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Editor in chief
M. Y. Taher
Founder Editors
Hilmy Abaza
Seham Abdel Reheem
Co-Editors
Ahmed Shawky
FathAlla Sidkey
Maher Osman
Mohamed Sharaf De Din
International Advisory Board
JP Galmiche France
A Sandeberg Sweden
X Rogiers Belgium
S Jensen Denmark
Des Verrannes France
Antonio Ascione Italy
S Brauno Italy
P Almasio Italy
National Advisory Board
Moustafa El Henawi
Amira Shams Eldin
Nabil Abdel Baki
Hoda E-Aggan
M Essam Moussa
Ahmed Bassioni
Saeid Elkyal
Abdel Fataah Hano
Khaled Madboli
Ezzat Aly
Contents Alexandria Journal of Hepatogastroenterology, Supplement (I) - January 2016
------------------------------------------- Manuscript Submission: For information and to submit
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Disclaimer: The Publisher, the Egyptian Society of
Hepatology Gastroenterology and Infectious Diseases in
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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
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Original Article:
Assessment of Clinical Significance of Serum
Angiopoietin-2 in Cirrhotic HCV Patients with and
without Hepatocellular Carcinoma
Ayman Farid EL Shayeb1, Mohamed Sobhy EL Shazly1, Akram Abdel moneim Deghady,2 Ehab Hassan Elkhouly1, Alaa
Mohamed Ragab Ahmed1; 1Tropical Medicine department, 2clinical pathology department, Faculty of Medicine, University
of Alexandria
--------------------------------------------------
Original Article:
Association of Type 2 Diabetes Mellitus and Liver Disease
in Elderly Patients
Suzanne Nashait Abou-Raya1, Abdel Aziz Elnekedy2, Maher
Mahmoud Abd ElNaby3, Fathya Mohamed Mohamed1; 1Department of Internal Medicine, Faculty of Medicine,
University of Alexandria, Egypt, 2Department of Radiology,
Faculty of medicine, University of Alexandria, Egypt, 3Department of Biochemistry, Medical Research Institute,
University of Alexandria, Egypt.
--------------------------------------------------
Original Article:
Combined Radiofrequency Ablation and Transcatheter
Hepatic Arterial Chemoembolization Versus
Radiofrequency Ablation Alone in Treatment of
Unresectable Hepatocellular Carcinoma
Mohamed Yousef el Hasafy1, Ehab Mustafa Hassona1, Hanaa
Mahmoud Nagdy1,Amr Mohamed el abed2, Omar Sameh Al
aesr2; 1Department of Internal Medicine; 2Department of
Radiodiagnosis; Faculty of Medicine; University of Alexandria.
--------------------------------------------------
Original Article:
Nano-ELISA in the Diagnosis of Experimental
Toxoplasmosis
Mervat Zakaria El Azzouni1, Lobna Abd El-Aziz El Zawawy1,
Doaa El-Said Said Ahmed1,Wegdan Ramadan Ahmed2, Maha
Mohamed Gomaa3; 1Professor of Medical Parasitology, 2Assistant
Professor of Physics, 3Assistant lecturer of Medical Parasitology
--------------------------------------------------
Original Article:
Study of The Association Between Vitamin A Level and
Uremic Pruritus in Chronic Renal Failure Patients in
Ismailia City
Roshdy Wasfi Mohamed, Nagwan Abd El Aziz Mohamed Sabek,
Yasser Salem NasrAllah, Omar Mostafa Mohamed Al Hashash.
Department of Dermatology and Venereology, Department of Medical Biochemistry, Faculty of Medicine, Suez Canal
University.
-------------------------------------------------- Original Article:
The Diagnostic Role of Serum Procalcitoninin
Differentiation Between Bacterial and Abacterial
Meningitis
Mohammed Kassem1, Akram Deghady2, Nasser Mohammed Abd
Alla1, Mahassen Hosny1; Departments of Tropical Medicine1 and
Clinical Pathology2, Faculty of Medicine, University of
Alexandria
--------------------------------------------------
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14
21
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Original Article
Assessment of Clinical Significance of Serum Angiopoietin-2 in Cirrhotic HCV
Patients with and without Hepatocellular Carcinoma
Ayman Farid EL Shayeb1, Mohamed Sobhy EL Shazly1, Akram Abdelmoneim Deghady2, Ehab Hassan
Elkhouly1, Alaa Mohamed Ragab Ahmed1; 1Tropical Medicine department, 2clinical pathology
department, Faculty of Medicine, University of Alexandria
ABSTRACT liver cirrhosis is characterized by remodeling leading to nodules that are difficult to discern from hepatocellular
carcinoma (HCC). HCC is one of the most common cancers in the world.The annual risk of developing HCC
following cirrhosis is between 1% and 6%.Thus diagnosis of HCC at an early stage is of utmost importance. HCC is
characterized by a high vascularity but the mechanisms of neovascularization that permit rapid growth have not been
defined. Angiopoietins (Ang-1 andAng-2) and vascular endothelial growth factor (VEGF) are endothelial cell-
specific vasculogenic and angiogenic growth factors, but their expression and roles in HCC have not been
extensively explored. Aim of the work: The aim of this study was to study the serum Ang-2in cirrhotic HCV patient
with and without HCC. Material and Methods: The study was done on 20 patients with cirrhosis, 20 patients with
HCC, and 20 healthy controls who were classified into three groups.Evaluation included full history taking, clinical
examination , laboratory investigations included; routine investigations, liver function tests and specific
investigations for patients such as alfa fetoprotein (AFP) and Ang-2 using ELISA assay. Abdominal
ultrasonography and then Triphasic CT was performed for patients with proven hepatic focal lesion. Results: The
mean value of Ang-2 was significantly higher in patients with HCC and patients with cirrhosis than in healthy
controls respectively(p= 0.013, p= 0.001, p<0.001).Also it was found to be significantly higher in patients with HCC
than those with cirrhosis. Moreover there was significant positive correlation between Ang-2 and the number of
HCC Foci (r=0.627*) (p=0.003), however no significant correlation was found between it and tumor size (r=0.250)
(p=0.287). Conclusion:Serum Ang-2 might be used as an early predictor of development of HCC in patients with
liver cirrhosis as well as a marker of progression and or invasiveness of cancer process.
Introduction HCC is the major form of primary liver cancer in
the world,(1) accounting for 662,000 deaths
worldwide per year.(2) HCC is frequently
diagnosed at an advanced stage, resulting in
rather poor survival rates. HCC typically starts
with a pre-existing liver disease caused by
infection with HBV or HCV, chronic aflatoxin
exposure or alcohol consumption. Chronic liver
damage associated with chronic exposure to
these agents results in cirrhosis, which can
eventually progress to liver cancer.(3). The role of
biomarkers for liver cancer related to early
detection, invasiveness, metastasis, and
recurrence has attracted great deal of research
interest leading to discovery and utilization of
several novel markers such as AFP, GPC3, GGT
and Ang-2.(4,5). Angiopoietin-2(Ang-2)destabilizes
the quiescent endothelium and primes it to respond
to exogenous stimuli, thereby facilitating the
activities of inflammatory cytokines (tumor
necrosis factor and interleukin-1) and angiogenic
(vascular endothelial growth factor). Intriguingly,
Ang-2 is expressed weakly by the resting
endothelium but becomes strongly up regulated
following endothelial activation.(6,7) Moreover,
endothelial cells store Ang-2 in Weibel-Palade
bodies from where it can be made available quickly
following stimulation, suggesting a role of Ang-2
in controlling rapid vascular adaptive
processes.(8,9). The vascular endothelium lines the
inside of all blood vessels, forming a non-
thrombogenic surface that controls the entry and
exit of plasma and white blood cells to and from
the bloodstream. It is one of the largest internal
surfaces of the body and can be considered
conceptually as a systemically disseminated
organ. The quiescent endothelium has turnover
rates of months to years, and proliferates only
following angiogenic activation. The molecular
mechanisms controlling the quiescent
endothelial-cell phenotype are poorly
understood.(10)
Patients and Methods
This study was conducted on 60 subjects who
were classified into three groups: group (I) 20
patients with HCV induced liver cirrhosis
without HCC, and group (II) 20 patients with
HCV induced liver cirrhosis and HCC and group
(III) 20 healthy controls .All patients were
subjected to the following: full history taking,
clinical examination , laboratory investigations
including ; routine investigations (Complete
blood picture, Serum urea and creatinine), Liver
function tests(ALT, AST, GGT, PT, TSB &
serum albumin) and specific investigations for
patients such as PCR for HCV RNA, as well as,
AFP and Ang-2 using ELISA assay and
ultrasonographic study of the abdomen.
Triphasic CT abdominal examination was
performed for patients with ultrasound proven
hepatic focal lesion.
Results
The mean value of Ang-2 was significantly high
in HCC and liver cirrhosis patients than in healthy
control (131.50 ± 89.06, 99.0 ± 10.87 and 86.25 ±
13.17) (p<0.001*, p= 0.001*, p<0.013*)
respectively. Moreover it was significantly higher
in HCC patients than in those with liver cirrhosis
alone . (Table I)
Table (I): Comparison between the studied groups according to Angiopeoitin-2 ng/l
Cirrhosis
(n=20)
HCC
(n=20)
Control
(n=20) KWχ2 p
Mean ± SD 99.0 ± 10.87 131.50 ± 89.06 86.25 ± 13.17 21.542 <0.001
Median 100.0 110.0 80.0
Significancebetween groups p1 = 0.013, p2= 0.001 , p3 <0.001
KW2: Chi square for Kruskal Wallis test
p1 : p value for comparing between Cirrhosis and HCC
p2 : p value for comparing between Cirrhosis and Control
p3 : p value for comparing between HCC and Control
*: Statistically significant at p ≤ 0.05
Regarding to CT findings; Size of focal lesion in
HCC patients it ranged from 1.20 – 8.0 cm with
mean range from 3.31 ± 2.31. However, no
significant correlation was found between serum
Ang-2 and tumor size (r=0.250)(p=0.287).
(Table II)
The number of focal lesion in HCC patients,
there were 13 patients with one lesion, 7 patients
with multiple lesions. There was a significant
positive correlation between serumAng-2 and the
number of HCC lesions(r =0.627*)(p=0.003).
(Table II)
Table (II):Correlation between Angiopeoitin-2 with CT Size of Focal Lesion, CT Number of Focal Lesion.
rs: Spearman coefficient *: Statistically significant at p ≤ 0.05
The mean value of AFP in liver cirrhosis patients,
HCC patients and control group were (55.38 ±
39.37), (654.53±376.15) and (3.35 ± 0.75) ng/ml
respectively. The mean value of AFP was
significantly high in HCC and liver cirrhosis
patients than in healthy control (p<0.001*,
p<0.001*, p<0.001*). HCC patients had a
significantly higher values of AFP than liver
cirrhosis patients only .(Table III)
Table (III): Comparison between the studied groups according to AFP
Cirrhosis
(n=20)
HCC
(n=20)
Control
(n=20) KWχ2 p
Alpha Feto Protein
Min. – Max. 10.0 – 137.0 20.50 – 1060.0 2.0 – 4.0
44.991* <0.001* Mean ± SD. 55.38 ± 39.37 654.53±376.15 3.35 ± 0.75
Median 46.50 785.50 3.50
Significance between groups p1 <0.001*, p2 <0.001*, p3 <0.001*
Angiopeoitin-2
rs p
CT Size of Focal Lesion 0.250 0.287
CT Number of Focal Lesion 0.627* 0.003
KW2: Chi square for Kruskal Wallis test
p1 : p value for Mann Whitney test for comparing between Cirrhosis and HCC
p2 : p value for Mann Whitney test for comparing between Cirrhosis and Control
p3 : p value for Mann Whitney test for comparing between HCC and Control
*: Statistically significant at p ≤ 0.05
Regarding to Ang-2 the cut off value was 110
ng/ml in HCC group with a sensitivity of 70%,
specificity of 95%, PPV 93.33%, NPV 76.0%
and accuracy 82.5% between HCC and other
groups.
Table (IV):Agreement (sensitivity, specificity and accuracy) for Angiopeoitinng/l with HCC cases
Control HCC
Sen
siti
vit
y
Sp
ecif
icit
y
PP
V
NP
V
Acc
ura
cy
Angiopeoitinng/l <110 19 6
70.0 95.0 93.33 76.0 82.50 ≥110 1 14
Figure (1):ROC curve for Angiopeoitin ng/l to diagnose HCC cases
Discussion
Hepatocellular carcinoma (HCC) is the most
common primary malignant tumor of the
liver.(11)Over a decade (1993-2002), there was
nearly a twofold increase of the proportion of
HCC among chronic liver disease (CLD)
patients in Egypt with a significant decline of
hepatitis B virus (HBV) and slight increase of
hepatitis C virus (HCV) as risk factors.(12).
Patients with chronic liver disease, and
particularly those with cirrhosis, have an
increased risk of having HCC development.
Several studies have shown that HCC develops
mainly in cirrhotic livers, with this association
being seen in more than 80% of cases in Western
countries.The incidence of HCC in patients with
cirrhosis is approximately 10 times higher than
in the non-cirrhotic population.(13-16). The first
serologic assay for detection and clinical follow up
of patients with HCC was AFP which has been the
standard tumor biomarker for HCC for many
years.(17). Angiopoietins are links between
angiogenesis and inflammation. The
angiopoietin Tie ligand-receptor system has a
key regulatory role in regulating vascular
integrity and quiescence. Besides its role in
angiogenesis, it is an important regulator in
numerous diseases including inflammation.(18).
The current study showed that the mean serum
levels of AFP in HCC group had the highest
level compared to other groups with statistically
significant difference (p≤0.001) This finding
came in agreement with previous studies of
many authors Mittal et al(19) and Guan et al(20).
Also comparable to Gad et al(21)who found a
significantly higher sensitivity of AFP in
Egyptian patients in comparison with Japanese
patients with HCC diagnosis (99% versus 67%
p< 0.001).Highest levels of Ang-2were detected in
HCC patients compared to cirrhosis patients (p<
0.001). Additionally, Diaz-Sanchez et al (22)
concluded that the Ang-2 seems to play an
important role in the angiogenic processes of HCC
and its serum levels are associated with tumor
characteristics and invasive behavior. Another
study done by Scholz et al (23) who found a
statistically highly significant elevation of Ang-2
serum levels in HCC patients when compared to
cirrhotic patients and also reported that Ang-2
mRNA was expressed in most of HCC
cryopreserved biopsies using in situ hybridization
in addition to also agree with a previous work done
by Hunter et al(24) who reported a statistically
highly significant elevation (p< 0.001) in the mean
serum Ang-2 in HCC group when compared with
both the control and cirrhosis groups.In the current
study, the results revealed that there was a
statistically highly significant elevation (p≤0.001)
in the mean serum Ang-2 in cirrhosis group when
compared to control group. These results are
consistent with Scholz et al(23) who reported a
statistically highly significant elevation of Ang-2
serum levels in cirrhotic patients when compared
to control subjects. In addition, in our work,
serum Ang-2 did not exhibit a significant
correlation with the size of focal hepatic lesions
which was also in agreement with Scholz et
al.(23) However, there was a significant
correlation between the number of hepatic focal
lesions and serum Ang-2 level. Zhang et
al(25)demonstrated that Ang-2is associated with
tumor size, portal vein invasion, metastases, and
the presence of a tumor capsule. Moreover, the
positive influence of Ang-2 in the microvessel
density and the differentiation of HCC were
observed in several other reports.(26-29). In the
current work Ang-2 had a cut off value 110
ng/ml in HCC group with sensitivity of 70%
specificity of 95%, PPV 93.33%, NPV 76.0%
and accuracy 82.5% between HCC and other
groups. Further studies are warranted in large
number of patient to determine the clinical value
of soluble Ang-2 as a prognostic factor, and a
surrogate indicator tor therapeutic response.
Conclusion
Serum Ang-2 might be used as a predictor of
development of HCC in patients with liver cirrhosis
as well as a marker of progression and /or
invasiveness of cancer process.
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Original Article
Association of Type 2 Diabetes Mellitus and Liver Disease in Elderly Patients
Suzanne Nashait Abou-Raya1, Abdel Aziz Elnekedy2, Maher Mahmoud Abd ElNaby3, Fathya Mohamed
Mohamed1; 1Department of Internal Medicine, Faculty of Medicine, University of Alexandria, Egypt, 2Department of Radiology, Faculty of medicine, University of Alexandria, Egypt, 3Department of
Biochemistry, Medical Research Institute, University of Alexandria, Egypt.
ABSTRACT Aging as a physiological process is associated with complex changes in all organs.By the age of 75, approximately
20% of the populations are afflicted with Type 2 diabetes mellitus (T2DM); about 10% of subjects aged 60 to 74
years have T2DM and do not know it. There is accumulating data demonstrating that obesity and insulin resistance
closely correlate with a more severe fibrogenic progression in different chronic liver diseases (CLDs), including
HCV-related hepatitis and alcoholic liver disease. Aim of the work: To investigate the prevalence and spectrum of
liver disease in elderly T2DM patients and to determine its relationship with metabolic control. Methods: The study
was conducted on 60 patients with T2DM aged above 65 years without any life-threatening condition. All patients
were subjected to the following: a) full history with particular stress on onset and control of DM, duration of illness,
history of hypertension, the presence of diabetic complications (micro or macro vascular), drug history, previous
operations, blood transfusions, history of schistosomiasis, hepatitis and jaundice.; b) comprehensive physical
assessment including measurement of blood pressure, peripheral pulsations, body mass index (BMI), presence of any
diabetic complications, abdominal examination and signs of liver disease and laboratory investigations which
included Fasting blood glucose (FBG), 2 hours postprandial glucose PPBG), glycated haemoglobin (HbA1C), Liver
function tests: aspartate aminotransferase (AST), alanine aminotransferase (ALT), serum bilirubin, albumin; viral
markers including Anti-HCV, α-fetoprotein assay and lipid profile including serum cholesterol and triglycerides.
