Download Diagnostics (HBV and HCV) - View the full AIDS 2016 programme

Screening for viral hepatitis:
Diagnostics (HBV and HCV)
Diana Hardie
Division of Medical Virology, Department of Pathology, University of
Cape Town, South Africa and National Health Laboratory Service
Biomarkers that identify infected individuals are fundamental to
advancing knowledge about infectious agents:
•1960’s
discovery of Australia antigen
•1990s
cloning a fragment of HCV genome
use of c100 peptide to detect antibody
Current status of laboratory testing for HBV and HCV
Promising new developments in POC technologies
Serology markers of HBV infection:
HBV antigens:
Surface Ag
e Ag
Core Ag
Marker
Normal interpretation
Limitation
Surface Antigen
Viraemia
Occult B infection
e Antigen
High infectivity
e minus mutants
Core IgM
Recent acute infection
Flare in chronic carrier (levels important)
Surface antibody
Immunity
S minus mutants
Core IgG
Exposure to HBV
Loss over time
Quantifying HBV: HBV DNA level or HBsAg?
cccDNA best marker of viral burden
Serum HBV DNA level
Realtime PCR:
accumulation of PCR product
Fluorescent probes
Standard curve
Threshold Cycle
sensitive
Dynamic range: 10-108 ge/ml
50
40
30
20
10
0
1
2
3
4
Concentration
WHO International Eurohep reference R1 standard
HBV DNA, IU/ml
5
6
log 10
Role in HBV diagnosis and management:
•Staging / work up of chronic hepatitis B virus infection
•Response to therapy,
•diagnosis of sero- negative HBV infection,
•blood safety (TMA)
CHB stage
Serum HBV DNA (IU/mL)
HBsAg log10 IU/mL
Immune tolerant
>2X106-7
4.5-5.0
Immune clearance (HBeAg
pos)
2X104-5
3.0-4.5
Inactive carrier
<2X103
1.5-3.0
immuno-active (HBeAg neg)
Fluctuating >2X103-4
2.5-4.0
From JABFM 2015, 28:6; 822-837
S Antigen quantification
Better measure of cccDNA
Interferon-a
Decline by week 12 predicts good response
Less value for NUC therapy
Commercial platforms:
Architect HBsAgQT
LOD of 0.2 ng/ml
dynamic range:
0.05 to 250 IU/ml
dilution
PNAS September 2014
Caveat: may under-quantify sAg variants, RT drug resistant mutants
Occult HBV infection
•sAg negative , but HBV DNA is present in blood or liver
•Other markers variably present
•Past infection, good control,
•low HBV DNA in blood <100 IU/ml
•Mutants with altered sAg: “a” determinant or
•alterations to preS causing impaired sAg secretion
“a” determinant of HBsAg
Sensitive HBV DNA assay needed
Genetic Variation of HBV:
Polymerase error 10-5 per nt position per year
Genotypes A to J
8% between genotypes,
4% between subtypes
D
Clinical utility: not routine, predicts response to IFN-a
Commercial methodologies:
- LiPA good for dual infections,
Whole genomes or pol gene
Monitoring genetic evolution within the host:
Immune and drug selection pressure
Immune escape (B cell)
PreS: deletion
MHR: K122I, T123N
G145R, etc.
