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Contents Alexandria Journal of Hepatogastroenterology,
Volume (XXII) - April 2017
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Disclaimer: The Publisher, the Egyptian Society of
Hepatology Gastroenterology and Infectious Diseases in
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Original Article
Analysis of Etiologies and Outcome of Cases of Fever of
Unknown (FUO) Origin Admitted to Alexandria Fever
Hospital
Mohiedeen KM, El Kady A, Elwazzan D, Helmy N
---------------------------------------------- Original Article
Cardiovascular Risk Assessment in Hemodialysis Patients:
Relation to Malnutrition, Inflammation and Body Fluid
Determined by Bioelectrical Impedance
Essam El Din Hassan El Kashef, Sameh Morsi Arab, Yasmine
Salah Naga, Shaimaa Elsayed Mohamed Mohamed, Montasser
Mohammed Hussein Zeid
---------------------------------------------- Original Article
Helicobacter Pylori CagA Line Test
Mohamed Alaa Eldin Nouh, Hossam Eldine Mostafa Saleam
and Bedair Haroun AbdAllah Fayed
---------------------------------------------- Original Article
Patterns of Peripheral Vascular Disease in Lower
Extremities as a Predictor of Disease Activity and Damage
in Systemic Lupus Erythematosus Patients
Manal Tayel, Magdy Megallaa, Nevine Mohannad and Mariam
Mostafa
---------------------------------------------- Original Article
Retinal Nerve Fiber Layer Thickness in Normal Egyptian
Population
Mohammad A.M. El-Hifnawy, Amir A. Abo-Samra, Mohsen A.
Abou-Shousha, Ehab M. Kassem
---------------------------------------------- Original Article
Role of Chromoendoscopy in Early Detection of Barrett's
Esophagus in some Egyptian Patients Suffering from Long
Standing Gastroesophageal Reflux Disease
Hanan Hosny Nouh, Hanan Yehia Tayel, Ahmed Ismail
Ellakany, Yara Mohamed Naguib Mohamed
---------------------------------------------- Original Article
Some Non Invasive Methods in Detection and Grading of
Oesophageal Varicse in Splenectomized Cirrhotic Patients
Alaa El-Din Mohamad Abdo, Akram Abd El-Moneim Deghady,
Ehab Hassan El-kholy, Abd El-Kader Hassan Abd El-Kader
---------------------------------------------- Original Article
Study of Serum Vitamin D3 Levels in Rheumatoid Arthritis
Patients and its Relation with Disease Activity and CD46
Activity
Ashraf El Zawawy, Eman Hassan, Hanaa Ali, Nehad Hussein
---------------------------------------------- Original Article
Study of the Proteomic Profile in Patients with
Inflammatory Bowel Disease, its Correlation with Diagnosis
and Disease Activity
Salah El-Din Ahmed Badr El-Din, Ezzat Ali Ahmed, Pacint El-
Saed Moez, Mohamed Eid Ibrahim, Doaa Abdou Mohamed
Header
---------------------------------------------- Original Article
Use of Early Lactate Clearance as a Predictor of Mortality
Rate after Initial Resuscitation in Patients with Severe
Sepsis or Septic Shock
Mohammed Mostafa Megahed, Dalia Abd Elmoaty, Haitham
Tammam, Islam Ahmed Saadallaah
---------------------------------------------- Case Report
Dunbar Syndrome (Median Arcuate Ligament Syndrome);
Case report
MY Taher M Rashed, Alexandria University HPB Unit, Egypt
----------------------------------------------
2
15
21
54
28
46
38
60
65
80
74
Original Article
Analysis of Etiologies and Outcome of Cases of Fever of Unknown (FUO)
Origin Admitted to Alexandria Fever Hospital
Mohiedeen KM1, El Kady A1, Elwazzan D1, Helmy N2; 1Department of tropical medicine, Faculty
of Medicine, Alexandria University, 2Alexandria GIT, Hepatology and Fever hospital
ABSTRACT Fever of unknown origin (FUO) is one of the most challenging diagnostic dilemmas in the field of infectious
diseases and tropical medicine. Petersdorf and Beeson defined FUO in 1961 as a temperature higher than
38.3°C on several occasions and lasting longer than 3 weeks, with a diagnosis that remains uncertain after
one week of investigation. Different definitions have been put forward to describe the difference in length of
diagnostic workup taking into account the outpatient setting. Aim of the work : The aim of this work was to
analyze final diagnosis and outcome of cases of FUO admitted to Alexandria Fever Hospital during the
period from 2014 to 2015 and to review the medical records of these cases in order to evaluate various
diagnostic approaches and to identify the extent of fulfillment of the basic diagnostic workup done for each
patient. Material and methods: The total number of cases included in this study was 275 cases, about half
of them that included did not satisfy the definition of FUO. Also 37 cases left the hospital upon their request
so they couldn't be reported. After the application of duration of fever according to the old definition, 105
cases only were selected to be focused in this study. In this study old definition was selected to give better
results. Basic investigations and procedures done during patient's stay in the hospital; Complete Blood Count
(CBC), Erythrocytes Sedimentation Rate (ESR), Anti Streptolysin O Titer (ASOT), C Reactive Protein
(CRP), serum Bilirubin, blood Sugar, blood Culture, urine analysis, stool examination, kidney functions
(serum urea and creatinine), liver enzymes (AST and ALT), ELISA for Brucella, widal test for typhoid,
Chest x-ray and pelvi-abdominal US. All of the previous investigations are routine procedures done for all
patients. Sputum culture, lumbar Puncture, tuberculin Test, Anti-Nuclear Antibodies (ANA), Rheumatoid
Factor (RF), Electrocardiogram (ECG), ELISA for Hepatitis A,B,C and HIV Viruses and other
investigations as Cytomegalo- virus (CMV) IgM , Epstien Barr virus (EPV) IgM, leptospira IgM, AFP, CT
chest, abdomen and pelvis, CT brain and thyroid and other miscellaneous investigations done for some of
the cases according to their complain and history. Results: In this study, diagnoses were grouped into 4
major categories. 79% of cases were diagnosed with an infectious disease, 10.5% of cases were diagnosed
with malignancies, 8.5% were diagnosed with an autoimmune disease, 2.0% of cases were diagnosed with
miscellaneous conditions. Conclusion: infectious diseases are still the most frequent cause of FUO in
Alexandria, Egypt, followed by malignant diseases.
Introduction
FUO is one of the most challenging
diagnostic dilemmas in the field of infectious
diseases and tropical medicine. Fever is a
cardinal manifestation of many diseases,
including both infectious and non-infectious
diseases. Petersdorf and Beeson.(1) defined
FUO in 1961 as a temperature higher than
38.3°C on several occasions and lasting
longer than 3 weeks, with a diagnosis that
remains uncertain after one week of
investigation.(1) Durack et al(2). have argued
for a more comprehensive definition of FUO
that takes into account medical advances and
changes in disease states, such as the
emergence of HIV infection and an
increasing number of patients with
neutropenia (2). The new definition proposed
in addition to the old definition criteria, to
include patients who are undiagnosed after
two outpatient visits within one week or
three days in hospital(2). Demographic and
geographic considerations need to be
factored into the diagnostic approach to
avoid needless or misdirected diagnostic
testing. With FUO patients, there are almost
always one or, more clues from the history
and physical examination or nonspecific
laboratory tests that suggest a disease
category in general, or more specifically, a
number of diagnostic possibilities.(2-5)
Geographic location has a major influence
on the distribution of the causes of FUO. For
example, visceral leishmaniasis in endemic
areas is a major diagnostic consideration
with FUO, whereas in non-endemic areas,
visceral leishmaniasis should not be
considered in the differential diagnosis of
FUO in HIV patients.(1) In the Mediterranean
area, adults’ infections (40% of cases) and
cancer (25% of cases) account for most of
FUO. Autoimmune disorders account for 10-
20% of cases, others (drugs, factitious, etc.)
account for 10% of cases and 10% of cases
remain undiagnosed.(6) While in children; 30-
70% of cases are due to infections, 5-10%
cancer and autoimmune disorders account for
10-20%. In Saudi Arabia, Infectious diseases,
especially TB, continue to be the leading
etiology of FUO.(4-5) In Egypt, Infections were
the commonest cause of FUO (41.94%)
followed by malignancies (30.11%). While
autoimmune diseases represented 15.05%
and in 12.9% of patients the diagnosis was
not established.( 5 ) Brucellosis and infective
endocarditis were the commonest infections,
while hematological malignancies were the
commonest oncological diseases and SLE
was the commonest auto-immune disease.
The authors recommended that Brucellosis,
infective endocarditis, hematological
malignancies and SLE must be considered in
the differential diagnosis of adult FUO in
Egypt.(6 )
Subjects and Methods All patients recordes were revised according
to demographic characteristics including;
age, gender, residence, occupation and
marital status. Risk factors and common
medical features presented in the patients of
FUO at the time of admission including: -
Myalgia, headache, lack of concentration,
rigor, back pain, sweating, arthralgia, rigor,
diarrhea, vomiting, burning micturition,
difficulty of breathing, loss of weight and
any other complaint not mentioned above. -
Items related to medical history including
number of febrile days before admission,
pregnancy, smoking, drug abuse or
addiction, travelling abroad, history of
jaundice, contact with animals, contact with
birds and any other relevant medical history
not mentioned above. - Associated medical
conditions including diabetes mellitus,
Hypertension, Bronchial Asthma, COPD,
Hepatitis B and C, HIV, Malignancy or other
associated medical condition. - General
examination of the patient at the time of
admission including vital signs; blood
pressure, respiratory rate, temperature in
Celsius and pulse; and common findings in
general examination of the patient like
general appearance, consciousness, icteric
sclera, cyanosis, clubbing, lower limb
odema, joint deformity, muscle or bone
deformity and the presence of rash. - Local
examination including head and neck, chest,
cardiac and abdominal examination. -
Assessment of fever pattern during patient's
stay in the hospital. - Empirical therapy with
antimicrobial drugs used were; Penicillin,
Cephalosporin, Quinolones, Amino-
glycosides, Macrolides, Tetracycline,
Vancomycine, antibiotics specific for TB,
Antiviral drugs and Antifungal drugs. - Basic
investigations and procedures done during
patient's stay in the hospital; - Routine lab
investigations; CBC, ESR, ASOT, C
Reactive Protein (CRP), serum Bilirubin,
blood Sugar, blood Culture, urine analysis,
stool examination, kidney functions (Serum
Urea and Creatinine), liver enzymes (AST
and ALT), ELISA for Brucella, Widal test
for typhoid. - Other specific investigations;
Sputum Culture, Lumbar Puncture,
Tuberculin Test, ANA, RF, ECG, ELISA for
Hepatitis A,B,C and HIV Viruses and other
investigation as CMV IgM, EPV IgM,
leptospira IgM, AFP and so on. -
Radiological investigations; Chest x-ray,
pelvi-abdominal US, CT chest, abdomen and
pelvis. Outcome and diagnosis includes data
about the fate of the patient such as; the
duration of hospitalization, mode of
discharge whether cured, died, transferred or
discharged at patient's request.
Statistical Analysis Statistical analyses were performed using the
Statistical Package for Social Science (SPSS)
version 18 (LEAD Technology Inc). Data
were presented as means with corresponding
standard error (SE). Comparisons among
different groups were performed by one way
analysis of variance (ANOVA). Qualitative
data were described using number and
percent and association between categorical
variables was tested using Chi-square test.
Correlation between variables was
determined using Pearson's or Spearman's
correlation test according to the variable. In
all tests, the level of significance was < 0.05.
Results
Section I: Demographic data of the patients.
Regarding to the age of total 105 patients
involved in this study, the age ranged from 1
year to 70 years. The age distribution of the
study population;69.5%of cases were adults,
14.3% were in adolescence period,11.4%were
children, old age were 2.9% and infants were
1.9% (Table 1).
Table (1): Age distribution among cases of FUO admitted to Alexandria fever hospital from 2014 to 2015 (n= 105) No. %
Age (years)
Infants (1 year) 2 1.9
Children ( > 1 - 9 ) 12 11.4
Adolescents (10 - 18 ) 15 14.3
Adults (19 - 65) 73 69.5
Elderly (>65) 3 2.9
As regards to the gender, occupation,
residence and marital status; table (2)
showed that 69.5% of the study population
were males and 30.5% were females. Of the
32 females included in the study, 18.75% of
them were pregnant. Occupations were
grouped into 2 major categories. These
categories were, non-workers including
children, Students, infants and house wives
(46.7%). Workers included bakers, butchers,
drivers, employee, farmers, garbage cans,
manual workers, teachers and medical rip
(53.3%). Residence was categorized into
urban (40.0%) and rural (60.0%). 43.8% of
the study population were married, 28.6%
were single, 27.6% were of under age of
marriage.
Table (2): Demographic criteria among cases of FUO.
No. %
Gender
Male 73 69.5
Female 32 30.5
Not pregnant 26 81.25
Pregnant 6 18.75
Occupation
Non worker 49 46.7
Infant 2 1.9
Child 11 10.5
Student 21 20.0
House wife 15 14.3
Worker 56 53.3
Baker 1 1.0
Butcher 2 1.9
Driver 2 1.9
Employee 14 13.3
Farmer 10 9.5
Sewer worker 2 1.9
Laborer 22 20.9
Teacher 2 1.9
Medical rep 1 1.0
Residence
Urban 42 40.0
Rural 63 60.0
Marital status
Married 46 43.8
Single 30 28.6
Under age 29 27.6
Section II: Risk factors and common medical
features in cases with FUO. A. Risk factors:
Smoking was detected in 43.8%, history of
jaundice in 39%, contact with animals in
26.7%and bronchial asthma in 26.7%,
travelling abroad in 21.9%, addiction in
20%,contact with birds in 17.1%, COPD in
16.2%, DM in 10.5%, hypertension in 8.6%,
HCV in 7.6%, HBV in 6.7%, pregnancy in
5.7%, HIV in 2.9%, while history of
malignancy was not reported. Figure (1)
0
5
10
15
20
25
30
35
40
45
50S
mo
kin
g
His
tory
of
jau
nd
ice
Bro
nch
ial a
sth
ma
Co
nta
ct w
ith
an
ima
ls
Tra
vel
ling
ab
roa
d
Ad
dic
tio
n
Co
nta
ct w
ith
bir
ds
CO
PD
Dia
bet
es m
ellit
us
Hy
per
ten
sio
n
HC
V
HB
V
Pre
gna
ncy
HIV
Ma
lign
an
cy
43.8
39.0
26.7 26.7
21.920.0
17.1 16.2
10.58.6
7.6 6.7 5.72.9
0.0
Per
cen
tag
e
Figure (1): Distribution of studied cases according to risk factors (n=105).
B. Clinical features of patients with FUO. :
1) Symptoms: In all cases admitted to
hospital the main complaint was fever for
long period more than 3 weeks, with other
complaint. 66.7% of patients involved in this
study complained from headache, 65.7%
complained from rigors, 63.8% complained
from sweating, 61.9% complained from
difficulty of breathing, 52% complained
from myalgia, 43.8% complained from
arthritis,40% complained from vomiting,
loss of weight in 35.2%, diarrhea in 32%,
burning micturition in 26.7% and lack of
concentration in 10.5%. No rash was
detected in the studied cases.
Table (3): Distribution of the studied cases according to presenting complaint No. %
Fever 105 100.0
Headache 70 66.7
Rigor 69 65.7
Sweating 67 63.8
Difficulty of breathing 65 61.9
Myalgia 55 52.4
Arthritis 46 43.8
Vomiting 42 40.0
Loss of weight 37 35.2
Diarrhea 32 30.5
Burning micturition 28 26.7
Lack of concentration 11 10.5
2) General examination: General
examination of patients involved in this
study revealed that most of them were
looking ill, conscious, non- icteric, not
cyanosed, without clubbing or lower limb
edema but coated tongue was detected in
62.9%, congested throat in 55.2% and
palpable lymph nodes in 55.2%. Table (4)
Table (4): Distribution of the studied cases of FUO according to general examination (n=105).
No. %
General appearance
Ill 49 46.7
Pale 17 16.2
Toxic 11 10.5
Pale ill 27 25.7
Pale ill toxic 1 1.0
Consciousness
Normal 101 96.2
Drowsy 3 2.9
Comatosed 1 1.0
Icteric sclera
No 91 86.7
Yes 14 13.3
Cyanosis
No 103 98.1
Yes 2 1.9
Clubbing
No 103 98.1
Yes 2 1.9
Lower limb edema
No 92 87.6
Yes 13 12.4
Tongue
Normal 39 37.1
Coated 66 62.9
Throat
Normal 47 44.8
Congested 58 55.2
Lymph Nodes
Not felt 47 44.8
Felt 58 55.2
Blood pressure
Systolic (85-180) 121.71 ± 35.93
Diastolic (45-110) 75.95 ± 24.98
Pulse rate beat/minute (70-110) 95.89 ± 11.11
Respiratory rate (15-31) 23.69 ± 7.83
Temperature in Celsius
Min. – Max. 38.30 – 40.0
Mean ± SD. 38.35 ± 0.20
Median 38.30
Section III: Empirical treatment and
antimicrobial drug selection: Regarding
empirical treatment used for the patients;
48.6% of cases received Penicillin or one of
its derivatives during their stay in the
hospital, followed by cephalosporin's
(57.1%), antiviral (31.4%), quinolones
(18.1%), corticosteroids (15.2%), drugs for
TB (12.4%), tetracycline's (10.5%),
macrolides (6.7%), antifungal (5.7%) and
vancomycine (2.9%),metronidazole was not
used in the study population (Fig 2).
0
10
20
30
40
50
60
Cep
halo
spor
in's
Peni
cilli
n
Ant
ivir
al
Qui
nolo
nes
Cor
ticos
tero
ids
Dru
gs f
or T
B
Tet
racy
clin
e's
Mac
rolid
es
Ant
ifun
gal
Van
com
ycin
e
57.1
48.6
31.4
18.115.2
12.410.5
6.75.7
2.9
Per
cen
tage
Figure (2): Distribution of the studied cases of FUO according to empirical treatment (n=105).
Section IV: Investigations done for the
patients: According to routine lab
investigations CBC,ESR, ASOT, CRP, urine
analysis, kidney functions, liver enzymes,
serum bilirubin, Elisa for Brucella, widal for
typhoid, blood sugar and blood culture, chest
X ray and pelvi-abdominal US done for all
patients. Stool analysis done for19.1%, CT
abdomen and pelvis in15.2%, lumber
puncture in 14.3%, CT chest in 14.3%,
leptospira IgM in 14.3%, RF 10.5%, ECG
9.5%, tuberculin test 6.7%, ANA 6.7% and
other investigations in 19% (Fig 3).
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
19.1%
19%
15.2%
14.3%
14.3%
14.3%
10.5%
9.5%
6.7%
6.7%
0 20 40 60 80 100 120
CBC
ESR
Urine examination
ASOT
CRP
Elisa for brucella
Blood sugar
Widal test for typhoid
Blood culture
Chest X ray
Ultra sound
Serum bilirubin
Kidney functions
Liver enzyme SGOT
Liver enzyme SGPT
Stool examination
Others
CT abdomen pelvis
Lumber Puncture
CT chest
leptospira IgM
RF
ECG
Tuberculin test
ANA
Basic Investigations
Figure (3): Investigations done for cases of FUO.
Section V: Diagnosis and outcome:
Regarding the duration of hospitalization,
47.6% of cases admitted to Alexandria fever
hospital less than one week, 30.5% admitted
for one week and 21.9% admitted more than
one week (Table 5) . According to type of
discharge and outcome; 67.6% were good at
the time of discharge, 28.6% were
transferred and 3.8% died (Table 5).
Table (5): Distribution of the studied cases according to duration of hospitalization,
discharge type and outcome (n=105).
No. %
Duration of hospitalization (weeks)
<1 week (5-6) days 50 47.6
One week 32 30.5
> One week 23 21.9
Discharge type and outcome
Died 4 3.8
Good 71 67.6
Transferred 30 28.6
Table (6) showed that infectious causes
represented 79% of different causes of FUO,
malignant causes were 10.5%, connective
tissue diseases were 8.57% and
miscellaneous causes were 2.0%.
Table (6): Distribution of the studied cases of FUO according to diagnosis and outcome (n=105).
No. %
Infections 83 79.04
UTI 8 7.6
Typhoid fever with UTI 2 2.0
UTI with DM 1 1.0
UTI with renal impairment 1 1.0
UTI with pregnancy 1 1.0
Total UTI 13 12.38
Respiratory tract infection 36 34.28
Acute bronchitis 24 22.9
Bronchopneumonia 1 1.0
URTI, ear effusion 1 1.0
Pneumonia 8 7.6
URTI 1 1.0
URTI with otitis media 1 1.0
Abscess 3 2.85
Psoas muscle abscess 1 1.0
RT femur abscess 1 1.0
Splenic abscess 1 1.0
HIV 3 2.9
Toxoplasmosis encephalitis with HIV 1 1.0
Encephalitis with HIV 1 1.0
Acute renal failure in HIV 1 1.0
Total HIV 6 5.6
Acute viral hepatitis A 1 1.0
Brucellosis 7 6.7
TB 5 4.76
CMV infection 1 1.0
EPV infection 2 2.0
Leptospirosis 5 4.8
Typhoid fever 2 1.9
Encephalitis 2 1.9
Malignancies 11 10.5
Renal carcinoma 1 1.0
Prostatic neoplasm 1 1.0
Uterine neoplasm 1 1.0
Liver metastasis 1 1.0
Pulmonary carcinoma 1 1.0
HCC 3 2.9
??Lymphoma 3 2.9
Connective tissue and autoimmune diseases 9 8.57
??SLE 1 1.0
?? rheumatoid arthritis 2 1.9
??Auto immune hemolytic anemia 1 1.0
Rheumatic fever 1 1.0
Auto immune hepatitis 1 1.0
Sarcoidosis 1 1.0
FMF 2 1.9
Miscellaneous 2 1.9
??Goiter 1 1.0
??Chronic appendisitis 1 1.0
?? Quarry diagnosis
Section VI: Correlations between the final
diagnosis & different demographic, medical
variables and risk of smoking. 1. Correlation
between final diagnosis and age: PUO in the
two infants who were included in this study
were diagnosed as infectious causes, children
had 10 cases with infectious cause from the
total 12 cases, one case was malignant cause
and the other case was due to connective
tissue disease. Infections represented
11.43%, connective tissue diseases 2.86% in
adolescence period. In adults; infections
were found in 55.2%, malignancies
represented 7.6%, connective tissue diseases
were 4.8%, and 1.9% represented
miscellaneous causes. Regarding elderly
included in the this study; 1 case had
infectious cause and the 2 other cases had
malignant causes (Fig 4).
0.1
1
10
100
Infant Children Adolescence Adulthood Elderly
1.9
9.5211.43
57.14
0.95
0.0
0.95
0.0
7.62
1.90
0.0
0.95
2.86
4.76
0.00.0 0.0 0.0
1.9
0.0
Per
cen
tag
e
The causes of the diseases
Infections
Malignancies
Connective tissue diseases
Miscellaneous
Figure (4): Correlation between different causes of FUO& age groups.
2. Correlation between final diagnosis and
sex: Table (7) showed the correlation
between sex &causes of FUO; infectious
causes were found in 80.8% of males'
patients, 12.3% malignant causes, 4.1% were
connective tissue diseases and 2.8% due to
miscellaneous causes from the total number
of males. Regarding females, infectious
causes represented 75%, 6.3% malignant
causes, 18.75% connective tissue diseases
from the total number of females.
Table (7): Correlation between the studied cases according to diagnosis and sex (n=105)
Male
(n = 73)
Female
( n = 32)
No. % No. %
Infections 59 80.8 24 75.0
UTI 4 5.5 4 12.5
Typhoid fever with UTI 1 1.4 1 3.1
UTI with DM 1 1.4 0 0.0
UTI with renal impairment 1 1.4 0 0.0
UTI with pregnancy 0 0.0 1 3.1
Respiratory tract infection
Acute bronchitis 17 23.3 7 21.9
Bronchopneumonia 1 1.4 0 0.0
URTI, ear effusion 1 1.4 0 0.0
Pneumonia 6 8.2 2 6.3
URTI 1 1.4 0 0.0
URTI with otitis media 0 0.0 1 3.1
Abscess
Psoas muscle abscess 1 1.4 0 0.0
RT femur abscess 0 0.0 1 3.1
Splenic abscess 1 1.4 0 0.0
HIV 2 2.7 1 3.1
Toxoplasmosis encephalitis with HIV 1 1.4 0 0.0
Encephalitis with HIV 1 1.4 0 0.0
Acute renal failure in HIV 1 1.4 0 0.0
Acute viral hepatitis A 1 1.4 0 0.0
Brucellosis 3 4.1 4 12.5
TB 4 5.5 1 3.1
CMV infection 1 1.4 0 0.0
EPV infection 2 2.7 0 0.0
Leptospirosis 4 5.5 1 3.1
Typhoid fever 2 2.7 0 0.0
Encephalitis 2 2.7 0 0.0
Malignancies 9 12.3 2 6.3
Renal carcinoma 1 1.4 0 0.0
Prostatic neoplasm 1 1.4 0 0.0
Uterine neoplasm 0 0.0 1 3.1
Liver metastasis 1 1.4 0 0.0
Pulmonary carcinoma 1 1.4 0 0.0
HCC 2 2.7 1 3.1
Lymphoma 3 4.1 0 0.0
Connective tissue and autoimmune diseases 3 4.1 6 18.75
SLE 0 0.0 1 3.1
Rheumatoid arthritis 0 0.0 2 6.3
Auto immune hemolytic anemia 0 0.0 1 3.1
Auto immune hepatitis 1 1.4 0 0.0
Rheumatic fever 1 1.4 0 0.0
Sarcoidosis 1 1.4 0 0.0
FMF 0 0.0 2 6.3
Miscellaneous 2 2.8 0 0.0
Goiter 1 1.4 0 0.0
Chronic appendicitis 1 1.4 0 0.0
3. Correlation between final diagnosis and
smoking. According to smoking, 80.4% of
smokers had FUO diagnosed as infectious
cause , chest infection was prominent cause
representing around half the causes in
smokers cases . Regarding non –smokers,
chest infection represented less than half of
cases for example pulmonary TB was found
in 3 smokers cases and was detected in only
one non-smoker case.All HIV infected cases
were smokers (Table 8).
