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Contents Alexandria Journal of Hepatogastroenterology,

Volume (XXII) - April 2017

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

manuscripts please contact the editors by e-mail at:

[email protected]

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Disclaimer: The Publisher, the Egyptian Society of

Hepatology Gastroenterology and Infectious Diseases in

Alexandria, and Editors cannot be held responsible for

errors or any consequences arising from the use of

information contained in this journal; the views and

opinions expressed do not necessarily reflect the those of

the Publisher, The Egyptian Society of Hepatology

Gastroenterology & Infectious Diseases in Alexandria,

Editors, neither dose the publication of advertisements

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and editors of the products advertised.

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


Helicobacter Pylori CagA Line Test

Mohamed Alaa Eldin Nouh, Hossam Eldine Mostafa Saleam

and Bedair Haroun AbdAllah Fayed


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


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


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


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


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


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


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

mailto:[email protected]

mailto:[email protected]

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

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sth

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ith

an

ima

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vel

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ab

roa

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Ad

dic

tio

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ith

bir

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CO

PD

Dia

bet

es m

ellit

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Hy

per

ten

sio

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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

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Cor

ticos

tero

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Dru

gs f

or T

B

Tet

racy

clin

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Mac

rolid

es

Ant

ifun

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Van

com

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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

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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

J Kidney Dis 2003; 42: 677–84.

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

dialysis patients: causes and consequences. Am J

Kidney Dis 2003;42:864-881.

5. Zoccali C, Benedetto FA, Mallamaei F.

Inflammation is associated with carotid

atherosclerosis in dialysis patients. J Hypertens

2000; 18: 1207-13.

6. London GM, Marchais SJ, Metivier F, Guerin

AP. Cardiovascular risk in end-stage renal disease:

vascular aspects. Nephrol Dial Transplant

2000;15(Suppl. 5):97–104.

7. Koc M, Toprak A, Tezcan H, Bihorac A,

Akoglu E, Ozener IC. Uncontrolled hypertension

due to volume overload contributes to higher left

ventricular mass index in CAPD patients. Nephrol

Dial Transplant 2002; 17(9):1661–6.

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: 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


(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


(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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https://www.ncbi.nlm.nih.gov/pubmed/?term=WOCN%20Clinical%20Practice%20Wound%20Subcommittee%2C%202005%5BCorporate%20Author%5D

https://www.ncbi.nlm.nih.gov/pubmed/?term=WOCN%20Clinical%20Practice%20Wound%20Subcommittee%2C%202005%5BCorporate%20Author%5D

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

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4036413/#B3

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.


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








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




had Barrett's with high grade dysplasia and 2

cases with adenocarcinoma. In conventional

biopsy, there was 7 patients showed normal








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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G. Re: Risk of malignant progression in Barrett's

esophagus patients: results from alarge

population-based study. Journal of the National

Cancer Institute. 2012; 104(22):1771.

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):

560-8.

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

the diagnosis of gastric intestinal metaplasia and

dysplasia. Gastrointestinal endoscopy. 2003 Apr

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HH, Stolte M, Kanzler S, Nafe B, Jung M, Galle

PR, Neurath MF. Methylene blue-aided

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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)


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


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


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


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



Fig. (4): ROC curve for serum zinc to differentiate



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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varices. Hepatology. 2008; 47(5):1587-94.

8. Camma C, Petta S, Di Marco V, Bronte F,

Ciminnisi S, Licata G, et al. Insulin resistance is a risk

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9. Erice E, Llop E, Berzigotti A, Abraldes JG,

Conget I, Seijo S, et al. Insulin resistance in patients

with cirrhosis and portal hypertension. American

Journal of Physiology-Gastrointestinal and Liver

Physiology. 2012; 302(12): G1458-G65.

10. Takahashi M, Saito H, Higashimoto M, Hibi T.

Possible inhibitory effect of oral zinc

supplementation on hepatic fibrosis through

downregulation of TIMP‐1: A pilot study.

Hepatology Research. 2007;37(6):405-9.

11. Matthews D, Hosker J, Rudenski A, Naylor B,

Treacher D, Turner R. Homeostasis model

assessment: insulin resistance and β-cell function

from fasting plasma glucose and insulin

concentrations in man. Diabetologia. 1985; 28(7):

412-9.

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12. Lann D, LeRoith D. Insulin resistance as the

underlying cause for the metabolic syndrome.

Medical Clinics of North America. 2007;91(6):1063-

77.

13. Antuna-Puente B, Disse E, Rabasa-Lhoret R,

Laville M, Capeau J, Bastard J-P. How can we

measure insulin sensitivity/resistance? Diabetes &

metabolism. 2011;37(3):179-88.

14. Gayoso-Diz P, Otero-González A, Rodriguez-

Alvarez MX, Gude F, García F, De Francisco A, et

al. Insulin resistance (HOMA-IR) cut-off values and

the metabolic syndrome in a general adult

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cross-sectional study. BMC endocrine disorders.

2013;13(1):1.

15. Camma C, Petta S, Di Marco V, Bronte F,

Ciminnisi S, Licata G, et al. Insulin resistance is a risk

factor for esophageal varices in hepatitis C virus

cirrhosis. Hepatology. 2009;49(1):195-203.

