Editor in chief

M.Y.Taher

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National Advisory Board

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Khaled Madboli

Ezzat Aly

Contents Alexandria Journal of Hepatogastroenterology, Volume (XV) - April 2015

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Original Article:

Cholecystobiliary Fistula : A Simple Surgical Approach

Maher Osman, Ahmad Shaban, Mohamad Y. Taher , Mekky F 3

Dept. of Surgery1, National Liver Institute, Menoufiya University,

Dept. of Surgery2 , High Institute for Research, and Dept of

Hepatobiliary Medicine3, and Dept. of Surgery , Faculty of

Medicine, Alexandria University

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

Original Article:

Diagnostic Accuracy of Ascitic Fluid Leucocyte Esterase

and Lactoferrin in Cirrohtic Patients with Spontaneous

Bacterial Peritonitis

Ayman El Shayeb1, Akram El Deghady2, Rania Abou Youssef 3,

Eman Elabsawy4

1:Professor of Tropical Medicine, 2:Professor of Clinical and

Chemical Pathology, 3:Lecturer of Tropical Medicine, 4:Instructor

of Tropical Medicine.

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

Original Article:

Interaction of Young and Adult Biomphalaria

Alexandrina Snails with Schistosoma Mansoni

Safaa Ibrahim Khedr 1, Hayam Abd El-Moniem Sadaka 2, Iman

Fathy Abou-El Naga2, Iman Hassan Diab 3, Eglal Ibrahim Amer

41 Assistant lecturer of Medical Parasitology, 2 Professor of

Medical Parasitology, 3 Professor of Medical Biochemistry, 4

Assistant Professor of Medical Parasitology.

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

Original Article:

Study of Ascitic Fluid Calprotectin in Cirrhotic Patients

with Spontaneous Bacterial Peritonitis

Ayman Mohamed El Lehleh MD1, Somaia Abdel Mohsen Shehab

El-deen MD1 Rania Azmi El shazly MD2 Amany Abas Amer

M.B.B.Ch1 Tropical Medicine department, Faculty of Medicine,

Menoufiya University.

2 Biochemistry department, Faculty of Medicine, Menoufiya

University.

------------------------------------------- Original Article:

The Prevalence of Insulin Resistance and Metabolic

Factors in Chronic Hepatitis C Patients with Genotype 4

Prof. Amira Amer, Prof. Manal Baddour, Prof. Mohamed El

Shazly, Prof. Gylan Fadaly, Assis. Prof Nesrine Hanafi, Assis.

Lect Sara Asser

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

2

13

18

24

33

Original Articles

Cholecystobiliary Fistula : A Simple Surgical Approach

Maher Osman, Ahmad Shaban, Mohamad Y. Taher , Mekky F 3

Dept. of Surgery1, National Liver Institute, Menoufiya University, Dept. of Surgery2 , High Institute for

Research, and Dept of Hepatobiliary Medicine3, and Dept. of Surgery , Faculty of Medicine, Alexandria

University

ABSTRACT Cholecystobiliary fistula (Mirizzi syndrome) is a very rare complication of long standing gallstone disease. Preoperative

diagnosis and surgical treatment are very challenging and still individualized. Aim of the work : We presented 3

patients with different stages of MS, presented within a 12 months period. Case 1 was MS type III (cholecystobiliary

fistula > 50% of bile duct circumference), case 2 was MS type II (fistula < 50% of bile duct circumference), while in

case 3 MS was type I (mere external compression of the cystic-CBD junction by a sizable stone impacted at the neck of

the gallbladder). Methods: The preoperative diagnosis and staging of our patients based on imaging pictures from

either ERCP or MRCP. All cases were treated by open cholecystectomy done through a right subcostal incision and a

retrograde (fundus-first) dissection of the gallbladder. After identification of the bile duct and extraction of the impacted

or eroding stone, the common bile duct was repaired over a T-tube of 18 0F diameter (ductoplasty). After complete

recovery and confirmation of CBD patency by a postoperative T-tube cholangiography done 2 weeks after hospital

discharge. The T-tube was removed 2 weeks later to confirm duct healing. Results: In 2 patients the postoperative

course was very uneventful, while in one patient its was a stormy from repeated vomiting and pre-renal failure. Hospital

stay ranged from 3-5 days. Serum bilirubin returned back to normal levels on the time of T-tube cholangiography. No

reported mortality or procedure-related complication. Conclusions: Cholecystobiliary fistula despite its rarity, is very

important clinical problem. Preoperative diagnosis is crucial to avoid intraoperative bile duct injury. MRCP is a non-

invasive tool for diagnosis, whereas CT is important to exclude malignancy. Staging is complicated and treatment is

challenging. Open cholecystectomy and ductoplasty over a T-tube seems to be a simple and safe procedure and can be

applied to almost all stages of Mirizzi syndrome.

Introduction

Cholecystobiliary fistula, Mirzzi Syndrome, is a

rare complication of long standing gallstone

disease, which results from impaction of a large

stone or multiple small stones in the cystic duct or

in the neck of the gallbladder (GB) causing

extrinsic compression and narrowing of the

common hepatic duct (CHD). These currently

bizarre complications, are being encountered in

0.7% to1.4 % of patients with symptomatic

cholelithiasis according to some recent series (1-3).

Pablo Luis Mirizzi, in 1948, was the first to

describe this syndrome that consisted of an

uncommon and benign cause of obstructive

jaundice, caused by a gallstone impacted at the

Hartmann’s pouch, or the cystic duct and believed

that the obstruction of the CHD was rather

functional from contraction of a “muscular

sphincter” located in the CHD. However, current

knowledge has dismissed the presence of any

sphincter, or even smooth muscle, in the CHD (4-

6). In 1982 McSherry et al (5) and in 1989 Csendes

et al(6), published seminal articles describing the

pathophysiological process involved in the

development of Mirizzi syndrome, including the

initial external compression of the bile duct by

impacted gallstones until the erosion of the

gallstones through the GB wall into the bile duct.

Both authors proposed similar classification of

this syndrome; where type I is due to simple

compression of the impacted stone on the CHD

and other types represent various degrees of

erosion and fistulation of the impacted stone

through GB wall into the lumen of the bile duct (5&6). The importance and implications of this

condition are related to their associated, and

potentially serious, surgical complications such as

bile duct injury, and to its modern management

when encountered during laparoscopic

cholecystectomy. The incidence of bile duct

injuries in patients operated on with Mirizzi

syndrome without preoperative diagnosis could be

as high as 17% (7). Therefore, the preoperative

diagnosis of this syndrome & its type is very

crucial for well-planned and safe surgical

intervention. Preoperative diagnosis of Mirizzi

syndrome is difficult and can be made in only 8%

to 62.5% of patients (8). In recent years, the use of

computed tomography(CT), endoscopic

retrograde cholangio-pancreatography (ERCP),

and magnetic resonance imaging of the bile duct

(MRCP) have allowed for a preoperative

diagnosis and, therefore, improve surgical results (7-9). The treatment of this syndrome is a surgical

challenge and considered as a trap for surgeons

because of the severe inflammatory process with

thick dense hard adhesions and associated

edematous tissues that distort the anatomy of the

cholecystohepatic triangle leading to an increase

in the risk of biliary duct injury. Furthermore, the

surgical treatment of Mirizzi syndrome avoids a

truly standardized approach and must be

individualized depending on the stage of the

disease and the expertise of the surgical team.

However, surgery for Mirizzi syndrome consists

of cholecystectomy, either classical or subtotal, as

well as drainage of the external bile ducts (10&11).

In this article, we present 4 cases of different

degrees of cholecystobiliary fistulas after precise

preoperative diagnosis and a simple surgical

strategy consisting of open cholecystectomy and a

direct repair of the bile duct on a T-tube.

Case Presentations

The preoperative clinical, laboratory and

radiological data of all 3 cases are presented in

table 1.

Table (1): The Preoperative clinical, serological and radiological data of all 3 cases

CASE 1 CASE 2 CASE 3

Age 60 years 62 years 63 years

Gender Male Male Female

Presentation Icterus

Itching

Deep colored urine

Upper abdominal discomfort

Known GB stone > 1 year

Icterus

Anorexia

S Bilirubin 5.4/3.2 mg% 11.5/9.5 mg/l 1.9/1.02 mg/l

Alk. Phos. 371/129 U/L 286/147 U/L 130/140 U/L

Gamma-GT 125/50 U/L 93/50 U/L 960/50 U/L

SGPT 69/41 U/L 49/41 U/L 144/42 U/L

SGOT 54/40 U/L 42/40 U/L 110/38 U/L

CA 19.9 7132/37 U/L >1000 U/L 89/37 U/L

US

Minimal IHBRD

Thick wall GB

Large stone in the CHD

Minimal ectasia of the IHBR.

Thick GB wall with echofree lumen.

CBD 8.3 mm with definite stones.

Large sized stone in the neck of

the GB.

No IHBRD

ERCP

Dilated biliary tree with

pattern of Mirizzi

syndrome (type III)

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

MRCP -------------

Marked IHBRD

Marked CBD dilatation 17 mm

A 9 mm stone entrapped in the

proximal part of the CBD (type II

MS)

GB distended with no stones

Large sized calculus at the neck

region of the GB exerting a

significant extrinsic compression

with subsequent mild to

moderate IHBRD.

Normal caliber of the CBD

Distended GB

Triphasic Spiral CT ---------------

10 mm stone in the CBD opposite the

cystic duct insertion associated with

marked IHBRD

CBD 15 mm

NO ductal mural lesion or thickening

GB stone 3 cm partially

impacted within the neck of the

GB with relative thickened

edematous walls, suggesting a

Mirizzi syndrome (type I)

IHBRD: intrahepatic biliary radicles dilatation. GB: gall bladder

All patients were admitted to hospital one day

before surgery for preparation by the

administration of intravenous fluids, antibiotics

and osmotic diuretics. Under general anesthesia,

all patients underwent laparotomy through a right

subcostal incision. The GB was markedly

contracted and thick walled in patients 1 & 2,

whereas it was distended in case 3. In all patients

the external bile ducts were thick walled &

dilated. A fundus-first dissection of the GB out

of its bed (retrograde cholecystectom) was done

reaching the Calot's triangle & cutting in the

assumed cystic duct, a large stone comes out with

bile flow noticed. Wash of the CBD with warm

saline was done. Distal CBD patency confirmed

by Pick's dilators and then a T-tube (18 0F) was

inserted into in the CBD defect and bile flow in

the T-tube confirmed. The CBD defect was

repaired (ductoplasty) over the T-tube using 3/0

vicryl sutures. A catheter drain was inserted in the

Morrison's pouch and the abdomen closed in

layers. The day after surgery, the patients started

oral fluids. On the 3rd day all patients underwent

US examination to exclude localized

intraperitoneal collection, and accordingly

patients discharged from hospital, after removal of

the abdominal catheter drain, while the T-tube left

in place for later radiologic examination. Two

weeks after surgery, a T-tube cholangiography

was done to confirm patency of the CBD. The T-

tube removed two weeks later on, after

intermittent clamping & de-clamping to ensure

patency and exclude cholangitis. Long-term

follow-up (within one year) was done by regular

clinical examination, serum bilirubin, and

Ultrasonography on 3 month basis. No procedure-

related complications nor mortality could be

detected.

