QQ

Early prediction of Gram
negative bacteremia in febrile

cancer patients: Correlation
between some inflammatory
mediators, exposure to gamma
radiation and severity of
infection
A Thesis
Submitted in Partial Fulfillment of the Requirements for the
Master degree
In Pharmaceutical Sciences
(Microbiology andImmunology)
By
Amira Abuzeid Abdelbaset

Bachelor of Pharmaceutical sciences, 2007
Pharmacist at National Centre for Radiation Research and

Technology (NCRTT)
2015
Early prediction of Gram negative bacteremia in

febrile cancer patients: Correlation between some
inflammatory mediators, exposure to gamma
radiation and severity of infection
A Thesis
Submitted in Partial Fulfillment of the Requirements for the
Master degree
In Pharmaceutical Sciences
(Microbiology andImmunology)
By
Pharmacist atNational Centre for Radiation Research and Technology
(NCRRT)
Under Supervision of
Dr. Mohammad Mabrouk Aboulwafa, PhD
Professor of Microbiology and Immunology,
Faculty of Pharmacy, Ain Shams University
Dr. Hala Abdallah Farrag, PhD
Professor of Medical Microbiology,
National Centre for Radiation Research and Technology (NCRRT)
Dr. Khaled Mohamed Anwar Aboshanab, Ph.D

Associate Professor of Microbiology and Immunology,
Faculty of Pharmacy, Ain-Shams University
2015
Acknowledgments
First, I thank "Allah" for granting me the power to accomplish this work.
My deepest gratitude and appreciation are expressed to Prof. Dr. Mohammad
Mabrouk Aboulwafa, Professor of Microbiology and Immunology, Faculty of
Pharmacy, Ain Shams University, for his divine support. His constructive criticism,
guided me immensely throughout the work and during the revision of the thesis.
I would like to express my deepest thanks to Prof. Dr. Hala Abdallah Farrag,
Professor of Medical Microbiology, National Centre for Radiation Research and
Technology (NCRRT), for suggesting the topic of research and for providing continuous
scientific supervision and follow up, for her valuable scientific supervision, constructive
advice and continuous guidance throughout the work.
I am also greatly indebted to Dr. Khaled Mohammed Aboshanab, Associate
Professor of Microbiology and Immunology, Faculty of Pharmacy, Ain Shams University.
His valuable time and big effort are greatly appreciated.
Finally, my deepest everlasting thanks and appreciation are for my beloved
parents and husband for their continuous support and encouragement throughout
my life.
........
Table of Contents
Table of Contents
ACKNOLEDGEMENT..
....
LIST OF ABBREVIATIONS........................................................................................I
TABLE OF CONTENTS...........................................................................................IV
LIST OF FIGURES...................................................................................................VI
LIST OF TABLES...................................................................................................VIII
ABSTRACT.................................................................................................................1
INTRODUCTION........................................................................................................4
LITERATURE REVIEW............................................................................................7
1. Bloodstream infection................................................................................
2. Overall picture of gram-negative bacteremia...........................................
11
3. What happens when microorganism enters body?...................................
12
4. Cytokines...................................................................................................
16
5. The role of chemokines in infectious diseases (Bacterial infections).........
19
6. The complement system............................................................................
22
7. Fever as a sign of infection.........................................................................
23
8. Blood cultures for identifying bacteremia.................................................
25
9. Antimicrobial resistance............................................................................
26
10. Ionizing Radiation.....................................................................................
27
11. Radiotherapy and inflammatory mediators............................................
34
12. The ROC curve analysis...........................................................................
35
MATERIALS AND METHODS....................................................................
39
MATERIALS.................................................................................................
39
1. Microorganisms .........................................................................................
39
2. Blood specimens .........................................................................................
39
3. Media .........................................................................................................
39
4. Kits..............................................................................................................
39
MSc Thesis 2015
Table of Contents
5. Blood culture bottles...................................................................................
40
6. Tubes used for plasma and sera separation................................................
40
7. Diffu-plate...................................................................................................
40
8. Antibiotic discs............................................................................................
40
9. Laboratory animals....................................................................................
40
10. Devices.......................................................................................................
41
METHODS.....................................................................................................
42
11. Collection and manipulation of specimens ...............................................
42
12. Plasma and serum samples collection.......................................................
42
13. Isolation of pathogenic bacteria................................................................
42
14. Identification of isolated organisms from blood specimens......................
43
15. Total leukocytic count assay......................................................................
44
16. Assay of serum C-Reactive Protein (CRP)................................................
44
17. Assay of serum Inteleukin-6(IL-6)............................................................
45
18. Assay of serum Interleukin-8(IL-8)...........................................................
46
19. Assay of serum complement C3................................................................
48
20. Antimicrobial susceptibility of some pathogenic bacterial isolates...........
48
21. Effect of in-vitro gamma irradiation on some selected isolates.................
50
22.Detection of Lipase and Protease enzymes production..............................
51
23. Effect of gamma radiation on IL-6 serum levels in rats with induced
fever of bacterial and non-bacterial origin.....................................................
51
24. Assay of IL-6 serum concentration in rats................................................
53
25. Statistical methods....................................................................................
54
RESULTS .....................................................................................................
55
1. Collection, isolation and identification of pathogenic bacteria recovered

from blood cultures.........................................................................................
55
2.Age, sex, white blood cells count and serum CRP levels of cancer and
non-cancer patients.........................................................................................
55
3. Interleukin-6 serum levels (sIL-6) in cancer and non-cancer patients.......
61
4.Interleukin-8 serum levels (sIL-8) in cancer and non-cancer patients........
63
5.Relationship between CRP, sIL-6 and sIL-8 in some selected cancer and
non-cancer patients with positive and negative blood cultures......................
64
6.Relationship between white blood cells count and each of CRP, sIL-6 and
67
MSc Thesis 2015
Table of Contents
sIL-8 in some selected cancer and non-cancer patients with positive blood
cultures............................................................................................................
7.Statistical analysis of CRP and IL-6 serum markers of some selected
cancer and non-cancer patients with positive blood cultures by Receiver
Operating Characteristic Curves (ROC).......................................................
70
8.Complement C3 serum levels (C3) in cancer and non-cancer patients.......
78
9. In-vitro effect of gamma irradiation on antimicrobial susceptibility of

some bacterial isolates recovered from cancer and non-cancer patients........
79
10.Activity profiles of lipase and protease enzymes of some selected

bacterial isolates recovered from cancer and non-cancer patients.................
91
11.Effect of gamma irradiation on IL-6 serum levels in rats with induced

fever of bacterial and non-bacterial origin.....................................................
92
12.Statistical analysis of IL-6 serum levels in bacteremic groups with and

without exposure to gamma radiation by Receiver Operating
Characteristic Curve (ROC)..........................................................................
97
DISCUSSION...........................................................................................100
SUMMARY..............................................................................................114
REFERENCES.........................................................................................120

MSc Thesis 2015
List of Abbreviations
ADH
Arginine dihydrolase
AK
Amikacin
AMC
AML
Amoxacillin/Clavulanic Acid
Acute Myeloid Leukemia
API
Analytical profile index system
AUC
Area under the curve
BAL
Broncho-alveolar lavage
BSI
Bloodstream infection
Chloramphenicol
C3
Complement 3
CAZ
Ceftazidime
CIT
Citrate
CN
Gentamicin
CRD
Carbohydrate recognition domain
CRP
C-reactive protein
CTX
Cefotaxime
DNA
Deoxyribonucleic acid
Ef
Efficacy
ELISA
Enzyme linked immunosorbent assay
FEP
FN
Cefepime
Febrile neutropenia
FUO
Fever of unknown origin
GEL
Gelatin
Gy
Gray
H2S
hydrogen sulfide
HIV
Human immunodeficiency virus
HRP
Horseradish peroxidase
i.p
Intraperitoneal
ICU
Intensive care unit
MSc Thesis 2015
IFN
Interferon
IL-1
Interleukin-1
IL-2
Interleukin-2
IL-6
Interleukin-6
IL-8
Interleukin-8
IPM
Imipenem
K2EDTA
Potassium salt of ethylene diamine tetraacetic

acid
LDC
lysine decarboxylase
LEV
Levofloxacin
LOS
Lipooligosaccharide
LPB
Lipoplopysaccharide binding protein
LPS
Lipopolysaccharides
LTA
Lipoteichoic acid
MBL
Mannose binding lectin
MCP-1
monocyte chemoattractant protein -1
mRNA
Messenger Ribonucleic acid
N2
Nitrogen gas
NO2
Nitrite
NPV
Negative predictive value
OD
Optical density
ODC
ornithine decarboxylase
OFX
Ofloxacin
PAMP
pathogen-associated molecular patterns
PBS
phosphate-buffered saline
PCT
Procalcitonin
PMNs
Polymorphnuclear leukocytes
PPV
Positive predictive value
ROC
Receiver Operating Characteristic
SAM
Ampicillin/ Salbactam
SIRS
Systemic inflammatory response syndrome
Sn
Sensitivity
MSc Thesis 2015
Sp
Specificity
SXT
Sulphamethoxazole/ trimethoprim
TLR
Toll like receptor
TMB
3,3,5,5-Tetramethylbenzidine
TNF-
Tumor necrosis factor-
TOB
Tobramycin
TR
Rectal temperature
TZP
Tazobactam
URE
Urease
UTI
Urinary tract infection
VP
Voges-Proskauer
WBCs
White blood cells
MSc Thesis 2015
List of Figures
List of Figures
Figure 1
Distribution of overlapped test results
36
Figure 2
The ROC curve distribution between Sensitivity and Specificity
38
Figure 3
BD BACTEC 9050 Blood culture System
41
Figure 4
Standard calibration curve of Human IL-6 serum concentration as

determined by ELISA technique
Figure 5
62
Relationship between IL-8 and CRP serum concentrations in (a)

cancer patients and (b) non-cancer patients both with gram
negative bacteremia. The numbers of patients in a and b were 6 and
5, respectively
Figure 6
66
Relationship between IL-8 and Il-6 serum concentrations in (a)

negative bacteremia.The numbers of patients in a and b were 6 and
5, respectively
Figure 7
66
Relationship between IL-6 and CRP serum concentrations in (a)

negative bacteremia. The numbers of patients in a and b were 15
and 9, respectively
Figure 8
67
Relationship between WBCs count and serum Levels of IL-6 both

simultaneously measured in (a) cancer patients and (b) non-cancer
patients both with positive blood cultures. The numbers of patients
in a and b were 15 and 9, respectively
Figure 9
69
Relationship between WBCs count and serum Levels of IL-8 both

measured simultaneously in (a) cancer patients and (b) non-cancer
patientsboth with positive blood cultures. The numbers of patients
in a and b were 6 and 5 respectively
Figure 10
69
Relationship between WBCs count and serum Levels of CRP both

measured simultaneously in (a) cancer patients and (b) non-cancer
patients both with positive blood cultures. The numbers of patients
Figure 11
in a and b were 15 and 9 respectively
69
ROC curve analysis showing the diagnostic performance of CRP
72
and IL6 for discriminating patients with positive culture from those
MSc Thesis 2015
List of Figures
negative among cancer patients

Figure 12

and IL6 for discriminating patients with positive culture from those
with negative culture among non- cancer patients
Figure 13
74

and IL6 and their combinations for discriminating patients with
positive culture from those negative (cancer and non- cancer
patients)
Figure 14
78
Frequency percentages of different susceptibility profiles (sensitive,

intermediate resistant and resistant)of some selected bacterial
isolates recovered fromcancer patients against some antimicrobial
agents inhibiting bacterial cell wall synthesisbefore and after
gamma irradiation
Figure 15
84

intermediate resistant and resistant) of some selected bacterial
agents inhibiting protein synthesis in bacteriabefore and after
gamma irradiation
Figure 16
85

agents inhibiting nucleic and folic acids syntheses before and after
gamma irradiation
Figure 17
86

isolates recovered from non-cancer patients against some
antimicrobial agents
inhibiting bacterial cell wall synthesis before and after gamma
irradiation
Figure 18
88

antimicrobial agents inhibiting protein synthesis in bacteria before
and after gamma irradiation
MSc Thesis 2015
89
List of Figures
Figure 19

antimicrobial agents inhibiting nucleic and folic acids syntheses
before and after gamma irradiation
Figure 20
90
ROC curve analysis showing the diagnostic performance of serum

IL-6 for discriminating rats with bacteremia (bacteremic fever) with
exposure to gamma radiation from those without exposure to
gamma radiation
MSc Thesis 2015
99
List of Tables
List of Tables
Table 1
Characters
of
antibiotic
discs
used for antimicrobial
susceptibilitytesting of collected bacterial isolates

Table 2
The interpretive standard (breakpoint) for all selected

antibiotics
Table 3
non-cancer patients
bacterial infection (positive blood cultures)
cultures)
bacterial infection (positive blood cultures)
blood cultures)
Table 10
58
Age, sex, white blood cells count and serum CRP measurements
of non-cancer patients without bacterial infection (negative
Table 9
57
Age, sex, white blood cells count serum CRP measurements and
bacterial species isolated from non-cancer patients with
Table 8
56
Age, sex, white blood cells count and serum CRP measurements
of cancer patients without bacterial infection (negative blood
Table 7
55
Age, sex, white blood cells count, serum CRP measurements

and bacterial species isolated from cancer patients with
Table 6
52
Number and frequency of positive and negative cases for

microbial growth of blood culture samples from cancer and
Table 5
49
Rat groups used for testing the effect of gamma radiation on

serum level of IL-6
Table 4
40
59
Numbers and percentages of different bacterial species

recovered from cancer and non-cancer patients
60
Levels of CRP of some cases of cancer and non-cancer patients
61
with positive and negative blood cultures selected for statistical
MSc Thesis 2015
List of Tables
Table 11
analysis
Levels of serum IL-6 in some cases of cancer and non-cancer
patients with positive and negative blood cultures selected for
statistical analysis
Table 12
Levels of serum IL-8 in some cases of cancer and non-cancer

patients with positive and negative blood cultures selected for
statistical analysis
Table 13
cultures:
Table 14
Mean values of tested serum markers (CRP, IL-6 and IL-8) in
Table 15
some selected cancer and non-cancer patients

Relationship between WBCs count and serum Levels CRP, IL-6
and IL-8 measured at the same time in some selected patients
with positive blood culture in cancer and non-cancer patients
72
73
74
Diagnostic validity test for serum CRP to discriminate positive

and negative blood cultures (cancer and non- cancer patients)
Table 21
71
Diagnostic validity test for serum IL-6 to discriminate positive

and negative blood cultures in non- cancer patients
Table 20
68

and negative blood cultures in non- cancer patients
Table 19
66

and negative blood cultures in cancer patients
Table 18
65

and negative blood cultures in cancer patients
Table 17
64
Summarization of Serum levels of CRP, IL-6 and IL-8 in cancer

and non-cancer patients with positive and negative blood
Table 16
63
75

and negative blood cultures (cancer and non- cancer patients)
MSc Thesis 2015
76
List of Tables
Table 22
Multi-ROC:determining the value of CRP with IL6 at 120.9
Table 23
Levels of serum C3 in some selected cancer and non-cancer
Table 24
patients with positive and negative blood cultures

Antibacterial susceptibilities of some selected bacterial isolates
recovered from cancer patients against different antimicrobial
agents before and after gamma irradiation exposure
Table 25
Table 26
Antibacterial susceptibilities of some selected bacterial isolates

recovered from non-cancer patients against different
antimicrobial agents before and after gamma irradiation
exposure
Profiles of lipase and protease enzymatic activities, resistance
prevalence against different tested antimicrobial agents as well
as serum levels of IL-6 and IL-8 of some selected bacterial
isolates recovered from cancer and non-cancer patients
77
79
81
82
92
Table 27
White blood cells count and IL-6 serum concentration in

control group
93
Table 28
White blood cells count and IL-6 serum concentration in

radiation control group
93
Table 29
Rectal temperatures, white blood cells count and IL-6 serum

concentrations infever of non-bacterial origin (yeast fever)
group
Table 30
concentrations in fever of non-bacterial origin (Yeast fever)

group with gamma radiation exposure
Table 31
aeruginosa) group
aeruginosa) group with gamma radiation exposure
95

concentrations in fever of bacterial origin (Klebsiella
pneumoniae) group
Table 34
95

concentrations in fever of bacterial origin (Pseudomonas
Table 33
94

concentrations in fever of bacterial origin (Pseudomonas
Table 32
94

concentrations in fever of bacterial origin (Klebsiella
MSc Thesis 2015
95
96
List of Tables
pneumoniae) group with gamma radiation exposure

Table 35
ROC analysis data of sIL-6 for bacteremic groups with and

without exposure to gamma radiation
MSc Thesis 2015
98
Abstract
Abstract
One hundred twenty four feverish (cancerand non- cancer) inpatients were enrolled in the study. Serum and plasma samples were
separated from collected blood samples at onset of fever for assay of
inflammatory biomarkers (using ELISA technique) and total leukocytic
count (using Beckman/Coulter semi automated), blood samples were
collected and cultured on blood culture media for isolation of gram
negative organisms which were identified by API 20E technique and
antibiotic susceptibility test was performed using disc diffusion method
as well as lipase and protease enzymatic activities were performed
(via Tween and Gelatin agar plates, respectively) for some selected
bacterial isolates through Tween- agar medium and gelatin- agar
medium. Assay of serum IL-6 in rats was also done using ELISA
technique. Cesium 137 (137 Cs) Gamma cell 40 located at National
Center for Radiation Research and Technology (Cairo, Egypt) was the
irradiation source used in the study.
IL-6 and IL-8 serum levels were higher in feverish patients with
gram negative bacteremia than in those with non-microbial fever for
both groups (cancer and nn- cancer) of patients. For cancer patients
with gram negative bacteremia and those without there was
significant difference in IL-6 and IL-8 serum levels (P=0.0001 and
0.0059, respectively) and similar resultswere also obtained for noncancer patients (P=0.0288 and 0.0059).
Also, serum levels of both mediators were higher in cancer
patients with gram negative bacteremia than in non-cancer patients
with gram negative bacteremia. While, CRP serum levels showed nonsignificant differences among all groups.
The Cutoff levels to distinguish between bacteremic (positive
blood cultures) and non-bacteremic (negative blood cultures) cases
MSc Thesis 2015
Abstract
were determined using receiver operating characteristic curves (ROC):

for CRP it was 29 mg/l for cancer patients, 119 mg/l for non- cancer
patients and with 60% and 100% specificity, 77.8% and 66.7%
sensitivity, NPV 60% and 62.5 %, PPV 77.8% and 100%, Efficacy
71.4% and 78.6% respectively. So the efficacy of CRP as marker to
discriminate between positive and negative blood cultures was higher
in cancer patients relative to non- cancer patients.
While, the cutoff level of serum IL-6 was 398.6 pg/ml for cancer
patients, 120.9 pg/ml for non- cancer patients and with 100%
specificity, 100% sensitivity, NPV 100%, PPV 100%, Efficacy 100% for
both groups. So the efficacy of IL-6 as marker to discriminate between
positive and negative blood cultures was higher than that of CRP.
The ROC curve analysis showing diagnostic performance of CRP
and IL-6 and their combination (via multi-ROC) could be used for
discriminating patients with positive cultures from those with negative
cultures (all cancer and non- cancer tested cases), We found that the
best cut-off value of IL-6 was 120.9 pg/ml with 60% specificity, 100%
sensitivity, NPV 100%, PPV 85.7%, Efficacy 88.2%, while for CRP the
best cut-off value was 85.9 mg/l with 50% specificity, 75% sensitivity,
NPV 45.5%, PPV 78.3%, Efficacy 67.6%, Using the multi-ROC for the
both markers to improve the results for CRP , we found that CRP best
cut-off value was 220 mg/ml at IL-6 of 120.9 pg/ml with improved
specificity, sensitivity, NPV, PPV and Efficacy of values 90%, 100%,
100%, 96% and 97.1% respectively. The AUC also improved using the
multi-ROC from 0.735 for CRP to 0.982 in combination with IL-6 which
showed AUC of 0.957.The usefulness of both markers (IL-6 and CRP)
was proved to distinguish between bacteremic and non- bacteremic
patients.
To study the role of measured serum IL-6 as marker of severity,
correlations were pointed with lipase and protease enzymatic activities
MSc Thesis 2015
Abstract
and antimicrobial susceptibility tests in some selected bacterial

isolates. Correlations were clear within bacterial isolates recovered
from cancer patients as E. coli isolates recovered from patients having
high values of serum IL-6 also showed high protease enzymatic
activity and antimicrobial resistance reaching 78.6%. While those
without protease activity and antimicrobial resistance reaching
71.4%were isolated from patients with low serum levels of IL-6.
Klebsiella pneumoniaeisolates recovered from patients having
high values of serum IL-6 also showed lipase activity and antimicrobial
resistance reaching 78.7%. For Pseudomonas species, isolates
recovered from patients having high values of serum IL-6 also showed
protease and lipase activities. For Acinitobacter baumannii, isolates
recovered from patients with high levels of serum IL-6 also showed
lipase enzymatic activity with antimicrobial resistance reaching 100%.
While, for bacterial isolates recovered from non-cancer patients, there
was no clear correlation between inflammatory mediators and
virulence factors, except for Pseudomonas fluorescence isolate no.94
there was positive lipase and protease activity.
In the animal model , (ROC) forIL-6 serum concentration
inbacteremic groups (due to Pseudomonas aeruginosa and Klebsiella
pneumoniae) with exposure to gamma radiation
and bacteremic
groups without exposure to gamma radiation showed a cutoff value of

25740 pg/ml with 75% specificity, 58.3% sensitivity, negative
predictive value 64.3% and positive predictive value 70% .
MSc Thesis 2015
Introduction
Introduction
The bloodstream was the second most frequent infection site,
representing
20%
of
all
infections
(Hugonnet
et
al.,
2004).
Bloodstream infection (BSI) continues to be a life threatening

condition.
Invading microorganisms induce the release of a large
number of humoral and cellular proinflammatory mediators, causing

systemic inflammatory response syndrome (SIRS) (Sungurtekin et al.,
2006). Nosocomial bloodstream infection (BSI) is a major complication
of intensive care unit (ICU) admission. Physiological features such as
fever, tachycardia and tachypnea have been proposed as indicators of
sepsis. These findings may be sensitive, but are less specific in the
diagnosis of systemic inflammation or infection (Vandijck et al., 2007).
Among various infections underlying sepsis, bacteremia is recognized
as a critical condition that influences the outcome of sepsis and is
reportedly associated with an attributable mortality of approximately
35% (Abe et al., 2010).People in good health with strong immune
systems rarely develop bacteremia. However, when bacteria are
introduced directly into the circulatory system, especially in a person
who is ill or undergoing aggressive medical treatment, the immune
system may not be able to cope with the invasion and symptoms of
bacteremia may develop.
Infections are still the major cause of treatment-related
morbidity and mortality in cancer patients. The malignant disease and
the intensive chemotherapy may cause an impaired host defence to
infection. Key factors are the intensity and duration of neutropenia, but
a decreased function of granulocytes and disturbances of natural
barriers may substantially add to the risk of serious infections (Diepold
et al., 2008).
MSc Thesis 2015
Introduction
BSI verified by a positive blood culture is a sign of poor

prognosis and predisposes patients to vascular hypotension and
shock, which are associated with high mortality rates. The clinical
symptoms of systemic inflammation associated with BSI, such as the
criteria defining systemic inflammatory response syndrome (SIRS),
derive from the hosts innate immune response to invading organisms.
This response is characterized by activation of phagocytes and
systemic release of soluble mediators of inflammation (Aalto et al.,
2004).
The differential diagnosis of infections is a daily problem in oncologic clinical
work. Symptoms typical of infection, such as fever and changes in the laboratory
parameters, can be caused equally well by the underlying malignancy or its treatment.
Thus, the use of different markers in terms of diagnosing infection in cancer patients is
limited and not quite reliable. These diagnostic difficulties lead to long periods of
hospitalization and empiric antimicrobial therapies, which are expensive and impair the
patients quality of life. Unnecessary empiric antimicrobial treatments also increase the
risk of developing resistant bacterial strains. Therefore, better diagnostic methods are
needed for the diagnosis of infections in cancer patients. Gram-negative bacteria
play an important role in bloodstream infections, about 30% of cases
in the ICU are caused by one or another species of klebsiella, E. coli,
Enterobacter species, and P. aeruginosa(Peleg and Hooper, 2010).LPS
in gram negative bacteria leads to increases in the expression of IL-6
and IL-8 (Sawa et al., 2008). A variety of laboratory markers of
systemic inflammation, such as interleukin- 6 (IL-6), interleukin- 8 (IL8), and C-reactive protein (CRP) have been used to identify patients
with infection. Of these markers, increased blood levels of IL-6 and IL-8
denote activation of monocytes/macrophages and both cytokines
induce CRP synthesis in the liver (Aalto et al., 2004).This makes IL-6
an interesting molecule to evaluate the early phase of infection and
sepsis (Gaini et al., 2006).
MSc Thesis 2015
Introduction
The induction of inflammation by bacterial and viral infections

increases cancer risk (de Martel; Franceschi, 2009). IL-6 is an early
indicator of inflammatory response to illness or injury. It rises within
hours of substantial injury or infection. With a half-life of 45 minutes,
IL-6 can be monitored to reveal if a patient is suffering an acute
response to surgery, trauma, or infection, and if the response is
waning slowly or rapidly, which can help to predict the patients risks
and prognosis (Kellum et al., 2007). IL-8 concentration increases
during different infections, such as bacteremia (Hack et al., 1992 and
Hynninen et al., 1997). In neutropenic patients, enhanced IL-8
production has been demonstrated in predicting bacteremia (Engel et
al., 2005). Recognition of bacteria by complement components is
likely to induce the activation complement pathways leading to the
formation of a C3 convertase and the generation of complement
activation products triggering diverse biological activities, such as
microbial opsonization, phagocyte recruitment and inflammation,
resulting in the elimination of pathogenic microorganisms (Petersen et
al., 2001; Reid
et al., 2002).
Early recognition of BSI and
administration of appropriate antimicrobial drugs play a crucial role in

reducing mortality in community-acquired infections (Aalto et al.,
2004). If physicians were able to rely on an early indicator of
bacteremia, they could restrict their antibiotic prescriptions to the right
indications, they could start therapy earlier and they could limit the
number of blood samples to be obtained for culture.
This
thesis
aimed
to
study
the
usefulness
of
serum
inflammatory mediatiors (IL-6 and IL-8) as early predictors of gram

negative bacteremia in cancer and non- cancer patients over other
markers like CRP and C3. Also, their usefulness to distinguish different
causes of fever in critically ill (especially cancer) patients which may
be bactremic fever, non-bacteremic fever due to either chemotherapy
or radiotherapy. This was achieved through the following steps:
MSc Thesis 2015
Introduction
1- Collection of blood samples from febrile cancer and non- cancer

patients.
2- Measurement of total leucocytes count, CRP, complement C3 as
well as some inflammatory mediators (IL-6 and IL-8)
in the
collected blood samples (plasma and serum samples)

3- Blood culture of the collected samples for detection of gram
4-
negative bacterial infection.

