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

Mohamed El Gendi

Moustafa El Henawi

Amira Shams Eldin

Nabil Abdel Baki

Hoda E-Aggan

M Essam Moussa

Ahmed Bassioni

Saeid Elkyal

Abdel Fataah Hano

Tarek Thabet

Ahmed Hussein

Khaled Madboli

Ezzat Aly

Contents Alexandria Journal of

Hepatogastroenterology, Volume IX ( III )

December 2011

------------------------------------------- Manuscript Submission: For information and to

submit manuscripts please contact the editors by e-

mail at

dryousrytaher@yahoo.com

fathallas@yahoo.com

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

info-rmation contained in this journal; the views and

opinions expressed do not necessarily reflect the those

of the Publisher, the Egyptian Society of Hepatology

Gastro-enterology and Infectious Diseases in

Alexandria, and Editors, neither dose the publication

of advertisements constitute any endorsement by the

Publisher, society, and editors of the products

advertised.

-------------------------------------------

Original Article:

Occult Hepatitis B Virus infection in Egyptian Hepatitis

C Virus positive patients: Prevalence and impact on

Hepatocellular Carcinoma development

Ibrahim Baghdady*, Sabry Shoeib*, Ehab Abdel-atti*, Ashraf G. Dala*, Emad M. Eed** and Ashraf A.

Zytoon****Internal Medicine, **Bacteriology

-------------------------------------------

Original Article:

Open Splenectomy For Hypersplenism-induced

Thrombocytopenia In Egyptian Patients With HCV-

related Liver Cirrhosis

Mahmoud A. Hemidaa, Mohamed MShamseyab, Ahmed M.

L. Bedewyca)Department of clinical and expermintal

surgery,b) Department of internal medicine,c.

-------------------------------------------

Original Article:

Use of Anticyclic Citrullinated Peptide Antibodies to

Distinguish Hepatitis C Virus (HCV) – Associated

Arthropathy From Concomitant Rheumatoid Arthritis in

Patients with Chronic HCV Infection

Khaled Mahmoud Mohiedeen* ,Akram Deghedy **Tropical Medicine

-------------------------------------------

Original Article:

Prevalence of Non-Organ-Specific Autoantibodies and its

Effect on response to Antiviral Therapy in Patients with

Chronic Hepatitis C Virus Genotype 4

Mohamed Abd El-Maksoud1, Hatem Elalfy1, Maha Ragab

Habeeb2, Abd-Elmohsen E. El-desoky2

-------------------------------------------

Original Article:

Non-traumatic intramural hematomas in patients on

anticoagulant therapy: report of three cases.

Mohamed Bekheit.

-------------------------------------------

Original Article:

Comparative Study of the Efficacy of Rifaximin in

Comparison with Lactulose for the Treatment of Hepatic

Encephalopathy

Fathia E Asal1, Ahmed Khalid Tawfik1-

-------------------------------------------

Original Article:

Effect of Rebamipide on Portal Hypertensive

Gastropathy and Proliferating Cell Nuclear Antigen in

Patients with Liver Cirrhosis

Mohamed Y. El-Hasafi(a), Suzan M. Helal(b), Marwa A. Madkour(c), Ahmed S.

-------------------------------------------

Original Article:

Accidentally discovered patients with Antibody to

Hepatitis C Virus: Clinical, Biochemical, Virologic,

ultrasonic and Histologic Features

Ahmed Faisal MD1; Abdel-Naser Gad allah MD2 , Ashraf zytoon MD3 Infectious and endemic diseases unit1, Internal

Medicine department, Suez Canal university;

-------------------------------------------

Original Article

Occult Hepatitis B Virus Infection in Egyptian Hepatitis C Virus Positive Patients:

Prevalence and Impact on Hepatocellular Carcinoma Development

Ibrahim Baghdady*, Sabry Shoeib*, Ehab Abdel-atti*, Ashraf G. Dala*, Emad M. Eed** and Ashraf A. Zytoon***

*Internal Medicine, **Bacteriology and ***Radiology Departments, Faculty of Medicine, Menofiya University

ABSTRACT

Occult Hepatitis B Virus infection (OBI) is characterized by positivity for HBV DNA in HBsAg-negative patients with or

without serological markers of previous HBV infection. The prevalence of OBI in different studies has been reported between

zero and 52.3% among patients with diverse liver disease due to Hepatitis C Virus (HCV) infection. The aim of the present

work is to study the prevalence of OBI among HCV-positive Egyptian patients and its impact on hepatocellular carcinoma

(HCC) development. Patients and methods: The study included 100 chronic HCV positive patients. There were 57 male and

43 females ranged between 41-77 years old. Full history taking and complete clinical examination were done for all patients.

A blood sample was withdrawn for CBC, AST, ALT, albumin, bilirubin, prothrombin activity (PT%) and alpha fetoprotein.

Diagnosis was done clinically, by abdominal ultrasonography and by assessing viral markers (HBsAg, HBsAb, HBcAb and

qualitative PCR for HBV-DNA). HCC lesions were further confirmed by triphasic CT of the liver and AFP. Results: Out of

the 100 examined chronic HCV patients, only 16 patients (16%) had OBI. Dually infected patients (OBI/HCV) had

significantly lower platelet count and PT% than HCV monoinfected patients (P=0.001 and 0.03 respectively). HCC were

significantly more common in OBI/HCV dually infected (31%) than HCV monoinfected patients (7%) (P=0.01). Dually

infected patients (OBI/HCV) had significantly higher serum transaminases (AST, ALT) than HCV monoinfected patients

(P=0.01 and 0.03 respectively). The mean value of AST/platelet ratio is significantly higher among OBI/HCV dual infection

than HCV monoinfection (P=0.03). Out of the 100 examined chronic HCV patients, only 11 patients (11%) had HCC.

Presence of OBI was significantly more common in HCV patients with HCC (45%) than in HCV patients without HCC

(12%) (P=0.01). All chronic HCV patients with HCC had shrunken liver (100%) which was significantly more common than

in HCV patients without HCC (58%) (P=0.0001). On doing multiple logistic regression analysis of risk factors of HCC in our

patients, we found that OBI and presence of shrunken liver are independent risk factors of HCC (P=0.04 and 0.03

respectively). Conclusion, occult HBV infection may influence the outcome of HCV infection leading to more hepatic

fibrosis and development of HCC. The persistent HBV infection may have a critical role in the development of HCC in

HBsAg-negative patients. So, occult HBV should be considered and evaluated by more sensitive PCR among HCV-infected

patients.

Introduction

Human hepatitis B virus (HBV) is a compact,

partially double-stranded, enveloped, deoxyribo-

nucleic acid (DNA) virus; member of the

Hepadnaviridae family. HBV infection is

worldwide spread and is considered a major

public health problem, with an estimation of 350

million people chronically infected. The HBV

replicates in the liver leading to hepatic

dysfunction. Persistent HBV infection is assoc-

iated with the development of chronic liver

disease, including cirrhosis and hepatocellular

carcinoma (HCC) (1). Occult hepatitis B virus

infection (OBI) is one of the most challenging

topics in the field of viral hepatitis with its

virological and clinical relevance being debated

for more than 30 years. Initially described in the

late 1970s, this form of hepatitis B infection has

now been further characterized. In particular, in

the last 10 years the application of highly

sensitive molecular biology techniques has

resulted in the elucidation of its virological

features and possible clinical implications (2).

Occult hepatitis B virus infection is characterized

by the persistence of HBV DNA in the liver and

serum of individuals negative for HBV-surface-

antigen (HBsAg). Recent data demonstrated that

occult HBV may exist in the hepatocytes as a

free genome, its molecular basis being related to

the long-term persistence in the hepatocyte

nuclei of the viral covalently-closed-circular

DNA (HBVcccDNA), a replicative intermediate

that serves as template for gene transcription

(3). On the basis of the HBV antibody profile,

OBI may be distinguished as: seropositive-OBI

(anti-HBc and / or anti-HBs positive) or

seronegative-OBI (anti-HBc and anti-HBs

negative). In seropositive-OBI subjects, serum

HBsAg may become negative either following

the resolution of acute hepatitis B (thus, after a

few months of HBsAg carriage) or after years of

chronic HBsAg positive infection (4). The

seronegative-OBI cases might have either

progressively lost the hepatitis B specific

antibodies or theoretically, the individual may

have been hepatitis B specific antibody negative

from the beginning of the infection (5). There is a

fairly general agreement in considering HCV

infected patients as the category of individuals

with the highest prevalence of occult HBV (6).

Occult HBV infection may have a significant

impact in several clinical contexts, since it

might favor the progression of liver fibrosis and

the development of hepatocellular carcinoma in

patients with coexisting additional causes of

liver damage, such as chronic hepatitis C virus

(HCV) infection (7). Moreover, occult HBV may

acutely reactivate when an immunosuppressive

status occurs, and it may be transmitted in the

case of blood transfusion and organ transp-

lantation, causing classic forms of hepatitis B in

newly infected individuals (3). Mainly, carriers

of occult infection may be a source of HBV

transmission in the event of orthotopic liver

transplantation as an obvious consequence of

the fact that the hepatocytes are the site of the

virus reservoir (8). The aim of the present work

is to study the prevalence of occult hepatitis B

among HCV-positive Egyptian patients and its

impact on HCC development.

Patients and method

The study included 100 chronic HCV positive

patients (previously diagnosed by PCR for

HCV-RNA) selected from internal medicine

department in Menofya university hospital.

They were 57 males and 43 females ranged

between 41-77 years old. All patients gave their

written informed consent before participating in

the study. Exclusion criteria included, HBsAg

positive patients, patients with concomitant

causes of chronic liver diseases and history of

alcohol consumption and the use of potentially

hepatotoxic drugs. Full history taking and

complete clinical examination were done for all

patients. A blood sample was withdrawn by

sterile venipuncture and divided into three parts:

EDTA was added to the first sample which was

used for complete blood count (in cell counter,

Pentra 80,France) .The second sample was put

in plain vacutainer tube which was left to clot at

37ºC, centrifuged in a plain container. Serum

was used to estimate AST, ALT, albumin,

bilirubin (on Synchron CX5 autoanalyser).

Third sample with sodium citrate was used to

estimate prothrombin activity. Diagnosis was

done clinically, by abdominal ultrasonography

(liver and spleen status and degree of ascites)

and by assessing viral markers (HBsAg,

HBsAb, HBcAb and qualitative PCR for HBV-

DNA). HCC is further confirmed by triphasic

CT of the liver and AFP.

Statistical analysis

Data were processed and analyzed using a

computer based program (SPSS software

version 10). The results were expressed as mean

and standard deviation. For comparison of two

means, the paired Student t-test was used for

normally distributed variables and Mann- Whit-

ney test (U-test) for not normally distributed

variables. Fisher Exact analysis was also used to

compare proportions between groups. Multiple

logistic regression analysis was used to detect

the independent risk factors of HCC. A P-value

<0.05 was considered significant.

Results

The study population consisted of 100 chronic

HCV positive patients (previously diagnosed by

PCR for HCV-RNA). They were 54 males and

46 females ranged between 41-77 years old. Out

of the 100 examined chronic HCV patients, only

16 patients (16%) had OBI. Comparison

between OBI/HCV dually infected and HCV

monoinfected patients (table 1) showed that

there was no significant difference with regard

to age, gender, history of upper GIT bleeding,

history of encephalopathy, size of the spleen and

the liver, presence of ascites, Hb level, WBCs,

blood, serum albumin, serum bilirubin, Child-

Pugh score and AFP level (P>0.05). Dually

infected patients (OBI/HCV) had significantly

lower platelet count and PT% than HCV

monoinfected patients (P=0.001 and 0.03

respectively). HCC were significantly more

common in OBI/HCV dually infected (31%)

than HCV monoinfected patients (7%) (P=0.01).

Dually infected patients (OBI/HCV) had

significantly higher serum transaminases (AST,

ALT) than HCV monoinfected patients (P=0.01

and 0.03 respectively). The mean value of

AST/platelet ratio is significantly higher among

OBI/HCV dual infection than HCV mono-

infection (P=0.03). Out of the 100 examined

chronic HCV patients, only 11 patients (11%)

had HCC. Comparison between HCV patients

with and without HCC (table 2) showed that

there was no significant difference with regard

to gender, presence of ascites, Hb level, WBCs,

serum albumin and serum bilirubin (P>0.05).

Chronic HCV patients with HCC are

significantly older than HCV patients without

HCC (P=0.02). Presence of OBI was

significantly more common in HCV patients

with HCC (45%) than in HCV patients without

HCC (12%) (P=0.01). All chronic HCV

patients with HCC had shrunken liver (100%)

which was significantly more common than in

HCV patients without HCC (58%) (P=0.0001).

Chronic HCV patients with HCC had signi-

ficantly larger splenic size than HCV patients

without HCC (P=0.0001). All chronic HCV

patients with HCC had history of hepatic

encephalopathy and upper GIT bleeding (100%)

which was significantly more common than in

HCV patients without HCC (52% and 49%

respectively) (P=0.002 and 0.0001 respectively).

Chronic HCV patients with HCC had

significantly lower platelet count than HCV

patients without HCC (P=0.01). Chronic HCV

patients with HCC had significantly higher

serum transaminases (AST, ALT) than HCV

patients without HCC (P=0.003 and 0.002

respectively). Chronic HCV patients with HCC

had significantly higher Child-Pugh score and

lower PT% than patients without HCC (P=0.03

for both). On doing multiple logistic regression

analysis of risk factors of HCC in our patients

(table 3), we found that Occult HBV and the

presence of shrunken liver size are independent

risk factors of HCC (P=0.04, 0.03 and 0.03

respectively)

Table 1. Comparison between (occult HBV/HCV dual infection) and (HCV monoinfection) (data expressed as mean±SD):

P-value HCV monoinfection (n=84) OBI/HCV dual infection (n=16)

0.6 52.5±7.9 51.5±8.9 Age (years)

0.4 55% (n=46) 69% (n=11) Gender (male %)

0.3 52% (n=44) 69% (n=11) GIT bleeding (%)

0.2 54% (n=45) 75% (n=12) Encephalopathy (%)

1 63%(n=53) 63%(n=10) Shrunken liver (%)

0.8 14.4±2 14.6±2 Spleen size (cm)

0.7 90%(n=76) 94%(n=15) Ascites (%)

0.01* 7%(n=6) 31%(n=5) HCC (%)

0.9 10.3±1.2 10.1±1.3 Hemoglobin (gm/dl)

0.9 6.91±2.45 7.01±2.69 WBCs (×103)

0.001* 80.2±17.6 65.9±10. 6 Platelet count (×103)

0.01* 58±19 82±36 AST (U/L)

0.03* 55±16 66±20 ALT (U/L)

0.8 2.65±0.45 2.66±0.47 Serum albumin(gm/dl)

0.9 2.5±0.9 2.5±0.9 Total bilirubin(mg/dl)

0.9 1.5±0.7 1.6±0.8 Direct bilirubin(mg/dl

0.03* 49.3±10.9 41.4±7.3 PT (%)

0.03* 1.64±2.21 3.61±5.59 AST/platelet ratio

0.3 10±1.8 10.6±2.3 Child-Pugh score (points)

0.2 30.3±77.8 88.4±166.5 AFP (ng/ml)

*=Significant, GIT=gastrointestinal, PT%=Prothrombin activity, WBCs=White blood count,

AFP=Alfa fetoprotein

Table 2. Comparison between HCV patients with and without HCC (data expressed as mean±SD):

P-value HCV patients without HCC

(n=89)

HCV patients with HCC

(n=11)

0.02* 51.6±7.6 57.4±9.4 Age (years)

1 57% (n=51) 56% (n=6) Gender (male %)

0.002* 52% (n=46) 100% (n=11) Encephalopathy (%)

0.0001* 58%(n=52) 100%(n=11) Shrunken liver (%)

0.0001* 14.2±1.9 16.5±1.3 Spleen size (cm)

1 88%(n=83) 100%(n=11) Ascites (%)

0.0001* 49%(n=44) 100%(n=11) GIT bleeding (%)

0.01* 12%(n=11) 45%(n=5) Occult HBV (%)

0.2 10.2±1.3 9.8±1.5 Hemoglobin (gm/dl)

0.2 7.02±2.4 6.14±2.93 WBCs (×103)

0.01* 79.8±17.3 66.8±15. 6 Platelet count (×103)

0.003* 59.2±21.5 87±30.4 AST (U/L)

0.002* 54.7±16.9 71.9±15.1 ALT (U/L)

0.1 2.68±0.45 2.46±0.45 Serum albumin(gm/dl)

0.1 2.39±0.84 2.87±1.1 Total bilirubin(mg/dl)

0.1 1.48±0.7 2±1 Direct bilirubin(mg/dl

0.03* 48.7±10.5 40.7±7.2 PT (%)

0.03* 9.9±1.7 11.5±2.2 Child-Pugh score (points)

0.0001* 13.4±11.2 251.5±199.4 AFP (ng/ml)

AFP=Alfa fetoprotein, *=Significant, n=number, GIT=gastrointestinal,

PT%=Prothrombin activity, WBCs=White blood count

Table3. Multivariate regression analysis for risk factors of HCC in HCV+ve patients

Risk factors P-value R

Age 0.3 0.001

Shrunken liver 0.03* 0.33

Splenic size 0.5 0.001

Occult HBV 0.04* 0.32

AST 0.5 0.001

ALT 0.9 0.001

PT% 0.3 0.001

Child-Pugh score 0.8 0.001

AFP=Alfa fetoprotein, PT%=Prothrombin activity, *=Significant, n=number

Discussion

Occult HBV infection is characterized by

positivity for HBV DNA in HBsAg-negative

patients with or without serological markers of

previous HBV infection (6). The prevalence of

occult HBV in different studies has been

reported between zero and 52.3% among

patients with diverse liver disease due to HCV

infection (9). Occult HBV infection has been

commonly reported among immunocompromised

patients and in patients undergoing anti-cancer

chemotherapy (10). Also, occult HBV prevalence

was significantly high among patients under

hemodialysis (11). In Egypt, occult HBV

infection is 1.26% in the blood donors (12).

Occult HBV infection may occur after complete

clinical recovery from acute self-limited

hepatitis so; HBsAg seroclearance does not

necessarily imply HBV eradication (13).

Therefore in endemic areas, the anti-HBc IgG

and anti-HBs as well as HBsAg were not

sufficient markers to exclude HBV-DNA

carriers (14) The frequency of occult HBV

infection may differ geographically, depending

on HBV prevalence. It is ranged from 2-16%

among general population in different countries (15). Reported occult HBV infection frequencies

have been as high as 70-90% of anti-HBc-

positive individuals in areas of high prevalence

but as low as 5-20% in areas of low prevalence (16). It is known that occult HBV infection is

frequent in those with chronic hepatitis C. This

is probably because the route of transmission of

hepatitis B and C is similar (17). In present study,

we found that 16 out of 100 patients (16 %)

positive for HBV-DNA by qualitative PCR. Out

of those 16 patients, there were 12 patients

(75%) were positive for HBcAb and 4 patients

(4%) were negative for HBcAb. The prevalence

of occult HBV infection did not differ with age

or sex. Overall, the prevalence of occult HBV

among chronic HCV patients was 16%. This

appears near to the rate reported in a study done

to detect Occult hepatitis B virus infection in

Lebanese patients with chronic hepatitis C liver

disease (16.3%). Although Lebanon is an area of

low endemicity for both HBV and HCV, occult

HBV infection is common in HCV-infected

patients (18). In contrast, other studies reported a

low prevalence occult hepatitis B infection in

chronic HCV patients. In study done by Emara

et al., (19) in Egyptian chronic HCV patients on

Pegylated interferon/ribavirin therapy, the

prevalence of occult hepatitis B infection was

3.9%. Also in recent study in France, Levast et

al., (20) studied HBV-DNA in 140 sera and 113

liver biopsies of HCV positive/HBsAg negative

patients, the results showed that 4.4% (5/113)

prevalence of occult hepatitis B was recorded in

liver samples and in none of the sera. On the

other hand, other studies reported higher

prevalence of occult hepatitis B infection in

chronic HCV patients. The prevalence of occult

HBV among chronic HCV patients was 19.4%

in Iran (21), 28% in USA (22), 24% In Brazil (23)

and 37.7% In Japan (24). The discrepancy in the

reported prevalence of HBV DNA in HBsAg

negative chronic hepatitis C patients might be

due to differences in the sensitivity of the

methods used for detection of viral genome,

different quantity of HBV viremia, geographical

variation in prevalence of HBV infection and

severity of liver disease (9). Occult HBV is more

common in cirrhotics than in patients with

chronic hepatitis. Moreover, a high prevalence

of OBI (32%) was detected in cryptogenic liver

cirrhosis. This suggests that occult HBV

infection alone may cause pathological disorders (25). Occult HBV may contribute to liver

inflammation through episodes of increased

viral replication, increased immune activity and

subsequent liver injury (26). In chronic HCV

infection, the presence of Occult HBV has been

associated with liver enzymes flare (26),

increased severity of liver disease towards

advanced fibrosis and cirrhosis and poor

response to standard interferon (27). In contrast,

in study, done by Emara et al., (19) in Egyptian

chronic HCV patients on Pegylated interferon

/ribavirin therapy, OBI/HCV dual infected

patients had less hepatic fibrosis than HCV

monoinfected patients and tend to affect

younger age patients. Also Silva et al., (28)

reported that occult HBV was not associated

with more severe grades of inflammation, liver

fibrosis or cirrhosis development. In the present

study, platelet count is significantly lower

among occult HBV/HCV dual infection than

HCV monoinfection which agrees with

Matsouka et al., (29) and Branco et al., (30). In our

study, serum transaminases (ALT and AST)

were significantly higher among occult HBV/

HCV dual infection than HCV monoinfection.

Our results agree with Shavakhi et al., (21) and

Kannangai et al., (26). Moreover, Selim et al., (31)

who studied the role of occult HBV infection in

chronic HCV patients with flare of liver

enzymes. It reported that in patients with normal

or slightly high ALT, HBV DNA was detected

in 13.3% patients, while in those with ALT

flare; HBV DNA was detected in 63.3%

patients. Therefore, presence of occult hepatitis

B, with its added deleterious effect, must always

be considered in chronic hepatitis C patients

especially those with flare in liver enzymes (31).

In contrast, Branco et al., (30) reported that there

was no significant difference between occult

HBV/ HCV dual infection and HCV

monoinfection regarding serum AST and ALT

levels. Additionally, it has been reported that

liver cirrhosis in chronic hepatitis C patients is

more common in those with occult HBV

infection and that their serum ALT levels and

histological activity are high (32). This suggests

the possibility that occult HBV infection is

synergistic with chronic hepatitis C in terms of

development of liver disease. On the other hand,

some studies have reported no difference in

serum ALT levels, histological inflammatory

activity, or degree of fibrosis between patients

with chronic hepatitis C accompanied by occult

HBV infection and those infected with HCV

alone (33). Prothrombin activity was significantly

lower in occult HBV/HCV dual infection than in

HCV monoinfection in the present study.

Similar results were observed by Branco et al., (30). AST/platelets ratio index (APRI) is a

noninvasive marker that has satisfactory

sensitivity and specificity together with a high

predictive value and it can be useful in assessing

hepatic structural changes (34). APRI is

calculated by formula of (serum AST/upper

normal of AST)/platelets x 100. APRI has two

cut-off values, a lower one is 0.5, and a higher

one is 1.5. If the APRI score is less than or equal

to 0.5, there is no fibrosis or just a little. If the

APRI score is 1.5 or above, there is probably

cirrhosis. APRI scores between 0.5 and 1.5 are

related to progressive fibrosis stages, like

Metavir F1-to-F4 (35). In our study, AST/

platelets ratio was significantly higher among

occult HBV/HCV dual infection than HCV

monoinfection. An explanation to increased

APRI among occult HBV/HCV dual infection

that co-infection leads to more progression of

liver fibrosis which may reduce AST clearance,

leading to increased serum levels, in addition,

liver disease may be associated with

mitochondrial injury, resulting in further AST

release from the hepatocytes (36). The platelet

count decreases, in inverse proportion to

progressive liver fibrosis, due to worsening of

portal hypertension with consequent increased

platelet sequestration and destruction in an

enlarging spleen (37). Hepatocellular carcinoma

is one of the most common malignant tumors

worldwide ranging between 3% and 9%

annually (38). In Egypt, HCC reports to account

for about 4.7% of chronic liver disease patients (39). HBV and HCV infections are strongly

associated with liver cirrhosis and HCC. The

occult HBV infection has frequently been

identified in patients with chronic HCV

infection, and in such patients, this occult

infection may be associated with more severe

liver damage and even the development of HCC (40). In present study, 31% of patients with dual

infection OBI/HCV have HCC and only 7% of

HCV monoinfection have HCC. Occult HBV is

detected in 45% (5/11) of patients with HCC

and it is an independent risk factor of HCC.

There were high prevalence of HCC among our

patients with occult HBV/HCV dual infection.