Ultrasonography of the abdomen and liver imaging was also done for all subjects. Results: This study showed that
there was an increase in liver enzymes in both male and female diabetic patients and positive correlation with the
increase in FBG, PPBG, HA1c. We found liver enzymes to be significantly increased in uncontrolled diabetes
compared to controlled diabetics. Triglycerides in uncontrolled diabetic patients were significantly higher than
controlled diabetics with a positive correlation between TG and the diabetic profile. Fatty liver was present in 36.7%
of cases. Conclusions: There is positive correlation between diabetes and liver functions. Fatty liver increases with
diabetes which increases the risk of cirrhosis and hepatocellular carcinoma in these patients.
Introduction
Aging as a physiological process is associated
with complex changes in all organs, and these
changes occur at varying rates. They affect the
body's functional reserve, leading to impaired
ability to maintain homeostasis and withstand
stressors. In many organs, function loss begins at
30 to 40 years of age and proceeds at a rate of
approximately 1% annually (1). Liver functions
are relatively well preserved in elderly
individuals (2). In old age there is a combination
of abnormal beta cell function with peripheral
insulin resistance leads to increased glucose
intolerance in normal aged persons (3). Diabetes
mellitus is a group of metabolic diseases
characterized by hyperglycemia resulting from
defects in insulin secretion, insulin action, or
both. The chronic hyperglycemia of diabetes is
associated with long term damage, dysfunction,
and failure of various organs, especially the eyes,
kidneys, nerves, heart, and blood vessels (4).
Diabetes is common in the elderly population.
By the age of 75, approximately 20% of the
populations are afflicted with this illness, about
10% of subjects aged 60 to 74 years have T2DM
and do not know it (5). T2DM is characterized by
impaired insulin secretion, insulin resistance,
excessive hepatic glucose production, and
abnormal fat metabolism. Obesity, particularly
visceral or central (as evidenced by the waist-hip
ratio (WHR), is very common in T2DM (6).
Elderly patients with diabetes may present with
non-specific symptoms only, which may be
dismissed as (normal aging), even typical mode
of presentation may be missed. (7). Accumulating
data has demonstrated that obesity and insulin
resistance closely correlate with a more severe
fibrogenic progression in different CLDs,
including HCV-related hepatitis and alcoholic
liver disease (8). In hepatocytes, the insulin
resistant state is brought about by at least one,
but more likely by a combination, of the
following metabolic pathological hormonal
alterations: hyperglycemia and hyper-
insulinaemia, formation of advanced glycation
end-products, increased free fatty acids and their
metabolites, oxidative stress and altered profiles
of adipocytokines (9). Hepatic insulin resistance is
an important underlying cause of the metabolic
syndrome that manifests itself in diseases such as
diabetes T2DM, atherosclerosis or non-alcoholic
fatty liver disease (NAFLD) (10). Insulin
resistance is present in the majority of the
patients with non-alcoholic fatty liver disease.
HOMA-IR index >4.5 or triglyceride levels
>180 mg/dl are predictors of the presence of
NASH. Insulin resistance may play" a role in the
pathogenesis of fibrosis progression in patients
with NAFLD. Drugs able to decrease insulin
resistance could be useful in the therapy of this
disease (11). Fibrosis and cirrhosis are the final
outcomes of all chronic liver disease; however,
some morphological and biological differences
distinguish fibrosis due to NASH from the one
secondary to other causes of liver damage (12).
The liver diseases seen in type 2 diabetes cover
virtually the entire spectrum of liver disease.
Cirrhosis is an important cause of death in
diabetes (13). The prevalence of hepatitis C virus
(HCV) is higher in patients with diabetes than in
the general population (14–22). Numerous studies
have confirmed a fourfold increased prevalence
of hepatocellular carcinoma in patients with
diabetes as well as an increased prevalence of
diabetes in patients with hepatocellular
carcinoma (23–26). The pathogenic sequence of
events leading to hepatocellular carcinoma
appears to be insulin resistance, increased
lipolysis, lipid accumulation in the hepatocytes,
oxidative stress, and cell damage followed by
fibrosis and cell proliferation, which are
procarcinogenic (27–30). Accordingly, the present
study was designed to investigate the prevalence
and spectrum of liver disease in elderly T2DM
patients and to determine its relationship with
metabolic control.
Subjects and Methods After approval of local ethics committee, this
study was conducted on sixty elderly type 2
diabetic patients with no life threatening
conditions. All subjects were subjected to full
history, comprehensive physical assessment,
laboratory investigations (FBG, 2hPPBG,
HbA1C, AST, ALT, Anti-HCV, α-FP, T.G,
cholesterol, CBC, urea and creatinine level) and
ultrasonography of the abdomen and liver
imaging.
Results
The patients included in this study were 30
males (50%) and 30 females (50%).Twenty one
subjects (35%) were controlled diabetic patients
and 39 subjects (65%) were uncontrolled
diabetic patients. Twenty six of the patients
(43.3%) in this study were on oral hypoglycemic
therapy while 34 subjects (56.7%) under insulin
therapy. Ultrasound findings of the patients
showed 31 patients had cirrhosis (51%), 22 had
fatty liver (36.7%), 10 had focal lesions (16.7%),
27 had ascities (30%), 27 had splenomegaly
(45%) and 10 subjects were normal (16.7%)
(Table 1). The ultrasonographic finding
indicated that, liver cirrhosis and splenomegaly
were significantly more abundant in diabetic
male patients than females ( p=0.020) while fatty
liver was significantly higher in diabetic female
patients than males (p=0.007) (Table 2).
Regarding liver function tests, it was found that
there was a statistical significant difference
between controlled and uncontrolled diabetic
patients. ALT, AST and total bilirubin were
significantly higher in the uncontrolled group
compared to the controlled group; on the other
hand, serum albumin level was significantly
lower in the uncontrolled group compared to the
controlled group p<0.001 (Table 3). The
triglycerides level was significantly higher in
uncontrolled group compared to the controlled
group p=0.001 (Table 4). We also found: 1-
Positive correlation between ALT activity and
the levels of fasting blood glucose (r=0.440,
p<0.001), post-prandial blood glucose (r=0.345,
p=0.007) and HbA1c (r=0.377, p=0.003) figure
1. 2- Positive correlation between AST activity
and the levels of fasting blood glucose (r=0.326,
p<0.011), and HbA1c (r=0.292, p=0.024) figure
2. 3- Positive correlation between total bilirubin
level and the levels of fasting blood glucose
(r=0.354, p=0.006), post-prandial blood glucose
(r=0.254, p=0.05) and HbA1c (r=0.415,
p=0.001)figure 3. 4- Positive correlation between
cholesterol level and the levels of triglycerides
(r=0.612, p<0.001), and HbA1c (r=0.364,
p=0.004) figure 4.
Table (1): Demographic and clinical history data of the study population (n= 60)
No. %
Sex
Male 30 50.0
Female 30 50.0
Age (years)
60 - <65 7 11.7
65 - <70 21 35.0
≥70 32 53.3
Diabetes
Controlled 21 35.0
Un controlled 39 65.0
Treatment
OHG 26 43.3
Insulin 34 56.7
Clinical history
HTN 39 65.0
Operation 18 30.0
Blood T.F 4 6.7
HCV 27 45.0
Jaundice 28 46.7
Ultrasound
Cirrhosis 31 51.7
Fatty liver 22 36.7
Focal lesion 10 16.7
Ascites 18 30.0
Splenomegaly 27 45.0
Normal 10 16.7
OHG=oral hypo glycemic, HTN=hypertension, HCV=hepatitis c virus
Table (2): Ultrasound findings of the study population according to their sex
Sex
2 P Male
(n = 30)
Female
(n = 30)
No. % No. %
Ultrasound
Cirrhosis 20 66.7 11 36.7 5.406* 0.020*
Fatty liver 6 20.0 16 53.3 7.177* 0.007*
Focal lesion 7 23.3 3 10.0 1.920 0.166
Ascities 12 40.0 6 20.0 2.857 0.091
Splenomegaly 18 60.0 9 30.0 5.455* 0.020*
Normal 5 16.7 5 16.7 0.0 1.000
2: Chi square test
FE: Fisher Exact test
*: Statistically significant at p ≤ 0.05
Table (3): Liver function tests in the study population according to their diabetic status.
Diabetes
Test of sig. P Control
(n = 21)
Uncontrolled
(n = 39)
ALT
Min. – Max. 16.0 – 95.0 40.0 – 122.0
Mean ± SD. 45.0 ± 28.35 66.79 ± 23.0 Z=2.645* 0.008*
Median 36.0 59.0
AST
Min. – Max. 32.0 – 215.0 46.0 – 300.0
Mean ± SD. 77.19 ± 71.96 79.49 ± 44.19 Z=2.255* 0.024*
Median 32.0 63.0
TB
Min. – Max. 0.40 – 13.50 0.20 – 11.65
Mean ± SD. 3.47 ± 3.74 4.53 ± 2.05 Z=2.794* 0.005*
Median 2.20 3.80
Albumin
Min. – Max. 2.80 – 4.80 0.70 – 3.40
Mean ± SD. 3.82 ± 0.71 2.01 ± 0.68 t =9.738* <0.001*
Median 3.90 2.0
ALT= alanine aminotransferase, AST= aspartate aminotransferase, TB=total billirubin
2: Chi square test
FE: Fisher Exact test
*: Statistically significant at p ≤ 0.05
Table (4): Lipid profile in the study population according to their diabetic status
Diabetes
Test of sig. P Control
(n = 21)
Uncontrolled
(n = 39)
Cholesterol
Min. – Max. 115.0 – 280.0 115.0 – 278.0
Mean ± SD. 183.76 ± 35.75 193.33 ± 48.66 0.868 0.389
Median 188.0 198.0
TG
Min. – Max. 68.0 – 170.0 65.0 – 256.0
Mean ± SD. 95.48 ± 22.83 130.18 ± 55.72 3.396* 0.001*
Median 89.0 112.0
FBG=fasting blood glucose, PPBG=post prandial blood glucose, HbA1c= glycated hemoglobin, TG= triglycerides
t: Student t-test
*: Statistically significant at p ≤ 0.05
0
20
40
60
80
100
120
140
0 100 200 300 400 500 600
AL
T
FPG
r = 0.440*
p <0.001
0
50
100
150
200
250
300
350
0 100 200 300 400 500 600
AS
T
FPG
r = 0.326*
p = 0.011
Figure (1): Correlation between FPG with ALT Figure (2): Correlation between FPG with AST
0
2
4
6
8
10
12
14
16
0 100 200 300 400 500 600
TB
FPG
r = 0.354*
p = 0.006
0
50
100
150
200
250
300
5 6 7 8 9 10 11
TG
HbA1c
r = 0.283*
p = 0.028
Figure (3): Correlation between FPG with TB Figure (4): Correlation between HbA1c with TG
Discussion
In the present study it was found that type 2
diabetes increases with age 11.7% were affected
between 60-65 years, 35% between 65-70 and
55% above 70 y. (table 1).This is in agreement
with the study done by Brown AF et al in 2003 (31). We found that cirrhosis was present in 31
patients (51%) while 27 patients (45%) only had
a history of HCV infection table (1) which
means that about 6% had cirrhosis due to another
cause; mainly NASH which end by cirrhosis also
and it is a common liver disease in diabetic
patients. These findings are in accordance with
previous studies which demonstrated that NASH
may be present with cirrhosis in late stages (32).
We also found that triglycerides in uncontrolled
diabetic patients were significantly higher than
the controlled group (table 4) and there was a
positive correlation between TG and the diabetic
profile (figure 56-58), this is in accordance with
the study done by Cassader M et.al (33). which
suggests that diabetes is accompanied by a
specific dyslipidemic profile characterized by
elevated plasma triglycerides, decreased HDL
cholesterol, and predominance of small LDL (33).
We also found that liver enzymes are
significantly increased in uncontrolled diabetes
(ALT 59-AST 63-T.B. 3.8 –Alp. 2) compared to
the controlled group (ALT 36-AST 32-T.B. 2.2-
Alp. 3.9) table 3 figures which suggest that
diabetes has a role in increasing liver enzymes.
There was also a positive correlation between the
increase in liver enzymes and the increase in
FBG, PPBG, HA1c figures 1, 2, 3. This is in
accordance with the study done by Lebovitz HE
et.al.in 2002 (34). In the present study we found
that fatty liver occurred significantly more in
diabetic females (46.2%) than males (19%) table
2 which is in contrast to the study done by
Amarapurkar D, Kamani P et al. (35) which
showed that NAFLD was more prevalent in
diabetic males than females (24.6% vs 13.6% ( p
< 0.001).(35) This difference may be attributed to
the small size of the sample. In the present study
we also found that Alfa fetoprotein was
increased in males more than females and focal
lesions were more common in males (23%) than
females-(10%) table 2. This is in accordance
with the findings in the study done in 2013 by
Hefaiedh R et al. (36) which showed that HCC is
more common in males than females. Recently a
study of HCC incidence was conducted in a
large cohort of VA patients (173,643 patients
with and 650,620 patients without diabetes). The
findings of this study indicated that HCC
incidence doubled among patients with diabetes
and was higher among those with a longer
duration of follow-up evaluation (37). This may
be due to the fact that a high concentration of
insulin could stimulate the IGF pathway in DM2
and molecular studies have shown that insulin
and insulin-like growth factor 1 (IGF-1) may
have carcinogenetic effects on liver and other
tissues(38-42). Moreover, chronic hyperglycemia
may cause oxidative stress and cellular damage(
43).
Conclusions
There is positive correlation between diabetes
and liver functions. Patients on insulin treatment
have significantly higher liver function tests;
(ALT, AST and total bilirubin) compared to
those patients under OHG agents. Fatty liver
increases with diabetes duration and severity
which increases the risk of cirrhosis and
hepatocellular carcinoma.
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Original Article
Combined Radiofrequency Ablation and Transcatheter Hepatic Arterial
Chemoembolization Versus Radiofrequency Ablation Alone in Treatment of
Unresectable Hepatocellular Carcinoma
Mohamed Yousef el Hasafy1, Ehab Mustafa Hassona1, Hanaa Mahmoud Nagdy1,Amr Mohamed el abed2,
Omar Sameh Al aesr2; 1Department of Internal Medicine; 2Department of Radiodiagnosis; Faculty of
Medicine; University of Alexandria.
ABSTRACT The incidence of hepatocellular carcinoma is increasing worldwide. Most hepatocellular carcinomas are diagnosed at
intermediate or advanced stages, and only 30% of patients benefit from curative therapies such as resection, liver
transplantation, or percutaneous ablation.combined interventional therapies are superior to any single therapy for
improving the prognosis and survival of patients with HCC. Trans-arterial chemoembolization (TACE) combined
with radiofrequency ablation (RFA) therapy has been used in treatment of medium size unrespectable HCC. Aim of
the work: This study was aimed to prospectively compare the effectiveness andsafety of combined trans-arterial
chemoembolization (TACE) plus radiofrequency ablation (RFA) versus RFA alone in patients with unresectable
hepatocellular carcinoma (HCC). Patients and Methods: Forty cirrhotic patients (Child-Pugh class A or B) with
solitary or oligo-nodular HCC. They were divided into two groups. Group І patients have been treated with RFA
alone. Group ІІpatients have been treated by combined TACE and RFA. Results: Twelve months after the ablation
treatment, 13/21 lesions (61.9%) had complete response, 2/21 lesions (9.5%) had partial response and 6/21 lesions
(28.6%) had local recurrence in group І patients. While in group ІІ patients, 25/25 lesions (100%) had complete
response. There was significant statistical difference between the two groups (p=0.001) as regard response to
treatment.The total number of deaths was 4/20(20%) in group І and the overall 1year survival rate was 80%. The
mean survival time was 11.55 months. While in group ІІ, there were no deaths and the overall survival rate was
100%. The mean survival time was 12 months. There was significant statistical difference between the two groups
(p=0.037) as regard overall one year survival rate.Conclusions: The combination of TACE and RFA is safe and
effective in treatment of unresectable HCC. It provides better local tumor control and better overall one year survival
than RFA alone for the treatment of patients with medium-sized HCC.