Screening antimicrobial susceptibility against some selected
bacterial isolates.
5- Studying the relation between serum IL-6 concentrations and
lipase as well as protease enzymatic activity as markers of
severity of bacterial infection.
6- Studying the effect of exposure to gamma radiation on the levels
of inflammatory mediators which was achieved through the
animal model using male Wister albino rats.
MSc Thesis 2015
Literature Review
Literature Review
1. Bloodstream infections
The bloodstream was the second most frequent infection site,
representing 20% of all infections (Hugonnet et al., 2004).
Bacteremia refers to a bacterial invasion into blood circulation.
Bacteremia can occur when you brush your teeth, pick a scab, or
squeeze a zit. Bacteremia may also result from any type of dental or
surgical procedure. Bacteremia may cause no symptoms and resolve
without treatment, or it may produce fever and other symptoms of
infection depending on whether the organism was able to replicate
themselves in the blood stream. For most people, the immune system
should "notice" the organisms immediately and respond with
specialized white blood cells to search out and destroy them. Of
course, it is possible for bacteremia to progress to septicemia,
especially if an individual has a weakened immune system(Young,
2012).
Septicemia, sometimes called sepsis, also refers to the presence
of bacteria in the blood with replication to cause an infection, but this
is an infection that moves rapidly and is life-threatening. Simply,
septicemia
is
bacteremia.
Septicemia
is
characterized
by
combination of different processes going on in the body, which are

toxemia, bacteremia, and systemic inflammatory response syndrome
(SIRS) .Septicemia can result from a kidney infection, pneumonia,
meningitis, endocarditis, osteomyelitis and other illnesses (Young,
2012).
Despite rapid progress in health care over the past decades,
sepsis continues as a major life-threatening condition in acute care
patients (Osuchowskiet al., 2006).
MSc Thesis 2015
Literature Review
Sepsis and its sequelae are the leading causes of death among
critically ill patients. Treatment is largely supportive following source
control and administration of antibiotics. Sepsis is associated with
uncontrolled and excessive cytokine release. However, potential
therapeutic intervention targeted at cytokines has failed to reduce
mortality in multiple clinical trials. These failures likely reflect the
molecular complexity and redundancy within the inflammatory
response and the extent to which an infecting organism dictates the
response. In this regard, distinct patterns of cytokine and leucocyte
gene expression have been shown for Gram-positive and Gramnegative organisms and activation of their respective receptor
pathways, Toll-like receptor TLR2 and TLR4. Understanding the cellular
mechanisms that account for these differences could, ultimately, aid
the development of novel and effective pharmacotherapies (Finney et
al., 2012).
Sepsis is a multi-step process that involves an uncontrolled
inflammatory response by the host cells that may result in multi organ
failure and death. Both gram-negative and gram-positive bacteria play
a major role in causing sepsis. These bacteria produce a range of
virulence factors that enable them to escape the immune defenses
and disseminate to remote organs, and toxins that interact with host
cells via specific receptors on the cell surface and trigger a
dysregulated immune response (Ramachandran, 2014).
Sepsis remains the most common cause of mortality among
patients in intensive care units (ICUs). Invading microorganisms induce
the release of a large number of humoral and cellular proinflammatory
mediators, causing systemic inflammatory response syndrome (SIRS)
(Sungurtekin et al., 2006). Sepsis is the second most common cause
of death in non-coronary intensive care units (ICU) and the tenth
overall cause of death in high income countries.
MSc Thesis 2015
Literature Review
The incidence of sepsis in the past two decades has annually

increased by 9% (Ramachandran, 2014).
Nosocomial bloodstream infection (BSI) is a major complication
of intensive care unit (ICU) admission. Physiological features such as
fever, tachycardia and tachypnea have been proposed as indicators of
sepsis. These findings may be sensitive, but are less specific in the
diagnosis of systemic inflammation or infection (Vandijck et al., 2007).
Despite recent advances in critical care medicine, the mortality
of sepsis in ICU remains high. Among various infections underlying
sepsis, bacteremia is recognized as a critical condition that influences
the outcome of sepsis and is reportedly associated with an attributable
mortality of approximately 35% (Abe et al., 2010).
People in good health with strong immune systems rarely
develop bacteremia. However, when bacteria are introduced directly
into the circulatory system, especially in a person who is ill or
undergoing aggressive medical treatment, the immune system may
not be able to cope with the invasion, and symptoms of bacteremia
may develop. For this reason, bacteremia is most common in people
who are already affected by or being treated for some other medical
problem. In addition, medical treatment may bring a person in contact
with new types of bacteria that are more invasive than those already
residing in that person's body, further increasing the likelihood of
bacterial infection (Young, 2012).
Common immediate causes of bacteremia include: drainage of
an abscess, including an abscessed tooth, urinary tract infection,
especially in the presence of a bladder catheter, decubitus ulcers
(pressure sores),intravenous procedures using unsterilized needles,
including IV drug use,prolonged IV needle placement, use of ostomy
tubes, including gastrostomy (surgically making a new opening into
the stomach), jejunostomy (surgically making an opening from the
MSc Thesis 2015
Literature Review
abdominal wall into the jejunum), and colostomy (surgically creating

an artificial opening into the colon). Symptoms of bacteremia may
include: fever over 101 oF (38.3C, chills, malaise, abdominal nausea,
vomiting, diarrhea, anxiety, shortness of breath and confusion(Young,
2012).
Not all of these symptoms are usually present. In the elderly,
confusion may be the only prominent symptom. Bacteremia may lead
to septic shock, whose symptoms include decreased consciousness,
rapid heart and breathing rates and multiple organ failures.
Historically, sepsis has often been defined as the presence of
pathogenic microorganisms or their toxins in the bloodstream, and the
term has been used more or less interchangeably with bacteremia.
More recently, we have developed the term sepsis syndrome, which
we define as the systemic response to infection. When sepsis
syndrome is accompanied by hypotension, it is referred to as septic
shock. If the hypotension does not respond to fluid therapy, it is
referred to as refractory septic shock (Bone, 1991).
Early and accurate recognition of bacterial infections is essential
for the treatment and prognosis of medical emergency admissions,
Short-term mortality was higher in patients with bacteremia even after
controlling for severity of illness, (Bates et al., 1995).
Conditions
which
increase
the
chances
of
developing
bacteremia include: immune suppression, either due to HIV infection

or drug therapy ,antibiotic therapy which changes the balance of
bacterial types in the body, prolonged or severe illness ,alcoholism or
other drug abuse, malnutrition, diseases or drug therapy that cause
ulcers in the intestines, e.g. chemotherapy for cancer (Young, 2012).
Patients with hematologic malignancies are at increased risk for
infection due to underlying disease and/or intensive chemotherapy. In
addition to duration and intensity of neutropenia, decreased functions
MSc Thesis 2015
Literature Review
and disturbances of natural barriers may substantially add to risk of

serious infections (Karan, 2002).
Bacterial and fungal infections are frequently noticed during
chemotherapy-induced neutropenia. The neutropenia and disruption of
the mucosal barrier as a consequence of the applied chemotherapy
regimen predispose patients to a bacterial infection (Tromp et al.,
2009).
Infections are still the major cause of treatment-related
morbidity and mortality in cancer patients. The malignant disease and
the intensive chemotherapy may cause an impaired host defence to
infection. Key factors are the intensity and duration of neutropenia, but
a decreased function of granulocytes and disturbances of natural
barriers may substantially add to the risk of serious infections (Diepold
et al., 2008).
Normally, clinical conditions during BSI are caused by pathogenassociated molecular patterns, which are components that bind to Tolllike receptor (TLR2) and (TLR4) on leukocytes, resulting in the
production of inflammatory cytokines (Abdallah et al., 2008).
condition. BSI verified by a positive blood culture is a sign of poor
prognosis and predisposes patients to vascular hypotension and
shock, which are associated with high mortality rates. The clinical
symptoms of systemic inflammation associated with BSI, such as the
criteria defining systemic inflammatory response syndrome (SIRS),
derive from the hosts innate immune response to invading organisms.
This response is characterized by activation of phagocytes and
systemic release of soluble mediators of inflammation (Aalto et al.,
2004).
The leading cause of treatment-related mortality among
patients with hematological malignancies undergoing chemotherapy is
MSc Thesis 2015
Literature Review
multiorgan failure due to systemic infection during neutropenia

(Buchheidt et al., 2003). Most neutropenic patients with infection
present with fever as the first symptom. However, there is a variety of
causes for febrile conditions. Apart from infection, reactions to drugs
or blood products as well as tumor-associated fever are all possible
underlying mechanisms. If the fever is not accompanied by clinical or
microbiological evidence of infection, it is classified as fever of
unknown origin (Link et al., 2003).
2.
Overall
picture
of
gram-negative
bacteremia
Most bacteremic infections are caused by Gram negative bacilli,
E. coli is the most commonly isolated pathogen, followed by Klebsiella,
Enterobacter species. While Pseudomonas species are somewhat less
frequently observed, P. aeruginosa have consistently been associated
with the highest mortality of all bacteremic infections (Bone, 1991).
Gram-negative bacteremia is essentially a disease with fewer
than 100 reported cases prior to 1920. Over the last 30 years, a
number of studies have looked at the incidence of gram-negative
bacteremia and noted a continuing increase (Lory, 1998).
E. coli is the most frequently isolated organism, with Klebsiella
species, and especially K. pneumoniae,as the second most common.
The Centers for Disease Control (CDC) in 1974 found that nosocomial
gram-negative bacteremias were most often caused by E. coli,
Enterobacter species, P. aeruginosa, and K. pneurnoniae, while also
noting an increasing rise in nosocomial bacteremia due to grampositive organisms.
Lory, (1998) assigned to direct the intensive-care unit at an
institution that treatedcancer patients with leukemia, lymphoma, or
advanced metastatic cancer with investigational cytotoxic agents, he
MSc Thesis 2015
Literature Review
rapidly became aware that the leading cause of death was infection.
Even the patients recognized that when one of them developed a
fever and was moved to the intensive care unit, usually because of
hypotension, the outlook was grim indeed, and the patients lived in
mortal fear of something known to them as "Pseudomonas."
It was noteworthy that the patient charts indicated that the site
of infection could not be identified in the vast majority of patients.
Antibiotic therapy usually included a combination of cephalothin plus
kanamycin (neither drug being active against P. aeruginosa) and was
usually not started until after a report of a positive blood culture for a
gram-negative rod had returned from the laboratory or not until the
patient developed signs of septic shock.
Although Gram-negative bacteria have often been implicated in
the pathogenesis of severe sepsis and septic shock, how they trigger
these often lethal syndromes is uncertain. In particular, the role played
by blood-borne bacteria is controversial. Two alternatives were
considered in the first, circulating Gram-negative bacteria induce toxic
reactions directly within the vasculature; in the second, the major
inflammatory stimulus occurs in local extravascular sites of infection
and circulating bacteria contribute little to inducing toxic responses.
Evidence for each alternative is found in the literature. Bacteremia and
severe sepsis are not so closely linked that the most striking cases can
be a model for the rest. Intravascular and extravascular triggers may
warrant different approaches to prevention and therapy (Munford,
2006).
E. coli is the most common gram-negative bacterium causing
neonatal sepsis, Gram-negative bacteria were more frequent than
gram-positive bacteria with a frequency of 54.6% and 45.4%
respectively (Muhammed et al., 2010). Gram-negative bacteria also
play an important role in bloodstream infections about 30% of cases in
MSc Thesis 2015
Literature Review
the ICU are caused by one or another species of klebsiella, E. coli,

Enterobacter speciesand P. aeruginosa (Smith, 2010).
3. What happens when microorganism enters

body?
It is traditional to organize host responses to infection into
separate arms or compartments, such as complement, cytokines, cellmediated immunity (monocytes, macrophages, and neutrophils), and
humoral immunity. A more current approach has been to consider 2
larger categories: innate immunity, incorporating the more rapid and
phylogenetically primitive nonspecific responses to infection, such as
surface defenses, cytokine elaboration, complement activation and
phagocytic responses (Janeway et al., 2002) and adaptive immunity,
involving more slowly developing, long-lived, and highly evolved
antigen-specific protective responses, such as antibody production and
cell-mediated immunity, that exhibit extraordinarily diverse ranges of
specificity (Padlan, 1994).
However, the components of innate and adaptive immunity
engage in a range of interactions that is remarkably diverse and
complex.
Determining the structural components of bacteria that are
responsible for initiating the septic process has been important not
only in understanding the underlying mechanisms, but also in
identifying potential therapeutic targets. These bacterial motifs, which
are recognized by the innate immune system, have been called
pathogen-associated molecular patterns (PAMPs), although it might be
more accurate to call them microorganism-associated molecular
patterns as it is by no means clear how the host distinguishes between
signals from pathogens rather than commensals (Janeway et al.,
1998).
MSc Thesis 2015
Literature Review
In order to cause disease, pathogens have to employ an array of

factors known as virulence factors that protect them from the host
innate immune system and enable them to cross mucosal barriers,
disseminate, and replicate in distant organs. Importantly, each stage
of infection involves the expression of different virulence factors
depending on the stage of infection. Some of the most important
bacterial virulence factors are toxins. These toxins include endotoxin
or lipopolysaccharide (LPS) that is present in the outer membrane of
the gram-negative bacterium and several other secreted exotoxins
and enterotoxins in other bacteria (Ramachandran, 2014).
In Gram-negative bacteria, lipopolysaccharide (LPS; known also
as endotoxin) has a dominant role. The outer membrane of Gramnegative bacteria is constructed of a lipid bilayer, separated from the
inner cytoplasmic membrane by peptidoglycan. The LPS molecule is
embedded in the outer membrane and the lipid A portion of the
molecule serves to anchor LPS in the bacterial cell wall.
Abe et al., (2010) reported that, Gram-negative (GN) bacteria
have often been implicated in the pathogenesis of severe sepsis and
septic shock, although the exact mechanism is uncertain. There is
evidence to support two different theories on how GN bacteria induce
harmful systemic responses. The intravascular stimulus hypothesis
posits that bacteria invade through a normal or damaged epithelium
and enter the bloodstream, inducing systemic inflammatory responses
(for example, increased vascular permeability, leukocyte-endothelial
adhesion, and activation of complement and clotting pathways) and
resulting in multiorgan failure.
A second theory suggests that the multiorgan dysfunction and
shock result from neuroendocrine dysregulation and mediators
released into the bloodstream from the infected tissues. Circulating
MSc Thesis 2015
Literature Review
bacteria or endotoxin are not needed as direct stimuli for intravascular

inflammation (Munford, 2006).
3. 1. Host recognition of microbial components
Carrigan et al., (2004) listed that, infections are fought in the
body
by
both
cellular
defenses,
including
monocytes,
macrophagesand neutrophils and humoral defenses incorporating

antibodies and the complement pathways. Recognition of pathogens
by extracellular CD14 and toll-like receptors 2 and 4 (TLR2 and 4) on
the membranes of monocytes and macrophages triggers the release
of cytokines to activate cellular defenses (Marshall
et al., 2003;
Haveman et al., 1999; Van Amersfoort et al., 2003).

The toll-like receptor (TLR-4) initiates a series of innate immune
mechanisms against various microorganism infections by sensing the
presence of pathogen-associated molecular patterns (PAMPs) like
lipopolysaccharide (LPS), which is the major component of the outer
surface of Gram-negative bacteria. The LPS stimulates leukocyte and
blood endothelium through the LPS recognition systems, binding with
CD14. In the blood endothelium, LPS leads to increases in the
expression of interleukin IL-6 and IL-8 (Sawa, 2008).
3. 2. Signal amplification
Following
the
initial
hostmicrobial
interaction
there
is
widespread activation of the innate immune response, the purpose of

which is to coordinate a defensive response involving both humoral
and cellular components. Mononuclear cells play a key role.
The first recognized cellular mechanism of host defense was the
accumulation of phagocytic host cells around a foreign body in starfish
observed by Metchnikoff (1905).
Polymorphonuclear leukocytes (PMNs), the most abundant
circulating phagocytes in the human host. These cells constitute a
MSc Thesis 2015
Literature Review
major line of defense against invading bacteria and fungi (Smith,

1994).
In response to invasive bacterial infection, circulating PMNs
engage in 3 major functions: (1) migration to the site of infection, (2)
recognition and ingestion of invading microorganisms, and (3) killing
and digestion of these organisms (Tosi, 2005).
Mononuclear phagocytes( including circulating monocytes ,
tissue macrophages, other phagocytic cells, and many epithelial cells)
play a key role, releasing the classic pro-inflammatory cytokines IL-1,
IL-6 and TNF-a, but in addition an array of other cytokines including IL12, IL-15 and IL-18, and a host of other small molecules. TNF-a and IL1 are the prototypic inflammatory cytokines that mediate many of the
immunopathological features of LPS-induced shock . They are released
during the first 3090 minutes after exposure to LPS and in turn
activate a second level of inflammatory cascades including cytokines,
lipid mediators and reactive oxygen species, as well as upregulating
cell adhesion molecules that result in the initiation of inflammatory cell
migration into tissues (Cohen, 2002). In concert, pro-inflammatory
stimuli (e.g. interleukins, tumour necrosis factor [TNF-) and microbial
antigens (e.g. lipopolysacharide [LPS] or lipoteichoic acid [LTA]) lead to
leukocyte recruitment at the site of infection, where they orchestrate
an
inflammatory
response.
Transmigrating
peripheral
blood
mononuclear cells, macrophages, parenchymal cells and various

intermittently or continuously released substances are included in the
host immune response. Mediators that are released have direct proand anti-inflammatory effects, as well as indirect effects by modulating
the release of secondary and further downstream substances.
Precursors, mature forms and degradation products of mediators with
and without bioactivity penetrate from the site of action into the
circulation where, theoretically, they can all be measured. As surrogate
MSc Thesis 2015
Literature Review
biomarkers they mirror the presence of, or more accurately the

inflammatory response to, an infection. Importantly, to act as
surrogates
for
bloodstream
infections,
circulating
biomarkers
absolutely depend on the presence and effects of inflammatory

triggers (Philippet al., 2007).
Lipopolysaccharide-binding protein (LBP) is an acute-phase
protein that has been suggested as a marker of infection. This protein
has
role
in
the
innate
immune
response.
It
binds
to
lipopolysaccharide and thereafter brings lipopolysaccharide to the

CD14 receptors on the monocyte-macrophage cell lineage. CD14
receptors then interact with Toll-like receptor-4, initiating cytokine
production. LBP has a longer half-life than the cytokines it induces.
These aspects make it interesting to evaluate LBP in infection and
sepsis. High levels of IL-6 have been associated with severe
inflammation and sepsis, IL-6 has a central role in inducing the
synthesis of acute-phase proteins such as CRP and LBP. IL-6 elevations
are seen earlier than the elevation of the aforementioned acute-phase
proteins. This makes IL-6 an interesting molecule to evaluate in the
early phase of infection and sepsis (Gaini et al., 2006).
The induction of inflammation by bacterial and viral infections

increases cancer risk (de Martel and Franceschi, 2009),recent work
has shown that in addition to being a tumor initiator by virtue of its
high carcinogen content.
4. Cytokines
Cytokines are largely inducible proteins or glycoproteins, which
can be secreted by any cell in the body, with the possible exception of
erythrocytes, and which can bind to and activate a range of cells. LPS
MSc Thesis 2015
Literature Review
is able to induce most cell populations to synthesize a range of

cytokines.
A heterogeneous group of soluble small polypeptide or
glycoprotein mediators, often collectively called cytokines, form part of
a complex network that helps regulate the immune and inflammatory
responses.
Today, the term cytokine encompasses interferons, the
interleukins, the chemokine family, mesenchymal growth factors and
the tumor necrosis factor family. Thirty three cytokines are called
interleukins. There are certainly over 100separate genes coding for
cytokine-like activities, many with overlapping functions and many still
unexplored(Dinarello, 2007).
4. 1. Cytokine Nomenclature
Being non-structural proteins, biological properties were and still
are the gold standards for defining a cytokine. The interleukin
nomenclature was invented to deal with the issue of multiple biological
properties of cytokines. At the time of the naming these molecules
with an interleukin number, primary amino acid sequences of the
active molecules were not known. The term IL-1 was used to define a
monocyte product and the term IL-2 was used to define a lymphocyte
product. But the nomenclature did nothing to resolve the broader
issue of multiple biological properties ascribed to a single molecule. IL1 was reported to cause fever, induce acute phase protein synthesis,
activate B-cells and act as a co-factor for T-cell proliferation in the
presence of antigens or mitogens. IL-2 was reported to expand T-cell
proliferation and also activate B-cells. IL-2 was initially termed T-cell
growth factor and expanded human T-cells in vitro (Dinarello, 2007).
4. 2. Pathological Actions of Cytokines
MSc Thesis 2015
Literature Review
Many components of bacteria have the capacity to stimulate a

range of cell populations to synthesize cytokines and thus tell the body
that it has been invaded. To date, most attention has focused on LPS
as the warning component. It was also proposed that commensal
bacteria induce the synthesis, by the host, of a range of cytokines
which either inhibit the inflammatory response to such commensal
organisms or induce a state of inflammation which is self-limiting and
recognized as healthy.
Thus, so far, the actions described for IL-1, IL-6, and TNF have all
been what one could term host protective and this is certainly one of
their key roles. However, these proinflammatory cytokines also have
activities which are damaging to the integrity of the organism, and this
can result from an accentuation of their normal protective function.
For example, if the pyrexia is too prolonged or the core
temperature rises too high, damage can ensue. As described above,
LPS can have lethal effects by activating leukocyte adhesion receptors
in the major organs, leading to vascular clogging, anoxia, and failure of
the affected organ. Indeed, administration of IL-1 or TNF to animals
can induce a septic shock-like state (Dinarello, 1994).
Many of the physiologic changes associated with gram-negative
sepsis can be reproduced by injecting experimental animals with these
cytokines in the absence of microorganisms. Depending on the doses
injected, these effects might include fever, hypotension, and either
neutrophilia or leukopenia. In the production of endotoxic shock
caused by gram-negative sepsis, IL-1 and TNF- are produced by
mononuclear phagocytes in response to activation of TLRs by bacterial
LPS (Tosi, 2005).
All evidence suggests that the administration of endotoxin or
bacteremia initiates the production and release of endogenously
produced cytokines such as TNF, ILI, IL6 and IFN in a complex cascade
MSc Thesis 2015
Literature Review
through which the lethal effects of endotoxin or septicemia are

mediated. The role of TNF as a central mediator of the lethal effects of
endotoxin has been supported by the findings that: Administration of
cytokines such as recombinant interleukin-I (IL-1) and tumor necrosis
factor (TNF), alone or in combination with each other, induces a shock
state in animals with hemodynamic and hematologic alterations that
are characteristic for septic shock in man (Hack et al., 1989).Passive
immunization against TNF protects mice from a subsequent lethal
endotoxin
challenge
and
prevents
hypotension
during
lethal
bacteremia in the baboon and circulating plasma TNF levels peak very
rapidly after endotoxin administration (Alexander, 1991). In surgical
and emergency departments and in critically ill febrile patients and
neonates, relatively small studies indeed have suggested some
predictive value for systemic microbial infection and its severity of
interleukin 6 (IL-6), the alarm cytokine, as an indicator of an activated
host defense. The factor may be superior to the commonly used
acute-phase reactant C1-reactive protein (CRP) in predicting microbial
infection (Groeneveld and Ailko, 2001).
Moreover, systemically elevated mediators of the primary
nonspecific host response to microbial infection, including cytokines
(IL-6) is of prognostic value, during established sepsis and septic
shock. Kellum et al., (2007); Kinasewitz et al., (2004) reported that IL-6
is an early indicator of inflammatory response to illness or injury. It
rises within hours of substantial injury or infection. With a half-life of 45
minutes, IL-6 can be monitored to reveal if a patient is suffering an
acute response to surgery, trauma, or infection, and if the response is
waning slowly or rapidly, which can help to predict the patients risks
and prognosis.Most studies on cytokine responses and bacteremia
have involved heterogenous patient groups with different primary
infection foci, from which bacteria have spread and reached the
bloodstream. Consequently, the local cytokine response, which
MSc Thesis 2015
Literature Review
precedes and conditions the systemic response, has not been

considered. In UTI, bacteria reach bladders and/or kidneys and elicit a
local host response that is common to the bacteremic and
nonbacteremic patient groups. Bacteremic infections may be expected
to increase the systemic cytokine responses and other IL-6mediated
host response parameters (Otto et al., 1999).
Groeneveld et al, (2001) suggested that, IL-6 can help to predict
infection at the onset of a new fever before microbiological culture
results are available and can alert the clinician that the patient has a
greater postsurgical sepsis risk before other signs and symptoms
appear. IL-6 levels can help predict if a patient with hospital-acquired
pneumonia is likely to progress to septic shock. Elevated IL-6 can
indicate late-onset neonatal sepsis 12 days before sepsis manifests
clinically. IL-6 levels are predictive of outcome (Dossow et al., 2005).
5. The role of chemokines in infectious diseases

(Bacterial infections)
Several different bacterial pathogens have been shown to be
potent inducers of chemokines of various classes and the level of
chemokine gene expression and protein production seems to be
affected by the particular bacterial species tested and the bacterial
component used as the stimulant (Wang et al., 2000). IL-8
concentration increases during different infections, such as bacteremia
(Hack et al. 1992; Hynninen et al. 1997) and meningococcal disease.
In
neutropenic
patients,
enhanced
IL-8
production
has
been
demonstrated in predicting bacteremia (Engel et al., 1999).
In
general, acute bacterial infections, such as Streptococcus pneumonia,

are characterized by the predominance of neutrophils in the
inflammatory reaction (Mizgerd et al., 1996).Several important
bacterial pathogens have been shown to stimulate chemokine
production
in
experimental
MSc Thesis 2015
systems.E.
coli,
P.
aeruginosaand
Literature Review
Staphylococcus aureus all often associated with severe acute illness

and
neutrophil
recruitment,
can
stimulate
IL-8
productionin
experimental systems (Balamayooran et al., 2010).Kinnaert and

colleagues (1996), studied the activation of human peritoneal
macrophages in response to stimulation with either heatkilled E. coli or
Staphylococci. Peritoneal macrophages are capable of producing IL-8
and demonstrated that in peritoneal macrophage monolayers,
bacterial products stimulated the production and release of a variety
of chemokines.
After 24 hours of incubation with heat-killed Staphylococcior E.
coli lipopolysaccharide (LPS), the production of various chemokines
was assessed. LPS from E. coli causedsharp increases in IL-8 levels as
measured by either enzyme-linked immunosorbent assay (ELISA) or
radioimmunoassay. In contrast, heat-killed Staphylococci caused an
increase in only IL-8 production, although not to nearly the same
degree as did the LPS from E. coli. These data suggest that peritoneal
macrophages can participate in the initiation and modulation of
intraperitoneal infections through the production of chemokines,
though varying degrees of inflammation are associated with different
bacterial pathogens. Similar differences in the ability of different
bacterial species to induce various chemokines have been shown for
the pathogenic bacteria responsible for oral disease (Balamayooran et
al., 2010). Peripheral blood mononuclear cells exposed to Candida
albicans,
orphyromonas
gingivalis,
and
Actinobacillus
actinomycetemcomitans produced high levels of both MCP-1 and IL-8,

whereas Streptococcus mutans induced mainly IL-8. Interestingly,
there was a dose response noted for the bacteria to monocyte ratio in
terms of gene expression, namely that an increased ratio resulted in
increased gene expression but not protein release, implying that
another signal is needed to increase the protein levels. In addition, a
role for chemokines in the development of the immune response in
MSc Thesis 2015
Literature Review
other organ systems has been shown. Elderly patients with lower
respiratory tract bacterial infections demonstrate elevated levels of IL8 in their sputum and this correlates with both the presence of
bacteria in the sputum and also with the presence, and amount, of
neutrophils in the sputum (Ponglertnapagorn et al., 1996). These
bacterial pathogens include P. aeruginosa.
Many potent pathological bacterial species, such as Yersinia,
Clostridium, Bacteroides and others, are able to cause potent
chemokine expression, (Schulte et al., 1996). In an interesting human
study, it has been demonstrated that the gastric levels of IL-8 correlate
with the clinical presence and degree of gastritis, which in turn has
been strongly associated with infection with Helicobacter pylori.
Human monocytes when stimulated with bacterial LPS release
IL-8 proteins. A substantial increase in IL-8 mRNA in human whole
blood cells within 30 minutes after treatment was observed .In
addition, secretion of IL-8 proteins was substantially higher 60 minutes
after stimulation with LPS. In contrast, expression of IL-6 mRNA and
protein was not increased within 60 minutes with any treatment.
Therefore, the response of IL-8 induction and production may be
earlier than that of IL-6 with treatment with bacterial antigens (Hairao
et al., 2000).
After chemotherapy, IL-8 has been found promising as an early
diagnostic marker in monitoring neutropenic patients for forthcoming
fever: IL-8 levels were elevated three days, IL-6 two days and CRP one
day before the onset of fever (Lindemann et al,. 1995). In a recent
study concerning patients with acute lymphoblastic or myeloid
leukaemia, IL-8 was found to be increased significantly more often in
patients with chemotherapy-related neutropenia and fever due to
bacteraemia than in neutropenic patients with fever of non-bacterial
origin (De Bont et al., 1999). This approach may warrant the early
MSc Thesis 2015
Literature Review
discharge of a defined group of neutropenic patients with fever who

are at low risk for septicaemia. High IL-8 has been shown to predict
organ failure in community-aquired septic shock (Takala et al., 1999b)
and a poor outcome in postoperative multi-organ failure (Hamano et
al., 1998) and nosocomial bacterial infections in neonates (Franz et al.,
1999a).
Patients with cytotoxic treatment have been shown to have
slightly increased values of CRP, presumably due to tumor degradation
(Milroy et al., 1989; Staal-van den Brekel et al., 1997; Senderowicz et
al., 1998). The determination of CRP has been used for years in the
diagnosis of infection as well as to monitor the outcome of infection
treatment in patients with haematological and pediatric malignancies
with profound neutropenia (Mackie et al., 1979; Rose et al., 1981;
Gozzard et al., 1985; Grutzmeier & von Schenck 1986; Katz et al.,
1992; Riikonen et al., 1993; Rintala 1994; Santolaya et al., 1994;
Manian 1995; Kinnunen et al., 1996; Engel et al., 1998; Lyytikainen et
al., 1998; Lehrnbecher et al., 1999). Serial measurements of serum
CRP levels have been shown to be helpful in determining the risk for
unidentified infections or poor outcome in neutropenic cancer patients,
whenever this marker has remained high for days, as well as for the
evaluation of the response to antibiotic treatment in patients with
severe bacterial infections and for identifying postoperative infections
(Santolaya et al., 1994; Manian 1995; Rintala et al., 1995; Hansson &
Lindquist 1997).
The CRP level typically exceeds 100 mg/l in patients with
bacteremia, and a stable or increasing CRP level after the first 34
days of the initiation of treatment usually indicates a treatment failure
(Hansson & Lindquist 1997). CRP can be detected in the plasma after
12 h and reaches a plateau after 2072 h.It decreases to its normal
value after 37 days.( Gabay and Kushner, 1999). Since bacterial
MSc Thesis 2015
Literature Review
infections are life-threatening for neutropenic patients, empiric

antibiotic therapy is started immediately when fever occurs, before
microbiological proof of an infection is obtained. In this setting, reliable
and readily available parameters to diagnose or rule out infection are
needed. The most common parameter is Creactive protein (CRP), an
acute-phase protein produced after proinflammatory cytokine release.
The CRP concentration rises within 24 h and is elevated in almost all
cases of microbial infection, but its reliability as a marker of infection is
hampered by very low specificity (Sudhoff et al., 2000).
Early
recognition
of
bloodstream
infections
(BSI)
and

reducing mortality in community-acquired infections. Although the
SIRS criteria were developed to improve the detection of infection,
they lack specificity for infection. In addition to clinical criteria, a
variety of laboratory markers of systemic inflammation, such as
(Interleukin- 6 (IL-6), Interleukin- 8 (IL-8), soluble IL-2 receptor (sIL-2R),
procalcitonin (PCT), and C-reactive protein (CRP) have been used to
identify patients with infection. Of these markers, increased blood
levels of IL-6 and IL-8 denote activation of monocytes/macrophages,
and both cytokines induce CRP synthesis in the liver (Aalto et al.,
2004).
IL-8 levels have been shown to increase much earlier than CRP
levels, increasing levels of IL-8 can frequently be detected even before
onset of fever (Miedema et al., 2011).
6. The complement system

Complement represents one factor of this significant host
defence system involved in the immediate protection of the host from
bacteria (Wolbink et al., 1998).
MSc Thesis 2015
Literature Review
Although its precise role in pathogenesis of sepsis is not clear,

activation of complement may contribute to the inflammatory
response via the release cytokines, via activation of clotting and of
neutrophils, and via the enhancement of vasopermeability (MullerEberhard et al., 1988).
Recognition of bacteria by complement components is likely to
induce the activation of three complement pathways, the classical, the
lectin and/or the alternative pathways. All three pathways lead to the
formation of a C3 convertase and the generation of complement
activation products triggering diverse biological activities, such as
microbial opsonization, phagocyte recruitment and inflammation,
resulting in the elimination of pathogenic microorganisms (Petersen et
al., 2001 and Reid et al., 2002).
MBL carbohydrate recognition domains (CRD) are able to bind,
in a calcium-dependent manner, to various carbohydrate structures
like
mannose,
N-acetyl-glucosamine,
l-fucose
and
N-acetyl-
mannosamine present on different microorganisms.

