time PCR Assays Impact on the Laboratory Increase ranges from 12 43% per year swine flu The introduction of real-time PCR for respiratory viruses has vastly increased demand but has provided significant additional benefits to our organisation Greater sensitivity & specificity of diagnostic tests Improved turn around times (3 24 hours) Reduced costs; particularly labour component Better patient care (positive diagnosis limits use of antibiotics/changed barrier nursing) Viruses in May, 2011

10 Common Viral Causes of Acute Respiratory Tract Infections We examined 8200 NPAs collected between from children with acute respiratory symptoms for common respiratory viruses Proportion of Viruses in Positive Specimens 16% 18% 23% 43% RSV PIV FLU ADV However, these viruses account only for approximately 25% of diagnoses leaving ~70% of cases undiagnosed. Using these assays, the majority of virus detections have typically been attributed to RSV 75% 69% 11% 5.5% 4% 4.5% Bacteria (6%) % Positive in All Samples Tested RSV PIV FLU ADV NEG

11 Other Viral Causes of Acute Respiratory Tract Infections Other known viruses have been implicated in ARTI, but clinically have been considered to be of limited significance. As a result they are often not tested for routinely. Some of these are: o rhinoviruses o coronaviruses OC43 and 229E o enteroviruses (parechovirus) o other adenoviruses o influenza C o parainfluenza 4 We sought to establish the epidemiology and clinical impact of these viruses as agents of ARTI Viruses in May, 2011

12 We examined 8200 NPAs collected between from children with acute respiratory symptoms for the other known viruses % RSV PIV FLU A & B ADV HRV HEV HCoV FLU C PIV 4 Neg Viruses in May, 2011

13 o 36% of patients with clinical symptoms of acute respiratory infection still remained undiagnosed. o This suggests that other as yet unknown respiratory viruses may be circulating in the community. o This notion was supported by the discovery of human metapneumovirus by the Osterhaus group in the Netherlands in 2001.

14 Discovery of Human Metapneumovirus Ab Osterhaus In 2001 van den Hoogen et al *, reported the existence of a new virus isolated from 28 children with respiratory tract disease (bronchiolitis) Symptoms attributed ranged from mild upper respiratory infection, severe cough to severe bronchiolitis (requiring mechanical ventilation) Clinical symptoms were indistinguishable from ARTI caused by RSV 500 nasopharyngeal swabs taken from well children were negative for the virus by culture and PCR * van den Hoogen, B. G., et al Nat. Med. 7:

15 Features of Human Metapneumovirus Isolated primarily during the winter months. Required prolonged cell culture Could induce mild respiratory disease in macaques, similar to human disease Morphology consistent with paramyxoviruses Showed sequence homology and gene organisation similar to avian pneumovirus (TRTV) genus metapneumovirus

16 Human Metapneumovirus in Queensland In 2003 the QPID laboratory commenced an NHMRC Project to investigate the epidemiology and clinical characteristics of hmpv in Queensland children. Tested 10,329 respiratory samples collected over 2001 to 2004 Specifically, we examined: the incidence of hmpv infection the seasonality of hmpv the molecular epidemiology of hmpv strains clinical impact of hmpv infection

17 Human Metapneumovirus in Queensland INCIDENCE OF hmpv 723 hmpv-positive specimens were detected giving an average incidence in our population of 7.0%. (Range 6.4% 7.6%) % % % % 82.8% of all positives were from children younger than 10 years of age. Subjects older than 40 years appear to be at greater risk

18 Human Metapneumovirus in Queensland TEMPORAL DISTRIBUTION OF hmpv INFECTION Infection with hmpv occurred primarily during late winter and spring. However there was considerable variation between years

19 Percentage positive Percentage positive Month Month summer autumn winter spring summer autumn winter spring IA IB PIV1 PIV 2 PIV 3 ADV RSV HMPV Month Month

20 MOLECULAR EPIDEMIOLOGY OF hmpv A A1 Amplification and sequencing of the HMPV P-gene was performed on 322 positive samples followed by phylogenetic analysis. HMPV showed the presence in our population of two genotypes A & B, each with two subtypes, A1,A2 & B1,B2 B A2 B1 B2

21 % of Total Positives Human Metapneumovirus in Queensland All four subtypes were detected in each of the years studied. Each year showed a predominant virus subtype present in the population hmpv Subtypes Detected A different subtype was dominant in successive years. (influenced by preexisting immunity?) A1 A2 B1 B2 In total, subtype B1 was most frequently detected over the 4 years