Table (8): Distribution of infectious causes in smokers and non-smokers
Smokers
(infections)
(n = 46)
Non Smokers
(infections)
(n =59)
No. % No. %
UTI 4 8.8 7 11.4
UTI with Typhoid fever Respiratory 1 2.2 1 2.2
Acute bronchitis 9 19.6 15 25.4
Pulmonary TB 3 6.5 1 1.7
Intestinal TB 0 0.0 1 1.7
Bronchopneumonia
Pneumonia
0
5
0.0
10.9
1
3 1.7
5.1
CMV infection 1 2.2 0 0.0
Typhoid fever 0 0.0 2 3.4
EPV infection 2 4.4 0 0.0
leptospirosis 3 6.5 2 3.4
brucellosis 2 4.3 5 8.5
toxoplasmosis encephalitis with HIV 1 2.2 0 0.0
encephalitis with HIV 1 2.2 0 0.0
Encephalitis 0 0.0 2 3.4
HIV 3 6.5 0 0.0
Splenic abscess 1 2.2 0 0.0
psoas muscle abscess 0 0.0 1 1.7
RT femur abscess 0 0.0 1 1.7
acute renal failure in HIV 1 2.2 0 0.0
acute viral hepatitis A 0 0.0 1 1.7
URTI 0 0.0 3 5.1
Total 37 80.4 46 78.0
Discussion
Fever is a cardinal manifestation of many
diseases, including both infectious and non-
infectious diseases, many authors suggested
that FUO at the present time should signify
prolonged fevers with temperatures of at
least 38.3°C, which remain undiagnosed
after a focused and appropriate laboratory
workup.(3-5) The age distribution in this study
can be attributed to the demographic
composition of Egypt, where a major
proportion of the population are from 1-65
years old. Which means all age groups of
patients in this study. Most studies
concentrates on special sub group of cases. A
study in France set the inclusion criteria to
adults older than 19 years of age,50.7% of
patients were females and 49.3% were males
.(7) Another study in USA concentrated on
the FUO in children(8). In the current work,
all patients were complained from fever,
66.7% complained from headache,
rigor65.7%, sweating 63.8%, difficulty in
breathing 65.7%, myalgia 52.4%, arthritis
43.8%, vomiting 40%, loss of weight 35.2%,
diarrhea30.5%, burning micturition 26.7%
and lack of concentration 10.5%. No rash
detected in this study population. In a
research done in Turkey, The most common
symptoms were fever (100%), fatigue (80%),
chills (67%), weight loss (62%), myalgia
(44%), and arthralgia (4%). Weakness and
arthralgia had observed significantly. (9) In
another study, in Iran, the most common
complaints were generalized weakness with
frequency of micturition 59.5%. Abdominal
pain came next with 27.4% of cases followed
by back pain (22.8%), sweating (21.4%),
headache (19%), arthritis (17.9%), rigor
(8.3%) and finally cough with 7.1% .(10 ) In
this research, 36.2% of cases had decreased
air entry, 3.8% had crepitation, 30.5% had
wheezes and 6.7% of cases had hemoptysis.
84.7% of cases had normal ranges of blood
pressure, 37.2% with tachycardia, 8 cases
were with abnormal heart sounds and 2 cases
had murmurs. 21% of cases presented with
enlarged liver, 11.4% of cases presented with
enlarged spleen, 5.7% of cases presented
with abdominal ascites. 9.5% of cases had
abnormal motor power but no cases were
reported with abnormal peripheral sensations
or had abnormal psychological status. 1.9 %
of cases had other neurological
manifestations like neck rigidity, 3.8%
abnormal reflexes, 1% positive Kerning's
sign and no case with positive Brudzinsky's
Sign reported. In another study in Iran,
Lymphadenitis was reported in 4% of cases,
cardiac murmurs were reported in 4.8% of
cases. Hepatosplenomegaly was reported and
required laparotomy to reach a diagnosis.
Abnormal chest findings were also reported
in many case series and usually attributed to
pneumonia or bronchitis with abnormal
presentation that is difficult to detect
especially in childhood age. Abnormal
sensations were found in 24% of cases in this
study mostly are attributed to diabetes
mellitus, which was reported in of the study
population. Other neurological findings
usually steer the physicians towards CNS
infections like encephalitis and meningitis.(10)
In this study, 57.1% of cases received
cephalosporin, followed by penicillin or one
of its derivatives during their stay in the
hospital (48.6%), 31.4% received antiviral
drugs, 18.1% received quinolones, 15.2%
received corticosteroids, 12.4% received
rifampicin and anti-TB drugs, 10.5%
received tetracycline group, 6.7% received
macrolides, 5.7% received antifungal and
2.9% received vancomycine. metronidazole
was not used in any of the study population.
In a recent study done in Egyptian university
hospital, 90.6% of cases received penicillin
or one of its derivatives during their stay in
the hospital, followed by cephalosporin
(50.8%). 36.6% received quinolones, 28.5%
received sulfa drugs, 19.6% received
aminoglycosides, 10.8% received
metronidazole, 8.8% received tetracycline
group, 8.8% received Rifampicin and anti-
TB drugs, 0.9% received antifungals, 0.9%
received antivirals and 0.2% received
macrolides group. Vancomycine was not
used in this study.(11) In the current work,
basic investigations were done for all
patients, which are CBC, ESR, CRP, ASOT,
measure of blood Sugar, urine analysis, Elisa
for Brucella , widal for typhoid , blood
culture, liver enzymes , kidney functions
and serum bilirubin. Chest x-ray, Abdominal
Ultrasound also were done for all cases. 19%
of cases had been examined for stool, 19% of
cases had Sputum Culture. 14.3% of cases
had Lumbar Puncture for Cerebrospinal
Fluid (CSF) examination, 6.7% had
tuberculin test, 9.5% of cases had ECG.
32.4% were tested for HAV, 21.9% HBV,
19% HCV, 9.5% HIV antibodies. 6.7 % of
patients had tested for ANA, 10.5% RF.
About 14.3% of cases had tested for
leptospira IgM, 19% of cases required other
investigations e.g CMV IgM, urine culture,
H1N1nasopharyngeal swab and so on. In a
research done in Saudi Arabia, the frequency
of using imaging and procedural techniques
were as following; Chest X-ray 96.9%, CT
chest, abdomen and pelvis 78.5%, US
abdomen 69.4%, BM aspirate/biopsy 59.2%,
Echocardiogram 51.0%,Tissue biopsy
49.0%, US pelvis 35.7%, FNA 34.7%, Fluid
aspirate 23.5%, CT-PET whole body 19.4%,
Upper endoscopy 19.4%, Bronchoscopy
16.3%, MRI chest, abdomen, pelvis 12.2%,
Colonoscopy 8.2%, Lumbar puncture 7.1%,
Laparoscopy 5.1% and Sigmoidoscopy
3.1%.(12) In this study, Duration of
hospitalization was about more or less than
one week. 47.6% of cases had been
hospitalized for less than 1 week. 30.5% of
cases stayed for one week in the hospital.
Only 21.9% required hospitalization for
more than one week. In a study in Turkey,
The mean interval between admission and
diagnosis was 32 ± 18 days (range 4 -90
days).(9) In the current research, 67.6% of
cases were discharged as improved cases.
28.6% of cases were transferred to more
specialized health care facility as university
hospitals. 3.8% died in the hospital. 91.4% of
cases were diagnosed appropriately, where
8.6% defied diagnosis. In a study in Turkey,
the number of undiagnosed cases reached
15.6% of cases (9). In a study in Iran, 16.7%
of cases remained undiagnosed (10). In
Netherland, in a multicenter study, the
number of undiagnosed cases reached 50%
of cases (13). This big difference between this
study and other studies, may be attributed to
the local socioeconomic status in Egypt,
where physicians may be rushed to reach a
probable final diagnosis even if it is
inaccurate and may be unsupported by
laboratory investigations and imaging
technique(14). This may be better visualized
taking into consideration that almost 13% of
patients were discharged at their request. In
this study, diagnoses were grouped into 4
major categories. 79% of cases were
diagnosed with an infectious disease, 10.5%
of cases were diagnosed with malignancies,
8.5% were diagnosed with an autoimmune
disease, 2.0% of cases were diagnosed with
miscellaneous conditions. In a study from
Netherland, infectious diseases were the cause
of the fever in 12 patients 17%, a neoplasm in
five patients 7%, noninfectious inflammatory
diseases in 16 patients 23%, metabolic
disorder within one case and drug fever in one
patient each. In 35 patients (50%), the cause
of the fever was not found. (15) In another
study, Out of 100 FUO patients, 50% were
found to have infectious diseases, 24% were
found to have connective tissue diseases, 8%
miscellaneous causes and 7% neoplastic
diseases. In 11 patients no definite cause for
FUO could be identified. Connective tissue
patients were: systemic lupus (33.3%),
Familial Mediterranean fever (20.8%),
rheumatoid arthritis (16.6%), Still’s disease
(12.5%) , Rheumatic fever and Behçet
syndrome/Crohn’s disease (4.3%).(16) In the
current work, the distribution of diagnoses
between genders showed that there is high
incidence of infections in both males and
females, among infections; HIV and TB
were common in males and rare in females,
also leptospirosis was prominent in males
because females less exposed to working as
sewer workers and farmers according to
distribution of final diagnosis. In this study,
females exceeded males in incidence rates of
autoimmune diseases. This difference
demonstrates that males having autoimmune
disease are misdiagnosed by the physician
more than females (17). As physician may
suspect the presence of an autoimmune
condition in females more than males, so
many males remain undiagnosed. (18)
Conclusion
Infectious diseases are still the most frequent
cause of FUO in Alexandria, Egypt,
followed by malignant diseases.
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PH. Fever of Unknown Origin: An Evidence-
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Original Article
Cardiovascular Risk Assessment in Hemodialysis Patients: Relation to
Malnutrition, Inflammation and Body Fluid Determined by Bioelectrical
Impedance
Essam El Din Hassan El Kashef1, Sameh Morsi Arab2, Yasmine Salah Naga1, Shaimaa Elsayed
Mohamed Mohamed3, Montasser Mohammed Hussein Zeid1; 1Department of internal medicine and
nephrology, Alexandria University , Egypt, 2Department of Cardiology and Angiology, Alexandria
University, Egypt.
ABSTRACT Cardiovascular disease prevalence increases as the renal function declines across the spectrum of chronic
kidney disease. In end-stage renal disease cardiovascular mortality may even account for 50 % of mortality.
The co-existence of malnutrition, inflammation and atherosclerosis (the so-called MIA syndrome) has been
observed in hemodialysis (HD) patients and is considered one of the cardiovascular risk factors unique to this
population. In addition, chronic fluid overload is frequently present in HD patients. Aim of the work : The aim
of the present study was to investigate the relationship between nutrition, inflammation, atherosclerosis, the
presence of overhydration measured by multi-frequency bioimpedance analysis (m-BIA) and the cardiac
condition as assessed by echocardiography on the other hand in HD patients. Material and methods: Thirty
HD patients (mean age 41.13 ± 12.77 years, 15 were males and 15 were females) were enrolled in the dialysis
unit of the Alexandria Main University Hospital. Serum albumin was used as a nutritional marker, and serum
C-reactive protein (CRP) was used as an inflammatory marker. Doppler ultrasonography was performed to
measure the carotid artery intima-media thickness (CA-IMT) was used to assess the presence of
atherosclerosis. Extracellular water (ECW), overhydartion (OH) and OH/ECW were measured by m-BIA to
detect overhydration.. Cardiac condition was determined by echocardiographic measurement of the left
ventricular mass index (LVMI) and ejection fraction (EF). Result: Only two (6.6%) of the studied patients
have a low albumin, 26 patients (86.6%) had a positive CRP, 23 patients (76.6%) had high CIMT and 9
patients (30%) had atherosclerotic plaques in the carotid artery. Bioimpedance showed overhydration in 15
patients (50%), who had an OH/ECW ratio of > 0.15. Echocardiography showed high LVMI in 55.5% and low
EF in 16.6% . The presence of inflammation as indicated by CRP was associated with higher CIMT (r=0.520,
p=0.003), higher interventricular wall thickness (r=0.469, p=0.007) and lower EF (r=-0.610, p=<0.001).
Overhydration as assessed by OH/ECW was associated with higher CRP (r=0.553, p=0.002), higher CIMT
(r=0.655, p=<0.001) and lower EF (r=-0.742, p=<0.001). Conclusion: Inflammation, atherosclerosis and fluid
overload are prevalent in ESRD patients on MHD. Both inflammation and overhydration are correlated with
increase CIMT and low EF implicating them in the cardiovascular diseases commonly found in ESRD patients
Introduction
In patients with chronic kidney disease, the
cardiovascular risk and mortality increase as
the GFR decreases due to multiple traditional
and non-traditional risk factors. (1) The risk is
highest in end-stage renal disease (ESRD)
patients on maintenance hemodialysis (HD), at
which stage cardiovascular disease (CVD) may
be responsible for as much as 50% of
mortality. (2) The 1-year mortality may even
rise to over 90% following a myocardial
infarction (MI) in the dialysis population.(3)
The co-existence of malnutrition,
inflammation and atherosclerosis has been
observed in HD patients and has been
collectively referred to as the MIA
syndrome. The MIA syndrome is considered
one of the main risk factors for mortality in
ESRD patients.(4) In particular,
inflammation, as indicated by serum C-
reactive protein (CRP) levels, remains an
integral risk factor for peripheral
atherosclerosis and cardiac ischemia. (5)
Another unique cardiovascular risk factor in the
dialysis population is chronic fluid overload
(FO).(6) Fluid overload can lead to hypertension
as well as cardiac volume and pressure
overload, which all contribute to the
development of cardiovascular disease,
including left ventricular hypertrophy, heart
failure, and pulmonary edema.(7,8) On the other
hand, chronic volume deficit can lead to
intradialytic hypotension, muscle cramps, and
shock.(9) Therefore, good fluid balance is
essential in dialysis patients to achieve ideal
blood pressure control and cardiac health. The
first step to reach that goal is to accurately assess
the hydration state of the patients. Despite its
importance, patients’ volume status is most
commonly clinically evaluated. Inter-dialytic
weight changes, the degree of edema and
blood pressure are the most frequently used
markers of hydration status in dialysis units,
but they are largely unreliable. (10) An
objective tool to measure body fluid
composition is the multi-frequency
bioelectrical impedance analysis technique
(m-BIA). This inexpensive, non-invasive
method can accurately determine the degree
of overhydration as well as the nutritional
status of patients. (11) The objective of the
present study was to examine how these
different factors affect each other and affect
the cardiac condition in hemodialysis
patients.
Patients and Methods After obtaining the approval of the ethics
committee of the Faculty of Medicine of the
Alexandria University, thirty ESRD patients
(50% male and 50% female) on maintenance
hemodialysis for at least six months were
recruited from the dialysis units of the
Alexandria University Hospitals. An
informed consent from the patients was
taken before conducting the study. Patients
with hypotension, known chronic
inflammatory disease including systemic
lupus erythematosis (SLE) and vasculitis,
malignancy, liver cirrhosis, gastrointestinal
diseases, history of a systemic infection
within one month before entry into the study
and patients with catheters as vascular access
were excluded. Data including name, age,
sex, past medical history, and data obtained
from clinical examination were recorded at
enrollment Laboratory investigations
included lipid profile, serum albumin and C-
reactive protein. To detect atherosclerosis, the
common carotid artery intima-media
thickness (CA-IMT) was assessed by using a
high-resolution color Doppler ultrasound unit.
After visualizing the double echogenic line of
the arterial wall, the CIMT was measured
including the inner echogenic line
representing the lumen–intima interface and
the adjacent hypoechoic and excluding the
outer echogenic line, which represents the
media–adventitia interface. M-mode
transthoracic echocardiography was performed
according to the recommendations of the
American Society of Echocardiography (11) just
before performing the m-BIA mainly to assess
the left ventricular mass index and ejection
fraction. Body fluid composition was assessed
using multi-frequency bioimpedance, which
relies on measuring the flow of current
through the body. (12) The flow of current
depends on the frequency applied. At low
frequencies, the current cannot bridge the
cellular membrane and will pass
predominantly through the extracellular
space. At higher frequencies, penetration of
the cell membrane occurs and the current is
conducted by both the extracellular water
(ECW) and intracellular water (ICW). The
impedance values were obtained at
frequencies of 5, 50, 100 and 200 kHz. The
following variables were measured: ECW,
OH and OH/ECW using a cut-off of > 0.15 as
an indicator of overhydration. (13,14) SPSS
Version 20.0 (Chicago, Illinois) was used for
statistical analysis. Data are presented as
mean ± standard deviation (SD). Proportions
were compared by Chi-square analysis.
Student t-test, analysis of variance and Mann–
Whitney tests were used for group
comparison. Correlation analysis was
performed using Spearman’s correlation
coefficient. A p-value of < 0.05 (two-sided)
was regarded as statistically significant.
Result
The study included 30 ESRD patients on
maintenance hemodialysis in the dialysis units
of the Alexandria University Hospitals. Fifty
percent of patients were males and 50% were
females. The studied variables are
summarized in Table 1. The mean age of the
patients was 41.13 ± 12.77 years. Total
cholesterol was high in only 3 patients (10%),
triglycerides were high in 16 patients (53.3%),
while the others had normal values. Serum
albumin as a marker of nutrition ranged from
3.40 – 4.90 g/dl with a mean of 4.03 ± 0.39
g/dl, with only two patients (6.6%) having
low serum albumin (<3.5g/dl). C-reactive
protein as a marker of inflammation ranged
from 1.05 – 47.54 with a mean of 12.64 ±
13.65. Twenty six patients (86.6%) had a
positive CRP. As for the lipid profile, total
cholesterol was high in only 3 patients (10%)
and triglycerides were high in16 (53.3 %).
The common carotid intima-media thickness
ranged from 0.10-0.95 with a mean of 0.70 ±
0.20 mm. Twenty-three patients (76.6%) had
increased CIMT. Carotid Doppler also showed
plaque in 30% of the examined patients. The
ejection fraction ranged from 35% to 87% with
a mean of 62.11 ± 12.80 %. Five patients
(16.7%) had low ejection fraction. LVMI
ranged from 63.0-178.0 with a mean of 118.40
± 31.49 g/m2, revealing that 20 patients
(66.7%) had left ventricular hypertrophy.
Bioimpedance measurements included the
extracellular water, which ranged from 10.70 –
62.30 with a mean of 20.23 ± 12.27 L, the
overhydration, which ranged from -2.70 – 6.0
with a mean of 2.10 ± 1.88 L, and the
OH/ECW ranged from -0.17–0.28 with a mean
of 0.11±0.10. According to the OH/ECW, 50
% of the patients are overhydrated at an
OH/ECW ratio of > 0.15.
Table 1: Summary of the data of the study subjects
Min. – Max. Mean ± SD. Median
Age (in years) 25.0 – 73.0 41.13 ± 12.77 40.0
Systolic blood pressure (mmHg) 100.0 – 200.0 140.0 ± 29.13 140.0
Diastolic blood pressure (mmHg) 70.0 – 110.0 83.67 ± 15.64 90.0
Cholesterol (mg/dl) 121.0 – 251.0 180.10 ± 27.92 181.0
Triglycerides (in mg/dl) 66.0 – 392.0 168.13 ± 79.93 160.0
LDL-cholesterol (in mg/dl) 31.0 – 153.0 108.29 ± 29.50 114.50
HDL-cholesterol (mg/dl) 23.0 – 69.0 41.40 ± 22.35 36.50
ALB (g/dl) 3.40 – 4.90 4.03 ± 0.39 4.0
CRP (mg/L) 1.05 – 47.54 12.64 ± 13.65 6.58
IVWT (cm) 1.03 – 10.0 1.65 ± 1.65 1.30
LVMI (g/m2) 63.0-178.0 138.40 ± 31.49 113.50
CIMT (mm) 0.10 – 0.95 0.70 ± 0.20 0.7
EF (%) 35.0 – 87.0 62.11 ± 12.80 62.0
ECW (L) 10.70 – 62.30 20.23 ± 12.27 17.30
OH (L) -2.70 – 6.0 2.10 ± 1.88 2.90
OH/ECW -0.17–0.28 0.11±0.10 0.13
Significant correlations: Due to the small
number of patients with low albumin (6.6
%), we could not analyze the effect of
malnutrition on inflammation, cardiovascular
disease and body composition. CRP was
positively correlated with IVWT (r=0.496,
p=0.007), CIMT (r=0.408, p=0.025, Figure 1),
and OH/ECW (r=0.553, p=0.002). It was
negatively correlated with EF (r=-0.610,
p=0.002) (Table 2).
Table 2: Significant correlations with CRP
CRP
r p
CIMT 0.408* 0.025*
IVWT 0.496* 0.007*
EF -0.610* <0.001*
LVMI -0.116 0.543
OH/ECW 0.553* 0.002*
There was a significant positive correlation
between CIMT (Table 3) and OH/ECW
(r=0.655, p=<0.001), ECW (r=0.392, p=0.032),
IVWT (r=0.499, p=0.007), and CRP (r=0.408,
p=0.025, Figure 1). Patients with carotid plaque
had significantly higher age (49.33 ± 11.65
versus 37.62 ± 11.78, p=0.018) and higher
LDL-C (127.67 ± 22.54 versus 100.0 ± 28.58,
p=0.016).
Table 3: Significant correlations with CIMT
CIMT
R P
CRP 0.408* 0.025*
OH/ECW 0.655* <0.001*
OH 0.619* <0.001*
ECW 0.392* 0.032*
IVWT 0.499* 0.007*
EF -0.457 0.078
LVMI 0.256 0.800
Figure 1: Correlation between CRP and CIMT
OH/ECW was positively correlated with CRP
(r=0.553, p=0.002, Figure 2), and the CIMT
(r=0.655, p=<0.001, Figure (3), and
negatively with the ejection fraction of the
heart (r=-0.742, p=<0.001) (Table 4).
Table 4: Significant correlations with OH/ECW
OH/ECW
r P
Age 0.024 0.899
Albumin 0.177 0.349
CRP 0.553* 0.002*
CIMT 0.655* <0.001*
EF -0.742* <0.001*
LVMI -0.214 0.256
Figure (2): Correlation between CRP and OH/ECW
Figure(2): Correlation between OH/ECW and IMT (n=30)
Discussion
ESRD patients on maintenance hemodialysis
are at increased cardiovascular risk secondary
to the complex interplay of multiple traditional
and non-traditional risk factors. The MIA
syndrome and fluid overload are among these
factors. (4,15) Fluid overload and overhydration
are common in HD patients, yet basing its
detection on clinical assessment is often
unsuccessful. Multi-frequency bioimpedance
to assess the body composition is a practical,
objective method for assessment of the
patients’ hydration status. (16 - 19) In this study,
the number of malnourished patients was small
and we could not analyze the effect of
malnutrition on inflammation, cardiovascular
disease and body composition. Other studies,
however, found a link between
hypoalbuminemia in dialysis patients and
inflammation and cardiovascular diseases. (20)
For example, Beddhu et al (21) showed an
association between serum albumin level and
cardiovascular disease in chronic hemodialysis
patients. This link is believed to be secondary
to albumin acting as a negative acute phase
reactant rather than a marker of poor nutrition.
Kaysen et al. (22) reported that albumin levels
are mainly correlated with inflammation, better
reflecting the presence of inflammation rather
than nutritional status in dialysis patients. In
the present study CRP was positive in 26
patients (86.6%). CIMT assessment was used
to detect atherosclerosis. CIMT was high in 23
patients (76.6%) and plaque was found in 9
patients (30%). CRP and CIMT were
significantly correlated (r=0.408, p=0.025).
Inflammation is a nontraditional risk factor
believed to play a role in mediating
cardiovascular risk in the general population as
well as in different stages of CKD. (5) Wanner
et al further identified an increased overall and
cardiovascular mortality in hemodialysis
patients associated with inflammation. (23)
CRP was also correlated to OH/ECW a marker
of fluid overload (p=0.002, r=0.553). Other
studies found a bidirectional relationship
between inflammation and extracellular fluid
overload. (24-26) Inadequate water and sodium
removal may act as a inflammatory stimulus,
while inflammation may lead to further
extracellular water accumulation. (27, 28) An
additional finding of the present study was the
presence of a significant negative correlation
between OH/ECW ratio and ejection fraction
(r=-0.742, p=<0.001). Other studies as
Schreiber BD and Collins AJ reported that
patients entering chronic dialysis display frank
symptoms of heart failure and because as much
as 48% of asymptomatic patients stabilized on
dialysis have compromised LV function. (29, 30)
However, LVMI did not significantly change
with the degree of overhydration (r=,-0.214,
p=0.256). In contrast, Fagugli et al(31) observed
an association between extracellular water and
left ventricular mass and hypertension in
haemodialysis patients. (31) Harnett JD et al
reported that heart failure is highly prevelant
complication in long term hemodialysis
Conclusion
Inflammation, atherosclerosis and fluid
overload are prevalent in ESRD patients on
maintenance hemodialysis. Fluid overload as
indicated by elevated OH/ECW measured by
m-BIA was significantly correlated with
markers of inflammation and atherosclerosis
and negatively correlated with cardiac function
in HD patients. The detection and appropriate
management of overhydration, inflammation
and atherosclerosis may allow the reduction of
cardiovascular risk in this high-risk population.
References 1. Sarnak MJ, Levey AS. Cardiovascular disease
and chronic renal disease: a new paradigm. Am J
Kidney Dis. 2000; 35(4 suppl 1):S117–S131.
2. Drey N, Roderick P, Mullee M, Rogerson M. A
population based study of the incidence and
outcomes of diagnosed chronic kidney disease. Am
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3. Herzog CA, Ma JZ, Collins AJ. Poor long-term
survival after acute myocardial infarction among
patients on long-term dialysis. N Engl J Med. 1998;
339:799 – 805.
4. Kalantar-Zadeh K, Ikizler TA, Block g, et al.
Malnutrition-inflammation complex syndrome in
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5. Zoccali C, Benedetto FA, Mallamaei F.
Inflammation is associated with carotid
atherosclerosis in dialysis patients. J Hypertens
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6. London GM, Marchais SJ, Metivier F, Guerin
AP. Cardiovascular risk in end-stage renal disease:
vascular aspects. Nephrol Dial Transplant
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Akoglu E, Ozener IC. Uncontrolled hypertension
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8. D'Amico M, Locatelli F. Hypertension in dialysis: pathophysiology and treatment. J Nephrol 2002; 15(4):438–45. 9. Leunissen KML, Kooman JP, Vander Sandle FM, Van Kuijik WH. Hypotension and ultrafiltration physiology in dialysis. Blood Purif 2000; 18(4):251-4. 10. Daugirdas, J. Chronic hemodialysis prescription. In: Daugirdas JT, Blake PG, Ing TS (eds). Handbook of dialysis. 4thed. Sydney: Lippincott Williams & Wilkins; 2007.146-69. 11. Kuhlmann MK, Zhu F, Seibert E, Levin NW. Bioimpedance, dry weight and blood pressure control: New methods and consequences. Curr Opin Nephrol Hypertens 2005; 14(6): 543-9. 12. Lang RM, Bierig M, Devereux RB. Chamber Quantification Writing Group. Recommendations for chamber quantification: a report from the American Society of Echocardiograpliy Guidelines and standards committee & the chamber quantification writing group, developed in corfunction with the European Association of echocardiography, a branch of the European society of cardiology. J Am Soc Echocardiogr 2001; 18:1440-63. 13. Lukaski HC. Validation of body composition assessment techniques in the dialysis population. ASAIOJ 1997; 43:261-5. 14. Wizemann V, Wabel P, Chamney P, Zaluska W, Moissl U, Rode C, et al. The mortality risk of overhydration in haemodialysis patients. Nephrol Dial Transplant 2009;24 (5):1574–9. 15. Paniagua R, Ventura MD, Avila-Díaz M, Hinojosa-Heredia H, Méndez-Durán A, Cueto-Manzano A, et al. NT-proBNP, fluid volume overload and dialysis modality are independent predictors of mortality in ESRD patients. Nephrol Dial Transplant 2010;25(2):551–7. 16. Konings CJ, Kooman jp, Schonck M, et al. Fluid status, blood pressure and cardiovascular abnormalities in patients on peritoneal dialysis. Perit Dial Int 2002:22:477-487. 17. Wabel P, Chamney P, Moissl U, Jirka T. Importance of whole-body bioimpedance spectroscopy for the management of fluid balance. Blood Purif 2009;27(1):75–80. 18. Moissl UM, Wabel P, Chamney PW, Bosaeus I, Levin NW, Bosy-Westphal A, et al. Body fluid volume determination via body composition spectroscopy in health and disease. Physiol Meas 2006;27(9):921–33. 19. Crepaldi C, Soni S, Chionh CY, Wabel P, Cruz DN, Ronco C. Application of body composition monitoring to peritoneal dialysis patients. Contrib Nephrol 2009; 163:1–6. 20. Wizemann V, Rode C, Wabel P. Whole-body spectroscopy (BCM) in the assessment of normovolemia in hemodialysis patients. Contrib Nephrol 2008;161:115–8.