Introduction
Hepatocellular carcinoma (HCC) is the fifth
most common cancer worldwide with over 600
000 deaths per year, HCC represents a major
health challenge with significant and increasing
global impact .It is the fifth most common
malignant disease in men and the seventh most
common in women. HCC is the second most
common cause of cancer-related death and the
leading cause of death after lung cancer among
patients with cirrhosis.(1)HCC incidence and
mortality are increasing, while mortality from
other cirrhosis-related complications appears to
be declining.(2). Although HCC is more prevalent
in Asian and African nations, there is rising
evidence that the incidence of HCC is increasing
in developed countries. During the last two
decades, an increase in HCC incidence has been
reported from Australia, France, the United
Kingdom, Japan and North America. This
increased incidence is attributable to more
accurate and earlier diagnosis as well as an
increase in immigration from high prevalence
regions.(3). HCC is commonly associated with
underlying chronic liver disease and cirrhosis
caused by persistent viral infection with HBV
and/or HCV, alcohol abuse, aflatoxin B1,
inherited metabolic diseases such as hereditary
hemochromatosis, α-1-antitrypsin deficiency and
hereditary tyrosinaemia. Obesity and diabetes
can lead to non-alcoholic steatohepatitis, which
is an established risk factor for HCC, mostly via
progression of the steato hepatitic disease to
cirrhosis and HCC (4). Increased hepatocyte
replication accompanied by DNA damage and
outgrowth of clonal cell populations appears to
underlie hepato carcinogenesis caused by known
HCC risk factors. However, it is unclear whether
the DNA damage that accompanies the increased
mitotic rate is the result of replication errors
imparted by abnormal and rapid progression
through the cell cycle and/or owing to
mutagenesis of the hepatocyte genome directly
by toxins or through oxidative stress induced by
inflammation or other mechanisms.(5). A
multidisciplinary approach is needed in the
management of patients with HCC as it involves
disciplines such as surgery, hepatology,
diagnostic and interventional radiology,
oncology and pathology.(6)
Aim of the work
The aim of the work is to evaluate the outcome,
necessity and feasibility of combined RFA and
TACE for unrespectable HCC versus RFA alone.
Subjects
Our study included 40 patients who will be
divided into two groups:Group I: 20 patients
with unresectable HCC who will be treated with
RFA. Group II: 20 patients with unresectable
HCC who will be treated with combined RFA
and TACE.Eligibility criteria were as follows: 1-
Unresectable HCC (according to the BCLC
classification). (6). 2-Three or fewer lesions, each
larger than 3 cm in greatest diameter; Monolobar
or Bilobar; Monofocal or Multifocal at the time
of treatment. 3-Lesions located at least 1cm
away from the hepatic hilum and the common
bile duct and 1 cm from the bowel. 4-No
previous HCC treatment. 5-Child-Pugh class A
or B. 6-There is no vascular invasion and no
extra-hepatic metastasis. 7-Randomization
method: closed envelope.Exclusion criteria: The
contraindication of this treatment regimen
included diffuse multifocal (more than 3 foci)
lesions in the whole liver, portal vein
thrombosis, Child-Pugh class C patients and
bleeding diathesis.
Methods
• Through history taking & clinical examination.
• Laboratory investigations. 1-Heamoglobin and
platelet count. 2- Liver enzymes:• Aspartate
transminase (AST) (30-35 IU/L).• Alanine
transminase (ALT) (30-40 IU/L).• Alkaline
phosphatase (ALP) (30-120 IU/L). 1- Serum
bilirubin total (0.2-1 mg/dl) and direct bilirubin.
2- Serum albumin (3.5-5 g/dl). 3- Prothrombin
activity (%) and INR. 4- AFP (Alfa-feto protein).
5- Viral markers:Serum HCV Abs using 3rd
generation ELISA.HBs Ag positive cases by
ELISA were excluded.• Assessment of severity
of liver disease according to Child-Pugh
classification. Only child (A) and (B) included in
the study. • Tumor staging using the Barcelona-
Clinic Liver Cancer Classification (BCLCC). •
Assessment of the patient’s performance using
the WHO performance status grades before and
after intervention done. • Treatment procedures:
Focal liver lesions were managed according to
AASLD 2010 guidelines.(7)1. Conventional
radio-frequency ablation (RFA) procedure.(8)
•RFA was performed per-cutaneously under
real-time US guidance and deep sedation in the
presence of an anesthetist and with continuous
non-invasive hemodynamic monitoring. Both
sub-costal and inter-costal approaches were
used. A fifteen cm long, 17 G, electrode with a 2
cm long exposed metallic tip was used to deliver
radiofrequency energy. A mono-polar
radiofrequency generator was used as the energy
source. A standard grounding pad was placed on
each of the patient’s thighs. The most
appropriate approach was determined to avoid
damaging large vessels near the targeted HCC,
and then a single electrode was directly inserted
through the skin and positioned at the center of
the HCC under US guidance. The tip of the
electrode was then further advanced to the
deepest margin of the tumor. RF energy was
applied for 8-12 min in each treatment session
according to the size of the tumor. To prevent
bleeding, bile leakage, and tumor seeding, the
intra-hepatic needle track was treated with
thermo-coagulation then the electrode was
removed.Tumors larger than 3.5 cm needed
multiple overlapping ablations.2. TACE
procedure:(9) After a catheter is inserted
percutaneously using Seldinger's technique, the
celiac artery and mesenteric artery will be
catheterized to assess the hepatic vascular
anatomy. Digital subtraction angiography will be
performed to identify the tumor blush and
arterial maps. Super-selective catheterization of
the tumor feeders using a micro-catehter shall be
performed followed by injection of a chemo-
emulsion mixture of (10 cc Lipiodol mixed with
50 mg of dissolved adriamycin) to occlude the
intra-hepatic tumor feeders and collateral arteries
supplying the tumor. This will be followed by
administration of an embolic agent (fractionated
Gel-Foam or Poly-Vinyl alcohol PVA) to
achieve complete ischemia of the tumor. 1- Tri-
phasic Ct abdomen was done 2-4 weeks after the
last session of either RFA or TACE to assess
response using Response Evaluation Criteria in
solid Tumors (mRECIST).(136). 2-Regular
follow up: Patients were evaluated regularly
every 3 months for 12 months by: 1- Complete
clinical examination. 2- Assessment of physical
performance status and child classification. 3-
Laboratory investigations. 4- Liver enzymes:
AST, ALT, ALP, Total and direct bilirubin,
Serum albumin, Prothrombin activity, AFP.
Figure (1): HCC before TACE(size 4.6-4.3cm).
Figure (2): HCC after TACE (SIZE 3.5-2.5cm) figure (3): HCC after a second TACE (size 2.8-2.3cm)
Figure (4): HCC after RFA following TACE showing complete response (12months later).
Results
There was no statistical difference between the
two groups as regards the demographic data. The
mean age was 55.05 ± 7.19 for group Ι, 54.05 ±
7.33 for group ΙΙ; the P value was 0.736.70% and
65% of the patients were males in group Ι and
ΙΙ, while 30% and 35% were females in group
Ι and ΙΙ, respectively. Figure (5)
Figure (5):Comparison between the two studied groups according to Gender
As regards BCLC, There was no significant
statistical difference between the two groups
(p=0.342). There were no very earl y BCLC
patients included in the study. In group І patients
12/20(60%) were early (A) while in group ІІ
patients 9/20(45%) were early (A). 8/20 (40%)
of group І and 11/20(55%) of group ІІ were
Intermediate (B), respectively.
Table (1): Comparison between the two studied groups according to BCLC
GroupI
(n=20)
Group II
(n=20) 2 p
No. % No. %
BCLC
Early (A) 12 60.0 9 45.0 0.902 0.342
Intermediate (B) 8 40.0 11 55.0
2: Chi square test
Tumor response: Two – four weeks following
ablation in group І patients, 21/28 lesions (75%)
had complete response and 7/28 lesions (25%)
had partial response. In group ІІ patients
21/25lesions (84%) had complete response and
4/25 lesions (16%) had partial response. there
was no significant statistical difference between
the two groups (p=0.420) .Three months after the
ablation treatment, 17/28 lesions (60.7%) had
complete response, 6/28 lesions (21.4%) had
partial response and 5/28 lesions (17.9%) had
local recurrence in group І patients. While in
group ІІ patients 25/25 lesions (100%) had
complete response. There was significant
statistical difference between the two groups
(p=0.001).Six months after the ablation
treatment, 15/28 lesions (53.6%) had complete
response, 4/28 lesions (14.3%) had partial
response and 9/28 lesions (32.1%) had local
recurrence in group І patients. While in group ІІ
24/25lesions (96%) had complete response. Only
one patient had local recurrence. There was
significant statistical difference between the two
groups (p=0.001).Nine months after the ablation
treatment, 14/25 lesions (56%) had complete
response, 2/25 lesions (8%) had partial response
and 9/25 lesions (36%) had local recurrence in
group І patients. While in group ІІ patients 25/25
lesions (100%) had complete response. There
was significant statistical difference between the
two groups (p=0.001).Twelve months after the
ablation treatment, 13/21 lesions (61.9%) had
complete response, 2/21 lesions (9.5%) had
partial response and 6/21 lesions (28.6%) had
local recurrence in group І patients. While in
group ІІ patients 25/25 lesions (100%) had
complete response. There was significant
statistical difference between the two groups
(p=0.001)
Male14
70.0%
Female6
30.0%
Table (2):Comparison between the two studied groups according to response to ablation
Response Group I Group II 2 p
2-4 Weeks N=28 N=25
CR 21 75.0 21 84.0 0.650 0.420
PR 7 25.0 4 16.0
3 Months N=28 N=25
CR 17 60.7 25 100.0
12.394* MCp
<0.001* PR 6 21.4 0 0.0
R 5 17.9 0 0.0
6 Months N=28 N=25
CR 15 53.6 24 96.0
12.748* MCp
<0.001* PR 4 14.3 0 0.0
R 9 32.1 1 4.0
9 Months N=25 N=25
CR 14 56.0 25 100.0
11.131 MCp
=0.001* PR 2 8.0 0 0.0
R 9 36.0 0 0.0
12 Months N=21 N=25
CR 13 61.9 25 100.0
11.131 MCp
=0.001* PR 2 9.5 0 0.0
R 6 28.6 0 0.0
2: Chi square test
MC: Monte Carlo test
*: Statistically significant at p ≤ 0.05
CR: complete response PR: partial responseR: local recurrence
Survival: The total number of deaths was
4/20(20%) in group І and the overall 1year
survival was 80%. The mean survival time was
11.55 months. While in group ІІ, there were no
deaths and the overall survival was 100%. The
mean survival time was 12 months. There was
significant statistical difference between the two
groups (p=0.037) i.e. Survival rates were
significantly better in the combined (TACE and
RFA) group than in the RFA group. In group І
the cause of death was liver failure in 2 patients
and multi-centric HCC (infiltrative) in 2 patients.
Figure (6):Kaplan Meier survival curve for overall survival rate.
Discussion
Local ablative therapy is using minimally
invasive techniques, preserving the uninvolved
liver parenchyma, has no systemic side effects
compared to systemic chemotherapy and also
avoids the morbidity and mortality of major
hepatic surgery. Local ablative therapy has
emerged in clinical practice to expand the pool
of patients considered for liver-directed
therapies. (10) Selecting the correct treatment
modality to suit individual patients with HCC
remains a matter of debate. This prompted us to
conduct the current study. The aim of our work
was to evaluate the safety, efficacy and outcome
of combination of RFA and TACE for
unrespectable hepatocellular carcinoma versus
RFA alone. To study the necessity and feasibility
of this combined modality.RFA is currently
performed widely due to the ease of use, safety,
reasonable cost and less side effects than other
ablative techniques. RFA can achieve a rate of
complete necrosis as 80-100% in small HCC.
However, the rate will drop to 71% in HCC of
3.1-5cm and 25% for HCC larger than 5cm.
However, the percentage of complete necrosis is
lower for PEI. Therefore, thermal ablation has
become popular and is the standard treatment in
many centers. However, for lesions that are close
to the main portal vein or intestine, thermal
ablation may not be feasible, and PEIT is
preferred. The efficacy of local ablation is also
dependent on the size of the treated lesion.
Tumors larger than 5 cm have a lower
percentage of complete necrosis and a higher
rate of recurrence. (11,12). In our work there was
no significant statistical difference between the
two groups all through the study duration as
regards; ALT, AST, total bilirubin, direct
bilirubin, s albumin, ALP and PA %.Before RFA
treatment in group І, 5/20 patients (25%) had no
ascites, 11/20 patients (55%) had mild ascites
and 4/20 patients (20%) had moderate ascites.
Three months later, the patients remain the same.
One year after RFA patients who had no ascites
decreased to 4/16(25%), 5/16(31.3%) had mild
ascites, moderate ascites patients decreased to
6/16(37.5.8%) and 1/16(6.3%) had sever ascites.
As regards performance status before RFA, 6/20
patients (30%) of was stage (0), 4/20 patients
(20%) was stage І, 7/20 patients (35%) was stage
ІІ and 3/20 patients (15%) was stage ІІІ and none
of the patients was stage ІV. Twelve months
later, 2/16 patients (12.5%) was stage 0, 3/16
patients (18.8%) was stage І, 4/16 patients (25%)
was stage ІІ and 5/16 patients (31.3%) was stage
ІІІ and two patients was stage ІV. The quality of
life decreased gradually all through the study.
After one year of RFA sessions, 13/21 lesions
(61.9%) had complete response, 2/21 lesions
(9.5%) had partial response and 6/21 lesions
(28.6%) had local recurrence. Number of
sessions ranged from two to six sessions. The
mean size of the lesion was 3.14±0.97 cm. As
we can see the number of sessions was more and
the mean size of the tumors was smaller than
group ІІ(combined TACE and RFA).The
combined technique started when Yamasaki et
al(13) improved the efficacy of RFA, with balloon
occlusion of the hepatic artery (balloon-occluded
RFA) and compared it to standard RFA. The
greatest dimension of the area coagulated by
balloon-occluded RFA was significantly larger
than that coagulated by standard RFA.
Cessation of blood flow in the treatment zone
decreases perfusion-mediated tissue cooling,
reducing the heat-sink effect. This increases the
lethal thermal coagulation zone. In addition, a
larger volume of sub-lethal hyperthermia is
exposed to synergistic high concentrations of
chemo-therapeutic drugs, particularly
doxorubicin. The increased volume of
coagulative necrosis including the lethal and
sub-lethal hyperthermic zones widens the
ablation margin, destroying microscopic satellite
lesions adjacent to the central tumor, ultimately
improving local control. Combined treatment
can control internal liver lesions as well as
extrahepatic metastases.(14). In our study, after a
year group ІІ patients (TACE with RFA), 25/25
lesions (100%) had complete response. There
was significant statistical difference between the
two groups (p=0.001).17 patient had no de novo
lesions, 1/20(5%) had one de novo lesion and
2/20(10%) had 2 de novo lesions. There was
significant statistical difference between the two
groups. (p=0.008).There were no deaths and the
overall one year survival was 100%. The mean
survival time was 12 months. There was
significant statistical difference between the two
groups (p=0.037).Cumulative meta-analysis of
the informative trials has positioned TACE as
the first-line option for BCLC (B) patients.(15)
Restrictive selection of candidates to exclude
those with decompensated cirrhosis, proper
techniques and an adequate policy to stop TACE
at the time of liver failure or lack of treatment
response results in median survival exceeding 4
years.(16248)
Conclusion
In our work we concluded that combination of
TACE and RFA for the treatment of patients
with medium-sized unresectable HCC is safe and
effective. It provides better local tumor control
and one year survival rate more than RFA alone.
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Original Article
Nano-ELISA in the Diagnosis of Experimental Toxoplasmosis
Mervat Zakaria El Azzouni1, Lobna Abd El-Aziz El Zawawy1, Doaa El-Said Said Ahmed1,Wegdan
Ramadan Ahmed2, Maha Mohamed Gomaa3; 1Professor of Medical Parasitology, 2Assistant Professor of
Physics, 3Assistant lecturer of Medical Parasitology
ABSTRACT Toxoplasmosis is a worldwide endemic disease. In congenitally infected infants and immunocompromised patients,
toxoplasmosis causes high rates of morbidity and mortality. In these cases antibody detection is non-valuable and
detection of parasite antigen could be a useful tool for proper diagnosis of acute infection.Gold nanoparticles can be
conjugated with antibodies affording them promising applications in signal enhancement of bio-chemical detection.
Aim of the work:Gold nano-ELISA was evaluated in in the detection of Toxoplasma SAG1 antigen for its potential
as an early sensitive diagnostic technique. Patient and Methods: 120 laboratory bred Swiss Albino mice were used.
A control group was formed of 60 mice; 30 mice were non-infected immunocompetent and the other 30 were non-
infected immunosuppressed. An experimental group was formed of 60 T. gondii RH HXGPRT (-) strain- infected
mice; 30 mice were immunocompetent and the other 30 were experimentally immunosuppressed by
cyclophosphamide.Six mice from each subgroup were sacrificed on days zero, 1, 2, 7 and 14 post infection
(PI).Blood samples were collected and sera were separatedto be assayed for T. gondii SAG1antigen by nano-ELISA
and capture ELISA as a golden standard assay. Results: T. gondii SAG1 antigen was detected in infected animal
subgroups whether immunocompetent or immunosuppressed as early as the 1st day PI by nano-ELISA with a
sensitivity of 83.3%. The antigen was detected till the end of the experiment with a sensitivity of 100%.No false
positive results were detected in control subgroups either by capture or by nano- ELISA (100% specificity).
However, capture ELISA detected the antigen only on the seventh and 14th day PI with a sensitivity of
100%.Conclusion: Nano-ELISA is a promising sensitive method for early diagnosis of experimental toxoplasmosis
under immunosuppressive conditions.
