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National Advisory Board
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Ezzat Aly
Contents Alexandria Journal of Hepatogastroenterology, Volume IVX ( I ), April 2014
------------------------------------------- Manuscript Submission: For information and to submit
manuscripts please contact the editors by e-mail at :
Disclaimer: The Publisher, the Egyptian Society of
Hepatology Gastroenterology and Infectious Diseases in
Alexandria, and Editors cannot be held responsible for errors
or any consequences arising from the use of information
contained in this journal; the views and opinions expressed
do not necessarily reflect the those of the Publisher, The
Egyptian Society of Hepatology Gastroenterology &
Infectious Diseases in Alexandria, Editors, neither dose the
publication of advertisements constitute any endorsement by
the Publisher, society, and editors of the products advertised.
Review Article:
HCV will Pass Away : Six Treatment Options for HCV
Genotype 4
Marwa Reda
Alexandria University Hepatobiliary unit
-------------------------------------------
Original Article:
Evaluation of Portal Hypertensive Duodenopathy Before
and After Band Ligation of Esophageal Varices in
Patients with Liver Cirrhosis
El-Said Hassan Ibrahim,1 Gamal Ahmed Amin,2 Mohammed
Mohammed Shamseya,2 Marwa Ahmed Madkour,2 Rita Makram
Tadros2 and Hala Khalil Maghraby.3
1Department of Internal Medicine; Faculty of Medicine;
2Department of Clinical and Experimental Internal Medicine; Medical Research Institute, 3Department of Pathology; Medical
Research Institute;University of Alexandria.
-------------------------------------------
Original Article:
HCV Genotypes & SubGenotypes and HBV Precore &
Core Mutations in Hepatocellular Carcinoma
Gamal Elden Ahmed Elsawaf1; Ola Abd El Kader Mahmoud1;
Mohamed Abd Elrahman Ahmed2; Mohamed Mohamed
Shamseya3 and Hanada Salem Salim Islim1
1Department of Microbiology, 2Department of Clinical
Pathology; Military Medical Academy, 3Department of Internal
Medicine; Medical Research Institute; University of Alexandria
-------------------------------------------
Original Article:
Impact of Schistosomal Peri-Portal Fibrosis on The
Results of Transient Elastography in Hepatitis C Virus
Patients
El-Kady A 1, Etaby A 2, Esmat G 3, Baddour N 4, Mohiedeen
K1, Abdel Halim A 5
1Department of Tropical Medicine Faculty of Medicine, Alexandria University, 2Department of Radiodiagnosis, Faculty
of Medicine, Alexandria University, 3Department of Tropical
Medicine Faculty of Medicine, Cairo University, 4Department of
Pathology, Faculty of Medicine, Alexandria University, 5National
Hepatology and Tropical Medicine Research Institute, Cairo
-------------------------------------------
Original Article:
Interleukin 23 p 19 Gene Expression in Patients with
Ulcerative Colitis and its Relation to Disease Severity
Hanan El-Bassat, Lobna AboAli, Sahar El Yamany, Hanan Al
Shenawy1, Rasha A. Al Din2 and Atef Taha3
Tropical Medicine, Pathology1, Microbiology and Immunology2
and Internal Medicine3 Departments, Faculty of Medicine, Tanta
University, Egypt.
-------------------------------------------
Original Article:
Nitric Oxide Level in the Ascitic Fluid of Cirrhotic
Patients with or without Spontaneous Bacterial Peritonitis
and its Relation to Hepatorenal Syndrome
1Mohamed A. El-Biali,2Mohamed Y. Elhasafi,1Marwa A.
Madkour,3RagaaA. Ramadan, 1Eman A.Abd El-Rahman 1Department of Clinical and Experimental Internal Medicine;
Medical Research Institute, 2Department of Internal Medicine;
Faculty of Medicine,3Department of Chemical Pathology;
Medical Research Institute;University of Alexandria.
-------------------------------------------
2
6
23
37
45
52
Review Article
HCV Will Pass Away : Six Treatment Options for HCV Genotype 4
Marwa Reda
Alexandria University Hepatobiliary unit
The hepatitis C virus genotype 4 (HCV-4) is prevalent in Egypt, the Middle East and Africa. The global epidemiology
of HCV-4 is difficult to establish because most epidemiological studies have focused on the prevalence and distribution
of HCV-4 in Egypt, the country with the highest worldwide incidence and prevalence of HCV, with rates of up to 13%,
where HCV-4 is the cause of 90% of HCV infections. (1–3) The prevalence of HCV-4 is 50% in the Kingdom of Saudi
Arabia, 30% in Syria, 76% in the Gaza Strip and 6% in Jordan. Recently, the epidemiology of HCV-4 has changed and
this genotype has begun to cross borders and spread to several regions in Europe through immigration and injection
drug use. HCV-4 has been considered a difficult-to-treat genotype based on the low sustained virological response
(SVR) rates obtained with conventional interferon (IFN)-based regimens. Pegylated interferons (PEG-IFN) plus
ribavirin therapy for chronic HCV-4 has been associated with increased SVR rates of more than 60%. Shorter treatment
of chronic HCV-4 patients with rapid and early virological responses has been associated with high SVR rates, better
compliance, fewer adverse events and lower costs. Despite this progress, the treatment of HCV-4 non-responders,
injection drug users, patients on haemodialysis and patients with HCV-4 recurrence after liver transplantation still
represents a significant therapeutic challenge. Treatment of HCV-4 has markedly improved, with higher sustained
response rates and the possibility of shorter regimens. Despite the recent progress in the treatment of HCV-4, more
research is required to optimize current therapy and include genotype 4 patients in clinical trials on emerging therapies
such as specifically targeted antiviral therapy for HCV with protease and/or polymerase inhibitors. (4)
Personalized medicine for hepatitis C virus
therapy: Currently, it is not clear whether
patients with chronic HCV-4 respond differently
to PEG-IFN-a and ribavirin therapy. A
retrospective analysis of SVR rates in French,
Egyptian and African patients with chronic HCV-
4 showed an overall better response in Egyptian
patients infected with the 4a subtype. (5) In
multivariate analysis, two factors were
independently associated with SVR: an Egyptian
origin of transmission and the absence of severe
fibrosis. It is not clear from this study whether the
difference in SVR was related to ethnicity, HCV-
4 subtype or the mode of transmission. Egyptian
patients acquired the infection through anti-
schistosomal therapy, while most of the French
and African patients acquired the infection
through illicit drug use. Another study showed
that treatment of patients with chronic HCV-4
infection by PEG-IFN-a2b and ribavirin results in
a more rapid decrease in HCV RNA level and a
better SVR rate (62 vs. 13%) in Egyptians than in
non-Egyptians. (6) In contrast, a Spanish study
evaluating the response of chronic HCV-4
treatment- naive Spanish patients to combination
therapy revealed a SVR of 55%. (7) Patients
infected with HCV-4 had a lower stage of fibrosis,
lower viraemia and a higher SVR rate compared
with those with genotype- 1. Despite these
interesting observations, it is not known why
Egyptians infected with HCV-4 respond better to
therapy than chronic HCV patients in the West or
sub-Saharan Africa. The difference could be due
to the mode of infection because genotype 4
infection is prevalent in special populations in
Europe and sub-Saharian Africa namely injecting
drug users, HIV-coinfected and homosexual men,
all of whom have been identified in several
studies as difficult-to-treat groups. The variations
in response to HCV therapy could be because of
different pharmacogenetics because individual
genetic make-up could influence the individual
response, resistance to therapy or the development
of potential side effects. Identifying individuals
with a high probability of response or upfront
resistance to therapy, determining the probability
of adverse events and understanding how certain
individuals metabolize drugs are the basis of
personalized medicine. It has been shown that in
non-responders, some IFN-stimulated genes were
highly expressed; thus, preactivation of the IFN
system in patients appears to limit the effect of
IFN antiviral therapy. (5, 6) This finding could help
develop personal treatment options in patients
with chronic HCV infection and explain some of
the apparent genetic differences in response to
treatment, for instance in African Americans,
Asians or Hispanics. Testing the patient’s
genotype to determine how likely they are to
respond to anti-HCV therapy would be a major
step in personalized medicine. Until the end of
2013, the combination of pegylated interferon
(IFN)-α and ribavirin for 24 or 48 weeks was the
approved treatment for chronic hepatitis C. With
this regimen, patients infected with HCV
genotype 1 had SVR rates of approximately 40-
50%. Higher SVR rates were achieved in patients
infected with HCV genotypes 2, 3, 5, and 6 (up to
about 80%, and higher for genotype 2 than for
genotypes 3, 5, and 6) and intermediate SVR rates
up to 60% were achieved in those with HCV
genotype 4. (8, 9) In addition to pegylated IFN-α
and ribavirin, three new HCV DAAs was licenced
in the EU in the first half of 2014, for use as part
of combination therapies for HCV infection.
Sofosbuvir, a nucleotide analogue inhibitor of
HCV RNA-dependent RNA polymerase, has been
approved in January 2014. Simeprevir, a second-
wave, first generation NS3/4A protease inhibitor
was approved in May 2014. Daclatasvir, an NS5A
inhibitor, is likely to be approved in August or
September 2014. Other drugs may be approved
later in 2014 or in 2015. New therapeutic
strategies aim towards higher efficacy, pan-
genotypic activity, shortened treatment duration,
easier administration and improved tolerability
and patient adherence. It is highly likely that IFN-
sparing and IFN-free regimens with or without
ribavirin, which are being evaluated in clinical
trials, will enter clinical practice in the next few
years. Decisions about the need for and timing of
antiviral treatment will need to take into account
this rapid rate of change. According to:
EASL 2014 Recommendations on Treatment of
Hepatitis C genotype 4, there are six treatment
options are available for patients infected with
HCV genotype 4, including IFN/ribavirin-
containing and IFN-free ones. (11)
The first option includes: a combination of
weekly pegylated IFN-α, daily weight-based
ribavirin (1000 or 1200 mg in patients <75 kg or
≥75 kg, respectively), and daily sofosbuvir (400
mg) 12 weeks (Recommendation B1). It appears
as the most efficacious and the easiest to use IFN
containing option, without the risk of selecting
resistant viruses in case of treatment failure. This
combination has been evaluated in the
NEUTRINO Phase III trial in treatment-naïve
patients. (10) The SVR rate in genotype 4 patients
was 96% (27/28). The patient who failed on this
regimen did not select HCV variants resistant to
sofosbuvir.
The second option includes: a combination of
weekly pegylated IFN-α, daily weight-based
ribavirin (1000 or 1200 mg in patients <75 kg or
≥75 kg, respectively), and daily simeprevir
(150mg) (Recommendation B1). Simeprevir
should be administered 12 weeks in combination
with pegylated IFN-α and ribavirin. Pegylated
IFN-α and ribavirin should then be administered
alone an additional 12 weeks (total treatment
duration 24 weeks) in treatment-naïve and prior
relapser patients, including cirrhotics, an
additional 36 weeks (total treatment duration 48
weeks) in prior partial and null responders,
including cirrhotics (Recommendation B1). HCV
RNA levels should be monitored on treatment.
Treatment should be stopped if HCV RNA level
is ≥25 IU/ml at treatment week 4, week 12 or
week 24 (Recommendation A2).
The third option includes: a combination of
weekly pegylated IFN-α, daily weight-based
ribavirin (1000 or 1200 mg in patients <75 kg or
≥75 kg, respectively), and daily daclatasvir (60
mg) 24 weeks (Recommendation B1). Daclatasvir
should be administered 12 weeks in combination
with pegylated IFN-α and ribavirin. Daclatasvir
should be continued in combination with
pegylated IFN-α and ribavirin an additional 12
weeks (total duration 24 weeks) in patients who
do not achieve an HCV RNA level <25 IU/ml at
week 4 and undetectable at week 10. Pegylated
IFN-α and ribavirin should be continued alone
between week 12 and 24 (total duration 24 weeks)
in patients who achieve an HCV RNA level <25
IU/ml at week 4 and undetectable at week 10
(Recommendation B1).
The fourth option includes: Patients infected
with HCV genotype 4 who are IFN intolerant or -
ineligible can be treated with daily weight based
ribavirin (1000 or 1200 mg in patients <75 kg or
≥75 kg, respectively), and daily sofosbuvir (400
mg) 24 weeks (Recommendation C2). Only
preliminary data is available (SVR at week 4 post-
treatment) in a small number of American patients
of Egyptian ancestry. The preliminary SVR rates
were 79% (11/14) and 100% (14/14) after 12 and
24 weeks of treatment, respectively, in treatment-
naïve patients, and 59% (10/17) and 93% (14/15)
after 12 and 24 weeks, respectively, in treatment-
experienced patients.
The fifth option includes: Patients infected with
HCV genotype 4 can be treated with an
interferon-free combination of daily sofosbuvir
(400 mg) and daily simeprevir (150 mg) 12 weeks
(Recommendation B2). There is no data on the
impact of adding ribavirin to this regimen.
However, adding daily weight-based ribavirin
(1000 or 1200 mg in patients <75 kg or ≥75 kg,
respectively) should be considered in patients
with predictors of poor response to anti-HCV
therapy, especially prior non-responders and/or
patients with cirrhosis (Recommendation B2).
The sixth option includes: Patients infected with
HCV genotype 4 can be treated with an
interferon-free combination of daily sofosbuvir
(400 mg) and daily daclatasvir (60 mg) 12 weeks
in treatment-naïve patients or 24 weeks in
treatment-experienced patients (pending data with
12 weeks of therapy in treatment-experienced
patients) (Recommendation B2). There is no data
on the impact of adding ribavirin to this regimen.
However, adding daily weight-based ribavirin
(1000 or 1200 mg in patients <75 kg or ≥75 kg,
respectively) should be considered in patients
with predictors of poor response to anti-HCV
therapy, especially prior non-responders and/or
patients with cirrhosis (Recommendation B2).
As regards fourth and fifth options, there are no
data with these combinations in patients infected
with HCV genotype 4. But, it is likely that the
results in patients infected with genotype 1 can be
extrapolated. In settings where none of these
options is available, the combination of pegylated
IFN-α and ribavirin remains acceptable.
Refrences
1. World Health Organization. Hepatitis C. WHO Fact
Sheet 164. Geneva, Switzerland: World Health
Organization, 2000 Available at
http://www.who.int/mediacentre/factsheets/
fs164/en/print.html (accessed 12 October 2008).
2. Egyptian Ministry of Health Annual report, 2007.
Available at http://www.mohp.gov.eg/Main.asp
(accessed 12 October 2008).
3. Ray SC, Arthur RR, Carella A, Bukh J, Thomas DL.
Genetic epidemiology of hepatitis C virus throughout
Egypt. J Infect Dis 2000; 182: 698–707.
4. Kamal SN. Hepatitis C virus genotype 4 therapy:
progress and challenges. Liver International 2011; 31:
45-52.
5. Roulot D, Bourcier V, Grando V, et al.
Observational VHC4 study group Epidemiological
characteristics and response to peginterferon plus
ribavirin treatment of hepatitis C virus genotype 4
infection. J Viral Hepat 2007; 14: 460–7.
6. Elefsiniotis IS, Vezali E, Mihas C, Saroglou G.
Predictive value of complete and partial early
virological response on sustained virological response
rates of genotype-4 chronic hepatitis C patients treated
with PEG-interferon plus ribavirin. Intervirology 2009;
52: 247–51.
7. Trapero-Marugan M, Moreno-Monteagudo JA,
Garcia-Buey L, et al. Clinical and pathological
characteristics and response to combination therapy of
genotype 4 chronic hepatitis C patients: experience
from a Spanish center. J Chemother 2007; 19: 423–7.
8. EASL Clinical Practice Guidelines: management of
hepatitis C virus infection. J Hepatol 2011; 55: 245-64.
9. EASL Clinical Practice Guidelines: Management of
hepatitis C virus infection. Journal of Hepatology
2014; 60: 392–420.
10. Grebely J, Dore GJ. What is killing people with
hepatitis C virus infection?
Semin Liver Dis 2011; 31: 331–339.
11. World Health Organization Guidelines for the
screening, care and treatment of persons with hepatitis
C infection. WHO 2014; 1-122.
Original Article
Evaluation of Portal Hypertensive Duodenopathy Before and After Band Ligation
of Esophageal Varices in Patients with Liver Cirrhosis
El-Said Hassan Ibrahim,1 Gamal Ahmed Amin,2 Mohammed Mohammed Shamseya,2 Marwa Ahmed Madkour,2 Rita
Makram Tadros2 and Hala Khalil Maghraby.3
1Department of Internal Medicine; Faculty of Medicine; 2Department of Clinical and Experimental Internal Medicine;
Medical Research Institute, 3Department of Pathology; Medical Research Institute;University of Alexandria.
ABSTRACT
In patients with liver cirrhosis and portal hypertension, gasteroesophageal varices and portal hypertensive gastropathy
(PHG) are well described. However, only few reports have investigated the duodenal lesions in patients with portal
hypertension in detail. Data about the effect of esophageal variceal eradication by band ligation (BL) on the presence and
severity of portal hypertensive duodenopathy are still conflicting. Aim of the work: This study aimed to assess the
endoscopic and histopathologic duodenal mucosal changes in patients with liver cirrhosis and portal hypertension before
and after BL of esophageal varices. Patients and methods: Twenty patients with liver cirrhosis, portal hypertension and
esophageal varices who were candidates for endoscopic BL were enrolled. All patients were subjected to clinical,
laboratory and ultrasound evaluation. Upper GIT endoscopy with BL was performed till eradication of esophageal varices;
with assessment of portal hypertensive duodenopathy (PHD) before and after band ligation both endoscopically and
histopathologically. Results: Before BL, endoscopic PHD was found in 45.0% of patients. After eradication of esophageal
varices by BL, 50.0% of patients had PHD; showing no statistically significant difference between both values. The
lesions identified included duodenal mucosal erythema, erosions, ulcers, varices, telangiectasia and mixed lesions. There
was a strong relation between the presence of PHD and the grade of PHG endoscopically, while there was no relation to
Child score or the size of esophageal varices. The histopathological findings of the duodenal mucosal biopsies were
categorized as vascular lesions (mucosal capillary congestion, extravasation and capillary angiogenesis) and non-vascular
changes (edema, fibrous proliferation, villous changes, and apoptotic figures). Histopathological features of PHD were
present in more patients than those identified endoscopically. Conclusion: PHD is a complication of portal hypertension
which is seen less frequently than gastroesophageal varices and PHG. Its prevalence is not affected by eradication of
esophageal varices by band ligation, the grade of liver dysfunction, or the size of esophageal varices, while it has a strong
relation to the grade of PHG. The endoscopic and histopathological changes of PHD do not go hand in hand. We recommend
that careful duodenal examination should be done routinely during upper GIT endoscopy screening of patients with
cirrhosis and portal hypertension for detecting lesions of PHD; as they are a potential source of occult and overt GIT
bleeding in these patients.
Introduction
Portal hypertension (PH) is known to be associated
with the development of mucosal changes in the
gastrointestinal tract (GIT) - the so called
“congestive gastroenteropathy” (1) or “portal
hypertensive syndrome”. By far, the most dreaded
of these changes is the development of esophago-
gastric varices with their clinically devastating
consequence of upper GIT bleeding.(2) McCormack
et al.(3) in 1985 gave a detailed pathological
description of gastric mucosal abnormalities
associated with portal hypertension. Thereafter, it
has been shown that PH changes can affect all parts
of the GIT and acquired names according to the
regions involved e.g. portal hypertensive gastro
pathy (PHG) , (2) duodenopathy (PHD),(4) entero
pathy (PHE), (5-7) and colonopathy (PHC). (8).
Variceal bleeding is a frequent and severe
complication of cirrhosis. Bleeding occurs in 30–
40% of patients with cirrhosis and oesophageal
varices.(9)In patients with cirrhosis, ruptured
oesophageal varices cause approximately 70% of
all upper digestive bleeding.(10) Mortality from
bleeding gastroesophageal varices remains at 15%-
20%.(11). Portal hypertensive gastropathy (PHG) is
a gastric mucosal change associated with portal
hypertension. The “mosaic pattern” (12) and the
“cherry red spots” are the most frequently observed
lesions in PHG. The former consists of multiple
erythematous areas outlined by a white reticular
network and is generally considered as “mild”
PHG. The latter are round, red lesions, slightly
raised over the surrounding hyperemic mucosa.
These carry a higher bleeding risk and are
considered to reflect “severe” PHG.(13) Overall,
during the course of cirrhosis, mild PHG may be
observed in up to 50–70% of patients and severe
PHG in 20–40%.(14) Histologically, the stomach in
PHG contains dilated, tortuous, irregular veins in
the mucosa and submucosa, sometimes with
intimal thickening, usually in the absence of
significant inflammation.(15). Portal hypertensive
duodenopathy is a known association of portal
hypertension. Its prevalence has been estimated to
be between 14 and 25% according to Gupta et al.(16)
However, varying figures for PHD prevalence have
been reported in literature. Its clinical significance
relies on its potential for being a source of upper
gastrointestinal bleeding either occult or overt.
