Khan et al.

Virology Journal 2011, 8:420

http://www.virologyj.com/content/8/1/420

RESEARCH Open Access

True prevalence of twin HDV-HBV infection in

Pakistan: a molecular approach
Asad U Khan1†, Muhammad Waqar1†, Madiha Akram2, Mehnaz Zaib2, Muhammad Wasim3, Shahzad Ahmad1,
Zeeshan Niaz1, Sajid Ali1, Haider Ali1, Muhammad Idrees4* and Mohammad A Bajwa5

Abstract
Hepatitis Delta Virus (HDV) infects only patients that are already infected by hepatitis B virus (HBV) because this is
sub satellite virus which depends on and propagate only in the presence of HBV. HDV causes co-infection or super
infection with sever complication as compared to only HBV infection. No study on molecular level on HDV is
available from this region; therefore, the aim of this study was to found out the molecular epidemiology of HDV
(as a co-infection with HBV) in different geographical regions of Pakistan.
Total 228 HBsAg positive samples were received for the study from different geographical regions of the country.
Only HBV DNA PCR positive samples were further utilized for the presence of HDV RNA. For this purpose, HDV RNA
and HBV DNA was extracted and amplified using reverse transcriptase polymerase chain reaction (RT-PCR), nested
PCR and real-time PCR.
Out of the total 228 HBsAg positive samples, HBV DNA was detected in total 190 (83.3%) samples belonged to
different patients. Of these 190 patients, HDV RNA was observed in 53 (28%) patients. Of the 53 HDV positive cases,
37 (69.8%) were males and 16 (30.2%) were female patients. The percentage of dual infection was found higher
significantly (p < 0.05) in male patients as compared to female patients. Total 41 (26.8%) patients were below 40
years and 13 (31.7%) were above 40 years of age. No significant difference was seen in patients with ages above or
below 40 years. In the provinces of Sindh, Khyber Pakhtoonkhaw and Punjab the observed prevalence of HDV was
67%, 6% and 4% respectively.
In conclusion, the HDV infection is not uncommon in Pakistan and its prevalence is higher significantly in the
Province of Sindh (p < 0.01) and male six (p < 0.05).

Introduction single-strand RNA, which is approximately 1700 nucleo-

Hepatitis Delta virus (HDV) infection is present globally tides in length [5]. These nucleotides is assemble with
and infects human being already infected by Hepatitis B two viral proteins HD Ag-S and HDAg-L to form a
virus (HBV). Hepatitis Delta is mostly present in Africa; ribonucleoprotein [6]. These Two Hepatitis D Ag pro-
South America, Romania, Russia and the Mediterranean teins are translated from viral mRNA and this process is
region included Southern Italy [1]. HDV was first dis- called RNA editing [7]. The dual infection of HBV and
covered by Rizzetto in the patients that were already HDV occurs in the form of co-infection or as a super-
infected by HBV in year 1980 [2]. HDV is a sub-satellite infection. The Super infection of HDV with HBV caused
virus associated with HBV to cause severe acute and a progressive chronic liver disease up to (80%), which
chronic forms of liver infection [3]. Tthe particle size of further enhances liver cirrhosis and hepatocellular carci-
HDV is about 36-nm that require hepatitis B surface noma (HCC). Co-infection by both HBV and HDV
antigen (HBsAg) for their enveloped and transmission viruses causes more severe acute liver disease and is a
[4]. The HDV genome is a circular, negative sense, higher risk for the development of fulminate hepatitis
compared to only HBV infected patients [8].
* Correspondence: [email protected] It has been estimated that approximately 5% of HBV
† Contributed equally carriers are co-infected with HDV, leading to an esti-
4
Division of Molecular Virology, CEMB, University of the Punjab Lahore-
53700, Pakistan
mated 15 million persons infected with HDV worldwide
Full list of author information is available at the end of the article [9]. HDV infection has a worldwide distribution, but its
© 2011 Khan et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Khan et al. Virology Journal 2011, 8:420 Page 2 of 5
http://www.virologyj.com/content/8/1/420