However, the binding of MBL to complex carbohydrates
structures is poorly understood. The bacterial capsule was reported to
reduce MBL binding of several bacteria (Van Emmerik et al., 1994),
whereas
the
structure
of
the
lipopolysaccharide
(LPS)
or
lipooligosaccharide (LOS) plays a role in the MBL attachment to

various gram-negative organisms (Devyatyarova-Johnson et al., 2000;
Jack et al., 2001a, b; Polotsky et al., 1996). Moreover, LPS isolated
from the outer cell wall of gram-negative bacteria has been shown to
interact with the complement system in different ways (Clas and Loos,
1980).
Many bacteria activate complement in-vitro and are therefore
considered to be the main activators of complement in patients with
sepsis. However,
MSc Thesis 2015
several
observations
indicate
that additional
Literature Review
mechanisms may contribute to complement activation in sepsis.

Administration of high doses of IL-2 to patients with cancer induces a
sepsis like syndrome, which is accompanied by activation of the
classical complement pathway, demonstrating the existence of a
cytokine-driven mechanism of complement activation in vivo (Thijs et
al., 1990).
7. Fever as a sign of infection:

Fever is the most frequent symptom of infection. When patients
present with fever, physicians must evaluate the risk of bacteremia, a
condition that is associated with a mortality rate as high as 30%.
However, unless there is clinical evidence of severe sepsis or septic
shock, clinical information obtained from physical examination cannot
reliably identify patients with bacteremia. Fever is a common cause of
childhood visits to emergency departments and pediatric offices, In
the majority of children, a benign infection is diagnosed after a good
history and a careful examination that reveal the site of infection .
In rare instances, especially in infants, infection is manifested
only by fever and vague or nonspecific signs and symptoms, and no
focus is evidenced after the clinical examination. Although most of
these children also have benign and self-limited illness, a few are at
risk of developing a severe bacterial infection such as bacteremia. The
missed diagnosis of which is a common source of malpractice suits.
The problem faced by the physician is to find clues that could
distinguish the few who have SBI from the vast majority of children
who have benign infection(Lacour et al., 2001).
Fever without localizing signs in young children remains a
difficult diagnostic problem, since clinical signs and symptoms are
often unreliable predictors of a serious bacterial infection which
requires rapid therapeutic intervention with intravenous antibiotic
therapy (Lacour et al., 2001).
MSc Thesis 2015
Literature Review
The leading cause of treatment-related mortality among

patients with hematological malignancies undergoing chemotherapy is
multiorgan failure due to systemic infection during neutropenia
(Buchheidt et al., 2003). Most neutropenic patients with infection
present with fever as the first symptom. However, there is a variety of
causes for febrile conditions. Apart from infection, reactions to drugs
or blood products as well as tumor-associated fever are all possible
underlying mechanisms. If the fever is not accompanied by clinical or
microbiological evidence of infection, it is classified as fever of
unknown origin (FUO) (Link et al., 2003).
Fever during chemotherapy-induced neutropenia may be the
first and only sign of bacterial infection, especially in children.
Therefore, standard care for patients with febrile neutropenia consists
of routine hospitalization and empirical treatment with intravenous
broad-spectrum antibiotics. Guidelines recommend that patients
should not be discharged until they are afebrile for at least 24 h.
However, in only 2030% of the children an actual bacterial infection is
documented during febrile neutropenia .In the majority of patients,
fever has other causes, like viral or fungal infections, the malignancy
itself, mucositis, drugs, or transfusions of blood products; all causes
that do not require antibiotics. Yet there is no generally accepted
method which can rapidly differentiate between patients with febrile
neutropenia who are at high risk for bacterial infection, thus needing
antibiotics, and in whom antibiotics can be withheld (Karin et al.,
2010).
8. Blood cultures for identifying bacteremia:

Results of blood cultures take 2448 h to become available; in
the meantime, physicians have to decide the patients needs for
antibiotic treatment.
MSc Thesis 2015
Literature Review
In doubtful cases, physicians would prefer to prescribe useless

or inappropriate antibiotics than to risk missing an indication for
antibiotic administration. However, such a policy participates in the
unnecessary increase in antibiotic prescription through so-called
empirical
or
preemptive
prescription,
which
has
well-known
consequences notably, the emergence of antibiotic-resistant bacteria

(Chirouze et al., 2002).
Hospitalization for empirical antibiotic therapy and monitoring
has been the accepted standard of care (Chanock, 1993). This has
resulted in exposing families to high costs in money, diagnostic studies
and disruption of family life. Antibiotics are generally given for a
minimum of 3 days but often longer, despite the inability to establish a
microbiological diagnosis.
Extended time in the hospital also subjects children to
acquisition of resistant organisms, a trend clearly encouraged by
liberal use of antibiotics on oncology wards.
Early
recognition
of
BSI
(Blood
stream
Infection)
and

reducing mortality in community-acquired infections (Aalto et al.,
2004).
If physicians were able to rely on an early indicator of
bacteremia, they could restrict their antibiotic prescriptions to the right
indications, they could start therapy earlier,and they could limit the
number of blood samples to be obtained for culture.
Attention has focused on identifying serum markers of the
immunologic response that may be useful for therapeutic intervention.
For example, laboratory measurement of C-reactive protein (CRP) in
serum has been investigated as a tool for the early diagnosis of a
bacterial or fungal infection and a possible marker for severity of
infection (Starke et al., 1984). Unfortunately, the increase in CRP is
MSc Thesis 2015
Literature Review
often delayed, and consequently, measurement of CRP may be of

limited value in the early recognition of life-threatening infections in
patients with neutropenia. Increases in circulating levels of the
cytokines IL-6 and IL-8 are apparent early in the course of infection,
indicating a rapid series of host response mechanisms, such as fever
and chemo-taxis of white blood cells (Waage et al., 1993). IL-6 levels
have been measured in several studies of patients with neutropenia.
The preliminary results are promising, yet definitive studies are lacking
.Fewer studies have been published addressing the usefulness of
measuring IL-8 to identify severe infection in febrile patients with
neutropenia (Engel et al., 1994).
Comparing the relative value of circulating inflammatory
variables versus clinical variables of the systemic host response in
predicting microbial infection in the blood-stream was evaluated at an
early stage when results of microbiological studies are not yet
available a large cohort of febrile patients was evaluated in whom
circulating levels of C3a and, IL-6 were measured serially. These
factors were chosen because of interrelationships in the primary
nonspecific host response to microbial infection(Groeneveld et al.,
2001).
9. Antimicrobial resistance
Bacterial pathogens have become increasingly resistant to
commonly used antibiotics and antimicrobial resistance has become a
major medical and public health problem as bacterial resistance often
result in treatment failure, which can have serious consequences,
especially in critically ill patients (Farrag et al., 2002; Tenover, 2006).
Some strains called (multi-drug resistant) showing high level
resistance to aminoglycosides, -lactam, and quinolones (Pitout et al.,
2005). The widespread use of broadspectrum antibiotics has led to
MSc Thesis 2015
Literature Review
emergence of antibiotic resistant strains of many gram-negative

organisms.
Extensive antibiotic resistance has developed in gram-negative
bacilli due to both innate resistance in some species and the fact that
they are highly adapts acquiring antibiotic resistant determinants from
each other. These conjugative plasmids are responsible for the
dissemination of resistance to other members of gram negative
bacteria in hospitals and in the community (Ananthan, 2002).lactamases are the most important mechanism of bacterial resistance
among the clinical isolates to - lactam antimicrobial agents (George
et al., 2005; Al-Jasser, 2006).
10. Ionizing Radiation

Ionizing radiation is energy that, during absorption, causes the
ejection of an orbital electron. A large amount of energy is associated
with ionization. The fundamental quantity necessary to describe the
interaction of radiation with matter is the amount of energy absorbed
per unit mass. This quantity is called absorbed dose, and the rad was
the most commonly used unit, i.e. the rad is a unit for the
measurement of the energy absorbed from ionizing radiation by the
matter through which the radiation pass. Absorbed dose is measured
in joules per kilogram; another name for 1J/kg is the gray (1Gy = 100
rad = 1 Joules/Kgm), which is now the recommended unit. A gray is
defined as the deposition of one joule per kilogram energy in tissue.
The value of absorbed dose depends on boh the photon energy of the
beam and on the type of absorbing medium. Dose can be expressed in
terms of total value, measured in rad or grays. However, it is
sometimes more convenient to express it in terms of dose rate, which
is the dose absorbed per unit time. Total dose = dose rate time
(George, 1975; Ehmann and Vance, 1991).
MSc Thesis 2015
Literature Review
Gamma rays are an electromagnetic radiation of short

wavelength, so they have relatively great penetrating power, and they
are produced intranuclearly, where they are produced artificially by
bombarding stable isotopes such as (Co59) with thermal neutrons. In
the course of bombardment the neutron is captured by (Co59) nuclei
leading to the formation of (Co60) isotope which is unstable, the excess
energy of the formed nucleus is liberated as gamma quanta.
The formation of (Co60) may be written as:
Co59 + n01 Co60 +
The most widely used gamma ray sources are:
137
Cs and
60
Co
(Maul and Ohara, 1989; Burcham and Jobes, 1995& Lowenthal and
Airey, 2001).
Moseley, (1984)reported that gamma rays used in radiation
therapy are produced by the decay of radioactive isotope.
10.
1.
Interaction
of
Ionizing
Radiation
with
Biological
Materials
Alabostro et al., (1978) reported that, among the methods
which can be used for changing the metabolic activities of living cells
is ionizing radiation. The biological effects of radiation occur as a result
of discrete changes in the nucleus and molecular structure of the
irradiated cells.
Radiation damage to cellular DNA is initiated by direct energy
deposition in the macromolecule and by attack of free radicals
produced in the surrounding milieu (Roots and Okada, 1975;
Hutchinson, 1985).
The interaction of ionizing radiation with irradiated material has
been reviewed by (Gentner and Paterson, 1994).
Radiation produces both direct and indirect injurious effects on
biological systems. The relative contribution of each mechanism
MSc Thesis 2015
Literature Review
depends on the system involved and a variety of other factors such as

the character of the radiation and the presence or absence of agents
known to protect against the effects of radiation(Anderson, 1985).
Chromosomal alterations resulting from the absorption of
ionizing radiations play a key role in the development of initial
molecular
lesions
to
their
eventual
expression
either
in
the
reproductive death of cells and subsequent tissue damage, or

permanent hereditary changes in surviving cells that may lead to
oncogenesis or to genetic damage affecting succeeding generations
(Hall et al., 1988).
Scala, (1995) suggested that, when ionizing radiation is incident
on biologic systems, energy is transferred into the system according to
fundamental physical principles. The effect on the biologic system
often is not so predictable. This effect, or endpoint, is related to
several factors, among which are total radiation dose, dose rate,
radiation quality, age of the system and several other environmental
factors. Nucleic acids found in living cells deoxyribonucleic acid (DNA)
and ribonucleic acid (RNA). They are complex macromolecules made
of purine and pyrimidine bases on a backbone of alternating sugar
and monophosphate molecules. There is considerable evidence
suggesting that nucleic acids, especially DNA, are the primary target
for cell damage from ionizing radiation. Breaks in the DNA chain
disrupt function of the molecule in several ways. In many cases breaks
in the double strand DNA can be repaired by the enzymes, DNA
polymerase, and DNA ligase. These enzymes function by detecting
breaks in the strand and correcting them. Before mitosis and during
transcription and replication when the DNA molecule exists in a single
strand, breaks are less likely to be repaired. Incorrect replication can
also occur. About chromosomes, which are the subcellular structures
of which the genetic material, DNA is a major component. Also
MSc Thesis 2015
Literature Review
contained in the chromosome structure are proteins called histones,

which bind to the DNA and are responsible for the varied morphology,
or shape, of the chromosome. This nucleoprotein complex is known as
chromatin. There is considerable evidence to support radiation
damage to the chromatin structure as the major factor in cell
reproductive death, as well as to mutations that lead to genetic
effects. In addition, radiation can cause structural aberrations with
pieces of the chromosomes break free and form aberrant shapes.
Tobias, (1985) suggested that, repair saturation invokes the
production by irradiation of a class of DNA damage that is accurately
and efficiently repaired at low radiation doses, but as the dose
increases the repair process becomes saturated and damage
fixation begins to complete. Damage fixation is the progression of the
damage to a state in which it can no longer be repaired. Misrepair
could occur either by an error-prone repair system or by repair
occurring on a template from which base sequence information has
been lost.
Hellman, (2001) reported that, radiation effects, whether direct
or indirect, are random, an important principle in the general nature of
cell killing. The major biologically important effects of radiation
therapy are those concerned with reproductive integrity. It is usually
assumed that DNA is the critical target for this radiation effect,
although it has not been proved with certainty. A cell that is damaged
by radiation and loses its reproductive integrity may divide once or
more often before all the progeny are rendered reproductively sterile.
This is an important consequence of radiation; it means that an
irradiated cell will not appear damaged until it faces at least the first
division.
When cells are irradiated, lethal damage can occur, or the
damage may be modified and not lead irrevocably to cell death. Such
MSc Thesis 2015
Literature Review
amelioration of radiation damage is called repair. Repair can be

divided into potentially lethal damage repair and sublethal damage
repair. Potentially lethal damage, under certain circumstances, leads
to cell death. If postirradiation conditions are modified to allow repair,
cells that would have died can be salvaged. In general, postirradiation
conditions that suppress cell division are the ones most favorable to
repair of potentially lethal damage (Hellman, 2001).
10. 2. Efect of Ionizing Radiation on Microorganisms
Exposure of cells to ionizing irradiation presents an additional
stress to the cells which tends to disturb their organization.
Inactivation of bacteria by radiation does not always cause the
immediate death of the organisms. Many biological functions may
persist for some hours after the bacteria have subjected to a dose that
prevents their multiplication. The ability to multiply thus becomes the
decisive
criterion
for
inactivation
in
relation
to
sterilization
(Christensen, 1970).
Gamma radiation induced three types of damage in DNA, single
strand breaks, double strand breaks and nucleotide damage which
include base damage and damage in the sugar moiety. The base
damage is a major component of damage induced by ionizing
radiation in prokaryotic as well as eukaryotic systems. Thus irradiation
produces damage which can cause mutations and disappearance of
some or all cell activities. After irradiation, bacterial cells die or lose
their ability to divide, some contain abnormal sets of chromosomes or
transmit their chromosomes abnormally, while others exhibit heritable
changes(Van der Schans, 1989)
The death of microorganisms is a consequence of the ionizing
action of high-energy radiation. Most studies indicate that lethal
damage of microbial DNA, resulting in loss of ability to reproduce, is a
MSc Thesis 2015
Literature Review
primary cause of lethality, but damage to other sensitive and critical

molecules may also have an effect (Ingram and Roberts, 1980).
Microorganisms have highly radiation resistance as compared
with multicellular organisms. The larger groups of microorganisms
were arranged according to increasing resistance, in the following
order, Gram Negative bacteria, Gram Positive bacteria (vegetative
form only), fungi and its spores, bacterial spores and viruses
(Christensen
et
al.,
1970).
Moreover,
radiation
sensitivity
of
microorganisms differs with species and even with strain.

The sensitivity of microorganisms to ionizing radiation is
described by its D10- value which is the absorbed radiation dose
required to reduce a population of microbes by one order of
magnitude (i.e. 90% of the population) (Sivinski, 1989).
The exposure of living cells to low doses of ionizing radiation
induce in response the activation of cellular protection mechanisms
against subsequent larger doses of radiation. This cellular adaptive
response may vary depending on radiation intensity and time of
exposure, and also on the testing probes used whether they were
yeast, bacteria and other organisms or cell types. Farrag et al., (2002)
suggested that, at a low dose of radiation Candida albicans cell
membranes are possibly as important target as DNA. They also
reported that, radiotherapy of cancer cervix patients had many effects
on the yeast fungal cells. The majority 75% of the isolated strains were
proteinase enzyme producers before irradiation, whereas, only 25% of
the isolates were enzyme producers after in-vitro irradiation.
Ionizing energy affects microorganisms in two ways, directly and
indirectly:
10.2. 1. The direct theory (the target theory)
MSc Thesis 2015
Literature Review
Direct action is the term used to describe the chemical events

occurring in the target molecule as a result of energy deposition by the
radiation in the target molecule, e.g. the ejection of electrons from
atoms in the DNA structure as a result of the passage of an ionizing
photon. The target theory is strictly a model, which is considered to be
applicable when the biological effect meets certain criteria in its
relation to dose. The target theory states that the effect of ionizing
radiation, in or very near to some particular molecules or structure is
responsible for the measured effect. The production of an effective
event in the target is often called a hit. Generally, the system studied
is a cell population in which the measured effect may be cell death or
inability to grow or divide. In the simplest form of the target theory,
one hit is sufficient to produce the measured effect in the associated
organism. This theory was further modified to the multi-hit target
theory and the multi-target theory. For the target theory to be
applicable,
destruction
must
be
influenced
by
concentration,
temperature or dose rate, these factors may affect the radiation

sensitivity of spores. With an exponential rate of death, then a single
hit on the sensitive site (presumably DNA) is responsible for cell death,
several hits (multi-hit theory) on DNA are necessary to bring about
inactivation (Russell, 1982). With a very small radiation doses, the
number of affected targets will be directly proportional to the amount
of radiation (Edwards, 1990).
10. 2. 2. The indirect theory (the difusion theory)
Living cells contain about 70 to 80% water and so it was
planned to give the explanation of the diffusion theory because it
plays an important role in the aqueous system, radiolysis of water is a
primary event in the initiation of biological damage (Potten, 1985). In
the diffusion theory when a cell is irradiated it is responsible to assume
that most of the irradiation energy will interact with water and that
MSc Thesis 2015
Literature Review
only a very small percentage of the incident radiation will be involved

as direct hit with a vital center as predicated by the target theory
(Scala, 1995).
10.3 Radiolysis of water
The indirect effect of radiation on target molecule is produced
by way of intermediary radiation products. The photon may interact
with water, to produce free radicals. These free radicals are relatively
short-lived; they can interact with the microbial cell causing a
detrimental effect; or, conversely, can react innocently to revert to
their former state. Molecular oxygen prolongs the life of free radicals
and prevents its reversion to their former state. By this mechanism,
oxygen may increase the effect of radiation (Hellman, 2001). Studies
usingfree radical scavengers suggest that ~ 65% of the damage
results from reactions of OH radicals produced by radiolysis. This
conclusion is sometimes questioned on the grounds of inadequate
access of diffusing water molecules to DNA. Because of the high
scavenging capacity close to DNA, the damaging OH radicals probably
originate in bound water (Hutchinson, 1985).
Because of the unique role of water in chemical and biological
systems, the irradiation of pure water is of special interest. Early
experiments demonstrated that many free radicals such as, H , O
and H2O2 etc. are produced which could play an important role
in the activity of cells (Granier and Gambini, 1993).
Induction of radiation injury has four phases (Scala, 995):
The first phase (physical phase) is the shortest, lasting about 10 16
second. During this phase, energy is deposited in the cell causing
ionization and excitation of ions and molecules. The absorption of

energy by a water molecule results in the ejection of an electron (e )
leaving a positively charged species H2O+.
MSc Thesis 2015
Literature Review
H2O ------------- H2O+ + e

e + H2O ------------- H2O
The second phase (physico- chemical phase) which lasts about 10-6
second, water ions produced in this way dissociate while forming free
radicals to yield a hydrogen ion, and hydroxyl free radical
H2O+ ------------- H+ + OH
H2O ------------- H + OH
The principle products of these two phases are hydrogen and
hydroxyl ions as free radicals. Hydrogen and Hydroxyl ions are
normally present, the free radicals are very reactive specters and may
undergo many reactions, where part of the molecular products are
framed by the recombination of the radicals:(H) + (OH)------------- H2O (recombination)
(H) + (H)-------------H2
(dimer)
(OH) + (OH)------------- H2O2 (peroxide dimer)
(OH) + (O)------------- HO2
Together with the interaction between the radicals and molecular
products:
(OH) + H2O2 ------------- HO2 + H2O
HO2 + (OH) ------------- H2O + O2
HO2 + H2O2 ------------- O2 + H2O + OH
The ratio of the reaction described above depends on the
condition of radiolysis and on the presence of other ions and
molecules dissolved in the water.
The third phase (chemical phase) lasting few seconds, the free
radicals can transfer and react with organic molecules within the cell
(proteins and DNA) which may produce changes in the secondary or
tertiary structure of proteins, as a result of disruption of hydrogen
bonds, or intermolecular cross-linking in the double strand (DNA)
molecules which may have particularly serious effects (Prasad, 1974).
(OH) + RH ------------- R + HOH (radical transfer)
MSc Thesis 2015
Literature Review
The fourth phase (biological phase) during which the chemical

changes are transformed into cellular changes. Three types of cellular
changes may be recognized, these are early cell death, prevention or
delay of cell division (mitosis) and the production of permanent
inheritable changes, which affect the whole organism. The phase of
development of radiation injury shows the longest and most variable
time scale. Some effects may develop within a few hours while others
for example cancer induction may take many years to be apparent.
The most important modifier of the biologic effect of ionizing
irradiation is molecular oxygen. If oxygen is present, it reacts with free
radicals produced by ionizing radiation and enabling the creation of
other free radical species with greater stability and lifetimes.
H + O2 ------------- HO2 (hydroperoxy free radical)
R + O2 ------------- RO2 (organic peroxy free radical)
The oxygen derived species such as hydroperoxy free radical
does not readily recombine into neutral forms. These more stable
forms have a lifetime long enough to migrate to the nucleus where
serious damage can occur. The transfer of the free radical to a biologic
molecule can be sufficiently damaging to cause bond breakage or
inactivation of key functions. In addition, the organic peroxy free
radical can transfer the radical from molecule to molecule causing
damage at each encounter. Thus a cumulative effect can occur,
greater than a single ionization or broken bond.
With no oxygen
present, this reaction cannot take place and many of the ionized target
molecules can repair or recover the ability of function normally (Hall,
1973).
11. Radiotherapy and inflammatory mediators

A long-standing paradigm in radiation biology has been that
most effects induced by ionizing radiation are the result of DNA
MSc Thesis 2015
Literature Review
damage arising from the actions of radiation in cell nuclei and

radiation transversal through the nucleus is a prerequisite to produce
genetic changes or biological response.
Recent evidence has indicated that exposure of cell populations
to ionizing radiation results in significant biological effects, such as
genetic alterations, change in gene expression and lethality, occurring
in both the irradiated and non-irradiated cells in the population (Pasi et
al., 2010).
Cytokines and chemokines produced by tumor and stromal cells
are centrally involved in tumor progression, vascularization, tumorassociated inflammatory processes, and anti-tumor immunity (Chen et
al., 2003) and (Strieter et al., 2004).
Animal experiments and clinical studies have been put forward
in which recombinant cytokines and cytokine genes were administered
as immunotherapeutics (Yang et al., 2006).Cytokine levels in serum as
well as bronchoalveolar lavage (BAL) fluid have been exploited as
markers for lung tumor progression, prognosis, and therapy associated
tissue damage. Taken together, cytokines are versatile and promising
agents in lung cancer research, diagnosis, and therapy. The
inflammatory process is a classical pathophysiological response to
ionizing radiation.Inflammatory lesions have been observed locally in a
number of tissues, such as skin, intestine or lung and in a wide range
of doses (between 5 and 40 Gy). Recent studies have shown early
changes such as an increase in the number of adherent and emigrated
leukocytes. An early and persistent cytokine production following local
exposure of rat intestine or lung has been observed and related to the
late appearance of fibrosis.In addition to these local effects, different
investigators have shown the release of proinflammatory cytokines in
peripheral blood. In humans, a total body irradiation of 10 Gy results in
a rapid increase in the serum levels of TNF-
MSc Thesis 2015
and IL-6, with a
Literature Review
maximum reached 4 h after radiation exposure. These increases were

substantial but transient, and returned to baseline levels 24 h after
irradiation (Van der Meeren et al., 1997).
Both IL-6 and IL-8 are involved in the inflammatory response.
They have been detected in the serum of patients with inflammatory
disorders and can be produced in response tovarious damaging stimuli
such as bacterial or viral infections or burns.
12.The ROC curve analysis

12. 1. Description
The ROC curve is a fundamental tool for diagnostic test
evaluation.
In a ROC curve the true positive rate (Sensitivity) is plotted in
function of the false positive rate (100-Specificity) for different cut-off
points of a parameter. Each point on the ROC curve represents a
sensitivity/specificity pair corresponding to a particular decision
threshold. The area under the ROC curve (AUC) is a measure of how
well a parameter can distinguish between two diagnostic groups
(diseased/normal).
12. 2.Theory summary
The diagnostic performance of a test, or the accuray of a test to
discriminate diseased cases from normal cases is evaluated using
Receiver Operating Characteristic (ROC) curve analysis (Metz, 1978)
and (Zweig & Campbell, 1993). ROC curves can also be used to
compare the diagnostic performance of two or more laboratory or
diagnostic tests (Griner et al., 1981).
When you consider the results of a particular test in two
populations, one population with a disease, the other population
without the disease, you will rarely observe a perfect separation
MSc Thesis 2015
Literature Review
between the two groups. Indeed, the distribution of the test results will
overlap, as shown in the following figure.
Figure 1: Distribution of overlapped test results.