Table (2) The postoperative data of the 3 patients are summarized:

CASE I

(MS Type III) CASE II

(MS type II) CASE III

(MS type I)

Postoperative course

Uneventful course, Mild

fever,

No collection detected by

US

Stormy course from

repeated vomiting &

prerenal failure (acute

tubular necrosis),

No collection detected by

US

Uneventful course,

Mild fever

No collection detected by US

Post. Op Hospital stay 3 days 5 days 3 days

T-tube cholangiography

Opacified bile ducts and

duodenum without

leakage.

No evidence of filling

defect or stricture

Non-obstructed and non-

impede flow of the contrast

along the entire course of

the extrahepatic ducts. Still,

mild dilatation of the biliary

ductal system

Mild IHBRD

Normal filling of the CBD till the

lower end with no evidence of filling

defect or stricture. Free flow dye to

the duodenum & jejunum in the

delayed films

S.Bilirubin (total/direct)

mg%

On discharge: 3.7/2.5

Before T-tube removal

0.86/0.34l

On discharge: 8.9/6.8

Before t-tube removal:

3.4/2.5

Normalized serum bilirubin 0.97/0.29,

on discharge & thereafter.

CA19.9 258.1/37 U/L 44/37 U/L

Histopathology

Chronic

xanthogranulomatous

cholecystitis

No malignancy detected

Chronic calcular

cholecystitis

No malignancy detected

Chronic calcular cholecystitis

No malignancy detected

CASE 1 (type III MS) ERCP revealed type III Mirizzi syndrome (> 50% of the BD circumference)

Intraoperative picture with contracted GB and markedly dilated bile duct

T-Tube cholangiography with free flow of dye to the duodenum, no filling defect nor stricture in the CBD

CASE 2 (type II MS)

MRCP Picture with MS type II with a sizable stone in the CHD

Triphasic CT Picture with IHBRD and a 9 mm stone in the CHD

Intraoperative picture showing a defect in the CBD

T-tube cholangiography showing free flow of the dye into the duodenum & jejunum

CASE 3 (type I MS) MRCP Picture for MS type I with 3 cm stone in the GB neck:

CT Picture of MS type I with a sizable 3 cm stone compressing the CHD

Intraoperative Picture for MS type I before (A) and after stone extraction (B)

Large stone and severely contracted, thick wall gall bladder

T-tube cholangiography with free flow of dye into the duodenum

Discussion

Chlecystobiliary fistula and its eponyme (Mirizzi

syndrome) is a rare complication of long standing

cholelithiasis, with an incidence rate ranging from

0.7 to 1.4% of all patient undergoing

cholecystectomy (12). The modern definition of

Mirizzi’s syndrome is thought to include four

components: anatomic arrangement of the cystic

duct at the gallbladder neck such that it runs

parallel to the common hepatic duct; impaction of

a stone in the cystic duct or neck of the

gallbladder; mechanical obstruction of the

common hepatic duct by the stone itself or by

secondary inflammation; and intermittent or

constant jaundice causing possible recurrent

cholangitis and, if longstanding, secondary biliary

cirrhosis (13). Therefore, MS and cholecystobiliary

fistulae appear to be different, evolving stages of

the same pathological condition, thus it is

reasonable that lubbers in 1983 (14) proposes that

the term MS can now be abandoned, since it is

only the first stage of a more complex process.

The importance of Mirizzi syndrome lies in that it

poses 3 clinical problems: firstly; the preoperative

diagnosis and staging are very difficult, secondly;

it is crucial to exclude malignancy in the gall

bladder and/or the biliary tree, and thirdly; the

surgical treatment is very challenging as it is

associated with a high degree of bile duct injury

and it depends largely on the surgeon's

experience. In the present report we described

three cases with different stages of Mirizzi

syndrome, encountered within one year. Taking in

consideration the rarity of the condition all over

the World and especially after the early diagnosis

and treatment of gall stones in the modern era, we

consider the 3 cases as good to describe.

However, most of the reports dealing with this

problem in the literature consist of case reports (3).

The preoperative diagnosis of Mirizzi syndrome is

of utmost importance for thoughtful surgical

planning and to avoid intraoperative bile duct

injuries. In the present article, we suspected the

presence of MS on the basis of MRCP in two

cases, while in the first case ERCP was the

diagnostic tool. Ultrasonography was an

important initial examination to detect a

contracted gall bladder in 2 cases, dilated

intrahepatic biliary radicles, while the common

bile duct was within normal size under the level of

obstruction in all cases. These later criteria are not

specific for Mirizzi syndrome and, therefore, we

could not rely on. However, the reported

diagnostic accuracy for ultrasonography in MS is

29%, with a reported sensitivity varying from

8.3% to 27% (8&9). The MRCP or ERCP-based

diagnosis of MS was accurate in all our 3 cases.

Some typical features of Mirizzi syndrome can be

shown by MRCP such as the extrinsic narrowing

of the common hepatic duct, a gallstone in the

cystic duct, dilatation of the intrahepatic and

common hepatic ducts, and a normal choledochus.

These features were evident in the 2 cases where

MRCP was the diagnostic tool (table 1). Magnetic

resonance imaging can also show the extent of the

inflammatory process surrounding the gallbladder

and has the advantage of avoiding the

complications associated with endoscopic

cholangiography. However, the diagnostic

accuracy for MRCP is 50% (7-9). In the first case,

ERCP was applied in an attempt to remove the

US-detected stone in the Cystic-CBD junction.

The endoscopic trial failed because of the large

sized stone (3 cm) and its location high-up in the

cystic-CBD junction. It is known that ERCP

besides being diagnostic allows the performance

of a sphincterotomy for stone extraction and

facilitates other interventions such as the

placement of stents or a nasobiliary tube or other

procedures (11 & 15). In all our 3 cases, a triphasic

spiral CT examination was performed in order to

exclude the presence of malignancy either in the

gall bladder or in the external bile duct. The main

utility of CT would be the exclusion of

malignancy in the porta hepatis area or in the

liver. However, the presence of periductal

inflammation can be misinterpreted as gallbladder

cancer (16). This was very important examination

because, in the three described cases, the serum

level of CA19.9 was markedly elevated especially

in the first (7132 U/L) and second (> 1000 U/L)

patient (table 1). CA19.9 is a carbohydrate antigen

of cellular surface and known as a tumor marker

measured in patients with malignant diseases of

the pancreas and biliary tree. The elevation of CA

19-9 in benign diseases is reported in literature,

both in biliopancreatic diseases as in other sites of

gastrointestinal system. Expressive levels,

however, are uncommon (17). Till nowadays,

only four cases are reported and presenting CA

19-9 > 20,000 U / ml. This elevation of CA19.9 in

association with MS may be explained by the

associated cholestasis and recurrent cholangitis

which is a hall mark in Mirizzi syndrome (17 & 18).

Classification and staging of Mirizzi syndrome

evolved continuously over time, where Mirizzi

described type I with external compression of the

common hepatic duct by the impacted stone in the

gallbladder neck or cystic duct. McSherry et al (5)

in 1982, classified the Mirizzi syndrome into two

types based on ERCP findings. Type I involves

the external compression of the bile duct by a

large stone or stones impacted in the cystic duct or

in the Hartmann’s pouch. Type II consists of a

proper cholecystobiliary fistula, caused by a

gallstone or gallstones that have eroded into the

bile duct. In 1989 Csendes et al (6), modified the

classification of McSherry by dividing Mirizzi

syndrome into four types. Csendes type I

corresponds to McSherry type I. Mirizzi

syndrome type II consists of a cholecystobiliary

fistula resulting from erosion of the bile duct wall

by a gallstone, the fistula must involve less than

one-third of the circumference of the bile duct.

Mirizzi syndrome type III consists of a

cholecystobiliary fistula involving up to two-

thirds of the bile duct circumference. Mirizzi

syndrome type IV is a cholecystobiliary fistula

with complete destruction of the bile duct wall

with the gallbladder completely fused to the bile

duct forming a single structure with no

recognizable dissection planes between both

biliary tree structures. In 2008, Beltran and

Csendes added one more type; the Mirizzi type V,

which includes the presence of a cholecysto-

enteric fistula together with any other type of

Mirizzi (19). However, on the light of its rarity (1-3),

the fact that over 50% of patients with Mirizzi

syndrome are diagnosed during surgery (8), these

reasons preclude the design of randomized trials

or large cohorts to validate these complicated on-

going classifications. Furthermore, type V MS

(with cholcysto-enteric fistula) mostly presents

without obstructive jaundice which is a hallmark

for Mirizzi's concept. This is because of drainage

of the biliary tree into the bowel. We may,

therefore, suggest a simple and more practical

diagnostic classification on the light of our 3

patients with different stages of Mirizzi syndrome.

We suggest type I MS for the mere external

compression with intact walls of the bile duct,

type II when there is a cholecystobiliary fistula

involving < 50% of the bile duct circumference,

and type III fistula whenever a fistula involves >

50% of the bile duct circumference can be

encountered. This suggested classification can

also guide preoperative treatment strategies as

will be shown below. The treatment of Mirizzi

syndrome is surgical, which is very demanding

and challenging for surgeons. The surgical

difficulties of this syndrome lie on three reasons:

firstly; the preoperative diagnosis is always

lacking, secondly; the surgical treatment of this

condition is associated with a significantly

increased risk of bile duct injury and finally; the

severe inflammatory process with thick dense

hard adhesions and associated edematous tissues

distort the anatomy (10 & 15).On the other hand,

there is no consensus for stage-related treatment

guidelines or algorithm because of the rarity of

the syndrome and lack of well designed trials.

Therefore, treatment must be individualized

depending on the stage of the disease and the

expertise of the surgical team (11). In the current

report, all cases underwent open surgery for

complete cholecystectomy (fundus-first

approach), extraction of the impacted stone (in all

cases it was solitary sizable stone), and repair of

the defect in the CBD (ductoplasty) over a T-tube 0F18. The technique was successful in all cases

with different stages of MS. Patency of the biliary

system was confirmed by postoperative T-tube

cholangiography, which proved the free flow of

contrast into the bowel in all cases. Our policy in

treatment was simple and applicable in all cases

especially when starting with fundus-first

approach of the gallbladder avoiding dissection

into the severely inflamed and dense Calot's

triangle, followed by simple ductal repair by

utilizing a thin rim of the gallbladder remnant (5-

10mm) and by using the traditional T-tube as a

stent until secure healing of the bile wall defect.

However, it has been assumed that Subtotal

cholecystectomy may be the best treatment for

MS type I and most cases of Mirizzi type II and

III (20). Subtotal cholecystectomy was described in

1985 by Bornman et al (21) for difficult open

cholecystectomy in patients with severe

cholecystitis associated with liver cirrhosis and

portal hypertension. Since then, this technique has

been also applied to cases of Mirizzi syndrome (10,

11 & 22). In our cases, formal cholecystectomy was

achieved on contrast to the suggested subtotal

cholecystectomy. Moreover, in the first patient

with type III Mirizzi ductoplasty over a T-tube

was applicable and we did not resort to a bilio-

enteric anastomosis as has been suggested in the

literature for stages III & IV (10 &11). The rationale

for a biliary-enteric anastomosis is that because of

the continued inflammation of the gallbladder

flap, strictures can develop, with an even greater

risk for large cholecystocholedochal fistulas or

badly damaged bile ducts (23 & 24).