frequency varies greatly throughout different geographic according to the procedures given in the kit protocols
regions. It is highly endemic in the Middle East, in the (Sacace Biotechnologies Italy). Sensitivity of the assay is
Mediterranean area, in the Amazonian region, and in 20 IU per ml blood sample. Specificity of the assay is
several African countries [10]. about 99%.
Several studies propose that most people acquired
HDV infections through parenteral and sexual routes Extraction of HDV RNA and complementary DNA (cDNA)
[11-13]. Furthermore the reported seroprevalence of synthesis
HDV infection among HBV carriers is significantly HDV RNA was extracted from 100 μL serum sample
higher in intravenous drug users (IDU) compared to the using Gentra RNA isolation kit (Life Technologies,
non-IDU population [14]. USA). About 50 ng of the extracted RNA was reverse
In Pakistan, the prevalence of chronic HBV infection transcribed into cDNA with Molony-murin leukemia
is estimated to be 16-57% in general population predo- virus reverse transcriptase enzyme (Gibco BRL, Life
minantly in younger males living in rural areas [15,16]. Technologies USA). The thermal cycling was carried out
However, in these studies enzyme linked immunosor- in thermal cycler for 50 minutes at 37°C. The reaction
bant assay (ELISA) based assays were utilized for the mixture for the preparation of cDNA, for a single reac-
estimation of this prevalence rate. For the true preva- tion contained 8 50 mM Tris-HCl (pH 8.3), 7.5 mM
lence of HDV-HBV co-infection, we used molecular KCl, 3 mM MgCl2, 0.1 M DTT, 10 mM dNTPs and 200
based methods such as PCR and real-time PCR for the U of MMLV reverse transcriptase enzyme. At the end of
detection of HBV DNA and HDV RNA in HBsAg posi- reaction, MMLV was heat inactivated at 95°C for 5
tive samples from patients’ different geographical minutes.
regions of Pakistan.
Qualitative detection
Methods The detection of the HDV cDNA was carried using
Source of Clinical Samples nested PCR. The Qualitative detection of hepatitis
A total of 228 HBsAg positive serum samples by enzyme Delta virus was done in two rounds. First round PCR
linked immunosorbant assay (ELISA) were received at was carried out in a PCR reaction tube that contained
Genome Centre for Molecular Based Diagnostics & 8 μL of mix I which having 2.5 mM MgCl2, 100 μM
Research (GCMBDR) Lahore, Pakistan from different concentration of each of the four deoxynucleotides, 10
geographical regions of the country for HBV DNA and/ pM of each outer sense nucleotides 695-718 {5’-
or HDV RNA detection from January 2011 to March CATGGTCCCAGCCTCCTCGCTGGC-3’} and outer
2011. GCMBDR is a state of the art molecular based anti-sense primers; nucleotides 873-896 {5’-
diagnostic facility that provides general public sensitive, CCGCGAGGAGGTGGAGATGCCATG-3’} [17], and 1
specific and more reliable diagnostic tests on cost-to- U of Taq DNA polymerase Enzyme. In the thermal
cost basis utilizing PCR and real-time PCR methods. All cycler, firstly the samples were incubated on 95°C for
sera were stored in aliquots at -70°C till was used for 2 min then it was followed by 35 cycles consisting of
nucleic acid (DNA/RNA) isolation. Demographic char- 95°C for 1 min, 64°C for 1 min and 72°C for 1 min.
acteristics, serological and biochemical data was avail- The second round (nested PCR) PCR was also carried
able for 190 patients (male 121; female 69, mean age 42 out in a reaction tube that contained 8 μL of mix II
years [range, 6-82 ± SD] years)) with chronic HBV which had 2.5 mM MgCl2, a 100 μM concentration of
infection and were thus included in the data analysis. each of the four deoxynucleotides, 10 pM of each
These samples belonged to different provinces/geogra- inner sense nucleotides 729-748 (5’-CAACATTCC-
phical regions of Pakistan. Seventy were from Sindh, 66 GAGGGGA CCGT-3’) and inner anti-sense primers
belonged to Khyber Pakhtoonkhaw (KPK) and 54 were nucleotides 846-865 (5’-GAAGGAAGGCCCTCGA-
from Punjab. There was no need for separate written GAACAAGA-3’) [17]. For standard PCR to seen con-
informed consent from subjects for this study, since this tamination we used the negative and reagent controls
analysis was a part of the original protocol in a routine along with samples in each run. Finally the PCR pro-
workup of Molecular Diagnostics. ducts were electrophoresis on a 2% agarose gel pre-
pared in 1× Tris-borate-EDTA (TBE) buffer, stained
HBV DNA extraction and real-time PCR amplification with ethidium bromide, and evaluated under UV tran-
Qualitative and quantitative detection of serum HBV silluminator. The sizes of PCR products were estimated
DNA in all patients were performed by SmartCycler II according to the migration pattern of a 50-bp DNA
real-time PCR (Cepheid, USA) with an internal RNA ladder (Fermentas Life Sciences). The sensitivity limit
standard derived from the 5’ UTR. The utilized kits for of this qualitative detection method was 10 copies per
HBV DNA extraction and real-time PCR were done ml blood sample.
Khan et al. Virology Journal 2011, 8:420 Page 3 of 5
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Statistical Analysis Table 1 Rate of HBV + HDV co-infection in male and

