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Contents Alexandria Journal of Hepatogastroenterology, Volume (XVII) - December 2015

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Original Article:

A Comparative Study Between Medical and Endoscopic Treatment of Esophageal Varices in Liver Cirrhosis

Abdel Fattah Fahmy Hanno, Akram Abdelmoneim Deghady, Hosam Fathy Abo El khair, Ayman Said Saad, Mohamed Taalab

------------------------------------------- Original Article:

Characterization of SHV Extended-Spectrum ß-Lactamases in Enterobacteriaceae

Ayman M. EL-Lehleh, Amira A. EL-Hendi, Abeer H. EL-Shalakany and SherenMosa.


Clinical Significance of Cerebrospinal Fluid Nitric Oxide Level in Patients with Meningitis

Ali Mahmmoud El-Kadi, Magdy Mamdouh El-Bardini, Ehab Hassan Abdelmaksood Elkholy, Mohamed Hamdy Abd-Esalam Ibrahim


Comparison of Azithromycin Plus Gentamicin versus WHO Recommended Therapy For The Treatment of Complicated and Uncomplicated Brucellosis

AlaaM.Abdo, NasserM. Abdullah, Ehab H. A. El-Khouly, Khaled Ebn El-Walid


Detection of SHV and TEM Genes in Escherichia Coli and Klebsiella Isolates

Azza Mohammed Abed-El Aziz, Ghada Rashad Hendawy, Tawfiek Mohammed Abed-El Motaleb, Amina Mohammed Elsaadany


Occult Hepatitis B Infection in Patients with Chronic Hepatitis C Associated Hepatocellular Carcinoma in Mansoura, Egypt

Mahmoud Abdel-Aziz, Azza Abdel-Aziz, Othman Zaki


Prevalence of Transfusion Transmitted Virus (TTV) Among HCC Patients and HCV Infection in Damietta Governorate

Samy Zaky , Mohamed Afify, Amgad Al Zahaby, Abd-Alla El-Bialy, Khalid El-Molla, Amin Abdel Baki, Ashraf Z. ElSayed.


Study of Prevalence of Spontaneous Bacterial Peritonitis in Patients with Liver Cirrhosis with Ascites in Menoufiya Governate

Atef Abo ELsoud Ali, Wafaa Ahmed Ibrahim Zahran, Fatma Mohamed Abdelfatahabdo


Study of The Diagnostic Role of Platelet Indices in Patients with Ulcerative Colitis

Hossam Ibrahim Mohamed, Abd Allah Said Essa, Enas Said Essa, Heba Hamdy.


Thyroid Diseases in Egyptian Patients with HCC: Case -Control Study

Essam S. Bedewy, Ahmed I. Ellakany.

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Original Article

A Comparative Study Between Medical and Endoscopic Treatment of Esophageal Varices in Liver Cirrhosis Abdel Fattah Fahmy Hanno*, Akram AbdelmoneimDeghady*, Hosam Fathy Abo El khair*, Ayman Said Saad*, Mohamed Taalab** Department of Tropical Medicine, Faculty of Medicine University of Alexandria

ABSTRACT Liver cirrhosis is the 14th most common cause of death in adults worldwide. The most common cause of cirrhosis worldwide is NASH,but in Egypt the most common cause is chronic HCV. Portal hypertension is the most common complication of liver cirrhosis. Oesophageal varices is the most common complication of portal hypertension. Treatment of portal hypertension includes medical treatment using non selective beta blockers ( NSBB e.g. propranolol or carvedilol), endoscopic treatment esophageal band ligation ( EVL) orendoscopic sclerotherapy ( EIS), or both. Carvedilol is two to four times more potent than propranolol as a β-receptor blocking drug. Recently it is used in treatment of portal hypertension either alone or mixed with EVL. Aim of the work :The purpose of this study was to compare between medical treatment and band ligation in the treatment of esophageal varices secondary to liver cirrhosis. Methods: The study was done on 60 patients with liver cirrhosis, classified into three groups :Group I: 20 patients with esophageal varices (OV).grade I and grade II ; received carvedilol. Group II: 20 patients with OV.grade III and IV; were subjected to EVL. Group III: 20 patients with OV.grade III and IV; were treated with combination of carvedilol and EVL. Evaluation included:Full history taking, clinical examination , laboratory investigations including; Complete blood count (CBC),renal function tests, liver function tests, markers of autoimmune hepatitis: Anti-nuclear antibodies (ANA), Anti-smooth muscle antibodies (ASMA) and Anti-mitochondrial antibodies (AMA), PCR examination for viral hepatitis. Imaging procedures: included ultrasonographic abdominal examination , Echo doppler study of the portal vein and upper gastrointestinal (GI.) endoscopy. Results: it was observed that there was positive correlation between pulse and both systolic and diastolic blood pressure in group I and group III , positive correlation between diameter of portal vein and grade of OV. and negative correlation between serum albumin and degree of ascites in the three studied groups.Conclusion: carvedilol was as effective as band ligation in the treatment of small and medium sized OV and the combination of carvedilol and EVL is more effective than EVL alone in the treatment of large sized OV. Introduction Cirrhosis is an increasing cause of morbidity and mortality in developed countries. It is the 14th most common cause of death in adults worldwide but the fourth in central Europe; it results in 1·03 million deaths per year worldwide(1); 170 000 per year in Europe,(2) and 33 539 per year in the USA.(3)the annual incidence was 15·3–132·6 per 100 000 people in studies in the UK and Sweden.(2) Cirrhosis is the main indication for 5500 liver transplants each year in Europe (2).The main causes in developed countries are infection with hepatitis C virus, alcohol misuse, and, increasingly, non-alcoholic liver disease; while infection with hepatitis B virus is the most common cause in subSaharan Africa and Asia. The prevalence of cirrhosis is di�cult to assess and probably higher than reported, because the initial stages are asymptomatic. The most common complication of liver cirrhosis is portal hypertension (PHT). Portal hypertension is the underlying cause of

most of other complications of liver cirrhosis. The most common complication of portal hypertension (PHT) is esophageal varices. esophageal varices are present in 40% of patients with Child–Pugh A cirrhosis and in 60% of patients Child–Pugh B and C.(4,5) varices are classified by their location as esophageal or gastric varices. Esophageal varices are graded by their size. In contrast, gastric varices are classified primarily by their location as follows; gastroesophageal varices (GEV) or isolated gastric varices (IGV). 7% of small varices (<5 mm) will bleed over a 2-year period compared to 30% of large varices.(5) The risk of subsequent hemorrhage is determined by the degree of underlying hepatic dysfunction, size of varices and presence of high risk stigmata at endoscopy.Carvedilol is a 3rd generation b-blocker with a nonselective beta-adrenoreceptor blocking effect. It is used in treatment of hypertension,coronary heart disease,hypertrophic cardiomyopathy, heart failure and portal

hypertension. Side effects of carvedilol include dizziness,fatigue,lowbloodpressure,diarrhea,weakness,bradycardia, and weight gain. Carvedilol has been a relatively newer addition to the NSBBs, in the treatment of portal hypertension and has demonstrated promising results in terms of clinical outcomes(6).Carvedilol has proven 2-4 times more potent than propranolol as a beta-receptor blocker.

Patients and Methods The study was conducted on 60 patients with liver cirrhosis, classified into three groups:Group I: 20 patients with OV grade I and grade II; received carvedilol. Group II: 20 patients with OV grade III and grade IV; were subjected to EVL. Group III: 20 patients with OV grade III and grade IV; were treated with combination of carvedilol and EVL. All patients were followed up by upper GI endoscopy for assessment every 3 months up to 12 months.All patients were subjected to the following: Full history taking, clinical examination , laboratory investigations including; Complete blood count (CBC),renal function tests,liver function tests,markers of autoimmune hepatitis: Anti-nuclear antibodies (ANA).Anti-smooth muscle antibodies (ASMA).Anti-mitochondrial antibodies (AMA), PCR testing for viral hepatitis. Imaging procedures :Ultrasonographic examination of the abdomen ,Doppler study of the portal vein and upper GI endoscopy and endoscopic Variceal band ligation according to the protocol. Results Group I included 20 patients, 11 males(55%) and 9 females(45%) with a mean age of 50.85 ± 8.65, while group II included 20 patients,15 males (75%) and 5 females(25%) with a mean age of 57.10 ± 6.07 and group III included 20 patients, 16 males(80%) and 4 females(20%) with a mean age of 55.90 ± 8.42.There was statistically significant difference between the three studied groups regarding to age (P= 0.033).As regards the systolic blood pressure, in group (I), in the 1st visit the mean value was 118.75 ± 5.59 and in the 4th visit the mean value was 110.40 ± 5.49. It was significantly lower in the 4th visit than the 1stvisit(P=<0.001).in group (II), in the 1st visit the mean value was 118.75 ± 5.59 and in the 4th visit the mean value was 119.30 ± 5.28. It was significantly higher in the 4th visit than the 1stvisit(P=<0.001). in group (III)

in the 1st visit the mean value was 116.40 ± 3.56, in the 4th visit the mean value was 108.70 ± 3.85. It was significantly lower in the 4th visit than the 1st visit(P=<0.001). Reregarding to the diastolic blood pressure, in group (I), in the 1st visit the mean value was 72.50 ± 5.50 and in the 4th visit the mean value was 64.05 ± 5.10. It was significantly lower in the 4th visit than the 1st

visit(P=<0.001).in group (II), in the 1st the mean value was 72.50 ± 5.50 and in the 4th visit the mean value was 72.25 ± 5.98. It was significantly lower in the 4th visit than the 1st

visit(P=<0.001). in group (III), in the 1st visit the mean value was 69.70 ± 5.60 and in the 4th visit the mean value was 61.65 ± 4.86. It was significantly lower in the 4th visit than the 1st

visit(P=<0.001). Regarding to the pulse, in group (I), in the 1st visit the mean value was 81.75 ± 5.15 and in the 4th visit the mean value was 74.55 ± 4.68. It was significantly lower in the 4th visit than the 1st visit(P=<0.001).in group (II), in the 1st visit the mean value was 81.75 ± 4.47 and in the 4th visit the mean value was 81.70 ± 4.43. iIt was significantly lower in the 4th visit than the 1st visit(P=<0.001). in group (III), in the 1st visit the mean value was 81.60 ± 4.89 and in the 4th visit the mean value was 74.50 ± 5.04. It was significantly lower in the 4th visit than the 1st

visit(P=<0.001).As regards Hb, in Group (I), the mean value was 11.84 ± 0.98. in group (II) the mean value was 10.85 ± 1.02 and in group (III), the mean value was 11.03 ± 0.91. Hemoglobin level (Hb )was significantly lower in group II than the other 2 groups (P=0.005). Regarding to platelets count, in Group (I) the mean value was 126.70 ± 15.66, in Group (II) the mean value was 96.65 ± 13.46 and in Group (III) the mean value was 98.20 ± 15.38. platelets count was significantly lower in group II than the other 2 groups (P=0.005). Regarding to the serum albumin level, in group (I), in the 1st visit the mean value was 3.18 ± 0.17 and in the 4th visit the mean value was 3.36 ± 0.12. It was significantly higher in the 4th visit than the 1stvisit (P=<0.001). In group (II), in the 1st visit the mean value was 2.49 ± 0.28 and in the 4th visit the mean value was 2.81 ± 0.26. It was significantly higher in the 4th visit than the 1stvisit (P=<0.001). Ingroup (III), in the 1st visit the mean value was 2.54 ± 0.30 and in the 4th visit the mean value was 2.90 ± 0.25. serum albumin was significantly higher in the 4th visit than the 1st visit (P=<0.001). Regarding to the

diameter of the portal vein, in the 1st visit in group I the mean value was 14.02 ± 0.30, in group II the mean value was 16.16 ± 0.63 and in group III the mean value was 15.52 ± 0.63. in the 4th visit in group I the mean value was 12.61 ± 0.14, in group II the mean value was 13.15 ± 0.25, in group III the mean value was 12.99 ± 0.22. the diameter of portal vein was significantly smaller in the 4th visit than the 1st visit in the 3 studied groups. the percentage of change between 1st& 4th visits in group II was significantly higher than the other 2 groups.Regarding to the degree of oesophageal varices (OV)in group (I), in the 1st visit 30.0% of patients had OV grade II and 70.0% had OV. grade I and in the 4th visit 100.0% of patients had no OV. In group (II), in the 1st visit 95.0% of patients had OV grade IV and 5.0% had OV grade III and in the 4th visit 80.0% of patients had OV grade I and 20.0% had no OV In group (III), in the 1st visit 20.0% of patients had OV grade IV and 80.0% had OV grade III and in the 4th visit 40.0% of patients had OV grade I and 60.0% had no OV . Varices were significantly smaller in the 4th visit than the 1st visit in the 3 studied groups.There was positive correlation between pulse and both systolic and diastolic BP in group I and group III. There was positive correlation between the diameter of the portal vein and the grade of OV in the three studied groups. There was significantly negative correlation between the degree of ascites and the level of serum albumin in the three studied groups. Discussion The aim of this work was to compare between medical treatment and band ligation in the treatment of esophageal varices secondary to liver cirrhosis.The present was conducted on 60 patients(42 males and 18 females) divided into three groups. Group I, 20 patients with esophageal varices grade I and grade II, received medical treatment with carvedilol ( in a dose of 6.25 mg carvedilol once daily, then increased to 6.25 mg twice a day after an interval of 1 week ), group II, 20 patients with esophageal varices grade III and grade IV, were subjected to EVL by endoscopy and group III, 20 patients with esophageal varices grade III and grade IV received combined therapy with carvedilol and EVL.In this study there was no statistical significant difference between the 3 studied

groups regarding to gender. this finding could be due to uneven distribution of the gender in this study. There was statistically significant difference between the 3 studied groups regarding to age. In this study, bleeding from esophageal varices in group II was significantly higher than group I and group III.that would be explained by that patients in group II had more advanced liver disease(Child C class ) than the other 2 groups. In the present study , systolic and diastolic blood pressure were significantly lower in the 4th visit than the 1st visit in group I and group III, but they were significantly higher in the 4th visit than the 1st visit in group II.that would be explained by the beta blockade effect of carvedilol which reduces the force of heart pumping, and the alfa blockade effect of carvedilol which leads to arterial vasodilatation. In the present study pulse was significantly lower in the 4th visit than the 1st visit in group I and group III. That would be explained by the beta blockade effect of carvedilol (on β1receptors) which slows the heart rate.There was positive correlation between pulse and both systolic and diastolic systemic blood pressure (BP) in group I and group III. that would be explained by the effect of carvedilol used in both groups which reduces pulse and BP. Smith J et al(7) in USA conducted a study on 200 patients having bleeding esophageal varices and reported that there was a positive correlation between pulse and blood pressure and hemoglobin concentration. This was in accordance with present study. In the present study, Hb and platelets count were significantly lower in group II than the other 2 groups. This would be explained by the higher variceal bleeding rate in group II (60% of patients were bleeders) than the other 2 groups, and the higher rate of hypersplenism in group II than the other 2 groups. Garcia- Tsao et al.(8) (180 patients), Pilette et al(9) (116 patients) and Thomopoulos et al.(10) (184 patients) found that platelet count <118 x 103 was good predictor for presence of varices and reported a low platelet count to be an independent risk factor for the presence of varices. Khuram et al . (11) (200 patients) found OV in 146 with 121 having thrombocytopenia (94.5%). In retrospective analysis of 143 patients with compensated cirrhosis, Schepis et al(12) reported OV in 63 patients (44%) with platelet count of <100,000 as predictor of OV. This was matched with present study. In the present study

serum albumin was significantly higher in the 4th visit than the 1st visit in the 3 studied groups.That would be attributed to the effect of carvedilol and EVL on reducing the rate of bleeding and rebleeding from esophageal varices leading to reducing blood loss and subsequently reducing albumin loss, and the good response of patients to the albumin infusions they received during the period of the study. There was significantly negative correlation between the degree of ascites and the level of serum albumin in the 3 studied groups. This was explained by the gradual elevation of the level of serum albumin in all patients leading to gradual increase of the plasma oncotic pressure with reduction of the amount of ascites. Gurubacharya DL et al(13) found that 25 of 32 (78.13%) patients had High SAAG and 7 of 32 (21.87%) had Low SAAG. Esophageal varices were present in 18 of 25 (72%) patients with High SAAG and in none of 7 (0%) patients with Low SAAG (p =< 0.001). As patients with high SAAG had low levels of serum albumin. This is corrletaed with present study. In the present study,100.0% of all patients in the three studied groups had liver cirrhosis. There was no statistically significant difference between the 3 studied groups regarding to viral hepatitis, but HCV was higher than HBV. Shah ha et al.(14), found that the etiology of liver cirrhosis was predominately HCV. This was similar to the present study. In the present study, the diameter of portal vein was significantly smaller in the 4th visit than the 1st visit in the 3 studied groups. The percentage of change between 1st& 4th visits in group II was significantly higher than the other 2 groups. This is explained by the good response of all patients to both carvedilol and EVL via reduction of portal blood pressure.In the present study, OV were significantly smaller in the 4thvisit than the 1st visit in the 3 studied groups. This is explained by the good response of all patients to both carvedilol and EVL leading to reduction of portal blood pressure and reduction of size of OV. In the present study, there was positive correlation between the diameter of the portal vein and the grade of OV thin is attributed to the good response of all patients to both carvedilol and EVL .. William F et al15) in Canada found a positive correlation between the diameter of portal vein and the success rate of EVL. This correlated with present study.Shah ha et al.(14) reported a high rate of variceal

eradication (75%) in the EVL arm. In the present study variceal eradication rate was (20%) in the EVL arm, but in carvedilol arm a variceal eradication rate was (100%) and in the mixed arm (carvedilol and EVL) variceal eradication rate of (60%) . This is explained by the larger number of patients in Shah ha et al.(14) study the present study ( 168 patients versus 60 patients). D’Amico et al(16) demonstrated that in patients with varices of any size, β-blockers reduced the risk of a first bleeding episode from 25% to 15% within 2 years. The absolute risk difference was 9 % (15% vs 24%) as compared to placebo. Moreover, the absolute risk reduction in mortality was found to be 4% (from 27% to 23%).Teran JC et al17) has reported the usage of Beta blockers as primary prophylaxis to be associated with a 40% reduction in bleeding risk and a trend towards improved survival. In Tripathi D et al(18) study Carvedilol has been compared with EBL and showed a significantly lower rate of first variceal bleeding in patients taking carvedilol 12.5 mg daily compared with EBL (10% vs 23%, HR = 0.41, 95%CI: 0.19-0.96). The lowest dose of carvedilol tested in this trial was 12.5 mg, which is known to cause a smaller reduction in hepatic venous pressure gradient ( HVPG )..Bernard B et al(19) conducted a study on secondary prophylaxis of variceal bleeding and showed that, the use of β-blockers was associated with an increase in mean percentage of patients with no re-bleeding (21% mean improvement rate and the mean survival rate (5.4% mean improvement rate), the mean percentage of patients free of death due to bleeding (7.4%). Lo et al(20), reported 17% rate of rebleeding with EBL vs. 33% with sclerotherapy, Villanueva et al.(21) reported 12% incidence rate for re-bleeding for EVL versus 21% for sclerotherapy. Conclusion Carvedilol is as effective as band ligation in the treatment of small and medium sized OV and The combination of carvedilol and EVL was more effective than EVL alone in the treatment of large sized OV.There was a positive correlation between diameter of portal vein and grade of OV. Dilatation of portal vein could be a predictor of portal hypertension and the minimal effective dose of carvedilol is 12.5 mg daily with which the side effects are minimal.

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Original Article

Characterization of SHV Extended-Spectrum ß-Lactamases in Enterobacteriaceae Ayman M. EL-Lehleh*, Amira A. EL-Hendi**, Abeer H. EL-Shalakany** and SherenMosa**, * Tropical department, faculty of Medicine, Menoufiya University, ** Clinical pathology department, faculty of Medicine, Menoufiya University

ABSTRACT Resistance to ß-lactam antibiotics is an increasing problem especially in Enterobacteriaceae. Therefore, detection of ESBLs in Enterobacteriaceae is crucial for optimal treatment of patients and to control the spread of resistance. The SHV-type ESBLs may be more frequently found in clinical isolates than any other type of ESBLs Aim of the work is to study the prevalence of SHV ESBLs in Enterobacteriaceae among patients conducted at Menoufiya University hospitals. Methods: The isolated strains of Enterobacteriaceaeare subjected to screening tests for ESBLs by disc diffusion then to confirmatory tests for ESBLs by double disc test. Finally, molecular detection of SHV gene by PCR. Results: 97 of 160 (60.6%) isolates were found to be ESBL producers by double disc test, Out of 97 ESBL producers, 45(28.1%) were E. coli, 28(17.5%) were Enterobacter and 22 (13.7%) were Klebsiella spp., Of the various clinical samples, The most frequent ESBLs isolates were from urine samples 16.9%, followed by sputum samples 11.3%. 70 of 97 (72.2%) ESBL-producing strains were positive for SHV gene by PCR. Conclusion: High prevalence of ESBL producers in our hospital (60.6%) calls for strict policies regarding antibiotic usage ,hospital-based clinical laboratories should screen isolates following hospitalization in patients in need for antibiotics to formulate effective antibiotic strategy and plan a proper hospital infection control strategy to prevent the spread of these ESBL strains.

Introduction Resistance to ß-lactam antibiotics is an increasing problem and ß lactamase production is the most common mechanism of drug resistance, especially in Enterobacte-riaceae(1).ESBL producing strains are a major problem in many hospitals leading to increased morbidity, mortality and health care costs. Cephalosporins are first line drugs in treatment of infections caused by Enterobacteriaceae family, however, the extensive use of third generation Cephalosporins has resulted in increased prevalence of ESBL and plasmid mediated AmpC among organism (2).The β-lactamases confer significant antibiotic resistance to their bacterial hosts by hydrolysis of the amide bond of the β-lactam ring. These enzymes are especially important in Gram-negative bacteria as they constitute the major defense mechanism against β-lactam-based drugs (3). The clinical and laboratory standard institute (CLSI) interpretive guidelines stated that, Enterobacteriaceae that produce ESBLs are resistant to therapy with Penicillin, Cephalosporins and Azetronam ,despite apparent in vitro susceptibility to some of these agents(4).Third-generation cephalosporin resistance is often mediated by TEM- and SHV-type b β-lactamases in Enterobacteriaceae(5).The

SHV-1 has approximately 68% amino acid sequence homology to TEM-1. The SHV-1 β-lactamase is often found in K. pneumoniae and is involved in ampicillin resistance. There have been 141 types of SHV at the end of May 2011(6).They are derivatives of SHV-1 enzyme and differ from the latter not only by the presence of point mutations but also by deletions (in SHV-9 and SHV-10) or inserts (in SHV-16) as well (7). The most prevalence enzymes of this group are SHV-5 and SHV-12 (8). Among the SHV type of β-lactamases, SHV-5 was found to be responsible for outbreaks of nosocomial infection in several countries (9). Patients and Methods Collection of samples: A total of 160 isolates from urine, sputum, stool, blood, pus, drain and other samples were collected from patients who referred to Menoufiya University hospitals, a referral center offering care to about 1/3 of population inhabiting the Nile Delta at North of Egypt.Isolation and identification: Colony isolates were obtained after culture of urine specimens on CLED ( Cystiene Lactose Electrolyte Deficient), while pus, blood and sputum specimens were cultured on blood agar and MacConkey agar and then incubated aerobically at 37ºc for 24 hours. The isolated

colony was considered for further studies and identification was done using standard conventional, morphological, cultural and biochemical tests. Detection of ESBL: 1-Screening for ESBL producers: According to CLSI 2011 recommendations to identify potential ESBL producing isolates using standard disk diffusion techniques. Ceftazidime (30 μg), Cefotaxime (30 μg) and Ceftriaxone (30 μg) are used, If a zone diameter of < 22 mm for Ceftazidime, < 27 mm for Cefotaxime and < 25 mm for Ceftriaxone were recorded, the strain was considered to be “suspicious for ESBL production”. The use of more than one antimicrobial agent for screening improves the sensitivity of detection. 2-Phenotypic confirmatory test for ESBL production: by Cephalosporin / clavulanate combination disks.All the strains which were suspicious for ESBL production were also subjected to confirmation by using the cephalosporin/ clavulanate combination disks, as recommended by the CLSI 2011. The Ceftazidime (30 μg) , Cefotaxime (30 μg)discs alone and in combination with Clavulanic acid (Ceftazidime + Cavulanic acid, 30/10 μg discs-Cefotaxime + Clavulanic acid, 30/10 μg) were applied onto a plate of Mueller Hinton Agar (MHA) which was inoculated with the test strain. An increase of ≥ 5-mm in a zone diameter for either antimicrobial agent tested in combination with Clavulanic acid versus its zone when tested alone = ESBL.Reference cultures used in this study were E. coli ATCC 25922 as negative control and Klebsiella pneumoniae ATCC 700603 as positive control.3-Molecular detection of SHV gene:The specific primers used for amplification of SHV gene:SHV/F: (5′-TCA GCG AAA AAC ACC TTG-3′) -SHV/R : (5′-TCC CGC AGA TAA ATC ACC-3′), were obtained commercially from the manufacturer.Amplification was carried out according to the following thermal and cycling condition for SHV gene: 1- Initial denaturation at 940C for 2 minute. 2- Then 30 cycles of: -Denaturation at 940C for 1 minute. -Annealing at 520C for 30sec . -Extension at 720C for 45 sec. 3- Final extension at 720C for 5 minutes.The amplified gene was visualized by 302 nm UV-Transilluminator. And the gel was examined for bands of 471 bp as determined by the molecular weight Ladder (100 bp ladder) run at the same time.