Similar results were reported by Squadrito et al., (41) and Obika et al., (42). Therefore, the

evaluation of HBV genomes in chronic hepatitis

patients appears to be a powerful tool for the

identification of individuals at higher risk of

HCC development. Moreover, it has been

reported that in patients with primary liver

cancer caused by HCV infection, the anti-HBc-

positive rate was 69.6% (43). Also, Hassan et al., (44) showed that prevalence of serum HBV-DNA

was 22.5% and prevalence of intrahepatic HBV-

DNA was 62.5% among HCC patients. This

suggests that if occult HBV infection accom-

panies chronic hepatitis C infection, the risk of

liver cancer may increase. These data confirm

reports that most HBsAg- and anti-HCV-

negative primary liver cancer patients are anti-

HBc-positive and that intrahepatic HBV DNA is

detected in most of these individuals. (33). There

was an elevated risk in HCV patients because

this silent infection can affect the progression

and HCC development (45). Occult HBV has

associated with more advanced fibrosis/cirrhosis (46). Our study shows that all HCC patients had

cirrhosis and shrunken liver is an independent

risk factor of HCC. Similar results were

observed in previous studies (47,48). In addition,

occult HBV infection may favor neoplastic

transformation in HCV-infected patients

through its contribution to cirrhosis. Many

epidemiologic and molecular studies indicate

that persistent HBV infection may have a

critical role in the development of HCC in

HBsAg-negative patients (49). The reason for the

increasing interest in occult HBV infection is

the increasing evidence of its clinical relevance.

Occult HBV infection is linked to chronic liver

diseases and development of hepatocellular

carcinoma (32). It will be necessary to perform

larger studies on the frequency of HBV occult

infection in chronic hepatitis C patients to

determine its effect on development of chronic

hepatitis C or primary liver cancers.

Conclusion

occult HBV infection may influence the

outcome of HCV infection leading to more

hepatic fibrosis and development of hepato-

cellular carcinoma and may have a strong

association with HCV in the carcinogenesis of

liver. The persistent HBV infection may have a

critical role in the development of HCC in

HBsAg-negative patients. So, occult HBV

should be considered and evaluated by more

sensitive PCR among HCV-infected patients.

Recommendation, we must search for occult

HBV in HCV+ve patients with HBs Ag –ve to

slow the progression of liver fibrosis and

decrease risk of HCC. Vaccination against HBV

is important in HCV+ve patients as well as in

general population.

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Original Article

Open Splenectomy for Hypersplenism Induced Thrombocytopenia in Egyptian

Patients with HCV Related Liver Cirrhosis

Mahmoud A. Hemidaa,Mohamed MShamseyab, Ahmed M. L. Bedewyca) Department of clinical and expermintal surgery,b)

Department of internal medicine,c) Department of Hematology.Medical Research Institute, Alexandria University, Egypt.

ABSTRACT

Egypt has the highest prevalence of antibodies to hepatitis C virus (HCV) in the world, estimated nationally at 14.7%. An

estimated 9.8% are chronically infected. Although, Pegylated-interferon (IFN) plus ribavirin remains the most effective

therapeutic regimen for patients with chronic hepatitis C infection, there are several limitations of this treatment because of its

side effects. Thrombocytopenia is a frequent side effect of antiviral therapy, and unfortunately many patients with HCV

related liver cirrhosis have a base line thrombocytopenia due to hypersplenism making them a poor candidate for interferon

therapy. A number of surgical procedures are available to manage the hypersplenism related thrombocytopenia. Splenectomy

is one of the oldest procedures used for this purpose however; the procedure may be hazardous in patients with poor liver

function. TheAimof this work isto evaluate the safety and efficacy of open splenectomy in the management of hypersplenism

induced thrombocytopenia in selected patients with HCV- liver cirrhosis (patients of Child-Pugh score A), so that they can

receive Pegylated Interferon & ribavirin therapy. Methods: 20 patients with HCV-related liver cirrhosis (Child-Pugh A) &

hypersplenism-induced thrombocytopenia were subjected to open splenectomy with follow-up for 6 months to determineits

safety and efficacy in treating the hypersplenism induced thrombocytopenia. Results:20 patients with HCV related liver

cirrhosis of Child Pugh score A, all had hypersplenism induced thrombocytopenia were subjected to elective open

splenectomy. All patients included in the study had a significant improvement in platelets counts from a mean value of

62.8(10)3± 19.16/mm3 preoperatively to 215(10)3± 41.73 /mm3 after three months (P= 0.003 ) and to 293(10)3±

47.01/mm3after six months ( P = 0.007 ) there were no deaths, the mean hospital stay was 5.3 ±1.05 days, the blood loss was

accepted , the mean loss was 250 ±67.2ml ( range :100-850ml) and the complications were in the form of subphrenic

collection ( one patient) , basal atelectasis (two patients), high fever (three patients) ,and one patient had portal vein

thrombosis. Conclusion: open splenectomy is a safe and effective procedure for management of hypersplenism induced

thrombocytopenia in patients with HCV related liver cirrhosis especially those with good liver functions (Child Pugh score

A)

Introduction

HCV is currently the most significant public

health problem in Egypt, while it infects 2% of

the world's population. With the percentage

positive ranges from 0.01% in Scandinavia to

3% in North Africa, Egypt represents a single

unique Exception(1). In 1992, when HCV

antibody testing became widely available, the

prevalence of HCV in Egypt was reported to be

10.8% among first-time blood donors (2). Since

this discovery, many prevalence estimates of

HCV have been reported, mostly from rural

communities located in the northern Nile Delta.

Two more recent prospective studies estimating

the incidence rate of new HCV cases have also

been published, suggesting ongoing transmission

in their respective communities (3, 4). For more

than a decade, Egypt has been widely regarded

as having an epidemic, with the highest

recorded prevalence of HCV in the world (1).

The recently published Egyptian Demographic

Health Survey (EDHS) in 2009 was a national

probability sample of the resident Egyptian

population. This report estimated an overall

anti-HCV antibody prevalence of 14.7% (5). The

number of Egyptians estimated to be chronically

infected was 9.8%. This report provides a

precise national prevalence estimate and

includes additional data on patterns of HCV

prevalence by gender, age, urban versus rural,

and between different regions of the country.

Pegylated interferon (IFN) plus ribavirin

remains the most effective therapeutic regimen

for patients with chronic hepatitis C infection.

However, this treatment often leads to anemia,

neutropenia, and thrombocytopenia that prevent

safe application in patients with baseline pancy-

topenia, due to hypersplenism related to cirrh-

osis (6). The term hypersplenism was first introd-

uced by Chauffaud (7). It is characterized by

splenomegaly and a significant reduction in one

or more of the cellular elements of the blood in

the presence of normocellular or hyper-cellular

bone marrow. The incidence of hypersplenism is

variable. It has been described in between 11%

and 55% of patients with cirrhosis and portal

hypertension in different series(8). Thrombocyt-

openia is the most frequent manif-estation of

hypersplenism (9).Although recombinant erythro-

poietin and filgrastim granulo-cyte-colony

stimulating factor raise red and white cell

counts, little can be done for thrombocytopenia

before or during interferon treatment(6). A

number of therapies are available for treating

thrombocytopenia due to hyper-splenism inclu-

ding spleenectomy, partial spleen-ctomy, partial

splenic embolization, TIPS etc. Splenectomy is

the definitive treatment for hypersplenism.

How-ever, the operation may be hazardous in

patients with poor liver function(9).

Aim of the work

To evaluate the safety and efficacy of open

splenectomy in the management of hyper-

splenism induced thrombocytopenia in selected

group of patients with HCV-related liver

cirrhosis ( those with Child-Pugh score A), so

that they can receive Pegylated Interferon and

ribavirin therapy.

Patients & methods

From all patients with serologically proven

HCV attending the hepatology unit in Medical

Research Institute, only patients with the

following criteria were included in the study: -

Patients with chronic hepatitis C. - Patients with

Child Pough score A. - Patients with throm-

bocytopenia. - Liver biopsy stage F2 and F3. –

Negative antiplatelet antibody(assayed by direct

platelet suspension immunofluorescence test). In

the period from October 2009 to March 2011,

twenty patients were eligible for the study and

they were subjected to the following: - Detailed

history taking. - Clinical examination. Investi-

gations: Complete blood picture, blood urea,

serum creatinine and fasting blood glucose.

Liver profile: serum albumin, serum bilirubin,

serum transaminases, and prothrombin time and

activity. HBsAg, HCV antibodies ( 3 rd genera-

tion ELISA). - Quantitative HCV-RNA by PCR.

-Ultrasound abdomen. -U/S guided liver biopsy.

- UGIT endoscopy. - Anti-platelet antibodies

(assayed by direct platelet suspension immune-

fluorescence test). - Bone marrow trephine

biopsy. These patients were referred to general

surgery unit to perform open splenectomy.

Procedure

After informed written consent, polyvalent

peumococcal polysaccharide vaccine was admi-

nistered two weeks before surgery. Open spleen-

ectomy was performed through left paramedian

incision. After opening the lesser sac, the

splenic artery was identified and ligated in

continuity above the pancreas when possible.

The posterior attachments were divided and the

spleen was delivered then the short gastric

vessels were divided and the stomach retracted

to the right. Hilar dissection progressed. The

splenic artery and vein were divided and suture

ligated ,and the spleen removed. Left gastric

ligation was done only in patients with varices.

Haemostasis was done and rubber drain was

placed routinely which was removed before

discharge and sutures were removed on post –

operative day ten (POD 10). Platelets were

administered only after clamping the splenic

inflow. Doppler U/S was performed on two

weeks post-operative to detect portal vein

thrombosis. Data about blood & platelet trans-

fusion, duration of surgery, complications (intra-

& post-operative), & hospital stay were repor-

ted. Follow up of the patients at 3 & 6 months

was done.

Statistical analysis

Statistical analysis was performed using SPSS

version 17.0. Normality test was done for

quantitative variables. Normally distributed

variables were presented as the mean ±standard

deviation (SD), while qualitative variables were

presented as the number and percent. For

normally distributed quantitative variables the

independent t- test was done for pre- and post-

intervention comparison. A value of P< 0.05

was considered statistically significant.

Results

This study was conducted on 20 patients with

HCV - related liver cirrhosis and hypersplenism

induced thrombocytopenia .The age of the

patients ranged between 38-56 years with a

mean value 47.0 ± 5.40 years. As regards

gender, most of patients were males, 14

patients,(70%) and only six patients were

females (30%). (Table 1)

Table (1): Demographic data of the studied patients.

Patients

n=20

Age (years)

Range 38.0 – 56.0

Mean ± SD 47.0 ± 5.40

Sex

Male n (%) 14 (70.0 %)

Female n (%) 6(30.0 %)

All patients were positive to HCV-Ab testing,

but negative to HBsAg. Quantitative HCV-RNA

ranged between50,000-750,000 IU/ml with a

mean value 321,252 ± 267,659 IU/ml.Liver

cirrhosis and splenomegaly detected by U/S

were present in all patients. The longitudinal

diameter of the spleen ranged between 13-17cm

with a mean value 14.87 ± 1.71 cm. Portal vein

diameter was increased in 14 patients (70%). It

ranged between 11-15 mm with a mean value

13.33 ± 1.40 mm. All patients were of Child

Pough class A.The liver profile of patients is

shown in Table (2); ALT ranged between 35-50

u / l with a mean value 42.73± 7.20, AST ranged

between 39-60 u/l with a mean value 49.73 ±

6.71, serum albumin ranged between 3.3- 4.0

g/dl with a mean value of 3.8 ± 0.2 g/dl, serum

bilirubin ranged between 0.8- 1.5 mg/dl with a

mean value 1.2 ± 0.21 mg/dl, and INR ranged

between 1.1-1.5 with a mean value 1.27 ± 0.19.

Table (2): Liver profile of the studied patients.

Parameter

ALT (u/l)

Range 35-50

Mean ± SD 42.73 ± 7.20

AST (u/l)

Range 39-60

Mean ± SD 49.73 ± 6.71

Serum albumin (g/dl)

Range 3.3-4.0

Mean ± SD 3.8 ± 0.2

Serum bilirubin (mg/dl)

Range 0.8-1.5

Mean ± SD 1.2 ± 0.21

INR

Range 1.1-1.5

Mean ± SD 1.27 ± 0.19

As regards the hematological investigations of

studied patients, all patients had thrombocy-

topenia, the platelets counts ranged between 48×

(10)3-122×(10)3 / mm3 with a mean value 62.8×

(10)3± 19.16 / mm3, the hemoglobin levels

ranged between 10-12.5 g/dl with a mean

value10.83 ± 1.7 g/dl, and the leucocytic count

ranged between 2.8× (10)3–4.3× (10)3 /mm3

with a mean value 3.2 × (10)3 ± 1.1 /mm3,

(Table 3)

Table (3): Hematologicalinvestigations of studied patients.

Parameter Result

Hb (g/dl)

Range 10-12.5

Mean ± SD 10.83 ± 1.7

WBCs (cells/mm3)

Range 2.8×(10)3–4.3(10)3

Mean ± SD 3.2×(10)3 ± 1.1

Platelet count (cells/mm3)

Range 48×(10)3-122(10)3

Mean ± SD 62.8(10)3± 19.16

UGIT endoscopy was done for all patients to

detect esophgogastric varices. Esophageal

varices of F1 grade were detected in five

patients (25%); these varices had no risk signs

of bleeding. No gastric varices were detected in

any patient. Mild portal hypertensive gastro-

pathy was detected in two patients (10%).

Histopathological analysis of U/S guided liver

biopsy according to METAVAIR score revealed

that five patients (25%) had F2, while 15

patients (75%) had F3. The bone marrow biopsy

was hypercellular in 16 patients (80%) and

normocllular in four patients (20%) (Table 4)

Table (4): Results of UGIT endoscopy, liver biopsy, and bone marrow biopsy

Table (5) .shows the perioperative data, the

meanoperative time was 96.4±18.2 minutes

(range:75-130 minutes) and there were no

deaths. As regard the intraoperative complic-

ations, intraoperative bleeding occurred in one

case due to ooze from splenic bed and

controlled by pack which was removed after 48

hours. In another patient there were severe

adhesions between the spleen and the diaphragm

and during sharp dissection injury to the

diaphragm and diaphragmatic pleura occurred

with lung collapse, repair of the tear was done

and the lung was inflated by positive pressure

ventilation by the anesthetist and the air entery

was good by auscultation and post-operative

plain x ray showed normally inflated lung. The

mean blood loss in our study was 250±67.2ml

(range: 100- 850ml) and five patients were in

need for blood transfusion, while platelets were

given empirically to all patients after splenic

artery ligation. As regard the postoperative

complications, there was sub-diaphragmatic

collection in one patient (patient with packing),

guided aspiration was done and the patient

improved. Two patients developed basal atelec-

tasis and three patients developed high fever.

The mean postoperative hospital stay in our

study was5.3 ±1.05 days (range:3-12days).

Duplex study was done two weeks post-

operative, there was one case (5%) with partial

portal vein thrombosis, and this case passed

unnoticed clinically.

Table (5) Theperioperative data.

Estimated blood loss

Range

Mean± SD

1oo-850ml

250± 67.2ml

Blood transfusion

RBCs packs:

Range

Mean± SD

n= 5 patients

1-3 packs

1.2±0.96 packs

Operative time (minutes)

Range

Mean± SD

75-130 minutes

96.4±18.2

Intra-operative complications

No. ( %)

2 (10%)

Post-operative complications:

Sub- phrenic collection

Basal atelectasis

High fever

Portal vein thrombosis

1(5%)

2 (10)

3 (15%)

1 (5 %)

Hospital stay (days)

Range

Mean ± SD

3-12days

5.3 ±1.05

Mortality 0

Parameter Number of patients (%)

Esophageal varices

Grade I

Grade II

Grade III

Portal hypertensive gastropathy

5(25%)

-

-

2(10%)

Liver biopsy ( METAVAIR score)

F2

F3

5(25%)

15(75%)

Bone marrow biopsy

Hypercellular

Normocellular

16(80%)

4(20%)

CBC was done 3 and 6 months post-operative,

there was significant improvement in platelets

count in all patients. The count was raised from

a mean value of 62.8(10)3 /mm3preoperatively to

215× (10)3 /mm3 after three months (p=0.03)

and to 293× (10)3 /mm3 after six months ( p =

0.007 ) table (7)

Table (7) Hematological changes 3 and 6 months postoperative

Pre-operative 3 months post-operative 6months post-operative

Platelet count 62.8×(10)3±19.16 215×(10)3± 41.73

P= 0.003*

293×(10)3± 47.01

P=0.007*

WBC count 3.2×(10)3 ± 1.1 7.4×(10)3± 1.92

P= 0.04*

8.2×(10)3± 1.61

P= 0.02*

HB 10.83± 1.7 gm.

12.3± 0.91 gm.

P= 0.04*

12.8±0.84gm.

P= 0.01*

*= significant

Discussion

Thrombocytopenia is a common manifestation

of chronic hepatic disease, although the exact

mechanisms remain incompletely understood.

Portal hypertension from cirrhosis diverts the

flow of blood from the portal circulation toward

the enlarged spleen, resulting in hypersplenism

and platelet sequestration. In healthy patients,

45% of platelet destruction occurs in the splee

(10). Studies utilizing radiolabeled platelets in

patients with chronic liver disease demonstrate

an accelerated destruction of platelets by the

spleen (11). Thrombocytopenia carries a risk of

bleeding complications including hematuria,

hematemesis, melena, and intracranial hemor-

rhage. The risk of spontaneous bleeding varies

with platelet count and the individual patient’s

clinical risk factors for hemorrhage. Previous

reports have demonstrated a dramatic increase

in the frequency and severity of hemorrhage in

patients with platelet counts below20 × (10)3/ml

(12). Moreover thrombocytopenia prevents pati-

ents with hepatitis C virus from receiving

potentially curative antiviral therapy. So, num-

erous surgical procedures have been proposed to

correct cirrhosis-associated thrombocytopenia.

Early attempts focused on the diversion of portal

flow from the spleen through portocaval anast-

omoses. Although these interventions were eff-

ective in decreasing splenomegaly, they did not

correct thrombocytopenia in cirrhotic pati-ents

with hypersplenism(13). Other forms of portal

decompression such as transjugular intrahepati-

cportosystemic shunting (TIPS) have also been

unsuccessful (14). Selective shunting procedures

such as the distal splenorenal shunt have

demonstrated success in both resolving the

thrombocytopenia associated with cirrhosis as

well as reducing the incidence of variceal-

hemorrhage (8, 15). Partial splenic embolization

(PSE) has been developed hoping to be an

effective and less invasive alternative however

the procedure was found to be associated with

many complications, Sakai et al.(16) reported that

the most frequent complications of PSE were

abdominal pain (82.4%)and fever (94.1%). In

addition, severe complications including splenic

abscess were observed in 16% of the patients

who underwent PSE (17). Splenic abscess leading

to death was observed in 6%(16). N’Kontchouet

al (17)concluded that the indication for PSE

should bevery limited and the extent of necrosis

of the spleens hould be strictly controlled during

the PSE procedure. Splenectomy has been

performed for hypersplenism since the 1950s,

(18) and it has been shown to correct the

manifestations of hypersplenism in patients

with cirrhosis (15, 19). However the operation is

hazardous in patients with poor liver functions.

Recently laparoscopic splenectomy has been

used successfully by Kercher and colleagues in

patients with Child Pough score Ato reverse the

thrombocytopenia and allow continuation of

anti-HCV therapy (20). In this study we tried

open splenectomy to raise platelet counts in

patients with Child Pough score A (patients

with good liver functions) to enable anti-viral

therapy and fortunately those are patients who

are candidate for interferon therapy. All patients

included in the study had significant improve-

ement in the platelet counts after splenectomy,

the mean platelet count raised from 62.8

×(10)3±19.16/ml preoperatively to 215× (10)3 ±

41.73 / ml three months post operatively and to

293 ×(10)3±47.01/ ml-six months postoper-

atively. In our study there were no deaths, and

the mean blood loss was 250±6.7.2ml, blood

transfusion was needed only in five patients and

the mean transfusion requirement was 1.2 ± 0.96

packs of RBCs.The mean hospital stay was 5.3

± 1.05 days. Of primary concern, portal vein

thrombosis (PVT) is a major potential compli-

cation of spleenec-tomy and has been reported

in up to 8% to 10% of patients treated with open

surgery(21-23). in our study Portal vein throm-

bosis occurred in one patient (5%), and the

patient was clinically asymptomatic. Other

complications were in the form of sub phrenic

collection in one patient, fever in three patients

and basal atelectasis in two patients. Taking into

consideration that, all our patients were

discharged in good health and showed a100%

efficacy regarding the improvement in platelet

count, this in addition to the reasonable

operative time, hospital stay and the acceptable

blood loss of the operation,we can conclude that

open splenectomy offer a safe and effective

procedure for management of hypersplenism

induced thrombocytopenia especially in selected

group of patients with chronic hepatitis C

infection. (Patients with Child Pugh score A),

however the major limitations of our study are

its small size and relatively shorter duration of

follow up so a larger studies may be needed to

draw a firm conclusion regarding the wide

spread applicability of this technique.

References

1. Alter MJ. Epidemiology of hepatitis C virus infection.

World J Gastroenterol 2007; 13:2436–2441.

2. Kamel MA, et al. High HCV prevalence in Egyptian

blood donors. Lancet 1992; 340:427.

3. Mohamed MK, et al. Intrafamilial transmission of

hepatitis C in Egypt. Hepato-logy 2005; 42:683–687.

4. Saleh DA, et al. Incidence and risk factors for hepatitis

C infection in a cohort of women in rural Egypt. Trans R

Soc Trop Med Hyg 2008; 102:921–928.

5. El-Zanaty, Fatma and Ann Way. 2009. Egypt

Demographic and Health Survey 2008. Cairo, Egypt:

Ministry of Health, El-Zanaty and Asso-ciates, and Macro

International.

6. Hayashi PH, Mehia C, Joachim Reimers H, et al.

Splenectomy for thrombocytopeniain patients with

hepatitis C cirrhosis. J ClinGastroenterol 2006;40:740–

744.

7. Chaufaud M. Apropos de la communication de M

Vaquez. Bulletins etMemoires de la SocieteMedicaledes

Hopitaux de Paris 1907; 24: 1201-1203.

8. El-Khishen MA, Henderson JM, MillikanWJ et al.

Splenectomy is contraindicated for throm-bocytopen-

iasecondary to portal hypertension. Surgery, Gynecology

Obstetrics 1985; 160: 233-238.

9. McCormick AP, MurphyMK. Splenomegaly, hyper-

splenism and coagulation abnormalities in liver cirrhosis.

Bailliere, sclinical Gastroenter-ology 2000;14,(6):1009-

1031.

10. Schmidt KG, Rasmussen JW, Rasmussen AD.

Kinetics of 111In-labelled platelets in healthy subjects.

Scand J Haemato l1985; 34:370–377.

11. Schmidt KG, Rasmussen JW, Bekker C, Madsen PE.

Kinetics and in vivo distribution of 111-In-labelled

autologous platelets in chronic hepatic disease:

Mechanisms of thrombocy-topenia. Scand J Haematol

1985;34:39–46.

12. Gaydos LA, Freireich EJ, Mantel N. The quantitative

relationbetween platelet count and hemorrhage in patients

with acuteleukemia. JAMA 1962;266:905–909.

13. Mutchnick MG, Lerner E, Conn HO. Effect of

portacavalanastomosis on hypersplenism. Dig Dis Sci

1980;25:929–938.

14. JabbourN,ZajkoA,Orons P, IrishW,FungJJ, Selby RR.

Doestransjugular intrahepatic port-osystemic shunt (TIPS)

resolve thrombocy-topenia associated with cirrhosis? Dig

Dis Sci1998;43:2459–2462.

15. Soper NJ, Rikkers LF. Effect of operations for

variceal hemorrhage on hypersplenism. Am J Surg

1982;144(6):700–703.

16. Sakai T, Shiraki K, Inoue H, et al. Compli-cations of

partialsplenic embolization in cirrhotic patients. Dig Dis

Sci2002;47:388–391.

17. N’Kontchou G, Sero O, Bourcier V, et al. Partial

splenicembolization in patients with cirrhosis: efficacy,

toleranceand long-term out-come in 32 patients. Eur J

GastroenterolHepatol 2005;17:179–184.

18. Lord JW Jr. The surgical management of secondary

hypersplenisms. Surgery 1951;29:407 –418.

19. Tomikawa M, Hashizume M, Akahoshi T,

Shimabukuro R,Gotoh N, Ohta M, Sugimachi K. Effects

of splenectomyon liver volume and prognosis of cirrhosis

in patients withesophageal varices. J Gastroenterol-

Hepatol 2002;17:77–80

20. Kercher KW, Carbonell AM, Heniford BT, et al.

Laparoscopicsplenectomy reverses throm-bocytopenia in

patients with hepatitis Ccirrhosis and portal hypertension.

J Gastrointest Surg. 2004;8:120–126.

21. Winslow ER, Brunt LM, Drebin JA, Soper NJ,

KlingensmithME. Portal vein thrombosis after

splenectomy. Am JSurg 2002;184:631–635; discussion

635–636.

22. Hassn AM, Al-Fallouji MA, Ouf TI, Saad R. Portal

veinthrombosis following splenectomy. Br J Surg

2000;87:362–373.

23. Chaffanjon PC, Brichon PY, Ranchoup Y, Gressin R,

Sotto JJ.Portal vein thrombosis follo-wing splenectomy

for hematologicdisease: Pros-pective study with Doppler

color flow imaging.World J Surg 1998;22:1082–1086.

