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Contents Alexandria Journal of Hepatogastroenterology,

Volume (XIX) - April 2016

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Original Article

A Study of Immunohistochemical Expression of

Hypoxia-Inducible Factor-1 Alpha and its

Correlation with The Expression of Homeodomain

Protein CDX2 in Colorectal Cancer

Suzan M F H, Nevine M F El Deeb, Shaimaa S M Omar.

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

Original Article

Alterations in Phosphatase and Tensin Homolog

(PTEN) Tumor Suppressor Gene in Egyptian

Patients with Hepatocellular Carcinoma

Mohamed H. Abdel-Rahman, Mohamed S. Kohla, Amr

M. Aziz, Sameera A. Ezzat.

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

Original Article

Atypical Manifestations of Viral Hepatitis A

Alaa Abdo, Nasser Abd Alla, Abu Rawy Ismail.

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

Original Article

Cerebrospinal Fluid Cortisol Level for

Differentiation Between Aseptic and Bacterial

Meningitis in Adults

Ayman Faried El-Shayeb, Soraya Abdel Fatah Hamoda,

Hossam Fathy Abo El-kheir, Iman Tayaa Elsayed, Iman Magdi Mohamed Mohamed.

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

Original Article

Clinical Use of Quantitative Serum (HBsAg) as New

Marker for Assessment of Improvement in Patients

with Chronic Hepatitis B Treated with Lamividine

Alaa-Eldin Mohamed Abdo, Essam Eldin Saeed

Bedewy, Sherine Mohamed Shawky, Salem Ramadan

Soleiman Khairalla.

------------------------------------------------ Original Article

Immunohistochemical Study of IL-8 And COX-2

Expression in Helicobacter Pylori Gastritis

Layla K Younis, Nevine M F El Deeb, Amany A

Elbanaa, Engy A A Shahtout.


Role of Gastric Varices Injection as a Method to

Prevent Predicted Hematemesis and its Impact on

Quality of Life of Hepatic Patients

Abdel Fattah Fahmy Hanno, Essam Eldin Saeed

Bedewy, Said Elsayed Ahmed Hammoda.


Serum Procalcitonin Level as a Marker for

Diagnosis of Bacterial Infections

Hossam Ibrahim Mohamed, Amira Maher Ahmed

Badawy, Safaa Ibrahim Tayel, Rasha Mohammed Abd-

Elmegeed Shetaya.


The Effect of the Cytochrome P450 (CYP450)

Genetic Polymorphism in Peptic Ulcer Therapy

Medhat Haroun, Abir Adel Abdel Razak, Nasser Abd

allah, Marwa kassem.

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

2

9

14

23

28

41

52

59

34

mailto:[email protected]

mailto:[email protected]

Original Article

A Study of Immunohistochemical Expression of Hypoxia-Inducible Factor-1

Alpha and its Correlation with The Expression of Homeodomain Protein

CDX2 in Colorectal Cancer

Suzan M F H*, Nevine M F El Deeb*, Shaimaa S M Omar**; *Department of Pathology, Faculty of

Medicine, University of Alexandria, Egypt, **Department of Pathology, Gamal Abd El Nasser Insurance

Hospital, Alexandria, Egypt.

ABSTRACT

Aim of the work: This work was designed to investigate the expression of CDX2 and HIF-1 α in colorectal

adenocarcinomas, and to evaluate their correlation with clinicopathological variables. Material Methods: Using

immunohistochemistry, we studied the expression of CDX2 and HIF-1α in tumor specimens obtained from 40

colorectal carcinoma patients. Marker expression was correlated to clinicopathological variables. Results: CDX2

immunostaining was observed in 80 % of cases. Higher CDX2 expression was significantly associated with lower

grade tumors (P = 0.040) and with negative lymph nodes status (P = 0.016). HIF-1α was expressed in 47.5 % of

cases. A significantly higher expression of HIF-1α was detected in higher grade tumors (P = 0.004) and with positive

lymph node status (P = 0.007). The relationship between the expression of the two markers and each of: patient age

and gender, tumor location, gross appearance and T stage was not significant (P > 0.05). A significant inverse

correlation was found between CDX2 and HIF-1α expression (P = 0.001). Conclusions: This study suggests that

hypoxia plays a role in the progression of colorectal cancer possibly through inactivation of CDX2 by HIF-1α.

Introduction

Colorectal cancer (CRC) is the third most

commonly diagnosed cancer worldwide and the

second leading cause of cancer mortality in the

developed world .(1) In Egypt, colorectal cancer

is one of the most common malignant

neoplasms, representing 6.5% of all cancers

according to the National Cancer Institute, Cairo

University. (2). CDX2 is a caudal-type homeobox

gene, encoding a transcription factor that plays

an important role in proliferation, differentiation,

adhesion and apoptosis of intestinal epithelial

cells.(3) It is expressed by the overwhelming

majority of colorectal carcinomas.(4,5) Different

lines of evidence suggest that CDX2 expression

is often lost or decreases and becomes

heterogeneous in colorectal cancers with high

tumor grade, advanced tumor stage,

microsatellite instability, poor prognosis and

reduced disease free survival.(6-13) Growing

evidence suggests that hypoxia plays a pivotal

role in disease progression and therapy

resistance in most solid tumors, including

colorectal cancer.(14,15) Many of the adaptations

to hypoxia are mediated by the activation of

specific genes through hypoxia-inducible factor

(HIF).(16,17) The first HIF described (HIF-1) is a

heterodimer of HIF-1 α and HIF-1β (18) and HIF-

1 activity is determined by HIF-1α.(19) Hypoxia

inducible factor 1- alpha (HIF-1 α) is a powerful

regulator for the adaption of tumour cells to a

hypoxic microenvironment that binds to the

hypoxia-response elements (HRE) of various

target genes and activates their transcription,

controlling glucose transport, angiogenesis,

erythropoiesis, vasomotor responses, and

potentially increasing the survival of tumour

cells. (20) As CDX2 gene is rarely mutated in

CRCs, it is suggested that its altered expression

in cancer cells depends on negative regulatory

pathway(s).(21) The search for these pathways has

revealed the importance of the micro-

environment surrounding the tumour cells and

because of the importance tumour hypoxia as a

micro-environmental determinant for tumour

progression, the present work designed to study

the correlation between CDX2 and HIF-1α

expression and their possible clinicopathological

significance in colorectal carcinoma.

Material and Methods

Patients and tissue specimens: A total of 40

specimens of primary tumor were collected from

patients with CRC who underwent radical

surgery during the period from January 2010 to

July 2015. Specimens were submitted to the

Pathology Department, Faculty of Medicine,

Alexandria University. The study was approved

by the Research Ethics Committee in Alexandria

Faculty of Medicine. Specimens included: right

hemicolectomy (14 cases), extended right

hemicolectomy (5 cases), transverse colectomy

(one case), left hemicolectomy (5 cases),

sigmoidectomy (4 cases), proctocolectomy (one

case), anterior resection (4 cases), subtotal

colectomy (2 cases) and total colectomy (4

cases). Clinical data (patient age, gender, tumor

location) were collected from the patient medical

records and the archives of the Pathology

Department, Faculty of Medicine, Alexandria

University. The histological features

(histological type and grade of tumor, depth of

invasion (T stage) and lymph node status) were

assessed on hematoxylin and eosin-stained tissue

sections. Inclusion criteria were:

histopathological diagnosis of conventional

adenocarcinoma and patients presenting with

primary colorectal tumors. Exclusion criteria

were: recurrent tumors and previous treatment

with radiation and/or chemotherapy. The

pathological staging of primary colorectal

carcinoma (pT) was performed according the 7th

edition of AJCC staging system. (22) All tissues

were fixed in 10% formalin and embedded in

paraffin wax. Immunohistochemistry: Immuno-

histochemical analysis of CDX2 and HIF-1α was

performed on 5 μm sections which were

prepared from the paraffin blocks. The

UltraVision ONE detection system (Thermo

FisherScientific, CA, USA) was used for

immunostaining. The staining was conducted by

the streptavidin-biotin-peroxidase complex

method. The sections were deparaffinized in

xylene and rehydrated in graded alcohols.

Endogenous peroxidase activity was blocked by

incubation with hydrogen peroxide for 15 min.

Antigen retrieval was performed by placing the

slides in citrate buffer (0.01 mol/l, pH 6.0) in a

microwave oven for 5 min twice. Slides were

allowed to cool to room temperature, and then an

ultra V block was applied for 3-5 min to block

nonspecific background staining. Thereafter,

tissue sections were incubated with the following

primary antibodies: CDX2, rabbit monoclonal

antibody (clone EPR2764Y, dilution 1:50;

Thermo Fisher Scientific, CA, USA) for one

hour at room temperature; and HIF-1α, mouse

monoclonal antibody (clone H1alpha 67, dilution

1:30; Thermo Fisher Scientific, CA, USA)

overnight at 4℃. Then, tissue sections were

incubated in UltraVision ONE HRP Polymer for

30 min. Between incubations, the sections were

washed in PBS (pH 7.00) for 3 min. Thereafter,

the sections were stained for 5 min with 3, 3’-

diaminobenzidine tetrahydrochloride (DAB),

and counterstained with hematoxylin. Sections

without primary antibodies served as negative

controls. Tissue sections of colon carcinomas

and placenta were used as a positive control for

CDX2 and HIF-1 α respectively. Evaluation of

immunohistochemical staining: For the

evaluation of immunohistochemical results, both

intensity and percentage of positively-stained

cells were taken into consideration. The scoring

criteria for staining intensity for both CDX2 and

HIF-1 α were: 0, no staining; 1, weak staining; 2,

moderate staining; 3, strong staining. The

percentage of positively-stained tumor cells was

divided into four grades, for CDX2: 0, <5%

positive tumor cells; 1, 6-25 % positive tumor

cells; 2, 26-50% positive tumor cells; 3, 51-75 %

positive tumor cells; and 4, 76-100% positive

tumor cells; and for HIF-1 α: 0, no positive

tumor cells; 1, 1-25 % positive tumor cells; 2,

26-50% positive tumor cells; 3, 51-75 % positive

tumor cells; and 4, 76-100% positive tumor cells.

The final score was calculated by adding the

tumor staining extent to the intensity score.

According to this method of assessment, staining

scores 0-2 and 3-7 were regarded as tumors with

negative expression and positive expression,

respectively.(23,24)

Statistical Analysis

Statistical analysis was performed using

Statistical Package for Social Sciences (SPSS)

software, version 20.0 (SPSS, Chicago, IL,

USA). Qualitative data were described using

number and percent. Quantitative data were

described using Range (minimum and

maximum), 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. For

normally quantitative variables, to compare

between two studied groups Student t-test was

used. The level of significance was set at p <

0.05.

Results

This study comprised 40 cases of CRC. The age

range of patients was 33-79 years, with a mean

of 53.72 (SD± 11.05). There were 15 males and

25 females. Clinicopathological data of the study

group (patient age, gender, tumor location, gross

appearance, tumor grade, T stage and lymph

node status) are mentioned in Table I.

Expression of CDX2. Immunostaining showed

that CDX2 was localized in the nuclei of normal

colonic epithelium adjacent to the tumor. In

tumor cells, immunoreactivity was nuclear

and/or cytoplasmic (Fig. 1 A-C). Thirty-two out

of the 40 studied cases (80%) expressed CDX2.

CDX2 staining score in positive cases (scores 3-

7) showed the following distribution: score 3,

one case (3%); score 4, 2 cases (6%); score 5, 5

cases (16%); score 6, 9 cases (28%); and score 7,

15 cases (47%). CDX2 expression was found to

be higher in tumors with lower tumor grade and

this relation was statistically significant

(P=0.040) (Table I). In addition, a significant

relation was found between CDX2 expression

and negative lymph node status (P=0.016) (Table

I). No significant correlation was found between

CDX2 expression and each of patient age, and

gender, the tumor location, gross appearance and

T stage (P=0.611, 1.000, 0.205, 0.689, and 1.000

respectively) (Table I). Expression of HIF1 α In

normal colonic epithelium adjacent to the tumor,

HIF1 α immunostaining was cytoplasmic. In

tumor cells, immunoreactivity was nuclear and

or cytoplasmic (Figure 1 C-F). Nineteen out of

the 40 studied cases (47.5%) expressed HIF1 α.

HIF1 α staining score in positive cases (scores 3-

7) showed the following distribution: score 3,

one case (5%); score 4, one case (5%); score 5, 8

cases (42%); score 6, 7 cases (37%); and score 7,

2 cases (11%). The HIF1 α expression score was

significantly higher in tumors with higher grade

(P=0.004). In addition, HIF1 α expression was

significantly higher in tumors with positive

lymph nodes (P=0.007) (Table I). No significant

correlation was found between HIF1 α

expression and each of patient age, and gender,

the tumor location, gross appearance and T stage

(P=0.689, 0.165, 0.415, 0.256, and 0.859

respectively) (Table I). Relation between CDX2

and HIF-1α expression. All of the 21 cases

(100%) with a negative HIF-1α expression were

positive for CDX2. Out of the 19 cases that were

positive for HIF-1α expression, 11cases (57.9%)

were positive for CDX2, whereas 8 cases

(42.1%) were CDX2 negative. A significant

negative correlation was detected between

CDX2 and HIF-

=0.001 (Table II).

Figure 1: Immunohistochemical staining with Anti-CDX2 (A-C) and Anti-HIF-1α (D-F) antibodies in colorectal carcinoma

(CRC). (A) A well differentiated CRC, with strong immunostaining for CDX2 (200×) (B) A moderately differentiated CRC

demonstrating strong immunostaining for CDX2 (200×) (C) A poorly differentiated CRC showing negative staining for

CDX2 (400×) (D) A well differentiated CRC, negative for HIF-1α (200×) (E) A moderately differentiated CRC

demonstrating moderate immunostaining for HIF-1α (200×). (F) A poorly differentiated CRC showing strong staining for

HIF-1α (400×).

Table I: Relationship between expression of CDX2 and HIF-1 alpha;

and clinicopathological features in colorectal carcinoma.

No. of pts.

CDX2 expression

p Value

HIF-1 alpha

p Value Negative Positive Negative Positive

No. (%) No. (%) No. (%) No. (%)

Age (years)

≤ 40 7 2 (28.5) 5 (71.5) FEp= 0.611

3 (42.9) 4 (57.1) FEp= 0.689

> 40 33 6 (18.2) 27 (81.8) 18 (54.5) 15 (45.5)

Gender

Male 15 3 (20.0) 12(80.0) FEp= 1.000

10 (66.7) 5 (33.3) 0.165

Female 25 5(20.0) 20(80.0) 11 (44.0) 14 (56.0)

Tumor location

Right sided

colon 18 3 (16.7) 15 (83.3)

MCp=0.205

10 (55.6) 8(44.4)

MCp=0.415

Transverse

Colon 4 2 (50.0) 2 (50.0) 1 (25.0) 3 (75.0)

Left sided

colon 12 1(8.3) 11 (91.7) 8 (66.7) 4 (33.3)

Rectum 6 2(33.3) 4(66.7) 2(33.3) 4 (66.7)

Gross appearance

Fungating 18 3 (16.7) 15 (83.3) MCp=0.689

11 (61.1) 7 (38.9)

0.256 Ulcerative 10 3 (30.0) 7 (70.0) 3 (30.0) 7 (70.0)

Annular 12 2 (16.7) 10 (83.3) 7(58.7) 5 (41.7)

Tumor grade

WD 15 1 (6.7) 14 (93.3) MCp=0.040*

13 (86.7) 2 (13.3)

0.004* MD 15 2 (13.3) 13 (86.7) 5 (33.3) 10 (66.7)

PD 10 5 (50.0) 5 (50.0) 3 (30.0) 7 (70.0)

T stage

T1 1 0 (0.0) 1 (100.0)

MCp=1.000

0 (0.0) 1 (100.0)

FEp= 0.859 T2 2 0 (0.0) 2 (100.0) 1 (50.0) 1 (50.0)

T3 36 8 (22.2) 28 (77.8) 19 (52.8) 17(47.2)

T4 1 0 (0.0) 1 (100.0) 0 (0.0) 1 (100.0)

LN status

Negative 15 0 (0.0) 15 (100.0) FEp= 0.016*

12 (80.0) 3 (20.0) 0.007*

Positive 25 8 (32.0) 17 (68.0) 9 (36.0) 16 (64.0)

WD: Well Differentiated; MD; Moderately Differentiated; PD: Poorly Differentiated; LN: Lymph Node

* Significant at p < 0.05

Table (II): Relation between HIF-1α and CDX2 immunostaining in the 40 cases of CRC studied

HIF-1α

Χ2 FEp Negative (n = 21) Positive (n = 19)

No. % No. %

CDX2

11.053* 0.001* Negative 0 0.0 8 42.1

Positive 21 100 11 57.9

Χ2: Chi square test, FE: Fisher Exact, *: Statistically significant at p < 0.05

Discussion

In the present work, immunohistochemical

analysis of CDX2 was carried out in 40 cases of

colorectal carcinoma. The immunoreactivity was

found to be nuclear and/or cytoplasmic. A

statistically significant relation was found

between CDX2 expression and the tumor grade,

where higher CDX2 expression was detected in

lower grade tumors. This finding is in agreement

with the findings reported by Bae et al (25) who

found that loss of CDX2 expression exhibited a

close association with poor differentiation.

Moreover, Olsen et al (26), Baba et al (11) and

Lugli et al (27) reported that a loss of CDX2

expression was correlated to high tumor grade.

On the other hand, Saad et al (28) showed that

CDX2 expression was not influenced by tumor

grade. Meanwhile, Werling et al (29) reported that

the expression of CDX2 did not appear to

correlate with the level of tumor differentiation.

The current work also showed that higher CDX2

expression was more frequent in tumors with

negative lymph nodes. This finding is in

agreement with the findings reported by Lugli et

al (27), Dawson et al (30) and Bae et al (25), who

found that the loss of CDX2 expression is

associated with a higher N stage. In the present

work, immunohistochemical study of HIF-1α

expression in CRC cases showed cytoplasmic

and/or nuclear immunoreactivity. Higher HIF-1α

expression was significantly associated with

higher tumor grade, suggesting that hypoxia

plays a role in the progression of CRC. This

finding is in agreement with the results reported

by Wang (31), who observed a significant

correlation between the differentiation status of

the tumor and expression of HIF-1α, with

increased HIF-1α expression in moderately and

poorly differentiated tumors. Moreover, Furlan

et al (32) and Xie et al (33) also found a significant

correlation with poor differentiation. On the

other hand, Cao et al (34) found no significant

relationship between the expression of HIF-1α

and the degree of histological differentiation.

The present study also showed that higher HIF-

1α expression was more frequent in tumors with

positive lymph nodes. This finding is in

agreement with the findings reported by Wang (31), who found that HIF-1α expression is closely

related to the advancement of lymphatic

metastasis. Moreover, Wu et al (35), Simiantonaki

et al (36) and Ding et al (37) found that the

expression of HIF-1α is significantly higher in

colon carcinoma tissue obtained from tumors

associated with lymph node metastasis.

Contradictory results have been reported by Xie

et al (33), who found no relation between

expression of HIF-1α and lymph node metastasis

in CRC. In the present study, the relation

between HIF-1α immunostaining, on one hand,

and patient’s age and gender, the tumor location,

gross appearance and T stage, on the other hand

was statistically insignificant. Similarly, Xie et al (33) reported that HIF-1α expression showed no

significant correlation with age, gender and

tumor localization in CRC. In the present study,

the finding that lymph node status (N stage) was

correlated negatively with CDX2 expression and

positively with HIF-1α expression, whereas no

correlation was found with tumor depth of

invasion (T stage) may be explained by the fact

that we had 36 cases in T3 category which

represents about 90% of the cases. In the present

study, a significant inverse correlation was found

between the expression of HIF-1α and CDX2,

suggesting that HIF-1α may contribute to the

progression of CRC by inactivating CDX2. This

finding supports the results reported by Derbal-

Wolfrom et al (38), who observed that hypoxia

downregulates the intestine-specific homeobox

gene CDX2.

Conclusions

Findings of the current study suggest that

hypoxia plays a role in the progression of

colorectal cancer. The detected inverse

correlation between HIF-1 α and CDX2 suggests

that hypoxia may result in colorectal cancer

progression through inactivation of CDX2 by

HIF-1α.

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Original Article

Alterations in Phosphatase and Tensin Homolog (PTEN) Tumor Suppressor

Gene in Egyptian Patients with Hepatocellular Carcinoma

Mohamed H. Abdel-Rahman1,2, Mohamed S. Kohla2,3, Amr M. Aziz4, Sameera A. Ezzat2,5; 1Department

of Pathology, 3Department of Hepatology, 4Department of Hepatobiliary Surgery, 5Department of Public

Health, National Liver Institute, Menoufia University, Egypt. 2National Liver Institute Sustainable

Sciences Institute Collaborative Research Center, Menoufia University, Egypt.

ABSTRACT

Phosphatase and tensin homolog (PTEN) is a tumor suppressor gene which is involved in the pathogenesis of

multiple cancers including hepatocellular carcinoma (HCC). Experimental evidence suggests that PTEN could be

utilized as an effective targeted therapy for multiple tumors. Aim of the work : The aim of this study was to

determine the frequency of PTEN alterations in Egyptian patients with HCC. Material and Methods: PTEN protein

expression was studied using immunohistochemistry in 42 unselected sporadic HCC who underwent hepatic

resection from Egyptian patients. Molecular genetic alterations in PTEN gene was studied in the same samples using

genotyping with two microsatellite markers flanking the PTEN gene and an intragenic marker within the gene.

Results: PTEN expression was successfully assessed in 37 tumors. Two of these tumors (5.4%) showed total loss of

PTEN by immunostaining, 16 (43.2%) showed weak or partial staining and the remaining 19 (51.4%) showed

staining similar to the normal internal controls. The strength of immunostaining correlated with tumor grade with

higher grade tumors showing more prominent PTEN loss. The loss of heterozygosity (LOH) in PTEN correlated with

loss of protein expression detected by immunostaining. Conclusion: Our results suggest that a small subset of

Egyptian patients with HCC have biallelic inactivation of PTEN. These patients are the ones likely benefit from

therapies targeting the AKT/mTOR (protein kinase-B/mammalian target of rapamycin) pathway. It is essential before

starting targeted based therapy to obtain biopsy from the patients in order to better select the appropriate therapy

based on genetic alterations in their tumors.

Introduction

PTEN (MMAC1/TEP1, phosphatase and tensin

homolog deleted on chromosome 10) is a tumor

suppressor gene located on chromosome sub-

band10q23.3. PTEN is variably mutated and/or

deleted in a variety of human cancers including

glioblastoma, melanoma, prostate cancer, breast

cancer, endometrial cancers and hepatocellular

carcinoma (1-3). PTEN encodes a dual-specificity

phosphatase and is the major lipid 3-phosphatase

which signals down the phosphatidylinositol 3-

kinase/Aktproapoptotic pathway and effects cell

cycle arrest and apoptosis (4-8). However, no

information is currently available on the role of

PTEN deficiency in hepatocellular carcinomas

from Egyptian patients. Several studies have

showed the utility of PTEN as potential targeted

therapy in different tumors. AKT inhibition has

remarkable activity against a wide range of

human cancers in vitro and in human tumor

xenograft models, especially when PTEN is

dysfunctional (reviewed in (9)).The clinical

challenge for the application of this class of

anticancer drugs is the ability to prospectively

identify which tumors will be sensitive to AKT

inhibition. The recent failure of the clinical trial

utilizing AKT inhibitor MK2206 on all-comer

with HCC, highlight the importance of pre-

selection of patients who could respond to target

therapy (clinicaltrials.gov, trialNCT01239355).

In Egypt liver cancer incidence rose dramatically

during the last two decades to become the most

common cancer in men and the second most

common cancer in women(10). The vast majority

of these are hepatocellular carcinoma (HCC)

caused by chronic hepatitis C virus infection.

This huge increase in liver cancer burden in

Egypt necessitates identifying novel strategies

for treatment and hopefully prevention of these

tumors. Targeted based therapy is a promising

avenue for treatment of many tumors. In HCC

Sorafenib Tosylate (Nexavar), a small molecule

Raf kinase and VEGF receptor kinase inhibitor is

currently the only clinically approved targeted

therapy in unresectable HCC(11). Sorafenib

therapy causes a modest 2-3 months but

significant improvement of the time-to-

progression in treated patients (11). However, the

high cost of the drug in Egypt and the modest

efficiency in the majority of patients limit its use

significantly. The aim of this study was to

investigate the frequency of alterations in the

PTEN, a major target for AKT inhibitors, in

HCC from Egyptian patients who underwent

hepatic resection to assess the potential utility of

these agents for treatment of HCC in preselected

patients.

