Hepatitis B Virus and Hepatitis C Virus infections are transmitted by parentral route. Early diagnosis and treatment can prevent cirrhosis of liver in HCV cases as drugs which can cure the infection are now available.

4. Hepatitis
• Hepatitis is a general term referring to inflammation of the liver
• Causes:
• Infectious
• Viral
• Bacterial
• Fungal
• Parasitic
• Non infectious
• Alcohol
• Drugs
• Autoimmune
• Metabolic diseases

5. Viral Hepatitis
Hepato-tropic viruses
• Hepatitis A Virus (HAV)
• Hepatitis B Virus (HBV)
• Hepatitis C Virus (HCV)
• Hepatitis D Virus (HDV)
• Hepatitis E Virus (HEV)
Other viruses
• Adenovirus
• Cytomegalovirus (CMV)
• Epstein Barr virus (EBV)
• Herpes simplex virus (HSV)
• Yellow fever virus (YFV)

6. Public Health Importance of Hepatitis B & C
• Major cause of acute and chronic liver disease
• Lead to 1.4 million death annually = death due to TB & > than HIV
• 248 million persons living with chronic HBV infection
• 100 million in South East Asia, 40 million Indians are infected
• 110 million persons living with HCV infection
• 30 million in South East Asia, 6 to 12 million Indians are infected
• Availability of DAA against HCV &
• Highly effective Tenofavir & Entecavir against HBV
• Have made WHO aim for Eradication of Viral Hepatitis by 2030

8. Viral Markers in the
Screening, Diagnosis & Monitoring of
Chronic Hepatitis B & C
Prof. Ashok Rattan,
MD, MAMS,
Common Wealth Fellow, INSA DFG Fellow, SEARO Temporary Advisor,
WHO Lab Director (CAREC/PAHO)

13. Serology for HBV Infection
Antigens
• HBsAg
• HBcAg *
• HBeAg
* Not detected in serum
Antibodies
• Anti HBs
• Anti HBc IgM & Anti HBc Total
• Anti HBe

17. HBV genotypes in India

19. HBV Genotype: The utility for the clinician
10 genotypes; 35 subgenotypes
• Disease severity & HCC development
• are influenced by genotype. HBV DNA level, sex, age, precore A1896 mutation & basal
core promoter T 1762/ A 1764 mutation.
• High in A1, C, D & F; low in A2, B
• Antiviral therapy is influenced by genotype especially IFN α; high in A1 & B;
poor in C,D & G
• Disease chronicity: B & C more common; Genotype A associated with acute
infection
• HBV transmission: horizontal in A1, D, E to I; vertical in A2, B & C
• Liver transplantation
• Occult HBV is infection where HBsAg is --ve but HBV DNA +ve
• Can be used for understanding epidemiology

20. HBV Genotyping Methods
Method Advantages Disadvatages
RFLP Easy, simple, rapid, low cost Low sensitivity for typing
isolates with low HBV load
Reverse Hybridization High sensitivity, automated High cost
Genotype specific PCR High sensitivity, easy, suitable
for mixed infections
High cost
Sequence analysis Gold standard Time consuming, technically
demanding, high cost

21. HBV infections : Who are at risk
• Infants born to an infected mother
• Persons who suffer from STDs
• Men who have sex with men
• Sex partner of an infected person
• Persons on haemodialysis
• Injected Drug users
• House hold contact of infected persons
• Health care workers

22. Concentration of Hepatitis B Virus
in various body fluids
High Moderate Low / not detected
Blood Semen Urine
Serum Vaginal fluid Faeces
wound Saliva Sweat
Tears
Breast Milk

25. • Serum anti HBc Ab is most useful & inexpensive marker for identification of
occult HBV infection in HBs Ag negative individuals

28. Immune Tolerant Immune Clearance Immune Control Immune Escape
High HBV DNA level High HBV DNA level Low HBV DNA level High HBV DNA level
Normal ALT Elevated ALT Normal ALT Elevated ALT
HBeAg +ve HBeAg +ve HBeAg –ve; anti Hbe +ve anti HBe +ve
Monitor every 6 to 12 months At risk of progression Monitor every 6 to 12 months At risk of progression
Consider treatment Consider treatment

