David Handojo Muljono,

Eijkman Institute, Jakarta, Indonesia

Molecular Diagnostics Symposium

Himpunan Kimia Klinik Indonesia (HKKI)Jakarta, 8 April 2017

2

Common Viral Hepatitis

Hepatitis E virus

3

Lead to chronicity

• About 2000 million people have been

infected with hepatitis B virus:

– more than 240 million are chronically

infected,

– 500 000-700 000 annual deaths due

to HBV-related diseases

– 75% in Asia-Pacific region

– Indonesia: • 2007: Moderate-to-high endemicity (9.4%)

• 2013: Moderate endemicity (7.1%)

• About 130–170 million people are

chronically infected with hepatitis C

virus:– more than 350 000 people die from

HCV-related liver diseases each year.

– Indonesia: • 2013: 1.0% (provisional data)Anti-HCV (+): 1.0%

(Provisional data, 2013)

HBsAg (+): 7.1%

(Provisional data, 2013)

Leading Causes of Infectious Disease Deaths Worldwide (2010)

Disease Estimated Deaths per Year

Lower respiratory tract infections ~2.8 million

HIV/AIDS ~1.5 million

Viral Hepatitis ~1.4 million

Diarrheal diseases ~1.4 million

Tuberculosis ~1.2 million

Malaria ~1.2 million

Meningitis ~422,000

Measles ~125,000

Lozano et al. Lancet. Vol 380. 2012

Hepatitis B

Hepatitis B virus (HBV)

Small DNA virus consisting of

• Outer protein envelope containing

HBsAg

• Inner protein core (nucleocapsid)

containing HBV DNA

Gene and protein products:

• circular, double-stranded DNA with

4 ORFs:

– Gene S → Envelope (HBsAg)

– Gene C → Core (HBcAg)

– Gene P → Polymerase

– Gene X → X protein

30–50 years

Acuteinfection

Chronic Carrier

Resolution

Chronichepatitis

Death

Stabilisation

Progression

Cirrhosis

Compensatedcirrhosis

Liver cancer

DecompensatedCirrhosis(Death)

•Host Virus

Manifestation of HBV Infection

is a result of Host and Virus interaction

• HBeAg status,

• HBV DNA titer

• HBV genotype,

• HBV mutants

• Gender

• Acquisition Age

• Immune system

Life cycle of hepatitis B virus

ICP = Intra cellular pathway

of cccDNA formation

No proof reading

Prone to mutation(2 x 10-5 / nucleotide / year)

Surviving variants/mutants will face

the host immune response/antivirus

Most mutants are lethal

Compact genomic structure(Overlapping ORF)

After selection by host immune system/

antivirus:

Variants / mutants

Life cycle of hepatitis B virus

Mutants in Gene S, P, X

and C

• Genotype: A to J

• Subtype: adw, adr, ayw, ayr

and many genotype/subtype

members

No proof reading

Distribution of HBV

genotype/subgenotype in ethnic populations of the Indonesian archipelago:

- B: dominant in the west

- C: dominant in the east

Genotype C

Genotype B

Genotype D

Genotype A

* Indonesian of Chinese ethnic origin

*

a

Balinese and Lombok

Sumbanese Flores

Alorese

Minahasa

and Talaud Ternate and Ambonese

C1

C5

BatakB2

B3

B7B9

C2

Malay

C2B3

B9

C1B2

B3

B8B9

C1C2

B3

B7

B5

B8B9

B3B5

B7B9

C2C1 Papuan

C1C2

C6

B3B7

Indonesian of Chinese ethnic origin

B2

B3B5

C1C2

B3B5

B8

B9

Nias and Mentawai

Minang

B3

C5

B7

b

Makasarese C1C2

B2B3B5B8B9

C1

C2

Javanese

B5

B3

B7

C1

B7B5

B8

B9

C1

C2

Torajan and Mandar

B3

B7

C1C2

B5

B3

B5B7B9

Genotype A B C D E-J

Clinical characteristics

• Modes of transmission Horizontal Perinatal/Vertical Perinatal/Vertical Horizontal Horizontal