Introduction
Toxoplasmosis is a zoonosis of worldwide
distribution. It is caused by T. gondii; an obligate
intracellular coccidian parasite that infects a
broad range of warm-blooded animals. (1) It
affects up to one third of the world’s population.
Approximately half billion humanshave
antibodies to T. gondii all over the world. (2,3)
The rate of human infection varies from 10-90 %
in different countries, depending on several
factors such as environmental conditions,
prevalence in animals and dietary habits.(4-6).
Toxoplasmic infection in immunocompetent
individuals is usually asymptomatic. Onlyfew
cases have self-limiting non-specific
lymphadenopathy and flu-like symptoms.
However, in immunocompromised hosts
including AIDS patients, cases with
malignancies and organ transplant recipients,
toxoplasmosis is severe and life-threatening if
untreated early.(7-10). The diagnosis of
toxoplasmosis is routinely based on serological
tests for detection of specific parasite IgG and
IgM antibodies. However, serology is inefficient
in immunocompromised patients because
antibody production significantly decreased due
to depletion of Th lymphocytes and/ or decrease
in the ability to stimulate B cell maturation and
differentiation.(11, 12) So, detection of T. gondii
antigen can provide specific diagnosis of acute
infection in those patients.(13)The major surface
antigen (SAG1 or P30) is one of the most
immunogenic T. gondii antigens. It is an
important candidate for development of
diagnostic techniques.(14)This antigen has no
cross reactivity with proteins of other
microorganisms and highly conserved in T.
gondii strains.(15,16). One of the most important
and widely used bio-chemical techniques is
enzyme linked immunosorbent assay (ELISA). It
can be used to detect either parasite antibodies or
antigens based on antigen-antibody immuno-
reactions. Although there are some highly
sensitive ELISA types, they are highly
complicated and expensive. Therefore, there is a
need to improve the sensitivity of the current
simple ELISA formats for detection of T. gondii
antigen, thus allows early diagnosis of the
disease.(17). The use of nanoparticles (1-100 nm
size) promises to promote in vitro diagnostics to
the next level of performance. Quantum dots,
gold nanoparticles (AuNPs), and super-
paramagnetic nanoparticles are the most
promising nanostructures in this context.(18)
AuNPs have high surface areas and unique
physicochemical properties such as resistance to
oxide formation under ambient conditions,
unusual optical properties, and the high atomic
number; a feature which renders AuNPs readily
detectable by electron microscopes or by X-
rays.(19) AuNPs can be conjugated with DNAs,
antibodies, enzymes and other bio-molecules,
which can afford them promising applications in
signal enhancement of bio-chemical
detection.(20,21) AuNPs functionalized with
specific antibodies were used for the
development of a microchannel immunoassay
that detected Eschericia coli and Helicobacter
pylori antigens. (22) In addition, an assay for
simultaneous detection of HBV and HCV
antibodies in human sera was developed using
AuNPs-labelled staphylococcal protein A. (23)
Interestingly, AuNPs are key components of the
bio-barcode assay which has been proposed as a
future alternative to PCR with promising
sensitivity. (24)Recently, some studies have
emphasized on the use of functionalized AuNPs
in the detection of parasite biomolecules either
for further understanding of protozoan
bionomics or for the diagnosis of parasitic
infection such as Cryptosporidium parvum,
Plasmodium falciparum (25) and Trypanosoma
cruzi. (26). In 2009, Jia et al. had developed a
sensitive protein detection method based on
nanoparticles and ELISA that is called “nano-
ELISA” in which the antibody-conjugated
AuNPs are used to detect a cancer protein
marker “P53” with very promising results.This
method combines the nanotechnology with the
sandwich ELISA. In nano-ELISA, the detector
antibody mixed with horseradish peroxidase
(HRP) at a certain ratio was immobilized on the
AuNPs to form Au nano probes that would
provide a signal amplification of ELISA assay. (27). The promising yields due to application of
nano assays in the diagnosis of cancer and
parasitic infectious diseases in the previous
studies, give us the challenge to use AuNPs in
the diagnosis of experimental toxoplasmosis. In
this context, nano-ELISA was applied in the
current work for possible early diagnosis of
acute toxoplasmosis especially in immune-
compromised animals by detection of T. gondii
SAG1antigen in serum samples.
Patients and Methods
T. gondii Strain: The virulent Toxoplasma RH
strainwas used in the present study. It was
maintained in the laboratory of Medical
Parasitology Department, Faculty of Medicine,
Alexandria University, Egypt.Tachyzoites were
harvested from the peritoneal exudates of
infected mice on the fifth day post infection (PI)
and kept at 4 0C to be used for animal infection
and Toxoplasma antigen preparation. Drug:
Cyclophosphamide (Endoxan) was used as an
immunosuppressive agent. It was IP injected
twice in a dose of 70 mg/kg body weight, one
week apart. (28). Experimental Design: This work
was carried out on 120laboratory bred, male
Swiss Albino mice, aged 5-6weeks and weighing
20-25 g each. All mice were supplied from the
animal house, Medical Parasitology Department,
Faculty of Medicine, Alexandria University,
Egypt. The animals were divided into two main
groups:- Control group (Group I) that included
60non-infected mice which was equally divided
into two subgroups; 30 immunocompetent mice
(I-a)and 30 experimentally immunosuppressed
mice (I-b). - Experimental group (Group II)that
involved 60mice and equally divided into two
subgroups: -Subgroup II-a (Infected,
immunocompetent subgroup): 30 mice were
intraperitoneally (IP) infected by T. gondii RH
HXGPRT (-) strain in a dose of 2 x 102
tachyzoites /mouse. -Subgroup II-b (Infected,
immunosuppressed subgroup): 30 mice were
immunosuppressed with Endoxan. Animalswere
infected with T. gondii RH HXGPRT (-)strain
the same as in subgroup II-a two days after the
second dose of Endoxan. (29,30). Six mice from
each subgroup were anaesthetized then sacrificed
on the following dates: days zero (the day of
infection), 1, 2, 7 and day 14 PI and a blood
sample was collected from each mouse.The
peritoneal exudate was withdrawn on days 7 and
14 from the experimental animals to be
microscopically examined for tachyzoites to
verify the infection. The control animals were
simultaneously sacrificed with their
corresponding experimental groups. Blood
samples were centrifuged at 1000 g for 20
minutes for serum separation. Sera were stored
at – 20 0C until tested for T. gondii SAG1
antigen by both capture and nano-ELISA.
● T. gondii Antigen (31,32):T. gondii tachyzoites
were centrifuged at 500 rpm for five minutes in
phosphate buffered saline (PBS pH 7.2). The
supernatant was centrifuged at 2000 rpm for 10
minutes to sediment tachyzoites then the
sediment was washed three times with PBS. The
sediment pellet was suspended in PBS and
tachyzoite concentration was adjusted to be 8 x
10 6 tachyzoites / ml using the haemocytometer.
The parasite suspension was sonicated
eighttimes for ten seconds eachwith ten seconds
interval then centrifuged at 10,000 rpm for 15
minutes. The supernatant was collected and
stored at – 20 0C until used as T. gondii
solubleantigen. The protein content of the
prepared antigen was measured by mass
spectrometry at the Central Laboratory, Faculty
of Agriculture, Alexandria University.●
Negative and Positive Control Sera: Diluted sera
of non-infected mice were used as a negative
control. The prepared T. gondii antigen was
added to the negative control to be used as a
positive control. ● AuNPs Synthesis: All used
glassware was cleaned by freshly prepared aqua
regia (3 parts hydrochloric acid + 1 part nitric
acid) to dissolve any residual particles.AuNPs
were prepared according to the citrate method of
Turkevich, 1985, in which 100 ml solution of 0.5
mM HAuCl4 (chloroauric acid)٭ was boiled. 2ml
of 3 mM trisodium citrate٭ solution was quickly
added to the refluxed HAuCl4 solution, resulting
in a rapid color change from pale yellow (Au+3)
to deep red (Au0) that indicated the formation of
AuNPs.After an additional 15 min continuous
reflux the solution was slowly cooled down to
the room temperature.(33) The solution was
purified by a syringe filter of 0.2 μm pore size
and stored at 4°C.● Preparation of Au nano
probes (34): The AuNps were conjugated with T.
gondii SAG1 antibodiesΔ and HRP٭ molecules
according to the method of Ambrosi et al. 2007
as follows: the pH of AuNPs solution was
adjusted to 8.2-8.5 with 0.2 M Na2 CO3. The
stock solutions (1mg/ml) of HRP and SAG1
antibodies were mixed at a ratio of 3:1 (protein
mixture). (27) One ml AuNPs solution was
centrifuged at 9000 rpm for 50 minutes and the
supernatant was removed. The protein mixture
was then added to the AuNPs pellet and the final
volume was adjusted to 100 μl by distilled water.
The solution was stirred for 10 minutes and
stood for two hours at room temperature,
followed by addition of 0.5% Poly-ethylene
glycol 20000 (PEG 20000) to stabilize and passivate ٭
the AuNPs. The prepared Au nano probes were
purified by centrifugation at 9000 rpm and three
time washes by a washing solution (10 mM PBS,
0.5% FCS, 2.5% sucrose0.1 ,٭% PEG 20000). The
Au nano probes were redispersed in the latter
washing solution (100 μl) and stored at 4 0C. ●
Characterization of AuNPs and Au nano-probes (27): The spectroscopic analysisof the prepared
AuNPs and nano probes was recorded by UV-
vis spectrophotometer.The morphological study
of both was done by transmission electron
microscopy (TEM).Determination of the mean
size and size distribution was performed by
particle size analyzer and the structural analysis
was carried out by X ray diffractometer
(XRD).●Capture ELISA Procedure: It was done
to detect T. gondii SAG1antigen in the sera of all
studied animal subgroups. Prior to its
performance, a checkerboard titration was
carried out in which different concentrations of
T. gondii SAG1antibodiesand SAG1 antibodies
conjugated with HRPΔwere titrated against serial
dilutions of the positive and negative control
sera.(35) The selected concentrations and dilutions
were: 6μg/ ml T. gondii antibodies, 1/5000
dilution of T. Gondii SAG1 antibody- HRP
conjugate, 1/10 dilution of negative control and
sera and 1/100 dilution for positive control. Each
polystyrene microplate٭ well was coated by 150
μl of T. gondii SAG1 antibody diluted in
carbonate-bicarbonate buffer pH 9.6 and
incubated at 4 0C overnight. The wells were
washed three times with PBS containing0.5 %
Tween 20٭ (washing buffer). The wells were
then blocked with 100 μl PBS containing 1 %
fetal calf serum (FCS)٭and 0.05 % Tween
20.The microplate was incubated at room
temperature for two hours after which the wells
were washed three times. The positive, negative
control sera and the serum samples to be tested
were diluted in incubating buffer (PBS
containing 15 % FCS and 0.01 % Tween 20)
then, 100 μl volume of each was added in
duplicate.The microplate was incubated at room
temperature overnight then, wells were washed
three times. 100 μl T. gondii SAG1 antibody-
HRP conjugate diluted in PBS containing 5 %
FCS and 0.01 % Tween 20 were added to each
well.The microplate was incubated at 37 0C for
two hours after which the wells were washed
threetimes.100 μl of ortho-phenylenediamine
(OPD)٭ substrate dissolved in phosphate-citrate
buffer pH 5 were added to each well and the
microplate was incubated at 37 0C for 45
minutes.The optical densities (OD) were read at
a wavelength of 492 nm using an ELISA reader.
Nano-ELISA: It was done as capture ELISA to
detect T. gondii antigen in both the experimental
and control animals’ sera but using the Au nano
probes instead of T. gondii antibody-HRP
conjugate. Prior to the performance of the nano-
ELISA, a checkerboard titration was carried on
whereby different concentrations of T. gondii
antibodies and Au nano probes were titrated
against serial dilutions of the positive and
negative control sera. (27) The selected
concentrations were: 6μg/ ml T. gondii
antibodies, 1/2000 dilution Au nano probes, 1/10
dilution negative control and sera to be tested
and 1/100 dilution positive control
serum..Calculation and Interpretation of the
Results: The OD values of the control and tested
sera were determined by calculating the mean
OD of the readings.The cut off value (COV) was
calculated as; the mean OD of the negative
control + twice standard deviation. (35)The
sample was considered positive for T. gondii if
its OD was equal to or higher than the COV
while that below the COV was considered
negative.
Statistical Analysis
Sensitivity and specificity of both capture and
nano-ELISA were calculated. Chi square (2)
and Fisher Exact (FE) testswere used for
comparison between the assays
sensitivity.Student t-test was used to analyze the
statistical difference between the OD492 values of
experimental subgroups. (49)
Results
Parasitological Verification of T. gondii
Infection: T. gondii tachyzoites were detected in
the peritoneal exudate of infected animals
sacrificed on days seven and 14 PI either in
subgroup II-aor II-b.. Characterization of AuNPs
and Nano Probes: A- Spectroscopic analysis:
Compared with AuNPs, The optical spectra
showed that the absorption peak of Au nano
probes shifted from 512 nm up to 592 nm with
broadening of the absorption peak of Au nano
probes (Fig. 1). B- Morphological study: The
morphological study of AuNPs and nano probes
by TEM revealed that they were spherical,
distinct and regular in shape with smooth surface
(Plate I). The mean size of AuNPs was around
17.2 nm and that of Au nano probes was 38.6
nm. Au nano probes showed a contrast in the
electron density between the dark spherical core
of the metal AuNPs and the surrounding lighter
shell of the immobilized antibodies and HRP
molecules.C- Particle size analysis: The average
AuNPs size was 24.7 nm andthat of Au nano
probes was 42.3 nm.A narrow size distribution
was also recorded.D- Structural analysis: The
calculated value of the particle size was around
21 nm for AuNPs and 35 nm for Au nano probes
(Fig. 2).
Fig. 1: UV-vis spectra of AuNPs and nano probes
Plate I: TEM of AuNPs and nano probes
Fig. a, b: TEM of AuNPs at scales 50, 100 nm
Fig. c, d: TEM of Au nano probes at scales 50, 100 nm
Fig. 4: XRD of AuNPs and nano probes
Results of Capture and Nano-ELISA: The cut off
value (COV) was estimated to be 0.075
therefore; the mean OD492 values equal to or
above the COV were considered positive
denoting the presence of T. gondii SAG1 antigen
in the sera. It was observed that there were no
false positive results in all control subgroups
neither by capture ELISA nor by nano-ELISA
formats. This means that the specificity was
calculated to be 100% for both assays.As regards
subgroup II-a, T. gondii SAG1 antigen was not
detected in sera of mice sacrificed before day
seven PI by capture ELISA. The antigen was
detected in five out of six serum samples on day
seven PI and in all samples on day 14 PI. The
antigen was also not detected in sera of mice
sacrificed on day zero PI by nano-ELISA.
However, it was detected in five out of six sera
on day one PI and in all samples on days two,
seven and 14 PI (six samples on each day). It
was observed that nano-ELISA had a statistically
significant higher sensitivity (83.3 % and 100 %)
on days one and two PI respectively than that of
capture ELISA (Table I). Meanwhile, there was
no statistically significant difference in the
sensitivity of capture ELISA (83.3%) and nano-
ELISA (100%) on day seven PI. Moreover, both
assays show a sensitivity of 100% on day 14 PI.
Both capture and nano-ELISA revealed an
increase in the mean OD492 values till the end of
the experiment (Fig. 3). It was noticed that there
was no statistically significant difference
between the mean OD492 values of animals
sacrificed on day seven and those sacrificed on
day 14 PI neither by capture nor by nano-ELISA
(Table II).Regarding animals of subgroup II-b,
the antigen was not detected in sera of mice
sacrificed on days zero, one and two PI by
capture ELISA. It was detected in all serum
samples on days seven PI and 14 PI (six
samples/ day). The antigen was also not detected
in sera of mice sacrificed on day zero PI by
nano-ELISA. However, it was detected in five
out of six sera on day one PI and in all tested
samples on days two, seven and 14 PI. It was
observed that nano-ELISA had a statistically
significant higher sensitivity (83.3% and 100%)
on days one and two PI respectively than that of
capture ELISA (Table III). Meanwhile, both
assays show a sensitivity of 100% on days seven
and 14 PI. Both capture and nano-ELISA
formats revealed an increase in the mean OD492
values after the second day PI till the end of the
experiment (Fig. 4). It was noticed that there was
a statistically significant increase in the mean
OD492 values in animals sacrificed on day 14 PI
compared to those sacrificed on day seven PI
either by capture or by nano-ELISA (Table
IV).Comparing animals of subgroup II-a to those
of subgroup II-b1, it was noticed that the mean
OD492 values by capture and nano-ELISA were
significantly increased in subgroup II-bin
relation to subgroup II-aeither on day seven or
on day 14 PI. However, the difference in the
mean OD492 values between these two subgroups
was statistically non-significant on days one and
two by nano-ELISA only (Table V).
a b
c d
Inten
sity a
.u
AuNps
Au nano
probes
2Ө (degree)
Table I: The sensitivity of capture and nano ELISA in T. gondii SAG1 antigen detection in virulent strain infected
subgroup (II-a)
Day PI Capture ELISA Nano-ELISA FEp
No of positive sera Sensitivity No of positive sera Sensitivity
0 0 0 0 0 -
1 0 0 5 83.3 % 0.015*
2 0 0 6 100 % 0.002*
7 5 83.3 % 6 100 % 1.000
14 6 100 % 6 100 % -
PI: Post infection, No: Number, FE: Fisher Exact test, *: Statistically significant at p ≤ 0.05
●Total number of sacrificed mice on each day is six
Fig. 3: The mean OD 492 values measured by capture and nano-ELISA in virulent strain- infected immunocompetent
subgroup II-a
Table II: Comparison between the mean OD492 values measured by capture and nano-ELISA in subgroup II-a on days 7
and 14 PI
Assay OD492 values Day 7 PI Day 14 PI p
Capture ELISA
Min.–Max.