Hence, it could bear important consequences on
mortality and morbidity in this condition.(1) A
consensus definition of PHD is not available at this
time but various workers have considered many
endoscopic and histological features to be
consistent with a diagnosis of the disease. These
endoscopic findings are: (17) (a) mucosal erythema
(patchy or diffuse), (b) mucosal edema, (c)
mucosal breaks (erosions or ulcers), and (d)
vascular lesions (varices or telangiectasia). Other
rare lesions such as duodenal polyps have also been
reported. (18). Bleeding from PHD is more
commonly related to erosions and/or ulcers, (17) but
erythematous duodenopathy and even polyps (18)
have been reported to cause bleeding as well.
Fortunately, most episodes of overt bleeding are
self-limited, although bleeding can be severe and
require intervention.(17-19)Medical treatment did not
solve the problem of chronic blood loss, so an
approach using the argon plasma coagulator (APC)
was described. Endoscopic treatment modalitie-
shave been classically known to be safer in the
stomach because the gastric wall is thick and it is
easier to work with a coagulator, whereas the
duodenal wall is thin and there is risk of
perforation.(20) However, the experience in using
APC to perform hemostasis of bleeding duodenal
ulcers shows that application of this device in the
duodenum can be also safe because the thermal
effect is quite superficial (thermal effect depth is 2
mm) and limited to the mucosa, and so APC is a
suitable treatment, which can be safely performed
and repeated in case of portal hypertensive
duodenopathy. (20). As regards the pathogenesis of
portal hypertensive duodenopathy, studies have
suggested that the determining factor for
development of PHD is not the high portal pressure
itself but the point at which this high pressure starts
to produce congestive changes. This point might be
individualized and varies according to the whole
collateralization pattern in every patient. When this
point is reached and the congestive state gives
spreading gastric lesions, then the process tends to
be more generalized also affecting intestinal
segments. (17, 21). The location of duodenal erosions
in patients with portal hypertension differs from
that in patients with ordinary duodenitis. The
lesions commonly observed in duodenitis are
speckle erosions mainly located in the duodenal
bulb.(22) In contrast, the most frequently seen form
of duodenal erosion among patients with portal
hypertension extended from the superior portion to
the descending portion, and tended to show a
circular alignment along the Kerckring's folds.(22)
Kunisakasi et al (23) reported the first case of
visualized duodenal varices by endoscopic
examination in 1973, and thence an increasing
number of case reports has been accumulated in the
literatures. (24-26) Although bleeding from duodenal
varices is extremely rare, hemorrhage following
rupture is massive and often fatal.(27,28).
Histopathologically, PHD presents as vascular and
nonvascular abnormalities. The vascular
abnormalities dominate as the main histologic
feature characterizing this condition; they
includecapillary congestion (engorgement and
dilation of previously normal capillaries as a result
of portal hypertension), and capillary angiogenesis
or neovascularization,which is an important
vascular phenomenon that mediates adaptation and
accommodation of the high portal pressure.(17,29)
Nonvascular abnormalities include Edema of the
lamina propria, fibrous proliferation, increased
apoptosis and changes in the villous appearance
(shortened villi with a decreased or reversed
villous/crypt ratio); all in a background of absent or
minimal inflammatory cells. (30). Edema of the
lamina propria results primarily from the increased
capillary hydrostatic pressure in portal
hypertension, but the decreased capillary osmotic
pressure resulting from hypoalbuminemia in
cirrhotic decompensated patients would also have
a share in edema formation.Both fibrous
proliferation and duodenal villous changes can be
explained by mucosal congestion and hypoxia.(17)
Apoptosis or programmed cell death is an essential
event in both normal life and disease states.(31) In
the normal gastrointestinal tract, where renewal of
epithelial cells occurs every few days, apoptosis of
old worn out cells exactly matches their
replacement by mitotic proliferation.(32) The excess
apoptosis in PHD can be explained by mucosal
suffering, resulting from the congestive state with
consequent decreased mucosal O2 levels. In fact,
decreased hemoglobin oxygen saturation has been
documented in the duodenal mucosa of patients
with liver cirrhosis.(28) According to Jonas et al(33)
portal hypertension also alters the digestive tract
mucosa and increases its susceptibility to injury.
Impaired oxygenation of the mucosa, and hence
ischemia, is the probable mechanism for this
increased susceptibilityof the digestive tract in
portal hypertension.(34)
Aim of the work
The aim of this work was to study the endoscopic
and histopathologic duodenal mucosal changes in
patients with liver cirrhosis and portal hypertension
before and after band ligation of esophageal
varices.
Patients and methods
The study was carried out on twenty patients with
liver cirrhosis, portal hypertension and esophageal
varices who were candidates for band ligation,
enrolled during the period from September 2012 to
December 2013 from the Hepatology and
Gastroenterology Unit of the Medical Research
Institute, Alexandria University, Egypt. Patients
with portal vein thrombosis, hepatocellular
carcinoma or history of surgical intervention for
portal hypertension were excluded from the study.
Patients suffering from cardiac, pulmonary, renal,
endocrinal and collagenic diseases were excluded
to avoid hemodynamic changes resulting from
these diseases. The diagnosis of liver cirrhosis and
portal hypertension was based on clinical
examination, laboratory investigations, ultra-sonographic and Doppler criteria and endoscopic
data. After obtaining their written consent, all
patients were subjected to the following: Detailed
history taking and clinical examination. Laboratory
investigations; which included routine laboratory
tests (Complete blood picture, blood urea, serum
creatinine and fasting blood glucose),(35) liver
profile (serum albumin, serum bilirubin, serum
transaminases, prothrombin time and activity,(35)as
well as serum HBsAg, andHCV antibodies.(36).
Assessment of the severity of liver disease; which
was done according to modified Child-Pugh
classification.(37). Abdominal ultras-onographic
examination; which were used to determine the
size and echo pattern of the liver,(38,39)the size and
echopattern of the spleen,(40)presence or absence of
ascites and its grade, as well as the diameter of the
portal veins.(41). Upper GIT endoscopy; which
included the following three steps: First step:
Diagnostic upper GI endoscopy session; which was
aimingto assess the presence of esophageal varices
(EV) in need for band ligation, the presence of
gastric varices, the presence of portal hypertensive
gastropathy (PHG), the presence of portal
hypertensive duodenopathy (PHD) and to exclude
helicobacter pylori infection by urease test. Four
biopsies were taken from the gastric mucosa (two
from the fundus and two from the body), and four
other biopsies were taken from the duodenal
mucosa (two from the bulb and two distal to
ampulla) for histopathological examination.
Esophageal varices were graded according to the
North Italian Endoscopic Club (NIEC)(42) as: Small
(F1; the varices can be depressed by the
endoscope), medium (F2; the varices cannot be
depressed by the endoscope) and large (F3; the
varices are confluent around the circumference of
the esophagus). Red color signs included red wale
markings, cherry red spots, hematocystic spots and
diffuse redness. The presence and location of PHG
were described (i.e. fundus, body and antrum).
PHG was graded according to NIEC
classification(43) as: Mild (mosaic or “snake skin”
pattern of erythema, and severe(variety of
morphological appearance including cherry red spots,
red point lesions and black or brown spots).(44). The
presence and location of PHD were described (i.e.,
first part, second part, or both), and the type of
duodenal lesion was described as: mucosal
erythema (patchy or diffuse), mucosal breaks
(erosions or ulcers), vascular lesion (varices or
telangiectasia), villous changes or exaggerated
villous pattern and mixed lesions (i.e. more than
one lesion in the same patient).(17,45) . Second step:
Band ligation sessions till eradication of all EV
indicated for intervention; which were determined
according to the indications of the British Society
of Gastroenterology (46,47)and implemented using
multi-band ligator device. Ligation sessions were
repeated until no or only F1 varices (which were not
eligible for ligation) were observed. Third step:
Follow up diagnostic upper GI endoscopy session;
which was done one month after eradication of EV
for all patients to assess changes in the grade of
PHG and PHD endoscopically. Four biopsies from
gastric and duodenal mucosa were reobtained to
reevaluate the grade of PHG and PHD
histologically.
Histopathological examination of gastric and
duodenal mucosal biopsies:
1. Duodenal biopsies were examined to assess the
severity of PHD; they were examined for: Vascular
lesions (including mucosal capillary congestion,
extravasation and capillary angiogenesis) and
nonvascular changes (including edema, fibrous
proliferation, villous changes, and apoptotic
figures).(2,48,49). 2. Gastric biopsies were examined to
assess the severity of PHG; which was graded by
counting the number of dilated ectatic capillaries in
three consecutive high-power fields (x400) of the
superficial and deep lamina propria in each part, and
the mean of these values was taken for statistical
calculation. The grade of PHG was scored according
to Misra et al(48) as: Absent (0; no ectatic capillaries
in superficial or deep lamina propria), mild (1; one
to three dilated ectatic capillaries in the deep
lamina propria), moderate (2; more than three dilated
ectatic capillaries in the deep lamina propria) and
severe (3; dilated ectatic capillaries even in
superficial lamina propria).PHG scores of the fundus
and the body of the stomach were summarized and
expressed collectively as ″the pathologic score of the
stomach″, from which ″the pathologic grade of the
stomach″ was classified as:Absent (0), mild (1, 2),
moderate (3, 4) and severe(5, 6).
Results
Clinical and demographic data: The age of the
studied patients ranged between 28-65 years with a
mean of 52.410.23years, mostly being males (15
patients; 75.0%) living in rural areas (14
patients;70.0%).11 patients (55%) reported
positive history of upper gastrointestinal bleeding;
it wasin the form of hematemesis in 5 of them
(25%), while melena was present in the remaining
6 patients (30%).History of hepatic
encephalopathy was reported by 6 patients only
(30.0%). Clinical examination revealed jaundice in
11 patients (55.0%) and lower limb edema in 7
(35.0%). Hepatomegaly was found in 5 patient
(25.0%), splenomegalyin 12 (60.0%) and ascites in
8 (40.0%).
Laboratory investigations: Blood urea, serum
creatinine, urine analysis and routine stool analysis
were normal in all patients.Fasting blood glucose
ranged between 80 and 161 with a mean value of
108.6 22.1 mg/dl.HCV antibodies were positive in
100% of patients, while HBs Ag was negative in all.
The mean value for hemoglobinlevel was 11.56
1.36 g/dl, WBCs mean value was 5040
1852.6cells/ mm3, whilePlatelets mean value was
112.75 28.9x 103 cells/ mm3.Liver profile
parameters were assessed for the studied patients
before and after variceal band ligation (BL),
showing no statistically significant difference
between their values; as summarized in table(1).
Table (1): Liver profile of the studied patients
Before BL After BL Test of Sig. P
AST (U/L) 44.7 18.9 --- --- ---
ALT(U/L) 34.75 15.98 --- --- ---
Serum albumin (g/dl) 2.59 0.87 2.46 0.68 0.69 0.61
Serum Bilirubin (mg/dl) 3.49 1.06 3.62 1.64 0.921 0.354
Serum Alkaline phosphatase (IU/L) 108.2 24.6 --- --- ---
Prothrombin time (seconds) 16.8 3.98 17.2 3.8 1.03 0.156
Prothrombin activity (%) 55.2 11.85 54.8 9.3 0.771 0.485
p: p value for comparison between the studied groups, *:Statistically significant at p ≤ 0.05, AST: aspartate transaminase,
ALT: alanine transaminase, BL: band ligation.
Child Pugh score and class: The mean value for
Child score was 10.5 2.11 before BL and 10.93
2.36 after BL, with no statistically significant
difference between both values. Before BL, 9
patients (45.0%) were Child class B, while 11
(55.0%) were Child class C. After band ligation 7
patients (35.0%) were Child class B and 13
(65.0%) were Child class C, with no statistically
significant difference between both values; as
shown in table (2).
Table (2): Child Pugh score and class before and after BL
Before BL After BL Test of Sig. P
Child score (mean ± SD) 10.5 2.11 10.93 2.36 t = 0.68 0.336
Child class
Class B
Class C
No. % No. %
X2 = 0.42
0.51 9
11
45.0
55.0
7
13
35.0
65.0
p: p value for comparison between the studied groups, *:Statistically significant at p ≤ 0.05, BL: band ligation, No: number
of patients, SD: standard deviation.
Abdominal ultrasonographic examination: The
size of the right lobe of the liver ranged between 12
and 17.7cm, with a mean of 14.98 2.65cm.The
echopattern was coarse in all patients.The size of
the spleen ranged between 14 and 20 cm, with a
mean value of 16.02 2.33cm.Ascites was absent
in 2 patients (10.0%), mild in 10 patients (50.0%),
and moderate to massive in 8 patients (40.0%)
before BL. After BL, all patients had ascites; it was
mild in 9 patients (45.0%), and moderate to
massive in 11 patients (55.0%). The difference was
statistically non – significant (p = 0.456).
Endoscopic findings: 1.Esophageal and gastric
varices: No patients had gastric varices before or
after band ligation (BL). F2 esophageal varices
were present in 10 patients (50.0%) and F3
esophageal varices were present in the other 10
(50.0%) before BL.Red color signs were absent in
3 patients (15.0%), mild in 4 (20.0%), moderate in
7 (35.0%), and severe in 6 (30.0%). BL was
performed for all patients till complete eradication
of esophageal varices; table (3), figures (1-3).
Table (3): Esophageal variceal grade and red color signs in the studied patients
Number Percent
Esophageal varices grade
F1 0 0.0
F2 10 50.0
F3 10 50.0
Red color signs
Absent 3 15.0
Mild 4 20.0
Moderate 7 35.0
Severe 6 30.0
Fig.1: Endoscopic picture of F3 esophageal varices
without risk signs before band ligation.
Fig.2: Endoscopic picture of F2 esophageal varices with
risk signs (red spots) before band ligation.
Fig.3: Endoscopic picture of F3 esophageal varices after performing band ligation.
2. Portal hypertensive gastropathy (PHG):
Before BL, PHG was mild in 8 patients (40.0%),
and severe in 12 patients (60.0%). After band
ligation, PHG was mild in 7 patients (35.0%), and
severe in 13 patients (65.0%), with no statistically
significant difference between the values before
and after BL. Fundal mucosal involvement was
noted in all the 20 patients with gastropathy
(100%), body mucosal involvement in 15 (75%),
and antral mucosal involvement in 2 (10%); table
(4), figures (4-7).
Table (4): Grade of PHG before and after band ligation (BL)
PHG Before BL After BL
No. % No. %
Absent 0 0.0 0 0.0
Mild 8 40.0 7 35.0
Severe 12 60.0 13 65.0
X2
p
0.69
0.36
p: p value for comparison between the studied groups, *:Statistically significant at p ≤ 0.05, PHG: portal hypertensive
gastropathy, BL: band ligation, No: number of patients.
Fig. 4: Distribution of patients according to grade of portal hypertensive gastropathy before and after band ligation (BL)
Fig.5: Endoscopic picture of mild portal hypertensive
gastropathy of the fundus with mosaic like pattern.
Fig.6: Endoscopic picture ofmild portal hypertensive
gastropathy of the body with mosaic like pattern.
Fig.7: Endoscopic picture of severe portal hypertensive gastropathy of the body with red point lesions.
3. Portal hypertensive duodenopathy (PHD):
Before BL, PHD (with its various mucosal lesions)
was identified in 9 patients (45.0%), while it was
absent in 11(55.0%). After BL, one more patient
developed PHD, so that a total of 10 patients
(50.0%) had endoscopic PHD. There was no
statistically significant difference between the
values before and after BL; table (5), figure (8).
Table (5): Presence of PHD before and after BL
PHD Before BL After BL
No. % No. %
Absent 11 55.0% 10 50.0%
Present 9 45.0% 10 50.0%
X2
P
0.25
0.725
p: p value for comparison between the studied groups, *:Statistically significant at p ≤ 0.05, PHD: portal
hypertensive duodenopathy, BL: band ligation, No: number of patients.
Fig.8: Distribution of patients regarding the presence of portal hypertensive duodenopathy before and after band ligation (BL).
4. Regional distribution of duodenal lesions:
Before BL, involvement of only the first part of the
duodenumwas seen in 4 patients (20%), while one
patient (5%) had lesions in the second part only.4
patients (20%) had extending lesions along both
parts of the duodenum.After BL, no change of
regional distribution was observed. The one patient
who developed de novo PHD had lesions restricted
to the second part of the duodenum; table (6) and
figure (9).
Table (6): Distribution of patients according to location of PHD lesions
Regional distribution of PHD Before BL After BL
Test of Sig. P No. % No. %
1st part only 4 20.0 4 20.0
0.28 0.868 2nd part only 1 5.0 2 10.0
Both parts
(extending lesions) 4 20.0 4 20.0
p: p value for comparison between the studied groups, *:Statistically significant at p ≤ 0.05, PHD: portal hypertensive duodenopathy,
BL: band ligation, No: number of patients.
0
10
20
30
40
50
60
Before BL After BL
Absent Present
Fig.9: Distribution of patients according to location of duodenal lesions (%) before and after band ligation (BL).
5. Types of duodenal lesions: Erythema (redness)
was seen in 6 out of the 9(66.7%) patients with PHD
(including 2 with mixed lesions).It was diffuse in 4
patients and patchy in 2. After BL, diffuse
erythema restricted to the second part was
identified in the patient who developed PHD de
novo.Mucosal erosions were found in 3 out of the
9 (33.3%) patients with PHD (including 2 with
mixed lesions). Two had multiple erosions at the
second part and only one patient had multiple
erosions at the first part.Only one patient (11.1%)
had a duodenal ulcer at the first part (with other
mixed lesions).Telangiectasia was present in one
patient (11.1%) with other mixed lesions.
Duodenal varices in the first part were encountered
in only one patient. None of the patients had villous
changes (exaggerated villous pattern).The term
″mixed lesions″ was reported in 3 patients (33.3%)
who had more than one type of lesions across the
first and second parts;tables (7,8) and figures (10-
16).
Table (7): Distribution of the studied patients according to types of duodenal lesions
1st part only (No.) 2nd part only (No.) Both parts (No.)
Erythema
Diffuse 1 1 1
Patchy 1 - -
Erosions and ulcers
Erosions 1 - -
Ulcers - - -
Telangiectasia - - -
Duodenal Varices 1 - -
Exaggerated Villous pattern - - -
Mixed lesions - - 3
No.: number of patients
Table (8): Patterns of mixed endoscopic lesions
Patient First part Second part
#1 Telangiectasia Diffuse erythema
#2 Patchy erythema Patchy erythema,erosions
#3 Ulcer Erosions
20 20
5
10
20 20
0
5
10
15
20
25
1st part only 2nd part only Both parts
Before BL After BL
Fig.10: Distribution of endoscopic duodenal lesions according to their types
Fig.11: Endoscopic picture of portal hypertensive
duodenopathy showing diffuse erythema of the 1st part
of the duodenum.
Fig.12: Endoscopic picture of portal hypertensive
duodenopathy showing diffuse erythema at the 2nd part
of the duodenum
Fig.13: Endoscopic picture of portal hypertensive
duodenopathy showing healing duodenal ulcer at the 1st
part of the duodenum
Fig. 14: Endoscopic picture of portal hypertensive
duodenopathy showing duodenal erosions at the 2nd
part of the duodenum.
Fig.15: Endoscopic picture of portal hypertensive
duodenopathy showing duodenal varix at the 1st part of
the duodenum
Fig.16: Endoscopic picture of portal hypertensive
duodenopathy showing duodenal telangiectasia at the
1st part of the duodenum
66.7
33.3
11.1 11.1 11.1
33.3
0
10
20
30
40
50
60
70
Perc
en
t
Erythema Erosion duodenal ulcer Telangectasia Varices mixed lesions
Fig.17 Relation between endoscopic
PHD and grade of PHG
Fig.18: Relation between endoscopic
PHD and esophageal variceal size.
Fig.19: Relation between PHD and
Child class
6. Relation of PHD to PHG: Before BL, only one
patient out of 8 (12.5%) with mild PHG was found
to have PHD, while 8 out of 12 (66.6%) with severe
PHG had PHD endoscopically.AfterBL, one out of
7 patients (14.2%) with mild PHGhad PHD, while 9
out of 13 (69.2%) with severe PHG had PHD.The
difference between values was statistically
significant (p=0.009);i.e. there was a strong relation
between the presence of PHD and the grade of
PHG; figure (17).
7. Relation of PHD to variceal size: Among the
10 patients having F2 esophageal varices, only 4
(40.0%) had endoscopic PHD, while 5 patients out
of 10 (50.0%) having F3 esophageal varices
showed features of PHD. The difference between
both values was statistically non-significant (p=
0.422); figure (18).
8. Relation of PHD to Child class: Among the 9
patients who were Child class B, only 4 (44.4%) had
endoscopic PHD, while5 out of 11 (45.5%) who
were Child class C had endoscopic PHD. The
difference between values was statistically non-
significant (p=0.442); figure (19).