Patients and Methods

Tumor Samples: Specimens were retrospectively

obtained from the archive of the pathology

department National Liver Institute, Menoufia

University. A total of 42 patients who underwent

hepatic resection were accrued, 38 of them were

males and four were female, Table 1. The

average age of the patients was 53 years (range

35-67). Thirty nine of the specimens were

surgical resections and three were explanted

liver. In 10 patients non-tumor tissue other than

liver tissue was available. In those 10 patients

genotyping was studied in both cirrhotic liver

tissue and tumor tissues. The research was

approved by an Institutional Review Board of

the National Liver Institute, Menoufia University

(IRB# 04/2008). Immunohistochemistry:

Immunohistochemistry was carried out at the,

Department of Pathology, The National Liver

Institute. Monoclonal antibody 6H2.1 against the

terminal 100 amino acids of human PTEN

(DAKO, Burlingame, CA) was used at a dilution

of 1:100 with an overnight incubation at 4 C°.

Deparaffinized tissue sections were heat

pretreated with, 0.01M sodium citrate buffer (pH

6.0). DAKO LSAB 2+ Kit was used for

detection of immunostaining using DAB

chromogen. After counterstaining with

hematoxyline and mounting, the slides were

evaluated under a light microscope. The

immunostaining signals were scored on a 0 to

300 scale with the intensity of the staining

scored from 0 to 3 and the percentage of the cells

stained was recorded from 1-100% (Figure 1).

The percentage of tumor cells showing nuclear

stain was scored from 1-100%. The final 0-300

score was derived from multiplication of the two

scores. Staining limited only to the periphery of

the tissues was excluded from the scoring

assessment. Staining of the endothelium of

normal blood vessels was used as an internal

positive control for PTEN, as previously

described (12). Staining intensity was normalized

to the intensity of staining of the internal

controls. The tumors were divided into

threegroups accordingto the degree of PTEN

cytoplasmic staining: normal staining was

assigned ++ when an overall score of ≥ 150 was

obtained, which represented tumors with staining

intensity equal to that of the matching internal

normal control (Figure 1); the group assigned +

had an overall immunostaining score ranging

from >15 to <150 representing tumors with

decreased staining intensity; and the group

assigned –ve had a total score ≤15 and was

considered null for PTEN immunostain.

Gentotyping for PTEN DNA were assessed for

LOH at three polymorphic markers flankingand

within the PTEN gene. The marker order, from

centromereto telomere is: D10S1765-IVS4 +

109ins/delTCTTA-D10S541. PTEN lies between

D10S1765 and D10S541. D10S1765 is within

500 kb upstream of the transcriptionalstart site of

PTEN and D10S541 is within 300 kb of the

translationalstop site. IVS4 + 109ins/delTCTTA

lies within PTEN. D10S1765and D10S541 were

screened as described previously22.

Polymerasechain reaction (PCR) conditions for

these markers are describedelsewhere 36. For

IVS4 + 109 ins/delTCTTA, we used forward 5’-

TGGGGGTGATAACAGTATCTA-3’ or 5’-

AGACATTATGACACCGCCAAAT-3’ and

reverse 5’-CTTTATGCAATACTTTTTCCTA-

3’ primers for a heterozygote PCR product

410/415 or 219/224 base pairs respectively. The

forward primer was labeled with the fluorescent

marker FAM. The PCR products were analyzed

using an ABI 377 sequencer and the GeneScan

and Genotyper softwares (Applied Biosystems,

Foster City, CA, USA). The genotyping was

carried out at least twice for all the samples.

Results

Genotyping: Out of the 42 studied tumors

informative results in at least one genotyping

marker was available in 37 samples. The

D10S1765 marker was informative in 32

patients, the D10S541 was informative in 24

patients and the IVS4 + 109ins/delTCTTA was

informative in 18 patients. Loss of

heterozygosity in at least one marker was

identified in 14/37 (37.8%). Out of those 14

patients with LOH, only 6 had evidence of large

deletion (LOH in 2 markers). In three tumors

multifocal cancer was observed and those tumor

tissues were dissected from individual cancer

foci. In two of them all the different foci showed

the same genotyping alteration while in one

tumor one focal mass showed evidence of LOH

in one marker while the other focal mass showed

LOH. None of the cirrhotic liver showed any

allelic imbalance in any of the studied markers.

For samples with LOH we didn’t observe total

loss of an allele in any of the samples and we

observed partial allelic imbalance, Figure 1.

Immunohistochemistry: Immunohistochemistry

was assessed in tumors with evidence of positive

staining of internal control (blood vessels) in the

tumor and non-tumor liver. Five tumors were

excluded because of negative staining of the

internal control of marked heterogeneity of the

internal control staining suggesting problem with

tissue fixation. In the assessed 37 tumors, 2

(5.4%) showed total loss of PTEN expression

from almost all the tumor tissue, 16 (43.2%)

showed decrease in PTEN expression in part of

the tumor tissue while the remaining 19 (51.2%)

tumors the expression of PTEN was similar in

the non-tumor liver to the tumor tissue, Figure 2.

The strength of immunostaining correlated with

tumor grade with higher grade tumors showing

more prominent PTEN loss. In 33 tumors both

immunostaining and genotyping were available,

of those 33, 16 had normal expression of PTEN

while 17 had no or decrease PTEN expression.

Out of the 16 with normal PTEN expression 2

tumors showed LOH of the gene while out of the

17 tumors with either noor decrease PTEN

expression 9 showed LOH. The difference was

statistically significant (p = 0.014).

Discussion

HCC is one of the major cancers in the world

with very limited systemic treatment options.

The progress in targeted based therapy for other

cancer has not been translated to HCC. Since

2007 years Sorafenib tosylate (Nexavar®) has

been the only targeted based therapy with proven

efficiency, although modest, for treatment of

advanced HCC(11, 13, 14). Several targeted based

therapy trials failed to show any significant

benefit for patients. The AKT inhibitor MK2206,

targeting the PTEN/AKT pathway, has shown

effect in several preclinical in-vitro studies.

Based on that a clinical trial (NCT01239355,

clinicaltrials.gov) has been conducted for

unselected adult HCC patients with advanced

unresectable tumors. However, the trial was

early terminated due to discouraging response.

Our study shed some light on potential

explanation based on the findings with PTEN,

which is considered one of the main tumor

suppressor genes in the AKT pathway. Our study

suggests that only very small subset of patients,

2/37 (5.4%), presenting with total loss of PTEN

expression would have maximum benefit from

the AKT inhibitor treatment based on their

PTEN status. These patients likely reflect

biallelic inactivation of PTEN in their tumors. In

a recent genome wide molecular genetics study

of 243 tumors, homozygous deletion of PTEN

was detected in only 5/243 patients (2%) which

is consistent with our findings (15). Consistent

with this explanation in HCC cell line SNU475

with total loss of PTEN protein expression,

suggesting biallelic inactivation, the MK2206

has a 10 folds better therapeutic response

compared with cell lines with normal or decrease

PTEN (16). Taken together these suggest that

patients with total loss of PTEN could have

maximum benefit from AKT inhibitors such as

MK2206. Future clinical trials with proper

selection of patients will be needed to explore

this hypothesis. In addition to the 2 cases with

total loss of PTEN protein expression in their

tumor, heterozygous LOH of PTEN and/or

partial loss of PTEN expression were identified

in the tumors of 20 (47.6%) additional patients.

In these patients the expression of PTEN protein

was not uniform across the tumor indicating

tumor heterogeneity. Also, we didn’t observe

total loss of PTEN allele in any of the tumors

with LOH which also indicate that some of the

tumor cells didn’t have LOH of PTEN which

supports tumor heterogeneity. In one of three

tumors with multifocal lesions one of the two

tumor foci showed LOH of PTEN while the

other tumor foci showed LOH of PTEN. Taken

together these data indicate that alterations in

PTEN are associated in the majority of tumors

with tumor progression rather than tumor

initiation. Also it suggests that targeted therapies

utilizing AKT inhibitors will impact only part of

the tumor which will result in partial tumor

regression with likely development of resistance

to therapy. Our result raises the importance of

biopsy based selection of patients who are

candidates for targeted based therapy in order to

maximize the benefit and minimize the risk of

these drugs. Out of the 42 tumors we were not

able to assess immunostaining status on 5

tumors. Failure of PCR amplification of at least

one genotyping marker on multiple attempts was

also observed in 6 tumors. Trouble shouting of

these experiments suggested prolonged fixation

and/or fixation in high concentration of formalin

as the cause of failure of immunostaining and/or

genotyping. The failure of molecular studies in

these samples highlights the importance of

proper processing and fixation of the samples in

order to obtain clinically applicable results.

Conclusion

Our results suggest that a small subset of

Egyptian patients with HCC could benefit from

AKT inhibitor therapy based on their PTEN

status. It is essential before starting targeted

based therapy to obtain biopsy from the patients

in order to better select the appropriate therapy

based on genetic alterations in their tumors.

Acknowledgments

This project was funded in part by a grant from

the Sustainable Sciences Institute.

Table 1. Summary of the clinical data of the patients included in the study

Clinicopathological factors Total 42

Age at diagnosis, mean (range) 52.9 (35-67)

Gender, n (%)

Male 38 (90.5 %)

Female 4 (9.5 %)

Hepatitis Virus Status(known on 32 patients)

HCV 28(87.5 %)

HBV 3 (9.4 %)

HCV+HBV 1 (3.1 %)

Treatment

Resection 39 (92.9 %)

Liver Transplantation 3 (7.1 %)

PTEN Genotyping (37/42 informative)

Loss of Heterozygosity (LOH) 14 (37.8 %)

Retention of Heterozygosity (ROH) 23 (62.2 %)

PTEN Protein expression (assessed 37of42)

0 2( 5.4 %)

1 16 (43.2 %)

2 19 (51.4 %)

*protein expression was not assessed on all samples with genotyping

Figure 1. Representative genotyping results: A) A sample with retention of heterozygosity in marker D10S541, note equal

heights of the two alleles. B) A sample with loss of heterozygosity of marker D10S1765. Note total loss of one allele in the

tumor (arrows). C) A multifocal tumor showing LOH/Allelic imbalance in all three tumor foci. Note the variation in allele

heights between peripheral blood the tumors. PB: peripheral blood, T: tumor.

Figure 2. Summary and representative samples of the PTEN immunostaining results

A) A sample with total loss of PTEN protein expression. Note positive staining of the blood vessels and the sinusoids

(arrows). B) A sample with weak staining of PTEN. Note the staining intensity in the tumor is lower than the staining in

the blood vessels (arrow). C) A sample with strong staining of PTEN. Note equeal staining in the tumor and blood vessels.

D) Summary of the PTEN protein expression and genotyping in the tumors studied. Note most of the tumors with

retention of heterozygosity showed normal protein expression.

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Original Article

Atypical Manifestations of Viral Hepatitis A

Alaa Abdo, Nasser Abd Alla (1), Abu Rawy Ismail (2); Department Of Tropical medicine, Faculty of

Medicine, University of Alexandria (1), Ministry of Health Hospitals (2)

ABSTRACT

Hepatitis A is a viral liver disease that can cause mild to severe illness. It is primarily spread from ingestion of food

or water contaminated with the faeces of an infected person. The disease is closely associated with a lack of safe

water, inadequate sanitation and poor personal hygiene. Unlike hepatitis B and C, hepatitis A infection does not

cause chronic liver disease and is rarely fatal, but it can cause debilitating symptoms and fulminant hepatitis (acute

liver failure), which is associated with high mortality. In Egypt, which is considered an area of high endemicity for

HAV infection, highly effective vaccines against hepatitis A have been available since the mid 1990s. Aim of the

work: was to investigate the clinical features and outcome of symptomatic viral hepatitis A with emphasis on the

incidence and clinical features of atypical presentation of the disease. Material and methods This study included

serologically confirmed 100 patients with acute viral hepatitis A and subjected to history taking (Name, age, sex,

occupation, and smoking) and asking for clinical manifestations (Fever, anorexia, vomiting, diarrhea, and others).

Laboratory investigations included viral hepatitis markers, complete blood count, liver and renal function tests.

Patients were subjected to ultrasound study of the abdomen stressing on the stigmata of liver disease. Results:

showed that 98 patients (98%) recovered without fulminant hepatitis. 11 patients (11%) showed atypical

manifestations: 7 (7%) prolonged cholestasis, 2 (2%) hemolytic anemia, 1 (1%) relapsing hepatitis, and 1 (1%)

pleural effusion. Conclusions :The overall case fatality was 1%, and none of our patients was indicated for liver

transplantation.

Introduction Hepatitis A virus (HAV) is the most common

form of acute viral hepatitis allover the world. It

is classified in the genus Hepatovirus within the

family Picornaviridae , isolated by Purcell in

1973. (1) HAV is among the smallest and

structurally simplest of the RNA animal viruses.

The virion is nonenveloped with a diameter of

27-32 nm. It is composed entirely of

viral protein and RNA. Due to its high

prevalence worldwide HAV alone accounts for

20–25% of clinically apparent hepatitis, with 1.5

million clinical cases reported worldwide

annually. (1) The likelihood of symptomatic

infection increases with the age of acquisition,

with most children under 6 years of age being

asymptomatic. The HAV-associated mortality of

0.2–0.4% of symptomatic cases is increased in

individuals older than 50 years or less than 5

years of age at acquisition. The significant

morbidity and mortality of HAV infection in

childhood are due to its high worldwide

prevalence , the development of fulminant

hepatic failure as well as hepatic and

extrahepatic complications. A significant change

in the epidemiology of HAV infection has

occurred over last decades. This is due to both

improved public health hygiene measures and

also the introduction of vaccination programs. (2)

Humans appear to be the only reservoir for this

virus. It is primarily spread from ingestion of

food or water contaminated with the faeces of an

infected person. The disease is closely associated

with a lack of safe water, inadequate sanitation

and poor personal hygiene. Since the application

of accurate serologic investigations in the 1980s,

the epidemiology, clinical manifestations, and

natural history of hepatitis A have become

apparent. (1) Hepatitis A can present as

symptomatic or asymptomatic infection, HAV

infection takes four clinical phases. First phase is

incubation period that varies from 15-45 days

(mean 30 days) , (3) where HAV excretion in the

stools continued for 1-2 weeks before the onset

of illness and at least one week afterward. The

prodromal period corresponds to second phase

and is characterized by nonspecific symptoms

followed by gastrointestinal symptoms such as

nausea, fatigue, abdominal pain, malaise,

anorexia, fever, vomiting and flu like

complaints. (4) These symptoms are usually short

lived and followed by complete recovery. Third

stage is mostly characterized by increase in

bilirubin level and jaundice becomes clinically

apparent when the total bilirubin exceeds 2.0-4.0

mg/dl. In half of the hepatitis A patients, clinical

signs such as hepatomegaly and hepatic

tenderness are prominent. The final phase is a

http://www.who.int/csr/disease/hepatitis/whocdscsredc2007/en/index7.html#virus

http://www.who.int/csr/disease/hepatitis/whocdscsredc2007/en/index7.html#virion

http://www.who.int/csr/disease/hepatitis/whocdscsredc2007/en/index7.html#protein

convalescent period during which the patient

recovers. Signs and symptoms usually last for

less than 2 months, although 10-15 percent of

symptomatic persons have prolonged or

relapsing illness lasting up to 6 months.

(5) Highly effective vaccines against hepatitis A

have been available since the mid 1990s. (6) HAV

is endemic in Egypt and represents a major cause

of acute viral hepatitis being responsible for

more than half of acute hepatitis cases. HAV

genotype IB is the prevalent genotype in Egypt.

Person-to-person transmission, consumption of

food contaminated in fields or through infected

food handlers is very common as well as

consumption of contaminated mollusks (clams,

oysters) and unsafe drinking water represent the

major routes of HAV transmission in Egypt. In

children, daycare centers contribute to HAV

spread among young children, siblings and

parents of daycare center attendees. (7, 8, 9) In the

past two decades, there has been significant

emphasis on improving sanitation measures and

hygiene in Egypt, coupled with extension of

municipal water, sewage, and solid-waste

management systems projects. Therefore, in high

social classes, children are infrequently exposed

to hepatitis A virus with subsequent decline in

herd immunity and a change to the epidemiology

of the illness with steady increases in the mean

age of occurrence of illness attributed to acute

hepatitis A virus infection. In urban Egypt,

despite the overall decrease in the incidence of

HAV infection, more HAV infections are

encountered at older age groups. (10) However,

the clinical manifestations of hepatitis A that

include atypical features were not studied in a

prospective manner, and detailed analysis of

such atypical presentation was limited.

Aim of The Work

Aim of this work was to investigate the clinical

features and outcome of symptomatic viral

hepatitis A with emphasis on the incidence and

clinical features of atypical presentation of the

disease.


This study included serologically confirmed 100

patients with acute viral hepatitis A , collected

from out patient clinic of Kafr El- dawar fever

hospital(Alexandria , Egypt). All age groups,

both sexes as well as the different grades of

severity of the disease were considered. An

informed consent was taken from every patient

included in the study. The diagnosis was made

by a positive result for anti-HAV IgM antibodies

and elevation of liver function tests (ALT, AST,

and GGT). All patients included in this study

were subjected to thorough and detailed history

taking stressing on the name, age, sex,

occupation, and smoking as well as history of

fever, anorexia, vomiting, diarrhea, abdominal

pain and itching. Physical examination stressed

on the presence of jaundice, hepato-

splenomegaly, anemia, chest , cardiac ,

pulmonary and neurological manifestations

together with the stigmata of liver disease. They

were subjected to laboratory investigations

including complete blood count ,ESR, CRP and

reticulocytes. Liver function tests included

serum transaminases (ALT, AST), Prothrombin

time, Serum albumin, Serum bilirubin (direct,

total), alkaline phosphatase and gammaglutamyl

transpeptidase (GGT). Renal function tests

included blood urea and serum creatinine. Urine

analysis, 24 hours urine, Micral test and

creatinine clearance were done. Autoimmune

markers were done to rule out autoimmune

hepatitis. Viral hepatitis markers by enzyme

immunoassay kits included HAV-IgM and IgG,

HCV Ab, HBs Ag, and HEV- IgM. All patients

were subjected to ultrasound study of the

abdomen to verify the predominant findings of

acute hepatitis as accentuated brightness and

more extensive demonstration of the portal vein

radicles walls and overall decreased echogenicity

of the liver which gives rise to the starry night

pattern. Ultrasound study was helpful also in the

examination of the other gross hepatic disease.

Chest X-ray was done to verify the presence of

pleural effusion and other pulmonary

complications . Statistical analysis of the results

was done data were fed to the computer using

the Predictive Analytics Software (PASW

Statistics 18).

Results

This study included serologically confirmed 100

patients with acute viral hepatitis A. The age of

the studied cases ranged from 2.5-34 years, with

a mean age of 11.24 ± 6.58 years .The most

prevalent age group ( ≤10 years) was 54% of

cases while only 9 cases were >20 years (9%).

49 cases were males (49%) and 51 cases were

females (51%), Table (1)

Table (1): Age and Sex distribution in the studied group of patients Age and Sex distribution No. %

Sex : Male 49 49.0

Female 51.0

Age :≤10 54 54.0

11 – 20 37 37.0

>20 9 9.0

Min. – Max. 2.50 – 34.0

Mean ± SD 11.24 ± 6.58

The major symptoms and signs included abdominal pain, vomiting, anorexia, fever and mild

splenomegaly as shown in table (2)

Table (2): Distribution of the clinical manifestations in the studied group of patients

clinical manifestations No %

Abdominal pain 75 75.0

Vomiting 61 61.0

Anorexia 59 59.0

Fever 47 47.0

Splenomegaly 40 40.0

Diarrhea 6 6.0

Pallor 2 2.0

Itching 1 1.0

Some patients (11) have been detected to have

underlying diseases (table 3). 4 patients had

favism, one of them had fulminant hepatitis with

spontaneous recovery and the other one

developed hemolytic anemia. One patient with

liver cirrhosis (on top of hemochromatosis)

developed prolonged cholestasis. The other 8

patients recovered within 13-25 days, including

2 patients with type I DM who developed

diabetic ketoacidosis. Antinuclear Antibodies

(ANA) were found positive in 40 patients (40%)

Table (3): Distribution of the underlying diseases in the studied group of patients

Underlying disease No %

Rheumatic heart disease 1 1.0

Diabetes Mellitus type I 2 2.0

Epilepsy 1 1.0

Favism 4 4.0

Bronchial asthma 2 2.0

Liver cirrhosis 1 1.0

The mean concentrations of hemoglobin, leucocytes, platelets and erythrocytes were 11.46 ± 1.51 (g/dl),

6.04 ± 2.25 (103/cm), 265.55 ± 75.43 (103/cm) and 4.9 ± 1.1 (106/cm) respectively. Table (4).

Table (4): The hematological findings in the studied group of patients

Hb (g/dl)

Mean ± SD 11.46 ± 1.51

WBC (1000/cm)

Mean ± SD 6.04 ± 2.25

Platelets (1000/cm)

Mean ± SD 265.55 ± 75.43

RBC (106/cm)

Mean ± SD 4.9 ± 1.1

The mean concentrations of serum creatinine and

blood urea were 0.72 ± 0.17 (mg/dl) and 25.90 ±

4.78 (mg/dl). The mean values of ALT, AST,

GGT, ALP, Total S. bilurubin , S.albumin and

prothrombine time were 924.64 ± 491.38

(IU/L), 676.53 ± 412.44 (IU/L), 88.35 ± 44.67

(IU/L), 184.88 ± 56.84 (IU/L), 7.08 ±

4.25(mg/dl), 3.92± 0.37(mg/dl) and 11.86 ±

1.72(sec) respectively as shown in figure (1).

0

100

200

300

400

500

600

700

800

900

1000

ALT AST GGT ALP

Mea

n

Figure (1): Liver function tests in the studied group of patients.

The clinical outcome of acute hepatitis A

showed that 98 patients (98%) recovered without

fulminant hepatitis. 11 patients (11%) showed

atypical manifestations: 7 (7%) prolonged

cholestasis, 2 (2%) hemolytic anemia, 1 (1%)

relapsing hepatitis and 1. (1%) pleural effusion.

Only 2 patients (2%) were complicated by

fulminant hepatitis; one patient died due to

hepatic failure and the other one recovered on

supportive management. The overall case fatality

was 1%, and none of our patients was indicated

for liver transplantation. Table (5) & Figure (2)

Table (5): The outcome of acute hepatitis A infection in the studied group of patients

Clinical outcomes No. %

Recovery without fulminant 98 98.0

Fulminant hepatitis 2 2.0

Spontaneous recovery 1 1.0

Death due to hepatic failure 1 1.0

Atypical manifestations: 11 11.0

Prolonged cholestasis 7 7.0

Hemolytic anemia 2 2.0

Relapse 1 1.0

Pleural effusion 1 1.0

Typical

8787%

Fulminant hepatitis

22%

Prolonged cholestasis

77%

Hemolytic anaemia

22%

Relapse

11%

Pleural effusion

11%

Atypical

1111%

Figure (2): The outcome of acute hepatitis A infection in the studied group of patients

Atypical manifestations of acute hepatitis A

infection: 1- Prolonged cholestasis: Among the

total 100 patients, 7 cases (7%) showed

prolonged cholestasis lasting for more than 4

weeks. The comparative analysis of these cases

according to antinuclear antibodies, demographic

data, clinical manifestations, laboratory

investigations and underlying diseases was

shown in tables (6,7, 8, 9 ,10). Patients with

prolonged cholestasis had higher frequency of

fever and anorexia, significantly low platelets

count, high white blood cells count and high

serum bilirubin (total and direct) level as well as

significantly high levels of ALT, AST, GGT,

and ALP. No significant difference was detected

on comparing the positivity of antinuclear

antibodies in the typical cases of acute hepatitis

A and those with prolonged cholestatic hepatitis.

Table (6): Age and sex distribution in the typical and prolonged cholestatic hepatitis A cases

Typical(n = 89)

Prolonged cholestasis (n = 7)

Test of sig.

No % No %

Sex: Male 41 47.0 5 71.4 FEp = 0.263

Female 46 52.9 2 28.6

Age: ≤10 46 52.9 3 42.9

MCp = 0.611 11 – 20 33 37.9 3 42.9

>20 8 9.2 1 14.3

Min. – Max. 2.50 – 34.0 6.0 – 23.0 p = 0.511

Mean ± SD 11.48 ± 6.64 12.86 ± 6.59

MCp: p value for Monte Carlo test FEp: p value for Fisher Exact test p:p value for Student t-test

Table (7): The distribution of the clinical manifestations in the typical and prolonged cholestatic hepatitis A cases

Typical

(n = 89)

Prolonged

cholestasis

(n = 7) Test of sig.