For every possible cut-off point or criterion value you select to
discriminate between the two populations, there will be some cases
with the disease correctly classified as positive (TP = True Positive
fraction), but some cases with the disease will be classified negative
(FN = False Negative fraction). On the other hand, some cases without
the disease will be correctly classified as negative (TN = True Negative
fraction), but some cases without the disease will be classified as
positive (FP = False Positive fraction).
Sensitivity: probability that a test result will be positive when the

disease
is
present
(truepositive
rate,
expressedas
percentage).= a / (a+b)
Specificity: probability that a test result will be negative when
the disease is not present (true negative rate, expressed as a
percentage).= d / (c+d)
Positive likelihood ratio: ratio between the probability of a
positive test result given the presence of the disease and the
probability of a positive test result given the absence of the
disease, i.e.= True positive rate / False positive rate = Sensitivity
/ (1-Specificity)
MSc Thesis 2015
Literature Review
Negative likelihood ratio: ratio between the probability of a

negative test result given the presence of the disease and the
probability of a negative test result given the absence of the
disease, i.e. = False negative rate / True negative rate = (1Sensitivity) / Specificity
Positive predictive value: probability that the disease is present
when the test is positive (expressed as a percentage). =
a / (a+c)
Negative predictive value: probability that the disease is not
present when the test is negative (expressed as a
percentage). = d / (b+d)
12.3. The ROC curve

In a Receiver Operating Characteristic (ROC) curve the true
positive rate (Sensitivity) is plotted in function of the false positive
rate (100-Specificity) for different cut-off points. Each point on the
ROC curve represents a sensitivity/specificity pair corresponding to
a particular decision threshold. A test with perfect discrimination (no
overlap in the two distributions) has a ROC curve that passes
through the upper left corner (100% sensitivity, 100% specificity).
Therefore the closer the ROC curve is to the upper left corner, the
higher the overall accuracy of the test (Zweig & Campbell, 1993) as
in Figure 2
MSc Thesis 2015
Literature Review
Figure 2: The ROC curve distribution between Sensitivity and

Specificity.
MSc Thesis 2015
Materials and Methods

Materials
1. Microorganisms: A number of different Gram negative isolates
were used in the present study. These isolates were recovered from
blood specimens and included E. coli, K.pneumoniae, A. baumannii
and Pseudomonas species.
2. Blood specimens: A total number of 124 blood specimens were
collected from 70 cancerous feverish patients and 54 noncancerous feverish patients. The specimens were withdrawn by
medical staff and obtained from Abbassia hospital for fever,
Radiotherapy unit at National Centre for Radiation Research and
Technology and Naser institute hospital. The blood specimens
were collected at onset of fever and before starting antibiotic
therapy.
3. Media: The readymade media used in the present study included
MacConkeys Medium Agar no.3, Nutrient agar and Nutrient
broth. The threeculture media were the products of Oxoid,
England. The media were prepared according to manufacturer
instructions and sterilized by autoclaving at 121oC for 20 minutes.
Also, blood agar, tween agar medium and gelatin agar medium
were prepared in the laboratory and used in the present study.
4. Kits:
4.1. Human
IL-6
ELISA
kit: The commercially available
RayBio(Raybiotech,
Inc,
USA),(Enzyme-Linked
Immunosorbent Assay) kit was used for quantitative

measurement of human IL-6 in serum. The measurement
was
performed
according
recommendations.
MSc Thesis 2015
48
to
manufacturers
4.2. Human IL-8 ELISA kit: Interleukin-8 (IL-8) serum levels

were measured quantitatively using Diaclone,SAS, France,
Human IL-8 ELISA (Enzyme-Linked Immunosorbent Assay)
kit.
The
measurement
was
performed
according
to
manufacturers recommendations.
4.3. API 20E BioMrieux ,France: API 20 E strips were used
for identification of microbial isolates collected from blood
specimens.
4.4. Rat IL-6 ELISA Kit: KOMA BIOTECH,Korea,Rat IL-6 ELISA
(Enzyme-Linked Immunosorbent Assay) kit was used for
quantitative measurement of rat IL-6 levels from serum
samples.
5. Blood culture bottles: Blood specimens were collected in blood
culture bottles (BACTEC Peds Plus/F).
6. Tubes used for plasma and sera separation: A vacutainer
blood collection tubes (Becton, Dickinson Company, USA)
containing either anticoagulant (K2EDTA) or thrombin which is a
rapid clot activator were used for plasma and sera separation,
respectively.
7. Difu-plate: Complement C3 concentration in patients sera was
measured using Radial immunodiffusion plate (DIFFU-PLATE),
Biocientifica S.A., Argentina.
8. Antibiotic discs: these discs containing fixed amounts of
different antibiotics and used for antimicrobial susceptibility
testing of collected isolates. The discs were the products of Oxoid,
England and their characteristics are shown in table 1.
Table 1: Characters of antibiotic discs used for antimicrobial
susceptibility testing of collected bacterial isolates
Disc
content(
g)
MSc Thesis 2015
Antimicrobial
Agent(s)/disc
49
Disc code
No.
30
20
30
30
120
5
5
10
10
10
30
30
25
30
Amoxycillin/ Clavulanic acid

2:1
Ampicillin/ Salbactam
Cefepime
Amikacin
Gentamicin
Levofloxacin
Ofloxacin
Imipenem
Tobramycin
Tazobactam
Chloramphenicol
Cefotaxime
Trimethoprim
Ceftazidime
AMC
SAM
FEP
AK
CN
LEV
OFX
IPM
TOB
TZP
C
CTX
SXT
CAZ
2
3
4
5
6
7
8
9
10
11
12
13
14
9. Laboratory animals: Forty-eight adult Wistar rats (weight range

from 80 to 135 g) were used for assay of serum IL-6 in rats.
10. Devices: Besides the basic equipment used in
the
microbiological laboratories, the following devices were used :

10.1. BD BACTEC 9050 Blood Culture System, Italy: the
BACTEC fluorescent series instrument
located at Naser
Institute hospital was used to determine if the blood culture

bottles were positive for microbial growth or not.
MSc Thesis 2015
50
Figure 3: BD BACTEC 9050 Blood Culture System

10.2. Beckman/Coulter
CBC-5
Semi
Automated,
Germany: was used for white blood cells count, the

instrument is located at Naser Institute hospital.
10.3. Turbiquick
reader, Accucare, India:
was used for
quantitative turbidimetric test for the measurement of CRP

in human sera samples andlocated at Naser Institute
hospital.
10.4. Cesium 137(137Cs) Gamma cell 40, Atomic Energy of
Canada Limited: is located at National center For Radiation
Research and Technology (Nasr City, Cairo, Egypt) and was
used for irradiation of some identified gram-negative
bacterial isolates and tested laboratory animals.
MSc Thesis 2015
51
Methods
11. Collection and manipulation of specimens: were carried
out according to Mackie and McCartney practical medical
microbiology (Collee et al., 1996), Manual of Clinical Microbiology
(Murry et al., 1999) andMicrobiology: A Laboratory Manual
(Cappuccino et al., 2004).
Blood samples were collectedby vein puncture for bacterial
culture in blood culture bottles (BACTEC Peds Plus/F). About 3
ml of blood from each patient were inoculated intoblood culture
bottle containing enriched Soybean-Casein Digest broth.The
blood culture bottle was used with the BACTEC fluorescent series
instrument for recovery of aerobic microorganisms (mainly
bacteria and yeast). In case of positive growth after incubation for
2-7 days at 37oC, CO2is produced when the organisms metabolize
the substrates present in the vials and the fluorescence of the
vials sensors increased and monitored by the BACTEC fluorescent
series instrument.
12. Plasma and serum samples collection: Blood samples
collected
in
tubes
containing
K2EDTA
were
centrifuged
immediately to sediment red cells and plasma samples were

gathered to be used for total leukocytic count analysis. While,
Blood samples collectedin thrombin containing tubeswere also
centrifuged immediately to sediment red cells and clotted
components, sera were gathered and frozen at -200C until use.
13. Isolation of pathogenic bacteria: After collection of blood
culture
bottles
positive
for
microbial
growth,
subsequent
cultivation was done on blood agar plates, then MacConkeys

agar plates for isolation of gram negative strains only.
Media were prepared according to manufacturers directions and
sterilized by autoclaving for 20 minutes at 121C.
MSc Thesis 2015
52
13.1. Blood agar medium preparation: Readymade Nutrient

agar was sterilized by autoclaving at 121oC for 20 minutes.
Sterile nutrient agar (900 ml) was then cooled to 45-47 oC
then about 100 ml of sterile whole animal (horse or sheep)
or human blood was added aseptically.
13.2. Tween
agar
medium
preparation:
Tween-agar
medium (Thaler et al., 1998), was prepared as follows:

molten
sterile
nutrient
agar
medium
(45-50oC)
was
supplemented with autoclaved solutions of CaCl2.H2Oand

Tween 80 at final concentrations of 0.01% w/v and 1%v/v
respectively (20 minutes at 121oC), after that the medium
was distributed in plates each containing 15 ml and after
solidification, wells were made in the prepared Tween- agar
platesusig sterile cork porer. For testing lipase activity,
100lof cell free culture supernatantto each well were added
and plates were then incubated at 37oC for 24 hours.
13.3
Gelatin
agar
medium
preparation:
Gelatin-agar
medium (Collee et al., 1996), was prepared as follows:

nutrient agar medium was supplemented with 5 grams of
KH2PO4, 1.5 grams of K2HPO4 and 4 g/L of gelatin and
autoclaved at 121 oC.After that the medium was distributed
in plates each containing 15 ml and after solidification, wells
were made in the prepared Gelatin- agar platesusig sterile
cork porer. For testing protease activity, 100lof cell free
culture supernatantto each well were added and plates were
then incubated at 37oC for 24 hours.
14. Identification
of
isolated
organisms
from
blood
specimens:Identification was done according to the following:
MSc Thesis 2015
53
a- Macroscopical examination which was carried out to observe

the colonial morphology, including shape, size, surface
structure, edge, color and opacity.
b- Microsopical examination which was done by Gram stain to
notice the shape, size, arrangement and staining reaction.
c- Biochemical examination which was carried out according to
the following references: Practical Medical Microbiology
(Collee et al., 1996),Manual of clinical Microbiology (Murray
et al.,
1999), Laboratory. Experiments in Microbiology
(Johnson and Case, 2003) and Microbiology: A Laboratory

Manual (Cappuccino et al., 2004).
d- Identification using API 20E BioMrieux
: The API 20 E strip
consists of 20 microtubes containing dehydrated substrates.

These microtubes were inoculated with suspension of the test
organism that was reconstituted in the supplied suspension
medium. During incubation, metabolism produces color
changes that were revealed either spontaneously or after the
addition of supplied reagents. The reactions results were read
and recorded. The identification was obtained according to
the analytical profile index. Oxidase test was not included in
the API 20E strips and it was done separately. The procedures
for both API 20E strip tests and oxidase test are briefly
described below.
Inoculums preparation: A single well isolated colony (18-24
hours old) from an isolation plate of the test organism was removed
using a loop, then it was emulsified carefully into the ampoule of API
suspension medium (5 ml) to achieve a homogeneous bacterial
suspension which was used immediately after preparation.
Inoculation of the strip: By using a single pipette, both tubes
and cupules of the tests CIT, VP and GEL and tubes of other tests were
MSc Thesis 2015
54
filled with the bacterial suspension. The ADH, LDC, ODC, H2S and URE
tests were carried out anaerobically by being overlayed with mineral
oil. Finally, the incubation box containing inoculated strip was closed
and incubated at 36C 2C for 18-24 hours.
Oxidase test: The oxidase test was carried out according to the
reagent manufacturer's recommendations.
Interpretation of the results: Identification was obtained with
the numerical profile. On the result sheet, the tests were separated
into groups of 3 and a value 1, 2 or 4 was indicated for each.By adding
together the values corresponding to positive reactions within each
group, a 7-digit profile number was obtained for the 20 tests of the API
20 E strip. The oxidase reaction constituted the 21st test.
In some cases, the 7-digit profile was not discriminatory enough
and the following supplementary tests were carried out: reduction of
nitrates to nitrites (NO2) and N2 gas production, motility test,oxidation
and fermentation of glucose.
15. Total leukocytic count assay: White blood cells count was
carried out using Beckman/Coulter CBC-5Semi Automated using
200l of separated blood sample.
16. Assay of serum C-Reactive Protein (CRP): It was carried out
by using CRP-turbilatex that was supplied by Turbiquick
reader.
A quantitative turbidimetric test for the measurement of CRP in

human serum was carried outaccording to manufacturers
recommendations.
17. Assay of serum Inteleukin-6 (IL-6)
The principle of the assay depends on employing an antibody
specific for human IL-6 coated on wells of 96-well plate. Standards
and samples were pipetted into the wells and IL-6 present in a sample
was bound to the wells by the immobilized antibody. The wells were
MSc Thesis 2015
55
then
washed
and
biotinylated
anti-human
IL-6
antibody
was
added.After washing away unbound biotinylated antibody, horseradish

peroxidase (HRP) conjugated streptavidin was pipetted to the wells.
The wells were again washed, then 3,3,5,5-tetramethylbenzidine
(TMB) substrate solution was added to the wells and color developed
was proportional to the amount of IL-6 bound. The stop solution
changed the color from blue to yellow and the intensity of the color
was measured spectrophotometrically at wavelength 450 nm.
17.1 Reagents preparation
All reagents and samples were brought to room temperature (1825C) before use.The supplied stock solution of Assay diluent B was
diluted 5-fold with deionized or distilled water to produce 1x assay
diluents B.The vial of IL-6 standard was spinand then 500l of assay
diluent A was added to the vial to prepare a 12,000 pg/ml
concentration.The powder was dissolved thoroughly by a gentle mix,
then40 l IL-6 standard from the vial were added into a tube with 440
l assay diluent A to prepare a 1000 pg/ml standard solution.
About 400 l of the above prepared standard solution were
pipetted into each tube for serial dilutions from 1000pg/ml to zero
pg/ml. Each tube was mixed thoroughly before the next transfer.
Assay dileunt A served as the zero pg/ml. Twenty ml of washing buffer
concentrate (20x) were diluted into deionized or distilled water to yield
400 ml of 1x washing buffer. The detection Antibody vial was spin
before use, then 100 l of 1x Assay diluent B was added into the vial
to prepare a detection antibody concentrate, then the vial was
pipetted up and down to mix gently. The detection antibody
concentrate should be diluted 80-fold with 1x assay diluent B. The
HRP-Streptavidin concentrate vial was spin and pipetted up and down
MSc Thesis 2015
56
to mix gently before use. HRP-Streptavidin concentrate should be

diluted 600-fold with 1x assay diluent B.
17.2Assay procedures
All reagents were prepared as mentioned before and according to
manufacturers manual. All previously prepared concentrations of the
standard and samples were applied in duplicates. One hundred l
aliquots of different dilutions of the standard and samples were added
into appropriate wells, then covered well and incubated over night at
4C with gentle shaking.The solution was discarded then the wells
were washed 4 times with the 1x washing solution and complete
removal of liquid at each step was applied. The plate was inverted and
blotted against clean paper towels. One hundred l of 1x prepared
biotinylated antibody were added to each well, the plate was
incubated for 1 hour at room temperature with gentle shaking,then
the solution was discarded and the plate was washed with the 1x
washing solution as mentioned above. One hundred l of prepared
streptavidin solution were added to each well, followed by incubation
for 45 minutes at room temperature with gentle shaking, then the
solution was discarded and the plate was washed with the 1x washing
solution as mentioned above.One hundred (100) l of TMB one-step
substrate reagent was added to each well, then the plate was
incubated for 30 minutes at room temperature in the dark with gentle
shaking.Finally, 50 l of stop solution were added to each well followed
by reading at 450 nm immediately.
18. Assay of serum Interleukin-8(IL-8)
The kit contain a mococlonal antibody specific for IL-8 coated
onto wells of microtiter plate. Samples, including standards of known
IL-8 concentrations, control specimens and unknowns were pipetted
into these wells.
MSc Thesis 2015
During the first incubation, the IL-8 antigen and
57
biotinylated polyclonal antibody specific for IL-8 were simultaneously

incubated. After washing, the enzyme (streptavidin-peroxidase) was
added. After incubation and washing to remove all unbound enzyme,a
substrate solution which was acting on the bound enzyme was added
to induce a colored reaction product. The intensity of this colored
product measured at wavelength 450nm was directly proportional to
the concentration of IL-8 present in the samples.
All
reagents
were
brought
to
room
temperature
before
use.Sufficient microwell strips were removed from the aluminum

pouch immediately prior to use.Each sample, standard, and control
was tested in duplicate. Washing buffer concentrate (200x) was
diluted 200 fold with distilled water to give a 1x working solution, then
mixed gently to avoid foaming, followed by transfer to a clean wash
bottle and stored at 25C. The standard diluents buffer (10x
concentrate) was prepared by adding225ml of distilled water before
use.Standard vials were reconstituted with the volume of standard
diluent shown on the vial immediately prior to use. This reconstitution
gave a stock solution of 2000pg/ml of IL-8,then the reconstituted
standard was mixed gently by repeated aspirations/ejections. Serial
dilutions of the standard were made directly in the assay plate to
provide the concentration range from 2000 to 62.5pg/ml. The
biotinylated anti-IL-8 was prepared immediately before use. It was
diluted with the biotinylated antibody diluent in an appropriate clean
glass vial using volumes appropriate to the number of required wells.
It was recommended to centrifuge the vial of streptavidin-HRPfor a few
seconds in a microcentrifuge to collect all the volume at the bottom.
Five l vial of streptavidin-HRP was diluted with 0.5ml of HRP diluent
immediately before use.
MSc Thesis 2015
58
Assay proceduresit was recommend that each vial was mixed

without foaming prior to use. All reagents were prepared as described
previously.One hundred l of each sample, standard and control were
added in duplicate to appropriate number of wells, then 50l aliquots
of diluted biotinylated anti-IL-8 were added to all wells, then the wells
were covered with a plastic plate cover and incubated at room
temperature for one hour.The cover was removed and the plate was
washed three times with 1x washing solution by aspirating the liquid
from each well, then 0.3ml of 1x washing solution was dispensed,
followed by aspiration of the contents of each well.
One hundred l aliquots of streptavidin-HRP solution were added
into all wells, then the plate was covered and incubated at room
temperature (18 to 25oC) for 30 minutes, then the solution was
discarded and the plate was washed with the 1x washing solution as
mentioned above.One hundred l aliquots of ready to-use TMB
substrate solution were added into all wells, then the plate was
incubated in the dark for 12-15 minutes at room temperature.Finally,
100l aliquots of H2SO4 stop reagent were added into all wells followed
by reading at 450 nm immediately.
19. Assay of serum complement C3
Complement C3 concentration was measured in patients sera
using
(Radial
immunodiffusion
plate)
DIFFU-PLATE,Biocientifica
S.A.,Argentina.
Principle of assay:the procedure depends on the occurrence
of an immunoprecipitation in agarose between an antigen and its
homologous antibody. It was performed by incorporating one of the
two immune reactants (usually antibody) uniformly throughout a
layer of agarose gel and then introducing the other reactant (usually
MSc Thesis 2015
59
antigen) into wells dully punched in the gel.Antigen diffused radially

out of the well into the surrounding gel-antibody mixture and a
visible ring of precipitation isformed when the antigen and antibody
reacted.While the precipitate was expanding,the ratio between ring
diameter and antigen concentration logarithm was approximately
linear.This relationship was useful when rapid estimations are
required.At reaction completion, the ratio between ring square
diameter and antigen concentration showed a linear ratio.Routine
determinationusing a reference table and end point measurements
were applied.
20. Antimicrobial susceptibility of some pathogenic bacterial
isolates
20.1Antimicrobial agents used
Some of the isolated pathogenic bacteria from blood samples
were subjected to antimicrobial susceptibility tests using 14 different
antimicrobial discs, all of them were used for pathogenic gramnegative bacilli. The interpretive standards breakpoints for the
selected antibiotics is shown in table 2
MSc Thesis 2015
60
Table 2: The interpretive standards breakpoints in mm for the

selected
antibiotics
that were applied for
antimicrobial
susceptibility testing
Susceptible
(S)
<20
<15
<18
<17
<10
<17
<16
<18
<15
<21
<18
<23
<16
<18
Intermediate
(MR)
14-17
12-14
15-17
15-16
7-9
14-16
13-15
14-17
13-14
18-20
13-17
15-22
11-15
15-17
Resistant
(R)
13
11
14
14
6
13
12
13
12
17
12
14
10
14
Disc Code
AMC
SAM
FEP
AK
CN
LEV
OFX
IPM
TOB
TZP
C
CTX
SXT
CAZ
All of these selected (14) antimicrobial discs together with zones

readings chart were supplied from Oxoid co. (England).
All of these commercially prepared discs were 6 mm in diameter
and the content of antibiotic discs were not varied more than the limits
set by the National Committee for Clinical Laboratory Standards
(NCCLS 2011).
20.2
Media used: Nutrient broth was used for inoculum

preparation of the test organism while, nutrient agar was
used for antimicrobial susceptibility testing.
20.3Disc difusion test: One of the most useful and widely

used laboratory test for antimicrobial susceptibility is the
antimicrobial disc-agar diffusion procedure, so called disc
diffusion method. This test (Kirby-Bauer disc susceptibility
test) was done according to (Bauer et al., 1966; Matsen and
Barry,
1974;
MSc Thesis 2015
Jorgensen
61
et
al.,
1999)
and
(National
Committee for Clinical Laboratory standards (NCCLS), 2011).

First pure 4-5 colonies were picked up from overnight growth
onto nutrient agar plate with a loop to be inoculated into a
sterile nutrient broth (about 3-4 ml/tube).The tubes were
incubated at 371oC for 2-4 hours until its turbidity were
equivalent to barium sulphate standard ( 0.5 McFerland). A
swab was immersed in the prepared suspension andthe
excess fluid was removed from the swab by rotating it
several times against the inside wall of the suspension tube,
before its withdrawal. The swab was then streakedon the
surface of the nutrient agar medium in 3 planes; horizontal,
vertical and diagonal according to Kirby-Bauer method
(Bauer et al., 1966). After the inoculum had been dried (3-5
min), the discs were placed individually on the agar surface
with a sterile forceps and were gently pressed down onto the
agar surface to provide uniform contact. Some of the
antimicrobial agents in disks diffuse almost immediately;
therefore once a disc contact to the agar surface, it should
not be moved.The plates were incubated aerobically for 1824 hours at 371oC. The diameter of the inhibition zone
around each disc was measured by a ruler from the back of
the plate.The results were compared with standardized chart
supplied
by
Oxoid-England,to
determine
whether
the
organism is susceptible(S) or of intermediate /moderate

resistance(MR) or resistant(R) to the antimicrobial agents
used.
21. Efect of in-vitro gamma irradiation on some selected
isolates
21. 1. Irradiation source
Cesium 137(137Cs) Gamma cell 40,Atomic Energy of Canada
Limited,Commercial products located at National center For Radiation
MSc Thesis 2015
62
Research
and
Technology(Nasrcity,Cairo,
Egypt)
was
used
for
irradiation of some identified gram-negative bacterial isolates.A low

radiation dose equal to 24.4 Gy which is biologically equivalent to invivo fractionated multiple therapeutic dose used in the protocol of
cancer treatment of some cancer patients was used for irradiation.
This source was operated at a dose rate of 0.88 rad/sec at the time of
experiments.
21. 2. Preparation of bacterial suspension for in-vitro
gamma irradiation
Each tested bacterial isolate was inoculated in 20ml nutrient
broth and incubated at 37oC for 24 hours. The cultures obtained were
divided under sterile conditions into 2ml aliquots in 2 groups. One of
them was exposed to gamma radiation dose level of 24.4 Gy and the
second
group
was
remained
as
control.Both
groups
of
microorganisms were subjected to antimicrobial susceptibility testing.

Antimicrobial susceptibility testing of some selected
isolates before and after in-vitro gamma irradiation: Both
control and gamma irradiated test groups of the selected isolates
earlier mentioned in 21.2 were subjected to antimicrobial susceptibility
testing by disc diffusion technique (mentioned before in 20.3) against
the 14 antimicrobial agents previously stated in table (2)
22. Detection of Lipase and Protease enzymes production
22.1. Detection of lipase production: was performed using
Tween-agar plates. Lipase was detected by formation of
opaque halo zones surrounding the wells due to precipitation
of water insoluble calcium salts of fatty acids liberated from
hydrolyzed Tween 80.
22.2. Detection of protease production: was performed
using Gelatin-agar plates. At the end of incubation period,
MSc Thesis 2015
63
gelatin hydrolysis was examined on the plates by flooding

them with 15% (w/v) HgCl2 in 20% conc. HCl. Protease
activity was detected by demonstrating clear zones around
the wells (indication of gelatin hydrolysis), non-hydrolyzed
gelatin was indicated by an opacity in the medium.
23.
Efect of gamma irradiation on IL-6 serum levels in rats
with induced fever of bacterial and non-bacterial origin: Fortyeight adult Wistar rats (weight range from 80 to 135 g) were used for
this experiment. All animals had free access to food and water
throughout the study. Before conducting the experiment, rats were
acclimatized to the animal house conditions (12:12 h light/dark cycle)
for
week.
Standard
pellet
feed
and
drinking
water
were
provided.Rectal temperatures (TR) were recorded by inserting a

lubricated digital thermometer (external diameter: 3 mm, 0.1C
precision) 2.8 cm into the rectum of rats. Animals presenting initial
rectal temperature between 34 and 37C were selected for the tests.
Stock
cultures
of
Klebsiella
pneumoniaandPseudomonas
aeruginosa were subcultured on MacConkey agar plates and

incubated at 37C for 18 h. Bacterial suspensions were prepared by
gently removing the growth from MacConkey agar plates and
suspending them in nonpyrogenic normal saline. The bacterial
density was adjusted to an optical density of 0.6 at wavelength of
600nm (approximates 1 X 109 organisms per ml).
The rats were divided randomly into 8 groups, each consisting of
6 animals and treated as described in table 3
Table 3: Rat groups used for testing the efect of gamma
radiation on serum level of IL-6.
Group
MSc Thesis 2015
Description
64
Group (1): control group
The animals received 1 ml intraperitoneal

(i.p) injection of 0.9% saline solution
Group
(2):
control group
Radiation
The animals were given 1ml intraperitoneal

(i.p) injection of 0.9% saline solution then
they were exposed to low (half of the group)
and high (the other group half) gamma
radiation doses of 2 and 7.5 grays,
respectively.
Group (3): Fever of nonbacterial origin (Yeast fever

group)
Fever was induced by subcutaneous

administration of 1 g/kg dried baker's yeast
(Matroh Co.) as a 20% suspension in 0.9%
saline solution. The rectal temperature of
each rat was measured at zero time (before
injection of the yeast ) and at 20 h following
yeast injection
Group (4): Fever of nonbacterial origin (Yeast fever

group)
with
gamma
radiation exposure
Fever was induced as described in group (3)

and
after
elevation
of
rats
rectal
temperature (18 h post injection of the
yeast),the animals were exposed to gamma
radiation of 2 (half of the group) and 7.5
grays (the other group half)
Group
(5):
Fever
of
bacterial
origin
(Pseudomonas aeruginosa)
Each rat received 1 ml intraperitoneal (i.p)

injection
of
Pseudomonas
aeruginosa
suspension. The rectal temperature of each
rat was measured at time, 0 and 4 h
following bacterial suspension injection.
Group
(6):
Fever
of
bacterial
origin
(Pseudomonas
aeruginosa)with
gamma
radiation exposure

and
after
elevation
of
rats
rectal
bacterial suspension),the animals were
exposed to gamma radiation of 2 (half of the
group) and 7.5 grays (the other group half)
Group (7):Fever of bacterial

origin
(Klebsiella
pneumoniae)
Each rat received 1ml intraeritponeal (i.p)

injection
of
Klebsiella
pneumoniae
suspension. The rectal temperature of each
rat was measured at time, 0 and 4 h
MSc Thesis 2015
65
following bacterial suspension injection

Group
(8):
Fever
of
bacterial origin (Klebsiella
pneumoniae) with gamma
radiation exposure

and
after
elevation
of
rats
rectal
bacterial suspension),the animals were
exposed to gamma radiation of 2 (half of the
group) and 7.5 grays (the other group half)
24. Assay of IL-6 serum concentration in rats

It was performed using KOMA BIOTECH,Korea,Rat IL-6 ELISA
(Enzyme-Linked Immunosorbent Assay) kit.
24.1Reagents preparation
All reagents and samples were brought to room temperature (1825C) before use.
IL-6 standard vials were reconstituted in 55l of sterile water to
prepare a concentration of 0.2 g/ml standard and this solution was
diluted with assay diluent at 1:2 serial dilutions as follows:20 l of IL-6
standard solution from the vial were added to 0.48 ml of assay diluent
to prepare 8000 pg/ml standard solution. Then, 0.25ml of assay
diluent were pipetted into each tube for preparation of two fold serial
dilutions of the standard from 8000pg/ml to 125pg/ml.
Each tube was mixed thoroughly before the next transfer and the
assay diluent served as the zero standard (0 pg/ml).Washing solution
(PBS) was prepared by resolving one pouch of PBS powder to sterile
water to make one liter then 1ml Tween 20 was added to that solution
and mixed well.
The detection antibody vial (biotinylated antigen-affinity purified
anti-Rat IL-6) was reconstituted in 275 l of sterile water to prepare a
detection antibody concentration of 8g/ml.Then, it was diluted with
assay diluents (1:20) to a concentration of 0.4g/ml.
MSc Thesis 2015
66
The HRP-Streptavidin conjugate vial(Color development enzyme)

was diluted (1:20) in the assay diluent.Finally, color development
solution was prepared by mixing 1 volume of color development
reagent A(TMB solution) and 2 volumes of color development reagent
B (H2O2 solution) (1:2) prior use.
24.2 Assay procedures
All reagents were prepared as mentioned above and according
to manufacturers manual.The reagents and samples were brought to
room temperature (18 - 25C) before use.Two hundred l of washing
solution were added to each well, the wells were aspirated to remove
liquid and the plate was washed three times using 300l of washing
solution per well.After the last wash the plate was inverted to remove
residual solution and blotted on paper towel.All previously prepared
concentrations of the standards and samples were applied in
duplicates. One hundred l of different dilutions of the standard and
samples were added into appropriate wells, covered and incubated for
2 hours at room temperature.Then,wells were aspirated to remove
liquidand washed 4 times as mentioned before. One hundred l of the
prepared diluted detection antibody were added to each well, then
the plate was covered with the plate sealer and incubated for 2 hours
at room temperature, then the wells were aspirated to remove liquid
then the plate was washed 4 times as mentioned before.
One hundred l of prepared color development enzyme were
added to each well, followed by incubation for 30 minutes at room
temperature, then liquid aspiration and washing 4 times were carried
out as mentioned above.
One hundred (100) l of color development solution were added
to each well, then the plate was incubated for 23-33 minutes at room
temperature for a proper color development.Finally, 100 l of stop
MSc Thesis 2015
67
solution were added to each well followed by reading at 450 nm

immediately.
25. Statistical methods:
The significance of differences between groups of the in-vivo
experiments was calculated with t-test and statistical significance
was designated at the 95% confidence level (two-sided P).The data
was analyzed using SPSS version 11.0 for determination of cut-off
levels,sensitivity
and
specificity
Characteristic (ROC) curves.
MSc Thesis 2015
68
through
Receiver
Operating
Results
Results
1.Recovery, Collection and identification of
pathogenicbacteria from blood cultures
In this study 124 blood samples were obtained from in-patients
of Abbassia fever hospital, Radiotherapy unit at National Centre for
Radiation Research and Technology and Naser institute hospital, all
located in Cairo, Egypt. The blood samples were collected from (a)
70cancer feverish patients and (b) 54 non-cancerous feverish patients
with relative percentages of 56% (a) and 44% (b).
The results of cases developed positive blood culture and those
with no detected growth with their relative percentages are shown in
table 4.
Table 4: Number and frequency of positive and negative cases
for microbial growth of blood culture samples from cancer and
non-cancer patients
Source of blood
culture samples
Total
cases
(%)
No. of
positive
cases
No. of
negati
ve
cases
% of
positive
cases
% of
negative
cases
Cancer patients
70
(56%)
34
36
49%
51 %
Non cancer
patients
54
(44%)
29
25
54%
46%
Total
124
63
61
The isolated pathogenic bacteria were identified on the bases of

their morphological characteristics (Gram stained films and colonies
grown on MacConkeys agar plates) and API System (20E) BioMeriex
France and the results are shown in tables 5 and 7
MSc Thesis 2015
Results
2.Age, sex, white blood cells count and serum