Histopathological findings
1. Gastric mucosal biopsies: The number of
dilated ectatic capillaries were counted and scored
in the superficial and deep lamina propria of the
gastric body and fundus. Before BL, the pathologic
score of PHG had a mean value of 3.8 0.65.After
BL, the mean value was 4.0 0.72.Consequently,
the pathologic grade of PHG of thestomach before
BL was assessed; it was mild in 4 patients (20.0%),
moderate in 8 (40.0%) and severe in 8
(40.0%).After BL, the grade was mild in 3 patients
(15%), moderate in 8 (40.0%), and severe in 9
(45.0%). There was no statistically significant
difference between the pathologic grade of PHG of
the stomach before and after BL (p= 0.365); table
(9) and figures (20-22)
Table (9): The pathologic grade of PHG and findings of the body and the fundus of the stomachbefore and after BL:
Body of stomach Fundus of stomach Whole stomach
Before After Before After Before After
No. % No. % No. % No. % No. % No. %
Absent 3 15.0 2 10.0 0 0.0 0 0.0 0 0.0 0 0.0
Mild 6 30.0 7 35.0 4 20.0 3 15.0 4 20.0 3 15.0
Moderate 7 35.0 6 30.0 8 40.0 8 40.0 8 40.0 8 40.0
Severe 4 20.0 5 25.0 8 40.0 9 45.0 8 40.0 9 45.0
Score range
Mean± SD
0-3
1.23±0.75
0-3
1.7±0.67
1-3
2.2±0.89
1-3
2.3±0.48
1-6
3.8±0.65
1-6
4.0±0.72
p 0.211 0.365 0.365
p: p value for comparison between the studied groups, *:Statistically significant at p ≤ 0.05, PHG: portal hypertensive
gastropathy, BL: band ligation, No: number of patients, SD: standard deviation.
8
12
Mild Sev er0
2
4
6
8
10
12
14
PHG PHD
10 10
F2 F30
2
4
6
8
10
12
EV PHD
9
11
Child B Child C0
2
4
6
8
10
12
Child class PHD
Fig.20: Distribution of patients according to the pathologic grade of PHG of the whole stomach before and after BL
Fig.21: Hematoxylin and eosin light microscopic picture
[x400] of the mucosa of the stomach body showing dilated
ectatic capillaries in the deep lamina propria (arrowed);
mild PHG.
Fig. 22: Hematoxylin and eosin light microscopic picture
[x400] of the mucosa of the stomach fundus showing dilated
ectatic capillaries in the superficial lamina
propria(arrowed); severe PHG.
2. Duodenal mucosal biopsies: In the first part
(bulb) of the duodenum; vascular abnormalities
included capillary congestion (as evidenced by the
presence of dilated capillaries filled with red blood
cells), capillary angiogenesis (giving abundant
small vascular spaces, with primarily subepithelial
location), with or without presence of hemorrhage
and red cells extravasation.Before BL, congestion
was detected in 60.0% of biopsies, while after BL
it was detected in 55.0%, with no statistically
significant difference between both values.There
was evidence of hemorrhage and extravasation in 3
patients (15.0%) before BL, with the same result
(15.0%) after BL. Non-vascular changes of the
duodenal bulb included edema, apoptotic figures
and fibrous proliferation. Edema was present in
75.0% of biopsies before BL, while after BL it was
detected in 65.0% , with no statistically significant
difference between values.Increased nuclear
apoptotic figures in the mucosal crypts, manifested
as pyknotic dark nuclei with extrusion of the cell
nucleus toward the crypt lumen, was reported in
20% of biopsies before BL, with the same result
after BL. Fibrous proliferation in the lamina
propria was detected in 10.0% of biopsies from the
first part before BL, with the same value after BL.
In the second part of the duodenum (distal to
ampulla); mucosal capillary congestion was
detected in 55.0% of biopsies before BL, while after
BL, it was found in 60.0%, with no statistically
significant difference between both values.There
was evidence of hemorrhage and extravasation in 2
patients only (10.0%) before BL, and in 3 patients
(15.0%) after BL, with no statistically significant
difference between values. Edema was present in
80.0% of biopsies before BL.After BL, it was
detected in 70.0%, with no statistically significant
difference between both values.Increased nuclear
apoptotic figures in the mucosal crypts were
reported in 20% of biopsies before BL, and in
25.0% after BL, with no statistically significant
difference between both values before and
after.Fibrous proliferation in the lamina propria
was seen before BL in 15.0% of biopsies from the
second part, with the same value after BL.Changes
in the villous appearance, including shortened villi
with a decreased or even reversed villous/crypt
ratio down to total villous atrophy, were only
evaluated in the second part of the duodenum(to
avoid misinterpretation in the transitional area of
the first part in which the villi might be less
developed as compared to the second part), and
they were found in 5% of biopsies before BL, with
the same findings after BL; table (10), figures (23).
0
5
10
15
20
25
30
35
40
45
Absent Mild Moderate Severe
Before After
Table (10): The histopathological changes of the duodenal mucosa before and after BL
1st part of duodenum 2nd part of duodenum
Before After Before After
No. % No. % No. % No. %
Vascular
changes
Congestion 12 60% 11 55% 11 55% 12 60%
p 0.336 0.652
Extravasation &hge. 3 15% 3 15% 2 10% 3 15%
p 1 0.71
Non
vascular
changes
Edema 15 75% 13 65% 16 80% 14 70%
p 0.231 0.225
Apoptosis 4 20% 4 20% 4 20% 5 25%
p 1 0.236
Fibrous proliferation 2 10% 2 10% 3 15% 3 15%
p 1 1
Villous changes not evaluated 1 5% 1 5%
p 1
p: p value for comparison between the studied groups, *:Statistically significant at p ≤ 0.05, BL: band ligation, hge.: hemorrhage,
No: number of patients.
Fig.23: Distribution of patients according to histopathological findings of the 1st part of duodenum before and after BL.
Fig. 24: Distribution of patients according to histopathological findings of the 2nd part of duodenum before and after BL.
0
10
20
30
40
50
60
70
80
Congestion Extravasation & hge oedema apoptosis Fibrous proliferation Villous changes
Vascular changes Non vascular changes
Before After
0
10
20
30
40
50
60
70
80
Congestion Extravasation & hge oedema apoptosis Fibrous proliferation Villous changes
Vascular changes Non vascular changes
Before After
Fig.25: Hematoxylin and eosin light microscopic picture
[x400] of the mucosa of the duodenal bulb showing capillary
congestion; dilated capillaries filled with red blood cells
(black arrows) seen within the lamina propria.
Fig.26: Hematoxylin and eosin light microscopic picture
[x400] of the mucosa of second part of duodenum showing
congested capillaries (black arrows) and edematous stroma.
Fig.27 (AandB): Hematoxylin and eosin light microscopic
picture [x400] of the mucosa of the 2nd part of the duodenum
showing apoptotic figures (pyknotic dark nuclei with
extrusion of the cell nucleus toward the crypt lumen)
(black arrows)
Fig.28: Hematoxylin and eosin light microscopic picture
[x400] of the mucosa of the 2nd part of the duodenum
showing new vessel formation (angiogenesis)
Relation of histopathologic to endoscopic
duodenopathy (table 11): The prevalence of
histopathological PHD was higher among our
patients than the prevalence of endoscopic PHD.
The histopathological changes of PHD (especially
capillary congestion) were not always associated
with endoscopicchanges of PHD; and there was no
statistical correlation between the presence of
capillary congestion in the 1st and 2nd parts of the
duodenum and the presence of endoscopic changes
of PHD. (P= 0.442, 0.365 respectively).
Table (11): Relation between endoscopic PHD and capillary congestion in the 1stand 2nd parts of the duodenum
Patients with endoscopic changes
(No=8)
Patients without endoscopic changes
(No=12) Test of sig.
No. % No. %
Capillary congestion in
the 1stpart of duodenum
5/8 62.5% 7/12 58% 0.442
Capillary congestion in
the 2ndpart of duodenum
3/ 5 60.0% 8/15 53.3% 0.365
p: p value for comparison between the studied groups, *:Statistically significant at p ≤ 0.05, No: number of patients.
A
B
Discussion
Among complications caused by portal
hypertension in the gastrointestinal tract (GIT),
duodenal affection has been reported less
frequently than gastroesophageal varices or portal
hypertensive gastropathy (PHG).(4,16) In the present
study, PHD was found in 45% of studied patients
before band ligation (BL). Varying figures for
PHD prevalence have been reported in the
literature, ranging from 8.4%(50) to 60%,(51)
probably representing differences in patient
selection criteria. Duodenal lesions were described
in relation to their location (i.e., first part, second
part, or both) and their type after Barakat et al(17) as
in some other studies.(22, 52). Diagnostic endoscopic
criteria of portal hypertensive duodenopathy
(PHD) in previous studies were not uniform.(53,54)
Lesions reported were erythema, scattered
petechiae, friable mucosa, erosions, ulcers and
edema. Histopathologically, although capillary
dilatation was considered the main feature,
capillary angiogenesis represented an additional
vascular change in PHD. Other non-vascular
duodenal mucosal changes were described,
including edema, fibromuscular proliferation,
apoptosis and decreased villous/crypt ratio.(17, 29) In
our patients, erythema was endoscopically detected
in 30%, while erosions were seen in 15%. Among
these, only one patient had multiple erosions at the
first part, while two patients had multiple erosions
at the second part. Similarly, a study by Shudo et
al(22) which included 440 patients with portal
hypertension reported that duodenal erosion were
found in 15% of them. They also stated that
duodenal erosion in case of portal hypertension
mostly extended from the superior portion to the
descending portion of the duodenum, and tended to
show a circular alignment along the Kerckring's
folds. In addition, one of our patients had duodenal
ulcer with negative helicobacter pylori test. In
justification of this finding, Corbishley CM et al
suggested that in the absence of a significant
association with helicobacter pylori,(55) duodenal
ulceration in cirrhotics could well be a part of
portal hypertensive vasculopathy. We were also
lucky to find one case with duodenal varices
among our patients, as it is known that endoscopic
evidence of duodenal varices in patients with portal
hypertension is uncommon.(56) The prevalence of
telangiectasia among our patients was 5%, while
the exaggerated villous pattern was not detected in
any of them. So, we can conclude that mucosal
erythema was the most common endoscopic
finding seen in PHD, which was also reported by
Desai et al(57) and others.(16, 17). In our study, we
assessed the relation between the diagnosis of PHD
endoscopically and other parameters; like grade of
liver dysfunction (Child class), variceal size and
grade of PHG. We found that PHD was present in
44.4% of Child class B and in 45.5% of Child class
C patients; i.e. almost equally distributed among
Child class B and C patients, and showing no
statistically significant relation between PHD and
the severity of liver disease.We also found that
among the nine patients with PHD, four patients
had F2 and five had F3 esophageal varices; with no
statistically significant relation between PHD and
variceal size. In addition, PHD was significantly
higher in patients having severe than mild portal
hypertensive gastropathy (frequency 66.6% versus
12.5%, respectively). These findings were in
agreement with Gupta et al(16) who assessed the
frequency and factors influencing PHD in cirrhotic
portal hypertension, and found no relation between
the frequency of PHD and the severity of liver
disease. They also found no relation between the
frequency of PHD and the size of esophageal
varices. Their findings were verified by two other
similar studies by Vigneri et al(4) and Oluyemi et
al.(58) A study by Barakat et al(17) described the
clinical, endoscopic, and histopathologic profiles
of PHD and showed that endoscopic duodenopathy
was significantly higher in patients having severe
(56.8%) than mild (23.5%) gastropathy, and that
there was no relation between endoscopic
duodenopathy and the size of esophageal varices.
Menchén et al(50) also demonstrated significant
correlation between presence of PHD and severe
PHG, while they could not prove a statistically
significant difference in the prevalence of
duodenopathy depending on the Child class. Data
concerning the relation between the presence and
severity of PHD and esophageal variceal
eradication have been conflicting. In our study,
endoscopic lesions of PHD were found in 45.0% of
patients before BL, compared to 50.0% after
esophageal eradication by BL, with statistically
non-significant difference between both values. In
contrast to our results, Menchén et al(50) in their
retrospective study found that PHD was more
frequent among those patients with prior BL of
esophageal varices. Also, EL- Khayat et
al(52)evaluated the effects of variceal obliteration on
portal hypertensive enteropathy (PHE) in thirty
patients and demonstrated higher frequency of
PHE after variceal obliteration. In agreement with
our results, however, Elnaser et al(59) studied the
effect esophageal varices eradication either by
endoscopic sclerotherapy or BL on PHD. Sixty
portal hypertensive patients were included, and the
results revealed no significant effect of varicael
eradication on the development of PHD. Another
study by Madkour et al(60) performed eradication of
esophageal varices by BL on thirty patients with
cirrhosis. Their results revealed that endoscopic
features of PHD were present in 13.3% of patients
before BL and in 16.7% of patients three months
after variceal eradication, showing no statistically
significant difference between both values.
Similarly, Gupta et al(16) prospectively evaluated 44
patients before sclerotherapy and one month after
eradication of esophageal varices for an increase or
decrease in the severity or extent of duodenopathy.
They found that six out of 44 patients (14%) had
PHD before sclerotherapy, while five had these
findings after variceal eradication; concluding that
no significant increase in PHD was noted after
variceal eradication. This information was also
supported by Nagral et al(5) who reported no
relation between PHD and previous sclerotherapy
or BL. The results of the histopathological
assessment of duodenal mucosa in our study were
presented as vascular changes (capillary
congestion and angiogenesis) and non-vascular
abnormalities (edema of the lamina propria, fibrous
proliferation, apoptosis and villous changes).
Similar findings were reported by Misra et al(29)
who described the histopathologic features of
duodenal and jejunal mucosal biopsy specimens
obtained from 58 patients with portal hypertension
and 30 healthy volunteers. They concluded that
thick-walled dilated vessels along with edema of
the lamina propria, fibromuscular proliferation,
decreased villous/crypt ratio, and thickened
muscularis mucosae form the characteristic picture
of portal hypertensive enteropathy. Shudo et al(22)
also described subepithelial edema and dilation of
mucosal and submucosal capillaries when
examining the biopsy specimens obtained from the
duodenal erosions among the patients with portal
hypertension. Similar to them and to Barakat et
al,(17) we found that the most common histological
change among our studied patients was edema of
the lamina propria. We also evaluated villous
changes only in the second part of the duodenum to
avoid misinterpretation in the transitional area of
the first part in which the villi might be less
developed as compared to the second part. As
regards the relation between histopathological and
endoscopic diagnosis of duodenopathy, many
patients in our study had histopathological changes
of PHD (especially capillary congestion), while
they were free from PHD changes endoscopically.
In other words, we found the prevalence of
histopathological PHD higher than the prevalence
of endoscopic PHD. This was verified by two
previously mentioned studies,(16, 17) which
demonstrated that the endoscopic changes of PHD
do not go hand in hand with histological changes.
From the present study we concluded that PHD is a
complication of portal hypertension which is seen
less frequently than gastroesophageal varices and
PHG. Endoscopic lesions of PHD include
erythema, erosions, ulcers, telangiectasia, varices,
exaggerated villous pattern and mixed lesions. Its
prevalence is not affected by eradication of
esophageal varices by band ligation, the grade of
liver dysfunction (Child score), or the size of
esophageal varices, while it has a strong relation to
the grade of PHG. Finally, endoscopic changes of
PHD do not go hand in hand with histopathological
changes of PHD. We recommend that careful
duodenal examination should be done routinely
during upper GIT endoscopy screening of patients
with cirrhosis and portal hypertension for detecting
lesions of PHD; as they are a potential source of
occult and overt GIT bleeding in these patients.
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Original Article
HCV Genotypes and SubGenotypes and HBV Precore and Core Mutations in
Hepatocellular Carcinoma Gamal Elden Ahmed Elsawaf1; Ola Abd El Kader Mahmoud1; Mohamed Abd Elrahman Ahmed2; Mohamed
Mohamed Shamseya3 and Hanada Salem Salim Islim1
1Department of Microbiology, 2Department of Clinical Pathology; Military Medical Academy, 3Department of Internal
Medicine; Medical Research Institute; University of Alexandria
ABSTRACT
HCC is the fifth common neoplasm in the world. It accounts for more than 500,000 new cases every year, and is the
third cause of mortality due to cancer. Known risk factors for HCC development are well documented: cirrhosis of any
etiology, hepatitis B (HBV), hepatitis C (HCV), hereditary liver disease, and exposure to carcinogens as aflatoxins, but
the synergism between these risk factors are still to be studied. HCV infection is a major risk factor for HCC. Markers
of HCV infection are found in a variable proportion of HCC cases varying from 27 up to 90% of cases. At least 6 major
HCV genotypes are identified. HCV genotype 4 is a very heterogeneous genotype showing significant genetic
divergence and more subtypes compared with other genotypes. Aim of the Work : to determine the HCV viral
genotypes and subgenotypes among HCC patients in Egypt, to investigate possible relation between any particular HCV
viral genotypes or subgenotypes and HCC, and to detect HBV precore and core mutations in HCC.
Patients and Methods: This study included 30 HCC patients. All relevant information was collected from each patient
including personal data, health data (history of blood transfusion, history of parenteral anti-schistosomal treatment
(PAT), previous surgical interference, and dentistry). Sera from the patients were tested for the following: liver
functions and virological studies including HCV-Ab, PCR for HCV-RNA, HCV genotypes and subgenotypes, HBsAg,
anti-HBc, anti-HBe, HBeAg, PCR for HBV-DNA, detection of HBV by SYBR Green Real Time PCR (ABI) using
specific primers for s, c and x genes and detection of HBV-DNA by conventional nested PCR using specific primers
for pol genes. Detection of precore-core promoter viral mutations in HBV-DNA positive, HBeAg negative and anti-
HBe positive patients by DNA sequencing. Results: In this study, anti-HBc was present in 18 (60%) among the 30
HCC patients, of whom 16 (88%) were anti-HCV positive which means that 16 (53.3%) of our HCC patients were both
anti-HCV and anti-HBc positive. No HBV-DNA could be detected in all the anti-HBc-positive HCC patients by
TaqMan probe technique and the conventional nested PCR. The 18 anti-HBc-positive HBsAg negative HCC cases were
tested for HBV genes (s, c and x) using Syber green Real time PCR. HBV DNA was detected in 18 cases (100%), x
gene was the dominant finding with 15 (83.3%) of the cases followed by s gene in 14 (77.7%) and core gene in 10
(55.5%) of the cases. In the current study both the highly conserved 5’UTR and NS5B regions of HCV genome were
used for the purpose of genotyping and subgenotyping. Out of the 26 positive HCV-RNA HCC cases only 23 cases
could be amplified. Amplification of NS5B region was only successful in 9 isolates out of the 23 positive HCV-RNA
HCC cases. 8 (88.8%) of the 9 isolates were successfully subgenotyped; 7 (77.7%) of them were of subgenotype 4a
followed by 1 (11.1%) of subgenotype 2c. Genotype 4 was diagnosed in 1 (11.1%) without accurate discrimination of
subgenotype. The 14 isolates, that couldn’t be amplified by primer encoding NS5B regions, were amplified by 5’UTR
primers. 5 (35.7%) of the 14 isolates region were successfully subgenotyped; 4 (28.6%) of them were of subgenotype 4a
followed by subgenotype 1g which represents only 1 (7.1%) of isolates. Genotype 4 was diagnosed in 9 (64.3%)
without accurate discrimination of subgenotype. Among the 23 genotyped isolates 2 (8.6%) were not HCV genotype 4.
One isolate belonged to 2c and other to 1g (4.3%). Conclusion : HCC in Egypt is strongly associated with HCV
infection; however, occult HBV infection might increase the risk of HCC development in chronic HCV patients.
Introduction
HCC is the commonest primary cancer of the
liver. Incidence is increasing and HCC has risen
to become the 5th commonest malignancy
worldwide and the third leading cause of cancer
related death, exceeded only by cancers of the
lung and stomach. The estimated incidence of
new cases is about 500 000-1000 000 per year,
causing 600,000 deaths globally per year. (1). The
distribution of liver cancer varies by region and
more than 80% of cases and deaths occur in
developing countries. In Africa, liver cancer has
been ranked as the fourth common cancer, and
most of liver cancers are HCC.(2) In most patients,
HCC is preceded by cirrhosis of the liver where
common causes of cirrhosis have been identified
as key risk factors for HCC. Of particular
importance is chronic infection with HBV or
HCV. It has been estimated that HBV is
responsible for 50–80% of HCC cases worldwide,
whereas 10–25% of cases are thought to be a
result of HCV infection.(3) In North Africa, the
higher HCV prevalence is expected to contribute
to the rising incidence of HCC over the next
decade in an aging cohort.(2). In Egypt liver cancer
constitutes 13% of all cancers(4) and is considered
the second most common malignant tumor after
bladder cancer in males and breast cancer in
females. (5). In HCV-infected patients, several host
and viral factors seem to accelerate progression to
cirrhosis and consequently HCC. These factors
include age, gender, heavy alcohol intake,
diabetes, obesity, co-infection with HIV or HBV,
level of HCV viremia and its genotypes.(6). In most
areas of the world, the incidence of HCC among
men is two to four times higher than the incidence
among women. The greatest differences between
male and female rates no longer occur among high-
risk HCC populations, but among the populations of
Central and Southern Europe. Typical among these
male: female ratios are the ones reported from
France (8.8:1), Switzerland (7:1) and Italy (4.8:1). In
contrast, typical ratios currently seen in high-risk
populations are those of China (3.7:1), Japan,
(4.0:1), Korea (3.6:1), and Vietnam (4.1:1). The
only registries in the world that report ratios at or
near 1:1 are in South America (Colombia, Ecuador
and Peru).(7). The more pronounced male:female
ratios currently found in low-to medium-rate areas
may be related to the changing rates in these
regions as male rates are increasing somewhat
faster than female rates in low-risk areas and
decreasing somewhat faster than female rates in
high-risk areas.(7). The reasons for higher rates of
liver cancer in males may relate to sex specific
differences in exposure to risk factors. Men are
more likely to be infected with HBV and HCV,
consume alcohol, smoke cigarettes, and have
increased iron stores. Androgenic hormones and
increased genetic susceptibility may also increase
risk among males.(8) . The global age distribution
of HCC varies by incidence, gender and, possibly,
also by etiology.(9) In almost all areas, female
incidence rates peak 5 years older than the peak
age of male rates. In low-risk populations, the
highest age-specific rates occur among persons
aged 80 years and greater.(7). A similar correlation
of risk and age is seen among most high-risk
Asian populations. Exceptions to these age
patterns occur among the high-rate populations of
Japan and Qidong, China. In Japan, male
incidence rates peak at age 65 and then, plateau,
while female rates plateau after age 70 years. In
China, the age-specific male incidence rates rise
until age 45 and then plateau. While the incidence
rates rise until age 60 before plateauing among
females in China.(7). In contrast, male rates in
high-risk African populations tend to peak
between ages 60 and 65 years before declining;
while female rates peak between 65 and 70 years
before declining.(7). The aim of this study was to
determine the HCV viral genotypes and
subgenotypes among HCC patients in Egypt, to
investigate possible relation between any
particular HCV viral genotypes or subgenotypes
and HCC, and to detect HBV precore and core
mutations in HCC.