Results The age of the studied cases ranged from 10 to 75 years with 55.72 ± 8.94 years as a mean ± standard deviation (SD) &45 years as a median.One hundred and sixty clinical samples were cultured from various sample sites from patients at Menoufiya University hospitals. The distribution of which (46.8%) were E. coli, (30.0%) Enterobacter, (21.8%) Klebsiella spp. and (1.3%) Serretia as shown in table (4).Most cases have been presented by Fever (73%) as the chief complaint, others were presented by persistent dysuria (15%), persistent wheezy chest (10%) and wound infection (2%).Clinically, 8 patients had ascites, 10 had jaundice, 8 had shrunken cirrhotic liver, 8 had splenomegaly, 4 had encephalopathy, 41 had pallor, 15 abdominal pain, 20 had wheezy chest, 3 had clinically infected wounds & C.V.P. lines, 18 had suprapubic pain & 9 had recurrent loin pain.Overall, 97 (60.6%) isolates were detected as ESBL producers by Phenotypic confirmatory test for ESBL production (Cephalosporin/Clavulanate combination disks) as shown in table (1) and include [45(28.1%) are E. coli, 28(17.5%) are Enterobacter, 22(13.7%) are Klebsiella spp. and 2 (1.3%) are Serretia] as shown in table (4).Among (60.6%) ESBL isolates, there were (45.6%) ESBL isolates with previous antimicrobial intake which are more than isolates without previous antimicrobial intake (15%) as shown in table (2). ESBLs isolates are higher in males (35.6%) than females (25.0%). The most frequent ESBLs isolates were from urine samples (16.9%), followed by sputum samples (11.3%), stool (7.5%) and pus (5.6%) while, the least frequent ESBLs isolates were from nasal swab and C.V.P line samples as shown in table (3).In the present study, we found that, SHV gene was found in (72.2%) of ESBLs isolates by PCR technique as shown in table (5), among them there were (51.6%) of SHV Positive ESBL isolates with previous antimicrobial intake which are more than SHV Positive ESBL isolates without previous antimicrobial intake (20.6%),the presence of SHV gene are higher in males than females. The highest percent of ESBLs isolates having SHV gene were among Enterobacter (26.7%) while, the lowest percent of ESBLs isolates having SHV gene were among Serratia (1.0%) as shown in table (6).

Discussion There is a problem that occurred among Extended-Spectrum β-lactamase (ESBL)–producing Enterobacteriaceae which have widely caused the spread of infections worldwide (10, 11,

12). Although, today hundreds variant of ESBLs have been described (13, 14), but, the most common of them are derivatives of TEM or SHV enzymes. Also, in recent years non-TEM and non-SHV ESBLs have been reported, mainly the CTX-M enzymes (11, 15, 16).Therefore, detection of ESBLs in Enterobacteriaceae is crucial for optimal treatment of patients and to control the spread of resistance (4).In the present study, the prevalence of ESBL was 60.6% which was near from result obtained by Olowe, et al. 2012, from Nigeria which show the prevalence of ESBL about (51.3%) (17)and also, by Dallal, et al., 2012 from Iran which show percent equal (57.5%) (18). In contrast to our study, many other recent studies showed prevalence of ESBLs ranged between 30-45% which were mild lower than our study like Aruna and Mobashshera;2012 from India-Grover, et al.,2013 from India-- Kaur And Aggarwal.,2013 from India Moosavian and Deiham .,2012 from Iran and Ahmad ,et al.,2013 from Iran and their ESBLs results were 34.71 %-- 40.07%-- 45.8%-- 30.5% and 46% respectively (19,20,21,22,23). The uncontrolled use of antibiotics at hospital could be a leading contributory factor to the high ESBL prevalence because third generation cephalosporins are usually first line against many severely infectious diseases. Justifiable use will be an effective means of controlling and decreasing spread of ESBLs strains.For certain reasons there is a wide variability in prevalence reporting of ESBLs. The possible factors could be different geographical locations, variable proficiency levels of microbiology trained technical staff, different antibiotic cut offs being used, different guidelines being followed (as they differ between different infectious diseases unions throughout the world) and different techniques being used for ESBL detection.In developing countries like India it is desirable to incorporate both ESBL detection and antimicrobial susceptibility testing. Different drugs have been used to detect ESBLs but no single antibiotic has been perfect at ruling -in or -out an ESBL producer (24).In several western studies, prevalence of ESBLs was less than that found in our study. The higher prevalence compared to

western countries can be explained by the fact that, western countries had strict infection control policies and practices, efficient and effective antibiotic audit systems, shorter average hospital stay, better nursing barriers and other important health care measures that are known to substantially decrease the chances of acquisition and spread of ESBL strains. This study showed that the highest rate of ESβL- producing Enterobacteriaceae were found in patients with history of antibiotic administration (45.6%)than other patients without antibiotic administration (15.0%) .Similar results were reported by Jain and Mondal;2008 in India, who reported that, the high percentage of ESβL-producing Enterobacteriaceaespp in their study were found in patients with history of antibiotic administration which may be due to the selective pressure imposed by extensive use of antimicrobials especially in intensive care units, in which antibiotic use was heaviest and the potential for patient-to-patient transmission of organisms was greatest (25).This also coincides with Graffunder, et al.; in the USA who found a correlation between the selective pressure of antimicrobial agents identified as risk factors for ESβL- producing organisms and the presence of related resistance genes residing on the plasmid (26).In the present study, The maximum ESBL production was seen among the isolates of E.coli (28.1%) , followed by Enterobacter (17.5%), Klebsiella spp.(13.7%) and Serretia (1.3%).Near results observed by Balan.,2013 From India who reported that, ESBLs were detected among the following species:24% Escherichia coli and 15% Klebsiella pneumoniae (2) . On the other hand another study in Chennai reported ESBL production was 47 % E. coli and 37 % Klebsiella pneumoniae in a tertiary care centre(27). But these results not similar to Kaur and Aggarwal.,2013 study which show The maximum ESBL production was seen among the isolates of Klebsiella pneumoniae (52.27%), followed by those of E.coli (46.43%) (21) which may be due to that Klebsiella pneumoniae have some virulence factors like hyperviscosity, polysaccharide capsule, presence of multidrug resistance efflux pump and production of endotoxine carbapenemases which make it more resistant.In the present study, The most frequent ESBLs isolates were from urine samples (16.9%), followed by sputum samples (11.3%), stool (7.5%) and pus (5.6%) while, the least

frequent ESBLs isolates were from Nasal swab and C.V.P line. These results were similar to some extent to other works like Ahmad, et al., 2013 from Iran who found that, most ESBLs isolates were from urine specimens, 4% from wounds and 3% from blood specimens (23). Also Dallal, et al., 2012 from Iran found that, ESBL-expressing strains of E. coli were more common in urinary samples (80%) (18). On the other hand, Olowe, et al.,2012 reported that most of the ESBL Producing Klebsiella species were from sputum followed by blood then urine as Klebsiella is less common in urinary tract infection than E.coli(17). Also Kaur and Aggarw.,2013 showed that, The ESBL production was quite high among the pus samples (51.37%), followed by urine samples (45.63%) (21).The rate of SHV-type β-lactamase produced by ESBLs isolates in our study (72.2%) was nearly similar to the rate of SHV-type β-lactamase produced by ESBLs isolates in study happened in India by Sharma, et al., 2010 in which the rate of SHV gene was 60.0% (28). In the present study, As regards to the isolated SHV Positive ESBLs organisms Enterobacter was the most predominant (37.1%) , followed by E. coli (32.9%), Klebsiella spp. (28.6%) and Serretia (1.4%). from above results, we found that, the most predominant isolated SHV Positive ESBLs organism was (Enterobacter) differ from the most predominant isolated ESBLs (positive and negative SHV) organism which was (E. coli) because SHV gene is more common in klebsiellaspp and Enterobacter than E. coli especially if it is associated with longer length of hospital stay, high severity of illness, longer time in the intensive-care unit; intubations and mechanical ventilation, urinary or arterial catheterization and previous exposure to antibiotics (especially cephalosporins). A recent study by Newire, et al., 2013 from Egypt reported that from 65 ESBL-producing K. pneumoniae strains, isolated from nosocomial

and community-acquired infections from 10 Egyptian University Hospitals distributed across seven governorates, 98% (64/65) of the total isolates carried the SHV core gene chromosomally which was higher than our study. 17% (11/65) of isolates carried SHV gene on the plasmid (29). In another recent study from Egypt by Afifi, et al.,2013 reported that SHV gene was the least frequent among Escherichia spp., SHV was the predominant gene among Klebsiella spp. and moderate in Enterobacter spp. by 6.9%, 72% & 29.4% respectively (30). In Garrec, et al.,2011 study, They mentioned that, the prevalence of SHV gene was (23.0%) of which (83.3%) was Enterobacter (31), similar to present study. But in Kaur And Aggarwal., 2013 study, the distribution of SHV gene among organisms was (53.6%) in Klebsiella spp. and (46.4%) in E. coli (21) , Also Moosavian and Deiham., 2012 study showed The distribution of SHV gene among organisms was (72.0%) in Klebsiella spp., (3.0%) in E.coli and (3.0%) in Enterobacter (22). Conclusion ESBL producing strains are a major problem in many hospitals leading to increased morbidity, mortality and health care costs. All members of the Enterobacteriaceaeare known to be potential ESBL producers, especially Escherichia Coli, Enterobacter and Klebsiella spp. High prevalence of ESBL producers in our hospital (60.6%) calls for strict policies regarding antibiotic usage and their screening methods.It is important for the clinical microbiology laboratory to implement one or more methods to detect ESBLs and hospital-based clinical laboratories should screen isolates from the community-based patients before hospitalization to prevent the spread of ESBL mediated resistance.The use of Cephalosporins should be discouraged for empirical therapy of infections and should only be used according to results of culture and sensitivity.

Table (1): Classification of Enterobacteriaceae into ESBLs and non ESBLs according to cephalosporin/clavulanate combination disks

ESBLs (%) Non- ESBLs (%) Total (%) 97(60.6) 63(39.4) 160 (100)

Table (2): Comparison of Enterobacteriaceae isolates according to ESBLs production and history of antimicrobial intake.

Medication Total (%) ESBL (%) Non-ESBL (%) With previous antimicrobial intake 96 (60%) 73 (45.6%) 23 (14.4%)

Without previous antimicrobial intake 64 (40%) 24 (15.0%) 40 (25%) Total 160 (100%) 97 (60.6%) 63 (39.4%)

Table (3) Distribution of Enterobacteriaceae isolates according to ESBLs production and according type of sample. Total ESBLs Non-ESBLs

Urine 42 (26.3) 27 (16.9) 15 (9.3) Sputum 29 (18.1) 18 (11.3) 11 (6.9)

Pus 17 (10.6) 9 (5.6) 8 (5.0) Stool 17 (10.6) 12 (7.5) 5 (3.1)

Ascetic Fluid 12 (7.5) 8 (5.0) 4 (2.5) Throat swab 11 (6.9) 6 (3.7) 5 (3.1)

Blood 9 (5.6) 5 (3.1) 4 (2.5) Drain 8 (5.0) 4 (2.5) 4 (2.5)

Pleural fluid 6 (3.8) 2 (1.3) 4 (2.5) Wound 5 (3.1) 2 (1.3) 3 (1.9) T-tube 2 (1.3) 2 (1.3) 0 (0)

Nasal swab 1 (0.6) 1 (0.6) 0 (0) C.V.P line 1 (0.6) 1 (0.6) 0 (0) TOTAL 160 (100) 97 (60.6) 63 (39.4)

Table (4) Distribution of Enterobacteriaceae isolates according to ESBLs production and type of organisms.

Organism Total (%) ESBLs (%) Non-ESBLs (%) E.COLI (%) 75 (46.8) 45 (28.1) 30 (18.8) ENTEROBACTER (%) 48 (30) 28 (17.5) 20 (12.5) KLEBSIELLA (%) 35 (21.8) 22 (13.7) 13 (8.1) SERRETIA (%) 2 (1.3) 2 (1.3) 0 (0) Total 160 (100) 97 (60.6) 63 (39.4)

Table (5): Classification of ESBLs isolates into ESBLs having SHV gene or not by PCR technique:

ESBL SHV ESBL not SHV Total No. (%) No. (%) No. (%) 70 (72.2) 27 (27.8) 97 (100)

Table (6): Distribution of ESBL producing Enterobacteriaceae isolates according to the presence of SHV gene and type of organism

Organism

TOTAL ESBL SHV Positive ESBL SHV Negative No. (%) No. (%) No. (%)

E.Coli 45 (46.4) 23 (23.7) 22 (22.7) Enterobacter 28 (28.8) 26 (26.7) 2 (2.1)

Klebsiella 22 (22.7) 20 (20.6) 2 (2.1) Serretia 2 (2.1) 1 (1.0) 1 (1.0) TOTAL 97 (100) 70 (72.2) 27 (27.8)

Figure (1): Gel electrophoresis showing 471 bp of bla SHV gene (Lane 1: ladder100bp - Lane 2: positive control – Lane 3, 4, 5, 6, 7, 8: positive cases)

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Original Article

Clinical Significance of Cerebrospinal Fluid Nitric Oxide Level in Patients with Meningitis Ali Mahmmoud El-Kadi*, Magdy Mamdouh El-Bardini*, Ehab Hassan Abdelmaksood Elkholy*, Mohamed Hamdy Abd-Esalam Ibrahim*, * Department of Tropical Medicine , Faculty of Medicine, University of Alexandria

ABSTRACT Meningitis is inflammation of the protective membranes covering the brain and spinal cord, known collectively as the meninges. The inflammation may be caused by infection with viruses, bacteria, or other microorganisms, and less commonly by certain drugs. Recent research suggests that nitric oxide (NO) may have some important pathophysiological effects during bacterial meningitis. However, the role of this molecule is unclear in other types of meningitis .Aim of the work : The aim of this work is to study the clinical significance of cerebrospinal fluid nitric oxide level in patients of meningitis. Methods: The study was conducted on forty patients with meningitis according to the etiology from all age groups, both sexes as well as different grades of severity. Evaluation include: full history taking, clinical evaluation, laboratory investigations including; routine investigations, CSF cytochemical analysis, Microbiological study of CSF sample, assessment of nitrite level in C.S.F at admission of patients and before discharge. Imaging studies include C.T brain and M.R.I brain if possible. Results: We observed that there was statistically significant relation between diagnosis and nitrite on admission. Nitrate levels in the CSF samples of the patients with bacterial and tuberculous meningitis were significantly elevated (p < 0.01), However, no significant elevations were found in patients with viral meningitis. The highest mean value of nitrite on admission (9.06 ± 5.88 and 9.89 ± 9.18 µmol/l) was detected in the tuberculous and bacterial meningitis respectively, while viral meningitis has the lowest mean (4.04 ± 1.61µmol/l). There was no statistically significant relation between nitrite and nitrate on admission with different parameters (Glucose, Protein, Lactate and WBC). Conclusion: Although the measurement of CSF nitrite levels is helpful for the differential diagnosis of meningitis, this parameter is not superior to other routine parameters. However, it may have a characteristic effect on prognosis. Introduction Meningitis is inflammation of the protective membranes covering the brain and spinal cord, known collectively as the meninges.(1)The inflammation may be caused by infection with viruses, bacteria, or other microorganisms, and less commonly by certain drugs.(2) Meningitis can be life-threatening because of the inflammation's proximity to the brain and spinal cord; therefore the condition is classified as a medical emergency.(1, 3). Bacterial or purulent meningitis is the most important form of infection in the United States in terms of incidence, sequela and ultimate loss of productive life.(4) Aseptic meningitis, usually caused by a virus, is also common, however significant sequela are rare and the disease is self-limiting. Strepto- coccus pneumoniae is a leading causative agent of diverse infections.(5) In Egypt, it was recently described as the leading cause of bacterial meningitis skewing the epidemiology from Neisseria meningitidis, which was previously reported as the major etiological agent.(6). Meningococcal disease occurs in epidemics in

areas where many people live together for the first time, such as army barracks during mobilization, college campuses.(7) Although the pattern of epidemic cycles in Africa is not well understood, several factors have been associated with the development of epidemics in the meningitis belt. They include: medical conditions (immunological susceptibility of the population), demographic conditions (travel and large population displacements), socioeconomic conditions (overcrowding and poor living conditions), climatic conditions (drought and dust storms), and concurrent infections (acute respiratory infections).(8, 9). The most common symptoms of meningitis are headache and neck stiffness associated with fever, confusion or altered consciousness, vomiting, and an inability to tolerate light (photophobia) or loud noises (phonophobia). Sometimes, especially in small children, only nonspecific symptoms may be present, such as irritability and drowsiness. If a rash is present, it may indicate a particular cause of meningitis; for instance, meningitis caused by meningococcal bacteria may be accompanied by a characteristic rash.(1). Although the etiologies

are different clinical symptoms and signs are similar in meningitis. Thus, macroscopic appearance, examination of white blood cells(WBC), biochemical tests (protein, glucose, lactate), serological tests, culture and direct microbiological examination of cerebrospinal fluid (CSF) are very important in differential diagnosis of meningitis.(10). Recent research suggests that nitric oxide (NO) may have some important pathophysiological effects during bacterial meningitis.(11) NO is produced from L-arginine by nitric oxide synthase (NOS) in neutrophils, macrophages,vascular endothelial cells, astrocytes, microglias and neurons in response to several immunological stimulations and cytokines . NO is not astable substance, and it converts into nitrite, nitrate in 5-10 seconds. It was established that there are roles of NO in microvascular injury, cerebral oedema, CSF pleocytosis, neurological injury and necrotic cell death in oligodendroglias.(12) Subjects and Methods This study was conducted on 40 patients with meningitis according to the etiology from all age groups,both sexes as well as different grades of severity. Patients subjected to any condition that has been reported to affect NO production (e.g diabetes, nitrate drugs) were excluded from the study. All patients were subjected to the following: full history taking, clinical evaluation, laboratory investigations including; routine investigations(Complete blood picture,E.S.R and C.R.P) and specific investigations for patients such as CSF cytochemical analysis: glucose, protein, WBC (neutrophils and lymphocyts) and RBC ,Microbiological study of CSF sample( no growth, G+ve bacilli, G-ve cocci, Staphylococci) and assessment of nitrite level in C.S.F at admission of patients and before discharge. Imaging studies include C.T brain and M.R.I brain if possible. The measurement of CSF nitrate and nitrite levels: The concentrations of nitrite and nitrate, the stable end products of NO in CSF, were measured using a colorimetric test. This assay determines nitric oxide concentrations based on the enzymatic conversion of nitrate to nitrite by nitrate reductase. The reaction is followed by colorimetric detection of nitrite as an azo dye product of the Griess Reaction. The Griess Reaction is based on the the two-step diazotization reaction in which acidified NO2 produces a nitrosating agent, which reacts with

sulfanilic acid to produce the diazonium ion. This ion is then coupled to N-(1-naphthyl) ethylenediamine to form the chromophoric azo-derivative which absorbs light at 540-570 nm. Results (45%) of cases were from 18y-40year. Males (65%) were more than females (35%) with ratio 1.9:1 in the study . (57.5%)were from rural area while (42.5%)were from urban area. Our results showed that fever was the main complaint (100%) of studied cases. Headache was the 2nd most frequent complaint, occurred in (85%).Vomiting reported in(67.5%)of cases. Lack of concentration reported in more than half percentage(55%) of cases. Blurring of vision and photophobia were present in equal percentage of cases (52.5%).Convulsion was reported in (40%) of cases. Poor suckling and squint reported in equal percentage (7.5%) while bulging anterior fontanel in (5%) of cases. Rigid neck and Kernig's Sign were present in equal percentage ( 82.5%)of cases.Brudzinsky sign was present in (55%)of cases. Reflexes was normal in (75%) of cases. Sensation and motor power were normal in (90% and 85% respectively).Cranial nerves and psychological status were normal in (92% and 95% respectively). CSF latex was streptococcal pneumonia in 5 cases by percentage (12.5%), while was N meningitides in 6 cases by percentage (15%).Diagnosis was viral in majority of cases by percentage (52.5%), while was bacterial in( 35%) of cases and tuberculous in (12.5%) of cases. Majority of cases cured by percentage (72.5%),while (17.5%) of cases cured with deficit and (10%) of cases died. Majority of neurological deficit was cognitive by percentage (20%),while motor deficit was recorded in (10%) of cases. Blindness, hearing loss and hydrocephalus were presented in equal percentage (2.5%). There was a statistically significant relation between some clinical presentations of studied cases and different age groups. Most of cases who had headache were in age group from 18y-40y.Most of cases suffered from vomiting ,convulsion and poor suckling were in age group <6y by percentage (100%),(88.9%) and (33.3%) respectively. There is no statistically significant relation between outcome of studied cases and different age groups. Also, there is no statistically significant relation between prognosis of studied cases and gram stain. There

was a statistically significant relation between outcome of studied cases and hospitalization in days. There was statistically significant relation between diagnosis and hospitalization in days. Tubercolous meningitis has the longest median duration of hospital stay (21 days) followed by bacterial and viral meningitis (14 days). There was statistically significant relation between diagnosis and nitrite on admission. Tubercolous and bacterial meningitis have the highest median of nitrite on admission (6.8 and 5.2) respectively,while viral meningitis has the lowest median (3.5). There was statistically significant relation between diagnosis and nitrate on admission. Tubercolous and bacterial meningitis have the highest median of nitrate on admission (12.3 and 8) respectively,while viral meningitis has the lowest median (5.4). Receiver Operating Characteristic curve (ROC curve) of the diagnostic performance for nitrite on admission with the diagnosis of viral meningitis versus bacterial/ TB meningitis shows that, using a cut point of 5.4, the values less than or equal to 5.4 are suggestive for viral meningitis and values more than 5.4 are suggestive of bacterial/TB meningitis. This test had a statistically significant Area under the Curve (AUC) of 0.772 (p<0.05) which can be classified from the traditional academic point system as a fair performance (class C). this cut point had the best tradeoff between the sensitivity and the specificity, with a sensitivity of 52.63 and a specificity of 85.71 with a positive predictive value(PPV) of 76.92 and a Negative Predictive Value (NPV) of 66.67 and an overall accuracy of 70. Receiver Operating Characteristic curve (ROC curve) of the diagnostic performance for nitrate on admission with the diagnosis of viral meningitis versus bacterial/ TB meningitis shows that, using a cut point of 5.4, the values less than or equal to 5.4 are suggestive for viral meningitis and values more than 5.4 are suggestive of bacterial/TB meningitis. This test had a statistically significant Area under the Curve (AUC) of 0.779 (p<0.05) which can be classified from the traditional academic point system as a fair performance (class C). this cut point had the best tradeoff between the sensitivity and the specificity, with a sensitivity of 94.74 and a specificity of 52.38 with a positive predictive value(PPV) of 64.29 and a Negative Predictive Value (NPV) of 91.67 and an overall accuracy of 72.50. There was no statistically significant

relation between outcome and nitrite on admission. The highest median of nitrite on admission was (5.80 ) with the cases which cure with deficit, while the lowest median was (3.90) with the cases which cure. Discussion Meningitis is an inflammation of the cerebrospinal membranes that may develop from infectious or noninfectious etiology.(1) Although the etiologies are different, clinical symptoms and signs are similar in meningitis. Thus, macroscopic appearance, examination of white blood cells (WBC), biochemical tests (protein, glucose, lactate), serological tests, culture and direct microbiological examination of cerebrospinal fluid(CSF) are very important in the differential diagnosis of the meningitis. In spite of these examinations, because of normal, almost normal or atypical CSF findings, difficulties in differential diagnosis are often confronted.(10). Recent research suggests that nitric oxide (NO) may have some important pathophysiological effects during bacterial meningitis.(11) However, the role of this molecule is unclear in other types of meningitis. It was established that there are roles of NO in microvascular injury, cerebral edema,CSF pleocytosis, neurological injury and necrotic cell death in oligodendroglias.(12). In this study,we aimed to investigate the role of NO in differential diagnosis and prognosis of meningitis by determining the CSF levels of nitrate and nitrite on fourty patients of meningitis who admitted to Alexandria Fever Hospital. In the present study, male patients represented (65%) , while females were (35%) i.e ,the ratio of males : females is nearly 1.9:1.In the study of Alif A. Allam et al(13) the ratio was 1.36:1.In Eman I. Mobarak et al(14) study in Alexandria the ratio was 1.8:1. Özden Turel et al(15) study in turkey the ratio was 2.17:1. Swarnali Jordar et al(16) study in Bangladesh the ratio was 1.5:1. Ragni Srivastava et al(17) study the ratio was 1.9:1. In the current study, higher percentage of cases in rural than urban areas.In the study of Alif A. Allam et al(13) percentage of cases in rural was found more than urban areas as our study. In Eman I. Mobarak et al(14) study in Alexandria percentage of cases in urban areas was more than rural.The difference may be attributed to variation in the categorization of rural and urban regions in Alexandria in both

studies. Swarnali Jordar et al(16) study in Bangladesh percentage of cases in rural was found more than urban areas as our study. According to age , the present study revealed that (45%) of cases were from (18-> 40 ys) and (22.5%) of cases were from (3m-<6ys) with mean age (22.50 ± 16.87yrs) similar to astudy done by Eman I. Mobarak et al(14) study in Alexandria between(1997-2006)revealed that (24.3%) of cases were below 6 yrs and mean age (22.26 ±19.16yrs).In the study of Ali Hassan Abro et al(18) which done in United Arab Emirates, from Jan 2005 to Dec 2007 the mean age was 33.73 ± 11.7 years (16-70).In the study of Alain Viallon et al(19) the mean age of cases divided according to the type of meningitis where mean age of bacterial meningitis was(55 ± 20yrs), while mean age of viral meningitis was (35 ± 18yrs). In the present study,we found that smoking and operation history were the most predisposing factors to meningitis by (35% )of cases followed by drug abuse (20%),otitis media,sinusitis(12.5%),immunocomprimised(10%) and lastly puerperal sepsis (2.5%).In the study of N. Proulx et al(20) the main predisposing factors for meningitis included sinusitis (20%), otitis media (20%), past neurosurgery (15%) and cerebrospinal fluid leak(14%).In the study of W.-N. Chang et al(21) the main predisposing factors for meningitis included DM(44%),liver disease(17%),alcoholism(15.3%),end stage renal disease, malignancy, otitis media and lastly drug abuse. In the results of CSF cultures in present study , most of cases (72.5%) showed negative cultures. In the study of Alif A. Allam et al(13),(33%) of cases had confirmed bacterial meningitis by culture. In Afifi et al(22) in Egypt (1998-2004), only 8% of cases had confirmed bacterial meningitis by culture. Ceyhan et al(23) found positive culture in 17% of cases, they confirmed the organism by latex agglutination test in 23% and by using PCR in 100%;%; while in Salih et al(24) study (54.7%) showed negative cultures ,(28.2%) showed positive culture and in (17%) the culture was not done. Pérez et al(25) showed BM of unknown etiology in 55.3%. Özden Turel et al(15) study in turkey found positive culture in 11.2% of cases. Most common complications in our study were cognitive deficit(20%),motor deficit(10%), blindness, hearing loss and hydrocephalus (1%). In Farag et al(26) study , epilepsy was the only complication reported in about 7 % of cases. In

