Download - Mci5004 biomarkers infectious diseases
![Page 1: Mci5004 biomarkers infectious diseases](https://reader036.vdocuments.net/reader036/viewer/2022062705/556c6ef3d8b42ad85e8b4ef4/html5/thumbnails/1.jpg)
MCI5004 MOLECULAR BIOMARKERS IN CLINICAL
RESEARCH Biomarkers in Infectious Diseases
![Page 2: Mci5004 biomarkers infectious diseases](https://reader036.vdocuments.net/reader036/viewer/2022062705/556c6ef3d8b42ad85e8b4ef4/html5/thumbnails/2.jpg)
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
![Page 3: Mci5004 biomarkers infectious diseases](https://reader036.vdocuments.net/reader036/viewer/2022062705/556c6ef3d8b42ad85e8b4ef4/html5/thumbnails/3.jpg)
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
![Page 4: Mci5004 biomarkers infectious diseases](https://reader036.vdocuments.net/reader036/viewer/2022062705/556c6ef3d8b42ad85e8b4ef4/html5/thumbnails/4.jpg)
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
![Page 5: Mci5004 biomarkers infectious diseases](https://reader036.vdocuments.net/reader036/viewer/2022062705/556c6ef3d8b42ad85e8b4ef4/html5/thumbnails/5.jpg)
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
![Page 6: Mci5004 biomarkers infectious diseases](https://reader036.vdocuments.net/reader036/viewer/2022062705/556c6ef3d8b42ad85e8b4ef4/html5/thumbnails/6.jpg)
Diagnostic markers
• MS protein profile used in MALDI-TOF system for bacterial identification
• (Matrix Laser Desorption/ Ionization Time of Flight Mass Spectrometry)
![Page 7: Mci5004 biomarkers infectious diseases](https://reader036.vdocuments.net/reader036/viewer/2022062705/556c6ef3d8b42ad85e8b4ef4/html5/thumbnails/7.jpg)
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
![Page 8: Mci5004 biomarkers infectious diseases](https://reader036.vdocuments.net/reader036/viewer/2022062705/556c6ef3d8b42ad85e8b4ef4/html5/thumbnails/8.jpg)
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
![Page 9: Mci5004 biomarkers infectious diseases](https://reader036.vdocuments.net/reader036/viewer/2022062705/556c6ef3d8b42ad85e8b4ef4/html5/thumbnails/9.jpg)
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?
![Page 10: Mci5004 biomarkers infectious diseases](https://reader036.vdocuments.net/reader036/viewer/2022062705/556c6ef3d8b42ad85e8b4ef4/html5/thumbnails/10.jpg)
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)
![Page 11: Mci5004 biomarkers infectious diseases](https://reader036.vdocuments.net/reader036/viewer/2022062705/556c6ef3d8b42ad85e8b4ef4/html5/thumbnails/11.jpg)
Pathogen virulence – lines of evidence
• Clinical– Impression – more severe disease– Complications– Q: How does this apply to influenza?
Dengue?
![Page 12: Mci5004 biomarkers infectious diseases](https://reader036.vdocuments.net/reader036/viewer/2022062705/556c6ef3d8b42ad85e8b4ef4/html5/thumbnails/12.jpg)
Pathogen virulence
• Epidemiology– Atypical age groups– Susceptible populations– ICU rates– Mortality rates; case fatality ratio– Q: what measures could be used for
pandemic influenza?
