Editor in chief

M.Y.Taher

Founder Editors

Hilmy Abaza

Seham Abdel Reheem

Co-Editors

Ahmed Shawky

FathAlla Sidkey

Maher Osman

Mohamed Sharaf De Din

International Advisory Board

JP Galmiche France

A Sandeberg Sweden

X Rogiers Belgium

S Jensen Denmark

Des Verrannes France

Antonio Ascione Italy

S Brauno Italy

P Almasio Italy

National Advisory Board

Moustafa El Henawi

Amira Shams Eldin

Nabil Abdel Baki

Hoda E-Aggan

M Essam Moussa

Ahmed Bassioni

Saeid Elkyal

Abdel Fataah Hano

Khaled Madboli

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 :

dryousrytaher@yahoo.com

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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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