Picture for the 3 MS types: A suggested classification

However, bilioenteric anastomosis in the setting

of infection and tissue edema is not without risk

of leakage. Moreover, it is a demanding technique

necessitating a great surgical experience in

contrast to the simple ductoplasty over the

traditional T-tube. Therefore, we preferred the

later approach taking into account the dilated duct

at the site of fistula, the utilization of a small

remnant of the gallbladder wall in repair and

keeping the integrity of the bile duct for further

postoperative leakage or stricture for endoscopic

or transhepatic approaches to treat expected

complications. Finally, the role of laparoscopic

cholecystectomy in the management of Mirizzi

syndrome is very debatable and requires very

advanced skill in laparoscopic surgery, yet,

whenever a preoperative diagnosis is solid, it is

preferable to start with direct open approach (25) .

On conclusion, the cholecystobiliary fistula is a

rare complication of longstanding gallstone

disease, and it preoperative diagnosis and staging

are mandatory for safe and well planned surgery.

On view of its rarity and the low rate of

preoperative diagnosis, the 3-stage classification

as suggested above (see illustration) is simple and

very practical. Surgery for Mirizzi syndrome is

very challenging and must be individualized

according to the fstula stage and experience of the

operating team. However, classical or subtotal

cholecystectomy are feasible and ductoplasty over

a T-tube is a very practical and accessible

technique allowing sound duct healing and

maintaining the duct integrity for further

manipulations whenever indicated.

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relationship of Mirizzi syndrome and cholecystoenteric

fistula: validation of a modified classification. World J

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P, Blumgart LH. Management of the Mirizzi syndrome

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Original Article

Diagnostic Accuracy of Ascitic Fluid Leucocyte Esterase and Lactoferrin in

Cirrohtic Patients with Spontaneous Bacterial Peritonitis

Ayman El Shayeb1, Akram El Deghady2, Rania Abou Youssef 3, Eman Elabsawy4

1:Professor of Tropical Medicine, 2:Professor of Clinical and Chemical Pathology, 3:Lecturer of Tropical

Medicine, 4:Instructor of Tropical Medicine.

ABSTRACT

SBP is the most frequent infectious complication (10-30%) of patients with cirrhosis and ascites, with high recurrence

rate (up to 70% in the first year).(1,2). Aim of the work: The aim of the present work was to validate the diagnostic

efficacy of the leukocyte esterase reagent strip test and ascitic fluid lactoferrin level for rapid, bedside diagnosis of SBP

in cirrhotic patients and to compare them to the classical way of diagnosis by using PMNL count. Subjects and

Methods: The study was conducted in 50 patients with cirrhosis and ascites divided into two groups. Group I consisted

of 25 patients without SBP,while group II had 25 patients with SBP. Ascitic fluid lactoferrin was measured using an

ELISA kit and leukocyte esterase was tested by reagent strips. Results: There was a significant difference between the

two studied groups as regard LERS as In group I,LERS was negative in 100% of patients, while in group II it was

negative in 52% ,and positive in 48% . The mean AFLAC was significantly higher in SBP patients than in patients

without SBP. significant positive correlation was found between LERS and ascitic fluid PMNLs. AFLAC had

Significant positive correlation with ascitic fluid PMNLs and significant negative correlation ascitic fluid Albumin.No

significant difference was found between the LERS results (++ and +++) as regard their relation with each of ascitic

fluid PMNLs, ascitic fluid albumin and AFLAC. Conclusions: Our findings suggest that AFLAC can serve as a

sensitive and specific diagnostic test for SBP patients.The positive correlation between each of AFLAC and LERS with

ascitic fluid PMNLs which indicates that their presence is proportional to the flux of neutrophils. LERS is rapid,

feasible and low-cost tests with high specificity, PPV and NPV for diagnosing of SBP in cirrhotic patients.

Introduction

SBP is considered the commonest infectious

complication in cirrhosis with ascites (1). the

diagnosis of SBP is confirmed based on a PMN

count in the ascites of > 250 cells/mm3 in the

absence of an intra-abdominal and surgically

treatable source of infection.(3). As a result of the

potential for delays in the diagnosis of SBP, there

has been interest in developing a surrogate test

that should have a high sensitivity and a low false-

positive rate (4). The use of leukocyte reagent

strips (LERS) has been proposed as a fast and

inexpensive method for diagnosing SBP (5,6).The

LERS test is based on the esterase activity of the

leucocytes.3-Hydroxy-5-phenyl-pyrrole esterified

with an amino acid is used as the substrate which

hydrolysis by the esterase releases 3- hydroxy-5-

phenyl-pyrrole, which in turn reacts with a

suitable diazonium salt, yielding a violet azo dye

in the relevant pad of the strip, the intensity of

which correlates to the leukocyte count(7). Of

growing interest,as a diagnostic marker is

measurement of Leukocyte-Derived Proteins such

as lactoferrin, released by activated PMNs which

are elevated in patients with SBP (8). The aim of

the present work was to validate the diagnostic

efficacy of the leukocyte esterase reagent (LER)

strip test and ascitic fluid lactoferrin level for

rapid, bedside diagnosis of SBP in cirrhotic

patients and to compare them to the classical way

of diagnosis by using PMNL count.

Patients and Methods

Patients and diagnosis: This study was

conducted on 50 cirrhotic ascitic patients admitted

to Tropical medicine department of Alexandria

Main University who were divided into two

groups. The first group consisted of 25 patients

without SBP, while group II contained 25 patients

with SBP. Liver cirrhosis and ascites were

diagnosed based on signs and symptoms of

chronic liver disease, abnormal laboratory

findings consistent with the disease as well as

evidence of cirrhotic changes and ascites by

ultrasound.SBP was diagnosed based on ascitic

fluid detection of PMNL count more than 250

cell/mm3 and ascitic fluid culture(9). Patients with

history of chronic diarrhea,active GIT

bleeding,chronic renal disease,abdominal surgery

within 3 months of study entry, diabetes mellitus

or hypertension were excluded from the study.

Estimation of ascitic fluid lactoferrin by

ELISA kit assay (10) : Centrifuge ascitic fluid for

10 minutes. Collect supernatants and store the

remaining samples at -20°C or below while the

assay is performed at temperature 20-25°C. This

assay employs a quantitative sandwich enzyme

immunoassay technique, which measures

lactoferrin in less than 4 hours.

Ascitic fluid detection of leukocyte esterase by

reagent strips(11): Immerse the reagent area of the

strip in the ascitic fluid sample and remove

quickly. Hold the strip horizontally and compare

the result on the strip with the colour chart on the

bottle label between 1-2 minutes after dipping

according to colorimetric scale readings(4-grade

scale (negative, 1+ to 3+)).Positive results(+ or

greater) are clinically significant.

Statistical Analysis

Data were fed to the computer and analyzed using

IBM SPSS software package version 20.0

(12).Qualitative data were described using number

and percent. Quantitative data were described

using range, mean, standard deviation and

median. Comparison between different groups

regarding categorical variables was tested using

Chi-square test. When more than 20% of the cells

have expected count less than 5, correction for

chi-square was conducted using Fisher’s Exact

test or Monte Carlo correction. The distributions

of quantitative variables were tested for normality.

If it reveals normal data distribution, parametric

tests was applied. If the data were abnormally

distributed, non-parametric tests were used. For

normally distributed data, comparison between

two independent population were done using

independent t-test. For abnormally distributed

data, comparison between two independent

population were done using Mann Whitney test.

Significance of the obtained results was judged at

the 5% level.

Results

Fifty cirrhotic patients with ascites were enrolled

in this study and divided into two groups.In group

I (without SBP) ,14 were female and 11 were

male with mean age of 52.68±7.16 years.In group

II (with SBP) 12 were female and 13 were male

with mean age of 52.88±7.21 years.

Ascitic fluid polymorphnuclear leukocytes

(PMNLcell/mm3): In group I, ascitic fluid

PMNLs ranged from 20 to 110 /mm3 with a mean

of 50±31.21/mm3, while in group II it ranged from

300 and 1000/mm3 with a mean of

663.84±220.64/mm3. The mean Ascitic fluid

PMNLs was significantly higher in group II than

in group I.

Ascitic fluid leukocyte esterase by leukocyte

esterase reagent strips(LERS): In group I,LERS

was negative in 100% of patients, while in group

II it was negative in 52% , positive(+) in 4%, (++)

in 28% and (+++) in 16% .There was a significant

difference between the two studied groups.

Sensitivity, specificity and accuracy for LERs

with SBP (Table 1).

Table (1): Agreement (sensitivity, specificity and accuracy) for LERs with SBP

Without

SBP With SBP

Sen

siti

vit

y

Sp

ecif

icit

y

PP

V

NP

V

Acc

ura

cy

LERs -ve 25 13

48.0 100.0 100.0 65.79 74.0 +ve 0 12

Ascitic fluid Lactoferrin (AFLACng/ml)

(Figure 1): In group I, AFLAC ranged from 2.5to

25.0 ng/ml with a mean of 10.14±8.10 ng/ml,

while in group II it ranged from 200to 2600 ng/ml

with a mean of 567.6±514.78 ng/ml. The mean

AFLAC was significantly higher in group II than

in group I. Sensitivity, specificity and accuracy

for AFLAC with SBP (Table 2).

Table (2):Agreement (sensitivity, specificity and accuracy) for AFLAC with SBP

Without

SBP With SBP

Sen

siti

vit

y

Sp

ecif

icit

y

PP

V

NP

V

Acc

ura

cy

Lactoferrin ≤25 25 0

100.0 100.0 100.0 100.0 100.0 >25 0 25

Figure (1): ROC curve for Lactoferrin to diagnose SBP

AUC p

Lactoferrin 1.000* <0.001

Correlation between LERS and AFLAC with

PMNLs and Ascitic fluid Albumin: In group I

(without SBP), AFLAC had Significant positive

correlation with ascitic fluid PMNLs (rs=

0.563)(p=0.003) and significant negative

correlation ascitic fluid Albumin(rs=-

0.417)(p=0.038). In group II (with SBP),AFLAC

had significant positive correlation with ascitic

fluid PMNLs (rs=0.401)(p=0.047), and significant

negative correlation with ascitic fluid Albumin

(rs= -0.565)(p=0.003). significant positive

correlation was found between LERS and ascitic

fluid PMNLs (rs=0.722)(p=<0.001) but no

significant correlation with ascitic fluid Albumin

(rs=-0.025) (p=0.906) in groupII.

Discussion

In the present work, the AFLAC in patients with

SBP was significantly higher than patients

without SBP.Moreover, we found a significant

positive correlation between AFLAC and ascitic

fluid PMNLs, which is in favor with our

hypothesis because Lactoferrin is released from

polymorphonuclear leukocytes on activation of

these cells, and its presence in body fluids is

proportional to the flux of neutrophils.