![Page 13: Mci5004 biomarkers infectious diseases](https://reader036.vdocuments.net/reader036/viewer/2022062705/556c6ef3d8b42ad85e8b4ef4/html5/thumbnails/13.jpg)
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
![Page 14: Mci5004 biomarkers infectious diseases](https://reader036.vdocuments.net/reader036/viewer/2022062705/556c6ef3d8b42ad85e8b4ef4/html5/thumbnails/14.jpg)
• 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
![Page 15: Mci5004 biomarkers infectious diseases](https://reader036.vdocuments.net/reader036/viewer/2022062705/556c6ef3d8b42ad85e8b4ef4/html5/thumbnails/15.jpg)
• Scanning for associations– e.g. influenza sequence changes and clinical
outcomes (like ICU cases, vaccine failure)
![Page 16: Mci5004 biomarkers infectious diseases](https://reader036.vdocuments.net/reader036/viewer/2022062705/556c6ef3d8b42ad85e8b4ef4/html5/thumbnails/16.jpg)
![Page 17: Mci5004 biomarkers infectious diseases](https://reader036.vdocuments.net/reader036/viewer/2022062705/556c6ef3d8b42ad85e8b4ef4/html5/thumbnails/17.jpg)
![Page 18: Mci5004 biomarkers infectious diseases](https://reader036.vdocuments.net/reader036/viewer/2022062705/556c6ef3d8b42ad85e8b4ef4/html5/thumbnails/18.jpg)
• “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)
![Page 19: Mci5004 biomarkers infectious diseases](https://reader036.vdocuments.net/reader036/viewer/2022062705/556c6ef3d8b42ad85e8b4ef4/html5/thumbnails/19.jpg)
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
![Page 20: Mci5004 biomarkers infectious diseases](https://reader036.vdocuments.net/reader036/viewer/2022062705/556c6ef3d8b42ad85e8b4ef4/html5/thumbnails/20.jpg)
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
![Page 21: Mci5004 biomarkers infectious diseases](https://reader036.vdocuments.net/reader036/viewer/2022062705/556c6ef3d8b42ad85e8b4ef4/html5/thumbnails/21.jpg)
Hua Yang 2010 PLOS Currents.Mutated D222G on reference virus – still maintain predominant affinity for alpha 2,6 receptors
![Page 22: Mci5004 biomarkers infectious diseases](https://reader036.vdocuments.net/reader036/viewer/2022062705/556c6ef3d8b42ad85e8b4ef4/html5/thumbnails/22.jpg)
Y Itoh et al. Nature 460, 1021-1025 (2009) doi:10.1038/nature08260
Pathological examination of the lungs of infected cynomolgus macaques.
![Page 23: Mci5004 biomarkers infectious diseases](https://reader036.vdocuments.net/reader036/viewer/2022062705/556c6ef3d8b42ad85e8b4ef4/html5/thumbnails/23.jpg)
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? …
![Page 24: Mci5004 biomarkers infectious diseases](https://reader036.vdocuments.net/reader036/viewer/2022062705/556c6ef3d8b42ad85e8b4ef4/html5/thumbnails/24.jpg)
PB2
• Jagger 2010 Mbio
• Herfst 2010 J Virol
• Zhu H 2010 Virology
• Using recombinant/ RG viruses – 627 mutation has no effect or attenuates
![Page 25: Mci5004 biomarkers infectious diseases](https://reader036.vdocuments.net/reader036/viewer/2022062705/556c6ef3d8b42ad85e8b4ef4/html5/thumbnails/25.jpg)
Virulence markers - approaches
• Need a combination of studies
• Theoretical modeling
• Cell culture studies
• Animal models
• Humans: tissue or autopsy
![Page 26: Mci5004 biomarkers infectious diseases](https://reader036.vdocuments.net/reader036/viewer/2022062705/556c6ef3d8b42ad85e8b4ef4/html5/thumbnails/26.jpg)
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
![Page 27: Mci5004 biomarkers infectious diseases](https://reader036.vdocuments.net/reader036/viewer/2022062705/556c6ef3d8b42ad85e8b4ef4/html5/thumbnails/27.jpg)
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?
![Page 28: Mci5004 biomarkers infectious diseases](https://reader036.vdocuments.net/reader036/viewer/2022062705/556c6ef3d8b42ad85e8b4ef4/html5/thumbnails/28.jpg)
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