22 Clinical Impact of Human Metapneumovirus We found a significant correlation between disease severity and genotype A1 Approximately 28% of cases were scored as severe ie. requiring ventilation, IV fluids and hospitalization for > 5 days. Have observed 13 proven cases of nosocomial infection at the RCH. (This has also been reported by others) Two deaths in immunocompromised adults following lung transplant and BM Tx. (Confirmed by Immuno-histochemical staining of hmpv in lung tissue). Sloots et al. Emerg Infect Dis (8):

23 Discovery of New Respiratory Viruses Lia van der Hoek, The Netherlands 2004 Described the presence of a new human coronavirus - NL63 in a 7-month-old child with bronchiolitis. Nat Med Apr;10(4): Patrick Woo, Hong Kong 2005 Discovered a novel coronavirus, HKU1, from a patient with pneumonia. J Virol Jan;79(2): Tobias Allander, Sweden 2005 Reported the presence of human bocavirus in respiratory tract samples. Proc Natl Acad Sci U S A Sep 6;102(36): Viruses in May, 2011

24 The New Human Coronaviruses Coronavirus NL63 NL63 has been found in respiratory samples from children with URTI & LRTI. (1.7%-9.3%) Studies in German, Canada and South Korea showed that NL63 infection alone was the cause of croup and bronchiolitis in children. Seasonal peaks have been described from January to March in temperate climates. Coronavirus HKU1 QPID was the first to show that HKU1 was more widespread than originally described. Samples from 2004 showed that 3.1% were positive for HKU1; most were children younger than 2 years of age. A study in USA (n = 1048) showed 1% of respiratory samples (negative for other viruses) were positive for HKU1 and that clinical manifestations included URT & LRT disease. Viruses in May, 2011

25 Human Bocavirus Respiratory Disease The initial Swedish study showed that 3.1% of NPAs from children hospitalized with respiratory symptoms were positive for human bocavirus. Additional evidence for infection early in life comes from a Swedish prospective study of a birth cohort, in which 4.5% of the first acute respiratory tract infection was attributed to human bocavirus. The virus has now been identified on several continents with incident rates ranging from 1.5 to 20%. Rates of co-infection have found to be very high (14-72%). Evidence has shown that bocavirus causes both upper and lower respiratory disease. Viruses in May, 2011

26 Human Bocavirus Other Disease Role in acute gastrointestinal disease Early studies at QPID showed that HBoV was present in 4.8% of stool samples collected from children with gastroentiritis. Other studies showed HBoV in stools (0.8% - 9.1%) of patients with GE. Recent discovery of new human bocaviruses causing GE in Pakistani & Scottish children Possible role in the development of Kawasaki disease. Study in France, samples from children with Kawasaki disease showed 31.2% of the samples positive for HBoV compared to 1.2% of controls. High viral loads present in adenoidal and tonsillar tissue Viruses in May, 2011

27 Average Annual Incidence over 4 years Study Samples Tested (N= 8200) ~24% of cases still remained undiagnosed RSV PIV FLU A & B ADV HRV HEV HCoV FLU C PIV 4 HMPV HBoV Neg % % Viruses in May, 2011

28 To answer this question we used a dual approach: Sought to identify new variants of known viruses Initiated a virus discovery program

29 In the QPID laboratory Ian Mackay (Post-doc) Peter McErlean (PhD scholar) QPID had an interest in typing rhinoviruses Used high throughput sequencing We noted an association of some HRV strains with LRTI Typing these, we identified a novel HRV variant These were mainly associated with asthma, bronchiolitis or pneumonia 5 UTR McErlean et al, JCV, March additional novel HRV strains were detected in 2500 specimens (1.4%) collected during These are now recognised as a new rhinovirus subtype HRV type C Mc Erlean et al. PLoS ONE, 3(4): e1847.

30 JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 2007, p PLoS ONE April 2008 Volume 3 Issue 4 e1847

31 Emerging Infectious Diseases Vol. 14, No. 6, June 2008

32 Human Rhinovirus C J Clin Virol Sep;46(1):85-9. Epub 2009 Jul 5.

33 Human Rhinovirus C and Asthma This study was a prospective population-based surveillance in 2 US counties among children <5 years of age hospitalized with acute RTI or fever from October 2001 to September children enrolled and tested for rhinoviruses by RT-PCR 167 (16%) had HRVs detected. o o 147 samples successfully sequenced, 77 HRV-Cs, 64 HRV-As, 6 HRV-Bs. Children with HRV-Cs significantly more likely than HRV-A to have underlying high-risk conditions, such as asthma (42% vs 23%, P=0.023) and to have had a discharge diagnosis of asthma (55% vs 36%, P=0.022). Suggests this novel group causes a substantial burden of paediatric disease.

