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