MCI5004 MOLECULAR BIOMARKERS IN CLINICAL RESEARCH  Biomarkers in Infectious Diseases
Biomarkers - aims <ul><li>To detect pathogen </li></ul><ul><ul><li>See (Gram stain, IF), grow (culture), PCR </li></ul></u...
Molecular diagnostic markers <ul><li>Assume  unique protein or nucleic acid  sequence </li></ul><ul><ul><li>Found in all t...
<ul><li>e.g.  Ngan GJ 2010 Research Microbiol </li></ul><ul><li>Salmonella genus:  ompC </li></ul><ul><li>S. Typhi or Para...
Problems with diagnostic markers <ul><li>Proteins in EIA serology : may lack sensitivity vs. whole cell EIA </li></ul><ul>...
Diagnostic markers <ul><li>MS protein profile used in MALDI-TOF system for bacterial identification </li></ul><ul><li>(Mat...
Markers of disease severity <ul><li>Non-specific  biochemical markers in use : CRP, procalcitonin </li></ul><ul><li>No spe...
Genetic susceptibility <ul><li>Apart from known functional associations e.g. sickle cell anaemia or immune deficiencies </...
Genetic susceptibility <ul><li>Khor CC 2010 </li></ul><ul><ul><li>8 402 patients; Kenya, Malawi, The Gambia, Hong Kong, Vi...
Genetic susceptibility <ul><li>Khor CC 2007 Nat Genet </li></ul><ul><ul><li>Mal  variant and invasive pneumococcal disease...
Pathogen virulence  – lines of evidence <ul><li>Clinical </li></ul><ul><ul><li>Impression – more severe disease </li></ul>...
Pathogen virulence <ul><li>Epidemiology </li></ul><ul><ul><li>Atypical age groups </li></ul></ul><ul><ul><li>Susceptible p...
Pathogen virulence  – molecular markers <ul><li>Start from what we know </li></ul><ul><ul><li>E.g. influenza </li></ul></u...
<ul><li>Follow up opportunistic finding </li></ul><ul><ul><li>IgG2 deficiency in pregnant women and severe H1N1 2009 infec...
<ul><li>Scanning for associations </li></ul><ul><ul><li>e.g. influenza sequence changes and clinical outcomes (like ICU ca...
 
 
<ul><li>“ D222G” (H1) mutation in severe cases </li></ul><ul><li>“ Based on currently available virological, epidemiologic...
D222G (H1) <ul><li>Found since April 2009 </li></ul><ul><li>Kilander 2010 Eurosurveillance </li></ul><ul><ul><li>11/61 (18...
D222G (H1) <ul><li>?statistical bias </li></ul><ul><li>?influenced by growth in eggs </li></ul><ul><li>Found in autopsy ti...
Hua Yang 2010 PLOS Currents. Mutated D222G on reference virus – still maintain predominant affinity for alpha 2,6 receptors
Y Itoh  et al.   Nature   460 ,  1021 - 1025  (2009) doi:10.1038/nature08 260 Pathological examination of the lungs of inf...
H1N1 2009 <ul><li>Itoh 2009 </li></ul><ul><ul><li>Replicate more effectively </li></ul></ul><ul><ul><li>More severe pathol...
H1N1 2009 <ul><li>Soundararajan 2009 Nat Biotech </li></ul><ul><li>Theoretical complex : bind mostly alpha 2,6 but also al...
Maines 2009 H1N1 2009 binding pattern similar to 1918 virus but lower affinity
PB2 <ul><li>Polymerase basic protein 2 </li></ul><ul><ul><li>Adaptation for replication in avian to human </li></ul></ul><...
PB2 <ul><li>Jagger 2010 Mbio </li></ul><ul><li>Herfst 2010 J Virol </li></ul><ul><li>Zhu H 2010 Virology </li></ul><ul><li...
<ul><li>Herfst 2010 : monitoring should not rely on virulence markers of the past </li></ul>
Virulence markers - approaches <ul><li>Need a combination of studies </li></ul><ul><li>Theoretical modeling </li></ul><ul>...
Virulence markers <ul><li>Instead of looking for likely markers based on past studies </li></ul><ul><li>Suggest generating...