Original Article

Use Of Anti-Cyclic Citrullinated Peptide Antibodies to Distinguish Hepatitis C

Virus (HCV) – Associated Arthropathy from Concomitant Rheumatoid Arthritis In

Patients with Chronic HCV Infection

Khaled Mahmoud Mohiedeen and Akram Deghedy*Tropical medicine and clinical pathology departments*

Alexandria faculty of medicine

ABSTRACT

Differentiating those patients whose symptoms are an extrahepatic manifestation of HCV from patients who have

concomitant Rheumatoid arthritis (RA) is essential for appropriate management. Objectives: To investigate that Cyclic

Citrullinated Peptide Antibodies (CCP antibodies), in contrast to Rheumatoid factor (RF), might be a candidate biomarker for

concurrent Rheumatoid arthritis in chronic HCV patients. Methods: Fifty non arthritic patients with chronic viral hepatitis C

were included. Testing for autoantibodies was performed using ELISA kits for IgG anti-CCP, IgG-RF and IgM-RF. Results:

CCP antibodies were positive in only two patients (4%), whereas, RF was elevated in the serum of 60% of the patients. IgG-

RF was detected more frequently (56%), followed by IgM-RF (30%). There was no statistically significant correlation

between CCP antibody level and serum IgG-RF (R2 = 0.030), or IgM-RF (R2 = 0.016). Conclusion: Anti-CCP may be more

useful than RF for the diagnosis or literally the exclusion of Rheumatoid arthritis in patients with chronic HCV infection.

Introduction

Chronic HCV infection is associated with extra

hepatic immune mediated conditions inclu-ding

vacuities, glomerulonephritis, thyroiditis, and

sialoadenitis(1, 2). Arthralgias are common, and

oligo or polyarthritis have been reported. (3, 4)

Arthralgia and arthritis in patients with chronic

hepatitis C can mimic rheumatoid arth-ritis

(RA), and discrimination is really difficult

without observing the erosions. (5) Differen-

tiating those patients whose symptoms are an

extra hepatic manifestation of HCV from

patients who have concomitant RA is essential

for appropriate management. Patients with virus

induced symptoms require antiviral therapy;

while patients with RA benefit from disease

modifying anti rheumatic drugs. (4) Fifty to

seventy percent of patients with HCV infection

may have rheumatoid factor (RF). The increased

prevalence of RF in patients with HCV infection

diminishes the diagnostic specificity of serum

RF for rheumatoid arthritis (RA) in this group of

patients. Therefore, the presence of RF mostly

does not help in distinguishing between RA and

HCV-associated rheumatic symptoms. (6)

Antibodies to cyclic citrullinated peptide (anti-

CCP) are recent serological marker available for

the diagnosis of RA. It is tested by commercially

available ELISA kit that has comparable sensit-

ivity and is more specific than Rheumatoid

factor for the diagnosis of RA. The clinical

usefulness of anti- CCP in diagnosis of early

arthritis is established and these antibodies

represent an important addition to the diagnostic

armamentarium in RA. (7, 8)

Patients and Methods

Patients. Fifty patients with chronic hepatitis C

infection were included in this study after

obtaining their consent. Patients who satisfied

the inclusion/exclusion criteria were recruited in

this study and selected from patients attending

the outpatient clinic or admitted to the inpatient

ward of Tropical Medicine Department, Alex-

andria Main University Hospital. Detailed

history taking and thorough clinical examination

were done for all patients. Patients were inclu-

ded after confirmation that HCV is the

etiological agent of their chronic liver disease

and obtaining an evidence of persistent infection

by either liver biopsy or abnormal transami-

nases. The exclusion criteria included concomi-

tant hepatitis B infection, known RA or the

presence of a history or clinically evident

arthritis, or current use of antiviral therapy.

Laboratory method: Serum samples were tested

for anti-hepatitis C antibodies by commercial

ELISA testing and were confirmed by HCV

RNA polymerase chain reaction amplification.

Testing for autoantibodies was performed using

ELISA kits for IgG anti-CCP, IgG-RF and IgM-

RF. All tests were performed according to the

manufacturer’s recommendations. Prediluted

control and diluted patient sera are added to the

microwell plates coated with the antigen.

Unbound sample was washed away and an

enzyme labeled anti-human IgG or IgM, was

added to each well to detect IgG-RFor IgM-RF

respectively, or enzyme labeled anti-human IgG

for CCP antibodies detection. The results of

anti- CCP ELISA were considered negative in

samples with results < 25 U/mL and are defined

as positive in samples with results ≥25 U/mL.

For RF, patients were considered to be

seropositive if serum RF was > 6 IU for all

isotypes. Comparison of CCP antibody and RF

concentrations was done. The study protocol

was approved by the ethics committee.

Results

Fifty HCV patients were included in the study.

The characteristics of the patients are presented

in Table 1. Subjects had a mean age of 45 years;

there were 34 men and 16 women. No patients

were receiving antiviral therapy. Sixty six

percent of patients were treatment-naive and

34% were treatment failures. RF was elevated

in the serum of 60% of the patients. IgG-RF

was detected more frequently (56%), followed

by IgM-RF (30%). CCP antibodies were

positive in only two patients (4%) (Figure.1).

Compared to CCP antibodies seropositivity,

there was a statistically significant increase in

the number of patients with elevations of IgG,

and IgM RF. Correlation of serum RF with

serum anti-CCP levels was examined by simple

regression. In this group of patients, there was

no statistically significant association between

CCP antibody level and serum IgG-RF (R2 =

0.306), IgM-RF (R2 = 0.016) (Figures 2,3).

Table.1 Patients demographic characteristics

Age Range: 39 - 54

Mean: 45 y

Sex Males: 34

Females: 16

Treatment

history

naive: 66%

Treatment failure: 34%

Figure 1 Prevalence of autoantibodies in HCV patients (% seropositivity)

0

10

20

30

40

50

60

IgG RF IgM RF ACCP

Figure 2: Correlation between ACCP and IgG RF

Figure 3: Correlation between ACCP and IgM RF

Discussion

Rheumatoid arthritis (RA) and hepatitis C virus

(HCV) infection are 2 distinct chronic diseases

that share several intriguing similarities. Each

illness is associated with immune system acti-

vation, autoantibody and cryoglobulin produ-

ction, secondary vasculitis and Sjögren’s

syndrome, and an increased risk of B cell lym-

phoma (9, 10) Although it is not always possible

to identify a specific pattern of HCV-related

arthropathy, two different clinical subsets of

arthritis have mainly been described; a mono

articular,oligoarticular intermittent arthritis aff-

ecting large and medium-sized joints and almost

invariably associated with the presence of mixed

cryoglobulinaemia and a polyarticular symm-

etrical arthritis closely resembling RA with met-

acarpophalangeal, proximal interphalangeal and

wrist joints involvement.(3,11) Arthritis associated

with mixed cryoglobulins or secondary to

immune complex deposition related to the

chronic viral infection might respond to

interferon- α. (4) However, induction or exace-

rbation of arthritis with interferon therapy in

some HCV patients has been noted. (12) These

patients might respond better to therapy directed

specifically to RA. Immunosuppressants are

generally avoided in chronic HCV infection due

to potential exacerbation of viral replication or

direct hepatotoxicity. (13) In this study, RF was

elevated in the serum of 60% of chronic HCV

patients. IgG-RF was detected more frequently

(56%), followed by IgM-RF (30%). This is in

accordance with other studies which reported

even higher percentages for the positivity of RF

in similar cohorts of patients.(14,15) This high rate

of RF seropositivity in patients with chronic

HCV infection presents diagnostic and thera-

peutic difficulties for the clinician. Recently,

Anti-cyclic citrullinated peptide (anti-CCP)

antibody testing was found to be particularly

useful in the diagnosis of rheumatoid arthritis,

with high specificity, presence early in the

disease process, and ability to identify patients

who are likely to have severe disease and

irreversible damage. (16) Unlike RF, CCP

antibody levels were not elevated in nonarthritic

patients with chronic hepatitis C included in this

study. In the two patients with CCP antibody

above the upper limit of normal, the level was in

the mild positive range. This is in agreement

with the results reported by Lienesch et al (17)

who detected a marginally elevated CCP

antibody in a single patient (2%). They also did

not find a correlation between anti CCP and RF

positivity which was also reported in the present

study. In a study on nonarthritic patients with

HCV, Orge et al(6) have found that, the

sensitivity, specificity and positive predictive

value of the anti-CCP test was superior to the

RF test and concluded that; Cyclic citrullinated

peptide antibody is a more useful test than RF

among patients with chronic HCV infection

without arthritis. Moreover, Ezzat et al (18)

reported that Anti-CCP antibodies were positive

in 4.5% in patients with HCV and poly-

arthropathy and in 83.3% of patients with RA.

RF was positive in 81.1% of HCV patients with

polyarthropathy and in 90% of RA patients.

They concluded that, anti-CCP antibodies are

reliable laboratory markers to differentiate

between RA and HCV-related polyarthropathy.

This observation may let us conclude that most

of the cases of chronic HCV associated poly-

arthropathy with positive RF testing are not

really due to Rheumatoid arthritis. Lienesch et

al explained that the HCV associated B cell

activity responsible for RF production does not

result in an increased production of antibodies

against CCP. Considering these data, a possible

interpretation of the presence of CCP antibodies

in HCV patients with arthritis could be that the

synovitis is secondary to concomitant RA.

Supporting this hypothesis is the lack of CCP

antibody production in other conditions ass-

ociated with inflammatory arthropathy.(18)

Similarly, measurement of anti-CCP antibodies

was also reported to be better than RF detection

to discriminate HBV-associated arthropathy

from concomitant RA in patients with chronic

HBV infection (19). The present study suggest

that, CCP antibodies unlike RF are not produced

in response to HCV infection, and therefore

Anti-CCP may be more useful than RF for the

diagnosis or literally the exclusion of Rheum-

atoid arthritis in this group of patients.

References

1. Ramos-Casals M, Trejo O, Garcia-Carrasco M, Font J.

Therapeutic management of extra-hepatic manifestations

in patients with chronic. hepatitis C virus infection.

Rheumatology 2003; 42: 818-28.

2. Pawlotsky JM, Roudot-Thoraval F, Simm-onds P, et al.

Extrahepatic immunologic manif-estations in chronic

hepatitis C and hepatitis C virus serotypes. Ann Intern

Med 1995;122:169-73

3. Lovy MR, Starkebaum G, Uberoi S. Hepatitis C

infection presenting with rheumatic manife-stations: a

mimic of rheumatoid arthritis. J Rhe-umatol 1996; 23:

979-83.

4. Zuckerman E, Keren D, Rozenbaum M, et al. Hepatitis

C virus related arthritis: characteristics and response to

therapy with interferon alpha. Clin Exp Rheumatol 2000;

18: 579-84.

5. Palazzi C, D'Angelo S, Olivieri I. Hepatitis C virus-

related arthritis. Autoimmunity Reviews 2008; 8: 48-51.

6. Örge J, Çefle A, Yazıcı A, Gürel-Polat N, Hulagu

S. The positivity of rheumatoid factor and anti-cyclic

citrullinated peptide antibody in nonarthritic patients with

chronic hepatitis c infection. Rheumatol Int 2010;30:485-

488

7. Jansen LM, van Schaardenburg D, van der Horst-

Bruinsma I, van der Stadt R, De Koning M, Dijkmans

BA. The predictive value of anti-cyclic citrullinated

peptide antibodies in early arthritis. J Rheumatol 2003;

30:1691-5

8. Khosla P, Shankar S, Duggal L. Anti CCP antibodies in

Rheumatoid arthritis. J Indian Rheumatol Assoc 2004 : 12

: 143 -46

9. Alter MJ. Hepatitis C virus infection in the United

States. J Hepatol 1999; 31 Suppl 1:88-91.

10. Lawrence RC, Hochberg MC, Kelsey JL, et al.

Estimates of the prevalence of selected arthritic and

musculoskeletal diseases in the United States. J

Rheumatol 1989; 16: 427-41.

11. Olivieri I., Palazzi C., Padula A. Hepatitis C virus and

arthritis. Rheum Dis Clin North Am 2003;29: 111–122

12. Nesher G, Ruchiemer R Alpha - interferon - induced

arthritis: clinical presentation, treatme-nt, and prevention.

Semin Arthritis Rheum 1998;27:360–5

13. Wener MH, Johnson RJ, Sasso EH, Gretch

DR.Hepatitis C virus and rheumatic disease. J

Rheumatol1996; 23: 953–9.

14. Clifford BD, Donahue D, Smith L, et al. High

prevalence of serological markers of autoi-mmunity in

patients with chronic hepatitis C. Hepatology

1995;21:613-9.

15. Nocente R, Ceccanti M, Bertazzoni G, Cammarota G,

Silveri NG, Gasbarrini G. HCV infection and extrahepatic

manifestations. Hep-atogastroenterology 2003; 50: 1149-

54.

16. Niewold TB, Harrison MJ, Paget SA Anti-CCP

antibody testing as a diagnostic and prog-nostic tool in

rheumatoid arthritis. QJM. 2007; 100 (4):193-201.

17. Lienesch D, Morris R, Metzger A, Debuys P,

Sherman K. Absence of Cyclic Citrullinated Peptide

Antibody in Nonarthritic Patients with Chronic Hepatitis

C Infection. J Rheumatol 2005;32:489–93

18. Niewold TB, Harrison MJ, Paget SA. Anti-CCP

antibody testing as a diagnostic and prognostic tool in

rheumatoid arthritis. QJM. 2007; 100(4):193-201.

19. Lim MK, Sheen DH, Lee YJ, Mun YR, Park M, Shim

SC. Anti-cyclic citrullinated peptide antibodies

distinguish hepatitis B virus (HBV)-associated

arthropathy from concomitant rheum-atoid arthritis in

patients with chronic HBV infection. J Rheumatol 2009;

36(4):712-6.

Original Article

Prevalence of Non-Organ-Specific Autoantibodies and Its Effect

on response to Antiviral Therapy in Patients with Chronic Hepatitis C Virus

Genotype 4

Mohamed Abd El-Maksoud1, Hatem Elalfy1, Maha Ragab Habeeb2, Abd-Elmohsen E. El-desoky2, Ziyad M. Tawhid3,

Basem S. Eldeek4

ABSTRACT

Immunological disorders have been frequently described in the course of hepatitis C virus (HCV)–related chronic hepatitis.

Our aim was to determine the prevalence of non-organ-specific autoantibodies (NOSAs) and to evaluate its impact on the

response to combined antiviral therapy in patients with chronic HCV genotype-4. Methods: A total of 134 adult patients with

chronic HCV genotype-4 were studied. Serum Antinuclear antibody (ANA), anti–smooth muscle antibody (SMA), and anti

liver/kidney microsomal antibody type 1 (LKM1) were detected by ELISA (Anova, Germany). 109 patients were treated

naive and received combined antiviral therapy (pegylated interferon–ribavirin). The presence of these autoantibodies was

related to patient’s characteristics and to the outcome of antiviral therapy. Result : Thirty-six (26.9%) patients were positive

for at least one autoantibody. Various autoantibodies were presented as follows: ANA in 29 (21.6%) patients, SMA in 9

(6.7%) and anti-LKM-1 in 2 (1.5%). In two patients, both ANA and anti-SMA were positive, and in other two cases both

ANA anti-LKM-1 were positive. Female patients had a higher prevalence of positive autoantibodies (P=0.005). Chronic

hepatitis C (CHC) patients with positive autoantibodies had higher serum ALT, AST and GGT levels. The rate of sustained

virological response to combined antiviral therapy was similar between autoantibody-positive and -negative groups (46.9%

vs. 53.2%). Conclusion: Autoantibodies can be induced in the course of CHC. Autoantibody-positive CHC patients are older

and have higher disease activity and severity. However, the presence of these autoantibodies did not influence the response to

combined antiviral therapy.

Introduction

Hepatitis C virus (HCV) is among the leading

causes of chronic liver disease worldwide and

affects approximately 170 million people (1).

Egypt has the highest prevalence of HCV infe-

ction in the world, ranging from 6% to 28%

with an average of approximately 13.8% in the

general population (2). Immunological disorders

have been frequently described in the course of

HCV-related chronic hepatitis, and non–organ-

specific autoantibodies (NOSAs) in particular

are common examples of autoreactivity associ-

ated with HCV infection (3). HCV has six major

genotypes according to its viral genome, num-

bered one to six. These viral types and sub-types

differ in their geographical distribution and anti-

genicity (4). Particular genotypes are associated

with different courses and outcome of liver

diseases, and also with different responsiveness

to interferon therapy. Results of the studies to

clarify the relationship between HCV genotype

and autoimmune manifestations are contrive-

rsial. A majority of them failed to confirm the

association between clinical course of HCV

infection, autoimmune disorders and particular

HCV genotypes. Genotype 4 is the predominant

genotype of HCV in Egyptian patients (up to

91%) (5). The currently recommended therapy

for chronic HCV infection is the combination of

pegylated interferon (PEG-IFN) and ribavirin (6).

The sustained virological response (SVR) rates

in patients treated with PEG-IFN and ribavirin

are 50% in HCV genotype 1 and 80-90% in

HCV genotype 2 or 3 (6- 9). The achievement of

the SVR in patients with chronic hepatitis C

(CHC) has been associated with improvements

in liver histology as well as a reduced risk of

hepatocellular carcinoma (HCC) and liver-

related mortality (10-12). However, several side-

effects have been published in patients treated

with IFN-α including the development or

exacerbation of underlying autoimmune

diseases and the development of a variety of

organ and non-organ specific autoantibodies

(NOSAs) .The association between these anti-

bodies and either HCV per se or IFN-a related

therapy is mainly based on epidemiological sur-

veys (13-18). Moreover, available data on the

relationship between autoantibody seropositivity

and the response to antiviral therapy in CHC

patients are limited and controversial (19, 20). In

this study, we aimed to assess the prevalence of

serum NOSAs in CHC patients. In addition, to

evaluate its impact on the response to com-bined

antiviral therapy (IFN or Pegylated IFN plus

ribavirin) in patients with HCV genotype 4-

related chronic hepatitis and to identify clinical,

biochemical, or immunological features predict-

ive of response to antiviral treatment.

Patients and Methods

The study was conducted into two stages: Stage

I: a comparative cross sectional study among

patients with chronic hepatitis C virus Genotype

4. Stage II: a case control study between patients

with chronic hepatitis C virus Genotype 4 and

healthy cross matched control. Sample size and

power of the study: The sample size was calcu-

lated by Medcalc program available at www.

Medcalc .be . At a level of 95% confidence

with alpha error 0.05. and the power of the

study was settled at 80 and beta error .02. The

prevalence of auto-antibodies was supposed to

be ranged from 20% to 10%. The estimated

sample is 86 patients. We try to increase the

sample of patients to 134 patients to increase the

power of the study. Limitation of our resources

enforce us to have a control group of 60

subjects. A total of 134 consecutive CHC pati-

ents were admitted to this study during the

period of July 2009 to January 2012 who visited

clinics of Tropical Medicine Unit (inpatients

and outpatients), Mansoura University Hospital.

They were 78 males and 56 females, with a

mean age of 48.4 ± 3.2 years and 60 healthy

controls with matched age and sex. All patients

had positive HCV antibody with enzyme-linked

immunosorbent assay (ELISA) ( Murex anti-

HCV (version 4.0) 7F51-06/-07, DiaSorin South

Africa (Pty) Ltd, Republic of South Africa) and

detectable HCV RNA ( Applied biosy stems,

Step One Real-time PCR system, USA) in the

serum. The exclusion criteria included human

immunodeficiency virus coinfection, hepatitis

other than hepatitis C (hepatitis B and autoim-

mune hepatitis), patients who showed evidence

of alcohol, illicit drug, or potentially hepatotoxic

medication use and major contraindications to

IFN or ribavirin therapy. Informed consent was

obtained from all patients, and the research

protocols were approved by the Medical Ethics

Committee of Mansoura University Hospital.

Detection of NOSA: Serum ANA was detected

by ELISA (ORG 538, ORGENTEC Diagnostika

GmbH, Germany), ASMA was detected by

ELISA (QUANTA LiteTM Actin IgG ELISA

708785, INOVA Diagnostics, Inc.USA) and

anti-LKM-1 was also detected by ELISA

(QUANTA LiteTM LKM-1 ELISA 708745,

INOVA Diagnostics, Inc.USA). Among the

laboratory parameters measured at baseline

serum levels of alanine- aminotransferase (ALT)

aspartate - aminotransferase (AST), total and

direct bilirubin, albumin, alkaline phosphatase,

γ-glutamyl transpeptidase (GGT) and α-

fetoprotein (AFP) were recorded and included in

the analysis. Samples positive for HCV-RNA by

real time PCR were subjected to genotyping of

HCV, by RT-PCR for the core domain using the

primers modified by Ohno et al. (21).

Histological assessment: Liver biopsy was done

for all patients before the initiation of therapy.

The histological evaluation was assessed using

the modified Knodell histology index and the

metavir scoring system reflecting the degree of

hepatic inflammation and fibrosis. Before

treatment, informed consent was obtained from

each patient. Treatment regimens and outcomes:

A total of 32 (88.9%) of 36 autoantibodies

positive patients and 77 (78.6%) of 98

autoantibodies negative patients had been

treated with a combination therapy ( either

Pegylated-IFN alfa-2a 180 µg subcutaneously

once a week or Pegylated IFN alfa-2b 1.5 µg/kg

subcutaneously once a week plus oral ribavirin

1000 or 1200 mg/day for subjects weighing <75

or ≥75 kg, respectively). According to HCV

genotype, the predetermined duration of

treatment was 48 weeks with a final efficacy

evaluation at week 24 of follow-up. Patients

were regularly followed–up for physical exam-

ination, blood tests and virological assays.

Treatment outcome was assessed as follows:

sustained virological response (SVR) was

defined as undetectable HCV RNA 24 weeks

after treatment discontinuation; relapse was

defined as HCV RNA clearance during treat-

ment and reappearance during follow-up; and

nonresponse was defined as a failure to clear

HCV RNA at any time during treatment (22.).

Statistical analysis: The data were collected and

entered the computer. The data were statistical

analyzed by using Statistical Package of Social

Science (SPSS). The qualitative data were pres-

ented in the form of number and percentage.

Chi-square with Yates correction was used as a

test of significance for qualitative data when the

expected cell less than 5. Chi-square test was

used as test of significance for qualitative data

when the expected cell more than 5. Signif-

icance was considered when p value less than

0.05. The quantitative data were presented in the

form of mean and standard deviation. Student t

test was used as a test of significance for

quantitative data of two groups. The fibrosis

score was presented in the form of median and

range. Mann Whitney u test was used as a test of

significance for fibrosis score. Significance was

considered when p value less than 0.05.

Result

Prevalence of NOSA in patients with chronic

hepatitis C: Table (1) shows the prevalence of

NOSAs in patients with chronic hepatitis C and

control groups. Among total of 134 patients

with chronic hepatitis C, thirty-six (26.9%) were

positive for at least one autoantibody. ANA was

present in twenty nine (21.6%) patients, anti-

SMA in nine (6.7%) patients , and anti-LKM-1

was found in two (1.5%) patients. In two of the

patients, both ANA and anti–SMA were

positive, and in other two cases both ANA anti-

LKM-1 were positive. The prevalence of serum

autoantibodies in patients with chronic hepatitis

C was significantly higher than in healthy

control (p < 0.05). Clinical significance of

NOSAs in patients with chronic hepatitis C:

Table (2) compares the clinical, laboratory and

histological parameters between CHC patients

with and without autoantibodies. As regard

demographic data, female patients had a higher

frequency of positive autoantibodies (P=0.005)

and age was significantly higher in the auto-

antibody-positive CHC patients (51.4 ±2.3) vs

(45.4 ±4.1) (P <0.001). The chronic hepatitis C

patients with and without serum autoantibodies

were analyzed with comprehensive clinical and

biochemical examinations: autoantibodies-

positive patients had significantly higher serum

levels of ALT (102±20.3) vs. (90±22.4)

(P=0.013), AST (96± 15.13) vs. (72±16.7)

(P=0.023), GGT (76.3± 15.2) vs. (50.9±12.7)

(P<0.001) and AFP (18± 4.5) vs. (13±3.9) (P=

0.012). Autoantibodies-positive patients had

also higher fibrosis scores and significantly

lower platelet counts (144 ±30.2) vs (186

±25.12) (P=0.004). No significant difference in

HCV viral load between both groups. Response

to combined antiviral therapy: Table (3) show

the response of chronic HCV- infected patients

to combined antiviral treatment (peg-IFN plus

ribavirin). In autoantibodies positive patients, 15

(46.9%) of 32 HCV-infected patients had a

sustained virological response (SVR), whereas 9

patients (28.1%) experienced nonresponse and 8

(25%) experienced relapse. In their counterpart,

autoantibodies negative HCV patients, the

response rate was as follow: 53.2% SVR, 24.7%

nonresponse and 22.1% relapse. The SVR rates

were comparable between autoantiboies positive

vs. autoantibodies negative patients (46.9% vs

53.2%). As regard the systemic autoimmune

manifestations, one patient with positive serum

autoantibodies developed hypothyrodism while

in autoantibodies negative group, one patient

developed diabetes mellitus, and another one

developed hypothyroidism. These complications

were con-trolled on therapy and did not required

withdrawal of combination therapy. Predictors

of response to antiviral therapy: In this study,

we compared patients with and without SVR

(Table 4) in order to predict the factors

associated with a favorable response to

combined antiviral therapy. Among the clinical,

biochemical, and histological parameters

studied, our results showed that younger age

(P<0.001), body mass index (BMI) (P<0.001),

higher serum ALT (P<0.001), lower GGT

(P<0.001), lower HCV viral load (P<0.001)

levels and lower fibrosis score were

significantly associated with SVR. In

comparison serum ANA, ASMA and LKM-1

were not significantly different between patients

with and without SVR.

