Transcript Viral Hepatitis - Laboratory Diagnostics

Laboratory Diagnostics in Hepatitis
T. Mazzulli, MD, FRCPC
Department of Microbiology
UHN/Mount Sinai Hospital
Objectives
Review the serologic diagnosis of viral hepatitis
Review the methodologies available for
molecular testing and describe some of the
advantages and disadvantages
Discuss the currently available commercial
assays that are available and those which are in
use in Toronto
Discuss the use of molecular methods for
genotyping and resistance testing
Hepatitis A - Diagnosis
Three serologic markers available:
1. Hepatitis A Total (IgG and IgM) antibody
2. Hepatitis A IgM
3. Hepatitis A IgG
First tests available since 1978
No antigen test
Antibody response is similar following
vaccination or wild type infection
Incubation time is 7 to 28 days
Hepatitis A Virus Infection
Typical Serologic Course
Symptoms
Total antiHAV
Titer
ALT
Fecal
HAV
0
1
IgM anti-HAV
2
4
5
6
3
Months after Exposure
1
2
2
4
Laboratory Tests for HBV
Serology:
– Many tests available – most common tests are
Enzyme Immunoassays (EIAs, MEIAs)
– First tests available in 1972
– For every rule, there is an exception/caveat
– No single test tells you everything
Molecular:
– HBV DNA (quantitative)
– HBV genotyping
– HBV resistance testing
Hepatitis B – Laboratory Tests
Serologic markers:
1) HBsAg (Hepatitis B surface antigen):
• if positive, person is infectious
• Sensitivity = 0.15 ng/ml
• Specificity = 99.5%
2) Anti-HBs (Antibody to HBV surface antigen):
• indicates immunity to HBV and protection
from disease
• Protective level is >10 IU/ml
Hepatitis B – Laboratory Tests
Serologic markers:
3) Anti - HBc (Antibody to HBV core antigen):
• Total - indicates past or active infection;
present whether person is immune or
chronic carrier
• Specificity = 99.8% to 99.9%
• IgM - early indicator of acute infection
• No antigen test
Hepatitis B – Laboratory Tests
Serologic markers:
4) HBeAg (Hepatitis Be antigen):
• indicates person is highly infectious
• Selecting patients for therapy
5) Anti-HBe (Antibody to HBVe antigen):
• prognostic for resolution of infection;
less infectious; spontaneous
seroconversion in 10 to 20% of
healthy adults per year
Acute Hepatitis B Virus Infection with Recovery
Typical Serologic Course
Symptoms
HBeAg
anti-HBe
Total anti-HBc
Titer
HBsAg
0
4
8
anti-HBs
IgM anti-HBc
12 16 20 24 28 32 36
Weeks after Exposure
52
100
Progression to Chronic Hepatitis B Virus
Typical Serologic Course
Acute
(6 months)
Chronic
(Years)
HBeAg
anti-HBe
HBsAg
Total antiHBc
Titer
IgM anti-HBc
0 4 8 12 16 20 24 28 32 36
52
Weeks after Exposure
Years
Virological and Biochemical Course
of Chronic Hepatitis B
Disease Phases in Chronic HBV Infection
Phase
HBsAg HBeAg AntiHBe
ALT
HBV DNA
range
Immune
Tolerant
+
+
-
Normal
>8 log IU/mL
Immune
Clearance
+
+
-
Normal or
elevated
3-8 log IU/mL
Inactive
Disease
+
-
+
Normal
<3 log IU/mL
HBeAgnegative
Chronic
HBV
+
-
+
Normal or
elevated
3-8 log IU/mL
Interpretation of Serologic Tests in
Hepatitis B
Hepatitis B – Laboratory Tests
Serologic markers – caveats:
Persistent HBsAg for >6 mos = chronic infection
HBsAg and anti-HBs may co-exist in up to 24% of
chronically infected individuals; likely due to
mutations in the “a” determinant of the S gene
– Surface antigen escape mutants described in infants
infected with HBV after HBIG + vaccination and in
Liver transplants after prolonged HBIG
Anti-HBc IgM may persist for up to 2 years in