CRP levels of cancer and non-cancer
patients
The data of age, sex, count of white blood cells, serum CRP
values as well as identified microbial species (in case of positive blood
cultures) in cancer and non-cancer patients are presented in tables 58.
Table 5: Age, sex, white blood cells count, serum CRP

measurements and bacterial species isolated from cancer
patients with bacterial infection (positive blood cultures)
Patients
no.
Age
(years)
Sex
Count
of
WBCs
(*103/cmm)
CRP
(mg/l)#
Isolated microorganism
26
male
0.65
15.7
E. coli
male
1.26
<3.3
K. pneumoniae
60
male
0.06
332
K. pneumoniae
male
1.88
68.1
P. fluorescence
male
0.3
373
K. pneumoniae
35
female
7.12
88.2
E. coli
60
male
0.21
260
K. pneumoniae
10
female
0.5
157
E. coli
10
female
0.16
71.4
E. coli
MSc Thesis 2015
Results
10
32
Female
0.38
205
E. coli
11
35
female
0.17
200
E. coli
12
female
0.2
60.1
K. pneumoniae
13
14
male
0.56
163
K.pneumoniae
14
27
male
0.67
200
E. coli
15
19
female
0.29
48.5
E. coli
16
33
male
0.18
20
E. coli
17
24
male
0.15
99.8
E. coli
18
male
1.28
<3.3
E. coli
19
male
2.42
28.6
E. coli
20
27
female
0.02
70.4
K. pneumoniae
21
male
0.89
<3.3
E. coli
22
10
female
0.56
90.8
E. coli
23
15
male
0.05
9.8
K. pneumoniae
24
37
female
0.05
373
A. baumannii
25
female
0.2
88.2
P. aeruginosa
26
42
female
0.14
282
A.baumannii
27
40
female
0.99
106
P. putida
MSc Thesis 2015
Results
28
19
female
0.56
122
P. putida
29
male
0.45
<3.3
P. fluorescence
30
male
0.87
30.1
P. aeruginosa
31
Male
0.45
120
P. putida
32
male
0.28
109
P. putida
33
male
0.32
202
P. aeruginosa
34
male
0.05
80
A. baumannii
WBCs count normal range (4-11)*103/cmm, CRP normal serum level less than or equal to 3.3 mg/l
MSc Thesis 2015
Results
Table 6: Age, sex, white blood cells count and serum CRP
measurements of cancer patients without bacterial infection
(negative blood cultures)
Patients no.
Age (years)
Sex
Count of
WBCs
(*103/cmm)#
CRP
(mg/l)#
35
35
Female
1.02
157
36
40
Male
0.34
206
37
53
Female
0.79
29
38
12
Male
0.08
<3.3
39
Male
1.39
16.4
40
23
Female
3.5
44.7
41
54
Male
0.89
82.9
42
15
Male
1.7
91.7
43
71
Male
0.02
102
44
57
Male
7.5
22
45
14
Male
2.8
150
46
32
Female
2.07
113
47
45
Male
1.98
119
48
23
Female
1.8
<3.3
MSc Thesis 2015
Results
49
67
Male
1.2
57
50
34
Male
1.7
28
51
23
Male
2.89
82.8
52
11
Female
0.08
57
53
Female
0.04
110
54
neonate
Male
7.08
67
55
12
Female
1.78
89
56
55
Female
1.05
32.8
57
29
Male
2.56
64.7
58
Female
5.09
<3.3
59
neonate
Female
8.09
140
60
66
Male
0.09
230
61
35
Female
10.5
<3.3
62
54
Male
25.3
22.6
63
32
Male
2.1
40.7
64
50
Female
5.4
<3.3
65
64
Female
4.3
59
MSc Thesis 2015
Results
66
59
Female
2.7
34.9
67
72
Male
2.3
23.8
68
65
Female
2.5
54.2
69
38
Female
2.2
45.8
70
49
Female
7.3
66.5
MSc Thesis 2015
Results
Table 7: Age, sex, white blood cells count serum CRP

measurements and bacterial species isolated from noncancer patients with bacterial infection (positive blood
cultures).
Patients
no.
Age
(years)
Sex
WBCs
(*103/cmm)
CRP
(mg/l)#
Type of microorganism
71
26
male
0.21
166
K. pneumoniae
72
Neonate
male
9.74
220
K. pneumoniae
73
24
male
0.01
89.5
E. coli
74
22
male
0.46
20.4
E. coli
75
72
female
162
E. coli
76
67
male
24.5
369
K. pneumoniae
77
Female
0.3
297
K. pneumoniae
78
20
female
10.3
26.5
K. pneumoniae
79
Neonate
female
8.9
186
E. coli
80
26
male
0.21
150
K. pneumoniae
81
28
female
15.29
20.9
K. pneumoniae
82
64
male
0.42
55.6
K. pneumoniae
83
24
male
0.01
129
E. coli
84
male
0.02
146
E. coli
MSc Thesis 2015
Results
85
67
male
11.8
117
E. coli
86
male
0.01
153
K. pneumoniae
87
14
male
0.09
119
E. coli
88
16
male
0.32
32.3
E. coli
89
55
female
11.4
136
K. pneumoniae
90
57
male
14.56
126.4
E. coli
91
Neonate
Male
170
K. pneumoniae
92
21
male
5.1
144
E. coli
93
16
female
5.6
102
K. pneumoniae
94
28
female
14.8
20.4
P. fluorescence
95
33
female
0.17
186
A. baumannii
96
27
female
9.7
47.1
P. fluorescence
97
24
male
11.5
47.4
P. putida
98
Neonate
female
9.5
15.4
A. baumannii
99
Neonate
male
13.5
121
P. putida
WBCs count normal range (4-110*103/cmm, CRP normal serum level less than or equal to 3.3 mg/l
MSc Thesis 2015
Results
Table 8: Age, sex, white blood cells count and serum CRP
measurements of non-cancer patients without bacterial
infection (negative blood cultures)
Patients no.
Age (years)
Sex
WBCs
(*103/cmm)#
CRP (mg/l)#
100
19
Male
0.14
119
101
23
Female
5.6
90.8
102
61
Male
16.5
40.2
103
30
Male
3.7
64.7
104
57
Male
6.5
85.9
105
18
Male
11
23.8
106
23
Male
5.7
45.9
107
22
Male
4.3
<3.3
108
52
Male
5.9
76.8
109
44
Male
6.4
110
110
29
Male
11.5
67.9
111
30
Male
14.6
33.9
112
31
Female
4.9
21.8
MSc Thesis 2015
Results
113
30
Male
15.6
89.9
114
27
Male
3.5
76
115
Male
11.4
41.9
116
31
Male
4.7
<3.3
117
56
Male
9.8
109
118
53
Female
14.9
35.7
119
22
Female
6.7
98.9
120
54
Male
8.7
54
121
63
Female
14.2
21.8
122
22
Male
7.6
18.9
123
56
Male
10.2
<3.3
124
32
Male
5.3
76.9
From table 5, it was found that all patients with different ages
were leukopenic except the case of patient number 6 whose total
WBCs count was 7.12*103/cmm (within normal range).
The numbers of different bacterial species and their relative
percentages isolated from cancer patients (34 cases) and noncancer patients (29 cases) are represented in table 9
MSc Thesis 2015
Results
Table 9: Numbers and percentages of diferent bacterial

species recovered from cancer and non-cancer patients
Bacterial isolate
Number and percentage (%) of recovered

isolates from
Cancer patients
(n=34)
Non-cancer
patients(n=29)
14 (41)
11 (38)
8 (23)
12 (41)
2 (6)
2 (7)
4 (12)
2 (7)
Pseudomonas
aeruginosa
3 (9)
Acinetobacter
baumnanii
3 (9)
2 (7)
Escherichia coli
Klebsiella
pneumoniae
Pseudomonas
fluorescence
Pseudomonas putida
The results of C-reactive protein (CRP) measurements revealed

that, in cancer patients with positive blood cultures (gram negative
bacteremia) (n=34) CRP was ranged from <3.3 mg/l (normal levels)
to 390 mg/l and in cancer patients with negative blood cultures (n=36)
it was ranged from <3.3 mg/l to 206 mg/l. In case of non-cancer
patients, CRP values ranged from 15.4 mg/l to 369 mg/l in positive
blood cultures cases (n=29) and from 40.2 mg/l to 119 mg/l in
negative blood culture cases (n=25).
The data of CRP for for two groups, with positive blood cultures
representing cancer and non-cancer patientseach of 9 cases as well as
5 cases with negative blood cultures from each group (cancer and
non-cancer patients) (Table 10) were statistically analyzed by T- test.
The results revealed that,there was non-significant difference in serum
MSc Thesis 2015
Results
CRP concentrations betweencancer patients with positive blood

cultures (number of samples evaluated were n=9) and non-cancer
patients with positive blood cultures (number of samples evaluated
were n=9) P- value = 0.985. Also, there was non-significant
difference in CRP serum concentration in cancer patients with positive
blood cultures (n=9) and patients with negative blood cultures (n=5)
P- value = 0.138. The same probability was also demonstrated in
non-cancer patients with positive blood cultures (n=9) and negative
blood cultures (n=5) where there was non-significant difference Pvalue = 0.052.
MSc Thesis 2015
Results
Table 10: Levels of CRP of some cases of cancer and noncancer patients with positive and negative blood cultures
selected for statistical analysis
Cancer patients
With positive
blood culture
Non-cancer patients
With negative
blood culture
With positive
blood culture
With negative
blood culture
Patien
ts no.
CRP
conc.
(mg/l)
Patient
s no.
CRP
conc.
(mg/l
)
Patien
ts no.
CRP
conc.
(mg/l
)
Patien
ts no.
CRP
conc.
(mg/l)
15.7
35
157
71
166
100
119
<3.3
36
206
72
220
101
90.8
332
37
29
73
89.5
102
40.2
68.1
38
<3.3
74
26.5
103
64.7
260
39
16.4
75
162
104
85.9
157
76
369
11
390
77
297
24
373
94
20.4
25
88.2
95
186
3.Interleukin-6 serum levels (sIL-6) in cancer

and non-cancer patients
MSc Thesis 2015
Results
For determination of sIL-6 concentration in different test samples,

standard
calibration
curve
was
constructed
between
IL-6
concentration and optical density (OD450nm) obtained under the assay

conditions (Figure 4). This was done using the standard supplied with
the kit and measuring the color developed with different prepared
dilutions
of
the
standard
under
the
assay
conditions.
The
concentrations of IL-6 in sample preparations were obtained by

measuring the OD at 450 nm of the color developed by reacting the
samples with kit reagents. The obtained optical densities (OD) were
then converted into IL-6 concentrations from the constructed standard
calibration curve using sigma plot software program version 10.
The results revealed that, IL-6 serum concentrations were above
normal values in gram negative bacteremia cases (positive blood
cultures) in cancer and non-cancer patients with high prevalence in
cancer patients. In most cases the values exceeded the normal values
recorded with healthy control samples (Table 11).While, in cancer and
non-cancer
patients
with
negative
blood
cultures,
serum
concentrations recorded dramatically lower values.

The data of sIL-6were statistically analyzed by T-test and the
results revealed that,IL-6serum concentrations for positive blood
cultures were significantly higher in cancer patients (n=9) than noncancer patients (n=9) P- value =0.0166.
Also, there was significant difference in IL-6 serum concentration
in cancer patients with positive blood cultures (n=9) and negative
blood cultures (n=5) P-value = 0.0001.
Furthermore, in non-cancer patients with positive blood cultures
(n=9) and negative blood cultures (non-microbial fever) (n=5) there
was significant difference P- value = 0.0288.
MSc Thesis 2015
Results
Figure 4: Standard calibration curve of Human IL-6 serum

concentration as determined by ELISA technique
MSc Thesis 2015
Results
Table 11: Levels of serum IL-6 in some cases of cancer and

non-cancer patients with positive and negative blood cultures
Cancer patients
With positive
blood culture
Non-cancer patients
With negative
blood culture
With positive
blood culture
With negative
blood culture
Patie
nts
no.
sIL-6
conc.
(pg/ml)
Patient
s no.
sIL-6
conc.
(pg/ml)
Patien
ts no.
sIL-6
conc.
(pg/ml)
Patient
s no.
sIL-6
conc.
(pg/ml)
2487
35
228.89
71
2267.96
100
74.6
2126.7
36
398.6
72
290.6
101
120.9
3083.4
37
59.17
73
491
102
28.3
2466.2
38
300.77
74
167.2
103
100.2
830.55
39
157
75
1617.6
104
49
2311.9
76
290.6
11
2743.9
77
2373.6
24
1200.97
94
880.3
25
1293.5
95
923.2
4.Interleukin-8 serum levels (sIL-8) in cancer

The concentrations of IL-8 in sample preparations were obtained
by measuring the OD at 450 nm of the color developed by reacting the
samples with kit reagents. The obtained optical densities (OD) were
MSc Thesis 2015
Results
then converted into IL-8 concentrations using sigma plot software

program version 10.
As shown in table 12, IL-8 serum concentrations were above
normal values in cancer and non-cancer patients with positive blood
cultures with high prevalence in cancer patients. In most cases the
values exceeded the normal values recorded with healthy control
samples (Table 12). While, in cancer and non-cancer patients with
negative
blood
cultures
IL-8
serum
concentrations
recorded
dramatically lower values.

The data of sIL-8 were statistically analyzed by T-test and the
results revealed that, there was non-significant difference in IL-8
serum concentration in cancer patients with positive blood cultures
(n=6) and non-cancer patients with positive blood cultures (n=5) Pvalue =0.85. Also there was significant difference in IL-8 serum
concentration in cancer patients with positive blood cultures (n=6) and
cancer patients with negative blood cultures (non-microbial fever)
(n=5) P- value = 0.0059.Meanwhile, in non-cancer patients with
positive blood cultures (n=5) and non- cancer patients with negative
blood cultures (non-microbial fever) (n=5) there was significant
difference P- value = 0.0059.
Table 12: Levels of serum IL-8 in some cases of cancer and
non-cancer patients with positive and negative blood cultures
Cancer patients
Non-cancer patients
With positive
blood culture
With negative
blood culture
Patien
ts no.
Patie
nts
sIL-8
conc.
MSc Thesis 2015
sIL-8
conc.
With positive
blood culture
Patien
t
sIL-8
conc.
With negative
blood culture
Patient
s no.
sIL-8
conc.
Results
(pg/
ml)
no.
(pg/ml
)
s no.
(pg/ml
)
(pg/ml
)
343.4
35
80.5
71
705.5
100
40.5
159.3
36
105
72
782.7
101
66.7
592.8
37
120.3
73
529.9
102
44
691
38
93.8
94
429.2
103
60.8
24
879.5
39
110
95
611.8
104
40
25
846.6
5.Relationship between CRP, sIL-6 and sIL-8 in

some selected cancer and non-cancer
patients with positive and negative blood
cultures
The data of CRP, sIL-6 and sIL-8 in some selected cancer and noncancer patients with positive blood cultures (microbial fever) and
negative blood cultures (non-microbial fever) are summarized in table
13. Relationship between CRP and IL-8, IL-6 and IL-8, CRP and IL-6 in
some selected cancer and non-cancer patients were statistically
analyzed by T-test and the results are shown in figures 5-7. While, the
mean values of these tested serum markers (CRP, sIL-6 and sIL-8) of
the selected cancer and non-cancer patients are presented in table 14.
MSc Thesis 2015
Results
Table 13: Summarization of Serum levels of CRP, IL-6 and IL-8 in cancer and non-cancer patients
with positive and negative blood cultures:
Cancer patients
Positive
blood cultures
Non-cancer patients
Negative
blood cultures
Positive
blood cultures
Pati
ent
s
no.
CRP
(mg/
l)
IL6(pg/
ml)
IL8(pg/
ml)
Patien
ts no
CRP(
mg/l)
IL6(pg/
ml)
IL8(pg/
ml)
Patien
ts no
CRP(m
g/L)
15.7
2487
343.4
35
157
228.89
80.5
71
166
<3.3
2126.7
159.3
36
206
398.6
105
72
220
332
3083.4
592.8
37
29
59.17
120.3
73
68.1
2466.2
691
38
<3.3
300.77
93.8
24
373
879.5
39
16.4
157
110
25
88.2
1200.9
7
1293.5
MSc Thesis 2015
846.6
IL6(pg/
ml)
Negative
blood cultures
IL8(pg/ml
)
Patie
nts
no
CRP
(mg/
l)
IL6(pg/
ml)
IL8(pg/
ml)
705.5
100
119
74.6
40.5
290.6
782.7
101
90.8
120.9
66.7
89.5
491
529.9
102
40.2
28.3
44
94
20.4
880.3
429.2
103
64.7
100.2
60.8
95
186
923.2
611.8
104
85.9
49
40
2267.9
6
Results
Table 14: Mean values of tested serum markers (CRP, IL6 and IL-8) in some selected cancer and non-cancer patients
Cancer patients
CRP
(mg/l)
IL-6
(pg/ml)
IL-8
(pg/ml)
Ratio of Positive
blood culture
/Negative blood
culture measured
parameter
IL-8
(pg/ml)
Negative blood
culture
IL-6
(pg/ml)
Positive blood culture
CRP
(mg/l)
Infection status
Non-cancer patients
120.2
2048.9
585.43
123.3
1033.6
611.8
69.3
228.89
101.9
56.5
74.6
50.4
1.73
8.95
5.7
2.2
13.86
12.14
The results in table 14 revealed that in the positive blood culture,

the mean values of different tested serum markers were higher than
those with negative blood culture with the most relative increase for
IL-6.
MSc Thesis 2015
Results
(a) cancer patients

400
200
0
200
400
600
800
1000
IL-8 serum concentration (pg/ml)
(b) non-cancer patients
CRP serum concentration (mg/l)
Il-8 serum concentration (pg/ml)
Figure 5: Relationship between IL-8 and CRP serum

concentrations in (a) cancer patients and (b) non-cancer
patients both with gram negative bacteremia. The numbers of
patients in a and b were 6 and 5, respectively.
MSc Thesis 2015
Results
(a) cancer patients

4000
2000
0
Figure 6: Relationship between IL-8 and Il-6 serum

patients in a and b were 6 and 5, respectively
MSc Thesis 2015
Results
(a) cancer patients

500
0
40 0
00
Figure 7: Relationship between IL-6 and CRP serum

patients in a and b were 15 and 9, respectively
6.Relationship between white blood cells

count and each of CRP, sIL-6 and sIL-8 in
some selected cancer and non-cancer
patients with positive blood cultures
The data of WBCs count, CRP, sIL-6 and sIL-8 in some selected
cancer and non-cancer patients with positive blood cultures (microbial
fever) presented in table 15 were statistically analyzed by T-test for
determining the relationship between the four tested parameters
[WBCs and sIL-6, WBCs and sIL-8, WBCs and CRP] and the results are
shown in figures 8-10.
MSc Thesis 2015
Results
MSc Thesis 2015
Results
Table 15: Relationship between WBCs count and serum Levels CRP, IL-6 and IL-8 measured at the
same time in some selected cancer and non-cancer patients with positive blood cultures
Cancer patients with positive blood culture
WBCs
Patient
CRP(mg/
sIL-6
sIL(*103/cm
s no.
l)
(pg/ml)
8(pg/ml)
m)
1
0.65
15.7
2487
343.4
2
1.26
<3.3
2126.7
159.3
3
0.06
332
3083.4
592.8
4
1.88
68.1
2466.2
691
5
0.3
373
830.55
6
7.12
88.2
2311.9
7
0.21
260
228.9
8
0.5
157
367.7
9
0.16
71.4
398.6
10
0.38
205
151.75
11
0.17
200
2743.9
24
0.05
373
1200.97
879.5
25
0.2
88.2
1293.5
846.6
26
0.14
282
799.8
27
0.99
106
136.32
MSc Thesis 2015
Non-cancer patients with positive blood culture

Patien
WBCs
CRP(
sIL-6
sILts
(*103/cmm)
mg/l)
(pg/ml)
8(pg/ml)
no.
71
0.21
166
2267.96
705.5
72
9.74
220
290.6
782.7
73
0.01
89.5
491
529.9
74
0.46
26.5
167.2
75
3
162
1617.6
76
24.5
369
290.6
77
0.3
297
2373.6
94
14.8
20.4
880.3
429.2
95
0.17
186
923.2
611.8
Results
(a) cancer patients

10
5
White blood cellscount (*103 )
0
2000
04000
IL- 6 serum concentration (pg/ml)

30
20
10
White blood cells count (*103)
0
4000
0
Figure 8: Relationship between WBCs count and serum Levels

of IL-6 both simultaneously measured in (a) cancer patients
and (b) non-cancer patients both with positive blood cultures.
The numbers of patients in a and b were 15 and 9,
respectively
MSc Thesis 2015
Results
10 0
00
White blood cellscount (*103 )
0 1 2
(a) cancer patients
20
IL-8 serum concentration(pg/ml)
Figure 9: Relationship between WBCs count and serum Levels

of IL-8 both measured simultaneously in (a) cancer patients
and (b) non-cancer patients both with positive blood cultures.
The numbers of patients in a and b were 6 and 5 respectively
MSc Thesis 2015
Results
(a) cancer patients

8
6
4
White bloodcells count (*103) 2

0
200
0 400
CRP(mg/l)

30
20
10
CRP(mg/l)
Figure 10: Relationship between WBCs count and serum

Levels of CRP both measured simultaneously in (a) cancer
patients and (b) non-cancer patients both with positive blood
cultures. The numbers of patients in a and b were 15 and 9
respectively
For the tested serum markers (CRP, IL-6 and IL-8) it was observed
that there was a pronounced difference between the values recorded
for IL-8 among positive and negative blood cultures in tested cases.
On the other hand, for the other two tested serum markers (CRP and
IL-6) the differences in recorded values among positive and negative
blood cultures tested cases overlapped in some cases. In such
conditions, a statistical test called Diagnostic validity test which was
illustrated by Receiver Operating Characteristic curve (ROC) was
MSc Thesis 2015
Results
applied to determine the best cut off value and as well as PPV, NPV,
specificity%, sensitivity% and efficacy.
7.Statistical analysis of CRP and IL-6 serum

markers of some selected cancer and noncancer patients with positive and negative
blood cultures by, Diagnostic validity test,
Receiver Operating Characteristic Curves
(ROC)
7.1. ROC curve analysis showing the diagnostic performance
of CRP and IL6 for discriminating patients with positive
culture from those with negative culture cancer patients:
The test was performed for 9 cases with positive blood cultures
and 5 cases with negative blood cultures of cancer patients and the
results are represented in tables 16 and 17 and figure 11.
of CRP and IL6 for discriminating patients with positive
culture from those with negative culture among non- cancer
patients:
The test was performed for 9 cases with positive blood cultures
and 5 cases with negative blood cultures among cancer patients and
the results are represented in tables 18 and 19 and figure 12.
of CRP and IL6 and their combinations for discriminating
patients with positive culture from those with negative
culture for both cancer and non- cancer patients:
The test was performed between 24 cases with positive blood
cultures (15 from cancer patients and 9 from non- cancer patients)
and 10 cases with negative blood cultures (5 from cancer patients and
MSc Thesis 2015
Results
5 from non- cancer patients) for both IL-6 and CRP. To improve the
results a multi- ROC was performed as shown in tables 20, 21 and 22
and figure 13.
Table 16: Diagnostic validity test results for serum CRP to
discriminate positive and negative blood cultures in cancer
patients.
CRP
3
15.7
16.4
29
68.1
88.2
157
206
260
332
373
TP
8
7
7
7
6
5
4
4
3
2
1
FN
1
2
2
2
3
4
5
5
6
7
8
FP
4
4
3
2
2
2
1
0
0
0
0
TN
1
1
2
3
3
3
4
5
5
5
5
Sp
20.0
20.0
40.0
60.0
60.0
60.0
80.0
100.0
100.0
100.0
100.0
Sn
88.9
77.8
77.8
77.8
66.7
55.6
44.4
44.4
33.3
22.2
11.1
P50.0
33.3
50.0
60.0
50.0
42.9
44.4
50.0
45.5
41.7
38.5
P+
66.7
63.6
70.0
77.8
75.0
71.4
80.0
100.0
100.0
100.0
100.0
Eff.
64.3
57.1
64.3
71.4
64.3
57.1
57.1
64.3
57.1
50.0
42.9
Shaded raw represents cutoff value of 29 mg/l with 60% specificity, 77.8% sensitivity,
60% NPV, 77.8% PPV and 71.4 Efficacy.
MSc Thesis 2015
Results
Table 17: Diagnostic validity test results for serum IL-6 to

discriminate positive and negative blood cultures in cancer patients
IL6
TP
FN
FP
TN
Sp
Sn
P-
P+
Eff.
59.17
20.0
100.0
100.0
69.2
71.4
157
40.0
100.0
100.0
75.0
78.6
228.89
60.0
100.0
100.0
81.8
85.7
300.77
80.0
100.0
100.0
90.0
92.9
398.6
100.0
100.0
100.0
100.0
100.
0
830.55
100.0
88.9
83.3
100.0
92.9
1200.9
7
100.0
77.8
71.4
100.0
85.7
1293.5
100.0
66.7
62.5
100.0
78.6
2126.7
100.0
55.6
55.6
100.0
71.4
2311.9
100.0
44.4
50.0
100.0
64.3
2466.2
100.0
33.3
45.5
100.0
57.1
2487
100.0
22.2
41.7
100.0
50.0
2743.9
100.0
11.1
38.5
100.0
42.9
Shaded raw represents cutoff value of 398.6 pg/ml with 100% specificity, sensitivity,
NPV, PPV and Efficacy.
AUC
IL6
MSc Thesis 2015
CRP
1.000
0.844
Results
Figure 11: ROC curve analysis showing the diagnostic

performance of CRP and IL6 for discriminating patients with
positive culture from those with negative culture among
cancer patients

discriminate positive and negative blood cultures in noncancer patients
CRP
20.4
26.5
40.2
64.7
85.9
89.5
90.8
119
162
166
186
220
297
Shaded
TP
8
7
7
7
7
6
6
6
5
4
3
2
1
FN
1
2
2
2
2
3
3
3
4
5
6
7
8
FP
5
5
4
3
2
2
1
0
0
0
0
0
0
TN
0
0
1
2
3
3
4
5
5
5
5
5
5
Sp
0.0
0.0
20.0
40.0
60.0
60.0
80.0
100.0
100.0
100.0
100.0
100.0
100.0
Sn
88.9
77.8
77.8
77.8
77.8
66.7
66.7
66.7
55.6
44.4
33.3
22.2
11.1
P0.0
0.0
33.3
50.0
60.0
50.0
57.1
62.5
55.6
50.0
45.5
41.7
38.5
P+
61.5
58.3
63.6
70.0
77.8
75.0
85.7
100.0
100.0
100.0
100.0
100.0
100.0
Eff.
57.1
50.0
57.1
64.3
71.4
64.3
71.4
78.6
71.4
64.3
57.1
50.0
42.9
raw represents cutoff value of 119 mg/l with 100% specificity, 66.7%
sensitivity, 62.5% NPV, 100% PPV and 78.6 Efficacy.
MSc Thesis 2015
Results