34 New Virus Discovery High-Throughput Sequencing Dave Wang Washington University, St Louis, USA Dave Wang Pan viral DNA microarrays (SARS project) High throughput sequencing (human genome project) In 2006, 10 NPA samples were dispatched for examination; all from children with LRTI and negative for known respiratory viruses Viral sequences showed 34-50% amino acid identity with known polyomaviruses 35% - JC virus STAg, 50% - BK virus LTAg, 34% - SV40 VP1,

35 New Virus Discovery High-Throughput Sequencing Dave Wang Washington University, St Louis, USA Dave Wang Pan viral DNA microarrays (SARS project) High throughput sequencing (human genome project) In 2006, 10 NPA samples were dispatched for examination; all from children with LRTI and negative for known respiratory viruses Virus detected was a new human polyomavirus Named WU polyomavirus. Gaynor et al. PLOS Pathogens 3(5): (9 February 2007) Viral sequences showed % amino amino acid identity acid with identity known with polyomaviruses known polyomaviruses 35% - JC virus STAg, 50% 35% -- BK JC virus LTAg, STAg, 34% 50% -- SV40 BK virus VP1, LTAg, 34% - SV40 VP1,

36 epublication 9 Feb 2007

37 Phylogenetic analysis over 4 genes confirmed that KI and WU polyomavirus were genetically distinct from other human polyomaviruses

38 Percentage Positive 2866 respiratory samples collected from patients with acute respiratory tract infection (Jan Dec 2003) were examined. 2.6% average annual prevalence of KI. (2009 = 3.8%) 4.5% average annual prevalence of WU (2009 = 5.8%) Little seasonal variation was observed for KI infection KIV WUV Both KI and WU polyomaviruses are more prevalent in children under 10 years of age Bialasiewicz et al, J Clin Virol Sep;40(1):15-8. Bialasiewicz et al, J Clin Virol Feb;41(2): J F M A M J J A S O N D Month

39 WU and KI polyomaviruses were frequently co-detected with another respiratory virus. N=1247 RSV IFAV PIV ADV HMPV HRV HCoV HBoV KIV WUV Total detections Single detections (% of total) % 74% 89% 52% 76% 75% 73% 39% 40% 27% Co-detections) (% of total) 28% 26% 11% 48% 24% 25% 27% 61% 60% 73% PIV was least frequently co-detected (53%) (80%) Out of 1247 samples tested, 53% had 1 or more viruses detected. (17%) 6 [1] (2%) 80% had 1 virus 20% had more than 1

40 Reports of New Human Polyomaviruses 22 FEBRUARY 2008 VOL 319 SCIENCE Emerging Infectious Diseases (2009), 15(3): Viruses in May, 2011

41 Reports of New Human Polyomaviruses 22 FEBRUARY 2008 VOL 319 SCIENCE July 2010 Discovery of a New Human Polyomavirus Associated with Trichodysplasia Spinulosa in an Immunocompromized Patient TS polyomavirus (TSV) Els van der Meijden, René W. A. Janssens, Chris Lauber, Jan Nico Bouwes Bavinck, Alexander E. Gorbalenya, Mariet C. W. Feltkamp Viruses in May, 2011

42 What is the Clinical Importance of These New Polyomaviruses? Are they new agents of respiratory disease?

43 Emerging Infectious Diseases Vol. 15, No. 8, August 2009 By age 5 years ~90% of the population is infected with WU and/or KI polyomavirus

44 Clinical Presentation of WU polyomavirus at RCH Medical records available for 98 of 128 (77%) WUV (+). 35% recorded URTI 57% recorded LRTI In 20 of 98 WUV (+) was the only viral pathogen detected. In 10 of these the diagnosis was recorded as pneumonia or bronchiolitis. Symptoms were not sufficiently different to clinically distinguish WUV from other respiratory viruses. Presenting Signs KIV (N= 51) WUV (N=98) Cough 56% 63% Fever 41% 45% Wheezing 38% 40% Malaise 39% 32% Bronchiolitis 11% 8% Pneumonia 0 2% Diarrhoea 8% 17%