29. HBs Ag
• Serological hallmark of HBV infection
• After acute exposure to HBV, HBsAg appears in serum in 1 to 10 wks
• Persistence beyond 6 M  chronic Hepatitis B (CHB)
• There is correlation between transcriptional activity of cccDNA in the
hepatocytes and serum HBsAg quantitation (qHBsAg)
• Monitoring of qHBsAg levels predict response to treatment esp peg-IFN
• Serum HBsAg titres are higher in HBeAg +ve CHB

30. Anti HBs
• Neutralizing antibodies that confer long term immunity
• After vaccination, anti HBs is the only serological marker detected
• Titres >10 mIU/ml confer immunity
• When a person recovers from natural infection, it is present along
with anti HBc IgG

31. HBe Ag & anti HBe
• In the past HBeAg and anti HBe levels have been used to know about
infectivity & viral replication.
• Now HBV DNA is used for that purpose
• Viral load is usually higher in HBeAg +ve CHB
• HBe Ag  anti HBe sero-convertion is related to remission

32. HBc Ag & anti HBc IgM & IgG
• HBcAg is only present within the hepatocytes and not detectable in
serum
• Antibodies to HBcAg are not neutralizing antibodies & so not
protective
• Presence of anti HBc indicate past infection
• IgM appear within 1 to 2 wks of acute infection and peter off within 6
months
• IgG continue to be detected in both patients & resolved HBV infected
CHB

33. HBV Viral Load
• HBV DNA is a direct measurement of viral load & reveals the replicative
activity of the virus
• HBV DNA is detectable at an early stage of infection & increases upto peak
levels of more than 108copies/ml approx. in 3 Months HBV DNA thereafter
either disappears (Cure) or decreases and persists (chronic)
• As prevalence of serological negative HBV infections (HBeAg –ve & occult
HBV infection) has increased, HBV DNA has become a reliable marker of HBV
infection
• Higher titres are associated with more rapid disease progression & higher
incidence of HCC

34. HBV Viral Load
• HBV DNA testing is useful in routine clinical setting
• To detect pts who need treatment
• Monitor those on treatment
• Two principle techniques
• 1. Signal amplification
• Hybrid capture
• Branched DNA
• 2. Target amplification
• qPCR (15 to 108 IU/ml)

35. HBV Viral Load Techniques
Method Assay Manufacturer Measurement range Limit of detection
Semi automated qPCR COBAS AmpliPrep
COBAS TaqMan
Roche 20 to 1.7 x 107 20
Semi automated RT PCR COBAS TaqMan HBV
Quant
Roche 29 to 1.1 x 107 6
RT PCR Abbott RT PCR Abbott 10 to 109 10
Branched DNA Versant HBV
3.0 assay
Siemens 2000 to 108 2000

36. 4. Monitoring treatment response for HBV ?

37. Viral load monitoring

38. Clinical significance of HBV markers
HBV marker Diagnostic category
Anti HBs antibodies Immunity
Anti HBc antibodies Exposure
HBs Ag &/or HBV DNA Infection
HBe Ag &/or HBV DNA Replication
IgM anti HBc &/or HBV DNA Disease

39. Liver Biopsy
Normal Cirrhosis

40. Liver Biopsy
Biliary cirrhosis Hepatocellular Carinoma

41. Non Invasive Methods
Fibroscan Liver Stiffness (kPa)

42. Tests for evaluation of hepatic fibrosis
Test Availability Formula Advantages Disadvantages
Liver biopsy In hospitals
/surgical centres/
labs
Pathological tissue evaluation Gold standard High cost, invasive, risk of
complications, painful,
sampling error
APRI Anywhere where
basic lab tests are
done
[(AST/ULN)/Plt] x 100 Cheap, non proprietary,
helpful for ruling in F3-F4
Low scores donot exclude
advanced fibrosis, suboptimal
for pts with CD4 < 250
FIB-4 “ (Age x AST)/(Pltx ALT) “, validated for pts with
CD4 < 250
Difficult to classify pts with
values in the mid range
Fibro Test /
FibroSure
Lab sent out Formula is proprietary,
components include α 2
macroglobulin, haptoglobin,
gamma globulin, apolipoprotein,
AL, GTT, total bilirubin, age, sex
Useful for distinguishing
between significant &
mild fibrosis
More expensive than APRI &
FIB-4, proprietary formula
HepaScore Lab send out Formula is proprietary
Transient
elastography
Only where
machines are
available
Measures shear wave velocity; a
50 MHz wave is passed into liver,
coverts liver stiffness score
Noninvasive, immediate
results, low values have
high NPV for F3-F4
Require machine, not
possible if there is ascites,
confounded by elevated ALT