All the data was analyzed and the summary statistic was female patients
carried out by SPSS version 10.0 (a statistical package). S.N Gender Total samples HBV Found positive Percentage
Variables are given in the form of rates (%). Chi Square positive with HDV
test was used for categorical variables that measured 1 Male 121 37 31%
association among categorical variables. Values of less 2 Female 69 16 23%
than 0.05 were considered significant Total 190 53 28%

Results
The study enrolments and patients’ disposition are (Table 2). In the provinces of Sindh, Khyber Pakhtoon-
shown in Figure 1. One hundred and ninety patients khaw (KPK) and Punjab the observed prevalence of
with chronic HBV who fulfilled the study criteria were HDV was 67%, 6% and 4% respectively. The co-infection
enrolled for this study out of total 288 consecutive of HDV with HBV was higher significantly in the Pro-
HBsAg positive patients. Total 98 patients samples were vince of Sindh as compared to Provinces of KPK and
excluded from the study either the volume of sera were Punjab. No significant difference was seen in the rates
not sufficient for testing (n = 19) or failed to meet inclu- of HBV+HDV co-infection in patients below or above
sion criteria of the study (n = 79) as they were HBV 40 years of ages (Table 3).
DNA negative by real-time PCR. Out of 190 enrolled
patients, 63.7% (n = 121) were males and 36.3% (n = 69) Discussion
were females. The mean age was 38 ± 11 years. The HDV can be caused either as a co-infection (concomi-
sensitivity of the assay was excellent that is 20 IU per tant) in persons infected with HBV or as a super-infec-
ml blood sample. tion (subsequent) in chronic HBV carriers. As it is now
Of the total 190 tested HBV positive cases, HDV RNA an established fact that HDV is a defective virus, con-
was detected in 58 (28%) patients. Out the HDV positive taining particles of RNA nucleoprotein in virion-like
patients, 37 were males and 16 were females. The rates form, present in patients with acute hepatitis B and
of HDV positivity in both sexes are shown in table 1. chronic hepatitis that requires the presence of a hepad-
The HDV RNA data was also analysed Province wise navirus (HBV) for full replication. That is why for its
penetration into the hepatocytes and assembly of virion,
it needs the help of HBV that provides the viral coat
surface antigen and can survive only in the presence of
HBV (co-infection or/and super-infection). Here we
report on the HDV-HBV co-infection or molecular epi-
demiology of HBV+HDV co-infection. Our study is the
first one to describe the molecular epidemiology of
HDV and its co-infection with HBV in different geogra-
phical regions of Pakistan. This is very important study
as it describes the dual HBV-HDV infection using mole-
cular methods. It has already been reported that in con-
trast to HBV mono-infection, HBV+HDV co-infection
leads to exacerbation and rapid progression of chronic
liver disease, hepatic failure, and deaths in patients
[18,19].
Figure 1 Study disposition and enrolments. Total 288 consecutive
HBsAg positive patient’s samples were received at Genome Centre Table 2 Rate of HBV + HDV co-infection in different
for Molecular based Diagnostics & Research. Of these total 98 provinces of Pakistan
patients samples were excluded from the study either the volume S.N province Total HBV DNA PCR Found Percentage
of sera were not sufficient for testing (n = 19) or failed to meet positive isolates positive with
inclusion criteria of the study (n = 79) as they were HBV DNA HDV
negative by real-time PCR. Total 190 patients with chronic HBV who 1 SINDH 70 47 67%
fulfilled the study criteria were enrolled for this study Out of 190
enrolled patients, 63.7% (n = 121) were males and 36.3% (n = 69) 2 KHYBER 66 4 6%
PUKHTUN
were females. Total 58(28%) samples were found positive with
KHWA
double infection of HBV &HDV. In the provinces of Sindh, Khyber
Pakhtoonkhaw (KPK) and Punjab the observed prevalence of HDV 3 PUNJAB 54 2 4%
was 67%, 6% and 4% respectively. Total 190 53 28%
Khan et al. Virology Journal 2011, 8:420 Page 4 of 5
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Table 3 Rate of HBV + HDV co-infection in different Also so far no epidemiological survey at molecular
patients age groups level has been documented in Pakistan to investigate the
S. Age Total HBV DNA PCR Found positive Percentage prevalence of double HBV plus HDV hepatitis infection
N group positive isolates with HDV hence this study provides new insights and dimensions
1 Below 149 40 26.8% for the molecular virologists to carry out investigations
40Y
on patients having this double hepatitis related liver dis-
2 40Y & 41 13 31.7% orders. Further we believe that the results of the current
above
study enrich the limited information on co-infection and
Total 190 53 27.9%
molecular epidemiology of HDV in the region.