Furthermore, there was a significant negative

correlation between AFLAC and ascitic fluid

Albumin, which is also in favor with our

hypothesis because it's prooved that patients with

low AF proteins (below 10 g/l)are more liable to

develop SBP during their hospital stay (13). So,it is

logic for AF Albumin to be lower in SBP and that

was confirmed by our work as AF albumin was

significantly lower in AF samples from SBP

patients than non SBP patients.The highest

combined sensitivity and specificity of AFLAC to

detect SBP was achieved at the cut-off level of 25

ng/mL. Our result is in accordance with Parsi MA

et al(10) who found that AFLAC concentration in

SBP samples was significantly higher than the

non-SBP samples. On the other hand, Parsi MA et

al showed that AFLAC, at the cut-off level of 242

ng/mL, had its highest combined sensitivity and

specificity.At this cut-off level, the sensitivity and

specificity of the test were 95.5% and 97%

respectively. The discrepancy between our result

and that of Parsi’s may be attributed the small

number of patients with SBP in our study (n=25)

and in Parsi’s study (n= 22) which makes it

difficult to determine a definitive cut-off value for

ascitic fluid lactoferrin. In the present study, there

was a significant difference between the two

studied groups as regard LERS.In groupI, LERS

was negative in 100% of patients.While in group

II it was negative in 52% ,1+ in 4% ,2+ in 28%

and 3+ in 16% .The sensitivity, specificity,

positive predictive value, negative predictive

value and Accuracy of leukocyte esterase dipstick

test to diagnose SBP were 48%, 100%,

100%,65.79, 74% respectively. There was

descripancy between most of the studies about

LERS which may be caused by several

factors.False positive results in some of them can

arise from detection of leukocyte esterase

originating other than from leukocyte such as

pancreas. While false negative resuts may be

attributed to that the PMN were not activated, and

also can result from alteration of pH, osmolality

and temperature of the specimens(14). With

Multistix®10SG (used in our study) , there were

many factors that influence the accuracy and

sensitivity of the dipsticks. False negative results

may be attributed to the smaller number of PMN

cells in these specimens.Also if any of the patients

received antibiotics prior to abdominal

paracentesis, that would result in false negative

result. Our study shows that the specificity of

reagent strips is very high in the diagnosis of

SBP(100%), so the strip may be helpful for the

clinician when the test is positive.In summary, our

study shows the excellent specificity of LERS in

the diagnosis of SBP, but reveals the poor

sensitivity of the test. Therefore, it should not

systematically replace standard ascitic fluid

analyses because of its weak sensitivity and

because it cannot rule out SBP.

Conclusions

Our findings suggest that ascitic fluid lactoferrin

(AFLAC) can serve as a sensitive and specific

diagnostic test for SBP in cirrhotic patients with

ascites with highest combined sensitivity and

specificity at the level of 25 ng/mL.The observed

significant negative correlation between AFLAC

and ascitic fluid albumin shows that patients with

SBP tend to have low ascitic fluid albumin

.Rreagent strips (LERS) are rapid, feasible and

low-cost tests with high specificity, PPV and NPV

for diagnosing of SBP in cirrhotic patients but

with poor sensitivity(48%). Therefore, it should

not systematically replace standard ascitic fluid

analysis for PMNLs.Significant positive

correlation between both AFLAC and LERS with

ascitic fluid PMNLs indicates that their presence

is proportional to the flux of neutrophils.

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Original Article

Interaction of Young and Adult Biomphalaria Alexandrina Snails with

Schistosoma Mansoni

Safaa Ibrahim Khedr 1, Hayam Abd El-Moniem Sadaka 2, Iman Fathy Abou-El Naga2, Iman Hassan Diab 3,

Eglal Ibrahim Amer 41 Assistant lecturer of Medical Parasitology, 2 Professor of Medical Parasitology, 3

Professor of Medical Biochemistry, 4 Assistant Professor of Medical Parasitology.

ABSTRACT

Schistosomiasis mansoni is one of the greatest health problems in Egypt. Biomphalaria alexandrina represents the

intermediate host snail of Schistosoma mansoni in Egypt. Targeting this snail by different means can for sure decrease

the risk of disease transmission. To make this goal a reality, snail bionomics should be thoroughly studied. Therefore,

Aim of the work: studying the impact of Biomphalaria alexandrina snails’ age on their compatibility to Schistosoma

mansoni infection, using different parasitological parameters. These included; pre-patent period, infection rate and total

cercarial production. Susceptible and resistant snails were reared singly for self-reproduction. Of their progeny, four

subgroups underwent our experiment. These are; young susceptible, adult susceptible, young resistant and adult

resistant subgroups. Young susceptible subgroup showed the highest infection rate being 92%, the shortest pre-patent

period and the highest total cercarial production of 151002. This was followed by the adult susceptible subgroup with

infection rate of 74% and total cercarial production of 41732. Young resistant subgroup possessed infection rate of 37%

with total cercarial production of 9877. While adult resistant subgroup contained only resistant members. Conclusion :

These results give a clue for the higher resistance found in adult aged Biomphalaria Alexandrina snails when compared

to their young peers even if they were obtained from the same parents. Identification of most susceptible snail’s age

determines best timing for applying molluscicides. Moreover, adult resistant snails could be beneficial in biological

snail control. Hence, these results provide potential implications in Biomphalaria control.

Introduction

Schistosomiasis is a major neglected tropical

disease. It is ranked second to malaria in terms of

its socioeconomic and public health impact in

tropical and subtropical areas. It represents a

great risk to health in developing countries,

including Egypt, with about 249 million people

infected all over the world.(1). The wide spread

distribution of Schistosoma mansoni (S. mansoni)

infection is permitted by the broad geographic

range of susceptible species of the pulmonate

freshwater snail, genus Biomphalaria. These

snails serve as obligatory hosts for the larval

stage.(2) There are more than 34 identified species

of Biomphalaria, among which Biomphalaria

alexan- drina (B. alexandrina) is the intermediate

host of S. mansoni in Egypt.(3). To control S.

mansoni transmission, many methods were

applied. Of these, targeting the snail host by

different means is very effective and at the same

time challenging goal. (1,2) To make this goal a

reality, snail bionomics should be thoroughly

studied. Natural populations of Biomphalaria are

polymorphic regarding their compatibility with S.

mansoni, with susceptible and resistant variants.(4)

Susceptible Biomphalaria snails, are those which

allow the invasion of S. mansoni miracidia, their

development into sporocysts with emergence of

their cercariae. In resistant ones there is a failure

of this life cycle of the parasite to complete.(5)

Many factors are known to affect Biomphalaria

susceptibility to S. mansoni, which are either

related to the parasite, or to the intermediate

host.(6) Snail related factors include, their genes

and age at the time of exposure to miracidia.(6-8).

Resistance of Biomphalaria glabrata to S. mansoni

infections appears to be age-dependant and

controlled by at least four genes, each with several

alleles. Snails resistant as juveniles usually remain

resistant throughout their life, although they may

show variable outcomes on becoming adults.

While snails susceptible as juveniles can become

resistant at maturity. (9) However, little is known

about the effect of B. alexandrina age on its

susceptibility to S. mansoni. The present work

aimed at studying the impact of B. alexandrina

snails’ age on the compatibility patterns to S.

mansoni using different parasitological

parameters.

Methods

Laboratory breeding of the snails: Snails were

bred in plastic containers, in well aerated aged

de-chlorinated tap water (DTW) that was

changed twice weekly. These were maintained at

room temperature under normal laboratory

illumination. Snails were fed on lettuce. Pieces of

foam were placed for egg deposition.(10)

Selection of susceptible and resistant snails:

Selection of resistant and susceptible isolates were

achieved according to Zanotti-Magalhães, et al.(11)

For selection of resistant isolate, snails that

remained uninfected after two parasitic expo-

sures (10 miracidia/snail) were isolated and reared

singly for selfing. While, for selecting susceptible

isolate, progeny of snails that yield high infection

frequencies, were isolated and reared singly for

selfing.(11) These snails were used as experimental

snails.

Maintenance of S. mansoni life cycle: S.

mansoni cycle was maintained in susceptible B.

alexandrina snails and laboratory bred male Swiss

strain albino mice. Those were kept under

standard living conditions. Each mouse was

infected by 100-120 cercariae using paddling

technique. Seven weeks after cercarial

penetration, S. mansoni eggs were collected from

livers of infected mice to be the source of

miracidia. Then, each snail was infected by 8-10

S. mansoni miracidia, in 3 ml of DTW, at room

temperature, at day light for 3-4 hours. Four

weeks later, snails were used to shed cercariae. (10)

Experimental design: This included two main

groups; susceptible and resistant groups.

Group I: Susceptible snails: Two hundred

young susceptible (subgroup Ia) and two

hundred adult susceptible (subgroup Ib) snails

were used in this work. Young snails were

infected on reaching the age of two months and

the size of 3-4 mm in diameter. Adult snails were

infected at the age of four months and the size of

8-10 mm in diameter.

Group II: Resistant snails: Two hundred young

resistant (subgroup II a) and two hundred adult

resistant (subgroup II b) snails were used in this

work. Young snails were infected on reaching the

age of two months and the size of 3-4 mm in

diameter. Adult snails were infected at the age of

four months and the size of 8-10 mm in diameter.

Snails of the previously mentioned subgroups

were exposed individually to 8-10 S. mansoni

miracidia under the previously mentioned condi-

tions. (10) Then, exposed snails of each subgroup

Ia, Ib, IIa and IIb were be put in a separate

container and were maintained for 4 weeks in

darkness. (10)

Determination of susceptibility and resistance

of the snails: Four weeks after snail exposure to

infection, snails were checked individually for

cercarial shedding for 2 hours in direct sunlight,

twice weekly for three successive weeks. (10)

Susceptible and resistant snails of each subgroup

were separated in different containers.

The following parasitological parameters were

determined for each of the subgroups:

A) Pre-patent period for each snail (PPP): (12)

Starting from the 28th day post exposure to

infection, all subgroups were examined twice

weekly for cercarial shedding till the 49th day post

infection.

B) Determination of the infection rate for each

subgroup (IR): (10) Percentage of susceptible and

resistant snails in each subgroup was determined.

All snails that died during the prepatent period

were crushed between two slides and inspected

under a microscope for immature parasite stages.

C) Mean cercarial output per each susceptible

snail (MCO): (10) Number of cercariae that were

shed from individual susceptible snails were

counted twice weekly for three successive weeks.

Results

In the present study the following results were

obtained:

A)Pre-patent period (PPP): Snails of each

subgroup were examined for cercarial shedding,

individually, twice weekly, starting from the 28th

day after infection till the 49th day. In Subgroup Ia

(Young susceptible) members, the majority of the

shedding snails were recorded by the 28th day

being 37 snails. 27 shed cercariae for the first

time at the 32nd day, 16 shed at the 36th day.