20%; chronically infected individuals may have
low titres which rise during acute flares
Hepatitis B – Laboratory Tests
Serologic markers – caveats:
Precore or HBeAg negative mutants:
– Due to mutation in precore (abolishes HBeAg
production) or core promoter region (downregulates HBeAg production)
– No effect on viral replication (may be enhanced)
– More difficult to treat; greater risk of cirrhosis
Co-infection with HCV may suppress both
HBeAg and HBsAg
HBV Viral Genome Organization
HBcAg
Hepatocyte
receptor
bindng site
HBeAg
Protein that
transactivates
transcriptional
promotors
HBsAg
3200 Base Pair Genome
HBV DNA Polymerase
Hepatitis B – Laboratory Tests
Serologic markers – caveats:
Isolated HBcAb may be due to:
– Remote infection (immune or chronic carrier)
– “Window” period between HBsAg and HBsAb
– Co-infection with HCV
– False positive test result – HBcAb is marker
most prone to false positives
HBV DNA may help sort this out
Laboratory Tests for HCV
Serology:
Detection of anti-HCV antibodies
Serologic test available since 1990
Molecular:
HCV RNA detection
Determination of HCV genotype
Viral load determination
Laboratory Tests for HCV
Serology:
Screening:
– 3rd generation EIAs measure antibodies directed
against recombinant peptides NS4, core, NS3,
and NS5 proteins
– Sensitivity = 97%
– Detects antibodies within 6 to 8 weeks
– No HCV IgM test available
Confirmatory/supplementary:
– RIBA, LiPA, Second EIA, HCV RNA
Serologic Pattern of Acute HCV Infection with
Progression to Chronic Infection
antiHCV
Symptoms +/-
Titer
HCV RNA
ALT
Normal
0
1
2
3
4
Months
5
6
1
2
3
Years
Time after Exposure
4
Rational Use of HCV Diagnostic Tests
TREATMENT
Diagnosis
Prognosis
Serological
assays
Qual HCV RNA
Liver
histology
Decision
to treat
Treatment
duration
ALT
Liver histology
Qual HCV RNA
Genotyping
Viral load
Response
and resistance
assessment
Qual HCV RNA
Viral load
Hepatitis D Virus - Diagnosis
• Anti-HDV Total (IgG & IgM) available
• Incubation time – similar to Hepatitis B
• High titres of HDV antibodies indicate ongoing
chronic infection
• Available only at National Microbiology Lab in
Winnipeg
Hepatitis E Virus - Diagnosis
• Both IgG and IgM antibody tests are
available
• Incubation period – 7 to 28 days
• No domestically acquired cases in Canada
• Available only at the National Microbiology
Lab in Winnipeg
Molecular Tests for Hepatitis
Hepatitis Virus – Molecular Tests
Molecular assays available as follows:
– Commercial assays for HBV DNA and HCV RNA
– In-house assays for HAV RNA & HDV RNA
– No molecular assay for HEV RNA
HCV RNA & HBV DNA, plasma or serum must
be separated from cells within 6 hrs and plasma
can be stored at 4oC for several days or -70oC
for long-term
No licensed tests for diagnostic purposes; all
tests are for monitoring or donor screening
– HCV RNA will be done in HIV or other
immunocompromised patients if requested
Hepatitis Virus – Molecular Tests
Lower limit of Detection (LLD) does not
equal dynamic (linear) range of
quantitative assays
– Determined by PROBIT analysis to determine
the value that is consistently detected 95% of
the time
Results of different assays may (HBV) or
may not (HCV) be interchangeable
Nucleic Acid Amplification Tests (NAAT)
for Detection of RNA/DNA
Quantitation of RNA or DNA may be reported as
copies/ml or IU/ml
Conversion factor for copies/ml to IU/ml is not
the same for different assays measuring the
same target or different targets
– HBV DNA: 5.82 copies/IU