21. Nehal Rachit Shah, Francis Dumler. Hypoalbuminaemia ; A Marker of Cardiovascular Disease in Patients with Chronic Kidney Disease Stages II – IV. Int. J. Med. Sci. 2008, 5 (6):366-70. 22. Beddhu S, Kaysen GA, Yan G, Sarnak M, Agodoa L, Ornt D, et al. HEMO Study Group. Association of serum albumin and atherosclerosis in chronic hemodialysis patients. Am J Kidney Dis 2002; 40: 721-7. 23. Kaysen GA, Dubin JA, Muller HG, Rosales LM, Levin NW.The acute-phase response varies with time and predicts serum albumin levels in hemodialysis patients: The HEMO Study Group. Kidney Int 2000; 58: 346–52. 24. Christoph Wanner, Josef Zimmermann, Susanne Schwedler, Thomas Metzger. Inflammation and cardiovascular risk in dialysis patients Kid Int 2002; 61( Suppl 80): S99–S102. 25. Vicenté-Martínez M, Martínez-Ramírez L, Muñoz R, et al. Inflammation in patients on peritoneal dialysis is associated with increased extracellular fluid volume. Arch Med Res 2004;35:220-224. 26. Woodrow G, Oldroyd B, Wright A, et al. Abnormalities of body composition in peritoneal dialysis patients. Perit Dial Int 2004;24:169-17. 27. Plum J, Schoenicke G, Kleophas W, et al. Comparison of body fluid distribution between chronic haemodialysis & peritoneal dialysis patients as assessed by biophysical & biochemical methods. Nephrol Dial Transplant 2001;16:2378-2385 28. Asghar RB, Green S, Engel B, et al. Relationship of demographic, dietary, and clinical factors to the hydration status of patients on peritoneal dialysis. Perit Dial Int 2004; 24:231-239. 29. Avila-Díaz M, Ventura MD, Valle D, et al. Inflammation & extracellular volume expansion are related to sodium and water removal in patients on peritoneal dialysis. Perit Dial Int 2006; 26:574-580. 30. Schreiber BD. Congestive heart failure in patients with chronic kidney disease and on dialysis. Am J Med Sci. 2003; 325(4):179-93. 31. Collins AJ. Cardiovascular mortality in end-stage renal disease. Am J Med Sci. 2003; 325(4):163-7. 32. Fagugli RM, Pasini P, Quintaliani G, Pasticci F, Ciao G, Cicconi B, et al.Association between extracellular water, left ventricular mass and hypertension in haemodialysis patients. Nephrol Dial Transplant 2003; 18(11):2332–8. 33. Harnett JD, Foley RN, Kent GM, Barre PE, Murray D, Parfrey PS.: Congestive heart failure in dialysis patients: Prevalence, incidence, prognosis and risk factors. Kidney Int 47: 884–890, 1995
Original Article
Helicobacter Pylori CagA Line Test
Mohamed Alaa Eldin Nouh MD1, Hossam Eldine Mostafa Saleam MD2 and Bedair Haroun
AbdAllah Fayed M.B.B.CH3; 1Tropical Medicine Department, Faculty of Medicine, Menoufiya
University,Egypt, 2Tropical Medicine Department, Faculty of Medicine, Menoufiya
University,Egypt, 3Shebin Elkioum fevers hospital,,Egypt
ABSTRACT Helicobacter pylori infection is one of the most widespread infections in humans. It is highly prevalent,
especially in developing countries Aim of the work : this study is to evaluate the efficacy of Helicobacter
pylori (H.Pylori) CagA line test as a new test for the detection of the virulent strains. Material and
Methods: 50 patients with H. pylori were enrolled in this study. Immunohistochemical analysis of CagA
was performed on paraffin-embedded sections of pretreatment endoscopic biopsy specimens. Visualization
was performed using an indirect immunoperoxidase method according to the manufacturer’s instructions.
Cell blocks of a CagA-translocated human B cell line served as a CagA-positive control. Results: Both
CagA and VacA tests showed ulcers in all patients 50 (100%), while endoscopy showed ulcer in 44 patients
(88.0%), while 6 patients (12.0%) had no ulcer. Statistically both CagA and VacA tests had a statistically
significant difference in detecting ulcer (P < 0.05). Conclusion: CagA line test is a simple non-invasive and
accurate test for diagnosis of H. pylori organisms.
Introduction
Helicobacter pylori infection is one of the
most widespread infections in humans. It is
highly prevalent, especially in developing
countries. In the western world, the infection
rate is gradually decreasing; however, the
prevalence still ranges from 25% to 50% (1).
Virtually everyone infected with H. pylori
develops lifelong chronic type B gastritis,
but only a minority of infected individuals
will develop a clinically relevant condition,
such as gastric cancer (GC).
Seroepidemiological studies have
demonstrated a three- to sixfold increased
risk for infected individuals of developing
GC (2). Based on these and other findings, H.
pylori has been classified as a class I human
carcinogen by the International Agency for
Research on Cancer, although the exact
nature and strength of its association with
GC has remained debatable. However, recent
meta-analyses (3) indicate a weaker RR
(approximately two) than originally reported
for seropositive individuals (2). Although it is
clear that H. pylori infection increases the
risk of these upper gastrointestinal diseases,
it is still not fully understood why infected
individuals develop one disease rather than
another, emphasizing the importance of the
host and other cofactors. It has been
suggested that both the possession of the
cytotoxin-associated gene A (CagA), and the
production of a vacuolating cytotoxin
encoded by the vacuolating cytotoxin A
(VacA) gene, are linked to increased
pathogenicity of H.pylori strains(4). There is
substantial evidence that H. pylori infection,
especially with the strains expressing the 128
kDa CagA protein, is associated with
enhanced gastric inflammatory response and
increased risk of developing atrophic
gastritis, peptic ulcer and even GC (3).
However, conflicting results regarding the
association between these virulence factors
and clinical disease have been reported, and
epidemiological papers suggest that not all
tumors are H. pylori-positive (5). The
objective of the present study was to
determine and evaluate the efficacy of H.
pylori CagA line test as a new test for the
detection of the virulent strains of
Helicobacter pylori. In this review we
attempt to categorize these tests, briefly
describe their advantages and disadvantages
and finally provide some hints regarding a
future oriented standard test.
Patients and Methods
This study comprised 50 patients diagnosed
with H. pylori CagA line test to have H.
pylori in a trail to evaluate the H. pylori
CagA test. They were 25 males and 25
females with age ranged from 28 to 68 years
old. They were selected according to the
inclusion and exclusion criteria from the
Internal Medicine Department, Faculty of
Medicine, Menofyia University over two
years period. History: personal, past history,
history of the present illness. Examination:
general and clinical examination, complete
cardiac and chest examination. Laboratory
investigation: Fasting serum glucose, serum
lipids, C-reactive protein and serum CagA
and VacA tests were measured.
Immunohistochemistry:
Immunohistochemical analysis of CagA
(A10; sc-28368, Santa Cruz Biotechnology,
Santa Cruz, CA, USA) was performed on
paraffin-embedded sections of pretreatment
endoscopic biopsy specimens. Visualization
was performed using an indirect
immunoperoxidase method according to the
manufacturer’s instructions. Cell blocks of a
CagA-translocated human B cell line served
as a CagA-positive control(6).
Results Data were coded, entered and statistically
analyzed by computer package (version 10).
Table (1): Age and sex distribution of the studied patients
Males Females Total
No. % No. % No. %
Gender 25 50.0 25 50.0 50 100
Age (years):
Range
Mean ± SD
29 – 68
44.84 ± 8.75
28 – 63
46.92 ± 9.97
28 – 68
45.88 ± 9.34
Table (2): Clinical presentation of the studied patients.
Clinical presentation Males Females Total Significance
No. % No. % χ2 P
Vomiting 8 32.0 15 60.0 23 46.0 5.381 <0.05
Epigastric pain 20 80.0 22 88.0 42 84.0 6.913 <0.05
Hematemesis 5 20.0 6 24.0 11 22.0 1.406 >0.05
Melena 1 4.0 3 12.0 4 8.0 2.977 >0.05
χ2 = Chi square
Table (3): Comparison between Cag A test and endoscopy in diagnosis of H. pylori.
H. pylori
diagnosis
Cag A Endoscopy Significance
No. % No. % χ2 P
Ulcer 50 100 44 88.0 0.561 <0.05
No ulcer 0 0.0 6 12.0 0.999 <0.001
Total 50 100 50 100
Table (4): Comparison between Vac A test and endoscopy in diagnosis of H. pylori.
H. pylori
diagnosis
Vac A Endoscopy Significance
No. % No. % χ2 P
Ulcer 50 100 44 88.0 0.561 <0.05
No ulcer 0 0.0 6 12.0 0.999 <0.001
Total 50 100 50 100
Table (5): Liver and renal functions among studied groups.
Liver & Renal
function testes
Males Females Total Significance
Mean ± SD Mean ± SD Mean ± SD F P
AST (u/L) 25.0 ± 9.6 26.2 ± 10.3 28.3 ± 11.6 1.012 >0.05
ALT (u/L) 27.2 ± 11.7 28.2 ± 1.4 29.3 ±12.9 0.627 >0.05
T. bilirubin (mg %) 0.9 ± 0.3 1.03 ± 0.4 1.1 ± 0.6 1.473 >0.05
Urea (mg/dL) 34.4 ± 9.34 33.3±10.6 36.6±13.98 1.25 >0.05
Creatinine (mg/dL) 0.7 ± 0.32 0.84 ±0.44 0.9 ±0.51 0.126 >0.05
Table (6): Diagnosis of H. pylori according to sex of the studied patients
H. pylori
diagnosis
Males Females Total Significance
No. % No. % No. % t-test P
Gastric ulcer 14 28.0 20 40.0 34 68.0 0.495 <0.05
Duodenal ulcer 7 14.0 3 6.0 10 20.0 0.664 <0.05
No ulcer 4 8.0 2 4.0 6 12.0 0.513 <0.05
Total 25 50.0 25 50.0 50 100
Discussion This study showed that both CagA and VacA
tests had ulcers in all patients 50 (100%),
while endoscopy showed ulcer in 44 patients
(88.0%), while 6 patients (12.0%) had no
ulcer. Statistically both CagA and VacA tests
had a statistically significant difference in
detecting ulcer (P < 0.05). Hirai et al. (7)
confirmed the presence of the East Asian
CagA genotype by performing two separate
rounds of PCR using specific primer pairs
(F2+EA-R and FA-F+R3). The specificity of
the detection method was confirmed by
using H. pylori strains with the East Asian
CagA genotype as a reference. The East
Asian cagA genotype was detected in 41 of
65 (63.1 %) genomic DNA samples of H.
pylori. The incidence of CagA -positive H.
pylori ranged from 40.0 to 100.0% across all
age groups. There was no significant
difference between the age groups, except
for the group comprising individuals aged
>60 years, because of the small sample
number of participants in this age group. The
present study also shows the gender
distribution of h. pylori ulcers of the studied
patients. Gastric ulcer was found in 34
patients (68%), they were 14 patients (28%)
in males and 20 patients (40%) in females.
They showed a statistically significant
difference (P <0.05) between both males and
females. Duodenal ulcer was found in 10
patients (20%), they were 7 patients (14%) in
males and 3 patients (6.0%) in females. They
showed a statistically significant difference
(P <0.05) between both males and females.
They showed also no ulcer was found in 6
patients (12%), they were 4 patients (8.0%)
in males and 2 patients (4.0%) in females.
They also showed a statistically significant
difference (P <0.05) between both males and
females. The results of the present study
indicated that 22.0% of the healthy
asymptomatic Japanese individuals
participating in the study may be infected
with the highly virulent H. pylori strain. A
considerably higher number of healthy
individuals were found to have infection
with the highly virulent East Asian cagA-
positive H. pylori in Japan than in Thailand
[where 2.8% (5/179) healthy asymptomatic
individuals were positive for the highly
virulent H. pylori infection; unpublished
data]. The cause of the highly virulent H.
pylori infection in a considerably high
number of asymptomatic Japanese
individuals is unknown. However, in a recent
report, it has been suggested that (1) the
geographical distribution of H. pylori strains
harboring a certain virulence factor genotype
and (2) the incidence of cancer are
responsible for the high incidence of H.
pylori infection among asymptomatic
Japanese individuals(8). The findings of
Yamaoka et al.(8) were based on genotype
analysis of H. pylori strains that were
clinically isolated from patients; however,
their finding of a high incidence of gastric
cancer in countries where the East Asian
CagA is predominant is in agreement with
the result obtained in our study. A relatively
recent study showed that the eradication of
H. pylori significantly suppressed the
development of metachronous gastric
cancer(9). The report does not directly
suggest that the eradication of H. pylori
infection in healthy asymptomatic
individuals will suppress the onset of gastric
cancer in the future, but it highlights the
significance of H. pylori infection in gastric
cancer development. Therefore, a silent
infection with a highly virulent strain of H.
pylori, such as one with the East Asian cagA
genotype, in healthy individuals may be a
critical public health issue in the prevention
of gastric cancer (7). H. pylori infection
causes both intestinal and diffuse types of
gastric adenocarcinoma. Its carcinogenic
effects have been attributed to induction of
inflammation. The phenotype of H. pylori
that expresses CagA causes higher degrees
of acute and chronic inflammation than the
CagA-negative condition. Recent serological
studies have shown that CagA and VacA
seropositivity is associated with an increased
risk for atrophic gastritis and GC (5). The H.
pylori strain possessing CagA-enhanced
gastric epithelial proliferation and apoptosis
induces tyrosine phosphorylation of the
CagA protein (10), causing gastric cells to
produce high levels of interleukin-8, which
plays a crucial role in the inflammatory cell
response to infection (11). Whereas some
studies on H. pylori patients were unable to
find an association between VacA and CagA
antibodies, and H. pylori, other studies
demonstrated a significant association
between VacA and CagA antibodies, and H.
pylori. Some authors found no difference in
the prevalence of anti-CagA antibodies
between H. pylori-positive duodenal ulcer
patients and patients without duodenal ulcer
and gastric ulcer (12). Although duodenal
ulcer patients tested negative for H. pylori at
the time of the present study, they tested
positive for serological CagA, VacA and IgG
antibodies. The presence of CagA, VacA and
IgG antibodies against H. pylori in these
patients likely indicates H. pylori infection
before the appearance of GC. In fact, CagA-
positive H. pylori strains seem to induce a
high immune response with a markedly
higher frequency of IgA. Although literature
is scarce, existing clinical data indicate that
CagA antibodies persist longer after
eradication treatment than antibodies
detected by IgG ELISA; a stronger
association emerged when antibodies to
CagA were used as markers of H. pylori
exposure (13). It therefore seems reasonable,
as other studies report (14), to assume that the
addition of immunoblotting would result in a
more correct representation of prior exposure
than the use of IgG ELISA alone (15). These
data were also confirmed by the Suriani et al. (5) study, in which patients with previous
DUs eradicated 10 years earlier, who were
known to be H. pylori-negative at the time of
the histological biopsy of the antrum and
corpus, and were followed up for a long
period of time (120±32 months) tested
positive for VacA and CagA proteins more
often than for IgG. This was also confirmed
by the high level of CagA and VacA than
IgG antibodies for H. pylori in the patients
with GC and in asymptomatic children (16).
These data reinforce the hypothesis that
CagA and VacA proteins induce a strong
mucosal and systemic immune response and
may represent an immunological memory
from previous contact with the bacteria. The
former DU patients showed seropositivity to
CagA and VacA without any difference of
prevalence versus GC patients, as some
studies report. Seropositivity for CagA,
VacA and IgG for H. pylori in GC patients
can be explained theoretically by a previous
infection that was cured. Because several
decades may pass between initiation and
detection of GC, and the precancerous
microenvironment promotes spontaneous
eradication, substantial misclassification of
relevant exposure to H. pylori is likely to
occur in case control and short-term follow-
up studies. A declining association between
H. pylori and GC risk with advancing age
could possibly be explained by increased
exposure misclassification with advancing
age (17). Continuous developments in both
invasive and non-invasive based methods for
detection of H. pylori infection will greatly
contribute to further improvement of the
health management of H. pylori associated
disorders. Although this mysterious
bacterium has been unanimously declared a
human pathogen, only a minority of infected
individuals develop an associated disease,
which seems mainly attributed to the
armament of virulence factors of H. pylori (18). Therefore, individual identification of
virulence factors of the bacterium for risk
stratification is discussed for risk assessment
in combination with histopathological
evaluation of gastritis. There are common
diagnostic methods for detection of the
infection and verification of the virulence
factors (19). However, while gold standard
methods are still mainly derived from
biopsy-based methods, the high prevalence
of the infection especially in areas with low
medical support suggests the urgent need for
introducing non-invasive and preferably high
throughput applicable procedures. The
selection of such a test depends on the
sensitivity, specificity, availability,
complexity, costs, and rapidity of results (20).
Unfortunately, none of the currently used
methods cover these criteria perfectly.
Although biopsy-based methods have a very
high specificity, only a moderate sensitivity
is observed in these assays mainly due to
factors that are difficult to control such as the
sampling site. Even in spite of correct
sampling, there are other factors such as PPI
treatment prior to sampling that possibly
affect the growth or presence of detectable
curved bacteria. PCR-based tests have slight
advantages compared to other tests (12). PCR,
as a highly sensitive and specific method, is
applicable not only in the detection of
infection with H. pylori but also in the
monitoring of treatment and therapy
efficiency. Kalali et al. (21) showed that with
the proper design of PCR, a single copy of
genomic DNA is sufficient to obtain a
positive signal indicating the presence of H.
pylori in the sample. Moreover, DNA
samples isolated from different sources such
as gastric biopsy, samples from the oral
cavity, and stool specimens could be
subjected to PCR assay. Accordingly,
precluding the possible chances of
contamination, PCR could be used as a gold
standard (22). The main disadvantage of PCR
is the level of diagnostic facilities and
implementation of this method in regions
with poor medical support. Since other
biopsy-based methods generally have
disadvantages such as the requirement of
endoscopic facilities or additional systems
such as RUT, they are not altogether suitable
as gold standards (21). Recently, most efforts
to introduce an adequate gold standard are
focused on non-invasive methods. In
addition to PCR, serological methods aiming
at the detection of H. pylori specific antigens
in different specimens have been
significantly improved. Bioinformatics
analysis greatly helps the selection of the
optimal antigen applied in serological assays.
Not only could the antigenicity of the antigen
be predicted in these analyses, more
importantly the homology of the antigen to
other relevant microorganisms can be
comprehensively analyzed (10). Through the
choice of a suitable antigen and the
application of advanced methods in
nanotechnology in assay design, new
serological tests have recently reached
relatively ideal performance (23). Although
the report of the Maastricht conference
emphasizes that antibodies against H. pylori
and especially against its most specific
antigen CagA, remain elevated for long
periods, for months or even years, there is
evidence that while CagA is a dominant
virulence marker, it is only present in around
70% of H. pylori strains depending on their
geographic origin (24). Recent studies
identified other antigens in all H. pylori
strains which possess not only high
immunogenicity but also elicit specific
antibodies which will fade away after a
relatively short time after eradication (25).
Advanced and simplified specific antibody
detection techniques like line assay and rapid
diagnostic tests have greatly improved the
application of serological tests for the
detection of H. pylori. These approaches
cover most of the criteria mentioned for H.
pylori diagnostic assays. Not only are they
applicable in high-through-put studies of
large cohorts but they are also highly cost
effective, bedside applicable, simple for
analysis and intelligible for medical
personnel (26). Less so, but still in an equally
acceptable relevant context, specific antigen
tracing tests are currently being developed.
In particular, noticeable progression has been
made in the area of stool tests. Indeed, when
a study focuses particularly on children of a
high prevalence area, antigen-tracing
systems like the stool antigen test are
advantageous (27). Finally, there is apparent
evidence suggesting that not every strain of
H. pylori is harmful and should be treated (28). In addition, there are accumulating data
on the beneficial aspects of infection with H.
pylori for its human host (29). Therefore,
conduction of risk stratification according to
the epidemiological data is highly
recommended. The feasibility of such
assessments is now exclusively conceivable
through serological assays, which can
indicate the infection with pathogenic strains
of H. pylori (21). Although the number of
patients included in the present study was
somewhat limited, it nevertheless reinforces
the idea that CagA and VacA induce a strong
mucosal and systemic immune response, and
may represent an immunological memory
from previous contact with the bacteria. H.
pylori can be considered a direct
carcinogenic agent of GC, although it is not
clear why some people develop cancer, and
others only develop DUs or no pathology at
all. Further studies are necessary for a better
understanding of the pathogenic mechanism (5). In conclusion, current available
diagnostic systems cannot meet the
requirements posed by such a widespread
and prevalent infection as H. pylori,
especially when more than simple detection
of positivity is required (i.e. risk
stratification, antibiotic resistance). We
suggest a collaborative effort from experts
and health organizations across the world to
strive for the development, validation, and
introduction of such multi-tasking diagnostic
tools which could not only become a new
recommended gold standard for the
screening of large infected populations but
would also assist in guiding the treatment
strategies for each infected individual.
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Original Article
Patterns of Peripheral Vascular Disease in Lower Extremities as a
Predictor of Disease Activity and Damage in Systemic Lupus
Erythematosus Patients
Manal Tayel1, Magdy Megallaa1, Nevine Mohannad2 and Mariam Mostafa3; 1Internal Medicine
Department, Faculty of Medicine, Alexandria University, Egypt, 2Alexandria University hospitals,
rheumatology Unit , Internal Medicine Department, Faculty of Medicine, Alexandria University,
Egypt, 3Amerreya hospital Ministry of health.
ABSTRACT Aim of the work is to analyze patterns of peripheral vascular disease (PVD) in lower extremities in
systemic lupus erythematosus patients (SLE) patients as a predictor of disease activity and damage.
Material and Methods: A cross sectional observational analysis of 40 SLE patients and 10 matched age
and sex controls, the patients were free of the following: diabetes mellitus or hypertension prior to disease
onset, smoking, overlap with other autoimmune disease, intake of vasodilator one week before assessment.
Through history taking and clinical examination were done. Routine investigations as well as lupus specific
laboratory analyses were done. An ankle brachial index (ABI) was done for all the subjects where ABI <0.9
or >1.3 was diagnostic of (PVD) ,Doppler study for dorsalis pedis and posterior tibial arteries in both
extremities was done as triphasic wave pulse excluded PVD,10 gram monofilament test was done to detect
peripheral neuropathy , disease activity was assessed by applying the score of SLE disease activity index
(SLEDAI)and damage by applying the score of SLE damage index (SLICC/ACR).Traditional and
nontraditional cardiovascular risk factors were assessed . Homocysteine was measured by competitive assay
method with normal range of 5-15µmol/L, as hyperhomocysteinemia is considered a predictor of endothelial
dysfunction. Results: Ankle brachial index was significantly lower in lupus patients than controls (
P<0.001). Prevalence of PVD in lupus patients was 32.5%,biphasic wave pulse was detected in 50%of
lupus patients ,homocysteine level was significantly higher in lupus patients than controls, also significantly
high homocysteine level was obsereved in abnormal ABI patients (P=0.005) . The following variables were
associated with abnormal ABI: older age (years) (P<0.001),disease duration (P=0.019), dyslipidemia
(P=0.006), ACL IgM(P=0.047), also increased serum level of the following: cholesterol (P=0.001), TG
(P=0.009), HDL (P=0.032).Another association with abnormal ABI was biphasic wave pulse in dorsalis
pedis artery bilaterally(P=0.004) and posterior tibial artery bilaterally(P=0.004). Conclusion: Prevalence of
PVD in lupus was higher than expected 32.5%, some lupus specific variable was associated with PVD;
disease duration, ACL IgM. Hyperlipidemia as an important traditional risk factor was also associated with
PVD in lupus, homocysteine as an indicator of endothelial dysfunction showed increased levels in PVD
lupus patients, mild PVD was the only detected form among lupus patients in this study.
Introduction
Systemic Lupus Erythematosus (SLE) is an
inflammatory autoimmune disorder that may
affect multiple organ systems. Many of its
clinical manifestations are secondary to
immune complex deposition in capillaries of
visceral structures or to autoantibody
mediated destruction of host cells. The
clinical course is characterized by remissions
and relapses.(1) Peripheral vascular disease
(PVD), also called peripheral arterial disease
(PAD) is defined as atherosclerotic disease
of infrarenal aorta and arteries of the lower
extremities. It is a frequent but under
diagnosed and undertreated disease with
substantial cardiovascular morbidity and
mortality. Accordingly, early recognition is
crucial to initiation of therapy. (2) Modifiable
risk factors for PVD are not different from
patients with coronary artery disease. Major
risk factors include smoking, hyperlipidemia,
hypertension, diabetes, and the metabolic
syndrome. (3) The clinical manifestations of
PVD depend upon the location and severity
of arterial stenosis or occlusion, and range
from mild extremity pain with activity (ie
claudication) to limb threatening ischemia as
demonstrated in Rutherford classification of
peripheral vascular disease table 1 .(4) In the
Third Report of the National Cholesterol
Education Program/Adult Treatment Panel
recommendations, peripheral arterial disease
is identified as a clinical atherosclerotic
disease equivalent that confers high risk of
coronary heart disease. When PVD is
identified, the low-density lipoprotein level
at which lipid-lowering medication is
recommended for CVD prevention is lower
than if PVD, or other CVD risk equivalent, is
not present.(5) Peripheral vascular disease
appears to be inherent in the pathogenesis
and clinical manifestations of SLE; its
severity ranging from mild cutaneous disease
to severe vasculitis, atherosclerotic cardio
vascular or cerebrovascular events, or
catastrophic antiphospholipid syndrome.
