Early prediction of Gram negative bacteremia in febrile cancer patients: Correlation between some inflammatory mediators, exposure to gamma radiation and severity of infectionA Thesis

Submitted in Partial Fulfillment of the Requirements for the

Master degreeIn Pharmaceutical Sciences(Microbiology andImmunology)ByAmira Abuzeid AbdelbasetBachelor of Pharmaceutical sciences, 2007Pharmacist 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 degreeIn Pharmaceutical Sciences(Microbiology andImmunology)

By

Amira Abuzeid AbdelbasetPharmacist atNational Centre for Radiation Research and Technology (NCRRT)

Under Supervision of

Dr. Mohammad Mabrouk Aboulwafa, PhDProfessor of Microbiology and Immunology,Faculty of Pharmacy, Ain Shams University

Dr. Hala Abdallah Farrag, PhDProfessor of Medical Microbiology,National Centre for Radiation Research and Technology (NCRRT)

Dr. Khaled Mohamed Anwar Aboshanab, Ph.DAssociate Professor of Microbiology and Immunology,Faculty of Pharmacy, Ain-Shams University

2015AcknowledgmentsFirst, 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.

........Amira Abuzeid Abdelbaset

Table of ContentsACKNOLEDGEMENT.......LIST OF ABBREVIATIONSITABLE OF CONTENTSIVLIST OF FIGURESVILIST OF TABLESVIIIABSTRACT1INTRODUCTION4LITERATURE REVIEW7Bloodstream infection7Overall picture of gram-negative bacteremia11What happens when microorganism enters body?12Cytokines16The role of chemokines in infectious diseases (Bacterial infections)19The complement system22Fever as a sign of infection23Blood cultures for identifying bacteremia25Antimicrobial resistance26Ionizing Radiation27Radiotherapy and inflammatory mediators3412. The ROC curve analysis35MATERIALS AND METHODS39MATERIALS391. Microorganisms 392. Blood specimens 393. Media 394. Kits395. Blood culture bottles406. Tubes used for plasma and sera separation407. Diffu-plate408. Antibiotic discs409. Laboratory animals4010. Devices41METHODS4211. Collection and manipulation of specimens 4212. Plasma and serum samples collection4213. Isolation of pathogenic bacteria4214. Identification of isolated organisms from blood specimens4315. Total leukocytic count assay4416. Assay of serum C-Reactive Protein (CRP)4417. Assay of serum Inteleukin-6(IL-6)4518. Assay of serum Interleukin-8(IL-8)4619. Assay of serum complement C34820. Antimicrobial susceptibility of some pathogenic bacterial isolates4821. Effect of in-vitro gamma irradiation on some selected isolates5022.Detection of Lipase and Protease enzymes production5123. Effect of gamma radiation on IL-6 serum levels in rats with induced fever of bacterial and non-bacterial origin5124. Assay of IL-6 serum concentration in rats5325. Statistical methods54RESULTS551. Collection, isolation and identification of pathogenic bacteria recovered from blood cultures552.Age, sex, white blood cells count and serum CRP levels of cancer and non-cancer patients553. Interleukin-6 serum levels (sIL-6) in cancer and non-cancer patients614.Interleukin-8 serum levels (sIL-8) in cancer and non-cancer patients635.Relationship between CRP, sIL-6 and sIL-8 in some selected cancer and non-cancer patients with positive and negative blood cultures646.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 cultures677.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)708.Complement C3 serum levels (C3) in cancer and non-cancer patients789. In-vitro effect of gamma irradiation on antimicrobial susceptibility of some bacterial isolates recovered from cancer and non-cancer patients7910.Activity profiles of lipase and protease enzymes of some selected bacterial isolates recovered from cancer and non-cancer patients9111.Effect of gamma irradiation on IL-6 serum levels in rats with induced fever of bacterial and non-bacterial origin9212.Statistical analysis of IL-6 serum levels in bacteremic groups with and without exposure to gamma radiation by Receiver Operating Characteristic Curve (ROC)97