Founder effect <ul><li>Be aware of “associations” which are actually due to founder effect </li></ul><ul><li>examples </li...
Molecular markers for epidemiology <ul><li>Tracking transmission, outbreaks, clusters </li></ul><ul><li>For bacteria, tren...
STEC outbreak, Germany <ul><li>Shiga-toxin producing E. coli (STEC) = Enterohaemorrhagic E. coli (EHEC) </li></ul><ul><li>...
Whole-genome sequencing <ul><li>New, cheaper and faster techniques for whole genome sequencing of microbes </li></ul><ul><...
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Mci5004 biomarkers infectious diseases

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Mci5004 biomarkers infectious diseases

  1. 1. MCI5004 MOLECULAR BIOMARKERS IN CLINICAL RESEARCH Biomarkers in Infectious Diseases
  2. 2. Biomarkers - aims <ul><li>To detect pathogen </li></ul><ul><ul><li>See (Gram stain, IF), grow (culture), PCR </li></ul></ul><ul><li>Antibody response (serology) </li></ul><ul><li>Marker of virulence </li></ul><ul><li>Marker of resistance </li></ul><ul><li>Marker of severity </li></ul><ul><li>Prognostication </li></ul><ul><li>Human immune response </li></ul><ul><li>Genetic susceptibility </li></ul>
  3. 3. Molecular diagnostic markers <ul><li>Assume unique protein or nucleic acid sequence </li></ul><ul><ul><li>Found in all target organisms </li></ul></ul><ul><ul><li>Not found in related non-target organisms </li></ul></ul><ul><ul><li>Preferably many copies in each organism </li></ul></ul><ul><li>Examples </li></ul><ul><ul><li>Enzyme immunoassay : syphilis EIA using recombinant protein </li></ul></ul><ul><ul><li>PCR : MPT64 for TB, MA for influenza A </li></ul></ul>
  4. 4. <ul><li>e.g. Ngan GJ 2010 Research Microbiol </li></ul><ul><li>Salmonella genus: ompC </li></ul><ul><li>S. Typhi or Paratyphi A: STY4220 </li></ul><ul><li>Intergenic region SSAPI </li></ul><ul><li>stgA fimbrial subunit protein in S. Typhi </li></ul>
  5. 5. Problems with diagnostic markers <ul><li>Proteins in EIA serology : may lack sensitivity vs. whole cell EIA </li></ul><ul><li>Mobile DNA elements may be lost (e.g. Chlamydia trachomatis ) </li></ul><ul><li>RNA sequence changes </li></ul><ul><li>MRSA DNA in GeneExpert system: variable ccr region used </li></ul><ul><li>Need fairly extensive clinical evaluation </li></ul>
  6. 6. Diagnostic markers <ul><li>MS protein profile used in MALDI-TOF system for bacterial identification </li></ul><ul><li>(Matrix Laser Desorption/ Ionization Time of Flight Mass Spectrometry) </li></ul>
  7. 7. Markers of disease severity <ul><li>Non-specific biochemical markers in use : CRP, procalcitonin </li></ul><ul><li>No specific molecular marker in routine application </li></ul><ul><li>For research (see later), helps in understanding pathogenesis </li></ul>
  8. 8. Genetic susceptibility <ul><li>Apart from known functional associations e.g. sickle cell anaemia or immune deficiencies </li></ul><ul><li>Genetic loci for intracellular infection </li></ul><ul><li>Example </li></ul><ul><ul><li>Khor CC 2010 NEJM. CISH alleles in malaria, tuberculosis, bacteremia </li></ul></ul><ul><ul><li>GRACE study in EU. 6000 samples from acute respiratory infections </li></ul></ul>