Vasculitis prevalence in SLE is reported to
be between 11 % and 36 %, it may manifest
in as high as 56% of lupus patients
throughout their life.(6)
Table (1): Rutherford classification of peripheral vascular disease. (7)
Grade Category Clinical Description
I 0 Asymptomatic
1 Mild claudication
2 Moderate claudication
3 Severe claudication
II 4 Ischemic rest pain
5 Minor tissue loss; nonhealing ulcer, focal gangrene with diffuse pedal ischemia
III 6 Major tissue loss extending above transmetatarsal level; foot no longer salvageable
Hyperhomocysteinemia was found to be
associated with an increased risk for ischemic
heart disease, stroke, peripheral arterial disease
and deep venous thrombosis, as well as for
neural tube defects and preeclampsia in
pregnancy. High concentrations of
homocysteine in blood induce endothelial
dysfunction, suggesting a causal role in
vascular disease. (8) Ankle brachial index is a
well-established reproducible method with
high sensitivity and specificity to assess the
patency of the lower limb arterial tree and to
detect the presence of peripheral arterial
disease. (9)
Patients and Methods
Patients: This study is a cross sectional
observational study it was carried out on 40
SLE patients who attended the rheumatology
clinic of Alexandria university hospital
diagnosed according to the American
College of Rheumatology revised criteria for
classification of SLE SLE patients were
included in this group.(10) Patients with
essential hypertension, DM type I or II
diagnosed prior to the onset SLE , patients
with overlap syndrome or with other
collagenic diseases, intake of vasodilators
less than one week prior to evaluation and
smokers were excluded from the study. Ten
age and sex matched healthy subjects were
selected as control group. They had no
family history of autoimmune illness and
were selected to be non-smokers. Methods:
All SLE patients fulfilled the criteria of
American College of Rheumatology (10)for
classification of SLE, they were subjected to
the following after informed consent: Full
history taking, thorough systemic physical
examination and full clinical examination of
both lower limbs, assessment of disease
activity by applying the score of SLE disease
activity index (SLEDAI)(11), assessment of
disease damage by applying the score of SLE
Disease Damage Index (SLICC/ACR)(12),
body mass index as measured by the
equation BMI=Mass (kg)/height (m)². (13)
Laboratory investigations included: complete
blood picture (CBC), erythrocyte
sedimentation rate (ESR),liver enzyme: ALT
and AST, fasting blood glucose, renal
function test: Urea and Creatinine, lipid
profile: HDL, Cholesterol and Triglycerides,
serum antinuclear antibodies (ANA) titer by
ELISA, complement factors: C3 and C4,
serum anti-double stranded DNA antibodies
(anti ds-DNA) titer, anti-cardiolipin (IgG,
IgM),lupus anti-coagulant and serum
homocysteine. -Serum homocysteine(14).
Principle of the Method: - Competitive
Assay : Bound homocysteine in the samples
was reduced to free homocysteine by the
action of dithiothreitol, and then converted
enzymatically to S-adenosyl-homocysteine
(SAH) in the next step. Conjugated S-
adenosyl-cysteine (SAC), added at the onset
of the reaction, competes with the SAH in
the sample for bonding by anti-SAH
antibodies bound to polystyrene particles. In
the presence of SAH, there was either no
aggregation or a weaker aggregation of
particles. In the absence of SAH in the
sample, an aggregation of the polystyrene
particles by the conjugated SAC occured.
The higher the SAH content of the reaction
mixture is, the smaller the scattered light
signal was seen. The result was evaluated by
comparison with a standard of known
concentration. (15). -Reference Intervals:
Reference interval for adult males and
females is between 5 and 15 µmol/L. Ankle
brachial index: The ankle-brachial index
(ABI) is the ratio of the systolic blood pressure
at the ankle to the systolic blood pressure at the
brachial artery. It is one of the most widely
available markers of atherosclerosis and least
expensive to perform, to determine the ABI
with the doppler method, the patient was left to
rest for five to 10 minutes in the supine
position. Using an 8 to 10 MHz Doppler probe
with gel applied over the sensor, the device
was placed in the area of the pulse at a 45 to 60
degree angle to the skin surface. The probe was
moved to find the clearest signal. To detect the
pressure, the cuff has been inflated
progressively to 20 mm Hg above the level of
flow signal disappearance and then slowly
deflated to detect signal reappearance. (16) The
ABI was reported separately for each leg,
and was calculated by dividing the higher of
the posterior tibial or dorsalis pedis blood
pressure by the higher of the right or left arm
systolic blood pressure. (17) The IWGDF
recommend the use of bedside non-invasive
tests to exclude PAD. No single modality has
been shown to be optimal. Measuring ABI
(with<0.9 considered abnormal) is useful for
the detection of PAD. Tests that largely
exclude PAD are the presence of ABI 0.9–
1.3 and the presence of triphasic pedal
doppler arterial waveform. (18). - Hand held
Doppler for lower limb peripheral arterial
system examination: Hand-held Doppler
ultrasound examination of pedal arteries
(posterior tibial and dorsalispedis arteries) is
the most frequently used non-invasive
vascular assessment modality utilised by
podiatrists for diagnosis and ongoing
monitoring of PAD(19). Podiatrists generally
use Doppler in two different ways, as part of
an ankle brachial index (ABI) or as a
standalone test. The presence of triphasic
pedal doppler arterial waveform excludes
PAD. Hand held doppler can be performed at
relatively low cost and is non-invasive (20). -
Semmes-weinstien monofilament (10 gram
Monofilament test): Monofilament testing is an
inexpensive, easy to use, and portable test for
assessing the loss of protective sensation, and it
is recommended by several practice guidelines
to detect peripheral neuropathy in otherwise
normal feet. (21)
Results
This study included 40 SLE patients and 10
age and sex matched controls.
Table (2): Comparison between patients and controls according to demographic data
Patients
(n=40)
Control
(n=10)
Test of sig. P
No % No %
Sex
Male 7 17.5 1 10.0 χ2= 0.335 FEp=1.000
Female 33 82.5 9 90.0
Age (years)
Min. – Max. 10.0 – 52.0 16.0 – 48.0
Z= 0.243 0.808 Mean ± SD. 30.22±11.36 31.50±11.09
Median 27.0 33.50
Marital status
Single 22 55.0 4 40.0 χ2= 0.721 FEp=0.490
Married 18 45.0 6 60.0
Residency
Urban 30 75.0 8 80.0 χ2=
0.110 FEp=1.000
Rural 10 25.0 2 20.0
BMI (kg/m2)
Underweight <18.5 2 5.0 0 0.0
χ2= 0.817 MCp=0.875 Normal (18.5 - 24.9) 15 37.5 3 30.0
Overweight (25 - 29.9) 17 42.5 5 50.0
Obese ≥30 6 15.0 2 20.0
Min. – Max. 15.83–40.50 22.60–30.50
t= 0.716 0.478 Mean ± SD. 25.69 ± 5.23 26.92 ± 2.84
Median 25.80 27.90
2: Chi square test MC: Monte Carlo for Chi square test
FE: Fisher Exact for Chi square test t: Student t-test
Z: Z value for Mann Whitney test *: Statistically significant at p ≤ 0.05
Table (2) shows that no statistically
significant difference was found between
patients and controls regarding sex, age,
marital status, residency and BMI.
Table (3): Comparison between patients and controls according to homocysteine level.
Patients
(n=40)
Control
(n=10) T P
Homocysteine (µmol/L)
Min. – Max. 8.10–14.90 5.0 – 8.10
7.699* <0.001* Mean ± SD. 10.67±1.65 6.45±1.02
Median 10.60 6.15
As shown in table (3) serum homocysteine
levels showed high significant difference
between patient group and control group it
ranged from 8.10 to 14.9 µmol/L in patients
while it ranged from 5 to 8.10 µmol/L in
control in group.
Table (4): Comparison between patients and controls according to ABI
Patients
(n=40)
Control
(n=10) Test of sig. P
No % No %
ABI
Normal 27 67.5 10 100.0 χ2= 4.392* FEp= 0.046*
Abnormal 13 32.5 0 0.0
Min. – Max. 0.84 – 1.0 0.94 – 1.0
t= 4.581* <0.001* Mean ± SD. 0.94±0.05 0.99 ±0.02
Median 0.94 1.0
As seen in table (4) ABI showed significant
difference between patients and controls,
32.5% of patients had abnormal ABI while all
the controls had normal ABI.The mean value
of ABI showed highly significant difference
between the two groups as it was 0.94±0.05 in
patients group and 0.99±0.02 in control
group(P<0.001).
Table (5): Relation between ABI and lipid profile in lupus patients.
Lipid profile
ABI
T P Normal
(n=27)
Abnormal
(n=13)
Cholesterol mg/dl
Min. – Max. 140.0– 320.0 189.0– 300.0
3.593* 0.001* Mean ± SD. 195.22±44.98 244.69±29.74
Median 180.0 248.0
TG mg/dl
Min. – Max. 68.0 – 170.0 69.0 – 190.0 2.750* 0.009*
Mean ± SD. 115.93±30.23 145.92± 36.42
Median 102.0 160.0
HDL mg/dl
Min. – Max. 31.0 – 72.0 25.0 – 62.0
1.176 0.247 Mean ± SD. 48.52±10.49 44.23± 11.46
Median 49.0 45.0
LDL mg/dl
Min. – Max. 60.0 – 260.0 58.0 – 190.0
2.231* 0.032* Mean ± SD. 118.81±41.16 149.31±38.98
Median 109.0 162.0
As regards patients’ lipid profile as seen in
table (5) a significant difference was found
between the two groups as the abnormal ABI
group showed increased level of serum
cholesterol, TG and LDL than the normal
ABI group, while HDL didn’t show any
significant difference between the two
groups.
Table (6): Traditional and nontraditional Risk factors
of peripheral vascular disease in SLE patients in relation to ABI
Risk factors of PVD
ABI
Test of sig. P Normal
(n=27)
Abnormal
(n=13)
No. % No. %
Traditional
Modifiable
Obesity 3 11.1 3 23.1 χ2= 0.985 FEp= 0.370
Dyslipidemia 6 22.2 9 69.2 χ2= 8.274* FEp=0.006*
Physical inactivity 21 77.8 12 92.3 χ2= 1.283 FEp= 0.393
Non modifiable
Sex
Male 6 22.2 1 7.7 χ2= 1.283 FEp= 0.393
Female 21 77.8 12 92.3
Age (years)
Min. – Max. 10.0 – 47.0 25.0 – 52.0
t= 4.036* <0.001* Mean ± SD. 25.96±9.7 39.08±9.5
Median 23.0 44.0
Family history of CVD
Negative 19 70.4 10 76.9 χ2= 0.189 FEp= 1.000
Positive 8 29.6 3 23.1
Non Traditional
High serum creatinine 8 29.6 8 61.5 3.723 0.086
Proteinuria ≥150mg 18 66.7 8 61.5 0.101 FEp= 1.000
Positive lupus
anticoagulant antibody 1 3.7 1 7.7 0.294 FEp= 1.000
Cs therapy 24 88.9 13 100 1.562 FEp= 0.538
ACL IgGU/ml
Min. – Max. 1.40-14.80 2.80–12.8
t= 1.213 0.233 Mean ± SD. 7.87±3.47 6.47±3.27
Median 8.10 5.30
ACL IgMU/ml
Min. – Max. 2.20 – 5.80 2.10 – 9.30
t= 2.051* 0.047* Mean ± SD. 3.82±1.12 5.11±2.11
Median 3.40 5.20
Nephritis 11 40.7 6 46.2 χ2= 0.105 0.746
Homocysteineµmol/l
Min. – Max. 8.10 14.90 3.20 13.70
t= 2.995* 0.005* Mean ± SD. 10.17±1.55 11.69±1.40
Median 10.20 12.0
As shown in table (6) dyslipidemia was
detected in 69.2% of lupus patients with
abnormal ABI this showed significant
difference (p=0.006) on the contrary other
modifiable risk factors(obesity, physical
inactivity) didn’t show any significant
difference between lupus patients with
normal ABI and others with abnormal ABI.
Among the three non-modifiable traditional
risk factors for PVD in lupus (age ,sex and
family history) only age showed significant
difference as it was increased in abnormal
ABI lupus patients (p<0.001) . Regarding
nontraditional risk factors for PVD in lupus
ACL IgM U/ml was significantly higher in
abnormal ABI lupus patients (p=0.0047) also
homocysteine showed higher levels in abnormal
ABI lupus patients (p=0.005) as seen in figure
(1), while none of the following risk factors
(high serum creatinine, proteinuria>150mg,
positive lupus anticoagulant antibody, Cs
therapy, nephritis, ACL IgG) showed any
significant difference between normal and
abnormal ABI patients.
Hom
ocy
stei
ne
level
µm
ol/L
Figure (1): Relation between ABI and homocysteine in patients groups
Table (7): SLEDAI and SLICC/ACR Damage Index in SLE patients in relation to ABI.
ABI
Test of sig. P Normal
(n=27)
Abnormal
(n=13)
No. % No. %
SLEDAI
Mild (4 - 8) 0 0.0 1 7.7
χ2= 4.177 MCp= 0.066 Moderate (9 - 12) 2 7.4 3 23.1
Severe (>12) 25 92.6 9 69.2
Min. – Max. 10.0 – 29.0 8.0 – 26.0
t= 0.743 0.462 Mean ± SD. 17.22±4.85 16.0 ±4.93
Median 16.0 16.0
SLICC/ACR Damage Index
Min. – Max. 0.0 – 4.0 0.0 – 3.0
Z= 0.061 0.952 Mean ± SD. 1.04 ± 1.02 1.15 ±1.28
Median 1.0 1.0
t: Student t-test Z, p: Z and p values for Mann Whitney test
2: Chi square MC: Monte Carlo for Chi square test
*: Statistically significant at p ≤ 0.05
Table (7) as regards SLEDAI in lupus
patients with abnormal ABI 7.7% had mild
disease activity, 23.1% had moderate activity
while the majority which represented 69.2%
had severe activity. Regarding SLICC/ACR
damage index no significant difference was
found between normal and abnormal ABI
lupus patients
Table (8): Relation between ABI and 10 gram monofilament test and doppler in lupus patients ABI
Test of sig. P Normal
(n=27)
Abnormal
(n=13)
No. % No. %
10 gram monofilament test
Min. – Max. 9.0 – 9.0 8.0 – 9.0
Z =1.441 0.150 Mean ± SD. 9.0 ± 0.0 8.92± 0.28
Median 9.0 9.0
Doppler dorsalispedis
Biphasic wave 7 25.9 13 100 χ2= 19.259* <0.001*
Triphasic wave 20 74.1 0 0.0
Doppler post tibial
Biphasic wave 7 25.9 13 100 χ2= 19.259* <0.001*
Triphasic wave 20 74.1 0 0.0
As seen in table (8) all patients with
abnormal ABI had biphasic waveform in
both dorsalis pedis and posterior tibial
arteries in both limbs this showed high
significant difference between the two
groups as all normal ABI patients had
triphasic waveform in both arteries.
Table (9): Distribution of lupus patients according to (RutherFord) classification of peripheral vascular disease in
the lower extremities (n=40)
No. %
I
Asymptomatic 34 85.0
Mild claudication 3 7.5
Moderate claudication 3 7.5
severe claudication 0 0.0
II
Ischemic rest pain 0 0.0
Minor tissue loss; no healing ulcer, focal gangrene 0 0.0
III
Severe ischemic ulcer with frank gangrene 0 0.0
Figure(1): Relation between ABI and homocysteine in patients groups
As shown in table (9),figure(2) 85% of
patients were asymptomatic, 7.5% had mild
claudications,7.5% had moderate
claudications while none had severe
claudications, ischemic rest pain ,minor
tissue loss, focal gangrene nor severe
ischemic ulcer. Regarding vasculitis 10% of
the patients had vasculitic rash in one or both
lower extremity at time of examination while
none had ulcers or gangrene, three of the
four patients with vasculitic rash in L.L
were firstly presented ,their mean age was
24.4 years ,by detailed history and
investigations they were negative for
nephritis ,their SLEDAI ranged from 20 to
26 ,this denotes that they had severe disease
activity, homocysteine levels ranged from
9.7 to 12.3 µmol/L which is relatively high
compared to other patients without
vasculitis.
Discussion
Cardiovascular risk factors seem to be more
prevalent among SLE patients, in the form of
hypertension, diabetes mellitus, sedentary
lifestyle, hyperlipidemia, hypertriglyceridemia,
hyperhomocysteinemia, and even premature
menopause.(22) However, after correcting for all
predisposing cardiovascular risk factors, lupus
still qualifies as an independent atherogenic
cardiovascular risk factor.(23) Premature
atherosclerosis has been repeatedly shown to
be prevalent in SLE patients when compared
to matched population; it has been primarily
related to traditional vascular risk factors.(24)
The development of non-invasive, simple
techniques with low intra-observer and inter-
observer variability, such as the ankle-
brachial index, has facilitated the detection
of subclinical PVD. The current study
reported abnormal ABI in 32.5% of lupus
patients, while Theodoridou et al. in 2003(25)
reported abnormal ABI in 37% of lupus
patients, this slightly different percentage
could be explained by the fact that they used
different cutoff point as Theodoridou et al
considered normal ABI range from 1 up to
1.3, while in this study normal ABI ranged
from <0.9 up to 1.3 according to the current
guidelines of IWGDF.(18) Rayford R.et al.
study in 2013 (26) reported a 33% prevalence
of PVD in lupus patient this agreed with our
results of 32.5% prevalence of PVD in SLE
patients. Female sex showed significant
association with abnormal ABI in
Theodoridou et al 2003(25) study, this was not
the same finding in a study done in Iraq in
2012(27) that reported significant association
between male sex and abnormal ABI, while
this study showed no significant association
with either male or female sex. The age of
patients showed significant difference
between normal and abnormal ABI groups
(p<0.001). In this study patients were
selected to be nonsmoker as we aimed to
avoid summing the effects of the traditional
risk factors of PVD such as DM, HTN,
smoking and to assess the nontraditional risk
factors effects on PVD in lupus patients. The
current study confirms the findings of
McDonald et al 1992(28) and Theodoidou A et
al (25) 2003as longer disease duration was
associated with abnormal ABI (p=0.019). In
the current study 37.5% of them were
dyslipidemic, where 69.2% of them had
abnormal ABI, significant difference was
seen as dyslipedimia was more common in
abnormal ABI patients, these results were
close to In Bhatt SP.et al study(29). We
reported significantly increased levels of
cholesterol, triglycerides and LDL in
abnormal ABI patients with p value of
(0.001,0.009,0.032) respectively, while HDL
didn’t show any significant difference. In
this study and also in agreement with
Rayford R. et al 2013(26)no correlation was
found between ABI and the following lupus
specific risk factors; nephritis, anti ds-DNA,
SLEDAI, SLICC, family history of CVS and
family history of CVD. This study reported
that homocysteine levels was significantly
increased in lupus patients than controls with
a mean 10.67±1.65 µmol/L in patients and
6.45±1.02 µmol/L in controls and this
showed highly statistical significant
difference (p<0.001),this means that
homocysteine levels are much higher in
lupus patients than normal population ,this
agreed with a study done in 2002 by Rizk M.
And Tayel M.(30), which also confirmed
correlation between high HCY levels and
increased age of patients. SLE associated
lower extremities vasculitis was observed in
four of our patients three of them were newly
diagnosed as lupus patients, vasculitis
appeared to be their first presentation, only
one of them had abnormal ABI, their age
ranged from 18 to 38 years, relatively
younger than other lupus patients, their
homocysteine levels ranged from 9.2 to 12.9
µmol/L which was relatively higher than
other lupus patients. Intermittent
claudications was reported in six lupus
patients (15%) in this study three of the six
patients had mild claudications only one of
these three patients had abnormal ABI while
moderate claudication was reported in three
other patients , two of them had abnormal
ABI.
Conclusion
This study demonstrated that PVD is an
under-recognized problem in lupus patients.
Peripheral vascular disease is more common
among lupus patients than normal
population. -The study revealed increased
prevalence of subclinical mild form of PVD
in lupus patients evidenced by abnormal ABI
and Doppler biphasic waveform. -Both
traditional and nontraditional risk factors are
important in the pathogenesis of
atherosclerosis in SLE, PVD in lupus
patients showed significant association with
the following risk factors: older age, disease
duration, high titre of Acl IgM, high serum
TG, cholesterol, LDL levels. -Higher than
expected levels of homocysteine is detected
in lupus patients. -Abnormal ABI was
associated with higher levels of
homocysteine. -Severe disease activity was
noticed in lupus patients with abnormal ABI,
but it didn’t reachthe level of statistical
significance. -According to this study PVD
of lower externities in lupus patients is
associated with high levels of serum
homocysteine. -Owing to multiple risk
factors either traditional or non-traditional
lupus patients are more liable to develop
PVD than healthy controls.
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Original Article
Retinal Nerve Fiber Layer Thickness in Normal Egyptian Population
Mohammad A.M. El-Hifnawy1, Amir A. Abo-Samra1, Mohsen A. Abou-Shousha1, Ehab M.
Kassem1; 1Department of Ophthalmology, Faculty of Medicine, Alexandria University, Egypt
ABSTRACT Aim of the work: The aim of this study was to collect normative retinal nerve fiber layer thickness
(RNFLT) data in the Egyptian population using the Spectralis SD-OCT and to assess its correlations with
demographic data and ocular parameters. Material and methods: A total of 100 healthy eyes of 100
Egyptian individuals were enrolled in this study. Body mass index (BMI) was calculated for each subject.
Uncorrected (UCVA) and best-corrected visual acuity (BCVA) were measured and converted to the
logarithm of the minimum angle of resolution (logMAR). Autorefraction, keratometric (K) readings and
axial length (AL) were obtained. Patients underwent a comprehensive ophthalmic examination, including
cup disc ratio (CD) and intraocular pressure (IOP) measurement. RNFLT measurement by Spectralis SD-
OCT scanning was done using circular scans of the RNFL of a diameter of 3.4 mm centered on the optic
nerve head. Results: The mean global (G) RNFLT was 101.74 ± 10.05 (range 79.0 – 123.0) µm. The mean
RNFL thickness was least at the T sector followed by N, NS, NI, TS and TI sectors respectively. Using
Pearson’s correlation: age showed a significant negative correlation with T and TI sectors. BMI showed a
significant positive correlation with the G RNFLT, NS and TI sectors. Square root of Log MAR of UCVA
showed a significant negative correlation with the G RNFLT, NI and TI sectors. Spherical equivalent (SE)
showed a significant positive correlation with the G RNFLT and N sector. K-readings showed a significant
positive correlation with the G RNFLT, N, NS, TS, NI and TI sectors. IOP showed a significant negative
correlation with NS sector only. CD ratio showed a significant negative correlation with the G RNFLT, NS,
TS and TI sectors. AL showed a significant negative correlation with the G RNFLT, N, NS and TI sectors.
Using regression analysis, RNFLT was directly related to better UCVA, hyperopic refractive error and
steeper cornea and inversely related to age, IOP and CD ratio. Conclusions: The mean RNFL thickness was
least at the T sector followed by N, NS, NI, TS and TI sectors respectively. RNFLT was directly related to
better UCVA, hyperopic refractive error and steeper cornea and inversely related to age, IOP and CD ratio.
Introduction
The examination of the retinal nerve fiber
layer (RNFL) is of high importance for the
diagnosis of optic nerve anomalies and
diseases (1). Examination of the RNFL may
even be helpful to get information about the
brain, since the retinal ganglion cells and
their axons have direct contact with the brain
and can be regarded as an outpost of the
brain outside the cranial cavity (2). The RNFL
can be assessed by conventional
ophthalmoscopy, on wide-angle fundus
photographs, scanning laser polarimetry and
optical coherence tomography (OCT) (3).
OCT has evolved over the past decade as one
of the most important tests in ophthalmic
practice. It is a noninvasive imaging
technique and provides high resolution,
cross-sectional images of the retina, the
RNFL and the optic nerve head. It provides
close to an in-vivo ‘optical biopsy’ of the
retina (4). OCT is used extensively for clinical
decision making and monitoring of many
posterior segment diseases based on macular,
optic nerve, RNFL and choroidal imaging (4).
A better understanding of complex diseases,
such as glaucoma, requires assessment of the
various demographic, environmental,
genetic, and ocular factors that are believed
to be involved in their occurrence. RNFL
thickness (RNFLT) measured noninvasively
by OCT is a reliable early marker of
glaucoma risk and can predict the
development of subsequent glaucomatous
visual field defects (5). The aim of the present
study was to collect normative data in the
Egyptian population of RNFLT by using
Spectralis OCT (Spectralis OCT, Heidelberg
Engineering, Heidelberg, Germany) and to
correlate it with different demographic and
ocular parameters.
Subjects and Methods
The study included 100 healthy eyes of 100
normal adult individuals who were recruited
from the Ophthalmology Outpatient Clinic at
Alexandria Main University Hospital,
Alexandria, Egypt. Inclusion criteria were
best corrected visual acuity (BCVA) of 6/12
or better, intraocular pressure (IOP) of 21
mmHg or less, cup to disc (CD) ratio of 0.5
or less with no optic disc changes suggestive
of glaucoma and normal macular
examination on slit lamp biomicroscopy with
90-diopter lens. Exclusion criteria were
subjects suffering from diabetes mellitus,
systemic inflammatory disease, ocular
abnormalities (media opacity, retinal
pathology, glaucoma, and uveitis), recent
ocular trauma, ocular surgery, laser treatment
and congenital anomalies. In subjects with
two eligible eyes, only one eye was
randomly selected for macular thickness
analysis. The study protocol adhered to the
tenets of the Declaration of Helsinki and was
approved by the local Institutional Review
Board. All patients or their legal guardians
signed an informed consent before being
enrolled in the study. All patients were
subjected to uncorrected visual acuity
(UCVA) and BCVA measurement, body
mass index (BMI) calculation, autorefraction
(SE) and keratometric recordings (K) using
autokerato-refractometer (Topcon KR-
8100PA, Topcon Corporation, Tokyo,
Japan), slit lamp biomicroscopy including
dilated fundus examination, IOP
measurement using Goldmann applanation
tonometry, axial length (AL) measurement
using A-scan ultrasound (E-Z Scan
AB5500+, Sonomed Escalon, NY, USA) and
retinal nerve fiber layer thickness
measurement by spectral-domain OCT
(Spectralis OCT, Heidelberg Engineering,
Heidelberg, Germany). The Spectralis SD-
OCT (Heidelberg Engineering, Heidelberg,
Germany) was used to measure the RNFLT
using circular scans of the RNFL of a
diameter of 3.4 mm centered on the optic
nerve head as shown in Fig. 1. Only the
scans with a numerical quality score of more
than 16/40 decibels (db), and in the blue
range of the quality bar were collected while
scans with significant image artifacts were
excluded. The pie chart represents the
classification results for the global average
of the circle scan “G”, which is displayed in
the center, and the six standard sectors:
temporal (T), temporal-superior (TS),
temporal-inferior (TI), nasal (N), nasal-
superior (NS) and nasal-inferior (NI) sectors
( Fig. 2).