Discussion

Patients chronically infected by HCV present

various immune mediated phenomena mainly

due to B lymphocyte dysfunction as mixed

cryoglobulinemia and non-organ-specific auto-

antibodies (NOSAs) production (23). Previous

studies have shown that serum autoantibodies

are commonly found in CHC patients (24). In this

study, the global prevalence of NOSAs among

patients with chronic hepatitis C was 26.9%.

ANA was the most commonly found

autoantibodies being present in 21.6% of

patients. The prevalence of ANA is higher than

that reported by studies from some countries (25),

while it is comparable to that reported from

some other countries. Lenzi et al., demonstrated

the occurrence of ANA in 16% of patients with

chronic hepatitis C(18). In Estonia, Zusinaite et

al., reported 14.4% prev-alence of ANA in

patients with chronic hepatitis C (26). As regard

the prevalence of ASMA in patients with

chronic hepatitis C, it was found to be 6.7%.

This result is lower than that reported in some

studies (18, 13, 26). Anti-LKM-1 autoantibodies are

detected worldwide in approximately 0-7% of

patients with chronic hepatitis C (27, 28).

Available data on the prevalence of anti-LKM-1

in Egyptian patients with CHC are relatively

uncommon. Here we reported that the positive

rate of anti-LKM-1 was 1.5%. These results

confirm that AIH related autoantibodies can

exist in CHC patients. Molecular mimicry

between the HCV polyprotein and "self"

proteins may account for the production of

autoantibodies in chronic HCV infection. A

sequence homology between the HCV

polyprotein and cytochrome p450 2D6 (CYP

2D6), the antigenic target of anti-LKM1, was

previously reported (29). The reactivity against

the viral protein would induce the production of

anti-LKM1 in HCV-related CLD. Gregorio and

colleagues documented molecular mimicry

between HCV polyprotein and three nuclear

host antigens including matrin, histone H2, and

replication protein as a mechanism for the

emergence of ANA (30). Polyclonal B cell

activation by persistent HCV infection has been

proposed as another mechanism for the prod-

uction of autoantibodies. In determining one of

the mechanisms for polyclonal B cell activation,

Pileri and colleagues documented that HCV

envelope protein (E2) represented a costimu-

latory signal to B cells by binding to CD 81

(tetraspanin) and thereby facilitated the produ-

ction of autoantibodies (31). B-lymphocyte acti-

vating factor (BAFF) appeared to play a crucial

role in HCV-induced autoimmunity (32). Varia-

tions in the prevalence of autoantibodies may be

attributed to several factors. First, there may be

differences in viral strains causing these differ-

ences (3). Secondly, the differences in detection

methods, ethnic background and geographic

distribution of the study cohort (33). In our study,

patients with positive autoantibodies were

significantly older. This is in agreement with the

findings of Squadrito et al., (13), who found that

NOSAs positive HCV patients were older than

those with negative autoantibodies. This phenol-

menon might result from functional defects in

suppressor T cells in older patients (34,35).

However, other studies found no age difference

between the two groups (36, 37). The positive

rate of autoantibodies was higher in females,

which is in accord with reports by other groups (25,38). This may reflect the difference in

autoimmune reactions between males and fema-

les after CHC infection, suggesting that horm-

ones, such as estrogen, may play an important

role in infection (39). As regard the biochemical

finding, this study showed that autoantibody-

positive CHC patients had significantly higher

serum ALT and AST levels than those without

autoantibodies. This is in agreement with

previous reports by Lenzi, et al., who reported

that NOSAs were significantly prevalent in

patients with HCV-related chronic liver disease,

and were especially so when the alanine aminot-

ransferase activity was higher (18). Moreover,

Cassani, et al., showed in a prospective series of

patients with HCV related chronic liver disease

who were positive for autoantibodies, a

biochemical and histological activity higher than

that of patients with no markers of autoim-

munity (40). In controversy, Stroffolini et al.,

showed no correlation between the positivity of

autoantibodies and liver damage (37). Muratori,

et al., showed that in the absence of active liver

disease the prevalence of non-organ specific

autoantibodies was similar in HCV positive

individuals and negative controls (3). This

suggests that the presence of non-organ-specific

autoantibodies is more likely associated with the

patient’s age and duration and severity of

chronic liver disease. Thus, reactivity against

self-antigens can be related to the severity of

liver damage without any independent patho-

genic role. Our finding also demonstrated that

NOSA-positive CHC patients had low platelet

count and more advanced fibrosis scores than

seronegative CHC patients. These finding are in

agreement with most published data, suggesting

that HCV-infected autoantibody- positive pati-

ents have higher disease activity and severity

than those who are autoantibody-negative ( 40,41).

IFN-alfa is the treatment of choice for patients

with chronic hepatitis C, but its imm-

unomodulatory activity may also favor the

appearance or amplification of autoimmune

reactions (42). The response to IFN-α in patients

with HCV infection and autoimmune markers

continue to be controversial (36). In our study,

we found that the presence of serum NOSA in

CHC patients did not influence the response to

combined antiviral therapy, which was similar

in both serum NOSA-positive and -negative

patients (46.9 % vs 53.2%). This results is in

agreement with other studies who reported that

the presence of autoantibodies such as ANA or

anti-LKM1 in patients with CHC is less likely to

affect the response to antiviral treatment (40,43).

In contrast, the favourable predictors of SVR

were younger age, body mass index (BMI),

higher serum ALT, lower GGT, lower HCV

viral load levels and lower fibrosis score. These

results are in agreement with other reports as in

all large prospective studies of (PEG) IFN and

RBV combination therapy younger age

correlated significantly with an SVR when

assessed by univariate and multivariate analyses

and patients younger than 40–45 years showed

the best response rates (44). GGT has been

identified as a prognostic factor in other studies (45,46). In this study, we found that low GGT

level had a favorable prediction of SVR. This is

in accordance with previous reports in which

low pre-treatment serum GGT levels were

significantly and independently associated with

SVR in multivariate regression analysis (47,48).

The pathogenetic background of GGT elevation

in chronic hepatitis C is not fully understood.

However a close relationship between serum

GGT levels and hepatic steatosis, advanced

fibrosis, and insulin resistance has been

described (49,50). Moreover, GGT levels are

related with an increased expression of TNFα in

the liver that seems to reduce the efficacy of

antiviral therapy (51). We also confirmed

previous reports signaling that a low viral load

is predictor of SVR. A low baseline viral load

(<600,000–800,000 IU/ ml or less) was shown

to be an independent predictor of SVR

regardless of genotype in numerous studies (44,47,52,53). In conclusion, serum NOSAS were

frequently found in HCV infected patients.

Patients with positive serum autoantibodies

were older, and have higher disease activity and

advanced fibrosis scores than their negative

counterparts. The positivity of autoantibodies

did not influence the response to combination

antiviral therapy. Combined antiviral treatment

is safe and effective in autoantibodies positive

patients with CHC. Routine testing of auto-

antiantibodies may be needed to monitors the

progress and severity of disease that might be

areas for further research. Limitation of the

study: Some limitations should be considered

when interpreting our findings. First, detection

of autoantibodies was based on ELIZA method,

and there was no record of the distribution type

of NOSAs. Whether the distribution type of

autoantibodies has clinical relevance is worthy

of future study. Second, the external validity of

this study is questionable, since the sample of

the patients may not be representative of all

Egyptian population due to cost variable, long

duration of follow up and the interferon therapy

is not available for most of Egyptian patients;

therefore, it is possible that our findings cannot

be extrapolated to all CHC patients in Egypt.

Table (1) Prevalence of NOSAs in patients with chronic hepatitis C

Case

(n)

Autoantibodies

(n (%))

ANA

(n (%))

ASMA

(n (%))

Anti-LKM-1

(n (%))

Patients with CHC 134 36 (26.9%) 29 (21.6%) 9 (6.7%) 2 (1.5%)

Control group 60 7 (11.7%) 7 (11.7%) 0 (0%) 0 (0%)

Test of significance 0.018* 0.098 .031* .47

Table (2) Clinical, laboratory and histological parameters of patients with chronic hepatitis C who did or not test positive for non-

organ specific autoantibodies

Parameters

Autoantibody positive

N= 36

Autoantibody negative

N= 98

P value

Gender

Male

Female

14

22

64

34

P=0.005**

Age (year) 51.4 ±2.3 45.4 ±4.1 <0.001***

Body mass index 26.1±2.1 27.2±2.4 .059

Hb level (g/dl) 13.6 ±1.2 13.9 ± 1.5 .28

WBCs 5.9±2.1 6.2 ±2.3 .19

/l)9(×10 Platlet count 144 ±30.2 186 ±25.12 0.004**

Albumin (g/dl) 4.03 ±0.75 4.2±0.32 .49

Total Bilirubin (mg/dl) 1.04±0.5 0.9±.41 .101

ALT (IU/ml) 102.3 ±20.3 90.5 ±22.4 0.013*

AST (IU/ml) 96.4 ± 15.13 72.9 ±16.7 0.023*

ALP (U/l) 247.08±42.3 238.67±36.8 .37

GGT (IU/l) 76.3±15.2 50.9±12.7 <0.001***

HCV RNA(×106 IU/ml) 0.58±.2 0. 66 ± .3 0.141

AFP 18.2 ±4.5 13.3 ±3.9 0.012*

Fibrosis score ( 0-2/3,4) 21/15

1(1-4)

70/28

2 (1-4)

<0.001***

*Significat p less than 0.05, ** highly significance less than .01, **** extremly significance less less than .001

Table ( 3) Response of chronic hepatitis C patients with and without autoantibodies to combined antiviral therapy

Autoantibodies positive

patients

N= 32

Autoantibodies negative patients

N=77 P value

SVR 15 (46.9%) 41 (53.2%)

.83 Non responder 9 (28.1%) 19 (24.7%)

Relapse 8 (25%) 17 (22.1%)

Table (4) Comparison between patients with SVR and Non-SVR

SVR

n= 56

Non-SVR

n= 53 P Value

Gender

Male/ Female

31/25

33/20

0.46

Age (year) 44±5.21 52±4.2 <0.001***

Body mass index 25.3±2.31 28.5±1.41 <0.001***

ALT (IU/ml) 116±25.7 86±50.1 <0.001***

AST (IU/ml) 17.2 87.7± 82.5±19.2 0.15

GGT (0-40IU/l) 47.7±5.7 82.3±6.2 <0.001***

ANA (+/-) 13/43 16/37 .41

ASMA (+/-) 4/52 5/48 .51

Anti-LKM-1 1/55 1/52 .96

HCV RNA (×106 IU/ml) 0.49±0.56 0.86±0.72 <0.001***

Fibrosis score 1

(1-3)

3

(2-4) 0.003**

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Original Article

Non-Traumatic Intramural Hematomas in Patients on Anticoagulant Therapy:

Report of Three Cases.

Mohamed Bekheit. MS, MSc, MRCS, MRCPS. a,b Mohamed Alaa Sallam. MS, FRCS. b

ABSTRACT

Non-traumatic intramural hematoma in the gastrointestinal tract is a rare event that could have diverse clinical presentation.

The small intestine is the site of predilection to this condition. Intestinal obstruction is the main presentation. Various degrees

of acute abdominal pain with peritoneal signs would make a cumbersome diagnosis. Patients and methods: Retrospective

review of the surgical emergency admissions database was conducted back to 1994. A literature overview was conducted.

Results: Three cases were retrieved and presented in this manuscript. More than 20 reports, with acute abdominal pain as a

main presentation, were found in the literature. Conclusions: Complete history taking is mandatory not to miss such an

uncommon complication. The INR level should be asked for in every case on oral anticoagulant presenting with acute

abdominal pain. CT scan is the main diagnostic tool. Conservative management is the standard therapeutic approach.

Introduction

In About 40% of the cases presenting with

abdominal pain; the diagnosis is uncertain. (1)

The primary goals of the management are to

diagnose the etiology of the abdominal pain and

then to ascertain the necessity for surgical inter-

vention. In order to avoid non indicated surgical

interference, meticulous workup is crucial. (2-4).

Meticulous differential diagnosis is not always

simple. Specific common patterns are usually

the guide for clinicians for further evaluation of

the patient condition. In at least third of the

cases the exhibited patterns are atypical, what

renders the diagnosis complex. (2,4). Accurate

history taking remains the most crucial initial

step in approaching these conditions. (5-10)

History of drug intake represents an essential

part of the history that might provide a direct

clue to the patient's problem. Anticoagulants are

commonly prescribed drugs for numerous surg-

ical and medical reasons. One of the rare repor-

ted complications of the drug is the occurrence

of intramural hematomas in the gastrointestinal

tract. (11). We report three cases of intramural

hematomas in various parts of the gastrointes-

tinal tract, who presented to the emergency dep-

artment with acute abdominal pain with or with-

out intestinal obstruction.

Cases presentations

Case 1: A 40 year-old male patient presented

with upper abdominal pain and food intolerance.

The patient had bronchial Asthma, COPD,

dilated cardiomyopathy, IHD and old MI, for

which he has been receiving the anticoagulant

therapy. The patient was on antihypertensive

medications plus the oral anticoagulant therapy.

His clinical examination showed minimal blee-

ding from the gum. The abdominal examination

revealed tender and rigid upper abdomen

(epigastric and Rt. Hypochondrium) with no

masses felt, bowel sounds were audible. Per

rectal examination (PR) showed normal color

stools. His admission profile showed mild

anemia (Hb 10 gm / dl), leucocytosis (WBCs

17,700 / dl), and deranged coagulation profile

(PT 163 sec and INR 17). An abdominal

ultrasound scan (U/S) revealed the presence of

18 mm thick hypoechoic gastric wall (Figure

1.a). UGI endoscopy (Figure 1.b) was done that

showed diffuse submucosal hemorrhage

involving the entire body and antrum of the

stomach down to the upper part of the

duodenum with no evidence of active bleeding.

CT scan of the abdomen confirmed the exact

extension of the hematoma (Figure 1.c). The

patient received fresh frozen plasma transfusion

and vitamin K. He was kept in the hospital for

13 days for correction of the coagulation deficits

and for stabilization of the heart condition.

Follow up ultrasound showed the reduced size

of the stomach thickness after five days of

admission. Follow up upper gastrointestinal

endoscopy after ten days of admission showed

significant improvement.

Case 2: A 62 year-old male patient presented to

the casualty complaining of abdominal pain

associated with constipation for two days. The

patient had ischemic heart disease and chronic

atrial fibrillation for which he required oral

anticoagulant therapy. His vital signs were

stable on presentation. His abdomen was

distended with diffuse tenderness, rebound

tenderness, sluggish intestinal sounds. His

digital rectal exam (P/R) was positive for mele-

na. Abdominal ultrasound scan (U/S) was done,

which reported thickened small bowel loops and

simple left renal cyst. Patency of the mesenteric

vessels was questioned by the U/S scan. Abd-

ominal CT scan with contrast showed thick-

ening of the proximal 20 cm of jejunum with

patent superior mesenteric artery and vein

(SMA and SMV) (figure 2). His initial laborato-

ry investigations demonstrated prolonged proth-

rombin time (PT 118 seconds) and an INR of

10.5. There were no significant derangements in

the rest of the laboratory work. There was a

dramatic cessation of pain on after correction of

the excessive anticoagulation with fresh frozen

plasma and fluids. The patient was discharged

after 4 days with adjusted dose of oral antico-

agulation.

Case 3: 65 year-old Male patient with diabetes

and ischemic heart disease for which he is on

regular warfarin treatment. The Pt. admitted to

our hospital through the E/R because of

abdominal pain. The pain was diffuse dull

aching with maximal tenderness at the Rt. Iliac

fossa. Clinical examination of the patient

showed tenderness in the right lower quadrant of

the abdomen, with no change in the stool color.

All laboratory investigations were within normal

except high blood sugar and abnormal

coagulation screening which showed elevated

INR 9. U/S abdomen was not conclusive. CT

scan of the abdomen with contrast showed

thickened terminal ileal loop about 10 cm with

normal mesenteric blood flow (figures 3.a, 3.b).

Fluid resuscitation and fresh frozen plasma were

given, the patient`s symptoms resolved by the

second day of admission. The patient was

discharged 2 days later.

Discussion and literature overview

Non-traumatic intramural hematoma of the

gastrointestinal tract complicating anticoagulant

therapy is mostly reported to take place with

Warfarin and less commonly reported with other

anticoagulants in the literature. (12,13) Warfarin

has a considerable variation in responsiveness

among patients. Factor VII polymorphism is

responsible for great portion of this variability. (14) Although the ambient temperature could be

another factor that would influence the response

to Warfarin, (15) the complete understanding of

the variability in responsiveness is not fully

elucidated. The high ambient temperature, where

our patients live, could be an implicated factor

in the development of excessive anticoagulation.

Intramural hematomas of the gastrointestinal

tract might have diverse presentations. Gastric

mural hematomas are very rare complications of

anticoagulant therapy that could present mainly

with upper abdominal pain and vomiting that

could be blood stained. (16,17) Jaundice and acute

pancreatitis have been reported as complications

of duodenal hematomas. (12,18-21) Intestinal

obstruction was the main presentation for

lesions occurring in the small intestine. (22-24)

Occasionally, these lesions could present with

bleeding from an orifice. (25,26) Acute abdominal

pain is not an uncommon presentation for the

Non-traumatic intramural hematoma. (27) All our

patients had acute abdominal manifestations on

presentation. Complete history taking remains

of paramount importance to the algorithm of

clinical diagnosis in patients with abdominal

pain. INR and bleeding profile would give direct

clue to the diagnosis. As a remarkably elevated

INR has been reported very often in the

published case reports, it is of benefit to screen

in anticoagulated patients presenting with

abdominal symptoms. (25,28) Despite that

endoscopy would allow direct visualization of

the mucosa; (28) the accessibility to the small

intestine is limited in most of the emergency

settings. CT scan would provide more discrete

information about the extent of the lesion and

the mural thickness. (29) Abdominal U/S usually

shows mural thickening of the affected region. (28). Bacterial translocation from the dysfun-

ctional mucosa due to the accumulation of blood

is related to the increase in the WBCs and septic

complications. (30) The increase in WBCs could

be as high as over 20 thousands, which could

hinder the diagnosis. The high WBCs count

could be related to the more extensive

hematomas. (30) Lethal out-comes occur if

extensive hematomas are associated with the

presence of significant comorbidities. (30)

Currently, the standard management for these

cases is the correction of the excessive

anticoagulation with resuscitation. (11, 12, 22, 28, 29,

31, 32). The necessity for surgical intervention in

these cases has diminished significantly and its

role now is reserved to those cases that do not

improve on resuscitative measures. The extent

of the mural hematoma, along with the WBCs

could predict those who might require surgical

intervention, (30) however, response to

resuscitation is governing factor for the decision

making. The importance of early recognition of

the condition is that surgical exploration could

be unnecessarily attempted with deleterious

outcomes. (33-35) Prevention of excessive

anticoagulation is a primary goal in patients who

require long term anticoagulant therapy. This

could be accomplished with tailored monitoring

approach.

Conclusions

Accurate detailed history taking is always

emphasized during assessment of acute abdo-

minal pain. Coagulation profile should be consi-

dered as a routine part of the evaluation of

patients on anticoagulant therapy who present to

with abdominal pain. The diagnosis of the

spontaneous intramural hematoma is achieved

through detailed history and clinical exami-

nation, abdominal CT scan along with the

deranged coagulation profile. The decision on

how to manage the clinical condition is

governed by the response to the conservative

management, which is the primary therapeutic

strategy for this clinical condition.

Acknowledgment

The authors would like to acknowledge the sincere efforts

of Prof. Khaled Katri FRCS, MD, professor of general

surgery, HBP surgery unit, department of surgery, faculty

of medicine, university of Alexandria, Egypt; in revising

the manuscript.

Figure 1.a: Abdominal ultrasound showing hypoechoeic

increased gastric mural thickness.

Figure 1.b: upper GI endoscopy demonstrating the swollen

congested mucosa of the stomach.

Figure 2: Abdominal CT scan showing increased thickness

of the proximal jejunum

Figure 3.a: Mural hematoma of the ileum.

Figure 3.b: mural hematoma of the ileum

Figure 1.c: Abdominal CT scan with thickened antrum of

the stomach.

Table1: Summary of cases presentations and management.

Case 1 Case 2 Case 3

Gender Male Male Male

Age 40 62 60

Presenting symptoms Upper abdominal pain Abdominal pain Diffuse abdominal pain

Associated symptoms Vomiting, constipation,

and fever

Constipation -

Indications for

anticoagulant

Ischemic heart disease Ischemic heart disease Ischemic heart disease

INR 17 10.5 9

Diagnostic tool U/S”, upper GI

endoscopy, CT

CT CT

Region Antrum of the stomach

and first part of

duodenum

Proximal jejunum Terminal ileum

Management FFP*, Vit K**, I.V***

Fluids

FFP, Vit K, I.V Fluids FFP, Vit K, I.V Fluids

*FFP: Fresh frozen plasma, **Vit K: Vitamin K, ***I.V: intravenous

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Original Article

Comparative Study of The Efficacy of Rifaximin in Comparison with Lactulose for

the Treatment of Hepatic Encephalopathy

Fathia E Asal1, Ahmed Khalid Tawfik1, Raafat A Salah1, Shimaa MMansour1, Amal H Abed-El Hameed2

Tropical Medicine & Infectious Diseases1, Clinical Pathology2departments, Faculty of Medicine, Tanta University, Egypt

ABSTRACT

Hepatic encephalopathy (HE) represents a broad spectrum of neuropsychological dysfunction. In cirrhotic patients, HE may

be clinically overt or minimal.Overt HE (OHE) may be further divided into episodic or persistent. Both episodic and

persistent HE may beinduced by a precipitating event or may occur apparently spontaneously.The most widely accepted

theory of the pathogenesis of HE is that nitrogenous substances derived from the gut adversely affect the cerebral function.

The main substance implicated is ammonia. Rifaximin is a derivative of rifamycin, which acts by inhibiting bacterial

ribonucleic acid (RNA) synthesis. Rifaximin is virtually unabsorbed after oral administration and exhibits broad spectrum

antimicrobial activity against both aerobic and anaerobic gram-positive and gram-negative microorganisms within the

gastrointestinal tract. Patients and Methods :The study populationincluded50 patients were diagnosed to have signsof the

first to third degree HE and classified into two groups:Group I: included 25 patients who had hepatic encephalopathy and

were treated with rifaximin (1200 mg daily divided into 3doses for 7 days).Group II: included 25 patients who had hepatic

encephalopathy and were treated with lactulose (90ml daily divided into 3doses for 7 days). Results :The results showed that

the both rifaximin and lactulose can decrease ammonia level by different mechanisms, but the response of patients regarding

improvement of symptoms of HE was more and rapid in rifaximin group than lactulose group. Conclusion :Patients treated

with rifaximin required shorter duration of hospitalization compared to lactulose, also rifaximin was better tolerated than

other pharmacologic treatments.

Introduction

Hepatic encephalopathy is a spectrum of neuro-

psychiatric abnormalities in patients with liver

dysfunction after exclusion of other known

brain disease(1). A number of theories have been

proposed to explain the development of hepatic

encephalopathy in patients with cirrhosis.Some

investigators contend that hepatic encepha-

lopathy is a disorder of astrocyte function. Ast-

rocytes account for about one third of cortical

volume. They play a key role in the regulation

of the blood-brain barrier. They are involved in

maintaining electrolyte homeostasis and in prov-

iding nutrients and neurotransmitter precursors

to neurons. They also play a role in the detox-

ification of a number of chemicals, including

ammonia(2). The current management of HE

requires prompt recognition of the disease state

particularly in its earliest or mildest stages,

careful identification and amelioration of precip-

itating factors, and judicious prescribing of a

therapeutic arsenal that is often multifaceted and

must be tailored to each patient(3,4).In the United

States, Non-absorbable dissaccharide lactulose

remains the mainstay of therapy for the majority

of patients with episodic or mild persistent HE.

Non-absorbable antibiotics particularly rifax-

imin with or without DS have become the

standard-of-care treatment for patients with

recurrent or persistent HE, after removal of und-

erlying precipitating factors where possible (3,4).

Rifaximin is a novel antimicrobial agent with a

wide spectrum of activity that has shown

promise as an alternative antimicrobial treat-

ment option for HE. It is a derivative of rifam-

ycin, which acts by inhibiting bacterial ribbon-

ucleic acid (RNA) synthesis. Rifaximin is virtu-

ally unabsorbed after oral administration (5).

Rifaximin is a gut–selective antibiotic with

negligible systemic absorption and broad–

spectrum activity in vitro against both gram–

positive and gram–negative pathogens.