Patients and Methods
This study was carried out during the period
between 2010- 2011. It included 30 HCC patients
who were admitted to the Hepatology Unit, MRI,
Alexandria University. All relevant information
were collected from each patient including
personal data as (age, sex, residence, smoking,
alcohol consumption ) as well as health data
(history of blood transfusion, history of PAT,
previous surgical interference, and dentistry).
Blood samples were collected from all patients,
left to clot. Serum was separated and stored in
small aliquot at -80°C and -20°C. Sera were tested
for the following investigations:
A- Biochemical studies: - Liver Functions: ALT,
AST, and bilirubin(10). - Prothrombin activity &
(INR) (10). - Platelet count (11).
B-Virological studies: - Detection of antibodies
against hepatitis C virus by ELISA (Abott Murex
Diagnostic Division).(12) - Detection of HCV-
RNA by conventional nested PCR amplifying the
5’UTR gene (GeneAmp PCR Systems 9700;
Applied Biosystems).(13) - Determination of
HCV genotypes and subgenotypes by Sequencing
of 5’UTR or NS5B genes.(13) - Detection of
hepatitis B virus surface antigen by ELISA
(DiaSorin HBsAg).(14) . - Detection of HBV
serological markers by ELISA.(14) - Antibodies
against hepatitis B core antigen (anti-HBc) (CTK
Biotech, Inc). - Antibodies against hepatitis Be
antigen (DiaSorin Anti-HBe). - Hepatitis Be
antigen (DiaSorin HBeAg). - Detection of HBV-
DNA by Real Time PCR (Artus).(15) - Detection
of HBV by SYBR Green Real Time PCR (ABI)
using specific primers for s , c and x genes.(16) .
-Detection of HBV-DNA by conventional nested
PCR using specific primers for pol genes.(17) -
Detection of precore-core promoter viral
mutations in HBV-DNA positive HBeAg negative
and anti-HBe positive patients by DNA
sequencing.(18)
Results
The present study group included 30 HCC
patients. Among the 30 HCC patients included in
this study, 23(76.7%) were males and 7(23.3%)
were females with a male to female ratio of 3.3:1..
(table. 1)
Table (1): Distribution of 30 HCC patients according to gender.
Gender Male Female Total n=30
No % No % No %
23 76.7 7 23.3 30 100
n=number of samples
Table (2): Distribution of 30 HCC patients according to age and gender.
Gender
Age
(years)
Male
n=23
Female
n=7
Total
n=30
No % No % No %
40 -49 2 8.7 0 0 2 6.7
50 -59 10 43.5 4 57.1 14 46.6
60 -69 9 39.1 2 28.6 11 36.7
≥ 70 2 8.7 1 14.3 3 10
Total 23 100 7 100 30 100
n=number of samples
Table (2) shows that among the 30 HCC patients
the highest percentages were in the age range
between 50-59 years (46.6%) followed by the age
group of 60-69 years (36.7%). The lowest
percentages were in the age group ≥ 70 year
(10%), followed by age group 40-49 years which
represented by only two patients (6.7%).
Table (3): Residence of 30 HCC patients included in this study.
Data
Egypt Governorates
No of Cases n=30
No %
Alexandria 12 40
EL-Beheira 15 50
Asyut 1 3.3
Suez 1 3.3
Dakahlia 1 3.3
n= number of sample
Table (3) shows that the highest percentage was encountered in El-Beheira governorate (50%), followed by
Alexandria (40%).
Table (4): Risk factors among the 30 HCC patients.
Risk factors No of patients n=30 %
PAT 15 50
Smoking 15 50
Diabetes Mellitus (DM) 12 40
Surgery 6 23.3
Blood transfusion (BT) 5 16.6
Dental intervention (DI) 5 16.6
Alcohol consumption (AC) 2 6.6
n= number of sample
Table (4) shows that among the 30 HCC patients,
PAT and smoking constituted the highest risk
factor (50%), followed by DM (40%) and surgery
(23.3%). History of Blood transfusion and dental
intervention were given by 16.6% of the patients,
whereas alcohol consumption was reported in
only 6.6% of cases.
Table (5): Biochemical laboratory investigations among the 30 HCC cases
Data Normal results No % Abnormal results No %
Bilirubin 11 36.7 19 63.3
Platelets count (PC) 6 20 24 80
Prothrombin % 3 10 27 90
ALT 5 16.7 25 83.3
AST 18 60 12 40
n= number of sample
Table (5) shows that among the 30 HCC patients,
only 3(10%) showed normal level of prothrombin
activity, 6(20%) had normal level of platelets
count, 5(16.7%) normal ALT and 11(36.7%)
normal bilirubin level, whereas the majority 60%
had normal level of AST.
Table (6): Distribution of anti-HCV positivity among the 30 HCC patients.
Anti-HCV No (n=30) %
Positive 28 93.3
Negative 2 6.7
Total 30 100
n=number of samples
Table (6) shows that 93.3% of the 30 HCC were anti-HCV positive.
Figure (1): Gel electrophoresis showing amplification of the 5'UTR of HCV genome
Figure (1) shows gel electrophoresis of the
amplified products of Nested RT-PCR of 5’UTR
region (237bp). From the twenty eight positive
anti-HCV results, twenty six showed bands at
237bp. Lane 16 and 22 were negative for HCV
5’UTR gene; LaneL:100bp ladder; Lane –C:
negative control, each lane had two negative
control; Lane +C: positive control used from
stored positive sample.
Table (7): Detection of HCV–RNA among the 28 anti-HCV positive HCC patients.
HCV-RNA No (n=28) %
Positive 26 92.8
Negative 2 7.2
Total 28 100
n=number of samples
Table (7) shows that 92.8% of the anti-HCV positive cases were HCV-RNA positive (Figure. 1)
Table (8): Distribution of HBV serological markers among the 30 HCC patients.
HBV marker No n=30 %
Anti-HBc 18 60
HBsAg 0 0
Anti-HBe 15 50
n=number of samples
Table (8) shows that 18(60%) of the 30 HCC
patients were anti-HBc positive and 15(50%) were
anti-HBe positive. HBsAg was not detected in any
of the studied cases.
Table (9): HBV serological profile of the 18 anti-HBc positive cases.
HBV marker No n=18 %
Isolated Anti-HBc 3 16.7
AntiHBc & Anti-HBeAg 15 83.3
HBV DNA (Artus) 0 0
HBV DNA (pol) 0 0
n=number of samples
Table (9) shows that among the 18 anti-HBc
positive HCC patients 15(83.3%) were also
positive for anti-HBe, whereas HBV-DNA could
not be detected in all cases by either conventional
nested PCR or TagMan probe technique.
Table (10): Distribution of anti-HCV and anti-HBc among the 30 HCC patients.
Anti-HBc positive Anti-HBc Negative Total
No % No % No %
Anti- HCV positive 16 53.3 10 33.3 26 86.6
Anti- HCV negative 2 6.7 2 6.7 4 13. 4
Total 18 60 12 40 30 100
Table (10) shows that among the 30 HCC patients
53.3% were positive for both anti-HBc and anti-
HCV, while 33.3% were positive only for anti-
HCV.
Table (11): Amplification of NS5B gene among the 26 HCV-RNA positive HCC patients.
No %
Amplified NS5B 10 38.5
Non-Amplified NS5B 16 61.5
Total 26 100
Table (11) shows that 10 (38.5%) out of the 26 HCV-RNA positive HCC patients showed NS5B
amplification. (Figure. 2)
Figure (2): Gel electrophoresis of NS5B Nested RT-PCR amplified products.
Figure (2) Shows gel electrophoresis of the
amplified products of Nested RT-PCR of NS5B
region (350bp). From the twenty six HCV RNA
positive cases only 10 samples (4,5,8,12,14,
17,19,21,23,26) gave NS5B band at 350bp. Lane
L: 100bp ladder; Lane -C: negative control.
Table (12): Amplified 5’UTR and NS5B genes used for HCV genotyping
and sub-genotyping in the 26 HCV-RNA positive HCC patients.
HCV genes No %
NS5B 10 38.5
5'UTR 16 61.5
Total 26 100
Table (12) Shows the 5'UTR of the 16 HCV-RNA positive patients which show no NS5B amplification were
used for sequencing.
Figure (3): Gel electrophoresis of purified products of 5’ UTR and NS5B genes
Figure (3) shows the twenty six purified amplified
products of NS5B and UTR genes. The above
section of the gel represented the bands of 10
samples of purified products of NS5b gene. The
below section represented the bands of 16 samples
of purified products of 5’UTR gene. L: 100bp
ladder
NS5B sequence results : After amplification of
NS5B region and purification step, samples were
sequenced using forward primer. Size of
sequenced NS5B region varies between 350 -
360bp.
Table (13): HCV genotyping and sub-genotyping of 9 isolates by direct sequencing of NS5B gene.
HCV genotype 4a 2c 4o/4a Total
No of patients 7 1 1 9
% 77.8 11.1 11.1 100
Table (13) shows that out of the 9 isolates 7
(77.8%) were subgenotyped as 4a and 1(11.1%)
was subgenotyped as 2c. Genotype 4 was present
in 11.1% without accurate discrimination of
subgneotype.
Sequence results of 5’UTR gene: After
amplification of UTR region and purification step,
samples were sequenced using forward primer.
Size of UTR region varies between 200 -210bp.
Table (14): HCV genotyping and sub-genotyping of 14 isolates by direct sequencing of 5'UTR gene.
HCV genotype 4a 1g 4a/4c 4g/4o Total
No of patients 4 1 8 1 14
% 28.5 7.2 57.1 7.2 100
Table (14): Shows that 5(35.7%) of the 14 isolates
which were subjected to sequencing of the 5’UTR
region were successfully subgenotyped; 4(28.5%)
of them were of subgenotype 4a followed by
subgenotype 1g (7.2%). Genotype 4 was
diagnosed in 64.3% without accurate
discrimination of subgenotype.
Table (15): HCV genotyping and sub-genotyping of 23 isolates by direct sequencing of NS5B or 5'UTR genes.
HCV genotype 4a 2c 1g 4a/4c 4g/4o 4a/4o Total
No of patients 11 1 1 8 1 1 23
% 48 4.3 4.3 34.8 4.3 4.3 100
Table (15): shows the distribution of the 23 cases
genotyped by direct sequencing. 56.6% of the
cases were successfully subgenotyped; 48% of
them were of subgenotype 4a followed by
subgenotype 1g and 2c that were equally detected
in 4.3% of isolates. Genotype 4 was detected in
43.4% without accurate discrimination of
subgenotype.
Table (16): Detection of HBV by SYBR Green Real time PCR in positive anti-HBc cases.
HBV genes n=18 No %
Surface antigen gene (s) 14 77.7
Core gene (c) 10 55.5
x gene 15 83.3
n=number of cases
Table (16) shows that x gene was detected in
15(83.3%) of the 18 antiHBc positive HCC cases
followed by s gene in 77.7%. Core gene was only
amplified in 10 (55.5%) of cases.
Table (17): Distribution of s,c,x genes among the 18 anti-HBc positive HCC cases.
HBV genes n=18 No %
s-c-x 5 27.7
s-c 3 16.6
s- x 6 33.3
c-x 2 11.1
C 0 0
S 0 0
X 2 11.1
n=number of cases
Table (17) shows that x gene was present either in
presence or absence of s and c genes in 15
(83.3%) out of the 18 anti-HBc positive HCC
cases.Detection of HBV by Syber green
technique :
Detection of s gene:
Figure (4): Amplification plot of s gene showing a CT of 16 cycles
Figure (5): Dissociation curve of s gene
A) Detection of c gene:
Figure (6): Amplification plot of c gene showing a CT of 18 cycles
Figure (7): Dissociation curve of c gene
B) Detection of x gene:
Figure (8): Amplification plot of x gene showing a CT of 20 cycles
Figure (9): Dissociation curve of x gene
Precore and Core Mutations
As all the cases studied were HBsAg negative, we couldn’t further investigate precore and core mutation.
Discussion
The present study group included 30 HCC
patients, 23(76.7%) were males and 7(23.3%)
were females. This ratio was consistent with
ratios of high risk populations like China, Korea
and Japan, but differed from that of El-Zayadi et
al (19) who showed a male to female ratio 7:1
which was closer to ratios reported from Europe.
In the present study, a different age pattern was
found since 46.6% of the HCC patients occur in
a younger age group (50-59 years), followed by
36.7% in the age group (60-69years). On the
other hand, only 10% were in the age group ≥ 70
years, and 6.7% were below 50years. Moreover,
both male and female rates peaked at the same age
group (50-59 years) before declining. El-Zayadi et
al (19) revealed that the highest percentage of HCC
was even among a younger age group (40-
59years). In North America and Western Europe
where HCC is rare before the age of 50 years, a
shift in incidence towards younger persons has
been also noted in the last two decades.(19). The
different age patterns of HCC incidence in
different areas are most likely related to the
dominant hepatitis virus in the population, the age
at viral infection, existence of other risk factors,
and cohort effects. In Japan, the dominant virus is
HCV, where most persons infected with HCV
were adults while in Qidong, China, the dominant
virus is HBV where persons became infected at a
very young age.(7). A unique invisible risk factor
for development of HCC in Egypt was attributed
to schistosomal infection and its parenteral
therapy, which played a role in transmission of
HBV and HCV through improperly sterilized
glass syringes.(20) PAT became possible in Egypt
in 1920 and these mass PAT campaigns
discontinued only in 1980. Mass PAT campaigns
to control schistosomiasis had great potential to
transmit HCV and HBV, because tartar emetic
was given in multiple doses by intravenous
injection and with insufficiently sterilized
injection equipment to people of all age groups.
PAT was a major risk factor of HCV
seropositivity which leads to HCC. High
incidence rates have been continued despite better
blood screening measures and better sanitization
practices within hospitals. In this study, among
the 30 HCC patients 15(50%) were heavy
smokers. Out of the 15 HCC smoker cases 14
(93.3%) were positive for HCV. In developed
countries, alcohol drinking seems to be the most
common source for HCC. Alcohol either directly
initiates HCC after its oxidation into
acetaldehyde, which is genotoxic, or indirectly
through the development of cirrhosis.
Epidemiological studies suggested a strong
synergistic effect of alcohol on both HBV and
HCV infections in developing HCC.(21). In the
current study, only 2 (6.7%) cases out of the 30
HCC patients were alcoholic. Although excess
alcohol consumption is a well- recognized risk
factor for HCC, this habit is relatively rare in
Egypt ruling out its role in the etiology of HCC in
Egypt. In Egypt HCV has been identified as a
cause of metabolic syndrome, a complex that
includes dyslipidemia, diabetes and insulin
resistance (IR). IR plays a crucial role in fibrosis
progression. HCV-associated IR may cause
hepatocarcinogenesis and proliferation of HCC.(22-
24). In a study done by Mohamed et al(25) on
Egyptian patients with HCV genotype 4 infection
found out that IR is induced by HCV-4
irrespective of severity of liver disease. IR starts
early in infection and facilitates progression of
hepatic fibrosis and HCC development. In the
current study, among the 30 HCC patients 12
(40%) patients were diabetic. Out of the 12
diabetic HCC cases 10 (83.3%) were positive for
HCV. Globally, HBV is the most frequent
underlying cause of HCC, with an estimated 300
million persons with chronic infection worldwide.
Case-control studies have shown that chronic
HBV carriers have a 5- to 15-fold increased risk
of HCC compared with the general population.
The great majority, between 70% and 90%, of
HBV-related HCCs develop in patients with
cirrhosis, however, HBV is a notorious cause for
HCC in the absence of cirrhosis.(8). Abe et al(26)
found a strong epidemiological evidence
correlating HCC to HBV infection. This was
shown by positive results in HCC patients for
both HBsAg and antiHBc or both together.
Seropositivity for the HBsAg is one of the most
important risk factors for HCC. The prevalence of
HBV infection in Egypt has been declining over
the last two decades. A single center study was
carried by El Zayadi et al (19) over a decade (1993-
2002) to identify any pattern changes of HCC in
hepatitis viruses markers in Egypt. They reported
a significant decline of HBsAg from 38.6% to
20.5%. A study carried by the Microbiology
Department of Faculty of Medicine and the
Medical Research Institute in Alexandria in
1999,(27) showed that the percentage of HBsAg
among HCC patients was 12%. A similar study
carried a decade later (2010) in the same institute
showed a significant decline of HBsAg from 12%
to 2%. In the present study, HBsAg was not
detected among our 30 HCC patients. In a study
conducted by Zhang et al (28) a large proportion of
HCV-infected patients in Japan had a past history
of HBV infection, as indicated by serum anti-HBc
positivity and HBsAg negativity. Marusawa et
al(29)showed that anti-HBc was detectable in
approximately 50% of patients with HCV-related
chronic liver disease who lacked HBsAg. Of note,
the proportion of patients who were positive for
anti-HBc increased in association with the
progression of liver disease, and accordingly the
prevalence of anti-HBc was substantially higher
in patients with HCC than in those with chronic
hepatitis or cirrhosis. The clinical presentation in
patients with anti-HCV positive and anti-HBc
positive was as severe as in patients with dual
HBV and HCV infection. In the present study,
anti-HBc was present in 18 (60%) among the 30
HCC patients. Out of the 18 positive anti-HBc, 16
cases were anti-HCV positive and 2 were
negative. The observation that anti-HBc-
positive/HBV-DNA negative patients show a
similar prevalence of severe liver disease to anti-
HBc/HBV-DNA positive patients and a
significantly higher prevalence than anti-HBc-
negative cases supports further the view that
isolated serum anti-HBc is a marker of clinical
significance.(30). The possibility of persistent HBV
infection in anti-HBc-positive individuals has
been supported by recent studies showing that
traces of HBV are often detectable in the blood
for many years after clinical recovery from acute
hepatitis. (31). The presence of HBV genomic
sequences distinguishes individuals at higher risk
of developing HCC among HBsAg negative
individuals, and so testing chronic hepatitis
patients for HBV genome appears to be an
important tool for identification of those who need
to be more carefully monitored for early diagnosis
of HCC. (32). The technical procedures used so far
have differed greatly from one study to another in
terms both of specificity and sensitivity and, as a
consequence, the results obtained have frequently
been contradictory. (33). Current technologies used
for DNA detection are nested-PCR and real-time
PCR. Primers must be specific for different HBV
genomic regions and complementary to highly
conserved (genotype shared) nucleotide
sequences.(34). In the present study, we tested three
technical procedures regarding their specificity
and sensitivity. Two real time PCR namely SYBR
Green (amplifying s, c and x genes), and TaqMan
probe technique (a commercial Artus kit targeting
a 134 base pairs) and a conventional nested PCR
with primers targeting the region encoding the pol
gene. No HBV-DNA could be detected in all the
anti-HBc-positive HCC patients by TaqMan probe
technique and the conventional nested PCR.
Using more sensitive techniques confirmed the
close correlation between occult HBV infection
and carcinogenesis. The incidence of occult HBV
infection varies significantly in HCC (12-63%).