Only 5 shed at the 40th day, no more snails

showed cercariae at the 44th day nor at the 49th

day. Regarding subgroup II a (Young resistant), 2

snails shed cercariae for the first time at the 28th

day, 3 shed at the 32 nd day, 5 shed at the 36th day,

7 shed at the 40th day, 9 shed at the 44th day , and

11 snails shed at the 49th day. Subgroup I b

(Adult susceptible) individuals showed the

following results; 11 snails shed cercariae for the

first time at the 28th day, 14 shed at the 32 nd

day,16 shed at the 36th day, 17 shed at the 40th

day, 9 shed at the 44th day and 7 snails shed at the

49th day. Completely resistant snails were

obtained from subgroup II b (Adult resistant), so,

no PPP was recorded. Statistical analysis of PPP

results revealed significant differences between

subgroup Ia members, subgroup IIa members at

all except the 40th day. Moreover, subgroup Ia

members showed significant differences with

those of Ib members at all days except the 36th

day. Moreover, subgroup Ia members showed

significant differences with those of IIb members

at the 28th, the 32 nd and the 36th days of shedding.

Significant differences were also noted between

subgroup IIa and subgroup Ib members at days

28th, 32 nd, 36th and 40th. Additionally, significant

differences were recorded between subgroups Ib

and IIb members at all days of shedding.

Subgroups Ib and IIb showed differences at the

40th, the 44th day and the 49th day. PPP results are

shown in figure (1)

Figure 1: The pre- patent period among the studied subgroups.

B) Infection Rate (IR): Percentage of

susceptible and resistant snails in each subgroup

was determined. Results showed that 92 snails of

subgroup Ia, 37 snails of subgroup IIa and 74 in

subgroup Ib were susceptible, whereas subgroup

IIb contained only resistant members. Statistical

analysis of the results of different subgroups

showed significant difference between subgroups

Ia – IIa, Ia – Ib, Ia–IIb , Ib- IIa, Ib – IIb and IIa –

II b. Infection rate results are shown in figure (2).

Figure 2: Infection rate among the different studied subgroups.

C) Total cercarial production (TCP) and mean

cercarial output (MCO) per susceptible snail in

each subgroup: TCP was 151002 from

subgroup Ia and 9877 from subgroup IIa. On

the other hand, TCP values of subgroup Ib was

41732, while subgroup IIb showed no cercarial

production at all. The highest mean cercarial

production per snail per shedding time was

recorded in subgroup Ia, followed by subgroups

Ib, and IIa, being 298.1 ± 132.13, 94.13 ± 44.17,

and 44.49 ± 32.35 respectively. MCO Results are

shown in figure(3).

Figure 3: Mean cercarial shedding over three successive weeks per susceptible snail among the different studied subgroups.

Discussion

In the current study, we investigated the effect of

snails’ age on their compatibility pattern. Results

revealed that younger snails whether belonging to

susceptible or resistant groups, showed higher

susceptibility when compared to their adult peers.

Young susceptible subgroup members possessed

the shortest range of PPP, where the majority of

the shedding snails were recorded by the 28th day

being 37. Moreover, the rest of shedding snails in

the same subgroup showed their first shedding to

be before the 40th day post infection, where 27

shed cercariae for the first time at the 32 nd day,

16 shed at the 36th day and 5 snails shed at the

40th day. Regarding Adult susceptible subgroup

(Ib) members, out of 74 shedding snails, only 11

snails shed for the first time at the 28th day post

infection. The maximum number of shedding

snails was 16 and 17 that were recorded by 36th

and 40th days respectively. Additionally, in

7snails, PPP extended to 49 th day. The

significant differences observed between

subgroups Ia and Ib in the shedding durations

indicate that subgroup Ia (Young susceptible)

carry higher susceptibility to S. mansoni infection,

with rapid development of the parasite inside the

snail tissue. (13). As for subgroup IIa (young

resistant) shedding members, the maximum

number of shedding snails for the first time was

11 that were recorded by 49th day, with only 2

snails that shed for the first time by the 28th day.

On the other hand, members belonging to adult

resistant subgroup IIb, displayed only resistant

phenotype, with no recoded PPP. When compared

to subgroup IIb (Adult resistant) that contained

only resistant members, subgroup IIa (Young

resistant) showed higher susceptibility. These

significant differences between subgroups (Ia) and

(Ib), and between (IIa) and (IIb) can be attributed

to the effect of age. The highest infection rate in

the present study was exhibited by the snails

belonging to the young susceptible subgroup Ia

members. 92% of subgroup Ia (young

susceptible), 74% of subgroup Ib (adult

susceptible), and 37% of subgroup IIa (young

resistant) were susceptible. As for subgroup IIb

(Adult resistant), all members were resistant.

Although our experiment was carried out on snails

resulting from self-reproduction, however

resistant snails were obtained in the progeny of

susceptible members indicating dominance of

resistance character in B. alexandrina. In their

study, Abou El Naga et al. 2010 observed the

appearance of resistant members in snails that

originated from crossing of susceptible

parents.(10). A probable explanation for the

resistant members obtained in the susceptible

subgroups, is that, although resistance alleles are

sometimes hidden from being shown in the snail

phenotype, they group together, forming resistant

phenotype to appear. Their parents carried

unexpressed resistance genes, and the resistance

alleles grouped among successive generations. In

contrary to Lewis et al. (2002), who found that

susceptible B. glabrata parents did not give rise to

any resistant progeny, susceptible subgroups in

the current study gave rise to 8%, 26% resistant

members at different age groups. (14) This could be

explained by the assumption proposed by Abou El

Naga et al. (2010) that, B. alexandrina may

contain more resistance alleles in their susceptible

population than those present in B. glabrata, thus

accounting for the appearance of resistant progeny

originating from completely susceptible parents. (10) In the current study, although the two

susceptible subgroups contained resistance

members, nevertheless the significant difference

noted between them, points to that the resistant

alleles obtained from (susceptible group) F1

parents were potentiated by the impact of age

resulting in the appearance of more resistant

members in the adult susceptible subgroup (Ib).

The highest susceptibility met in young

susceptible subgroup was also evidenced by

showing the highest TCP among the four studied

subgroups, being 151002. This was followed by

the adult susceptible subgroup that produced

41732 cercariae over the three weeks of shedding.

Coming next, the young resistant subgroup had

TCP of 9877 over the shedding weeks. According

to Frandsen classification, the three subgroups

laid in classes 4, 2 and 1 respectively, being well

compatible, poorly compatible and not very

compatible. Regarding adult resistant subgroup,

no cercariae were produced at all, putting this

subgroup in Frandsen class 0 or resistant

snails.(12). These results provide potential

implications in Biomphalaria control.

Identification of most susceptible snail’s age

determines best timing for applying

molluscicides. Moreover, adult resistant snails

could be beneficial in biological snail control.

References

1-WHO. World Health Organization. Schistosomiasis

Fact Sheet, 2014. No.115. http:// www. who.int/

mediacentre/factsheets/fs115/en/.

2- Morgan JA, Dejong RJ, Snyder SD, Mkoji

GM, Loker ES. Schistosoma mansoni and

Biomphalaria: past history and future trends.

Parasitology 2001; 123(l):211-28.

3- DeJong RJ, Morgan JA, Paraense WL, Pointier

JP, Amarista M, Ayeh-Kumi PF, et al. Evolutionary

relationships and biogeography of Biomphalaria

(Gastropoda: Planorbidae) with implications regarding

its role as host of the human bloodfluke, Schistosoma

mansoni. Mol Biol Evol 2001; 18(12):2225-39.

4- Morand S, Manning SD, Woolhouse ME. Parasite-

host coevolution and geographic patterns of parasite

infectivity and host susceptibility. Proc Biol Sci 1996;

263(1366):119-28.

5- Coelho PM, Carvalho OS, Andrade ZA, Martins-

Sousa RL, Rosa FM, Barbosa L. Biomphalaria

tenagophila/Schistosoma mansoni interaction: premises

for a new approach to biological control of

schistosomiasis. Mem Inst Oswaldo Cruz 2004;

99(1): 109-11.

6- Anderson RM, Mercer JG, Wilson RA, Carter N.

Transmission of Schistosoma mansoni from man to

man: Experimental studies of medical survival and

infectivity in relation to larval age, water temperature,

host size and host age. J Parasitol 1982; 85:339-60.

7- Richards CS, Knight M, Lewis FA. Genetic of

Biomphalaria glabrata and its effect on the outcome

of Schistosoma mansoni infection. Parasitol Today

1992; 8:171-4.

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Biomphalaria glabrata hemocytes. Mem Inst Oswaldo

Cruz 2006; 10(1):213-8.

9- Richards CS. Influence of snail age on genetic

variations in susceptibility of Biomphalaria glabrata for

infection with Schistosoma mansoni. Malacologia

1984; 25:493-502.

10- El Naga IF, Eissa MF, Mossallam FS and Abd El-

Halim SI. Inheritance of Schistosoma mansoni

infection incompatibility in Biomphalaria alexandrina

snails. Mem Inst Oswaldo Cruz 2010; 105(2): 149-54.

11- Zanotti-Magalhães EM, Magalhães LA, Carcalho

JF. Relationship between pathogenicity of Schistosoma

mansoni in mice and the susceptibility of the vector

mollusk. IV--Infectiousness of miracidia. Rev Saude

Publica 1997; 31(5):488-94.

12- Frandsen F. Discussion of the relationships

between Schistosoma and their intermediate hosts,

assessment of the degree of host-parasite

compatibility and evaluation of schistosome

taxonomy. Parasitol Research 1979; 58: 275-96.

13- Abou-El-Naga IF, El-Nassery SMF, Allam SR,

Shaat EA and Mady RFM. Biomphalaria species in

Alexandria water channels. Parasitol International

2011; 60: 247–54

14- Lewis FA, Patterson CN and Gizywarz C. Parasite

susceptibility phenotypes of F1 Biomphalaria glabrata

progeny derived from interbreeding of Schistosoma

mansoni resistant and susceptible snails. Parasitol Res

2002; 89: 98-101.

Original Article

Study of Ascitic Fluid Calprotectin in Cirrhotic Patients with Spontaneous

Bacterial Peritonitis

Ayman Mohamed El Lehleh MD1, Somaia Abdel Mohsen Shehab El-deen MD1 Rania Azmi El shazly MD2

Amany Abas Amer M.B.B.Ch1 Tropical Medicine department, Faculty of Medicine, Menoufiya University.

2 Biochemistry department, Faculty of Medicine, Menoufiya University.

ABSTRACT

The aim of the present work was to study ascitic fluid calprotectin in cirrhotic patients with spontaneous

bacterial peritonitis. Spontaneous bacterial peritonitis (SBP) is an important cause of morbidity and mortality

in cirrhotic patients with ascites. The diagnosis of SBP is based upon the polymorphonuclear (PMN) leukocyte

cell count exceeding 250 cell/mm3 in ascitic fluid but, PMN is usually performed by a manual method

operator-dependent and lysis of PMN cells during laboratory transport may occur leading to false-negative

results and delay in diagnosis of SBP. Aim of the work: was to evaluate Calprotectin as a surrogate marker

for routine screening and diagnosis of SBP. Methods: 45 patients with cirrhotic ascites with spontaneous

bacterial peritonitis (G1) and 45 patients with cirrhotic ascites without spontaneous bacterial peritonitis (G2)

were included in this study. Ascitic fluid calprotectin measured by enzyme-linked immunosorbent assay.

Results: There was highly significant increase in ascitic fluid calprotectin in SBP group when compared with

non SBP group (528.02±17.47 & 31.56.±2.04) respectively. Conclusion: Ascitic fluid calprotectin may be

used as a valuable tool for screening and diagnosis of SBP in cirrhotic patients with ascites.