No % No %

Fever 37 42.5 6 85.7 P = 0.001*

Anorexia 47 54.0 7 100.0 FEp = 0.019*

Vomiting 53 60.9 5 71.4 FEp = 0.704

Diarrhea 6 6.9 0 0.0 FEp = 1.000

Abdominal pain 65 74.7 7 100.0 FEp = 0.194

Itching 1 1.1 0 0.0 FEp = 1.000

Mild splenomegaly 31 35.6 4 57.1 FEp = 0.418

p: p value for Chi-square test & FEp: p value for Fisher Exact test *: Statistically significant at p ≤ 0.05

Table (8): Comparison between the typical and prolonged cholestatic cases according to CBC

Typical (n=89) Prolonged cholestasis (n = 7) Test of sig.

Hb (gm/dl): Mean ± SD 11.59 ± 1.24 11.50 ± 1.80 p = 0.856

WBC(1000/cmm): Mean ± SD 5.52 ± 1.69 9.36 ± 2.42 MWp <0.001*

Platelets(1000/cmm): Mean ± SD 276.95 ± 73.10 170.29 ± 38.58 MWp <0.001*

RBCs9106): Mean ± SD 5.1 ± 0.6 4.9 ± 0.56 p = 0.755

p:p value for Student t-test & MWp: p value for Mann Whitney test *: Statistically significant at p ≤ 0.05

Table (9): Comparison between the typical and prolonged cholestatic cases according to liver function tests

Typical (n = 89) Cholestasis (n = 7) Test of sig.

ALT(u/ml) :Min. – Max. 120.0 – 2248.0 953.0 – 1858.0 MWp = 0.001*

Mean ± SD 852.08 ± 419.15 1428.57 ± 325.80

AST(u/ml): Min. – Max. 89.0 – 2212.0 509.0 – 1780.0 MWp = 0.037*

Mean ± SD 718.68 ± 353.28 1037.14 ± 414.02

GGT(u/ml): Min. – Max. 40.0 – 99.0 30.0 – 482.0 MWp = 0.002*

Mean ± SD 82.31 ± 11.50 170.0 ± 147.93

ALP (u/ml):Min. – Max. 105.0 – 200.0 20.0 – 490.0 MWp = 0.006*

Mean ± SD 177.14 ± 12.95 286.0 ± 179.65

Proth. Time(%): Min. Max 10.60-14.70 11.20 – 12.70 MWp = 0.126

Mean ± SD 11.65 ± 0.54 11.94 ± 0.56

S. albumin(gm/dl): Min. –Max 3.20 – 4.80 3.20 – 4.30 p = 0.854

Mean ± SD 3.94 ± 0.36 3.91 ± 0.41

T.S.Bil.(mg/dl): Min. – Max 2.60-16.20 3.90-15.0 MWp = 0.003*

Mean ± SD 6.28 ± 2.50 10.90± 3.83

D.S.Bil.(mg/dl): Min. – Max 1.90-12.50 3.30-13.10 MWp = 0.002*

Mean ± SD 5.15 ± 2.12 9.23± 3.27

p:p value for Student t-test &MWp: p value for Mann Whitney test&*:Statistically significant at p ≤ 0.05

Table (10): The Distribution of the underlying disease in the typical and prolonged cholestatic cases

Typical

(n = 89)

Prolonged cholestasis

(n = 7) Test of sig.

Underlying disease No % No %

Rheumatic heart disease 1 1.1 0 0.0 MCp = 0.264

diabetes mellitus type I 2 2.3 0 0.0

Epilepsy 1 1.1 0 0.0

Favism 2 2.3 0 0.0

Bronchial asthma 2 2.3 0 0.0

Liver cirrhosis 0 0.0 1 14.3

MCp: p value for Monte Carlo test

2- Hemolytic anemia: Among the total 100

patients, 2 cases (2%) showed hemolytic anemia.

The first one was male, 5 years old, presented

initially with pallor and jaundice with positive

combs test and antinuclear antibodies. The

ultrasound study revealed mild splenomegaly.

He recovered within 15 days after blood

transfusion. The second one was male; 6 years

old, presented with pallor and jaundice. He had

a history of favism (glucose 6 phosphate

dehydrogenase deficiency) .The combs test and

antinuclear antibodies were negative. The

ultrasound study revealed mild splenomegaly.

He recovered within 13 days after blood

transfusion. 3- Pleural effusion: Among the total

100 patients, 1 case (1%) showed pleural

effusion. She was 11 years female, presented

with fever, jaundice, abdominal pain and

dypsnea. The laboratory investigations revealed

leucocytosis, hypoalbuminemia, hyperbiliru-

binemia, high liver function tests (ALT, AST,

GGT, and ALP) and positive antinuclear

antibodies. The chest X-ray study revealed

closure of costophrenic sinus, but the ultrasound

examination showed mild hepatomegaly without

ascites. She recovered within 15 days without

complication. 4- Relapsing hepatitis: Among the

total 100 patients, 1 case (1%) showed relapsing

hepatitis. This case was characterized by a

biphasic peak of serum aminotransferase

elevation with 3 weaks intervals between the

first and second peak. During the relapse, there

were hypoalbuminemia, hyperbilirubinemia,

high serum aminotransferases (ALT, AST) and

prolonged prothrombin time. Recovery occurred

within 28 days after plasma infusion

Discussion

Hepatitis virus A (HAV) is the etiologic agent of

acute viral hepatitis. Following initial studies on

the clinical features of the disease and

subsequent development of safe and effective

vaccines in the early 1990s, research on acute

hepatitis A faded away. (1) However, HAV

remains an important cause of hepatitis

outbreaks and fulminant hepatitis. (11). Our study

showed that the mean age of patients was 11

years and 91% of patients were <20 years. This

was compatible with the report on hepatitis A in

Egypt from 2001 to 2004 (1684 patient) by M.

Talaat et al(12) in which the mean age of patients

was 9 years .(12)In the report on hepatitis A in

Qatar from 2002 to 2006 by Bener A et al

hepatitis A was more prevalent in children below

15 years (72.3%). (13) Also with the report in

Saudi Arabia from 2000 to 2005 (1214 patient)

by Al-Tawfig JA and Anani as HAV infection

predominates in children (1-20 years). (14) On the

other hand, these findings were not compatible

with the report on hepatitis A (595 patients) by

Youn Mu Jung et al in Korea in which the

median age of patients were 30

years. (15) Frequency of fever, vomiting and

diarrhea were 47%, 61% and 6% respectively

which were compatible with the report on

hepatitis A (595 patients) by Youn Mu Jung et al

in Korea in which frequency of fever, vomiting,

and diarrhea were 56%, 61%, 4%

respectively. (15). The present study showed that

the case fatality rate was 1% which was not

compatible to previous results showing a case

fatality rate of 0.2% in Egypt from 2001 to 2004

(1684 patient) by M Talaat et al. (12) The

incidence of fulminant hepatitis in our study was

2%, the same reported by Youn Mu Jung et

al. (15) In our study, high bilirubin levels were

significantly associated with fulminant

hepatitis, which was compatible to previous

results by Rezende et al. This suggests that

HAV-related liver failure is due to a vigorous

host immune response with a marked reduction

in viral load, rather than a direct viral effect.

(16) The first patient associated with

encephalopathy and deterioration of renal

function died. The second patient with favism

(without encephalopathy) recovered within 28

days with normal renal function. This was

compatible to the previous results by Ileana et al

which reported that creatinine >2 mg/dl is the

best index of sensitivity and specificity for

detecting fulminant hepatitis and mortality,

without overlapping and 66.2% patients died,

while 32.4% patients recovered from the total of

73 patient. (17). The atypical manifestations in our

study were 11% (7% prolonged cholestasis, 2%

hemolytic anemia, 1% relapsing hepatitis and

1% pleural effusion). The atypical

manifestations reported by Youn Mu Jung et al

in Korea (595 patient) were 12.6%

(4.7% prolonged cholestasis, 6.4% delayed anti-

HAV IgM seroconversion, and 1.5% acute

kidney injury). (15). The mean peak bilirubin level

in our study was 11mg/dl, while the non-

cholestatic group showed a mean peak bilirubin

level of 6mg/dl, These findings were compatible

with the report on cholestatic hepatitis A by

Tong et al. (18) Higher frequency of fever in our

study was compatible with the report on

cholestatic hepatitis A by Youn Mu Jung et

al. (15) The low platelet count in our study was

compatible with the report on cholestatic

hepatitis A by Hyoung Su Kim et

al. (19) Although the detailed mechanisms of

cholestasis are not exactly known (20), this may

be caused by viral infection which probably

induces inhibition of bile salt transporter

function by proinflammatory cytokines. On the

other hand, secretion of HAV across the apical

canalicular membrane of hepatocytes into the

biliary system may involve vectorial cellular

vesicular transport mechanisms. (1) Complete

recovery of all cases was compatible with the

report on cholestatic hepatitis A by Ileen L et

al (21). Hemolytic anemia is an extrahepatic

manifestation of viral hepatitis, including

hepatitis A. The results of our study showed that

2 cases developed hemolytic anemia, G6PD

deficient, direct Coombs’ test was negative in

the first one which was compatible with the

report on hemolytic anemia following hepatitis

A infection by Ozbay Hosnut F et

al (22) but G6PD normal-direct Coombs’ test was

positive, and antinuclear antibodies were

positive in the second one which was

compatible with the report on autoimmune

hemolytic anemia following hepatitis A infection

by Nafiye Uranci et al (23). The pathogenic

mechanism of hemolytic anemia during hepatitis

A infection has not been elucidated completely.

It could be associated with shortened red cell

survival, or it might be autoimmune in nature (24,

25). Massive hemolysis has been reported in

patients with infection with hepatitis A, B or E

viruses and a concomitant glucose-6- phosphate

dehydrogenase (G6PD) deficiency.(26, 27). Pleural

effusion is a rare complication of acute hepatitis.

To the best of our knowledge, few cases

associated with HAV have been previously

reported. (28, 29, 30, 31, 32) The first case was reported

in 1971. (33) In our study, 1% of hepatitis A

patients showed pleural effusion.

Hypoalbuminemia and fever in this case were

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compatible with the report on pleural effusion by

Ela Erdem et al. (34) Leucocytosis in our study

was compatible with the report on pleural

effusion by Ponnurangam et al (35) but

leucocytosis was not compatible with the report

on pleural effusion by Ela Erdem et al in which

leucocytes were normal. (34) The exact

pathogenesis of the effusion is unknown. It

seems likely that it is related to associated

inflammatory reaction caused by immune

complexes. Other serosal surfaces were involved

and there was evidence of glomerular

involvement. (36). Our study showed that 1% of

hepatitis A patients was associated with

relapsing hepatitis but other study by Samanta

T et al showed 4.7% of hepatitis A patients were

associated with relapsing hepatitis. (37) In our

study, one case without any underlying disease

developed relapse after 3 weeks of normalization

of ALT, AST, serum bilirubin, and improvement

of clinical manifestations. In relapse, there were

elevations of ALT, AST, serum bilirubin and

appearance of clinical manifestation such as

abdominal pain, and persistence of HAV

antibodies (IgM and IgG). These findings were

compatible with the report on relapsing hepatitis

A by Schiff (38) who described that a biphasic or

relapsing form of viral hepatitis A occurred in 6-

10% of acute hepatitis A cases and that the full

duration of illness was 16-40 weeks in these

cases (38). These findings were also compatible

with the report on relapsing hepatitis A

by Vildan et al (39), but not compatible with the

report on relapsing hepatitis A by Mikata R et al

in which IgM anti-HAV and HAV RNA were

negative during relapse. (40). From this study , we

concluded that the clinical outcome of acute

hepatitis A showed 1% case fatality rate and

11% atypical manifestations. Abdominal pain,

vomiting, anorexia, fever, and mild

splenomegaly were the major symptoms and

signs. High bilirubin levels were significantly

associated with fulminant hepatitis and serum

creatinine >2 mg/dl is the best index of

sensitivity and specificity for

detecting fulminant hepatitis and mortality.

Prolonged cholestasis had higher frequency as

compared to fever , anorexia, thrombocytopenia,

and leucocytosis .Hhigh serum bilirubin level

and high levels of ALT, AST, GGT, and ALP.

were common also Patients with hemolytic

anemia had reticulocytosis , Jaundice and pallor.

Patients with pleural effusion had leucocytosis

and low serum albumin level. During the relapse,

there were hypo-albuminemia, hyperbiliru-

binemia, high liver enzymes and prolonged

prothrombin time. We recommend formulation

of cost-effective strategies for the use of hepatitis

A vaccine as a part of a comprehensive plan for

the prevention and control of viral hepatitis A

and this should involve careful economic

evaluations and considering alternative or

additional prevention methods, such as improved

sanitation, and health education for improved

hygiene practices. Patients with hepatitis A

infection should be followed to avoid missing

relapse as well as evaluation of atypical

manifestations.

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3.

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http://www.ncbi.nlm.nih.gov/pubmed?term=Al-Tawfiq%20JA%5BAuthor%5D&cauthor=true&cauthor_uid=18160946

http://www.ncbi.nlm.nih.gov/pubmed?term=Anani%20A%5BAuthor%5D&cauthor=true&cauthor_uid=18160946


http://www.ncbi.nlm.nih.gov/pubmed?term=Ozbay%20Hosnut%20F%5BAuthor%5D&cauthor=true&cauthor_uid=18320220

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Original Article

Cerebrospinal Fluid Cortisol Level for Differentiation Between Aseptic and

Bacterial Meningitis in Adults

Ayman Faried El-Shayeb(1), Soraya Abdel Fatah Hamoda(1), Hossam Fathy Abo El-kheir(1), Iman Tayaa

Elsayed(2), Iman Magdi Mohamed Mohamed(1); Tropical Medicine(1)and Clinical pathology(2)department,

Faculty of Medicine, University of Alexandria.

ABSTRACT

Meningitis is inflammation of the protective membranes covering the brain and spinal cord, known collectively as

the meninges. The inflammation may be caused by infection with viruses, bacteria, or other microorganisms, and less

commonly by certain drugs. Aim of the work : The purpose of this work was to study the utility of cerebrospinal

fluid (CSF) cortisol level as a simple, rapid marker for discriminating between acute bacterial meningitis and acute

aseptic meningitis on admission and whether intrathecal level of this marker had prognostic value. Material and

Methods: 40 patients with meningitis. Evaluation included Complete blood picture, complete urine analysis, lumbar

puncture for Cerebrospinal fluid analysis , cerebrospinal fluid gram stain, serology and culture for bacteria , Cranial

tomography of the brain , Cerebrospinal fluid Cortisol using ELISA test. Results: we observed significantly higher

concentration of cortisol in Cerebrospinal fluid of patients with bacterial meningitis (46.55 ± 17.43 ng/ml) as

compared to the aseptic meningitis group (23.50 ± 5.25 ng/ml) (p<0.001*). There was marked decrease in CSF

cortisol level in response to treatment in both groups. Conclusion: The higher CSF cortisol levels in bacterial

meningitis may suggest its use as a valuable, rapid, relatively inexpensive diagnostic marker in discriminating

between bacterial and aseptic meningitis. Moreover, The significant reduction in CSF cortisol levels following

treatment in both groups reflecting its validity in monitoring treatment response.

Introduction

Meningitis is inflammation of the protective

membranes covering the brain and spinal cord,

known collectively as the meninges.(1)

Meningitis can be life-threatening because of the

inflammation's proximity to the brain and spinal

cord; therefore, the condition is classified as a

medical emergency.(1, 2) Meningitis is typically

caused by an infection with microorganisms.

Most infections are due to viruses,(3) with

bacteria, fungi, and protozoa being the next most

common causes. It may also result from various

non-infectious causes.(4) The term aseptic

meningitis (AM) refers loosely to all cases of

meningitis in which no bacterial infection can be

demonstrated. This is usually due to viruses but

it may be due to bacterial infection that has

already been partially treated. Other causes as

tuberculous meningitis,(5) brucellosis,

mycoplasma and fungal meningitis is seen in

people with immune deficiency.(6, 7) Bacterial

meningitis is a life-threatening condition that

requires prompt recognition and treatment. The

incidence of meningitis varies according to the

specific etiologic agent, as well as in conjunction

with nation’s medical resources. The incidence

in developing countries has been reported to be

10 times higher than that in developed countries.

Before the era of antibiotics, meningitis had a

case fatality rate of almost 100%.(8)Today,

despite the availability of nontoxic and

affordable antibiotics worldwide, the case

fatality rate and neurological sequelae among

survivors are still high, ranging from 15 to 70%

and 10 to 35%, respectively. Early antibiotic

therapy is crucial for optimizing the outcome of

bacterial meningitis. Therefore, it is important to

distinguish bacterial meningitis from aseptic

meningitis during the acute phase of the disease,

when clinical symptoms are often similar.(8, 9)

Current microbiological tests are highly specific,

but they lack sufficient sensitivity(10). Use of

various biological markers in blood (C-reactive

protein, white blood cell count [WBC], and

procalcitonin) or cerebrospinal fluid (CSF; for

instance, protein, glucose, WBC, lactate,

inflammatory cytokines and combinations

thereof) has been suggested to improve

sensitivity in determining the etiological

diagnosis(6, 11-14). However, a sensitive laboratory

test that is easy to perform is still required, so

that all patients with bacterial meningitis can be

identified reliably on admission. Cortisol is the

major glucocorticoid in humans, maintaining the

stress reaction of the body to all kinds of

physical and psychological discomfort. Increase

http://en.wikipedia.org/wiki/Infection

http://en.wikipedia.org/wiki/Microorganisms

http://en.wikipedia.org/wiki/Bacteria

http://en.wikipedia.org/wiki/Fungi

http://en.wikipedia.org/wiki/Protozoa

http://en.wikipedia.org/wiki/Aseptic_meningitis

http://en.wikipedia.org/wiki/Aseptic_meningitis

http://en.wikipedia.org/wiki/Fungal_meningitis

in cortisol secretion takes place very quickly,

within minutes in acute stress conditions, and

can stay at high levels for long periods,

sometimes days, months, and even years.(15) In

normal circumstances, the balance between CSF

cortisol and blood cortisol levels is controlled by

active efflux of the hormone from the

brain;(14) however, disturbance of this

mechanism by inflammation along with reduced

ability of brain cells to metabolize sterol

molecules, may lead to persistent increase in

CSF cortisol.(11)

Aim of The Work

The aim of this work was to study the utility of

cerebrospinal fluid (CSF) cortisol level as a

simple, rapid marker for discriminating between

acute bacterial meningitis and acute aseptic

meningitis on admission and whether intrathecal

level of this marker had prognostic value.

Subjects and Methods The study included 40 patients with meningitis

admitted to Alexandria fever hospital classified

into two groups: Group I: Included twenty

patients diagnosed as bacterial meningitis.

Group II: Included twenty patients diagnosed as

aseptic meningitis. The Diagnostic criteria of

meningitis were presence of more than five

leukocytes in cubic millimeter of cerebrospinal

fluid. Diagnostic criteria of bacterial meningitis

were positive culture or positive gram staining or

positive CSF serology or dominance of

polymorphnuuclear cells, low sugar, high protein

and high lactate in CSF. Diagnostic criteria of

aseptic meningitis were dominance of

lymphocytes, almost normal sugar and protein in

CSF and negative gram staining, serology and

culture of CSF. All patients underwent clinical

examination and lumbar puncture. The resultant

CSF was analyzed and culture was done. CT

brain was also done. All patients underwent

routine laboratory tests (CBC, Complete urine

analysis) and Cerebrospinal fluid cortisol level.

Samples of CSF were centrifuged and stored at –

20°C for not more than one month until the

examination was performed. Quantitative

measurement was done with general cortisol,

ELISA Kit (Eiaab, China)(16) in the Clinical

Pathology department of the Alexandria

University Hospital.

Results

The present study revealed that there was no

statistically significant difference between the

two studied groups regarding symptoms like

fever, headache, vomiting, convulsions and

disturbed conscious level, while there was

statistically significant difference as regard neck

rigidity and kerning's sign as they were more

observed in the bacterial group. Our study

showed that total WBCs count and absolute

neutrophilic count were significantly higher in

the bacterial group (leukocytosis with absolute

neutrophilia). Current study showed that there

was a statistically significant difference between

bacterial and aseptic groups as regard CSF

aspect. In the bacterial group, aspect was turbid

in (75%) of patients. In the aseptic group, it was

turbid in only (15%) of patients. CSF WBCs

count was significantly higher in the bacterial

group(6252.0 ± 5632.73) (c/mm3) compared to

the aseptic group(179.25 ± 155.39) (c/mm3) with

p value <0.001*. In the present study CSF gram

stain was positive in 25% of patients with

bacterial meningitis and CSF serology was

positive in 30% them. CSF culture was positive

only in 10% of bacterial meningitis patients. In

patients with bacterial meningitis, the mean CSF

cortisol level was significantly higher than in

those with aseptic meningitis (46.55 ± 17.43 and

23.50 ± 5.25 ng/ml) respectively (p value

<0.001*) ( t=5.662*). (table 1)

Table (1):Comparison between the two studied groups according to C.S.F. cortisol level (on admission)

C.S.F. analysis

(on admission)

Group I

(n= 20)

Group II

(n = 20) Test of sig. p

Cortisol level (ng/ml)

Min. – Max. 20.0 – 75.0 16.0 – 38.0

t=5.662* <0.001* Mean ± SD. 46.55 ± 17.43 23.50 ± 5.25

Median 44.50 23.0

Before discharge the mean C.S.F. cortisol in

bacterial meningitis cases was significantly

lower than C.S.F. cortisol on admission (17.53 ±

4.59 and 46.55 ± 17.43 ng/ml) respectively (p

value <0.001*). (table 2). Furthermore, in aseptic

meningitis patients, the C.S.F. cortisol before

discharge was significantly lower than C.S.F.

cortisol on admission (15.78 ± 2.73 and 23.50 ±

5.25 ng/ml) respectively (p value <0.001*). (table

2)

Table (2): Comparison between the two studied groups according to C.S.F. cortisol

C.S.F. analysis Group I Group II

Admission Discharge Admission Discharge

Cortisol level (ng) 46.55 ± 17.43 17.53 ± 4.59 23.50 ± 5.25 15.78 ± 2.73 tp1 <0.001* <0.001*

The AUROC was calculated =0.919* at a cut off

value of 28 ng/ml, with sensitivity and

specificity (80% and 90%) respectively for

discriminating bacterial meningitis patients from

aseptic meningitis patients.(figure 1)

Figure (1): ROC curve for cortisol level (ng/ml) on admission to diagnose group I

Among the bacterial group, 80% of patients

improved, 15% developed neurological

complications and one patient (5%) with a

comorbid disease (DM) and complications

(pneumonia and septic shock) died. In the

aseptic group, 75% of patients improved, 20%

developed neurological complications and

2(10%) patients died; one of them had co morbid

diseases and the other developed complications.

The overall mortality in both groups was

associated with high CSF cortisol levels on

admission.

Discussion The present study aimed to determine the CSF

cortisol levels in acute bacterial meningitis as

compared to aseptic meningitis and to evaluate

its role as a diagnostic and prognostic marker in

acute bacterial meningitis. Apart from bacterial

meningitis, increased CSF cortisol levels have

also been reported in multiple sclerosis,

Alzheimer's disease, depression, and

posttraumatic stress disorder.(17-19) In the present

study there was no statistically significant

difference as regard gender and age between the

two studied groups, this matches the study of

Viallon et al(20) and Linder et al(21). Our study

showed that there was a statistically significant

difference between the two groups as regard total

WBCs count and neutrophil count. These results

were in agreement with Hassan et al(22) and

Makoo et al(23). In the present study there was a

statistically significant difference between


glucose, protein and lactate levels with p value

<0.001* for all. It means that CSF glucose was

significantly lower in the bacterial group, while

CSF protein and lactate were significantly higher

among the bacterial group. In our study there

was a statistically significant difference between


WBCs count with p value <0.001*. Moreover,

there was a statistically significant difference

between the two studied groups as regard CSF

lymphocyte% and PMN% with p value <0.001*

for both. This means that CSF WBCs count and

PMN% were significantly higher among the

bacterial group, while lymphocytes% was

significantly higher among the aseptic group.

These results were in agreement with Makoo et

al(23), Alavi et al(24), Nouh et al(25), Fouad et al(26),

Viallon et al(20), and Junior et al(27) who stated

that, there was significant difference in CSF

analysis of bacterial and aseptic groups

regarding white blood cell count, protein and

glucose. Fouad et al(26), Viallon et al(20) and Nouh

et al(25) found that significantly higher CSF

leukocyte count with marked increase in the

polymorphnuclear leukocyte count was present

in bacterial meningitis group. In our study CSF

cortisol concentrations were significantly

elevated in patients with bacterial meningitis as

compared with concentrations in patients with

aseptic meningitis, our results were matched to

that of Holub et al(28) and Mehta et al(29). The

AUROC was calculated =0.919* at a cut off

value of 28 ng/ml, with sensitivity and

specificity (80% and 90%) respectively for

discriminating bacterial meningitis patients from

aseptic meningitis patients. In the study of Holub

et al(28), the median (interquartile range) cortisol

concentration in cerebrospinal fluid (CSF) was

133 (59 to 278) nmol/l in the bacterial group

comparing to 17 (13 to 28) nmol/l in the aseptic

group. After setting the threshold of 46.1 nmol/l

using ROC analysis, they identified a sensitivity

of 82% and a specificity of 100% for the CSF

cortisol test for discriminating bacterial

meningitis patients from aseptic meningitis

patients, with an AUC of 0.94. We reported

significant decrease in CSF cortisol level in

response to treatment. Our results matched

Mehta et al(29). Our study was in agreement with

Viallon et al(20) who showed that CSF lactate was

significantly higher in bacterial meningitis cases.

