Upload
others
View
0
Download
0
Embed Size (px)
Citation preview
Diagnosis of Occult Diagnosis of Occult ggHepatitis B Infection Hepatitis B Infection
(OBI), and (OBI), and TroubleshootingTroubleshooting
Dr Seyed Mohammad Dr Seyed Mohammad JazayeriJazayeriDr Seyed Mohammad Dr Seyed Mohammad JazayeriJazayeri
MD, PhD Clinical VirologistMD, PhD Clinical Virologist
Hepatitis B LabHepatitis B Lab DeptDept Virology Virology
MD, PhD Clinical VirologistMD, PhD Clinical Virologist
Hepatitis B LabHepatitis B Lab DeptDept Virology Virology Hepatitis B LabHepatitis B Lab--DeptDept Virology Virology
Tehran University of Medical SciencesTehran University of Medical Sciences
Hepatitis B LabHepatitis B Lab--DeptDept Virology Virology
Tehran University of Medical SciencesTehran University of Medical Sciences
IntroductionIntroductionApplication of Real Time PCR for OBI Di iDiagnosisNested PCR for OBI DiagnosisMutational Analysis for OBIModel Prediction for OBI pathogenesisModel Prediction for OBI pathogenesis
2 billion people have been infected by HBV ld idworldwide.
350‐400 million suffer from chronic HBV infection of whom 25% will die of chronic liver disease or HCC.Currently over 1/000/000 people die each year from the consequences of HBV y qinfection (Fulminant hepatitis, Cirrhosis and Hepatocellular Carcinoma).p )
The World Health Organization places h i i B i (HBV) i h hepatitis B virus (HBV) in the top 10 causes of death worldwide.Each year there are estimated to be
illi f HBV >350 million new cases of HBV infection worldwide.
A, G A
B, CF, H E
D D
EC
B
F
A
Perinatal/ Adult
A SUMMARY OF OUTCOME OF HBV INFECTION
9510-70% Perinatal/childhood
Acute Infection
AdultAcute
InfectionRecovery Recovery
95%
Chronic Infection
<5%30-90%
Mild ? Moderate ? Severe? I ti
20-30%
Mild ? Moderate ? Severe?Chronic Hepatitis
InactiveCarrier State
Cirrhosis
Decompensation TransplantationDeath HCC
Perinatal/ Adult
A SUMMARY OF OUTCOME OF HBV INFECTION
9510-70% Perinatal/childhood
Acute Infection
AdultAcute
InfectionRecovery Recovery
95%
Chronic Infection
<5%30-90%
OBIOBI
Mild ? Moderate ? Severe? I ti
20-30%
Mild ? Moderate ? Severe?Chronic Hepatitis
InactiveCarrier State
OBICirrhosis
OBIOBI
Decompensation TransplantationDeath HCC
OccultOccult HepatitisHepatitis BB infectioninfection isis defineddefined asas::“Detectable“Detectable HBVHBV DNADNA AmongAmong PatientsPatientsNegativeNegative ForFor HBsAgHBsAg””..OccultOccult HBVHBV infectioninfection hadhad notnot beenbeen wellwellOccultOccult HBVHBV infectioninfection hadhad notnot beenbeen wellwell
studiedstudied untiluntil HBVHBV polymerasepolymerase chainchainreactionreaction (PCR)(PCR) becamebecame availableavailablereactionreaction (PCR)(PCR) becamebecame availableavailable..
HBsAg detection by immunoassay is the basis for diagnosis of HBV infection infection. This is dependent on excess HBsAg p gin blood. Th d t t bilit f HB A i The non‐detectability of HBsAg in serum may arise because of several serum may arise because of several mechanisms:
HBV mutants with defective li i i i h i f S replication activity or synthesis of S
proteins respectively due to proteins respectively due to mutations in the Pol gene or in the
hS promoter genomic region, thus escaping detection by routine testsescaping detection by routine tests.(Immune/Diagnostic escape)
Diagrammatic presentation of HBV life cycleTaken from Chisari, 1997.
Major Hydrophilic Region (MHR) and “a” determinant of Hepatitis B Virus Surface Ag, Jazayeri et al, 2012.
Proposed model of the major hydrophilic region (MHR) of HBsAg
١٥
Alternatively, the quantity of (HBsAg in serum is decreased (co‐
infections low carrier state) and infections, low carrier state), and might just be enough for viral assembly but below the sensitivity of standard serologic assay of standard serologic assay.
One of the reasons could be due to f i f i l formation of immune complexes between circulating HBsAg particles g g pand anti‐HBs, leading to high frequency of HBV DNA in the absence frequency of HBV DNA in the absence of HBsAg.
1. Window period (in the post acute phase h HB i d li i when HBs antigen declines or immune
complexes are present)2. Resolving infection (under the detection
limit of the assay in chronic infection who limit of the assay in chronic infection who eliminate HBsAg for many years).HBV ith HDV/HCV/HIV i f ti3. HBV with HDV/HCV/HIV co‐infection.