Salih et al(24) study, the most common complication was hemiplegia (3.4%), deafness(1.7%),epilepsy (1.7%),cerebral palsy (0.9%) and hydrocephalus (0.9%).. In Chinchankar et al(27) study, Hemiparesis occurred in 6.4% of cases and cranial nerve palsy in 3.7%. In Franco- Paredes et al(28) study , the most common complication identified was epilepsy in 37% of patients followed by hearing loss (32%). In our study , headache, vomiting, convulsion and poor suckling had a stastically significant relation with age . Most of cases who had headache were in age group from 18y-40y.Most of cases suffered from vomiting ,convulsion and poor suckling were in age group <6y by percentage (100%),(88.9%) and (33.3%) respectively. bulging anterior fontanel was identified in 22.2% of children aged less than 6 months. In the study of Alif A. Allam et al(13) stated that bulging of the fontanel, a sign of increased intracranial pressure, is of great diagnostic value. They also reported that vomiting is more marked in infants and young children. François et al(29), reported that seizures were more frequent among infants than in older age groups with a highly significant difference between them and they also reported that headache was more prominent with adults than in the younger age with statistically significant difference too. Silva et al(30) also noted the higher occurrence of seizures in younger age groups while headache was more prominent in older age groups. Franco-Paredes et al(28) study, focal neurologic findings were more commonly seen in children over 5 years of age, seizures were most commonly seen in children aged 6 to 12 months and bulging anterior fontanel was identified in 27% of children aged less than 6 months. In the present study, there was no statistically significant relation between prognosis of studied cases and age . In Afify et al(22) study , mortality was relatively higher among children <5 years of age and those >40 years of age.In Pérez et al(25) study ,the highest death were observed mainly in infants below 1 year . In Erleena et al(31) most complications occur in children less than five years old. In Eman I. Mobarak et al(14) study in Alexandria, mortality was higher among those between 18-60 yrs old and also similar to our study , there was no statistically significant relation between prognosis of studied cases and age. In current study,there was no statistically significant

relation between age and CSF latex. Streptococcal pneumonia and N. meningitides were the highest percentage in the age group (18-40) by (80% and 83.3%) respectively. In the study of Alif A. Allam et al(13) H. Influenza was the leading cause of meningitis in the age group below 1 year and N. meningitides with Staph aureus were the second leading cause of meningitis in the age group 1 years to less than 5 years old (9.5%) & (9.5%) respectively, while S. pneumoniae was the leading cause above this age (35.3%). Silva et al(30) found in their study that the most common etiologic agent of bacterial meningitis in infants under the age of 1 year was H. influenzae (42.2%). In current study, there was statistically significant relation between diagnosis and W.B.C's count of C.S.F on admission. The highest median of W.B.C’s count of C.S.F on admission was bacterial meningitis followed by T.B meningitis then viral meningitis. A Viallon et al(19) found that the highest median of W.B.C’s count of C.S.F was bacterial meningitis followed by viral meningitis. In the present study, there was statistically significant relation between diagnosis and nitrite on admission.Nitrite levels in the CSF samples of the patients with bacterial and tuberculous meningitis were significantly elevated (p < 0.01), However, no significant elevations were found in patients with viral meningitis. The highest mean value of nitrite on admission (9.06 ± 5.88 and9.89 ± 9.18 µmol/l) was detected in the tuberculous and bacterial meningitis respectively,while viral meningitis has the lowest mean (4.04 ± 1.61µmol/l).In K ÇETİN et al(32) study, The highest mean value of nitrite (3.9±2.0 µmol/l) was detected in the bacterial meningitis group similar to our study,also as our study Nitrite levels in the CSF samples of the patients with bacterial and tuberculous meningitis were significantly elevated (p < 0.01). E Murawska-Ciałowicz et al(33) study, Tsukahara H et al(34) and Kornelisse R et al(35) reported elevated CSF nitrite levels in patients with bacterial meningitis. In our study, There was no statistically significant relation between nitrite and nitrate on admission with different parameters(Glucose, Protein, Lactate and WBC). In K ÇETİN et al(32) study, apositive correlation was found between nitrite levels and WBC counts (r = 0.567, p < 0.001), and nitrite levels and protein levels (r = 0.548, p < 0.001). A negative correlation was found between

nitrite and glucose levels (r = -0.271, p < 0.05). G Uysal et al(36) study, There was no correlation between CSF nitrate/:nitrite levels and CSF white blood cell count (r-0.217), protein (r-0.263) or TNF-alevels (r-0.046), but there was a moderate negative correlation with CSF glucose levels (r-0.46,p-0.057) and this similar to our study. In the present study, There was no statistically significant relation between nitrite on admission and outcome. In K ÇETİN et al(32) study,there was no significant difference was found between the CSF nitrite levels of the patients who died and those who recovered (p > 0.05) similar to our study. In our study,the finding that ahigher proportion of patients with CSF nitrite above the normal range had neurologic or audiologic sequelae (or both) in contrast to the patients with normal levels suggests that NO may be responsible for neurologic damage in humans. Kornelisse R et al(35) reported the same finding as our study. Conclusion Although CSF nitrite measurement is no more useful than other routine examinations in the differential diagnosis of meningitis, enhanced NO production may have an important effect on prognosis and may contribute to the pathophysiology of bacterial and tuberculous meningitis. Referrence 1. Bendszus M, Hähnel S. Inflammatory diseases

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Original Article

Comparison of Azithromycin Plus Gentamicin versus WHO Recommended Therapy For The Treatment of Complicated and Uncomplicated Brucellosis Alaa M. Abdo*, Nasser M. Abdullah*, Ehab H. A. El-Khouly*, Khaled Ebn El-Walid*, *Department of Tropical Medicine, Faculty of Medicine, Alexandria University, Egypt

ABSTRACT Brucellosis is the commonest zoonotic infection worldwide. More than 500 000 new cases occur annually but with an uneven global distribution.Human brucellosis is a multisystemic disease characterized by a prolonged clinical course and relapses. Severe complications involving musculoskeletal, neurological, genitourinary, and cardiovascular systems may be encountered during the course of the disease.Aim of this work was to perform a non-blinded treatment trial comparing an azithromycin based regimen (azithromycin 20 mg/kg/d up to 1000 mg orally plus Gentamicin 5 mg/kg/d IM/IV for 2 weeks) with the WHO recommended regimen (Doxycycline 00mg orally twice daily for 6 weeks plus rifampin 600 mg/d for 6 weeks) and to testan alternative shorter treatment course for uncomplicated and complicated brucellosis to determine if it is as effective as the recommended treatment. In addition, the secondary objectives of this work was to search for prediction of relapse, chronicity, resistance to the drugs used in the study, possible complications during course of treatment and assessment of possible side effects regarding gastrointestinal, renal, hepatic or neurological side effects. Methods: The present study was conducted on 150 persons divided into four groups: group one included 60 patients with uncomplicated Brucellosis treated with azithromycin for one month plus Gentamicin for 2 weeks, group two included 60 patients with uncomplicated Brucellosis treated with WHO recommended therapy for 6 weeks, group three included 15 patients with complicated Brucellosis treated with azithromycin for one month plus Gentamicin for 2 weeks and group four included15 patients with complicated Brucellosis treated with WHO recommended therapy for 6 weeks.Conclusion: A regimen that is shorter could significantly improve the compliance resulting in fewer long term complications. Azithromycin, antibiotic with excellent intracellular penetration and long half-life is a very potential for use as apart of a regimen that would require a shorter duration of therapy. Gentamicin, another aminoglycoside with a wide spectrum of antibacterial activity, may be an active in vitro against clinical Brucella isolates and less toxic than streptomycin.Our findings also introduce new considerations on the potential role of new therapeutic regimen such as gentamycin plus azithromycin for treatment of human brucellosis especially among uncomplicated cases. Their therapeutic application in earlier stages of human brucellosis might prove useful in the clinical management of human brucellosis before complication or chronic form of the disease develops. Our results do not favor the use of gentamycin plus azithromycin to treat complicated brucellosis in humans. Introduction Brucellosis is a systemic disease that may involve any organ or system in the body and mimic various multisystem diseases showing a wide clinical polymorphism. Clinically it progress as acute, subacute or chronic infection. Complications of the disease are relatively common and widespread; the most common affected systems are the osteoarticular, gastrointestinal, hematological, genitourinary, cardiovascular, respiratory and central nervous systems.(1-4). Brucella species are small, non-motile, non-spore-forming, encapsulated Gram-negative coccobacilli. There are seven species, of which only four can cause human brucellosis: Brucella abortus, Brucella melitensis, Brucella suis, and Brucella canis.(5,6). Brucellosis was first reported in Egypt in 1939 and is now considered endemic in most parts of the country.Despite its

economic and public health importance, in recent years, the official Egyptian Brucellosis Control program does not appear to have been fully implemented.(7,8). The goals of treatment of brucellosis include control of acute illness, Prevention of associated complications, and prevention of relapse.(9) More than 50 years after active drug therapy was defined, there are still questions regarding the choice of antibiotics and the duration of treatment of brucellosis. The selected antibiotics must be active in vitro. But in vitro activity may poorly predict efficacy, since brucellae survive in compartments that are inaccessible or hostile to antimicrobial activity, so the antibiotic must also be one that diffuses readily into tissues and cells.(10) Duration of treatment must also be long enough to eliminate the organism. The presence of local disease, underlying patient conditions (immune status,

pregnancy, etc.), and age must also be considered.(11,12). Azithromycin, an azalide (macrolide) antibiotic with excellent intracellular penetration and an extremely long half-life is a very intriguing candidate as a part of a regimen that would require a shorter duration of therapy. Streptomycin, an aminoglycoside, has the advantage of possessing synergistic action with tetracyclines. On the other hand it has very low phagocytic penetration and decreases the penetration of tetracyclines.(13) Gentamicin, another aminoglycoside with a wide spectrum of antibacterial activity, may be more active in vitro against clinical Brucella isolates than streptomycin.(14). When compared to other macrolides, azithromycin accumulates better and has higher intracellular half-life than erythromycin.(15) The half-life of azithromycin after one dose of 12 mg / kg was estimated to be 49.7 ± 34 hours and after multiple doses to be 69.8 ± 43 hours.(16) Consequently, azithromycin’s pharmacokinetics makes it good therapeutic option for Brucellosis, which survives inside of host’s cells. Landinez et al (17) found that the sensitivity of azithromycin and tetracycline to three different strains of Brucella melitensis was about equal. The dosing for azithromycin of 30 mg/ kg of body weight for children and 1.5 g total dose for adults is well tolerated up to 12% of patients report any symptoms, <10% report gastrointestinal symptoms and 0.7% to 1.3% discontinue therapy. Such tolerability compares favorably to that of other antibiotics.(18,19) The safety and tolerability of the long-term use of azithromycin was summarized in a review article by Ruuskanen.(20) Forty-one children with cystic fibrosis received either azithromycin 250 mg or 500 mg according to body weight or placebo for 6 months. Side effects were assessed by audiometry, and liver enzymes before, during and on completion of therapy, no side effects were recorded and treatment was well tolerated. There is a marked public health burden attributed to Brucella in Egypt. Due to the long, multiple-drug regimens required for the treatment for brucellosis, compliance is often inadequate. Patients tend to discontinue the treatment course, and hence relapses and complications become further public health concerns. To more effectively deal with brucellosis, a shorter regimen promoting compliance is required. Furthermore, since Brucella is considered a

potential bio-warfare agent, multiple tested regimens are needed to provide appropriate treatment options in the event of a shortage of resources due to a large attack or due to programmed antibiotic resistance. As this study is one of the first trails ever to evaluate the effectiveness of azithromycin for the treatment of brucellosis, the duration of treatment was selected to be 4 weeks in addition to treatment with Gentamicin (WHO recommended dosing and duration). If this trial is successful, it could be the start of a series of trails to determine the shortest effective course of treatment. The purpose of this study is to test an alternative shorter treatment course for uncomplicated brucellosis to determine if it is an effective as the recommended treatment. Patients with complicated brucellosis will not be enrolled in this study. Patients and Methods The present study was conducted on 150 persons divided into four groups: group one included 60 patients with uncomplicated Brucellosis treated with azithromycin for one month plus Gentamicin for 2 weeks, group two included 60 patients with uncomplicated Brucellosis treated with WHO recommended therapy for 6 weeks, group three included 15 patients with complicated Brucellosis treated with azithromycin for one month plus Gentamicin for 2 weeks and group four included15 patients with complicated Brucellosis treated with WHO recommended therapy for 6 weeks. Patients were selected from those admitted to the inpatient ward of Alexandria Fever Hospital. Patients were excluded if they had age less than 18 years, pregnancy, patients presenting with complications or focal disease, like arthritis, spondylitis, sacroilitis, neurobrucellosis, unusual gastrointestinal manifestations like peritonitis, acute cholecystitis or acute abdomen, splenic or liver abscess or endocarditis in group I and II. history of treatment with any antibiotic which may be effective against brucellosis in the last 4 days taken for a duration of at least in week in appropriate doses, significant underlying illness other than brucellosis, know allergy to any of the antibiotics used in the study, inability to swallow oral medications, toxic patient (defined as respiratory rate greater than 30, blood pressure less than 85/50, heart rate >120 beats/minute),refusal to sign a consent form and

refusal to adhere to study follows ups after discharge from the hospital. Severe liver and/ or kidney function tests impairment, severe liver function tests impairment is set at the elevation of the transminases to more than three times the normal. The normal value for the creatinine clearance is 95±20 mL/min in women and 120± 25 mL/min in men. Thus, these levels suggest the loss of approximately 30 percent of glomerular filtration rate (GFR).Detailed history taking and thorough clinical examination were done for all patients including heart rate and mean blood pressure .Blood samples were collected from all patients to assess the following parameters: routine laboratory investigations; Complete blood count, complete liver profile, renal functions,fasting and post prandial blood glucose level, complete stool and urine analysis, urine pregnancy test will be performed on post-pubertal females before entry into the study. Imaging was done for all patients including Plain X ray chest and Plain X-ray of lumbo-sacral region, ultrasound abdomen was done to assess the size of liver, longitudinal diameter of the spleen, MRI of lumbo-sacral region whenever indicated. Serology was done including Standard tube agglutination (STA) serology and ELISA antibody testing. Blood culture and bone marrow culture were done for all patients. Follow up after a week of therapy comparison between the four groups of patients was performed according to the following definitions: - Clinical cure: all symptom resolved by day 8 after initiation of therapy. - Clinical improvement: not all symptoms resolved but decreased as compared to admission. - Clinical failure: persistence or appearance of new symptoms with or without positive blood culture or patients developing complications which can be directly attributable to brucellosis beginning after at least 7 days of the therapy. Microbiological cure: negative blood cultures 2 and 6 weeks after beginning of therapy.

Microbiological failure: Brucella isolated from week 2 or 6 blood culture. - Relapse: patient with initial clinical cure that again develops signs and symptoms of brucellosis and a positive blood culture. - Failure due to side effects attributable to the study medication. Statistical Analysis Data were fed to the computer and analyzed using IBM SPSS software package version 20.0. Qualitative data were described using number and percent. Quantitative data were described using range (minimum and maximum) mean, standard deviation and median. Comparison between different groups regarding categorical variables was tested using Chi-square test. For normally distributed data, comparison between the different groups were done using F-test (ANOVA) to be used and Post Hoc test (Tukey).Significance of the obtained results was judged at the 5% level. Results Table 1: shows distribution of the studied sample according to demographic characteristics. The study enrolled 150 cases of brucellosis that represent the number of Brucella i cases admitted to Alexandria Fever Hospital from 1/1/2014 till 31/8/2014 from them was 115 (76.7%) were males and 35 cases (23.3%) were females. As regard age distribution was from 18 to 50 years with the mean age was 30.05 ± 9.45 in group I, 29.63 ± 8.93 in group II, 30.13 ± 8.37 in group III and 27.0 ± 8.08 in group IV. Most of the cases were living in rural areas, 23.3% were shepherds, 23.3% were house wives, 25.3% were farmers, 18% were butchers, 1.3% teachers, 4% were workers, 0.7% was fisherman, 0.7% was agriculture engineer, 0.7% was veterinarian and 0.7% was milk man. Most of the cases (88.7%) were living in rural areas while (11.3%) were living in urban areas.

Table (1):Socio-demographic characteristics among studied groups

Group I (n = 60)

Group II (n = 60)

Group III (n =15 )

Group IV (n = 15) Test of sig. MCp

No. % No. % No. % No. % Residence Urban 7 11.7 5 8.3 2 13.3 3 20.0 χ2=

1.725 0.585

Rural 53 88.3 55 91.7 13 86.7 12 80.0 Sex Male 44 73.3 48 80.0 12 80.0 11 73.3 χ2=

1.014 0.835

Female 16 26.7 12 20.0 3 20.0 4 26.7

Age (years) Min. – Max. 18.0 – 50.0 18.0 – 50.0 18.0 – 45.0 18.0 - 42.0

F=0.483 0.694 Mean ± SD. 30.05 ± 9.45 29.63 ± 8.93 30.13 ± 8.37 27.0 ± 8.08 Median 27.0 27.0 29.0 25.0 Weight (kg) Min. – Max. 69.0 ± 101.0 59.0 – 104.0 65.0 – 95.0 66.0 – 105.0

F=4.581* 0.004* Mean ± SD. 85.20 ± 8.23 78.46 ± 11.44 79.90 ± 9.98 81.47 ± 11.69 Median 86.0 77.0 82.0 79.0 Sig.bet. Grps. p1= 0.002* p2=0.974 Occupation Shepherd 17 28.3 12 20.3 2 13.3 4 26.7

χ2= 21.397

0.852

Butcher 10 16.7 11 18.6 3 20.0 3 20.0 Farmer 14 23.3 17 28.8 4 26.7 3 20.0 House wife 16 26.7 12 20.3 3 20.0 4 26.7 Teacher 1 1.7 1 1.7 0 0.0 0 0.0 Worker 1 1.7 3 5.1 1 6.7 1 6.7 Fisherman 1 1.7 0 0.0 0 0.0 0 0.0 Agriculture engineer

0 0.0 2 3.4 1 6.7 0 0.0

Veterinarian 0 0.0 1 1.7 0 0.0 0 0.0 Milk man 0 0.0 0 0.0 1 6.7 0 0.0 2: Chi square test MCp: Monte Carlo test F: F test (ANOVA), Sig. bet. grps was done using Post Hoc test (Tukey) p1: p value between group I and group II p2: p value between group III and group IV *: Statistically significant at p ≤ 0.05 Table 2: Illustrate the presenting symptoms and signs of the studied cases of brucellosis. The most common symptoms were fever (100%), Profuse sweating (100%), fatigue (92%), body ache (92%), headache (91.3%), chills (89.3%),

joint pain (67.3%) and abdominal pain (38.7%). In addition, it shows that 100% of cases had positive history of animal contact and consumptions of unpasteurized milk

Table (2): History and clinical signs and symptoms among studied cases

Group I (n = 60)

Group II (n = 60)

Group III (n =15 )

Group IV (n = 15) �� P

No. % No. % No. % No. % Animal contact 57 95.0 56 93.3 13 86.7 14 93.3 1.339 0.724 Consumption of unpastarized milk 55 91.7 56 93.3 14 93.3 13 86.7 1.152 0.900 Fever 60 100.0 60 100.0 15 100.0 15 100.0 - - Chills 53 88.3 51 85.0 15 100.0 15 100.0 3.906 0.239 Headache 51 85.0 56 93.3 15 100.0 15 100.0 4.575 0.157 Vomiting 6 10.0 7 11.7 8 53.3 5 33.3 16.593* <0.001*

Sig.bet. Grps. p1=0.769, p2<0.001*, p3=0.037*, p4<0.001*, p5=0.055, p6=0.269 Nausea 10 16.7 5 8.3 9 60.0 2 13.3 17.686* <0.001 *

Sig.bet. Grps. p1=0.168, p2=0.001*, p3=0.037*, p4=0.753, p5=0.622, p6=0.008* Abdominal Pain 29 48.3 14 23.3 9 60.0 6 40.0 11.202* 0.011* Sig.bet. Grps. p1=0.004*, p2<0.001*, p3=0.419, p4=0.006*, p5=0.207, p6=0.273 Body Aches 55 91.7 53 88.3 15 100.0 15 100.0 2.527 0.416 Fatigue 55 91.7 53 88.3 15 100.0 15 100.0 2.527 0.416 Joint Pains 55 91.7 16 26.7 15 100.0 15 100.0 78.330* <0.001*

Sig.bet. Grps. p1<0.001*, p2=0.576, p3=0.576, p4<0.001*, p5<0.001* Profuse sweating 60 100.0 60 100.0 15 100.0 15 100.0 - - Anorexia 9 15.0 3 5.0 4 26.7 3 20.0 7.448* 0.040* Sig.bet. Grps. p1=0.068, p2=0.279, p3=0.697, p4=0.026*, p5=0.090, p6=1.000 Diarrhea 6 10.0 6 10.0 4 26.7 4 26.7 5.679 0.106 Conistipation 25 41.7 3 5.0 5 33.3 3 20.0 25.056* <0.001*

Sig.bet. Grps. p1<0.001*, p2=0.556, p3=0.121, p4= 0.007*, p5= 0.090, p6=0.682 Cough 11 18.3 8 13.3 4 26.7 2 13.3 1.910 0.609 Dyspnea 0 0.0 0 0.0 1 6.7 0 0.0 5.549 0.205 Convulsions 0 0.0 0 0.0 2 13.3 0 0.0 8.220* 0.018* Sig.bet. Grps. p1 = - ,p2=0.038*, p3 = - p4=0.038*, p5 = - p6=0.483

2: Chi square test MCp: Monte Carlo test p1: p value between group I and group II p2: p value between group I and group III p3: p value between group I and group IV p4: p value between group II and group III p5: p value between group II and group IV p6: p value between group III and group IV *: Statistically significant at p ≤ 0.05 This table 3 shows the vital science findings of the studied cases and there is a significant difference regarding the temperature and heart

rate between complicated and uncomplicated cases.

Table (3): Vital signs among studied cases

Group I (n = 60)

Group II (n = 60)

Group III (n =15 )

Group IV (n = 15)

F p

Temperature Min. – Max. 37.80 – 40.0 37.80 – 40.20 38.60 – 40.50 38.40 – 40.20

7.723* <0.001* Mean ± SD. 38.88 ± 0.61 39.04 ± 0.64 39.65 ± 0.57 39.37 ± 0.54 Median 38.90 39.10 39.70 39.50 Sig.bet.Grps. p1=0.454 p2=0.575 Heart rate Min. – Max. 80.0 – 99.0 78.0 – 100.0 85.0 – 198.0 86.0 – 110.0

15.300* <0.001* Mean ± SD. 90.57 ± 4.69 90.63 ± 4.82 107.93 ± 26.83 96.33 ± 6.65 Median 90.0 90.0 102.0 95.0 Sig.bet.Grps. 1.000 0.006* F: F test (ANOVA), Sig. bet. grps was done using Post Hoc test (Tukey) p1: p value between group I and group II p2: p value between group III and group IV *: Statistically significant at p ≤ 0.05 Table 4: shows comparison between the different groups according to some clinical signs. There is a significant difference between complicated and uncomplicated cases regarding spleenomegally

and hepatomegally which was higher in the uncomplicated cases. There is no significant difference between complicated and uncomplicated cases regarding skin rash.

Table (4): Some clinical signs among studied cases

Group I (n = 60)

Group II (n = 60)

Group III (n =15 )

Group IV (n = 15) 2 MCp

No. % No. % No. % No. % Neck rigidity 0 0.0 0 0.0 2 13.3 3 20.0 15.046* <0.001* Sig.bet. Grps. p1= -,p2=0.038*, p3= 0.007*, p4=0.038*, p5=0.007*, p6=1.000 Spleen Palpable 12 20.0 7 11.7 6 40.0 6 40.0 9.572* 0.018* Sig.bet. Grps. p1= 0.211, p2=0.173, p3=0.173, p4=0.018*, p5=0.018*, p6=1.000 Liver palpable 5 8.3 7 11.7 6 40.0 6 40.0 14.322* 0.002* Sig.bet. Grps. p1= 0.543, p2=0.006*, p3=0.006*, p4=0.018*, p5=0.018*, p6=1.000 Skin Rash 5 8.3 3 5.0 1 6.7 1 6.7 0.874 0.995 2: Chi square test MCp: Monte Carlo test p1: p value between group I and group II p2: p value between group I and group III p3: p value between group I and group IV p4: p value between group II and group III p5: p value between group II and group IV p6: p value between group III and group IV *: Statistically significant at p ≤ 0.05 This table 5 shows comparison between the different groups according to different clinical findings. There is pulmonary involvement in two cases in group IV. The heart was clinically free among the four groups. The joints were clinically free in groups I and II while there is

inflammation of sacroiliac joint in 66.7% of cases in group III and 53.3% of cases in group IV. Spinal affection was reported in one case of group III. Knee joint affection was reported in one case of group IV. Spinal abscess was reported in one case in group IV.

Table (5): Different clinical findingsamong studied cases

Group I (n = 60)

Group II (n = 60)

Group III (n =15 )


No. % No. % No. % No. % Chest Free 60 100.0 60 100.0 15 100.0 13 86.7

8.220* 0.018* Mild bronchitis 0 0.0 0 0.0 0 0.0 2 13.3 Sig.bet. Grps. p1=- p2=0.483 Heart Free 60 100.0 60 100.0 15 100.0 15 100.0 - - Joints Free 60 100.0 60 100.0 4 26.7 6 40.0

10.573* <0.001* Inflammation of SI joint

0 0.0 0 0.0 10 66.7 8 53.3

Spinal affection 0 0.0 0 0.0 1 6.7 0 0.0 Knee joint affection 0 0.0 0 0.0 0 0.0 1 6.7 Sig.bet. Grps. p1=- p2=0.568 Spine Normal 60 100.0 60 100.0 14 93.3 0 0.0

92.661* <0.001* Spinal affection 0 0.0 0 0.0 1 6.7 0 0.0 Spinal abscess 0 0.0 0 0.0 0 0.0 1 6.7 Sig.bet. Grps. p1=- p2=<0.001* 2: Chi square test MCp: Monte Carlo test p1: p value between group I and group II p2: p value between group III and group IV *: Statistically significant at p ≤ 0.05 Table 6 shows the comparison between the different groups according to the clinical response in visit 1 (1 week after discharge from the hospital) there was clinical cure in 91.7% in group I, 95% in group II, 40% in group III and 60% in group IV. There is clinical improvement in 40% of cases of group IV. There was clinical failure in 8.3% of cases of group I, 5% of cases of group II and 60% of cases of group III. Visit 2 (2 weeks after discharge from hospital) there was clinical cure in 100% of cases of group I, 95% of

cases of group II and 100% of cases of group III. There was clinical improvement in 100% of the cases of group IV. Visit 3 (4 weeks after discharge from hospital) there was clinical cure in 100% of cases of group I, 95% of cases of group II, 66.7% of cases of group III and 100% of cases of group IV. There was clinical improvement in 33.3% of cases of group III. There was clinical failure in 5% of cases of group II.