43. Non Invasive marker cut offs for prediction of
stages of fibrosis
Test Stage of
fibrosis
No. of patients Cut off AUROC Sensitivity Specificity PPV NPV
Fibroscan F3
F4
560 HCV +ve
1855 HCV +ve
10 kPa
13 kPa
0.83
0.90
72%
72%
80%
85%
62%
42%
89%
95%
ARFI (VTQ) F3
F4
2691 including
1428 HCV +ve
1.60 m/s
2.19 m/s
0.94
0.91
84%
86%
90%
84%
n.a. n.a.
Aixplorer F3
F4
379 HCV +ve 9 kPa
13 kPa
0.91
0.93
90%
86%
77%
88%
n.a. n.a.
Fibrotest F4 1579 0.74 0.82 63% 81% 40 93
FIB 4 F4 2297 HCV +ve 1 – 45
3.25
0.87 90%
55%
58%
92%
n.a. n.a.
APRI F4 16,694 HCV
+ve
1.0
2.0
0.84 77%
48%
75%
94%
n.a. n.a.
APRI= aspartate aminotransferase to platelet ration index ARFI = acoustic radiation force impulse;
AUROC= Area under the receiver operating characteristic curve; FIB-4=fibrosis 4;

44. Follow up of Patients not on antiviral treatment
• HBeAg +ve with high HBV DNA but normal ALT (Immune Tolerant)
• Monitored at 3 to 6 M interval, more frequently if ALT levels rise
• If HBV DNA > 20,000 IU/ml & HBeAg +ve & ALT 2 X the upper limit of
normal (.50 U/L for women & > 70 U/L for men)  consider for anti
viral treatment
• Consider liver biopsy if persistently borderline ALT
• If > 40 years and acquired infection many years back
• Pts with moderate to severe inflammation and / or fibrosis (F2 or
higher)  consider for antiviral treatment
• Noninvasive methods can be used instead of liver biopsy

45. Inactive CHB
• HBeAg –ve, anti HBe +ve, ALT normal, HBV DNA < 2,000 IU/ml
• Moniotred with ALT every 3 months during first year
• Thereafter every 6 months; more frequently if ALT becomes elevated
• qHBsAg and HBV DNA in HBeAg –ve cases can help if pt is in gray zone
between inactive & immune active CHB
• qHBsAg < 1,000 IU/ml & HBV DNA < 2,000 IU/ml  inactive CHB
• Sensitivity 71%, Specificity 85%

46. Screening for Hepatocellular Carcinoma
• All HBsAg +ve pts with cirrhosis should be screened
• Ultra Sound with or without
• Alpha fetoprotein
• Every 6 months

47. Strategy for Screening
• HBsAg & anti HBs should be used for screening
• Or anti HBc can be used for screening
• Those +ve are further tested for by both
• HBsAg and anti HBs
• To differentiate between current & previous HBV exposure
• HBV vaccination doesnot lead to anti HBc antibody production

48. Common Testing Strategies for HBV infection
• Blood Screening: HBsAg, multiplex NAAT for HBV DNA [HIV-1 RNA, HCV RNA]
• Diagnostic testing strategies:
• Antenatal : HBsAg, if possible also anti HBs, to assess need for vaccination
• Preoperative : HBsAg
• Health check up : HBsAg, if possible also anti HBs, to assess need for vaccination
• Protective immunity : anti HBs antibodies, should be > 10 IU/ml for protection
• HCW pre employment: anti HBs antibodies, should be > 10 IU/ml for protection
• Diagnosis of acute infection : HBsAg, anti HBs, anti HBc IgM
• Diagnosis of chronic HBV infection : HBsAg, anti HBs, anti HBc
• Degree of infectivity when HBsAg positive: HBeAg, anti Hbe, HBV DNA
• Monitoring of therapy : Quantitative HBV DNA, HBsAg, HBeAg, anti HBs, anti HBe