Mean ± SD
Median
0.200 – 0.260
0.234 ± 0.024
0.241
0.253 – 0.270
0.268 ± 0.095
0.255
0.304
Nano-ELISA
Min.-Max.
Mean ± SD
Median
0.215 – 0.263
0.240 ± 0.019
0.243
0.253 – 0.270
0.268 ± 0.095
0.256
0.311
PI: Post infection, Min: Minimum, Max: Maximum, SD: Standard deviation, *: Statistically significant at p ≤ 0.05 for Student t-
test, COV = 0.075
Table III: The sensitivity of capture and nano-ELISA in T. gondii SAG1 antigen detection in virulent strain-infected
subgroup (II-b)
Day PI Capture ELISA Nano-ELISA FEp
No of positive sera Sensitivity No of positive sera Sensitivity
0 0 0 0 0 -
1 0 0 5 83.3 % 0.015*
2 0 0 6 100 % 0.002*
7 6 100 % 6 100 % -
14 6 100 % 6 100 % -
PI: Post infection, No: Number, 2: Chi square test, FE: Fisher Exact test, *: Statistically significant at p ≤ 0.05
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0 1 2 7 14
Mean
OD
49
2 v
alu
es
Days post infection
Capture ELISA
Nano-ELISA
Cut off value
=0.075
●Total number of sacrificed mice on each day is six
Fig. 6: The mean OD 492 values measured by capture and nano-ELISA in RH strain-infected subgroup (II-b)
Table IV: Comparison between the mean OD492 values measured by capture and nano-ELISA in RH strain-infected
subgroup (II-b) on days 7 and 14 PI
Assay OD492 values Day 7 PI Day 14 PI p
Capture ELISA
Min. – Max. 0.205 – 0.365 0.671 – 0.998
<0.001* Mean ± SD 0.311 ± 0.062 0.825 ± 0.122
Median 0.324 0.816
Nano-ELISA
Min. – Max.
Mean ± SD
Median
0.206 – 0.367
0.313 ± 0.061
0.333
0.674 – 0.999
0.828 ± 0.121
0.819
<0.001*
PI: Post infection, Min: Minimum, Max: Maximum, SD: Standard deviation,*: Statistically significant at p ≤ 0.05for Student t-
test, COV = 0.075.
Table (V): Comparison between the positiveOD492 values of subgroups II-a and II-b measured by capture and nano-
ELISA
Day PI Subgroup II-a Subgroup II-b p
Capture ELISA
Day 7/ Min. – Max.
Mean ± SD
Median
0.200 – 0.260
0.234 ± 0.024
0.241
0.205 – 0.365
0.311 ± 0.062
0.324
0.018*
Day 14/ Min. – Max.
Mean ± SD
Median
0.253 – 0.510
0.334 ± 0.095
0.303
0.671 – 0.998
0.825 ± 0.122
0.816
<0.001*
Nano-ELISA
Day 1/ Min. – Max.
Mean ± SD
Median
0.065 – 0.080
0.078 ± 0.005
0.079
0.057 – 0.083
0.078 ± 0.010
0.082
1.000
Day 2/ Min. – Max.
Mean ± SD
Median
0.077 – 0.084
0.083 ± 0.002
0.081
0.078 – 0.093
0.086 ± 0.006
0.087
0.083
Day 7/ Min. – Max.
Mean ± SD
Median
0.215 – 0.263
0.240 ± 0.019
0.243
0.206 – 0.367
0.313 ± 0.061
0.333
0.019*
Day 14/ Min. – Max.
Mean ± SD
Median
0.253 – 0.510
0.334 ± 0.095
0.303
0.674 – 0.999
0.828 ± 0.121
0.819
<0.001*
PI: Post infection, Min: Minimum, Max: Maximum, SD: Standard deviation, *: Statistically significant at p ≤ 0.05 for Student t-
test, II-a: RH- infected immunocompetent mice, II-b: RH- infected immunosuppressed mice, COV = 0.075
Discussion
The worldwide epidemiology, the high
prevalence of T. gondii infection among humans
together with the poor obstetric outcomes in
infected pregnant women and its lethal
manifestations among immunocompromised
patients; all these should draw our attention to
address toxoplasmosis as a serious public health
problem and to improve the diagnosis in these
high risk groups.The most commonly used
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0 1 2 7 14
Mea
n O
D4
92
valu
es
Days post infection
Capture ELISA
Nano-ELISA
Cut off value
=0.075
serological tests for the diagnosis of
toxoplasmosis are detecting T. gondii IgG and
IgM antibodies but they are not enough for the
diagnosis of acute infection. This was due to life-
long persistence of IgG, persistence of IgM for
more than one year, in addition to false positive
and false negative results for IgM. The false
positive results could occur in patients having
rheumatoid factor or antinuclear antibodies,
while false negative results might be due to high
serum levels of IgG that competitively inhibit the
reaction with IgM. Moreover, the
immunosuppressive drugs are widely used
nowadays for treatment of malignancies,
autoimmune disorders and in organ
transplantation cases. (36) These drugs markedly
decrease antibody production and its serum level
giving negative results with the commonly used
diagnostic techniques with missing the diagnosis
of toxoplasmosis. (15) Similarly, in
immunocompromised patients, there is
impairment of antibody production which guards
against the serological diagnosis. (13) Currently,
detection of pathogen proteins in sera of patients
with an acute / active infection is an important
diagnostic tool. This is valuable especially in
those with impaired immune response for whom
a rapid and accurate diagnosis is critical.
Therefore, the current study highlighted the
value of detection of T. gondii antigen instead of
antibodies by a new assay “nano-ELISA” for an
early and accurate diagnosis of active
toxoplasmosis. According to our knowledge, this
is the first time to apply a newly emerging assay
“nano-ELISA” in the diagnosis of
toxoplasmosis. It was evaluated in comparison to
capture ELISA in possible early diagnosis of
experimental toxoplasmosis under immune-
suppressive conditions . The immunosuppressive
drug used in this study was cyclophosphamide
which is one of the cytotoxic drugs belonging to
the family of alkylating agents.(50) It inhibits the
synthesis of DNA in the T cells particularly Th
ones, thereby thwarting the stimulus for cell
proliferation. (36) In addition, cyclophosphamide
inhibits the clonal expansion and differentiation
of B cells to plasma cells, thus inhibiting the
antibody production. (36) it was selected in the
present work for its specific immunosuppressive
activity on both the humoral and cell mediated
immunity. (36,50) Moreover, it acts rapidly after
administration, which was essential especially in
RH strain-infected mice.(50) Regarding its
administration schedule in the present work, it
was given two days before the infection in two
doses, seven days apart. As it was proved that
the humoral immunosuppressive effect of this
drug was proved to be the greatest when it was
given before the infection. Repeated drug
administration would induce better
immunosuppression and avoid recovery of the
immune response. It is well known that the
membrane surface antigens of Toxoplasma
parasite are the key of successful parasite entry
into host cells.(37) T. gondii major surface
antigen “SAG1 or P30” was the selected antigen
to be detected in the current experimental work.
SAG1 (30 KDa) is a glycoprotein detected in T.
gondii tachyzoites and sporozoites. It was
proven to be a superior candidate for the
development of diagnostic reagents or subunit
vaccines that induce a dominant immune
response. (15) This antigen is abundant on the
surface of both extracellular and intracellular
tachyzoites. (15) It was reported thatSAG1 has no
cross reactivity with proteins from other
microorganisms. (16) Moreover, this molecule is
involved in parasite attachment and could be an
important indicator of active infection. (38,39) The
gene coding SAG1 occurs as a single copy
without introns and it is highly conserved in T.
gondii strains; either virulent or avirulent ones. (40,41). In the current work, ELISA was applied
for detection of circulating T. gondii SAG1
antigen in mice sera by employing monoclonal
anti-SAG1 as the capture antibody. This was in
consistent with a study performed by Chaves-
Borges et al.1999 which detected soluble
Toxoplasma antigen in human CSF by the use of
the avidin-biotin system of sandwich ELISA
format involving the monoclonal T. gondii P30
antibodies as capture antibodies. Moreover,
Emelia et al. 2010 had developed an ELISA that
employed monoclonal anti-Toxoplasma SAG1
as the capture antibody to detect T. gondii
circulating antigens in patients’ serum samples.
(32) Monoclonal antibodies of defined specificity
are a powerful tool for serological detection of
antigens, and false-positive results due to
rheumatoid factor was not reported to occur
when they were used in the assay. (42) Previous
studies that used polyclonal antibodies in
ELISAs to detect circulating T. gondii proteins
reported relatively low positive rates. (Van 1977, 43)
Furthermore, Emelia et al. 2010 had tried using
polyclonal anti- T. gondii IgG as the capture
antibody in two different ELISA formats and
their findings showed that the assays were
unable to discriminate between positive and
negative samples. They attributed that to the
non-specific binding of antigens to polyclonal
antibodies, since a lot of other interfering
proteins might exist in the serum samples or the
polyclonal antibodies might be cross-reacting to
multiple epitopes. (32). In the present study, T.
gondii antigen was added to diluted sera of non-
infected mice to be used as a positive control for
ELISA. It was better to prepare the Antigen from
tachyzoites of the virulent T. gondii RH
HXGPRT (-) strain as it was cleaner and less
contaminated by tissue cells Furthermore, T.
gondii SAG1 antigen was detected only in
tachyzoites and sporozoites so; it is not reliable
to be prepared from bradyzoites. (15). One of
protein detection methods based on nanoparticles
and ELISA, named nano-ELISA emerged
recently. The basic principle of nano-ELISA
used in this study was illustrated as: The detector
antibody and HRP were immobilized on the
AuNPs, the capture monoclonal T. gondii SAG1
antibody was immobilized onto microtiter plate,
the T. gondii SAG1 antigen in serum samples
was sandwiched by capture and detector
antibodies and HRP molecules on Au nano
probes. The immobilized HRP molecules
represented the signal amplification moieties that
catalyzed the oxidation of chromogenic substrate
into colorful products indicating the presence of
the target antigen. The amount of the target
antigen is proportional to the absorbance value
of the emerging colorful products that was
measured by the ELISA reader. Since a single
enzyme-conjugated antibody is used and one
antibody molecule can only eventually bind one
HRP molecule, the sandwich system has more
limited sensitivity. While for the nano-ELISA,
one detector antibody can be linked with many
HRP molecules through nanoparticle bridge,
which can substantially cause signal
amplification and increase of the detection
sensitivity. Nano-ELISA included only two
reaction steps and three wash steps, which can
significantly simplify the whole detection assay
and shorten its duration. (27). In the present work,
the AuNPs were used in nano-ELISA because
they were considered as ideal candidates for bio-
sensing assays. That is due to their unique
characteristics; namely high solubility in water,
easily tailored synthesis for suitable morphology,
size dispersion and surface functionalities. (44).
AuNPs could be prepared either by citrate-
reduction method or by seed-mediated growth
method. (19,33) In this study, AuNPs were
prepared by citrate-reduction method as it was
easier than the other method. Moreover, the
produced colloidal solution had excellent
stability and uniform particle size of 10-15 nm in
diameter. (33). The size of AuNPs and Au nano
probes could significantly affect the assay
sensitivity. In this study, we used AuNPs of ≈15
nm in diameter depending on the TEM
measurements. It was proven that the detection
signal of antigen at the same concentration of Au
nano probes prepared at different size of AuNPs
was stronger with 15 nm AuNPs than that with
larger AuNPs. (27) That difference might be
attributed to the variance in the specific surface
area of AuNPs. Small AuNPs have the higher
surface-to-volume ratio than larger ones. (34).
Nano-ELISA that was applied in this study had
evolved functionalization of AuNPs with
detector T. gondii SAG1 antibody and HRP
molecules. The success of the immobilization of
both biological moieties was confirmed by four
different characterization techniques. These were
TEM images, UV–vis spectrophotometer
measurements, particle size analyzer peaks and
XRD records. TEM images show that antibodies
and HRP molecules were conjugated to AuNPs.
Au nano probes had a contrast in the electron
density between the dark spherical core
indicating the metal AuNPs and the surrounding
lighter shell representing the immobilized
antibodies and HRP molecules. Moreover, TEM
revealed small fuscous spots around the AuNPs
that could be iron metals present in the heme
group of HRP molecules.(34) These findings were
similar to that found by Jia et al. 2009, who
succeeded in functionalization of AuNPs with
detector antibody and streptavidin–HRP for
detection of a common cancer protein marker
“P53” by nano-ELISA. (27) Furthermore, the
optical spectra of AuNPs and Au nano probes
were recorded by an UV–vis spectrophotometer.
The absorption peak of Au nano probes show a
red shift of the plasmon band in addition to its
broadening in comparison to AuNPs. These
results indicated the growth in the particle size
that was attributed to the coating of AuNPs with
antibody and HRP molecules. (34,43) A similar red
shift of gold plasmon resonance was also
observed by Jia et al. 2009. (27) In addition,
Pissuwan et al. in 2007 revealed a red shift by
UV- vis spectroscopic analysis of gold nano
spheres that were conjugated with antibodies
specific for murine macrophage cells (CD11b).
This bio-conjugate was used in attaching to live
cells for hyperthermal therapeutic application. (19) The immobilization of HRP was furtherly
confirmed by adding the chromogenic substrate
(OPD) with subsequent production of visible
signal when nano-ELISA was performed. For
collaborative verification of bio-
functionalization of AuNPs, both particle size
and structural analysis were performed in the
current study. The obvious difference in the
measurement values obtained either from the
particle size analyzer or XRD of AuNPs and
nano probes revealed the immobilization of
antibodies and HRP molecules on AuNPs. Both
particle size analyzer and XRD provided a well-
detailed characterization of AuNPs and Au nano
probes. In this context, particle size analysis
show information about the particle size
distribution in the present study; the intensities
of peak one that was recorded for AuNPs and Au
nano probes were about 88 % and 86%
respectively. This finding indicated the narrow
size distribution and mono-dispersion of either
AuNPs or Au nano probes. Furthermore, XRD
provided data about the phase purity of
nanoparticles in the present work. XRD patterns
of both AuNPs and Au nano probes were
corresponding to the pure metallic gold with
monophasic nature of nanoparticles. This result
was similar to that recorded by Ahmed et al.
2013 who studies the size and properties of
AuNPs by different characterization methods. It
was noticed that the difference between values
obtained from TEM, XRD and particle size
analyzer was within the accepted error between
different characterization techniques. The
detector antibody immobilized onto Au nano
probes acts as a “bridge” between the detected
protein (antigen) and the detection signal (HRP).
In nano-ELISA process that was applied in the
present study, different proportion of these kinds
of molecules (HRP and detector antibody) could
affect the detection signal significantly. Three
different ratios (2:1, 3:1 and 4:1) of these two
molecules were tried. The results showed that
the detection signal was the best when the ratio
was 3:1 and this was similar to the results of a
previous study performed by Jia et al, 2009. (27)
Based on quantification of HRP molecules
immobilized on the AuNPs functionalized with
HRP and antibodies at the ratio 3:1, the number
of HRP molecules was calculated on each ≈15
nm AuNPs. The concentration ratio of HRP and
AuNPs was 5.8 which meant that there were
about five to six HRP molecules on each 15 nm
AuNPs. The sandwich complex would show
reaction relative to five to six HRP molecules
once the target antigen was bound to one Au
nano probe through the immunoassay, which can
amplify the detection signal. Ambrosi et al. 2007
had developed a novel double-codified gold
nano-label. It was based on AuNPs modified
with anti-human IgG-HRP conjugate for
spectrophotometric and electrochemical
detection of human IgG as a model protein.
There were about ten anti-human IgG–HRP
molecules on each 13 nm AuNPs with promising
results. (34) Furthermore, Yang et al. 2009 had
employed the double-codified gold nano-label
for detection of serum alfa fetoprotein (marker of
hepatocellular carcinoma). They reported that
about nine HRP molecules could be conjugated
with each 15 nm AuNPs with marked
enhancement of the immunoassay sensitivity.
Therefore, there was a good correspondence
between the findings of the present work and the
previous studies’ results.In sandwich ELISA, the
detector antibody modified with biotin could be
detected either by streptavidin coupled HRP, or
by a secondary antibody linked with HRP. (45,46)
In the present study, a modified sandwich
ELISA (capture ELISA) was applied; it involved
the use of the detector antibody that was directly
conjugated with HRP through covalent bond. It
included two reaction steps and three wash steps
only, which reduces the assay duration.
Meanwhile, in conventional sandwich ELISA,
labeling the detector antibody with biotin
eventually limits the versatility of the assay.
Furthermore, the use of HRP-labelled secondary
antibody includes three reaction steps and four
washes that take more time. (45,46). T. gondii
infection was verified in all animal subgroups.