Table (6): Clinical response in different visits among studied cases

Group I (n = 60)

Group II (n = 60)

Group III (n =15 )


No. % No. % No. % No. % Visit 1 (1 week) Clinical cure 55 91.7 57 95 6 40.0 9 60.0

124.94* <0.001* Clinical improvement 0 0.0 0 0.0 0 0.0 6 40.0 Clinical failure 5 8.3 3 5.0 9 60.0 0 0.0 Sig.bet.Grps. p1=0.717, p2<0.001* Visit 2 (2 weeks) Clinical cure 60 100.0 57 95.0 15 100.0 0 0.0

88.807* <0.001* Clinical improvement 0 0.0 0 0.0 0 0.0 15 100.0 Clinical failure 0 0.0 3 5.0 0 0.0 0 0.0 Sig.bet.Grps. p1=0.244 p2<0.001* Visit 3 (4 weeks) Clinical cure 60 100.0 57 95.0 10 66.7 15 100.0

23.625* <0.001* Clinical improvement 0 0.0 0 0.0 5 33.3 0 0.0 Clinical failure 0 0.0 3 5.0 0 0.0 0 0.0 Sig.bet.Grps. p1=0.244 p2=0.042* 2: Chi square test MCp: Monte Carlo test p1: p value between group I and group II p2: p value between group III and group IV *: Statistically significant at p ≤ 0.05

Table 7 shows the comparison between the different groups according to follow up results. There was no relapse in all cases of groups I and II but there was a relapse in two cases (13.3%) in group III in follow up period after 1 month, 2 months, 3 months and 4 months respectively. There was a relapse of 7 cases (11.7%) in group

I and 4 cases (6.7%) in group II which was significant in a follow up period from 4 to 8 months. There was a relapse in 8 cases (13.3%) in group I and 4 cases (6.7%) in group II in the follow up period from 8 to 13 months which was significant.

Table (7): Follow up results among studied cases

Group I (n = 60)

Group II (n = 60)

Group III (n =15 )


No. % No. % No. % No. % Follow up (1 – 4 month) Relapse 0 0.0 0 0.0 2 13.3 2 13.3

88.757* <0.001* No relapse 60 100.0 60 100.0 0 0.0 13 86.7 Clinical improvement 0 0.0 0 0.0 0 0.0 0 0.0 Clinical cure 0 0.0 0 0.0 13 86.7 0 0.0 Sig.bet. Grps. p1<0.001* p2<0.001* Follow up (4 -8 month) Relapse 7 11.7 4 6.7 0 0.0 0 0.0 No relapse 53 88.3 56 93.3 12 80.0 13 86.7

24.103* 0.002* Clinical improvement 0 0.0 0 0.0 2 13.3 2 13.3 Clinical cure 0 0.0 0 0.0 1 6.7 0 0.0 Sig.bet. Grps. p1=0.343 p2=0.0232 Follow up(8 – 13 month) Relapse 8 13.3 4 6.7 0 0.0 0 0.0 No relapse 52 86.7 56 93.3 13 86.7 13 86.7

14.789* 0.007* Clinical improvement 0 0.0 0 0.0 0 0.0 0 0.0 Clinical cure 0 0.0 0 0.0 2 13.3 2 13.3 Sig.bet. Grps. p1=0.224 p2=1.000 2: Chi square test MCp: Monte Carlo test p1: p value between group I and group II p2: p value between group III and group IV *: Statistically significant at p ≤ 0.05

Table 8 shows comparison between the different groups according to different radiological findings. There were abnormal findings in X-ray lumbo-saccral in 9 cases (60%) in group III denoting sacroiliac affection and 6 cases (40%) in group IV. There was abnormal findings in

MRI in 9 cases (60%) denoting sacroiliac affection in group III and 6 cases (40%) in group IV. There was abnormal brain finding in 2 cases (13.3%) in group III and 3 cases (20%) in group IV.

Table (8): Different radiological findings among studied cases

Group I (n = 60)

Group II (n = 60)

Group III (n =15 )


No. % No. % No. % No. % X-ray Chest Normal 60 100.0 60 100.0 15 100.0 15 100.0 - - X-ray lumbo-sacral Normal 60 100.0 60 100.0 6 40.0 9 60.0

66.234* <0.001* Abnormal finding 0 0.0 0 0.0 9 60.0 0 0.0 SI affection# 0 0.0 0 0.0 0 0.0 6 40.0 Sig.bet.Grps p1=- p2<0.001* MRI@ Normal 60 100.0 60 100.0 4 26.7 6 40.0

96.462* <0.001* Abnormal SI finding# 0 0.0 0 0.0 9 60.0 6 40.0 Abnormal brain finding 0 0.0 0 0.0 2 13.3 3 20.0 Sig.bet.Grps p1=- p2=0.608 2: Chi square test MCp: Monte Carlo test p1: p value between group I and group II p2: p value between group III and group IV *: Statistically significant at p ≤ 0.05 #: Sacro- iliac joint @:Magnetic resonance imaging

This table 9 shows the comparison between the different groups according to U/S abdomen findings. There was spleenomegally in 11 cases (18.3%) in group I and 6 cases (40%) in group III. There was hepatomegally in 5 cases (8.3%) in group I. Both hepatomegally and

spleenomegally recorded in 7 cases (11.7%) in group 2 and 6 cases (40%) in group IV. There was a significant difference regarding size of the liver and spleen among the different studied groups.

Table (9): Ultrasonographic abdominal findings among studied cases

Group I (n = 60)

Group II (n = 60)

Group III (n =15 )

Group IV (n = 15) Test of sig. p

No. % No. % No. % No. % U/S abdomen Normal 44 73.3 53 88.3 9 60.0 9 60.0

2=47.320* MCp<0.001*Splenomegally 11 18.3 0 0.0 6 40.0 0 0.0 Hepatomegally 5 8.3 0 0.0 0 0.0 0 0.0 Both 0 0.0 7 11.7 0 0.0 6 40.0 Sig.bet.Grps. p1<0.001*, p2=0.200, p3<0.001*, p4<0.001*, p5=0.018*, p6=0.001* Size of the Spleen Min. – Max. 9.0 – 15.0 9.0 – 15.0 9.0 – 15.10 9.0 – 15.70

F=2.941* 0.035* Mean ± SD. 11.91 ± 1.72 11.58 ± 1.55 12.70 ± 1.82 12.72 ± 2.13 Median 12.0 11.50 12.50 13.0 Sig.bet.Grps. p1=0.713,p2=0.379,p3=0.357,p4=0.108,p5=0.099,p6=1.000 Size of the Liver in M.C.L Min. – Max. 14.0 – 18.10 14.0 – 18.20 14.0 – 17.90 15.10 – 17.60

F=4.252* 0.007* Mean ± SD. 15.31 ± 1.03 15.36 ± 1.12 15.99 ± 1.31 16.23 ± 0.86 Median 15.0 15.0 16.0 15.90 Sig.bet.Grps. p1=0.993,p2=0.133,p3=0.019*,p4=0.188,p5=0.031*,p6=0.930 2: Chi square test MCp: Monte Carlo test p1: p value for comparing between group I and group II p2: p value for comparing between group I and group III p3: p value for comparing between group I and group IV p4: p value for comparing between group II and group III p5: p value for comparing between group II and group IV p6: p value for comparing between group III and group IV *: Statistically significant at p ≤ 0.05 This table 10 illustrates comparison between the different groups according to standard tube agglutination test (STA): there was no

significant difference between the different studied groups.

Table (10): Standard tube agglutination test (STA) among studied cases

Group I (n = 60)

Group II (n = 60)

Group III (n =15 )

F p

STA Min. – Max. 320.0 – 1280.0 320.0 – 1280.0 640.0 - 1280.0

1.129 0.326 Mean ± SD. 1040.0 ± 380.12 1072.0 ± 357.53 1194.67 ± 225.2 Median 1280.0 1280.0 1280.0 F: F test (ANOVA) This table 11 illustrate the comparison between the different groups according to serology and culture results, blood culture was positive in 49 cases (81.7%) in group I, 51 cases (85%) in group II, 13 cases (86.7%) in group III and 12

cases (80%) in group IV. Bone marrow culture was positive in 56 cases (93.3%) in group I, 57 cases (97%) in group II, 14 cases (93.3%) in both groups III and IV. ELISA was positive in all cases (100%) in all the groups.

Table (11): Serology andculture results among studied cases

Group I (n = 60)

Group II (n = 60)

Group III (n =15 )


No. % No. % No. % No. % Blood culture Negative 11 18.3 9 15.0 2 13.3 3 20.0

0.599 0.943 Positive 49 81.7 51 85.0 13 86.7 12 80.0 ELISAIgM Negative 0 0.0 0 0.0 0 0.0 0 0.0

- - Positive 60 100.0 60 100.0 15 100.0 15 100.0 BM culture Negative 4 6.7 3 5.0 1 6.7 1 6.7

0.709 1.000 Positive 56 93.3 57 95.0 14 93.3 14 93.3 2: Chi square test MCp: Monte Carlo test This table 12 shows the comparison between the different groups according to CBC findings. There was no significant difference between the different groups as regard HB, total WBCs and platelets. However, there was a significant

difference between complicated group of patients and uncomplicated group of patients as regard lymphocytes % which was higher in complicated groups of patients.

Table (12): CBC findings among studied cases

Group I (n = 60)

Group II (n = 60)

Group III (n =15 )

Group IV (n = 15)

F p

Hb Min. – Max. 11.0 – 15.20 11.0 – 15.10 11.40 – 15.0 11.10 – 15.10

0.143 0.934 Mean ± SD. 13.13 ± 1.14 13.05 ± 1.01 13.14±1.06 13.25 ± 1.27 Median 13.05 13.0 13.10 13.50 Total W.B.Cs (×103) Min. – Max. 3.95 - 10.64 4.10 - 10.75 3.78 – 12.0 5.86 – 10.25

2.063 0.108 Mean ± SD. 6.79 ± 1.83 7.18 ± 1.67 7.55 ± 2.71 8.01 ± 1.53 Median 6.80 7.18 7.56 8.17 Lymphocytes % Min. – Max. 18.0 – 42.0 13.0 - 44.0 26.0 – 63.0 24.0 – 62.0

25.544* <0.001* Mean ± SD. 29.65 ± 6.20 29.75 ± 6.99 44.80 ± 12.45 43.60 ± 12.87 Median 29.0 29.50 44.0 45.0 Sig.bet. Grps p1=1.000,p2<0.001*,p3<0.001*,p4<0.001*,p5<0.001*,p6=0.978 Platelets Min. – Max. 150.0 – 411.0 118.0 – 396.0 187.0 – 367.0 186.0 – 410.0

0.931 0.427 Mean ± SD. 265.78 ± 68.21 276.42 ±65.80 284.07 ± 56.19 295.07 ± 74.75 Median 261.0 282.0 295.0 290.0

F: F test (ANOVA) p1: p value for comparing between group I and group II p2: p value for comparing between group I and group III p3: p value for comparing between group I and group IV p4: p value for comparing between group II and group III p5: p value for comparing between group II and group IV p6: p value for comparing between group III and group IV *: Statistically significant at p ≤ 0.05 This table 13 shows the comparison between the different groups according to their need to chagefron the studied treatment to the WHO recommended treatment. The studied treatment was changed to WHO recommended treatment

in 3 cases (5%) in group I and 10 cases in group III. There was clinical failure due to side effect in 2 cases (3.3%) in group I, 3 cases (5%) in group II and 1 case (6.7%) in group III.

Table (13):Comparison between the different groups according to their need to change from the studied treatment to the WHO recommended treatment

Group I (n = 60)

Group II (n = 60)

Group III (n =15 )


No. % No. % No. % No. % Need to change 3 5.0 0 0.0 10 66.7 0 0.0 40.707* <0.001* Sig.bet.Grps. p1=0.244, p2<0.001* Clinical failure due to side effect 2 3.3 3 5.0 1 6.7 0 0.0 1.183 0.917 2: Chi square test MCp: Monte Carlo test p1: p value between group I and group II p2: p value between group III and group IV *: Statistically significant at p ≤ 0.05 This table 14 illustrates comparison between the different groups according to time of defervescence of fever. Table (14): Time of defervescence of fever among studied cases

Group I (n = 60)

Group II (n = 60)

Group III (n =15 )

Group IV (n = 15)

F p

Time of defervescence of fever Min. – Max. 3.0 – 10.0 3.0 – 9.0 3.0 – 12.0 3.0 – 12.0

5.180* 0.002* Mean ± SD. 5.07 ± 1.80 6.0 ± 2.18 7.20 ± 2.98 6.67 ± 2.82 Median 5.0 6.0 6.0 6.0 Sig.bet.Grps 0.022* 0.508 F: F test (ANOVA), Sig. bet. grps was done using Post Hoc test (Tukey) p1: p value between group I and group II p2: p value between group III and group IV *: Statistically significant at p ≤ 0.05 Table (15): Number of cases of Brucellosis admitted to Alexandria Fever Hospital from 1997 to 2014

Year Number 1997 59 1998 79 1999 84 2000 111 2001 91 2002 132 2003 227 2004 184 2005 176 2006 208 2007 202 2008 269 2009 201 2010 224 2011 199 2012 267 2013 216 2014 230

Discussion The residence of the studied patients in our research illustrates that most of the cases (88.7%) were living in rural areas while (11.3%) were living in urban areas.This may be attributed to more exposure to risk factors for infection by Brucella species beside occupational exposure in rural areas. Sex difference in the present study found that males were more infected by brucellosis than females(76.7%malesversus23.3% females).

This result is constant with Akritidiset al,(2002),(21) who reported that 62.5 % of cases were males and 37.5 % were females. Other researchers also reported higher male percentage (74.8% versus 25.2%)(22).While several authors reported that both sexes were affected more equally (21,23,24). The present study declared that age distribution was from 18 to 50 years with the mean age was 30.05 ± 9.45 in group I, 29.63 ± 8.93 in group II, 30.13 ± 8.37 in group III and 27.0 ± 8.08 in group IV.

Most of the cases in our study were living in rural areas, 23.3% were shepherds, 23.3% were house wives, 25.3% were farmers, 18% were butchers, 1.3% teachers, 4% were workers, 0.7% was fisherman, 0.7% was agriculture engineer, 0.7% was veterinarian and 0.7% was milk man. In addition, our work showed that 95% of cases had positive history of animal contact in group I, 93.3% in group II, 86.7% in group III, and 93.3% in group IV. Moreover, 91.7% of patient in group I, 93.3% in group II and III, 86.7% in group IV had positive history of consumptions of unpasteurized milk.We investigated whether there is a high risk of occupational exposure of Brucella spp. among our studied patients. The finding of present study concluded that presence of contact with cattle, goats and sheep especially, among shepherds, butchers and farmers (71.9% of our patients) was the main risk factor for our infected patients. Assisting in delivery or abortion of animals was another risk factor particularly among house wives. There is also the potential for the exposure through consumption of milk and dairy products processed from raw milk that are regularly consumed in most houses in such rural areas. Our results agreed with Holt et al,,(25)showed that Brucellosis is endemic at high levels among the large ruminant population of the studied village in the Nile Delta region of Egypt, they added that the main risk factor for cattle and buffalo seropositive status is the presence of sheep or goats in the same household. Animals suspected of having brucellosis are much more likely to be sold to the butcher or in the market than reported to the necessary authorities. Given the observed seroprevalence and reported knowledge, attitudes and practices, in the studied population, there is a high risk of occupational exposure of Brucella spp. in livestock keepers, whilst assisting in parturition or abortion of ruminants. There is also the potential for human exposure through consumption of dairy products processed from raw milk that are regularly consumed in most households. The present study illustrated the presenting symptoms and signs of the studied cases of Brucellosis. The most common symptoms were fever (100%), Profuse sweating (100%),fatigue (92%), body ache (92%), headache (91.3%), chills (89.3%), joint pain (67.3%) and abdominal pain (38.7%).In addition, it shows that 91% of cases had positive history of animal contact and 92% of cases had

positive history of consumptions of unpasteurized milk. Our results were in agreement with the study done by Koseet al, (2014),(26) they found that the average age was determined to be 44.8 ± 18 years, and 40 of the patients were female (55.6%). The most frequent complaints were joint pain, high fever, weakness, low back pain, and gastrointestinal symptoms, whereas the most frequent physical examination findings were fever, osteoarticular involvement, splenomegaly, hepatomegaly, and lymphadenopathy. All of the patients were positive for Rose Bengal testing. The standard tube agglutination titer was 1/160 or higher in 64 (88.9 isolated from blood %) patients. Brucella melitensis was cultures in 18.1% of patients and bone marrow cultures in 9.7% of patients.In the current research found that there is a significant difference between complicated and uncomplicated cases regarding splenomegally and hepatomegally which was higher in the uncomplicated cases. However, there is no significant difference between complicated and uncomplicated cases regarding skin rash. The joints were clinically free in groups I and II while there is inflammation of sacroiliac joint in 66.7% of cases in group III and 53.3% of cases in group IV. Spinal affection was reported in one case of group III. Knee joint affection was reported in one case of group IV. Spinal abscess was reported in one case in group IV. Hasanogluet al,,(27)Supported this finding as they declared that the disease has a broad spectrum of clinical manifestations. The musculoskeletal system involvement is frequent and, rarely, arthritis can be the only clinical feature of the disease. Our research found that there is 2(13.3%) of group III have evidence of neurobrucellosis in the form of meningitis and three cases (20%) of group IV have affection of C.N.S due to brucellosis in the form of 2 cases of meningitis and one case of meningioencephalitis. All the 5 cases have both clinical, laboratory and radiological evidences of neurobrucellosis. Mermutet al,(2012),(28) found that there is 3.7% of direct invasion of the central nervous system of his study is slightly less than the 5–6.6% reported in other studies.(29-32) Acute or chronic meningitis is the most common manifestation of neurobrucellosis but meningoencephalitis, brain abscess, epidural abscess and demyelinating disorders and meningovascular syndromes have also been reported. 3.7% of their studied patients

manifested a direct invasion of the central nervous system, 6 had meningitis and the other had a brain abscess. The finding of the present study concluded that the results among the different groups according to serology and culture results, blood culture was positive in 49 cases (81.7%) in group I, 51 cases (85%) in group II, 13 cases (86.7%) in group III and 12 cases (80%) in group IV. Bone marrow culture was positive in 56 cases (93.3%) in group I, 57 cases (97%) in group II, 14 cases (93.3%) in both groups III and IV.ELISA was positive in all cases (100%) in all the groups. The present study results were in consistence with And riopouloset al,(2015),(33)who declared that rapid and low-cost tests as RBT and STA are still very useful in diagnosing acute brucellosis; however, every positive test must be examined together with clinical symptoms and occupational history. The tests can be used as screening tests in endemic populations to rule out acute Brucellosis. One can argue that the treatment of human brucellosis is controversial. Another purpose of this study was to search published clinical trial papers to provide a simple and effective treatment in brucellosis and try to find out a regimen that is of shorter duration could significantly improve compliance, resulting in fewer long term complications. Our research found that the rate of clinical failure was 8.3% of cases of group I, 5% of cases of group II, 60% of cases of group III and 40% of cases of group IV. Previous researches carried out in this field showed that Regarding the comparison between the different groups according to the clinical response in visit 1 (1 week after discharge from the hospital) there was clinical cure in 91.7% in group I, 95% in group II, 40% in group III and 60% in group IV. A study in Iran by Roushanet al, (34) recorded a therapeutic failure of 3.6%,9.3%,7.1% and 23% in their various groups of combinations. Soleraet al. (1995)(35) reported 8% therapeutic failure in doxycycline-rifampicin combination and 2% therapeutic failure in doxycycline-streptomycin combination. Akovaet al, ,(36) reported 3.2% therapeutic failure in ofloxacin-rifampicin combination group and 3.2% in doxycycline-rifampicin group.However,AL-Sibaiet al, (1992)(37) had reported a 0% therapeutic failure.Adverse reaction of the drug used in treatment of brucellosis is an important issue as it is used for a long period. In the current study, the side effects of treatment were

emphasized in clinical interviews conducted during the treatment period. In general, both therapeutic regimens were well tolerated with the exception of only two cases (3.3%) that were treated with the studied regimen in group I that had adverse effects severe enough to discontinue therapy, one in the form of allergic reaction, and the other in the form of abscess formation at the site of drug injection. three cases (5%) that were treated with the WHO recommended regimen in group II that had adverse effects severe enough to discontinue therapy, one in the form of gastric ulcer , another one in the form of severe gastritis and third one in the form of jaundice .One case in group III had adverse effect in the form of renal impairment. Other treated cases in our study complained of some side effects that were not considered severe enough to shift to other therapeutic regimen. As regard cases treated by conventional regimen 5 cases (3.3%) complained of gastrointestinal tract (GIT) troubles, mainly gastritis, 1 case (0.66%) suffered from tremors.As regard the new studied regimen cases (2.6%) suffered from mild renal impairment which is a reported side effect of gentamycin.Soleraet al, (1995), (35) reported mild adverse effects in 31% of cases treated by doxycycline plus rifampicin (DR) & 23%in doxycycline plus streptomycinegroup, but they reported only 4% and 2% severe adverse effect. Akovaet al, (1993),(36)reported a ratio of 6.5% gastric irritation in a combination of rifampicin and ofloxacin but they recorded 43.3% gastric discomfort in doxcycycline- rifampicin combination. This difference is attributed to doxcycycline which is well known to cause gastric discomfort and irritation. AL-Sibaiet al.(1995),(37) reported that ciprofloxacin was well tolerated and that all patients in their study complied with the course of therapy, from them 2 patients (12.5%) complained of mild gastrointestinal disturbance, one patient (6.25%)developed a skin reaction in a form of maculopapular rash, however, all of these adverse reactions were self- limiting and did not necessitate interruption orcessation of therapy. Arizaet al, (1992)(38) compared between two groups treated by doxycycline – rifampicin and doxycycline-streptomycin, they reported that no major adverse effects were associated with either combination. Mild gastric complaints were frequent and were attributed to the doxycycline. They also reported that 3 cases developed

ototoxicity in the form of dizziness, and vertigo with doxycycline plus streptomycine combination. Gilbert, (2000)(39) reported that nephrotoxicity and hyper sensitivity reactions can follow the administration of gentamycin. Results of the present work disagreed with Soleraet al, (2001)(40) that reported ten patients with brucellosis were treated with azithromycin and Gentamycin to assess the treatment’s safety and efficacy.70% of patients had an excellent therapeutic response at the end of therapy; however, relapse was noted in 30% of patient. When relapse was considered in combination with an initial lack of efficacy, 50% of patients (95% confidence interval, 18.7%–81.3%) did not respond to therapy; these results do not favor the use of azithromycin to treat brucellosis in humans.Soleraet al.(2001)(40) argued thatazithromycin may be an effective therapy for treatment of brucellosis, but, to their knowledge, its use has not been assessed in humans. They reported the findings of a pilot investigation that studied treatment with azithromycin for 21 days plus Gentamicin for the first 7 days of therapy among 10 patients with acute brucellosis. They concluded that a regimen of azithromycin of 3 weeks’ duration plus Gentamicin given during the first week results in a high rate of therapeutic failures and relapses. These results do not favor the use of azithromycin to treat brucellosis in humans. However, in our patients who were treated with azithromycin for 30 days [versus 21 days in Soleraet al.(2001)](40) and with Gentamicin for the first 15 days [versus 7 days in Soleraet al.(2001)] in this study, the overall failure rate of was lower than reportedin Soleraet al.(2001)(40) in uncomplicated group of patients .However,, the overall failure rate of was higher than reported in Soleraet al.(2001)(40) in our complicated group of patients. Soleraet al.(2001)(40)declared that these findings are especially notable not only for the relapse of brucellosis within only a few weeks of stopping azithromycin therapy in 42.8% of patients who completed 21 days of therapy but, also, for the apparent lack of a significant treatment effect in 20% of patients, 10% of whom developed sternoclavicular arthritis during the second week while undergoing azithromycin therapy. This suggests that azithromycin could not prevent the development of osteoarticular complications.The goals of treatment of brucellosis include control of acute illness, prevention of associated

complications, and prevention of relapse. There are still questions regarding the choice of antibiotics and the duration of treatment of brucellosis. Duration of treatment must also be long enough to eliminate the organism. Because the WHO recommended course of therapy is so long compliance is a significant concern. A regimen that is shorter could significantly improve the compliance resulting in fewer long term complications. Azithromycin, antibiotic with excellent intracellular penetration and long half-life is a very potential for use as apart of a regimen that would require a shorter duration of therapy. Gentamicin, another aminoglycoside with a wide spectrum of antibacterial activity, may be an active in vitro against clinical Brucella isolates and less toxic than streptomycin. Our findings also introduce new considerations on the potential role of new therapeutic regimen such as Gentamicin plus azithromycin for treatment of human brucellosis especially among uncomplicated cases. Their therapeutic application in earlier stages of human brucellosis might prove useful in the clinical management of human brucellosis before complication or chronic form of the disease develops. Our results do not favor the use of Gentamicin plus azithromycin to treat complicated brucellosis in humans References 1. Young EJ. Brucella species; in Mandell GL,

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Original Article

Detection of SHV and TEM Genes in Escherichia Coli and Klebsiella Isolates Azza Mohammed Abed-El Aziz**, Ghada Rashad Hendawy*, Tawfiek Mohammed Abed-El Motaleb**, Amina Mohammed Elsaadany***, *medical Microbiology and Immunology Department, Faculty of Medicine ,Menoufia university,**medical Microbiology & Immunology Department, National Liver Institute, Menoufia university,***menoufia health directorate- ministry of health