DISCUSSION100SUMMARY114REFERENCES120

Table of Contents

XIMSc Thesis 2015List of Abbreviations

ADHArginine dihydrolase

AKAmikacin

AMCAMLAmoxacillin/Clavulanic AcidAcute Myeloid Leukemia

APIAnalytical profile index system

AUCArea under the curve

BALBroncho-alveolar lavage

BSIBloodstream infection

CChloramphenicol

C3Complement 3

CAZCeftazidime

CITCitrate

CNGentamicin

CRDCarbohydrate recognition domain

CRPC-reactive protein

CTXCefotaxime

DNADeoxyribonucleic acid

EffEfficacy

ELISAEnzyme linked immunosorbent assay

FEPFNCefepimeFebrile neutropenia

FUOFever of unknown origin

GELGelatin

GyGray

H2Shydrogen sulfide

HIVHuman immunodeficiency virus

HRPHorseradish peroxidase

i.pIntraperitoneal

ICUIntensive care unit

IFNInterferon

IL-1Interleukin-1

IL-2Interleukin-2

IL-6Interleukin-6

IL-8Interleukin-8

IPMImipenem

K2EDTAPotassium salt of ethylene diamine tetraacetic acid

LDClysine decarboxylase

LEVLevofloxacin

LOSLipooligosaccharide

LPBLipoplopysaccharide binding protein

LPSLipopolysaccharides

LTALipoteichoic acid

MBLMannose binding lectin

MCP-1monocyte chemoattractant protein-1

mRNAMessenger Ribonucleic acid

N2Nitrogen gas

NO2Nitrite

NPVNegative predictive value

ODOptical density

ODCornithine decarboxylase

OFXOfloxacin

PAMPpathogen-associated molecular patterns

PBSphosphate-buffered saline

PCTProcalcitonin

PMNsPolymorphnuclear leukocytes

PPVPositive predictive value

ROCReceiver Operating Characteristic

SAMAmpicillin/ Salbactam

SIRSSystemic inflammatory response syndrome

SnSensitivity

SpSpecificity

SXTSulphamethoxazole/ trimethoprim

TLRToll like receptor

TMB3,3,5,5-Tetramethylbenzidine

TNF-Tumor necrosis factor-

TOBTobramycin

TRRectal temperature

TZPTazobactam

UREUrease

UTIUrinary tract infection

VPVoges-Proskauer

WBCsWhite blood cells

List of Abbreviations

List of FiguresFigure 1Distribution of overlapped test results 36

Figure 2The ROC curve distribution between Sensitivity and Specificity38

Figure 3 BD BACTEC 9050 Blood culture System41

Figure 4Standard calibration curve of Human IL-6 serum concentration as determined by ELISA technique62

Figure 5Relationship 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 66

Figure 6Relationship between IL-8 and Il-6 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, respectively66

Figure 7Relationship between IL-6 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 15 and 9, respectively67

Figure 8Relationship 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, respectively69

Figure 9Relationship 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 respectively69

Figure 10Relationship 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 69

Figure 11ROC curve analysis showing the diagnostic performance of CRP and IL6 for discriminating patients with positive culture from those negative among cancer patients72

Figure 12ROC curve analysis showing the diagnostic performance of CRP and IL6 for discriminating patients with positive culture from those with negative culture among non- cancer patients74

Figure 13ROC curve analysis showing the diagnostic performance of CRP and IL6 and their combinations for discriminating patients with positive culture from those negative (cancer and non- cancer patients)78

Figure 14Frequency 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 84

Figure 15Frequency percentages of different susceptibility profiles (sensitive, intermediate resistant and resistant) of some selected bacterial isolates recovered fromcancer patients against some antimicrobial agents inhibiting protein synthesis in bacteriabefore and after gamma irradiation 85

Figure 16Frequency percentages of different susceptibility profiles (sensitive, intermediate resistant and resistant) of some selected bacterial isolates recovered fromcancer patients against some antimicrobial agents inhibiting nucleic and folic acids syntheses before and after gamma irradiation 86

Figure 17Frequency percentages of different susceptibility profiles (sensitive, intermediate resistant and resistant) of some selected bacterial isolates recovered from non-cancer patients against some antimicrobial agents inhibiting bacterial cell wall synthesis before and after gamma irradiation 88

Figure 18Frequency percentages of different susceptibility profiles (sensitive, intermediate resistant and resistant) of some selected bacterial isolates recovered from non-cancer patients against some antimicrobial agents inhibiting protein synthesis in bacteria before and after gamma irradiation 89

Figure 19Frequency percentages of different susceptibility profiles (sensitive, intermediate resistant and resistant) of some selected bacterial isolates recovered from non-cancer patients against some antimicrobial agents inhibiting nucleic and folic acids syntheses before and after gamma irradiation 90

Figure 20ROC 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 radiation99

List of Figures

List of TablesTable 1Characters of antibiotic discs used for antimicrobial susceptibilitytesting of collected bacterial isolates

40

Table 2The interpretive standard (breakpoint) for all selected antibiotics

49

Table 3Rat groups used for testing the effect of gamma radiation on serum level of IL-6

52

Table 4Number and frequency of positive and negative cases for microbial growth of blood culture samples from cancer and non-cancer patients

55

Table 5Age, sex, white blood cells count, serum CRP measurements and bacterial species isolated from cancer patients with bacterial infection (positive blood cultures)

56

Table 6Age, sex, white blood cells count and serum CRP measurements of cancer patients without bacterial infection (negative blood cultures)

57

Table 7Age, sex, white blood cells count serum CRP measurements and bacterial species isolated from non-cancer patients with bacterial infection (positive blood cultures)

58

Table 8Age, sex, white blood cells count and serum CRP measurements of non-cancer patients without bacterial infection (negative blood cultures)

59

Table 9Numbers and percentages of different bacterial species recovered from cancer and non-cancer patients

60

Table 10Levels of CRP of some cases of cancer and non-cancer patients with positive and negative blood cultures selected for statistical analysis61

Table 11Levels of serum IL-6 in some cases of cancer and non-cancer patients with positive and negative blood cultures selected for statistical analysis