This verification allowed the reliability of the
results of both capture and nano-ELISA for
detection of Toxoplasma antigen in mice sera.
This allowed the proper judgment of the
sensitivity of both techniques.As regards the
specificity of the both ELISA assays applied in
the present work, it was 100%. There were no
false positive results obtained in serum samples
of non-infected animals neither by capture nor
by nano-ELISA. This finding was similar to that
reported by previous studies using sandwich
ELISA for detection of Toxoplasma antigen in
experimentally infected mice sera. However,
Araujo et al. 1980 noticed that sandwich ELISA
used for the diagnosis of recently acquired acute
toxoplasmosis through antigen detection
reported false positive results in patients with
rheumatoid factors. (42). In the current work,
capture ELISA was able to detect T. gondii
SAG1antigen in the sera of both RH HXGPRT (-
) strain-infected immunocompetent (II-a) and
immunosuppressed (II-b) subgroups only on
days seven and 14 PI with a sensitivity of 100%.
The antigen level, which was reflected by the
measured OD492 values was not significantly
increased from the seventh day PI till the end of
the experiment (day 14 PI) in the
immunocompetent subgroup. However, there
was a statistically significant increase in the
mean OD492 values on the 14th day PI in the
immunosuppressed subgroup as compared to that
on the seventh day PI. Moreover, the antigen
level was significantly increased in subgroup II-
bas compared to subgroup II-aeither on day
seven or on day 14 PI. Several studies had
applied the conventional sandwich ELISA in
detection of Toxoplasma antigens in the sera of
experimentally virulent strain-infected mice. For
example, Hassl and Aspöck ,1990 had diagnosed
acute infection in mice experimentally infected
with four different virulent strains of T. gondii.
Circulating antigens were detectable in the
serum from the 1st day PI till the end of the
experiment (five days PI). Also, Lee et al. 1995
had detected the antigen two days PI with
progressive increase of its concentration till the
experiment end (five days PI). (47) In addition,
Awadalla et al. 1998 were able to detect T.
gondii antigen in the sera of mice either
receiving cyclophosphamide or not on the third
day PI. Furthermore, Hammouda et al. 1998 had
detected Toxoplasma antigen on the second day
PI with steady increase of the antigen level until
the 6th day PI then it decreased gradually till the
9th day PI.The earlier detection of Toxoplasma
antigen in the previous studies might be
attributed to the high infection doses; 5 x 106, 2.2
x 107, 5 x 105, 10 x 103 and 5 x 103 tachyzoites
per mouse respectively, while in the present
study, a much lower infection dose (2 x 102
tachyzoites/ mouse) was used. It was observed in
the previously mentioned studies that, there was
continuous increase in the detectable antigen
levels in sera within the first six days PI. The
most obvious explanation was the continuous
parasite proliferation and the increasing
liberation of antigenic material from the infected
organs and cells into the circulation.The gradual
decrease of serum Toxoplasma antigen level
after the 6th day PI in the study performed by
Hammouda et al. in 1998 could be attributed to
the development of the immune response and
formation of immune complex between the
circulating antigens and the specific antibodies
that usually appear in the second week PI. (42)
The formation of immune complex might also
give an explanation for non-significant increase
in toxoplasma antigen level on day 14 PI in
subgroup II-ain the current work. Contrairily,
there was statistically significant increase in the
serum antigen level in the second week PI in
subgroup II-b. This significant increase could
be attributed to the experimental
immunosuppression of the mice thus hindering
the specific antibody production and allowing
the progressive parasite multiplication and
liberation of parasite antigens. Regarding nano-
ELISA format used in the current research, it
was able to detect T. gondii antigen in the same
animal subgroups (II-a and II-b) much earlier
than capture ELISA. It detected the antigen as
early as the first day PI with a sensitivity of
83.3% and 100% on days one and two PI
respectively. In nano-ELISA, one detector
antibody can be linked with many HRP
molecules through a nanoparticle bridge, which
can substantially cause signal amplification and
thus increased the detection sensitivity. (27)
While in capture ELISA, a single enzyme-
conjugated antibody is involving one antibody
molecule that can eventually bind to one HRP
molecule, thus it has more limited sensitivity. (27)
the results of the current study were in
consistence with that of Azami SJ et al. (48) they
had studied the antigenaemia in Toxoplasma-
infected mice using two ELISA formats:
sandwich ELISA and dot-ELISA. Antigenaemia
was detected from day two PI by dot – ELISA in
22% of mice infected with RH strain, 33% by
day three, 77% by day four and 100% of mice
sera from day five up to the end of experiment
(seven days PI). While by capture ELISA, it was
detected from day three PI in 40% of mice sera
and 100% by day four up to day seven. (48)
Inspite of higher dose of infection used in the
previous study, an easy sensitive dot-ELISA
could not detect Toxoplasma antigenaemia as
early as the 1st day PI. However, in our study the
antigen was detected on the first day PI by nano-
ELISA.Both capture and nano-ELISA had the
advantage of less reaction steps than that of
conventional type, in turn both of them could be
considered as time-saving techniques. High
assay specificity was recorded either by capture
or by nano-ELISA formats. Nano-ELISA had
reported much earlier diagnosis with much
higher sensitivity than capture technique in all
studied infected animal groups whether
immunocompetent or immunosuppressed. Even
by immunoblotting which is considered as a
sensitive method for the diagnosis of acute
toxoplasmosis, , Azami SJ(48) had detected
Toxoplasma antigens not earlier than the fourth
day PI in sera of RH strain-experimentally
infected mice by immunoblotting. (48). In
conclusion, nano-ELISA is a promising sensitive
method for early and specific diagnosis of acute
phase of toxoplasmosis in animal model
especially when the immunity is suppressed.
This technique can be used as a potential
alternative to conventional ELISA especially in
immunosuppressed patients.
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Original Article
Study of The Association Between Vitamin A Level and Uremic Pruritus in
Chronic Renal Failure Patients in Ismailia City
Roshdy Wasfi Mohamed, Nagwan Abd El Aziz Mohamed Sabek, Yasser Salem NasrAllah, Omar Mostafa
Mohamed Al Hashash. Department of Dermatology and Venereology, Department of Medical
Biochemistry, Faculty of Medicine, Suez Canal University.
ABSTRACT Pruritus is a common symptom in advanced chronic kidney disease (CKD) patients with significant impact on quality
of life; increased levels of vitamin A in these patients have been suggested as an etiological factor.Aim of the work:
is to correlate the presence and severity of pruritus in patients with advanced CKD and hemodialysis (HD) patients
with serum vitamin A level. Materials and Methods: This is a descriptive cross-sectional study where 70 advanced
CKD patients were recruited from Suez Canal University Hospital, Egypt divided into three groups; the first group
without pruritus complains, the second with Pruritus and not on hemodialysis, and the third with Pruritus and on
hemodialysis . These patients results were compared to a fourth age and sex matched healthy control subjects. Serum
vitamin A levels were determined and the subjective severity of pruritus was assessed using the visual analogue scale
(VAS) score. Results: A significantly higher serum vitamin A concentrations among CKD patients with pruritus
(14.7 ± 5.3 µg/dl) than those patients without pruritus (12.3 ± 3.9 µg/dl) and high in hemodialysis group (15.2
±5.0µg/dl). And all those groups are higher than healthy controls (5.2 ± 1.2 µg/dl). Moreover, correlation studies
have identified a significant positive correlation between the visual analogue scale (VAS) score and the serum levels
of vitamin A in chronic renal disease (r= 0.466; p= 0.027). This correlation means that whenever there is a rise in the
level of vitamin A in the serum of renal patients, there's a similar correlated rise in the severity of pruritus assessed
by VAS and vice versa. Conclusion: The difference in the serum levels of vitamin A in advanced CKD patients
suffering from uremic pruritus and CKD patients without pruritus symptom further suggest the role of vitamin A in
the pathogenesis of uremic Pruritus. And also mandate adequate monitoring of vitamins and trace elements among
those patients having pruritus not improving on hemodialysis. In these patients Serum level of vitamin A was
increased even with hemodialysis.
Introduction
Pruritus is a common symptom in chronic
hemodialysis patients, with significant impact on
quality of life. Despite improvements in dialytic
technology, the incidence of uremic Pruritus
remains elevated in dialysis patients. A sharp
increase in the prevalence of Pruritus was
observed in advanced chronic kidney disease
(CKD) patients on dialysis.(1)Although the exact
pathophysiologic mechanisms leading to pruritus
in uremic patients are still under investigations,
increased levels of vitamin A in these patients
have been suggested as an etiological
factor.(2)Pruritus is an unpleasant cutaneous
sensation prompting a desire to scratch. It is a
common and disturbing symptom among
patients receiving hemodialysis. The percentage
of HD patients suffering from Pruritus varies
reportedly from 50% to 90%.(3)Preformed
vitamin A (retinol) is the most active form in
humans; it is usually used in supplements in the
form of retinyl esters. High intake of synthetic
vitamin A over a prolonged period can lead to
toxicity, but toxicity from food sources is rare.
Periodic supplementation should not cause
serious adverse effects.(4) Elevated serum levels
of vitamin A have been routinely reported in
patients undergoing dialysis; the recommended
values found in the specialized literature have
ranged from3.50 µmol/L to 10.50 µmol/L.(5) It
has been suggested that the elevated serum
vitamin A in uremic patients due to the
decreased renal clearance of retinol-binding
protein (RBP) by proximal tubules can lead to
accumulation of retinol in the skin as a
result.(6)Material and methods : This is a
descriptive cross-sectional study,which was
carried out on end-stage renal disease patients on
dialysis with and without symptoms of Pruritus.
This study was carried out in the dermatology
outpatients’ clinics, biochemistry department and
renal dialysis unit at Suez Canal University
Hospital in Ismailia city. Study was carried out
on 70 CKD patients and 20 healthy
controls.Patients were classified into :Group
(1):25 CKD patients without symptoms of
pruritus.Group (2):25 CKD patients with
symptoms of pruritus.Group (3): 20 CKD
patients on hemodialysis with symptoms of
pruritus.Group (4) included the 20 healthy
controls.Inclusion criteria was:Gender: male or
female,any age.Patients having end-stage renal
disease based on clinical and laboratory data and
on renal dialysis. Exclusion criteria included:
Patients with itching due to other skin conditions
including: allergic contact dermatitis, bullous
pemphigoid, mycosis fungoides, eczema,
dermatitis herpitiformis, folliculitis, pediculosis,
psoriasis, xerotic eczema and lichen
planus.Patients with history of psoriasis or lichen
planus will not be included to avoid overlap of
symptoms.Patients with other systemic causes of
pruritus such as cholestatic liver disease,
thyrotoxicosis and diabetes mellitus.Also patient
with scabies were excluded. Methods
included:Detailed Clinical examination.Pruritus
was diagnosed on the basis of the presence of
associated dermatitis, the distribution of itching
or rash or a history of recent exposure to
exogenous causes.The skin was examined for
evidence of any recognizable disorder.
Scratching (causing excoriations) or rubbing
(producing papules, nodules, and lichenified
plaques).The skin was examined for evidence of
parasitic infestation, especially scabies and lice.
Examination of the skin, hair, and genitalia with
surveillance scrapings can identify either
disorder. Examination of clothing seams can
identify body lice. 1- Grading of pruritus
severity: (visual analogue scale "VAS"):
Visual analogue scale (VAS),was used to assess
the severity of Pruritus , It provides an easy and
rapid estimation of itch degree. It was developed
originally to assess the intensity of pain, but
subsequently it was also adopted for pruritus
evaluation. The VAS is a 10 point scale for the
severity of itching, with (0) being the lowest
possible value meaning the absence of itching
and (10) being the worst possible itching. The
mean, median and mode of the VAS scoring for
each VAS score (rounded to the whole) was used
to define cut-offs values of the VAS
scoring.(7).Laboratory investigations included :
Estimation of serum vitamin A levels.Venous
blood samples were obtained following
sterilization with proper antiseptics for
assessment of serum levels of vitamin A using
enzyme linked immunosorbant essay using
(human vitamin A, VA ELISA kit; USCN Life
science Inc,. Export Processing Zone, Economic
& Technological Development Zone, Wuhan
430056, China).Serum separator tubes were used
and samples were allowed to clot for 30 minutes
before centrifugation for 15 minutes at
approximately 1000 rpm. Samples were stored at
-20oC with avoidance of repeated freeze-thaw
cycles.Assessment of patients conditioninvolved
:Renal function assessment, electrolyte
evaluation ,serum albumin level.
Results
There was no statistically significant difference
in the mean and S.D. of age and sex in different
studied groups with (P value >0.05). The study
showed that there was no statistically significant
difference of the duration ofCKD between the
studied groups.Serum vitamin A among studied
groups :Table (1) and figure (1)showed that there
was higher statistically significant difference in
level of vitamin A in all patients compared to
control.( P<0.001) Also we found higher
significant difference in level of vitamin A
inpatients on hemodialysis (group 3) as
compared to other groups (15.2 ±5.0) µg/dl .
Table (1) Vitamin A in all patients
Group 1 Group 2 Group 3 Group 40
2
4
6
8
10
12
14
16
18
20
22
24
**
**
****
Se
rum
vit
am
in-A
(µ
g/d
l) **
Figure 1: Vitamin A in all patients
Present study showed that there was higher
statistically significant difference in serum
vitamin A and theseverity of Pruritus .Also that
there was statistically significant differences in
the mean serum creatinine level in controls
compared to other groups. Also there was
statistically significant differences in the mean
value of creatinine clearance level in group 1
compared to group 3 (on hemodialysis) and
othergroups.Correlationbetween VAS score and
the serum vitamin A level in the CKD
patient:Present study results showed that there
was statistically significant difference in severity
of pruritus and the serum vitamin A level in
CKD patients with pruritus ascompared to
patients onhemodialysis without pruritus . Table
(2) and fig.(2).
Table 2. Correlation between VAS Score and Serum vitamin-A among CKD patients with pruritus
Correlation Coefficient (p-value)
Group 2 (n = 25)
Group 3
(n = 20) Total
(N = 45)
Serum vitamin-A (µg/dl) 0.736 (<0.001**) 0.820 (<0.001**) 0.862 (<0.001**)
Group 2: CKD wit pruritus, Group 3: Hemodialysis patients. **. Statistically significant at p<0.01; Pearson's Correlation
Table 1. Serum vitamin A among studied participants
Serum vitamin A Mean ± SD
Study Groups
p-value Group 1
(n = 25) Group 2
(n = 25) Group 3
(n = 20) Group 4 (n = 20)
Serum vitamin-A(µg/dl) 9.6 ±2.2 * 14.0 ± 4.7 15.2 ±5.0 5.7 ± 1.0 * <0.001**
Group 1: CKD without pruritus, Group 2: CKD wit pruritus, Group 3: Hemodialysis patients, Group 4: healthy controls. **. Statistically significant at p<0.05; One-way ANOVA *. Significantly different from other two groups; Bonferroni Post-Hoc test a. Kruskal Wallis test (Non-parametric test)
Figure 2
Discussion
uremic pruritus remains a frequent and
disturbing problem in patients with end-stage
renal disease, mainly because of the lack of
knowledge of the underlying pathophysiological
mechanisms(1)Hemodialysis patients who had
moderate to extreme itch reported a significantly
greater chance of remaining awake at night,
feeling sleepy during the day. In the largest
population-based study to date, severe CKD-
associated pruritus was associated independently
with death in Japanese hemodialysis
patients(8).Pruritus is a common symptom in
chronic hemodialysis patients, with significant
impact on quality of life.(1) Despite
improvements in dialytic technology, the
incidence of uremic pruritus remains elevated in
dialysis patients..(2) The present study aimed to
correlate serum vitamin A levelwith the severity
of pruritus in patients with advanced CKD on
hemodialysis using VAS score in patient with
and without pruritus .The study showed
significantly higher serum vitamin A level
among all groups of CKD patients compared to
controls. Moreover, there was statistically
significantly higher serum vitamin A levels in
the patients with pruritus compared to the
patients without pruritus. These results agree
with Cabral et al., .(5)whofound thatelevated
vitamin A serum levels on 55 haemodialysis
patients as compared to 28 controls.(5)This study
have identified a higher significant positive
correlation between the visual analogue scale
(VAS) score and the serum levels of vitamin A
in the CKD patients with pruritus.Statistically
significant high serum vitamin A And VAS
score was higher in patients with severe pruritus
compared to mild and moderate types
(P=0.001).Similar ,(9)results was reported by
Penniston and Tanumihardjo(9)who suggested
that the elevated serum vitamin A in uremic
patients due to decreased renal clearance of
retinol-binding protein (RBP) by proximal
tubules can lead to accumulation of retinol in the
skin and pruritus as result.(1)Abahusain ,Al-
Nahedh(10)found that serum vitamin A was
significantly higher in CKD patients with severe
pruritus. Berne and colleagues noted that the
decreased content of epidermal retinol following
UV therapy was associated with a relief in
pruritus in these patients, which explain the role
of vitamin A as a contributing factor in the
pathogenesis of uremic pruritus.(11)Present study
revealed a significant relation between pruritus
in CKD on hemodialysis and serum level of
vitamin A and serum level of calcium which
agree with Germain M, et al . (12)who showed
that vitamin A toxicity can contribute to
hypercalcemia in patients undergoing
hemodialysis.(12)
Conclusion this study using quantitative analysis of pruritus
in chronickidney disease patients with or without
hemodialysis showed that etiology of severe
pruritus is multifactorial , and that elevated
serum vitamin A is one of the important
etiological factors .Blood urea nitrogen, serum
calcium level , inorganic phosphorus,
parathormone might be involved also .