– HCV RNA: PCR - 2.4 copies/IU; bDNA: 5.2 copies/IU
Coefficient of variation (COV) may range from
15 to 50%
HBV DNA Quantification Assays
Assay
Versant bDNA v3.0
(Siemens)
Hybrid Capture II
(Digene)
Liquid Hybridization
(Abbott)
Cobas Amplicor
Monitor (Roche)
Sensitivity
(pg/ml)*
LLD
(copies/ml)*
2.1
2 x 103
2 x 103 to
1 x 108
15 - 37%
0.02 to 0.5
5 x 103
5 x 103 to
6 x 107
10 – 15%
1.6
6 x 105
5 x 105 to
1 x 1010
12 – 22%
0.001
2 x 102
2 x 102 to
2 x 105
14 – 44%
Cobas Taqman
(Roche)
RealArt HBV PCR
(artus/Qiagen)
*283,000 copies/pg; 5.26 copies/IU
Linearity
Coefficient
(copies/ml) of Variation
35 (Manual)
2 x 102 to 1
x 1010
70 (Automated)
10
16 – 54%
1 to 4 x 108
A. Lok et al. Hepatology 2001;34; J. Servoss et al. Infect
Dis Clin N Am 2006;20; B. Weber. Future Drugs 2005
Measuring HBV DNA
Gish and Locarnini, Clin Gastro Hep 2006
Comparison of Quantitative HBV DNA Assays
Versant 3.0 vs. Versant 1.0:
R2 = 0.9001
Versant vs. Cobas:
R2 = 0.7711
Versant vs. Digene:
R2 = 0.9849
Yao J et al. J Clin
Microbiol 2004:42(2)
HCV RNA Detection Assays
Assay
Method
LLD*
(IU/ml)a
Linearity
(IU/ml)
TMA
5 - 10
NA
Amplicor Qualitative v2.0 (Roche)
RT-PCR
50
NA
Ampliscreen (Roche)
RT-PCR
50
NA
Amplicor Monitor v2.0 (Roche)
RT-PCR
600
600-800,000
Cobas Taqman (Roche)
RT-PCR
15
15 – 1 x 108
Abbott RealTime (Abbott)
RT-PCR
12 - 30
10 – 1 x 107
Versant Quantitative v3.0
(Siemens)
bDNA
615
615 7,700,000
Versant Qualitative (Siemens)
*LLD = Lower Limit of Detection;
aConversion
factor IU/ml to copies/ml varies with
each assay (e.g. PCR: 1 IU/ml = 2.4 copies/ml;
bDNA: 1IU/ml = 5.2 copies/ml)
S. Chevaliez et al. World J Gastro 2007;13;
J Scott et al. JAMA 2007;297; A. Caliendo
et al. J Clin Microbiol 2006;44
HBV DNA in Clinical Practice
Routine monitoring on therapy to assess
response to treatment
– Every 3 months X years on oral agents
– Every 1 month X 6-12 on PEG/IFN
Routine monitoring off therapy to estimate
prognosis and to evaluate need for
treatment
– Every 6 –12 months normally
– Diagnosis of occult HBV infection
Laboratory Tests for HCV
Molecular:
Both qualitative and quantitative HCV RNA
assays available
Used for treatment monitoring (and in some
circumstances for confirmation of positive or
indeterminate serology)
HCV RNA is detectable 2 to 14 days after an
exposure
Ministry of Health
and Long-Term Care
Ministère de la Santé
et des Soins de longue durée
Laboratories Branch
P.O. Box 9000, Terminal “A”
(81 Resources Road, Etobicoke)
Toronto, Ontario M5W 1R5
Direction des Laboratoires
C.P. 9000, Terminal “A”
(81 Chemin Resources, Etobicoke)
Toronto (Ontario) M5W 1R5
Telephone: (416) 235-5737
Facsim ile: (416) 235-6197
Téléphone: (416) 235-5737
Télécopieur: (416) 235-6197
PHL LAB INFORMATION FORM
FOR HEPATITIS C RNA AND HEPATITIS B VIRAL LOAD
THIS IS NOT A DATA S HEET, please attach to PHL test requisition
Minimum volume 2.5 mL serum, removed from clot within 4 hours and submitted frozen to the lab.
PATIENT'S NAME: ___________________________________
PHL LAB#: _________________
DR’s NAME ______________________________
HEPATITIS C (HCV) RNA - QUANTITATIVE
Baseline and Genotyping (Pre-Treatment)
Quantitative Viral Load - Week 12 of treatment
Quantitative Viral Load – due to change in treatment/dose ______ weeks
HEPATITIS C (HCV) RNA - QUALITATIVE
Query the presence of active HCV infection
(HIV immunocompromised, infant of HCV positive mother, patient with anti HCV
indeterminate result, 8-10 weeks post exposure, etc).