63

Table 12Levels of serum IL-8 in some cases of cancer and non-cancer patients with positive and negative blood cultures selected for statistical analysis

64

Table 13Summarization of Serum levels of CRP, IL-6 and IL-8 in cancer and non-cancer patients with positive and negative blood cultures:

65

Table 14Mean values of tested serum markers (CRP, IL-6 and IL-8) in some selected cancer and non-cancer patients66

Table 15Relationship 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

68

Table 16Diagnostic validity test for serum CRP to discriminate positive and negative blood cultures in cancer patients

71

Table 17Diagnostic validity test for serum IL-6 to discriminate positive and negative blood cultures in cancer patients

72

Table 18Diagnostic validity test for serum CRP to discriminate positive and negative blood cultures in non- cancer patients

73

Table 19Diagnostic validity test for serum IL-6 to discriminate positive and negative blood cultures in non- cancer patients

74

Table 20Diagnostic validity test for serum CRP to discriminate positive and negative blood cultures (cancer and non- cancer patients)

75

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

76

Table 22Multi-ROC:determining the value of CRP with IL6 at 120.9

77

Table 23Levels of serum C3 in some selected cancer and non-cancer patients with positive and negative blood cultures79

Table 24Antibacterial susceptibilities of some selected bacterial isolates recovered from cancer patients against different antimicrobial agents before and after gamma irradiation exposure

81

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

82

Table 26Profiles 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

92

Table 27White blood cells count and IL-6 serum concentration in control group

93

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

93

Table 29Rectal temperatures, white blood cells count and IL-6 serum concentrations infever of non-bacterial origin (yeast fever) group94

Table 30Rectal temperatures, white blood cells count and IL-6 serum concentrations in fever of non-bacterial origin (Yeast fever) group with gamma radiation exposure

94

Table 31Rectal temperatures, white blood cells count and IL-6 serum concentrations in fever of bacterial origin (Pseudomonas aeruginosa) group

95

Table 32Rectal temperatures, white blood cells count and IL-6 serum concentrations in fever of bacterial origin (Pseudomonas aeruginosa) group with gamma radiation exposure

95

Table 33Rectal temperatures, white blood cells count and IL-6 serum concentrations in fever of bacterial origin (Klebsiella pneumoniae) group

95

Table 34Rectal temperatures, white blood cells count and IL-6 serum concentrations in fever of bacterial origin (Klebsiella pneumoniae) group with gamma radiation exposure

96

Table 35ROC analysis data of sIL-6 for bacteremic groups with and without exposure to gamma radiation

98

List of Tables

AbstractOne hundred twenty four feverish (cancerand non- cancer) in-patients 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 non- cancer 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 non-significant differences among all groups. The Cutoff levels to distinguish between bacteremic (positive blood cultures) and non-bacteremic (negative blood cultures) cases 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 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% .

Abstract

3MSc Thesis 2015IntroductionThe 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).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 (IL-8), 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).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: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 negative bacterial infection.4- 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.

Introduction

18MSc Thesis 2015Literature Review1. 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 a 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).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 Gram-negative 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. 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 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 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 pathogen-associated molecular patterns, which are components that bind to Toll-like receptor (TLR2) and (TLR4) on leukocytes, resulting in the production of inflammatory cytokines (Abdallah et al., 2008).Bloodstream infection (BSI) continues to be a life threatening 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 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 gram-positive 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 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 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, cell-mediated 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).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 Gram-negative 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 bacteria or endotoxin are not needed as direct stimuli for intravascular inflammation (Munford, 2006).3. 1. Host recognition of microbial componentsCarrigan 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 amplificationFollowing 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 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 IL-12, IL-15 and IL-18, and a host of other small molecules. TNF-a and IL-1 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 pro- and 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 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 a 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).Department of Morphological Biology (YS,ET) and Department of Oral Growth & Development (TU,MH,KI,HK,HI), Fukuoka Dental College, Fukuoka, Japan, and Department of Oral Functional Science, Graduate School of Dental Medicine, Hokkaido University, Sapporo, Japan (SY)Correspondence to: Yoshihiko Sawa, Department of Morphological Biology, Fukuoka Dental College, 2-15-1 Tamura, Sawara-Ku, Fukuoka, 814-0193, Japan. E-mail: ysawa@college.fdcnet.ac.jp

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. CytokinesCytokines 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 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. IL-1 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 CytokinesMany 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 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 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 systems.E. coli, P. aeruginosaand 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 other organ systems has been shown. Elderly patients with lower respiratory tract bacterial infections demonstrate elevated levels of IL-8 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 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 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 administration of appropriate antimicrobial drugs play a crucial role in 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 systemComplement represents one factor of this significant host defence system involved in the immediate protection of the host from bacteria (Wolbink et al., 1998). 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 (Muller-Eberhard 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, several observations indicate that additional 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).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.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 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. 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 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 resistanceBacterial 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 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). 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: 137Cs and 60Co (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 MaterialsAlabostro 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 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 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 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. Effect of Ionizing Radiation on MicroorganismsExposure 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 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)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 diffusion 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 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 waterThe 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. Earl