Date post: | 01-Jun-2015 |
Category: |
Health & Medicine |
Upload: | r-lin |
View: | 536 times |
Download: | 0 times |
MCI5004 MOLECULAR BIOMARKERS IN CLINICAL
RESEARCH Biomarkers in Infectious Diseases
Biomarkers - aims
• To detect pathogen– See (Gram stain, IF), grow (culture), PCR
• Antibody response (serology)• Marker of virulence• Marker of resistance
• Marker of severity• Prognostication• Human immune response• Genetic susceptibility
Molecular diagnostic markers
• Assume unique protein or nucleic acid sequence– Found in all target organisms– Not found in related non-target organisms– Preferably many copies in each organism
• Examples– Enzyme immunoassay : syphilis EIA using
recombinant protein– PCR : MPT64 for TB, MA for influenza A
e.g. Ngan GJ 2010 Research Microbiol
• Salmonella genus: ompC
• S. Typhi or Paratyphi A: STY4220
• Intergenic region SSAPI
• stgA fimbrial subunit protein in S. Typhi
Problems with diagnostic markers
• Proteins in EIA serology : may lack sensitivity vs. whole cell EIA
• Mobile DNA elements may be lost (e.g. Chlamydia trachomatis)
• RNA sequence changes
• MRSA DNA in GeneExpert system: variable ccr region used
• Need fairly extensive clinical evaluation
Diagnostic markers
• MS protein profile used in MALDI-TOF system for bacterial identification
• (Matrix Laser Desorption/ Ionization Time of Flight Mass Spectrometry)
Markers of disease severity
• Non-specific biochemical markers in use : CRP, procalcitonin
• No specific molecular marker in routine application
• For research (see later), helps in understanding pathogenesis
Genetic susceptibility
• Apart from known functional associations e.g. sickle cell anaemia or immune deficiencies
• Genetic loci for intracellular infection
• Example– Khor CC 2010 NEJM. CISH alleles in
malaria, tuberculosis, bacteremia– GRACE study in EU. 6000 samples from
acute respiratory infections
Genetic susceptibility• Khor CC 2010
– 8 402 patients; Kenya, Malawi, The Gambia, Hong Kong, Vietnam
– Bacteremia; malaria; tuberculosis– CISH (cytokine-inducible SRC homology 2
domain protein) – negative regulator of IL2 immune response
– 5 alleles– Each allele increases risk by 18%– Potential therapeutic target?
Genetic susceptibility
• Khor CC 2007 Nat Genet– Mal variant and invasive pneumococcal
disease (OR 2.39)
• Ladhani 2010 CID– 1992 to 2005: 175 families of children with
invasive H. influenzae b disease after immunization (vaccine failure)
– SNP in Mal/TIRAP and interleukin-10 genes (OR 5.6)
Pathogen virulence – lines of evidence
• Clinical– Impression – more severe disease– Complications– Q: How does this apply to influenza?
Dengue?
Pathogen virulence
• Epidemiology– Atypical age groups– Susceptible populations– ICU rates– Mortality rates; case fatality ratio– Q: what measures could be used for
pandemic influenza?
Pathogen virulence – molecular markers
• Start from what we know– E.g. influenza
• Role of HA, tissue tropism (alpha 2,3 or alpha 2,6 receptors)
• Polymerase PB2 gene E627K
• Follow up opportunistic finding– IgG2 deficiency in pregnant women and
severe H1N1 2009 infection (Gordon CL 2010, CID)
– Started with one unusual case– Systematically studied 39 cases (pregnant/
non-pregnant)– Still not sure what it means
• Scanning for associations– e.g. influenza sequence changes and clinical
outcomes (like ICU cases, vaccine failure)
• “D222G” (H1) mutation in severe cases
• “Based on currently available virological, epidemiological and clinical information, the D222G substitution does not appear to pose a major public health issue.” (WHO)
D222G (H1)
• Found since April 2009
• Kilander 2010 Eurosurveillance– 11/61 (18%) severe cases in Norway, 0/205
mild
• Observed elsewhere but lower <10%
• Also found in asymptomatic
• Mutation in receptor binding domain
D222G (H1)
• ?statistical bias
• ?influenced by growth in eggs
• Found in autopsy tissue from Ukraine ?selective bias from lung
• Not sure still!
• No strong virological or experimental support
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/nature08260
Pathological examination of the lungs of infected cynomolgus macaques.
PB2
• Polymerase basic protein 2– Adaptation for replication in avian to human– Previous pandemic strains had E627K
mutation
• Taubenberger 2005 : important for adaptation and pathogenicity
• Mutation absent from H1N1 2009, but what if? …
PB2
• Jagger 2010 Mbio
• Herfst 2010 J Virol
• Zhu H 2010 Virology
• Using recombinant/ RG viruses – 627 mutation has no effect or attenuates
Virulence markers - approaches
• Need a combination of studies
• Theoretical modeling
• Cell culture studies
• Animal models
• Humans: tissue or autopsy
Founder effect
• Be aware of “associations” which are actually due to founder effect
• examples– Influenza, pandemic H1N1-2009– Enterovirus 71 genotypes– Severe adenovirus disease
• Sometimes the new strain has new biological properties ? Significance
• Nowadays, easy to blame new “virulent strain” when something bad happens– e.g. enterovirus, EHEC, group A streptococus
STEC outbreak, Germany
• Shiga-toxin producing E. coli (STEC) = Enterohaemorrhagic E. coli (EHEC)
• >3000 cases, 25% HUS, some deaths• Unusual serotype O104• Whole-genome sequence showed
– EAEC genome with stx-2 prophage– stx-2 possibly turned on by quinolone antibiotics
• But is it more virulent than previous EHEC? What does virulence in this setting mean?
Whole-genome sequencing
• New, cheaper and faster techniques for whole genome sequencing of microbes
• Other areas which can be investigated– Human metagenome– Microbial phylogeny and evolution– Genomic islands, recombination, horizontal
gene transfer, virulence diversity, antigen discovery for vaccines