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.

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