49. High Risk Groups Who should be screened for
HBV infection
• Persons born in regions of high HBV prevalence ( HBsAg prevalence > 2%)
• Persons who have ever injected drugs
• Men who have sex with men
• Persons needing immunosuppressive therapy including chemotherapy,
immunosuppression for organ transplant
• Individuals with elevated ALT or AST of unkown etiology
• Donors of blood, plasma, organs, tissue or semen
• Persons with end stage renal disease, on haemodialysis
• All pregnant women
• Infants born to HBsAg positive mothers
• Persons with chronic liver disease eg. HCV or with HIV
• Persons with multiple sex partners
• Health care workers
• Unvaccinated persons with diabetes

50. Interpretation of screening tests for HBV infection
HBsAg Anti HBc Anti HBs Interpretation Management
+ + -- Chronic hepatitis B Additional testing &
management
-- + + Post HBV infection,
resolved
No further management
unless
immunocompromised
-- + -- Post HBV infection,
resolved or false
positive
HBV DNA testing if
immunocompromised
-- -- + Immune No further testing
-- -- -- Uninfected and not
immune, susceptible
No further testing

51. Diagnostic criteria & Definitions for chronic Hepatitis B
Test Chronic Hep B Immune
Tolerant
Immune Active Inactive CHB
HBsAg +ve, > 6 M +ve, >6 M +ve, > 6 M +ve, > 6 M
HBeAg a. +ve
b. --ve
a. +ve
b. --Ve
a. +ve
b. --Ve
--ve
Anti Hbe +ve
HBV DNA Varies from
undetectable to
several billion IU/ml
Very high
(typically 106IU/ml)
a. >20,000 IU/ml in HBeAg
+ve
b. < 2,000 IU/ml when
HBeAg --ve
< 2,000 IU/ml
ALT Normal or elevated Normal or minimally
elevated
Persistently elevated Persistently normal
Liver biopsy Chronic hepatitis with
variable fibrosis
Minimal
inflammation
no fibrosis
Chronic hepatitis moderate
to severe inflammation with
or without fibrosis
Absence of
inflammation
Variable levels of
fibrosis

52. Management of HBsAg +ve without cirrhosis
who are HBeAg +ve

53. Management of HBsAg +ve without cirrhosis
who are HBeAg --ve

55. End points of therapy for
chronic hepatitis B infection
End point Criteria
Biochemical Normal ALT levels
Serologic HBeAg loss & sero conversion to anti HBe
HBsAg loss , with/or without sero conversion to anti HBs
Virologic Sustained decrease in serum HBV DNA to undetectable level
Histologic Reduction in fibrosis stage
No worsening of fibrosis
Reduction of inflammatory activity

59. HBV Drug Resistance Testing
• Primary Resistance: Failure of antiviral treatment to reduce HBV DNA level
> 1 x log 10 IU/mL within 3 months of initiating therapy
• Secondary Resistance: Rebound of HBV DNA of > 1 log 10 IU/mL from the
nadir in persons with an initial antiviral treatment effect (> 1 x log 10 IU/mL
decrease in serum HBV DNA)
• Confirmation of antiviral drug failure can be established by sequencing HBV
DNA polymerase & identifying specific genetic markers
• Of the 6 NAs (lamivudine, adefovir, entecavir, telbivudine, tenofovir,
emtricitabine), lamivudine is associated with highest rate of drug
resistance, entecavir with very low rates & tenofovir with no resistance

61. Cumulative incidence of resistance

63. cccDNA acts as a template for viral replication & is responsible for persistence even after HBsAg
clearance & HBV DNA SVR, HBcrAg (core related) biomarker can provide clinicians with reliable
information on HBV infected patients in a non invasive manner. Serum levels of HBcrAg level
reflect cccDNA in hepatocytes in both HBeAg +ve or – ve patients. LUMIPULSE G HBcrAg