Statistical Analysis
Statistical analysis was done using IBM
SPSS statistics program (SPSS I, IBM SPSS
Statistics for Windows version 21.
International Business Machines Corporation
2012, Armonk, NY, USA) and Medcalc
program (Medcalc Software bvba, Ostend,
Belgium; https://www.medcalc.org; 2016).
Quantitative data were described by mean
and median as measures of central tendency
and Standard deviation, minimum and
maximum, while categorical variables were
summarized by frequency and percent. Pair-
wise comparisons of the mean RNFLT in the
different sectors of the RNFL map were done
using paired t-test. Pearson's correlation
coefficient was used due to large sample size
(>30. A multivariate stepwise linear
regression analysis was done after univariate
analysis to estimate the magnitude of the
association between each predictor and
different zones of retinal nerve fiber layer.
Model fit was tested by AVOVA test. R2
was calculated to explain the amount of
variance in outcome accounted by the
predictors in model and non standardized
and standardized regression coefficients (b)
were calculated to estimate the change in
outcome for one unit change in the
predictors. All statistical tests were done at
0.05 significance level.
Results
This study included 100 normal subjects.
These comprised 47 right eyes and 53 left
eyes. There were 51 males and 49 females.
The mean age of all subjects was 36.15
±14.7 (range 10-67) years. The mean BMI
was 27.8 ± 5.96 (range 19-49) kg/m2. The
majority of cases (71%) were overweight or
obese. The log MAR of UCVA was 0.14 ±
0.2 (range 0-1). The log MAR of BCVA was
0.04 ± 0.196 (range 0-0.1). The mean SE
was +0.1 ± 0.75 (range -2.75 to +2.75) D.
The mean K- readings was 43.37 ± 1.74 D
(range 40.50 – 48.31 D). The mean IOP was
14 ± 2.41 mmHg (range 10-20 mmHg). The
mean CD ratio was 0.3 ± 0.06 (range 0.2-
0.5). The mean AL was 22.98 ± 0.77 mm
(range 21.57 – 24.5 mm). The mean RNFLT
of each sector is shown in Table 1 and Fig. 3.
The mean global RNFLT (G) was 101.74 ±
10.05 µm (range 79.0 – 123.0 µm). The
mean RNFLT was least at the T sector
followed by N, NS, NI, TS and TI sectors,
respectively. However, 20 % of the cases
followed this rule precisely. On the other
hand, in the majority of cases (80 %), it was
found that one sector didn’t follow this rule.
Table (1): Retinal nerve fiber layer thickness.
Sector Range Mean ± SD. Median Lower limit
(mean -2 SD)
Upper limit
(mean +2 SD)
G 79.0 – 123.0 101.74 ± 10.05 105.0 81.64 121.84
N 53.0 – 119.0 80.0 ± 14.1 81.0 51.8 108.2
T 51.0 – 137.0 73.72 ± 11.8 71.0 50.12 97.32
NS 67.0 – 166.0 105.79 ± 21.4 102.0 62.99 148.59
TS 107.0 – 181.0 137.8 ± 17.04 138.5 103.72 171.88
NI 57.0 – 172.0 114.8 ± 26.15 112.0 62.5 167.1
TI 107.0 – 179.0 147.9 ± 19.13 152.0 109.64 186.16
G = global , N = nasal sector, T = temporal sector, NS = nasal superior sector, TS = temporal superior sector, NI = nasal
inferior sector, TI = temporal inferior sector.
Comparison of the thickness between the
different sectors showed that the temporal
sector was thinner than the nasal one.
Comparing superior and inferior sectors
showed that the nasal superior sector was
thinner than the nasal inferior one and the
temporal superior sector was thinner than the
temporal inferior one. It should be noted that
the superior sectors were significantly
thinner than the inferior ones and in the
superior and inferior sectors, the nasal part
was significantly thinner than the temporal
part (Table 1 and Fig. 3). The average RNFL
was thicker in females than in males,
however, the difference was not statistically
significant (P=0.054). The average RNFLT
was not statistically significantly different
between the right and left eyes (P=0.329).
However, in the NS sector, the RNFL was
significantly thicker in the left eyes than in
the right eyes (P=0.003). By applying
Pearson’s correlation as shown in table 2,
age showed a significant negative correlation
with the T and TI sectors. BMI showed a
significant positive correlation with the
average RNFLT, NS and TI sectors. Log
MAR of UCVA showed a significant
negative correlation with the average
RNFLT, NI and TI sectors. SE showed a
significant positive correlation with the G
RNFLT and N sector. K-readings showed a
significant positive correlation with the G
RNFLT, N, NS, TS, NI and TI sectors. IOP
showed a significant negative correlation
with NS sector only.CD ratio showed a
significant negative correlation with the G
RNFLT, NS, TS and TI sectors. AL showed
a significant negative correlation with the G
RNFLT, N, NS and TI sectors.
Table (2): Correlations between RNFLT and different personal and ocular parameters.
Parameters G N T NS TS NI TI
AGE r -0.11 0.108 -0.21 -0.035 -0.103 -0.025 -0.23
P 0.27 0.286 0.036* 0.73 0.307 0.805 0.02*
BMI r 0.25 0.157 0.097 0.214 0.058 0.092 0.285
p 0.01* 0.118 0.338 0.03* 0.56 0.364 0.004*
UCVA r -0.26 -0.139 -0.051 -0.048 -0.054 -0.33 -0.22
P 0.01* 0.167 0.613 0.637 0.59 0.001* 0.026*
BCVA r 0.056 0.147 -0.117 0.036 0.117 -0.047 0.004
P 0.578 0.058 0.245 0.725 0.247 0.639 0.97
SE r 0.21 0.3 -0.128 0.109 0.129 0.162 0.137
P 0.035* 0.003* 0.204 0.282 0.202 0.108 0.173
K r 0.41 0.332 -0.087 0.37 0.25 0.235 0.372
P 0.001* 0.001* 0.389 0.00* 0.011* 0.018* 0.000*
IOP r -0.04 0.076 0.149 -0.22 -0.181 -0.099 0.11
P 0.69 0.455 0.138 0.027* 0.071 0.325 0.276
CD r -0.27 -0.022 0.157 -0.27 -0.24 -0.187 -0.295
P 0.006* 0.828 0.118 0.007* 0.015* 0.063 0.003*
AL r -0.29 -0.33 0.101 -0.29 -0.07 -0.17 -0.26
P 0.003* 0.001* 0.317 0.003* 0.461 0.088 0.009*
G = global, N = nasal sector, T = temporal sector, NS = nasal superior sector, TS = temporal superior sector, NI = nasal
inferior sector, TI = temporal inferior sector. BMI= body mass index, UCVA = log MAR of uncorrected visual acuity,
BCVA = log MAR of best corrected visual acuity, SE = spherical equivalent, K = average keratometery, IOP =
intraocular pressure, CD = cup disc ratio, AL = axial length.
* Significant at p ≤ 0.05 level, r= Pearson’s coefficient.
By using linear regression analysis as shown
in Table 3, an increase in age by a decade
was associated with a decrease in the T
RNFLT by 1.68 µm and the TI RNFLT by
4.26 µm. An increase in square root of Log
MAR of UCVA by 0.1 (decrease in UCVA)
was associated with a decrease in the G
RNFLT by 0.969 µm and the NI RNFLT by
3.59 µm. An increase in SE by 1 D was
associated with an increase in the N RNFLT
by 3.997 µm. An increase in K-readings by 1
D was associated with an increase in the G
RNFLT by 2.5 µm, N RNFLT by 2.1 µm,
NS RNFLT by 4.74 µm, TS RNFLT by 2.05
µm, NI RNFLT by 4.48 µm and TI RNFLT
by 4.88 µm. An increase in IOP by 1 mmHg
was associated with a decrease in the NS
RNFLT by 2.5 µm. An increase in CD ratio
by 0.1 was associated with a decrease in the
TS by 5.73 µm.
Table (3): Multiple linear regression analysis.
Parameters G N T NS TS NI TI
AGE B - - -0.168 - - - -0.426
P - - 0.036* - - - <0.000*
UCVA B -9.69 - - - - -35.9 -
P <0.000* - - - - <0.000* -
SE B - 3.997 - - - - -
P - 0.034* - - - - -
K B 2.5 2.1 - 4.74 2.05 4.48 4.88
P 0.006* 0.01* - <0.000* 0.036* 0.002* <0.000*
IOP B - - - -2.5 - - -
P - - - 0.009* - - -
CD B - - - - -57.3 - -
P - - - - 0.048* - -
G = global, N = nasal sector, T = temporal sector, NS = nasal superior sector, TS = temporal superior sector, NI = nasal
inferior sector, TI = temporal inferior sector. BMI= body mass index, UCVA = square root oflog MAR of uncorrected
visual acuity, SE = spherical equivalent, K = average keratometery, IOP = intraocular pressure, CD = cup disc ratio, AL
= axial length.
* Significant at p ≤ 0.05 level, B= change in outcome per unit change in predictor.
Discussion
RNFL measurement is of utmost importance
in diagnosing and following up both ocular
and neurological diseases. OCT has
revolutionized the sensitivity and specificity
of diagnosis, follow up and response to
treatment in almost all fields of clinical
practice involving primary ocular
pathologies like glaucoma and secondary
ocular manifestations to systemic diseases
like multiple sclerosis (6). In the present
study, the mean RNFLT was 101.74 ± 10.05
(range79.0 – 123.0) µm. The mean RNFLT
was least at the T sector followed by N, NS,
NI, TS and TI sectors, respectively.
Comparison of the thickness between the
different sectors showed that the temporal
sector was significantly thinner than the
nasal one. The superior sectors were
significantly thinner than the inferior ones
and in the superior and inferior sectors, the
nasal part was thinner than the temporal part.
This indicates that the results of the present
study obeys the “ISNT” rule. Similar results
were found in several previous studies
including Zhao et al (using Spectralis SD-
OCT) (7), Budenz et al (using Stratus OCT) (8), Malik et al (using Stratus OCT III) (9),
Wang et al (using iVue-100 OCT) (10) and
Zhu et al (using iVue-100 OCT) (11) where
the mean RNFLT was 100-103 µm in these
studies. The study of Budenz et al (8) and
Malik et al (9) respected the ISNT rule while
in the study of Zhao et al (7), Wang et al (10)
and Zhu et al (11) the nasal sector was thinner
than the temporal sector. However, in the
studies of Abou Shousha and Ibrahim (using
Cirrus OCT) (12) and Pakravan et al (using
3D-OCT) (13), the mean RNFLT was thinner
than in the present study (94±7 μm and
75.50±8.38 μm respectively) and the ISNT
rule was respected in both studies. In the
study of Mansoori et al (using Spectral
OCT/SLO) (14), the mean RNFLT was thicker
than in the present study (114.03 ± 9.6 μm)
and the ISNT rule was also respected. This
difference between studies reflects difference
between machines in segmentation and
indicates that the results of different
machines should not be compared in the
same patient during follow up. It also reflects
ethnic variation, which indicates the need to
determine the normative data base for each
population. In addition, most of these studies
have included both eyes of the same patient
which could result in bias of statistical
analysis. In the present study, the RNFL was
thicker in females than in males in all
sectors, however, the difference was not
statistically significant. Similar findings were
reported by Budenz et al (8), Pakravan et al (13), Malik et al (9), Hirasawa et al (15),
Mansoori et al (14) and Rao et al (using
RTVue OCT) (16). However the RNFL was
found to be significantly thicker in females
than males in Zhao et al [7] and Abou
Shousha and Ibrahim studies (12). In the study
of Zhu et al (using iVue-100 OCT) (11) that
was carried out on children between 10-16
years, the mean RNFLT was thicker in girls
than in boys by 1.90 μm. These gender-
specific differences were statistically
significant in the temporal (2.89 μm, P <
0.0001) and inferior (3.82 μm, P < 0.0001)
quadrants but not in the superior (0.28 μm, P
= 0.42) and nasal (0.60 μm; P = 0.37)
quadrants (11). In the present study, the mean
RNFLT was not statistically significantly
different between the right and left eyes.
However, in the NS sector, the RNFL was
significantly thicker in the left eyes than in
the right eyes. Pakravan et al (13) and Budenz
et al (8) found that there was no statistically
significant difference between right and left
eyes. In the present study, the age had a
significant negative correlation with the T
and TI sectors. On multiple regression
analysis, it was found that an increase in age
by a decade was associated with a decrease
in the T RNFLT by 1.68 µm and the TI
RNFLT by 4.26 µm. A statistically
significant negative correlation between the
average RNFLT and age was noted in many
previous studies, (7-10, 12, 14, 15) in which the
RNFLT decreased by 1.16 -3.9 μm per
decade of life. In the study of Abou Shousha
and Ibrahim, The RNFL was thickest in the
age group less than 30 years, and was
thinnest in the age group more than 59 years (12). However, Pakravan et al (13), Rao et al (16)
and Zhu et al (11) found that there was no
statistically significant correlation between
average RNFLT and age. In the present
study, the BMI showed a significant positive
correlation with the average RNFLT, NS and
TI sectors. However, this correlation was
insignificant on multiple linear regression
analysis. Similar findings were reported by
previous studies (7, 10). However, Dogan et al
found that the RNFLT was significantly
thicker in type III obesity (BMI ≥ 40) than
normal weight subjects (BMI 18.5-24.99) (17). In the present study, the log MAR of
UCVA was 0.14 ± 0.2 (range 0-1). The log
MAR of BCVA was 0.04 ± 0.196 (range 0-
0.1). Log MAR of UCVA showed a
significant negative correlation with the
average RNFLT, NI and TI sectors. On
multiple regression analysis, an increase in
square root of Log MAR of UCVA by 0.1
(decrease in UCVA) was associated with a
decrease in the average RNFLT by 0.969 µm
and the NI RNFLT by 3.59 µm. In the
present study, the mean spherical equivalent
was +0.1 ± 0.75 (range -2.75 to +2.75) D. it
showed a significant positive correlation
with the average RNFLT and N sector. On
multiple linear regression analysis, an
increase in SE by 1 D was associated with an
increase in the N RNFLT by 3.997 µm.
Similar results were reported by previous
studies including Zhao et al (7), Zhu et al, (11)
and Budenz et al (8). Higher RNFLT
remained significantly associated with more
hyperopic refractive error. However, in other
studies including Pakravan et al (13) and
Wang et al (10) the RNFLT was not
significantly associated with refractive error.
In the present study, the mean keratometry
was 43.37 ± 1.74 D (range 40.50 – 48.31 D).
K-readings showed a significant positive
correlation with the average RNFLT, N, NS,
TS, NI and TI sectors. On multiple
regression analysis, an increase in K-
readings by 1 D was associated with an
increase in the average RNFLT by 2.5 µm, N
RNFLT by 2.1 µm, NS RNFLT by 4.74 µm,
TS RNFLT by 2.05 µm, NI RNFLT by 4.48
µm and TI RNFLT by 4.88 µm. Similar
results were reported by Zhao et al (7). On the
contrary, Wang et al (10) found that the
RNFLT had a significantly positive
correlation with flatter anterior corneal
curvature. However, in the study of Abou
Shousha and Ibrahim (12), the RNFLT
showed no statistically significant correlation
with the k-readings. In the present study, the
mean IOP was 14 ± 2.41 mmHg (range 10-
20 mmHg). IOP showed a significant
negative correlation with the NS sector only.
After applying linear regression analysis, an
increase in IOP by 1 mmHg was associated
with a decrease in the NS RNFLT by 2.15
µm. On the contrary, several studies
including Zhao et al (7) and Wang et al (10)
found that there was no statistically
significant effect of IOP on RNFLT. In the
present study, the mean CD ratio was 0.3 ±
0.06 (range 0.2-0.5). CD ratio showed a
significant negative correlation with the G
RNFLT, NS, TS and TI sectors. On multiple
regression analysis, an increase in CD ratio
by 0.1 was associated with a decrease in the
TS sector by 5.73 µm. On the contrary, Zhao
et al (7) found that there was no statistically
significant effect of CD ratio on RNFLT. In
the present study, the mean axial length was
22.98 ± 0.77 mm (range 21.57 – 24.5 mm).
AL showed a significant negative correlation
with the average RNFLT, N, NS and TI
sectors. On multiple regression analysis, AL
had no statistically significant effect on
RNFLT. Similar results were reported by
previous studies (7, 12, 15, 16). However, other
studies showed that the RNFLT was
inversely correlated with the axial length (8,
10, 11). Budenz et al (8) and Wang et al (10)
found that 1 mm increase of the AL was
associated with 2.2- 2.4 µm decrease in the
RNFLT.
Conclusion
The mean RNFLT was least at the T sector
followed by N, NS, NI, TS and TI sectors,
respectively. The results of the present study
obeys the “ISNT” rule. RNFLT was directly
related to better UCVA, hyperopic refractive
error and steeper corneas and inversely
related to age, IOP and CD ratio.
Figure 1: The RNFL thickness map by the Spectralis SD-OCT. The graph on the lower right of the window
presents the RNFL thickness detected along the circular scan (black curve), compared to values from the
normative database (green curve). The gray curve indicates the values of the chosen reference scan. The black
values in the pie chart (lower left) give the average RNFL thickness value for each sector as well as the global
average (G).
Figure 2: The Pie chart. The black numbers display
the measured mean RNFL thickness for global and
for each sector. The green numbers in parentheses
represent the values of the normative database. The
color-coding of the pie chart indicate whether a
specific area is “within normal limits” (green),
“outside normal limits” (red) or “borderline”
(yellow). The color-coding of the bar below the pie
chart indicates the overall classification.
Figure 3: Schematic topography of RNFL.
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Original Article
Role of Chromoendoscopy in Early Detection of Barrett's Esophagus in
some Egyptian Patients Suffering from Long Standing Gastroesophageal
Reflux Disease
Hanan Hosny Nouh1, Hanan Yehia Tayel2, Ahmed Ismail Ellakany3, Yara Mohamed Naguib
Mohamed4; 1Professor of Internal Medicine, 2 pathology department , 3 Internal Medicine
Department , 4 Internal Medicine Department ; Faculty of Medicine, University of Alexandria.
ABSTRACT Barrett's esophagus is a premalignant condition for adenocarcinoma of the esophagus. Early detection of
Barrett's metaplasia and dysplasia is very important to decrease morbidity and mortality of adenocarcinoma.
Aim of the work : : The aim of the work is to evaluate the significance of the use of chromoendoscopy in
detection of Barrett's esophagus in patients with long standing Gastroesophageal reflux disease. Material
and Methods: the study was conducted on 75 patients who gave long history of GERD or history of
Barrett's esophagus. The patients were divided into two groups: Group I: 45 patients were selected for
conventional and biopsies were taken by 4 quadrant technique then they underwent chromoendoscopy after
which biopsies were taken from stained and unstained areas. Group II: 30 patients were selected for
conventional endoscopy and biopsies were taken by 4-quadrant technique. Conventional and
chromoendoscopic assessment were compared with histopathologic examinations. Results: There was no
significant statistical difference as regards age, gender & duration of symptoms between both groups. The
sensitivityof chromoendoscopy for Barrett's epithelium was superior to that of conventional endoscopy.
Conclusion: The diagnostic accuracy of the Methylene blue directed biopsy technique was superior to that
of the random biopsy technique for identifying specialized intestinal metaplasia, but not dysplasia or
carcinoma.
Introduction
Gastroesophageal reflux disease (GERD) is a
chronic disease that is associated with a
range of troublesome symptoms such as
heartburn and regurgitation or complications
such as erosive esophagitis which can in turn
have a significant impact on health-related
quality of life and work productivity. (1)
Barrett's oesophagus (BE) is the condition in
which a metaplastic columnar mucosa (of
intestinal type), replaces an esophageal
squamous mucosa damaged by
gastroesophageal reflux disease. (2) Barrett's
esophagus is pre-dominantly seen in the age
group 55-65, with males being affected twice
as frequently as females. The disease is more
prevalent in the white population. Obesity,
smoking and alcohol intake being further
risk factors.(3) Endoscopically, the gastro-
esophageal junction is identified as the most
proximal extent of gastric folds, and the
columnar mucosa is salmon-colored and
coarse, in contrast to the pale, glassy
esophageal squamous mucosa. The extent of
esophageal columnar metaplasia determines
whether long-segment or short-segment
Barrett's esophagus (≥3 cm or <3 cm of
columnar metaplasia, respectively) is
diagnosed. (4) BE is considered a
premalignant condition because it is
associated with an increased risk of
esophageal cancer (more specifically,
adenocarcinoma). The metaplastic columnar
cells may be of two types; gastric or colonic.
A biopsy of the affected area will often
contain a mixture of both. Colonic-type
metaplasia is the type of metaplasia
associated with risk of malignancy in
genetically susceptible people. (5) The
reported prevalence of BE has been
estimated widely to be between 1.6% and
6.8% in the western hemisphere. (6) Incidence
of oesophageal adenocarcinoma (EAC) in
BE was reported in earlier studies to be
around 0.5%, but more recently it has been
found to be much lower ranging from 0.12%
to 0.39%.(7) The most appropriate method for
both diagnosis and surveillance of BE is
endoscopy. Its sensitivity is higher than other
comparative techniques, such as barium
based studies, Computerized Tomography
(CT) or Magnetic Resonance Imaging
(MRI). Endoscopic screening programs can
be beneficial in both highlighting patients
with BE from those with chronic GERD, as
well as monitoring patients with established
disease who are at risk of progressing to
adenocarcinoma of the esophagus. (8)
Although screening for BE relies largely on
established endoscopic techniques, it remains
an area of controversy for several reasons
including low prevalence and the
invasiveness of endoscopy, as well as a lack
of an easily identifiable demographic group. (8) Chromoendoscopy refers to the
topicalapplication of stains or dyes at the
time of endoscopy in an effort to enhance
tissue characterization, differentiation, or
diagnosis. The stains that are used for
chromoendoscopy are classified as
absorptive (Lugol's solutions and methylene
blue), vital (as indigo carmine), and reactive
stains (as congo red). (9) Methylene blue is a
vital stain that is readily taken up by
absorptive epithelium, primarily that of the
small bowel and colon, but not by normal
squamous or gastric epithelium. Most
chromoendoscopic studies in BE have
evaluated the role of methylene blue.
However, the use of this agent, either for the
diagnosis of Barrett’s metaplasia or for the
detection of Barrett’s dysplasia and early
cancer, remains controversial because of a
wide range of reported diagnostic
sensitivities (32%-98%) and specificities
(23%-100%).(10) It has been used primarily in
BE. (11) and, to a lesser extent, for the
detection of gastric intestinal metaplasia (12)and dysplasia in chronic ulcerative colitis. (13) Positive staining for Barrett’s intestinal
metaplasia is defined as the presence of dark
blue– stained mucosa that persists despite
vigorous irrigation, whereas staining pattern
heterogeneity and decreased stain intensity
suggest Barrett’s high-grade dysplasia or
cancer. (14) The use of MB staining in
conjunction with magnification or high-
resolution endoscopy may improve the
diagnostic yield, whereas inadequate staining
technique and inflammation may contribute
to errors in interpretation.(15)
Aim of the work
The aim of the study is to evaluate the
significance of the use of chromoendoscopy
in detection of Barrett's esophagus in patients
with long standing Gastroesophageal reflux
disease.
Subjects & Methods
This study was conducted on 75 patients who
gave long history of GERD or history of BE.
They were recruited from the
gastroenterology unit, Internal Medicine
Department at Alexandria University
Hospitals. A written informed consent was
taken from all patients and the study was
approved by local ethical committee at
Alexandria Faculty of Medicine. The
patients were divided into two groups: Group
I: 45 patients were selected for conventional
and biopsies were taken by 4 quadrant
technique then they underwent
chromoendoscopy after which biopsies were
taken from stained and unstained areas.
Group II: 30 patients were selected for
conventional endoscopy and biopsies were
taken by 4 quadrent technique. All patients
were subjected to thorough history taking as
regards age, sex, duration of symptoms,
previous ablative therapy on esophagus, or
any associated diseases,Reflux diagnostic
questionnaire and complete clinical
examination. A clean container labeled with
the patient namewas used for collecting
biopsies. Method of taking biopsies:
Principle: MB is a blue dye that is readily
taken up by intestinal-type absorptive cells in
the GIT. Chromoendoscopy of the distal
esophagus with 1% MB was performed on
45 patients and biopsies were taken from
stained and unstained areas. In other 30
patients, unstained columnar epithelium
lined esophagus was sampled by obtaining 4-
quadrant biopsy specimens at 2 cm intervals.
Procedure: All the patients were sedated
during endoscopicexamination. Removal of
surface mucus with an agent such as a 10%
solution of N-acetylcysteine by spraying it
on the Barrett's mucosa with a special
washing catheter that creates a fine mist.
Next, a 0.5% solution of MB is sprayed on
the columnar lined epithelium (CLE) before
vigorous washing with tap water. A 1- to 2-
minute wait was needed to allow the
mucolytic agent to work and also for the dye
to be absorbed. The volumes of mucolytic
agent and methylene blue dye required vary
according to the length of the columnar
mucosa being stained. The original technique
involves the use of approximately 10 mL of
acetylcysteine and 20 mL of methylene blue
dye for every 5 cm of circumferential CLE.
The endpoint of staining is the point at which
the surrounding or adjacent squamous
epithelium is free of dye and the staining
pattern within the CLE appearsstable.
Positive staining is defined as the presence of
blue-stained, non-eroded mucosa that
persists despite vigorous water irrigation.
MB staining adds an average of 5 to 7
minutes to the procedure time. All of the
biopsies were fixed immediately with 80%
alcohol. Biopsies are embedded with
paraffin. Serial sections were made and
stained with H&E for histopathological
analysis. These slides were coded and
evaluated by the pathologist, and the code
was broken after all of the histopathological
analyses were completed. Histopathological
diagnoses and evaluations were made
according to the cellular morphological
changes and tissue architecture. The
epithelium was graded as normal,
esophagitis, Barrett's Esophagus
(intestinalmetaplasia), dysplasia and
carcinoma (adenocarcinoma or squamous
cell carcinoma). Conventional endoscopic or
chromoendoscopic diagnoses were compared
with histopathologic diagnosis. The study
was conducted in accordance with the ethical
guidelines of the 1975 Declaration of
Helsiniki and informed consent was obtained
from each patient.
Statistical Analysis
Data were checked, entered, and analyzed
using the SPSS 18 software package (SPSS
Inc., Chicago, Illinois, USA). The normally
distributed data were expressed as mean ±
SD. Multiple group comparisons were
performed by one-way analysis of variance.
Univariate correlations between study
variables were calculated with Spearman's
rank correlation coefficients (r). P-values
less than 0.05 were considered significant.
