1. Distribution of Hepatitis B Virus
genotypes in Libya using PCRbased diagnostics
Presented By:
Milud.A.Salem

2. 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

3. ≥8% - high
2-7% - intermediate
< 2 - low
Terrault and Wright 1998. Sleisenger and fordtran's gastrointestinal and liver Disease, pp.1123-1170

4.  Hepadnaviridae family.
 Partially double-stranded, circular DNA.
 4 partially overlapping ORFs.
 Genome size: 3.2 kb.
Norder et al. Intervirology. 2004; 47:289–309.

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

6. Genotypes of HBV
 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.

7.  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.

8. 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.

9. 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 prevalence .
4
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

10. 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

11. Materials and Methods
HBV-DNA quantification
clinical plasma specimens
DNA purification
RoboGene® HBV quantification kit
DNA Quantification
Rotor-Gene 3000™

12. BV
H
e ® kit
Gen tion
ob o i fi c a
R nt
qua
H
RC
SEA
RE 0
T
ET e 300
B
OR -Gen
C or
Rot

13. Materials and Methods
HBV Genotyping
clinical plasma isolates
DNA purification
Wizard® SV genomic DNA purification system, Promega
Outer amplification
INNO-LiPA primer
Gel electrophoresis
2% agarose gel
+
Genotyping using
INNO- LiPA assay
-
Nested amplification
INNO-LiPA primer

14. mic m,
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Pro
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TT l Cycle
RBE rma
CO The
RBE RCH
CO EA
RES

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ing
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16. Materials and Methods
INNO-LiPA major steps
Denaturation
5 min
Denaturation of amplified
Biotinylated DNA
Hybridization
60 min
Hybridization with specific
oligonucleotide probes
immobilized on membrane-based
strips
Stringent wash
30 min
Remove unbound
DNA
Color development
60 min
Incubate with conjugate
and substrate resulting in
purple brown precipitate

17. Results and discussion
HBV-DNA quantification
121 clinical plasma isolates
DNA purification
RoboGene® HBV quantification kit
DNA Quantification
Rotor-Gene 3000™
DNA quantity in samples ranged from
50 IU/ ml and 3.6x10ˉ IU/ml

18. Results and discussion
HBV-DNA purification and amplification
clinical plasma isolates
DNA quantity > 1000 IU/ml (85/121 samples)
DNA purification
Wizard® SV genomic DNA purification system, Promega
Outer amplification
INNO-LiPA primer
54 samples
-
Gel electrophoresis
Nested amplification
2% agarose gel
INNO-LiPA primer
31 samples +
+ 33 samples
Genotyping using
INNO- LiPA assay

19. 456
411 415
1
3
5´
CATC CTGCTGCTATGCCTCATC TTCTTG TTGGTTCTTCTGGATTA TCAAGGTATGTTGCCCG TTTGTCCTCTAATTCC AGGATCAACAACAACCAGTACG
GTAG GACGACGATACGGAGTAGAAGAACAACCAAGAAGACCTAAT AGTTCCATACAACGGGCAAACAGGAGATTAAGGTCCTAGTTGTTGTTGGT CATGC
GGACCATGCAAAACCTGCACGACTCCTGCTCAAGGCA ACTCTATGTTTCCCTCATGTTGCTG TACAAAACCTACGGATGGAAATTGCACCTGTATTCCCAT
CCTGGTACGTTTTGGACGTGCTGAGGACGAGTTCCGTTGAGATACAAAGGGAGTACAACGACATGTTTTGGATGCCTACCTTTAACGTGGACATAAGGGTA
CCCATCGT CCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCCTCAGTCCGTTTCTCT TGGCTCAGTTTACTAGTGCCATT TGTTCAGTGGTTCGTAGGG
GGGTAGCAGGACCCGAAAGCGTTTTATGGATACCCTCACCCGGAGTCAGGCAAAGAGAACCGAGTCAAATGATCACGGTAAACAAGTCACCAAGCATCCC
798
CTTTCCC CCACTGTTTGGCTTTCAGCTATATGGATGATGTGGTATTGGGGGCCAAGACTGTACAGCATCGTGAGTCCCTTTATACCG CTGTTACCAATTTT
GAAAGGGGGTGACAAACCGAAAGTCGATATACCTACTACACCATAACCCCCGGTTCTGACATGTCGTAGCACTCAGGGAAATATGGC GACAATGGTTAAAA
4
824
2
3´
837
CTTTTGT CTCTG GGTATACATTTAA END OF HbsAg
GAAAACAGAGAC CCATATGTAAATT
Outer amplification fragment
Nested amplification fragment
Osiowy and Giles. Journal of clinical Virology; 2003: 5473-5477.
1= sense outer primer sequence
2= anti-sense outer primer sequence
3= sense nested primer sequence
4= anti-sense nested primer sequence