• Tendency of chronicity Higher Lower Higher Lower ND

• Positivity of HBeAg Higher Lower Higher Lower ND

• HBeAg Seroconversion Earlier Earlier Later Later ND

• HBsAg seroclearance More More Less Less ND

• Histologic activity Lower Lower Higher Higher ND

• Clinical outcomes (LC and HCC) Better Better Worse Worse Worse in F

• Response to interferon α Higher Higher Lower Lower Lower in G

• Response to nucleos(t)ide analogues No significant differences among genotypes A to D ND

Virological characteristics

• Serum HBV DNA level ND Lower Higher ND ND

• Frequency of precore A1896 mutation Lower Higher Lower Higher ND

• Frequency of BCP T1762/A1764 mutation Higher Lower Higher Lower ND

• Frequency of preS deletion mutation ND Lower Higher ND ND

Comparison of clinical and virological features

among hepatitis B virus genotypes

‘a’ determinant (124-147)

Detection failure in regular blood donor

Thedja et al., 2010

HBV Genotyping Methods

1. Sequencing and phylogenetic analysis

– The definitive method for HBV genotyping

– defined by an inter-group divergence of >8% in the complete genome

sequence, and of >4% in the S gene

2. Restriction fragment length polymorphisms (RFLP),

– based on the amplification of an S gene amplicon followed by restriction

digestion

3. Polymerase Chain Reaction (PCR)

– PCR with specific primers for single genotypes

– Multiplex-PCR for many HBV genotypes,

4. Hybridization technologies

– HBV DNA is mplified using PCR primers complementary to a conserved

region in the PreS1 region of the HBV genome, then hybridised with

genotypic specific oligonucleotides on membrane strips.

5. Serological Method

– Enzyme-linked immunosorbent assay (ELISA) by identifying immune epitopes within the pre-S2 region using monoclonal antibodies

Hepatitis C

Hepatitis C Virus

• Single-shelled RNA Virus of

Flaviviridae family

10 protein products

- Structure: C, E, (p7)

- Function: NS2, NS3, NS4,

NS5

Genetic structure

• 9600 bp RNA,

• No RNA polymerase proofreading

ability

• Each new viral particle is coded by

various RNAs

1.4 - 1.9 x 10-3 mutation per nucleotide

per year.

3-18 mutations per year for the whole

genome

Prone to mutation

1012 virions/day

HCV Life cycle

Multiple variants

Genotype 1-6

1 3

2

4 5

6

c

a da

lb

10a

ac

cb

bc

ad

ef

a

ceg

b a6a

6b c

ba

11a

6a6b

9c9b9a

d7

Six genotypes and hundreds of subgenotypes

Simmonds et al, 2005

Genotype 1 (and 4)

• Resistance to Interferon treatment

(G1 and 4 > G2 and 3)

• Higher replication

• Associated with more severe disease

Genotype 3:

• High relapse rate

• Associated with obesity and lipid

profile

Role of genotypes:

Distribution of HCV Genotypes in the World

WHO 2014. Guidelines for the screening, care and treatment of persons with

hepatitis C infection

Historical epidemiology of hepatitis C

virus (HCV) in select countries –

volume 3

Journal of Viral Hepatitis, 2015, 22 (Suppl. 3), 4–20

Comparison of Viral Kinetic in Chronic

Infections with HIV, HBV and HCV

Virus HIV HBV HCV

Half life (Tm1/2) 4-8 h 19-38 h 2-5 h

Turnover 87-98% 35-58% 97-99%

Production (day) 108-1010 1011-1013 1010-1013

Mutation Rate 105 104 105

(Nucleotide/site/year)

• Millions of antigenic epitopes are formed

(some have similarities to host proteins =

molecular mimicry) – Genotype D>A; C>B

The host’ response

• Millions of antibodies are generated, including

auto antibodies autoimmune diseases

(extra-hepatic manifestation))

No RNA polymerase proofreading ability

Another Manifestation of HCV infection

Hadziyannis SJ. J Eur Acad Dermatol Venereol. 1998;10:12-21.