Rifaximin has a similar tolerability profile to

that of placebo. Rifaximin tablets 200 mg are

approved in over 30 countries worldwide (6). In

2004, rifaximin was approved by food and drug

administration (FDA) in the United States for

the treatment of travelers' diarrhea. In 2005, it

received orphan drug status as a treatment for

hepatic encephalopathy (7). In March 2010,

rifaximin was approved by the FDA to reduce

recurrence of hepatic encephalopathy (7). During

the past decade, several European studies have

proved the efficacy of rifaximin for the

treatment of HE in Caucasian patients (8).

Aim Of The Work

The purpose of this study is to evaluate the

efficacy of rifaximin in comparison to lactulose

for the treatment of hepatic encephalopathy

Patients and Methods

This study was carried out at Tropical medicine

department (Tanta University Hospital). The

study population included 50 patients who were

diagnosed to have signs of the first to third

degree HE, according to the criteria suggested

by Conn HO, et al. (9), and classified into two

groups: Group I: included 25 patients who had

hepatic encephalopathy and were treated with

rifaximin capsules 1200 mg daily divided into

3doses for 7 days. According to the dose

suggested by Festi D., et al (10) & Williams R ,et

al (11). Group II: included 25 patients who had

hepatic encephalopathy and were treated with

lactulose syrup (laxolac) 90ml daily divided into

3doses for 7 days.According to the dose sugg-

ested by Cash WJ, et al (12). No other measures

were done for the patients. Inclusion criteria: 1-

All patients affected by decompensated liver

cirrhosis. 2- Patients showing signs of the first

to third degree HE. Exclusion criteria: 1- The

presence of a major neuropsychiatric illness. 2-

Presence of intestinal obstruction or inflamm-

atory bowel disease. 3-Hypersensitivity to rifa-

mycin or disaccharides. 4-A serum creatinine

level > twice normal. 5-Patients with correctable

cause of HE as gasterointestinal bleeding. Infor-

med consent was obtained from all patients

enrolled in the study. All patients were

subjected to: A) Full history taking. B) Full

clinical examination; including: 1-General exa-

mination: to detect the grade of mental state

according to West Haven criteria (9) 2-Local

Abdominal examination: assessment of liver and

spleen state, detection of ascites. C) Laboratory

investigations including :Liver function tests,

Coagulation profile, renal functions, Serum

electrolytes, ascetic fluid analysis. Ammonia

level: blood samples were collected from a

stasis-free vein (that is, without using a

tourniquet and taking care not to cause

turbulence or hemolysis) and immediately

transported on ice to the laboratory to be

analyzed within 20 min. Norm-al ammonia

level was considered < 75 µmol/L (14). D)

Imaging evaluation: 1-Chest x-ray to exclude

chest infection. 2- Pelvi-abdominal ultrasono-

graphy. Conn score (grade of mental state) &

grade of flapping tremor & blood ammonia level

were done before beginning the treatment in the

first day and on days 3,5,7 of the trial period (13).

Results

The results showed that the both rifaximin and

lactulose can decrease ammonia level by

different mechanisms, but the response of

patients regarding improvement of symptoms of

HE was more and rapid in rifaximin group than

lactulose group. After the end of treatment 21

patients (84%) in rifaximin group became in

grade 0 but only 8 patients (32%) in lactulose

group became in grade 0. These results showed

that the response of patients regarding

improvement of mental status was more and

rapid in rifaximin group than lactulose group.

Table (1): Shows liver function tests, platelets count &base line ammonia (NH3) in group I & group II

Lab. Investigation Group I

Rifaximin (n=25)

Group II

Lactulose (n=25)

Pvalue

S e r u m a l b u m i n ( g / d l ) 2.788+0.3407 2.668+0.3326 0.214

T o t a l b i l i r u b i n ( m g / d l ) 3.844+5.6092 3.420+5.0661 0.780

P l a t e l e t s ( x 1 0 3 / µ l ) 89.64+37.443 99.68+88.464 0.604

H a e m o g l o b i n ( m g / d l ) 9.852+1.1399 9.436+0.7064 0.127

C r e a t i n i n e ( m g / d l ) 1.148+0.2275 1.400+1.0145 0.231

N H 3 ( µ m o l / l ) 182.4+36.436 162.2±60.4 0.279

Table (2): Shows Similar attacks of HE

Similar attacks of HE

GROUP I

Rifaximin (n=25)

GROUP II

lactulose (n=25) P value

no. of patients % no. of patients %

Present 20 80 21 84 0.713

Absent 5 20 4 16

Table (3): mean level of blood ammonia (µmol/l) pre & post treatment in both groups.

P r e - t r e a t m e n t Post treatment p v a l u e $

GroupIRifaximin (n=25) 182.4+36.436 120.8+44.8 <0.001*

Group IILactulose (n=25) 162.2±60.4 118.7±50.7 <0.001*

P v a l u e $ $ 0 . 2 7 9 0 . 8 8 1

P < 0.001** highly significant test.P value $pre treatmentVS post treatmentin the same group.

P value $$pre treatment and post treatment in group I VS group II.

Table (4): Shows changes in the grading of mental status in both groups in 1st&3rd&5th&7th days of treatment in both groups

Grade of

Mental status

Group I

Rifaximin(n=25)

Group II

Lactulose(n=25)

0 1 2 3 0 1 2 3

No of cases in the 1st day (before

treatment)

0

0%

6

24%

11

44%

8

32%

0

0%

3

12%

15

60%

7

28%

No of cases in the 3rd day

12

48%

10

40%

3

12%

0

0%

1

4%

10

40%

11

44%

3

12%

No of cases in the 5th day

19

76%

5

20%

1

4%

0

0%

1

4%

16

64%

7

28%

1

4%

N o o f c a s e s i n t h e 7 t h d a y 2 1

8 4 %

3

1 2 %

1

4 %

0

0 %

8

32%

1 4

5 6 %

2

8 %

1

4 %

Table (5): Shows changes in the grading of flapping tremors in both groups in 1st&3rd&5th&7th days of treatment

GroupI

Rifaximin(n=25)

GroupII

Lactulose(n=25)

0 1 2 3 4 0 1 2 3 4

No of cases in the 1st

day(before treatment)

0

0%

0

0%

9

36%

15

60%

1

4%

0

0%

1

4%

6

24%

16

64%

2

8%

No of cases in the

3rd day

9

36%

6

24%

10

40%

0

0%

0

0%

0

0%

3

12%

10

40%

12

48%

0

0%

No of cases in the

5th day

15

60%

5

20%

5

20%

0

0%

0

0%

1

4%

7

28%

12

48%

4

16%

1

4%

No of cases in the 7 t h day 1 8

7 2 %

4

1 6 %

3

1 2 %

0

0 %

0

0 %

3

1 2 %

1 2

4 8 %

7

2 8 %

2

8 %

1

4 %

Figure (1) mental status at the 1st day before beginning of treatment in both groups

Figure (2) mental status at the 7th day of treatment in both groups

Discussion

Hepatic encephalopathy (HE) is a metabolically

induced, potentially reversible, functional distu-

rbance of the brain. While HE may be a com-

plication of acute or chronic liver disease, it is

most commonly associated with cirrhosis (15).

Depending on its cause, HE can be categorized

as either type A, which occurs in patients with

acute liver failure; type B, which occurs in

patients with bypass shunts; or type C, which

occurs in patients with chronic liver disease(16).

In this study the cause of the liver disease was

HCV infection with percentage 100% in both

groups. In Egypt, the main cause of decom-

pensated liver disease is HCV infection, because

Egypt developed the world's highest rates of

HCV infection over a short period of time. The

majority of infections among individuals aged

30 years and older can be explained by

parenteral anti-Schistosomal therapy (PAT) and

other iatrogenic exposures. Since Schisto-

somiasis was a greater problem in rural regions,

these populations were more affected by the

PAT campaign, and consequently, HCV

transmission(17). Regarding laboratory investti-

gations (albumin level, Total bilirubin, Platelets

count, Haemoglobin level, and Serum creatinine

level) there was no significant difference

between both groups. In this study we found that

protein overload was the precipitating factor in

9/25(36%) patients in group I & 15/25(60%)

patients in group II. Infection like (spontaneous

bacterial peritonitis, chest infection, urinary tract

infection or gastroenteritis) was present in 9/25

patients in group I & 3/25 patients in group II.

Constipation was present in 3/25 patients in

group I & 2/25 patients in group II. Four from

25 patients in group I & 5/25 patients in group II

were presented with unknown precipitating

factor of hepatic encephalopathy, which means

that protein overload is one of the common

precipitating factor of HE. These data were in

agreement with Cash WJ., et al (12), Chung Rt et

al., 18)and Sundaram V., et al(19), who reported

that consumption of large amounts of protein

was one of the common precipitating factor of

HE as it led to increase ammonia level in blood

which is the most widely accepted theory of the

pathogenesis of H(20). Also Yong-Han Paik et al,

(13)reported in their study that 8/32(25%)

patients presented by HE which was precipitated

by protein over load. But Antoni Mas et al.

(21)reported in their study of comparison

between rifaximin and lactitol that only 1

(2%)in rifaximin group &1(1.9%) in lactitol

group were precipitated by protein overload .

Heyman JK et al. (22) showed that protein

restriction (0–40g protein/day) in all patients

with cirrhosis who developed encephalopathy

has no impact on encephalopathy grade and that

it may even worsen their nutritional status. The

consensus of opinion nowadays is that protein

restriction is avoided in all patients, and protein

be maintained between 1.2 and 1.5g of proteins

per kg of body weight per day. but in severely

protein intolerant patients, particularly in

patients in grades III-IV HE, protein may be

reduced for short periods of time (23). In this

study 4/25 (16%) patients in group I and 5/25

(5%) patients in group II were presented with

unknown precipitating factor of hepatic encep-

halopathy. That was similar to the findings of

Cash WJ. ,et al(12) who found that in some cases

, no clear cause for an attack can be found. Also

Yong-Han Paik et al, (13) concluded that

7(21.9%) patients in rifaximin group &

5(22.9%) patients in lactulose group were

presented by HE of unknown precipitating

factor. So, many patients suffer from episodic

HE, with episodes being either precipitated or

spontaneous(24). There was no significant

statistical difference between both groups as

regard presence of similar attacks. Hepatic

encephalopathy was presented for the first time

in 5/25(20%) in group I Vs 4/25(16%) in group

II. With no significant difference between both

groups. this finding means that the most of

patients presented by recurrent attacks of HE.

This meets the finding of Bajaj JS et al 2010(24)

who found that many patients suffer from

intermittent or “episodic HE,”. Episodes of HE

may be isolated events, but more commonly

they are recurrent, with patients having seem-

ingly normal cognitive functioning between

episodes. Regarding blood ammonia level,there

was significant statistical difference pretreatm-

ent and post treatment in group I(rifaximin)

with p value <0.001*. also there was significant

statistical difference pretreatment and post

treatment in group II (lactulose) with p value

<0.001*.but in rifaximin group the decrease in

ammonia level was faster than in lactulose

group as shown in the results figure. These

results were similar to results reported by Bucci

and Palmieri(25) who reported in their study that

ammonia concentrations decreased in both

groups. AlsoYong Han Paik et al 2005(13) found

that ammonia concentrations decreased in both

groups. Mas A, et al(15)in their study that comp-

ared between rifaximin and lactitol found that

ammonia concentrations decreased for rifaximin

superior to lactitol. This means that all patients

in both groups showed increase in ammonia

level before beginning of both treatment then

ammonia level decreases after treatment by

rifaximin & lactulose this support that increase

ammonia level in blood is the most widely

accepted theory of the pathogenesis of HE, and

it is thought to be a central player in the

development of HE (20). Both rifaximin and

lactulose can decrease ammonia level by

different mechanisms but rifaximin is more

rapid than lactulose. This can be explained by

the fact that rifaximin acts by inhibiting

bacterial ribonucleic acid (RNA) synthesis and

it is unabsorbed after oral administration and

exhibits broad spectrum antimicrobial activity

against both aerobic and anaerobic gram-

positive and gram-negative microorganisms

within the gastrointestinal trac(26), So rifaximin

lowers the bacterial load 100–1000-folds(27). As

regard grading of mental status, the result

showed that after 3 days, 12 patients became in

grade 0 in the group treated with rifaximin vs 1

patients only in the group treated with lactulose.

After the end of treatment 21 patients (84%) in

rifaximin group became in grade 0 but only 8

patients (32%) in lactulose group became in

grade 0. These results showed that the response

of patients regarding improvement of mental

status was more & rapid in rifaximin group than

lactulose. These data were in agreement with

Neff G et al(28) who reported in their study that

the response to rifaximin ranged from 80% to

90%, with a faster onset of action by up to 3

days. Also Loguercio et al(29) documented faster

improvement in hepatic encephalopathy and

higher percentages of normalized mental state in

patients treated with rifaximin. Alsothese

resultsare similar to that reported by Bucci and

Palmieri(25) who found in their study that the

improvement in the rifaximin group was

significantly superior to that of the lactulose

group as regard mental status. Mas A, et al(15)

reported in their study that improvement in

grading of mental status for rifaximin was

superior to lactitol. Two recent trials confirmed

that it is a useful alternative to disaccharides in

patients with grades I–III HE in improving

mental status(10,11). Also HinaYasmeen et al(30)

reported that rifaximin was effective in im-

proving behavioral, laboratory, mental status

and intellectual abnormalitiesassociated with

hepatic encephalopathy and patients treated with

rifaximin also required shorter duration of hosp-

italization ,compared with lactulose. It may be

due to rifaximin is unabsorbed by the gut,

thereby allowing the antibiotic to reach high

concentrations in the intestinal tract and to

remain in the feces in its active form. Due to

these properties (high antibacterial activity with

null absorption), rifaximin decreases ammonia

plasma levels and improves the symptoms relat-

ed to HE rapidly(31). Regarding the grading of

flapping tremors, the results showed that after 3

days of treatment with rifaximin 9 patients had

no flapping tremors and this number increased

to 18 patients after the end of treatment. But as

regard lactulose group no patients became in

grade 0 flapping tremors after 3 days of treat-

ment and only 3 patients had no flapping

tremors after end of treatment. These results

showed that the response of patients regarding

improvement of flapping tremors was more &

rapid in rifaximin group than lactulose

group.These data were in agreement with Neff

G et al(28)who reported in their study that the

response to rifaximin ranged from 80% to 90%,

with a faster onset of action by up to 3 days.

Loguercio et al (29) documented in their study

faster improve-ement of asterixis in patients

treated with rifaximin.As 80% to 90% of

rifaximin taken by mouth concentrates in the

gut. This accum-ulation enh-ances the

effectiveness of rifaximin in the treatment of

intestinal infections. Due to its distribution in

the gut, rifaximin is excreted almost entirely in

the feces and its half-life is approximately six

hours. so its action more rapid than lactulose. As

regard side effects in this study only one

patients (4%) developed mild abdominal pain

after treatment with rifaximin, 5 patients (20%)

developed abdominal discomfort and flatulence

& 2 patients (8%) developed nausea after

treatment with lactulose so rifaximin was better

tolerated than cathartics and these data were in

agreement with David C Wolf, (32)and Iadevaia

et al,(33) and Hina Yasmeen et al, who concluded

that rifaximin is generally well tolerated, and

has a better profile in terms of side effectswhen

compared with other systemic antibiotics &

cathartics and the adverse effects of rifaximin

were mostly minor gastro-intestinal complaints

and this is because rifaximin is a synthetic

antibiotic that has a very low rate of systemic

absorption (0.4%), and has an excellent safety

profile than neomycin and the non- absorbable

disaccharides(34).

Conclusion: So rifaximin can be used in the

treatment of episodic HE alone or in

combination with lactulose. Competing

Interests: None

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Original Article:

Effect of Rebamipide on Portal Hypertensive Gastropathy and Proliferating Cell

Nuclear Antigen in Patients with Liver Cirrhosis

Mohamed Y. El-Hasafi(a), Suzan M. Helal(b), Marwa A. Madkour(c), Ahmed S. Saher(a) (a) Internal Medicine Department,

Faculty of Medicine, University of Alexandria (b) Pathology Department, Faculty of Medicine, University of Alexandria (c) Clinical and Experimental Internal Medicine Department, Medical Research Institute, University of Alexandria

ABSTRACT

Portal hypertensive gastropathy (PHG) is a complication of advanced liver cirrhosis and portal hypertension, in the form of

mucosal and submucosal vascular ectasia without significant inflammatory changes. It is a frequent cause of acute and

chronic upper gastrointestinal hemorrhage. Another sequel of PHG is increased susceptibility of gastric mucosa to injury by

noxious factors, with impaired mucosal healing response. Rebamipide is a gastroprotective drug used for mucosal protection

and promoting healing of gastroduodenal ulcers. The proliferating cell nuclear antigen (PCNA) is an antigen expressed in the

nuclei of actively dividing cells, and is considered to be a useful tool for assessing the proliferative activity of gastric mucosa

during the ulcer healing process. Aim: To evaluate the effect of Rebamipide on PHG and on the gastric mucosal expression

of PCNA in patients with liver cirrhosis and portal hypertension. Methods: 60 patients with liver cirrhosis and endoscopic

evidence of PHG were divided into 3 groups: Group І patients with PHG and non-ulcerated gastric mucosa receiving

Rebamipide for 3 months, Group П patients with PHG and gastric mucosal ulcers receiving Rebamipide for 3 months and

Group Ш patients with PHG and intact non-ulcerated gastric mucosa receiving Placebo for 3 months. Clinical and laboratory

assessment, Doppler study of the portal vein, endoscopic examination of the stomach, histopathologic and

immunohistochemical assessment of mucosal biopsies for PCNA were all performed before and after drug therapy in all 3

groups. Results: Clinical and laboratory assessment and Doppler study of the portal vein showed no significant difference

after drug administration in all patients. The platelet count showed a significant drop after drug administration in patients’

Group П only, while in Groups І and Ш it showed no similar drop. Endoscopically, there was also no significant change in

PHG score after drug therapy in any of the three groups. In Group II, however, the gastric ulcer score showed significant

improvement after drug therapy (p<0.001). There was no significant difference in the histopathologic stomach score of PHG

after drug therapy in all groups. However, there was a significant rise in PCNA score of the stomach in Groups І and П

(Rebamipide groups, p<0.001), while there was no similar change in Group Ш (Placebo group). Conclusions: Rebamipide

has no effect on the grade of PHG. However, it significantly increases the healing capacity of the ulcerated as well as non-

ulcerated gastric mucosa in patients with PHG, as proved by the significant rise in the gastric mucosal expression of PCNA

after Rebamipide therapy. Further research is needed to clarify the effect of Rebamipide on platelet count in cirrhotic

patients.

Introduction

Liver cirrhosis is a consequence of chronic liver

disease characterized by loss of architecture

with replacement of liver tissue by fibrosis and

regenerative nodules, ultimately leading to loss

of liver function. Portal hypertension is an

increase in the blood pressure within the portal

venous system. Normal portal pressure is

generally defined between 5 and 10 mm Hg.

However, once the portal pressure rises to 12

mm Hg or greater, complications can arise.

These include ascites, spontaneous bacterial

peritonitis, hepatorenal syndrome, hepatopulmo-

nary syndrome, portopulmonary hypertension

and bleeding from ruptured esophageal varices. (1) Portal hypertensive gastropathy (PHG) has

emerged during the last two decades as a clin-

ical and pathological entity among the compli-

cations of advanced liver cirrhosis, in the form

of mucosal and submucosal vascular ectasia

without significant inflammatory changes.(2) The

pathogenesis of portal hypertensive gastropathy

has not been completely cleared up. Clinical and

experimental studies suggest that chronic inc-

rease of pressure in the portal vascular system is

the main prerequisite for this condition. How-

ever, the involvement of humoral and autocrinal

factors has been recently implicated in its path-

ogenesis, as nitric oxide, tumor necrosis factor

alpha and several growth factors.(3,4) The diag-

nosis of PHG is fundamentally endoscopic. The

classic mosaic-like pattern (MLP) of the gastric

mucosa consists of multiple rectangular or

diamond-shaped erythematous areas, outlined

by a delicate white or yellowish network. Red

marks in the form of red point lesions, cherry

red spots or black-brown spots can also be

found. These changes are prominent in the

gastric body and cardia, but can be present in all

parts of the stomach.(5) In a classification

proposed by the North Italian Endoscopic Club

(NIEC), PHG was categorized into mild and

severe grades. Mild PHG is diagnosed when

only mosaic-like pattern (MLP) lesions are

found. MLP can be mild, moderate or severe.

Severe PHG is diagnosed when any of the red

marks, with or without MLP, are found.(6) The

incidence of PHG fluctuates between 30% and

45% among patients with cirrhosis undergoing

endoscopy.(7) In fact, PHG is a frequent cause of

acute and chronic upper gastrointestinal (GI)

hemorrhage: 10-20% of bleeding episodes occu-

rring in cirrhotic patients are caused by this

condition, with high incidence of morbidity and

mortality.(8) Another sequel of PHG is increased

susceptibility of gastric mucosa to injury by

noxious factors as non-steroidal anti-inflamm-

atory drugs, alcohol and helicobacter pylori

infection. Experimental evidence has also demo-

nstrated an impaired gastric mucosal healing

response to such injuries.(9) Excessive nitric

oxide production, in addition to increased gene-

ration of reactive oxygen species and lipid

peroxidase in the portal hypertensive gastric

mucosa are the main mechanisms incriminated

for these phenomena.(10) At the moment, no

single line of pharmacotherapy is able to prevent

or cure PHG. Beta-blockers seem to be an

effective treatment for this condition, which act

by decreasing portal pressure, resulting in decre-

ased gastric blood flow and reduced bleeding

from gastric mucosa.(11) Rebamipide is a gastro-

protective drug used for mucosal protection,

healing of gastroduodenal ulcers, and treatment

of gastritis. It works by enhancing mucosal

defense, scavenging free radicals and stimuli-

ting prostaglandin generation in gastric mucosa,

which improves not only the speed but also the

quality of ulcer healing. In addition, it protects

the gastric mucosa against acute injury caused

by various noxious and ulcerogenic factors.

After being tested in several experimental and

clinical trials it is now approved in Japan for

therapeutic use in patients with gastric ulcers

and acute gastritis.(12) A recent study by Kinjo et

al. demonstrated a new application of Rebami-

pide in PHG, by achieving a significant decrease

in lipid peroxidase and nitrotyrosine (which are

both indicators of oxidative stress) in portal

hypertensive rats. In addition, this drug succeed-

ded in restoring the integrity of mucosal

enzymes and proliferating cell nuclear antigen

(which are both responsible for gastric mucosal

healing) back to normal state.(10) The prolif-

erating cell nuclear antigen (PCNA) is an

antigen expressed in the nuclei of cells during

the DNA synthesis phase of cell cycle. It was

also proved to be an important mediator of DNA

repair in response to damage.(13) Its particular

role in the process of gastric ulcer healing was

identified and proved in more than one study,

which lead some authors to consider the

immunohistochemical staining of PCNA in the

gastric mucosa to be a useful tool for assessing

the proliferative activity of gastric mucosa

during the ulcer healing process. (14-16) Intere-

stingly, a decreased expression of PCNA in the

gastric mucosa of portal hypertensive rats was

recently described by two studies and hence was

added to the possible mechanisms of impaired

healing power in portal hypertensive gastric

mucosa.(9,17) This finding was further confirmed

by Kinjo et al. who demonstrated an improved

PCNA labeling index back to normal levels and

enhanced ulcer healing in the portal hypert-

ensive gastric mucosa of rats after the use of

Rebamipide.(10) However, whether Rebamipide

can achieve similar results among human

subjects with PHG, this needed further eluci-

dation. Therefore, the aim of our study was to

evaluate the effect of Rebamipide on portal

hypertensive gastropathy (endoscopically and

histopathologically ) and on the gastric mucosal

expression of proliferating cell nuclear antigen

(PCNA) in patients with liver cirrhosis and

portal hypertension.