We found a statistically significant relation

between CSF protein and CSF cortisol level.

This matched the study of Holub et al.(28)

Conclusion

The higher CSF cortisol levels in bacterial

meningitis may suggest its use as a valuable,

rapid, relatively inexpensive diagnostic marker

in discriminating between bacterial and aseptic

meningitis. Moreover, The significant reduction

in CSF cortisol levels following treatment in

both groups reflecting its validity in monitoring

treatment response.

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BA, Roos KL, Scheld WM, et al. Practice guidelines

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at onset of systemic inflammatory response

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B, Venet C, et al. High sensitivity and specificity of

serum procalcitonin levels in adults with bacterial

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B, Horn PS, Bednarik L, et al. Higher levels of basal

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Etiology of Aseptic Meningoencephalitis. Prague

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L, Christensson B. Heparin-binding protein: A

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Study of Meningitis Regarding: Causative Agent,

Risk Factors and Characteristic Features of Patients

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Lacinová Z, Příhodová J, et al. Cortisol levels in

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bacterial origin of meningitis. Critical Care.

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http://www.eiaab.com/

Original Article

Clinical Use of Quantitative Serum (HBsAg) as New Marker for Assessment of

Improvement in Patients with Chronic Hepatitis B Treated with Lamividine

Alaa-Eldin Mohamed Abdo, Essam Eldin Saeed Bedewy, Sherine Mohamed Shawky, Salem Ramadan

Soleiman Khairalla; Department of Tropical Medicine, Faculty of Medicine, University of Alexandria

ABSTRACT

Approximately one third of the world’s population has experienced HBV infection, and nearly 350-400 million of

them are HBV surface antigen (HBsAg) carriers (1). HBsAg is a protein surrounding hepatitis B virus, and the main

marker in both acute and chronic HBV infection diagnosis (2). Recently, there was an evidence indicating that

quantitative HBsAg levels can be used to define inactive carriers (3) and to be used to evaluate response to treatment.

Aim of the work : The Aim of The study was to assess clinical use of quantitative serum (HBsAg) as new marker

for assessment improvement in patients with chronic hepatitis B treated with lamivudine. Material and Methods:

The study was done on 50 chronic hepatitis B infected patients over 18 years old with PCR more than 2000 IU /ml,

SGPT more than two fold of normal, negative pregnancy for females, no evidence of cirrhosis or HCV, HIV, HDV

co infection . All were treated with 150 mg lamivudine for 24 weeks. Included: Full history taking, clinical

evaluation, laboratory investigations including, quantitative PCR for HBV DNA, quantitative HBsAg, ALT, AST

.Ultrasound abdomen, follow up period was 24 week. And all these parameters reevaluated during 3, 24 weeks.

Results: there was, improvement regarding SGPT level as it was reduction during treatment especially after 24

weeks, significant reduction of HBV viral load and HBsAg during different phases of the study, Statically

significant positive correlation between PCR value and HBsAg after 3 weeks and after 24 weeks (p value:0.001).

Conclusion: The combined use of HBsAg and HBV DNA assessment in patients with CHB treated with lamivudine

can guide the clinician to evaluate the chances of treatment response with the possibility to individualize therapy

strategies, such as (continue and follow up), (add on, switch or stop and follow up).

Introduction

Hepatitis B infection is caused by the hepatitis B

virus (HBV), an enveloped DNA virus that

infects the liver and causes hepatocellular

necrosis and inflammation (4). It is estimated that

worldwide, 2 billion people have evidence of

past or present infection with HBV, and 240

million are chronic carriers of HBV surface

antigen (HBsAg) (5). Hepatitis B surface antigen

is the first HBV protein used to show ongoing

HBV infection. In various studies, it has been

reported that serum HBsAg titer is related to

intrahepatic covalently HBV. (6). High HBV

DNA levels have been shown to be related to

severe liver diseases. If patients with HBV

DNA<2000 are followed up for 1 year, and their

alanine aminotransferase (ALT) levels are

normal, then they are accepted as inactive

carriers. Currently, there is some evidence

indicating that quantitative HBsAg levels can be

used to define inactive carriers (7). Lamivudine is

a nucleotide analogue inhibiting the viral

polymerase, lamivudine has been associated with

a four log reduction of the viral load.

Lamivudine treatment (150mg/dl) has been

associated with 16-18% seroconversion rate

from HBeAg to HBeAb, 30-33% rate of HBeAg

loss, 40-50 % normalization of the value of

aminotransferases, and 1-2 % HBsAg

seroconversion rate (8).

Subjects and Methods

The study was conducted on 50 chronic HBV

patients, all of them were over 18 years old,

alanine aminotransferase (ALT)level more than

two folds the upper limit of normal (ULN) and

HBV DNA above 2,000 IU/ml, negative

pregnancy test for female patients.no evidence of

cirrhosis, HCC or HCV, HDV or HIV

coinfection. All Patients were treated with

lamivudine (150mg/dl) for 24 weeks. All

patients was subjected to the following,

Thorough history taking, Clinical examination

with particular stress on stigmata of chronic liver

disease, Laboratory investigations, Complete

Blood Count (CBC), Liver enzymes ALT (U/L),

AST (U/L), quantitative PCR for HBV (IU/ML),

Quantitative HBsAg (IU/ML), Serum pregnancy

test for females,HCV antibodies. Ultrasound

examination of the abdomen initially and during

follow up. (To exclude cirrhosis and HCC) and

all these parameters reevaluated during 3, 24

weeks.

Results

The present study conducted on 50 patient

infected with HBV, mainly HBeAg negative and

all of them was HBsAg psitive.treated with

lamivudine for 24 weeks.

Table (1): prevalence of hepatitis Be Ag among studied patients

HBeAg

Negative Positive

No % No %

44 88.0 6 12.0

Table (2): distribution of liver enzymes among studied patients

Liver functions Phase

F (P) Baseline After 3 wks. After 24 wks.

SGPT

Minimum

Maximum

Mean

SD

80.0

140.0

95.0

15.1

20.0

90.0

41.8

17.7

15.0

45.0

27.6

7.3

85.6 (0.001)*

SGOT

Minimum

Maximum

Mean

SD

80.0

160.0

101.6

17.9

25.0

100.0

47.2

18.1

15.0

50.0

29.4

7.6

76.2 (0.001)*

Figure (1): changes of SGPT

levels during different study phases

This figure showed that, there was improvement in SGPT

level especially after 24 weeks

Figure (2): changes of SGOT

levels different during study phases

This figure showed that, there was improvement in SGOT level

especially after 24 weeks

Figure (3): This figure showed significant improvement of HBV

Viral load and HBsAg during different phases of the study.

After 24 wksAfter 3 wksBaseline

Phase

100.00

80.00

60.00

40.00

20.00

SG

PT

After 24 wksAfter 3 wksBaseline

Phase

100.00

80.00

60.00

40.00

20.00

SG

OT

Table (3): Correlation matrix of HBV load and HBsAg quantity for studied HBVpatients.

Phase Item Q HBsAg

rho P

Baseline

After 3 wks.

After 24 wks.

PCR

PCR

PCR

0.78 0.001*

0.79 0.001*

0.90 0.001*

The above mentioned table showed that, there was statically significant positive strong correlation between PCR value and

HBsAg. After 3 weeks and after 24 weeks (p value: 0.001)

Figure (4): scatter diagram for studied HBV patients. This

figure showed significant positive strong correlation

between viral load and hepatitis HBsAg especially in

low count.

Figure (5): Bland-Altman plot for agreement between HBV

load and HBs Ag This graph showed that, there was a poor

quantitative agreement between HBV viral load and

HBsAg for HBV patients especially with high viral

load which is best at very low viral load.

Discussion

In the present study significant results was,

statically improvement decline of live enzymes

(SGPT and SGOT) during treatment with

lamivudine. the changes was statically

significant (P value: 0.001), regarding to HBV

DNA, Quantitative PCR level decline during

therapy (This change showed to be statically

significant (P value: 0.001),and regarding to

quantitative HBsAg, there was a significant

reduction regarding to HBsAg at 0, 3 and 24

weeks of therapy and (This change showed also

to be statically significant (P value: 0.002).

0zgur Gunal ,et, al (9) . This study was aimed to

investigate whether quantitative HBsAg and

alanine aminotransferase (ALT) levels correlated

with Hepatitis B Virus (HBV) DNA levels in

patients with chronic HBV infection. in

conclusion, ALT level was decline

(improvement)during study, Also, a significant

correlation was also found between levels of

HBV DNA (log) and HBsAg (log) (r: 0.503,

p=0.0001). This study match with present study.

In present study, there was also decline

(improvement) in ALT level (P value: 0.001)

and significant correlation was also found

between levels of HBV DNA and HBsAg (P

value: 0.001). Kaan et ,al.(10) aimed to investigate

the clinical importance of quantitative levels of

serum hepatitis B surface antigen (HBsAg) and

hepatitis B e antigen (HBeAg), and to detect

their correlation with hepatitis B virus (HBV )

DNA load, alanine aminotransferase (ALT)

levels, hepatic activity index (HAI) and fibrosis

scores. A total of 56 HBeAg-positive patients

with chronic hepatitis B (CHB) were included in

the study. Results was Significant differences

were found between groups of pre and post

treatment quantitative levels of HBsAg, HBeAg,

HBV DNA, and ALT. Conclusion, HBsAg and

HBeAg levels significantly decreased during

treatment and that HBeAg correlated with HBV

DNA load. Quantitative HBeAg and HBsAg

assays could therefore have an important role in

treatment of CHB. In present study, there was

statically improvement in ALT and AST levels

especially after 24 weeks and the improvement

was statically significant (p value:0.001) and

there was statically positive strong correlation

for HBsAg and H B V DNA and the patient

mostly HBeAg negative (R-value was after 3

weeks 0.79 and after 24 weeks was 0.90). In a

Guner et al. (11). Reported that there was no

significant correlation between serum HBsAg

levels and HAI. However, serum HBsAg levels

correlated to fibrosis scores. Kaan et,al.(10) , there

was no correlation between HBsAg levels and

HAI or fibrosis scores. In conclusion, our study

indicated that HBsAg, HBeAg, HBV DNA, and

ALT levels significantly decreased during the

treatment. There was a positive correlation

between HBeAg and HBV DNA levels,

Quantitative HBeAg and HBsAg assays are less

expensive than HBV DNA assays and may be

used as suitable indicators of a response guided

therapy approach in CHB. HBsAg quantification

may be used for determination of the optimal

dose and duration of therapy in the future.

Johannes et, al. (12) within this study, 23 liver

transplant patients and 18 heart transplant were

retrospectively analyzed. In this study expected

to demonstrate a high correlation between HBV

DNA and HBsAg. However, only a moderate

correlation for HBsAg and HBV DNA. In

present study, there was statically positive

correlation for HBsAg and H B V DNA (R-

value was after 3 weeks 0.79 and after 24 weeks

was 0.90) which is much higher than the R-value

of ≥0.5 w h i c h i s usually accepted as

correlation and the patient mostly HBeAg

negative. Conclusions, Quantitative HBsAg

cannot substitute for HBV DNA quantification

during the assessment of antiviral therapy;

however, the decline of HBsAg does predict

eventual HBsAg clearance. A 2 log10 drop to

below 100 IU/ml is associated with a high

likelihood of HBsAg clearance. Jing Lai, et, al (13). In this study Fifty-nine nucleoside-naive

patients ACLF were enrolled and treated with

150 mg of lamivudine daily. The dynamics of

serum levels of HBsAg and HBV DNA were

analyzed at baseline and at week 12, there was

no significant difference in HBV DNA levels

between survivors and those who died, and no

correlation was observed between HBV DNA

and HBsAg (all P > 0.05). In the present study,

there was statically positive s correlation for

HBsAg and H B V DNA and the patient mostly

HBeAg negative and all were survivors (R-value

was after 3 weeks 0.79 and after 24 weeks was

0.90). Li et al. (14) .reported that the level of

HBsAg was not directly correlated with the

serum level of HBV DNA or with the severity of

the disease in HBV related ACLF. To date this

has remained controversial and only a few

studies have investigated the role of HBsAg

levels during lamivudine monotherapy in

HBeAg negative ACLF. This study showed that

serum levels of HBV DNA decreased

significantly after short-term lamivudine therapy

(all p < 0.05). No significant difference was

found in HBV DNA levels between the patients

who survived and those who died, both pre and

post treatment (all p > 0.05). Neither was a

difference found among the patients with

different pretreatment HBsAg levels (all p >

0.05). However, serum levels of HBsAg did not

decrease significantly after treatment in either

the patients who survived or those who died.

Conclusions, Changes in HBsAg level could be a

useful parameter for predicting the short term

outcome of lamivudine monotherapy in HBeAg

negative ACLF. Azita Ganji,et, al (15) .in this

study determined whether quantitative HBsAg

levels correlate with HBV DNA levels in CHB.

Conclusions, HBeAg negative patients have

higher levels of HBsAg and lower levels of HBV

DNA. By electrochemiluminescence assay,

HBsAg has no significant correlation with HBV

DNA levels in CHB. (16). HBsAg is a classical

marker of infection with hepatitis B virus, and

serological assays to detect HBsAg have guided

its diagnosis. We hypothesized that HBsAg can

be used to manage and monitor patients as well.

In present study the patients were mainly HBeAg

negative and there was positive correlation

between HBV DNA count and quantitative

HBsAg .Based on the results of the two studies,

quantitative HBsAg level cannot be used as a

surrogate marker for replicative state in HBeAg

negative patients in Iran. We recommend

performing larger studies in different groups of

CHB and patients who are being treated for CHB

separately. HBeAg negative patients have higher

levels of HBsAg and lower levels of HBV DNA.

By electrochemiluminescence assay do not

correlate significantly with HBV DNA level in

HBeAg positive or negative patients in Iran,

necessitating additional studies to standardize

quantification assays and define thresholds of

HBsAg that have clinical predictive value.

zeeshan ali.et, al (17) .The aim of the study was to

assess the short term outcomes (03 months) of

lamivudine in adults aged 14–41 years with

chronic hepatitis-B (CHB) patients. The results

showed that at the end of three months treatment,

lamivudine produced a reduction in ALT and

also the reduction in Hepatitis virus detection.

The result of the systematic review Conclusion,

Treatment of chronic hepatitis patients with

lamivudine shows reduction in serum ALT with

no adverse effects as reported during 03 months

of treatment. In present study , the use of

lamivudine in treatment of chronic hepatitis B

viral infection improve ALT,AST,HBV DNA

level and QHBsAg level during 6 months of

treatment with minimal side effects, Jian Liang,

et, al (18) .The aim of this study was to

systematically review the efficacy and safety of

entecavir versus lamivudine for the treatment of

chronic hepatitis B (CHB). Conclusion, clinical

evidence suggests that despite of short or long

term use, entecavir appears to be more effective

than lamivudine in reducing serum HBV DNA

load, improving liver histology, and normalizing

ALT in patients with CHB. However, the

probability for patients to experience HBeAg

loss or HBeAg seroconversion, or the risk for

adverse events seems to be similar between

entecavir and lamivudine regimens.

Conclusion

We found that there was ,reduction

(improvement) in liver enzymes(SGPT and

SGOT levels) during therapy with lamivudine,

statically significant positive correlation between

quantitative PCR for HBV value and HBsAg,

there was a poor quantitative agreement between

HBV viral load and HBsAg for HBV patients

especially with high viral load which is best at

very low viral load and the combined use of

HBsAg and HBV DNA assessment in patients

with CHB treated with lamivudine can guide the

clinician to evaluate the chances of treatment

response with the possibility to individualize

therapy strategies, such as continue and follow

up, add on, switch or stop and follow up.

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Original Article

Immunohistochemical Study of IL-8 And COX-2 Expression in Helicobacter

Pylori Gastritis

Layla K Younis 1, Nevine M F El Deeb 1, Amany A Elbanaa 2, Engy A A Shahtout 3; Departments of

Pathology1 and Internal Medicine2, Faculty of Medicine, University of Alexandria, Alexandria, Egypt,

Department of Pathology3 , Alexandria Police Hospital, Alexandria, Egypt.

ABSTRACT

This study aimed to evaluate immunohistochemical expression of IL-8 and COX-2 in Helicobacter pylori gastritis

and to correlate the findings with the different grades of gastritis, atrophy, intestinal metaplasia, dysplasia and gastric

cancer. Material and Methods: The study comprised gastric endoscopic biopsy specimens (antral specimens)

obtained from 37 patients with helicobacter pylori gastritis (15 prospective cases confirmed by positive rapid urease

test; and 22 retrospective cases). Ten helicobacter pylori negative endoscopic gastric biopsies were used as control.

Ten cases of gastric carcinoma were included for comparison of the immunohistochemical results. Results: A

significantly higher expression of IL-8 was observed in the H.Pylori gastritis group compared to the control group

(F=15.907, P<0.021) and in the gastric cancer group compared to the H.Pylori gastritis group ( F=15.907, P<0.001).

In addition, A significant higher expression of Cox-2 was detected inthe H.Pylori gastritis group compared to the

control group (F=15.471, P<0.001) and in the gastric cancer group compared to the H.Pylori gastritis group

(F=15.471, P<0.043). Conclusions: our study confirmed the presence of high expression of COX-2 and IL-8 in

patients with H. pylori-induced gastritis.

Introduction

Gastritis refers to a group of diseases

characterized by inflammation of the gastric

mucosa. In clinical practice, the role of the

pathologist who evaluates a gastric biopsy for

gastritis is to find the cause of gastritis because

that will provide direct targets toward which

therapeutic measures can be directed. (1,2).

Helicobacter infection is the most frequent cause

of chronic gastritis, where HP inhabits the

stomach of more than 50% of humans and is the

most frequent cause of chronic gastritis

worldwide. (3, 4) H pylori are gram negative rods

that have the ability to colonize and infect the

stomach. The bacteria survive within the mucous

layer that covers the gastric surface epithelium

and the upper portion of the gastric foveolae.

The infection is usually acquired in childhood. (5)

During progression of gastritis over the years,

the gastric mucosa undergoes a sequence of

changes that may lead to glandular atrophy,

intestinal metaplasia, increased risk of gastric

dysplasia and carcinoma, (6,7) and mucosa-

associated lymphoid tissue lymphoma. (8.9)

Interleukine -8, a member of the CXC

chemokine family, was originally identified as a

potent chemoattractant for neutrophils and

lymphocytes. Subsequent studies confirmed that

IL-8 could also induce cell proliferation and

migration, as well as angiogenesis (10, 11, 12).

COX-2 is a highly inducible gene, expressed in

response to cytokines, growth factors, oncogenes

and chemical carcinogens.(13) Unlike COX-1,

which is expressed constitutively in many tissues

including the normal esophageo-gastrocolonic

mucosa., COX-2 is not detected in most normal

tissues, but its expression is rapidly induced by

both inflammatory and mitogenic stimuli

resulting in increased synthesis of prostaglandins

(PGs) in the inflamed and in the neoplastic tissue (14, 15). It has been reported that H. pylori could

induce the production of inflammatory mediators

such IL-8 (16) and secondary high COX-2

expression, which could directly or indirectly

damage the surface mucosal epithelial cells (16,17).

The aim of this study was to evaluate the

immunohistochemical expression of IL-8 and

COX-2 in Helicobacter pylori gastritis and

correlate the findings with the different grades of

gastritis, atrophy, intestinal metaplasia, dysplasia

and gastric cancer.

Material and Methods

Study population: This study comprised 37

patients (15 men and 22 women; age range 11-

80 years; mean 42.46 ± 16.49 years) who

presented at Alexandria University Hospital for

upper gastrointestinal symptoms. Fifteen cases

were prospective, in whom positivity for H.

pylori was determined by rapid urease test, and

was subsequently confirmed by

histopathological examination of the biopsy

specimens. Twenty two cases were retrospective,

obtained from the archives of the Department of

Pathology, Faculty of Medicine, Alexandria

University. In addition, 10 patients (8 men and 2

women; age range 19-68 years; mean 39.7±

15.64years) whose gastric endoscopic biopsies

showed non H. pylori gastritis constituted the

control group for the microscopic investigations.

Endoscopy was performed for the patients using

Fujinon EPX-2200 (Fujinon-Saitana, Japan) after

an overnight fast. None of the patients had

previous history of treatment with anti-H. pylori

antibiotic therapy, and none had received

medications for gastrointestinal disease for at

least one month prior to endoscopy. At the time

of routine upper gastrointestinal endoscopy,

from every patient biopsy specimens were taken

from the antrum of the stomach. Furthermore, 11

retrospective cases of gastric carcinoma who had

underwent gastrectomy (6 men and 5 women;

age range 24-81 years; mean 53.18 ± 16.15

years) were included in the study for comparison

of immunohistochemical marker expression. The

tissues were used with the approval of the ethics

committee of Alexandria Faculty of Medicine.

From formalin fixed, paraffin embedded tissue

blocks, 5 μm thick sections were cut, four

sections from each case: one section was stained

by Haematoxylin and Eosin (H&E) stain to

define the basic histopathological parameters

required for determination of the severity of H.

pylori gastritis activity according to the Updated

Sydney Classification. The studied cases were

assessed as regards: (18) (a) chronic inflammation,

where cases were graded according to the

density of lymphocytes and plasma cells in the

lamina propria into mild, moderate, and severe;

(b) activity, which signifies neutrophil

infiltration of the lamina propria, pits or surface

epithelium. Cases were graded as: none:

polymorph difficult to find, mild: less than 1/3

pit and surface infiltrated, moderate:1/2 - 2/3 pit

and surface infiltrated and severe: more than 2/3

pit and surface infiltrated; (c) atrophy, where

there is loss of specialized glands, graded as:

none: absent, mild: mild loss, moderate:

moderate loss and severe: severe loss; (d)

intestinal metaplasia of the foveolar or surface

epithelium, graded as: none: no metaplasia,mild:

less than 1/3 of mucosa involved, moderate:1/2-

2/3 of mucosa involved and severe: more than

2/3 of mucosa involved; (e) H.Pylori density

overlying the epithelium, graded as: none: no

curved bacilli, mild: organisms covering less

than 1/3 of the surface, moderate: intermediate

numbers and severe: organism clusters over

more than 2/3 the surface. Another section was

stained with Giemsa stain to diagnose the

presence or absence of H. Pylori bacilli. The

remaining two sections were immunostained

using IL-8 and COX-2 antibodies. Evaluation of

immunohistochemical staining: For the

evaluation of COX-2 immunohistochemical

results, both intensity and percentage of

positively-stained cells were taken into

consideration(19). The scoring criteria for staining

intensity were: 0, no staining; 1, weak staining;

2, moderate staining; 3, strong staining.The

percentage of positively-stained cells was

divided into four grades: 0, no or rare positive

cells; 1, <10 % positive cells; 2, 10-50% positive

cells; and 3, >50 % positive cells.The final score

was calculated by adding the percentage score (0

to 3) to the intensity score (0 to 3). The total

score ranged from 0 up to6. IL-8

immunostaining was assessed as for COX-2

except for the percentage of positively-stained

cells, which was divided into five grades: 0, no

or rare positive cells; 1, <25 % positive cells; 2,

>25-50% positive cells; and 3, >50-75 %

positive cells; and 4,>75%.The final score was

calculated by adding the percentage score (0 to

4) to the intensity score (0 to 3). The total score

ranged from 0 up to 7. (20)

Statistical Analysis Statistical analysis was performed using

Statistical Package for Social Sciences (SPSS)

software, version 20.0 (SPSS, Chicago,

IL,USA). Qualitative data were described using

number and percent. Quantitative data were

described using range (minimum and

maximum), mean, standard deviation and

median. Significance of the obtained results was

judged at the 5% level. The used tests were Chi-

square test, for categorical variables, to compare

between different groups, Fisher’s Exact or

Monte Carlo correction, correction for chi-

square when more than 20% of the cells have

expected count less than 5 and Student t-test, for

normally quantitative variables, to compare

between two studied groups.

Results

Clinical data: Out of the 37 studied patients with

H pylori gastritis, 15 patients (40.5%) presented

with epigastric pain, 11 patients (29.7%) with

persistent vomiting, 9 patients (24.3%) with

dyspepsia and 2 patients (5.4%) with anaemia.