Introduction

Liver cirrhosis is the clinical end-stage of different

entities of chronic liver diseases when patients

suffer from substantial mortality and morbidity (1,2).

Ascites is the most common complication and

around 60% of patients with liver cirrhosis develop

ascites within 10 years of disease onset (3).

Spontaneous bacterial peritonitis is an important

cause of morbidity and mortality in cirrhotic

patients with ascites (4). Many of SBP patients are

asymptomatic and therefore, it is recommended

that, all patients with ascites undergo paracentesis

at the time of hospital admission to confirm the

SBP status (5). The diagnosis of SBP is based upon

the polymorph nuclear (PMN) leukocyte cell count

exceeding 250 cell/mm3 in ascitic fluid (6,7).

Currently, (PMN) is usually performed by a

manual method using light microscopy and

counting chambers, operator dependent and lysis of

PMN cells during transport to the laboratory may

lead to false-negative results. Also, ascitic fluid

culture is insensitive and leads to delay in diagnosis

for several days and delay in starting antibiotic

therapy which, entails a high mortality rate, this is

a major drawback as rapid diagnosis of SBP and

immediate initiation of antibiotic treatment is of

paramount importance (7). Alternative methods

using automated PMN counting (8), reagent strips

(urine dipsticks) (9) or ascitic lactoferrin (10) have

been developed; unfortunately, their diagnostic

accuracies are limited and their use is dependent

upon availability of laboratory personnel and

reagents/components from the commercial source.

Calprotectin, a calcium and zinc-binding protein is

detected almost exclusively in neutrophils and its

presence in body fluids is proportional to the influx

of neutrophils (11). Faecal calprotectin is a well-

established marker of inflammation and is used to

monitor inflammatory bowel disease (12). A rapid

bedside test has been developed to measure

calprotectin in faeces; systematic comparison with

the established enzyme-linked immunosorbent

assay (ELISA) technique showed good correlation

between the two tests’ results (13) and this rapid

bedside test has been suggested as an equally

valuable tool for diagnosing inflammatory bowel

disease (14). It is possible that, such test may be

useful for measuring ascitic fluid calprotectin and

may serve as a surrogate marker for routine SBP

screening and diagnosis especially when measured

by a rabid bedside test (15).

Aim of the Work

The aim of this work was to study ascitic fluid

calprotectin in cihrotic patients with spontaneous

bacterial peritonitis.

Patients and Methods

The study was carried out on 90 patients with

decompensated chronic liver diseases and ascites

with and without spontaneous bacterial

peritonitis.They were selected from 125 patients

admitted to Tropical Medicine department,

Menoufiya university hospital and National Liver

Institute from September 2013 to May 2014 , 35

patients excluded due to presence of exclusion

criteria(Patients with ascites due to any cause other

than liver cirrhosis, Patients with evidence of

active infection other than ascitic fluid infection,

Pre-hospitalization antibiotic administration and

Abdominal surgery within 3 months of the study).

An informed consent was obtained before patients

enter the study. They were divided into two groups:

Group 1 (SBP group): Included 45 patients with

cirrhotic ascites with spontaneous bacterial

peritonitis. Group 2 (non SBP group): Included 45

patients with cirrhotic ascites without spontaneous

bacterial peritonitis. Sixty one of them (67.8%)

were males and twenty nine (32.2%) were females.

Their ages ranged from 49 to 68 years with a mean

All patients were subjected to:

Full and detailed history taking, complete clinical

examinations, routine laboratory investigations

as(CBC, LFT, Prothrombin time,serum creatinine,

ESR, Random blood sugar and viral markers),

abdominal ultrasonography,diagnostic abdominal

paracentesis (ascitic fluid total analysis: Physical,

biochemical, cytological and microbiological

cultures) and ascitic fluid calprotectin

measurement of by ELISA assay specific for

calprotectin by laboratory blinded to the

patients"clinical information and other laboratory

tests" LEGEND MAX human MRP8/14 ( Human

calprotectin) enzyme–linked immunosorbent assay

kit 2012, supplied by Biolegend inc . Results were

collected, tabulated, statistically analyzed by IBM

personal computer and statistical package SPSS

version 11.

Results

The present study revealed that, SBP was common

in males (71.1%) than females (28.9%) and not

influenced by the age. - Clinical presentations of

the studied groups revealed that, there was a

statistical significant difference between the two

groups regarding fever, abdominal pain, hepatic

encephalopathy which were the most commmen

clinical manifistations of SBP group and history of

previous SBP episode was more frequent in SBP

group and there was no statistical significant

difference between the two groups regarding

history of haematemesis,melena and bleeding

tendency as shown in table (1).

Table 1 :Clinical presentations of the studied groups:

P value

Chi

square

test

G2(non

SBP)

N=45

G1(SBP)

N=45

Clinical

presentati

ons % N % N

< 0.05 6.48 42.2 19 68.9 31 Fever

<0.001 10.08 37.8 17 71.1 32 Abdomina

l pain

<0.05 4.45 40.0 18 62.2 28

Previous

SBP

episode

> 0.05 1.607 60.0 27 46.7 21

Haematem

esi&

melena

< 0.001 10.601 55.6 25 86.7 39

Hepatic

encephalo

pathy

> 0.05 2.96 31.1 14 48.9 22 Bleeding

tendency

Examination of both groups by ultrasound revealed

that, there was a statistical significant difference

between the two groups regarding amount of

ascites as all patients with SBP were presented by

moderate to tense ascites and there was no

statistical significant difference between the two

groups regarding internal echoes , liver

examination and spleen examination as shown in

table (2).

Table 2 :Abdominal ultrasound findings of the studied groups:

P

value

Chi

square

test

G2(non

SBP)

N=45

G1(SBP)

N=45

U/S

% N % N

>0.05

1.68

4.4

53.4

42.2

2

24

19

4.4

40.0

55.6

2

18

25

Liver size :

Hepatomegaly

Average

Shrunken liver

> 0.05

2.69

48.9

44.4

6.7

22

20

3

33.3

53.3

13.4

15

24

6

Spleen size:

Splenomegaly

Average

Splenectomy

< 0.05

>0.05

10.04

1.34

20

33.6

44.4

0.0

9

20

16

0

0.0

57.8

42.2

6.6

0

26

19

3

Ascites :

Mild

Moderate

Tense

Internal echos:

Laboratory investigations of the studied groups

revealed that, there was a statistical significant

difference between the two groups regarding

WBCs count, ALT and AST levels which were

higher in SBP group and there was no statistical

significant difference between the two groups

regarding other parameters as shown in table (3).

Table3: Laboratory investigations of the studied group P value T test G2(non

SBP)

N=45

(X±SD)

G1(SBP)

N=45

(X±SD)

Parameters

<0.01 0.491 9.56±

9.24

12.81±

4.58

WBCs (103)

>0.05 0.68 9.04±

1.87

9.39±

2.90

Haemoglobin

(gm%)

>0.05 2.65 4.16±

1.25

3.51±

1.06

RBCs (106)

>0.05 0.936 68.34±

24.59

73.71±

29.59

Platelets (103)

<0.05 *2.14 82.71±

32.87

104.22

±58.87

ALT

<0.05 *2.80 73.08±

41.92

92.40±

45.97

AST

>0.05 *1.256 6.21±

3.98

7.56±

4.41

Total bilirubin

(mg%)

>0.05 *1.211 4.01±

4.38

5.56±

0.05

Direct bilirubin

(mg%)

>0.05 1.35 2.31±

0.69

2.13±

0.57

Serum albumin

(gm%)

>0.05 0.263* 43.16±

15.08

51.73±

45.20

Prothrombin

time (sec)

>0.05 1.39* 1.07±

0.76

2.08±

1.97

Creatinine

>0.05 0.35* 20.40±

5.94

49.53±

27.66

ESR

>0.05 0.723* 185.84±

118.65

230.51±

310.09

Random blood

sugar (mg%)

Mann Whitney U test *

In this study, SBP group contained 40 HCV Ab

positive patients (88.9%),3 HBsAg positive

patients (6.6%) and two coinfected patients by

HCV and HBV (4.4%). Non SBP group contained

38 HCV Ab positive patients (84.4%), 4 HBsAg

positive patients (8.9%) and 3 coinfected patients

by HCV and HBV (6.6%) . Ascitic fluid total

analysis revealed that, in physical examination

there was highly significant turbidity in the aspect

of ascitic fluid in SBP group and no significant

difference between two groups regarding the

colour of ascitic fluid, there was a statistical high

significant difference between the two groups in

biochemical analysis of ascitic fluid regarding

TLC, PMN count which were higher in SBP group

and total protein which was lower in SBP group

and there was no statistical significant difference

between the two groups regarding LDH and

glucose and cytological analysis was negative in

both groups as shown in table (4).

Table 4: Ascitic fluid total analysis of the studied groups

P value T

test

G2(non

BP)

N=45

G1(SBP)

N=45

Ascitic

fluid

analysis

<0.001

#23.

47

% N % N Aspect:

Clear

Turbid

Physical

analysis

88.9

11.1

40

5

40.0

60.0

18

27

>0.05

#1.5

4

% N % N Colour:

Yellowis

h

Whitish

Serosan

genous

Bloody

91.1

0.0

6.7

2.2

41

0

3

1

82.2

0.0

13.3

4.4

37

0

6

2

< 0.001 *6.7

79

209.11±6

7.14

4175.96±2

725.21

TLC

Biochem

ical

analysis

< 0.001 *8.0

84

91.04±13

.56

425.02±37

.87

PMNLs

> 0.05 0.93

6

68.34±24

.59

73.71±29.

59

LDH

< 0.001 3.40 86.20±22

.36

70.22±22.

27

Total

protein

>0.05 0.15

2

1.91±0.3

2

1.90±0.36 Glucose

>0.05 0.16

8

1.65±0.3

6

1.66±0.28 SAAG

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

--

0 0 Maligna

nt cells

Cytologi

cal

analysis

Ascitic fluid bacterial culture was positive in 36

cases (80%) of SBP group and negative in all cases

of non SBP group, the isolated organisms in SBP

group were E.coli in 27 cases (60%), Klebsiella in

8 cases (17.8%) and pseudomonas in one case

(2.2%), the isolated organisms were

monomicrobial, gram negative aerobic pathogens

as shown in table (5).

Table 5: Ascitic fluid culture of the studied groups

P value Chi

squar

e test

G2(non

SBP)

N=45

G1(SBP)

N=45

Ascitic fluid

culture

% N % N

<0.001

60.00 100.

0

0.0

0.0

0.0

45

0

0

0

20.0

60.0

17.8

2.2

9

27

8

1

Negative

E coli

Klebseilla

pneumonia

Pseudomon

as

- Ascitic fluid calprotectin was detected in both

groups, there was highly significant increase in

ascitic fluid calprotectin in SBP group when

compared with non SBP group (528.02±17.47 &

31.56.±2.04) respectively as shown in table (6).