Cli i l S tti OHB P l (%)Clinical Setting OHB Prevalence (%)
Blood Donors 0.05-13HIV 0- 89HIV 0 89HCV 6.7-91,HCC 12-80Immunosuppression 3.3-37.8Immunosuppression 3.3 37.8Dialysis 0-58Chronic HBV carriers 5-55Cryptogenic cirrhosis 4.8-40yp gTransplantation
LiverStem Cell
36-640-50
Kidney 0-3.3HBV vaccinated 2.7-28
Family contact of HBsAg positive carriers 8 8 28 8Family contact of HBsAg positive carriers 8.8- 28.8
General Healthy Population 0.7- 34Haemophilia 5.3- 51.2
The percentage of OBI among different p g gclinical settings depends on:
1. Methods of DNA detection2. Patient recruitment3. Rate of HBV endemicityNature of biological material 4. Nature of biological material tested
Carriers of occult infection may be aCarriers of occult infection may be a source of HBV transmission in the case of blood transfusion with the consequent development of a typicalconsequent development of a typical type B hepatitis in the recipients.
Immunosuppression induced by:HIV i f i1. HIV infection
2. Organ transplantation3. Any patient with occult HBV receiving
systemic chemo- radio- or immun otherapy.y py4. Hemato-oncologic patients
leading to the development of a typicalleading to the development of a typical hepatitis B that often has a severe – and
ti f l i t li i lsometimes even fulminant – clinical course.
Occult HBV infection is a risk factor for HCC developmentHCC development.
Three follow up studies showed the Three follow up studies showed the role of OBH in HCC development .Th l f lt HBV The prevalence of occult HBV among HCC patients varies from 12% to 80% d di th t ddepending on the study.OBI increases the rate of HCC up to
%3.7%.
HBV DNA is detected 3 to 5 weeks after f d d b f hinfection and up to 6‐15 days before the
appearance of HBsAg.pp gIt rises slowly at low levels (102 to 104
copy/ml) during the early HBsAg copy/ml) during the early HBsAg seronegative window period.I d / l f d i h It exceeds 109 cop/ml often during the prodromal, acute, or chronic phase.
HBV DNA detection is the key to di i f OBI
ydiagnosis of OBI
HBV DNA is the only reliable diagnostic marker of OBI.The HBV DNA detection rate varies between different groups:
1. It is highest in in subjects who are anti‐HBcpositive but anti‐HBs negative.
2. It is intermediate in who are positive for both anti‐HBc and anti‐HBs.
3. It is lowest in sero‐negatives.
During occult infection HBV viral load i ll l ith l th 4is usually low with less than 104
copies/ml.However, the occult HBV viraemia seems to fluctuate over time and seems to fluctuate over time and remains at a higher level in the liver versus serum in comparison to HBV chronic carrierschronic carriers.
The gold standard to test for occult HBV is the analysis of DNA extracts HBV is the analysis of DNA extracts from liver (if not possible) serum samples performed by:samples performed by:Real Time technique (as screening t l)tool)NestedPCR and the use of oligonucletide primers specific for at least two different HBV genomic at least two different HBV genomic regions.
HBsAg Screening
HBsAgpositive
HBsAgNegative
Biopsy and/or serum
Overt HBV
I f i
Real Time PCR
Infection
OBIHBsAg and other
ProteinsNested
PCR
Proteins
Sequencing
Mutational Analysis
90%
80 9%
60%
70%
80%
66%
80.9%
CR(%
)
40%
50%
60% 57%
ntag
e of Pos
itive PC
20%
30%
4
19%
Per
cen
0%
10%
Surface Core, Pre‐Core Pre‐S X
DNA should be isolated using the most efficient gextraction procedure. DNA should be extracted from at least 1 ml of DNA should be extracted from at least 1 ml of serum and that serially collected samples be testedtested.It is mandatory to include appropriate internal and external controls of specificity and sensitivity and external controls of specificity and sensitivity and for contamination in each assay run. M i l i f li i Moreover, sequencing analysis of amplicons is recommended.
Clinical Setting OHB Prevalence (%) Prevalence of OHB in Anti‐g ( )HBc positive patients (%)
Blood Donors 0.05%‐13% 0%‐17%
HIV % 8 % % %HIV 0%‐ 89% 9%‐44%HCV 6.7%‐91 1%, 28%‐71%HCC 12%‐80% 28.8%‐64%
Immunosuppression 3 3% 37 8% 37 8% 62 3%Immunosuppression 3.3%‐37.8% 37.8%‐62.3%
Dialysis 0%‐58% 6.4%‐64.7%Chronic HBV carriers 5%‐55% 7%‐60%
C t i i h i 8% % 8 %Cryptogenic cirrhosis 4.8%‐40% 17.8‐100%
TransplantationLiverStem Cell
36%‐64%
0%‐50%
3%‐100%
4.4%‐100%Stem CellKidney 0. %‐3.3% 3%‐10%
HBV vaccinated 2.7%‐28% 6.5%‐100%
Family contact of HBsAg positive 8.8%‐ 28.8% 23.6%‐ 96.4%Family contact of HBsAg positive carriers
8.8% 28.8% 23.6% 96.4%
General Healthy Population 0.7%‐ 34% 6.1%‐ 51%.