Endoscopic findings: At endoscopy, a total of 32

patients (86.48%) had gastritis without

ulceration: 29 patients (90.62%) had antral

gastritis and 3 patients (9.3%) had pangastritis.

In addition, 5 patients (13.51 %) had active

peptic ulcer disease: they were prepyloric.

Pathological findings: H&E-stained sections

revealed the typical histological signs of chronic

active gastritis, including infiltration of the

lamina propria by lymphocytes, plasma cells as

well as variable numbers of neutrophils.

Intraepithelial neutrophils were seen, with

formation of glandular microabscesses in some

cases (Fig.1a). Lymphoid aggregates and

lymphoid follicles were observed occasionally.

According to the Updated Sydney classification

of gastritis (1996), in the H.pylori positive cases,

the chronic inflammation was mild in (12 cases,

32.4%) and moderate in (25 cases, 67.56%). In

the H.pylori negative gastritis, the chronic

inflammation was mild in (6 cases, 60%) and

moderate in (4 cases, 40%). In the included

H.Pylori positive cases. there was no activity in

19 cases (51.35 %), mild activity in 15 cases

(40.54%) and moderate activity in 3 cases

(8.1%). Meanwhile, all the H.pylori negative

gastritis cases showed no activity. In H.Pylori

positive cases, atrophy was present in 7cases

(18.9%), graded as mild in 2 cases (5.4%),

moderate in 3 cases (8.1%) and severe in 2 cases

(5.4%); while the rest of the cases (30 cases,

81.1%) showed no atrophy. All of the H.pylori

negative gastritis cases showed no atrophy. In

H.Pylori positive cases, intestinal metaplasia was

present in 7 cases (18.9%), graded as mild in 2

cases (5.4%), moderate in 3 cases (8.1%) and

severe in 2 cases (5.4%). while the rest of the

cases (30 cases, 81.1%) showed no metaplasia.

Meanwhile, no intestinal metaplasia was

detected in H.pylori negative cases. In the

included H.Pylori positive cases, the H.pylori

density was mild in (19 cases, 51.4%) and

moderate in 18 cases (48.6%). All H.pylori

negative gastritis cases were confirmed for the

absence of the bacilli. Dysplasia was noted in 4

out of the 37 studied cases of H pylori gastritis

(10.8%). No dysplasia was detected in any of the

H.pylori negative cases. In Giemsa-stained

sections, H. pylori organisms could be detected

within the mucus layer that covers the apical side

of surface epithelial cells (Fig.1b) and also in the

lumens of the gastric mucosal glands, closely

related to the surface of the epithelial cells. IL-8

expression: All of the 37 cases of H.Pylori

gastritis (100%) expressed IL-8. The total IL-8

score ranged from 3 to 7 (M= 4.65, SD= 0.89).

In the ten control cases, the total IL-8 score

ranged from 3 to 5 (M=3.80, SD= 0.79).

Statistical analysis showed a significantly higher

expression of IL-8 in the H.Pylori gastritis group

as compared to the control group (F=13.198,

P<0.012). In the 11 gastric cancer cases, the total

IL-8 score ranged from 4 to 7 (M= 5.82, SD=

1.08). Significantly higher expression of IL-8

was detected among the gastric cancer group as

compared to the H.Pylori gastritis group

(F=15.907, P<0.001). IL-8 expression in

H.Pylori gastritis showed a significant direct

relationship with degree of activity and degree of

H.pylori density (p=0.001& ˂0.001

respectively).On the other hand, the relationship

between the expression of IL-8 in H.Pylori

gastritis and each of age, gender, chronic

inflammation, atrophy, intestinal metaplasia and

dysplasia was not significant (p=0.170, 0.921,

1.291, 0.676, 0.676 & 0.410 respectively). COX-

2 expression: All of the 37 cases of H.Pylori

gastritis (100%) expressed Cox-2. The total Cox-

2 score ranged from 4 to 6 (M= 5.08, SD= 0.92).

In the ten control cases, the total Cox-2 score

ranged from 2 to 5 (M=3.60, SD= 1.07).

Statistical analysis showed a significantly higher

expression of Cox-2 in the H.Pylori gastritis

group compared to the control group (F=15.471,

P<0.001). In the 11 gastric cancer cases, the

total Cox-2 score ranged from 4 to 6 (M= 5.73,

SD= 0.65). Significantly higher expression of

Cox-2 was found among the gastric cancer group

compared to the H.Pylori gastritis group

(F=15.471, P<0.043). Cox2 expression in

H.Pylori gastritis showed a significant direct

relationship with presence of atrophy, intestinal

metaplasia and of dysplasia (p=0.029, 0.029 &

˂0.001 respectively). On the other hand, the

relationship between the expression of Cox2 in

H.Pylori gastritis and each of: age, gender,

chronic inflammation, activity and H.pylori

density was not significant (p=0.083, 0.174,

0.427, 0.054 & 0.212 respectively).

Figure (1): (a) Active superficial gastritis, with infiltration of the mucosa and lamina propria by acute and chronic

inflammatory cells. A crypt abscess is noted (arrow) (H&E, x200); (b) Rod-shaped bacilli of Helicobacter pylori are seen

on the surface epithelium (Gemisa stain, x400).

Figure (2): Cox2 immunostaining : (A) Chronic full

thickness active gastritis showing a strong expression of

COX-2 which appear as brown cytoplasmic stain of the

gastric epithelial cells in the deep antral glands and in

the monocytic cells in the lamina propria. (antiCox2,

x200); (B) Chronic moderate gastritis with intestinal

metaplasia showing a strong intensity of COX-2 which

appear as brown cytoplasmic stain. (antiCox2, x100);

(C) Moderately differentiated gastric adenocarcinoma

intestinal type showing a strong intensity of COX-2

which appear as brown cytoplasmic stain of the

neoplastic cells. (antiCox2, x200).

Figure (3): IL8 immunostaining: (A) Moderate active

chronic gastritis with intestinal metaplasia showing a

strong intensity of IL-8 which appear as brown

cytoplasmic staining in the foveolar glandular

epithelium and mononuclear inflammatory cells;

lymphocytes, monocytes and plasma cells in the lamina

propria. (anti-Il8, x100); (B Chronic moderate gastritis

with intestinal metaplasia showing a moderate intensity

of IL-8 which appear as brown cytoplasmic staining.

(anti-Il8, x200); (C) Moderately differentiated

adenocarcinoma, intestinal type showing a strong

intensity of IL-8 which appear as brown cytoplasmic

staining in the neoplastic cells. (anti-Il8, x100).

b a

A A

B B

C C

Discussion

In the present study, cases with H.pylori positive

gastritis showed a significantly higher IL-8

expression than the control group. This is in

agreement with Yoshino et al (21), who reported

that H. pylori infection significantly increased

IL-8 production in the gastric mucosa. In

contrast to our findings, Lopes et al.,(22) reported

that the epithelial IL-8 staining did not differ

significantly between H. pylori positive and H.

pylori-negative cases and this difference between

the current study results and his results may be

due to the small number of the cases included in

his study which were 10 H. pylori positive

gastritis and 10 H. pylori negative gastritis in

pediatric. Current study showed that gastric

cancer cases revealed IL-8 expression more than

H.pylori positive cases (p ˂0.001) in agreement

with Lee KH et al.,(23) who reported that that

IL-8 induced by H. pylori plays a major role in

gastric cancer and that targeting IL-8 may be a

promising strategy for the treatment of cancer.

Macrì et al.,(24) reported that the serum levels of

IL-8 act as markers of gastric cancer. Lee KH et

al.,(25) reported that increased expression of IL-8

mRNA in tissue extracts from gastric cancer

patients has been associated with some

clinicopathological aspects of the disease,

including poor prognosis. In contrast to our

findings, Bartchewsky et al.,(26)reported that the

IL-8 levels decreased significantly from chronic

gastritis to gastric cancer. In the current study,

and in agreement with Siddique et al.,(27) and

Holck et al.(28) , a significant relationship was

found between IL-8 score and H.pylori gastritis

activity. In addition, and in agreement with

Holck et al.(28), a significant relationship was

detected between IL-8 score and H.pylori

gastritis density. On the other hand, Siddique et

al.,(27) found no significant relationship between

the density of H. pylori and the IL-8 level in

H.pylori gastritis. The use of Hematoxylin and

eosin-stained sections for detecting H.pylori has

limitations. It may fail to detect organisms

because of patchy distribution of bacteria and/or

sampling error. Low density infections may,

therefore, be missed and these may be the cause

of variation in results among the different

studies. Other specific stains, like Giemsa, have

proved better for detecting H.pylori (29) and thus

Giemsa stain was used in our study. The present

study, in agreement with Siddique et al.,(27) ,

showed that there was no significant relationship

between IL-8 score and the degree of glandular

atrophy or intestinal metaplasia in H.pylori

gastritis. In addition, there was no significant

relationship between the absence and presence of

dysplasia and IL-8 score in H.pylori gastritis in

the present study. In the present study, and in

accordance with Kim et al.,(30) and Bhandari et

al.,(31) COX-2 expression was localized to the

gastric epithelial cells in the deep antral glands

and in the monocytic cells in the lamina propria

in both H. pylori positive and negative cases.

COX-2 is induced in numerous processes such as

cellular growth, differentiation, inflammation

and tumorogenesis( 32). H. pylori infection causes

chronic gastritis and induces COX-2

expression(33). In agreement with Forones et

al.,(34), Cho et al.,(35) and Erkan et al.,(36) the

current study showed that H. pylori positive

gastritis cases showed significantly higher COX-

2 staining scores than those of H. pylori negative

gastritis. The current study showed that there

was no relationship between COX-2 score in

H.pylori gastritis and the grade of chronic

inflammation and activity. In contrast, Hokari et

al.,(37) found that the level of lipocalin-type

prostaglandin D synthase mRNA expression (a

specific enzyme that synthesizes prostaglandin

D2 from precursor prostaglandin H2, synthesized

by COX-2) decreased as gastritis became more

severe, this study investigated the expression of

PGD synthase, a key molecule for PGD2

synthesis, in the gastric mucosa. Expression of

both types of PGDS (L-PGDS and H-PGDS) in

the gastric mucosa was shown by the

quantitative RT-PCR method. In addition,

Rossolymos et al (38) reported a significant link

between the severity of gastritis and COX-2

expression scores, where the COX-2 expression

scores increased with the severity of gastritis

from mild to moderate, but was found to

decrease from moderate to severe gastritis. This

difference between the current study results and

his result may be due to the large number of

cases included in his study 153 cases and/or

different scoring system used by the both

studies, as in his study immunohistochemical

staining was scored by on a scale of 0-3, based

on the number and intensity of stained cells. In

the present study, a significant relationship was

detected between COX-2 score and atrophy,

intestinal metaplasia and dysplasia in H.pylori

gastritis. Erkan et al.,(36) reported a steady

increase in COX-2 expression from normal

http://www.ncbi.nlm.nih.gov/pubmed/?term=Lee%20KE%5Bauth%5D

mucosa to chronic active gastritis to intestinal

metaplasia. Bhandari et al.(31) reported a stepwise

increase in the expression of COX-2 as mucosal

damage progressed from normal to gastritis to

gastric ulcer. Moreover, a significant steady

increase in COX-2 starting at atrophic gastritis to

intestinal metaplasia and gastric carcinoma was

reported by Ding et al.,(39). This was confirmed

by the findings of Kim et al.(40), Cho et al. (35) and

Erkan et al. (36) who reported that COX-2 plays

an important role in the stepwise process that

eventually leads to gastric cancer. Collectively,

these findings come in agreement with the

current study in which gastric cancer cases

significantly expressed COX-2 more than

H.pylori positive gastritis cases. In this study, we

have shown that COX-2 is expressed in the

epithelial lining of the stomach in the whole

spectrum of H. pylori-associated gastric

carcinogenesis pathway from gastritis, to atrophy

and intestinal metaplasia, and finally to gastric

cancer, suggesting that COX-2 might be

involved in the early stages of gastric

carcinogenesis. Similar phenomena have been

reported in colonic adenoma and colorectal

cancer as well as Barrett’s esophagus and

esophageal cancers.(41-42). Thus, in the present

work, IL-8 and COX-2 were expressed at higher

levels in gastric mucosa from H. pylori infected

patients, these findings are consistent with the

study of Bartchewsky et al.(26) It has been

suggested that H. pylori infection might activate

nuclear factor-kappaB (NF-kappaB), a

transcriptional regulator of IL-8 and COX-2 and

increase their levels leading to chronic gastritis

and subsequent more advanced lesions (40).

Conclusion: our study confirmed the presence of

high expression of COX-2 and IL-8 in patients

with H. pylori-induced gastritis.

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Original Article

Role of Gastric Varices Injection as a Method to Prevent Predicted

Hematemesis and its Impact on Quality of Life of Hepatic Patients

Abdel Fattah Fahmy Hanno, Essam Eldin Saeed Bedewy, Said Elsayed Ahmed Hammoda; Department of

Tropical Medicine, Faculty of Medicine, University of Alexandria

ABSTRACT Liver cirrhosis is the 14th most common cause of death in adults worldwide. The most common cause of cirrhosis

worldwide is non-alcoholic steatohepatitis, but in Egypt the most common cause is chronic hepatitis C virus (HCV).

Portal hypertension is the most common complication of liver cirrhosis and varices are the most common

complication of portal hypertension. Gastric varices are found in 20% of patients with portal hypertension and gastric

variceal bleeding is severe and is associated with high mortality. Health-related quality of life (HRQOL) is a

multidimensional concept that reflects a patient’s perceived well-being and functioning in physical, psychological

and social domains of health. Aim of the work: We compared the efficacy of endoscopic cyanoacrylate injection and

beta-blockers in primary prophylaxis of gastric variceal bleeding and hence in improvement in quality of life (QOL)

of hepatic patient. Material and Methods: The study was done on 50 cirrhotic patients with large gastroesophageal

varices type 2 or large isolated gastric varices type 1, classified into two groups group I: 25 patients subjected to

histoacryl injection, group II: 25 patients with gastric varices received propranolol. Evaluation included: Full history

taking, clinical evaluation, laboratory investigations including; Complete blood count (CBC), renal function tests,

liver function tests. Imaging investigations: Ultrasound abdomen, doppler study of the portal vein. Upper G.I.

endoscopy over a mean follow up period of 6 months. Results: we observed that the actuarial probability of bleeding

from gastric varices over a median follow-up of 6 months was decreased in group I than in group II, also the actuarial

improvement of QOL domains was higher in the cyanoacrylate group compared to those on beta blockers.

Conclusion: Primary prevention is recommended in patients with large and high risk gastric varices to reduce the

risk of first bleeding. Cyanoacrylate injection is more effective than beta-blockers therapy in preventing first gastric

variceal bleeding and in improvement of QOL. Beta-blockers even if portal hypertension (PHT) fell, did not reduce

the incidence of first gastric variceal bleeding or mortality.

Introduction

Cirrhosis is an increasing cause of morbidity and

mortality in developed countries. It is the 14th

most common cause of death in adults

worldwide. It results in 1.03 million deaths per

year worldwide.(1) 170 000 per year in Europe(2)

and 33 539 per year in the USA.(3) The annual

incidence was 15·3–132·6 per 100 000 people in

studies in the UK and Sweden.(2). Cirrhosis is the

main indication for 5500 liver transplants each

year in Europe.(2) The most common

complication of liver cirrhosis is PHT. PHT is

the underlying cause of most of other

complications of liver cirrhosis. The most

common complication of PHT is varices. Gastric

varices are found in 20% of patients with portal

hypertension.(4) Upper gastrointestinal bleeding

(UGIB) is common, costly, and potentially life

threatening. It must be managed promptly and

appropriately to prevent adverse outcomes. More

people are admitted to the hospital for upper GI

bleeding than for congestive heart failure or deep

vein thrombosis. In the United States, UGIB

results in over 300,000 hospital admissions

annually, at a cost of $ 2.5 billion. Mortality

after an index hemorrhage in patients with

cirrhosis had been previously reported to be as

high as 50%. Varices are the most common

complication of PHT. Varices are classified by

their location as esophageal or gastric varices.

Esophageal varices are graded by their size. In

contrast, gastric varices are classified primarily

by their location, as follows; gastroesophageal

varices (GOV) or isolated gastric varices (IGV).

(5,6) The bleeding from GV is more severe, and

has a higher mortality rate than esophageal

variceal bleeding. (4) GV could be associated

with esophageal varices (along the lesser curve,

gastroesophageal varices type 1 (GOV1); or

along the fundus, type 2 (GOV2) or could

present as isolated GV (IGV): in the fundus

(IGV1), or at ectopic sites in the stomach or the

first part of the duodenum (IGV2).(4) The 2-year

incidence of variceal bleeding from IGV1 and

GOV2 type of varices is more frequent and

profuse (78% and 54%, respectively) than the

lesser curve (GOV1) varices (28%).(7) Risk

factors for gastric variceal hemorrhage include

the size of fundal varices (large(>10

mm)>medium(5-10 mm)>small(<5 mm), Child

class (C>B>A), and endoscopic presence of

variceal red spots. The mortality from first

variceal bleeding remains as high as 20 %.(4,7)

Propranolol is a nonselective beta-

adrenoreceptor blocker (NSBB). It is used in

treatment of hypertension, coronary heart disease

and portal hypertension. Side effects of

propranolol include dizziness, fatigue, postural

hypotension, impotence, bronchospasm.

Endoscopic gastric variceal obturation (EVO)

with cyanoacrylate (CA) injection is used as the

first-line treatment for bleeding GV. (8-10)

HRQOL is a multidimensional concept that

reflects a patient’s perceived well-being and

functioning in physical, psychological and social

domains of health.(11) Chronic diseases are

associated with a significant burden of

psychosocial problems including impaired

HRQOL, depression, anxiety, and other

psychological impairments.(12)

Subjects and Methods

The study was conducted on 50 cirrhotic patients

with GOV2 or IGV1, classified into two groups:

Group I: 25 patients subjected to endoscopic

cyanoacrylate injection. Group II: 25 patients

received beta-blockers. All patients were

followed up by upper G.I. endoscopy for

reassessment after one and 6 months. All

patients were subjected to the following: Full

history taking, clinical evaluation, laboratory

investigations including; Complete blood count

(CBC), renal function tests, liver function tests.

Imaging investigations (Ultrasound abdomen)

before therapy and at one and 6 months, doppler

study of the portal vein before therapy and at 6

months, upper G.I. endoscopy initially then

followed at one and 6 months by another upper

G.I. endoscopies , assessment of Child Pugh

before and after therapy and HRQOL

measurement after 6 months of therapy in both

groups.

Results All of the patients had gastric varices (32

patients with IGV 1, 18 patients with GOV 2),

the majority of gastric varices were IGV 1

(32/50, 64.00 %). Patients with esophageal va-

rices had achieved eradication of esophageal va-

rices before therapy. Patients were randomized

to be subjected to cyanoacrylate injection (Group

I, n =25), and to be received beta-blockers

(Group II, n = 25).

Table (I): Comparison between the two studied groups regarding to demographic data

Characteristics

Group

MCP Group I Group II

No % No %

Age (years)

0.402

<50 7 28.0 3 12.0

50-54 4 16.0 8 32.0

55-59 5 20.0 5 20.0

>60 9 36.0 9 36.0

Gender

0.248! Male 13 52.0 17 68.0

Female 12 48.0 8 32.0

MCP: Mont Carlo exact probability.

!: Fisher exact probability.

https://en.wikipedia.org/wiki/Dizziness

https://en.wikipedia.org/wiki/Fatigue_(medical)

https://en.wikipedia.org/wiki/Hypotension

https://en.wikipedia.org/wiki/Hypotension

Figure (I): Comparison between the two studied groups regarding to pulse in the three different visits

Figure (II): Comparison between the two studied groups regarding to systolic blood pressure in the three different visits

Figure (III): Comparison between the two studied groups regarding to diastolic blood pressure in the three different visits

0

10

20

30

40

50

60

70

80

90

1st visit 2nd visit 3rd vsit

7880.8 80.9

77.1

67.8 63.9P

uls

e r

ate

Visit

Group I

Group II

0102030405060708090

100110120

1st visit 2nd visit 3rd visit

111.6 114.5 114.2118.6

102.6100.8

SBP

Visit

Group I

Group II

0

10

20

30

40

50

60

70

80


75.2 72.275.2

68.2 65.862.8

DB

P

Visit

Group I

Group II

Table (II): Comparison between the two studied groups according to haemoglobin in the three different visits.

Lab

investigations

Group t (P)

Group I Group II

Min Max Mean SD Median Min Max Mean SD Median

Hb

1st visit 10 14 12.3 1.5 12.5 9.9 15 11.4 1.2 10.9 0.018*

2nd visit 10 16 12.6 1.5 12.9 9 14 11.0 1.2 10.8 0.001*

P1 0.703 0.186

3rd visit 10 15 13.1 1.6 13.9 8 12.4 10.0 1.2 10.0 0.001*

P2 0.009* 0.006*

p1: p value for comparing between the 1st and 2nd visits.

p2: p value for comparing between the 1st and 3rd visits.

t: student t-test #: Mann-Whitney test * P < 0.05 (significant).

Table (III): Comparison between the two studied groups according to platelets in the three different visits

Lab investigations Group

t (P) Group I Group II

Platelets1st visit

Minimum 93000 89000

0.002*# Maximum 199000 185000

Mean 155200 124200

SD 40733.9 25594.6

Platelets 2ndvisit

Minimum 95000 90000

0.001*#

Maximum 199000 180000

Mean 167000 108000

SD 38047.1 38063.5

P1

0.074 0.036*

Platelets 3rd visit

Minimum 99000 65000

0.001*# Maximum 200000 170000

Mean 162400 100120

SD 36779.1 38063.5

P2 0.121 0.003*

t: student t-test #: Mann-Whitney test * P < 0.05 (significant).


p2: p value for comparing between the 1st and 3rd visits.

Figure (IV): Comparison between the two studied groups regarding to S. Creatinine in the three different visits.

00.10.20.30.40.50.60.70.80.9

11.11.21.31.41.5


0.97 0.96 0.950.99

1.13

1.27

Cre

tin

ine

Visit

GroupI

Table (IV): Comparison between the two studied groups according to Liver function tests

Liver functions

Group


Min Max Mean SD Min Max Mean SD

ALT

1st visit 9 150 56.6 28.9 10 90 47.4 18.9 1.3 (0.191)

2nd visit 11 140 55.1 27.5 9 95 51.5 18.1 0.54 (0.588)

3rd visit 9 100 53.6 22.0 11 105 57.1 17.9 0.62 (0.538)

P1

0.644

0.001*

AST

1st visit 12 130 60.6 27.5 11 120 55.8 22.1 0.68 (0.496)

2nd visit 14 128 59.8 26.2 11 122 57.4 21.9 0.34 (0.736)

3rd visit 12 120 56.6 22.8 12 139 64.5 23.0 1.2 (0.227)

P2

0.311

0.001*

t: student t-test. P: Paired t-test. * P < 0.05 (significant)

p1: P value for comparing between the 1st and 2nd visits regarding AST.

p2: P value for comparing between the 1st and 3rd visits regarding ALT.

Table (V): Comparison between the two studied groups according to serum albumin



Albumin 1st visit

Minimum 2.1 2.1

0.95 (0.342) Maximum 4.1 3.7

Mean 3.2 3.1

SD 0.6 0.4

Albumin 2ndvisit

Minimum 2.2 2.1

2.1 (0.042)*

Maximum 4.1 3.5

Mean 3.2 3.0

SD 0.5 0.4

P1

0.856 0.106

Albumin 3rd visit

Minimum 2.3 2.0

5.4 (0.001)* Maximum 4.2 3.1

Mean 3.4 2.7

SD 0.5 0.4

P 2 0.396 0.028*

Table (VI): Comparison between the two studied groups according to prothrombin time



PT 1st visit

Minimum 12.5 12.4

0.45 (0.655) Maximum 17.0 16.5

Mean 14.2 14.3

SD 1.3 1.2

PT 2nd visit

Minimum 12.3 12.8

2.0 (0.050)* Maximum 16.5 16.7

Mean 14.1 14.6

SD 1.1 1.2

P1

0.763 0.083

PT 3rd visit

Minimum 12.0 14.0

6.2 (0.001)* Maximum 15.0 17.5

Mean 13.6 15.4

SD 0.8 1.1

P2 0.086 0.074

t: independent samples t-test. * p < 0.05 (significant).


p2: P value for comparing between the 1st and 3rd visits.

Figure (V): Comparison between the two studied groups regarding ascites in the three different visits.