Table 6: Ascitic fluid calprotectin of the studied groups:

P

value

Mann

Whitney

U test

G2(non

SBP)

N=45

X±SD

G1(SBP)

N=45

X±SD

<

0.001

8.171 31.56±

12.04

528.02±

174.47

Calprotectin

In child classification B there was a high statistical

significant difference in calprotectin level in both

groups and in child classification C there was a

high statistical significant difference in calprotectin

level in both groups and there was no difference in

the same group between child classification B and

C in calprotectin level as shown in table (7).

Table 7: Calprotectin of the studied groups in relation to child classification

Child classification Calprotectin

C B

540.25±188.32

29.46±12.06

479.11±94.27

36.73±12.75

G1(SBP)

G2(non SBP)

1.98 1.37 T test

<0.001 <0.001 P value

There was a high statistical significant difference between calprotectin and outcome of SBP group as shown

in table (8).

Table 8: Relation between calprotectin level and outcome of SBP group

P value T test Outcome of SBP

Calprotecti

n

Dead

N=7

Cured

N=38

< 0.001 5.55 572.01±

74.36

470.37±

97.83

(X±SD)

The sensitivity of the ascitic fluid calprotectin was

100%, the specificity was 97.8%, PPV was 98%,

NPV was 100% and accuracy was 99%. So, it is a

good negative test (if calprotectin is below 55.4

ng/ml we can conclude that, the patient does not

have SBP) as shown in table (9).

Table 9 :Evaluation of calprotectin as a screening test for SBP

Interpretation Roc curve

100 % Area under the curve(AUC)

55.4ng/ml Cutoff point

100% Sensitivity

97.8% Specificity

98% PPV

100% NPV

99% Accuracy

Discussion

Ascites is one of the most common complications

of patients with cirrhosis and its development

carries a relatively poor prognosis (16).

Spontaneous bacterial peritonitis (SBP) is a

common and serious infection occurring in 8 – 27

% of hospitalized patients with cirrhosis and ascites (4). It is also one of the potential life- threatening

complications in cirrhotic patients with ascites with

a mortality rate ranging between 30 and 50% (17),

so, it requires a rapid and accurate diagnosis in

addition to prompt effective therapy (18). The

present study was conducted on 90 patients with

decompensated chronic liver disease and ascites

with and without spontaneous bacterial

peritonitis,Sixty one of them (67.8%) were males

and twenty nine (32.2%) were females. Their ages

ranged from 49 to 68 years with a mean age of

two groups: Group I: Included 45 patients with

cirrhotic ascites with spontaneous bacterial

peritonitis (SBP) with mean age57.86±11.32 with

32 males (71.1%) and13 females(28.9%) and

Group II: Included 45 patients with cirrhotic ascites

without spontaneous bacterial peritonitis (non

SBP) with mean age59.47±9.57 with 29 males

(64.4%) and 16 females(35.6%) and there was no

significant difference between the two groups

regarding age and sex indicating no bias in this

study also,SBP was common in males (71.1%) than

females (28.9%) and it was not influenced by the

age. This result was in agreement with the study

done by Angelli et al (19) who stated that, SBP was

frequent in males and was not affected by the age.

Regarding the clinical presentations of the patients

analysis of the results showed that, fever,

abdominal pain, bleeding tendency and previous

SBP episode were the only significant clinical

presentations in patients with SBP compared to non

SBP group (68.9%, 71.1%,48.9% and 62.2%

respectively). This difference could be explained

by Rimola and Navasa (20) who said that, the

clinical picture of SBP is extremely broad and very

variable and a very high degree of clinical

suspicion is required for diagnosis. Regarding the

clinical examination, all patients with SBP were

presented by moderate to tense ascites (57.78% and

42.22% respectively). This result was in agreement

with that reported by Runyon (6) who stated that,

ascitic fluid infection usually developed when the

volume of ascites was at its maximum. The

ultrasonographic findings in the present study

detected no significant difference between both

groups regarding to internal echoes and this

correlates with data obtained by Al-Sharif (21) who

concluded that, ultrasonographic detection of

echoes and/or adhesions is neither diagnostic nor

prognostic and there was no single imaging test is

totally sensitive or specific for the detection of

infected fluid collection. Regarding the parameters

of laboratory investigations , there was significant

difference between both groups in total leucocytic

count, leucocytosis was present in 62.2% of SBP

patients and this result was in agreement with that

reported by Rodriguez et al (22) who detected

leucocytosis in their SBP cases with significant

difference when compared to non SBP cases.

Regarding liver function tests, there was a highly

statistical significant difference between SBP cases

and non SBP cases in ALT and AST levels.

otherwise, no statistically significant differences

was recorded in the other parameters. Casafont et

al (23) found no significant difference in liver

biochemistry, Also, these results were in agreement

with those reported by Runyon et al (24) who stated

that, prolonged prothrombin time and

hypoalbuminaemia are not related to SBP per se,

but rather to the underlying liver disease. The

relation between occurrence of SBP and severity of

liver disease showed that, 22.2% and 77.8% of

SBP cases had Child Pugh class B and C

respectively ,This result came in accordance with

that reported by Rodriguez et al (22) who elicited

that, SBP patients usually had Child-Pugh class B

or C. Physical examination of ascitic fluid revealed

turbid fluid in 60.0% of patients of SBP with a

statistical significance when compared to the non

SBP group in which 88.9% of patients had clear

fluid. In agreement with this result, Albillos et al (25) observed that, the appearance of ascitic fluid in

patient with SBP is usually turbid. Moreover,

Runyon (6) reported that, the opacity of many

cloudy ascitic fluid specimens is caused by

neutrophils. Moreover, Chijung et al (26) in a

retrospective review of 916 outpatient ascitic fluid

samples showed that, abnormal ascitic fluid

appearance had a sensitivity of 98.1% and a

specificity of 22.7% in the detection of SBP. Total

protein concentration in ascitic fluid showed

significant decrease in SBP group than non SBP

group. Runyon (6) denoted that, patients with lowest

ascitic protein concentrations are the most

susceptible to SBP. Ascitic fluid WBCs and PMN

count were significantly higher in SBP group than

non SBP group. Jansen (27) stated that, although

ascitic total WBC count increases in SBP cases, it

suffers from low specificity because a large

proportion of patients with sterile ascites have

increased white blood cell count. Also, diuretic

therapy has been shown to increase the total white

blood cells count but does not alter the PMN count.

Ascitic fluid bacterial culture was positive in 36

cases (80%) of SBP group and negative in all cases

of non SBP group. The isolated organisms in SBP

group were E.coli in 27 cases (60%), Klebsiella in

8 case (17.8%) and pseudomonas in one case

(2.2%), Liovet et al (28) detected positive ascitic

fluid bacterial culture in 71.7% of cases of SBP

which was close to the result of the present study.

In SBP group, the isolated organisms were

monomicrobial, gram negative aerobic pathogens.

This was in agreement with that reported by Liovet

et al (29) who stated that, single gram negative

organisms (specially E.coli and Klebsiella) cause

most episodes of SBP. In SBP group, ascitic fluid

culture was negative in 20% of patients,the culture

negativity may be due to low concentration of

bacteria in ascitic fluid. This was in agreement with

that reported by Navasa et al (30) who stated that,

inspite of using good culture techniques; cultures

are still negative in approximately 30-50% of

patients with an increased ascites PMN count. In

this study, ascitic fluid calprotectin was detected in

both groups, there was highly significant increase

in ascitic fluid calprotectin in SBP group when

compared with non SBP group (528.02±17.47 &

31.56.±2.04) respectively, the sensitivity of the test

was 100% with specificity of 97.8%, with the cut-

off level was 0.554 µg/ml (55.4ng/ml). The results

of this study were close to that reported by Burri et

al (15) who stated that, the sensitivity and specificity

of the test were 94.8% and 98.2% respectively with

the cut-off level 0.63µg/ml.The results of this study

were in disagreement with that reported by RITO

NOBRE et al (9) who stated that, the diagnostic

accuracy of calprotectin are limited in ascitic fluid

infection also, the results of this study were in

disagreement with that reported by Sydora et al

(14) who stated that, calprotectin is a valuable tool

for diagnosing IBD especially when used as a rapid

bedside test and the role of calprotectin as a

marker for PMN count and routine SBP screening

and diagnosis still limited. In this study, the out

come of SBP group was cure of 38 patients out of

45 (84.4%). This result was in agreement with that

elicited by Fernandez et al (31) who found that, SBP

cure rate ranged between 60-90%. In this study,

there was statistical high significant difference

between calprotectin and outcome of SBP ,38

patients cured with calprotectin mean

470.37±97.83 and7 patients dead with calprotectin

mean 572.01±74.36. This result was in agreement

with that elicited by Fernandez et al (31) who found

that, there was statistical significant difference

between calprotectin and outcome of SBP.

Conclusion : Ascitic fluid calprotectin may be

used as a valuable diagnostic tool for routine

screening and diagnosis of SBP in cirrhotic patients

with ascites.

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Original Article

The Prevalence of Insulin Resistance and Metabolic Factors in Chronic

Hepatitis C Patients with Genotype 4

Prof. Amira Amer, Prof. Manal Baddour, Prof. Mohamed El Shazly, Prof. Gylan Fadaly, Assis. Prof Nesrine

Hanafi, Assis. Lect Sara Asser

ABSTRACT

Egypt has been widely regarded as having an epidemic, with the highest recorded HCV prevalence in the

world. There is strong epidemiological evidence linking HCV and diabetes. The association between HCV

infection and glucose abnormalities is true if, instead of looking at the occurrence of overt T2D, prediabetic

conditions, such as insulin resistance (IR) should be considered. Aim of the work : was to evaluate the

prevalence of insulin resistance in Egyptian patients infected with chronic hepatitis C virus genotype 4 and to

assess factors associated with insulin resistance. Insulin resistance was detected in 31 of the 100 non diabetic

CHC patients infected with genotype 4 (HOMA-IR >3.0). HOMA-IR was positively correlated with age,

baseline viral load, BMI, TG, fibrosis and steatosis. Results : Relationship between elevated HOMA-IR and

baseline viral load and degree of fibrosis was statistically significant. Out of 29 liver tissue sections, 14 had

low level of expression of IRS-1 by immunohistochemical studies. This study showed that patients with high

HOMA-IR had higher basal viral load, and higher incidence of fibrosis. Also patients with high HOMA-IR

had high levels of triglycerides, high BMI and steatosis. HOMA-IR was negatively correlated with

cholesterol, LDL, HDL and total lipids. Conclusion: This study suggested that viral load remained the only

independent factor associated with elevated HOMA-IR levels.

Introduction

Egypt has been widely regarded as having an

epidemic, with the highest recorded HCV

prevalence in the world. The latest published

Egyptian Demographic Health Survey (EDHS) in

2009 estimated an overall anti-HCV antibody

prevalence of 14.7%. The number of Egyptians

estimated to be chronically infected was 9.8%.(1).

