Distribution of Hepatit is B Virus genotypes in Libya using PCR-

based diagnostics

Presented By:Milud.A.Salem

Introduction to Hepatitis B Virus

350 million people are infected with HBV worldwide¹.

HBV infection develop to CHB, cirrhosis or HCC¹.

Libya has an intermediate prevalence of the HBV infection reaching around 2.2%².

¹Hu and Hirshfield, 2006. Advanced techniques in diagnostic microbiology, pp. 342-343

²Elzouki et al. Journal of Gastroenterology. 2006; 21 (2):114

≥8% - high

2-7% - intermediate

< 2 - low

Terrault and Wright 1998. Sleisenger and fordtran's gastrointestinal and liver Disease, pp.1123-1170

Hepadnaviridae family.

Partially double-stranded, circular DNA.

4 partially overlapping ORFs.

Genome size: 3.2 kb.

Norder et al. Intervirology. 2004; 47:289–309.

Regmi S, 2004. PhD thesis, Tribuwan University, Nepal.

Classified into 9 genotypes to date (A-I) based on 8% or more of DNA sequence differences.

HBV genotypes were found to have varied geographic distributions.

Schaefer S. World Journal of Gastroenterology. 2007, 13(1):14-21.

Genotypes of HBV

HBV genotypes have clinical significance:1) Disease severity:

genotypes C and D are more associated with sever liver disease than genotypes B and A¹. genotype D has a high rate of developing HCC².

2) Induction of chronicity:

CHB is more often induced in patients with genotype A than with genotypes B and C³

¹Chan et al. Journal of Clinical Microbiology. 2003; 41:1277–1279 ²Thakur et al. Journal of Gastroenterology and Hepatology. 2002; 17:165–170 ³Schaefer. Journal of Viral Hepatitis. 2005; 12:111-124.

3) Prevalence of seroconversion: genotypes A and B have a higher seroconversion

rate than genotypes D and C, respectively¹.

4) Mutations variants: precore variant is common with genotypes D and

B in comparison with genotypes A and C, respectively. the core promoter mutation is more frequent in individuals with genotype C than genotype B ²

¹Mahtab et al. Hepatobiliary and Pancreatic Diseases International. 2008; 7(2): 457-464. ²Lindh et al. Journal of Infection Disease. 1999; 179:775–782.

5) Viraemia levels: high DNA levels in HBV genotype D patients with

HBeAg negative, and in HBV genotype C patients with HBeAg positive.¹

6) Response to anti-viral drugs and vaccines: The response to IFN is higher with genotypes A and

B than genotypes D and C, respectively², while the response don’t vary between genotypes A and D with lamivudine³ but higher with genotype C than B³.

The immune escape after vaccination occurred in region with genotype D prevalence4.

¹Westland et al.Gastroenterology. 2003; 125:107-116. ²Zhang et al.Journal of Medical Virology. 1996; 48:8–16. ³Buti et al. Journal of Hepatology. 2002; 36:445-446. 4Carman et al. Lancet. 1990; 336 (8711): 325–329

Objective of this study

To determine genotypes of HBV among Libyan patients who were referred to the St.James clinical Laboratory, Tripoli, using INNO-LiPA HBV genotyping assay

Materials and Methods HBV-DNA quantification

clinical plasma specimens

DNA purification RoboGene HBV quantification kit

DNA QuantificationRotor-Gene 3000™

CORBETT RESEARCH

Rotor-Gene 3000

RoboGene HBV

quantification kit

Materials and Methods HBV Genotyping

clinical plasma isolates

DNA purificationWizard SV genomic DNA purification system, Promega

Outer amplificationINNO-LiPA primer

Gel electrophoresis2% agarose gel

Genotyping usingINNO- LiPA assay

-

+

Nested amplificationINNO-LiPA primer

CORBE CORBETT

RESEARCH Thermal Cycler

Wizard SV genomic

DNA purification system,

Promega kit

Mini gel electrophoresis

system (Gel X

L Plus)

INNO-LiPA HBV Genotyping kit

Materials and Methods INNO-LiPA major steps

Denaturation Hybridization Stringent wash Color development

5 min Denaturation of amplified

Biotinylated DNA

60 min Hybridization with specific

oligonucleotide probes immobilized on membrane-based

strips

30 min Remove unbound

DNA

60 min Incubate with conjugate

and substrate resulting in purple brown precipitate

Results and discussion HBV-DNA quantification

121 clinical plasma isolates

DNA purification RoboGene HBV quantification kit

DNA QuantificationRotor-Gene 3000™

DNA quantity in samples ranged from 50 IU/ ml and 3.6x10ˉ IU/ml

Results and discussion HBV-DNA purification and amplification

clinical plasma isolatesDNA quantity > 1000 IU/ml (85/121 samples)

DNA purificationWizard SV genomic DNA purification system, Promega

Outer amplificationINNO-LiPA primer

Gel electrophoresis2% agarose gel

Genotyping usingINNO- LiPA assay

-

+

Nested amplificationINNO-LiPA primer

31 samples

54 samples

+ 33 samples

4565´CATC CTGCTGCTATGCCTCATC TTCTTG TTGGTTCTTCTGGATTA TCAAGGTATGTTGCCCG TTTGTCCTCTAATTCC AGGATCAACAACAACCAGTACGGTAG GACGACGATACGGAGTAGAAGAACAACCAAGAAGACCTAAT AGTTCCATACAACGGGCAAACAGGAGATTAAGGTCCTAGTTGTTGTTGGT CATGC