Table (VII): Comparison between the two studied groups according to doppler study for portal vein diameter

Echo doppler of portal vein size Group


Echo doppler of portal vein 1st visit

Minimum 13.2 13.0

0.64 (0.524)

Maximum 16.0 16.0

Mean 14.3 14.1

SD 0.7 0.9

Median 14.1 14.0

Echo doppler of portal vein 2nd visit

Minimum 13.2 11.6

8.1 (0.001)*

Maximum 16.0 14.5

Mean 14.5 12.8

SD 0.6 0.8

Median 14.5 12.8

P

0.718 0.045*

U: Mann-Whitney test. P: Wilcoxon test.

* P < 0.05 (significant). P: p value for comparing between the 1st and 3rd visits.

Table (VIII): Comparison between the two studied groups regarding to the size of gastric varices

Upper GIT endoscopy Group

U P Group I Group II

Upper GIT endoscopy size 1st visit

Minimum 10.0 10.0

0.57 (0.566)

Maximum 23.0 23.0

Mean 16.5 15.8

SD 4.3 4.1

Median 17.0 16.0

Upper GIT endoscopy size 2nd visit

Minimum 0.0 10.0

6.1 (0.001)*

Maximum 7.5 23.0

Mean 1.3 15.8

SD 2.3 4.14

Median 0.0 15.8

Upper GIT endoscopy size 3rd visit

Minimum 0.0 10.0

5.7 (0.001)*

Maximum 5.0 27.0

Mean 0.6 16.1

SD 1.2 4.68

Median 0.0 15.5

P 0.001* 0.487

12.0

64.0

24.0

12.0

68.0

20.0

72.0

16.012.0

16.0

52.0

32.0

16.0

60.0

24.0

12.0

68.0

20.0

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

No

Mild

Mo

der

ate

No

Mild

Mo

der

ate

No

Mild

Mo

der

te


%

Group I

Group II

Figure (VI): Comparison between the two studied groups regarding child pugh in the two different visits.

Figure (VII): Comparison between the two studied groups according to the bleeding rate

after one (1st visit) and 6 months (2nd visit).

Figure (VIII): Comparison betweenthe two studied groups according to Quality of life after 6 months

Discussion In this study there was no statistical significant

difference between the 2 studied groups

regarding to gender and age. This finding could

be due to uneven distribution of the gender and

sex in this study. There was positive correlation

between pulse and both systolic and diastolic BP

in our study. That would be explained by the

4436

20

72

20

08

20

4436

0

48

32

20

0

10

20

30

40

50

60

70

80

A B C A B C C +Bleeding

Child pugh 1st visit Child pugh 2nd visit

%

Group I

0%

5%

10%

15%

20%

1st visit 2nd visit

4%

8%

4%

20%

Ble

ed

ing

rate

Visit

Group1Group2

effect of beta blockers in group (II) which

reduces pulse and BP. Smith J et al(2013)(13) in

USA conducted a study on 200 patients having

bleeding esophageal varices on beta blockers and

reported that there was a positive correlation

between pulse and blood pressure and

hemoglobin concentration. This was in

accordance with our study. Haemoglobin (Hb)

was significantly lower in group (II) than in

group (I) in the 3rd visit (P=0.001). That would

be attributed to the more bleeding occurred in

group taking beta blockers. Also in our study,

there was a positive correlation between

haemoglobin levels and physical functioning,

vitality, mental health and general health as we

found bad QOL in group with lower Hb level. In

agreement with our study, Afsar et al (2009)(14)

in assessment of quality of life in hemodialysis

patients with HBV found positive correlation

between haemoglobin levels and the following

SF-36 subscales: physical functioning, role-

physical limitation, bodily pain, general health

perception, vitality, social functioning, role

emotional and mental health subscales. In this

study we found that platelets was significantly

lower in group (II) than in group (I) in the 3rd

visit (P=0.011). That would be attributed to the

more bleeding incidence in group taking beta

blockers. The association of low platelet count to

the presence of varices is probably a reflection of

the degree of portal hypertension.

Thrombocytopenia may be due to splenic

sequestration, antibody-mediated destruction of

platelets or reduced hepatic production of liver-

derived thrombocytopoietic growth factor

thrombopoietin. Garcia- Tsao et al (1997).(15)

(180 patients), Pilette et al(1999).(16) (116

patients) and Thomopoulos et al(2003).(17) (184

patients) reported a low platelet count to be an

independent risk factor for the presence of

varices. Prothrombin time significantly was

longer in group (II) than in group I in the 2nd and

3rd visits (P=0.011). That would be explained by

the higher variceal bleeding rate in group (II),

and hence more loss of coagulation factors.

Serum albumin was significantly lower in group

II than in group I in the 2nd and 3rd visit

(P=0.011). That would be attributed to the effect

CA injection reducing the rate of bleeding and

rebleeding from gastric varices leading to

reducing blood loss and subsequently reducing

albumin loss. There was significantly negative

correlation between the degree of ascites and the

level of serum albumin in the two studied

groups. That would be explained by that the

gradual elevation of the level of serum albumin

in all patients leaded to gradual increase of the

plasma oncotic pressure leading to reduction of

the amount of ascites. Ascites was improved in

both studied groups in the 3rd visit but improved

significantly in group (I) than in group (II)

(P=0.001). This would be attributed to less

bleeding and hence less albumin loss and

increase in plasma oncotic pressure in group (I)

but would be attributed to reduction occurred in

portal hypertension by beta blockers in group

(II). We found in our study that the use of NSBB

might also be beneficial for other outcomes, such

as ascites, spontaneous bacterial peritonitis

(SBP), hepatorenal syndrome (HRS), hepatic

encephalopathy (HE) and overall survival. A

landmark study by Abraldes et al (2012)

documented a positive effect of NSBB in the

prevention of the development of ascites, SBP

and hepatic encephalopathy. Likewise,

Hernandez-Gea (2012) and colleagues

demonstrated that in patients with compensated

cirrhosis and large varices treated with NSBB,

even an HVPG decrease >10% was able to

significantly reduce the risk of developing

ascites and other complications such as

refractory ascites and HRS.(18,19) These findings

were similar to our findings. Portal vein diameter

significantly decreased in group (II) on the other

hand increased minimally in group (I) (P

=0.001). This could be explained by the lowering

effect of beta blockers on portal hypertension in

group (II).In our study we demonstrated increase

in the diameter of the portal vein in injection

group. Avgerinos et al (2004).(20) performed

repeated hepatic venous pressure gradient

(HVPG) measurements before and immediately

after endoscopic treatment and every 24 hours

for a 5-day period. They found that endoscopic

injection sclerotherapy (EIS) causes a sustained

increase in HVPG than ligation group related to

leaving a less number of portal-systemic

collaterals that account in part for the differences

in HVPG after treatment. These findings were

similar to those in our study. Size of gastric

varices significantly decreased in group (I) on

the other hand increased in group (II), this means

that endoscopic injection of cyanoacrylate is

significantly more effective than beta blockers in

the prevention of first bleeding from high risk

G.Vs. In this study injection procedure success

was 96% with one patient bled within 48 hours

and this patient needed another session of

cyanoacrylate injection. In this present study, we

had documented successful eradication of non-

bleeding gastric varices with CA. CA is safe

method for elective gastric variceal eradication.

Kind R, et al (2000)(21) carried a study on174

case with glue over a mean follow up period of

36 months, demonstrated that initial control of

bleeding was 97%. In our study success rate was

96% and all patients who bled in group II

achieved 100% hemostasis after application of

CA injection, so results of this study was

matched with our study. In this study 72%

and76% were the eradication rates at one and 6

months respectively with 18 patients needed one

session to eradicate varices, 3 patients needed 2

sessions, 3 patients needed 3 sessions and one

patient needed more than 3 sessions. In our study

recurrence rate was 8% after a mean follow up

period of 6 months (2patients experienced small

sized varices after 6 months). Kang et al,

performed EVO with cyanoacrylate in 127

patients with GVs (100 active bleeding and 27

prophylactically) and reported a primary

hemostasis rate of 98.4% (1 session-98 patients,

2 sessions- 25 patients, ≥ 3 sessions-4 patients),

with a recurrent bleeding rate of 18.1 % at 1

year. (22) In our study an early and a late bleeding

was determined in two patients (8%) in injection

group; one patient bled within 48 h and

subjected to TIPS operation, the other patients

bled after 4 months and this patients subjected to

endoscopic injection again and achieved

hemostasis. While the probability of bleeding

from GV in the beta blockers group was 20% (5

patients) (P=0.041). Those patients were

resuscitated and subjected to endoscopic

cyanoacrylate injection (hemostasis was 100%

after injection). This means cyanoacrylate

injection is more effective than beta-blockers in

preventing first gastric variceal bleeding even if

threse was reduction in portal hypertension. In

our study we reported probability of bleeding as

8% in cyanoacrylate group compared to 20% in

beta blocker group (P=0.041) over a follow up

period of 6 months. Mishra, (2010)(23) conducted

a randomized study on eighty-nine cirrhotics

with GV (GOV2/IGV1) without EV, who had no

history of GV bleeding, patients were

randomized to receive cyanoacrylate injection

(Group I, n = 30), beta-blockers (Group II n =

29) or no treatment (Group III, n = 30). Mishra

reported the probability of bleeding from GV as

13% in the cyanoacrylate group compared to

28% in the beta blocker group (p = 0.039) and

45% in the no-treatment group (p= 0.003) over a

median follow-up period of 26 months.

However, between Group II and III, no

significant difference in GV bleeding was

observed (p = 0.374). These finding matched

with our study. In our study most of bleeders

were from child C, a strong association was

found between bleeding from gastric varices and

Child-Pugh class, also a strong association was

found between gastric varices size and bleeding

from them. The North Italian Endoscopic Club

for the Study and Treatment of Varices.(24) In

prediction of the first variceal hemorrhage in

patients with cirrhosis of the liver in a

prospective multi-center study demonstrated that

the main factors predicting risk of variceal

bleeding are the size of varices, the presence of

red wale marks and the severity of liver disease

as expressed by the Child Pugh score. In our

study we found that there was significantly a

strong positive correlation between ages of

cirrhotic patients and the HRQOL items in the

two studied groups as we found that low QOL

scales was associated with old ages.

Sobhonslidsuk et al (2006), found that old age

had a negative impact on HRQL, the elderly is

associated with less favorable appraisal of

personal health due to their health concerns,

pessimistic health appraisal, social isolation and

unemployment.(25) Physical quality of life was

significantly better in group I than in group II, as

when patients in beta blockers group were asked

about their feeling of fatigue, tiredness, sleep

disturbances, weakness, low energy and

dizziness, the majority of them felt tired all time,

the majority of them also felt weak, sleepy, and

had low energy most of time secondary to

bleeding, more than in injection group.

Dienstag&Isselbacher (2005), asserted that the

client with cirrhosis often experiences severe

fatigue, leading to activity intolerance related to

bed rest, fatigue, lack of energy secondary to

ascites and bleeding.(26) In relation to the pain

and discomfort, the majority of the patients in

the study sample felt abdominal distension

secondary to ascites, pain and discomfort in beta

blockers group, while in injection group minority

of them felt it little of time, This coincides with

the available literature which shows that

symptoms of cirrhosis may develop gradually,

when symptoms do occurs they can include

abdominal bloating, abdominal indigestion or

pain, nausea and vomiting, and ascites, vomiting

blood, or blood in stool, weakness and weight

loss (Garcia, 2009).(27) In relation to the domain

of emotional function, the majority of patients in

group (II) felt anxious, unhappy most of time,

irritable about complication of portal

hypertension and had insomnia most of time. As

regards to mood swings and inability to sleep at

night, most of patients in group (II) felt it most

of time, while patients in group (I) felt anxious

little of time. This coincides with Stein, et al

(2001), who emphasized that patients who learn

that he or she has contracted chronic liver

disease or HCV may exhibit a range of

emotional responses, including worry, fear,

hopelessness, depression, and anger. (28) In our

study social quality of life was significantly

better in group (I) than in group (II) (P=0.001),

as when we asked patients in beta blockers group

about their sex life we found that most of them

had problems in their sex life as impotence and

this could be explained by the most common

side effects of propranolol that include

impotence, postural hypotension and

bronchospasm. In agreement with our findings

Sinagra E et al (2014) (29), De BK et al (2002) (30)

and Lin HC et al (2004)(31) had conducted

randomized trials on comparison of carvedilol to

propranolol for portal hypertension. These

studies favored carvedilol against propranolol, in

terms of: (1) acute effects on reduction in HVPG

(2) long term effects and (3) overall effects,

additionally the same metaanalysis showed that

carvedilol had a lower relative risk of failure to

achieve hemodynamic response than

propranolol. The most common reported side

effects of propranolol include bronchospasm,

heart failure, hypotension and impotence.

Overall quality of life was significantly better in

group (I) than in group (II) (P=0.001). This was

attributed to that cyanoacryle injection was a

good modality in management of gastric varices.

Bianchi et al (2003) (32), Cordoba et al (2005) (33),

stated that chronic liver disease had negative

effect on QOL and QOL worsened as the

severity of disease increased or as the

complications as bleeding were not managed.

Conclusion We found that bleeding rate was significantly

lower (8%) in cyanoacrylate injection group than

in beta blocker group (20%) in elective

management of gastric varices. This means CA

is more effective than beta-blocker therapy in

preventing first gastric variceal bleeding even if

there was reduction of portal hypertension. Also

we demonstrated lack of portal hypertension

response in predicting bleeding from G.Vs in

contrast to that for esophageal variceal bleeding,

where portal hypertension response to NSBBs

has been shown to predict both bleeding and

varices formation. There was a strong

association between child classification of liver

disease and first bleeding from gastric varices as

we documented that all patients who had bled

were child C. Also prevention of first gastric

variceal bleeding had a good prognosis on child

grade. Finlay we concluded favorable outcomes

in elective management of gastric varices with

cyanoacrylate injection in terms of quality of life

and general health. Primary prophylaxis reduces

the risk of first bleeding and hence improves

general health and quality of life of hepatic

patients.

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Original Article

Serum Procalcitonin Level as a Marker for Diagnosis of Bacterial Infections

Hossam Ibrahim Mohamed1, Amira Maher Ahmed Badawy1, Safaa Ibrahim Tayel2, Rasha Mohammed

Abd-Elmegeed Shetaya3; 1Tropical Medicine department, Faculty of Medicine, Menoufiya University, 2Biochemistry department, Faculty of Medicine, Menoufiya University, 3Zawiet Elnaoura fever hospital.

ABSTRACT

Bacterial infections are the major cause of morbidity & mortality. Diagnosis of bacterial infections is sometimes

challenging, there are several markers for diagnosis of bacterial infection & inflammation such as Total leucocytic

count, Erythrocyte sedimentation rat, C reactive protein & Procalcitonin.Aim of work : this study was to evaluate the

role of serum procalcitonin level as a marker for diagnosis of bacterial infection. Material and Methods: This study

was conducted on 55 patients with confirmed bacterial infections (bacterial group), 20 patients with viral infections

(viral group) &15 healthy persons (control group). These patients were subjected to full history taking, complete

clinical examination, Complete blood picture, Erythrocyte sedimentation rat, C reactive protein, bacteriological,

serological & imaging studies & measurement of serum procalcitonin (PCT) level. Results There was highly

significant increase in the mean values of serum Procalcitonin levels in bacterial group (1501.1±641.2 Pg/ml) in

comparison to viral (541.8±236.1 Pg/ml) & control (195.01±38.2 Pg/ml) groups & in viral group when compared

with control group. Serum Procalcitonin at cut off point (774.83 Pg/ml) could differentiate between bacterial & viral

etiologies with sensitivity (85.5%), specificity (70.0%), Negative predictive value (63.6%) & accuracy (81.3%), but

when combined with C reactive protein at cutoff point (12.5 mg/L), the sensitivity, Negative predictive value &

accuracy increase to 94.5%, 76.9% & 82.7% respectively. Conclusion: PCT is valuable, simple, non invasive, time

saving, cost effective serum marker in evaluating patients with infection especially bacterial infection.

Introduction

Infectious agents are the most common causes of

febrile illness (30%–40%). Therefore, in the first

contact with febrile patient doctors have doubts

concerning the etiology of the disease & the

need for giving empirical antibiotic therapy.

Among these infectious agents, bacterial

infections are the major cause of morbidity &

mortality (1,2). Diagnosis of bacterial infections is

sometimes challenging, because clinical

presentation of infections from different

causative agents can be similar, for example, it

may be difficult to differentiate viral from

bacterial infections in certain instances (3). The

Bacteriological culture method is the gold

standard for the diagnosis of bacterial infection,

but a definitive result can take 24 hours or more

before a conclusive diagnosis which favors the

use of another marker that is more rapid. The

identification of markers for the early

recognition of bacterial infections could guide

treatments, reduce misuse of antibiotics, &

possibly improve long-term outcomes (4). Several

serum biomarkers have been identified that have

the potential to help diagnosis of infections.

Among these markers, procalcitonin (PCT) is the

most extensively studied biomarker (5). PCT has

been proposed as a marker of bacterial infection

in critically ill patients & is related to the

severity of infection. PCT is a more sensitive &

specific marker of bacterial infection compared

with CRP & WBC count (6). Under normal

metabolic conditions, PCT is synthesized by C-

cells of the thyroid gl&. Whereas in systemic

inflammation, particularly in bacterial infections

under the influence of inflammatory cytokines &

bacterial endotoxin, PCT is produced in a

number of tissues (lung, liver, kidney, adipose

tissue) & goes in circulation. PCT is a stable

marker, whose concentration is not affected by

neutropenia, immunodeficiency conditions & the

use of non steroid & steroid anti-inflammatory

drugs, which is not the same with CRP (7). The

major advantage of PCT compared to other

parameters such as CRP is its early & highly

specific increase in response to bacterial

infections & sepsis (8).

Methods

This study was conducted on seventy five

patients with clinical, laboratory, serological,

imaging & bacteriological evidences of infection

selected from inpatients & outpatients of

Menoufiya fever hospitals in the period from

April 2014 to December 2014. They were

classified into Bacterial group: Included 55

patients with clinical, laboratory, serological,

imaging & bacteriological evidence of confirmed

bacterial infection. They were 30 males (54.5 %)

& 25 females ( 45.5 %). Their ages ranged

between 5 & 60 years with mean value of

31.65±17.38 years. Bacterial etiology was

brucellosis ( in 13 patients), enteric fever ( in 12

patients), bacterial meningitis ( in 10 patients),

urinary tract infection ( in 7 patients), acute

follicular tonsillitis (in 7 patients) & bacterial

pneumonia (in 6 patients). Viral group: Included

20 patients with viral infection diagnosed

clinically with or without laboratory or

serological investigations. They were 11 males

(55.0 %) & 9 females (45.0 %). Their ages

ranged between 5 & 63 years with mean value of

22.60±17.62 years. Viral etiology was aseptic

meningitis ( in 6 patients), chicken pox (in 5

patients), mumps ( in 5 patients) & acute

hepatitis A infection ( in 4 patients). Control

group: Included 15 healthy persons. Oral

consents were obtained from all participants in

the study. All patients & controls were subjected

to thorough history taking, complete clinical

examination, laboratory investigations including

(CBC, ESR, CRP & complete urine & stool

analysis), bacteriological study (culture)( was

done for 55 patients of bacterial group),

serological studies {Widal test was done for 12

patients of bacterial group, Brucella

agglutination test was done for 13 patients of

bacterial group, HBs Ag, Hbc Ab (Ig M & total)

& anti-HAV IgM were done for 4 of viral group

patients with jaundice.}, lumbar puncture & CSF

analysis (were done for 10 of bacterial group

patients & 6 of viral group patients with

meningeal signs), imagining study as indicated

{thyroid ultra sound, abdominal & pelvic ultra

sound, plain x – ray chest & plain x – ray

urinary tract} & measurement of serum PCT

level by ELISA method Was done for cases &

control subjects (9). Results were collected,

tabulated & analyzed by SPSS (statistical

package for social science) version 17.0 on IBM

compatible computer (SPSS Inc., Chicago, IL,

USA) (10). The final diagnosis of bacterial group

was based on the presence of fever & subjective

clinical features depending on the type of

infection which was confirmed by appropriate

investigation. Bacterial etiology was brucellosis

(13 patients), that was diagnosed by positive

blood culture in 6 patients & positive Brucella

agglutination test in 7 patients, enteric fever (12

patients) diagnosed by positive blood culture in

4 patients or positive Widal test in 8 patients,

bacterial meningitis (10 patients) diagnosed by

presence of meningeal signs, lumbar puncture &

CSF findings {turbid CSF aspect with increased

pressure, decrease CSF glucose, increase CSF

(protein, total leucocytic count(TLC) with

polymorphnuclear cells predominance), presence

of organism in gram stain & positive CSF

culture}, urinary tract infection (7 patients)

diagnosed by urinary symptoms, pyuria more

than 100/mm3 with positive urine culture, acute

follicular tonsillitis (7 patients) diagnosed by

local throat examination with positive throat

swab culture & bacterial pneumonia (6 patients)

diagnosed by pulmonary manifestations, positive

sputum culture & pneumonic patches on chest x

ray. The final diagnosis of viral group included:

Aseptic meningitis (6 patients) diagnosed by

presence of meningeal signs, lumbar puncture &

CSF findings ( clear CSF aspect with normal or

increased pressure, normal CSF glucose &

protean, increased CSF TLC lymphocytes

predominance, absence of organism in Gram

stain, negative CSF culture & self limited

disease without antibiotic), Chicken pox (5

patients) diagnosed by fever & characteristic

skin rash, Mumps (5 patients) diagnosed by

fever & parotid enlargement & acute hepatitis A

infection (4 patients) diagnosed by prodrome,

jaundice, tender hepatomegaly with positive

HAV IgM antibody.

Results

There was no significant difference between

studied groups as regard age & sex distribution.

Fever was present in all patients & was

associated with other signs & symptoms of

infection in various proportion based on the type

of infection. Differentiation between various

infection on clinical bases was difficult except in

cases with pathognomonic signs such as

exanthema of Chicken pox, parotid enlargement

in Mumps & jaundice in hepatitis patients. There

were highly significant ( p value >0.001)

increase in the mean values of total leucocytic

count (11.69±4.03) & decrease in mean values of

Hb concentration (11.65±0.75) in bacterial

group, in comparison with viral group ( mean

TLC was 8.21±2.57 & mean Hb concentration

12.51±0.99) & control group (mean TLC

7.11±1.63 & mean hemoglobin (Hb)

concentration 13.35±1.23). Highly significant ( p

value >0.001) decrease in mean values of

platelet count in viral group (200.95± 36.97) in

comparison to bacterial (235.4±31.6) & control

(286.13±96.47) groups & in bacterial group in

comparison with control group. No significant

difference between viral & control groups as

regard mean values of Hb level & TLC (table

1). There were highly significant increase ( p

value >0.001) in the mean values of CRP &

ESR (first hour) in bacterial group (31.22±18.56

& 44.05±5.84 respectively) in comparison to

viral (4.11±1.01&6.10±1.62respectively) &

control (3.86±0.92 &4.73±1.44 respectively)

groups, while there was non significant

difference between viral & control groups as

regards ESR & CRP (table 2). There were highly

significant increase in the mean values of serum

PCT levels in bacterial group (1501.1±641.2

Pg/ml) in comparison to viral (541.8±236.1

Pg/ml) & control (195.01±38.2 Pg/ml) groups &

in viral group when compared with control group

(table 3). There was highly significant positive

correlation between serum PCT level & other

parameters (TLC, CRP & ESR) (figure 1,2&3).

Cut off point of CRP (figure 4) with highest

sensitivity (78.2%) & specificity (70%) was

12.5, while cut off point of serum PCT (figure 5)

with highest sensitivity (85.5%) & specificity

(70%) was 774.83 Pg/ml. Serum PCT at cut off

point (774.83 Pg/ml) could differentiate between

bacterial & viral etiologies with sensitivity

(85.5%), specificity (70.0%), NPV (63.6%) &

accuracy (81.3%), but when combined with CRP

at Cutoff point (12.5 mg/L ), the Sensitivity,

NPV & accuracy increased to 94.5%, 76.9% &

82.7% respectively (table 4).

Discussion

Statistical analysis revealed highly significant

increase in the mean values of TLC in bacterial

group in comparison with viral & control groups

in this study This result was in agreement with

Lopez et al.,(11) who studied PCT in pediatric

emergency departments for the early diagnosis

of invasive bacterial infections in febrile infants

& found that TLC, total neutrophils & immature

neutrophils were significantly higher in the

bacterial infections group {included lower

urinary tract infections, gastroenteritis in

children <3 months of age & otorhinolaryngeal

infections} than in viral infection group{ viral

bronchiolitis, rotavirus gastroenteritis, Epstein-

Barr virus infection, viral meningoencephalitis,

Herpes zoster infection & Herpes type 6

viruses}. In contrast, ChiLin et al.,(12) & Limper

et al.,(6) reported that there was no significant

difference in TLC between bacterial & non

bacterial infection groups in their studies on

febrile patients. Also Callaham (13), reported that

The WBC is an insensitive & non specific test.