Therefore it is recommended to avoid high
vitamin A supplementation inCKD patients
especially those on hemodialysis
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Sahan H, et al ;An epidemiology study of patients
with uremic pruritus. J Eur Acad Dermatol
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2. Patel TS, Freedman BI, Yosipovitch G An update
on pruritus associated with CKD. Am J Kidney
Dis , 2007; 50:11-20
3. Weening RH, Sewell LD, Davis MD
Calciphylaxis: natural history, risk factor analysis,
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79.
4. Hathcock JN, Rader JL, Micronutrient safety. Ann
of NY AcadSci 1990; 587;257-266.
5. Cabral PC, Diniz AS, de Arruda IK. Vitamin A
and zinc status in patients on maintenance
haemodialysis. Nephrology 2005; 10:459-63.
6. Frey SK, Nagl B, Henze A, et al. Isoforms of
retinol binding protein 4 (RBP4) are increased in
chronic diseases of the kidney but not of the liver.
Lipids Health Dis 2008; 7:29.
7. Reich A, Heisig M, Phan NQ, Taneda K,
Takamori K, Takeuchi S, et al (2012): Visual
Analogue Scale: evaluation of the instrument for
the assessment of pruritus. ActaDermVenereol
2012; 92:497-501.
8. Namer B, Carr R, Johanek LM, et al. Separate
peripheral pathways for pruritus in man. J
Neurophysiol. 2008; 100(4):2062–2069.
9. Penniston KL, Tanumihardjo SA. The acute and
chronic toxic effects of vitamin A. Am J ClinNutr
2006; 83:191-201.
10. Abahusain MA, Al-Nahedh NN. The biochemical
status of vitamin A and alpha-tocopherol during
different stages of renal disease and its
relationship to diabetes. Saudi J Kidney Dis
Transpl.2002; 13(1):18-23.
11. Berne B, Vahlquist A, Fischer T, Danielson BG,
Berne C. UV treatment of uraemic pruritus
reduces the vitamin A content of the skin. Eur J
Clin Invest 1984; 14:203-6.
12. Germain M, et al. Correlation Between Uremic
Pruritus Intensity and Quality of Life: A Report
from the ITCH National Registry. Abstract
American Society of Nephrology. 2008,TH-
PO798.
Original Article
The Diagnostic Role of Serum Procalcitonin in Differentiation Between
Bacterial and Abacterial Meningitis
Mohammed Kassem1, Akram Deghady2, Nasser Mohammed Abd Alla1, Mahassen Hosny1; Departments
of Tropical Medicine1 and Clinical Pathology2, Faculty of Medicine, University of Alexandria
ABSTRACT
Meningitis is defined as an inflammation of the lining of the brain and spinal cord. It is caused when the
protective membranes around the brain and spinal cord known as the meninges become infected by
viruses, bacteria, fungi, and parasites. Aim of the work: The purpose of this study was to study the
diagnostic role of serum procalcitonin in differentiation between bacterial and abacterial meningitis.
Patients and Methods: 40 patients with clinical manifestations of suspected meningitis. Evaluation
included Complete blood picture, random blood sugar, lumbar puncture for cerebrospinal fluid analysis
including chemical and cytological analysis as well as culture and sensitivity, erythrocyte sedimentation
rate, C reactive protein, liver function tests, renal function tests, serum procalcitonin and Cranial
tomography or Magnetic resonance imaging of the brain. Results: We observed significantly higher
concentration of serum procalcitonin of patients with bacterial meningitis as compared to the abacterial
group. Conclusion: Serum procalcitonin assay might be considered as a simple, easy, sensitive and
specific marker for the diagnosis of acute bacterial meningitis. Serum procalcitonin can be used as useful
marker to differentiate between bacterial and abacterial meningitis, as it was significantly higher in
bacterial than abacterial meningitis.
Introduction
Meningitis is defined as an inflammation of the
lining of the brain and spinal cord.(1) It is
caused when the protective membranes around
the brain and spinal cord known as the meninges
become infected by viruses, bacteria, fungi, and
parasites.(1,2)Suspected bacterial or viral
meningitis is a medical emergency; thus,
immediate steps must be taken to establish the
specific diagnosis and empirical antimicrobial
treatment must be started rapidly.(3,4) Early
initiation of appropriate therapy, particularly
antibiotics for bacterial meningitis, improves
outcome.(5) It is a major cause of morbidity and
mortality among children and elderly.(4,6) The
mortality of untreated bacterial meningitis
approaches 100% and even with optimum
treatment mortality and morbidity might
happen.(3,7) In Egypt, the case fatality rate ranged
from 8.5 to 55%(6).Meningitis is typically caused
by an infection with microorganisms. The
etiology of bacterial meningitis is affected by
the age of the patient. In neonates, the most
common etiologic agents are group B
Streptococci subtype III, which normally inhabit
the vagina and gram-negative enteric
bacilli(8)including Escherichia coli carrying K1
antigenwhich normally inhabit the digestive tract
and other Gram-negative enteric bacilli,
including Klebsiella, Enterobacter, and
Salmonella.(9,10) Other pathogens that
occasionally cause meningitis in neonates
include Listeria monocytogenes and
Enterobacter. Other rare causes of meningitis in
neonates include Staphylococci, Enterococci,
and Viridans streptococci.(8,10) In infants and
young children, Streptococcus pneumoniae
serotypes 6,9,14,18 and 23, Neisseria
meningitides serogroups B, C, Y, and W-135,
and Haemophilus influenzae type b are the most
common causes of bacterial meningitis.(3,11-
13)Among children older than 5 years of age,
adolescents and adults, S. pneumoniae and N.
meningitidis are the predominant causes of
bacterial meningitis.(13,14). The term abacterial
meningitis refers loosely to all cases of
meningitis in which no bacterial infection can be
demonstrated. This is usually due to viruses
including Enteroviruses, herpes simplex virus
(HSV) type 2 and less commonly type 1,
Varicella Zoster virus, Mumps virus, human
immune deficiency virus (HIV) and
Cytomegallo virus.(15) But, it may be due to
bacterial infection that has already been partially
treated. Other causes as tuberculous
meningitis,(16) brucellosis, mycoplasm and fungal
meningitisare seen in people with immune
deficiency.(1,17) Non-infectious causes of
abacterial meningitis such as malignancies with
brain metastasis or some medications notably
sulphamethoxazole and non-steroidal anti-
inflammatory drugs have also been
identified(18).The key to the diagnosis of
meningitis is the evaluation of CSF.(19) Lumbar
puncture is a safe procedure, although
postprocedure headache occurs in about one
third of patients.(20) The peripheral white blood
cell count alone is not helpful in distinguishing
bacterial from abacterial meningitis, particularly
in young children as normal white blood cell
count does not rule out bacterial
meningitis.(21)After CSF is obtained, the Gram
stain results, white blood cell counts, glucose
levels and protein levels should be evaluated
immediately.Although no single measure is
diagnostic, a combination of abnormal CSF
findings is highly suggestive of meningitis and
helpful in determining the likely etiology.
Rarely, patients with bacterial meningitis may
present with normal or near-normal white blood
cell counts, glucose levelsand protein levels.
During the initial evaluation of a patient with
suspected meningitis, diagnostic and therapeutic
maneuvers should begin concomitantly. If a
computed tomography scan is required before a
lumbar puncture, blood cultures should be
obtained, followed by prompt initiation of
empiric antimicrobial therapy before the scan.
Adjunctive therapy with dexamethasone should
be added in adults with suspected S.pneumoniae
infection(22). Procalcitonin (PCT), the calcitonin
precursor propeptide, is a 13 kDa protein that is
synthesized in C cells of the thyroid gland and
secreted from leukocytes of the peripheral
blood.(23) Its gene is located on the short arm of
chromosome 11 (11p15.4).(24) The blood of
healthy individuals contains only low levels of
PCT.(23,25) The secretion of PCT was found to
increase up to several thousand-fold in the
presence of bacterial sepsis but remains normal
or slightly increases in viral infections and
inflammatory reactions that are not
infectious.(26,27) In contrast to CRP levels that rise
between 12 and 18 h after bacterial challenge,(28)
serum PCT level rises within 2–3 h after
infection with a peak value at 6–12 h, and
normalizes within 2 days.(29,30) PCT is stable in
plasma with plasma half-life of approximately
22 h.Its production is not impaired by neutropenia
or other immunosuppressive states. PCT levels
parallel the severity of the inflammatory insult or
infection meaning those with more severe disease
have higher levels. Furthermore, procalcitonin has
some utility as a prognostic indicator with higher
serum concentrations related to the risk of
mortality (30-33). Procalcitonin was initially
described as a potential marker of bacterial
diseases by Assicot et al(34) , and assumed to be a
protein of the acute phase of inflammation with
kinetics faster than that of CRP.(29) Although the
performance of PCT was nearly similar to that of
CRP in predicting sepsis, the rapid kinetics and
the more specificity of PCT made it superior to
CRP in that aspect. Indeed, the current
hypothesis on this marker in sepsis has gained a
solid scientific basis as many researches
demonstrated that quantitative evaluation of PCT
is superior to the other used biomarkers which
can markedly reduce antibiotic usage and as
being a hormokine mediator, immune-
neutralisation of it might offer new hope for
more effective treatment options.(35-38)
Subjects and Methods
This study is a prospective cohort study which
was conducted in Alexandria Fever Hospital, on
40 subjects who were divided into two
groups.Group I: Included twenty patients with
clinical manifestations and positive CSF criteria
of suspected bacterial meningitis.Group II:
Included twenty patients with clinical
manifestations and positive CSF criteria of
suspected abacterial meningitis.After taking
informed consent ,all patients were subjected to
the following:Detailed history taking , thorough
clinical examination , routine laboratory
investigations; Complete blood picture, random
blood sugar,ESR 1st, 2nd hour, C-reactive protein
(CRP), liver function tests and renal function
tests. Lumbar puncture for CSF analysis
including chemical and cytological analysis as
well as culture and sensitivity,estimation of
serum procalcitonin and radiological
investigations including CT brain or MRI brain
as needed.
Results
Diagnosis of meningitis was established on the
basis of clinical picture, CSF analysis and
culture for bacterial infection, as well as imaging
studies of the brain.In our study, as regard the
clinical presentations of patients with meningitis,
the current study proved that fever (90%) was
the most frequent symptom of the studied groups
followed by disturbed level of
consciousness(DLC) (65%), GIT symptoms
(65%), neck stiffness (32.5%), convulsions
(30%) and lastly signs of meningeal irritation
(25%). Cases presented by the classic triad of
meningitis (fever, neck rigidity and DLC)
constituted about 32.5% of the meningitic cases
(both bacterial and abacterial).Table (1,2). We
observed that there was no statistically
significant difference was found between the two
studied groups as regard gender, whereas as
regard age there was statistically significant
difference between the three studied groups.
Bacterial meningitis was more common in
younger patients.
Table (1):Comparison between The two studied groups according to history taking
Bacterial (n = 20) Abacterial (n = 20) Test of Sig. P
No. % No. %
Fever 19 95.0 17 85.0 1.111 FEp= 0.605
GIT symptoms 12 60.0 14 70.0 0.440 0.507
Convulsions 10 50.0 2 10.0 7.619* 0.006*
Past history of chronic diseases other than
CNS disorders 4 20.0 3 15.0 0.173 FEp= 1.000
Delayed speech 3 15.0 6 30.0 1.290 FEp= 0.451
Headache 2 10.0 7 35.0 3.584 FEp= 0.127
Abnormal gait 1 5.0 2 10.0 0.360 FEp= 1.000
Eye symptoms 1 5.0 2 10.0 0.360 FEp= 1.000
Cough 1 5.0 1 5.0 0.0 FEp= 1.000
Vesicular rash 1 5.0 0 0.0 1.026 FEp= 1.000
Tremors 0 0.0 0 0.0 - -
2: Value for chi square test FE: Fisher Exact *: Statistically significant at p ≤ 0.05
Table (2):Comparison between the two studied groups according to clinical examination
Bacterial (n = 20) Abacterial (n = 20) Test of Sig. P
No. % No. %
Days of fever
Min. – Max. 0.0 – 15.0 0.0 – 15.0 Z= 0.014 0.989
Mean ± SD. 4.35 ± 4.39 4.75 ± 4.97
Clinical examination
Not free 9 45.0 5 25.0 0.185
Free 11 55.0 15 75.0
Conscious level
Consious 7 35.0 7 35.0
1.566 MCp= 0.894 Drowzy 10 50.0 12 60.0
Semi conscious 1 5.0 0 0.0
Comatosed 2 10.0 1 5.0
Neck rigidity
Rigid 7 35.0 6 30.0 0.736
Lax 13 65.0 14 70.0
Kernig's sign
Negative 14 70.0 16 80.0 0.465
Positive 6 30.0 4 20.0
Brudziniski’s sign
Negative 16 80.0 16 80.0 FEp= 1.000
Positive 4 20.0 4 20.0
Muscle tone
Normal 20 100.0 19 95.0 FEp= 1.000
Abnormal 0 0.0 1 5.0
Muscle power
Normal 20 100.0 19 95.0 FEp= 1.000
Abnormal 0 0.0 1 5.0
Reflexes
Normal 20 100.0 19 95.0 FEp= 1.000
Abnormal 0 0.0 1 5.0
2: value for chi square test &MC: Monte Carlo FE: Fisher Exact &Z: Z for Mann Whitney test
Haematological profile:The Hb level (gm/dl) in
the bacterial group ranged from 7.90 to 15.10
(gm/dl) with a mean of 10.92± 2.23. In the
abacterial group, it ranged from 8.60 to 13.50
with a mean of 11.75 ± 1.37. WBCs count (x
1000/ul) in the bacterial group ranged from4.07
to 25.20 , with a mean of 12.02 ± 5.11. Among
the abacterial group it ranged from 4.32 to 17.63,
with a mean of 8.06 ± 3.31. Theplatelet count (x
1000/ul) in the bacterial group ranged from
118.0 to 590.0, with a mean of 332.95 ± 120.91.
In the abacterial group, it ranged from 150.0 to
460.0, with a mean of 309.40 ± 95.47. The
neutrophils % in the bacterial group ranged from
29.10 to 83.0% of the total WBCs count with a
mean of 57.01 ± 17.24. In the abacterial group, it
ranged from 21.90 to 70.20 % with a mean of
56.52 ± 18.06.The lymphocytes % in the
bacterial group ranged from 13.0 to 30.50 % of
the total WBCs count with a mean of 26.92 ±
6.57. In the abacterial group, it ranged from 3.20
to 41.60% with a mean of 27.0 ± 12.11. The
monocytes % in the bacterial group ranged from
0.50 to 19.30 % of the total WBCs count with a
mean of 4.59 ± 4.14. In the abacterial group, it
rangedfrom 1.60 to 8.90 % with a mean of 4.23
± 2.38.There was no statistically significant
difference as regard haemoglobin level and
platelet count in bacterial and abacterial
meningitic cases with p value = 0.275 and 0.685
respectively among the two groups. There was a
statistically significant difference as regard total
WBCs count with p value = 0.009, with a mean
of (12.02 ± 5.11) and (8.06 ± 3.31) in bacterial
and abacterial meningitic groups respectively.
This means that WBCs count were higher in the
bacterial group. As regard neutrophils %,
lymphocytes % and monocytes % there were no
statistically significant difference as p values
were (0.807, 0.956 and 0.882) among the
bacterial and abacterial meningitic groups
respectively. Table(3).
Table (3):Comparison between two studied groups according to CBC
Bacterial (n = 20) Abacterial (n = 20) Test of Sig. P
HB (gm/dL)
Min. – Max. 7.90 – 15.10 8.60 – 13.50 t= 1.111 0.275
Mean ± SD. 10.92 ± 2.23 11.57 ± 1.37
WBCs *1000/ul
Min. – Max. 4.07 – 25.20 4.32 – 17.63 Z= 2.625* 0.009*
Mean ± SD. 12.02 ± 5.11 8.06 ± 3.31
Platelate *1000/uL
Min. – Max. 118.0 – 590.0 150.0 – 460.0 Z= 0.406 0.685
Mean ± SD. 332.95 ± 120.91 309.40 ± 95.47
Neutrophils %
Min. – Max. 29.10 – 83.0 21.90 – 70.20 Z= 0.245 0.807
Mean ± SD. 57.01 ± 17.24 56.52 ± 18.06
Lymphocytes %
Min. – Max. 13.0 – 30.50 3.20 – 41.60 Z= 0.055 0.956
Mean ± SD. 26.92 ± 6.57 27.0 ± 12.11
Monocytes %
Min. – Max. 0.50 – 19.30 1.60 – 8.90 Z= 0.149 0.882
Mean ± SD. 4.59 ± 4.14 4.23 ± 2.38
t: Student t-test Z: Z for Mann Whitney test *: Statistically significant at p ≤ 0.05
C reactive protein:Among the bacterial group,
there were 18 cases out of 20 had CRP level
(mg/dl) ≥ 6 with serum procalcitonin levels
ranged from 0.12 – 20.50 ng/ml, while there
were 2 cases with CRP levels (mg/dl) < 6 with
serum procalcitonin level ranged from 0.50 –
2.50 ng/ml. Among the abacterial group, there
were 19 out of 20 cases had CRP level < 6 with
serum procalcitonin level ranged from 0.02 –
0.42 , while there was only 1 case with CRP
level ≥ 6 with serum procalcitonin level 0.02
ng/ml. There was a statistically significant
difference between bacterial and abacterial
meningitic groups as regard CRP with p value (<
0.001*) as CRP was significantly higher among
the bacterial group. Table (4)
Table (4):Comparison between the two studied groups according to CRP
Bacterial (n = 20) Abacterial (n = 20) P
No. % No. %
CRP (mg/dl) < 6 2 10.0 19 95.0 28.972* <0.001*
CRP (mg/dl) ≥ 6 18 90.0 1 5.0
2: Chi square test*: Statistically significant at p ≤ 0.05
Erythrocyte sedimentation rate (ESR): Among
the bacterial group, ESR first hour (mm/hr)
ranged from 4.0 to 125.0 with a mean of (33.85
± 33.05) while in the abacterial group, it ranged
from 5.0 to 80.0 with a mean of (40.90 ± 23.33).