ABSTRACT Study the TEM& SHV genes in ESBLs producing Enterobacteriaceae isolated from National Liver Institute Patients. Resistance to ß-lactam antibiotics is an increasing problem and ß lactamase production is the most common mechanism of drug resistance , especially in Enterobacteriaceae. The clinical and laboratory standard institute (CLSI) interpretive guidelines stated that, Enterobacteriaceae that produce ESBLs are resistant to therapy with penicillin , cephalosporins and azetronam ,despite apparent in vitro susceptibility to some of these agents. Aim of the work :To study the detection of ESBLs in Enterobacteriaceae and impact to control the spread of resistance.Methods:The isolated strains of Enterobacteriaceaeare subjected to phenotype detection by screening tests for ESBLs By disc diffusion then to confirmatory tests for ESBLs by double disc test.Results: 78 (56.5%)of 138 (100%) isolates were found to be ESBL producers by the CLSI confirmatory method, Out of 78 ESBL producers, 47(60.3%)were E. coli and 31(39.7%) were Klebsiella., Of the various clinical samples, The most frequent ESBLs isolates were from stool (23.9%) followed by urine (16.7%). Conclusion: High prevalence of ESBL producers in our hospital (60.6%) calls for strict policies regarding antibiotic usage and their screening methods and hospital-based clinical laboratories should screen isolates following hospitalization in patients in need for antibiotics to formulate effective antibiotic strategy and plan a proper hospital infection control strategy to prevent the spread of these ESBL strains. Introduction Resistance to ß-lactam antibiotics is an increasing problem and ß lactamase production is the most common mechanism of drug resistance , especially in Enterobacteriaceae(1) .ESBL producing strains are a major problem in many hospitals leading to increased morbidity, mortality and health care costs. Cephalosporins are first line drugs in treatment of infections caused by Enterobacteriaceae family, however, because of extensive use of third generation cephalosporin has resulted in increased prevalence of ESBL among organisms(2).The most important mechanism of resistance to β-lactam antibiotics is the production of β-lactamase enzymes. The β-lactamases confer significant antibiotic resistance to their bacterial hosts by hydrolysis of the amide bond of the β-lactam ring. Classes A, C, and D include enzymes that hydrolyze their substrates by forming an acyl (penicilloic or cephalosporoic) enzyme through an active site serine, whereas, class B β-lactamases are metalloenzymes that utilize at least one active-site zinc ion. These

enzymes are especially important in Gram-negative bacteria as they constitute the major defense mechanism against β-lactam-based drugs(3). ß-lactamasesare remarkably diversified due to their continuous mutation(1). The clinical and laboratory standard institute (CLSI) interpretive guidelines stated that, Enterobacteriaceae that produce ESBLs are resistant to therapy with penicillin , cephalosporins and azetronam,despite apparent in vitro susceptibility to some of these agents(4). Third-generation cephalosporin resistance is often mediated by TEM- and SHV-type L-lactamases in Enterobacteriaceae(5). Aim of theWork Study the TEM& SHV genes in ESBLs producing Enterobacteriaceae isolated from National Liver Institute Patient. Patients and Methods Collection of samples: the study was carried on 138 isolates of E coli and klebsiella from cultures of different microbiological specimens

including urine, sputum, pus, stool, ascetic fluid, throat swab, blood, drain, wound, T tube, nasal swab and C.V.P line. From patient with hospital acquired infection.Isolation and identification: Colony isolates were obtained after culture of urine specimens on CLED (Cystiene Lactose Electrolyte Deficient), while pus and sputum specimens were cultured on blood agar and MacConkey’s (MAC) agar, then were all incubated aerobically at 37ºc for 24 hours. Blood samples were inoculated by blood culture bottles, then subcultures were done also on blood and MacConkey’s (MAC) agar. A single isolated colony was considered for further studies and identification was done using standard conventional, morphological, cultural and biochemical tests(6).Detection of ESBL: 1-Screening for ESBL producers: According to CLSI 2011 recommendations to identify potential ESBL producing isolates using standard disk diffusion techniques.ceftazidime (30 μg), cefotaxime (30 μg) and ceftriaxone (30 μg) are used, If a zone diameter of < 22 mm for ceftazidime, < 27 mm for cefotaxime and < 25 mm for ceftriaxone were recorded, the strain was considered to be “suspicious for ESBL production”. The use of more than one antimicrobial agent for screening improves the sensitivity of detection.2-Phenotypic confirmatory test for ESBL production: by cephalosporin/ clavulanate combination disks.All the strains which were screened out for ESBL production were also subjected to confirmation by using the PCDDT, as recommended by the CLSI 2011. The ceftazidime (30 μg) , cefotaxime (30 μg) discs alone and in combination with clavulanic acid (ceftazidime + cavulanic acid, 30/10 μg discs- cefotaxime + clavulanic acid, 30/10 μg) were applied onto a plate of Mueller Hinton Agar (MHA) which was inoculated with the test strain.An increase of ≥ 5 mm in a zone diameter for either antimicrobial agent tested in combination with clavulanic acid versus its zone when tested alone denoted ESBL producing strains.Reference strains used in this study were E. coli ATCC 25922 as negative control and Klebsiellapneumoniae ATCC 700603 as positive control.Statistical Methods:The (t) test is used to asses the statistical significance of difference between two means. By knowing the (t) test and the degree of freedom, the (P) value is calculated from special tables, and so, the significance of

the results was determined from the “t” distribution tables.P <0.05 = insignificant difference, P >0.05 = significant difference.P > 0.01 = highly significant difference, P > 0.001 = very highly significant difference. Results The age of the studied cases ranged from 10 to 75 years with 55.72 ± 8.94 years as a mean ± standard deviation (SD) & 45 years as a median. One hundred and thirty eight clinical samples were cultured from various sample sites from patients admitted at National Liver Institute. The distribution of isolates were 79(57.2%)E. coli and 59(42.8%)Klebsiella spp.Most cases have been presented by chronic liver diseases Clinically, 19 patients had ascites, 10 had jaundice, 8 had shrunken cirrhotic liver, 8 had splenomegaly, 4 had encephalopathy,8 had septicemia, 15 abdominal pain, 9 had wheezy chest, 4 had clinically infected wounds & C.V.P. lines, 12 had suprapubic pain & 9 had recurrent diarrhea.Overall, 78(56.5%)isolates were detected as ESBL producers by phenotypic confirmatory test for ESBL production (cephalosporin/ clavulanate combination disks) and include45 (57.6%) E. coli and 31(39.7%)Klebsiella spp.Among 56.5% ESBL isolates, there were 73 (52.9%)ESBL isolates from patients with previous antimicrobial intake which were more than isolates from patients without previous antimicrobial intake 5(3.6%)ESBLs isolates are higher in males 40(29%) than females 38(27.5%). The most frequent ESBLs isolates were from stool (23.9%) followed by urine (16.7%) while, the least frequent ESBLs isolates were from nasal swabs and C.V.P line samples (0.7%). Discussion The increasing incidence of antibiotic resistanceis getting more global attention.Antibioticmulti-resistant Gram negative bacteria pose a risk to public health(7). Extended spectrum ß-lactam antibiotics have been widely used for treatment of serious Gram-negative infections since the 1980s. However, bacterial resistance has emerged quickly due to the production of ESBLs(8). Enterobacteriaceae producing ESBL are emerging as a threatening cause of both hospital andcommunity acquired infection as they are often resistant to standard

antimicrobial choices (9-10). It is generally thought that patients infected by an ESBL-producing organism are at an increased risk of treatment failure (11). Recently, an increasing percentage of ESBL-producing K. pneumonia strainshave been detected worldwide with a significant impact on the clinical course of disease and expenditure of resources (12). As it is shown in a meta-analysis study that infections with ESBL-producing bacteria are associated with nearly twice the mortality rate compared to that of non-ESBL producers(13). Extended spectrum ß-lactamases are enzymes produced by some K. pneumonia and E coli species thatinactivatepenicillin’s, expanded- spectrum cephalosporin’s, monobactams including older beta-lactam antimicrobial agents and are inhibited by clavulanic acids, sulbactam or tazobactam(14-15). The first ESBLs have evolved by genetic mutation from native β-lactamases TEM and SHV. The epidemiology of ESBL-producing K pneumonia is complex and their prevalence among clinical isolates varies from country to country, institution to institution and therapeutic choices for infections caused by ESBL-producing bacterial strains remain limited (14). In Egypt, a high prevalence of infections caused by ESBL-producing E. coli and Klebsiella spp. has been reported (16-17). Suggested approach to the management of patients with serious infections due to ESBL producing Enterobacteriaceae: First-line therapy: - carbapenems. - piperacillin-tazobactam (low inoculum). - fosfomycin (oral formulation for simple urinary tract infections). Second-line therapy: - tigecycline (not in urinary tract infections). –fluoroquinolone. – colistin(18). In the present study, the prevalence of ESBL producing Enterobacteriaceae isolates was 60.6%. Nearly same results were obtained by (Olowe, et al.; 2012) from Nigeria which showed that the prevalence ofESBL producing Enterobacteriaceae isolates was about 51.3%14 (19) and also by(Dallal, et al.; 2012) from Iran who found that57.5% of isolates were ESBL-producing Enterobacteriaceae(20).In several western studies, prevalence of ESBLs was less than that found in our study. The uncontrolled use of third generation cephalosporin’s at our hospital could be a leading contributory factor to the high ESBL prevalence observed in this study.In the present study, The maximum ESBL

production was seen among the isolates of E. coli (34.1%) followed by klebsiella (22.5%). Another study in Chennai reported that ESBL production were among 47 % E. coli and 37 % Klebsiella pneumoniae in a tertiary care centre(21).But these results disagreed with that of Kaur And Aggarwal, 2013 study which showed that the maximum ESBL production found among the isolates of Klebsiella pneumoniae (52.27%), followed by those of E.coli (46.43%)(22).Based on the results of this study, all the ESBL positive isolates that were selected for multiplex PCR for TEM and SHV genes showed that in case of E coli (26.9%) were positive for TEM gene alone, (7.7%) were positive for SHV gene, (25.6%) were positive for both TEM and SHV genes.In case of K. pneumoniae(12.8%) were positive for both TEM and SHV genes, (14.1%)for SHV gene alone and (12.8%)were positive for TEM gene alone. Nearly same results were obtained from Kashan, Iran showed that The rates of blaTEM and bla SHV in E coli isolates were 44(63%) and 5(7%), respectively. In 9 (13%) of the isolates bothblaTEM and blaSHV were detected(23).In the Rastegar Lari study, ESBLsproducing E. coli had 85.6% TEM, 69.2% SHV and 53.8% in both genes that were much higher than in our results (24). In another study in Turkey, the ESBLs producing organisms (E. coli and K. pneumoniae) werealso positive for SHV (52.7%) and TEM genes (30); and the frequency of SHV (32.4%)gene was higher than TEM gene(25) that was different from our results. Conclusion There is high prevalence of ESBL producers among Enterobacteriaceae isolates in our hospital (60.6%). This calls for strict policies regarding antibiotic usage and their screening methods. It is important for the clinical microbiology laboratory to implement one or more methods to detect ESBLs and hospital-based clinical laboratories should screen isolates from the community-based patients before hospitalization to prevent the spread of ESBL mediated resistance.The use of cephalosporin’s should be discouraged for empirical therapy of infections and should only be used according to results of culture and sensitivity.

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Original Article

Occult Hepatitis B Infection in Patients with Chronic Hepatitis C Associated Hepatocellular Carcinoma in Mansoura, Egypt Mahmoud Abdel-Aziz *, Azza Abdel-Aziz **, Othman Zaki***, *Tropical medicine department, faculty of medicine, Mansoura university, **Pathology department, faculty of medicine, Mansoura University, ***Clinical Pathology department, Faculty of medicine, Fayoum University.

ABSTRACT Hepatocellular carcinoma (HCC) incidence is rising Worldwide with high prevelance in Egypt. Hepatitis B virus (HBV) and hepatitis C virus (HCV) are major causes of HCC.The concept of occult hepatitis B virus infection was introduced with growing studies on its role in liver diseases and carcinogenesis. Aim of the work : In this study we report occult HBV in HCV associated HCC. Methods: The study included 118 newly diagnosed HCV associated HCC patients in Mansoura University Hospitals Variables including liver function tests, Alfa fetoprotein (AFP), quantitative assay of HCV in serum and tumor grade were evaluated. Detection of HBV DNA was performed by PCR on liver tissue biopsy and serum as well as. Determination of serological markers of previous HBV infection. Results: Tissue HBV DNA was detected in 14 cases (11.87%), All of them were under 60 years of age, presented by high grade HCC and higher level of AFP. Conclusions: Significant clinical, laboratory and pathologic differences exist among patients with underlying Occult HBV positive HCV related HCC vs. HCV only related HCC. These differences may impact eligibility for more sophisticated screening programs, potentially-curative therapy and prognosis in special patient groups. Introduction Hepatocellular carcinoma (HCC) is the fifth most common malignancy and the second most frequent cause of cancer-related mortality in the world(1). In Egypt, hepatocellular carcinoma (HCC) is the first most common malignancy in men and the third most common malignancy in women (2). HCC is one of the rapidly increasing causes of cancer mortality for both men and women. The increased incidence of HCC has been partially attributed to the emergence of the hepatitis C virus (HCV), an established risk factor for developing HCC (3,4). Egypt has the highest prevalence of HCV in the world, ranging from 6 to 28%(5-8).In the clinical setting, the viral status in patients with HCC is based on serologic testing rather than DNA detection. Negative serologic status, however, is not always consistent with absence of HBV and HCV in the liver. According to the international expert meeting held in Taormina (Italy) in 2008, occult infection with hepatitis B virus (HBV) is defined as presence of HBV DNA in the liver (with detectable or undetectable HBV DNA in the serum) of individuals testing hepatitis B surface antigen (HBsAg) negative by currently available assays (9). This is not a novel concept yet described since the early 1980's(10) following the

development of highly sensitive diagnostic tests able to detect minute quantities of HBV genome(10-12). However, several other explanations for this condition, such as HBV DNA sequence alteration by mutation(13) and viral DNA integration in the host genome, (14) have also been proposed. Occult HBV infection has been considered to play a role in some clinical phenomena including cryptogenic liver disease, (15) poor response to antiviral treatment(16) and development of hepatocellular carcinoma(17).Furthermore, chronic hepatitis C virus (HCV) infection is frequently reported with occult HBV infection(18).Taking into consideration the fact that HBV and HCV infections are common in Egyptian HCC patients, we investigate the occult HB infection in HCV associated HCC in Mansoura. Materials and Methods A total of 118 newly diagnosed HCC cases from Mansoura University Hospitals from Jan, 2013 to Jun 2015 were included in the study. All serologic markers for HBV infection (HBsAg, anti-HBs, anti-HBc Ig M, and anti-HBc Ig G) were detected with current standard assays (Adalts, Italy) and antibodies to HCV with HCV EIA version 3.0 according to the manufacturer’s

instructions (Adalts, Italy). All cases were positive for serum anti-HCV and serum HCV-RNA and negative for serum HBs-Ag and Anti HBc Ig M.Dynamic imaging (CTwith intravenous contrast) was done for all cases showing hypervascular solid liver mass with features characteristic for HCC. In all cases, the diagnosis of HCC was based upon histopathology following surgical resection or biopsy. Demographic data including age, sex were recorded upon initial HCC diagnosis together with clinico-pathologic features including the following: tumor grade, tumor size, presence of coexisting cirrhosis, presence distant metastases and serum biochemistries including liver function tests, serum alpha-fetoprotein. The diagnosis of cirrhosis was made based on imaging features and confirmed by histologic findings (in resection cases).Exclusion criteria were established before commencement of the study. Any patient with chronic liver disease due to other etiologies rather than HCV or patients with decompensated liver cirrhosis were excluded from the study.PCR for HBV DNA was performed on patient’s serum and Formalin fixed Paraffin embedded tissue (FFPE). FFPE tissue sections were prepared and digested with proteinase K, and DNA was isolated by phenol and chloroform. The first targeted a 228 bp region of the HBV X gene (forward primer, 5′-CTG GAT CCT GCG CGG GAC GTC CTT-3′ and reverse primer, 5′-GTT CAC GGT GGT CTC CAT-3′).The second assay was a nested PCR designed to amplify a 239 bp sequence of a highly conserved region of the HBV gene (forward primer, 5′-AGG TAT GTT GCC CGT TTG TCC TC-3′ and 5′-AAT TCT TTG ACA TAC TTT CCA ATC AAT-3′ and reverse primer, 5′-AYT GCA CYT GTA TTC CCA TCC CAT-3′ and 5′-biotin-TYA AAT GTA TAC CCA AAG ACA AAAGAA A-3′). Thirty

cycles of DNA amplification were performed in 50μl PCR reaction mixture. The conditions for each cycle were: denaturing at 94°C for 30s, primer annealing at 55°C for 30 s, and elongation at 72 C for 30s, followed by a final 10-min elongation at 72°C. DNA marker, proteinase K, Taq DNA polymerase, and dNTP were purchased form TaKaRa Co., Dalian. Primer was synthesized by BioAsia Co., Shanghai. According to the results of HBV DNA detection by PCR in liver tissue and / or serum the studied patients were further classified to two main groups, the first was Occult HBV positive HCV associated HCC group which included14 cases and the rest of patients (104 cases) formed the second group which was Occult HBV negative HCV associated HCC group.The study was conducted following the guidelines of the 1975Declaration of Helsinki, and all patients gave their written informed consent to participate in it. Statistical Analysis Data are expressed as mean value ± standard deviation (SD). All the data were edited and processed using the SSPS version16. Statistical analyses were evaluated using the Student t-test and the chi-square method(χ2 test), P value of ≤0.05 was considered statistically significant. Results A total of 118patients with hepatocellular carcinoma were included in this study.All cases were positive for serum anti-HCV and serum HCV-RNA and negative for serum HBs-Ag and HBcIg M Table (1) summarizes the demographic characteristics of the patients. The studied cases were 72 males and 46 female with age ranging (48-73 years) with no statistically significant difference between male and female groups regarding the age

Table (1) Age sex distribution

Group Age Range t P Male (72) 57.57±6.69 48-73 years

0.81

P>0.05 Female (46) 58.59±6.47 48-70 years

Diagnosis of occult hepatitis B was established by detection of HBV DNA in liver tissue. Out of these 118 participants with negative HBs Ag, 14 cases (11.86%) revealed positive HBV DNA in biopsy specimen on the other hands, only 11

cases (9.32%) were positive for HBV DNA in serum, Moreover, HBV viraemia levels were relatively low ranging between 120 to 2450 IU/ml with mean501.82±662.25 IU/ml.Different serological patterns for HBV infection were

shown in table (2).Out of these 118 patients with serological positive HCV infection confirmed by quantitative assay of HCV in the serum, 25 patients (21.18%) had positive HBc Ig G and only 17 patients (14.4%) had positive Anti HBs Ab. Occult HBV infection was 10/17 (58.82%)

in cases positive for both anti-HBc Ig G and anti-HBs Ab. The overall prevalence was lower for those positive for anti-HBc Ig G alone 10/25 (40%)and the same for anti-HBs Ab positive patients which was significantly higher than for those negative for all markers (P≤0.001)

Table (2) Serological patterns for HBV infection in the studied groups

Group Occult HBV positive group (14) Occult HBV negative group (104) χ2 HBc IgG positive 10 (8.47%) 15 (12.71%) 20.72

***P≤0.001 HBc IgG negative 4 (3.4%) 89 (75.42%) HBs Ab positive 10 (8.47%) 7 (5.93%) 36.8

***P≤0.001 HBs Ab negative 4 (3.4%) 97 (82.2%) *** P significant if ≤0.001

Table (3) summarizes the clinical, laboratory and histopathologic characteristics of the two patient groups. There were statistically significant difference regarding the age, being much younger in occult HBV positive group. There were no significant difference between both groups regarding sex distribution, Alt, Ast, serum bilirubin or the level of HCV in patient’s serum detected by real time PCR. AFP level was significantly higher in occult HBV positive group than the negative group being 676.71±1167.25 and 192.59±651.71 respectively

P≤0.05.Histopathological grading of HCC included 2 cases (1.69%) GII and12 cases (10.17%) GIII in occult HBV positive Group and 21 cases (17.8%) GI, 42 cases (35.59%) GII and 41 cases (34.75%) GIIIHCC in occult.HBV negative group. Cirrhosis was evident almost all cases except for 4 cases (3.4%) in occult HBV negative group as proved by either ultrasound examination or histopathological examination of surrounding liver tissue in surgically resected cases

Table (3) Clinical, Laboratory and Histopathologic characteristics of the two patient groups

Group Variable

Occult HBV Positive group (14)

Occult HBV negative group (104)

Test P

Age 53.86±4.0 58.52±6.69 t: 2.54 **P≤0.01 Sex: Male Female:

9 (7.63%) 5 (4.24%)

63 (53.39%) 41 (34.74%)

χ2 : 0.01

P>0.05

Alt 66.07±11.71 61.7±14.41 t: 1.09 P>0.05 Ast 65.07±19.04 63.4±15.31 t: 0.37 P>0.05 S. Bilirubin 1.74±0.58 1.55±0.54 t: 1.25 P>0.05 AFP 676.71±1167.25 192.59±651.71 t: 2.34 *P≤0.05 PCR HCV 1079082.36±

1564163.11 1556242.9± 2492996.24

t: 0.7

P>0.05

HCC Grade: Grade I: Grade II: Grade III:

0 2 (1.69%) 12 (10.17%)

21(17.8%) 42 (35.59%) 41 (34.75%)

χ2 : 10.97

**P≤0.01

* P significant if ≤0.05 ** P significant if ≤0.01

Discussion Occult HBV infection is best defined as positivity for HBV DNA in HBsAg-negative patients with or without serological markers of previous HBV infection(19).The failure of assays to detect HBsAg in occult HBV infection could be attributable to mutations of the S and pre-S genes which may cause modifications to hepatitis B surface antigenicity (20,21). In our study, HBV DNA in liver tissue was tested by

PCR assays amplifying two distinct areas of the viral genome. This meets the criteria for the detection of occult HB infection by some previous studies (22-24). In Egypt, a study was performed on 712 blood donors detected 9 persons with occult HBV infection (1.26%) (25).Similarly, previous studies reported prevalence of Occult HBV in blood donors in different countries where, HBV DNA was detected in 0.1%-1.59% (26,27). Few studies

reported occult HBV in the general population, serum HBV DNA was detected in different countries ranging 2% upto12% (28-30).The presence of occult HBV during chronic HCV infection is well described, Cacciola et al (10) published the first study of prevalence of occult HBV in patients with chronic hepatitis C; in that study tissue HBV DNA sequences were detected in 66 out of the 200 (33%) HCV infected patients. Other studies from different countries reported wide variation of prevalence ranging (4.8%- 41.9%)(31-36).HBV is a major etiological agent of HCC, and chronic HBV infection is estimated to be the cause of about 55%-60% of all HCC in the world (37).Hernandez et al (38) studied liver tissue viral genome in 61 HCC cases in US. In that study HBV DNA was detected in 10% of HBV seronegative cases irrespective to HCV status. In our study, occult HBV infection was detected in , 14 cases (11.86%) of the studied HCV positive HCC cases from Mansoura, Egypt. Lok et al (39) reported occult HBV prevalence in US reaching 10.7% of HCV associated HCC cases and higher prevalence of 73% was reported by Momosaki et al (21) who studied 30 cases. The variability in prevalence may be related to different protocol of PCR amplification sites and also different studied population.A significant difference in serum alpha-fetoprotein (AFP) production was observed in HCC’s associated with underlying occult HBV as opposed to those associated with HCV alone In their study, Hiotis et al (40) observed that the proportion of HCC producing higher amount of AFP (>100 ng/ml) was greater in HBV-associated HCC so they suggested a higher sensitivity of serum AFP as a tumor marker when used in patients with HBV, in relation to less so in screening patients with HCV for cancer. In the last twenty years, epidemiological studies from different geographic areas have shown that occult HBV infection is associated with severe forms of liver disease in HCV-infected patients, thus suggesting that occult HBV might favor or accelerate the progression of HCV-related chronic liver disease and that the hepatocarcinogenic risk increases synergistically also in these patients (41-43). In the studied cases, all the fourteen cases with occult HBV were below the age of 60 years. Histopathologic examination revealed high grade HCC in 12 0ut 0f 14 occult HBV associated cases. This may be

explained by the synergistic oncogenic effect of Occult HBV infection with early onset of HCC and more aggressive behavior. The study of Cacciola et al (10) demonstrated that occult HBV was significantly correlated with cirrhosis among HCV infected patients; where 33% of patients with HCV infection and occult HBV had cirrhosis as compared with 19% of patients with HCV infection and no occult HBV, proposing that occult HBV can accelerate the evolution to cirrhosis in HCV infected patients. Other studies suggested increased association of cirrhosis in patients with HCV and occult HBV (11, 44,

45).However, these observations have not been confirmed by other investigations. Kao et al.(46) reported that in patients with chronic HCV infection, the prevalence of occult HBV did not parallel the severity of liver disease. Other studies reported that there was no significant association between occult HBV and degree of liver necroinflammation and fibrosis (32, 47).In our study, cirrhosis was recorded in all cases of occult HBV positive with HCV associated HCC compared with 100 cases (87.72%) of HCV associated HCC with statistically insignificant difference between both groups. Our results require its performance on larger number of HCC patients from multiple medical centers in Egypt with liver tissue and serologic information. In addition, further research is needed to understand the clinical significance of occult HBV on the development, progression, and recurrence of HCC. References 1. Jemal A, Bray F, Center MM, Ferlay J, Ward E,

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Original Article

Prevalence of Transfusion Transmitted Virus (TTV) Among HCC Patients and HCV Infection in Damietta Governorate Samy Zaky 1, Mohamed Afify2, Amgad Al Zahaby2, Abd-Alla El-Bialy3, Khalid El-Molla4, Amin Abdel Baki5, Ashraf Z. ElSayed 6.1Tropical Med., Al Azher university, Damaitta, 2Tropical Med., Al Azher university, Cairo, 3Clinical pathology, Zakzik university, Zakazik, 4Tropical Med., Al Azher university, Damietta, 5Tropical Med., National Hepatology and Tropical Medicine Research Institute, 6Al Azher university, Cairo, 7Tropical Med., Ministry of health, Damaitta, Egypt

ABSTRACT TTV has been identified as a causative agent of non-A to non-G hepatitis. However, its exact role in the pathogenesis of chronic liver disease (CLD) and development of hepatocellular carcinoma (HCC) remains controversial. Aim of the work: The current study was performed aiming to assess the prevalence of TTV and its impact on the severity of liver disease and development of HCC among Egyptian patients with chronic HCV Infection.Methods: one hundred chronic HCV patients were enrolled in this study; 50 cases (Group A), chronic hepatitis C only and 50 cases (Group B) Chronic hepatitis C associated with HCC. They were diagnosed by clinical, biochemical, ultrasonographic and triphasic spiral CT examination of the abdomen. Serum samples were collected from all patients. All samples were serologically tested for routine lab, liver functions, markers of hepatitis B virus (HBV) and hepatitis C virus (HCV) infection and AFP. HCV RNA and TTV DNA were detected by RT-PCR assays.Results: Coinfection with TTV was significantly higher (80.0%) in group B(HCC) than that of group A (30.0%). HCV coinfection with TTV was significantly associated with high alpha fetoprotein levels and HCV viremia (54.5%) than HCV monoinfection (28.5%). Prevalence of TT virus co-infection was significantly higher among advanced and end stages HCC cases than early or intermediate stage according to the BCLC staging system.Conclusion: Prevalence of TTV/HCV co-infection was significantly higher among HCC patients than HCV patients without HCC and more associated with advanced HCC stages. Introduction TTV was first detected in subjects with post-transfusion hepatitis and indicated as a possible an etiologic agent of non A-non C hepatitis (1). Its prevalence and clinical significance are being assessed worldwide, however its relationship with aggravation and progression to severe liver disease and HCC remain controversial (4). TTV is a parenterally transmitted virus common within groups at high risk for contracting blood-borne viruses, such as individuals with chronic liver disease, intravenous drug users and recipients of blood and its products . TTV DNA is present at higher titre in liver than in corresponding serum sample indicating a hepatic site for virus replication (3). A high prevalence of TTV infection in patients with acute and chronic liver disease of unknown etiology has been reported (2). The significance and interaction of TTV with other hepatitis viruses remain controversial .several studies on HCV patients coinfected with TTV suggested that the association of both viruses increased severity of liver damage 8).