discriminate positive and negative blood cultures in noncancer patients
IL6
28.3
49
74.6
100.2
120.9
167.2
290.6
491
880.3
923.2
1617.6
2267.96
TP
9
9
9
9
9
8
6
5
4
3
2
1
FN
0
0
0
0
0
1
3
4
5
6
7
8
FP
4
3
2
1
0
0
0
0
0
0
0
0
TN
1
2
3
4
5
5
5
5
5
5
5
5
Sp
20.0
40.0
60.0
80.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
Sn
100.0
100.0
100.0
100.0
100.0
88.9
66.7
55.6
44.4
33.3
22.2
11.1
Shaded raw represents cutoff value of 120.9 pg/ml
P100.0
100.0
100.0
100.0
100.0
83.3
62.5
55.6
50.0
45.5
41.7
38.5
P+
69.2
75.0
81.8
90.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
Eff.
71.4
78.6
85.7
92.9
100.0
92.9
78.6
71.4
64.3
57.1
50.0
42.9
with 100% specificity, 100%
sensitivity, 100% NPV, 100% PPV and 100 Efficacy.
AUC
CRP
IL6
0.875
1.000

performance of CRP and IL6 for discriminating patients with
MSc Thesis 2015
Results
positive culture from those with negative culture among noncancer patients.
MSc Thesis 2015
Results

discriminate positive and negative blood cultures among
cancer and non- cancer patients
CRP
15.7
16.4
20.4
26.5
29
40.2
64.7
67.1
68.1
71.4
85.9
88.2
89.5
90.8
106
119
157
162
166
186
200
205
206
220
260
282
297
332
369
373
Shaded
TP
23
23
22
21
21
21
21
20
19
18
18
17
16
16
15
15
14
13
12
11
10
9
9
8
7
6
5
4
3
2
FN
1
1
2
3
3
3
3
4
5
6
6
7
8
8
9
9
10
11
12
13
14
15
15
16
17
18
19
20
21
22
FP
10
9
9
9
8
7
6
6
6
6
5
5
5
4
4
3
2
2
2
2
2
2
1
1
1
1
1
1
1
1
TN
0
1
1
1
2
3
4
4
4
4
5
5
5
6
6
7
8
8
8
8
8
8
9
9
9
9
9
9
9
9
rawrepresents cutoff value of
Sp
0.0
10.0
10.0
10.0
20.0
30.0
40.0
40.0
40.0
40.0
50.0
50.0
50.0
60.0
60.0
70.0
80.0
80.0
80.0
80.0
80.0
80.0
90.0
90.0
90.0
90.0
90.0
90.0
90.0
90.0
Sn
95.8
95.8
91.7
87.5
87.5
87.5
87.5
83.3
79.2
75.0
75.0
70.8
66.7
66.7
62.5
62.5
58.3
54.2
50.0
45.8
41.7
37.5
37.5
33.3
29.2
25.0
20.8
16.7
12.5
8.3
85.9 mg/l with
sensitivity, 45.5% NPV, 78.3% PPV and 67.6 Efficacy.
MSc Thesis 2015
P0.0
50.0
33.3
25.0
40.0
50.0
57.1
50.0
44.4
40.0
45.5
41.7
38.5
42.9
40.0
43.8
44.4
42.1
40.0
38.1
36.4
34.8
37.5
36.0
34.6
33.3
32.1
31.0
30.0
29.0
P+
69.7
71.9
71.0
70.0
72.4
75.0
77.8
76.9
76.0
75.0
78.3
77.3
76.2
80.0
78.9
83.3
87.5
86.7
85.7
84.6
83.3
81.8
90.0
88.9
87.5
85.7
83.3
80.0
75.0
66.7
Eff.
67.6
70.6
67.6
64.7
67.6
70.6
73.5
70.6
67.6
64.7
67.6
64.7
61.8
64.7
61.8
64.7
64.7
61.8
58.8
55.9
52.9
50.0
52.9
50.0
47.1
44.1
41.2
38.2
35.3
32.4
50% specificity, 75%
Results

discriminate positive and negative blood cultures among cancer and
non- cancer patients
IL6
28.3
49
59.17
74.6
100.2
TP
24
24
24
24
24
FN
0
0
0
0
0
FP
9
8
7
6
5
TN
1
2
3
4
5
Sp
10.0
20.0
30.0
40.0
50.0
P+
72.7
75.0
77.4
80.0
82.8
Eff.
73.5
76.5
79.4
82.4
85.3
120.9
24
60.0
85.7
88.2
23
85.7
85.2
85.3
22
91.7
75.0
84.6
82.4
22
21
2
3
70.0
70.0
91.7
87.5
77.8
70.0
88.0
87.5
85.3
82.4
21
80.0
87.5
72.7
91.3
85.3
2
2
8
8
80.0
80.0
83.3
75.0
66.7
57.1
90.9
90.0
82.4
76.5
90.0
75.0
60.0
94.7
79.4
17
70.8
56.3
16
10
66.7
55.6
491
15
10
62.5
52.6
799.8
14
10
10
58.3
50.0
830.5
5
13
11
10
54.2
47.6
880.3
12
12
10
50.0
45.5
923.2
11
13
10
45.8
43.5
10
14
10
41.7
41.7
15
10
37.5
40.0
16
10
33.3
38.5
17
10
29.2
37.0
18
10
25.0
35.7
19
10
20.8
34.5
20
10
16.7
33.3
94.4
100.
0
100.
0
100.
0
100.
0
100.
0
100.
0
100.
0
100.
0
100.
0
100.
0
100.
0
100.
0
100.
76.5
398.6
90.0
100.
0
100.
0
100.
0
100.
0
100.
0
100.
0
100.
0
100.
0
100.
0
100.
0
100.
0
100.
0
100.
136.3
2
151.7
5
157
167.2
228.8
9
228.9
290.6
300.7
7
367.7
1200.
97
1293.
5
1617.
6
2126.
7
2267.
96
2311.
9
2373.
Sn
100.0
100.0
100.0
100.0
100.0
100.
0
P100.0
100.0
100.0
100.0
100.0
100.
0
60.0
95.8
60.0
3
3
7
7
20
18
4
6
18
MSc Thesis 2015
76.5
73.5
70.6
67.6
64.7
61.8
58.8
55.9
52.9
50.0
47.1
44.1
41.2
Results
6
2466.
2
21
10
2487
22
10
2743.
9
23
10
0
100.
0
100.
0
100.
0
12.5
32.3
8.3
31.3
4.2
30.3
0
100.
0
100.
0
100.
0
38.2
35.3
32.4
Shaded raw represents cutoff value of 120.9 pg/ml with 60% specificity, 100%
sensitivity, 100% NPV, 85.7% PPV and 88.2 Efficacy.
MSc Thesis 2015
Results
Table 22: Multi-ROC test resultsof CRP with IL6 at 120.9 to

discriminate positive and negative blood cultures among
cancer and non- cancer patients
CRP
15.7
16.4
20.4
26.5
29
40.2
64.7
67.1
68.1
71.4
85.9
88.2
89.5
90.8
106
119
157
162
166
186
200
205
206
TN
6
6
7
7
7
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
FP
4
4
3
3
3
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
TP
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
FN
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Sp
60.0
60.0
70.0
70.0
70.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
80.0
Sn
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
220
24
90.0
100.0
260
282
297
332
369
373
9
9
9
9
9
9
1
1
1
1
1
1
24
24
24
24
24
24
0
0
0
0
0
0
90.0
90.0
90.0
90.0
90.0
90.0
100.0
100.0
100.0
100.0
100.0
100.0
Shaded
raw represents cutoff value of
P+
85.7
85.7
88.9
88.9
88.9
92.3
92.3
92.3
92.3
92.3
92.3
92.3
92.3
92.3
92.3
92.3
92.3
92.3
92.3
92.3
92.3
92.3
92.3
Eff.
88.2
88.2
91.2
91.2
91.2
94.1
94.1
94.1
94.1
94.1
94.1
94.1
94.1
94.1
94.1
94.1
94.1
94.1
94.1
94.1
94.1
94.1
94.1
96.0
97.1
96.0
96.0
96.0
96.0
96.0
96.0
97.1
97.1
97.1
97.1
97.1
97.1
220 mg/l with 90% specificity, 100%
sensitivity, 100% NPV, 96% PPV and 97.1 Efficacy.
MSc Thesis 2015
P100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.
0
100.0
100.0
100.0
100.0
100.0
100.0
Results
AUC
IL-6
Multi-ROC
CRP
0.957
0.982
0.735
Figure 13:ROC curve analysis showing the diagnostic

performance of CRP and IL6 and their combinations for
discriminating patients with positive culture from those with
negative cultureamong cancer and non- cancer patients
From ROC curves results, serum IL-6 showed higher specificity,
sensitivity, positive predictive value and negative predictive values
than serum CRP in bacteremic (positive blood culture) than nonbacteremic (negative blood culture) patients as described
8.Complement C3 serum levels (C3) in cancer

The complement C3 serum levels of some selected tested
cases which included 9 cancer patients (6 with positive blood
cultures and 3 with negative blood cultures) and 7 non-cancer
patients (4 with positive blood cultures and 3 with negative blood
cultures) were determined as described in materials and methods
and the results are shown in table 23.
MSc Thesis 2015
Results
C3 serum concentrations were below normal values during

bacteremia in cancer patients (n=6), most values were between 24.8
and 72.5 mg/dl with mean value of 40.25mg/dl. Also, in non-cancer
patients with bacteremia (n=4) C3 serum concentrations values were
lower than the normal values and ranged between 8.7 and 72.5
mg/dl with mean value of 45.7 mg/dl.
By statistical analysis, it was found that, there was non-significant
difference in C3 serum concentration in cancer patients with positive
blood cultures (n=6) and non-cancer patients with positive blood
cultures (n=4) P- value =0.85.Also there was non-significant
difference in C3 serum concentration in cancer patients with positive
blood cultures (n=6) and cancer patients with negative blood culture
(non-microbial fever) (n=3) P- value = 0.3004. Meanwhile, in noncancer
patients
with
positive
blood
culture
(Gram
negative
bacteremia) (n=4) and non- cancer patients with negative blood

culture (non-microbial fever) (n=3) there was significant difference Pvalue = 0.026.
Table 23: Levels of serum C3 in some selected cancer and noncancer patients with positive and negative blood cultures.
Cancer patients
Non-cancer patients
With positive
With negative
With positive
With negative
blood culture
blood culture
blood culture
blood culture
Patie
C3
nts
conc.
no.
(mg/dl)
Patie
C3
nts
conc.
no.
(mg/dl)
8.7
100
140
72
29.1
101
90.5
73
72.5
102
110
94
72.5
Patien
C3 conc.
Patient
C3 conc.
ts no.
(mg/dl)
s no.
(mg/dl)
24.8
35
120
71
24.8
36
60
24.4
37
40
47.3
MSc Thesis 2015
Results
24
47.3
25
72.5
*Normal range of Complement C3 80-160 mg/dl
9.In-vitro efect of gamma irradiation on

antimicrobial
susceptibility
of
some
bacterial isolates recovered from cancer
The tested isolates were exposed to a dose level of 24.4 Gy of
gamma radiation which is biologically equivalent to the fractionated
multiple therapeutic dose used in the treatment protocol in some
cancer
patients.
After
gamma
irradiation
exposure,
the
susceptibilitiesof the tested isolates against different antimicrobial

agents were determined and the results are shown in tables 24 and
25.
In this study, Out of 34 bacterial isolates recovered from cancer
patients 15 were selected for antimicrobial susceptibility test before
and after exposure to gamma irradiation. These 15 isolates were
recovered from cancer patients whom their sIL-6 levels were
determined.
The relative percentages of resistant and susceptible bacterial
isolates against different antimicrobial agents which are classified
according to mechanism of action are presented in Figure 14-16.
Figure 14 showed the results of 7 tested antimicrobial agents acting
on the inhibition of cell wall synthesis. It is clear that, the highest
relative resistance percentage for - lactam antibiotics was observed
with:
ampicillin/sulbactam (SAM) 80%, amoxicillin/clavulanic acid
(AMC) 66.66%, cefotaxime (CTX) 60%, then cefepime (FEP) 46.66%,

ceftazidime (CAZ) 46.66%, tazobactam/piperacillin (TZP) 40%. On the
other hand, imipenem (IPM) showed no resistant phenotype for the
MSc Thesis 2015
Results
tested isolates but intermediate resistant reaction 26.66%. Some other

antibiotics play an important role in the inhibition of protein synthesis,
Figure13, showed that most of the tested strains were resistant to
gentamycin (CN) and tobramycin (TOB) 73.33% followed by 33.33%
were resistant to amikacin (AK),all previous antibiotics belonged to
aminoglycosides group. Meanwhile, resistance to chloramphenicol (C)
was represented by 40%.
MSc Thesis 2015
Results
Table 24: Antibacterial susceptibilities of some selected bacterial isolates recovered from cancer patients against different
antimicrobial agents before and after gamma irradiation exposure.
Patient
s no.
Recovered
bacterial isolate
Irradiation
status
Mean inhibition zone diameter (mm)/ Susceptibility profile against *

AK
SAM
FEP
CTX
CAZ
AMC
IPM
OFX
SXT
TOB
TZB
LEV
B
18/S
9/R
12/R
6/R
10/R
20/S
12/R
6/R
26/S
6/R
6/R
6/R
20/IR
6/R
A
18/S
8/R
12/R
6/R
10/R
20/S
9/R
6/R
26/S
6/R
6/R
6/R
20/IR
6/R
B
17/S
6/R
28/S
23/S
21/S
6/R
15/IR
15/S
28/S
6/R
6/R
15/I
20/IS
6/R
6
E. coli
A
18/S
10/R
26/S
25/S
22/S
9/R
12/R
18/S
28/S
6/R
6/R
17/S
23/S
6/R
B
21/R
12/IR
18/S
12/R
18/S
22/S
6/R
11/R
26/S
31/S
11/IR
6/R
9/R
6/R
8
E. coli
A
17/R
13/I
18/S
9/R
16/I
22/S
6/R
10/R
27/S
29/S
6/R
8/R
10/R
7/R
B
22/S
8/R
26/S
29/S
22/S
25/S
15/I
20/S
26/S
27/S
6/R
22/S
25/S
30/S
9
E. coli
A
22/S
8/R
29/S
23/S
20/S
24/S
13/R
20/S
28/S
28/S
6/R
20/S
24/S
24/S
B
17/S
8/R
13/R
8/R
12/R
23/S
14/IR
6/R
28/S
6/R
6/R
9/R
21/S
8/R
10
E. coli
A
16/IS
8/R
13/R
8/R
12/R
22/S
12/R
6/R
25/S
6/R
6/R
8/R
21/S
8/R
B
20/S
9/R
29/S
25/S
20/S
14/IR
18/S
21/S
28/S
6/R
6/R
20/S
22/S
6/R
11
E. coli
A
19/S
9/R
25/S
25/S
21/S
12/R
14/IR
18/S
27/S
6/R
6/R
18/S
20/IS
6/R
B
18/R
9/R
22/S
18/I
10/R
9/R
15/IR
10/R
25/S
6/R
6/R
11/R
21/S
9/R
2
K. pneumoniae
A
17/R
6/R
23/S
18/I
10/R
9/R
15/IR
10/R
25/S
6/R
6/R
13/IR
21/S
9/R
B
14/I
6/R
6/R
6/R
6/R
18/S
6/R
6/R
18/S
6/R
11/IR
6/R
9/R
6/R
3
K. pneumoniaee
A
14/I
6/R
6/R
6/R
6/R
19/S
6/R
6/R
19/S
6/R
6/R
8/R
10/R
7/R
B
17/S
6/R
12/R
6/R
18/S
25/S
11/R
6/R
25/S
18/S
6/R
6/R
20/IS
21/S
5
K. pneumoniae
A
15/IR
6/R
10/R
6/R
19/S
26/S
11/R
6/R
25/S
20/S
6/R
6/R
20/IS
21/S
B
19/S
6/R
27/S
16/IR
20/S
20/S
6/R
8/R
18/S
6/R
9/R
10/R
9/R
8/R
7
K. pneumoniae
A
20/S
6/R
18/S
15/IR
19/S
22/S
6/R
6/R
19/S
6/R
14/IS
10/R
11/R
6/R
B
19/S
6/R
21/S
10/R
16/I
27/S
6/R
12/R
17/IS
30/S
6/R
12/R
15/R
32/S
4
P. fluorescence
A
18/S
6/R
24/S
10/R
18/S
26/S
6/R
I2/R
20/S
30/S
6/R
10/R
14/R
32/S
B
25/S
6/R
27/S
16/IR
25/S
6/R
6/R
18/S
30/S
20/S
15/IR
23/S
31/S
20/S
25
P. aeruginosa
A
22/S
6/R
26/S
16/IR
25/S
6/R
6/R
18/S
30/S
20/S
12/IR
21/S
30/S
20/S
B
6/R
14/IS
6/R
6/R
6/R
9/R
6/R
12/R
15/IR
6/R
6/R
11/R
15/R
10/R
27
P. putida
A
6/R
10/R
6/R
6/R
6/R
6/R
6/R
9/R
15/IR
6/R
6/R
8/R
14/R
8/R
B
6/R
17/S
6/R
6/R
6/R
6/R
6/R
6/R
17/IS
6/R
6/R
6/R
16/S
8/R
24
A. baumennii
A
6/R
11/R
6/R
6/R
6/R
6/R
6/R
6/R
15/R
6/R
6/R
6/R
13/R
6/R
B
6/R
11/R
6/R
6/R
6/R
6/R
6/R
12/R
14/IR
6/R
6/R
12/R
6/R
6/R
26
A. baumennii
A
6/R
6/R
6/R
6/R
6/R
6/R
6/R
9/R
11/R
6/R
6/R
11/R
12/R
6/R
*
Susceptibility profile was interpreted as R (Resistant), IR (Intermediate resistant) and S (Sensitive) according to table 3 recommended by NCCLS 2011, before (B) and after (A) exposure to gamma
irradiation
1
E. coli
MSc Thesis 2015
Results
AK,Amikacin(30g); CAZ, Ceftazidime(30g); IPM, Immipenem10g); TZP, Tazobactam/Piperacillin (10g); SAM, Ampicillin/Sulbactam(20g); C, Chloramphenicol(30g); OFX,
Ofloxacin(5g); LEV, Levofloxacin(5g);FEP, Cefepime (30g); AMC, Amoxycillin/Clavulanic acid (30g); SXT, Sulphamethoxazole/Trimethoprim (25g);CTX, Cefotaxime (30g); CN,
Gentamycin (120g); TOB, Tobramycin(10g)
Table 25: Antibacterial susceptibilities of some selected bacterial isolates recovered from non-cancer patients against different
antimicrobial agents before and after gamma irradiation exposure
Irradiation
Mean inhibition zone diameter (mm)/Susceptibility profile against *
AK
SAM
FEP
CTX
CAZ
C
AMC
CN
IPM
OFX
SXT
TOB
TZB
LEV
status
B
16/IS
6/R
6/R
6/R
6/R
20/S
8/R
9/R
24/S
6/R
6/R
6/R
17/R
6/R
A
16/IS
6/R
6/R
6/R
6/R
6/R
8/R
9/R
25/S
6/R
6/R
6/R
14/R
6/R
74
E. coli
B
15/IR
9/R
12/R
6/R
12/R
6/R
16/IR
10/R
26/S
6/R
6/R
14/IS
20/IS
6/R
A
17/S
10/R
11/R
6/R
10/R
6/R
15/IR
I0/R
23/S
6/R
6/R
9/R
21/S
6/R
75
E. coli
B
16/IS
9/R
22/S
20/IS
19/S
25/S
12/R
6/R
29/S
6/R
6/R
9/R
21/S
9/R
A
13/R
9/R
22/S
15/IR
18/S
23/S
12/R
6/R
26/S
6/R
6/R
6/R
20/IS
8/R
71
K. pneumoniae
B
15/IR
6/R
6/R
6/R
6/R
25/S
6/R
20/S
10/R
6/R
6/R
11/R
9/IR
6/R
A
19/S
6/R
6/R
6/R
8/R
22/S
6/R
20/S
12/R
6/R
6/R
11/R
9/IR
6/R
72
K. pneumoniae
B
12/R
6/R
10/R
6/R
6/R
21/S
6/R
16/S
10/R
16/S
20/S
9/R
9/R
15/I
A
9/R
6/R
10/R
6/R
6/R
21/S
6/R
12/R
12/R
12/R
9/R
9/R
9/R
15/I
76
K. pneumoniae
B
17/S
6/R
8/R
6/R
8/R
20/S
9/R
21/S
25/S
6/R
15/IS
8/R
11/R
9/R
A
19/S
6/R
10/R
6/R
6/R
18/S
6/R
20/S
24/S
6/R
11/IR
13/IR
18/IR
6/R
77
K. pneumoniae
B
16/IS
6/R
14/R
8/R
6/R
6/R
11/R
12/R
25/S
18/S
6/R
12/R
15/R
21/S
A
15/IR
6/R
8/R
8/R
6/R
6/R
9/R
10/R
22/S
19/S
6/R
6/R
16/R
21/S
94
P. fluorescence
B
24/S
6/R
20/S
6/R
12/R
6/R
6/R
6/R
12/R
13/IR
6/R
15/S
12/R
17/S
A
20/S
6/R
15/IR
6/R
10/R
6/R
6/R
6/R
17/IS
14/I
6/R
20/S
13/R
17/S
95
A. baumannii
B
6/R
11/R
13/R
6/R
6/R
15/I
6/R
15/S
17/IS
6/R
6/R
17/S
16/R
8/R
A
6/R
16/S
13/R
6/R
6/R
8/R
6/R
13/IR
12/R
6/R
6/R
17/S
13/R
6/R
*
Susceptibility profile was interpreted as R (Resistant), IR (Intermediate resistant) and S (Sensitive) according to table 3 recommended by NCCLS 2011, before (B) and after (A) exposure to gamma
irradiation
AK,Amikacin(30g); CAZ, Ceftazidime(30g); IPM, Immipenem(10g); TZP, Tazobactam/Piperacillin (10g); SAM, Ampicillin/Sulbactam(20g); C, Chloramphenicol(30g); OFX,
Ofloxacin(5g); LEV, Levofloxacin(5g);FEP, Cefepime (30g); AMC, Amoxycillin/Clavulanic acid (30g); SXT, Sulphamethoxazole/Trimethoprim (25g); CTX, Cefotaxime (30g); CN,
Gentamycin (120g); TOB, Tobramycin(10g)
Patients
no.
73
Recovered
bacterial isolate
E. coli
MSc Thesis 2015
Results
In case of antibiotics which act on inhibition of nucleic acid

synthesis, the results in figure 15 showed a high percentage of
resistance
among
the
isolated
gram
negative
bacilli
against
levofloxacin (LEV) 73.33% then ofloxacin (OFX) 66.66%. Susceptibility

profile
of
sulphamethoxazole/trimethoprim
(SXT)
as
type
of
antimicrobial agents act on inhibition of folic acid synthesis 80% of the

isolates were resistant and 20% were intermediately resistant (Figure
16).
It was clear that there was significant difference in antibiotics
resistance before and after irradiation for Amikacin (AK) and
sulphamethoxazole/trimethoprim (SXT) with p-value = 0.05 and
0.0448 respectively.
Also, there was significant p-value = 0.0086, 0.0198 and 0.05 for
AMC in E. coli strains, for TOB in Pseudomonas strains andSAM in
Acinetobacter strains, respectively.
The frequency of resistance to antibiotics acting on inhibition of
cell wall synthesis after in-vitro exposure to gamma irradiation at a
dose level of 24.4 Gy increased against ampicillin /sulbactam (SAM)
from 80% to 93.3%, amoxicillin/clavulanic acid (AMC) from 66.66% to
86.66%,
tazobactam/piperacillin
(TZP)
from
40%
to
46.66%.
Meanwhile, for imipenem (IPM) 26.66% intermediate resistance

changed to 6.66% intermediate resistance and 13.33% resistance.
However, resistance against Ceftazidime (CAZ), cefepime (FEP) and
cefotaxime (CTX) were not changed with radiation, (Figure14).
While, for antibiotics inhibiting protein synthesis, tobramycin
(TOB) showed decreased resistance from 73.33% to 66.66%, amikacin
(AK) showed increased intermediate resistance from 6.66% to 19.98%
but resistance against gentamycin (CN) remained unchanged.
MSc Thesis 2015
Results
Resistance to chloramphenicol (C) increased from 40% to 46.66%,

(Figure15).
In case of antibiotics acting on inhibition of nucleic acid synthesis,
ofloxacin (OFX) and levofloxacin(LEV) showed unchanged resistance.
While,sulphamethoxazole/trimethoprim (SXT) resistance increased
from 80% to 86.66% after exposure to in-vitro gamma irradiation as
shown in figure 16.
(a) before gamma irradiation
Antimicrobial agent
Sensitive
MSc Thesis 2015
(IP
M
)
ne
e(
C
Intermediate Resistant
Resistant
Im
ip
e
m
ef
ta
zi
di
C
Am
ox
yc
i
lli
n
/C
la
vu
l
an
ic
ac
id
Relative percentage
AZ
(A
M
C
100
80
60
40
20
0
Results
(b) after gamma irradiation
Sensitive
M
)
m
(IP
Im
ip
e
ne
zid
im
e(
Ce
fta
Am
ox
yc
ill
in
/C
la
vu
la
n
ic
ac
id
(
Relative percentage
CA
Z
AM
C)
100
80
60
40
20
0
Antimicrobial agent
Resistant
Figure 14: Relative percentages of diferent susceptibility profiles

(sensitive, intermediate resistant and resistant) of some selected
bacterial isolates recovered from cancer patients against some
antimicrobial agents inhibiting bacterial cell wall synthesis before
(a)before gamma irradiation
Antimicrobial agent
Sensitive
MSc Thesis 2015
)
C
ni
co
l(
ph
e
hl
or
am
C
ik
ac
in
(A
yc
in
(T
O
B)
To
br
am
en
ta
ic
in
(C
Relative percentage
K)
80
60
40
20
0
Resistant
Results
C)
l(
AK
)
en
ic
o
ci
n(
Ch
lo
r
am
ph
Am
ik
a
ra
m
yc
in
(
To
b
G
en
ta
m
ic
in
(
CN
)
Relative percentage
TO
B)
80
60
40
20
0
Antimicrobial agent
Sensitive
Resistant

and after gamma irradiation.
Relative percentage
40
80
Levofloxacin(LEV) Ofloxacin(OFX)
Antimicrobial agent
Sensitive
MSc Thesis 2015
Intermediate resistant
Resistant
(SXT)
Results
Relative percentage
40
80
Levofloxacin(LEV)
Ofloxacin(OFX)
(SXT)
Antimicrobial agent
Sensitive
Resistant

Among the bacterial isolates recovered from non-cancer patients,

nine (9) isolates out of
29
were selected for antimicrobial
susceptibility test before and after exposure to gamma irradiation.