Conclusion
Several important findings emerged from the results of
Our study showed a higher prevalence of HDV in HBV
this study. The first finding of the current study showed
DNA positive patients that highlights the importance of
that more than 83% of the HBsAg positive patients by
screening for HDV infection in HBV carriers. Its preva-
ELISA were also positive by HBV DNA PCR. HBV
lence is higher significantly in the Province of Sindh and
+HDV co-infection was observed in 28% HBV DNA
male six. Clinicians particularly gastroenterologist, hepa-
positive patients that is very alarming. This is the true
tologists and other medical and practitioners should be
prevalence of active HDV infection in HBV DNA posi-
made aware of the danger of twin infection with HDV
tive cases. The previously conducted studies on the ser-
and HBV. Further studies, with full genome characteri-
oprevalence of HDV infection in Pakistan have reported
zation of different HDV strains are required.
the prevalence of antibodies against HDV (HDV-Ab) of
16% and 57%, respectively [15,16]. However these stu-
dies were based on ELISA methods that might give both Acknowledgements
false positive and false negative results. In spite of this All the HBsAg positive samples were provided by Genome Centre for
Molecular Based Diagnostics & Research (GCMBDR). The molecular diagnosis
high prevalence, no data on molecular epidemiology of was done at GCMBDR free of charges. Also thanks to all the clinicians and
HDV in Pakistan are available. The prevalence of HDV patients for their cooperation in the study.
positive cases obtained in this study is extremely high.
Author details
More recently a high endemicity of HDV in Cameroo- 1
Department of Microbiology, Hazara University Mansehra, Pakistan.
nian populations was reported by Foupouapouognigni 2
Genome Center for Molecular Diagnostics & Research, 945 J-II Johar Town
and colleagues [20]. Lahore, Pakistan. 3Khyber Medical University, Hayatabad Road, Peshawar,
Khyber Pakhtoonkhaw, Pakistan. 4Division of Molecular Virology, CEMB,
Next important finding of the study is the observa- University of the Punjab Lahore-53700, Pakistan. 5Department of
tion that HDV rate is significantly high (p < 0.001) in Gastroenterology, Sheikh Zayed Medical Complex, Lahore, Pakistan.
male (69.81%) as compared to female (30.18%)
Authors’ contributions
patients. However the chances of this co-infection is AUK and MW conceived the study. AUK and MW collected the samples and
same in all age groups as no significant difference was performed PCRs and other the molecular analysis. MA and MZ helped in
seen for the rates of co-infection in patients with ages molecular analysis. MW, SA, ZN, SA, HN and MAB helped in literature search
and drafting manuscript. MI critically reviewed the manuscript and revised it.
below or above 40 years. This finding of the current All the authors read and approved the final manuscript.
study is contrary to the finding of another study from
Pakistan where HDV was predominant in young popu- Competing interests
The authors declare that they have no competing interests.
lation [15].
Another important finding of the present study is the Received: 25 July 2011 Accepted: 4 September 2011
observation that rates of HDV is significantly high (P < Published: 4 September 2011
0.001) in ethnic group Sindhi as compared to Punjabi
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doi:10.1186/1743-422X-8-420
Cite this article as: Khan et al.: True prevalence of twin HDV-HBV
infection in Pakistan: a molecular approach. Virology Journal 2011 8:420.

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