Its predictive value for bacterial disease is low

regarding accuracy & making urgent clinical

decisions. The present study, revealed highly

significant increase in the mean values of CRP &

ESR (first hour) in bacterial group (CRP

31.22±18.56 mg/L, ESR 44.05±5.84 mm/h) in

comparison to viral group (CRP 4.11±1.01

mg/L, ESR 6.10±1.62 mm/h) & control group

(CRP 3.86±0.92 mg/L, ESR 4.73±1.44 mm/h)

while there was no significant difference

between viral & control groups as regard CRP &

ESR. This was in agreement with the findings of

Lopez et al., (11) & Limper et al.,(6) who reported

that there was highly significant difference (P

>0.001) in CRP between the bacterial & non-

bacterial infection groups, in their studies on

febrile patients in emergency departments. In

contrast, ChiLin et al., (12) who reported that CRP

showed no significant difference between the

bacterial & non-bacterial infection groups, CRP

level (P values of 0.55). The present study

revealed highly significant increase in the mean

values of serum PCT levels in bacterial group

patients (1501.1±641.2 Pg/ml = 1.501±0.6412

ng/mL) in comparison to viral (541.8±236.1

Pg/ml = 0.5418±0.2361 ng/mL) & control

(195.01±38.2 Pg/ml = 0.19501 ± 0.0382 ng/mL)

groups & also in viral group when compared

with control group. The elevation of serum PCT

in bacterial versus non bacterial & control

groups in the present study agreed with previous

reports. However, there were variability in the

level of these elevations in various studies. In a

multicenter study, Lopez et al.,(11) assessed the

utility of PCT as a rapid qualitative test for early

diagnosis of invasive bacterial infections in

febrile infants in pediatric emergency

departments. The authors reported that the mean

value of PCT in localized bacterial infection

group (n=80) (ENT infection, lower urinary tract

infections, acute bacterial gastroenteritis without

systemic involvement) was 35.2 ng/ml, the mean

value of PCT in the invasive or severe bacterial

infection group (n=230) (meningitis, sepsis,

bone or joint infections, acute pyelonephritis,

lobar pneumonia, bacterial enteritis & occult

bacteremia) was 15.9 ± 47.7 ng/ml, the mean

value of PCT in viral group(n=122)(viral

gastroenteritis, Epstein-Barr virus infection, viral

meningoencephalitis, Herpes zoster infection &

Herpes type 6 viruses) was 0.26 ± 0.17 ng/ml,

while in control group, the mean PCT value was

0.15 ng/ml. The authors reported that the mean

PCT was significantly higher in bacterial than

viral & control group & in the invasive bacterial

than in the non invasive bacterial group, & in

viral group when compared with control group.

Andreola et al.,(14) studied PCT as marker of

severe bacterial infections in febrile infants &

children in emergency department. The authors

classified studied patients into severe bacterial

infections group (94 patients), non severe

bacterial infections group (314 patients), proved

viral infection group (36 patients), probable viral

infection group (213 patients). The authors

reported highly significant elevation in the mean

value of PCT in severe bacterial infections group

(1.9 ng/mL with range 0.5–10.7ng/mL) when

compared with non severe bacterial infections

group (0.2 ng/mL 0.1-0.5 ng/mL). ChiLin et

al.,(12) studied PCT level in febrile infant &

young children & the authors reported that PCT

showed a significant difference between the

bacterial (PCT mean value was 5.34±7.17

ng/mL) & non-bacterial infection (PCT mean

value was 0.55±0.99 ng/mL) groups (P

value=0.002). Limper et al.,(6) studied PCT in

febrile Afro-Caribbean patients at the emergency

department & reported that there was highly

significant difference in PCT with P value of

>0.001 between the bacterial & non-bacterial

infection groups. The authors concluded that

PCT level was a potent marker of bacterial

infection & it could be differentiated well

between infectious & non infectious fever &

between confirmed bacterial & confirmed viral

infection. Prasad et al., (15) in a study of serum

PCT in septic meningitis reported that the mean

value of serum PCT level was significantly

higher in children with septic meningitis

(22,669.21 ± 7,656.45 pg/ml) than those with

aseptic meningitis (14,451.24 ± 4,266.15 pg/ml)

or in controls (p<0.001). In the present study,

serum PCT at cut off point (774.83 Pg/ml =

0.77483 ng/mL) could differentiate between

bacterial & viral etiologies with sensitivity

(85.5%), specificity (70.0%), PPV (88.7%), NPV

(63.6%) & accuracy (81.3%), but when

combined with CRP at cutoff point (12.5 mg/l ),

the sensitivity, NPV & accuracy increased to

94.5%, 76.9% & 82.7% respectively. Several

studies assessed the cutoff points of serum

PCT&CRP in differentiation between bacterial

& non bacterial infection & various cutoff points

were reported. Lopez et al.,(11) reported that

serum PCT at optimum cutoff, 0.53 ng/ml &

CRP at optimum cutoff, 27.5 mg/l could

differentiate between bacterial & viral infection

with sensitivities & specificities ( 65.5% &

94.3% respectively) for PCT(area under curve

0.82 ) & (63.5% & 84.2% respectively) for CRP

(area under curve 0.78). Moreover differentiation

between invasive & non invasive bacterial

infection could be done by serum

PCT(sensitivity 91.3% & specificity 93.5%) &

CRP (sensitivity 78% & specificity 75%) at

cutoff point (0.59 ng/ml ) for serum PCT(area

under curve was 0.95) and < 27.5 mg/l for CRP

(area under curve was 0.81). Also the authors

stated that serum PCT is more specific than CRP

in differentiation between bacterial & viral

infection & between invasive & non invasive

bacterial infection. Also Andreola et al.,(14)

studied serum PCT, CRP, total WBC & absolute

neutrophil count (ANC) as diagnostic markers of

severe bacterial infections in febrile infants &

children in the emergency department. The

optimum statistical cutoff value for detecting

sever bacterial infection was 0.8 ng/mL for PCT

(sensitivity, 69.1%; specificity, 85.3%), 32 mg/L

for CRP (sensitivity, 84.0%; specificity, 75.5%),

10,470/mm for WBC (sensitivity, 84.9%;

specificity, 47.4%), & 6,450/mm for ANC

(sensitivity, 81.8%; specificity, 62.3%). Limper

et al.,(6) reported that PCT at cut off point 0.24

ng/mL could differentiate between confirmed

bacterial & confirmed viral infections with

sensitivity 85%, specificity 69%, (AUC 0.82),

while at cut off point 0.21 ng/mL, PCT could

differentiate not only between confirmed

bacterial infection & non-infectious fever but

also between all bacterial infections (confirmed

& suspected) & non infectious fever with

sensitivity (90% & 85%) & specificity (71% &

71%) respectively. Moreover, the authors

concluded that CRP levels were less accurate

when comparing the same groups. ChiLin et

al.,(12) studied PCT, CRP, WBC in febrile infant

& young children & the authors reported that

PCT at cutoff point 0.4 ng/mL could

differentiate between bacterial & non bacterial

infections with sensitivity of 73% & specificity

of 72%. Moreover PCT level was significantly

higher in bacterial group & prevailed over CRP

& WBC. Prasad et al.,(15) in a study of serum

PCT in patients with meningitis (septic &

aseptic), reported that serum PCT at cut off

level≥5,000 pg/mL could differentiate between

septic & aseptic meningitis, with sensitivity

(98.5 %), specificity (93.5 %), PPV (98.6 % ) &

NPV (93.3 %), while at cutoff level 15,000

pg/ml, the sensitivity, specificity, PPV & NPV

decreased to 92 %, 67 %, 91.4 % & 71.4 %

respectively. Hatherill et al.,(16) in a study of

diagnostic markers of infection, compared serum

PCT with CRP & TLC, the authors reported that

PCT > 2 ng/ml identified all patients with

bacterial meningitis or septic shock & at cutoff

concentration > 20 ng/ml, PCT could predict

septic shock. In the present study, highly

significant positive correlation between serum

PCT level & other markers of infection (TLC,

CRP & ESR). This was in agreement with the

findings of Ibrahim et al.,(17) who studied

diagnostic value of serum PCT levels in children

with meningitis: in comparison with WBC &

CRP, & the author reported that there was a

positive correlation between serum PCT, TLC &

CRP in bacterial & non bacterial meningitis

cases. Lastly, a systematic review found 30

articles on the topic of PCT & concluded that

PCT has value as a diagnostic & prognostic tool

in patients with febrile neutropenia (18). Studies

have confirmed that the inclusion of PCT in

therapeutic guidelines of bacterial infections

effectively & safely reduces unnecessary

administration of antibiotics & duration of

antibiotic treatment, with no adverse effects on

the ultimate outcome of the disease (19).

Persistently elevated daily plasma PCT

concentrations > 2 μg/L are an indication that the

infectious process is not under control. Such a

condition is associated with a poor prognosis &

could trigger modifications in patient care,

additional investigations, or even justify changes

in treatment. Effective antibiotic treatment is

reflected by declining PCT values (20). One major

advantage of PCT compared to other parameters

is its early & highly specific increase in response

to bacterial infections & sepsis (21). Moreover

PCT is produced ubiquitously in response to

endotoxin or mediators released in response to

bacterial infections (that is, IL - 1b, TNF -α, &

IL-6) & strongly correlates with extent &

severity of bacterial infections (22). Because up

regulation of PCT is attenuated by interferon

(INF)-Ɣ, a cytokine released in response to viral

infections, PCT is more specific for bacterial

infections & may help to distinguish bacterial

infections from viral illnesses (7).Also the

production of PCT seems not to be attenuated by

corticosteroids unlike CRP(23) & does not rely on

white blood cells(24). Therefore, PCT may be a

promising candidate marker for diagnosis & for

antibiotic stewardship in patients with systemic

infections. Importantly, as with any diagnostic

tool, PCT should be used embedded in clinical

algorithms adapted to the type of infection & the

clinical context & setting (19).

Conclusion

PCT is valuable, simple, non invasive, time

saving, coast effective serum marker in

evaluating patients with infection especially

bacterial infection. Serum PCT at cut off point

(774.83 Pg/ml) could differentiate between

bacterial & viral etiologies. with sensitivity

(85.5%), specificity (70.0%), NPV (63.6%) &

accuracy (81.3%), but when combined with CRP

at Cutoff point (12.5 ), the Sensitivity, NPV and

accuracy increased to 94.5%, 76.9% & 82.7%

respectively.

Table (1):- Results of CBC in the studied groups. Groups

Variable

The studied groups t-test

P value

Bacterial group

N = 55

Viral group

N = 20

Control group

N = 15

Hb (gm/dl)

X±SD

Range

11.65±0.75

10.3 – 13.5

12.51±0.99

11.5 – 13.7

13.35±1.23

12 – 15

4.02

6.70

2.24

<0.001 ⃰

<0.001 ⃰ ⃰

0.033 ⃰ ⃰ ⃰ Platelets (10^3/cmm)

X±SD

Range

235.4±31.6

195 – 300

200.95±36.97

145 – 276

286.13±96.47

100 – 435

3.99

3.35

3.25

<0.001 ⃰

0.001 ⃰ ⃰

0.005 ⃰ ⃰ ⃰ TLC (10^3/mm3)

X±SD

Range

11.69±4.03

3.6 – 19

8.21±2.57

5 – 13.3

7.11±1.63

4 – 10

3.60

4.29

1.45

<0.001 ⃰

<0.001 ⃰ ⃰

0.15 ⃰ ⃰ ⃰ N = number. X = mean.

SD = stander deviation. * = Comparison between bacterial and viral groups.

** = Comparison between bacterial and control groups. *** = Comparison between viral and control groups.

Table (2) :- Results of ESR and CRP in the studied groups.

Groups

Variable

The studied groups Test of sig.

P value

Bacterial group

N = 55

Viral group

N = 20

Control group

N = 15

CRP (mg/L)

X±SD

Range

31.22±18.56

2 – 65

4.11±1.01

2 – 6

3.86±0.92

2 – 5

U

5.11

4.55

0.75

<0.001 ⃰

<0.001 ⃰ ⃰

0.51 ⃰ ⃰ ⃰ ESR (first hour) (mm/h)

X±SD

Range

44.05±5.84

31 – 50

6.10±1.62

5 – 12

4.73±1.44

2 – 6

t-test

28.50

25.72

2.59

<0.001 ⃰

<0.001 ⃰ ⃰

0.01 ⃰ ⃰ ⃰ N = number. U = Mann Whitney.

X = mean. SD = stander deviation.

* = Comparison between bacterial and viral groups. ** = Comparison between bacterial and control groups.

*** = Comparison between viral and control groups.

Table (3) :- Comparison between serum PCT level in the studied groups.

Groups

Procalcitonin level

The studied groups U P value

Bacterial group

N = 55

Viral group

N = 20

Control group

N = 15

Serum procalcitonin (Pg/ml)

X±SD

Range

1501.1±641.2

704.4 –

2944.36

541.8±236.1

260 – 899.7

195.01±38.2

149.85 – 280

5.39

5.90

4.93

<0.001 ⃰

<0.001 ⃰ ⃰ <0.001 ⃰ ⃰ ⃰

N = number. U = Mann Whitney.

X = mean. SD = stander deviation.

* = Comparison between bacterial and viral groups. ** = Comparison between bacterial and control groups.

*** = Comparison between viral and control groups.

Table (4):- Validity of assay of serum PCT and CRP

separately or in combination in discrimination between bacterial and viral infection.

Combination serum procalcitonin CRP Combined test

AUC 90.9 85.2 -----

P value <0.001 <0.001 ------

Cutoff point 774.83 (Pg/ml) 12.5 (mg/L) ------

Sensitivity 85.5% 78.2% 94.5%

Specificity 70.0% 70% 50.0%

PPV 88.7% 87.8% 83.9%

NPV 63.6% 53.8% 76.9%

Accuracy 81.3% 76% 82.7%

AUC = area under curve. PPV = positive predictive value.

NPV = negative predictive value.

Figure Legends

Figure 1 :- Correlation between serum PCT level

and TLC, Figure 2 :- Correlation between serum

PCT level and CRP, Figure 3 :- Correlation

between serum PCT level and ESR, Figure 4 :-

ROC Curve of CRP to discriminate bacterial

cases from viral cases, Figure 5 :- ROC Curve of

serum PCT to discriminate bacterial cases from

viral cases.

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Original Article

The Effect of the Cytochrome P450 (CYP450) Genetic Polymorphism in Peptic

Ulcer Therapy

Medhat Haroun *, Abir Adel Abdel Razak *, Nasser Abd allah ** , Marwa kassem *; Department of

Biochemistry , Institute of graduate study and research * And Department Of Tropical medicine**,

Faculty of medicine , Alexandria university

ABSTRACT

Peptic ulcer disease (PUD) embraces both gastric and duodenal ulcers. There are several factors involved in the

development of gastric and duodenal mucosal injury such as use of non-steroidal anti-inflammatory drugs (NSAIDs)

& Helicobacter pylori infection. This disease had a tremendous effect on morbidity & mortality until the last decades

of the 20th century. As a result of pharmacological development and discovery of effective and potent acid

suppressants as proton pump inhibitors, management of PUD has changed dramatically. Proton pump inhibitors

(PPIs) such as Omeprazole and Pantoprazole are widely used as first line acid inhibitors. They are extensively

metabolized in the liver by cytochrome P450 (CYP450) system. The principal enzyme involved in the metabolism is

CYP2C19.This enzyme exhibits single nucleotide polymorphism. There are inter- individual differences in the

activity of CYP2C19 which in turn affects the clinical outcomes of PPI-based PUD therapy. Two mutant alleles

CYP2C19*2 and CYP2C19*3 were recently found responsible for genetically deficient metabolic activity of

CYP2C19. Aim of the work: in the current study the effect of genetic polymorphism of CYP2C19 on PUD therapy

outcome has been investigated in a group of Egyptian patients. Material and methods: The study population

included 91 PUD patients with ages ranging between 18 and 70 years. Polymerase chain reaction–Restriction

fragment length polymorphism (PCR-RFLP) method was used to analyze the two mutant alleles CYP2C19*2,

CYP2C19*3 and the wild type CYP2C19*1 gene in peripheral blood samples of all patients. Conclusion: The

current study confirmed that most Egyptians are fast metabolizers and increasing PPIs dose twice daily would result

in a much better cure rate for PUD patients.

Introduction

Peptic ulcer disease (PUD) embraces both

gastric & duodenal ulcers. It has decreased in

incidence dramatically over the past 30 years.(1)

This is largely due to improvement in

pharmacological management of dyspeptic

symptoms & a greater understanding of the

aetiology. Whereas surgery was previously a

frequent solution for chronic peptic ulcer

disease, this is now almost exclusively reserved

for emergency situations & hemorrhage .(2) The

complex & multifactorial pathogenesis of peptic

ulcer has been studied over several decades. It

was found to result from an imbalance between

aggressive gastric luminal factors, acid & pepsin

& defensive mucosal barrier function. Several

environmental & host factors contribute to ulcer

formation by increasing gastric acid secretion or

weakening the mucosal barrier(3). Gastric

hypersecretion associated with gastrinoma in

Zollinger-Ellison syndrome, antral G-cell

hyperplasia, an increase in parietal-cell mass & a

physiological imbalance between the

antagonistic gastric hormones gastrin &

somatostatin, is still an important issue in peptic

ulcer disease(2). Moreover, it is known that

cholinergic hypersensitivity & parasympathetic

dominance are related to the stimulation not only

of hydrochloric acid but also pepsin, which is

often neglected as a cofactor in the development

of erosive injury to the gastric mucosa.

Psychologic stress, cigarette smoking, alcohol

consumption, use of non-steroidal anti-

inflammatory drugs (NSAIDs) including aspirin,

oral bisphosphonates, potassium chloride,

immunosuppressive medications & an age-

related decline in prostaglandin levels have all

been shown to contribute to peptic ulcer disease.

(1) Several factors are known to be involved in

the development of gastric & duodenal mucosal

injury Among these, the use of non-steroidal

anti-inflammatory drugs (NSAIDs) & infection

with Helicobacter pylori (H.pylori) were shown

to play, by far, the biggest roles. (4) Reporting of

the Campylobacter-like organism, H. pylori , by

Warren & Marshall in 1984,(5) has marked a

giant leap in medical understanding of this

condition. This Gram-negative, helical,

microaerophilic, flagellated bacterium has since

been recognized to be responsible for up to 95%

of duodenal & 70% of gastric ulcers.(6) H. pylori

possesses a urease enzyme that converts urea to

ammonia & carbon dioxide, buffering gastric

acid in its vicinity & facilitating its survival in

the acidic gastric environment(4). More than 50%

of the world¡¦s population has a chronic H. pylori

infection of the gastric mucosa, yet only 5¡V10%

of these develop ulcers.(3) H. pylori colonises the

entire gastric epithelium, from the prepylori

cantrum to the cardia(3). In patients with

duodenal ulcer, density of infection & severity of

inflammation are greatest in the distal antral

region while sparing the acid-secreting body

mucosa. After H.pylori eradication, gastric

mucosal changes are usually fully reversible. In

gastric ulcer, inflammation affects the body &

antral mucosa to a similar degree, although it

varies depending on ulcer location. (7) H. pylori

infection impairs negative feedback regulation of

gastrin release & thus acid secretion. (8,9) Neural

pathways are also affected by H. pylori infection

causing functional disruption of antral fundic

neural connections that down regulate acid

production. (10) Specific H. pylori genotypes are

associated with severe morbidity.The most

prevalent H pylori genotypes in patients with

peptic ulcerations are vacA-positive & cagA-

positive ones.(11) H. pylori-derived vacuolating

cytotoxin (VacA), an 87-kDa protein, causes

vacuolar degeneration in cultured gastric-cell

preparations & gastric ulceration in laboratory

animals. Although present in all H.pylori strains,

the vacA gene, dependent on its allelic form, is

expressed in only 60% of cases.(12( The

cytotoxin-associated gene A (cagA), restricted to

cytotoxin-producing strains of H.pylori, is within

an island of 31 genes defined as CagA

pathogenicity island. CagA encodes a 120¡V160

kDa immunodominant protein that is a marker of

increased virulence & that enhances the local

inflammatory response.(13) Despite their well-

accepted anti-inflammatory & analgesic benefits,

NSAID use is one of the common causes of

gastrointestinal mucosal injury. NSAIDs

including aspirin, significantly increase the risk

of adverse gastrointestinal events, particularly

those related to gastric and/or duodenal mucosal

injury such as erosions, ulcers & ulcer

complications, especially bleeding.(14) Although

not always the case before, the majority of

patients with peptic ulcer disease can now be

treated perfectly well with medication. The

development of histamine (H2)-receptor

antagonists in the 1970s led to a dramatic shift,

with rates of elective ulcer surgery decreasing by

up to 80% in the 1980s.(2) Medical treatment

improved further, in the late 1980s, when the

proton-pump inhibitor (PPI) omeprazole was

introduced. Although H2-receptor antagonists

are effective at reducing acid secretion, with

treatment leading to healing of 80-90% of peptic

ulcers, they do not block it completely as gastrin

& vagal stimulation can still induce parietal cell

acid secretion.(2) Moreover, lack of tachphylaxis

to PPI therapy ensures very high healing rates

for both duodenal & gastric ulcers.(15) Over the

past 20 years, H. pylori eradication therapies

have mainly consisted of antimicrobial agents

combined with antisecretory drugs. There is now

a worldwide consensus that the first-line

treatment should be triple therapy with a PPI

twice daily plus clarithromycin 500 mg twice

daily & either amoxicillin 1 g twice daily (PPI-

CA) or metronidazole 500 mg twice daily (PPI-

CM) for 7-14 days. Successful eradication with

first-line treatment varies from 70-95%. (1) The

aim of this study was to investigate the effect of

genetic polymorphism of cytochrome P4502C19

(CYP2C19) on the metabolism of proton pump

inhibitor & its effect on peptic ulcer disease

outcome in Egyptians.


Ninety one patients with peptic ulcer disease

with or without Helicobacter pylori infection

were included in this study. They were recruited

from the out-patients Clinic of the Tropical

Medicine Department, Alexandria Main

University Hospital. All patients were subjected

to physical examination, gastroduodenoscopy

and laboratory tests including complete blood

picture, occult blood in stool, Helicobacter pylori

antigen in the stool , rapid urease test in the

endoscopic biopsy specimens and urease breath

test. Patients were also asked about whether or

not they had Helicobacter pylori infection

before, if they had been treated and how many

times they were infected and treated. Patients

were then given 40mg omeprazole twice daily

with antibiotic therapy in H. pylori positive case.

They were asked to come after 4 and 6 weeks

treatment for follow up. Informed consent was

obtained from all patients. Diabetic patients as

well as those with chronic liver or kidney

diseases & those with malignancy were all

excluded from this study. Sample collection:

Three milliters of venous blood were collected

from every patient in tubes containing

EDTA.1ml of each blood sample was divided

into 500£gl aliquots in Eppindorf tube and kept

at-20 oC until required. Leukocyte genomic

DNA isolation from whole blood was carried out

manually. The extracted DNA was stored at -20

oC until analysis. Isolation of DNA from whole

blood samples: DNA isolation from whole blood

samples was carried out manually(16). Freshly

collected 500ul blood aliquot was transferred to

a clean sterile 1.5 ml Eppindorff's tube. Then

900ul of red blood cells lysis solution

(Ammonium chloride 155mm, Potassium

bicarbonates 10mm, EDTA 1mm) was added &

the mixture was incubated at room temperature

for 10 minutes. After the incubation period,

centrifugation was carried out at 13,000 rpm

using micro centrifuge at room temperature for

30 seconds to precipitate the WBCs. The

supernatant that contained the RBCs lysates was

then poured off leaving behind about 25ul of

lysate on the collected WBCs. White blood cells

precipitate was then vortexed in this residual

lysate to re-suspend the white blood cells

thoroughly. This process was followed by adding

300 ul of WBCs lysis solution (EDTA 25mm,

SDS 2%). The tubes were inverted several times

to lyse WBCs. Proteins were then precipitated by

the addition of 100ul protein precipitation

solution (Ammonium acetate 7-10M) & the

tubes were left for 5 minutes. Precipitated

proteins & cell debris were collected by

centrifugation at 7,000 rpm for 5 minutes using a

micro centrifuge at room temperature. The above

clear supernatant was then transferred to clean

1.5 ml tubes & the DNA was precipitated using

equal volume of isopropanol (400ul).

Precipitated DNA was then collected by

centrifugation at 10,000 rpm for 10 minutes at

room temperature. DNA pellets were washed

twice with 1 ml 75% ethanol & left at room

temperature in an inverted position for 15

minutes to evaporate the minimal residues of

ethanol. DNA pellets were re suspended in TE

buffer (Tris-Cl 25mM, PH=8; EDTA 10Mm).