  9. 9. Genetic susceptibility <ul><li>Khor CC 2010 </li></ul><ul><ul><li>8 402 patients; Kenya, Malawi, The Gambia, Hong Kong, Vietnam </li></ul></ul><ul><ul><li>Bacteremia; malaria; tuberculosis </li></ul></ul><ul><ul><li>CISH (cytokine-inducible SRC homology 2 domain protein) – negative regulator of IL2 immune response </li></ul></ul><ul><ul><li>5 alleles </li></ul></ul><ul><ul><li>Each allele increases risk by 18% </li></ul></ul><ul><ul><li>Potential therapeutic target? </li></ul></ul>
  10. 10. Genetic susceptibility <ul><li>Khor CC 2007 Nat Genet </li></ul><ul><ul><li>Mal variant and invasive pneumococcal disease (OR 2.39) </li></ul></ul><ul><li>Ladhani 2010 CID </li></ul><ul><ul><li>1992 to 2005: 175 families of children with invasive H. influenzae b disease after immunization (vaccine failure) </li></ul></ul><ul><ul><li>SNP in Mal/TIRAP and interleukin-10 genes (OR 5.6) </li></ul></ul>
  11. 11. Pathogen virulence – lines of evidence <ul><li>Clinical </li></ul><ul><ul><li>Impression – more severe disease </li></ul></ul><ul><ul><li>Complications </li></ul></ul><ul><ul><li>Q: How does this apply to influenza? Dengue? </li></ul></ul>
  12. 12. Pathogen virulence <ul><li>Epidemiology </li></ul><ul><ul><li>Atypical age groups </li></ul></ul><ul><ul><li>Susceptible populations </li></ul></ul><ul><ul><li>ICU rates </li></ul></ul><ul><ul><li>Mortality rates; case fatality ratio </li></ul></ul><ul><ul><li>Q: what measures could be used for pandemic influenza? </li></ul></ul>
  13. 13. Pathogen virulence – molecular markers <ul><li>Start from what we know </li></ul><ul><ul><li>E.g. influenza </li></ul></ul><ul><ul><ul><li>Role of HA, tissue tropism (alpha 2,3 or alpha 2,6 receptors) </li></ul></ul></ul><ul><ul><ul><li>Polymerase PB2 gene E627K </li></ul></ul></ul>
  14. 14. <ul><li>Follow up opportunistic finding </li></ul><ul><ul><li>IgG2 deficiency in pregnant women and severe H1N1 2009 infection (Gordon CL 2010, CID) </li></ul></ul><ul><ul><li>Started with one unusual case </li></ul></ul><ul><ul><li>Systematically studied 39 cases (pregnant/ non-pregnant) </li></ul></ul><ul><ul><li>Still not sure what it means </li></ul></ul>
  15. 15. <ul><li>Scanning for associations </li></ul><ul><ul><li>e.g. influenza sequence changes and clinical outcomes (like ICU cases, vaccine failure) </li></ul></ul>
  16. 18. <ul><li>“ D222G” (H1) mutation in severe cases </li></ul><ul><li>“ Based on currently available virological, epidemiological and clinical information, the D222G substitution does not appear to pose a major public health issue.” (WHO) </li></ul>
  17. 19. D222G (H1) <ul><li>Found since April 2009 </li></ul><ul><li>Kilander 2010 Eurosurveillance </li></ul><ul><ul><li>11/61 (18%) severe cases in Norway, 0/205 mild </li></ul></ul><ul><li>Observed elsewhere but lower <10% </li></ul><ul><li>Also found in asymptomatic </li></ul><ul><li>Mutation in receptor binding domain </li></ul>
  18. 20. D222G (H1) <ul><li>?statistical bias </li></ul><ul><li>?influenced by growth in eggs </li></ul><ul><li>Found in autopsy tissue from Ukraine ?selective bias from lung </li></ul><ul><li>Not sure still! </li></ul><ul><li>No strong virological or experimental support </li></ul>
  19. 21. Hua Yang 2010 PLOS Currents. Mutated D222G on reference virus – still maintain predominant affinity for alpha 2,6 receptors
  20. 22. Y Itoh et al. Nature 460 , 1021 - 1025 (2009) doi:10.1038/nature08 260 Pathological examination of the lungs of infected cynomolgus macaques.