Many authors demonstrated the relationship
between occult HBV infection and hepatocellular
carcinogenesis. Shiota et al (35) reported in their
case studies without a control group that serum of
18 out of 26 HCC patients without HBsAg and
anti-HCV were positive for either S, C or X
region on PCR and southern blotting. Pollicino et
al (36) described that viral DNA was detected in 68
of 107 cases of HCC tissue (63.5%) and in 63 of
192 cases of chronic hepatitis tissue (32.8%), and
concluded that occult HBV is a risk factor for
development of HCC. Sagnelli et al (37) performed
their study on 185 chronic HCV patients and
showed an occult HBV prevalence of 88 (47.6%)
patients who had anti-HBc positive sera. Of these
88 patients, 68.1% were anti-HBe positive and
36.3% were found positive for serum HBV DNA
by PCR. Occult HBV strain populations harbor a
genetic heterogeneity in viral regions (Pre-S/S,
Pre-Core/Core; X, Polymerase) and regulatory
elements (Core promoter, Enhancer I and II)
potentially involved in viral replication and/or
gene expression.(38). In the study carried in the
Faculty of Medicine and MRI in 2010, the HBV–
DNA was tested for s, c and x genes. HBV DNA
was detected in 18 (60%) out of the 30 anti-HBc
positive HBsAg negative HCC cases. Detection of
Core gene alone in 6 (20%) cases, or with Surface
and/or x gene in 12 (38.7%) cases was the
dominant finding. Surface gene was detected
together with Core and/or X in 8 (25.8%) cases. x
gene was detected in 6 (19.3%) cases together
with Core and/or s gene. In the present study, the
18 anti-HBc positive HBsAg negative HCC cases
were tested for HBV genes (s,c,x) using SYBR
green real time PCR. HBV DNA was detected in
the entire 18 anti-HBc positive. Our results differ
from the previous study. Detection of x gene was
the dominant finding with 15 (83.3%) followed by
s gene in 14 (77.7%) and core gene in 10 (55.5%)
of the cases. Integration of the viral DNA into the
host genome was suggested to be the initiating
factor for HBV-induced carcinogenesis.(39)
Integration of HBV DNA, however, has been
found in varied regions of the host chromosomes
and no preferential and specific site has been
identified. It was suggested that integration of
HBV DNA could also induce carcinogenesis via
transactivation of other oncogenes. Both HBx
protein and the truncated pre-S/S protein are
potent transactivators and are commonly found in
HCC tissue. (40). HBx is a well-known viral non-
structural gene that has roles as a multifunctional
regulator modulating gene transcription, as well as
controlling cell responses to genotoxic stress,
protein degradation, apoptosis, and several
signaling pathways. Its critical role in liver
malignant transformation has been demonstrated
in studies of transgenic mice with HBx
overexpression. HBx protein has been shown to
complex the tumor suppressor p53 protein and to
suppress its function.(41-43). Shetty et al (44)
prospectively examined the rate of HCC in
patients with HCV-associated cirrhosis and found
that those patients with occult HBV infection had
a significantly higher rate of HCC (59%)
compared to patients without occult HBV (36%).
In another large prospective study, Matsuoka et al (45) investigated the influence of occult HBV
infection on clinical outcomes of 468 HBsAg-
negative patients with chronic HCV. The authors
found a higher probability of developing HCC in
patients with occult HBV. Tamori et al (46) found
that patients with CHC who achieved SVR and
developed HCC had a higher rate of OBI than a
control group of 50 patients with CHC without
OBI. Miura et al (47) found that OBI was an
important independent factor affecting the
appearance of HCC. HCV is an important risk
factor for HCC in Western European and North
American countries, since epidemiological studies
have shown up to 70% of patients with HCC have
anti-HCV in the serum. Markers of HCV infection
are found in a variable proportion of HCC
patients; for example, 44%–66% in Italy, 27%–
58% in France, 60%–75% in Spain, and in 80%–
90% of HCC patients in Japan.(48) In Egypt unlike
most other parts of the world in where HBV and
heavy alcohol consumption are major causes for
HCC, chronic HCV infection is exceedingly
common. Egypt has the highest prevalence of
HCV in the world (14.7%).(49) Even higher HCV
infection rates, up to 60%, have been reported in
older individuals, in rural areas such as the Nile
delta, and in lower social classes. An association
between HCV-4 and the high rates of HCC in
Egypt has been speculated. Data from the
National Cancer Registry of Egypt, the National
Cancer Institute, and the Middle East Cancer
Consortium, as well as several published studies
show a close association between HCC and HCV-
4 in addition to a significant annual increase of
newly diagnosed patients with HCC. (50, 51). Our
findings agree with the previous studies since 21
out of the 23 HCV isolates were found to be
genotype-4. In our study it appears that HCC in
Egypt is strongly associated with HCV infection,
however; occult HBV infection increases the risk
of HCC development in chronic HCV patients.
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Original Article
Impact of Schistosomal Peri-Portal Fibrosis on The Results of Transient
Elastography in Hepatitis C Virus Patients
El-Kady A 1, Etaby A 2, Esmat G 3, Baddour N 4, Mohiedeen K1, Abdel Halim A 5
1 Department of Tropical Medicine Faculty of Medicine, Alexandria University, 2 Department of Radiodiagnosis,
Faculty of Medicine, Alexandria University, 3 Department of Tropical Medicine Faculty of Medicine, Cairo University,
4 Department of Pathology, Faculty of Medicine, Alexandria University, 5 National Hepatology and Tropical Medicine
Research Institute, Cairo
ABSTRACT
Both HCV and Schistosomiasis are highly endemic in Egypt and cases of coinfection are frequently encountered.
Transient eleastography (TE) is a promising tool for the rapid and non-invasive assessment of disease progression in
viral and non-viral chronic liver disease, which is currently subjected to extensive validation. Therefore, the aim of this
work was to evaluate the impact of schistosomal peri-portal fibrosis on the measurements of transient elastography in
HCV patients and to compare elastography with liver biopsy and conventional ultrasound findings. Patients and
Methods: This study was conducted on 150 chronic HCV patients diagnosed by seropositivity for HCV antibodies and
detection of HCV RNA by PCR. According to the results of anti-schistosomal antibody serology, they were categorized
into two equal groups. Routine work-up was done. Real-Time Ultra-sonography was performed including grading of
peri-portal fibrosis (PPF) and evaluation of liver condition. Liver stiffness measurements using Fibroscan and reference
needle-liver biopsy were done. Results: No significant difference was found between the two groups regarding Metavir
stages of fibrosis (P > 0.05). No significant differences were found among the two groups regarding Fibroscan results (P
> 0.05). Fibroscan tends to overestimate fibrosis in positive schistosomal serology patients with higher grades of PPF.
Significant agreement between Fibroscan reading and the liver biopsy was more obvious in patients with stage F0-1 and
patients with stage F4 in either negative or positive schistosomal serology patients. There was a statistically significant
agreement between the results of Fibroscan and the Metavir scoring among both groups; however this agreement was
not obvious among F2 and F3 fibrosis stage. Conclusions: Fibroscan tends to overestimate fibrosis in positive
schistosomal serology patients with higher grades of PPF. In addition, schistosomal infection decrease the sensitivity of
the Fibroscan to detect fibrosis stages (F2 and F3) as assessed by the Metavir scoring system. Hence, Fibroscan may be
considered a good tool for detection of fibrosis stages (F0-F1 and F4) but it is less sensitive in detection of fibrosis
stages (F2 and F3) in Egyptian patients with schistosomiasis and HCV coinfection.
Introduction
Viral hepatitis C is a serious liver disease
affecting 180 million people worldwide. The
severity of the disease associated with Hepatitis C
Virus (HCV) infection varies from asymptomatic
chronic infection to cirrhosis and hepatocellular
carcinoma. (1) . Schistosoma mansoni infection is a
chronic helminthic disease that develops primarily
because of chronic granulomatous inflammation
against parasite eggs in the liver, which results in
hepatic periportal fibrosis, portal hypertension and
sometimes death by bleeding of esophageal
varices, present in the severe hepatosplenic
clinical form.(2). Both HCV and schistosomiasis
are highly endemic in Egypt and cases of
coinfection are frequently encountered. Some
authors postulated an evidence of the association
between the schistosomiasis treatment campaigns
and the high HCV sero-prevalence rates in Egypt. (3) In chronic HCV infection, hepatocellular
damage occurs when the infected cell is
recognized by the immune system and destroyed.
This process is extremely variable and dynamic,
resulting in different intensities of hepatic ne-
crosis and inflammation. Thus, this continuous
inflammatory process is responsible for
fibrogenesis. (4) . Fibrosis is a wound healing
response in which damaged regions are
encapsulated by an extracellular matrix or scar. It
develops in almost all patients with chronic liver
injury at variable rates depending in part upon the
cause of liver disease and host factors. In contrast,
for unclear reasons, patients with self-limited
injury (such as fulminant hepatitis) do not develop
scarring despite an abundance of fibrogenic
stimuli; unless they go on to develop chronic
injury. (5). Liver biopsy is still recommended in
the majority of patients with chronic viral
hepatitis for fibrosis evaluation and treatment
indication. However, it is a painful and invasive
procedure, with rare but potentially life -
threatening complications, and prone to sampling
errors. Thus many patients with chronic viral
hepatitis are reluctant to undergo liver biopsy and
may be discouraged to start therapy for this
reason. These limitations have stimulated the
search for new noninvasive approaches. Ideally, a
noninvasive marker of liver fibrosis should be
liver-specific, easy to perform, reliable and
inexpensive. It should in addition be accurate not
only for the staging of fibrosis, but also for the
monitoring of disease progression and antiviral
therapy efficacy. (6). Considering all these facts,
noninvasive methods for the evaluation of liver
fibrosis were developed in the last few years, in
order to replace liver biopsy, among them the
liver stiffness (LS) evaluation by means of
transient elastography (TE) using a FibroScan
device (EchoSens, Paris, France). (7). Transient
elastography (TE) using Fibroscan is a novel
noninvasive method for assessment of liver
fibrosis, showing high concordance with values
acquired by the golden standard which is liver
biopsy. (8). Therefore, the present study was held
to find the impact of previous exposure to
schistosomiasis evidenced by positive
schistosomal serology on the results of Fibroscan
as a reliable non-invasive method for staging of
fibrosis in chronic HCV patients.
Patients and methods
This study was conducted in National Hepatology
& Tropical Medicine Research Institute
(NHTMRI), Cairo. The study was done on 150
chronic HCV patients diagnosed by seropositivity
for HCV antibodies and detection of HCV RNA
by PCR. According to the results of anti-
schistosomal antibody serology, they were
categorized into two groups: group I containing
75 patients with negative anti-schistosomal
antibody and group II including 75 patients with
positive anti-schistosomal antibody. An informed
consent was signed by every patient prior to start
of the research. Full history taking and clinical
evaluation was done for all patients. Blood
samples were collected from all patients to assess
the following parameters: Complete blood picture,
complete liver profile, AFP, viral markers
including: HBsAg assay, HBc-antibodies (IgM,
IgG), HCV Ab and detection of HCV RNA by
PCR. Schistosomiasis was screened by detection
anti-schistosomal antibodies using the indirect
haemagglutination test (IHAT). Real-Time
Ultrasonography for evaluation of liver size,
surface and texture was done for all patients.
Grading of peri-portal fibrosis (PPF) for patients
in group II was also done. Ultrasound guided liver
biopsy was performed using a semi-automatic
true-cut needle (16 G). Liver biopsy was fixed in
formalin and embedded in paraffin. H&E and
masson trichrome sections were prepared. Liver
fibrosis staging was evaluated according to the
Metavir scoring system. (9). Fibrosis was staged on
a 0 – 4 scale as follows: F0: No fibrosis. F1:
Portal fibrosis without septa. F2: Portal fibrosis
with rare septa. F3: Numerous septa without
cirrhosis. F4: Cirrhosis. Liver stiffness was
measured on the same day as liver biopsy using
the ultrasound TE FibroScan device (Echosens,
Paris, France), which consists of a 5-MHz
ultrasound transducer probe mounted on the axis
of a vibrator. TE measures liver stiffness in a
volume that approximates a cylinder 1 cm wide
and 4 cm long, between 25 and 65 mm below the
skin surface. The classification used for Fibroscan
stiffness was that described by Castera et al. (7).
F0–F1 ≤ 7 kPa, F2 = 7.1 – 9.4 kPa, F3 = 9.5 –
12.4 kPa and F4 ≥12.5 kPa. Patients unable or
unwilling to provide informed consent, as well as
patients with confirmed diagnosis and /or history
of malignancy or other terminal disease were
excluded from the study. Similarly, patients with
ascites, marked obesity and patients known to
have other chronic liver disease including
Wilson’s disease, alpha 1 antitrypsin deficiency,
autoimmune liver diseases, cholestatic liver
disease, hemochromatosis or hepatitis B virus
infections were excluded from the study.
Statistical analysis of data was performed using
SPSS 17 (Statistical Package for Scientific
Studies) for Windows.
Results
The current study revealed the following findings:
No significant difference was found between the
two groups of patients regarding age and gender
(table 1). No significant difference was detected
between the two groups regarding the laboratory
tests (complete blood count, liver function tests,
and AFP).
Ultrasonographic findings: There were no
statistically significant differences between both
groups concerning the liver size, surface or
texture. Ultrasonographic estimation of the
periportal fibrosis grades, in group (I) revealed
that all patients (100%) had no periportal fibrosis.
In group II, 31 patients (41.3%) had no periportal
fibrosis, 30 patients (40%) had grade I periportal
fibrosis, 9 patients (12%) had grade II periportal
fibrosis and 5 patients (6.7%) had grade III
periportal fibrosis. There was a highly significant
difference between both groups regarding
periportal fibrosis lesions (p<0.001).
Liver biopsy findings: No significant difference
was found between the two groups regarding
Metavir grades of inflammatory activity or stages
of fibrosis in liver biopsy samples.(p 0.094 &
0.763 respectively).
Fibroscan results in both groups: Regarding the
transient elastography results, the stiffness range
was 3.3-39.8 kpa among Schistosoma negative
patients (group I) with a mean of 8.68±7.86 kpa,
and 2.5-34.8 kpa among positive schistosomal
serology patients (group II), with a mean of
9.10±7.07 kpa. There was no statistically
significant difference between both groups as
shown in (Figure1).
Fibroscan results in relation to liver texture by
ultrasonography: There was a significant
positive correlation between Fibroscan results and
liver echotexture in either negative or positive
schistosomal serology patients. The mean
Fibroscan score was 5.35 kpa for normal liver,
6.80 kpa for bright liver and 14.63 kpa for coarse
liver in negative schistosomal serology (group I),
while it was 5.33 kpa for normal liver, 9.57 kpa
for bright liver and 11.8 kpa for coarse liver in
positive schistosomal serology patients (group II).
Fibroscan results in relation to
ultrasonographic grading of PPF In patients of
group II: There was a significant positive
correlation between Fibroscan results and
different grades of PPF. The mean Fibroscan
score was 4.93 kpa for patients with no PPF, 8.45
kpa for patients with PPF grade I, 15.46 kpa for
patients with PPF grade II and 27.32 kpa for
patients with PPF grade III as shown in (Table 2).
In patients of group II with no PPF, Metavir
scores were similar to Fibroscan results in patients
with F0-F1 as well as patients with F3 fibrosis
stages. There was a discrepancy between Metavir
scoring and Fibroscan results in patients with F2
fibrosis stage by Metavir. Fibroscan results for
those patients gave a lower value F0-F1 as shown
in (Tables 3, 4). In patients of group II with PPF
grade I, significant association and agreement
were found between fibroscan results and Metavir
fibrosis stages (F0-F1, F2 and F3) (Tables 5, 6).
The sensitivity of fibroscan for detection (F0-F1
and F3) among patients with no PPF was better
than that in patients with grade I PPF. In patients
of group II with PPF grade II, no significant
association was found between Fibroscan score
and Metavir fibrosis stage as shown in (Table 7).
Fibroscan tends to overestimate fibrosis in group
II positive schistosomal serology patients with
higher grades of PPF, as all 5 patients with grade
III PPF have F 4 Fibroscan result and F3 Metavir
stage.
Fibroscan results in relation to fibrosis stage
according to Metavir score in both Groups: There was a significant positive correlation
between Fibroscan score and liver biopsy
(Metavir) in either negative or positive
schistosomal serology patients i.e. irrespective to
the presence of schistosomiasis (P < 0.05).
Significant agreement between Fibroscan reading
and the liver biopsy was more obvious in patients
with stage F0-1 and patients with stage F4 in
either negative schistosomal serology patients or
positive schistosomal serology patients. However
this agreement was not obvious among F2 and F3
fibrosis stage. (Tables 8, 9). Fibroscan was able to
detect F2 and F3 stages with 38.4% (5/13) and
44.4% (4/9) sensitivity among negative
schistosomal serology patients, while these values
were reduced to15.4% (2/13) and 21.5% (3/14),
respectively, in positive schistosomal serology
patients.
Table (1): Demographic data of patients
Groups Group I Group II P value
Age Range
Mean + SD
18 -58
36.80±11.49
22 – 59
39.99± 8.39
0.56
Gender Males
Females
54 (72%)
21 (28%)
58 (77.3%)
17 (22.7%) 0.453
Table (2): Fibroscan results in relation to ultrasonographic grading of PPF In patients of group II
Peri-Portal fibrosis
grades
Group II
P value N %
Fibroscan
Mean SD
No PPF 31 41.3 4.935 2.072
0.00
Grade I 30 40.0 8.453 3.798
Grade II 9 12.0 15.467 7.076
Grade III 5 6.7 27.320 6.685
Table (3) Fibroscan results according to castera cut-off among patients
with no PPF (in group II) in relation to liver biopsy (Metavir)
Table (4): The agreement between Fibroscan and the liver biopsy (Metavir) in patients with no PPF (in group II)
Metavir Agreement
Total P value Yes No
F0 – F1 25 (100%) 0(0%) 25 0.00
F3 2 (100%) 0 (0%) 2
Table (5): Fibroscan results according to castera cut-off in patients with PPF grade I in relation to liver biopsy (Metavir)
Metavir Fibroscan by Castera cut-off
P value F0-F1 F2 F3 F4 Total
F0- F1 13 5 0 1 19
0.001
F2 3 2 1 0 6
F3 0 0 1 4 5
F4 0 0 0 0 0
Total 16 7 2 5 30
Table (6): The agreement between Fibroscan and liver biopsy (Metavir) in patients with PPF grade I (in group II)
Metavir Agreement
Total P value Yes NO
F0-F1 13 (68.4%) 6(31.6%) 19
0.00
F2 2 (33.4%) 4(66.6%) 6
F3 1 (20%) 4(80%) 5
Total 16 14 30
Table (7): Fibroscan results according to castera cut-off in patients with PPF grade II in relation to liver biopsy (Metavir)
Metavir Fibroscan by castera cut-off P value
F0- F1 F2 F3 Total
0.188
F0- F1 0 1 1 2
F2 2 0 1 3
F3 0 0 2 2
F4 0 0 2 2
Total 2 1 6 9
Metavir Fibroscan by castera cut-off
Total P value
F0-F1 F2 F3
F0-F1 25 0 0 25
0.00
F2 4 0 0 4
F3 0 0 2 2
Total 29 0 2 31
Table (8): The agreement between Fibroscan and liver biopsy (Metavir) in group I
Metavir Agreement
Total P value Yes NO
F0-F1 40 81.6% 9 18.3% 49
0.00
F2 5 38.4% 8 61.6% 13
F3 4 44.4% 5 55.6% 9
F4 3 75% 1 25% 4
Table (9): The agreement between Fibroscan and liver biopsy (Metavir) in group II
Metavir Agreement
Total P value Yes NO
F0-F1 38 82.6% 8 17.4% 46
0.00
F2 2 15.4% 11 84.6% 13
F3 3 21.5% 11 78.5% 14
F4 2 100% 0 0% 2
Figure (1) : Fibroscan score for Metavir fibrosis stages among group I & group II.
Discussion
Staging liver fibrosis is considered to be an
essential part in the management of patients with
chronic hepatitis C, because it provides prognostic
information and in many cases, assists in
therapeutic decisions. (10). Many parameters for
noninvasive diagnosis of liver fibrosis have been
studied extensively in the past, but none has yet
replaced liver biopsy as the gold standard. (11)
Measurement of liver stiffness by TE (FibroScan)
is widely used nowadays as a validated non-
invasive method for the assessment of liver
fibrosis. (12). The present study showed that, there
was no significant difference between both groups
regarding Metavir stages of fibrosis (p>0.05).