Results
The demographic data of the 45 patients of
group I showed that 32 (71.1%) were males
and 13 (28.9 %) were females with mean (±
SD) age of 45.84 ± 11.24 years. The
demographic data of the 30 patients of group
II showed that 20 (66.7 %) were males and
10 (33.3 %) were females with mean (± SD)
age of 44.53 ± 7.93 years. (Table 1). As
regards duration of symptoms;In group I, the
mean duration of symptoms of GERD (±
SD) was 3.89 ± 0.78 years. In group II, the
mean duration of symptoms of GERD (±
SD) was 3.77 ± 0.68 years. There was no
statistically significant difference between
both groups as regards duration of symptoms
(p.value0.529)(Table 2). As regards
symptoms; In group I 35 patients had heart
burn, 35 patients had regurgitation, 7 patients
had dysphagia, 13 had nausea, 3 patients had
upper GI bleeding and 12 patients had extra-
esophageal symptoms. In group II 20
patients had heart burn, 22 patients had
regurgitation, 3 patients had dysphagia, 10
had nausea, 2 patients had upper GI bleeding
and 10 patients had extra-esophageal
symptoms. Comparison between all groups
as regards symptoms was statistically
insignificant. (Table 3). As regards
endoscopic finding, 50 patients from the
studied 75 patients had Incompetent GEJ 23
patients had sliding hiatal hernia the 75
patients had different grades of reflux
esophagitis from grade A to D according to
LA classification, three patient was
presented by lower esophageal ulceration.
Comparison between studied groups
regarding endoscopic findings was
statistically insignificant. (Table 4). As
regards histopathological data;Two cases of
esophageal adenocarcinoma were found
during this study. In comparison between
group I and group II, we found that in group
I there was 4 patients showed normal
stratified squamous epithelium, 12 had
esophagitis with normal normal stratified
squamous epithelium, 10 patients had
esophagitis with metaplastic columnar
epithelium without goblet cells, 8 patients
had Barrett's metaplasia, 5 patients had
Barrett's with low grade dysplasia, 4 patients
had Barrett's with high grade dysplasia. In
group II, there was 6 patients showed normal
stratified squamous epithelium, 11 had
esophagitis with normal normal stratified
squamous epithelium, 5 patients had
esophagitis with metaplastic columnar
epithelium without goblet cells, 4 patients
had Barrett's metaplasia, 2 patients had
Barrett's with low grade dysplasia, 2 patients
had Barrett's with high grade dysplasia.
(Table 5). In comparison between methylene
blue target biopsy and conventional biopsy
in the same group I, we found that in
methylene blue target biopsy there was 4
patients showed normal stratified squamous
epithelium, 12 had esophagitis with normal
stratified squamous epithelium, 10 patients
had esophagitis with metaplastic columnar
epithelium without goblet cells, 8 patients
had Barrett's metaplasia, 5 patients had
Barrett's with low grade dysplasia, 4 patients
had Barrett's with high grade dysplasia and 2
cases with adenocarcinoma. In conventional
biopsy, there was 7 patients showed normal
stratified squamous epithelium, 17 had
esophagitis with normal normal stratified
squamous epithelium, 8 patients had
esophagitis with metaplastic columnar
epithelium without goblet cells, 5 patients
had Barrett's metaplasia, 4 patients had
Barrett's with low grade dysplasia, 2 patients
had Barrett's with high grade dysplasia and 2
cases of adenocarcinoma. (Table 6). In group
(I), MB targeted biopsies showed a higher
sensitivity (100%) than conventional
biopsies (68.42%). while specificity of the
two endoscopies was the same
(100%).(Table 7) The diagnostic accuracy of
the Methylene blue directed biopsy
technique was superior to that of the random
biopsy technique for identifying specialized
intestinal metaplasia, but not dysplasia or
carcinoma.
Table 1: Comparison between the two studied groups according to demographic data
Group I (n = 45) Group II (n = 30)
Test of Sig. p No. % No. %
Sex
Female 13 28.9 10 33.3 2= 0.683
Male 32 71.1 20 66.7
Age
Min. – Max. 26.0 – 73.0 29.0 – 59.0
t= 0.553 0.582 Mean ± SD. 45.84 ± 11.24 44.53 ± 7.93
Median 46.0 44.0
Table 2: Comparison between the two studied groups according to duration of symptoms
Group I (n = 45) Group II (n = 30) MW p
Duration of symptoms (Years)
Min. – Max. 3.0 – 5.0 3.0 – 5.0
0.630 0.529 Mean ± SD. 3.89 ± 0.78 3.77 ± 0.68
Median 4.0 4.0
Table (3): Comparison between the two studied groups according to symptoms
Symptoms Group I (n = 45) Group II (n = 30)
p No. % No. %
Heart Burn 35 77.8 20 66.7 1.136 0.286
Regurgitation 35 77.8 22 73.3 0.195 0.659
Dysphagia 7 15.6 3 10.0 0.481 FEp=0.731
Nausea 13 28.9 10 33.3 0.167 0.683
GI Bleeding 3 6.7 2 6.7 0.000 FEp=1.000
Extra esophageal 12 26.7 10 33.3 0.386 0.534
Table (4): Comparison between the two studied groups according to endoscopic finding
Endoscopic finding Group I (n = 45) Group II (n = 30)
2 p No. % No. %
Incompetent GEJ 33 73.3 17 56.7 2.250 0.134
Hiatal hernia 18 40.0 5 16.7 4.609* 0.032*
LA-A 13 28.9 11 36.7 0.500 0.479
LA-B 22 48.9 13 43.3 0.223 0.637
LA-C 9 20.0 6 20.0 0.000 1.000
LA-D 1 2.2 1 3.3 0.086 FEp=1.000
Esophageal ulcer 2 4.4 1 3.3 0.058 FEp=1.000
Esophageal stricture 0 0.0 0 0.0 - -
Table (5): Comparison between the two studied groups according to histopathology:
Histopathology Group I (n = 45) Group II (n = 30)
2 p No. % No. %
Normal str.sq.epi 4 8.9 6 20.0 FEp=0.185
Esophagitis with normal st.sq.epi 12 26.7 11 36.7 0.358
Esophagitis with metaplastic columnar
epithelium (without goblet cells) 10 22.2 5 16.7 0.347 0.556
Barrett's metaplasia 8 17.8 4 13.3 0.265 0.607
Barrett's with low grade dysplasia 5 11.1 2 6.7 0.420 FEp=0.695
Barrett's with high grade dysplasia 4 8.9 2 6.7 0.121 FEp=1.000
Adenocarcinoma 2 4.4 0 0.0 1.370 FEp=0.514
Table (6): Comparison between methylene blue target biopsy and four quadrant biopsy in the same group I
Histopathology
Methylene blue
target biopsy
(n = 45)
Conventional
biopsy (n = 45) 2 p
No. % No. %
Normal str.sq.epi 4 8.9 7 15.6 0.334
Esophagitis with normal st.sq.epi 12 26.7 17 37.8 0.259
Esophagitis with met aplastic
columnar epithelium 10 22.2 8 17.8 2.000 0.157
Barrett's metaplasia 8 17.8 5 11.1 0.809 0.368
Barrett's with low grade dysplasia 5 11.1 4 8.9 0.123 FEp=1.000
Barrett's with high grade dysplasia 4 8.9 2 4.4 0.714 FEp=0.677
Adenocarcinoma 2 4.4 2 4.4 0.000 FEp=1.000
Table (7): Comparison between sensitivity of conventional and chromoendoscopy in the same group I.
Histopathology
Sensitivity Specificity PPV NPV Non Barrett (n=26) Barrett (n=19)
Methylene blue target biopsy
Non Barrett 26 0 100.0 100.0 100.0 100.0
Barrett 0 19
Conventional biopsy
Non Barrett 26 6 68.42 100.0 100.0 81.25
Barrett 0 13
Discussion In Barrett's esophagus, the stratified
squamous epithelium that normally lines the
distal oesophagus is replaced by an abnormal
columnar epithelium that has intestinal
features. It is found in 6% to 18% of patients
undergoing upper GI endoscopy for
symptoms of reflux disease. The abnormal
epithelium (called specialized intestinal
metaplasia) usually shows evidence of DNA
damage that predisposes to malignancy.(16)
After the first destruction of squamous
mucosa by gastric acid or bile, the second re-
epithelization of the lower esophagus may be
fulfilled by pluripotent basal cells,which may
be the progenitors of Barrett's epithelium.(17)
The groups at high risk for BE mainly
consist of patients with GERD especially
those with long duration and increased
frequency of symptoms due to esophageal
dysmotility, patients above 50 years, obesity,
alcohol use, smokers, and patients with large
hiatal hernia.(18)These are well known
precursors of esophageal adenocarcinoma
with a risk of 18% for low- grade dysplasia
and 34% for high grade dysplasia.(19) In our
study, a total of 75 patients were enrolled,
they are divided into two groups: Group I: 45
patients were selected for conventional and
biopsies were taken by 4 quadrant technique
then they underwent chromoendoscopy after
which biopsies were taken from stained and
unstained areas. Group II: 30 patients were
selected for conventional endoscopy and
biopsies were taken by random 4 quadrant
technique. Conventional and chromo
endoscopic assessments were compared
according to histopathological examination.
We found that the sensitivity of chromo
endoscopy for Barrett's epithelium was
superior to that of conventional endoscopy,
while there were no statistical difference
between specificity of the twomethod. These
finding are in agreement with (N
Ormeci,et.al 2008) that was a performed
study 109 patients and the sensitivity of
chromoendoscopy for Barrett’s epithelium
was superior to that of conventional
endoscopy (p < 0.05). However, there was
no statistical difference between the two
methods in the diagnosis of esophagitis or
esophageal carcinoma (p > 0.05). Stained
biopsies were superior to unstained biopsies
in terms of sensitivity for Barrett’s
epithelium and esophageal carcinoma (p <
0.001).(20) Multiple studies KiesslichR,et.
Al.(21); Kouklakis GS,et.al. (22); Ragunath
K,et.al.(23) reported high sensitivity (91%-
98%) and variable specificity (43%-97%),
whereas 2 small studies Breyer HP, et.al (24);
Dave U,et.al. (25) reported unsatisfactory
results (sensitivity 53%-70% ; specificity
32%-51%). Difference in the study design,
the technique of MB staining, the
interpretation of staining pattern, and the
endoscopist experience with vital staining
have contributed to the difference in the
results. To improve the technique,
endoscopists have used high-magnification
endoscopy, together with MB staining to
improve the characterization of the
esophageal mucosal pit pattern and to
increase the specificity for the specificity for
detection of BE to 92%to 100% according to
Endo T, et.al . (26) Kiesslich et.al. (21) found
that the sensitivity and specificity of MB
were 98% and 61% respectively. However
wo et al 2001 (27) found no statistical
difference in sensitivity and specificity
between conventional and chromoendoscopy
examination for diagnosis of esophageal
carcinoma. In this study, we found that the
sensitivity of MB staining for diagnosis of
Barrett's epithelium was 100% compared to
sensitivity of conventional biopsies that was
68.42% in group I. Even more controversial
is the role of MB chromoendoscopy for
improving the diagnosis of dysplasia in BE.
Two studies (canto MI et.al. 2000 (28);
GossnerL,et.al (29) showed MB directed
biopsy to be significantly better than random
biopsy for the diagnosis of dysplasia and
require fewer biopsies, but 2 others (
Ragunath K et.al. (23); Wo J, et.al (27) ) did
not confirm these results as these two studies
reported that MB directed biopsy is similar
conventional biopsy in detection of SIM. So,
our study confirm the finding of (Ragunath
K et.al. (23) and Wo J, et.al . (27)We found,
among the studied 45 patients in group I, 8
cases of barrett's esophagus without
dysplasia , 5 cases of low-grade dysplasia , 4
cases of high-grade dysplasia and 2 cases of
adenocarcinoma in histopathological
examination of MB targeted biopsies taken
during chromoendoscopy, while 4 cases of
barrett's esophagus without dysplasia , 2
cases of low-grade dysplasia, 2 cases high-
grade dysplasia and 2 cases of
adenocarcinoma in histopathological
examination of 4-quadrant biopsies taken
during conventional endoscopy which was
statistically insignificant (p > 0.05) We
found that both techniques were equally
effective in identifying histological BE with
slightlyhigher sensitivity of chromo
endoscopy than conventional endoscopy.
Sharma p et.al (30) reported the result of a
study that compared methylene blue-directed
biopsy versus conventional four-quadrant
biopsyfor detection of IM in patients with
suspected SSBE (columnar-appearing
mucosa <3 cm in length). It stated that MB
chromoendoscopy significantly increases the
detection of IM and requires fewer biopsies
in patients with suspected SSBE with greater
than 1 cm of columnar-appearing mucosa. It
does not appear to be beneficial in patients
with irregular Z lines (<1 cm). Horwhat et.al (31) reported the result of a study that
compared methylene blue-directed biopsy
versus conventional four-quadrant biopsy for
the detection of intestinal metaplasia and
dysplasia in patients with long-segment
Barrett's esophagus (LSBE). The
investigators have found that the sensitivity
of MB for SIM and dysplasia was 75.2% and
83.1%, respectively. The yield of 4QB for
identifying non dysplasia SIM was 57.6%
and for dysplasia was 12%. It stated that MB
may help to define areas to target for biopsy
during surveillance endoscopy in patients
with LSBE. Ngamruengphang et.al (32)
evaluated the diagnostic yield of methylene
blue chromoendoscopy for detecting
specialized intestinal metaplasia & dysplasia
in Barrett's esophagus. It stated that The
technique of MB chromoendoscopy has only
a comparable yield with RB for the detection
of SIM and dysplasia during endoscopic
evaluation of patients with BE. But there was
limitation for the study that only data on MB
were analyzed because of limited availability
of data for other chromoendoscopy dyes,
minor variations in inclusion and exclusion
criteria, & the small sample size, & because
differences in application technique could
have led to an underestimation of the
diagnostic yield of MB chromoendoscopy. In
conclusion, The diagnostic accuracy of the
Methylene blue directed biopsy technique
was superior to that of the random biopsy
technique for identifying specialized
intestinal metaplasia, but not dysplasia or
carcinoma. Conflicts of interest: There are no
conflicts of interest.
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population-based study. Journal of the National
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8. Ramus JR, Gatenby PA, Caygill CP,
WinsletMC, Watson A. Surveillance of Barrett's
columnar-lined oesophagus in the UK:
endoscopic intervals and frequency of detection
of dysplasia. European journal of
gastroenterology &hepatology. 2009 Jun 1;
21(6):636-41.
9. Trivedi PJ, Braden B. Indications, stains and
techniques in chromoendoscopy. QJM. 2012 Oct
24:hcs186.
10. Amano Y, Kushiyama Y, Ishihara S, Yuki T,
Miyaoka Y, Yoshino N, Ishimura N, Fujishiro H,
Adachi K, Maruyama R, Rumi MA. Crystal
violet chromoendoscopy with mucosal pit pattern
diagnosis is useful for surveillance of short-
segment Barrett's esophagus. The American
journal of gastroenterology. 2005 Jan 1;
100(1):21-6.
11. Canto MI, Setrakian S, Willis J, Chak A,
Petras R, Powe NR, Sivak MV. Methylene blue–
directed biopsies improve detection of intestinal
metaplasia and dysplasia in Barrett's esophagus.
Gastrointestinal endoscopy. 2000 May 31; 51(5):
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12. Dinis-Ribeiro M, da Costa-Pereira A, Lopes
C, Lara-Santos L, Guilherme M, Moreira-Dias L,
Lomba-Viana H, Ribeiro A, Santos C, Soares J,
Mesquita N. Magnification chromoendoscopy for
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13. Kiesslich R, Fritsch J, Holtmann M, Koehler
HH, Stolte M, Kanzler S, Nafe B, Jung M, Galle
PR, Neurath MF. Methylene blue-aided
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Ohtsu A, Yoshida S. Association of multiple
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Original Article
Some Non Invasive Methods in Detection and Grading of Oesophageal
Varicse in Splenectomized Cirrhotic Patients
Alaa El-Din Mohamad Abdo1, Akram Abd El-Moneim Deghady2, Ehab Hassan El-kholy1, Abd
El-Kader Hassan Abd El-Kader; 1Department of Tropical Medicine; Faculty of Medicine,
University of Alexandria, 2Department of clinical and chemical pathology; Faculty of Medicine,
University of Alexandria
ABSTRACT Gastroesophageal varices are the most relevant portosystemic collaterals. It would be impossible to perform
endoscopic examinations at regular interval for all patients with liver cirrhosis and it may be more cost-
effective to routinely screen only cirrhotic patients at high risk for the presence of varices. Aim of the work
is to study the value of serum zinc and homeostasis model assessment of insulin resistance (HOMA-IR), as
non- invasive markers in detection and grading of esophageal varices in splenectomized cirrhotic patients.
Material and Methods: This study was done on 50 cirrhotic patients divided into group I: 25
splenectomized and group II: 25 non-splenectomized patients. All patients were subjected to full history
taking, physical examination, laboratory investigations (complete blood count, liver function tests, serum
zinc, fasting plasma glucose and fasting insulin with calculation of (HOMA-IR)), abdominal
ultrasonography and Esophagogastroduodenoscopy (EGD). Results: A HOMA-IR cut off value (COV) of
3.3 could differentiate between cirrhotic with no EV and cirrhotic with EV with a sensitivity of 73% and a
specificity of 71% in splenectomized patients and a cut off value of 3.4 with a sensitivity of 71% and a
specificity of 82% in non splenectomized patients. Serum zinc cut off value (COV) of 61.5μg/dl could
differentiate between cirrhotic with no EV and cirrhotic with EV with a sensitivity of 63% and a specificity
of 65% in splenectomized patients and a cut off value of 59.5μg/dl with a sensitivity of 64% and a
specificity of 73% in non splenectomized patients. Conclusions: Serum zinc and HOMA-IR could be useful
non-invasive markers for detection of the presence of esophageal varices.
Introduction
Cirrhosis results from different mechanisms of
liver injury that lead to necroinflammation and
fibrogenesis; histologically it is characterized by
diffuse nodular regeneration surrounded by
dense fibrotic septa with subsequent
parenchymal extinction and collapse of liver
structures, together causing pronounced
distortion of hepatic vascular architecture.(1) This
distortion results in increased resistance to portal
blood flow and hence in portal hypertension and
in hepatic synthetic dysfunction. Cirrhosis often
is a silent disease, with most patients remaining
asymptomatic until decompensation occurs.(2)
Complications of cirrhosis are Portal
hypertension, Varices, variceal bleeding, ascites,
hepatorenal syndrome, hepatic encephalopathy,
and spontaneous bacterial peritonitis. The most
common prognostic tool used in patients with
cirrhosis was the Child-Turcotte-Pugh (CTP).(3)
Portal hypertension is defined as elevation of
hepatic venous pressure gradient
(HVPG).(4) Clinically significant portal
hypertension is defined as a hepatic venous
pressure gradient of 10 mm Hg or more.(5)
This threshold is required for the
development of complication of PHT, such
as porto-systemic collaterals, varices, ascites
and circulatory dysfunction. EV are Porto-
systemic collaterals i.e., vascular channels that
link the portal venous and the systemic venous
circulation. Upper endoscopy remains the gold
standard for diagnosis of esophageal varices. It
is recommended that all patients undergo
endoscopy to assess the presence and the size
of varices at the time of the diagnosis of
cirrhosis. Endoscopic examination is
considered an invasive procedure. Moreover,
sedation of a cirrhotic patient to perform
endoscopy may be hazardous.(6) To reduce the
number of endoscopies, many studies were
carried out to identify features that may
noninvasively predict the presence of EV of
any size, or at least of medium-large size at
higher risk of bleeding.(7) Different published
clinical studies had suggested that IR is able to
independently predict the presence of
gastroesophageal varices in cirrhosis.(8,9) The
authors attributed this effect to a possible
pathophysiological link between IR and PH.
The homeostatic model assessment (HOMA)
is a validated method to measure insulin
resistance from fasting glucose and insulin. In
patients with chronic liver disease (CLD), the
blood zinc concentrations were lower in
patients with liver cirrhosis or hepatocellular
carcinoma than in patients with chronic
hepatitis, leading to hypothesize that the
reduced blood zinc value could be associated
with the formation and progression of
varices.(10)
Patients and Methods
In the present study cases were selected from
the Endoscopy unit of the medical Research
Institute during the year 2015. The extensive
exclusion criteria were as follow: presence of
diabetes mellitus, hypertension, renal
disease, haemochromatosis, history of
esophageal bleeding, sclerotherapy or band
ligation, and hepatocellular carcinoma. All
patients were subjected to full history taking,
physical examination, laboratory
investigations (complete blood count, liver
function tests, serum zinc, fasting plasma
glucose and fasting insulin with calculation
of (HOMA-IR)), abdominal ultrasonography
and Esophagogastroduodenoscopy (EGD).
HOMA-IR calculated using the following
equation: HOMA-IR=Fasting insulin
(μU/mL) ×fasting glucose (mg/dl)/405.(11)
Results
In the present study, group I without EV
included 12 (85.7%) males and 2 (14.3%)
females, their age ranged between 39-
61years with a mean of 50.6 ± 6.7.while
group I with EV included 9 (81.8%) males
and 2 (18.2%) females, their age ranged
between 40-61 years with a mean of 50.1±
6.3. Group II without EV included 6 (54.5%)
males and 5 (45.5%) females, their age
ranged between38-63 years with a mean of
50.2 ±7.8. while group II with EV included
11(78.6%) males and 3 (21.4%) females,
their age ranged between 40-62 years with a
mean of 51.8 ± 6.0. There was no
statistically significant difference between
the patients without or with EV in both
group I and group II according to age and
sex. According to the clinical data (Jaundice,
hepatic encephalopathy, ascites, spider
angioma, and palmer erythema) there was no
statistically significant difference between
patients without or with EV in both group I and
group II. There was statistically significant
relation between BMI, portal vein diameter,
child class, low platelet count, low serum
albumin and esophageal varices presence in
both group I and group II. The Homeostasis
model assessment of insulin resistance
(HOMA-IR) ranged between 1.2 to 4.2 with a
mean of 2.6 ± 1 and from 2.2 to 5.1 with a
mean of 3.7 ± 0.9 in group I without and with
EV respectively. There was statistically
significant difference between the patients
without and with EV of group I. Also it ranged
between 1.7 to 4.4 g/dl with a mean of 2.7 ±
0.8 and from 2 to 6.2 with a mean of 3.9± 1.2
in group II without and with EV respectively.
There was statistically significant difference
between patients without and with EV of group
II.
Table (1): Comparison between the studied groups according to fasting blood sugar, fasting insulin, HOMA-IR
and serum Zinc
Parameter
Group I
t (P)
Group II
t (P) Esophageal varices Esophageal varices
No Yes No Yes
FBS(mg/dl)
1.2 (0.239)
1.0 (0.323)
Minimum 70.0 69.0 78.0 68.0
Maximum 107.0 107.0 115.0 110.0
Mean 86.3 92.3 93.4 97.9
SD 11.9 12.8 11.4 11.1
Insulin(µU/mL)
2.7 (0.013)*
2.4 (0.024)*
Minimum 6.0 9.0 8.0 8.0
Maximum 19.0 20.0 21.0 26.0
Mean 12.3 16.5 11.8 16.2
SD 4.1 3.5 3.7 5.4
HOMA_IR
2.8 (0.010)*
2.7 (0.012)*
Minimum 1.2 2.2 1.7 2.0
Maximum 4.2 5.1 4.4 6.2
Mean 2.6 3.7 2.7 3.9
SD 1.0 0.9 0.8 1.2
Zinc(μg/dl)
2.0 (0.050)*
2.1 (0.048)*
Minimum 50.0 43.0 49.0 43.0
Maximum 89.0 71.0 87.0 72.0
Mean 65.9 58.5 65.0 57.1
SD 10.9 9.4 11.0 10.1
t: Independent sample t-test * P < 0.05 (significant)
The diagnostic performance of HOMA-IR was
compared to that of endoscopy which is
considered the gold standard in diagnosis of
esophageal varices. The receiver operator
characteristic (ROC) curve analysis generated
a cut off value (COV) of 3.3 that could
differentiate between cirrhotic with no EV and
cirrhotic with EV with area under the curve of
0.792 with a sensitivity of 73% and a
specificity of 71% in splenectomized patients
and a cut off value of 3.4 with area under the
curve of 0.799 with a sensitivity of 71% and a
specificity of 82% in non splenectomized
patients.
Fig. (1): ROC curve for HOMA-IR to differentiate
between cirrhotic with no EV and cirrhotic with EV in
splenectomized group.
Fig. (2): ROC curve for HOMA-IR to differentiate
between cirrhotic with no EV and cirrhotic with EV in
non splenectomized group.
Serum zinc level ranged between 50 to 89μg/dl
with a mean of 65.9 ± 10.9μg/dl and from 43 to
71μg/dl with a mean of 58.5 ± 9.4 mg/dl in
group I without and with EV respectively. There
was statistically significant difference between
patients without and with EV of group I. Also it
ranged between 49 to 87 μg /dl with a mean of
65 ± 11μg/dl and from 43 to 72μg/dl with a
mean of 57.1± 10.1μg/dl in group II without and
with EV respectively. There was statistically
significant difference between both patients
without and with EV of group II. The receiver
operator characteristic (ROC) curve analysis
generated a cut off value (COV) of 61.5μg/dl
that could differentiate between cirrhotic with
no EV and cirrhotic with EV with area under the
curve of 0.7with a sensitivity of 63% and a
specificity of 65% in splenectomized patients
and a cut off value of 59.5μg/dl with area under
the curve of 0.7 with a sensitivity of 64% and a
specificity of 73% in non splenectomized
patients.
Fig. (3): ROC curve for serum zinc to differentiate
between cirrhotic with no EV and cirrhotic with EV in
non splenectomized group.
Fig. (4): ROC curve for serum zinc to differentiate
between cirrhotic with no EV and cirrhotic with EV in
non splenectomized group.