The spectrum of severity of liver disease

associated with HCV varies widely and depends

on both viral and host factors. Age, male gender,

alcohol consumption, immune status and co-

infections are defined as risk factors for a

progressive course of CHC.(2) One of the co-

factors is type 2 diabetes (T2D).(3,4). There is

strong epidemiological evidence linking HCV and

diabetes. Patients with CHC are more likely to

develop T2D and diabetic patients are more likely

to be infected with HCV. (5) Type 2 diabetes has

been recognized to worsen the course of hepatitis

C.(6) The association between HCV and IR has

significant clinical consequences. Mounting

evidence indicates that HCV-associated insulin

resistance may cause accelerated fibrogenesis,

reduced response to IFN-based therapy and

hepatocellular carcinoma.(7). Prediabetic

conditions, such as insulin resistance (IR) should

be considered to evaluate the association between

HCV infection and glucose abnormalities. Insulin

resistance is defined as a condition in which

higher than normal insulin levels are needed to

achieve normal glucose metabolism. (8). During

the past years, basic research, clinical trials and

epidemiological studies have provided evidence

that HCV can independently contribute to IR. (9,10)

Adding to this growing body of evidence, it is

now suggested that HCV interferes with insulin

signaling pathway. (11) Insulin carries its biological

effects through phosphorylation of the substrate of

the insulin receptor 1 (IRS-1) and 2 (IRS-2). (12,13)

Thus research has focused on IRS-1 and IRS-2 as

a locus for insulin resistance. Our aim was to

evaluate the prevalence of insulin resistance in

Egyptian patients infected with chronic hepatitis

C virus genotype 4 and to assess factors

associated with insulin resistance (viral,

metabolic, histopathologic including steatosis,

fibrosis and necroinflammatory changes).

Subjects and Methods

A total of 100 adult patients with chronic hepatitis

C (CHC) genotype 4 infection were enrolled

randomly from the “Center for treatment of

hepatitis viruses” in Sharq ElMadina hospital

(being one of the centers established by the

Ministry of Health for treating CHC patients as a

part of the viral hepatitis National Treatment

Program). All patients were treatment-eligible and

non-diabetic. All patients were asked to give their

informed consent before being included in the

study.A control group comprised of 60 healthy

HCV-negative individuals, was included in the

study for comparing their HOMA scores with

those of our patients. Clinical and demographic

data were collected from patients’ files, including:

age, sex, height, weight, waist circumference and

blood pressure. Body mass index (BMI) was

calculated. The metabolic syndrome was

diagnosed according to the revised WHO

definition as the presence of 3 or more of the

following criteria: central obesity (waist

circumference >102 cm, [male] and > 88 cm,

[female}, hypertension (blood pressure> 135/85

mm Hg), fasting plasma glucose > 110 mg/dl,

triglycerides > 150 mg/dl, high density lipoprotein

(HDL) cholesterol < 40 mg/dl (male) or < 50

mg/dl (female). [25] Venous blood samples were

collected after an overnight fast of 12 hours to test

the lipid profile and to determine serum levels of

glucose and insulin. Insulin resistance was

assessed using homeostasis model assessment for

insulin resistance (HOMA-IR) method, using the

following equation: HOMA-IR = fasting insulin

(μU/mL) X fasting glucose (mmo/L)/22.5 (using

Insulin kitTM, Cobas e analyzerTM, Roche

diagnostic, Germany). (14) A HOMA –IR score of

more than 3.0 was considered as the criterion of

insulin resistance. (15). Assessment of the HCV

viral load of the 100 patients included in our study

was done by quantitative measurement of RNA

using real-time PCR (COBAS

AmpliprepTM/COBAS TaqManTM, Roche

Molecular Systems, Pleasonton, CA, USA). Level

of viremia was classified as high, intermediate

and low according to viral load being >106, 105 -

106 or <105 IU/ml respectively. (16) Determination

of the genotype of the virus was done using a real-

time PCR kit (AmpliSens®/HCV-FRT PCR kit,

InterLabService Ltd, Moscow, Russia). All

patients underwent an ultrasound guided

percutaneous liver biopsy prior to the start of

treatment and patients with hemochromatosis or

primary biliary cirrhosis were excluded. The

degree of necroinflammatory activity and of

fibrosis were scored based on the Metavir score. (17) Hepatic steatosis was scored as the percentage

of hepatocytes containing macrovesicular fat

droplets and was graded from 0 to 3. (18) Paraffin-

embedded liver sections from selected patients

were deparaffinized and subjected to

immunohistochemical staining using an anti-

human IRS1 (Ultravision Detection System

Antipolyvalent, HRP/DAB kitTM,Thermo Fischer

Scientific, UK) to examine the protein expression

levels of IRS1.

Results

Among the one hundred chronic HCV patients

included in the study, 40 were males and 60 were

females, with a mean age of 42.82 ± 10.23 years.

The mean BMI was 27.06 ± 3.44. Fifteen patients

fulfilled the criteria of the metabolic syndrome.

According to the metavir score,

necroinflammation was moderate-severe in 45%

of patients and fibrosis was significant in 49% of

cases. Steatosis was moderate in 20.7% of cases

and severe in 10.3% of cases. The distribution of

the 100 chronic HCV patients included in this

study with respect to their baseline viral load was

as follows: 22% had low level viremia, 43% had

intermediate level viremia and 35% had high level

viremia. The median was 487.3 ×103 IU/ml with

mean and standard deviation of

2400.89±6854.84×103 IU/ml. Insulin resistance

was detected in 31 of the 100 non diabetic CHC

patients infected with genotype 4 (HOMA-IR

>3.0). When HOMA scores were categorized into

three groups (<2, 2-4,and >4), a highly significant

difference existed between values of patients and

those of controls p= 0.001(table 1).

Table (1): HOMA-IR of the selected chronic HCV patients and the control subjects

Controls

(n=60)

Cases

(n=100) Test of sig. p

No. % No. %

HOMA-IR

<2 44 73.3 49 49.0

χ2=9.168* 0.009* 2 – 4 13 21.7 40 40.0

>4 3 5.0 11 11.0

Min. – Max. 0.03 – 6.81 0.23 – 15.17

Mean ± SD. 1.61 ± 1.29 2.55 ± 2.36 Z=3.322* 0.001*

Median 1.41 2.05

Data on the relationship between HOMA-IR and

clinical and biological variables are shown in

table 2. HOMA-IR was positively correlated with

age, baseline viral load, BMI, TG, fibrosis and

steatosis. HOMA-IR was negatively correlated

with cholesterol, LDL, HDL and total lipids.

Relationship between elevated HOMA-IR and

each of baseline viral load (fig.2) and degree of

fibrosis mounted to a statistical significance

(r=0.218, p=0.029 and r=0.223, p=0.026

respectively). When the data were analyzed by

multivariate linear regression, results suggested

that viral load remained the only independent

factor associated with elevated HOMA-IR levels

(p=0.001).

Table (2): Univariate analysis of correlations between HOMA-IR and different variables in patients with genotype 4 chronic

hepatitis C infection

Variables HOMA-IR

sr P

Age 0.101 0.315

Viral load *0.218 0.029

BMI 0.151 0.133

Cholesterol -0.098 0.338

TG 0.118 0.246

HDL -0.081 0.430

LDL -0.120 0.238

Total Lipids -0.037 0.716

Fibrosis grade *0.223 0.026

Steatosis grade 0.336 0.075

The expression of IRS-1 was estimated by

immunohistochemical staining of 29 liver tissue

sections of chronic HCV cases included in the

study, the results were as follows: 9 cases grade 0

(10% positive cells), 6 cases 1+ (10–50% positive

cells with weak staining), 10 cases 2+ (10–50%

positive cells with strong staining or 50% positive

cells with weak staining) and 4 cases 3+ (50%

positive cells with strong staining) (Figure 1). (19).No statistical significant difference was found

between any grades of immunohistochemistry and

HOMA-IR before therapy (p= 0.942). (table 3)

Table (3): Relation between IHC and HOMA IR

IHC

Z p

-ve

(n = 9)

+ve

(n = 20)

HOMA IR

Min. – Max. 0.40 – 10.52 0.37 – 15.17

0.141 0.888 Mean ± SD. 2.82 ± 3.08 3.08 ± 3.58

Median 1.90 1.58

Z: Z for Mann Whitney test

Figure 1 shows grade 3+ immunostaining

Discussion

This study was conducted on 100 chronic HCV

patients, to evaluate the prevalence of insulin

resistance in Egyptian patients infected with

chronic hepatitis C virus genotype 4 and to assess

factors associated with insulin resistance in those

patients. In this study, insulin resistance was

detected in 31 of the 100 non diabetic CHC

patients infected with genotype 4 (HOMA-IR

>3.0) with mean 2.55 ± 2.36. In another study by

Khattab et al conducted also on patients with

genotype 4, the mean pre-treatment HOMA-IR >2

was 2.82 ± 1.19. (20) Similarly, Ezzat et al studied

HOMA-IR of CHC patients with genotype 4, the

mean was 2.6 and 31(40.7%) patients had insulin

resistance >2. (21) Also Moucari et al studied CHC

genotype 4 patients and HOMA-IR >3 was 3.7 ±

4.0. (22).Asselah et al also conducted their study on

CHC patients genotype 4, the HOMA-IR>3 was

32.4%.(23). In this study, the correlational

analysisof factors affecting pre-treatment HOMA-

IR revealed that baseline viral load proved to be

statistically significant (p=0.029) and the major

independent factor associating high HOMA-IR by

linear regression analysis (p=0.001). Similarly,

Asselah et al found by univariate analysis that

insulin resistance associated significantly with

basal viral load (p=0.008) as well as by multiple

logistic regression analysis (p=0.02).(23) Moucari

et al also had similar results where the univariate

analysis of insulin resistance showed statistically

significant correlation with serum HCV RNA

(p<0.001) and also by multiple logistic regression

analysis (p=0.002). (22) This finding supports that

HCV has a direct effect on insulin resistance

progression in chronic patients. Alternatively, in

another study by Ezzat et al, their results showed

that insulin resistance had no impact on early

virological response of combined therapy, viral

load or necroinflammation. (21) The contradiction

in the current study and Ezzat et al may be

because they assessed HOMA-IR before therapy

and at 12 weeks after therapy only and did not

measure HOMA-IR at the end of treatment. In

this study, 29 liver tissue sections from the

selected cases were tested by

immunohistochemistry for expression of insulin

receptor substrate-1 to assess viral role in

induction of insulin resistance state. Fourteen

cases showed high level of IRS-1 expression with

grades 2+ and 3+ and 15 cases had low level of

expression zero and 1+ with no statistical

significance. Kawaguchi et al demonstrated a two

and three fold increase in intensities of IRS1 and

IRS2 staining, respectively, after antiviral therapy.

They identified mechanisms for HCV-associated

insulin resistance postulating that HCV core down

regulates hepatic expression of IRS 1/2, and thus

decreases downstream signaling effect of insulin

on glucose uptake by cells. (24) Almost half of the

cases in our study showed low level of expression

of IRS-1 which also supports the postulation of

the direct role of the virus on cells. Considering

HCV infection on the causal side of insulin

resistance may have important implications. From

the management point of view; should patients

with CHC be monitored regularly for IR? A

practical and also well-accepted method of

measuring IR is the HOMA-IR (homeostasis

model assessment of IR) which-being a

noninvasive, non expensive test–can be

implemented easily in routine clinical practice.

Should lifestyle modification and anticipation of

diabetic condition be considered in chronic HCV

patients to warrant against occurrence of more

severe complications and treatment failure? Some

points that. need to be elucidated.

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