Patients and Methods

This prospective interventional, single-blind,

placebo-controlled study was carried out on 60

patients with liver cirrhosis and portal hyper-

tension (based on clinical, laboratory and

ultrasonographic findings) and with endoscopic

evidence of portal hypertensive gastropathy

(PHG). Patients were obtained from Hepatology

and Gastroenterology Unit of the Clinical and

Experimental Internal Medicine Department,

Medical Research Institute, Alexandria Univer-

sity during the period from January 2010 till

March 2011. The patients were divided into

three groups: Group І included 20 patients with

PHG and intact non-ulcerated gastric mucosa

receiving Rebamipide 100 mg tablets three times

daily (tds) before meals for a period of three

months, Group П included 20 patients with PHG

and gastric mucosal ulcers receiving Rebamipide

100 mg tablets tds before meals for a period of

three months and Group Ш included 20 patients

with PHG and intact non-ulcerated gastric mucosa

receiving Placebo tablets tds before meals for a

period of three months. Exclusion criteria of the

patients included those with a history of

hypersensitivity to any ingredient of the drug,

past history of sclerotherapy or band ligation of

esophagogastric varices (due to their potential

effect on the grade of PHG,(18,19) patients having

grade 2 or 3 esophageal varices with risk signs

of bleeding or with active variceal hemorrhage

(due to their need for immediate endoscopic

intervention by sclerotherapy or band ligation),

patients using β-blockers, mononitrates, proton

pump inhibitors or H2- blockers which may

affect the grade of gastropathy or ulcer healing (20), patients with gastric mucosal infection by

helicobacter pylori(21) and finally patients with

hepatocellular carcinoma. Therapeutic regimen:

After obtaining their written consent, patient's

Group І and П were instructed to blindly take

Rebamipide (Mucosta®; Otsuka pharmaceutical,

Tokushima, Japan) 100 mg tablets tds before

meals for a period of three months, while

patient's Group Ш was instructed to take place-

bo for a period of three months. Clinical and

laboratory assessment: All patients were

subjected to detailed history taking, clinical exa-

mination and Child-Pugh-Turcotte scoring to

assess the severity of liver disease before and

three months after continuous drug therapy.(22)

Routine laboratory investigations included

complete blood picture, blood urea, serum creat-

inine and fasting blood sugar. Liver profile was

assessed in the form of serum albumin, biliru-

bin, serum transaminases and prothrombin time

and activity. Also, baseline serum HBs antigen,

HCV antibodies (3rdgeneration ELISA) and

ocult blood in stool were tested.(23, 24) Abd-

ominal ultrasound was performed at the beg-

inning of the study using a Siemens G50

sonograph, equipped with a 3.5 mHz sector

transducer scanner to verify diagnosis of cirrh-

osis, diameter and patency of portal vein,

splenomegaly as well as the presence and grade

of ascites. Moreover, Doppler ultrasound of

portal circulation was performed before and

three months after drug therapy for assessing the

portal vein congestion index, which was

calculated as the ratio of cross-sectional area of

portal vein (cm2) and mean flow velocity

(cm/sec).(25) Upper gastrointestinal tract endo-

scopy: The procedure was performed for all

patients using a video-endoscopy system (Oly-

mpus GIF-240 and Olympus GIF-260) in two

sessions: before and three months after drug

therapy. In each of the two sessions, the follow-

ing was assessed: - The endoscopic grade

of portal hypertensive gastropathy, which was

classified according to the North Italian Endos-

copic Club (NIEC) classification into: mild

PHG (mosaic pattern of variable degrees but

with no red signs) and severe PHG (any degree

of mosaic pattern in addition to any of the three

red signs: red point lesions, cherry red spots and

black-brown spots).(6) - The presence or absence

of esophageal or gastric varices, their grade and

risk signs. - The presence or absence of gastric

mucosal ulceration and their stage according to

Sakita-Miwa classification as follows.(26) (table1)

Table (1): Sakita-Miwa classification of gastric mucosal ulceration(26)

Stages Manifestation

Active stage A1

(6 points)

The surrounding mucosa is edematously swollen and no regenerating epithelium is

seen endoscopically

Active stage A2

(5 points)

The surrounding edema has decreased, the ulcer margin is clear, and a slight

amount of regenerating epithelium is seen in the ulcer margin. A red halo in the

marginal zone and a white slough circle in the ulcer margin are frequently seen.

Usually, converging mucosal folds can be followed right up to the ulcer margin

Healing stage H1

(4 points)

The white coating is becoming thin and the regenerating epithelium is extending

into the ulcer base. The gradient between the ulcer margin and the ulcer floor is

becoming flat. The ulcer crater is still evident and the margin of the ulcer is sharp.

The diameter of the mucosal defect is about one-half to two-thirds that of A1

Healing stage H2

(3 points)

The defect is smaller than in H1 and the regenerating epithelium covers most of the

ulcer floor. The area of white coating is about a quarter to one-third that of A1

Scarring stage S1

(2 points)

The regenerating epithelium completely covers the floor of ulcer. The white coating

has disappeared. Initially, the regenerating region is markedly red. Upon close

observation, many capillaries can be seen. This is called “red scar”

Scarring stage S2

(1 point)

In several months to a few years, the redness is reduced to the color of the

surrounding mucosa. This is called “white scar”

Gastric mucosal infection with helicobacter

pylori was screened among all patients by rapid

urease test kit, using a gastric mucosal biopsy,

and positive cases were excluded . (21) - Two

gastric mucosal biopsies were taken for histop-

athologic and immunohistochemical study from

gastric fundus and antrum before and three

months after drug therapy.

Histopathological examination of gastric

mucosal biopsies

The two biopsies from gastric mucosa (fundus

and antrum) obtained from each of the patients

before and three months after Rebamipide or

placebo intake were stained by Hematoxylin and

Eosin (H&E) for identification of inflammatory

infiltrate, whereas Periodic Acid Schiff stain

(PAS) was used for better visualization of

mucosal capillaries.(27) Microscopic examination

of the gastric mucosa was carried out by using a

computer-assisted image analysis system. The

severity of portal hypertensive gastropathy was

graded by counting the number of dilated ectatic

capillaries in three consecutive high-power

fields (X400) of the superficial and deep lamina

propria, and the mean of these values was taken

for statistical calculation. The grade of portal

hypertensive gastropathy was scored for the

antrum and the fundus biopsy separately

according to Misra et al(28) as follows: - No PHG

(0): No ectatic capillaries in superficial or deep

lamina propria. - Mild (1): 1-3 dilated ectatic

capillaries in the deep lamina propria - Moderate

(2): More than 3 dilated ectatic capillaries in the

deep lamina propria - Severe (3): Dilated ectatic

capillaries even in the superficial lamina propria

The histopathologic whole stomach score and

grade of PHG were calculated as the sum of

antrum and fundus score for each patient, so that:

score = 0 (No PHG), score 1-2 (mild PHG),

score 3-4 (moderate PHG) and score 5-6 (severe

PHG).

Immunohistochemical study of gastric

mucosal biopsies

Fundus and antrum mucosal biopsies obtained

before and three months after drug therapy were

stained and studied immunohistochemically for

assessing cellular proliferation. Primary mono-

clonal antibodies against proliferating cell nu-

clear antigen (PCNA) as a cell proliferation

marker were used. The antibody was provided

by Lab Vision Corporation (Neo Markers,

Fremont, USA). PCNA-labeled cells and the

total number of cells in standardized rectangles

were counted at 200x magnification. The PCNA

-labeling index (PCNA score) was expressed as

the percentage of labeled cells per all counted

cells.(29)

Statistical analysis

Qualitative data were analyzed using number,

percent and 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 median, 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 quantita-

tive 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

The demographic data of patients showed male

predominance in gender, rural predominance in

residence and an age range from 36 to 71 years.

There was no statistically significant difference

between the three groups as regards sex, age or

residence. HCV was positive in 100% of

patients in Groups І and П, and in 95% of Group

Ш, while HBV was positive in 15% of patients

in Group І, 10% of Group П and 5% of Group

Ш. Occult blood in stool was positive in 55% of

patients in Group І, 35% of Group П and 25%

of Group Ш. Blood urea, serum creatinine and

fasting blood sugar values showed no

statistically significant difference from before to

after drug administration. There was a statist-

ically significant improvement of hemoglobin

level in Group І only, its mean value increased

from 8.76 g/dl before drug administration to

9.67 g/dl after drug administration. The platelet

count showed a statistically significant drop

from a mean value of 169.75x103/mm3 before

drug administration to a mean value of

165.05x103/mm3 after drug administration in

patients in Group П only. The platelet count in

Groups І and Ш, however, showed no

statistically significant difference (table 2).

Table (2): Comparison between the three studied groups according to complete blood picture laboratory results before and after

Rebamipide / Placebo

Group I Group II Group III

Hb

(g

/dl)

Before Rebamipide / placebo

Range 6.50 – 11.90 6.80 – 13.0 7.60 – 12.30

Mean ± SD 8.76 ± 2.11 9.99 ± 1.88 9.97 ± 1.43

Median 8.05 9.65 10.20

After Rebamipide / placebo

Range 7.20 – 12.60 7.30 – 113.0 8.0 – 12.60

Mean ± SD 9.67 ± 2.09 15.23 ± 23.08 10.30 ± 1.40

Median 9.05 10.40 10.40

pWX *<0.001 0.210 0.304

WB

C

)3

/mm

3(c

ou

nt

x1

0

Before Rebamipide / placebo

Range 3.60 – 9.30 2.60 – 8.40 2.30 – 9.60

Mean ± SD 5.56 ± 1.40 5.31 ± 1.83 6.08 ± 2.09

Median 5.60 5.15 6.40

After Rebamipide / placebo

Range 2.80 – 8.20 2.90 – 7.80 2.80 – 9.60

Mean ± SD 5.35 ± 1.29 5.61 ± 2.01 6.0 ± 1.91

Median 5.50 6.25 5.90

pt 0.310 0.328 0.783

Pla

tele

t

)3

/mm

3(c

ou

nt

x1

0

pide / placeboBefore Rebami

Range 63.0 – 345.0 88.0 – 397.0 67.0 – 314.0

Mean ± SD 121.90 ± 65.23 169.75 ± 79.82 153.35 ± 62.96

Median 97.0 145.50 138.50

After Rebamipide / placebo

Range 83.0 – 350.0 91.0 – 402.0 54.0 – 386.0

Mean ± SD 125.50 ± 62.31 165.05 ± 82.42 155.50 ± 72.34

Median 102.0 137.0 140.50

pWX 0.167 *0.038 0.925

Hb = Hemoglobin WBC = White blood cells tp:p value for Paired t-testWXp: p value for Wilcoxon signed ranks test between before and after

rebamipide/placebo in each group *: Statistically significant at p ≤ 0.05

Child-Pugh class C patients represented the

majority of all three groups before drug

administration; ranging from 50% to 80%, while

after drug administration it ranged from 45% to

85%. However, the results showed no

statistically significant difference in Child score

or class from before to after drug administration.

Also, the other liver profile parameters showed

no statistically significant difference from

before to after drug administration, (table 3). Table (3): Comparison between the three studied groups according to Child score and class before and after Rebamipide / Placebo

Group I Group II Group III

No % No % No %

Ch

ild

sco

re &

cla

ss Before Rebamipide / placebo

Class A (score 5 – 6) 0 0.0 0 0.0 0 0.0 Class B (score 7 – 9) 8 40.0 10 50.0 4 20.0 Class C (score 10 – 15) 12 60.0 10 50.0 16 80.0

After Rebamipide / placebo Class A (score 5 – 6) 0 0.0 0 0.0 0 0.0 Class B (score 7 – 9) 3 15.0 11 55.0 4 20.0 Class C (score 10 – 15) 17 85.0 9 45.0 16 80.0

P 0.063 1.000 1.000

p: p value for McNemar test *: Statistically significant at p ≤ 0.05

Doppler ultrasound of portal circulation reve-aled that the portal vein flow direction, portal vein diameter, cross sectional area, mean flow velocity and congestion index showed no statistically significant difference from before to

after drug administration. The congestion index of the portal vein ranged from 0.11 to 0.81 in all patients of the three groups before drug therapy, and ranged from 0.12 to 0.76 after drug therapy, (table 4).

Table (4): Comparison between the three studied groups according to Doppler study (PV diameter, cross sectional area, mean

velocity and congestion index of the portal vein) before and after Rebamipide / Placebo

Group I Group II Group III

PV

Dia

met

er (

mm

)

Before Rebamipide / placebo

Range 12.70 – 17.80 12.10 – 17.70 12.10 – 17.30

Mean ± SD 15.59 ± 1.82 14.41 ± 1.77 14.55 ± 1.52

Median 16.0 13.95 14.50

After Rebamipide / placebo

Range 12.80 – 17.70 12.30 – 18.10 12.40 – 17.0

Mean ± SD 16.56 ± 1.75 14.53 ± 1.83 14.46 ± 1.32

Median 15.85 14.25 14.30 tp 0.690 0.071 0.560

Cro

ss s

ecti

onal

are

a

(cm

2)

Before Rebamipide / placebo

Range 1.28 – 2.49 1.24 – 2.44 1.22 – 2.23

Mean ± SD 1.94 ± 0.44 1.67 ± 0.41 1.70 ± 0.32

Median 2.01 1.59 1.78

After Rebamipide / placebo

Range 1.31 – 2.56 1.28 – 2.60 1.28 – 2.19

Mean ± SD 1.93 ± 0.42 1.69 ± 0.42 1.70 ± 0.30

Median 1.98 1.59 1.79

P 0.672 0.339 0.525

Mea

n v

eloci

ty

(cm

/sec

)

Before Rebamipide / placebo

Range 3.10 – 6.50 3.20 – 7.40 2.90 – 7.30

Mean ± SD 4.60 ± 1.05 5.79 ± 1.37 4.96 ± 1.53

Median 4.30 6.10 4.70

After Rebamipide / placebo

Range 3.0 – 6.70 3.50 – 7.60 2.80 – 7.70

Mean ± SD 4.49 ± 1.16 5.79 ± 7.44 5.06 ± 1.47

Median 4.10 6.10 4.90

P 0.360 0.958 0.245

Co

nges

tio

n i

ndex

Before Rebamipide / placebo

Range 0.21 – 0.81 0.18 – 0.77 0.11 – 0.73

Mean ± SD 0.47 ± 0.20 0.33 ± 0.18 0.45 ± 0.21

Median 0.47 0.27 0.42

After Rebamipide / placebo

Range 0.19 – 0.74 0.18 – 0.76 0.12 – 0.75

Mean ± SD 0.47 ± 0.19 0.33 ± 0.19 0.47 ± 0.19

Median 0.48 0.27 0.43

P 0.614 0.525 0.263

PV = Portal vein,tp: p value for Paired t-testp: p value for Wilcoxon signed ranks test between before and after rebamipide/placebo in each

group*: Statistically significant at p ≤ 0.05

Upper gastrointestinal tract endoscopy at the

beginning of the study revealed that 15 patients

(75%) in Groups I and II, and 17 patients (85%)

in Group III had esophageal varices, all without

risk signs of bleeding. The number of columns

of esophageal varices showed no significant

change after drug therapy. There was a

significant rise in the grade of esophageal

varices from before to after drug in both Group І

and Group Ш, while there was no similar

change in Group П. There was also a significant

rise in esophageal varices risk signs only in

Group П after drug therapy, while the difference

was non-significant in both Groups І and Ш,

(table 5).

Table (5): Comparison between the three studied groups according to endoscopic study of varices before and after Rebamipide /

Placebo

Group I Group II Group III

No % No % No %

Eso

ph

agea

l v

aric

es

nu

mb

er

Before Rebamipide / placebo

Range 0.0 – 2.0 0.0 – 2.0 0.0 – 2.0

Mean ± SD 1.15 ± 0.81 0.95 ± 0.69 1.15 ± 0.67

Median 1.0 1.0 1.0

After Rebamipide / placebo

Range 0.0 – 3.0 0.0 – 3.0 0.0 – 2.0

Mean ± SD 1.30 ± 1.03 1.05 ± 0.76 1.50 ± 0.69

Median 1.0 1.0 2.0

P 0.257 0.157 0.439

Eso

ph

agea

l v

aric

es

gra

de

Before Rebamipide / placebo

Range 1.0 – 2.0 1.0 – 2.0 1.0 – 2.0

Mean ± SD 1.67 ± 0.49 1.87 ± 0.35 1.71 ± 0.47

Median 2.0 2.0 2.0

After Rebamipide / placebo

Range 1.0 – 3.0 1.0 – 3.0 1.0 – 3.0

Mean ± SD 2.0 ± 0.65 1.69 ± 0.79 2.0 ± 0.69

Median 2.0 1.50 2.0

P *0.025 0.527 *.0340

Eso

ph

agea

l

var

ices

ris

k s

ign

s

Before Rebamipide / placebo

No 15 100.0 15 100.0 17 100.0

Yes 0 0.0 0 0.0 0 0.0

After Rebamipide / placebo

No 14 93.3 9 60.0 14 82.3

Yes 1 6.7 6 40.0 3 17.7

pM 0.317 *0.014 0.0833

Mp: p value for McNemar test ,p: p value for Wilcoxon signed ranks test between before and after Rebamipide/placebo in each group,*: Statistically

significant at p ≤ 0.05

Endoscopic signs of portal hypertensive

gastropathy (mosaic pattern and mucosal red

signs) showed no significant change after drug

administration. Portal hypertensive gastropathy

NIEC grade was mild in the majority of patients

before drug therapy, ranging from 50% to 80%,

while after drug therapy 40% to 80% of all

patients had mild grade of PHG. There was no

significant change in PHG NIEC score from

before to after drug therapy in any of the three

groups, (table 6, figures 1, 3-5).

Table (6): Comparison between the three studied groups according to the endoscopic study of PHG before and after Rebamipide /

Placebo

Group I Group II Group III

No % No % No %

PH

G N

IEC

sco

re a

nd

gra

de

Before Rebamipide/ placebo

None (0) 0 0.0 0 0.0 0 0.0

Mild (1) 10 50.0 16 80.0 16 80.0

Severe (2) 10 50.0 4 20.0 4 20.0

Range 1.0 – 2.0 1.0 – 2.0 1.0 – 2.0

Mean ± SD 1.50 ± 0.51 1.20 ± 0.41 1.20 ± 0.41

Median 1.50 1.0 1.0

After Rebamipide / placebo

None (0) 2 10.0 0 0.0 0 0.0

Mild (1) 8 40.0 16 80.0 14 70.0

Severe (2) 10 50.0 4 20.0 6 30.0

Range 0.0 – 2.0 1.0 – 2.0 1.0 – 2.0

Mean ± SD 1.40 ± 0.68 1.20 ± 0.41 1.30 ± 0.47

Median 1.50 1.0 1.0

P 0.414 1.000 0.414

PHG = Portal hypertensive gastropathy ,NIEC = North Italian Endoscopy Club,p: p value for Wilcoxon signed ranks test between before and after

rebamipide/placebo in each group *: Statistically significant at p ≤ 0.05

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

Group I Group II Group III

Mea

n o

f P

HG

NIE

C

Before rebamipide / placebo

After rebamipide / placebo

Figure (1): Comparison between the three studied groups according to PHG NIEC score of the stomach before and after

Rebamipide / Placebo

In patients’ Group II, 90% of gastric ulcers were

present in the antrum and 10% were in the body

of the stomach. The gastric ulcer score ranged

from 4 to 6 before drug therapy, with a median

value of 5.0. After drug therapy, the ulcer score

ranged from 1 to 5, with a median value of 2.0.

The drop in endoscopic ulcer score after drug

therapy was statistically significant. Active ulcer

stages represented the majority of presenta-

tionsat the beginning of the study; with 25% of

patients at stage A1 and 40% of patients at stage

A2. After drug therapy, most ulcers were classi-

fied as scarring stage; with 30% of patients at

stage S1 and 45% of patients at stage S2. The

drop in endoscopic ulcer stage after drug

therapy proved to be significant, (table 7, figures

2, 6-8).

Table (7): Comparison of the endoscopic gastric ulcer score and stage according to Sakita Miwa classification (26)

in study Group II before and after Rebamipide/Placebo

Group II

No %

En

do

sco

pic

Ulc

er s

tag

e (s

core

)

Before Rebamipide / placebo

A1 (6 points) 5 25.0

A2 (5 points) 8 40.0

H1 (4 points) 7 35.0

After Rebamipide / placebo

A1 (6 points) 0 0.0

A2 (5 points) 1 5.0

H1 (4 points) 1 5.0

H2 (3 points) 3 15.0

S1 (2 points) 6 30.0

S2 (1 point) 9 45.0

Before Rebamipide / placebo

Range 4.0 – 6.0

Mean ± SD 4.90 ± 0.79

Median 5.0

After Rebamipide / placebo

Range 1.0 – 5.0

Mean ± SD 1.95 ± 1.15

Median 2.0

P <0.001*

p: p value for Wilcoxon signed ranks test between before and after Rebamipide/placebo in each group

*: Statistically significant at p ≤ 0.05

0

5

10

15

20

25

30

35

40

45

50

A1 A2 H1 H2 S1 S2

Mea

n o

f E

nd

osco

pic

Ulc

er s

tage

Before rebamipide / placebo

After rebamipide / placebo

Figure (2): Comparison of the endoscopic gastric ulcer score and stage according to Sakita Miwa classification(26)

in study Group II before and after Rebamipide/Placebo

Figure (3): Upper gastrointestinal tract endoscopy

showing mild PHG (Mild mosaic pattern)

Figure (4): Upper gastrointestinal tract

endoscopy showing mild PHG (Moderate

mosaic pattern)

Figure (5): Upper gastrointestinal tract endoscopy

showing severe PHG -red spots of the fundal

mucosa on background of mosaic pattern

Figure (6): Upper gastrointestinal tract endoscopy

showing stage H1- multiple small gastric ulcers at the

antrum of the stomach

Figure (7): Upper gastrointestinal tract endoscopy

showing stage A2- large gastric ulcer surrounded

by multiple smaller linear ulcers at the body of the

stomach

Figure (8): Upper gastrointestinal tract

endoscopy showing stage H2- linear gastric

ulcer at the antrum of the stomach

Histopathologic examination of gastric mucosal

biopsies revealed that there was no significant

change in the histopathologic score or grade of

PHG from before to after drug therapy, neither

in the fundus, nor the antrum, nor in the “whole

stomach” score, which was calculated as the

sum of fundus and antrum, (table8, figures 9-12)

Table (8): Comparison between the three studied groups according to histophathologic all over stomach score and grade of PHG

before and after Rebamipide / Placebo

Group I Group II Group III

No % No % No %

His

top

atho

log

ic S

tom

ach

sc

ore

& g

rad

e

of

PH

G

Before Rebamipide/ placebo

Mild (score 1-2) 6 30.0 6 30.0 3 15.0

Moderate (score 3-4) 7 35.0 9 45.0 14 70.0

Severe (score 5-6) 7 35.0 5 25.0 3 15.0

After Rebamipide / placebo

Mild (score 1-2) 7 35.0 8 40.0 4 20.0

Moderate (score 3-4) 6 30.0 9 45.0 10 50.0

Severe (score 5-6) 7 35.0 3 15.0 6 30.0

Before Rebamipide/ placebo

Range 2.0 – 6.0 1.0 – 6.0 2.0 – 6.0

Mean ± SD 3.60 ± 1.35 3.30 ± 1.42 3.75 ± 1.02

Median 3.50 3.0 4.0

After Rebamipide / placebo

Range 1.0 – 6.0 1.0 – 6.0 1.0 – 6.0

Mean ± SD 3.55 ± 1.79 2.95 ± 1.47 3.75 ± 1.29

Median 3.50 3.0 4.0

P 0.977 0.244 1.000

p: p value for Wilcoxon signed ranks test between before and after Rebamipide/placebo in each group

*: Statistically significant at p ≤ 0.05

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

Group I Group II Group III

Mea

n o

f H

isto

path

olo

gic

Sto

mach

sco

re o

f P

HG

Before rebamipide / placebo

After rebamipide / placebo

Figure (9): Comparison between the three studied groups according to histopathologic stomach score of PHG

Figure (10): Light microscopic picture (X 400) of

gastric mucosa with special stain (PAS) showing a

few ecstatic capillaries in the deep lamina propria, a

case of mild PHG.

Figure (11): Light microscopic picture (X 400) of

gastric mucosa with special stain (PAS) showing

multiple ectatic capillaries in the deep lamina

propria, a case of moderate PHG.

Figure (12): Light microscopic picture (X 400) of

gastric mucosa with special stain (PAS) showing

multiple ectatic capillaries in the superficial and

deep lamina propria, a case of severe PHG.

Immunohistochemical study of gastric mucosal

biopsies showed a significant rise in PCNA

scores of the antrum and the fundus mucosa in

Groups І and П, while there was no similar

change in Group Ш. The PCNA score of whole

stomach was calculated as the mean value of the

antrum and fundus PCNA scores for each

patient, demonstrating a significant rise in

PCNA expression by the stomach mucosa in

Groups І and П, while no similar change was

measurable in Group Ш, (table 9, figures 13-

17).

Table (9): Comparison between the three studied groups according to PCNA score of the antrum, fundus and whole stomach

before and after Rebamipide / Placebo

Group I Group II Group III

PC

NA

sco

re (

antr

um

)

Before Rebamipide / placebo

Range 24.0 – 98.0 0.0 – 89.0 24.0 – 86.0

Mean ± SD 67.75 ± 22.04 45.95 ± 27.44 50.0 ± 21.26

Median 72.50 47.50 41.0

After Rebamipide / placebo

Range 31.0 – 98.0 43.0 – 98.0 20.0 – 97.0

Mean ± SD 81.40 ± 20.99 75.85 ± 17.72 50.80 ± 19.90

Median 87.50 79.0 45.0

P 0.001* <0.001* 0.285

PC

NA

sco

re (

fund

us)

Before Rebamipide / placebo

Range 24.0 – 87.0 0.0 – 88.0 15.0 – 94.0

Mean ± SD 55.35 ± 20.98 44.40 ± 24.18 50.90 ± 26.02

Median 52.50 41.50 44.50

After Rebamipide / placebo

Range 22.0 – 98.0 26.0 – 98.0 18.0 – 96.0

Mean ± SD 76.40 ± 22.27 69.90 ± 21.73 56.20 ± 24.50

Median 86.0 68.0 58.50

P <0.001* <0.001* 0.073

PC

NA

sco

re (

sto

mac

h)

Before Rebamipide / placebo

Range 27.0 – 88.0 0.0 – 86.0 21.0 – 83.0

Mean ± SD 61.75 ± 20.15 45.35 ± 24.51 50.75 ± 21.02

Median 69.50 45.50 51.0

After Rebamipide / placebo

Range 27.0 – 98.0 35.0 – 98.0 20.0 – 97.0

Mean ± SD 79.05 ± 20.84 72.75 ± 18.27 53.90 ± 20.11

Median 87.0 78.50 52.50

P <0.001* <0.001* 0.150

p: p value for Wilcoxon signed ranks test between before and after Rebamipide/placebo in each group

*: Statistically significant at p ≤ 0.05

0

10

20

30

40

50

60

70

80

90

100

Group I Group II Group III

Mea

n o

f P

CN

A s

core

sto

ma

ch

Before rebamipide / placebo

After rebamipide / placebo

Figure (13): Comparison between the three studied groups according to PCNA score stomach

Figure (15): Light microscopic picture (X 400) with

PCNA immunostain showing rare positive (brown)

stain of nuclei before drug therapy (+ve), indicating

low mucosal cellular proliferation.