This is in consistent with Ahmad et al. (13) who
showed that schistosomiasis coinfection with
HCV and / or non-alcoholic steatohepatitis had no
significant impact on fibrosis stage. The present
results agree with the study conducted by Abdel-
Rahman et al. (14) which demonstrated that positive
schistosomal serology has no effect on fibrosis
staging. Moreover, Shiha and Zalata (15) found
that, schistosomal hepatic affection does not alter
or interfere with assessment of fibrosis in mixed
HCV schistosomal liver affection. On the other
hand, Kamal et al. (16) reported that Egyptian
patients with co-infections had higher HCV RNA
titers, more advanced liver disease, more hepatic
complications, and a greater mortality rate than
those with HCV mono infection. Regarding the
ultrasonographic findings, there were no
statistically significant differences between both
groups concerning the liver size, surface or
texture. Regarding the grades of PPF, there was a
statistically significant association between the
presence of schistosomiasis and the PPF. This
finding agrees with Berhe et al. (17) who concluded
that prevalence of periportal thickening and
fibrosis (PPT/F) increased significantly with
increasing intensity of S. mansoni infection. There
was a significant positive correlation between
Fibroscan results and different grades of PPF by
ultrasound in patients with positive schistosomal
serology. The Fibroscan stiffness increase with
increased grade of PPF and this may be explained
by the influence of schistosomal infection which
is responsible for some degree of fibrosis
especially periportal fibrosis and so affecting the
reading of Fibroscan. In group II patients with
positive schistosomal serology, there was a
statistically significant association between
Fibroscan score and the Metavir fibrosis stage
with statistically significant agreement especially
in (F0-F1) and F3 fibrosis stages for those with no
PPF or grade I PPF. However this relation was not
obvious in grade II PPF due to its small number
relative to other groups. In patients with grade III
PPF, Fibroscan tends to overestimate their fibrosis
stage to be F4 although it was given a score of F3
by Metavir scoring. In the present study,
Fibroscan was able to detect F0-1 and F3 stages
with 100% (25/25) and 100% (2/2) sensitivity
among patients with no PPF in group II, while
these values were reduced to 68.4% (13/19) and
20% (1/5), respectively in patients with grade I
PPF. This is in consistent with Esmat et al. (18)
who reported that the change in the sensitivity of
Fibroscan in different fibrosis stages and the
overestimation among patients with positive
schistosomal serology may be attributed to the
added effect of schistosomiasis on the rate of
fibrosis progression among coinfected patients
and the schistosomal periportal fibrosis, which
affects the interpretation of the liver stiffness
measurements by Fibroscan. The present study
revealed that there is a significant positive
correlation between Fibroscan results and liver
echotexture among both groups. Tchelepi et al. (19)
proved that ultrasound can assess hepatic
parenchyma composition qualitatively but it is
both subjective and operator dependent and that
liver fibrosis and steatosis can have similar
appearances and can be present at the same time
in a fatty fibrotic pattern. Moreover Zheng et al.
(20) reported that in patients with liver cirrhosis,
US could not detect the abnormalities caused by
histological changes such as fine and sparse
fibrotic septa or small and uniform pseudolobular
nodules, thus false negative diagnosis might be
made. As they found that eighteen patients who
were predicted as mild or moderate fibrosis by US
were finally diagnosed as cirrhosis by histology
(false negative results), while twenty patients who
were predicted as liver cirrhosis by US were
finally diagnosed as mild and moderate fibrosis by
histology (false positive results). On the other
hand, Afdhal et al. (21) found that a proper US
examination can identify patients with cirrhosis
when the biopsy findings are equivocal, or at
variance with the clinical impression. This study
showed no significant difference between the two
groups in terms of transient elastography results.
This can be explained by several factors affecting
liver stiffness as only a minority of the individuals
infected with schistosoma mansoni may develop
hepatic fibrosis or be more sensitive to
infection(s). As the majority of patients included
in this study had no significant fibrosis attributed
to schistosomiasis (31 patients showed no PPF
and 30 patients with minimal grade I PPF).
Moreover, frequency of exposure is directly
correlated with the presence and amount of
fibrosis. In addition, several clinical and
pathological studies have shown that schistosomal
hepatopathy is a reversible condition and that
resolution of the schistosomiasis disease is
accompanied by subsequent fibrosis resorption.
Also praziquantel is believed to exert antifibrotic
effects by affecting (decreasing) activation of
hepatic stellate cells through inhibition of
profibrotic gene expression. (14). In the present
study, there was a significant correlation between
Fibroscan results and liver fibrosis stage in liver
biopsy samples by (Metavir) in either negative or
positive schistosomal serology patients (group I &
II). This finding agrees with Esmat et al. (18) who
stated that there was a statistically significant
concordance between histopathology results
(Metavir) and Fibroscan results in either negative
schistosomal serology patients or positive
schistosomal serology patients. The present study
showed that, the agreement between the Fibroscan
and the liver biopsy was similar in patients with
negative schistosomal serology and in patients
with positive schistosomal serology. On the other
hand , Esmat et al. (18) found that, the agreement
between the Fibroscan and the liver biopsy was
slightly better in patients with negative
schistosomal serology than in those with positive
schistosomal serology but this difference may be
attributed to the smaller number of patients
included in this group in the present study. In the
present study, the agreement between Fibroscan
readings and the liver biopsy, is more obvious in
patients with stage F0-1 and patients with stage
F4 (P value <0.01). This finding agrees with
Esmat et al. (18) who revealed that fibrosis stages
(F0–F1 and F4) were the most independent factors
that were associated with that agreement. The
present study showed that there was a statistically
significant disagreement between the results of
liver biopsy (F2 and F3 fibrosis stages) and
Fibroscan in either negative schistosomal serology
patients or positive schistosomal serology patients
(P value <0.01). However, Esmat et al. (18)
reported that this relation was not statistically
significant among those with negative
schistosomal serology. The sensitivity of
Fibroscan for detection of fibrosis stages (F 2 and
F3) decreased from 38.4% , (5/13) and 44.4%
(4/9) in group I to 15.4% (2/13) and 21.5%
(3/14), respectively in group II and this was in
agreement with the study conducted by Esmat et
al. (18) which revealed that previous exposure to
schistosoma was assumed to impair the sensitivity
of Fibroscan for the detection of fibrosis stages
(F2 and F3). The sensitivity of Fibroscan for
detection of significant fibrosis in the whole
patients of the present study was 26.9% and
these results were different from the study
conducted by Castera et al. (5) which revealed
that the sensitivity of Fibroscan for detection of
significant fibrosis among studied patients was
67% . Moreover, results were reported by
Shaheen et al. (22) in his meta analysis in
assessing Fibroscan for prediction of significant
fibrosis at a cut off level of 7.1-8.8 kpa with
sensitivity of 63.8% . Results involving
differences in sensitivity of Fibroscan may be
explained by; the present study was done on
Egyptian population but castera studied other
population. This is in consistent with Friedman (5)
who reported that host genes that regulate the risk
of fibrosis progression, could represent an
important insight into the pathogenesis of fibrosis
in humans. From the present study, it may be
concluded that Fibroscan tends to overestimate
fibrosis in positive schistosomal serology patients
with higher grades of PPF. The sensitivity of
Fibroscan for detection of (F0-F1 and F3) among
patients with no PPF was better than that in
patients with grade I PPF. In addition
schistosomal infection decrease the sensitivity of
the Fibroscan to detect fibrosis stages (F2 and F3)
as assessed by the Metavir scoring system. Hence,
Fibroscan may be considered a good tool for
detection of fibrosis stages (F0-F1 and F4) but
less sensitive in detection of fibrosis stages (F2
and F3) in Egyptian patients.
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Original Article
Interleukin 23 p 19 Gene Expression in Patients with Ulcerative Colitis and Its
Relation to Disease Severity
Hanan El-Bassat, Lobna AboAli, Sahar El Yamany, Hanan Al Shenawy1, Rasha A. Al Din2 and Atef Taha3
Tropical Medicine, Pathology1, Microbiology and Immunology2 and Internal Medicine3 Departments, Faculty of
Medicine, Tanta University, Egypt.
ABSTRACT
This study was to determine whether mucosal expression of IL 23 p 19 has a role in the pathogenesis of ulcerative
colitis and its relation to disease severity. Patients and methods: This study was carried out on 50 patients with
ulcerative colitis and 10 individuals with normal colonoscopy and histopathology as control. They were divided
according to endoscopic and histopathological findings into: Group I: 27 patients with mild to moderate ulcerative
colitis disease. Group II: 23 patients with severe ulcerative colitis disease. Group III: 10 individuals whose colonoscopic
and histopathologic findings were normal as control. All patients and control were subjected to histopathological study,
IL-23p19 immunohistochemical staining, IL-23 R expression by flowcytometry and serum IL-23 by ELISA. Results:
Significant increased expression of IL-23p 19 gene and IL-23 R in patients with ulcerative colitis compared with
control. Patients with severe disease have significantly increased expression of IL-23 p19 gene; IL-23 R and high serum
level of IL-23 compared with patients have mild to moderate disease. Significant positive correlation were detected
between increased expression of IL-23 p 19 gene, IL-23 R, high serum IL-23 and severity of the disease. Conclusion:
Increased expression of IL-23 p 19 gene has a role in the pathogenesis of ulcerative colitis and that targeted therapy
directed against IL-23 p 19 may be effective in the treatment of the disease. Increased expression of IL-23p19 gene and
IL-23 receptors together, with high serum level of IL-23 correlate positively with disease severity.
Introduction
Inflammatory bowel disease (IBD) consists of two
distinct diseases, Crohn's disease (CD) and
ulcerative colitis (UC). Both diseases are thought
to arise due to combination of genetic variations
and alteration in bacterial flora which can
subsequently drive a dysregulated immune
response that results in chronic intestinal
inflammation [1, 2]. Interleukin 23 (IL-23) is a
member of a small family of proinflammatory
cytokine, consisting of a p19 subunit and a
common p40 subunit [3]. The receptor for IL-23
(IL-23 R) consists of the IL-12R beta 1 subunit
and a novel component termed IL-23R [4], which
is expressed predominantly on T, NK, and NKT
cells and to a smaller extent on monocytes,
macrophages and DCs [5]. IL-23 plays a crucial
role in the pathogenesis of a number of immune-
mediated inflammatory diseases by recruitment of
several inflammatory cells and Th17 cells [6, 7]. IL-
23 promotes Th17 cells producing TNFα, IL-17,
IL-6, IL-22, GM-CSF, and other novel factors,
which are associated with the induction of
autoimmune inflammation [6, 8, 9].
Patients and Methods
This study was carried out on 50 patients with
ulcerative colitis and 10 age and sex matched
individuals whose colonoscopic and
histopathologic findings were normal as control.
The studied subjects were selected from the
inpatient and outpatient clinics of tropical
medicine and internal medicine departments,
Tanta University Hospital, Tanta, Egypt, in the
period between November 2012 and October
2013. Ulcerative colitis patients were diagnosed
on the basis of clinical, endoscopic and
histological manifestations according to the
criteria of American Gastroenterology
Association [10]. They were divided according to
endoscopic and histopathological findings into:
Group I: 27 patients with mild to moderate
ulcerative colitis disease. Group II: 23 patients
with severe ulcerative colitis disease. Group III:
10 individuals whose colonoscopic and
histopathologic findings were normal as control.
Exclusion criteria: Pregnancy, malignancy, heart
failure, renal failure, thyroid disorders, acute
infection and stroke. Patients with
immunosuppressive drugs were excluded. All
patients and control were subjected to complete
history taking and thorough clinical examination.
Laboratory investigations including, complete
blood picture, blood urea and serum creatinine,
erythrocyte sedimentation rate and stool
examination to exclude bacterial causes of colitis.
Colonoscopy was performed in all groups and the
severity of the disease was determined. An
endoscopic scoring system for UC of Pineton de
Chambrun et al., 2010[11] was used. Score O:
Normal or inactive disease. Score 1: Mild disease
(erythema, decreased vascular pattern and mild
friability). Score 2: Moderate disease (marked
erythema, increased vascular pattern, friability
and erosion). Score 3: Severe disease
(spontaneous bleeding and ulceration).
Endoscopic findings were recorded and multiple
biopsies were taken for histopathology, IL-23 p
19 immunohisto-chemical staining and IL-23 R
expression by flowcytometry.
Histopathological study: 4-μm-thick serial
sections of formalin fixed, paraffin - embedded
tissue were cut and stained by Hematoxylin and
Eosin for histopathological evaluation and grading
of the groups. A six grade classification system
for inflammation was used. The grades were:
0, structural change only; 1, chronic
inflammation; 2, lamina propria neutrophils;
3, neutrophils in epithelium; 4, crypt destruction;
and 5, erosions or ulcers [12].
IL-23p19 Immunohistochemical staining: 4-
μm-thick serial sections of formalin fixed,
paraffin-embedded tissue were cut and mounted
on positively charged glass slides. After
incubation at 60°C overnight and
deparaffinization, sections placed in 0.01 M
sodium citrate buffer (pH 6.0) and heated twice
for 5 minutes in a microwave oven. After
inactivation of endogenous peroxidase with 0.5%
metaperiodic acid in phosphate-buffered saline
(PBS) for 10 minutes, sections were incubated
with 10% horse serum in PBS for 1 hour Sections
were incubated at 4°C overnight with 100×
diluted primary goat anti - mouse IL-23 p19
antibody (R&D Systems, Inc.). The standard
avidin-biotinperoxidase complex (ABC)
technique was performed using the LabVision
Secondary Detection Kit (Ultra Vision Detection
System Anti-polyvalent, HRP). The color was
visualized by incubation with chromogen 3, 3'
diamino-benzidine for 5 minutes. The slides were
then counterstained with Mayer hematoxylin and
cover slipped with Permount (StatLab, McKinney,
TX). Negative controls were set for each test
without the primary antibodies.
Immunohistochemical evaluation: Results were
expressed semi - quantitatively. Positively stained
cells were counted by examining at least 10
random fields (X200) in each section and
expressed as the percentage of positive cells over
total cell number [13].
IL-23 R expression by flowcytometry:
Peripheral blood mononuclear cells (PBMC).
CD4+ T isolation from blood samples:
Lymphocytes were isolated from peripheral blood
by incubation with Rosette Sep Human CD4+T
cells enrichment cocktail (Stem Cells
Technologies, Grenoble, France) followed by
centrifugation on a density gradient (Lymphoprep,
PAA, Pasching, Austria). Lymphocytes were
purified by centrifugation through Lymphoprep.
For peripheral CD4+: For cellular surface staining
the following antibodies and secondary reagents
were used in different combinations: biotinylated
goat anti-human IL-23R (BAF1400, R&D
System, Minneapolis, MN) Streptavidin-APC
(BD Bioscience, San Jose, CA), CD3-FITC
(eBioscience, San Diego, CA), CD4 PE-Texas
Red (Invitrogen, Carlsbad, CA), CD45RO-FITC
(Dako, Glostrup, Denmark), CD45RO-Pacific
Blue (BioLegend, San Diego, CA), CCR6-PE
(BD Bioscience), CD45RA-PE (Invitrogen),
CD45RA - PE - Cy7 ( eBioscience ), plus
matched isotypes as controls. Cells were acquired
on a BD FACSAria II (BD Bioscience). Analysis
of FACS data was performed by FlowJo
(TreeStar, Ashland,OR) software.
Lamina propria mononuclear cells (LPMC).
Isolation of intestinal lymphocytes: For IEL
isolation, endoscopic procedure was done, tissue
(dissected mucosa) was placed and stirred for 15
minutes at room temperature in prewarmed
(37°C) 1× HBSS containing 10% fetal bovine
serum (Gibco catalog no. 10082), 0.015 M
HEPES, and 5 mM EDTA and stirred for 15
minutes at 37°C, followed by three 15-minutes
washes with buffer adjusted to room temperature.
The supernatant from each wash was pooled and
poured through a nylon wool column to enrich for
T cells and remove mucus. The resulting cell
suspension was used to analyze IEL. For IEL: The
IEL suspensions containing approximately 4 × 106
cells each were resuspended in cold phosphate-
buffered saline and stained with Aqua Live/Dead
cell discriminator (Invitrogen catalog no. L34597)
according to the manufacturer's protocol. Cells
were then stained for 1 hour in the dark at 4°C
with optimized concentrations of anti-CD3
Alexa750-APC (eBioscience clone 17A2), anti-
CD8 Alexa700 (eBioscience clone 53-6.7), anti-
CD4 Pacific Blue (eBioscience clone RM4-5),
anti-TCR GD R-PE (BD Pharmingen clone GL3),
and biotinylated polyclonal anti-IL-23R (BAF
1686; R&D Systems). Cells were washed twice
with PBS containing 1% bovine serum albumin
(fluorescence-activated cell sorting [FACS]
buffer). Cells were then stained for 1 hour with
streptavidin-conjugated Qdot 605 (Q10101 MP;
Invitrogen). Stained cells were washed once in
FACS buffer and subsequently fixed in 4%
formalin for 1 hour. Cells were then washed once
and resuspended in FACS buffer and analyzed
using an LSR II flow cytometer (Becton
Dickinson, San Jose, CA). The data were
analyzed by using FlowJo software (Treestar, Inc.,
Ashland, OR). Gates were set on singlets and then
on live lymphocytes. Subsequent gates were based
on Fluorescence-Minus-One & unstained controls.
Serum IL-23 by ELISA. IL-23 cytokine level
was assayed using a commercially available IL-22
ELISA kit (R&D Systems) according to
manufacturers' instructions. All patients gave their
informed consents and the study was approved by
Ethical, and Research Committee, Tanta Faculty
of Medicine, Tanta, Egypt.
Statistical Analysis
The statistical data are reported as the mean ± SD,
frequencies (number) and percentages when
appropriate. A comparison of numerical variables
between the study groups was performed using
Mann–Whitney U test to compare independent
samples from two groups. One-way analysis of
variance test was used to compare between more
than two groups when data were normal and the
Kruskal–Wallis test when the data were not
normal while qualitative data compared with chi
square test. Spearman’s rank correlation was used
to quantify the association between continuous or
ordered categorical variables. A P-value less than
0.05 were considered statistically significant. All
statistical calculations were performed using the
computer program SPSS (Statistical Package for
the Social Science; SPSS, Chicago, IL, USA)
version 15 for Microsoft Windows.
Results
Table (1) shows clinical and laboratory data in all
groups. Significant higher levels of ESR and
disease duration were detected in patients with
severe disease compared to patients with mild to
moderate ulcerative colitis disease P < 0. 05.
Abdominal pain, diarrhea, blood in the stool and
ESR were significantly higher in ulcerative colitis
patients compared with control. Histopathological
evaluation and correlation with endoscopic groups
(Table2). The studied 50 cases of UC showed
different grades of severity 5 cases were grade 0
(10%); 12 cases were grade 1 (24%); 8 cases were
grade 2 (16%), 15 cases were grade 3 (30%) (Fig
1a); 6 cases were grade 4 (12%) %) (Fig 1b) and
only 4 cases were grade 5 (8%) %) (Fig 1c). In
correlation with the endoscopic grouping; in
group I; the cases distributed among grades 0-3;
while in group II; the cases were distributed
among grades 2-5 with statistical significance
(P=0.02). Table (3) shows significant increase in
serum level of IL-23and IL-23R expression in
both peripheral blood and lamina propria
lymphocyte (CD4) in ulcerative colitis patients
compared with control group. This increase was
more in patients with severe disease compared
with patients with mild to moderate disease. IL-23
p 19 Immunohisto-chemical evaluation showed
that IL23p19 was expressed in the lamina propria
macrophages. The mean number of the cells
expressing IL23p19 in group I was 11.6±3.4
(Fig2a) while in group II it was 19.6± 5.5 (Fig 2b)
with statistical difference between the two groups.
On the other hand, it was only 3.5± 1.6 in group
III (control group) (Fig 2c) with statistical
difference between the three groups. Table (4)
shows significant positive correlation between
both endoscopic severity and histological grading
of ulcerative colitis with each of IL-23p19
expression ( r=0.591, p=0.038) and , (r=0.612,
p=0.047) respectively, IL-23 R expression
(r=0.542, p=0.015) and (r=0.526, p=0.031)
respectively, serum level of IL-23 (r=0.637,
p=0.024) and (r=0.551, p=0.019) respectively.
Table (1): Clinical and laboratory data of the studied groups.
Parameters
Mild-moderate UC
Group I
(n =27)
Severe UC
Group II
(n =23)
Control Group
Group III
(n =10)
P-value
Age (ys)
Sex (m/f)
Disease duration(ys)
Abdominal pain
Diarrhea
Blood in stool
ESR (mm/hr)
Hb (gm/dl)
36.1 ± 13.4
16/11
2.6± 1.7
20/27
18/27
19/27
21.8± 7.9
12.1± 1.3
41.5± 12.7
14/9
b4.2±3.1
18/23
19/23
21/23
b33.6± 8.5
11.6± 1.6
35.2± 12.8
6/4
-
a3
a2
-
a10.9± 1.9
12.6±1.5
0.270
1.000
b0.042
0.083
0.017*
0.111
<0.001*
0.197
P < 0.05*significant between all groups; asig between patients & control;b sig between GI & GII. UC, ulcerative colitis.
Table (2): Correlation between histological grading and endoscopic severity in the UC patients.
Studied groups Histopathological grading
P -value Grade 0 Grade 1 Grade 2 Grade 3 Grade 4 Grade 5
Mild-moderate UC
Group I
(n=27)
5 12 6 4 0 0
0.02* Severe UC
Group II
(n=23)
0 0 2 11 6 4
P < 0.05 *significant; UC, ulcerative colitis.
Table (3): IL-23 R expression, serum IL-23 and IL-23p19 expression in the studied groups.
Parameters
Mild-moderate UC
Group I
(n =27)
Severe UC
Group II
( n =23)
Control Group
Group III
( n =10)
P-value
-IL-23 R expression %
.PBMC
.LPMC
-S IL-23 (pg/ml)
-IL-23 p 19 expression
Immunohistochemestry
% of +ve cells
6
20
48.9±7.6
11.6±3.4
12
29
137.4±45.5b
19.6± 5.5b
1c
6a
243.2±90.4a
3.5± 1.6a
0.037*
0.001*
<0.001*
<0.001*
P < 0.05*significant between all groups; a sig between patients & control; b sig between GI & GII. c sig between GII & control; UC,
ulcerative colitis; PBMC, peripheral blood mononuclear cells (lymphocyte) LPMC, lamina propria mononuclear cells (lymphocyte).