Haemophilia 5.3%‐ 51.2% 6%‐100%
1. HBsAg False Positivity2. Mutations in HBV surface protein.3 Window period (in the post acute phase 3. Window period (in the post acute phase
when HBs antigen declines or immune complexes are present)complexes are present)
4. Resolving infection (under the detection limit of the assay in chronic infection who eliminate HBsAg for many years).g y y )
False positive results are observed with: False positive results are observed with:
1. Heparinised samples 2. Interferences with Hgb or bilirubine2. Interferences with Hgb or bilirubine3. During pregnancy
A i di4. Autoimmune diseases5. Chronic liver diseases5
no.Real-Time PCRno.Real-Time PCR
28%28%
PosNegPosNeg
72%72%
Real Time PCR (Real Time PCR (TaqManTaqMan) was positive in ) was positive in 21 21 ((2828%)%)Real Time PCR (Real Time PCR (TaqManTaqMan) was positive in ) was positive in 21 21 ((2828%)%)
Sample Code Ageα Sex* Anti‐HBc Anti‐HBs Titer(mIU/mL) HBV DNA (copy/mL)14 16 2 + >100 210040 15 1 ‐ 30 200040 15 1 30 200042 61 1 ‐ 28 5546 128 1 ‐ 18 7752 17 2 ‐ >100 127056 18 1 ‐ >100 8156 18 1 >100 8165 32 1 ‐ 95 380067 38 2 ‐ 38 41572 37 1 ‐ >100 22384 57 1 ‐ 36 924084 57 1 36 924086 63 2 ‐ >100 474103 12 1 ‐ >100 468106 66 2 ‐ >100 1920108 35 2 ‐ >100 347108 35 2 >100 347110 10 1 + >100 500112 22 1 ‐ 47 450115 10 1 + 38 1200116 64 2 25 4560116 64 2 ‐ 25 4560616 23 1 ‐ 47 2330122 12 2 + >100 2300125 72 2 + 94 395
“a” determinanta determinant
Mutations Causing Undetectability of sera by Serology
Clinical Presentation No OBI Prevalence No NotesClinical Presentation No
Samples
OBI Prevalence No
Anti-HBc
Notes
Children born to HBsAg- 75 21 (28%) 5 (23%)
Positive Mothers
Cryptogenic Cirrhosis 29 11 (38%) 2 (18%)
( ) T b C ti dChildren Autistic
Disorders
53 1 (1.8%) ?? 1 To be Continued….
Health Care Workers 120 4 (3.3%) 0Health Care Workers 120 4 (3.3%) 0
Vaccinated Children 100 27 (27%) ?? 0 To be Continued….
Behcet Syndrome 36 2 (5 5%) NI To be Continued….Behcet Syndrome 36 2 (5.5%) NI
HIV-Positive 172 31 (18%) 20 (64.5%)
HTLV 1 P iti 109 1 (0 9%) 1HTLV-1 Positive 109 1 (0.9%) 1
Dentists 55 (1628) 1 (1.8%) 1
Serodiscordant Chronic 5 5 1 (20%)
ItIt isis suggestedsuggested thatthat HBsAgHBsAg negativitynegativity isis notnotsufficientsufficient toto completelycompletely excludeexclude HBVHBV DNADNAsufficientsufficient toto completelycompletely excludeexclude HBVHBV DNADNAcarrierscarriers..
TheseThese resultsresults highlightshighlights thethe factfact thatthat antiantiTheseThese resultsresults highlightshighlights thethe factfact thatthat antianti--HBcHBc,, antianti--HBsHBs andand HBsAgHBsAg maymay notnot bebeeffectiveeffective toolstools forfor diagnosisdiagnosis ofof HBVHBV infectioninfectioneffectiveeffective toolstools forfor diagnosisdiagnosis ofof HBVHBV infectioninfectioninin somesome patientspatients..
The use of sensitive molecular tests such as real‐time PCR would be helpful in solving these problemsthese problems.
Appropriate application, quality control and Appropriate application, quality control and standardization of serologic assays are issues that must also be addressedissues that must also be addressed.
These patients must be followed upcarefully as chronically infected individualscarefully, as chronically infected individualswith or without vaccine escape mutants are
t i li di i l diprone to various liver diseases, includingcirrhosis and hepatocellular carcinoma laterin life.
These findings highlight the need for an g g galternative regimen, such as the administration of boosters or a more administration of boosters or a more effective HBV vaccine (a third-generation or HBV DNA vaccine) especially for high-or HBV DNA vaccine), especially for high-risk children.
Please Turn Please Turn Hepatitis Off !Hepatitis Off !