72. HCV Genotypes in India

73. Classification of HCV Genotype

74. Risk Factors

80. HCV POCT
Test Manufacturer Nature of
device
Matrix Volume
needed
Time to
result
CE
marked
FDA
approved
WHO
prequalified
OraQuick OraSure Lateral flow Oral fluid,
whole blood,
serum,
plasma
Oral fluid 40
ul, others 5 ul
20 – 40
min
Yes Yes Yes
Toyo Turklab,
Turkey
Lateral flow WB, S, P 60 ul WB or
30 ul S, P
5 - 15 Yes No No
Signal HCV
ver 2.0
Span, India Flow through S, P 100 10 Yes No No
Labmen
HCV
Turklab,
Turkey
Lateral flow WB, S, P 10 15 No No No
MultiSure
HCV
MPBiomedicals,
Singapore
Lateral flow WB, S, P 25 15 Yes No No
Assure HCV “ Lateral flow WB, S, P 50 ul WB
5 ul S, P
15 No No No
First
Response
Premier
Medical, india
Lateral flow WB, S, P 35 15 No no No
SD Bioline Standard Lateral flow WB, S, P 10 5 - 20 No No yes

81. HCV RNA automated systems available
Roche
Molecular
System
Abbott
Diagnostics
Siemens Sacace
Biotechno
logies
Quaigen Hologenic
Inc
Qualitative
assay
COBAS Ampli Prep
HCV Qualitative
(LLOD: 15 IU/ml)
Quantitative COBAS Quant
Ver.2
Abbott RT HCV
Assay
VERSANT kPCR
HCV RNA
HCV RT TMA
Quant Dx
Artus HCV QS-
RCQ kit
RT-TMA
Technology
Linearity
Range of
quantification
IU/ml
15 to 108 12 to 108 15 to 108 13 to 108 35 to 1.77 x 106 --
Sample
volume
0.650 ml 0.5 0.5 1 ml 1 ml 0.24 ml
Sample type P, S P, S P, S P P P
Cost per test 35 to 45 15 100 20 40 10

83. Interpretation of HCV assays
Anti HCV HCV RNA interpretation
+ + Acute or chronic HCV depending upon clinical
context
+ -- Resolution of HCV
-- + Early acute HCV, chronic HCV in
immunosuppressed states
-- -- Absence of HCV

89. Directly Acting AntiVirals (DAA)
Pan Genotype Active

93. Timing of laboratory testing for treatment of HCV

94. Optimal approach to detection of
HCV infection
• Screen persons for a history of risk of exposure to the virus
• Test selected individuals who have identifiable risk factor
• IV drug abuser
• Received blood component transfusion
• Haemodialysis
• Children born to HCV positive mothers
• Exposure to an infected sexual partner
• Needle stick injury in HCW

95. Who should be tested for HCV ?
• Persons who have received medical or dental interventions in health-care settings where
infection control practices are substandard
• Persons who have received blood transfusions prior to the time when serological testing
of blood donors for HCV was initiated or in countries where serological testing of blood
donations for HCV is not routinely performed
• Persons who inject drugs (PWID)
• Persons who have had tattoos, body piercing or scarification procedures done where
infection control practices are substandard
• Children born to mothers infected with HCV
• Persons with HIV infection
• Persons who have used intranasal drugs
• Prisoners and previously incarcerated persons

97. How to test for HCV ?
• One quality assured serological test for antibodies to HCV
• ELISA
• CLIA
• RTD
• Alternate strategy (under investigation)
• HCV core Ag (p22) appearance parallel HCV RNA

98. Viral Load in HCV infection
• Useful for confirmation of current infection
• Titre doesnot correlate with severity of disease
• Useful for monitoring response to DAA
• Baseline titre
• SVR after 12 wks
• SRV on 24 wk follow up

99. Monitoring treatment response for HCV ?
Manufacturer Test Technique LLOD Linearity Range
Roche Cabas TaqMan RT PCR 20 copies/ml 20 – 10 7 copies/ml
Siemens Versant HIV RNA RT PCR 37 37 – 10 6
Abbott Molecular Abbott RT HIV-1 RT PCR 40 40 – 10 6
Siemens Versant HIV RNA 30
assay (bDNA)
b DNA 65 50 --500,000
Booteries Nucleisens Easy Q NASBA 250 25 – 7,900,000
Viral Load quantitation
Number of copies Log 10
10 1.0
50 1.7
100 2.0
500 2.7
1,000 3
10,000 4
50,000 4.7
100,000 5
1,000,000 6

104. “Woods are lovely, dark and deep,
But I promises to keep, and miles to go
Before I sleep” Robert Frost