Table (4): Correlation between endoscopic severity, histopathological grading and studied parameters in UC patients.
Parameters IL-23p19 gene expression IL-23 R expression Serum IL-23
R P-value R P-value R P-value
-Endoscopic severity
-Histopathological grade
0.591
0.612
0.038 *
0.047*
0.542
0.526
0.015 *
0.031*
0.637
0.551
0.024 *
0.019*
P < 0.05 *significant; UC, ulcerative colitis.
a(x200) b(x200) c(x100)
Fig (1): Histopathological grades of ulcerative colitis showing (a): grade 3 with neutrophilic infiltrate in the epithelial cells,
(b): grade 4 showing crypt destruction and (c): grade 5 showing erosions and ulcers. [H&E]
a(x200) b(x400) c(x400)
Fig (2): Immunohistochemical expression of IL23p19 in ulcerative colitis groups showing mild expression in group I(a),
extensive expression in group II (b) and very minimal expression in group III (c). [IHC]
Discussion
Patients with UC are at increased risk of
inflammation. IL-23 is a newly identified cytokine
with increased expression in inflamed biopsies of
colon mucosa in patients with CD; however there
is inconsistent evidence on its role in ulcerative
colitis [14]. IL-23 is a heterodimeric cytokine that
shows similar function to IL-12 in promoting
cellular immunity and enhancing lymphocyte
proliferation [15]. This study showed significant
increased expression of IL-23p 19 gene in patients
with UC compared with control. This increase
was significantly higher in patients with severe
UC disease compared with mild to moderate
disease. This was similar to the results reported by
Schmidt et al [16], Zhanju et al [13] and Kobayashi
et al [17]. We observed that IL-23 p19 positive
cells by immunohistochemistry were mainly
macrophages. This was in agreement with Zhanju
et al [13].These findings suggest that IL-23 is
produced by intestinal mucosal macrophages in
inflamed mucosa of ulcerative colitis patients [13].
Upon stimulation by bacterial ligand, IL-23 is
produced by antigen presenting cells. After
binding to appropriate receptor (IL-23 R), this
cytokine can stimulate the production of IL-17,
TNF alpha and IL-6 from T cells. IL-17 stimulates
the expression of adhesion molecules like ICAM-
1 on endothelial cells, as well as the release of IL-
6 and IL-8 from myofibroblast and epithelial
cells. IL-8 act as chemotactic factor for neutrophil
influx into the intestine. Inflammatory neutrophil
release inflammatory mediators like matrix
metalloproteinase and inducible nitric oxide. This
sequel of pathogenic events leads to the chronic
inflammation and epithelial cell damage
associated with the disease [18]. Therefore, IL-23
was proposed to play an integral role in the
pathogenesis of IBD [19]. This study showed the
increased expression of IL-23 R in both peripheral
blood and mucosal biopsy of ulcerative colitis
patients were significant compared with control
group. This increased expression was higher in
patients with severe disease compared with those
have mild to moderate disease. Also, Zhanju et al [13] demonstrated significant increased expression
of IL-23 R in peripheral blood and mucosal
lamina propria cells. In accordance with the
findings reported by Mohammadi et al [14] and
Zheng et al [20], we demonstrated increased serum
level of IL-23 in ulcerative colitis patients
compared with control. In addition, the increased
serum levels of IL-23 were more in patients with
severe disease compared with mild to moderate
disease. Moreover, this study reported significant
positive correlation between high serum IL-23
levels and endoscopic severity of the disease.
These findings support the hypothesis that,
increased IL-23 levels reflect the activity of T
helper 17 in patients with ulcerative colitis and
that IL-23 participate in the pathogenesis of the
disease [20].This study noticed, the increased
expression of IL-23p19 and IL-23 R were
significantly positively correlated with endoscopic
severity of the disease. These were consistent with
Schmidt et al [16] and Zhanju et al [13].In consistent
with experimental studies of Daniel et al [21] and
Ando et al [22], there were significant positive
correlation between histopathological severity and
both IL-23p19, IL-23R expression in the mucosa
of ulcerative colitis patients. Furthermore the
severity of histopathological lesions was
significantly correlated with serum IL-23, similar
to an experimental study of Sheikh et al [23].
Conclusion
The increased expression of IL-23p19 has a role
in the pathogenesis of ulcerative colitis; therefore
targeted therapy directed against IL-23p19 may
have a therapeutic role for the disease. The
increased expression of IL-23p19, IL-23 R and
serum IL-23 correlate with disease severity.
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Original Article
Nitric Oxide Level in The Ascitic Fluid of Cirrhotic Patients With or Without
Spontaneous Bacterial Peritonitis and Its Relation to Hepatorenal Syndrome 1Mohamed A. El-Biali,2Mohamed Y. Elhasafi,1Marwa A. Madkour,3RagaaA. Ramadan, 1Eman A.Abd El-Rahman 1Department of Clinical and Experimental Internal Medicine; Medical Research Institute, 2Department of Internal
Medicine; Faculty of Medicine,3Department of Chemical Pathology; Medical Research Institute;University of
Alexandria.
ABSTRACT
Ascites, spontaneous bacterial peritonitis (SBP) and hepatorenal syndrome (HRS) are among the common
complications of liver cirrhosis. Nitric oxide (NO) is a potent vasodilating molecule that plays a major role in
splanchnic arteriolar vasodilatation and the development of ascites and HRS in patients with portal hypertension. Aim
of the work: Assessment ofNO level in the serum and ascitic fluid of cirrhotic patients with or without SBP and its
relation to HRS. Patients and methods: Sixty four patients with liver cirrhosis, portal hypertension and ascites were
enrolled. They were divided into: Group I (fifteen patients with sterile ascites and no HRS), group II (seventeen patients
with sterile ascites and HRS), group III (seventeen patients with SBP and no HRS) and group IV (fifteen patients with
SBP and HRS). Analysis of ascitic fluid sample obtained by paracentesis was performed;a polymorphonuclear
leucocytic (PMNL) count more than 250 cells/mm3 was considered diagnostic for SBP. Bacteriologic cultures from
ascitic fluid were done for patients with SBP. Nitric oxide metabolites (nitrate and nitrite) were measured in serum and
ascitic fluid; as an index of NO level using a colorimetric assay kit. Results: Ascitic fluid culture was positive in 29.4%
of group III patients and in 26.7% of group IV. The mean value for NO level in ascitic fluid was 57.45 ± 23.01 μmol/L;
while serum NO level was32.90 ± 9.55 μmol/L. Both demonstratedsignificant difference among the studied groups (p <
0.001). Levels werelowermost in patients with sterile ascites and normal renal functions,they became higher in patients
with isolated SBP or isolated HRS, and they were highest in patients having both SBP and HRS. Serum and ascitic fluid
NO levels showed positive correlation to each other. Ascitic fluid NOlevel correlated positively with Child score, serum
creatinine and PMNL count, while it correlated negatively withthe mean arterial pressure of patients. Higher levels of
ascitic fluid and serum NO were detectedin HRS vs. non-HRS patients, and also in SBP vs. non-SBP patients. No
difference was found in serum or ascitic fluid NO levels between culture-positive and culture-negativeSBP patients.
Conclusion: NO seems to play a complex role between SBP and HRS in the setting of liver cirrhosis and ascites. It is
probably the main precipitating factor for HRS among patients with SBP. However, isolated HRS without SBP canby
itself induce increased levels of NO.
Introduction
Liver cirrhosis is defined as a diffuse irreversible
process characterized by fibrosis and conversion
of normal liver architecture into nodules lacking
lobular organization.(1-3)While hepatitis viruses are
the most common causes of cirrhosis in Egypt,
cirrhosis can be caused by other conditions
including fatty liver disease, autoimmune
diseases, excess alcohol use, drug-induced injury,
bile duct disorders and inherited disorders. Major
complications of cirrhosis include ascites,
spontaneous bacterial peritonitis, hepatic
encephalopathy, portal hypertension, variceal
bleeding, and hepatorenal syndrome.(4) Ascites
refers to the retention of abnormal amounts of
fluid in the peritoneal cavity, which occurs in
cirrhotic patients as a result of factors like portal
hypertension and low albumin level. Due to
inadequate defense mechanisms, cirrhotic patients
with ascites have an increased susceptibility to
infections, the most frequent and the most severe
one being spontaneous bacterial peritonitis (SBP)
which refers to infection of the ascitic fluid.
Because SBP is often asymptomatic, its diagnosis
is based on laboratory testing of the ascitic fluid
obtained by paracentesis. A polymorphonuclear
cell count of more than 250 cells/mm3 of ascitic
fluid is considered diagnostic for SBP. SBP is
90% monomicrobial.(5)Gram negative enteric
bacilli are responsible for 60% of all SBP
episodes, mostly in the form of Escherichia coli
and Klebsiella species. Anaerobes and fungi are
very unlikely to produce SBP; their presence or
the presence of polymicrobial infection should
raise the suspicion of secondary bacterial
peritonitis rather than SBP.(6,7). Hepatorenal
syndrome (HRS) is the development of renal
impairment in patients with end-stage liver
cirrhosis and ascites in the absence of any
identifiable renal pathology. It is a functional
rather than structural disturbance in renal
function. (8)When the combination of portal
hypertension and splanchnic arterial vasodilata-
tion (mediated mainly by nitric oxide) alters the
intestinal capillary pressure and permeability,
accumulation of fluid within the abdominal cavity
occurs and ascites develops. As the disease further
progresses, there is marked impairment in renal
excretion of free water and renal vasoconstriction
- changes that lead to dilutional hyponatremia and
hepatorenal syndrome, respectively.(9, 10). Nitric
oxide (NO) is an important cellular signaling
molecule involved in many physiological and
pathological processes.(11) It is a powerful vaso-
dilator; also known as the “endothelium-derived
relaxing factor”. NO contributes to vessel
homeostasis by inhibiting vascular smooth muscle
contraction and growth, platelet aggregation, and
leukocyte adhesion to the endothelium.(12)In the
cirrhotic liver, an overactivation of the
cyclooxygenase pathway 1 (COX-1) causes
increased production of vasoconstrictor
endoperoxides, while the synthesis of the
vasodilating NO becomes insufficient to
compensate for these activated vasoconstrictor
systems. This inadequate hepatic NO generation
plays a major role in increasing intrahepatic
vascular resistance in cirrhosis, thereby worsening
portal hypertension.(13) NO is also considered to be
a messenger molecule with important biological
functions that include suppression of pathogenic
microorganisms.Its production by activated
macrophages has been implicated in killing
various microorganisms by inhibiting the
respiratory cycle and damaging the DNA of these
pathogens. Increased NOproduction by activated
ascitic fluidmacrophages ishence expected to
occur in the setting of SBP.(14)
Aim of the Work
The aim of this work was to study nitric oxide
level in the ascitic fluid and serum of cirrhotic
patients with or without spontaneous bacterial
peritonitis andits relation to hepatorenal
syndrome.
Subjects and Methods
This study was carried out on sixty four patients
with liver cirrhosis, portal hypertension and
ascites enrolled from the Hepatology and
Gastroenterology Unit of the Medical Research
Institute, Alexandria University, Egypt during the
period from October 2012 to January 2014. They
were divided into four groups: Group I included
fifteen cirrhotic patients with sterile ascites and no
hepatorenal syndrome (HRS), group II included
seventeen cirrhotic patients with sterile ascites
and HRS, group III included seventeen cirrhotic
patients with spontaneous bacterial peritonitis
(SBP) and no HRS and group IV included fifteen
cirrhotic patients with SBP and HRS. The
diagnosis of cirrhosis was verified on the basis of
clinical examination, laboratory investigations and
abdominal ultrasonography. SBP was diagnosed
based on a polymorphonuclearleucocyte (PMNL)
cell count of more than 250 cells/mm3 of ascitic
fluid.(15)HRS was identified based on the criteria
set by the International Ascites Club.(16)After
obtaining their written consent, all patients were
subjected to detailed history taking and clinical
examination.Abdominal ultrasound examination
was performed to confirm the diagnosis of liver
cirrhosis andportal hypertension. Also, the grade
of ascites was determined (mild, moderate or
massive) and scored as 1,2 or 3, respectively.(17)
Modified Child–Pugh score and classification
were used for assessing liver disease severity.(18)
Laboratory investigationsincluded complete blood
picture, blood urea and serum creatinine, serum
sodium and potassium levels, liver profile
parameters, fasting blood glucose, complete urine
analysis and hepatitis viral markers (hepatitis C
serum antibodies; HCV Abs and hepatitis B
surface antigen; HBs Ag).(19)Analysis of ascitic
fluid sample obtained by paracentesis was
performed to confirm the diagnosis of SBP and to
calculate serum ascites albumin gradient (SAAG).
A SAAG more than 1.1 g/dL indicated that the
ascitic fluid type was transudate, while a SAAG
less than 1.1 g/dL denoted exudate.(20)Also,
routine aerobic and anaerobic bacteriologic
cultureof ascitic fluid samples was done for all
patients with SBP.(21)Nitric oxide metabolites
(nitrate and nitrite) were measured in serum and
ascitic fluid; as an index of NO levelusing a
colorimetric assay kit. Direct determination of NO
radical is difficult because it decomposes rapidly
in biologic solutions into nitrite and nitrate.
Therefore, the stable end products of NO radical
(nitrite; NO2−and nitrate; NO3
−) were measured
with the Griess reagent which consists of
sulfanilamide and N-1-napthyl ethylenediamine.
The first step was the conversion of nitrate to
nitrite using nitrate reductase, while the second
step was the addition of Griess reagent, which
converts nitrite to a deep purple azo compound.
Measurement of the absorbance at 540 nm
accurately determines nitrite concentration
(sodium nitrate is used as a standard). Protein
interference is avoided by treating the reacted
samples with zinc sulphate followed by
centrifugation for 10 min at 2000 × G.(22)
Statistical Analysis
Statistical Package for Social Sciences software
(SPSS, Windows version release 18.0; SPSS Inc.;
Chicago, IL, USA) was used for analyzing
data.Qualitative data were analyzed using number,
percentand Chi-square test. When more than 20%
of the cells had expected count less than 5,
correction for chi-square was conducted using
Monte Carlo correction. Quantitative data were
described using minimum, maximum, mean and
standard deviation. For normally distributed data
comparisons between more than two populations
were conducted by F-test (ANOVA), while Paired
t-test was used to analyze paired data. For
abnormally distributed data, Mann-Whitney test
was used to analyze two independent populations,
and Kruskal Wallis test was used for more than
two populations. Correlations between two
quantitative variables were assessed using
Spearman coefficient. Significance test results
were quoted as two-tailed probabilities.
Significance of the obtained results was judged at
the 5% level.
Results
Demographic and clinical data: The age of the
studied patients ranged between 40 and 71 years;
with a mean value of58.27 ± 8.03 years, and male
patients represented the predominant gender
(78.2%),with no statistically significant difference
among the four groups as regards age or gender
(p= 0.297 and p = 0.338, respectively).Medical
history of large volume paracentesis (>5L/ day)
was reported by 20% to 66.7% of patients, being
lowest among patients of group I and highest in
group VI. Diuretic refractory ascites was reported
in 13.3% to 64.7% of patients, being highest
among HRS patients (groups II and IV).History of
gastro-intestinal tract bleeding was reported in 20-
60% of patients; being highest among SBP
patients (groups III and IV). However, the
difference between the four groups proved to be
statistically significant only for diuretic refractory
ascites (p = 0.007).Clinical examination revealed
fever and abdominal tenderness only in SBP
patients (groups III and IV), with statistically
significant difference among the studied groups (p
value < 0.001), while splenomegaly,
jaundice,bleeding tendency (in the form of easy
bruising and epistaxis), palmar erythema and
lower limb edema werepresent in all 4 groupswith
no statistically significant difference. The mean
arterial blood pressure (MAP)ranged from 70.0 to
93.30 mmHg in all patients; being lowest in
groups II and IV i.e. the HRS groups with
statistically significant difference (p= 0.001);
table (1).
Child-Pugh score and class: All patients
belonged to Child class B and C; the latter
representing the majority (82.8%) of patients. The
Child score ranged from 8.0-15.0; withno
statistically significant difference among the
studied groups (p = 0.456); table (2).
Table (1): Comparison between the studied groups according to medical history and clinical examination
Group I Group II Group III Group IV
Test of Sig. P (n=15) (n=17) (n=17) (n=15)
LVP: n(%) 3 (20%) 8 (47.1%) 6 (35.3%) 10 (66.7%) 7.21 0.066
DRA: n(%) 2 (13.3%) 11 (64.7%) 3 (17.6%) 6 (40%) 12.196* 0.007*
GIT Bleeding: n(%) 3 (20%) 4 (23.5%) 7 (41.2%) 9 (60%) 6.767 0.08
Fever: n(%) 0 (0%) 0 (0%) 10 (58.8%) 11 (73.3%) 32.017* <0.001*
Abd. tenderness: n(%) 0 (0%) 0 (0%) 8 (47.1%) 11 (73.3%) 29.658* <0.001*
Splenomegaly: n(%) 14 (93.3%) 15 (88.2%) 16 (94.1%) 12 (80%) 2.003 0.636
Jaundice: n(%) 3 (20%) 10 (58.8%) 7 (41.2%) 9 (60%) 6.554 0.088
Bleeding tendency: n(%) 11 (73.3%) 12 (70.6%) 10 (58.8%) 10 (66.7%) 0.887 0.856
Palmar erythema: n(%) 10 (66.7%) 11 (64.7%) 11 (64.7%) 10 (66.7%) 0.027 0.999
Lower limb edema: n(%) 8 (53.3%) 9 (52.9%) 10 (58.8%) 9 (60%) 0.259 0.968
MAP
Range 80.0 – 93.30 70.0 – 90.0 70.0 – 93.30 70.0 – 90.0 6.494* 0.001*
Mean ± SD. 87.54 ± 4.62 78.61 ± 6.45 81.55 ± 8.82 77.11 ± 7.44
p1 0.001* 0.020* <0.001*
p2 0.229 0.549
p3 0.08
GIT: gastro-intestinal tract, DRA: diuretic refractory ascites, LVP: large volume paracentesis>5L/day, Abd.: abdominal,MAP:
mean arterial blood pressure, n: number, SD: standard deviation, p: p value for comparing between the four studied groups, p1:
p value for comparing between group I and each other group, p2: p value for comparing between group II with group III and
group IV, p3: p value for comparing between group III and group IV, *: Statistically significant at p ≤ 0.05.
Table (2): Comparison between the studied groups according to Child-Pugh score and class
Group I Group II Group III Group IV Test of
Sig. p
(n= 15) (n=17) (n=17) (n= 15)
Child class
2.945 0.456 B: n(%) 4 (26.7%) 2 (11.8%) 4 (23.5%) 1 (6.7%)
C: n(%) 11 (73.3%) 15 (88.2%) 13 (76.5%) 14 (93.3%)
Child score
1.096
0.358 Range 8.0 – 13.0 9.0 – 15.0 9.0 – 15.0 9.0 -15.0
Mean. ± SD. 11.33 ± 1.72 12.20 ± 1.84 11.88 ± 2.18 12.60 ± 2.16
SD: standard deviation, n: number, p: p value for comparing between the four studied groups, *: Statistically significant at p ≤ 0.05
Abdominal ultrasonographic data: The diameter of
the right hepatic lobe ranged between 9.40 and 17.80
cm with a mean of 13.36 ± 2.06 cm, the spleen
diameter ranged between 12.0 and 21.50 cm with a
mean of 16.28 ± 1.94 cm andthe portal vein diameter
ranged between 9.80 and 16.50 mm with a mean of
14.24 ± 1.08 mm in all patients; with no statistically
significant difference between the four groups.
Massive ascites (grade 3) was found in all patients.
Laboratory data:All patients were positive forserum
hepatitis C virus antibodiesand negative for hepatitis B
surface antigen (HBs Ag).As regards complete blood
count, there was no statistically significant difference
among the studied groups in hemoglobin level (ranging
from 5.60-12.80 g/dl), red blood cell count (ranging
from 1.90–4.50 x106 /mm3) or platelets count (ranging
from 25.0 – 327.0 x103 /mm3). There was a statistically
significant difference among the studied groups only as
regards white blood cell count (ranging from 1.04 –
19.20 x103 /mm3) being significantly higher in groups
III and IV i.e. the SBP groups (p=0.003). The
biochemical laboratory investigations revealed no
statistically significant difference among the studied
groups as regards fasting blood glucose (FBG) and
serum potassium level. There was, however, a
statistically significant difference among the studied
groups in serum urea and creatinine levels; being
highest in groups II and IV i.e. HRS groups. Also, a
statistically significant difference among the studied
groups was detected in serum sodium level; being
lowest in groups II and IV i.e. HRS groups; table
(3).The liver profile parameters – on the other hand –
demonstrated no statistically significant difference
among the studied groups as shown in table (4).