Hafez et al (17), studied the prevalence of transfusion transmitted virus (TTV) genotypes among HCC patients in Qaluobia governorate, Egypt, and found that TTV is commonly present in adult patients with HCC and liver cirhosis as well as healthy individuals. The most prevalent TTV genotype is genotype 1. It seems that the infection neither contribute to the severity of liver disease nor to the causation of HCC. It was suggested that, high TTV viremia significantly associated with the occurrence of HCC in patients with HCV-related chronic liver disease . Aim of the Study The aim of the present study was to assess the prevalence of transfusion transmitted virus (TTV) infection among HCC Egyptian patients and HCV infection in Damietta Governorate. Patients and Methods One hundred chronic hepatitis C patients were selected from the tropical medicine department, Al-Azhar University and Tropical Medicine

Department of National Hepatology and Tropical medicine Research Institute (NHTMRI)., during the period from March 2009 till the end of April 2011. They were divided into two groups;Group A (50 cases) chronic hepatitis C only: were chosen according to the following inclusion criteria (adult male and female 18-60 years with documented chronic hepatitis C by investigations as HCV Ab by ELISA technique and HCV RNA by PCR.Group B (50 cases) Chronic hepatitis C associated with HCC were chosen according to the following inclusion criteria (adult male and female 18-60 years with documented chronic hepatitis C and associated with HCC documented by alpha fetoprotein ,abdominal sonar and triphasic spiral CT abdominal examination . Ethical Considerations : The objectives of the study and the possible side effects were explained to all patients included in the study and they were asked to sign a formal consent.Diagnostic tools and methods: Alpha-Feto-Protein (AFP) was determined by the method of Abelev et al(8).Principle: Biosource AFP ELISA Kit is a solid phase enzyme-linked immunosorbent assay (ELISA) based on the sandwich principle. The microtiter wells are coated with a monoclonal antibody directed towards a unique antigenic site on an AFP molecule. An aliquot of a case sample containing endogenous AFP is incubated in the coated well with enzyme conjugate, which is an anti-AFP antiserum conjugated with horseradish peroxidase. After incubation the unbound conjugate is washed off with water. The amount of bound peroxidase is proportional to the concentration of AFP in the sample. Having added the substrate solution, the intensity of color developed is proportional to the concentration of AFP in the case sample. Normal values of Alpha fetoprotein were from 5- 10 ng/ml; high if more than 200ng/dl (9).Qunatitative detection of HCV virus by PCR. Quantitatation of HCV-RNA was carried out by a commercially available kit (Amplicov HCV Monitor Assay, Version 2-0; Nippon Roche, Tokyo, Japan) using Taqman real time PCR (rotor Gene 6000). The detection level: 1.5 IU/ml; Low viremia: 1.5 to 100000 IU/ml. Moderate viremia: from 100000 to 1000000 IU/ml. High viremia: more than 1000000IU/ml

(Le Guillou-Guillemette and Lunel-Fabiani, (10).Detection of TTV DNA: The kits used for detection of TTV virus were High Pure Viral Nucleic Acid Kit (Roche Diagnostics GmbH, Mannheim, Germany .) Detection of TTV DNA was carried out by the nested PCR method described by Nishizawa, et al. (4).Briefly, DNA was extracted from 100 ml serum using DNA ZOL Reagent (Life Technologies, Grand Island, NY). After washing with 95% ethanol, the DNA pellet was allowed to dry for 15 min; it was then resuspended in 50 ml of water, and stored at -20oC until use. The PCR reaction mixture of the first PCR contained 3 ml of DNA, 1 mmol of each primer of the outer primer pair, and 47 ml of Super Taq Premix Kit (Omgen Co. Ltd., Tokyo, Japan). The primer sequences were 50-GCA GCA GCA TAT GGA TAT GT-30 (RD 037; sense), and 50-TGA CTG TGC TAA AGC CTC TA-30 (RD 038; antisense) for the outer primer pair (270 bp), and 50- CTA ACA CAT GAA TGC CAG GC-30 (RD 051, sense), and 50-GTA CTT CTT GCT GGT GAA AT-30 (RD 052; antisense) for the inner primer pair (197 bp). The thermal cycle condition was carried out in a Perkin- Elmer Model 9600 Gene AMP PCR system (Norwalk, CT). The conditions of PCR for the ®rst reaction were 50 cycles of 94oC for 20 sec, 55oC for 20 sec and 72oC for 1 min. For the second PCR, the PCR reaction mixture contained 3 ml of the first PCR product, 1 mmol of each primer of the inner primer pair, and 47 ml of Super Taq Premix Kit (Omgen Co. Ltd., Tokyo, Japan). The thermal cycle condition of the second PCR reaction was the same as that of the first PCR reaction. Then, the second PCR product was analyzed by electrophoresis on a 2% agarose gel and stained with ethidium bromide. A sample was considered to be positive for TTV DNA if a DNA fragment of 197 base pairs was visible. We classified all included cases into three groups according to modified Child’s Pugh classification A, B and C (Pugh et al., 1973)(11).Patients of group B, were classified into 4 stages according to modified Barcelona-Clinic Liver Cancer (BCLC) classification: The BCLC classification has emerged as the standard classification for clinical management of HCC. We used the modified Barcelona classification that divided liver cancer into 4 stages

Table (1): Barcelona Clinic Liver Cancer Staging system: BCLC stage PST Tumor stage Okuda stage Liver Function Early Stage HCC A1 0 Single, <5 cm I No portal hypertension and normal bilirubin A2 0 Single, <5 cm I Portal hypertension and normal bilirubin A3 0 Single, <5 cm I Portal hypertension and abnormal bilirubin A4 0 3 tumors <3cm I-II Child-Pugh A–B Intermediate HCC Stage B 0 Large multinodular I-II Child-Pugh A–B Advanced HCC

Stage C

1-2 Vascular invasion or extrahepatic spread

I-II Child-Pugh A–B

End stage HCC Stage D 3-4 III Child-Pugh C

Statistical Analysis Data entry and analysis was accomplished with the aid of computer's SPSS program version16. The results were represented in tabular forms and charts then interpreted. Mean, standard deviation, range, frequency and percentage were used as descriptive statistics .Chi square test were used as test of significance for non parametric data and t test were used as test of

significance for parametric data .The significant was adjusted when P< 0.05. Results The present study included 100 cases; presented with chronic hepatitis C. they were divided into two groups: Group A (50 cases): chronic hepatitis C only. Group B (50 cases) Chronic hepatitis C associated with HCC

Table (2): Basic Characteristics of the studied groups.

History

Group A Group B x² p

No % No % Sex:

0.000 1.000 NS Male 29 58.0% 29 58.0% Female 21 42.0% 21 42.0% Residence

6.11 0.01 (S) Rural 25 50.0% 37 74.0% Urban 25 50.0% 13 26.0% Age (Mean+ SD) years 38.44 12.27 51.56 4.63 0.001(S) Smoking 13 37.1% 22 62.9% 3.56 0.06(NS) History of blood transfusion 6 12.0% 7 14.0% 0.088 0.7(NS) Jaundice 12 24.0% 39 78.0% 29.17 0.0001(S) Ascites 20 40.0% 23 46.0% 0.367 0.5 (NS) Hepatomegaly 20 40.0% 40 80.0% 36.86 0.0001(S) splenomegaly 20 40.0% 45 90.0% 27.47 0.0002(S)

S= significant NS= not significant Table (3): Basic laboratory data among studied groups.

laboratory investigations Group A Hepatitis C virus

Group B Hepatitis C virus &HCC t P

Mean SD Mean SD ALT (U/ml) 43.90 18.75 48.00 22.50 0.99 0.3 (NS) AST (U/ml) 52.72 22.45 51.32 30.75 0.260 0.8 (NS) Bilirubin (mg/dl) 1.33 0.90 3.61 3.29 4.73 0.001 (S) Serum albumin (g/dl) 3.88 1.04 3.77 1.04 0.512 0.6 (NS) GGT (U/L) 46.60 22.03 63.00 23.37 3.61 0.0001 (S) Alkaline phosphatase (U/L) 231.68 111.04 346.30 100.34 5.42 0.0002 (S) WBCs x 103 (cell/ml) 7.21 2.35 6.51 2.326 1.49 0.1 (NS) HB% (g/dl) 11.12 2.00 10.41 1.93 1.80 0.07 (NS) Platelet countx103 (cell/ml) 189.58 131.80 141.82 80.94 3.42 0.001 (S) Serum creatinine (mg/dl) 0.60 0.18 0.57 0.15 0.833 0.4 (NS) Blood urea (mg/dl) 2.80 4.11 21.36 4.81 0.491 0.6 (NS) Alpha fetoprotein (ng/ml) 14.17 10.84 618.46 598.67 7.14 0.0001 (S)

PC % 74.14 18.06 64.44 19.07 2.61 0.01 (S) HCV-RNA level

2.144 0.3 (NS) Low viremia 15 30.0% 13 26.0% Moderate viremia 17 34.0% 12 24.0% High viremia 18 36.0% 25 50.0%

S= significant NS= not significant ##= Standard deviation Quantitative detection of HCV virus by PCR:Low viremia: less than100000 IU/ml, moderate viremia: from 100000 to 1000000 IU/ml and high viremia: more than 1000000IU/ml Table (4): Prevalence of TT virus infection among the studied groups

TT virus

Group A (HCV)

Group B (HCV +HCC) x² P

No % No % Positive (55) 15 30.0% 40 80.0%

25.25 0.0001(S) Negative (45) 35 70.0% 10 20.0% Total (100) 50 100.0% 50 100.0%

Group A: 50 patients with chronic HCV. Group B: 50 patients with chronic HCV and HCC Coinfection with TTV was significantly higher (80.0%) in group B than that (30.0%) of group A. Table (5): Association between detection of TT virus and HCV viral load.

TTV by PCR PCR for HCV

TT virus Positive

TT virus Negative

No % No % Low viremia 8 14.5% 20 44.4% Moderate viremia 17 30.9% 12 26.7% High viremia 30 54.5% 13 28.9% Total n=100 55 100.0% 45 100.0% x² 11.84 P 0.003 S

Quantitative detection of HCV virus by PCR:Low viremia: less than100000 IU/ml, moderate viremia: from 100000 to 1000000 IU/ml and high viremia: more than 1000000IU/ml HCV coinfection with TTV was significantly associated with high HCV viremia (54.5%) than HCV monoinfection (28.5%). Table (6): Association of PCR for TT virus and alpha fetoprotein levels

TTV by PCR Alpha fetoprotein

TT virus Positive

TT virus Negative

x² P

No % No %

Normal 6 10.9% 22 48.9% 9.65 0.002 High (≤ 200 ng/ml) 20 36.4% 16 35.6% 0.0 0.94 High (> 200 ng/ml) 29 52.7% 7 15.6% 11.2 0.001 Total n= 100 55 100.0% 45 100.0%

Alpha fetoprotein levels were significantly higher among HCV patients co-infected with TTV than HCV monoinfection Table (7):prevalence of TT virus among group B according to BCLC staging system

TTV by PCR BCLC staging

TT virus Positive (40)

TT virus Negative (10)

No % No % A (early; 13 cases) A1 A2 A3 A4

6 2 2 1 1

15.0% 5.0% 5.0% 2.5% 2.5%

7 3 2 1 1

70.0% 30.0% 20.0% 10.0% 10.0%

B(Intermediate; 6 cases) 6 15.0% 0 0.0% C (advanced; 10 cases) 9 22.5% 1 10.0% D (end stage; 21 cases) 19 47.5% 2 20.0% x² 12.87 P 0.005 S

BCLC: Barcelona Clinic of Liver Cancer. S: significant BCLC staging system has come to be widely accepted in clinical practice. Prevalence of TT virus coinfection was significantly higher among advanced and end stages HCC cases than early or intermediate stage according to the BCLC staging system

Discussion Hepatitis C virus (HCV) infection is a worldwide health problem because of its incidence and pathogenicity. It might evolve into chronic disease, cirrhosis, and/or hepatocellular carcinoma (HCC) and the outcome is mainly determined by the host immune response (13). Transfusion transmitted virus (TTV) is a discovered virus which was suspected to be a causative agent of non-A to non-E hepatitis. TTV was first identified in the serum of a patient who was hospitalized with post transfusion hepatitis of unknown etiology in 1997 Nishizawa et al.,(1). Since its discovery, it has become clear that TTV infection is present orldwide among blood donors and is common in patients with liver diseases, including cryptogenic cirrhosis and fulminant hepatic failure .(14).In some studies, TTV-DNA has been found more frequently in patients with liver cirrhosis and hepatocellular carcinoma than in those with chronic hepatitis. However, TTV DNA is not integrated in tumor cells, which may suggest that the virus is a passenger rather than a cause of the tumor. And therefore, further studies are required to determine the role of TTV. In fact, the significance of TTV infection in liver disease is, at present, analogous to that of HGV (14). Thus, the present study was designed to examine the relationship between the extent of TTV viremia and the occurrence of HCC among patients with chronic hepatitis C. The present study was conducted from the first of March 2009(12) till the end of 2011. The study was conducted at Damietta University Hospital in the tropical medicine department, in new Damietta city and Tropical Medicine Department of National Hepatology and Tropical medicine Research Institute (NHTMRI).We selected 100 patients admitted to the tropical medicine department and diagnosed as chronic hepatitis C. These cases were divided into two groups: Group A: (50 cases) chronic hepatitis C only; Group B (50 cases) Chronic hepatitis C associated with HCC. In the present study, age ranged from 18 to 60 with a mean of 45.0±11.34 years and the mean age of group A were (38.44) years and group B were (51.56) years and there is a significant difference between groups. In the present study, the majorities of cases among both group A and B were males (58.0%) and live in rural areas but the minorities of cases among both group A and B having history of blood

transfusion (12.0% and 14.0% respectively) and there is no significant difference. In addition, the majority of cases among group A were Compensated liver (60.0%) but the majority of cases among group B were Decompensated liver (62.0%) with statistically significant difference. Pellicano et al. (15) reported that, 64.8% of cases were males, and these results supports that of the present work in that, males are largely affected by HCV and HCC in comparison to females, but their percent is slightly higher.Although TTV DNA was found at high concentrations in liver tissue and in serum of patients with liver disease, several studies also reported a high endemicity of infection in subjects with no evidence of hepatitis. Therefore, the role of TTV as a cause of liver disease is controversial (16).Zein et al. (5) reported that TTV infection was more prevalent among patients with advanced HCV-associated liver disease (decompensated cirrhosis and HCC) than in those with stable disease (chronic hepatitis and compensated cirrhosis). In the present study, 30.0% of cases in group A were positive for TT virus compared to 80.0% of group B with statistically significant difference. These results reflect the association of TT virus with both HCV and HCC, and association is marked with HCC. In contradiction to results of the present study, Hafez et al. (17) reported that, in their study, the rate of TTV infection did not significantly differ between patients with liver disease, with or without HCV infection, and healthy blood donors. The high prevalence of TTV in general population, may complicate linking TTV to hepatic disease and other pathologic states.Our results of the present study were supported by those reported by Colombatto et al. (18) who reported that, in a study in part including a population of subjects with different liver diseases living in Italy (n = 28), TTV–DNA was found in 32% of patients with chronic hepatitis C.In our study, all cases in group B had palpable liver; 90% had palpable spleen; lower limb edema (58.0%), ascites in 46%, pallor in 56% and jaundice in 78% and there was statistically significant difference between groups as regard liver, spleen, LL edema, and jaundice. In addition, 30%, 34% and 36.0% of group A had low, moderate and high viremia respectively; compared to 26.0%, 24% and 50% of group B had low, moderate and high viremia respectively with statistically insignificant difference between groups; 52% of cases in

group A had normal alpha fetoprotein, compared to 4% in group B with statistically significant difference between groups; ALT, Bilirubin, GGT, Alkaline Phosphatase, urea and alpha fetoprotein were significantly higher in HCC group; the majority of patients in group A were child A (60.0%), while the majority of group B were child C (42.0%) and there was statistically significant difference between groups; the majorities (42.0%) of group B were end stage (D) according to Barcelona classification.Kato et al. (19) found that among patients with HCC, those who were TTV-positive had a significantly higher blood transfusion history (p=0.02) compared with TTV-negative patients. This supports the fact that TTV is transferred by blood transfusion. They also investigated whether there is a correlation between TTV and the severity of chronic liver disease with concomitant HCC. Infection with TTV in HCV-positive liver disease has no effect on the severity of the liver disease, and neither does it have a significant effect on serum transaminase levels, platelet count or hepato-carcinogenesis. TTV infection also has no effect on HCV-RNA load. In our study, the majority (56.4%) of positive TTV cases among female and live in rural areas but the minority of them (12.7%) having history of blood transfusion and there is no significant difference between TT-positive and TT-negative cases. In addition, the majority of TT-positive cases (65.5%) had a decompensated liver and there was a statistically significant difference. In addition, the main findings when comparing TT-positive with TT-negative cases were as the following: all clinical presentations of liver disease were statistically higher in cases with positive TT-virus infection; The majority (54.5%) of positive TTV cases had high HCV viremia; the majorities of positive TTV cases (46.7%) had coarse cirrhotic liver with mild splenomgaly and ascites and there is no significant difference; the majority of positive TTV cases (47.5%) had more liver cirrhosis, moderate splenomgaly and portal hypertension, ascites and single or multiple focal lesions; the majority of positive TTV cases (47.5%) were among patients with end stage group (D) according to Barcelona classification; majorities of positive TTV cases (52.7%) had high alpha fetoprotein (> 200); TT- positive cases showed statistically significant association with the hepatic function reserve as evidenced by fact

that, the majority of cases with TT-positive were child C. Moriyama et al. (20) reported that, TTV DNA was detected in the sera of 62 (34.4%; 35 males and 27 females; mean age, 50.6 years) of the 180 patients with type C chronic liver disease. They reported blood transfusion in 66.1% of TT-positive cases. These results are quite different from those reported in the present work, as regard the percentage of positive cases were mainly in females in the present work but in males in their work. In addition, they reported higher percentage of blood transfusion in comparison to the present study. On the other hand, they agreed with the present work as regard the fact that there was statistically insignificant difference between TT-positive and negative cases as regard clinical or laboratory parameters. They also reported that, the degree of irregular regeneration of hepatocytes in the liver of the TTV DNA-positive patients was more severe than that in the TTV DNA-negative patients, although the difference was not statistically significant. When the patients were classified according to the fibrosis (F) stage, however, the irregular regeneration of hepatocytes score of the TTV DNA-positive patients was significantly higher than that of the TTV DNA negative patients only among those in the F4 stage (20). Dai et al. (21) reported that, the positive rate of TTV DNA was significantly higher in the patients with history of blood transfusion than those who were never transfused (24/41, 58.5 vs. 45/124, 36.3%; P<0.01). No other clinical and virological factor was related to positive TTV DNA. These results are quite different than that of the present work especially for blood transfusion and this may be attributed to the small number of cases who had blood transfusion in our study. Uchida (1995, 1994)(22,23) reported that irregular regeneration of hepatocytes induced by repeated cycles of necrosis and regeneration of hepatocytes in patients with hepatitis is the most important background factor for hepatocarcinogenesis. Acute viral hepatitis is characterized by necrosis in the liver that recovers to the normal parenchyma with uniform regeneration of hepatic cells, whereas chronic viral hepatitis is characterized by endless repeated cycles of necrosis and regeneration. New necro-inflammatory reactions often occur in newly regenerated cell areas, and as repeated cycles of regeneration of hepatocytes occur, it gradually

ignores the acinar structure; this is defined as irregular regeneration of hepatocytes. Irregular regeneration of hepatocytes is characterized by a mosaic distribution of hepatocytes with different proliferating ability. A typical feature of liver cirrhosis is hepatic nodulation with irregular regeneration of hepatocytes. The onset of carcinogenesis may represent the selection of a subclone with strong proliferating capacity from such a lesion. Shibata et al. (24) reported that in type C chronic hepatitis and liver cirrhosis patients, severe irregular regeneration of hepatocytes is the strongest risk factor for the development of HCC. Moriyama et al. (27) compared the irregular regeneration of hepatocytes scores of the non-cancerous region of the liver of HCC patients and the irregular regeneration of hepatocytes scores of the liver biopsy specimens of chronic liver disease patients [and it was concluded that the degree of irregular regeneration of hepatocytes in the HCC patients was significantly more severe than that in the chronic liver disease patients in each etiological type of chronic liver disease. A high irregular regeneration of hepatocytes score contributed to hepatocarcinogenesis irrespective of the viral etiology, as the mean irregular regeneration of hepatocytes score increased with the advance in the F stage of fibrosis among the chronic liver disease patients. High irregular regeneration of hepatocytes may reflect the histological expression of genetic instability, namely a carcinogenic state. The degree of irregular regeneration of hepatocytes may be used to assess the risk for hepatocarcinogenesis in nearly all liver diseases. Moriyama et al. (20) reported that, among the patients with the F4 stage of fibrosis, the degree of irregular regeneration of hepatocytes in the TTV-coinfected patients was significantly more severe than that in the patients infected with HCV alone; therefore, TTV coinfection may influence the development of HCC in patients with type C chronic hepatitis. Conclusions high TTV viremia was significantly associated with the occurrence of HCC in patients with HCV-related chronic liver disease in the present study. In addition, TTV coinfection seems to modify the biochemical features of type C chronic liver disease.

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Original Article

Study of Prevalence of Spontaneous Bacterial Peritonitis in Patients with Liver

Cirrhosis with Ascites in Menoufiya Governate

Atef Abo ELsoud Ali*, Wafaa Ahmed Ibrahim Zahran**, Fatma Mohamed Abdelfatahabdo *Tropical Medicine department, Faculty of Medicine, Menoufiya University, **Microbiology department,

Faculty of Medicine, Menoufiya University.

ABSTRACT The aim of the present work was to study the Prevalence of Spontaneous Bacterial Peritonitis in Patients with Liver

Cirrhosis with Ascites. Aim of the work: the aim of this work is to study the prevalence of SBP among

patients with liver cirrhosis and ascites. Methods: Ninty patients with decompensated chronic liver disease and

ascites, admitted to (Tropical Medicine Department in Menoufiya University Hospital, Shebin Elkom and Tala Fever

Hospital) during the period from August 2014 to April 2015. Sixty of them (66%) were males and thirty (34%) were

females. All patients had abdominal paracentesis done within 48 hrs of admission under aseptic condition and the

data obtained were analyzed. Results: The prevalence of neutrocytic bacterascites was 35.6% (in clinically

suspected SBP 40.8% and in not clinically suspected 29.3%). Conclusion: It is therefore imperative to do diagnostic

abdominal paracentesis for cell count and culture in any patient with onset of ascites or cirrhotic patients with ascites

and suggestive symptoms compatible or suggestive of SBP.

Introduction

Spontaneous bacterial peritonitis (SBP) is a very

common bacterial infection in patients with

cirrhosis and ascites requiring prompt

recognition and treatment (1-3). It was first

described by Conn and Fessel in 1971 as a

syndrome of infected ascetic fluid in patients

with hepatic cirrhosis, which they named SBP (4).

SBP is by definition an infection of previously

sterile ascetic fluid, without any apparent intra-

abdominal source of infection (5). The infecting

organisms are usually those found among the

normal intestinal flora. When first described, its

mortality exceeded 90% but it has been reduced

to approximately 20% with early diagnosis and

treatment (6-7).All patients with cirrhosis and

ascites are at risk of SBP and the prevalence of

SBP in outpatients is 1.5-3.5% and about 10%-

30% in hospitalized patients (8-9). Half of the

episodes of SBP are present at the time of

hospital admission while the rest are acquired

during hospitalization (1). In-hospital mortality

for the first episode of SBP ranges from 10% to

50%, depending on various risk factors (10-11).

One-year mortality after a first episode of SBP

has been reported to be 31% and 93% (12).The

pathophysiology of SBP is not completely

understood. Translocation of bacteria and

endotoxins from the gastrointestinal tract to

peritoneal fluid is believed to be a key

mechanism behind the development of SBP, and

is facilitated by impaired defensive mechanisms

in cirrhotic patients (13-17). Proteins of the

complement cascade have lower levels in

cirrhotic patients and the opsonic and phagocytic

properties of neutrophils are decreased in

patients with cirrhosis. Bacteremia from the

urine or the respiratory tract can also lead to

infection of the ascetic fluid. SBP may also be

iatrogenic (18), such as after endoscopic treatment

of esophageal or gastric varices.Patients with

SBP may have one of the following(1,8) 1) local

symptoms and/or signs of peritonitis: abdominal

pain, abdominal tenderness, vomiting, diarrhea,

ileus; 2) signs of systemic inflammation: hyper

or hypothermia, chills, altered white blood cell

count, tachycardia, and/or tachypnea; 3)

worsening of liver function; 4) hepatic

encephalopathy; 5) shock; 6-renal failure; and 7-

gastrointestinal bleeding. However, it is

important to point out that SBP may be

asymptomatic in about 10-32% of cases,

particularly in outpatients (8,19-21).Nigerian data is

very scanty regarding the prevalence of SBP in

patients with liver cirrhosis and ascites.