Confirm successful treatment at 4 weeks (genotypes 2 and 3)
On Treatment
_________ weeks
Post Treatment
_________ weeks
(2 samples less than the detection limit (<50 IU/mL) and 6 months apart are required to confirm
successful treatment. No follow up required unless there is a new exposure).
HEPATITIS B (HBV) VIRAL LOAD
(NOTE: Test is not u seful for diagnosis. Research use only)
Pre-Treatment for HBV
On Treatment
_________ weeks
Post Treatment
LFTs/Clinical Information:
ALT
___________
AST
__________
Genotyping
Used for:
– Detection of mutations that confer resistance to
antiviral agents
– Genotyping of isolates for epidemiological purposes;
categorizes patient isolates into 8 different HBV
genotypes (A to H) and 6 different HCV genotypes (1
to 6 with 24 subtypes)
Methods include:
– Sequencing
– Hybridization (Line Probe Assay, Trugene Assay)
Laboratory Diagnosis of Resistance
Sequencing
Line Probe
Pros
Cons
Discovers
Labor-intensive
new mutations
Low sensitivity
(15-20% pop.)
High throughput
Detects known
High sensitivity
(5-10% pop.)
mutations only
InnoLiPA Principle
Chromogen
(NBT/BCIP)
Marker line
Conj.cont.
Amp.cont.
Purple
precipitate
Alkaline
Phosphatase
L180
Streptavidin
M180
Biotin
M204
V204
Amplified
target
I204
V207
L207
M207
I207
DNA-probe
Nitrocellulose
strip
InnoLIPA HBV Drug Resistance
M a r k e r lin e
1 -
C o n j. C o n tr o l
2 -
A m p .C o n t ro l
3 -
L80 W T
4 -
V 8 0 M u ta n t
5 -
I 8 0 M u ta n t
6 -
V173 W T
7 -
G 173 W T
8 -
L 1 7 3 M u ta n t
9 -
L180 W T
10 -
M 1 8 0 M u ta n t
11 -
A181 W T
12 -
T 1 8 1 M u ta n t
13 -
V 1 8 1 M u ta n t
14 -
M 204 W T
15 -
V 2 0 4 M u ta n t
16 -
I 2 0 4 M u ta n t
17 -
S 2 0 4 M u ta n t
18 -
N 236 W T
19 -
T 2 3 6 M u ta n t
HBV Resistance Testing
HBV Resistance Testing
InnoLiPA vs. Sequencing
Hussein et al, J Clin Micro 2006
Drug Resistance Report
LAMIVUDINE
Present
Mixed
L80V
V173L
Absent
X
X
L180M
X
M204V
X
M204I
X
M204S
X
ADEFOVIR
Present
Mixed
Absent
A181V
X
A181T
X
N236T
X
Interpreting HBV DR Reports
Resistance Mutation
Sensitive
Resistant or
Reduced
Susceptibility
Lamivudine
Resistance
Adefovir
Resistance
Adefovir
Resistance
L180M +M204V
N236T
A181V/T
Adefovir
Tenofovir
Lamivudine
Entecavir
Telbivudine
Emtricitabine
Lamivudine
Tenofovir
Entecavir
Emtricitabine
Lamivudine
Entecavir
Telbivudine
Emtricitabine
Clevudine
Adefovir
Tenofovir
Clevudine
Adefovir
 L180M + M204V/I are the key lamivudine-resistant mutations
 A181V/T leads to 4-fold increase in IC50
 N236T leads to 7-fold increase in IC50
Diagnostics in Viral Hepatitis: Summary
Serology remains the cornerstone for diagnosis
and screening
NAAT is critical to patient management
Of the many NAAT tests available, PCR, bDNA
and TMA remain most popular
– Sensitivity and dynamic range varies between assays
– Standardization allows (to some degree)
interchangeability of the results with different assays
Resistance/Genotyping requires amplification
first
– Increasing role in making treatment decisions as
more drugs become available for HBV