These 9 isolates were recovered from non-cancer patients whom their
sIL-6 levels were determined. The frequency of resistant and
susceptible bacterial isolates against different antibiotics by mode of
action is shown in figures 17-19. In figure 17,
which showed the
results of 7 antibiotics acting on the inhibition of cell wall synthesis, it

was clear that, the highest percentage of antibiotic resistance of the
nine tested bacterial strains was against ampicillin/sulbactam (SAM)
100%, amoxicillin/clavulanic acid (AMC) 88.88%, cefotaxime (CTX)
88.88%, ceftazidime (CAZ) 88.88% then cefepime (FEP) 77.77%,
tazobactam/piperacillin (TZP) 66.66%, finally imipenem (IPM) showed
resistant percentage 33.33%.
Some other antibiotics play important role in the inhibition of
protein synthesis. It is clear from figure 18 that most of the isolates
were resistant to tobramycin (TOB) 66.66% , gentamycin (CN) 55.55%
and 22.22% were resistant to amikacin (AK),all previous antibiotics
MSc Thesis 2015
Results
belonged to aminoglycosides group. Resistance percentage to

chloramphenicol (C) was represented by 33.33%.
In case of antibiotics which act on inhibition of nucleic acid
synthesis, the results in figure 19 showed a high percentage of
resistance among the tested strains against levofloxacin (LEV) and
ofloxacin (OFX) 66.66%.
sulphamethoxazole/trimethoprim
(SXT)
as
type
of
chemotherapeutic agents act on inhibition of folic acid synthesis

77.77% of the treated isolates were resistant and 11.11% were
intermediately resistant to it.
cell wall synthesis after in-vitro exposure to gamma radiation at a dose
level of 24.4 Gy decreased against ampicillin /salbactam (SAM) and
tazobactam/piperacillin (TZP) from 100% to 88.88% and from 66.66%
to 55.55%, respectively. However, intermediate resistance against
cefepime (FEP) and cefotaxime (CTX) increased to 11.11%.Resistance
against amoxicillin/clavulanic acid (AMC), imipenem (IPM) and
ceftazidime (CAZ) were not changed with radiation (figure 17).
MSc Thesis 2015
Results
(IP
M
)
em
e(
C
en
ip
Im
Am
ox
yc
i
lli
n
/C
la
vu
l
an
ic
ef
ta
zi
di
ac
id
Relative percentage
AZ
(A
M
C
100
80
60
40
20
0
Antimicrobial agent
S ens itive
Intermediate Res is tant
Res is tant
(IP
M
)
em
e(
C
Im
ip
en
im
ef
ta
zi
d
C
Am
ox
yc
i
lli
n/
la
vu
la
n
ic
ac
id
Relative percentage
AZ
(A
M
C
100
80
60
40
20
0
Antimicrobial agent
Sensitive
Resistant

bacterial isolates recovered from non-cancer patients against some
antimicrobial agents inhibiting bacterial cell wall synthesis before
MSc Thesis 2015
Results
C)
l(
AK
)
en
ic
o
ci
n(
Ch
lo
r
am
ph
Am
ik
a
To
b
G
en
ta
ra
m
yc
in
(
m
ic
in
(
CN
Relative percentage
TO
B)
100
80
60
40
20
0
Antimicrobial agent
Sensitive
Resistant
ic
o
AK
)
Resistant
en
ci
n(
am
ph
Ch
lo
r
ra
m
yc
in
(
To
b
m
ic
in
(
ta
G
en
Am
ik
a
TO
B)
CN
Relative Sensitive
percentage
100
80
60
40
20
0
Antimicrobial agent

MSc Thesis 2015
Results

100
80
60
Relative percentage
40
20
0
Levofloxacin(LEV)
Ofloxacin(OFX)
(SXT)
Antimicrobial agent
Sensitive
Resistant

100
80
60
Relative percentage
40
20
0
Levofloxacin(Lev)
Ofloxacin(Ofx)
(SXT)
Antimicrobial agent
Sensitive
Resistant

Meanwhile, for antibiotics inhibiting protein synthesis, gentamicin

(CN), chloramphenicol (C) and amikacin (AK) showed increased
resistance
to
66.66%,55.55%
and
33.33%,
respectively.
While
resistance against tobramycin (TOB) remained unchanged, Figure18

ofloxacin
(OFXshowed
levofloxacin(LEV)
MSc Thesis 2015
increased
showed
resistance
unchanged
to
77.77%
resistance.
and
Also,
Results
forsulphamethoxazole/trimethoprim (SXT) resistance increased from

77.77% to 88.88% after exposure to in-vitro gamma radiation as
shown in figure 19.
10.
Activity
profiles
of
lipase
and
protease enzymes of some selected
bacterial isolates recovered from cancer
The summarized data for lipase and protease enzymatic activities,
resistance prevalence of each test organism against different tested
antimicrobial agents as well as IL-6 and IL-8 serum levels are
presented in table 26. For Klebsiella pneumoniae (n=4), isolates
recovered from patients having high values of serum IL-6 also
showed high lipase enzymatic activity.
For Pseudomonasspecies
(n=3), isolates recovered from patients having high values of serum

IL-6 also showed high protease and lipase enzymatic activities. Also,
there was significant difference in antibiotics sensitivity before and
after radiation for strain no.25 with P-value=0.019, while strain no.27
having low serum level of IL-6 with lipase enzymatic activity and pvalue=0.006 for antibiotics resistance before and after irradiation. For
Acinetobacter baumannii (n=2), isolates recovered from patients with
high levels of serum IL-6 also showed high lipase enzymatic activity
with p-value= 0.04 for significant difference in antibiotic sensitivity
before and after irradiation for strain no.24. It was clear that IL-8
serum levels were correlated to lipase enzyme activity in all strains
except for E.coli. While, for bacterial isolates recovered from noncancer patients, there was noclear correlation between inflammatory
mediators
and
virulence
factors,
except
for
Pseudomonas
fluorescence no. 94 there was positive lipase and protease activity.

Table 26: Profiles of lipase and protease enzymatic activities,
resistance prevalence against diferent tested antimicrobial
agents as well as serum levels of IL-6 and IL-8 of some
MSc Thesis 2015
Results
selected bacterial isolates recovered from cancer and noncancer patients
1
6
8
9
10
11
2
3
5
7
4
25
27
26
24
71
72
76
77
73
74
75
95
94
Recovered isolate
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
K. pneumoniae
K. pneumoniae
K. pneumoniae
K. pneumoniae
Pseudomonas sp.
Pseudomonas sp.
Pseudomonas sp.
Acinetobacter
baumannii
Acinetobacter
baumannii
K. pneumoniae
K. pneumoniae
K. pneumoniae
K. pneumoniae
E. coli
E. coli
E. coli
Acinetobacter
baumannii
Pseudomonas sp.
Lipas
e
Proteas
e
Patie
nts
no.
Resista
nce
prevale
nce (%)*
Cancer patients
+
+
+
+
+
+
+
+
+
+
+
-
Serum IL6 (pg/ml)
Serum
IL-8
(pg/ml)
78.6
50
64.3
14.3
71.4
35.7
64.3
78.7
50
64.3
50
35.7
85.7
100
2487
2311.9
367.7
398.6
151.75
2743
2126.7
3083.4
830.55
228.9
2466.2
1293.5
136.32
799.8
343.4
71.4
1200.97
879.5
Non-cancer patients
85.7
64.3
64.3
71.4
+
78.6
78.6
50
71.4
2267.96
290.6
290.6
2373.6
491
167.2
1617.6
923.2
705.5
782.7
880.3
429.2
71.4
159.3
592.8
691
846.6
529.9
611.8
(*)
represents the organism resistance before exposure to gamma radiation to some

antimicrobial agents relative to the total tested antimicrobial agents and expressed as
percentage
11.
Efect of gamma irradiation on IL-6
serum levels in rats with induced fever of
bacterial and non-bacterial origin
It was clear that IL-6 serum level ranged from 873.4 to 1104
pg/ml in normal rats received saline only. However, the effect of
gamma radiation exposure caused an increase in rat serum levels of
MSc Thesis 2015
Results
IL-6 reaching 1197 and 1245 pg/ml at radiation doses of 2 and 7.5
gray. In yeast fever group representing fever of non-bacterial origin,
rat IL-6 serum levels ranged from 965 to 1426.6 pg/ml, while when
rats were exposed to radiation, their IL-6 serum levels elevated to
1470 pg/ml. In bacteremic fever groups there were very high
elevations in rat IL-6 serum levels reaching 27284 and with exposure
to gamma radiation the IL-6 serum levels reached 29753.9 pg/ml. For
the different treated rat groups as well as control groups, the rectal
temperature, white blood cells count and IL-6 serum concentrations
determined at the end of the experiment from blood samples of
sacrificed animals and the results are presented in tables 27-34.
Table
27:
White
blood
cells
count
and
IL-6
serum
concentration in control group

Sample No.
WBCs count (*109/L)
IL-6 serum
concentration (pg/ml)
7.1
1011.6
9.4
919.5
5.3
1104
5.3
873.4
8.1
1056
9.2
1011.6
Table 28: White blood cells count

concentration in radiation control gp.
and
IL-6
serum
Sample No.
WBCs count
(*109/L)
IL-6 serum concentration

(pg/ml)
1 (2 Gy)
5.2
1197
2 (2 Gy)
4.9
1059
3 (2 Gy)
1103.9
MSc Thesis 2015
Results
4 (7.5 Gy)
5.3
1245
5 (7.5 Gy)
3.9
1059
6 (7.5 Gy)
4.5
1103.9
Table 29: Rectal temperatures, white blood cells count and IL6 serum concentrations in fever of non-bacterial origin (yeast
fever) group.
Rectal temp.
20 h after
infection
WBCs
count
(*109/L)
IL-6 serum
concentrati
on (pg/ml)
37.5
36.9
9.4
965
35.2
37
9.5
919.5
35
36.6
4.2
1196.1
36.1
38.3
2.5
1334.4
36.3
37
3.5
1242.2
34.6
37.3
4.6
1426.6
Sample
No.
Zero time
Table 30: Rectal temperatures, white blood cells count and IL6 serum concentrations in fever of non-bacterial origin (Yeast
fever) group with gamma radiation exposure
Rectal temp.
Zero time
20 h after
infection
WBCs
count
(*109/L)
1 (2 Gy)
36.4
37.4
4.2
1288.3
2 (2 Gy)
37
37.7
4.5
1470
3 (2 Gy)
34.9
37.6
1243
4 (7.5 Gy)
34.9
37.6
5.6
1102
5 (7.5 Gy)
35.1
35.6
2.6
1426.6
6 (7.5 Gy)
37.2
37.9
1243
Sample No.
MSc Thesis 2015
IL-6 serum
concentratio
n (pg/ml)
Results
Table 31: Rectal temperatures, white blood cells count and IL6 serum concentrations in fever of bacterial origin
(Pseudomonas aeruginosa) group.
36.3
33.9
36.9
IL-6
serum
concentra
tion
(pg/ml)
26667.9
34.2
34.9
36
5.3
25740
34.7
35.2
36.1
2.1
24506
34.7
34.7
36.5
5.2
26667.9
35.9
34.5
36.1
3.5
25126
34.1
34.2
34.7
2.1
23736
Rectal temp.
Sampl
e No.
Zero
time
2 h after 4 h after
infection infection
WBCs
count
(*109/L)
Table 32: Rectal temperatures, white blood cells count and IL6 serum concentrations in fever of bacterial origin
(Pseudomonas aeruginosa) group with gamma radiation
exposure.
Sample
No.
Zero
time
1 (2 Gy)
2 (2 Gy)
3 (2 Gy)
4 (7.5 Gy)
5 (7.5 Gy)
6 (7.5 Gy)
35.5
34.6
36.2
34.9
36.2
35.4
Rectal temp.
2h
after
4 h after
infectio infection
n
36.1
37.1
35.5
35.2
34.1
36.6
34.4
34.4
36.4
37.1
36.9
37.3
WBCs
count
(*109/L
)
IL-6 serum
concentrati
on (pg/ml)
7.6
5.1
6.2
9.3
11
7.9
26000
26202
25800
28208
28200
27304
Table 33: Rectal temperatures, white blood cells count and IL6 serum concentrations in fever of bacterial origin (Klebsiella
pneumoniae) group.
Rectal temp.
Sampl
e No.
1
2
Zero
time
35.3
34.9
2h
after
infectio
n
34.3
36.4
MSc Thesis 2015
4 h after
infection
WBCs
count
(*109/L)
IL-6 serum
concentration
(pg/ml)
36.3
35.7
1.4
4.5
26050.7
1472.6
Results
3
4
5
6
36
34.6
35.7
34.8
35.7
34.4
34.2
36.4
36.9
32.1
34
36.8
4.8
2.3
4.2
1.5
27284
26359.4
21268
22454
Table 34: Rectal temperatures, white blood cells count and IL6 serum concentrations in fever of bacterial origin (Klebsiella
pneumoniae) group with gamma radiation exposure
Rectal temp.
Sample No.
WBCs
count
(*109/L)
IL-6 serum
concentratio
n (pg/ml)
Zero
time
2 h after
infection
4h
after
infectio
n
1 (2 Gy)
35.3
34.6
33.9
1.8
19262
2 (2 Gy)
34.6
34.8
35.6
1.1
29753.9
3 (2 Gy)
34.9
35.3
35.3
1.5
22347
4 (7.5 Gy)
35.3
35.4
36.7
4.8
24971
5 (7.5 Gy)
34.7
34.4
35.5
6.5
26823
6 (7.5 Gy)
34.9
34.1
37.3
1.7
29753.9
In gamma radiation exposure groups (a) bacteremic fever (P.

aeruginosa and K. pneumoniae) and (b) non-bacteremic fever, it was
observed
thatthere
was
significant
difference
in
IL-6
serum
concentration in bacteremic fever group and non-bacteremic fever

group P-value = 0.0001 (for P. aeruginosa group) and 5.9*10-8 (for
K.pneumoniae group). Similarily, ingroups that were not exposed to
gamma radiation (a) bacteremic fever (P. aeruginosa and K.
pneumoniae) and (b) non-bacteremic fever (yeast) group, there was
significant difference in IL-6 serum concentrations between group of
bacteremic fever due to P. aeruginosa and non-bacteremic fever group
due to yeast with P-value = 0.0001 and between group of
bacteremic fever due to K. pneumoniae and non-bacteremic fever
group due to yeast with P-value=0.000657. For
MSc Thesis 2015
P. eaeruginosa
Results
bacteremic group with exposure to gamma radiation and bacteremic

group without exposure to gamma radiation there was significant
difference in serum IL-6 concentration P-value = 0.0412and for K.
pneumoniae bacteremic group with exposure to gamma radiation and
bacteremic group without exposure to gamma radiation there was
non-significant difference in serum IL-6 concentration P-value =
0.27. In studying the effect of gamma radiation exposure,
IL-6
serum concentration in group exposed to gamma radiation was

significantly different from bacteremic group and bacteremic group
with exposure to gamma radiation.P-value = 0.0001 between
gamma radiation exposure group and bacteremic group due to P.
aeruginosa while for K. pneumoniae group P- value = 0.0006518.
Also, there was significant difference in IL-6 serum concentration
betweengamma radiation exposure group and bacteremic group due
to P. aeruginosa and K. pneumoniaewith exposure to gamma radiation
P-value=0.0001 and 5.5*10-8respectively. For yeast group (nonbacteremic fever without exposure to gamma radiation) and nonbacteremic fever with exposure to gamma radiation there was nonsignificant difference in IL-6 serum concentration P-value = 0.2726.
12.
Statistical analysis of IL-6 serum
levels in bacteremic groups both with and
without exposure to gamma radiation by
Receiver Operating Characteristic Curve
(ROC)
Diagnostic validity test Receiver Operating Characteristic Curve
(ROC) was done between bacteremic group (due to P. aeruginosa and
K. pneumoniae) with exposure to gamma radiation and bacteremic
group without exposure to gamma radiation [group 6 and 8]for IL-6
serum concentration as presented in table 35 and figure 20 as follows:
MSc Thesis 2015
Results
MSc Thesis 2015
Results
Table 35: ROC analysis data of sIL-6 for bacteremic groups

with and without exposure to gamma radiation.
Cutof
1472.6
19262
21268
22347
22454
23736
24506
24971
25126
25740
25800
26000
26050.
7
26202
26359.
4
26667.
9
26823
27284
27304
28200
28208
TP
1
1
2
2
3
4
5
5
6
7
7
7
FN
11
11
10
10
9
8
7
7
6
5
5
5
FP
0
1
1
2
2
2
2
3
3
3
4
5
TN
12
11
11
10
10
10
10
9
9
9
8
7
Sp
100.0
91.7
91.7
83.3
83.3
83.3
83.3
75.0
75.0
75.0
66.7
58.3
Sn
8.3
8.3
16.7
16.7
25.0
33.3
41.7
41.7
50.0
58.3
58.3
58.3
P52.2
50.0
52.4
50.0
52.6
55.6
58.8
56.3
60.0
64.3
61.5
58.3
P+
100.0
50.0
66.7
50.0
60.0
66.7
71.4
62.5
66.7
70.0
63.6
58.3
Ef.
54.2
50.0
54.2
50.0
54.2
58.3
62.5
58.3
62.5
66.7
62.5
58.3
58.3
66.7
63.6
61.5
62.5
50.0
66.7
60.0
57.1
58.3
50.0
75.0
66.7
60.0
62.5
11
50.0
91.7
85.7
64.7
70.8
11
12
12
12
12
1
0
0
0
0
7
7
8
9
10
5
5
4
3
2
41.7
41.7
33.3
25.0
16.7
91.7
100.0
100.0
100.0
100.0
83.3
100.0
100.0
100.0
100.0
61.1
63.2
60.0
57.1
54.5
66.7
70.8
66.7
62.5
58.3
*Shaded raw represents cutoff value of 25740 with 75% specificity, 58.3% sensitivity,
negative predictive value 64.3 and positive predictive value 70
MSc Thesis 2015
Results

performance of serum IL-6 for discriminating rats with
bacteremia (bacteremic fever) with exposure to gamma
radiation from those bacteremic without exposure to gamma
radiation
MSc Thesis 2015
Discussion
Discussion
condition. Bacterial sepsis is a major cause of fatality worldwide.One of
the most serious medical complications causing significant morbidity
and mortality among cancer patients is bacteremia. Therefore, rapid
diagnosis of this kind of bacterial infections can lead to better treatment; as a result, the morbidity and mortality of patients will decrease.
Many investigations have reported that among blood born infections,
Gram-negative bacteria are associated with more mortality than
Gram-positive bacteria. On the other hand, many scientists also report
that the causative agents of bacteremia are changing; therefore,
obtaining a better understanding of the spectrum of pathogens
causing bacteremia is vital for prompt treatment (Kalantar et al.,
2014).
Reyes et al., (2013) reported that prevalence of bacteremia for all
cancer, neutropenia and fever events was 24.3% and Gram-negative
bacteria were predominant (65%), the ratio of isolates of Gram
negative and Gram-positive was 2.2. The most frequently isolated
bacteria were Pseudomonas sp. (21.6%).
Cancer patients who are leukopenic due to chemotherapy are
susceptible to bacteremia. The mechanism of this inflammatory
response in cancer patients with diminished number of leukocytes is
not completely clear (Abdallah et al., 2008).
Blood stream infections (BSI) are a major cause of fever in
children with chemotherapy-induced neutropenia, accounting for 30%
of all episodes of fever and neutropenia (FN) in this population. High
mortality rates (up to 80%) in FN patients with Gram-negative
bacteremia have been previously reported. Therefore, over the past 2
MSc Thesis 2015
Discussion
decades, hospitalization and immediate empirical treatment with

broad-spectrum intravenous antibiotics are considered mandatory in
all pediatric oncology patients with FN episodes (Hazan et al., 2013).
Many studies associate bloodstream infection in cancer patients
with Gram-negative bacteria. Amongst Gram-negative organisms, E.
coli was the most common isolate; in cancer patients, twice as many
BSIs were caused by Pseudomonas aeruginosa and E. cloacae (Bos et
al., 2013).
Overall, from 2007 to date, the rate of Gram-negative bacteria
recovery ranged from 24.7 to 75.8% (mean 51.3%) in cancer patient
cohorts. E. coli represented the most common species (mean
frequency of isolation 32.1%) among the Gram-negatives, followed by
P. aeruginosa (mean frequency of isolation 20.1%). An increasing
frequency of Acinetobacter sp. and Stenotrophomonas maltophilia
was also reported (Trecarichi and Tumbarello, 2014).
In this study for cancer feverish patients the prevalence of E. coli
was 41% followed by K.
pneumoniae 23% then Pseudomonas sp.
27%, while for non-cancer feverish patients K.
pneumoniae
prevalence was 41% followed by E. coli 38% then Pseudomonas sp.

14%.
Culturing microorganisms is the most definitive way to confirm
bacterial infections.
Unfortunately, this gold standard is time consuming and may be
influenced by several factors including previous antibiotic usage.
Currently used conventional infection markers such as CRP level, WBC
count and the erythrocyte sedimentation rate have relatively poor
discriminatory capacity in distinguishing patients with bacterial
MSc Thesis 2015
Discussion
infections versus patients with nonbacterial infections (De jager et al.,

2010).
Raynor et al., (2012) reported that,Cytokines have been proposed
as promising biomarkers because some of them rise very early in the
course of bacteremia (PC and Lam, 2010) and thus are more sensitive
for detecting the early phase of sepsis than acute-phase proteins such
as C-reactive protein (CRP) (PC et al., 1997). This was demonstrated
previouslyin a prospective study of preterm infants in which blood
levels of interleukin IL-6 and IL-1 receptor antagonist (IL-1ra) rose 12
d before the clinical presentation of blood cultureproven sepsis (Kuster
et al., 1998).
Treatment of leukaemia and other malignant tumors
chemotherapy
susceptibility
suppresses
to
infection.
the
In
immune
most
system
cases,
fever
and
is
by
increases
an
early
andimportant sign of infection, but clinical signs are non-specific and

fever can be attributed to other causes. Furthermore, the presently
available laboratory techniques such as CRP and procalcitonin (Prat et
al., 2008), determination, do not have high sensitivity or specificity for
early diagnosis of infection in these patients. Although bacterial
culture with sensitivity testing using a variety of specimens, such as
blood, sputum, secretions, and body fluids is the most important
method for guiding antibiotic selection, the positive rate is low,
especially in patients with haematology/oncology malignancies, who
have usually been heavily treated with antibiotics (Peters et al., 2004).
Thus,
the
current
management
of
febrile
episodes
in
immunocompromised children is primarily based on empirical broadspectrum antibiotics. However, widespread use of broad-spectrum
antibiotics may be associated with the emergence of resistant strains
and increases in treatment costs. Recently, attention has been paid to
the role of cytokines in the diagnosis of infection. Most studies have
MSc Thesis 2015
Discussion
focused on IL-6, IL-8, or both and measurement of these cytokines by

ELISA (Tanget al., 2011).
Neutropenic cancer patients with bacteremia, in particular those
with gram-negative bacteremia, appear to have a relatively high risk
of complications and lethal outcome which is clearly higher than the
risk
of adverse outcomes
associated with
unexplained fever.
Accordingly, there have been several attempts to predict bacteremia

or gram-negative bacteremia by inflammatory markers measured in
serum. Other markers such as IL-6 and IL-8 may be more
discriminative than CRP (Engel et al., 2005).
Increases in circulating levels of the cytokines IL-6 and IL-8 are
apparent early in the course of infection, indicating a rapid series of
host response mechanisms, such as fever and chemotaxis of white
blood cells (Lehrnbecher et al., 1999).
In this study, a total of 124 in-patients with fever onset were
included. Seventy of them (70) were cancer patients and 54 were noncancer patients suggesting that inflammatory cytokines are already
released in febrile patients before positive reports of microbiological
cultures. We evaluated the usefulness of CRP, IL-6, IL-8 and
complement C3values as early markers of Gram negative bacteremia
in addition to commonlyused clinical variables such as WBCs count.A
valuable marker of infection with gram negative bacteria in this setting
would be associated with high sensitivity and specificity for predicting
infection.
Fever as described in previous studies which showed that CRP,
which is commonly used in practice, was a poor tool for distinguishing
bacterial infection from other causes of fever (Miedema et al.,
2010).The CRP concentration rises within 24 h and is elevated in
almost all cases of microbial infection, It was described that levels of
MSc Thesis 2015
Discussion
CRP rose, especially in the patients with bacteremia due to gramnegative organisms (Lehrnbecher et al., 1999) but its reliability as a
marker of infection is hampered by very low specificity (Von LilienfeldToal et al., 2004). CRP is relatively slow to develop, and several
sequential determinations are necessary for the most accurate
diagnosis of infection. Moreover, these values can be influenced by
underlying malignant disease and tissue damage (Kitanovski et al.,
2008). Measured levels of CRP, displayed a broad overlap among the
different groups.
Both IL-6 and IL-8 serum levels have been shown to increase
much earlier than CRP and increasing levels can frequently be
detected even before onset of fever (Engel et al., 1999). CRP serum
concentrations in our study showed non-significant difference between
bacteremic groups and groups of non-microbial fever. Cutoff levels to
distinguish between bacteremic (positive blood cultures) and nonbacteremic (negative blood cultures) cases were chosen using receiver
operating characteristic curves : 29 mg/l for cancer patients, 119 mg/l
for non- cancer patients and with 60% and 100% specificity, 77.8%
and 66.7% sensitivity, NPV 60% and 62.5 %, PPV 77.8% and 100%,
Efficacy 71.4% and 78.6% , respectively. So the efficacy of CRP as
marker to discriminate between positive and negative blood cultures
was higher in cancer patients.
Levels of IL-6 in serum were measured because this cytokine is
thought to have a central signaling function in the inflammatory
response to microbial infection, i.e., induction of acute phase reaction
(Groeneveld et al., 2001).
In the present study, IL-6 serum concentrations for positive blood
cultures were significantly higher in cancer patients (n=9) than noncancer patients (n=9) P- value = 0.0166. Also, there was significant
MSc Thesis 2015
Discussion
difference in IL-6 serum concentration in cancer patients with positive

blood cultures (n=9) and negative blood cultures (n=5) P-value =
0.0001. Furthermore, in non-cancer patients with positive blood
cultures (n=9) and negative bloodcultures (non-microbial fever) (n=5),
there was significant difference P- value = 0.0288.
Our results found that IL-6 was discriminatory marker between
bacteremic and non-bacteremic fever in cancer and non-cancer
patients. Cutoff levels to distinguish between bacteremic (positive
blood cultures) and non-bacteremic (negative blood cultures) cases
were chosen using receiver operating characteristic curves : 398.6
pg/ml for cancer patients, 120.9 pg/ml for non- cancer patients and
with 100% specificity, 100% sensitivity, NPV 100%, PPV 100%, Efficacy
100% for both groups. So the efficacy of IL-6 as marker to discriminate
between positive and negative blood cultures was higher than that of
CRP.Using the ROC curve analysis showing diagnostic performance of
CRP and IL-6 and their combination (via multi-ROC) for discriminating
patients with positive cultures from those with negative cultures (all
cancer and non- cancer tested cases), We found that the best cut-off
value of IL-6 was 120.9 pg/ml with 60% specificity, 100% sensitivity,
NPV 100%, PPV 85.7%, Efficacy 88.2%, while for CRP best cut-off value
of was 85.9 mg/l with 50% specificity, 75% sensitivity, NPV 45.5%, PPV
78.3%, Efficacy 67.6%, Using the multi-ROC for the both markers to
improve the results for CRP , We found that CRP best cut-off value was
220 mg/ml at IL-6 of 120.9 pg/ml with improved specificity, sensitivity,
NPV, PPV and Efficacy of values 90%, 100%, 100%, 96% and 97.1%
respectively. The AUC also improved using the multi-ROC from 0.735
for CRP only and 0.957 for IL-6 to 0.982.The usefulness of both
markers (IL-6 and CRP) was proved to distinguish between bacteremic
and non- bacteremic patients.Also, there was direct proportionality
between IL-6 serum levels and WBCs count, meaning that IL-6 playing
MSc Thesis 2015
Discussion
role in chemotaxis of white blood cells to site of infection as described

before in figure7 for cancer patients, A plausible explanation for the
higher levels of IL-6 in patients with neutropenia may be related to the
fact that gram-negative bacteria release high concentrations of
endotoxin,
which
directly
activates
monocytes
via
the
CD14
receptor(Lehrnbecher et al., 1999). When febrile neutropenic children

were assessed for studies, IL-6 values peaked 24-48 h before CRP
values and there was a positive correlation of IL-6 with the presence of
fever, therefore, plasma IL-6 may be a more sensitive marker than CRP
of acute infection and should prove usefulness in the assessment of
fever (Heney et al., 1992), as relations between serum concentrations
of IL-6 and CRP was described in for both cancer patients showing
inverse proportionality P- value = 0.006 and non-cancer patients
with direct proportionality P- value = 0.0149.
In a previous study, IL-8 was analyzed in serial serum samples
from 6 patients undergoing chemotherapy for AML, these patients had
sepsis with bacterial infection. Case 1 showed significant elevation in
serum IL-8 concentration preceding onset of fever (>37C) and
elevation of CRP level, the other 5 cases showed the same sequence.
Elevation
of
IL-8
concentration
preceded
elevation
of
IL-6
concentration and CRP level, and onset of fever in patients with

leukemia during septicemia. Release of IL-8 in human whole blood
cells were challenged with LPS at time zero and IL-8 protein levels
were determined at 0.5, 1, and 3 hours. All of these antigens
significantly (P<.05) induced IL-8 production at 1 h after exposure and
continued to do so at 3 h (Reid et al., 2002).
The data of sIL-8 in our study revealed that, there was significant
difference in IL-8 serum concentration in cancer patients with positive
blood cultures (n=6) and cancer patients with negative blood cultures
(non-microbial fever) (n=5) P- value = 0.0059. Similarily, in non-
MSc Thesis 2015
Discussion
cancer patients with positive blood cultures (n=5) and non- cancer
patients with negative blood cultures (non-microbial fever) (n=5) there
is also significant difference P- value = 0.0059.When measuring IL-8
serum concentrations, there were strict differences in concentrations
between bacteremic and non-bacteremic fever groups reaching 100%
specificity and sensitivity.
When kinetics of IL-8, IL-6, CRP and WBCs count were studied, no
significant correlation was seen between IL-8 concentrations and
numbers of WBCs. However, IL-8 correlated positively with circulating
CRP and IL-6 in patients with sepsis syndrome (Hirao et al., 2000).
In a previous study IL-8 serum levels appeared to be similar to IL6 in being significantly different between patients with and without
documented infection (Engel et al., 1999). In the present study, for
cancer and non-cancer patients with Gram negative bacteremia, there
were significant relationships between IL-8 serum levels and CRP with
P- value = 0.0114 and 0.00017,respectively. Also, there was
significant correlation between IL-8 and IL-6 serum levels with Pvalue = 0.0026 in cancer patients, while in non-cancer patients the
relationship between both markers was non-significant with P- value
= 0.336. Meanwhile, there was non-significant relationship between
IL-8 and WBCs with P- value = 0.18 and significant relationship
between IL-6 serum levels and WBCs count with P- value =
0.000025.
Tromp et al.,(2009) reported that, the lowest median value for
serum IL-8 is found in patients with unexplained fever, high IL-8 levels
are correlated with Gram-negative bacteremia, based on these
findings it appears that serum IL-8 measurement may be valuable to
predict early complicated courses of bacteremic infection in the
neutropenic host (Engel et al., 2005). In comparison to CRP, the IL-8
MSc Thesis 2015
Discussion
level seems to be more worthwhile for discriminating infectious from

non-infectious causes in neutropenic febrile patients, especially at the
start of febrile episode. Lehrnbecher et al., (1999) found that either IL6 or IL-8 might be useful parameters in a febrile child with cancer and
neutropenia at the time of admission. IL-6 and IL-8 levels were higher
in patients with bacteremia due to Gram-negative organisms than in
patients with febrile episodes without an identifiable source(Diepold et
al., 2008) and also found that IL-6 and IL-8 were highly correlated.
The peak C3a did not contribute to prediction of a positive culture
(Von Lilienfeld-Toal et al., 2004). Ongoing activation of the complement
pathway may be a risk factor for poor prognosis, and persistently
depressed serum complement protein levels may be an indirect
marker for this (Sungurtekin et al., 2006).Serum levels of complement
C3 were decreased in bacteremic patients meaning there was
activation of the complement system by Gram negative bacteremia
with non-significant differences between the studied groups.
Bloodstream infections (BSIs) and antimicrobial resistance
(AMR) are worldwide health care problems causing substantial patient
morbidity and mortality. (Saied et al., 2011) reported that Gramnegative bacteria accounted for 61.7% of total pathogens isolated
from blood cultures. One major concern associated with Gramnegative pathogens is the emergence of extended-spectrum betalactamas(ESBL) producing strains. These strains are resistant to all lactam antimicrobial agents except cephamycins and carbapenems.
Isolation of multidrug-resistant ESBL-producing K. pneumoniae strains
has been described, especially in ICUs (Rebuck et al., 2000).Increased
rates
of
multidrug-resistant
Gram-negative
strains
have
been
highlighted among Enterobacteriaceae and nonfermenting Gramnegative rods, despite discontinuation of fluoroquinolone-based
antibacterial
prophylaxis
MSc Thesis 2015
for
neutropenic
patients.
In
addition,
Discussion
antimicrobial resistance and/or the inadequacy of empirical antibiotic

treatment have been frequently linked to a worse outcome in cancer
patients with bloodstream infections caused by Gram-negative isolates
(Trecarichiand Tumbarello, 2014).
Drug resistance has become an important issue for Gram
negative pathogens as well. The use of fluoroquinolones for
prophylaxis in high-risk patients with neutropenia has been associated
with the emergence of resistance among E. coli and P. aeruginosa
isolates (Carratala et al., 1995; Kern et al., 1994; Rolston, 1998).
Resistance to ciprofloxacin among P. aeruginosa isolates has been
>20% at some institutions (Jacobson et al., 1999; Rolston et al., 2003).
The
use
of
older
quinolones
(norfloxacin,
ofloxacin
and
ciprofloxacin) may also have led to the increased frequency of

infections caused by drug-resistant, nonfermentative Gram-negative
bacilli, such as Alcaligenes species, Pseudomonas species other than
P. aeruginosa and Stenotrophomonas maltophilia. Rates of ESBLproducing isolates among E. coli and Klebsiella species are increasing
(Paterson and Rice, 2003).
ESBL-producing
K.
pneumonia
isolates
will
render
most
cephalosporins and some combinations of -lactam and -lactamase

inhibitors ineffective (Bradford, 2001). They may also acquire
mechanisms of resistance to quinolones and some aminoglyosides.
Resistance of ESBL-producing organisms to the combination of
piperacillin and tazobactam has been reported and is of concern
(Babini and Livermore, 2000).
Plasmid-mediated,
AmpCtype
-lactamases
produce
similar
patterns of resistance, as well as resistance to some carbapenems