Discussion
The current study assessed insulin resistance
and serum zinc as noninvasive parameter for
detection and grading of esophageal varices
in splenectomized cirrhotic patients. The
HOMA-IR has proved to be a potent tool for
the assessment of IR.(12,13) and it is the most
frequent technique both in clinical practice
and in epidemiological studies due to its
simplicity in the determination and
calculation of insulin resistance. However,
there is great variability in the threshold
HOMA-IR levels to define IR. Population
based studies for defining cut-off values of
HOMA-IR for the diagnosis of IR had been
conducted in different geographic areas.(14) In
our study HOMA-IR score for measuring
insulin resistance was found higher in patients
with varices than patients without varices in
splenectomized and non splenectomized
patients. Statistically there was significant
relation between high HOMA-IR score and
esophageal varices presence (p = 0.010 in
splenectomized group and p = 0.012 in non
splenectomized gruop). This result agreed with
Camma` et al(15) who concluded that in patients
with Child A cirrhosis because of hepatitis C
virus, the platelet/spleen ratio and insulin
resistance as measured by the homeostasis
model assessment of insulin resistance,
regardless of the presence of diabetes,
significantly predict the presence of esophageal
varices, outweighing the contribution given by
transient elastography. Camma` et al (15)
validated cut off value to predict the presence
of EV was ˃3.5 with specificity of 76% and
sensitivity of 61%. A multi-centered study was
done by Eslam et al (16) who concluded that in
cirrhotic patients HOMA score correlated with
HVPG and independently predict clinical
outcomes. Also concluded that platelet count,
IR assessed by HOMA-IR and adiponectin
significantly predicted the presence of
esophageal varices. This study validated a cut
off value > 4 for HOMA-IR to predict the
portal hypertension and the variceal bleeding
with a sensitivity of 89.7% and a specificity of
71%. In a study done by Erice et al(17) to
investigate the potential relationship between
IR and hepatic and systemic hemodynamics in
patients with cirrhosis, they found that non
diabetic patients diagnosed with liver cirrhosis
and CSPH had increased HOMA-IR and the
HOMA-IR may have potential for the
noninvasive prediction of CSPH and as a
consequence of gastroesophageal varices. The
liver plays an important role in zinc
homeostasis and different zinc compartments
have been recognized to explain zinc kinetics
in humans; the liver represents a fast-
exchangeable zinc pool with an important
role in the metabolism of zinc and other trace
elements.(18) Interestingly, supplementation
with zinc has been shown to improve the
prognosis of cirrhotic patients, as well as the
cirrhosis-related symptoms.(19,20) In patients
receiving oral supplementation with zinc,
maintenance of the serum zinc concentration
at more than 80 μg/dl was the most important
factor associated with cancer-free
survival.(20) In the present study serum zinc
was significantly lower in cirrhotic patients
with E.V than cirrhotic patients without
esophageal varices in both splenectomized and
non splenectomized patients. Iwata et al.(20)
conducted a study on 75 patients with
compensated cirrhosis Similar to our result
they found that the zinc value was associated
with the histological progression of liver
fibrosis and the severity of esophageal varices
in virus related compensated cirrhosis and
validated that the serum zinc cut off value of
59.0 μg/dl for differentiation between patients
with or without EV and serum zinc cut off
value of 56.0 μg/dl for differentiation between
patients with or without high risk EV. Atia et al (21) studied 100 adults, to observe the
association of serum zinc level with liver
cirrhosis. They found that Serum zinc level
was low in 72% of patients. Mean ±SD of
serum zinc levels (μg/L) were 610.32 ± 169.60
and 827.66 ± 267.32 in cases and controls
respectively. In cirrhotic patients serum zinc
level was significantly lower than that of
healthy controls (P<0.001).
Conclusion
In conclusion, Serum zinc and HOMA-IR
could be useful non-invasive markers for
detection of the presence esophageal varices.
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cirrhosis. Hepatology. 2009;49(1):195-203.
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Original Article
Study of Serum Vitamin D3 Levels in Rheumatoid Arthritis Patients and
its Relation with Disease Activity and CD46 Activity
Ashraf El Zawawy,1 Eman Hassan,1 Hanaa Ali,2 Nehad Hussein1; Department of internal
medicine,1 Department of clinical pathology,2 Faculty of Medicine, University of Alexandria.
ABSTRACT Vitamin D deficiency has been found to be associated with disease activity in patients with RA (Rheumatoid
Arthritis). Vitamin D also can exert a direct effect on T cells, and may be beneficial in several pathologies,
including RA. Calcitriol affects the CD46 pathway, and that it promotes anti-inflammatory responses
mediated by CD46. Aim of the work is to measure the level of Vitamin D3 in the serum of patients with
Rheumatoid Arthritis and correlate it with disease parameters specially disease activity and CD46 activity.
Material and methods : We included 40 RA patients fulfilling the American College of Rheumatology /
European League Against Rheumatism (ACR/EULAR) 2010 classification criteria of RA and 20 age ,sex,
diet intake and sun exposure duration matched healthy individuals. We excluded Patients on vitamin D
supplement. Results t serum vitamin D was lower in the rheumatoid cases group than in the control group.
There was statistically significant negative correlation between vitamin D and tender joint count, swollen
joint count, DAS28 score and CD 46 activity on lymphocytes and monocytes. Conclusions: Vitamin D
deficiency is highly prevalent in patients with RA which indicates that it has role in its
pathogenesis. . Vitamin D level is significantly correlated with disease activity as expressed by
DAS 28 (ESR) score. . CD46 expression is increased in rheumatoid arthritis patients who have
lower levels of vitamin D.
Introduction Rheumatoid arthritis (RA) is a chronic
systemic autoimmune inflammatory disease
that affects all ethnic groups throughout the
world. Females are 2.5 times more likely to
be affected than males (1). It results from a
complex interaction between genes and
environment, leading to a breakdown of
immune tolerance and synovial inflammation
in a characteristic symmetric pattern.
Distinct mechanisms regulate inflammation
and matrix destruction, including damage to
bone and cartilage.(2) The complement
system, when not regulated, can cause tissue
damage. This condition can be induced by
pro-inflammatory mechanisms, such as
cytokines and chemokines. These are usually
up-regulated in RA, indicating that these
patients are at increased risk to damage
mediated by the complement system. To
counteract or contain self-damage, the
complement system has a variety of
regulators which can be membrane-bound or
secretory proteins, which appear to be more
or less efficient in distinct conditions.
Normal cells resist complement-mediated
lysis by several mechanisms such as specific
membrane-bound proteins. Examples of
these are the decay accelerating factor
(CD55), the membrane inhibitor of reactive
lysis or protectin (CD59), the membrane
cofactor protein (CD46) and the complement
receptor type I (CD35).(3)Importantly, the
CD46 pathway is altered in an increasing
number of human pathologies. A defect in
IL-10 production was first identified in
patients with MS, followed by a report of
defective IL-10 production in patients with
asthma and altered cytokine production was
shown in patients with rheumatoid arthritis.(4)
Vitamin D is a steroid hormone that is
produced in the skin from 7-
dehydrocholesterol under the influence of
sunlight as well as intake from the diet. In
the liver, vitamin D is converted to 25-
hydroxyvitamin D [25(OH)D], which is the
specific vitamin D metabolite that is
measured in serum to determine a person’s
vitamin D status.(5) In the kidneys and
extrarenal tissues, 25(OH)D is converted into
calcitriol, i.e. 1,25(OH)2D3, the biologically
active form of vitamin D(6)which mediates its
biological effects by binding to the vitamin
D receptor. Circulating 25 (OH) D
concentrations decreased with the evolution
of the systemic inflammatory response.(7)
Vitamin D is known to induce immunologic
tolerance.(8) Thus, vitamin D deficiency may
perturb immune tolerance and induce the
development of autoimmune diseases, such
as RA. Vitamin D has immunomodulatory
properties, acting on the immune system
both in an endocrine and in a paracrine
manner.(9) Vitamin D deficiency may
increase the risk for the development of RA.
Recently, the role of vitamin D deficiency in
the pathogenesis of RA, as well as the
relationship between vitamin D deficiency
and the activity of RA is discussed.(10)
Important new research into the function of
CD46 has demonstrated the existence of a
calcitriol-mediated Th1–Tr1 switch
controlling the delicate homeostatic balance
between pro-inflammatory and anti-
inflammatory states.CD46 and its murine
analog complement receptor 1-related
protein Y (Crry) are transmembrane
glycoproteins that bind complement
fragments C3b andC4b,and function as T-
cell co-stimulatory molecules.(11)
Subjects and Methods
This study was conducted on 40 patients
fulfilling the American College of
Rheumatology / European League Against
Rheumatism (ACR/EULAR) 2010
classification criteria of RA and 20 age and
sex matched healthy individuals. we
excluded Patients on vitamin D supplement.
All patients were subjected to detailed
history taking through physical examination,
Disease activity was assessed by Disease
Activity Score 28 (DAS28), laboratory
investigations including CBC, ESR, CRP,
RF, ACPA, ionized calcium and
Radiological Evaluation. We also measured
vitamin D3 and Flow Cytometric Analysis of
CD46 expression to all subjects. Vitamin D3
by enzyme linked immunosorbent assay
(ELISA): The first step, samples have to be
pretreated in separate vials with denaturation
buffer to extract the analyte, since most
circulating 25-OH Vit D is bound to VDBP
in vivo. After neutralization, biotinylated 25-
OH Vitamin D (enzyme conjugate) and
peroxidase-labeled streptavidin- (enzyme
complex) are added. After careful mixing,
the solution is transferred to the wells of
microtiter plate. Endogenous 25-OH Vitamin
D of a patient sample competes with a 25-
OH Vitamin D3-biotinconjugate for binding
to the VDBG that is immobilized on the
plate. Binding of 25-OH Vitamin D –biotin
is detected by peroxidase-labeled
streptavidin. Incubation is followed by a
washing step to remove unbound
components. The color reaction is started by
addition enzyme substrate and stopped after
a defined time. The color intensity is
inversely proportional to the concentration of
25-OH Vitamin D in the sample. Flow
Cytometric Analysis of CD46 expression on
peripheral blood leucocytes: The expression
of CD46 on peripheral blood leucocytes
(Granulocytes, monocytes, and lymphocytes)
was analyzed using flow cytometric analysis.
EDTA whole blood was stained with
Phycoerythrin (PE) - conjugated anti CD46
monoclonal antibody (clone TRA-2-10)
(Biolegend, San Diego, CA, USA). Briefly,
100 μL of whole blood were stained with
PE-conjugated anti CD46. After incubation,
2.0 mL of lysing solution was added and
lysis was allowed for 10 min at room
temperature. Samples were then washed
twice and resuspended in PBS. Cells were
analyzed on a FACS Calibur flow cytometer
using Cell Quest software (BD Biosciences,
San Diego, CA, USA). Membrane
fluorescence intensity was estimated by the
relative mean fluorescence intensity (MFI).
Results
In our study, RA patients were subdivided to
3 categories: patients had mild disease
activity (4 patients), patients had moderate
disease activity(15 patients) and patients had
severe activity (21patients). The results of
vitamin D3 level among the cases group
were lower than that in the control group and
this difference was of high Statistically
significance (p<0.024) (figure.1) According
to our results, the proportions of CD46 and
Mean Fluorescence Intensity (MFI) of CD46
in lymphocytes, monocytes and granulocytes
did not show significant differences between
RA and controls. (figure.2). In the present
study there was a negative correlation
between vitamin D level and CD46 activity
in lymphocytes and in monocytes and such
correlation was statistically significant
(p<0.04).(figure3) We found also a negative
correlation between vitamin D and DAS 28
score and such correlation was highly
statistically significant (p<0.001).(figure.4)
Figure(1):Comparison between the two groups
according Vitamin D3 level.
Figure(2):Comparison between two groups according
to CD46 activity.
Figure (3): Relation between vitamin D and Monocytes
on cases group
Figure(4): Relation between vitamin D and DAS 28
score in cases group.
Discussion
One potential environmental factor for RA
that has been studied. Extensively in the past
decade is vitamin D. This focus is due, in
part, to the accumulating evidence
suggesting that a worldwide deficiency in
vitamin D might be linked with common
health problems in humans.(12) Recent studies
showed that 25(OH)D not only regulates
calcium and phosphorus metabolism, but
also plays a role in regulating immune and
anti-inflammatory activities by adjusting
growth and differentiation of macrophages,
dendritic cells, T lymphocytes, and B
lymphocytes, inhibiting inflammatory factors
such as TNF-𝛼 and promoting generation of
anti-inflammatory factors such as IL-4 and
IL-10(13) Our results, serum vitamin D was
lower in the cases group than in the control
group and this difference was of high
statistical significance (p <0.024).We did not
observe any statistically significant
difference in CD46 expression in
granulocytes, monocytes and lymphocytes
from peripheral blood of patients with RA
compared with healthy controls. Our results
showed a statistically significant negative
correlation between vitamin D and tender
joint count, swollen joint count, DAS28
score and CD 46 activity on lymphocytes
and monocytes, and positive correlation
between vitamin D and ionized calcium.
0
20
40
60
80
100
120
Lymphocytes Monocytes Granulocytes
Mea
n o
f C
D46 A
ctiv
ity
Cases
Control
0
100
200
300
400
500
600
700
800
0 20 40 60 80 100
Mo
no
cyte
s
Vitamin D
r = -0.326*
p = 0.040
0
1
2
3
4
5
6
7
8
0 20 40 60 80 100
DA
S 2
8 S
core
Vitamin D
r = -0.562*
p <0.001
There was no significant correlation with
ESR, CRP and x ray changes. Qiong Hong e
t a l,(14) in their study of included 130
patients with RA and 80 healthy controls
stated that Serum level of 25(OH)D was
markedly lower in the RA group than in the
control group. In RA patients, 25(OH) D
levels were significantly and negatively
associated with clinical parameters of disease
activity including swollen joint count, tender
joint count and joint pain degree, and
laboratory measure including platelets and
ESR While 25(OH)D levels were not
associated with radiographic bone erosions
of RA. Kostoglou - Athanassiou I,
Athanassiou P et al,(15) In the cohort of 44
patients with RA 25(OH)D3 levels were
found to be low compared With the control
group. Levels of 25(OH)D3 were found to be
negatively correlated to the DAS28, Levels
of 25(OH)D3 were also found to be
negatively correlated to CRP and ESR. Cen
X, Liu Y et al,(16)suggested that serum
25(OH)D in RA groups has significant lower
level (35.99 ±12.59 nmol/L) than that in the
normal groups (54.35 ± 8.20 nmol/L, <
0.05). Based on the DAS28, patients with
RA were divided into four subgroups, and no
differences were found in the four groups (>
0.05). This variability between different
studies regarding the correlation between
serum vitamin D level and different disease
parameters may be explained by several
causes: First, is the difference in the
exclusion criteria of each study as did not
exclude patients on vitamin D supplement.
Second, vitamin D influenced by many
factors, such as region, season, BMI, sun
exposure duration, nutritional status and
gender. Third, The accuracy of clinical SJC
and TJC assessment has issues of
reproducibility and may not differentiate
between tender joints in fibromyalgia and the
swelling of OA, fibrous thickening or
obesity. Patient assessment may be confused
by co morbid symptoms and fluctuations of
mood. In our study, we did not observe any
statistically significant difference in CD46
expression in granulocytes, monocytes and
lymphocytes from peripheral blood of
patients with rheumatoid arthritis compared
with healthy controls. Piccoli AK et al. (17)About CD46 they did not observe any
statistically significant difference in CD46
expression in granulocytes, monocytes and
lymphocytes from peripheral blood of
patients with RA compared with healthy
controls. Current evidence may provide the
rationale for vitamin D supplementation in
the treatment of RA. However, little is
known about how vitamin D intake the risk
and activity of RA, modifies although
increased vitamin D intake has been shown
to be associated with a lower risk of
contracting other autoimmune diseases. (18)
Conclusions The following conclusions can be withdrawn
from our study: 1. Vitamin D deficiency is
highly prevalent in patients with RA which
indicates that it has role in its pathogenesis.
2. Vitamin D level is significantly correlated
with disease activity as expressed by DAS 28
(ESR) score. 3. CD46 expression is
increased in rheumatoid arthritis patients
who have lower levels of vitamin D.
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10. Kim T, Choi S, Lee Y, Song G, Ji J.
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Hypotheses 2012; 79: 757–60.
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217(2):169-75.
12. Holick MF. Vitamin D deficiency. N Engl
J Med 2007; 357:266–81.
13. Holick MF. Sunlight and vitamin D for bone
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cancers, and cardiovascular disease. Am J Clin
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Original Article
Study of the Proteomic Profile in Patients with Inflammatory Bowel
Disease, its Correlation with Diagnosis and Disease Activity
Salah El-Din Ahmed Badr El-Din1, Ezzat Ali Ahmed1, Pacint El-Saed Moez2, Mohamed Eid
Ibrahim3, Doaa Abdou Mohamed Header1; 1Departments of Internal Medicine, 2Clinical
Pathology, 3Radiodiagnosis Faculty of Medicine, Alexandria University
ABSTRACT Inflammatory bowel disease (IBD) comprises primarily 2 disorders: ulcerative colitis (UC) and Crohn's
disease (CD). The hallmark of IBD is chronic, uncontrolled inflammation of the intestinal mucosa. Although
major advances have enhanced the understanding of the multifactorial influence of genetic, environmental,
microbial, and inflammatory determinants of IBD, the etiology of the disease remains elusive. The diagnosis
is based on a combination of disease history, colonscopy, inflammatory biomarkers, radiological and
histological evaluation. Most biomarkers used are not reliable and not disease specific, but reflect
generalized inflammation. Aim of the work: The aim of the work is to identify serum proteomic profiles of
IBD cases and correlating this profile with the other diagnostic markers and activity of the disease. Patients
and methods: We performed a study with 101serum samples collected from patients classified in 3 groups
(31 Crohn's, 37 ulcerative colitis, 33 healthy controls) according to accredited criteria. They were subjected
to: complete history taking, thorough clinical examination, Lab investigations ( routine, fecal calprotectin,
ANCA, ASCA), endoscopy (upper, lower), histopathology, imaging were done. plasma proteomic pattern of
IBD patients and control subjects was determined using matrix-assisted laser desorption/ionization
(MALDI) TOF MS analysis, all serum samples were subjected to solid-phase extraction (SPE). We analyzed
the spectra obtained from all the samples using ClinProTool. Results: There was a statistical significant
difference of the serum proteome profiles of UC group in comparison to health volunteers, Also there was a
statistical significant difference of the serum proteome profiles of crohn's group in comparison to health
volunteers, we used Support Neural Network (SNN), Genetic algorithms (GA) to analyse proteomic profile
of UC and CD cases versus control cases respectively. (64, 76) signals were identified by the ClinProt
software with a statistically different area for UC, Crohn's disease respectively. There was a statistical
significant difference between active versus inactive UC group and Crohn's disease group. Conclusion:
Advances in mass spectrometry and bioinformatics have allowed capturing the signal of thousands of small,
low molecular weight peptides. The pattern of these peptides holds the promise of distinguishing disease
states and providing clinically important information such as prognosis, response to therapy, or perhaps
targets of therapy.
Introduction
Inflammatory bowel disease (IBD)
comprises primarily 2 disorders: ulcerative
colitis (UC) and Crohn's disease (CD). The
hallmark of IBD is chronic, uncontrolled
inflammation of the intestinal mucosa.(1) The
etiopathogenesis has not been fully
elucidated but involves a multifactorial
influence of genetic, environmental,
microbial, and inflammatory factors. (2,3) The
cardinal symptom of ulcerative colitis is
bloody diarrhoea. Associated symptoms of
colicky abdominal pain, urgency, or
tenesmus may be present.(4) Symptoms of
Crohn’s disease are more heterogeneous, but
typically include abdominal pain, diarrhea
for more than 6 weeks and/or weight loss,
systemic symptoms as malaise, anorexia, or
fever are more common.(5) The diagnosis of
IBD is confirmed by clinical evaluation and
a combination of haematological,
endoscopic, histological, or imaging-based
investigations. (6,7,8) Biological markers
potentially useful in IBD include proteins of
inflammation such as C-reactive protein
(CRP), fecal calprotectin and several
antibodies. (9) However, these biomarkers
have many limitations. Acute inflammatory
markers, such as CRP or fecal calprotectin
cannot differentiate between infectious
colitis and flare of IBD. (10,11,12) Anti-
saccharomyces antibodies (ASCA) and
perinuclear anti-neutrophil cytoplasmic
antibody (pANCA) are the only available
commercial tests that can be helpful for CD
and UC discrimination. Although, they show
a quite good specificity, their sensitivity is
rather low and they are therefore not
recommended for broad clinical practice. (13,
14,15) initial diagnosis still relies on the
combination of several biological and
morphological tests, including
gastrointestinal endoscopies and histology,
and is based on standardized validated
diagnostic criteria. (16,17) However, even
using these invasive methods, differential
diagnosis between IBD and self-limited
colitis as well as between the two main
forms of IBD is still difficult. (18,19) Advances
in genomic, proteomic and metabolomic
array-based technologies are facilitating the
discovery of new biomarkers for IBD,
especially proteomic advancement. During
the last two decades, advances in MS
techniques and instrumentation
revolutionized protein chemistry and
basically changed the analysis of proteins.(20)
Proteomics is the study of the set of proteins
encoded by the genome including its
isoforms, modifications, interactions, and
structure.(21) Mass spectrometry (MS) has
gained popularity because of its ability to
handle the complexities associated with the
proteome. The three primary applications of
MS to proteomics are cataloging protein
expression, defining protein interactions, and
identifying sites of protein modification.(22,23)
In this paper, we present a study based on
serum proteomic profiles of IBD cases where
we compared profiles from IBD versus
healthy controls. We correlated this profile
with other diagnostic markers. The same
samples were tested for comparing active
versus inactive disease in both UC and CD.
Patients and Methods A total of 101 subjects, including 33 healthy
donor volunteers as a control group, 31
Crohn’s disease patients and 37 ulcerative
colitis patients were admitted to
Gastroenterology Unit at Alexandria main
University Hospital after obtaining informed
consent from all subjects. From each subject
10 mL of blood sample were collected in
EDTA tubes, 7 ml for routine laboratory
investigations and 3 ml for proteomics
analysis, they were carried in ice box and
centrifuged at 4000 rpm for 10 min at 4oc.
The obtained plasma samples were
distributed into aliquots and stored frozen in
plastic vials at -80°C until use. All patients
with diabetes mellitus, hepatic and renal
diseases were excluded from the study.
Thorough history was taken as regard
demographic data, medical history, drug
history, complete physical examination.
Routine laboratory investigations and routine
markers for IBD as Fecal calprotectin level,
ANCA, ASCA were performed for all
samples according to manufacturers'
recommendations. Determination of the
activity indices clinically, endoscopically
and histopathological for both Crohn’s
disease and ulcerative colitis patients were
done. CD was considered as clinically active
or inactive according to Harvey-Bradshow
index (HBI).(24) UC was considered active or
inactive according to activity index (Seo
index).(25) All plasma samples were subjected
to solid-phase extraction (SPE). Two
different SPE procedures were used: MB-
HIC 8 is based on reverse phase interaction,
whereas MB-WCX is a weak cation
exchanger.
Data Analysis
For the proteome analysis, we used a linear
MALDI-TOF mass spectrometer
(ultraflextreme; Bruker Daltonics) with the
following settings: ion source 1, 25.23 kV;
ion source 2, 23.87 kV; lens, 6.86 kV; pulsed
ion extraction, 300 ns, Laser type smart
beam 2. For matrix suppression, we used a
high gating factor with signal suppression up
to 800 Da. Mass spectra were detected in
linear positive mode. Mass calibration was
performed with the calibration mixture of
peptides and proteins in a mass range of
1074.18–16 952 Da. We measured 4 MALDI
preparations (MALDI spots) from each
magnetic bead fraction. For each MALDI
spot, 3000 spectra were acquired (50 laser
shots at 6 different spot positions). All
signals with a signal-to-noise (S/N) ratio >3
in a mass range of 900–20 000 Da were
recorded. We used the ClinProTools
bioinformatics software (Ver. 3.0; Bruker
Daltonics) for proteome pattern recognition. (26)
Data Processing
The MS spectra for peaks of 900-20,000 m/z
were generated by summating 3000 laser
shots (500 shots at 6 different spot
positions). We exported all unprocessed
spectra from the ultraflextreme MS in
standard format. We normalized all spectra
to their own total ion count by summation of
peak areas using the most prominent peaks,
followed by baseline subtraction, peak
defining, and calculation of peak intensities,
peak numbers and areas. In order to evaluate
the degree of variation among different
proteome spectra, we first defined the peaks
and carefully checked each corresponding
peak throughout all of the spectra. Then, the
program calculates the mean value of peak
intensity, SD and CV (%) for each
corresponding peak in our study. The degree
of variation on the basis of the whole
spectrum was thus determined by calculating
the CV values for all of the peaks of the
tested spectra.
Statistical Analysis
Statistical analysis of the demographic data
were performed with IBM SPSS software
package (version 20.0) for Windows.
Qualitative data were presented as numbers
(n) and percentages (%). Quantitative data
were presented as means and standard
deviation (SD). Testing for distributional
assumption for numerical data using One-
Sample Kolmogorov-Smirnov Test then
Comparison between the means of
quantitative variables was performed using
the one-way ANOVA (F-test). The
correlations between different variables were
evaluated by Mont Carlo exact test and
Fishers exact test according to the
distribution of variables (continuous or
discontinuous quantitative variables
respectively). P value ≤ 0.05 was accepted
as statistically significant. The statistical
analysis of the proteomic profiles were
performed using ClinProTools which offers
an automatic detection mode to determine
and restrict the best number of peaks to be
integrated in a model to be between 1 and
25 peaks. The use of individual peaks as
diagnostic biomarker was addressed using 3
types of algorithms: Genetic Algorithm
(GA), Quick Classifier (QC) and Support
Neural Network (SNN) analysis.
Results
First we conducted a pilot study on 10 UC
cases, 10 Crohn's cases & 10 controls cases
using MB- HIC8 and MB-WCX beads to
assess the solid phase extraction
functionality with better performance. The
decision to use C8 beads in this study instead
of WCX chelating beads was made by
comparing the basic parameters of the total
peak number, peak area and peak height and
the ability to analyze the differences in peaks
using ClinProt software. For the
reproducibility of the protein profiling,
Within- and between-run reproducibility of 2
samples were determined with the MB-
HIC8 fractionation and MALDI-TOF MS
analysis. In each profile, 3 peaks with
different molecular masses were selected to
evaluate the precision of the assay. Despite
varying peptide masses and spectrum
intensities, the peak CVs were all <4% in the
within-run and <10% in the between-run
assays. These values were consistent with the
reproducibility data for the Protein Biology
System reported by the manufacturer (Bruker
Daltonics). To determine the accuracy of the
class prediction, Twenty percent of model
construction group (control= 16, UC= 18,
CD= 15) were randomly selected as a test
set, and the rest of the samples were taken as
a training set. In the class predictor algorithm
all detected peaks were analysed by ClinProt
3.0 to generate cross-validated classification
models. Then, the samples of external
validation group (control=17, UC=19,
CD=16) were classified by the classify
peptidome patterns constructed by the 3
algorithms. a- Plasma proteome profiles of
UC patients: Differences of the plasma
proteome profiles of UC versus health
volunteers, about 64 peptide peaks were
identified by the ClinProt software with a
statistically significant different areas
(P<0.05 by Wilcoxon analysis) in model
construction population, including 22 up-
regulated and 42 down-regulated peptides.
Representative Mass Spectrum in figure
1(a,b).
Figure 1(a)
Figure 1(b)
Figure 1: View of the aligned mass spectra of the Plasma protein profile of model construction group (green: 18
health subjects , red 19 UC Patients ) obtained by MALDI-TOF after purification with C8magnetic beads in
Stack view Figure 1(a) and in gel view Figure 1 (b).
Model construction on clinprot Classification models were developed to
classify samples between UC and controls
cases. The peptide pattern for UC was
addressed using Support Neural Network
(SNN) analysis which gave the best results.
First, we conducted comparison between UC
and controls. Second, all detected peaks were
analysed by ClinProt 3.0 to generate cross-
validated classification models. The
optimized SNN model resulted in the
following correct classification of samples.