Aim of the work

The aim of this Work is to study the prevalence

of SBP among patients with liver cirrhosis and

ascites.

Patients and methods

This study was carried out on 90 patients

withdecompensatedchronic liver diseases and

ascites.An informed consent wasobtained before

patients enter the study.They were divided into

two groups:Group 1 : comprised 49 patients

clinically suspected SBP .Group 2: comprised 41

patients not clinically suspected SBP.Patients of

this study were selected from patients admitted

to Tropical Medicine department, Menoufiya

University Hospital Shebin Elkom and Tala

Fever Hospital during the period from August

2014 to April 2015, Sixty of them (66%) were

males and thirty (34%) were females. Their age

ranged from 46 to 69 years with a mean age of

All patients were subjected to: Full

and detailed history taking, complete clinical

examinations, routine laboratory investigations,

viral markers, abdominal ultrasonography,

diagnostic abdominal paracentesis (ascetic fluid

total analysis: Physical, biochemical, cytological

and microbiological cultures) Results were

collected, tabulated, statistically analyzed by

IBM personal computer and statistical package

SPSS version 11.

Results The present study revealed that, clinically

suspected SBP was common in males (73.5%)

than females (26.9%) and it was not influenced

by the age.- Clinical presentations of the studied

groups revealed that, there was high a statistical

significant difference between the two groups

regarding fever, abdominal pain, haematemesis

and melena which were the most commmen

clinical manifistations of SBP group and there

was no statistical significant difference between

the two groups regarding bleeding tendency

while there was significant difference regarding

hepatic encephalopathy as shown in table (1).

Table 1 :Clinical presentations of the studied groups:

Clinical history

Clinically suspected SBP

(no=49)

Not Clinically suspected

SBP(no=41)

χ2 test P value

No % No %

Fever Negative

Positive

12

37

24.5

75.5

41

0

100

0

52.57

0.001

HS

Abdominal pain

Negative

Positive

9

40

18.4

81.6

41

0

100

0

60.25

0.001

HS

Haematemesis&melena Negative

Positive

24

25

48.97

51

25

16

60.97

39

7.22

0.007

HS

Bleeding tendency

Negative

Positive

15

34

30.6

69.4

5

36

12.2

87.8

4.38

0.03

S

Hepatic

Encephalopathy

Negative

Positive

27

22

55.1

44.9

21

20

51.2

48.8

0.14

0.71

NS

- Examination of both groups by ultrasound

revealed that, there was a no statistical

significant difference between the two groups

regarding amount of ascites examination and

spleen examination as shown in table (2).

Table 2 :Abdominal ultrasound findings of the studied groups

Ultrasound

findings

Clinically suspected SBP (no=49) Not Clinically suspected SBP(no=41) χ2 test P value

No % No %

Liver

Average size

Hepatomegally

shrunken

26

3

20

53.1

6.1

40.8

21

2

18

51.2

4.9

43.9

0.13*

0.94

NS

Spleen

Average size

Splenomegally

Splenectomy

17

22

10

34.7

44.9

20.4

19

13

9

46.3

31.7

22

1.78

0.41

NS

Ascitis

Mild

Moderate

Tense

7

23

19

14.3

46.9

38.8

5

23

13

12.2

56.1

31.7

0.75

0.68

NS

Laboratory investigations of the studied groups

revealed that, there was a statistical significant

difference between the two groups regarding,

ALT , AST, serum creatinine and total bilirubin

which were higher in clinically suspected SBP

group and there was no statistical significant

difference between the two groups regarding

other parameters as shown in table (3).

Table3: Laboratory investigations of the studied group

Laboratory

investigations


(no=49)


SBP(no=41)

Mann-

Whitney

test

P value

Mean±SD Mean±SD

Hb% 9.01±1.73 9.13±1.87 0.29* 0.77

Platelet(cell/cmm)×103 73.59±26.71 73.07±25.96 0.16 0.87

WBCs(cell/cmm)×103 9.67±8.44 7.86±3.4 0.05 0.95

RBCs(cell/cmm)×103 3.69±1.17 4.08±1.22 1.5* 0.14

AST(IU/L) 91.85±69.43 68.36±63.73 2.58 0.01

ALT(IU/L) 105.00±84.42 79.68±68.79 2.16 0.03

Total bilirubin(mg/dl) 4.1±2.1 2.8±2.1 1.48* 0.03

Albumin(gm/dl) 2.84±2.76 2.35±0.71 0.36 0.72

PT 42.39±11.98 46.56±11.83 1.65* 0.10

Creatinine(mg/dl) 2.81±4.22 1.66±1.08 1.14* 0.04

RBS 199.1±118.77 177.7±114.59 0.77 0.43

Mann Whitney U test *

In this study, clinically suspected SBP group

contained 46 HCV antibody positive patients

(93.9%) , 1 HBsAg positive patients (2%) and.

Not clinically suspected SBP group included 33

HCV positive patients (80.5%), 7 HBsAg

positive patients (17.1%) . Ascetic fluid total

analysis revealed that, in physical examination

there was highly significant turbidity in the

aspect of ascetic fluid in SBP group and no

significant difference between two groups

regarding the colour of ascetic fluid, there was a

statistical high significant difference between

the two groups in biochemical analysis of ascetic

fluid regarding TLC, PMN count which were

higher in clinically suspected SBP group and

there was no statistical significant difference

between the two groups regarding total protein

SAAG, glucose and cytological analysis was

negative in both groups as shown in table (4,5).

Table 4: Ascitic fluid total analysis of the studied groups

Ascitic fluid

examination

Clinically suspected SBP (no=49) Not Clinically suspected SBP(no=41) Fisher's

Exacttest

P value

No % No %

Physical analysis

Colour

Yellow

Red

42

7

85

15

39

2

95

5

4.1

>0.05

Aspect

Clear

Turbid

20

29

40

60

39

2

95

5

13.7

<0.001

Table 5: Biochemical analysis of ascitic fluid

Ascitic fluid examination


(no=49)


SBP(no=41)

Mann-

Whitne

y

test

P value

Mean±SD Mean±SD

TLC 3036.73±4519.72 600.48±756.65 2.8 0.005 HS

PML 514.1±823.82 256.9±263.26 2.48 0.01 S

SAAG 1.69±0.31 1.61±0.33 1.21* 0.23 NS

Glucose 86.77±20.69 87.41±19.53 0.76* 0.88 NS

LDH 136.28±89.41 112.27±84.21 2.21 0.02 S

Total protein 1.93±0.33 1.87±0.36 0.76* 0.45 NS

Cytological analysis

Malignant cells

0

0

--

--

Ascetic fluid bacterial culture was positive in 22

cases (44.9%) of clinically suspected SBP group

and 13 cases of not clinically suspected SBP

group, the isolated organisms in SBP group were

E.coli in 15 cases (42.8%), Klebsiella in 1 cases

(2.8%) and streptococcus in five case

(14.28%),in clinically suspected SBP group

while the isolated organisms in not clinically

suspected SBP group were E-coli in 12 cases

(34.2%) and streptococcus in 1 case (2.8%) , the

isolated organisms were mono -microbial Gram

negative aerobic pathogens as shown in table (6).

Table 6:Ascitic fluidculture of the studied groups

Causative organism

Positive culture group (n=35)

Clinically suspected SBP NotClinicallysuspectedSBP

No. % No. % %

E.coli 15 42.8 12 34.2 77.2

Klebsiella 1 2.8 - - 2.8

Klebsiella-pneumococci 1 2.8 - - 2.8

Streptococcuspneumonii 5 14.28 1 2.8 17.1

- there was no significant difference between the studied groups as regard child classification.

Table 7: Child classification of studied groups

Child classification


(no=49)


SBP(no=41)

χ2 test P value

No % No %

A

B

C

0

8

41

0

16.3

83.7

0

14

27

0

34.1

65.9

3.83

0.05

Discussion

Ascites is one of the most common

complications of patientswith cirrhosis and its

development carries a relatively poor

prognosis(22).Spontaneous bacterial peritonitis

(SBP) is a common and serious infection

occurring in 10 – 30 % of hospitalized patients

with cirrhosis and ascites (23). It is also one of the

potential life- threatening complications in

cirrhotic patients with ascites with a mortality

rate ranging between 30 and 50% (24), so, it

requires a rapid and accurate diagnosis in

addition to prompt effective therapy (25).The

present study was conducted on 90 patients with

decompensatedchronic liver diseases and ascites,

they were selected from 120 patients admitted to

(Tropical Medicine Department in Menoufiya

University Hospital, Shebin Elkom and Tala

Fever Hospital) during the period from August

2014 to April 2015, Sixty of them (66%) were

males andthirty of them (34%) were females.

Their ages ranged from 46 to 69 years witha

.These patients were

classified into two groups.Group I: comprised

49 patients clinically suspected SBP. Group2:

comprised 41 patients not clinically suspected

SBP.The present study revealed that, SBP was

common in males (73.5%) than females (26.5%)

and it was not influenced by the age. This result

was in agreement with the study done by Angelli

et al (26) who stated that, SBP was frequent in

males and was not affected by the age.Regarding

the clinical presentations of the patients ,

analysis of the results showed that, fever,

abdominal pain and bleeding tendency were the

only significant clinical presentations in patients

with clinically suspected SBP ( 75.5%, 81.6%

and 61% respectively) compared to not clinically

suspected SBP group This difference could be

explained by Rimola and Navasa(27) who said

that, the clinical picture of SBP is extremely

broad and very variable and a very high degree

of clinical suspicion is required for diagnosis.

Regarding the clinical examination, all patients

with SBP were presented by moderate to tense

ascites (46.9% and 38.8% respectively). This

result was in agreement with that reported by

Runyon (28) who stated that, ascetic fluid

infection usually developed when the volume of

ascites was at its maximum.The ultrasonographic

findings in the present study detected no

significant difference between both groups

regarding to internal echoes and this correlates

with data obtained by Al-Sharif (29) who

concluded that, ultrasonographic detection of

echoes and/or adhesions is neither diagnostic nor

prognostic and there was no single imaging test

is totally sensitive or specific for the detection of

infected fluid collection.Regarding the

parameters of laboratory investigations , there

was significant difference between both groups

in total leucocytic count, leucocytosis was

present in 62.2% of SBP patients and this result

was in agreement with that reported by

Rodriguez et al (30) who detected leucocytosis in

their SBP cases with significant difference when

compared to non SBP cases.Regarding liver

function tests, there was a highly statistical

significant difference between SBP cases and

non SBP cases in ALT and AST levels.

otherwise, no statistically significant differences

was recorded in the other parameters. Casafont

et al (31) found no significant difference in liver

biochemistry, Also, these results were in

agreement with those reported by Runyon et al (32) who stated that, prolonged prothrombin time

and hypoalbuminaemia are not related to SBP

per se, but rather to the underlying liver

disease.The relation between occurrence of SBP

and severity of liver diseases howed that,(16.3%

and 83.7%)of clinically suspected SBP cases

had (Child Pugh class B and C cirrhosis

respectively).(43.1% and 65.9%) of not

clinically suspected SBP cases had Child Pugh

class B and C cirrhosis respectively. Physical

examination of ascetic fluid revealed turbid fluid

in 60.0% of patients of SBP with a statistical

significance when compared to the non SBP

group in which 96.6% of patients had clear fluid.

In agreement with this result, Albillos et al (33)

observed that, the appearance of ascitic fluid in

patient with SBP is usually turbid. Moreover,

Runyon (28) reported that, the opacity of many

cloudy ascetic fluid specimens is caused by

neutrophils. Moreover, Chijung et al (34) in a

retrospective review of 916 outpatient ascetic

fluid samples showed that, abnormal ascitic fluid

appearance had a sensitivity of 98.1% and a

specificity of 22.7% in the detection of

SBP.Total protein concentration in ascetic fluid

showed decrease in both groups of clinically and

not clinically suspected SBP with no significant

difference with mean value of .45 gm/dl .Runyon

(1986) denoted that, patients with lowest ascetic

protein concentrations are the most susceptible

to SBP. Ascetic fluid WBCs and PMN count

were significantly higher in SBP group than non

SBP group. Jansen (35) stated that, although

ascetic total WBC count increases in SBP cases,

it suffers from low specificity because a large

proportion of patients with sterile ascites have

increased white blood cell count. Also, diuretic

therapy has been shown to increase the total

white blood cells count but does not alter the

PMN count. Ascetic fluid bacterial culture was

positive in 22 cases (44.9%) of clinically

suspected of SBP group and 13 cases (31.7%) of

not clinically suspected SBP The isolated

organisms in clinically suspected SBP group

were E.coli in 15 cases (42.8%), Klebsiella in

one case (2.8%) , Klebsiella pneumia in one

case (2.8%) and streptococcus pneumonii in five

cases (14.28%) . while The isolated organisms in

not clinically suspected SBP group were E.coli

in 12 cases (34.2%) and streptococcus

pneumonii in one cases (2.8%).Liovet and co-

workers (2000) detected positive ascetic fluid

bacterial culture in 41.7% of cases of SBP which

was close to the result of the present study. In

SBP group, the isolated organisms were mono

microbial, gram negative aerobic pathogens.

This was in agreement with that reported by

Liovet et al. (1997) who stated that, single gram

negative organisms (speciallyE.coli and

Klebsiella) cause most episodes of SBP.Culture

negative neutrocytic ascites (CNNA) which is

considered as a variant of SBP was detected in

12.2% of SBP cases in this study.(in clinically

suspected SBP 18.4% and in not clinically

suspected 11%)Culture negativity may be due to

low concentration of bacteria in ascetic

fluid.This was in agreement with that reported

by Navasa and coworkers(1996) who stated that,

inspite of using good culture techniques; cultures

are still negative in approximately 30-50% of

patients with anincreased ascites PMN count.The

prevalence of non neutrocytic bacterascitis

which is considered as avariant of SBP was

detected in 3.3% (in clinically suspected SBP

4.1% and in not clinically suspected 2.4%).The

prevalence of SBP group with negative culture

and neutrophil Count <250 cell/mm3 was 48.9%

(in clinically suspected SBP 36.7% and in not

clinically suspected 63.4%).The prevalence of

neutrocytic bacterascites was 35.6% (in

clinically suspected SBP 40.8% and in not

clinically suspected 29.3%). This result was in

agreement with that reported by tendon p et al

(2008) .Who stated that the prevalence of SBP in

hospitalized patients with cirrhosis and ascites is

between 10% and 30%.Even in case of not

clinically suspected SBP theres found that 29.3%

was Culture positive neutrocytic ascites.

Conclusion

High prevalence and mortality rate of SBP

among patients in our study. Ascetic fluid

bacterial culture was positive in not only

clinically suspected SBP but it was also found

that there was positive cases in not clinically

suspected SBP patients with present of isolated

organisms.

Ascetic fluid culture revealed that, ecoli is the

predominant organism isolated from ascetic fluid

of clinically and not clinically group.

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Original Article

Study of The Diagnostic Role of Platelet Indices in Patients with Ulcerative

Colitis

Hossam Ibrahim Mohamed*, Abd Allah Said Essa*, Enas Said Essa**, Heba Hamdy***. *Tropical Medicine department, Faculty of Medicine, Menoufiya University, **Clinical pathology

department, Faculty of Medicine, Menoufiya University, ***Menouf fever hospital.

ABSTRACT Ulcerative Colitis (UC) is a chronic inflammatory disease. Many serum biomarkers have been studied for diagnosis

and monitoring of disease activity in UC. Platelets play an important role in inflammation. Aim of the work:The aim

of the present study is to determine whether platelet indices; mean platelet volume (MPV), platelet distribution width

(PDW) and platelet-crit (PCT) could be useful, non-invasive biomarkers for diagnosis and determining severity and

activity of UC. Methods: The studied groups consisted of 30 patients with UC (UC group), 20 patients with IBS

(IBS group)and 20 healthy subjects as control group (Control group). Disease activity was evaluated clinically and

by endoscopy with biopsy. Platelet indices and inflammatory parameters were measured for all study participants.

Results: In patients with active UC, there was a statistically significant decrease in MPV, PDW levels and increase

in PCT level when compared to healthy controls. All platelet indices; MPV (r:-0.39, p= 0.02), PDW (r: –0.42, p=

0.01) and PCT (r: 0.45, p= 0.006) have a significant correlation with UC disease activity. Conclusions: The present

work revealed that changes of platelet indices in UC could be reliable inflammatory markers, especially to monitor

disease activity.

Introduction

The pathogenesis of UC likely involves genetic,

environmental and immunological factors(1). The

assessment of UC is based on a combination of

symptoms, clinical examination, laboratory

indices, radiology and endoscopy with histology.

There is no single gold standard test or

examination. The search for a non-invasive

biomarker that determines the type, disease

activity, prognosis and response to therapy of

UC has been a focus in IBD research(2-4). The

main biomarkers in UC are C-reactive protein

(CRP), erythrocyte sedimentation rate (ESR),

white blood cells (WBC), acid glycoprotein, and

platelet count, albumin with fecal and serologic

markers(5-7). All these biomarkers have little

accuracy in predicting UC activity. Therefore

new, cheap and non-invasive additional serum

biomarkers are needed for diagnosis and

determination of disease activity. It has been

shown that decreased mean platelet volume

(MPV) may be an indicator for increased disease

activity in patients with ulcerative colitis (UC)(8).

Recent studies also revealed the association of

platelet indices (PCT, PDW and MPV) with

WBC count and CRP which underline the wide

relation between platelets and inflammation(9).

But the relation between these platelet indices

and UC has not been well investigated. The

primary objective of this study was to determine

whether PDW, PCT and MPV could be used for

assessment of disease activity and as follow up

markers in patients with UC.

Materials and Method

50 patients with lower gastrointestinal symptoms

indicated for colonoscopy were recruited for this

study after the approval of the local ethical

committee; those patients were selected from the

endoscopy unit and the outpatient clinic of

Tropical Medicine Department in Menoufia

University Hospital, Egypt, between July 2013

and June 2014. Patients were classified into the

following groups: patients with UC (UC

group),patients with symptoms and criteria

suggesting IBS(10) (IBS group). 20age and sex

matched healthy volunteer were enrolled as a

control group (Control group).Patients who have

history of any platelet disorder (e.g.

thrombocytosis) or drug known to affect

platelets (e.g.acetyl salicylic acid) were excluded

from the study, also any patient with positive

family history for IBD were excluded from IBS

group. Informed consents were obtained from all

participants before enrolment in the study. All

study participants were subjected to: full history

taking, clinical examination and routine lab

investigations. Patients (UC and IBS groups)

included in the study were subjected to

colonoscopy and biopsy with histopathological

examination to confirm diagnosis, assess the

extent and disease severity of UC.The activity of

UC was classified according to Mayo scoring

system into minimal, mild, moderate or

severe(12). Scoring system for the assessment of

histopathological severity in UC which takes

granularity, vascular pattern, and mucosal

vulnerability also used to assess disease activity

in UC patients(13).Collection of blood samples:

For patients and controls, 2 ml of blood were

collected on EDTA for complete blood picture

(CBC), 2 ml on plain tubes for serum separation

and C-reactive protein (CRP) measurement, 1.6

ml on sodium citrate for erythrocyte

sedimentation rate (ESR) determination.

Laboratory methods: Determination of CRP:

This was done by immunoturbidimetric

measurement using Quik Read CRP kit ( Orion

Diagnostica, Espoo, Finland). Measurement of

CBC: This was done using an automatic cell

counter; ERMA PCE-210 (Yushima, Bunkyo-

ku, Tokyo, Japan). Measurement principle:

Measurement method of PCE-210 is electric

resistance. It applies electric pulse method for

counting blood cells. The instrument recognizes

frequency of changes in voltage and count it as

blood cell quantity. Particle analysis is a function

to analyze particle size (volume) by the blood

cell type. Particle distribution graph is a

histogram obtained by particle sizing which was

detected by measurement of voltage variation

when cells go through the orifice. Hemoglobin is

measured by measuring the absorbance of

Cyanmethemoglobin solution.PCE-210 uses

LED for hemoglobin is measurement. Photo Cell

is Photo Diode. Prior to measurement, back

ground noise was checked by measuring diluent

solution only. Measurement accuracy was

checked by hematology control (Para 12 Extend,

Multi-Parameter Assayed Hematology Control,

Streck, Omaha, USA). Measurement was

performed within 2 hours of blood sampling

because MPV increases over time as platelets

swell in EDTA(11).

Statistical Analysis

Quantitative data were expressed as mean ±

standard deviation (X±SD) and analyzed by

applying student t-test, Mann Whiteny test or

Kruskal–Wallis test. Qualitative data were

expressed as number and percentage (No and %)

and analyzed by applying chi-square test (χ2).

Correlation co-efficient test (r-test) was used for

correlation between two quantitative variables.

All tests were performed using Statistical

Package for the Social Sciences, version 17.0

(SPSS Inc., Chicago, IL, USA). P<0.05 was

considered significant.

Results

The present study was conducted on 50 patients

with lower gastrointestinal symptoms indicated

for colonoscopy. They were 24 males (48%) and

26 females (52%), their age ranged from 23 to

62 years. In addition to 20 healthy subjects of

matched age and sex were recruited as a control

group. According to colonoscopic and

histopathological evaluation, all patients were

assigned to UC group: 30 patients (14 males and

16 females) with colonoscopic and

histopathological findings of UC, IBS group: 20

patients (10 males and 10 females) met ROME

III criteria for IBS diagnosis in addition to

negative endoscopic and histopathological

findings compatible with UC diagnosis or any

organic colonic disease. Control group:

comprised 20 normal healthy subjects (9 males

and 11 females).As regard clinical presentation,

rectal bleeding was the main presenting

manifestation in UC group (90%) which differs

according to the severity of the disease (e.g.

streaks, obvious or blood alone), however rectal

bleeding was absent in all patients of IBS group.

Measurement of ESR, CRP in all groups

revealed highly significant increase (P < 0.001)

in UC patients in comparison to IBS and control

groups. Significant decrease (p < 0.002) in the

mean values of Hb concentration was found in

UC patients in comparison to IBS and control

groups. A significant increase (p < 0.04) in the

mean value of platelet count in UC group when

compared to IBS and control groups was also

noted. Measurement and analysis of the previous

inflammatory parameters revealed non-

significant difference (p > 0.05) between IBS

group and control group as regard ESR, CRP,

WBC& platelet count. Colonoscopic

examination revealed inflammatory picture of

different severity and extent in all UC patients

while there was mild inflammation in 5 patients

within IBS group (Table 1).Architectural

changes and chronic inflammatory infiltration

(diagnostic for UC) were present in all patients

included in UC groupand were associated with

other histopathological findings denoting activity

in different grades according to Mayo score

(UCAI). Histopathological study of IBS patients

suggests low grade chronic inflammation with T-

lymphocytes, none of histopathological findings

characteristic for diagnosis of UC were found in

this group (Table 2& 3). Measurement of

platelets indices (MPV, PDW and PCT) in all

studied groups revealed highly significant

decrease (p < 0.001) in the mean values of MPV

&PDW and highly significant increase (P

<0.001) in the mean values of PCT% in UC

patients in comparison to IBS and control

groups. While there was no significant difference

between IBS patients and control group as regard

previous platelet indices (Table 4).A receiver

operating characteristic (ROC) curves were

generated for each platelet index in relation to

disease activity (UCAI) in ulcerative colitis

group. MPV, PDW and PCT at different cut-off

points can distinguish patients with severe UC

from those with lesser grades of disease activity

with sensitivity (59, 54.5 and 75%) and

specificity (75, 75 and 72.7%) respectively

(Figure 1, 2 and table 5). In UC group a

significant correlation was revealed between

MPV, PDW, PCT and disease activity index

(UCAI, Mayo score); this correlation was

negative as regard MPV, PDW; where it was

positive as regard PCT (Figure 3, 4, 5). A

significant negative correlation was also noted

between MPV, PDW and both ESR, CRP

(Figure 6, 7).

Discussion UC is disorder that primarily affects the

gastrointestinal tract. The immune system, with

its active components, dominates UC

pathogenesis, but many genetic and

environmental factors have been also implicated.

A growing number of studies are high lighting

the importance of non-immune cells like

endothelial, mesenchymal, and nerve cells, as

well as platelets, as key players in the UC

inflammatory cascade (14).PLT dysfunction is

expected to participate in UC pathogenesis,

although the existing evidence is rather weak.

On the other hand, there is a solid evidence

supporting PLT as potent pro inflammatory cells

in addition to their role in homeostasis. Several

studies have shown that PLT constitute a crucial

link between inflammation and coagulation in

UC (15). Many PLT changes have been described

in UC, including morphological alterations

(MPV, PDW, PCT), count increase, micro

particles (MPs) release, over excretion of

granular content, and increased formation of

PLT-PLT and PLT-leukocyte aggregates ,which

are all linked to PLT activation induced by

inflammatory agonists(16).Chronic inflammatory

disorders are connected to several morphological

changes in PLT indices calculated in whole

blood count, such as MPV, PDW, and PCT. The

most widely-studied PLT parameter in humans is

MPV. PLT volume decreases when an

inflammatory process is present, which is mainly

attributed to thrombopoiesis abnormalities and

increased PLT consumption. Inflammatory

mediators stimulate bone marrow precursors to

enhance PLT generation at the cost of maturation

time, delivering smaller PLT in circulation,

while at the same time larger and more active

PLT are consumed at inflammatory sites, as is

proposed in the intestinal microvasculature of

UC patients(9).Many studies observed a decrease

in MPV in IBD patients(17), which has been

inversely correlated with endoscopic and disease

activity indices, such as C-reactive protein and

erythrocyte sedimentation rate(18).This MPV

reduction can be attributed to the decreased

circulating reticulated PLT number that was

found in patients with active UC compared to

inactive and healthy control subjects (17).Many

studies have reported an inverse relationship

between extent of intestinal inflammation and

MPV in UC patients. Öztürk et al suggested that

all PLT parameters (PDW, PCT, MPV) proved

to be useful markers for IBD follow-up, as they

represent strong relationship with activity indices (19). In the present study, we investigated the

changes in platelet indices in patients with UC

with different grades of severity to figure out if

these changes are related to the etiopathogensis

of ulcerative colitis and more importantly the

relation of these changes to disease extent&

activity and subsequently following up of

patients with UC whether in remission or

exacerbation. Moreover; the correlation between

these platelet indices and other inflammatory

parameters was assessed. 3 groups were studied

(UC, IBS and control groups), UC was

diagnosed based on clinical endoscopic, and

histopathological evidences. The result of the

present study revealed significant decrease in

mean value of MPV in UC group compared to

IBS and control groups. There was highly

significant difference between UC group and

IBS group (<0.001),also there is highly


control group (<0.001), while there was no

significant difference between IBS and control

groups regarding MPV (0.08). Also this study

revealed a significant negative correlation

between MPV and disease activity (in this study

determined by mayo score).These findings were

in agreement with Kapsoritakis(18) who conducted

a study on 93 patients with UC and 38 healthy

blood donors. Disease activity was assessed by

the Clinical Colitis Activity; this study revealed

that; mean platelet volume was significantly

decreased in patients with active compared to

inactive ulcerative colitis or healthy controls.