(Odeh et al., 2002). Among Enterobacter isolates, resistance to
ceftazidime is mediated by a Bush group 1 cephalosporinase and has
MSc Thesis 2015
Discussion
been >30% (Jones, 1999). Resistance to carbapenems among P.

aeruginosa isolates is increasing (15%20% in some institutions).
Some of these isolates are truly multidrug resistant and are
susceptible only to such agents as colistin and polymyxin B (Toleman
et al., 2003 and 2004; Nguyen et al., 2004; Bodey, 1984). However,
like the aminoglycosides, colistin and polymyxin B have suboptimal
clinical efficacy in patients with neutropenia when used as single
agents, and need to be combined with other agents (preferably lactams) that are active against the offending pathogen (Bodey,
1984).
In this study, Out of 34 bacterial isolates recovered from cancer
patients 15 were selected for antimicrobial susceptibility test before
and after exposure to gamma irradiation. These 15 isolates were
recovered from cancer patients whom their sIL-6 levels were
determined.
The frequency percentages of resistant and susceptible bacterial
isolates against different antimicrobial agents which are classified
according to mechanism of action are presented in table 24. It was
clear
that,
the
highest
resistance
percentage
for
lactam
antibioticswas observed with:SAM 80%, AMC 66.66%, CTX 60%, then

FEP 46.66%, CAZ 46.66%, TZP40%. On the other hand, IPM showed no
resistant phenotype for the tested isolates but intermediate resistant
percentage of 26.66%.
Some other tested antibiotics which play an important role in the
inhibition of protein synthesis, showed that most of the tested strains
were resistant to CN and TOB 73.33% followed by 33.33% were
resistant to AK,all previous antibiotics belonged to aminoglycosides
group. Meanwhile, resistance to Cwas represented by 40%.In case of
antibiotics which act on inhibition of nucleic acid synthesis, the high
MSc Thesis 2015
Discussion
percentage of resistance among the isolated Gram negative bacilli was

demonestrated against LEV 73.33% then OFX 66.66%.SXT as a type of
chemotherapeutic agents act on inhibition of folic acid synthesis 80%
of the isolates were resistant and 20% were intermediately resistant to
it.It was clear that there was significant difference in antibiotics
resistance before and after irradiation for AK and SXTwith p-values of
= 0.05 and 0.0448,respectively.Also, there was significant p-value
= 0.0086, 0.0198 and 0.05 for AMC in E. coli strains, TOB in
Pseudomonas strains and SAM in Acinetobacter strains, respectively.
cell wall synthesis after in-vitro exposure to gamma irradiation at a
dose level of 24.4 Gy increased against SAM from 80% to 93.3%, AMC
from 66.66% to 86.66%, TZP from 40% to 46.66%. Meanwhile, for IPM
26.66% intermediate resistance was changed to 6.66% intermediate
resistance and 13.33% resistance. However, resistance against CAZ,
FEP and CTX unchanged with radiation.While, for antibiotics inhibiting
protein synthesis, TOBshowed decreased percentage of resistant
isolates from 73.33% to 66.66%, AK showed increased percentage of
resistant isolates from 6.66% to 19.98% but resistance percentage of
isolates against CN remained unchanged.
Percentage of resistant
isolates to to C increased from 40% to 46.66%.In case of antibiotics

acting on inhibition of nucleic acid synthesis, OFX and LEVshowed no
change in percentage of resistant isolates. While, for SXTresistant
isolates increased from 80% to 86.66% after exposure to in-vitro
gamma radiation.
Among the bacterial isolates recovered from non-cancer patients,
9isolates out of 29 were selected for antimicrobial susceptibility test
before and after exposure to gamma irradiation. These 9 isolates were
recovered from non-cancer patients whom their sIL-6 levels were
determined. With 7 antibiotics acting on the inhibition of cell wall
MSc Thesis 2015
Discussion
synthesis, it was clear that the order of
percentage of antibiotic
resistance of the nine bacterial strains was against SAM 100%, AMC
88.88%, CTX 88.88%, CAZ 88.88% then FEP77.77%, TZP 66.66%,
finally IPMshowed resistant
percentage of 33.33%.Some other
antibiotics that play important role in the inhibition of protein

synthesis, it was clear that the isolates order of resistancewas TOB
66.66%, CN 55.55% and 22.22% were resistant to AK, all previous
antibiotics belonged to aminoglycosides group. Resistance to C was
represented by 33.33%.In case of antibiotics which act on inhibition
of nucleic acid synthesis, the results showed a high percentage of
resistance of the tested strains against LEV and OFX 66.66%. SXT
recorded
77.77%
and
11.11%for
resistant
and
intermediate
resistant isolates.The frequency of resistance to antibiotics acting on

inhibition of cell wall synthesis after in-vitro exposure to gamma
radiation at a dose level of 24.4 Gy decreased for SAM and TZP from
100% to 88.88% and from 66.66% to 55.55%, respectively. However,
intermediate resistance against FEP and CTX increased to 11.11%.
Resistance against AMC, IPMand CAZ remain unchanged with radiation
treatment.
Meanwhile, for antibiotics inhibiting protein synthesis, increased
isolates resistance was recorded for CN, C and AK, while resistance
against TOB remained unchanged as shown in fig.18.
OFX showed increased percentage of resistant isolates to 77.77% and
LEV showed no change inpercentage of resistant isolates.Also, for SXT
percentage of resistant isolates increased from 77.77% to 88.88%
after exposure to in-vitro gamma radiation.
So, with increasing antimicrobial resistance, it is important to find
antibiotics with enhanced activity against resistant organisms.
MSc Thesis 2015
Discussion
Antibiotics combinations are used to enhance antibacterial efficacy

and prevent the development of resistance.Extracellular or surface
localization of virulence determinants is an important attribute of
pathogenic microorganisms. The past decade has seen significant
research advances in defining the steps and identifying the necessary
machinery for protein secretion from bacterial cells (Lory, 1998).
Wensink et al., (2014) reported that Granzymes are
serine proteases released by cytotoxic lymphocytes and
induce
cell
death
in
virus-infected
cells
and
tumor
cells.However, granzymes also exist extracellularly in the

blood circulation of patients with autoimmune diseases and
infections and may contribute to inflammation. Human
granzyme K (GrK) binds to Gram-negative bacteria and to
lipopolysaccharide (LPS), a Gram negative bacterial cell wall
component. The data indicate that GrK lowers the threshold
for monocyte activation by LPS, in that GrK synergistically
increases LPS-induced release of proinflammatory cytokines in
vitro and in vivo.
In conclusion, GrK modulates the innate
immune response againstLPS and Gram-negative bacteria

and
may
contributeto
the
pathogenesis
of
diseases
associatedwith a local or systemic bacterial infection.

In an attempt to study the role of measured serum IL-6 as marker
of severity, correlations were pointed with lipase and protease
enzymatic activities and antimicrobial susceptibility tests in some
selected bacterial isolates. Correlations were clear within bacterial
isolates recovered from cancer patients as E.coli isolates recovered
from patients having high values of serum IL-6 also showed high
protease enzymatic activity and antimicrobial resistance reaching
78.6%. While those without protease activity were isolated from
patients with low serum levels of IL-6 and antimicrobial resistance
MSc Thesis 2015
Discussion
reaching 71.4%.K. pneumoniaeisolates recovered from patients having

high values of serum IL-6 also showed lipase activity and antimicrobial
resistance reaching 78.7%. For Pseudomonas species, isolates
protease and lipase activities. For A. baumannii, isolates recovered
from patients with high levels of serum IL-6 also showed lipase
enzymatic activity and antimicrobial resistance reaching 100%. While,
for bacterial isolates recovered from non-cancer patients, there was no
clear correlation between inflammatory mediators and virulence
factors, except for P. fluorescence(isolate no. 94) there was positive
lipase and protease activity. The role of serum IL-8 was not clear and
may be due to small sample size in both groups.
Concern regarding radiation effects on human health continues to
increase worldwide. Given that infection is a major cause of morbidity
and mortality after exposure. Pecaut et al.,(2014), recorded that when
mice were exposed to total-body irradiation (TBI) with 3 Gy protons
(70 cGy/min). One, 2, 4, 8 or 16 days later, subsets of mice were
injected intraperitoneally with live Escherichia coli, the results showed
that there were no unexpected effects of radiation or E. coli alone.
Despite
dramatic
radiation-induced
decreases
in
all
leukocyte
populations in both the blood and spleen, irradiated mice were still
able to respond to an immune challenge based on capacity to
generate an oxidative burst and secrete inflammatory cytokines, i.e.,
TNF- and IL-6. However, these responses were generally elevated
above control values.
In this study evaluation of the effect gamma irradiation on serum
levels of IL-6 in bacteremic and non-bacteremic rats groups was done.
IL-6 serum concentration was higher in bacteremic groups with
exposure to gamma irradiation compared to the other groups. The
ROC analysis carried outbetween bacteremic groups (due to P.
MSc Thesis 2015
Discussion
aeruginosa and K. pneumoniae) with exposure to gamma irradiation

[group 6,8] and bacteremic groups without exposure to gamma
radiation [group 5, 7] for IL-6 serum concentration showed
good
values.
We concluded that serum IL-6 concentration can be used as a
good marker for differentiation between bacteremic fever and nonbacteremic
fever
either
for
radiation
exposed
or
unexposed
individuals.
Conclusion
Serum IL-6 and IL-8 are good markers for early prediction of
Gram negative bacteremia in cancer patients and other patients.

Serum IL-6 can be used as good marker for detection of
bacteremic fever in cancer patients even for patients under
radiotherapy .
Severity of infection can be determined upon the determination
of different enzymatic activities and correlate them to values of
serum IL-6 as shown for GNB

Cytokines have been proposed as promising biomarkers
because some of them rise very early in the course of bacteremia
and thus are more sensitive for detecting the early phase of sepsis
than acute-phase proteins such as CRP.

Combination between serum CRP
and
IL-6
gives
high
discriminatory value among bacteremic and non-bacteremic
feverish patients.
Increases in circulating levels of the cytokines IL-6 and IL-8 are
apparent early in the course of infection, indicating a rapid series of
host response mechanisms, such as fever and chemotaxis of white
blood cells.
Serum levels of complement C3 can not be used as an early
marker of bacteremia due to non-significant differences between
the studied groups.
MSc Thesis 2015
Discussion
Continuous monitoring of antimicrobial resistance should be

carried out regularly upon cancer patients pathogens to decrease
the risk of antimicrobial resistance, sepsis and high morbidity/
mortality.
Optimal therapy for these antibiotic-resistant pathogens still
remains to be determined for cancer patients.
MSc Thesis 2015
Summary
Summary
condition. The systemic host response to microbial infection involves
clinical signs and symptoms of infection including fever. In addition,
inflammatory biomarkers are released, including C-reactive protein
(CRP), Interleukin-6 (IL-6), Interleukin-8 (IL-8) and Complement C3.
In this study we assessed the value of serum inflammatory
mediators levels(IL-6 and IL-8) at fever onset in comparison with
routinely used markers as WBCs, CRP and C3 to predict gram negative
bacteremia in cancer and non-cancer patients.
Severity of infection was also studied comparing enzymatic
activity and antimicrobial susceptibility of some selected isolated
bacterial strains with measured serum inflammatory marker mainly IL6. Also, the effect of gamma irradiation on IL-6 serum levels in rats
with induced fever of bacterial and non-bacterial origin was
investigated through the animal model on forty-eight adult Wister rats
and comparisons between groups were performed.
In this study,One hundred twenty four feverish in-patients were
enrolled in the study. Serum and plasma samples separated from
blood specimens at onset of fever were used for assay of inflammatory
biomarkers (IL-6 and IL-8) using ELISA technique, CRP using turbilatex
and C3 ussig diffu-palte. Total leukocytic count was determined by
coulter counter. Blood culture was carried out for isolation of Gram
negative organisms which were identified by API 20E technique and
subjected to antibiotic susceptibility test that was performed using
disc diffusion method. Lipase and protease enzymes detection was
performed via tween and gelatin agar media, respectively. Assay of
serum IL-6 in rats was also done using ELISA technique. Cesium 137
MSc Thesis 2015
Summary
(137 Cs) Gamma cell 40 located at National Center for Radiation

Research and Technology (Cairo, Egypt) was the irradiation source
used in the study.
It was found that out of the 124 blood samples collected there
were 70 samples cancer feverish patients and 54 noncancerous
feverish patients with relative percentages of 56% and 44%,
respectively. About 49% of blood culture samples collected from
cancer patients showed positive bacterial growth and 51% showed
negative growth. While, blood samples showing positive growth in
non- cancer patients were 54% against 46% negative growth.
In this study for cancer feverish patients the prevalence of E. coli
was 41% followed by K. pneumoniae 23% then Pseudomonas sp 27%,
while for non-cancer feverish patients K. pneumoniae prevalence was
41% followed by E. coli 38% then Pseudomonas sp. 14%.
Results showed that, IL-6 and IL-8 serum levels were higher in
patients with Gram negative bacteremia than in those with nonmicrobial fever for both groups of patients (cancer and non- cancer
patients). For cancer patients with Gram negative bacteremia and
those with out there was significant difference in IL-6 and IL-8 serum
levels (P=0.0001 and 0.0059, respectively) and for non-cancer
patients (P=0.0288 and 0.0059).
CRP serum concentrations in our study showed non-significant
difference between bacteremic groups and groups of non-microbial
fever. Cutoff levels to distinguish between bacteremic (positive blood
cultures) and non-bacteremic (negative blood cultures) cases were
chosen using receiver operating characteristic curves : 29 mg/l for
cancer patients, 119 mg/l for non- cancer patients and with 60% and
100% specificity, 77.8% and 66.7% sensitivity, NPV 60% and 62.5 %,
PPV 77.8% and 100%, Efficacy 71.4% and 78.6%, respectively. So the
MSc Thesis 2015
Summary
efficacy of CRP as marker to discriminate between positive and

negative blood cultures was higher in cancer patients.
While, Cutoff levels of serum IL-6 to distinguish between
bacteremic (positive blood cultures) and non-bacteremic (negative
blood cultures): 398.6 pg/ml for cancer patients, 120.9 pg/ml for noncancer patients and with 100% specificity, 100% sensitivity, NPV
100%, PPV 100%, Efficacy 100% for both groups. So the efficacy of IL6 as marker to discriminate between positive and negative blood
cultures was higher than that of CRP.The AUC was 0.844 and 1.000 for
CRP and IL-6 respectively in cancer patients. While, it was 0.875 and
1.000 for non- cancer patients.The diagnostic performance of CRP and
IL-6 and their combination (via multi-ROC) for discriminating patients
with positive cultures from those with negative cultures (all cancer and
non- cancer tested cases) resulted in that the AUC was improved from
0.735 for CRP only and 0.957 for IL-6 to 0.982 using the both
markers.The usefulness of both markers (IL-6 and CRP) was proved to
distinguish between bacteremic and non- bacteremic patients.On the
other hand IL-8 showed 100% values of positive predictive value,
negative predictive value, specificity and sensitivity.
For complement C3 there was non-significant differences among
the tested groups.
Also, there was direct proportionality between IL-6 serum levels
and WBCs count, meaning that IL-6 playing role in chemotaxis of white
blood cells to site of infection cancer patients.
For these critically ill patients (immunocompromised cancer
patients) continuous monitoring of antimicrobial resistance must be
considered.
MSc Thesis 2015
Summary
It was clear that, the highest resistance percentage for - lactam

antibiotics was observed with:
ampicillin/sulbactam (SAM) 80%,
amoxicillin/clavulanic acid (AMC) 66.66%, cefotaxime (CTX) 60%, then

cefepime
(FEP)
46.66%,
ceftazidime
(CAZ)
46.66%,
tazobactam/piperacillin (TZP) 40%. On the other hand, imipenem (IPM)

showed
no
resistant
phenotype
for
the
tested
isolates
but
intermediate resistant reaction 26.66%. An increase in resistance

phenotype was demonestrated after exposure to in-vitro gamma
irradiation for SAM, AMC, TZP. Meanwhile, for imipenem (IPM) 26.66%
intermediate
resistance
was
changed
to
6.66%
intermediate
resistance and 13.33% resistance. However, resistance against

ceftazidime (CAZ), cefepime (FEP) and cefotaxime (CTX) did not
change with radiation. While, for antibiotics inhibiting protein
synthesis, Tobramycin (TOB) showed decreased resistance from
73.33% to 66.66%, amikacin (AK) showed increased intermediate
resistance from 6.66% to 19.98% but resistance against gentamycin
(CN) remained unchanged 73.33%. Resistance to chloramphenicol (C)
increased from 40% to 46.66%.
In case of antibiotics acting on inhibition of nucleic acid
synthesis, ofloxacin (OFX) and levofloxacin(LEV) showed unchanged
resistance
73.33%
and
66.66%
respectively.
While,
for
sulphamethoxazole/trimethoprim (SXT) resistance increased from 80%

to 86.66% after exposure to in-vitro gamma radiation.
Also, there was significant p-value = 0.0086, 0.0198 and 0.05 for
AMC in E. coli strains, TOB in Pseudomonas strains and SAM in
Acinetobacter strains, respectively before and after exposure to invitro gamma irradiation.
Antimicrobial resistances of some selected bacterial strains
isolated from non- cancer patient was showed high percentage of
MSc Thesis 2015
Summary
resistance reaching 100% for SAM.So, with increasing antimicrobial

resistance, it is important to find antibiotics with enhanced activity
against resistant organisms. Antibiotics combinations are used to
enhance antibacterial efficacy and prevent the development of
resistance.
In cancer patients, it was clear that there are correlations
between lipase and protease enzymatic activity, antimicrobial
susceptibility and serum levels of IL-6 of some selected bacterial
isolates
Correlations were clear within bacterial isolates recovered from
cancer patients as E.coli isolates recovered from patients having high
values of serum IL-6 also showed high protease enzymatic activity and
antimicrobial resistance reaching 78.6%. While those without protease
activity were isolated from patients with low serum levels of IL-6 with
antimicrobial resistance reaching 71.4%. K. pneumoniae isolates
lipase activity and antimicrobial resistance reaching 78.7%. For
Pseudomonas species, isolates recovered from patients having high
values of serum IL-6 also showed protease and lipase activities. For
Acinetobacter baumannii, isolates recovered from patients with high
levels of serum IL-6 also showed lipase enzymatic activity with
antimicrobial resistance reaching 100%. While, for bacterial isolates
recovered from non-cancer patients, there was no clear correlation
between inflammatory mediators and virulence factors, except for P.
fluorescence isolate no.94 there was positive lipase and protease
activity.Accordingly, IL-6 serum concentration can be used as marker
of severity of infection in cancer patients.
In the animal model, for the different treated rat groups as well as
control groups, the rectal temperature, white blood cells count and IL-6
MSc Thesis 2015
Summary
serum concentrations were determined at the end of the experiment

from blood samples. Forty eight Wister rats were involved and divided
into 8 groups as follows:
Group (1): control group as the animals received 0.9% saline solution
Group (2): Radiation control group as the animals were exposed to low
(half of the group) and high (the other group half) gamma
radiation doses of 2 grays and 7.5 grays respectively.
Group (3): Fever of non-bacterial origin (Yeast fever group)
Group (4): Fever of non-bacterial origin (Yeast fever group) with
gamma radiation exposure
Group (5): Fever of bacterial origin (Pseudomonas aeruginosa)
Group (6): Fever of bacterial origin (Pseudomonas aeruginosa) with
Group (7): Fever of bacterial origin (Klebsiella pneumoniae)
Group (8): Fever of bacterial origin (Klebsiella pneumoniae) with
Measuring IL-6 serum concentrations comparisons between the
groups were studied.
In gamma radiation exposure groups (a) bacteremic fever (6,8)
and (b) non-bacteremic fever (4), it was observed that there was
significant difference in IL-6 serum concentration P-value = 0.0001for
group 6 and 4 and 5.9*10-8for group 8 and 4. While, in groups that
were not exposed to gamma radiation (a) bacteremic fever (5 and 7)
and (b) non-bacteremic fever(3) groups, there was significant
difference in IL-6 serum concentrations with P-value = 0.0001 and Pvalue = 0.000657
for groups 5,3 and 7,3,
respectively. For P.
aeruginosa bacteremic group with exposure to gamma radiation

(Group 6) and bacteremic group without exposure to gamma radiation
MSc Thesis 2015
Summary
(Group 5) there was significant difference in serum IL-6 concentration

P-value = 0.0412. For K. pneumoniae bacteremic group with exposure
to gamma radiation (Group 8) and bacteremic group without exposure
to gamma radiation (Group 7) there was non-significant difference in
serum IL-6 concentration, P-value = 0.27. There was significant
difference in IL-6 serum concentration between gamma radiation
exposure group (Group 2) and bacteremic group due to P. aeruginosa
and K. pneumoniae with exposure to gamma radiation (Group 6 and 8)
P- value = 0.0001 and 5.5*10-8 , respectively. For yeast group (Group 3)
and the same group with exposure to gamma radiation (Group 4)
there was non-significant difference in IL-6 serum concentration Pvalue
0.2726.
In
the
animal
model,
(ROC)forIL-6
serum
concentration inbacteremic groups (due to P. aeruginosa and K.

pneumoniae) with exposure to gamma radiation
and bacteremic
groups without exposure to gamma radiation showed a cutoff value of

25740 pg/ml with 75% specificity, 58.3% sensitivity, negative
predictive value 64.3% and positive predictive value 70%.
These results showed the potential usefulness of IL-6 and IL-8 as
early predictors of gram negative bacteremia than commonly used
markers such as CRP and complement C3. Also, usefulness of serum
IL-6 as marker of severity of infection in cancer patients to differentiate
causes of fever especially in patients under radiotherapy treatment.
MSc Thesis 2015
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MSc Thesis 2015

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MSc Thesis 2015
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MSc Thesis 2015
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MSc Thesis 2015

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) :( ) (7 , 5 )(
)) 3 IL-6 P = 0.0001 P
= 0.000657 53 73 .
) (6
) (5 IL-6P = . 0.0412
) (8
) (7
IL-6 P = . .0.27 IL-6

P . = 0.0001
) (2
) (5 )
(7 .P- = 0.0006518 IL-6
) (2
) 6 (8
P- = 0.0001 8-10 * 5.5 . ) (3
) (4
IL-6 P = .0.2726
MSc Thesis 2015
(ROC ) IL-6
) (
25740 /
75 58.3 64.3
.70
IL-6 IL-8
CRP .C3 IL-6

.
MSc Thesis 2015
"
:

"

) (

2007 ,

:
/

-
/

-
2015

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Early prediction of Gram

negative bacteremia in febrile

Amira Abuzeid Abdelbaset

Pharmacist at National Centre for Radiation Research and

Early prediction of Gram negative bacteremia in

Dr. Khaled Mohamed Anwar Aboshanab, Ph.D

2. Overall picture of gram-negative bacteremia...........................................

3. What happens when microorganism enters body?...................................

5. The role of chemokines in infectious diseases (Bacterial infections).........

6. The complement system............................................................................

7. Fever as a sign of infection.........................................................................

8. Blood cultures for identifying bacteremia.................................................

10. Ionizing Radiation.....................................................................................

11. Radiotherapy and inflammatory mediators............................................

12. The ROC curve analysis...........................................................................

MATERIALS AND METHODS....................................................................

2. Blood specimens .........................................................................................

MSc Thesis 2015

5. Blood culture bottles...................................................................................

6. Tubes used for plasma and sera separation................................................

11. Collection and manipulation of specimens ...............................................

12. Plasma and serum samples collection.......................................................

13. Isolation of pathogenic bacteria................................................................

14. Identification of isolated organisms from blood specimens......................

15. Total leukocytic count assay......................................................................

16. Assay of serum C-Reactive Protein (CRP)................................................

17. Assay of serum Inteleukin-6(IL-6)............................................................

18. Assay of serum Interleukin-8(IL-8)...........................................................

19. Assay of serum complement C3................................................................

20. Antimicrobial susceptibility of some pathogenic bacterial isolates...........

21. Effect of in-vitro gamma irradiation on some selected isolates.................

22.Detection of Lipase and Protease enzymes production..............................

24. Assay of IL-6 serum concentration in rats................................................

25. Statistical methods....................................................................................

1. Collection, isolation and identification of pathogenic bacteria recovered

3. Interleukin-6 serum levels (sIL-6) in cancer and non-cancer patients.......

4.Interleukin-8 serum levels (sIL-8) in cancer and non-cancer patients........

MSc Thesis 2015

8.Complement C3 serum levels (C3) in cancer and non-cancer patients.......

9. In-vitro effect of gamma irradiation on antimicrobial susceptibility of

10.Activity profiles of lipase and protease enzymes of some selected

11.Effect of gamma irradiation on IL-6 serum levels in rats with induced

12.Statistical analysis of IL-6 serum levels in bacteremic groups with and

MSc Thesis 2015

Analytical profile index system

Area under the curve

Carbohydrate recognition domain

Enzyme linked immunosorbent assay

Fever of unknown origin

Human immunodeficiency virus

Intensive care unit

MSc Thesis 2015

Potassium salt of ethylene diamine tetraacetic

Lipoplopysaccharide binding protein

Mannose binding lectin

monocyte chemoattractant protein -1

Messenger Ribonucleic acid

Negative predictive value

pathogen-associated molecular patterns

Positive predictive value

Receiver Operating Characteristic

Systemic inflammatory response syndrome