Genotyping of CYP2C19 wild type (wt) & two

mutants: Genotyping of extracted DNA for

CYP2C19*1 (wt), CYP2C19*2 (m1) in exon 5,

& CYP2C19*3(m2) in exon 4 was done using

polymerase chain reaction (PCR) restriction

fragment length polymorphism (RFLP)

technique. PCR was set up with minor

modifications to the reaction described by

Goldstein & Blaisdell, to amplify CYP2C19*2 &

CYP2C19*3 alleles (17(. Detection of

CYP2C19*2 mutation was performed using

PCR. The PCR was carried out in 30 £gl reaction

mixture containing 15£gl master mix (GoTaqR

Green MasterMix ,Promega),1£gl forward

primer,1£gl reverse primer,10£gl nuclease free

water & 3£gl extracted DNA.PCR amplification

was performed with Primus 25 advancedR

Peqlab thermocycler & consisted of initial

denaturation step at 94oC for 5 min, followed by

35 cycles consisting of denaturation at 94oC for

45 seconds, annealing at 50oC for 1min,

extension at 72oC for 1 min, & final extension at

72oC for 5 min. Detection of CYP2C19*3

mutation was performed using PCR. The PCR

reagents used were the same. The PCR

conditions were initial denaturation at 94oC for 5

min, followed by 35 cycles consisting of

denaturation at 94oC for 45 seconds, annealing

at 48oC for 45 seconds, extension at 72oC for

45seconds & final extension at 72oC for 5 min.

The amplified PCR products of CYP2C19 exon

5 were digested with fast digest restriction

enzyme SmaI. While the amplified PCR

products of CYP2C19 exon 4 were digested with

fast digest restriction enzyme BamHI . DNA

detection: The extracted DNA samples were

analyzed by 1% agarose gel electrophoresis &

amplified PCR products & digested PCR

products were analysized by 2% agarose gel

electrophoresis & were visualized by staining

with ethidium bromide under UV

transilluminator & photographed.

Statistical Analysis

The observed genotype frequencies of CYP2C19

were compared with expected according to the

Hardy-Weinberg equilibrium law. P<0.05 was

considered statistically significant. The

frequency of each allele in the study population

was analyzed using the chi-square test with 95%

confidence interval (CI). Statistical analysis of

the data was performed using SPSS software

version 18.

Results Demographic characteristics: This study was

conducted on 91 patients with peptic ulcer

disease, 66 males & 25 females with ages

range between 18 & 70 years. 26 patients were

H.pylori negative while 65 patients were

H.pylori positive. DNA extraction results: The

extracted DNA samples were analyzed on 1%

agarose gel electrophoresis. (Figure 1)

Genotyping of exon 5 (m1) of CYP2C19 gene: The amplified PCR products of CYP2C19 exon 5 produced

a band at 321 bp and were analyzed on 2% agarose gel electrophoresis. (Figure 2)

Genotyping for exon 4 (m2) of CYP2C19 gene:

Genotyping for exon 4 (m2) of CYP2C19 gene:

The amplified PCR products of CYP2C19 exon

4 produced a band at 271 bp and were analyzed

on 2% agarose gel electrophoresis.(figure:3).

PCR product of CYP2C19 exon 5 after restriction

with SmaI: The PCR product of CYP2C19 exon

5 was digested with the restriction enzyme SmaI.

Since the restriction site is absent in the mutant

allele (CYP2C19*2), therefore the samples

containing CYP2C19*1 allele produced 212 and

109 bp after digestion with SmaI, while those

containing CYP2C19*2 allele were not

digested.(figure:4)

PCR product of CYP2C19 exon 4 after restriction

with BamHI: The PCR product of CYP2C19

exon 4 was digested with the restriction enzyme

BamHI. Since the restriction site is absent in the

mutant allele (CYP2C19*3), therefore the

samples containing CYP2C19*1 allele produced

175 and 96 bp after digestion with BamHI,

while those containing CYP2C19*3 allele were

not digested.( Figure 5,6)

Statistical data results : Two patterns were noted

in 91 patients: 52 patients were homozygous

for wild type allele CYP2C19*1/*1 in both

exons 5 and 4. 39 patients were heterozygous

for CYP2C19*1/*2 i.e.CYP2C19 m1 mutation

(mutation in exon 5) without CYP2C19 m2

mutation. There were no statistical differences

in mean age or male and female ratio among

these two genotype groups (table1).

Table (1): Distribution of the CYP2C19 Genotype polymorphism in the studied peptic ulcer patients.

CYP2C19 Genotype polymorphism

(n=91) Sig.

OR (95% CI) CYP2C19*1 (n=52) CYP2C19*2

(n=39) No. % No %

Age:

Min-Max Mean±SD

18-70

41.9±12.1

18-70

38.9±12.8

Gender Males Females

35

17

67.3

32.7

31

8

79.5

20.5

X2

=1.659

P=0.198

0.53

(0.18-1.54)

t: t-test & X2: Chi-Square test & significant at P≤0.05

The most frequently identified allele in these

Egyptian patients was wild type allele

CYP2C19*1 with a frequency 0.79, while the

frequency of CYP2C19*2 allele was 0.21. The

frequency of CYP2C19*1/*1 genotype

(homozygous extensive metabolizer) was 57.1%

while that of CYP2C19*1/*2 genotype

(heterozygous extensive metabolizer) was 42.9 %

(table 2).

Table ( 2): Genotype distribution and allele frequencies for PCR gene polymorphism in peptic ulcer patients.

ALLELE FREQUENCY GENOTYPES

2* 1* 2*/2* 1*/2* 1*/2*

0.21 0.79 0 39 52 N Patients (N=91)

0 (42.9%) (57.1%) %

H.Pylori infection and CYP2C19 Genotype

polymorphism among the studied peptic ulcer

patients. Frequency of CYP2C19*1 genotype

was 38.5% in H.pylori negative patients in

comparison to 15.4 % for frequency of

CYP2C19*2. Meanwhile, the frequency of

CYP2C19*1 was 61.5 % in H.pylori positive

patients in comparison to 84.6% for

CYP2C19*2. The difference was significant

where P= 0.016, OR = 3.44, 95% CI (table 3).

Table (3): H.Pylori infection and CYP2C19 Genotype polymorphism among the studied peptic ulcer patients.

. Pylori

CYP2C19 polymorphism

(n=91)

Sig.

OR (95% CI) CYP2C19*1 (n=52) CYP2C19*2 (n=39)

No. % No. %

H. Pylori -ve

H. Pylori +ve

2 0

3 2

38.5

61.5

6

33

15.4

84.6 X2

=5.815 P=0.016* 3.44 (1.1-1.1)*

1

X2

: Chi-Square test *significant at P≤0.05

Healing of patients after 4 weeks of treatment

according to their CYP2C19 Genotype

polymorphism: Frequency of partially healed

CYP2C19*1 genotype patients was 34.6% in

comparison to 25.6% for CYP2C19*2

genotype ones. Moreover, frequency of

completely healed CYP2C19*1 patients was

65.4% in comparison to 71.8% for

CYP2C19*2 ones. On the other hand, frequency

of CYP2C19*2 non-healed patient was 2.6%

(one patient). Figure 7.

Healing of patients after 6 weeks of treatment

according to their CYP2C19 Genotype

polymorphism: The number of patients who

needed to continue treatment for six weeks was

18 for CYP2C19*1 and 11 for CYP2C19*2.

Frequency of completely healed patients was

66.7% for CYP2C19*1 genotype and 90.9%

for CYP2C19*2 genotype with a significant

difference at P= 0-037, OR= 0.2 (0.01-2.3),

95%CI.Frequency of partially healed

CY2C19*1 patients was 33.3%.In contrast,

frequency of the non-healed patient was 9.1%

for CYP2C19*2 genotype (one patient). Figure

8.

Frequency of the total healed patients during

this study was 88.5% for CYP2C19*1

genotype in comparison to 97.4% for

CYP2C19*2 genotype. Frequency of no/partially

healed patients in this study was 11.5% for

CY2C19*1 genotype (six patients) compared to

2.6% for CY2C19*2 genotype (one patient)

.figure 9.

Correlation of peptic ulcer healing with the

presence of H pylori infection after 4 weeks

treatment: The frequency of completely healed

patients was 69.2% for those without H.Pylori

infection in comparison to 67.7% for infected

ones. The frequency of partially healed cases

was 30.8% for both those with and those

without H. Pylori infection. The frequency of

non- healed patient was 1.5% for H.Pylori

infected patients (one patient). There was no

significant difference between them. (table 4).

Correlation of peptic ulcer healing with the

presence of H pylori infection after 6 weeks

treatment: The number of patients who

continued till the 6 week was 8 without

H.Pylori infection and 21 with H.Pylori

infection. The frequency of completely healed

patients was 75% for those without infection

and 76.2% for H.Pylori infected ones. The

frequency of partially healed patients was 25%

for cases without and 19% for those with

H.Pylori infection. The frequency of non-healed

patients was 4.8% for the H.Pylori infected case.

(one patient). There was no significant

difference between them (table 4). Correlation

between the allele frequencies in peptic ulcer

patients showed healing at 4 weeks. The number

of healed patients after 4 weeks treatment was

sixty two (62) of which thirty four (34)

carried genotype CYP2C19*1/*1 with allele

frequency 0.77 while twenty eight (28)

patients carried genotype CYP2C19*1/*2 with

allele frequency 1.23. After 4 weeks treatment,

the number of the non- healed or partially

healed patients was twenty nine (29) of which

eighteen (18) were carrying the genotype

CYP2C19*1/*1 with allele frequency 0.81 and

eleven (11) were carrying the genotype

CYP2C19*1/*2 (37.9%) with allele frequency

0.19

Table (4): Timing of healing of the studied peptic ulcer patients according to their infection with H.Pylori.

Timing of healing

Infection with H. Pylori (n=91)

Significance OR (95% CI) No infection (n=26) Infection (n=65)

No. % No. %

Healing at 4 weeks

Not healed

Partially healed

Complete healing

0

8

18

0.0

30.8

69.2

1

20

44

1.5

30.8

67.7

MCP=1.0

0.0 (0.0-44.7)

2.3 (0.7-7.9)

1.1 (0.4-3.2)

Healing at 6 weeks (n=29)

Not healed

Partially healed

Complete

healing

(n=8) (n=21)

MCP=1.0

0.0 (0.0-50.9)

1.42 (0.1-13.6)

0.94 (0.1-9.4)

0

2

6

0.0

25.0

75.0

1

4

16

4.8

19.0

76.2

significant at P≤0.05 & MC

P: Monte Carlo test

Discussion

Peptic ulcer disease embraces both gastric and

duodenal ulcers and has been a major threat to

the world’s population over the past two

centuries, with a high morbidity and substantial

mortality. The discovery of Helicobacter pylori

switched the notion from an acid-driven state to

an infectious disease as a suggested mechanism

of pathogenesis. In both conditions, ulcer is

associated with an imbalance between protective

and aggressive factors, with inflammation being

a leading cause of this imbalance. (3) Eradication

of H.pylori infection is now the mainstay of

treatment for peptic ulcer disease. It has resulted

in very high ulcer healing rates along with

dramatic drop of recurrence rates, especially in

individuals with duodenal ulcers. Current

treatment strategies for the cure of H pylori

infection have included a triple therapy with a

proton pump inhibitor, amoxicillin and/or

clarithromycin or metronidazole with high

eradication rate (greater than 80%). However,

there still remains incomplete eradication with

therapy, even though considerable dosage of

antimicrobials are used(18). Proton pump

inhibitors (PPIs) produce a profound suppression

of gastric acid secretion by inhibition of H+/K+-

ATPase (proton pump) activity in gastric parietal

cells(19) and are used as potent antiulcer

agents(20). PPIs such as omeprazole and

lansoprazole (a benzimidazole derivative) are

metabolized mainly in the liver by the same

genetically determined enzyme, S-mephenytoin

4-hydroxylase (CYP2C19) which exhibits single

nucleotide polymorphisms (SNPs)(21). This

pharmacogenetic entity has shown a marked

interethnic difference in the incidence of poor or

deficient metabolizers: the poor metabolizer.

frequency is much greater in Japanese

persons(18 to 23%) than that in American or

European whites (3 to 5%)(22). Beside the wild

type allele CYP2C19*1, two different mutation

events associated with the poor metabolizer

genotypes of CYP2C19 were determined by De

Morais et al. They concluded that CYP2C19m1

in exon 5 (m1) and CYP2C19m2 in exon 4 (m2)

account for 100% of the available Japanese poor

metabolizers and that comparable detection of

m1 and m2 predicts the phenotypes of

CYP2C19(23,24). These genotype could be

reasonably classified into three groups: the

homozygous of CYP2C19*1, the heterozygous

of CYP2C19*1, and the combination of mutant

alleles, because the metabolic activity from

CYP2C19*2 and CYP2C19*3 was almost

perfectly deficient(25). CYP2C19 polymorphism

influences pharmacokinetics and

pharmacodynamics of PPIs(26) beside affecting

the efficacy of H. pylori eradication therapy with

a PPI (omeprazole, pantoprazole or

lansoprazole) together with amoxicillin or

clarithromycin(27). Accordingly, the aim of this

research was to investigate the effect of genetic

polymorphism of cytochrome P4502C19

(CYP2C19) on the metabolism of proton pump

inhibitor drugs and its effect on peptic ulcer

disease outcome in Egyptians. In the current

study, 91 Egyptian peptic ulcer patients were

included in the study; 26 of them were H.pylori

positive. After physical examination,

gastroduodenoscopy and laboratory tests to

detect presence or absence of H.pylori, patients

were then given 40mg omeprazole twice daily

with antibiotic therapy in H. pylori positive case.

They were asked to come after 4 and 6 weeks

treatment for follow up. Many scientists have

reported a correlation between CYP2C19

genotype and cure rates of PUD patients In the

present research, patients were given 40 mg

omeprazole twice daily with antibiotic therapy in

H. pylori positive case. Out of 91 PUD patients,

62 healed after 4 weeks treatment, 34 of them

carried CYP2C19*1/*1(54.8%) and 28 patients

carried CYP2C19*1/*2(42.2%) indicating that

the use of the higher dose of omeprazole for fast

metabolizer PUD cases resulted in a much better

healing rate where about 68.13% of the total fast

metabolizer PUD patients (sum of homoEM and

heteroEM) healed after 4 weeks treatment with

40 mg omeprazole. So, increasing the dose of

PPIs twice daily seemed to lead to a better

healing rate for fast metabolizer PUD patients. In

the present study, two combination patterns were

observed in the 91 studied PUD patients: 52

patients (57.1%) were homozygous for wild type

CYP2C19*1/*1 with a mean age of 41.9±12.1

years. Beside 39 patients (42.9%) were

heterozygous for CYP2C19*1/*2 with a mean

age of 38.9±12.9 years. The most frequently

identified allele in these Egyptians was the wild

type allele CYP2C19*1 with frequency 0.79

whereas the frequency of CYP2C19*2 was 0.21.

These results were in concordance with those

obtained by Samar, et al.(2002) (28).This study

was performed on two hundred and forty-seven

unrelated Egyptians to determine the frequencies

of important allelic variants of CYP2C9,

CYP2C19, CYP2E1 and DPYD in the Egyptian

population and compare them with their

frequencies in other ethnic populations. For

CYP2C19, the frequencies of the wild type

(CYP2C19*1) and the nonfunctional (*2 and *3)

alleles were 0.888, 0.110 and 0.002,

respectively. CYP2C19*3, which is considered

an Asian mutation, was detected in one subject

(0.40%) who was heterozygous (*1/*3). Two

subjects (0.80%) were homozygous for *2/*2,

while no compound heterozygotes (*2/*3) or

homozygotes for *3 were detected. Accordingly,

it was concluded that, most Egyptians are fast

metabolizers (having the wild type

CYP2C19*1/*1). In other words, they

metabolize PPIs very fast and thus shorten the

time needed for the drug to carry out its function.

Since the aim of therapy is to inhibit the

production of HCl long enough to allow the

regeneration of damaged gastric cells and so in

the case of the fast metabolizer PUD patient, the

intragastric pH will be low and will affect the

stability and activity of antibiotics and thus

affect eradication of H.pylori. Hence, dose

adjustment is needed for these patients.

Futhermore, in a study performed on Swedish

Caucasians, high concentration of omeprazole in

poor metabolizers (PMs) resulted in great gastric

acid suppression as compared with extensive

metabolizers(Ems).(29) In another work done on

Japanese population in which PM is the most

frequent allele, dual therapy with omeprazole

and amoxicillin was given for H.pylori infection.

Cure rates in these patients were found to be

28% in homozygous EMs (CYP2C19*1/*1),

60% in heterozygous EMs (CYP2C19*1/*2 and

*1/*3), and 100% in PMs (CYP2C19*2/*2 and

*2/*3), indicating the importance of dose

adjustment in cases of EMs.(30) In the current

study, two genotypes were detected;

CYP2C19*1 and CYP2C19*2 while

CYP2C19*3 genotype was absent. Regarding

the frequency of CYP2C19*3 recorded by

Samar, et al. the present finding could be

attributed to the fact that it was not easy to find

this allele in a rather small population (91

patients). Moreover, both mutations are similar

in the outcome as both genotypes give non-

functional enzyme i.e. poor metabolizer(25). In

the present research, the number of CYP2C19*1

patients having H.pylori infection was 32

(61.5%) while infected CYP2C19*2 ones were

33 cases (84.6%) with a significant difference.

This indicates that patients with CYP2C19*2

genotype were more susceptible to H.pylori

infection than the other genotype. However, this

did not affect healing of H.pylori infected PUD

patients carrying CYP2C19*2 after 4 weeks

treatment. On the contrary, this seemed to be in

their favor, since it is considered that enzyme

activity of CYP2C19*2 subjects (hetero EM) is

almost half that in CYP2C19*1ones (homoEM).

Thus that PPIs were not metabolized by

heteroEM patients at the same rate as in

homoEM ones thus allowing PPI to carry out its

action, permitting the medium to be less acidic

for antibiotic effect to take place. Although there

was no significant difference between the ratio

of healed patients for both genotypes(65.4% for

CYP2C19*1/*1 and 71.8% for CYP2C19*1/*2)

after 4 weeks treatment, yet, there was still a

significant difference between the two genotypes

after 6 weeks treatment (66.7% for

CYP2C19*1/*1 and for 90.9% for

CYP2C19*1/*2) indicating that heteroEM

(CYP2C19*1/*2) heals much better than

homoEM (CYP2C19*1/*1) due to the presence

of non-functional allele.The difference between

homoEM and heteroEM are too slight compared

to that of PM as confirmed by the following

studies. Toshiyuki, et al.(2001) carried out a

randomized study with various PPIs on 18

Japanese healthy volunteers, classified into

homozygous, heterozygous extensive

metabolizer and the poor metabolizer based on

the CYP2C19. Each subject received a single

oral dose of 20 mg omeprazole, 30 mg

lansoprazole or 20 mg sodium rabeprazole, with

at least 1 week washout period between different

PPIs. Plasma concentrations of PPIs and their

metabolites were monitored until 12 hours after

medication. Pharmacokinetic profiles of

omeprazole and lansoprazole were well

correlated with the CYP2C19 genotype. The

heterozygous extensive metabolizer was slightly

different from the homozygote, with no

statistically significant difference. The CYP2C19

genotype dependence detected with lansoprazole

was not obvious compared to that recorded with

omeprazole. As for rabeprazole, the

pharmacokinetic profile was independent of the

CYP2C19 genotype. (25) Takaski, et al.(2008)

performed a randomized double blind parallel

study on 74 PUD Japanese patients divided into

2 groups; one group was given 10 mg

rabeprazole daily for 1 week and the other one

was given omeprazole daily for 1 week.

Endoscopic examination was done to evaluate

healing rate beside genotyping for CYP2C19 for

both groups. The overall improvement ratios for

both rabeprazole and omeprazole showed no

significant difference between both groups. (31)

Chii-Shyan et al (2010) conducted a study on 95

consecutive patients with cirrhosis and H. pylori-

infected active peptic ulcers. Patients were given

2-weeks course of oral amoxicillin 1,000 mg

b.i.d., rabeprazole 20 mg b.i.d. and

clarithromycin 500 mg b.i.d. Subsequently, all

patients received oral rabeprazole 20 mg once

daily until week 8. The CYP2C19 genotype

status for 2 mutations associated with the

extensive metabolizer phenotype was determined

by PCR-RFLP. Cure rates for H. pylori infection

were 80.9%, 89.8%, and 100% in the rapid-,

intermediate-, and poor-metabolizer groups,

respectively. Healing rates for duodenal and

gastric ulcers in the 3 groups were roughly

parallel with cure rates for H. pylori infection. (32) Furuta, et al. (2001) reported that eradication

rates for H. pylori infection using triple therapy

with daily doses of omeprazole 40 mg or

lansoprazole 60 mg, amoxicillin 1500 mg, and

clarithromycin 600 mg for 1 week were 72.7% in

RMs, 92.1% in IMs, and 97.8% in PMs. The

above authors increased both PPIs and

antibiotics doses to achieve higher eradication

rates in such short time as one week. (33) In the

current study, the choice of giving 40 mg

omeprazole was decided depending on the fact

that most Egyptians are fast metabolizers beside

some studies stated that 20 mg omeprazole was

not efficient for treating fast metabolizers.

Therefore, 40 mg omeprazole was chosen being

the highest dose found on the Egyptian market. (34) Furuta, et al. (1999) (34) have reported that

when 20 mg of omeprazole was given in a single

dose, plasma omeprazole concentrations differed

among the three different CYP2C19 genotype

(RM, IM, and PM) groups. Plasma omeprazole

levels in the PM group were sustained for a long

time after dosing. Plasma levels of 5-

hydroxyomeprazole, which is formed from

omeprazole via CYP2C19, in the PM group were

lower than those detected in the RM and IM

groups. This could be attributed to the fact that,

in PMs the sulfoxidation of omeprazole is the

main metabolic pathway and omeprazole sulfone

cannot be metabolized to 5-hydroxyomeprazole

sulfone by CYP2C19 in this group, because PMs

lack CYP2C19. Therefore, plasma omeprazole

sulfone levels are sustained for a long time after

dosing in the PM group. The same results were

obtained by Saitoh et al (2002)(35) on a single

oral dose of 20 mg omeprazole as the mean 24-

hour intragastric pH level in the RM group was

the lowest, and highest in the PM group. The

acid inhibition achieved by 20 mg omeprazole,

the so-called standard dose, seemed to be

therapeutically insufficient in the RM group. In

spite of this, the difference in acid inhibitory

effect of a PPI among different CYP2C19

genotype status becomes smaller with repeated

PPI dosing. (26) Tomohiko et al (2007)(36) carried

a study on Japanese H. pylori-negative

CYP2C19 homozygous extensive metabolizers

for the strongest effect on cure rate for PUD

using omeprazole as 10, 20, and 40 mg once and

twice daily. He concluded that, for gastric acid

suppression, omeprazole 20 mg bid is superior to

40 mg od and omeprazole 20 mg bid was

superior to 10 mg bid , so, giving a small dose

twice daily is much better than giving a large

dose once daily. In the present study ,it was

observed that most Egyptians are fast

metabolizers either being homoEM

(CYP2C19*1/*1) or heteroEM (CYP2C19*1

/*2). Therefore, dose adjustment is needed to

achieve better cure rates for fast metabolizers

PUD patients. Hence, the 91 PUD patients

involved in this study were given 40 mg

omeprazole twice daily with antibiotic therapy in

case of H.pylori infection. The omeprazole dose

was increased while antibiotics doses were kept

as usual. The overall cure rate during the present

research was 88.5% for CYP2C19*1 genotype in

comparison to 97.4% for CYP2C19*2 genotype.

Conclusion

Most Egyptians are fast metabolizers either

being homoEM (CYP2C19*1/*1) or heteroEM

(CYP2C19*1/*2). Giving small dose of PPIs

twice daily is much better than giving one large

dose once daily. As well, using higher doses of

proton pump inhibitor drug twice daily while

keeping the antibiotic dose as usual would seem

to result in a much better cure rates for fast

metabolizer peptic ulcer disease patients. All

PPIs are of equal efficiency and efficacy.

Patients carrying genotype CYP2C19*2 are

susceptible to have H.pylori infection more than

that of CYP2C19*1 bearing ones. We may

recommend to carry out a genotyping of

CYP2C19 before using PPIs for treating PUD

for dose adjustment and subsequent better cure

rates. Also, the usual high dose of PPIs twice

daily while keeping antibiotics would result in a

much better cure rates for H.pylori infected for

fast metabolizer PUD patients.

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Editor in chief - eshgid.comeshgid.com/images/2018June1100PM_574786627.pdf · Hilmy Abaza Seham Abdel-Reheem Co-Editors Ahmed Shawky Fathalla Sidky Maher Osman Mohamed Sharaf El-Din