20. te r
Ou
t
Nes
pli
d am
e
n
atio
f ic
nds
ba
pli
am
atio
fic
nds
ba
n

21. Results and discussion
HBV genotypes
Denaturation
5 min
Denaturation of amplified
Biotinylated DNA
Hybridization
60 min
Hybridization with specific
oligonucleotide probes
immobilized on membrane-based
strips
Stringent wash
30 min
Remove unbound
DNA
Color development
60 min
Incubate with conjugate
and substrate resulting in
purple brown precipitate

22. HBPr 216 (F)
411
CAGGATCCACGACCACCAG
5´ CATCCTGCTGCTATGCCTCATCTTCTTGTTGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGGATCAACAACA ACCAGTACG
CTCTACTTCCAGGAACAT HBPr 193 (A)
HBPr 153 (C)
HBPr 77 (A)
HBPr 140 (A)
GGACCATGCAA
AACCTGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTTGCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCATC
CTGCACGATTCCTGCT
TACGGACGGAAACTGC
ACTCTATGTATCCCTCCT
HBPr 154 (C)
HBPr 165 (D)
GCTGTACCAAACCTTCGGAC
HBPr 78 (B)
GGACCCTGCCGAAC
HBPr 208 (D)
HBPr 172 (E)
CCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCCTCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCT
AGTGGTTCGCCGGGCT
HBPr 213 (E)
HBPr 101 (G)
TTTCAGCTATGTGGATGA
TTCCC CCACTGTTTGGCTTTCAGCTATATGGATGATGTGGTATTGGGGGCCAAGACTGTACAGCATCGTGAGTCCCTTTATACCG CTGTTACCAATTTTCT
TCAGTTATATGGATGAT
GGCCAAATCTGTGCAGC
HBPr 98 (B)
HBPr 186 (F)
TTTGT CTCTG GGTATACATTTAA 3´ END OF HbsAg
837
Osiowy and Giles. Journal of clinical Virology; 2003: 5473-5477.

23. G
A
pe
oty
en
G en
pe E
oty

24. e
typ
eno
G
D
eno
G
D
pe
ty

25. d
Mixe D/E
e
otyp
Ge n
G
ixed /E
M
ype D
enot

26. Results and discussion
HBV genotypes
(60 samples) Genotypable using INNO- LiPA assay
Genotype A
1 patient
Genotype E
1 patient
Genotype D
54 patients
1.7%
1.7%
90%
Mixed
genotype D/E
4 patients
6.6%

27. Conclusion
These 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.

28. Author
Study
place
Detection
method
Samples
.No
%Genotypes
A
B
C
D
E
109
91
84.6
Italy
LiPA
272
Sharara et al.
((2004
Lebanon
/
67
Zekri et al.
((2007
Egypt
Type-specific
primers
70
10
Al ashgar et al.
((2008
Saudi
Arabia
INNO-LiPA
54
5.6
85.2
Basaras et al.
((2007
Spain
INNO-LiPA
14
28.6
64.3
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
D/E
100
RFLP
A/E
Alavian et al.
((2006
Iran
Ezzikouri, et al.
((2008
Morocco
Dal Molin et al,
((2006
INNO-LiPA
26
73
98
25.7
8.6
2
15.7
37.1
5.6
3.6
7.1
6.6

29. 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.