16. Eslam M, Ampuero J, Jover M, Abd-Elhalim

H, Rincon D, Shatat M, et al. Predicting portal

hypertension and variceal bleeding using non-

invasive measurements of metabolic variables. Ann

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17. Erice E, Llop E, Berzigotti A, Abraldes JG,

Conget I, Seijo S, et al. Insulin resistance in patients

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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)


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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arthritis. Autoimmune Rev 2004; 3 (Supp11):

S20-2.

2. Lee DM, Weinblatt ME. Rheumatoid

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4. Cardone J, Le Friec G, Vantourout P,

Roberts A, Fuchs A, Jackson I, et al.

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5. Pittas AG, Chung M, Trikalinos T, Mitri J,

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7. Vieth R. Why ‘’vitamin D’’ is not a

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https://www.ncbi.nlm.nih.gov/pubmed/?term=Jackson%20I%5BAuthor%5D&cauthor=true&cauthor_uid=20694009

vitamin D, its analogs or deltanoids. J Steroid

Biochem Mol Biol 2004; 89-90:571-3.

8. Weiss S. Bacterial components plus vitamin

D: the ultimate solution to the asthma

(autoimmunedisease) epidemic? J Allergy Clin

Immunol 2011; 127: 1128-30.

9. Hewison M. Vitamin D and immune

function: autocrine, paracrine or endocrine?

Scand J Clin Lab Invest Suppl 2012; 243: 92-

102.

10. Kim T, Choi S, Lee Y, Song G, Ji J.

Combined therapeutic application of m TOR

inhibitor and vitamin D(3) for inflammatory bone

destruction of rheumatoid arthritis. Med

Hypotheses 2012; 79: 757–60.

11. Ni Choileain S, Astier AL. CD46 processing:

a means of expression. Immunobiology 2012;

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

health and pre vention of autoimmune diseases,

cancers, and cardiovascular disease. Am J Clin

Nutr 2004; 80(6):1678-88.

14. Hong Q, Xu J, Xu S, Lian L, Zhang M, Ding

C. Associations between serum 25-

hydroxyvitamin D and disease activity,

inflammatory cytokines and bone loss in patients

with rheumatoid arthritis. Rheumatology

(Oxford) 2014; 53(11):1994-2001.

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Lyraki A, Raftakis I, Antoniadis C. Vitamin D

and rheumatoid arthritis. Ther Adv Endocrinol

Metab 2012; 3(6):181-7.

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Association between Serum 25-Hydroxyvitamin

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PS, Brenol CV, Xavie RM, et al. Expression of

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Blood Cells from Rheumatoid Arthritis Patients.

OJRA 2014; 4:69-73.

18. Szodoray P, Nakken B, Gaal J, Jonsson R,

Szegedi A, Zold E, et al. The complex role of

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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 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.


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


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)


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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sepsis campaign: international guidelines for

management of severe sepsis and septic shock

2008; 36:296-327.

6. Okorie N, Dellinger RP. Lactate: Biomarker

and potential therapeutic target. Crit Care Clin

2011; 27:299-326.

7. Bakker J, Gris P, Coffernils M, et al. Serial

blood lactate levels can predict the development

of multiple organ failure following septic shock.

Am J Surg. 1996;171(2):221-226.

8. Bone RC, Balk RA, Cerra FB, et al.

Definitions for sepsis and organ failure and

guidelines for the use of innovative therapies in

sepsis. The ACCP/SCCM Consensus Conference

Committee. American College of Chest

Physicians/Society of Critical Care Medicine.

Chest. 1992;101(6):1644-1655.

9. Levy MM, Fink MP, Marshall JC, et al. 2001

SCCM/ESICM/ACCP/ ATS/SIS International

Sepsis Definitions Conference. Crit Care Med.

2003;31(4):1250-1256.

10. Nguyen HB, Rivers EP, Knoblich BP, et al.

Early lactate clearance is associated with

improved outcome in severe sepsis and septic

shock. Crit Care Med. 2004;32(8):1637-1642.

11. Shapiro NI, Howell MD, Talmor D, et al.

Serum lactate as a predictor of mortality in

emergency department patients with infection.

Ann Emerg Med. 2005;45(5):524-528.

12. Trzeciak S, Dellinger RP, Chansky ME, et al.

Serum lactate as a predictor of mortality in

patients with infection. Intensive Care Med.

2007;33(6):970-977.

13. Bakker J and Jansen TC. Don’t take vitals,

take a lactate. Intensive Care Med.

2007;33(11):1863-1865.

14. Aduen J, Bernstein WK, Khastgir T, et al.

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specific electrode for rapid determination of

blood lactate concentrations. JAMA.

1994;272(21):1678-1685.

15. Dellinger RP, Levy MM, Carlet JM, et al.

Surviving Sepsis Campaign: international

guidelines for management of severe sepsis and

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exogenous lactate clearance as an early indicator

of mortality in normolactatemic critically ill

septic patients. Crit Care Med 2003; 31:705– 710.

32. Bakker J, Gris P, Coffernils M, et al: Serial

blood lactate levels can predict the development

of multiple organ failure following septic shock.

Am J Surg 1996; 171:221–226.

33. Abramson D, Scalea TM, Hitchcock R, et al:

Lactate clearance and survival following injury. J

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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)

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3865839/#i1524-5012-13-4-561-b01

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.




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