Table (3): Comparison between the studied groups according to biochemical laboratory investigations
Group I Group II Group III Group IV Test of
Sig. p
(n=15) (n=17) (n=17) (n=15)
FBG (mg/dl)
Range 76.0 – 223.0 75.0 – 165.0 63.0 – 203.0 78.0 – 255.0 0.192 0.908
Mean ± SD. 100.0 ± 35.54 97.59 ± 26.52 106.18 ±42.67 114.87 ± 56.36
Urea (mg/dl)
Range 19.0 – 40.0 70.0 – 240.0 24.0 – 40.0 91.0 – 320.0 66.037 <0.001*
Mean ± SD. 31.40 ± 6.61 139.88 ±45.38 33.12 ± 4.92 198.33 ± 66.86
p1
<0.001* 0.904 <0.001*
p2
<0.001* <0.001*
p3
<0.001*
Creatinine (mg/dl)
Range 0.80 – 1.20 1.80 – 3.50 0.70 – 1.20 2.20 – 4.90 102.167* <0.001*
Mean ± SD. 1.04 ± 0.14 2.37 ± 0.49 0.96 ± 0.20 3.51 ± 0.79
p1
<0.001* 0.655 <0.001*
p2
<0.001* <0.001*
p3
<0.001*
Na+ (mEq/L )
Range 127.0 – 138.0 112.0 – 139.0 112.0 –135.0 112.0 – 132.0 7.078* <0.001*
Mean ± SD. 132.07 ± 3.35 123.76 ± 6.99 127.65 ± 6.95 122.80 ± 6.57
p1
<0.001* 0.049* <0.001*
p2
0.073 0.663
p3
0.032*
K+ (mEq/L )
Range 3.20 – 5.10 3.80 – 5.50 3.1 – 6.0 3.10 – 5.70 0.929 0.432
Mean ± SD. 4.19 ± 0.53 4.43 ± 0.41 4.14 ± 0.76 4.37 ± 0.63
FBG: fasting blood glucose, Na: serum sodium, K: serum potassium, SD: standard deviation, *: Statistically significant at p ≤
0.05, p: p value for comparing between the studied groups, p1: p value for comparing between group I and each other group, p2: p
value for comparing between group II with group III and group IV, p3: p value for comparing between group III and group IV
Table (4): Comparison between the studied groups according to liver profile parameters
Group I Group II Group III Group IV Test of
sig. p
(n=15) (n=17) (n=17) (n=15)
AST (IU/L)
Range 26.0 – 611.0 29.0 – 273.0 21.0 – 162.0 16.0 – 251.0 3.953 0.139
Mean ± SD. 130.2 ± 143.23 102.0 ± 74.39 66.53 ± 43.44 88.47 ± 64.65
ALT (IU/L)
Range 17.0 – 124.0 12.0 – 160.0 13.0 – 170.0 9.0 – 106.0 1.432 0.489
Mean ± SD. 43.20 ± 27.88 51.88 ± 42.93 40.35 ± 40.21 37.87 ±23.77
Prothrombin time
(sec.)
Range 12.50 – 33.10 12.30 – 33.0 12.0 – 33.0 11.90 – 20.60 0.279 0.87
Mean ± SD. 16.93 ± 4.87 16.68 ± 4.64 16.82 ± 4.93 16.66 ± 3.16
Albumin (g/dl)
Range 1.40 – 3.50 1.70 – 3.0 1.70 – 2.90 1.60 – 2.90 2.069 0.114
Mean. ± SD. 2.51 ± 0.56 2.21 ± 0.46 2.19 ± 0.37 2.18 ± 0.32
Bilirubin (mg/dl)
Range 0.90 – 9.30 0.90 – 15.70 0.80 – 13.40 0.90 – 11.10 4.873 0.087
Mean. ± SD. 3.01 ± 2.01 7.51 ± 5.45 5.04 ± 3.76 5.21 ± 3.67
AST: serum aspartate transaminase, ALT: serum alanine transaminase, sec.: seconds, SD: standard deviation, p: p value for
comparing between the studied groups, *: Statistically significant at p ≤ 0.05
Ascitic fluid analysis:The biochemical analysis
of ascitic fluid samples revealed no statistically
significant difference among the studied groups as
regards serum ascites albumin gradient (SAAG);
it was found to be transudate in all patients.On the
other hand, the polymorphonuclearleukocytic
(PMNL) count in ascitic fluid was significantly
higher in groups III and IV i.e. spontaneous
bacterial peritonitis groups; table (5). Ascitic fluid
culture was positive in 29.4% of group III patients
and in 26.7% of group IV,andit was 100%
negative in groups I and II. Among the culture
positive patients, the microorganisms isolated
were predominantly Escherichia coli (17.6% in
group III and 26.7% in group IV), Enterococcus
Fecalis in 5.9% of patients of group III and
Staphylococcus aureus in 5.9% of patients of
group III.
Table (5): Comparison between the studied groups according to analysis of ascitic fluid
Group I
(n= 15)
Group II
(n=17)
Group III
(n=17)
Group IV
(n= 15)
Test of
sig. p
SAAG (g/dL)
Range 1.30 – 2.70 1.30 – 2.0 1.20 – 1.90 1.2 – 1.90 0.863 0.426
Mean. ± SD. 1.88 ± 0.41 1.52 ± 0.21 1.59 ± 0.23 1.42 ± 0.19
PMNL (cells/mm3)
Range 10.0 – 90.0 30.0 – 205.0 253.0 – 3600.0 255.0 – 6910.0 36.794* <0.001*
Mean. ± SD. 49.40 ± 26.48 116.12 ± 61.96 1063.0±1120.3 2171.0±1950.8
SAAG: serum ascites albumin gradient, PMNL: polymorphonuclearleukocytic count , SD: standard deviation, p: p value for
comparing between the studied groups, *: Statistically significant at p ≤ 0.05
Serum and ascitic fluid nitric oxide levels:
Nitric oxide (NO) level in ascitic fluid ranged
between 9.0 and 118.0 μmol/L with a mean value
of 57.45 ± 23.01μmol/L; while serumNO level
ranged between 7.0 and 54.0μmol/L with a mean
of 32.90 ± 9.55 μmol/L. Both demonstrateda
progressive elevation from group I to group IV
and statistically significant difference among the
studied groups (p < 0.001), and both serum and
ascitic fluid levels showed a significant positive
correlation to each other; table (6) and figures (1-
3).
Table (6): Comparison between the studied groups according to ascitic fluid and serum nitric oxide level
Group I
(n= 15)
Group II
(n=17)
Group III
(n=17)
Group IV
(n= 15)
Test of
significance p
Ascitic NO level
(μmol/L)
Range 9.0 - 83.0 12.80 – 87.0 33.0 – 88.0 35.0 – 118.0 9.132* <0.001*
Mean ± SD. 36.66 ± 25.77 55.53 ± 25.76 56.59 ± 15.17 81.0 ± 25.34
p1 0.026* 0.019* <0.001*
p2 0.895 0.003*
p3 0.004*
Serum NO level
(μmol/L)
Range 7.0 – 44.30 22.0 – 47.0 8.0 – 54.0 17.0 – 54.0 8.319* <0.001*
Mean ± SD. 24.33 ± 10.35 32.62 ± 7.41 32.71 ± 11.29 41.93 ± 9.16
p1 0.018* 0.017* <0.001*
p2 0.979 0.008*
p3 0.009*
NO: nitric oxide, SD: standard deviation, p: p value for comparing between the studied groups, *: Statistically significant at p ≤ 0.05,
p1: p value for comparing between group I and each other group, p2: p value for comparing between group II with group III and
group IV, p3: p value for comparing between group III and group IV
Figure (1): Comparison between the studied groups
according to serum nitric oxide level Figure (2): Comparison between the studied groups
according to ascitic fluid nitric oxide level
Figure (3): Correlation between ascitic fluid nitric oxide (NO) and serum NO in all studied cases.
0
5
10
15
20
25
30
35
40
45
Group I Group II Group III Group IV
Seu
m N
O (
μm
ol/
L)
0
10
20
30
40
50
60
70
80
90
Group I Group II Group III Group IV
Asc
itic
flu
id N
O (
μm
ol/
L)
0
10
20
30
40
50
60
0 20 40 60 80 100 120 140
Seru
m N
O (μ
mo
l/L
)
Ascitic fluid NO (μmol/L)
r = 0.526
p < 0.001*
A significant positive correlation existed between
ascitic fluid NO and Child score, history of
hepatic encephalopathy, serum creatinine level,
serum urea level, ascitic fluid PMNL as well
asserumbilirubin, while a significant negative
correlation was found between ascitic fluid NO
and MAP as well as serum sodium concentration;
table (7).Ascitic fluid NOwas also significantly
related to history of large volume paracentesis (p
= 0.002)andhistory of GIT bleeding(p = 0.002),
whereas no relation was proved between ascitic
fluid NO and history of diuretic refractory ascites
(p = 0.106).
Table (7): Correlation between ascitic fluid NO and different studied parameters in all studied cases.
Ascitic fluid NO
Coefficient p
MAP r= -0.381 0.002*
Child score r= 0.364 0.003*
HE rs= 0.316 0.011*
Creatinine r= 0.513 <0.001*
Urea r= 0.493 <0.001*
Sodium r= -0.251 0.045*
PMNL rs=0.433 <0.001*
Bilirubin rs= 0.268 0.032*
MAP: mean arterial blood pressure,HE: hepatic encephalopathy, NO: nitric oxide, r: Pearson coefficient, rs: Spearman coefficient,
p: p value for comparing between the studied groups, *: Statistically significant at p ≤ 0.05
When comparing the patients with HRS (groups II
and IV) with those without HRS (groups I and
III), a statistically significant difference was found
as regards ascitic fluid NO and serum NO; being
higher in HRS groups. Serum creatinine level
correlated positively with both ascitic fluid
(r=0.493, p=0.004) and serum levels of NO
(r=0.458, p=0.008) among HRS patients.
Furthermore, when comparing patients with SBP
(groups III and IV) with those without SBP
(groups I and II), a statistically significant
difference was proved between both groups as
regards ascitic fluid and serum levels of NO;
being higher in SBP groups. In this case, however,
ascitic fluid PMNL count correlated neither with
ascitic fluid (r=0.234, p=0.198) nor with serum
level of NO (r=0.263, p=0.146) among SBP
patients. Also, no statistically significant
difference was found between culture positive and
culture negativeSBP patients as regards serum or
ascitic fluid NO levels; table (8) and figures (4-6).
Table (8): Comparison of ascitic fluid and serum NO levels among patientsaccording to presence or absence of HRS, SBP and
culture positive results.
Ascitic NO level (μmol/L) Serum NO level (μmol/L)
HRS(n=32)
Range 12.80 – 118.0 17.0 – 54.0
Mean. ± SD. 67.47 ± 28.27 36.98 ±9.41
Non-HRS(n=32) (n=32)
Range 9.0 – 88.0 7.0 – 54.0
Mean. ± SD. 47.25 ± 22.82 28.78 ± 11.50
p 0.003* 0.003*
SBP (n=32)
Range 33.0 – 118.0 8.0 – 54.0
Mean. ± SD. 68.03 ± 23.71 37.03 ± 11.21
Non-SBP(n=32)
Range 9.0 – 87.0 7.0 – 47.0
Mean. ± SD. 46.68 ± 27.09 28.73 ± 9.72
p 0.001* 0.002*
Culture +ve SBP(n=9)
Range 40.0 – 115.0 17.0 – 50.0
Mean. ± SD. 65.67 ± 26.45 37.56 ± 10.11
Culture -veSBP (n=23) (n=32)
Range 33.0 – 118.0 8.0 – 54.0
Mean. ± SD. 68.96 ± 23.12 36.83 ± 11.82
p 0.730 0.872
NO: nitric oxide, HRS: hepatorenal syndrome, SBP: spontaneous bacterial peritonitis, SD: standard deviation, p: p value for
comparing between the two studied groups, *: Statistically significant at p ≤ 0.05
Figure (4): Comparison between HRS versus non- HRS groups
according to ascitic fluid and serum nitric oxide (NO) level Figure (5): Comparison between SBP versus non-SBP groups
according to ascitic fluid and serum nitric oxide (NO) level
Figure (6): Correlation between serum creatinine with ascitic fluid NO and serum NO in HRS groups.
Discussion
Nitric oxide (NO) is a potent vasodilating
molecule that plays a major role in splanchnic
arteriolar vasodilatation and the development of
ascites in patients with portal hypertension. In the
present work, sixty four patients with liver
cirrhosis, portal hypertension and ascites were
enrolled. NO was assessed in all patients,
revealing a significant positive correlation
between ascitic fluid and serum levels of NO.
Moreover, we found a significant positive
correlation between ascitic fluid NO and different
parameters reflecting the severity of liver
cirrhosis; namely hepatic encephalopathy (HE)
and Child score. In other words; the higher the
NO level, the worse were HE and Child score.
Similar to us; Park et al(23) demonstrated a
significant direct correlation between ascitic fluid
and serum nitric oxide levels in cirrhotic patients.
El-Sherif et al(24) showed that in patients with
chronic active hepatitis, Child class “A” patients
had no significant increase in serum NO levels
compared to normal control subjects. Child class
0
10
20
30
40
50
60
70
80
Ascitic fluid NO Serum NO
NO
lev
el (
μm
ol/
L)
HRS
Non HRS
0
10
20
30
40
50
60
70
80
Ascitic fluid NO Serum NO
NO
lev
el (
μm
ol/
L)
SBP
Non SBP
0
20
40
60
80
100
120
140
1.5 2.5 3.5 4.5 5.5 6.5
NO
(μ
mol/
L)
Serum creatinine (mg/dl)
Asceticfluid NO(r=0.493*,p=0.004)
Serum No(r=0.458*,p=0.008)
“B” patients, however, had significantly higher
levels of serum NO, while in Child class “C”
patients the highest levels were observed. They
also reported significant positive correlation
between serum NO levels and hepatic
encephalopathy among their patients. In the
setting of liver cirrhosis and portal hypertension,
inappropriate sodium retention is an important co-
factor in the pathogenesis of ascites. The first
abnormality that eventually leads to fluid and
sodium retention, according to underfill theory, is
peripheral arterial vasodilatation that is mediated
mainly by nitric oxide. According to Schrier et
al,(25) arterial vasodilatation results in a reduction
of effective arterial blood volume and a decrease
in systemic arterial blood pressure. In fact, El-
Sherifet al(24) demonstrated in their study a highly
significant negative correlation between serum
nitric oxide level and mean arterial blood pressure
among patients with cirrhosis. This drop in
systemic arterial blood pressure will eventually
lead to the activation of the rennin - angiotensin -
aldosterone system (RAAS) which leads to
hyponatremia and sodium and water retention.As
the state of vasodilatation worsens, renal function
deteriorates and plasma levels of vasoconstrictors
and sodium-retentive hormones increase.
Hepatorenal syndrome is the extreme end of the
spectrum, according to Schrieret al.(25)In
accordance with these statements, our results
demonstrated a highly significant negative
correlation between ascitic fluid NO and MAP as
well as serum sodium levels. When we compared
NO levels between HRS and non-HRS patients;
both ascitic fluid and serum levels of nitric oxide
were also found to be significantly higher in HRS
patients. Moreover, a significant positive
correlation was found between ascitic fluid NO
and renal function parameters (serum urea and
creatinine) in all studied groups. In other words;
the higher the NO levels, the worse were renal
function tests. The association between SBP and
HRS has been well established quite a while ago.
A study by Fasolato et al(26)stated that a
progressive form of HRS occurs only as a
consequence of SBP. Also, the results of Folloet
al(27) indicated that renal impairment is a frequent
event in cirrhotic patients with spontaneous
bacterial peritonitis.Nitric oxide is considered to
be a messenger molecule with important
biological functions that include suppression of
pathogenic microorganisms.NO produced by
activated macrophages has been implicated in
suppressing the respiratory cycle and damaging
the DNA of these pathogens. Increased NO
production by activated ascites macrophages
ishence expected to occur in the setting of SBP.(14)
However, the question remains whether or not this
infection-induced rise of ascitic fluid NO (with its
known vasodilating effect) is the true link
between SBP and development of HRS.In our
work, we compared SBP and non SBP groups as
regards ascitic fluid and serum NO; demonstrating
that their levels were significantly higher in
patients with SBP. Moreover, a positive
correlation existed between ascitic fluid PMNL
count and ascitic fluid NO levels among the four
groups. These findings have been a subject of
debate between investigators. Contrary to our
results, Park et al(23) demonstrated that ascitic
fluid NO levels in patients with SBP were not
different from those in patients with sterile
ascites, and that there was no significant change
of NO levels in sequential ascites samples during
antibiotic treatment. In agreement with our
results, however, a study by Natarajan et al(28)
demonstrated significant increase in ascitic fluid
nitric oxide in patients with SBP when compared
to control patients (cirrhotic patients without SBP)
and concluded that ascitic fluid nitrate may be a
marker for diagnosing SBP and a useful index in
determining therapeutic response to antibiotic
treatment. Also, Such et al(11) showed that SBP
was associated with high serum and ascitic fluid
levels of proinflammatory cytokines. A subset of
patients in this situation showed high levels of
serum and ascitic fluid NO levels and these
patients seemed to be predisposed to the
development of renal impairment. They
concluded that the increased NO synthesis and
associated aggravated vasodilatation may be the
reason why patients with SBP show high levels of
plasma renin activity. In accordance with Suchet
al (29), we demonstrated in the present work a
significant increase of ascitic fluid and serum NO
levels from patients with sterile ascites and
normal renal function (group I) to patients with
SBP and renal impairment (group IV); as well as a
significant increase of NO levels from patients
with SBP alone (group III) to patients with SBP
and HRS (group IV). At this point,however,
another important question is raised , which is
whether this rise in serum and ascitic fluid NO is
actually the precipitating factor for developing
HRS, or rather the result of impaired urinary
excretion of NO in the setting of HRS. In fact, our
results demonstrated a significant rise of NO
levels in group II (HRS and sterile ascites)
compared to group I (normal renal function and
sterile ascites); indicating that HRS alone can
induce increased NO levels. Likewise, Campillo
et al(30) conducted a study to investigate the
relationship between nitric oxide production,
endotoxemia, renal function and hyperdynamic
circulatory syndrome in patients with cirrhosis.
Their results revealed that creatinine and nitrate
clearances by the kidneys were lower in ascitic
patients than in non-ascitic patients, and that
serum nitrate levels did not correlate with
endotoxemia. They concluded that renal
impairment actually accounts for the increased
levels of serum nitrate in ascitic patients. In
contrast to their assumption, however, a study by
Such et al(29) stated that patients with infected
ascites showed increased serum and ascitic-fluid
levels of nitric oxide metabolites compared with
patients with sterile ascites. They carried out a
multivariate analysis that identified ascitic-fluid
nitric oxide metabolites as an independent
predictor of renal impairment. From their results,
They concluded that the increased serum and
ascitic fluid nitric oxide found in patients with
infected ascites might induce a deterioration of the
increased peripheral vasodilation found in this
setting, leading to the development of renal
impairment in a series of patients with
spontaneous bacterial peritonitis.Also, a study by
Abd El-Azeezet al(31) showed that NO may be a
potentially useful predictor of renal impairment in
cirrhotic patients with SBP. Furthermore,
Grangéet al (32) stated that in patients with SBP,
serum and ascitic fluid levels of NO levels were
higher than those of patients with sterile ascites,
and that renal impairment might be caused by a
decrease in effective arterial blood volume as a
result of the infection. In accordance with the last
three mentioned groups of authors, the statistical
analysis of our results also demonstrated a
significantly increased serum and ascitic fluid NO
from group I to group III, indicating that SBP per
se can cause the rise of NO without the presence
of actual renal impairment. Moreover, the
significant progressive rise of serum and ascitic
fluid NO levels from group I through group III till
group IV also strongly suggests that NO is the
cause rather than the result of HRS. It is; however,
fair to admit that the role of NO level between
SBP and HRS in the setting of cirrhosis seems to
be a complex one. Finally, one question remains;
whether or not a particular microorganism
involved in SBP might be responsible for
significant rise of ascitic fluid NO and for
precipitating HRS. In our work, SBP ascitic fluid
cultures were positive only in 29.4% of patients of
group III and in 26.7% of patients of group IV
(mostly Escherichia coli); with no significant
difference in ascitic fluid and serum NO levels
between culture positive and culture negative
groups. Unfortunately, the number of culture
positive patients turned to be too small for proper
statistical evaluation; which invites for further
investigation of this point on a larger number of
patients in the future. From this study we can
conclude that a positive correlation exists between
serum and ascitic fluid levels of nitric oxide (NO)
in patients with liver cirrhosis and ascites.Ascitic
fluid NO correlates positively with liver disease
severity (in terms of Child score) as well as renal
function parameters (namely serum urea and
creatinine). Serum and ascitic fluid NO levels are
lowest in patients with sterile ascites and normal
renal functions,they become significantly higher
in patients with isolated SBP or isolated HRS, and
they are highest in patients with both SBP and
HRS. NO seems to play a complex role between
SBP and HRS in the setting of liver cirrhosis and
ascites. It is probably the main precipitating factor
for HRS among patients with SBP. However,
HRS by itself can induce increased levels of NO.
We recommend that further investigation on a
large number of patients with SBP is needed to
determine whether or not a particular
microorganism might be responsible for
significant rise of ascitic fluid NO and for
precipitating HRS.
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