They concluded that; MPV is significantly

reduced in active inflammatory bowel disease

and is negatively correlated with the known

inflammatory bowel disease activity markers and

the platelet activation products. Similarly, Yükse

l2 1who conducted a studyon a total of 61 UC

patients (male/female: 41/20) , and 27 healthy

subjects (male/female: 18/9). For all subjects, the

following tests were performed; ESR, CRP,

white blood cell count and mean platelet volume.

Their study revealed a statistically significant

decrease in MPV in patients with UC (8.29 +/-

1.02 fL) compared with healthy controls (8.65

+/- 0.79 fL). MPV of active UC (8.06 +/- 1.19

fL) patients were significantly lower than that of

inactive UC (8.45 +/- 0.87 fL). Overall accuracy

of MPV in determination of active UC was 71%

(with sensitivity 67%, specificity 73%). A

negative correlation was found between MPV

and endoscopic activity index. They concluded

that decreased MPV may be an indicator for

increased disease activity in patients with UC.

Another study conducted by Polinska(22)

concluded that; the decrease in MPV reflects the

activation and participation of platelets in the

inflammatory process of colon mucosa, and so

MPV may be a useful marker of active ulcerative

colitis. On the other hand ÖZTÜRK (19) in a

study conducted on 103 patients with UC and a

control group included 40 healthy subjects;

disease activity was evaluated both by

endoscope and clinically, platelet indices and

inflammatory parameters were measured for all

study participants. Patients were checked in both

active and remission phase of the diseases. Their

study revealed that, MPV levels were lowest in

remission and highest in control group. Also this

study did not find any correlation between MPV

levels and UC activity. In the present study,

statistical analysis revealed a highly significant

difference between UC group and IBS group as

regard PCT (<0.001). Also there was a highly


IBS group, while, there was no significant

difference between IBS and control groups

regarding PCT. Also this study revealed positive

significant correlation between PCT and disease

activity (in this study determined by mayo

score). These findings were in agreement with

ÖZTÜRK(19) who found that PCT percent was

lowest in control group and highest in active

phase of UC and PCT was positively correlated

with disease activity. In the present study, it has

been found that PDW significantly decreased in

UC group in comparison to IBS and control

groups(<0.001).While there was no significant

difference between IBS and control groups

regarding PDW. Also this study revealed

negative significant correlation between PDW

and disease activity (in this study determined by

mayo score).These findings were in agreement

with ÖZTÜRK(19)who revealed that PDW was

significantly lower in active phase of UC than

control group. In the present work, MPV has

negative correlation with ESR, CRP, WBCs,

platelet count, and Mayo score. These findings

were in agreement with Kapsoritakis(18) who

found that mean platelet volume was inversely

correlated with the white blood cell count, C-

reactive protein and erythrocyte sedimentation

rate. No significant correlations were found

between mean platelet volume and serum

thrombopoietin or erythropoietin levels;

however, a strong negative correlation between

mean platelet volume and β-thromboglobulin

and platelet factor 4 was observed. Similarly,

Polińska(22)showed significant negative

correlation between clinical, pathological and

colonoscopic activity indices of ulcerative colitis

disease and MPV (p<0.001). However as ex-

pected in any systemic inflammatory response,

in our ulcerative colitis clinically active patients,

we found serum albumin, which is known as

negative acute phase reactant, as low as well as

the increase in sedimentation, platelet number

and CRP. In our study, there was an inverse

proportional relation between MPV and the in-

crease in platelet number, sedimentation, and

direct proportional relation with the decrease in

albumin. Also ÖZTÜRK(19) found that, MPV was

weakly negatively correlated with ESR and CRP.

On the other hand Yüksel(21) revealedthat, MPV

did not correlate with ESR, CRP and white blood

cell. Our data suggests that platelet indices MPV,

PDW and PCT are non-invasive biomarkers for

evaluation of patients of UC and patient's

activity correlates well with other modalities in

UC such as UCAI, ESR and CRP. It should not

be considered a stand-alone test for this use

owing to non-specificity with other diseases.

However further studies are needed to establish

the relation between platelet functions and UC.

Table (1): Colonoscopic findings in UC and IBS patients.

UC group (30) IBS group (20)

Presence of inflammation

1. No 0 0% 15 75%

2. Mild 18 60% 5 25%

3. Moderate 6 20% 0 0%

4. Sever 6 20% 0 0%

Extent of inflammation

1. No 0 0% 15 75%

2. E1 18 60% 5 25%

3. E2 6 20% 0 0%

4. E3 6 20% 0 0%

Table (2): Histopathological findings in UC patients.

Histopathological findings UC group (30)

Grade 0: architectural changes 30 100%

Grade 1: chronic inflammatory infiltration 30 100%

Grade 2: Lamina propria neutophils&eosinophil 20 66.66%

Grade 3: Neutophils in epithelium 12 40%

Grade 4:Crypt destruction 6 20%

Grade 5: Erosions or ulceration 6 20%

Table (3): Mayo score for detecting severity in UC group.

Mayo score UC group (30)

(0-2) Remission or minimal active disease 3 10%

( 3 - 5 ) Mild active disease 15 50%

(6-10) Moderate active disease 6 20%

(11-12) Sever disease 6 20%

Table (4): Platelet indices (MPV, PDW, PCT) in the studied groups.

The studied groups

t- test P value UC group N = 30

IBS group

N = 20 Control group

N = 20

MPV(fl)

X ± SD

Range

7.89±1.36

5 – 10.9

11.1±2.1

10 – 17

10.01±1.76

9 – 17

6.56

4.81

1.77

<0.0011

<0.0012

0.083

PDW(fl)

X ± SD

Range

11.13±0.98

9.5 – 11.5

13.1±0.91

12 – 14

13.51±1.04

11.5 – 14.5

7.16

8.21

1.33

<0.0011

<0.0012

0.193

PCT %

X ± SD

Range

0.29 ±0.5

0.22 –0. 38

0.22±0.47

0.15 –0. 26

0.18±0.57

0.13 – 0.28

7.09

7.07

0.89

<0.0011

<0.0012

0.383

X = Mean, SD = standard deviation 1 = comparison between GI and GII groups

2 = comparison between GI and GIII groups 3 = comparison between GII and GIII groups

Table (5):Validity of platelet indices in evaluation of activity of UC [UCAI].

AUC 95%CI Cutoff point Sensitivity Specificity Accuracy

MPV 0.79 0.62 – 0.96 9.05 fL 59% 75.0% 67

PDW 0.53 0.30 – 0.75 11.9 fL 54.5% 75% 64.75

PCT 0.74 0.52 – 0.96 0.272% 75% 72.7% 73.85

AUC: area under curve

Figure (1): ROC curve of MPV and PDW in UC group in

relation to UC activity index UCAI (Mayo score).

ROC Curve

Diagonal segments are produced by ties.

1 - Specificity

1.00.75.50.250.00

Se

nsitiv

ity

1.00

.75

.50

.25

0.00

Source of the Curve

Reference Line

PDW

MPV

Figure (2): ROC curve of PCT % in UC group in

relation to UC activity index UCAI (Mayo score)

ROC Curve of PCT

Diagonal segments are produced by ties.

1 - Specificity

1.00.75.50.250.00

Se

nsitiv

ity

1.00

.75

.50

.25

0.00

Figure (3): Correlation between MPV & UCAI (Mayo

score) in UC group.

r = - 0.39 P value = 0.02

Mayo score

876543210

MP

V(f

l)

13

12

11

10

9

8

7

6

5

Figure (4): Correlation between PDW & UCAI

(Mayo score) in UC group.

r = - 0.42 P value = 0.01

Mayo score

876543210

PD

W(f

l)

15

14

13

12

11

10

9

8

Figure (5): Correlation between PCT % & UCAI (Mayo

score) in UC group.

r = + 0.45 P value = 0.006

Mayo score

876543210

PC

T%

.4

.3

.2

.1

Figure (6): Correlation between MPV and both

ESR &CRP in UC group.

MPV (fl)

1312111098765

140

120

100

80

60

40

20

0

ESR

MPV (f l)

CRP

MPV (f l)

MPV & ESR (r:-0.35& p: 0.03)

MPV & CRP (r: -0.32 & p: 0.04)

Figure (7): Correlation between PDW and both ESR

&CRP in UC group.

706050403020100

140

120

100

80

60

40

20

0

PDW

ESR

CRP

PDW

PDW &ESR (r: -0.29 & p: 0.04)

PDW & CRP (r:-0.38 & p: 0.02)

Figure (8): Correlation between PCT% and both

ESR &CRP in UC group.

.4.3.2.1

140

120

100

80

60

40

20

0

ESR

PCT%

CRP

PCT%

PCT% &ESR (r: 0.48 & p: 0.004)

PCT% & CRP (r: 0.57 & p: <0.001)

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Original Article

Thyroid Diseases in Egyptian Patients with HCC: Case -Control Study

Essam S. Bedewy*, Ahmed I. Ellakany**, *Department of Tropical Medicine, **Department of Internal Medicine, Faculty of Medicine, University of Alexandria, Egypt

ABSTRACT Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide, Chronic hepatitis C virus (HCV) infection and its underlying cirrhosis were suggested to be the reasons for this increase. Patients with HCV are susceptible to autoimmune thyroid diseases before and after completion of interferon therapy suggesting an association between thyroid diseases and HCV. Hypothyroidism has been observed in 5%–12% of patients with HCV. Aim of the : The aim of this article was to study the association between thyroid dysfunction and HCC development in Egypt by comparing HCC patients with healthy controls. We also aimed to describe the relationship between thyroid diseases and established risk factors for HCC, particularly chronic hepatitis virus infection, diabetes, and obesity. Methods: The current study included 80 subjects divided into two groups. Fifty patients with newly diagnosed HCC and 30 healthy subjects of matched age and sex were enrolled in the study as (Control group). Patients with HCC and controls were recruited simultaneously and were personally interviewed. We used a structured and validated questionnaire to collect information on demographic features and HCC risk factors such as personal smoking history, medical history, and family history of cancer. Cases and controls were interviewed for prior history of thyroid disorders including hypothyroidism, hyperthyroidism, inflammatory and autoimmune thyroid diseases. Subjects with a history of any thyroid condition were asked for age at diagnosis, treatment modality (whether medical, surgical or radioactive iodine therapy) and duration of the disorder. Blood samples from cases and controls were tested for HBV and HCV. HCV antibodies (anti-HCV), hepatitis B surface antigen (HBsAg) were detected by use of a third-generation enzyme-linked immunosorbent assay (Abbott Laboratories, North Chicago, IL). Positive results prompted repeated confirmatory enzyme-linked immunosorbent assay testing. Results: There was male predominance in both patients and control groups (70%) and (63.3%) respectively. Multivariate logistic regression analyses indicated that having a history of HCV, HBV, cigarette smoking, or having a family history of cancer (including liver cancer), or diabetes mellitus were significantly associated with HCC development. Most of the HCC patients and the healthy controls were non-smokers (58%) and (60%) respectively. Similarly, there was no family history of cancer in most of the HCC patients (88%) and in the control subjects (93.3%). There was a significant association between infection with HCV or HBV and HCC. Most of the HCC patients have been infected by HCV or HBV or both. The results were (30%) with HBV infection, (24%) with HCV, (16%) with both and 30% of HCC patients had no HCV or HBV infection. The majority of the control subjects had no hepatitis viral infection (73.3%). A total of 32 patients with HCC (64.0%) and 7 controls (23.4%) recalled a history of thyroid disease. Among all studied subjects, significantly more cases (44%) than controls (16.7%) reported hypothyroidism (P = 0.0123). A significant relationship in HCC patients was observed with long-term thyroid dysfunction (>2 years), (p= 0.033). Most of HCC patients having thyroid dysfunction (32 patients) were HBV/HCV infected. The results showed significant association between thyroid dysfunction and HBV/HCV infection (p = 0.0021). The majority in the control group (90.9%) have no HBV/HCV infection, and no thyroid dysfunction (76.7%). Conclusion: This study can conclude that long-term hypothyroidism is associated with HCC, independent from other major HCC risk factors. Screening and proper management of thyroid diseases in patients with diabetes or HCV or HBV infection may help prevent HCC. Further studies among different populations are warranted to confirm the association between hypothyroidism and HCC .

Introduction Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide,(1) Chronic hepatitis C virus (HCV) infection and its underlying cirrhosis were suggested to be the reasons for this increase.(2,3) Several extra hepatic conditions have been associated with HCV infection as mixed cryoglobulinemia, porphyria cutaneatarda and membrano-proliferative glomerulo-nephritis.(4)In addition, patients with HCV are susceptible to autoimmune thyroid

diseases before and after completion of interferon therapy suggesting an association between thyroid diseases and HCV. (5, 6). Hypothyroidism has been observed in 5%–12% of patients with HCV and is the most common thyroid disorder in the adult population, particularly among older women. (7) . Thyroid hormones play an essential role in lipid mobilization, lipid degradation, and fatty acid oxidation. (8)Accordingly, thyroid hormone deficiency may cause hyperlipidaemia and may

play a role in the pathogenesis of non-alcoholic steatohepatitis (NASH).(9)In addition, patients with hypothyroidism may experience a 15% to 30% weight gain(10)and associated insulin resistance, (11,12) which are significant factors in NASH. In one study, (9) the prevalence of hypothyroidism was significantly higher in patients with NASH (15.0%) than in controls (7.2%) (P =0.001). This finding was later supported by Reddy and his colleagues(13) at the Mayo Clinic, who reported an odds ratio (OR) of 6.8 (95% confidence interval [CI], 1.1 to 42.1) of hypothyroidism in patients with HCC with unknown aetiology compared to patients with HCC with underlying HCV infection or alcoholic liver diseases after adjusting for several confounding factors. Overall, these studies have suggested a clinical association between thyroid dysfunction, chronic liver diseases and HCC. In the present study, our goal was to determine the association between thyroid dysfunction and HCC development in Egypt by comparing HCC patients with healthy controls. We also aimed to describe the relationship between thyroid diseases and established risk factors for HCC, particularly chronic hepatitis virus infection, diabetes and obesity. Subjects and Methods The current study is a hospital-based case-control study, included 80 subjects divided into two groups. Fifty patients with newly diagnosed HCC who were evaluated and treated at Alexandria Main University Hospital, Faculty of Medicine, Alexandria University. The inclusion criterion was pathologically confirmed diagnosis of HCC. The exclusion criteria were the presence of other types of primary liver cancer (such as cholangiocarcinoma or fibrolamellar hepatocarcinoma), unknown primary tumours, and concurrent or past history of cancer at another organ site ( HCC group). Also, 30 healthy subjects of matched age and sex with the HCC group were enrolled in the study as (Control group). The control subjects were genetically unrelated family members (i.e., spouses and in-laws). The reason for excluding family members was to prevent the introduction of selection bias connected with shared environmental and genetic factors that are highly associated with HCC, e.g., alcohol consumption, smoking, family history of cancer, and hepatitis virus infection. A written informed consent was

taken from every subject participated in this study. Patients with HCC and controls were recruited simultaneously and were personally interviewed. We used a structured and validated questionnaire (14) to collect information on demographic features and HCC risk factors such as personal smoking history, medical history, and family history of cancer. (15, 16). Cases and controls were interviewed for prior history of thyroid disorders including hypothyroidism, hyperthyroidism, and inflammatory and autoimmune thyroid diseases. Subjects with a history of any thyroid condition were asked for age at diagnosis, treatment exposure, and duration of the disorder. Blood samples from cases (n = 50) and controls (n = 30) were tested for HBV and HCV. HCV antibodies (anti-HCV), hepatitis B surface antigen (HBs Ag) were detected by use of a third-generation enzyme-linked immuno-sorbent assay (Abbott Laboratories, North Chicago, IL). Positive results prompted repeated confirmatory enzyme-linked immuno-sorbent assay testing. Statistical Methods For continuous variables, group differences were tested by analysis of variance; when this was significant at the P≤0.05 level, between-group comparisons were carried out by the Bonferroni-Dunn test. Frequencies were compared with the chi-squared test. A P value≤0.05 was considered significant. Results Table 1: shows the demographic characteristics of HCC patients and control subjects. Most study subjects were males. Overall, control subjects were slightly older than patients; the mean age ± S.D was 52.6 ± 10.3 years for patients with HCC and 53.6 ± 9.89 years for controls. There was male predominance in both patients and control groups (70%) and (63.3%) respectively. Multivariate logistic regression analyses indicated that having a history of HCV, HBV, cigarette smoking, or having a family history of cancer (including liver cancer), or diabetes mellitus were significantly associated with HCC development. In our study most of the HCC patients and the healthy controls were non smokers (58%) and (60%) respectively. Similarly, there was no family history of cancer in most of the HCC patients (88%) and in the control subjects (93.3%).

Table (1): Demographic and clinical data of the two studied groups:

HCC Group n=50

Control Group n=30

P

Age Range Mean S.D.±

44 – 72 52.6 10.3

45 – 71 53.6 9.89

0.365

Sex Male Female

35 15

70.0 30.0

19 11

63.3 36.7

0.412

Medical history D.M Hypertension Cardiac disease Cancer

6 12 7 4

12.0 24.0 14.0 8.0

2 3 4 1

6.7 10.0 13.3 3.3

0.106

Smoking Yes No

21 29

42.0 58.0

12 18

40.0 60.0

0.685

Family history of cancer Yes No

6 44

12.0 88.0

2 28

6.7 93.3

0.221

Table 2: shows that there is a significant association between infection with HCV or HBV and HCC. Most of the HCC patients have been infected by HCV or HBV or both. The results were (30%) with HBV infection, (24%) with

HCV, (16% ) with both and 30% of HCC patients had no HCV or HBV infection .While, the majority of the control subjects had no hepatitis viral infection (73.3%).

Table (2): Distribution of the two studied groups regarding hepatitis virus

Hepatitis virus

HCC Group n=50

Control Group n=30

P

No. % No. % None 15 30.0 22 73.3

0.001* HCV 12 24.0 2 6.7 HBV 15 30.0 6 20.0 Both HCV and HBV 8 16.0 0 0.0

Table 3: A total of 32 patients with HCC (64.0%) and 7 controls (23.4%) recalled a history of thyroid disease. Among all studied subjects, significantly more cases (44%) than

controls (16.7%) reported hypothyroidism (P = 0.0123). A significant relationship in HCC patients was observed with long-term thyroid dysfunction (>2 years), (p= 0.033) .

Table (3): Incidence of thyroid diseases in the two studied groups

Thyroid diseases HCC Group n=50

Control Group n=30

P

No. % No. % None 18 36.0 23 76.7

0.0123* Hypothyroidism 22 44.0 5 16.7 Hyperthyroidism 10 20.0 2 6.7 Duration of thyroid disease < 2 years 8 25.0 3 42.9

0.033* 2-5 years 14 43.8 4 57.1 > 5 years 10 31.3 0 0.0

Table 4: shows that most HCC patients having thyroid diseases (29 patients) were HBV/HCV

infected. The results show significant association between thyroid diseases and HBV/HCV

infection (p = 0.0021).

Table (4): Relation between incidence of hepatitis virus and thyroid diseases in HCC group

HCC Patients

“n=50” None HCV HBV Mixed None 12 80.0 3 25.0 3 20.0 0 0.0 Hypothyroidism 2 13.3 7 58.3 9 60.0 4 50.0 Hyperthyoridism 1 6.7 2 16.7 3 20.0 4 50.0 Total 15 12 15 8 P 0.0021*

Table 5: Shows that the majority in the control group (90.9%) have no HBV/HCV infection, and no thyroid disease (76.7%). Table (5): Relation between incidence of hepatitis virus and thyroid diseases in the control group

Control “n=30”

None HCV HBV Mixed None 20 90.9 1 50.0 2 33.3 0 0.0 Hypothyroidism 2 9.1 0 0.0 3 50.0 0 0.0 Hyperthyoridism 0 0.0 1 50.7 1 16.7 0 0.0 Total 22 2 6 0 P 0.039*

Discussion In the present study, the prevalence of thyroid involvement in patients with HCC compared with control group was evaluated. Hypothyroidism was more frequent in patients with HCC than in control group (44%) versus (16.7%), (P= 0.0123). Our findings are supported by early results from the Mayo Clinic, which showed a prevalence of hypothyroidism among patients with HCC of 11.3 %.( 13). This relationship between hypothyroidism and HCC is not a surprising fact actually for two reasons. First, hypothyroidism is associated with the pathogenesis of NASH,(9) one of the most common chronic liver diseases characterized by liver inflammation and fibrosis and hence a predisposing condition for HCC development

(17,18). Second reason is the conjunction of thyroid disorders along with the major HCC risk factors. For example, hypothyroidism was previously reported to be more common in patients with HCV infection and more common in HCV patients with severe fibrosis. (7, 19) The association between thyroid cancer and HCV was previously reported in literature.(20) In addition, diabetic patients which is considered as another risk factor for HCC, tend to have a high prevalence of thyroid disorders.(21-

24) Endogenous glucose production and

utilization rates are enhanced by thyroid hormones and could be affected by thyroid hormone deficiency.(25) . Whether or why hypothyroidism causes HCC are not clear. However, the association between hypothyroidism and NASH could be explained by the underlying hyperlipidaemia, decreased fatty acid oxidation, insulin resistance and lipid peroxidation in patients with hypothyroidism. All of these conditions may enhance the susceptibility to chronic inflammation, DNA damage and HCC development. Moreover, concurrent thyroid dysfunction among patients with diabetes may exacerbate the coexisting diabetes-induced dyslipidaemia and may explain our observation of HCC risk modification among patients with hypothyroidism and diabetes. (9). Hypothyroidism-induced obesity may have promoted the development of steatohepatitis, fibrosis, and cirrhosis in these patients and increased their susceptibility to HCC development. Moreover, obesity and hyperinsulinemia may increase level of insulin-like growth factor 1, which in turn may reduce hepatic synthesis and blood concentration of sex hormone–binding globulin (SHBG), a glycoprotein produced in the liver with high binding affinity for testosterone and lower affinity for estradiol.(26,27) . There is sufficient

evidence that thyroid hormones have a positive effect on hepatic SHBG synthesis and that patients with hypothyroidism may experience a lower level of SHBG (28). Thus, a decreased level of SHBG may lead to increased plasma testosterone and estradiol, both of which may promote cellular proliferation and inhibit apoptosis. Elevated levels of serum testosterone and testosterone-to-estradiol ratio have been proposed to be predictive of HCC development in Japanese men with cirrhosis.(29) Nevertheless, the fact that the association between hypothyroidism and HCC continued to be significant after adjustment for prior history of obesity at different ages suggested that other mechanisms of hepatocarcinogenesis were involved, especially among women. Liver is a major target tissue for the proliferative effect of growth hormones, growth hormone receptors, and growth hormone binding protein, and many evidence suggesting that such hormones and hormone receptors are associated with hepatocellular carcinoma development.(30) In addition, an experimental study in rats with hypothyroidism,(31)showed that the levels of hepatic growth hormone receptors and growth hormone binding protein messenger RNA were increased in the female group and decreased in the male group as compared to euthyroid controls (P < 0.001); this may partially explain the susceptibility of females with hypothyroidism to develop HCC than males with hypothyroidism. In the present study, in order to ensure that the observed association between HCC and hypothyroidism was not related to the cancer diagnosis, patients with HCC with prior or concurrent head and neck cancer or cancer in other sites were excluded to minimize hypothyroidism misclassification due to cancer chemotherapy or radiotherapy. Interview data indicated no prior history of neither chemotherapy nor radiation exposure among HCC cases or control subjects. (32). One possible limitation in the present study was that thyroid diseases were self-reported by patients and controls during personal interviews. Therefore, patients with HCC might be more prone to recall previous diseases than are healthy controls. In the current study, questions of prior history of thyroid diseases along with other chronic medical conditions were part of a long list of questions. The questionnaire included year and age at diagnosis, duration, and treatment intake.

It is reasonable to assume that subjects who had received a definite diagnosis and had been treated could accurately report their prior history of medical conditions and recalled the condition duration. Control subjects were selected to represent the population from which the patients with HCC were ascertained. Only Egyptian patients and controls were included, and the geographic distribution of their residential areas was similar. Therefore, it is unlikely that our finding of the positive relationship between hypothyroidism and HCC was confounded by selection bias of HCC patients or controls. In conclusion, the prevalence of HCC is more common in patients with thyroid dysfunction than normal individual .Our results suggested that long-term hypothyroidism is associated with HCC, independent from other major HCC risk factors. Although hypothyroidism-associated weight gain (overweight or obesity) may partially explain the association between hypothyroidism and HCC, hypothyroidism independent from obesity can also contribute to HCC development. Screening and proper management of thyroid diseases in patients with diabetes or HCV or HBV infection may help prevent HCC. Further studies among different populations are warranted to confirm the association between hypothyroidism and HCC and to identify the underlying biological mechanisms and the genetic predisposition factors that may contribute to susceptibility to HCC development in the presence of thyroid disorders. The significant association of chronic hepatitis C with hypothyroidism and thyroid autoimmune phenomena implies that these patients should be screened for thyroid function on a periodic basis. References 1. Parkin DM, Bray F, Ferlay J, Pisani P. Global

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