Figure (14): Light microscopic picture (X 400) with

PCNA immunostain showing negative stain of all

nuclei before drug therapy, indicating absence of

mucosal cellular proliferation.

Figure (17): Light microscopic picture (X 400) with

PCNA immunostain showing diffuse strong positive

(brown) stain of all nuclei after drug therapy

(+++ve).

Figure (16): Light microscopic picture (X 400) with

PCNA immunostain showing positive (brown)

nuclear stain involving moderate amount of nuclei

(++ve), indicating moderate mucosal cellular

proliferation.

A strong negative correlation was found

between occult blood in stool and hemoglobin

level in Groups І and П. On the other hand,

there was a strong positive correlation between

endoscopic portal hypertensive gastropathy

NIEC score and occult blood in stool in all three

groups. Strong positive correlations were also

found between the Child score and portal vein

congestion index in the three groups, and

between the Child score and PHG NIEC score

in Groups І and П. In addition, significant

positive correlations were found between the

portal vein congestion index and portal

hypertensive gastropathy NIEC score in the

three groups, and between the portal vein

congestion index and histopathologic stomach

score of PHG in Groups І and П. Lastly there

was no correlation between the histopathologic

stomach score of PHG and the PCNA score of

the stomach in any of the three studied groups,

neither before nor after drug therapy except for

a weakly significant one in Group Ш before

drug therapy, (table 10).

Table (10): Correlation of portal vein congestion index with PHG NIEC score and histopathologic stomach score of PHG in the

different studied groups at the beginning of the study

Portal vein congestion index

Group I Group II Group III

Rho P Rho p rho p

PHG NIEC score 0.729 <0.001* 0.694 0.001* 0.488 0.029*

Histopathologic stomach score of

PHG 0.574 0.008* 0.567 0.009* 0.404 0.078

R : Spearman coefficient*: Statistically significant at p ≤ 0.05

A significant positive correlation was detected

between the inflammatory infiltrate and PCNA

score of the antrum and the fundus in all three

groups, both before and after drug therapy. The

higher the PCNA score, the higher was the value

of the inflammatory infiltrate, (table 11).

Table (11): Correlation between inflammatory infiltrate and PCNA score of the antrum and fundus in the different studied groups

before and after Rebamipide / Placebo administration

Inflammatory infiltrate

Group I Group II Group III

Rho P Rho p rho p

PC

NA

sco

re

Antrum

Before Rebamipide /

Placebo 0.773 <0.001* 0.576 0.008* 0.759 <0.001*

After Rebamipide /

Placebo 0.916 <0.001* 0.716 <0.001* 0.488 0.029*

Fundus

Before Rebamipide /

Placebo 0.746 <0.001* 0.520 0.019* 0.499 <0.001*

After Rebamipide /

Placebo 0.797 <0.001* 0.753 <0.001* 0.306 0.190

Rho ( : Spearman coefficient*: Statistically significant at p ≤ 0.05

Discussion

Portal hypertensive gastropathy (PHG) has

emerged as a new clinical and pathological

entity among the complications of advanced

liver cirrhosis. It has been identified as a frequ-

ent cause of acute and chronic upper gastro-

intestinal hemorrhage; 10-20% of bleeding

episodes occurring in cirrhotic patients are

caused by this condition, with high incidence of

morbidity and mortality.(8) The patients included

in the present work were chosen to have positive

endoscopic evidence of PHG. Occult blood in

stool was tested for all patients at the beginning

of the study; it was positive in up to 55% of

patients, and it showed a positive correlation

with the endoscopic score of PHG. Also, there

was a significant negative correlation between

occult blood in stool and hemoglobin level in

patients of Groups І and П. In other words; the

higher the severity of PHG, the more likely did

the patients test positive for occult blood in

stool, and the more severe was their anemia. In

accordance to these findings, Primignani et al.

stated in their study that the main clinical

presentation of PHG was in the form of severe

anemia and chronic gastrointestinal (GI) blood

loss, manifested by accidental discovery of

occult blood in stool, rather than frank GI

bleeding (incidence 12% versus 2.5% among

PHG patients, respectively).(30) Several factors

were suggested to affect the grade of PHG; like

the severity of the liver disease and the severity

of portal hypertension. In the present study, a

significant positive correlation was found betw-

een PHG NIEC score and Child-Pugh score in

all patients. The higher the Child-Pugh score,

the more severe was the grade of PHG. Another

significant positive correlation was found betw-

een portal hypertensive gastropathy NIEC score

and the portal vein congestion index in all our

patients. The higher the value of portal vein

congestion index, the more severe was the grade

of gastropathy. In the same context, Sarin et al.

investigated factors affecting the severity of

PHG in 107 patients with different etiologies of

portal hypertension. Their results demonstrated

a significant influence of liver disease severity

on the development and grade of gastropathy.(18)

Kim et al. also found that the hepatic venous

pressure gradient (HVPG), which is a classic

method for portal venous pressure determin-

ation, was significantly higher in patients with

severe PHG than in those with mild or no

PHG.(31) An important sequel of PHG is incre-

ased susceptibility of gastric mucosa to injury

by noxious factors as non-steroidal anti-inflam-

matory drugs, alcohol and helicobacter pylori

infection. Experimental evidence has also dem-

onstrated an impaired gastric mucosal healing

response to such injuries.(9) So far, beta-blockers

seem to be the only effective treatment for this

condition; acting by decreasing portal pressure. (32) Therefore, the aim of our work was to

evaluate (endoscopically and histopathology-

ically) the benefits and/or hazards of a new

therapeutic agent, Rebamipide, on PHG among

cirrhotic patients. The patients enrolled in our

study were divided into three groups: Patient's

Group І and П were instructed to take

Rebamipide, while patient's Group Ш was instr-

ucted to take placebo for a period of three

months. As regard the Child- Pugh score and

other liver profile parameters, our results

showed no significant change after drug admini-

stration, except for an isolated drop of ALT

level in Group П patients. Only few studies have

attempted to evaluate the effect of Rebamipide

on liver functions. Taniguchi et al. studied the

protective effect of anti-ulcer agents on ethanol-

induced gastric mucosal lesions and D-

galactosamine-induced hepatitis in rats, and

showed that Rebamipide had only little effect in

the form of some insignificant decline of serum

ALT and AST levels.(33) Other routine labo-

ratory results were also evaluated in the course

of our work. Baseline fasting blood sugar level,

blood urea and serum creatinine were measured

for all patients and showed no significant

change after drug administration. In comparison,

a study by Ha et al. investigated whether

Rebamipide could ameliorate the pathoph-

ysiology associated with experimental diabetes

in vivo. They found that Rebamipide showed no

effect on the glycemic level and that it atten-

uated high glucose-induced nephropathy by its

antioxidative properties.(34) Complete blood

picture of our patients demonstrated a signi-

ficant improvement of hemoglobin level after

drug administration in Group І only, while no

similar change was found in Groups П and Ш.

This isolated change in Group I could be

attributed to the fact that some patients received

blood transfusion over the course of the study

rather than the effect of Rebamipide itself. In

fact, the absence of similar hemoglobin level

improvement in Group II, who also received

Rebamipide for three months, suggests that this

hemoglobin level improvement is probably

Rebamipide independent. On the other hand,

thrombocytopenia was detected in most of our

patients at the beginning of the study, and a

statistically significant drop in platelet count

occurred after drug administration in patients’

Group П only (p=0.038). The platelet count in

Groups І and Ш, however, showed no similar

change. These results raise the important

question of whether the drop of platelet in

Group П was Rebamipide induced or not.

Indeed, thrombocytopenia has been reported as

an infrequent side effect of Rebamipide on

www.drugcite.com, a web site of the American

Food and Drug Administration (FDA) Adverse

Event Reporting System (AERS) which is

specialized in keeping a record on the adverse

effects of FDA approved drugs as reported by

customers.(35) However, no clinical studies have

been conducted so far to prove or deny the

significance of this finding. Besides, the absence

of similar drop of platelet count in patients’

Group І, who also received Rebamipide,

suggests that this drop might have been a

coincidental feature of liver disease progression

due to either hypersplenism, platelet-associated

immunoglobulins, or decreased serum thrombo-

poietin level, rather than the effect of Reb-

amipide itself.(36) Portal hypertension has been

agreed upon by most authors as a prerequisite

for the development of PHG.(37) In fact, the

reduction in portal pressure is the effective

mechanism behind the success of beta-blockers

in the treatment of PHG.(11) Therefore, we

assessed the portal vein hemodynamic para-

meters using pulse Doppler technique before

and after drug administration, where no

significant change was detected in any of the

three groups in response to drug administration.

These results seem to be expected, because

Rebamipide has never been reported to exert

any action on blood vessel wall or to affect the

hemodynamics of the portal circulation. Never-

the less, no comparable studies are available up

till now to prove or contradict these results.

Upper GI endoscopy revealed that esophageal

varices were a finding in many of our patients at

the beginning of the study. However, no patients

with variceal risk signs of bleeding were

included due to their need for urgent endoscopic

intervention. After three months of Rebamipide/

placebo administration, the grade of esophageal

varices showed significant rise in Groups І and

Ш, while Group П showed no change. Esop-

hageal varices risk signs of bleeding also

showed a significant rise in Group II only,

whereas Groups I and III showed no such

change. Again, no literature is available so far to

support or contradict these results. So, the

question is raised whether or not these findings

were Rebamipide induced. The significant rise

of variceal grade in Groups І and IП versus its

absence in Group II suggests that the drug is

probably innocent. Similarly, the appearance of

variceal risk signs of bleeding in Group П

versus their absence in Groups І and Ш suggests

that this might not be the effect of Rebamipide

but rather a coincidental feature of progressing

portal hypertension.(38) The endoscopic signs of

PHG were assessed in all patients before and

after drug administration, and they were

expressed in the form of PHG NIEC score. The

majority of patients had mild grade of PHG

before drug therapy, ranging from 50% to 80%,

while after drug therapy 40% to 80% of all

patients had mild grade of PHG. There was no

statistically significant change in PHG NIEC

score to after drug therapy in any of the three

groups. Similar to our results, a study by

Kijdamrongthum et al. was performed on eight

patients with endoscopic evidence of PHG.

Despite admitting the very small sample size,

they concluded that Rebamipide did not show

any improvement of PHG in this study. In fact,

their results actually demonstrated worse PHG

scores in the Rebamipide group compared to the

placebo group, but the difference was not

statistically significant. Also, no statistically

significant difference occurred in Child-Pugh

score or blood hemoglobin levels in the two

groups after Rebamipide administration for 12

weeks.(39) In the present work, study Group П

was chosen from patients with PHG and gastric

mucosal ulcers, which were endoscopically

assessed according to Sakita Miwa classify-

cation. Ninety percent of ulcers were present in

the antrum and ten percent were in the body of

the stomach. This finding was not different from

the results of Chiba et al, who stated in their

work that 60% of gastric disorders induced by

NSAIDs or other noxious agents affect the

antrum of the stomach.(40,26) The gastric ulcer

score according to Sakita-Miwa classification

showed a median value of 5.0 before drug

therapy, which significantly dropped to a

median value of 2.0 after Rebamipide therapy.

This beneficial effect of Rebamipide on gastric

ulcer healing is not novel; it has been already

well investigated and even compared to proton

pump inhibitors by various authors.(26,41,42)

However, almost no studies have attempted to

evaluate this effect microscopically. In our

work, a histopathologic evaluation of PHG was

performed before and three months after

Rebamipide/placebo intake. The histopathologic

whole stomach score of PHG was calculated as

the sum of antrum and fundus score for each

patient, and no significant change was recorded

in any of the three groups after drug admini-

stration. Similar to our endoscopic NIEC score

results of PHG, the histopathologic stomach

score of PHG showed a significant positive

correlation with the portal vein congestion index

at the beginning of the study. The higher the

value of congestion index, the higher was the

histopathologic stomach score of PHG. These

findings can be compared to the results of a

study by Madkour et al, who reported a

significant positive correlation between the

histopathologic and endoscopic grades of PHG,

with a specificity of 81.7% of the histopatho-

logic grading system.(43) Moreover, we perfo-

rmed immunohistochemical study of gastric

mucosal biopsies of our patients before and

three months after drug therapy for assessing the

proliferating cell nuclear antigen (PCNA)

expression in the gastric mucosa, which is

considered to be a useful tool for assessing the

proliferative activity of gastric mucosa during

the ulcer healing process.(16) In fact, studies

have shown a close relation between PCNA

score and both, inflammatory process and

gastric ulcer healing.(44) Similarly, our results

showed a significant positive correlation

between the inflammatory infiltrate and PCNA

score of the antrum and fundus in all three

groups, both before and after drug therapy. On

the other hand, there was no significant

correlation between histopathologic stomach

score of PHG and the PCNA score of the

stomach in any of the three studied groups,

neither before nor after drug therapy. From

these two findings we could conclude that the

PCNA score of the stomach is a good reflection

of the inflammatory process as well as healing

process of the gastric mucosa, whereas it is

completely irrelevant to the grade of PHG.

Interestingly, a decreased expression of PCNA

in the gastric mucosa of portal hypertensive rats

was recently described by two studies, and

hence was added to the possible mechanisms of

impaired healing power in portal hypertensive

gastric mucosa.(9,17) In our work, we assessed the

PCNA expression by the antrum, fundus and

whole stomach mucosa for all patients before

and after drug administration. After drug admin-

istration, there was a significant rise in PCNA

score in Groups І and П, while there was no

similar change in Group Ш. From these finding

we concluded that Rebamipide could achieve a

significant improvement of gastric epithelial

proliferation, as indicated by PCNA expression,

in both non-injured (Group І) and injured

(Group П) gastric mucosa of patients with PHG,

in contrast to failure of epithelial proliferation in

patients receiving placebo (Group Ш). Similar

results were demonstrated so far only on rat

models in a study by Kinjo et al. In their study,

the use of Rebamipide as a gastroprotective drug

and oxygen free radical scavenger succeeded to

normalize the oxidative state and to completely

reverse the impaired PCNA expression back to

normal, hence improving mucosal healing.(10)

From the results of the present work we

concluded that Rebamipide does not have a

significant effect on the grade of PHG, neither

endoscopically, nor microscopically. However,

Rebamipide significantly increases the healing

capacity of the ulcerated as well as non-

ulcerated gastric mucosa in patients with PHG,

as proved by the significant rise in the gastric

mucosal expression of PCNA after Rebamipide

therapy. The use of Rebamipide is, therefore,

recommended for the treatment of PHG-assoc-

iated gastric ulcers and mucosal injuries.

However, its use as monotherapy in the treat-

ment of PHG is not recommended, as it has no

effect on the grade of PHG. Further research is

also needed to clarify the effect of Rebamipide

on platelet count in patients with liver cirrhosis

and portal hypertension.

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Original Article

Accidentally Discovered Patients with Antibody to Hepatitis C Virus: Clinical,

Biochemical , Virologic , Ultrasonic and Histologic Features

Ahmed Faisal MD1; Abdel-Naser Gad allah MD2 , Ashraf zytoon MD3 ,Infectious and endemic diseases unit1, Internal

Medicine department, Suez Canal university; Internal Medicine department, Al Monofyia university,2 Radiology department3

Al Monofyia university.

ABSTRACT

To assess the clinical significance of antibody to hepatitis C virus (anti-HCV) in volunteer blood donors. Prospective cohort

study in blood bank of Suez general hospital and Menoyifia University Hospital. Patients: One hundred twenty four

accidentally discovered positive HCV-antibody, most of them are volunteer blood donors (identified as positive for anti-

HCV by first- or second-generation enzyme immunoassay (EIA-1 or EIA-2 according to availability they were followed up

for a minimum two years. Measurements: Medical history, results of laboratory and virologic testing, and percutaneous

liver biopsy findings. Results: Participants with normal alanine aminotransferase levels were older and more often female

than those with abnormal levels. The source of infection, duration of disease, symptom score, and amount of alcohol

consumed were similar in the three groups. Hepatitis C virus RNA was detectable in 85% of participants, more commonly in

the groups with elevated alanine aminotransferase levels (95%) than in the group with normal levels (65%); however, titers

were similar in all groups. Examination of liver biopsy specimens showed chronic hepatitis in 54 participants (90%) and

cirrhosis in 1 participant. The only normal liver biopsy specimens (n = 3) were those from participants who were HCV RNA

negative and had normal alanine aminotransferase levels. Conclusions : Most blood donors with anti-HCV antibodies have

chronic hepatitis C regardless of their serum alanine aminotransferase levels. Donors with normal alanine aminotransferase

levels and no HCV RNA in their serum generally have normal liver histologic findings or minimal changes and have

probably recovered from HCV infection.

Introduction

The discovery of hepatitis C virus (HCV) by

molecular techniques (1) was soon followed by

the development of sensitive and specific assays

for the detection of antibody to HCV (anti-

HCV) in serum (2).These assays have shown that

HCV causes most cases of post transfusion

hepatitis and that screening blood donations for

anti-HCV eliminates 80% to 90% of cases of

hepatitis C (3) . In the United States, approx-

imately 0.4% of blood donors and 1.4% of the

general population test positive for anti-HCV (4).

The initiation of routine screening of blood

donors for anti-HCV has led to a marked

decrease in the incidence of post-transfusion

hepatitis C (3) . The initiation of routine

screening of blood donors has also led to the

identification of many persons with anti-HCV

who are asymptomatic, have no history of liver

disease, and deny any risk factors for exposure

to viral hepatitis. These blood donors who test

positive for anti-HCV are informed by blood

bank personnel of their serologic status and are

advised to consult their private physicians for

evaluation . However, the clinical significance

of finding anti-HCV in these otherwise healthy

persons is unclear. Previous studies have shown

that 60% to 80% of blood donors with anti-HCV

have elevated serum aminotransferase levels; in

most cases, these elevations are persistent,

indicating the presence of chronic hepatitis.

Furthermore, most anti-HCV-positive blood

donors can be shown by molecular techniques to

harbor HCV RNA in serum and can transmit

hepatitis C regardless of whether serum

aminotransferases are elevated (5,6) . Studies of

liver histologic findings in healthy blood donors

with anti-HCV indicate that most of these

donors have chronic hepatitis, despite the

absence of symptoms or abnormal serum

aminotransferase levels (7). Thus, the clinical

significance of the presence of anti-HCV in

volunteer, apparently healthy blood donors

remains uncertain. To better define the natural

history of chronic hepatitis C in this population,

we have initiated a long-term study of volunteer

blood donors found to have anti-HCV on routine

testing. An initial cohort of these donors has had

extensive medical evaluation, including liver

biopsy.

Aim of work

Long-term, accurate natural history data are

needed to better understand the pathogenesis of

the disease and to determine which patients need

treatment now, which ones can wait for more

effective treatment, and which ones will pro-

bably never need treatment. The high prevalence

of HCV antibody among asymptomatic volun-

teer blood donors, and the contradictory views

about the outcome of HCV infection motivated

us to study the natural history of HCV infection

among this cohort group, to answer these ques-

tions: What is the significance of the presence of

anti-HCV antibodies in these asymptomatic

individuals? What are the risk factors associated

with the infection? What proportion of antibody

-positive individuals has viremia and chronic

infection? What proportion has significant liver

disease? What insights can be derived from

asymptomatic donors in assessing the natural

history of HCV infection? In the spectrum of

HCV associated disorders, what proportions are

associated with extrahepatic disorders? What

proportion are represented by relatively benign

disease seen in donors, what proportion progress

to severe disease?

Patients and Methods

Participants: The studied sample included one

hundred and twenty four accidentally discovered

positive HCV-antibody, most of them are

volunteer blood donors, of both sexes, at the age

group 15-50 years. Subjects who had evidence

of decompensated chronic liver disease, HBs Ag

positive, chronic exposure to hepatotoxic agents

in the last 6 months, or received anti-viral

therapy for HCV infection before one year of

follow up were excluded from the study. All

subjects who proved to have anti-HCV antibody

and agreed to enter the study were exposed to

the following baseline assessment; which

included complete history taking and clinical

examination . Abdominal U/S, liver function

tests, ALT, AST, prothrombin time, serum

bilirubin, serum albumin, random blood glucose

, and HCV-RNA by polymerase chain reaction

(PCR) were done. Every 3-4 months, clinical

history and examination, laboratory investti-

gation as ALT, AST, PT, serum bilirubin, and

serum albumin were done. Abdominal U/S was

done every 6 months. Liver biopsy was done in

86 patients, and repeated at least after one year

of follow-up in 31 patients. Biopsy specimens

were histopathologically evaluated to determine

the grade of necroinflammatory changes, acc-

ording to histologic activity scoring system, and

the stage of fibrosis, according to The META-

VIR scoring system. Mixed Cryoglobulins and

serum ferritin were done in 80 patients, and

bilharzial serology in 103 subjects. Statistical

Analysis: Continuous variables were analyzed

by one-way analysis of variance with post hoc

Student t-tests. Discrete variables were analyzed

by the chi-square test with Yates' correction and

by the Fisher exact test.

Results

The present study was done on 124 anti-HCV-

positive accidentally discovered subjects (about

90% of them discovered that during blood

donation) , None of the participants was aware

of having hepatitis before attempting to donate

blood. Basic demographic data: The demo-

graphic and clinical characteristics of the sub-

jects are shown in table 1. Out of them 88

(71.0%) male, and 36 (29.0%) female, their ages

ranged between (18-53y) with a mean of (36.3

+7.4y). Seventy-one of them live in urban area,

the rest live in rural areas. About 77.5% of

subjects were married, and 19.3 were single.

They were of different educational levels (16.1

% illiterates, 16.1% read and write, 41.9% basic,

16.9% secondary, and 8.9% university). Most of

them have sedentary work. About 31% of

subjects were smokers, and only 2.4% were

consuming alcohol. Medical history and

baseline clinical data & specialized tests: Table

1 shows medical history and baseline clinical

data in the study subjects, including some of risk

factors expected to be associated with HCV

infection. Among 124 subjects; positive history

of dental procedure or surgical intervention was

the most frequent risk factor as was present in

83 (66.6%), followed by past history of tartar

emetic injection 57(46 %), and family history of

HCV 24(19.4%). History of blood transfusion

was present in only 17(13.7%), while common

sharing of razors, and toothbrushes was the least

frequent risk factor 4(3.2)%. Past history of

schistosomiasis was present 72(58.1%), positive

history of exposure schistosomal infection in 40

(32.3%), past history of jaundice was present in

about 8%, 58.9%were asymptomatic, and high

body mass index (> 25) in 86 (69.4%). Although

all subjects denied any symptoms at time of

blood donation, or discovering anti-HCV anti-

body, many of them noted symptoms on inquiry

by the researcher after being tested positive for

HCV antibody, and during the subsequent

follow up. PCR for HCV RNA qualitatively was

positive in 102/111(91.9%) subjects, mixed

Cryoglobulins was positive in 5/80 subjects,

serum ferritin was high in 9/80subjects, bil-

harzial serology was positive in 36/103(35%).

Clinical manifestation & Extrahepatic mani-

festations: Table 2 shows the frequency of

different symptoms which elicited on the first

visit, The most common symptoms were hyper-

somnia in 25(20.1%) subjects, fatigue 23

(18.5%), bleeding per gum after teeth brushing

10(8.0%), right quadrant pain 16(12.9%), abd-

ominal distention after meals 16(12.9%), pass-

ing deep yellow urine 9(7.2%), weight loss 4

(3.2), anorexia 4(3.2%), dizziness 4(3.2%), and

Pruritus 3(2.4%). No one was complaining of

fever, lower limb edema, disturbed menstru-

ation, impotence, or clinical jaundice, and most

participants reported that the symptoms were

mild and did not interfere with daily activities or

work. None of these participants was seeing a

physician for these symptoms, and none had a

concurrent disease or were regularly taking any

medications. At the end of 24 months follow

up. The most common symptoms were hyper-

somnia 32 (25.8%), fatigue 30 (24.2%), blee-

ding per gum after teeth brushing 18 (14.5%),

right quadrant pain 18 (14.5%), abdominal

distention after meals 21(16.9%), passing deep

yellow urine 16(12.9%), weight loss 6 (4.8%),

anorexia 5 (4%). Fever, dizziness, and Pruritus

were uncommon, each 4(3.2%), and only one

subject was complaining of clinical jaundice,

and most participants reported that the symp-

toms were mild and did not interfere with daily

activities or work, and these symptoms had not

related to concurrent disease. Table 2 shows the

frequency of different signs in the study group;

on first presentation, pallor was present in

5(4.0%), the liver was found to be enlarged in

28(22.6%) of the subjects, splenomegaly in 4

(3.2%), and skin rash in 5 (4%), each of these

signs; lower limb edema, palmar erythema,

finger clubbing, spider naive, jaundiced sclera,

ascites , encephalopathy or flapping tremors

were not detected in the study subjects. After 24

months, pallor was present in 7 (5.6%), the liver

was found to be enlarged in 35(28.2%) of the

subjects, splenomegaly in 11(8.9%), and skin

rash in (5/124), each of these signs; lower limb

edema , palmar erythema, finger clubbing, spid-

er naevie, jaundiced sclera were present in less

than 4 (3.2%), no case of ascites , encephalo-

pathy or flapping tremors were detected in the

study subjects. Extrahepatic manifestations

appeared in the study group on first presentation

were; joint pain was the most frequent extra-

hepatic symptom present in 18(14.5%) subjects,

vasculitic skin lesions in 3(2.4%), each of,

vetiligo, and neuropathy in 2 (1.6%), and lichen

planus in only 1(0.8%) subjects. The extra-

hepatic manifestations appeared in the study

group after 24 months follow up: Thirty six out

of 124 subjects developed, one or more of

studied extrahepatic manifestations, as joint pain

was the most frequent extrahepatic symptom

present in 23(18.5%) subjects, vasculitic skin

lesions in 5 (4%), and each of lichen planus,

vetiligo, and neuropathy in 2 (1.6%) subjects.