  21. 23. H1N1 2009 <ul><li>Itoh 2009 </li></ul><ul><ul><li>Replicate more effectively </li></ul></ul><ul><ul><li>More severe pathology in lungs of mice, ferrets, primates </li></ul></ul><ul><ul><li>Transmit among ferrets </li></ul></ul><ul><li>Maine 2009 Science: worse disease in ferrets and mice, but lower transmissibility (!) </li></ul><ul><li>Increase affinity for alpha 1,3 receptors (lungs) appeared supported by modeling and glycan studies </li></ul>
  22. 24. H1N1 2009 <ul><li>Soundararajan 2009 Nat Biotech </li></ul><ul><li>Theoretical complex : bind mostly alpha 2,6 but also alpha 1,3 </li></ul>
  23. 25. Maines 2009 H1N1 2009 binding pattern similar to 1918 virus but lower affinity
  24. 26. PB2 <ul><li>Polymerase basic protein 2 </li></ul><ul><ul><li>Adaptation for replication in avian to human </li></ul></ul><ul><ul><li>Previous pandemic strains had E627K mutation </li></ul></ul><ul><li>Taubenberger 2005 : important for adaptation and pathogenicity </li></ul><ul><li>Mutation absent from H1N1 2009, but what if? … </li></ul>
  25. 27. PB2 <ul><li>Jagger 2010 Mbio </li></ul><ul><li>Herfst 2010 J Virol </li></ul><ul><li>Zhu H 2010 Virology </li></ul><ul><li>Using recombinant/ RG viruses – 627 mutation has no effect or attenuates </li></ul>
  26. 28. <ul><li>Herfst 2010 : monitoring should not rely on virulence markers of the past </li></ul>
  27. 29. Virulence markers - approaches <ul><li>Need a combination of studies </li></ul><ul><li>Theoretical modeling </li></ul><ul><li>Cell culture studies </li></ul><ul><li>Animal models </li></ul><ul><li>Humans: tissue or autopsy </li></ul>
  28. 30. Virulence markers <ul><li>Instead of looking for likely markers based on past studies </li></ul><ul><li>Suggest generating a variety of possible genotypes and studying biological effects </li></ul>
  29. 31. Founder effect <ul><li>Be aware of “associations” which are actually due to founder effect </li></ul><ul><li>examples </li></ul><ul><ul><li>Influenza, pandemic H1N1-2009 </li></ul></ul><ul><ul><li>Enterovirus 71 genotypes </li></ul></ul><ul><ul><li>Severe adenovirus disease </li></ul></ul><ul><li>Sometimes the new strain has new biological properties ? Significance </li></ul><ul><li>Nowadays, easy to blame new “virulent strain” when something bad happens </li></ul><ul><ul><li>e.g. enterovirus, EHEC, group A streptococus </li></ul></ul>
  30. 32. Molecular markers for epidemiology <ul><li>Tracking transmission, outbreaks, clusters </li></ul><ul><li>For bacteria, trend toward sequencing specific loci </li></ul><ul><li>Examples </li></ul><ul><ul><li>MLST, Spa typing for MRSA: hospitals have ST239 and ST22, community cases have ST30 (Western Samoan) </li></ul></ul><ul><ul><li>US300 is ST8, not found here </li></ul></ul><ul><ul><li>From pigs in Netherlands, ST398 </li></ul></ul>
  31. 33. STEC outbreak, Germany <ul><li>Shiga-toxin producing E. coli (STEC) = Enterohaemorrhagic E. coli (EHEC) </li></ul><ul><li>>3000 cases, 25% HUS, some deaths </li></ul><ul><li>Unusual serotype O104 </li></ul><ul><li>Whole-genome sequence showed </li></ul><ul><ul><li>EAEC genome with stx-2 prophage </li></ul></ul><ul><ul><li>stx-2 possibly turned on by quinolone antibiotics </li></ul></ul><ul><li>But is it more virulent than previous EHEC? What does virulence in this setting mean? </li></ul>
  32. 34. Whole-genome sequencing <ul><li>New, cheaper and faster techniques for whole genome sequencing of microbes </li></ul><ul><li>Other areas which can be investigated </li></ul><ul><ul><li>Human metagenome </li></ul></ul><ul><ul><li>Microbial phylogeny and evolution </li></ul></ul><ul><ul><li>Genomic islands, recombination, horizontal gene transfer, virulence diversity, antigen discovery for vaccines </li></ul></ul>

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