Sixteen peptide ion signatures (m/z11681.03,
11721.3511094.04, 7764.48, 11627.71,
3882.38, 7467.18, 2379.07, 1077.04,
4963.75, 9287.42, 8140.65, 949.45, 1098.78,
12689.45&11076.02Da) were provided as a
class prediction for a cross-validation set to
discriminate UC disease from control cases
as shown in table 1. Cross Validation was
82.68 % and the Recognition Capability was
100%.
Table 1: Integration Regions used for UC Classification.
Index Mass Start Mass End Mass Weight
54 11681.03 11643.39 11704.77 0.232
55 11721.35 11704.77 11804.25 0.100
50 11094.04 11084.49 11151.3 0.091
31 7764.48 7729.13 7794.56 0.079
53 11627.71 11605.71 11636.32 0.075
16 3882.38 3865.15 3895.03 0.049
28 7467.18 7424.96 7485.3 0.040
12 2379.07 2370.96 2387.96 0.038
8 1077.04 1072.03 1079.86 0.038
19 4963.75 4944.62 4973.82 0.036
43 9287.42 9244.98 9320.75 0.035
34 8140.65 8104.2 8159.35 0.034
3 949.45 943.9 956.61 0.032
9 1098.78 1093.46 1106.43 0.027
56 12689.45 12664.34 12718.48 0.018
49 11076.02 11024.77 11084.49 0.018
Figure 2: ROC curve of peaks selected for model generation of UC patients vs. healthy volunteers, Peak 54 and 55
with m/z 11681&11721 respectively and AUC=0.885&0.830 respectively
External validation: To verify the accuracy
of the established SNN classification model
with the adopted peptides for UC training
set versus control training set as shown in
(table 2), with 100% sensitivity and 84.4%
specificity.
Table 2: External validation of UC and control training sets.
Group Name Sensitivity(%) Sepecificity(%)
1 UC training set vs. External validation set 100%
2 Control training set vs. external validation set 84.4%
a- Plasma proteome profiles of Crohns'
patients: Differences of the serum proteome
profiles of CD versus health volunteers,
about 76 peptide peaks were identified by the
ClinProt software with a statistically
different area (P<0.05 by Wilcoxon analysis)
in model construction population, including
41up-regulated and 35 down-regulated
peptides. Representative Mass Spectrum in
Figure 3 (a,b)
Figure 3(a) Figure 3(b) Figure 3: View of the aligned mass spectra of Plasma protein profile of model construction group (green: 19
health subjects, red 19 Crohn’s Patients) obtained by MALDI-TOF after purification with C8magnetic beads in
both Stack view in figure 3a & pseudo-gel view in figure 3b.
Model construction on clinprot Classification models were developed to
classify samples between CD and healthy
volunteers. The peptide pattern for for CD
was addressed using Genetic algorithms
(GA) analysis which gave the best results in
table 3.
Table 3: ClinProt Model List between CD and healthy volunteers using the 3 algorithms.
Name Algorithm
Validation
XVal X1 X2 Rec
Cap
Crohn’s vs. Control GA 93.2 % 93.6 % 92.9 % 100 %
Crohn’s vs. Control SNN 76.1 % 80.9 % 71.4 % 93.2 %
Crohn’s vs. Control QC 79.3 % 87.2 % 71.4 % 88.6%
The optimized GA model resulted in the
following correct classification of samples.
Five peptide ion signatures were provided as
a class prediction for a cross-validation set to
discriminate Crohn’s disease from control
cases
(m/z4282.18,6447.44,3475.77,13289.15&13
878.44 Da) and were termed “Integration
Regions”, with a recognition capacity of
100% and a cross-validation of 93.24% in
table 3.
Table 4: Integration Regions used for CD Classification.
Index Mass Start Mass End Mass Weight
26 4282.18 4274.99 4291.65 0.552
36 6447.44 6443.49 6458.11 0.353
21 3475.77 3467.49 3484.19 0.241
70 13289.15 13224.9 13303.75 0.803
73 13878.44 13850.2 13923.14 0.040
Figure 4: ROC curve of Peak 70 and 71 with m/z 13289 &13312 and AUC=0.828 & 0.764 respectively.
External validation : To verify the accuracy
of the established GA classification model
with the adopted peptides for CD training set
versus control training set as shown in (table
5), with 91.7%% sensitivity and 78.6%
specificity.
Table 5: External validation of CD and control training sets.
Group Name Sensitivity(%) Specificity(%)
1 CD Training set vs. validation set 91.7 %
2 Control training set vs. external validation set 78.6 %
Discussion
We report the first proteomic study using
serum profiling with MALDI-TOF-MS in
IBD patients in Egypt. The study was
performed on 101 samples. Patients were
recruited from our University hospital and
are representative of IBD populations
affected in our region. The major finding of
the present study was that the external
validation of the proteome profiles of UC in
comparison to health volunteers showed a
sensitivity of 100% and specificity of 84.4%
and the proteome profiles of CD in
comparison to health volunteers showed a
sensitivity of 91.7% and somewhat lower
specificity of 78.6%. The mass spectra
analysis revealed a large number of
potentially interesting protein peaks with
significant P value. In agreement with our
results a study presented by Vermeulen et al (27) at Digestive Disease Week DDW, they
used commercial human protein arrays to
profile serum IBD biomarkers from a very
small cohort of subjects (10 UC, 15 CD, and
5 healthy controls). Seventy-five proteins
were found to react more strongly with IBD
sera than those from healthy controls, while
reactivity of another 88 proteins was just the
opposite Meuwis et al (28) published the first
proteomic serum profiling study using
SELDI-TOF MS in IBD, a variation of
MALDI-TOF MS. The study included 30
patients with CD, 30 patients with UC, 30
inflammatory controls and 30 healthy
controls. The group was able to differentiate
CD from UC with sensitivity of 85% (51/60)
and specificity of 95% (57/60). Several of
the unidentified signals were subsequently
identified by MALDI-TOF MS, western
blotting, and ELISA assay. The study
highlighted the potential of serum profiling.
Nanni et al (29) performed a study using
MALDI-TOF-MS (Matrix-Assisted Laser
Desorption/Ionization Time of Flight-Mass
Spectrometer). The study, which involved a
small cohort (15 CD, 26 UC and 22 healthy
controls), found the revered-phase extraction
and selection of 20 m/z value gave the best
overall predictive value (96.9%). In another
study, reported at Digestive Disease Week
(DDW) Subramanian et al ,(30) analyzed sera
from a cohort of 62 UC and 48 CD by
SELDI-TOF MS. Biostatistical analysis
identified 12 discriminative peaks, with
specificity and sensitivity approximately
95% (compared to 80.9% of the sensitivity
of ASCA for CD and 64.5% of pANCA for
UC), suggesting the utility of serum
proteomic profiling in IBD. M’Koma et al
(2011),(31) performed a study on 51 patients
with inflammatory colitis (n=24 Crohn's
Colitis (CC) and n=27 UC), they identified
several signals in the mass spectra at discrete
m/z values in the inflamed and uninflamed
mucosal and submucosal layers of CC and
UC specimens. The optimal classification
between inflamed vs. uninflamed CC
submucosa was achieved with two
differentiating signals at m/z 6445 and
12692, (p=0.0003 and p=0.003 respectively).
When analyses were performed on inflamed
vs. uninflamed UC submucosa, four
differentiating m/z signatures were
identified: 4627, 4024, 27848 and 25289. P-
values ranged from p=0.001 to p=0.005.
Seeley EH et al (2013),(32) collected samples
from Crohn's Colitis CC (n = 26) and UC (n
= 36) patient (total n = 62) from the mucosal
and submucosal colon tissues layers, out of
312 total peaks in the averaged spectra, 114
were found to have p-values of less than
0.05, this indicates there is considerable
difference in the protein expression levels
between the two diseases. Wasinger VC et al (33) identified two proteins that were able to
differentiate IBD patients and health
controls.
Conclusion
Use of proteomic profiles gave high
sensitivity and specificity for IBD diagnosis.
Advances in mass spectrometry and
bioinformatics have allowed for advances in
proteomics by capturing the signal of
thousands of small, low molecular weight
peptides.
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Original Article
Use of Early Lactate Clearance as a Predictor of Mortality Rate after
Initial Resuscitation in Patients with Severe Sepsis or Septic Shock
Mohammed Mostafa Megahed1, Dalia Abd Elmoaty2, Haitham Tammam1, Islam Ahmed
Saadallaah1; 1Department of Critical Care Medicine, 2Department of Clinical and Chemical
Pathology; Faculty of medicine, University of Alexandria, Egypt.
ABSTRACT A high lactate clearance in 6 hours after initial resuscitation supposed to be associated with decreased
mortality rate. Aim of the work: to evaluate the prognostic value of lactate clearance and lactate production
in severely ill septic patients to determine disease severity in the intensive care unit. Material and
methods: Prospective observational study in an urban emergency department and intensive care unit over a
1-yr period. Included ninety patients with severe sepsis or septic shock who had blood lactate concentration
<3 mmol/L. Therapy was initiated immediately in the emergency department (ED) and continued in the
intensive care unit, including central venous and arterial catheterization, antibiotics, fluid resuscitation,
mechanical ventilation, vasopressors, and inotropes when appropriate. Material Methods: vital signs,
laboratory values and sequential organ failure assessment (SOFA) score were obtained at 0 hour, after 6 hrs
and over the first 72 hrs of hospitalization. Lactate clearance was calculated as percent of serum lactate
change from ED presentation to hour 6. We compared hospital days, vasopressor days, mechanical
ventilation period, mortality at day 7 and mortality at day 28 between patients who cleared lactate and those
who did not. Results: 90 patients were included. There were 43 patients in the clearance group and 47
patients in the non-clearance group. The 28-day mortality rates were 9.3% in the lactate clearance group and
87.23% in the non-clearance group (p<0.01). Vasopressor support was initiated in all the non-clearance
group (100%) than in the clearance group (42.2%, p<0.01). Conclusion: Patients with higher lactate
clearance after 6 hrs of emergency department intervention have improved outcome compared with those
with lower lactate clearance because this reflect resolution of global tissue hypoxia and is associated with
decreased mortality rate.
Introduction
Sepsis remains a major cause of morbidity
and mortality in hospitalized patients. (1) It is
the second leading cause of death among
patients in non-coronary intensive care units
(2) Furthermore, sepsis is associated with a
reduced quality of life in those who survive
their acute illness. (3) Early and aggressive
quantitative resuscitative care is
recommended for the treatment of septic
shock and meta-analytic data have shown its
efficacy at reducing mortality. (4) The
Surviving Sepsis Campaign recommends the
use of many parameters to assess perfusion
in sepsis: central venous pressure (CVP),
mean arterial pressure (MAP), urine output,
central venous oxygen saturation (ScvO2)
and arterial lactate as resuscitation goals. (5)
Lactate is the metabolic end-product of
anaerobic glycolysis. In condition of low
flow or cellular hypoxia, pyruvate cannot
enter the mitochondria and is preferentially
reduced to lactate, causing arterial lactate
concentration to increase. The daily
production of lactate is around 1300
mmol/day and the concentration of arterial
lactate is a reflection of net production and
clearance, and is generally around 2mmol/L.
Metabolism and clearance of lactate is
primarily via the liver and kidneys, and
dysfunction of these organs has been
associated with varying levels of reduced
clearance. (6) Blood lactate concentration is
widely used in intensive care units as a
reliable diagnostic tool for global tissue
hypoxia and hypoperfusion, therefore can
serve in identifying patients with severe
sepsis or septic shock. (7-9) However, in recent
years there has also been recognition of the
prognostic value of serum lactate
measurement. (10-12) Increased initial lactate
values have been associated with mortality
among all-comers with sepsis. (13, 14) Based
upon these findings, international sepsis
guidelines now suggest routine measurement
of lactate among patients with severe sepsis
and immediate resuscitation for septic
patients whose serum lactate measurement is
greater than 4 mmol/L. (15) Relatively few
studies, however, have examined the role of
lactate clearance or serial lactate
measurements as endpoints among patients
with severe sepsis or septic shock patients
presenting in the emergency department
(ED). (16-18) Our aim was to evaluate the
predictive value of lactate clearance on 28-
day in-hospital mortality and to investigate
secondary outcomes such as need for
particular treatments and interventions like
mechanical ventilation or vasopressor. We
hypothesized that patients with severe sepsis
or septic shock who present to the ED and
have evidence of lactate clearance within 6
hours after resuscitation would have lower
in-hospital mortality rates than those who did
not clear initial lactate levels.
Subjects and Methods
After approval of local ethics committee, this
study was conducted on ninety patients with
no life threatening conditions. We also
exclude pregnant females and patients less
than 18 years. All subjects were subjected to
full history, comprehensive physical
assessment, laboratory investigations (CBC,
urea and creatinine level, liver function tests,
arterial blood gases, C-reactive protein,
cultures according to source of sepsis, lactate
levels on 0 hour of presentation and another
one after 6 hours of resuscitation). We
calculated lactate clearance as: lactate at ED
presentation (hour 0) minus lactate at hour 6,
divided by lactate at ED presentation, then
multiplied by 100. A positive value denotes a
decrease or clearance of lactate, whereas a
negative value denotes an increase in lactate
after 6 hours of ED intervention.(19) Lactate
clearance= [(Lactate 0 hour – Lactate6
hours)/ Lactate 0 hour] X 100.(19) We
compared the 28-day in-hospital mortality
rate among patients who cleared lactate with
those who showed an increase in lactate
levels at the time of admission. We also
calculated sequential organ failure
assessment (SOFA) score at presentation and
after 6 hours between clearance and non-
clearance groups and its relation to outcome
of the patients.
Results
The patients included in this study were 56
males (62.2%) and 34 females (37.8%) were
enrolled over a 1-yr period with mean age of
62.66 ± 12.29 in total patients. 72% of them
have diabetes mellitus, 63% have
hypertension, 46% have ischemic heart
diseases (IHD), 31% have chronic kidney
disease, 18% have chronic obstructive
pulmonary disease (COPD) and 15% have
chronic hepatic diseases. In our study we
find that high mortality rates were found in
critically ill septic patients with chronic liver
and renal diseases (p=0.048 and p=0.004)
respectively. This may be due to their low
immunity status in response to the septic
condition they had.
Table 1: Comparison between the two studied groups according to demographic data
Total (n=90) Survived (n=45) Died (n=45) Test of
Sig. P
No. % No. % No. %
Sex
χ2 = 0.756 0.384 Male 56 62.2 26 57.8 30 66.7
Female 34 37.8 19 42.2 15 33.3
Age (years)
Min. – Max. 28.0 – 87.0 35.0 – 87.0 28.0 – 83.0
T = 0.333 0.740 Mean ± SD. 62.66 ± 12.29 62.22 ± 12.92 63.09 ± 11.75
Median 64.0 62.0 65.0
χ2: Chi square test t: Student t-test
Source of sepsis in our study was varying
including chest (42%), urosepsis (25%),
abdominal (18%), skin and soft tissue (22%)
blood stream infections (31%). Patients with
abdominal infections and blood stream
infections had significant high mortality rates
(p<0.001) in both groups. On the other side,
patients with skin and soft tissue infections
had low mortality rates.
Table 2: Comparison between the two studied groups according to source of sepsis
Source of sepsis Total (n=90) Survived (n=45) Died (n=45)
χ2 P No. % No. % No. %
Chest 42 46.7 18 40.0 24 53.3 1.607 0.205
UTI 25 27.8 11 24.4 14 31.1 0.498 0.480
Abdominal 18 20.0 2 4.4 16 35.6 13.611* <0.001*
Skin 22 24.4 16 35.6 6 13.3 6.016* 0.014*
BL stream 31 34.4 7 15.6 24 53.3 14.221* <0.001*
χ2: Chi square test *: statistically significant at p ≤0.05
Directing to our aim of our study about
lactate, from ninety patients of the study 43
patients showed decrease in their lactate
level after 6 hours of resuscitation (lactate
clearance group) from them 39(90.7%) were
survivors at day 28 of hospital stay and only
4 (9.3%) patients died indicating low
mortality with early lactate clearance group.
However, 47 patients showed increase
lactate levels, although resuscitation, (non-
lactate clearance group) from them 6 only
survived (12.8%) at day 28 of hospital stay
and 41 (87.2%) patients died indicating
higher mortality rates with non- lactate
clearance group. The previous results had
high significant values (p <0.001). this
means that there is significant inverse
relationship between early lactate clearance
and sepsis.
Figure 1: Relation between change of lactate level and morality at day 7
Change of lactate level
χ2 FEP
Decreased (n=43) In decreased (n=47)
No. % No. %
Morality at day 7
No 41 95.3 39 83.0 3.479 0.093
Yes 2 4.7 8 17.0
χ2, p: χ2 and p values for Chi square test FE: Fisher Exact for Chi square test
According to the need of vasopressor, as an
initial resuscitative measure, we found that
from the 43 lactate clearance patients, 18
(41.9%) need vasopressor and 25 (58.1%)
ones did not need it while in the 47 non
clearance patients, only one patient escaped
the need of vasopressor and 46 patients
(97.9%) need it. The previous results were
significant (p<0.001). The duration of
vasopressor therapy in clearance group is
significantly lower than the duration in non-
clearance group with the mean of duration
was (5.83 ± 3.94 and 9.54 ± 5.75)
respectively.
Figure 2: Relation between change of lactate level and morality at day 28
Change of lactate level
χ2 P
Decreased (n=43) In decreased (n=47)
No. % No. %
Morality at day 28 – 60
Survived 39 90.7 6 12.8 54.552* <0.001*
Died 4 9.3 41 87.2
χ2, p: χ2 and p values for Chi square test *: Statistically significant at p≤0.05
As regard mechanical ventilation, as an
important invasive intervention, our results
show significant need of mechanical
ventilation in lactate non clearance group (42
from 47) (89.4%) more than lactate clearance
group (21 from 43) (48.8%) (p<0.001).
When we calculated the SOFA score of the
both two groups, there is significant increase
of SOFA score in lactate non clearance
(13.55 ± 5.09) higher than lactate clearance
group (5.40 ± 3.53) (p<0.001).
Figure 3: Relation between change of lactate level and hospital (days)
Change of lactate level
t P Decreased (n=43) In decreased (n=47)
Hospital (days)
Min. – Max. 4.0 – 24.0 5.0 – 31.0
4.468* <0.001* Mean ± SD. 10.95 ± 5.07 16.89 ± 7.42
Median 10.0 20.0
t, p: t and p values for student t-test *: Statistically significant at p≤0.05
Discussion There is great association between severe
sepsis or septic shock and tissue hypoxia,
morbidity and mortality. Lactate is the end
product of glycolysis under hypoxic
conditions and represents a useful and
clinically obtainable surrogate marker of
tissue hypoxia and disease severity,
independent of blood pressure. (20) The cause
of elevated lactate in patients with sepsis is
multifactorial. Lactate elevation may result
from acute tissue hypo perfusion and
anaerobic metabolism. (21) Other possible
causes may include sepsis induced inhibition
of pyruvate-dehydrogenase enzyme activity
(22), increased lactate production by
catecholamine- driven pathways (23-25), and
decreased lactate clearance due to liver
dysfunction occurring in sepsis. (26, 27).
However, regardless of etiology of an
elevated serum lactate, lactate elevation in
sepsis has been consistently linked to
increased mortality (10, 28). Although initial
studies of lactate clearance in sepsis were
published more than 15 years ago (29), just
recently after the third consensus of sepsis
(sepsis-3) lactate became an integrated
component of sepsis definition referring to
organ dysfunction occurring in sepsis. (30)
Therefore, these data suggest that serial
lactate level measurement may provide
unique and important information on
resuscitation effectiveness. Moreover, serial
lactate level measurement is more reliable
predictor of mortality than single lactate
value as early lactate clearance is associated
with better outcome than lactate non-
clearance which is responsible about high
rates of in-hospital death. (29, 31) Bakker et al. (32) provided a new term; “lactime” as the
time in which lactic acid remains >2 mmol/L
and confirmed that this duration of lactic
acidosis was predictive of organ failure and
survival. Trauma patients whose lactate
normalized in 24 hours were shown to have
100% survival. (33) whereas persistent lactate
elevation >6 hours is associated with
increased mortality rate in septic patients. (34)
A goal of resuscitation is then to minimize
lactime. Our present study confirms the
concept of lactate normalization during early
therapeutic interventions. Our findings
suggest that lactate clearance, as defined by
the percentage of lactate cleared over the
first 6-hr period of disease presentation, is
associated with decreased mortality rate.
Patients with lactate clearance required less
vasopressor therapy, less mechanical
ventilation needs, less hospital stays and
their SOFA score was much better than
patients without lactate clearance.
Conclusion Early lactate clearance seems to be an
important predictor of survival in patients
with severe sepsis or septic shock. In this
study, lactate non-clearance during the
resuscitation phase of therapy was a strong
independent predictor of in-hospital death. In
the context of quantitative resuscitation,
assessment of lactate clearance provides
unique information on resuscitation
effectiveness. It is more valuable to use
serial lactate levels measurements than use
of single lactate level in predicting outcome.
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Early lactate clearance is associated with
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Case Report
Dunbar Syndrome (Median Arcuate Ligament Syndrome) MY Taher M Rashed, Alexandria University HPB Unit, Egypt
ABSTRACT Postprandial abdominal pain that does not have a clearly established etiology may be due to median arcuate
ligament syndrome (MALS) .Usually in such a case the median arcuate ligament. lies too low on the aorta,
the ligament may cause symptoms of abdominal pain related to compression of the celiac artery.Normally it
passes superior to the origin of the celiac artery and is a continuation of the posterior diaphragm that wraps
over the aorta.Computed tomography angiography of the abdomen detect stenosis of the origin of the celiac
artery and confirms the diagnosis,Surgical release of the median arcuate ligament resulted in relief of the
patient's symptoms, We present a case of Dunbar syndrome presented with unexplained severe abdominal
pain after meals for 2 years in a lady aged 23 years.
Introduction The median arcuate ligament is a fibrous
arch that unites the diaphragmatic crura on
either side of the aortic hiatus. The ligament
usually passes superior to the origin of the
celiac artery near the first lumbar vertebra. In
the general population, 10-24% of people
may have indentation caused by an
abnormally low ligament.1 Few of these
patients have hemodynamically significant
stenosis that would cause symptoms. We
present the case of a patient with median
arcuate ligament syndrome that caused
abdominal pain associated with nausea,
emesis, and bloating.(1)
Case Presentation
A 23-year-old woman with no significant
past medical history presented with a 2-year
history of intermittent epigastric abdominal
pain. The pain was associated with nausea,
nonbilious vomiting, and bloating. The pain
became worse when she ate large meals; the
nausea worsened with any oral intake and
relieved with fasting. Diarrhea occurs rarely
She had lost 10 kilgtams over 2 years.
Clinical examination revealed epigastric
tenderness to palpation with faint bruit but
no other abnormalities after extensive
evaluation of the gastrointestinal tract by
endoscopy as well as gallbladder evaluation
by ultrasound examination. Computed
tomography angiography (CTA) of the
abdomen showed stenosis involving the
origin of the celiac axis without significant
atherosclerotic plaque or calcification These
findings clinched the diagnosis.
Figure 1
Figure 2
Figure 3
CT angiographic findings in MALS: Focal
narrowing of proximal celiac artery with
poststenotic dilatation, Indentation on
superior aspect of celiac artery, Hook-shaped
contour of celiac artery. (Figure 1,2,3)
Discussion
Median arcuate ligament syndrome (also
known as Dunbar syndrome or celiac artery
compression syndrome) was first described
by Harjola in 1963.(1) A patient who
presented with postprandial abdominal pain
and an epigastric bruit was found to have his
celiac artery encased with thick ganglionic
tissue at the time of surgery. The patient
experienced full relief of symptoms
following removal of this thick fibrotic tissue
from the celiac artery. It is estimated that in
10-24% of normal, asymptomatic individuals
the median arcuate ligament crosses in front
of (anterior to) the celiac artery, causing
some degree of compression. Approximately
1% of these individuals exhibit severe
compression associated with symptoms of
MALS. The syndrome most commonly
affects individuals between 20 and 40 years
old, and is more common in women,
particularly thin women.(2). The
pathophysiology of the disease is external
compression of the celiac artery by an
abnormally low lying ligament. The
compression worsens with expiration as the
diaphragm moves caudally during expiration,
causing compression of the celiac trunk. This
compression leads to visceral ischemia and
postprandial abdominal pain.(3) Some also
claim that this causes a steal phenomenon
from blood flow being diverted away from
the superior mesenteric artery via collaterals
to the celiac axis, causing midgut
ischemia.(4). Sustained compression of the
celiac axis may lead to changes in vascular
layers such as intimal hyperplasia,
proliferation of elastic fibers in the media,
and disorganization of the adventitia.
Epigastric pain may be present, and physical
examination may reveal epigastric bruit in as
many as 83% of patients.(2) This bruit may
increase on expiration. Epigastric pain may
be present, and physical examination may
reveal epigastric bruit in as many as 83% of
patients.(2) This bruit may increase on
expiration.(4) Angiography has largely been
supplanted by multidetector CT scanners
with 3-dimensional software, allowing
reconstructions at various anatomical planes.
A CT scan will be able to detect focal
narrowing of the celiac axis, particularly in
sagittal views. This narrowing has a
characteristic hooked appearance similar to
that seen in our patient's CT. Collateral
vessels may also be noted. Surgical median
arcuate ligament release has been the
mainstay of treatment. Decompression of the
celiac artery is the general approach to
treatment of MALS.(2) The mainstay of
treatment involves an open surgical approach
to divide, or separate, the median arcuate
ligament to relieve the compression of the
celiac arteryA laparoscopic approach may
also be used to achieve celiac artery
decompression however, should the celiac
artery require revascularization, the
procedure would require conversion to an
open approach. Endovasular methods such as
percutaneous transluminal angioplasty (PTA)
have been used in patients who have failed
open and/or laparoscopic intervention. PTA
alone, without decompression of the celiac
artery, may not be of benefit.(5)
References 1. Duffy AJ, Panait L, Eisenberg D, Bell RL,
Roberts KE, Sumpio B. Management of median
arcuate ligament syndrome: a new paradigm. Ann
Vasc Surg. 2009 Nov-Dec;23 (6):778–784. Epub
2009 Jan 6.]
2. Horton KM, Talamini MA, Fishman EK.
"Median arcuate ligament syndrome: evaluation
with CT angiography". (2005) Radiographics. 25
(5): 1177–82.
3. Duncan AA (April 2008). "Median arcuate
ligament syndrome". Curr Treat Options
Cardiovasc Med. 10 (2): 112–6
4. A-Cienfuegos J, Rotellar F, Valentí V, et al.
The celiac axis compression syndrome (CACS):
critical review in the laparoscopic era. Rev Esp
Enferm Dig. 2010 Mar;102 (3):193–201.
5. Matsumoto AH, Tegtmeyer CJ, Fitzcharles
EK, et al. (1995). "Percutaneous transluminal
angioplasty of visceral arterial stenoses: results
and long-term clinical follow-up". J Vasc Interv
Radiol. 6 (2): 165–74.