Biochemical findings: Table 3- shows baseline

Liver functions tests, and after 24 months follow

up among 124 subjects:. seventy five (60.5%)

subjects has normal level (AST >48u/l), 31

(25%) subjects has mild elevation (AST= >

48:100u/l), and 18(14.5%) has moderate elev-

ation (AST=>100: 400 u/l). ALT levels ranged

from 3-290u/l. seventy one (57.3%) subjects has

normal ALT level, 36(29%) has mild elevation,

and 17(13.7%) has moderate elevation, 8(6.5%)

has total serum bilirubin > 1.1mg/dl, no one has

serum albumin < 3.5mg/dl, and 4 (3.2%)

subjects has prothrombin <80%. After 24

months of follow up; it was found that AST

level ranged from 8- 400u/l. seventy seven

(62.1%) subjects had normal levels, 30(24.2%)

subjects had mild elevation, and 17(13.7%) has

moderate elevation. ALT levels ranged from

10-290 u/l, 73(58.9%) subjects had normal

level, 35(28.2%) had mild elevation, and 16

(12.9%) had moderate elevation, 10/124(8.1%)

has total serum bilirubin > 1.1mg/dl, and only

one had serum albumin < 3.5 mg / dl, and 5/124

(4%) subjects had prothrombin <80%. Table 3

shows the pattern of liver transaminases through

the 24 months of follow up, ALT level was

persistently normal in 51(41.1%) subjects,

Fluctuating in 36(29.1%), and persistently in

37(29.8%), while AST level was persistently

normal in 50/124(40.3%) subjects, Fluctuating

in 36(29.1%), and persistently in 38(30.6%)

subjects. Blood picture of study group: Table 2-

shows the baseline blood profile among study

group (n.109), it was found that WBCs ranged

from 2550-12900 with mean value = 5480.2

±1713, leucopoenia (WBCs < 4000) was present

in 19(17.4%) subjects. Neutrophils ranged from

25%-67% with mean value = 51.8 %±9.8%,

lymphocytes ranged from 24%-68% with mean

value = 40.2%+9.9%, basophiles ranged from 0-

5% with mean value = 0.4±0.7, platelets count

ranged from 12000-342.000 with mean value=

173.558 ± 7133. Thrombocytopenia ( platelet

count> 150.000 ) was present in 30(27.5%),

hemoglobin level ranged from 8.2-16.9; anemia

( hemoglobin > 13g/dl ) was present in 37

(33.9%) subjects. Abdominal ultrasound find-

ings: Table 4- shows the baseline U/S findings

among 124 subjects, it was found that the liver

size ranged from 9.4-16.5cm with mean value =

11.4±2.6cm. Twenty - eight (22.6%) subjects

has hepatomegally (liver right lobe span >

13cm), the liver texture was normal in 29

(27.4%) subjects, bright texture in 83(66.9%)

subjects, and coarse texture in 12(9.7%)

subjects. Intra-hepatic veins were attenuated in

10(8.1%) subjects. The portal vein diameter

ranged from 9-17mm with mean value =

11.0±2.2, and dilated in 3(2.9%) subjects. The

splenic vein diameter ranged from 5-13mm with

mean value = 6.6±1.6, splenic vein diameter

was dilated in 4(3.2%). splenomegaly

(longitudinal splenic span >13cm) was found in

4(3.2%) subjects. According to suggested

criteria of CLD evide-nce by abdominal

ultrasonic examination, there was no evidence

of CLD in 84(67.7%); possible evidence of

CLD in 24(19.4%), and probable evidence of

CLD in 15(12.1%) subjects, and only one has

definitive evidence of CLD. U/S findings among

124 subjects after 24 months of follow up: It

was found that the liver size ranged from 8-17.5

cm with mean value = 11.8±3.2 cm, 35(28.2%)

subjects had hepatomegaly, the liver texture was

normal in 9(7.3%)subjects, bright texture in

85/124(68.5) subjects, and coarse texture in

30(24.2%) subjects. Intrahepatic veins were

attenuated in 31(25%) subjects. The portal vein

diameter ranged from 10-16cm with mean value

= 10.3±2.1, and dilated in 16 12.9% subjects.

The splenic vein diameter ranged from 4-13cm

with mean value = 6±1.3. Splenic vein dilated in

14(11.3%). Splenomegaly ( longitudinal splenic

span >13cm) was found in 11(8.9%) subjects.

According to suggested criteria of CLD

evidence by abdominal ultrasonic examination,

there was no evidence of CLD in 59 (47.6%),

possible evidence of CLD in 15 (12.1%), and

probable evidence of CLD in 31 (25%) subjects,

and 19(15.3) had definitive evidence.

Histopathologic findings: Table 5- shows

baseline histopathological activity according to

Knodell’s scoring system in 86 patients

underwent liver biopsy on start of follow up, it

was found that the hepatitis activity index of

subjects ranged from 0-18 with mean value =

8.2±5.3, piecemeal/bridging hepatic necrosis

ranged from 0-10 with mean value = 4.5±3.5,

Intralobular degeneration and focal hepatic

necrosis was not present in 10(11.6%) subjects,

mild in 28(32.6%), moderate in 24(27.9%),

severe in 16(18.6), and marked in 8(9.3%).

Portal inflammation was not present in 8(9.3%)

subjects, mild in 30(34.9%) pt., moderate in

21(24.4%), severe in 19(22.1%), marked in

8(9.3%). the hepatic fibrosis was not present in

50(58.1%), stage one (Fibrous Portal Expa-

nsion) in 16(18.6%pt., stage 2 ( Minimal Bridg-

ing Fibrosis) in 4(4.7%), stage 3 (marked Porto-

portal, or central linkage fibrosis) in 3(3.5%)pt.,

and stage 4 (Cirrhosis in 13(15.1%). Histologic

activity (intensity of necroinflammatory lesion)

was classified according to the new classify-

cation proposed by Desmet and colleagues for

chronic hepatitis(8), it was found that normal or

minimal active hepatitis was present in 18

(20.9%)pt., mild active hepatitis was present in

32(37.2%), moderate active hepatitis in 11

(12.8%), and severe active hepatitis in 25

(29.1%).

Table 1 Characteristics of the study sample (n=124)

333 Frequency Percent Frequency Percent

Age (years) Age (years)

Range 18.0-53.0 Range 18.0-53.0

Mean ± SD 36.3±7.4 Mean ± SD 36.3±7.4

Residence: Gender:

Urban 88 71.0 Male 88 71.0

Rural 36 29.0 Female 36 29.0

Marital status: Education:

Single 19 15.3 Illiterate 20 16.1

Married 96 77.4 Read / write 20 16.1

Widow 5 4.0 Basic 52 41.9

Divorced 4 3.2 Secondary 21 16.9

University 11 8.9

Special habits: Job:

None 83 66.9 Not working 36 29.0

Smoking 38 30.9 Sedentary 82 66.1

Alcohol/smoking 3 2.4 Manual 6 4.8

Table 2 Baseline and after 24 months clinical manifestations.

Symptoms 1st month 24th month Signs

1st month 24th month

Freq. % Freq. % Freq. % Freq. %

Hypersomnia 25 20.1 32 25.8 Hepatomegaly 28 15.0 35 28.2

Fatigue 23 18.5 30 24.2 Pallor 5 4.0 7 5.6

Abdominal distension 16 12.9 21 16.9 Skin rash 5 4.0 5 4.0

Right quadrant pain 16 12.9 18 14.5 Splenomegaly 4 3.2 13 10.5

Bleeding gum or nose 10 8.0 18 14.5 Jaundiced sclera 0 0.0 1 0.8

Deep yellow urine 9 7.2 16 12.9 L.L oedema 0 0.0 3 2.4

Dizziness 4 3.2 4 3.2 Muscle wasting 0 0.0 3 2.4

Weight loss 4 3.2 6 4.8 Pulmar erythema 0 0.0 3 2.4

Anorexia 4 3.2 5 4.0 Spider naevi 0 0.0 2 1.6

Pruritis 3 2.4 4 3.2 Clubbing finger 0 0.0 1 0.8

Fever 3 2.4 3 2.4 Shrunken liver 0 0.0 1 0.8

Hypersomnia 25 20.1 32 25.8 Ecchymosis 0 0.0 3 2.4

Table 3. Baseline and 24-month follow-up liver function tests of study subjects (n=124)

Baseline 24-month

Freq. % Freq. %

AST grade:

Normal (<48 IU) 75 60.5 77 62.1

Mild (48-<100 IU) 31 25.0 30 24.2

Moderate (100-400 IU) 18 14.5 17 13.7

ALT grade:

Normal (<48 IU) 71 57.3 73 58.9

Mild (48-<100 IU) 36 29.0 35 28.2

Moderate (100-400 IU) 17 13.7 16 12.9

Total bilirubin (>1.1) 8 6.5 10 8.1

Direct bilirubin (>0.4) 5 4.5 5 4.5

Prothrombin time (<80%) 4 3.2 5 4.5

Serum albumin (<3.5) 0 0.0 1.0 0.8

Table 4. Baseline U/S findings of study subjects (n=124)

Baseline

24-month

follow-up

No. % No. %

Hepatomegaly(Rt.Lobe span>13cm) 28 22.6 35 28.2

Liver texture:

Normal 29 23.4 9 7.3

Bright 83 66.9 85 68.5

Coarse 12 9.7 30 24.2

Intra-hepatic veins attenuation:

No 114 94.4 93 75.0

Yes 10 8.1 31 25.0

Dilated portal vein (>13 mm) 3 2.9 16 12.9

Splenomegaly 4 3.2 11 8.9

Dilated splenic vein diameter (>8 mm) 4 3.2 14 11.3

Ultrasonographic diagnosis:

Normal 84 66.7 59 47.6

Probable CLD 24 19.4 15 12.1

Possible CLD 15 12.1 31 25.0

Definitive CLD 1 0.8 19 15.3

Table 5. Baseline liver biopsy data of study subjects (n=87)

Frequency Percent

Intralobular degeneration and HC necrosis:

None 10 11.6

Mild 28 32.6

Moderate 24 27.9

Severe 16 18.6

Marked 8 9.3

Portal inflammation:

None 8 9.3

Mild 30 34.9

Moderate 21 24.4

Severe 19 22.1

Marked 8 9.3

Fibrosis by Metavir:

None 50 58.1

Mild 16 18.6

Moderate 4 4.7

Severe 3 3.5

Marked 13 15.1

Cirrhosis:

None 73 84.9

Cirrhosis 13 15.1

Desmet’s classification:according to knodell score

Normal/minimal (1-3) 18 20.9

Mild (4-8) 32 37.2

Moderate (9-13) 11 12.8

Severe (14-18) 25 29.1

Discussion

Community-acquired HCV infection makes up

the bulk of all HCV infections. However, kno-

wledge of the natural history in this setting

remains limited, primarily as a result of the

methodological difficulties associated with con-

ducting these studies. Such difficulties include

the problems of accurately identifying those

with incident infections. In this study, we traced

and followed up a cohort of volunteer blood

donors and accidentally discovered subjects who

were testing positive for HCV antibody by

second generation ELISA. The present study

was carried on 124 subjects who were acid-

entally discovered to be anti-HCV seropositive,

most of them during screening for blood

donation at the blood bank of Suez general

hospital. They were apparently healthy, and

denied any disease at that time. Initial liver

biopsy was done in most of patients, as a gold

standard for staging HCV-related liver disease

in terms of fibrosis and cirrhosis. Additionally,

the entire cohort did not take any specific anti-

viral treatment throughout the follow-up. The

selection of HCV seropositive patients in this

study was based on 2nd generation ELISA test,

which offered much greater sensitivity, and

remains the most commonly used assay used in

blood donation centers for detecting anti-HCV.

The second-generation test reduces the "wind-

ow phase" to seroconversion, with a dramatic

reduction in the number of false positive reac-

tions seen with the first-generation test (9). The

fact that PCR for HCV-RNA qualitatively was

positive in 91.9% of subjects tested provides

confidence that the vast majority of them had

HCV infection, but also indicates that some of

them might not be vireamic. Nonetheless, it

does not preclude the possibility of inclusion of

few false positive anti-HCV subjects. Baseline

Chronic Liver disease in asymptotic accidentally

discovered HCV antibody positive cases:

Symptoms and signs: Although the patients

included in this study were apparently healthy

and denied any diseases at the time of

screening, the questionnaire elicited that Just

over 40% had one symptom or more.. The most

frequent symptoms were hypersomnia, fatigue,

right quadrant pain, abdominal distention after

meals, with less frequent symptoms as passing

deep yellow urine, weight loss, anorexia,

dizziness, and pruritus. However most of these

symptoms are considered subjective, non-

specific, and common in the general population.

Meanwhile, none of our patients had specific

liver symptoms like jaundice, lower limb

edema, or ascites. These foregoing findings are

in agreement with Martinot et al (10) who

documented that most patients with chronic

hepatitis C have few if any symptoms; the most

common being fatigue, which is typically

intermittent, and hypersomnia. Right upper qu-

adrant pain, nausea, and poor appetite do occur

in some patients. In this respect, Shakil et al (11)

have evaluated a cohort of over 100 patients

with chronic HCV infection and no clinical

signs of cirrhosis and in 100 healthy blood

donors without HCV using self administered

symptom. Fatigue was the most common

complaint in both groups and occurred with

similar frequency (62 percent in those with

HCV versus 70 percent of controls). Abdominal

pain, itching, and dark urine were the only

complaints that were significantly more com-

mon among the HCV group members, although

they were present in only a small number of

patients. None of the participants studied in the

present work, including a participant with

cirrhosis, had symptoms that were obviously

related to liver disease. All through the follow-

up, most of the participants reported that the

symptoms were fluctuating, mild, and did not

interfere with daily activities or work. At the

end of follow-up, only one subject was

complaining of clinical jaundice. These findings

underscore the silent nature of chronic hepatitis

C, and the absence of symptoms and history of

liver problems in many patients with this

disease. As regard clinical sings, like other

investigators (12,13,14,15) hepatomegaly was the

most frequent sign observed on physical

examination, as it was detected in 22.6%

increased to 28.2% of our subjects. Splen-

omegaly was observed in 3.2% increased to

10.5% of our subjects , after 24 months of

follow up, this was in line with (15-18) who found

splenomegaly in (5%) of 756 anti-HCV positive

blood donors, no cases of ascites, encepha-

lopathy, or flapping tremors have been found in

the physical examination, indicating a low

degree of hepatic compromising in accidentally

discovered HCV patients

Risk factors

At least one or more risk factor was found in

each patient in the present study. The most

frequent risk factor was dental procedures,

which was reported by 66% subjects. The mode

by which HCV is transmitted during dental

procedure is through contaminated blood or

saliva. The role of saliva in HCV transmission

was verified by (19). These authors have

respectively reported that HCV-RNA was found

in 30% and 52.4 % of saliva specimens. It was

unknown whether these traces of genetic

material in the saliva could infect another

individual, but that it was not impossible. The

presence of HCV-RNA in saliva might make the

toothbrushes infected after brushing, as (19-33)

have reported that forty percent of the rinsing

water of the toothbrushes tested positive for the

virus. This makes the common sharing of

toothbrushes a risk factor for HCV transmission

frequent. The second most frequent risk factor

was previous tartar emetic injection, which was

also reported The EDHS study estimated that

29.6% of anti-HCV antibody positives (25.3%

of women and 31.5% of men) received injec-

tions to treat schistosomiasis (34). The Egyptian

HCV epidemic could be the result of a wide-

spread treatment campaign against schistose-

miasis, From the 1920s to the 1980s, the

government administered parenteral antischis-

tosomal therapy (usually 6-12 injections) with

reusable syringes. With a course of injections

taking two to four weeks, an individual infected

early in treatment could then spread HCV on a

sub-sequent injection to others who used the

same syringe. Current risk factors for acquiring

or having acquired hepatitis C in blood donors

include illegal intravenous drug use, which was

found only in two subjects in our study. The

finding in the present study that only 14% of

subjects reported a history of blood transfusion,

is in agreement with what was reported by (35,36),

claimed that , blood transfusion was identified

in 24.3% and needle reuse in 20.6% of the

HCV-positive cases in this nationwide sample.

It is clear that the identification of a certain

practice as a risk factor is related to the

frequency of exposure to that practice in the

community. Extrahepatic manifestation:

Chronic hepatitis C virus can directly or

indirectly affect a number of organs other than

the liver. A minority of patients with chronic

hepatitis develops clinically extrahepatic

features of disease. The specific viral and host

features that predispose patients to extrahepatic

expressions of disease have not been elucidated.

In studying extrahepatic manifestations in the

present work, the most common symptoms

involved the joints and skin. HCV infection was

associated with joint pains (19%), skin changes

(17%), dry mucous membranes (12%), and

sensory nerve changes (9%). Serum transami-

nase levels: Baseline measurement of serum

transaminases levels in our study revealed that

57.3% of subjects had normal ALT level, while

60.5% had normal AST level. At the 24-month

follow up, only 40.1%, 40.3% remained

persistently normal for ALT, AST respectively.

These results are similar to those reported by

Jamal (1999) who concluded that a single

normal ALT at the time of first evaluation is

common in volunteer blood donors with chronic

HCV patients. However, most of them did not

have persistently normal values on serial

assessment. Only 39% of normal ALT patients

at first assessment had persistently normal ALT.

Moreover, (37) have reported that serum alanine

aminotransferase (ALT) levels were elevated in

approximately 60 to 70% of chronic patients,

but these levels are highly variable and may

fluctuate over time. The remaining 30-40% of

patients had persistently normal ALT values,

with positive anti-HCV and HCV-RNA results.

In the present study, fluctuating transaminases

over serial determinations occurred mostly in

those with normal or mildly elevated levels at

initial measurement. Those with moderately

elevated levels remained unchanged. None of

the patients had ALT level >400u/l. In this

respect, (38) found that ALT level rarely exceeds

5 folds in asymptomatic blood donors.

Unexpectedly, mixed Cryoglobulins (CG+) in

this study were found in only 5/80 (6.25%) in

the text of the study subjects. In contrast, (39)

found (CG+) in 42.5% of 207 patients with

CHC. This can be explained by the fact that

CG+ might be present in more advanced stages

of the disease, as it is documented from many

studies (40,41). Mixed Cryoglobulins are often

seen in patients who have advanced histologic

lesions of bridging fibrosis or cirrhosis in liver

biopsy specimens, with hepatomegaly, and

splenomegaly (42). Other studies have shown that

mixed cryoglobulinemia was associated with

female gender, older age, longer disease

duration, and liver cirrhosis. The stage of

fibrosis was significantly higher in CG+

patients, whereas the activity grades and mean

ALT levels did not seem to be associated with

CG+. Mixed cryoglobulinemia was also

associated with the titer of rheumatoid factor,

and the prevalence of extrahepatic manife-

stations such as CG-related vasculitis, and some

hematological and autoimmune syndromes (39).

Blood picture: Study of blood profile among

109 subjects revealed presence of anemia,

thrombocytopenia, and leukopenia, in 33.9%,

27.5%, and 17.4% consequently, confirming the

haemocytopathic effects of HCV infection. The

pathogenic role of HCV in hepatitis-associated

aplastic anaemia develop-ment has not been

confirmed. The thrombocytopenia has been

observed more frequently during chronic

hepatitis C than during infections with other

hepatotropic viruses (43). However, a direct viral

megakaryocyte infection or an immune

mechanism could explain this thromb-

ocytopenia, as this disorder may be associated

with antiplatelet autoantibodies production, low

thrombopoietin production may play a role,

along with hyper-splenism, in the development

of thrombocytopenia in patients with liver

cirrhosis (44), Histopathological findings: In the

present work, hepatitis activity index (HAI) for

liver biopsy was studied in 86 accidentally

discovered HCV antibody positive patients.

Normal or minimal chronic hepatitis (HAI: 0-3)

was found in 20.9% of patients, mild chronic

hepatitis (HAI: 4-8) in 37.2%, moderate chronic

hepatitis (HAI: 9-12) in 12.8%, and severe

chronic hepatitis (HAI: 13-18) in 29.06% of the

cases. Meanwhile, assessment of fibrosis stagi-

ng resulted in fibrosis stage 0 in 58%, stage 1 in

18.6%, stage 2 in 4.7%, stage 3 in 3.5%, and

cirrhosis or stage 4 in 15.1% of the cases. These

results are near to those were reported by (43)

This author applied the histological evaluation

based upon the new class, the hepatitis activity

index (HAI) for liver biopsy, in 106 patients

who were positive for HCV antibody by a

second-generation ELISA. Minimal chronic

hepatitis (HAI: 1-3) was found in 13.2%, mild

chronic hepatitis (HAI: 4-8) in 65.09%, and

moderate chronic hepatitis (HAI: 9-12) in

21.69% of the cases. Meanwhile, assessment of

fibrosis (staging) resulted in fibrosis 0/1 in

41.5%, fibrosis 2 in 13.2%, fibrosis 3 in 34.9%,

and cirrhosis or fibrosis 4 in 10.37% of the cases

(43). The anti-HCV-positive subjects in the

present study were categorized into three groups

according to levels of serum ALT levels prior to

liver biopsy for analysis of the relation of

histopathologic activity to ALT levels. Althou-

gh most of the subjects with minimal or mild

histologic activity had normal ALT, and most

subjects with severe histologic activity had

elevated ALT levels, there was no statistically

significant association between ALT level and

severity of histopathologic activity by using

Desmet’s classification of chronic hepatitis.

This might be due to the small number of

subjects in certain categories, especially the

severe one. Nonetheless, the results might point

to the utility of the measurement of serum ALT

levels as a readily available way to assess

chronic hepatitis disease activity. However, it is

clear from this study and others, that the

presence of normal serum ALT levels even on

multiple occasions does not rule out the

presence of chronic hepatitis. Indeed, 40.1% of

participants with repeatedly normal serum ALT

levels in our study had chronic hepatitis on liver

biopsy. Six of these participants had cirrhosis,

but several (39) subjects, had chronic hepatitis

with minimal and mild inflammatory activity

(HAI: 0-8). These findings are similar to those

reported by other groups who have studied anti-

HCV-positive blood donors in Europe, Aus-

tralia, and Asia (27,43,44). Abdominal Ultra-

sonographic findings: Abdominal ultrasound at

baseline assessment of patients in the present

study revealed the presence of hepatomegaly in

22.6%, while splenomegaly was present in only

3.2%. After 24 months of follow-up, hepatom-

egaly was present in 28.2%, and splenomegaly

in 10.5%. Ultrasound assess-ment was similar

to that elicited by clinical examination. (44,45,36)

Ultrasound assessment revealed presence of

hepatomegaly in 23.4%, and splenomegaly in

10.5% throughhout the 24 months of follow-up.

Splenomegaly was parallel to dilated splenic

vein diameter, which occurred in 3.2% of

subjects, and/or dilated portal vein diameter.

This might support the view that splenomegaly

is not an early sign of the disease, and its

presence means complication rather than

activity of the disease. Abdominal imaging at

baseline revealed no evidence of CLD in 67.7%

of the total cohort, inspite of presence of

histologic or biochemical evidence of liver

insult. Hence, abdominal U/S at early stages of

HCV disease does not offer an advantage in

early detection of liver insult. Meanwhile, all

patients with evidence of CLD by U/S had

severer histologic activity. Nonetheless, it is

important to bear in mind that the interpretation

of these tests depends on probabilities and

likelihoods. Thus, it is more likely that a patient

will have more advanced liver disease if they

have if they have ultrasonographic evidence of

chronic liver disease. However, there are still

probabilities, although smaller, of not having

such grade of the disease. This is why it still is

generally accepted among hepatitis specialists

that liver biopsy is the only way to most

accurately assess a person's stage of liver

disease. It is well established that liver biopsy is

the most reliable way to assess the activity and

stage of liver disease, and should be

recommended in anti-HCV-positive subjects

who are HCV-RNA positive and have elevated

ALT levels. Nonetheless, the resulting data

might support the view of liver biopsy should be

routinely recommended also in those who are

HCV RNA - positive and have persistently

normal ALT levels. The aim is to evaluate them,

and to provide a base for assessment of

progression and eligibility to antiviral treatment.

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