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46 Sero-prevalence studies show first infection occurs in children younger than 5 years of age. WU and KI polyomaviruses are found in (6.8%) respiratory secretions of patients with ARTI and not in controls Prevalence of WUV is 6x greater in immunocompromised children with ARTI compared with immunocompetent children with ARTI. WUV found in patients with progressive multifocal encephalopathy (PML) in absence of other polyomaviruses. MCV found in 24% of Merkel cell carcinoma biopsies (integrated genome) MCV also found in respiratory secretions (6%)

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48 Jan-05 Feb-05 Mar-05 Apr-05 May-05 Jun-05 Jul-05 Aug-05 Sep-05 Oct-05 Nov-05 Dec-05 Jan-06 Feb-06 Mar-06 Apr-06 May-06 Jun-06 Jul-06 Aug-06 Sep-06 Oct-06 Nov-06 Dec-06 Jan Mar May Jul- Sep Nov Jan-08 Feb-08 Mar-08 Apr-08 May-08 Jun-08 Jul-08 Aug-08 Sep-08 Oct-08 Nov-08 Dec-08 % Positive of Total Tested Influenza A Su Au Wi Sp Su Au Wi Sp Su Au Wi Sp Su Au Wi Sp S Influenza B

49 Until April 2009, specimens for clinical diagnosis of Influenza were tested using a consensus duplex real-time PCR assay for INF A & INF B targeting the gene coding for the matrix protein. * This assay was previously validated against a large number of influenza A strains (human, avian) including H5N1**. ** Vietnamese; Indonesian strains Any Influenza A-positive samples are then screened further with a H5N1-specific assay targeting the haemagglutinin gene. All positive specimens are forwarded to the QH Public Health Virology laboratory for Influenza typing and confirmation of any H5N1 result *Whiley et al. Diagnostic Microbiology and Infectious Disease 53 (2005)

50 The Australian National Incident Room (NIR) was notified by WHO on 24 April 2009 of outbreaks in Mexico & USA of a novel strain of A/H1N1 influenza (Swine flu). H1N1 influenza 09 was a novel influenza A virus infecting humans. H1N1 influenza 09 emerged after reassortment of human and swineorigin influenza strains, creating a virus against which humans had little or no immunity.

51 1. Can the Matrix consensus screening assay detect swine flu? 2. Need to develop a new real-time PCR assay for swine flu H1 TaqMan (2009 A/H1N1 specific) N1 TaqMan (2009 A/H1N1 specific) Positive control: Auckland influenza H1N strain. (kindly provided by WHO-Melbourne, FSS-Brisbane) Range of Flu Isolates: H3N2 (n=18) H1N1 (n=14) years (kindly provided by FSS-Brisbane) Wild-type porcine H1N1 (kindly provided by AAHL,Geelong ) Clinical samples: n = 152, from years Influenza A - not detected n = 91 Influenza A - DETECTED n = 61 (H1N1 = 6; H3N2 = 23; Not typed = 32) (kindly provided by Pathology Queensland, Brisbane)

52 1. Can the Matrix consensus screening assay detect swine flu? 2. Need to develop a new real-time PCR assay for swine flu H1 TaqMan (2009 A/H1N1 specific) N1 TaqMan (2009 A/H1N1 specific) RESULTS: Matrix screening assay detected ALL subtypes including human swine flu Specific human swine flu assays ONLY detected pandemic influenza H1N1 (2009) The H1 assay was slightly more sensitive than the N1 assay (3 copies vs 8 copies/reaction) Assay protocol was transferred to Pathology Qld and introduced as a routine diagnostic procedure on 6 May 2009

53 First Australian case was detected by the Pathology Queensland laboratory on the 7 May, This result was confirmed by the WHO Reference Laboratory on the 9 May,

54 First Australian case was detected by the Pathology Queensland laboratory on the 7 May, This result was confirmed by the WHO Reference Laboratory on the 9 May, (June 6)

55 The integrated strategy for the detection of respiratory viruses in Queensland, delivers rapid, sensitive and specific results to the clinicians in Queensland Health The extended screening of respiratory viruses provides comprehensive up to date epidemiological data about respiratory viruses circulating in our population. Sophisticated molecular characterisation has resulted in the detection of new viruses, and previously unknown strains of known virus families. Our current strategy allows a rapid response to new significant viral respiratory pathogens such as swine flu.

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