In the bacterial group, ESR (second hour)
(mm/hr) ranged from 12.0 to 155.0 with a mean
of (55.25 ± 39.86), while in the abacterial group,
it ranged from 12.0 to 120.0 with a mean of
(71.90 ± 35.71). There was no statistically
significant difference between bacterial and
abacterialmeningitic groups as regard ESR 1st
hours, ESR 2nd hours with p values (0.095 and
0.093) respectively. Table (5)
Table (5):Comparison between the two studied groups according to ESR
Bacterial
(n = 20)
Abacterial
(n = 20) Z P
ESR 1st hour(mm/hr)
Min. – Max. 4.0 – 125.0 5.0 – 80.0 1.668 0.095
Mean ± SD. 33.85 ± 33.05 40.90 ± 23.33
ESR 2nd hour(mm/hr)
Min. – Max. 12.0 – 155.0 12.0 – 120.0 1.681 0.093
Mean ± SD. 55.25 ± 39.86 71.90 ± 35.71
Z: Z for Mann Whitney test
Renal function tests: Blood urea (mg/dl) in the
bacterial group ranged from 13.0 to 68.0 with a
mean of 27.09 ± 16.76. In the abacterial group, it
ranged from 10.0 to 59.0 with a mean of 27.42 ±
12.56. Serum creatinine (mg/dl) in the bacterial
group ranged from 0.16 to 1.0 with a mean of
0.49 ± 0.18. In the abacterial group, it ranged
from 0.30 to 1.16 with a mean of 0.59 ±
0.21.There was no statistically significant
difference between bacterial and abacterial
meningitic groups as regard blood urea and
serum creatinine with p values0.597 and
0.150.The Random blood sugar :The random
blood sugar (mg/dl) in the bacterial group ranged
from 79.0 to 193.0 with a mean of 115.53 ±
34.45, while in the abacterial group, it ranged
from 88.0 to 229.0with a mean of 116.0 ±
34.71.There was no statistically significant
difference between bacterial and abacterial
meningitic groups as regard random blood sugar
with p value = 0.797.Liver function tests:The
prothrombin time (PT) (seconds) in the bacterial
group ranged from 10.80 to 17.30 with a mean
of11.90 ±1.61. In the abacterial group, it ranged
from 10.0 to 17.30with a mean of 11.41 ± 1.59.
The prothrombin activity (PA) (%) in the
bacterial group ranged from 48.10 to 100.0 with
a mean of91.66 ± 15.37. Among the abacterial
group, it ranged from 48.10 to 100.0 with a mean
of 93.153 ± 11.55. The international normalized
ratio (INR) in the bacterial group ranged from
1.0 to 1.59, with a mean of 1.08 ± 0.15. Among
the abacterial group, it ranged from 0.90 to 1.59
with a mean of 1.04 ± 0.14. The total bilirubin
(mg/dl) in the bacterial group ranged from 0.20
to 0.70 with a mean of 0.44 ± 0.22. In the
abacterial group, it ranged from 0.20 to 1.50with
a mean of 0.48 ± 0.35. Direct bilirubin (mg/dl) in
the bacterial group ranged from 0.03 to 0.50 with
a mean of 0.11 ± 0.14. In the abacterial group, it
ranged from 0.0 to 0.50with a mean of (0.09 ±
0.10) . Serum alanine transferase (ALT) (IU/L)
in the bacterial group ranged from 15.0 to
49.0with a mean of25.50 ± 12.40. Among the
abacterial group, it ranged from 12.0 to 72.0 with
a mean of 34.25 ± 14.62. Serum aspartate
transferase (AST) (IU/L) in the bacterial group
ranged from 14.0 to 43.0 with a mean of 23.50 ±
9.75. In the abacterial group, it ranged from 20.0
to 60.0 with a mean of 27.45 ± 8.74. Serum
albumin (g/dl) in the bacterial group ranged from
2.80 to 4.30with a mean of 3.52 ± 0.49. Among
the abacterial group, it ranged from 2.0 to 3.70
with a mean of (3.28 ± 0.37).There were no
statistically significant differences between
bacterial and abacterial meningitic groups as
regard prothrombin time, prothrombin activity,
INR, total bilirubin, direct bilirubin, ALT, AST
and serum albumin as p value was 0.089, 0.473,
1.000, 0.943, 0.795, 0.106, 0.053 and 0.104
respectively. Cerebrospinal fluid examination
(CSF): Table (6). 1-Aspect: In the bacterial
group, the aspect of CSF was clear in 8 (40.0%)
patients, turbid in 7(35.0%) patients, slightly
turbid in 4 (20.0%) patients, and bloody with
clear supernatant in 1 (5.0%) patient. Among the
abacterial group, it was clear in 11 (55.0%)
patients, turbid in 3 (15.0%) patients, slightly
turbid in 2 (10.0%) patients, and opalescent in 4
(20.0%) patients.2- Cerebrospinal fluid glucose:
CSF glucose (mg/dl) ranged in the bacterial
group from 5.0 to 55.0 with a mean of 38.45 ±
19.14. Among the abacterial group, it ranged
from 25.0 to 110.0 with a mean of 60.50 ± 18.79.
There was a statistically significant difference
between bacterial and abacterial meningitic
groups with p value = 0.001*.3- Cerebrospinal
fluid protein: CSF protein (mg/dl) in the
bacterial group ranged from 100.0 to 541.0
(mg/dl) with a mean of .267.60 ± 142.37. In the
abacterial group, it ranged from 12.0 to 90.0
with a mean of 56.85 ± 31.29. There was a
statistically significant difference between
bacterial and abacterial meningitic groups with p
value <0.001*.4- Cerebrospinal fluid WBCs
count: CSF WBCs (c/mm3) in the bacterial group
ranged from 6.0 to 80050.0 with a mean of
5784.0 ± 17923.91. In the abacterial group, it
ranged from 7.0 to 1340.0 with a mean of 253.50
± 397.67. There was a statistically significant
difference between bacterial and abacterial
meningitic groups as with p value 0.021*.
Lymphocytes % in the bacterial group ranged
from 0.0 to 50.0 (%) with a mean of 16.91 ±
14.21. It ranged in the abacterial group from 60.0
to 95.0 % with a mean of 77.43 ± 11.28
(%).There was a statistically significant
difference between bacterial and abacterial
meningitic groups with p value <0.001*.PMN (%
) among the bacterial group ranged from 28.0 to
100.0 (%) with a mean of 81.99 ± 17.40 (%). It
ranged in the abacterial group from 5.0 to
40.0(%) with a mean of 22.56 ± 11.27(%).There
was a statistically significant difference between
bacterial and abacterial meningitic groups with p
value <0.001*.5- Cerebrospinal fluid lactate:
CSF lactate (mg/dl) ranged in the bacterial group
from 43.0 to 192.0 with a mean of 156.84 ±
37.83. In the abacterial group, it ranged from
15.0 to 52.0 with a mean of 26.10 ± 9.01.There
was a statistically significant difference between
bacterial and abacterial meningitic groups with p
value <0.001*.6- Cerebrospinal fluid gram stain
and culture: Among the bacterial group, CSF
gram staining and culture showed no growth in 2
(10.0%) patients, gram positive diploccoci intra
and extracellular in 15 (75.0%) patients and
Haemophilus influenzae in 3 (15%) patients.
CSF gram staining and culture showed no
growth in abacterial meningitic group.
Table (6):Comparison between the two studied groups according to CSFexamination
Bacterial (n = 20) Abacterial (n = 20) Test of sig. p
No. % No. %
CSF aspect
Clear 8 40.0 11 55.0
7.299 MCp= 0.082
(NS)
Turbid 7 35.0 3 15.0
Slightly turbid 4 20.0 2 10.0
Opalescent 0 0.0 4 20.0
Bloody with clear Supernatant 1 5.0 0 0.0
Glucose (mg/dl)
Min. – Max. 5.0 – 55.0 25.0 – 110.0 Z= 3.413* 0.001*
Mean ± SD. 38.45 ± 19.14 60.50 ± 18.79
Protein (mg/dl)
Min. – Max. 100.0 – 541.0 12.0 – 90.0 Z= 5.448* <0.001*
Mean ± SD. 267.60 ± 142.37 56.85 ± 31.29
WBCs /mm
Min. – Max. 6.0 – 80050.0 7.0 – 1340.0 Z= 2.301* 0.021*
Mean ± SD. 5784.0 ± 17923.91 253.50 ± 397.67
Lymphocytes(%)
Min. – Max. 0.0 – 50.0 60.0 – 95.0 t= 14.916* <0.001*
Mean ± SD. 16.91 ± 14.21 77.43 ± 11.28
Polymorph nuclear leukocytes(%)
Min. – Max. 28.0 – 100.0 5.0 – 40.0 Z= 5.235* <0.001*
Mean ± SD. 81.99 ± 17.40 22.56 ± 11.27
Lactate (mg/dl)
Min. – Max. 43.0 – 192.0 15.0 – 52.0 Z= 5.395* <0.001*
Mean ± SD. 156.0 ± 37.83 26.10 ± 9.01
CSF culture
No growth 2 10.0 20 100.0
MCp<0.001* Gram +ve diplococcic 15 75.0 0 0.0
Haemophilus influenza 3 15.0 0 0.0
2: value for chi square MC: Monte Carlo test Z: Z for Mann Whitney test
t: Student t-test *: Statistically significant at p ≤ 0.05 NS= non significant
Serum procalcitonin:In the bacterial group the
value of PCT ranged from 0.12 - 20.50 (ng/ml)
with a mean of 8.27 ± 7.08, while in the
abacterial group, it ranged from 0.02 - 0.42 with
a mean of 0.13 ± 0.11. It was noticed that the
level of serum procalcitonin among the bacterial
group was significantly higher than that of the
abacterial meningitic group (p value
<0.001*).Table (7) There was a positive
correlation between serum PCT, total WBCs
count and CRP in bacterial and non-bacterial
meningitis cases but this relation becomes highly
significant with bacterial meningitis positive
group. (Fig.1). There was a positive correlation
between serum PCT and neutrophils % and a
negative correlation with lymphocytes % in the
bacterial meningitic group, while in the
abacterial group, there was a negative correlation
between serum procalcitonin with WBCs and
neutrophils %, and a positive correlation with
lymphocytes.
Table(7): Comparison between two the studied groups according to serum procalcitonin
Bacterial (n = 20) Abacterial (n = 20) Z P
Serum Procalcitonin (ng/ml)
Min. – Max. 0.12 – 20.50 0.02 – 0.42 4.792* <0.001*
Mean ± SD. 8.27 ± 7.08 0.13 ± 0.11
Z: Z for Mann Whitney test *: Statistically significant at p ≤ 0.05
Figure (1):Comparison between two studied groups according to serum procalcitonin
Agreement (sensitivity, specificity and accuracy) for procalcitonin to diagnose bacterial cases:
The cut off value of serum procalcitonin to
differentiate between bacterial and abacterial
meningitic groups is ≥ 0.5 ng/ml. The sensitivity
and specificity of serum procalcitonin were
(75.0% and 100%) respectively, PPV=100%,
NPV=80.0% and accuracy= 87.50%. Also the
area under the curve was calculated AUC =
0.943* with P value <0.001*.The following
curve in figure (2) shows that, there was a
statistically significant difference as regard
serum procalcitonin level to differentiate
between bacterial and abacterial meningitis with
AUROC=0.943* and P value <0.001*.
Figure (2):ROC curve for serum procalcitonin to diagnose bacterial cases
Discussion
Procalcitonin was initially described as a
potential marker of bacterial diseases(34), and
assumed to be a protein of the acute phase of
inflammation with kinetics faster than that of
CRP.(29) Indeed, the current hypothesis on this
marker in sepsis has gained a solid scientific
basis as many researches demonstrated that
quantitative evaluation of PCT is superior to the
other used biomarkers which can markedly
reduce antibiotic usage and as being a hormokine
mediator, immunoneutralisation of it might offer
new hope for more effective treatment
options.(35,38)The aim of this work was to study
the diagnostic value of serum procalcitonin in
differentiation between bacterial from abacterial
meningitis.However, on reviewing the results of
different studies carried out all over the world to
evaluate PCT discriminative ability in detecting
septic meningitis, controversial was clear. Prasad
et al(39) found that, serum PCT level was
significantly higher in children with septic
meningitis than in those with aseptic meningitis
or in controls and it may be used to differentiate
between septic and aseptic meningitis. Another
Egyptian study by Ibrahim et al(40) who reported
that, serum PCT levels were significantly higher
in bacterial meningitis compared with non
bacterial meningitis. On contrary, Hoffmann et
al(41)reported normal PCT serum concentrations
in 12 adults suffering from bacterial meningitis,
where PCT on admission was ≤ 0.5 ng/ml. In
another German study of Schwarz et al(42) neither
the sensitivity nor the specificity of the test using
the PCT were significantly better than those
using the CRP test.Although the performance of
PCT was nearly similar to that of CRP in
predicting sepsis, the rapid kinetics and the more
specificity of PCT made it superior to CRP in
that aspect.(43) It is important to notify that the
increase of PCT and CRP in bacterial meningitis
is due to extracellular multiplication in the
bloodstream which induce a strong systemic
inflammatory response.(44) This is supported by
the study of Jereb et al(44) where two cases of
bacterial meningitis with low serum PCT
occurred due to non-encapsulated bacteria that
multiply intracellularly, these were proved to be
caused by borreliosis and tuberculosis.Our
results showed that the level of serum
procalcitonin among the bacterial group was
significantly higher than abacterial
meningiticgroup.Our results was matched to
those of Carrol et al(37), Prasad et al(39), Ibrahim
et al(40)andJereb et al(44).We found that,
peripheral WBCs and CRP were significantly
higher in the bacterial group as compared to
abacterial group. Our results were matched
toHoffman et al(41), Mayah et al(43), Viallon et
al(45) andOnal et al(46) studies.Our results showed
that, in the bacterial meningitic group there was
a positive correlation between serum
procalcitonin with CSF protein, CSF WBCs,
CSF polymorph nuclear leukocytes, and CSF
lactate and a negative correlation with CSF
glucose and CSF lymphocytes, while in the
abacterial group, there was a positive correlation
between serum procalcitonin with CSF glucose,
CSF protein and CSF lymphocytes and a
negative correlation with CSF WBCs, CSF
polymorph nuclear leukocytes and CSF lactate.
In the study ofDubos et al(47), among the 167
patients , 21 had bacterial meningitis showing
that PCT (≥0.5 ng/mL) and CSF protein (≥0.5
g/L) were the best biologic tests, with 89% and
86% sensitivity rates, 89% and 78% specificity
rates, and areas under the ROC curves of 0.95
and 0.93 respectively, denoting that PCT and
CSF protein had the best predictive value to
distinguish between bacterial and aseptic
meningitis in children.Our results showed that,
among the bacterial group, there were 16 cases
out of 20 were recovered with serum
procalcitonin level ranged from 0.12 –
20.50ng/ml, while there were 4 cases died with
serum procalcitonin level ranged from (0.32 –
17.20 ng/ml,while among the abacterial group,
there were 16 out of 20 cases were recovered
with serum procalcitonin level ranged from 0.02
– 0.42 ng/ml, and 3 cases were referred to a
neurosurgery with serum procalcitonin level
ranged from 0.02 – 0.03 ng/ml, while there was
only 1 case died with serum procalcitonin level
0.16 ng/ml. In the study of El- Shami et al(48),
serum PCT levels were significantly higher in
non-survivors than in survivors with bacterial
meningitis, it can be concluded that serum PCT
levels might have a prognostic in addition to its
diagnostic value in patients with acute bacterial
meningitis.
Conclusion
Finally, it may be conculded that serum
procalcitonin assay might be considered as a
simple, easy, sensitive and specific marker for
the diagnosis of acute bacterial meningitis. It can
be used as useful marker to differentiate between
bacterial and abacterial meningitis, as it was
significantly higher in bacterial than abacterial
meningitis. However, serum procalcitonin
should not replace the conventional tests for
meningitis, but should be used as a
complementary tests to increase the precision of
diagnosis.
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