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Contents Alexandria Journal of Hepatogastroenterology,
Volume (XIX) - April 2016
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and editors of the products advertised.
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.
------------------------------------------------ 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.
------------------------------------------------ Original Article
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.
------------------------------------------------ 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.
------------------------------------------------
2
9
14
23
28
41
52
59
34
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
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.
Patients and Methods
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
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.
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
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
bacterial and aseptic groups as regard CSF
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
bacterial and aseptic groups as regard CSF
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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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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6. Frosner GG, Schomerus H, Wiedmann KH,
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7. Werle-Lapostolle B, Bowden S, Locarnini S,
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in patients with chronic hepatitis B,turk J
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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
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.
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
1st visit 2nd visit 3rd visit
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).
p1: p value for comparing between the 1st and 2nd visits.
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
1st visit 2nd visit 3rd visit
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
t (P) Group I Group II
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
Lab investigations Group
t (P) Group I Group II
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
Lab investigations Group
t (P) Group I Group II
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).
p1: p value for comparing between the 1st and 2nd visits.
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
t (P) Group I Group II
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
1st visit 2nd visit 3rd visit
%
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.
Patients and Methods
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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