GGACCATGCAAAACCTGCACGACTCCTGCTCAAGGCA ACTCTATGTTTCCCTCATGTTGCTG TACAAAACCTACGGATGGAAATTGCACCTGTATTCCCATCCTGGTACGTTTTGGACGTGCTGAGGACGAGTTCCGTTGAGATACAAAGGGAGTACAACGACATGTTTTGGATGCCTACCTTTAACGTGGACATAAGGGTA

CCCATCGT CCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCCTCAGTCCGTTTCTCT TGGCTCAGTTTACTAGTGCCATT TGTTCAGTGGTTCGTAGGGGGGTAGCAGGACCCGAAAGCGTTTTATGGATACCCTCACCCGGAGTCAGGCAAAGAGAACCGAGTCAAATGATCACGGTAAACAAGTCACCAAGCATCCC

798 CTTTCCC CCACTGTTTGGCTTTCAGCTATATGGATGATGTGGTATTGGGGGCCAAGACTGTACAGCATCGTGAGTCCCTTTATACCG CTGTTACCAATTTTGAAAGGGGGTGACAAACCGAAAGTCGATATACCTACTACACCATAACCCCCGGTTCTGACATGTCGTAGCACTCAGGGAAATATGGC GACAATGGTTAAAA 4 824 3´ CTTTTGT CTCTG GGTATACATTTAA END OF HbsAgGAAAACAGAGAC CCATATGTAAATT

1= sense outer primer sequence 2= anti-sense outer primer sequence 3= sense nested primer sequence 4= anti-sense nested primer sequenceOsiowy and Giles. Journal of clinical Virology; 2003: 5473-5477.

411

837

4151

2

Outer amplification fragment

Nested amplification fragment

3

Outer amplification bands

Nested amplification bands

Results and discussion HBV genotypes

Denaturation Hybridization Stringent wash Color development

5 min Denaturation of amplified

Biotinylated DNA

60 min Hybridization with specific

oligonucleotide probes immobilized on membrane-based

strips

30 min Remove unbound

DNA

60 min Incubate with conjugate

and substrate resulting in purple brown precipitate

HBPr 216 (F) CAGGATCCACGACCACCAG 5´ CATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGGATCAACAACA ACCAGTACG CTCTACTTCCAGGAACAT

HBPr 153 (C)

HBPr 140 (A)GGACCATGCAA

AACCTGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTTGCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCATC ACTCTATGTATCCCTCCTGGACCCTGCCGAAC HBPr 172 (E)

CCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCCTCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCT AGTGGTTCGCCGGGCT HBPr 213 (E)

HBPr 101 (G) TTTCAGCTATGTGGATGA TTCCC CCACTGTTTGGCTTTCAGCTATATGGATGATGTGGTATTGGGGGCCAAGACTGTACAGCATCGTGAGTCCCTTTATACCG CTGTTACCAATTTTCT GGCCAAATCTGTGCAGC HBPr 186 (F)

TTTGT CTCTG GGTATACATTTAA 3´ END OF HbsAg

Osiowy and Giles. Journal of clinical Virology; 2003: 5473-5477.

HBPr 193 (A)

TACGGACGGAAACTGC

HBPr 78 (B)

TCAGTTATATGGATGATHBPr 98 (B)

CTGCACGATTCCTGCTHBPr 154 (C) HBPr 165 (D) GCTGTACCAAACCTTCGGAC

HBPr 208 (D)

HBPr 77 (A)

411

837

Genotype A Genotype E

Genotype D

Genotype D

Mixed

Genotype D/E Mixed

Genotype D/E

Results and discussion HBV genotypes

(60 samples) Genotypable using INNO- LiPA assay

Genotype A1 patient

Genotype E1 patient

Genotype D54 patients

Mixed genotype D/E

4 patients1.7% 1.7% 90% 6.6%

ConclusionThese results showed that HBV genotype D is

the most prevalent genotype in Libya.

This study agree with other studies in Mediterranean and Middle East countries where HBV genotype D is predominates.

HBV D/E hybrid may represent CHB patients or IDUs.¹

¹ Chen et al.Journal of Medical Virology. 2004; 74: 536-542.

Author Study place

Detection method

Samples No.

Genotypes %

A B C D E A/E D/E

Alavian et al. (2006(

Iran INNO-LiPA 109 100

Ezzikouri, et al.(2008(

Morocco RFLP 91 84.6

Dal Molin et al,(2006(

Italy LiPA 272 26 73

Sharara et al. (2004(

Lebanon / 67 98 2

Zekri et al. (2007(

Egypt Type-specific primers

70 10 25.7 8.6 37.1 15.7

Al ashgar et al. (2008(

Saudi Arabia

INNO-LiPA 54 5.6 85.2 5.6 3.6

Basaras et al. (2007(

Spain INNO-LiPA 14 28.6 64.3 7.1

Bahri et al. (2006(

Tunisia RFLP 138 8 80 9

Khelifa and Thibault (2009(

Algeria sequencing 75 5 93 2

This study (2009)

Libya INNO-LiPA 60 1.7 90 1.7 6.6

Recommendation Further studies are needed to explore the

nucleotide sequences of HBV genotype D isolates in Libya.

*This work may open new avenues for further studying the molecular virology of HBV.

*HBV genotype D may need to be broken down into sub-genotype.