Increased viral replication
Decreased HBeAg
Expression
Immune escape (T cell)
BCP: A1762 and G1764A
preC: G1896A
Core gene: CTL epitope
Drug resistance
RT: V173L, L180I/V,
A181V/T, T184S,
S202I, M204I/V,
N236T, F249A,
M250I/V, etc
Tumorgenesis:
Truncated mutant
C1653T, T1753V, etc
Screening for these is mainly of research interest
NUCs
Population (Sanger) sequencing:
• Only the majority sequence
• Minority populations double peaks
• <20% not detected
• Cloning and sequencing of multiple clones
• Not practical for routine diagnostics
Hepatitis C
9.6kB
11608–11613 | PNAS | July 12, 2011 | vol. 108 | no. 28
HCV antibody testing
c100 peptide
3rd generation EIA
Recombinant antigens: NS3, core, NS4 and NS5
Better sens, spec
Automated platforms
Point of care tests:
Lateral flow, recombinant antigens
Blood, saliva
OroQuick HCV antibody
Limitations:
Delayed sero-conversion
Immuno-compromised, dialysis patients, MSM may
remain sero-negative
HCV nucleic acid testing: qualitative vs quantitative
Qualitative: PCR or TMA
•viraemia in sero positives
•Incident infections
•Blood products
RNA dynamics:
8-10 days post infection
Intermittently positive before
Challenge for blood safety
Glynn et al., 2005 Transfusion
Viral load:
Inter assay variation at low RNA
levels (<log 3)
RealTime PCR:
LOD 5-15 IU/mL
wide dynamic range
Inter assay variance at <log 3 IU/ml
Therapeutic monitoring:
Important for Peg/IFN and Ribavirin:
Baseline and RGT
Med Microbiol Immunol.
2015; 204(4): 515–525
DAA monitoring?
- Baseline levels don’t affect duration of therapy
- Viral kinetics during therapy don’t necessarily predict outcome
- assay specific decision points
Monitoring for relapse, re-infection can be done with cheaper tests
HCV core antigen testing
Indicates viraemia
Less sensitive than RNA
Levels correlate with RNA levels
Precede antibody detection
Abbott ARCHITECT CMEIA
LOD = 3 fmol/L ~ 1000 IU/ml RNA
Mixon-Hayden et al.,
JCV 66 (2015) 15-18
Specificity:
Good in HCV antibody negative samples,
but false positives in HCV antibody pos RNA negative samples!
Potential role:
Confirming HCV positive serology
Diagnosis of incident infections
Therapeutic monitoring with core antigen?
Comparing cAg and RNA at weeks 2, 4, end of therapy:
•Core Ag undetectable more rapidly
•Both had similar predictive values for treatment outcome
Genetic variation of HCV:
1-5%
within host
15-25% within sub-genotype
30-50% between
E1, E2 p7
NS5A
Essential for therapeutic workup
Commercial options
NS5B sequence
Lowest variation 5’NCR
-Pan-genotypic PCR
Viruses 2015, 7, 5018-5039
Coding region
HCV drug resistance testing
Genotype specific PCR and Sanger sequencing , online analysis
V36
F43
T54
V55
Y56
Q80
S122 I132
R155
A156
D168
V170
N174
NS3/4A
M28
Q30
L31
P58
Y93
NS5A
L159
NS5B
S282
C289
C316
V321
L326
A395
N411
S368 M414
N444
C445
Y448
C451
P495
A553
G554
S556
G557
G558
D559
Y561 S565 I585
Lontok et al., 2015
Clinical utility:
Previous failure of DAA containing regimen
Targetted testing for particular RAVs in patients with specific genotypes
In the pipeline for HBV and HCV...
Next generation sequencing:
Gastroenterology 2012;142:1303-1313
•Simultaneous sequencing of millions of targets
•Whole genomes
•Quasispecies
•Relative abundance of particular SNPs
•Even minor populations
•Clinical significance of minor populations?
Improving point of care diagnostics
most HBV and HCV infected individuals are unaware of their status
Viral nucleic acid
Isothermal
amplification
Naked eye detection
RNA or DNA
•Robust
•Simple equipment
•Tolerates inhibitors
•Masses of product
Loop Mediated amplification is particularly robust and in diagnostic
development for HBV and HCV POC assays
Biosensors for POC testing
•High specificity
•Tiny sample
•Robust device
Rafael Vargas-Bernal, Esmeralda Rodríguez-Miranda and Gabriel Herrera-Pérez
DOI: 10.5772/46227
Thank You