HCV Infection: Extrahepatic Manifestations

Hematologic• Mixed cryoglobulinemia• Aplastic anemia• -cell lymphoma

Dermatologic• Porphyria cutanea tarda• Lichen planus• Cutaneous necrotizing

vasculitis

Renal• Glomerulonephritis• Nephrotic syndrome

Endocrine• Anti-thyroid antibodies• Diabetes mellitus

Salivary• Sialadenitis

Ocular• Corneal ulcer• Uveitis

Vascular• Necrotizing vasculitis• Polyarteritis nodosa

Neuromuscular• Weakness/myalgia• Peripheral neuropathy• Arthritis/arthralgia

Autoimmune Phenomena•CREST syndrome

Patients my come with these diseases

Source: WHO 2014. Guidelines for the screening, care and

treatment of persons with hepatitis C infection

Manifestation of HCV infection

April 2016

April 2016

April 2016

April 2016

2014 2017Perhimpunan Peneliti Hati Indonesia (PPHI)

Available DAAs (in Indonesia)

Terapi Hepatitis C (DAA):

Tanpa Sirosis

GTPegIFN,

RBVPegIFN,RBV,SOF

PegIFN,RBV,SIM

SOF,RBV

SOF/LED

SOF,DAC

SOF,SIM

ELB/GRA

SOF/VEL

1

Response guided

12 24-48* - 12 12 1212**

16 (+RBV)***12

2 12 - 12 12 12 - - 12

3 12 - 24 - 12 - - 12

4 12 24-48* - 12 12 1212

16 (+RBV)***12

5 12 - - 12 12 - - 12

6 12 - - 12 12 - - 12

Terapi Hepatitis C (DAA):

Sirosis Kompensata

GTPegIFN,

RBV

PegIFN,RBV,SOF

PegIFNRBV,SIM

SOF,RBV

SOF/LED

SOF,DAC

SOF,SIM

ELB/GRA

SOF/VEL

1

Response guided

12 24-48* -12 (+RBV)24 (-RBV)

12 (+RBV)24 (-RBV)

12 (+RBV)24 (-RBV)

12**16 (+RBV)***

12

2 12 - 16-24 12 12 - - 12

3 12 - 24 -24

(+/- RBV)- - 12 (+RBV)

4 12 24-48* -12 (+RBV)24 (-RBV)

12 (+RBV)24 (-RBV)

12 (+RBV)24 (-RBV)

12**16 (+RBV)***

12

5 12 - -12 (+RBV)24 (-RBV)

12 (+RBV)24 (-RBV)

- - 12

6 12 - -12 (+RBV)24 (-RBV)

12 (+RBV)24 (-RBV)

- - 12

Terapi Hepatitis C (DAA):

Sirosis Dekompensata

GTPegIFN,

RBVPegIFN,RBV,SOF

PegIFN,RBV,SIM

SOF,RBV

SOF,LED

SOF,DAC

SOF,SIM

ELB/GRA

SOF,VEL

1

KontraIndikasi

KontraIndikasi

KontraIndikasi

-12 (+RBV)24 (-RBV)

12 (+RBV)24 (-RBV)

KontraIndikasi

KontraIndikasi

12 (+RBV)24 (-RBV)

2 16-20 -12 (+RBV)24 (-RBV)

12 (+RBV)24 (-RBV)

3 - - 24 (+/-RBV) 24 (+/-RBV)

4 -12 (+RBV)24 (-RBV)

12 (+RBV)24 (-RBV)

12 (+RBV)24 (-RBV)

5 -12 (+RBV)24 (-RBV)

12 (+RBV)24 (-RBV)

12 (+RBV)24 (-RBV)

6 -12 (+RBV)24 (-RBV)

12 (+RBV)24 (-RBV)

12 (+RBV)24 (-RBV)

1. Sequencing and phylogenetic analysis

• The best genotyping method, by sequencing the most conserved regions

– The 5’ untranslated region (5’ UTR),

– The 3’untranslated region.

2. Several commercial assays:

• TruGene 5’ NC HCV Genotyping kit (Siemens, Tarrytown, NY, USA):

– based on direct sequence analysis of the 5’UTR (untranslated region);

• VERSANT HCV Genotype Assay (LiPA) 2.0 (Siemens, Tarrytown, NY, USA),

reverse hybridization line-probe assay targeting the 5’ UTR and core regions

• Abbott m2000 Real-Time HCV Genotype II assay (Abbott Molecular Inc., Des

Plaines, IL, USA):

– PCR-based assay targeting specific regions of the 5’NCR and the NS5B genes

3. Newly developed methods:

• Liquid Microarray

– based on liquid microarray technology (xMAP Technology, Luminex Corp, Texas).

• Next generation sequencing (NGS)

– Perform rapid and mass sequencing, and distinguish viral population from a single host

HCV Genotyping Methods

Thank you