Detection of anti-Hepatitis A and E IgG, IgM and Nucleic Acid among Chemical Bombardment Survivors in Kurdistan
A thesis Submitted to the Council of Faculty of Science and Science Education School of Science at the University of Sulaimani in partial fulfillment of the requirements for the degree of Master of Science in Biology (Microbiology)
By Shawnm Ahmed Aziz B.Sc. Biology (1995), University of Salahaddin H.D. Microbiology (2011), University of Sulaimani
Supervised by Dr. Salih Ahmed Hama Lecturer
May, 2015
Jozardan, 2715
Supervisor Certification I certify that the preparation of thesis titled “Detection of anti-Hepatitis A and E IgG, IgM and Nucleic Acid among Chemical Bombardment Survivors in Kurdistan” accomplished by (Shawnm Ahmed Aziz), was prepared under my supervision in the School of Science, Faculty of Science and Science Education, at the University of Sulaimani as partial fulfillment of the requirements for the degree of Master of Science in Biology (Microbiology).
Signature: Supervisor: Dr. Salih Ahmed Hama Title: Date:
Lecturer 20 / 2 / 2015
In view of the available recommendations, I forward this thesis for debate by the examining committee.
Signature: Name:
Dr. Huner Hiwa Arif
Head of the Biology Department Date:
20 / 2 / 2015
Examining Committee Certification
We
certify
that
we
have
read
this
thesis
entitled
“Detection of anti-Hepatitis A and E IgG, IgM and Nucleic Acid among Chemical Bombardment Survivors in Kurdistan” accomplished by (Shawnm Ahmed Aziz), and as examining Committee, we examined the student in its content and in connected with it, and in our opinion it meets the basic requirements toward the degree of Master of Science in Biology- Microbiology. Signature:
Signature:
Name:
Dr. Gaza F. Salih
Name:
Dr. Sahand K. Arif
Title:
Assistant Professor
Title:
Assistant Professor
Date:
25 / 5 / 2015
Date:
(Chairman)
Signature:
25 / 5 / 2015 (Member)
Signature:
Name:
Dr. Dlnya Asad Mohamad
Name:
Dr. Salih Ahmed Hama
Title:
Assistant Professor
Title:
Lecturer
Date:
25 / 5 / 2015
Date:
25 / 5 / 2015
(Member)
(Supervisor- Member)
Approved by the Dean of the Faculty of Science and Science Education. Signature: Name:
Dr. Bakhtiar Q. Aziz
Title:
Professor
Date:
/
/ 2015
(The Dean)
بسم اه ا رحمن ا رحيم ِ ِ ُه َو ا َ ض ِفي ِستَ ِة َر اْ و ات او م س ا ق ل خ ي ذ َ أ َ َ ََ َ أ َ َ أََي ٍ استََوى َعلَى ا أ َع أر ِ ش َي أعلَ ُم َما َيِل ُج ِفي م ث ام ُ َ أ أاْ أَر ِ ض َو َما َي أخ ُر ُج ِم أن َها َو َما َي ِ نز ُل ِم َن سماء وما يعرج ِ يها َو ُه َو َم َع ُ أم أ أَي َن َما ف ا َ َ َ َ َ أُ ُ َ ون ب ِ ُ نتُم َوا لَ ِ ير ٤ ص ل م ع ت ا م ب ه ُ َ ُ َ َ أ ٌ أ َ َ صدق اه ا عظيم
سورة الحديد اأية ٤
Dedication
This thesis is dedicated with profound gratitude to my parents and to all chemical bombardment victims in Kurdistan Region
Shawnm
---------------------------------------------------------------------------------Acknowledgements
ACKNOWLEDGEMENTS Thanks to ALLAH before and after 1.
First I would like to express my deepest gratitude and appreciation to my supervisor, Dr. Salih Ahmed, for his sincere advice, meticulous supervision, his assistance and suggestions that have proven crucial in the realization of this study.
2.
I would like to thank Kurdistan Regional Government, Ministry of Higher Education and Scientific Research, Ministry of Health and the Presidency of Sulaimani University for providing me the opportunity and support to accomplish this work. I also would like to offer my thanks to the School of Science and Biology Department for their help and facilities required for this research.
3.
My sincere thanks go to all the chemical survivors in Halabjah, Goptapa, Sewsenan, Shanakhse , Balisan and
Sheikh Wasan who
gratefully helped me in the collection of blood, I hope all will be in a better health . 4.
Many thanks are extended to the Rehabilitation Center for Victims of Chemical Attacks / Halabja and to the entire laboratory staff in Halabja General Hospital and to the Clinical Hospital of Halabja, especially Emad Fayroz and Intisar Ibrahim.
5.
My specially thanks are also to those who helped me on this project; especially Nyan Salih in Biology Department who provided me with training in molecular virology technical skills necessary for the project.
6.
Many thanks go to Maryam Salih in Shahid Hadi Laboratory Hospital and Paiman Ali in Hiwa Hospital also to Hajy Ari and Ismail Xalil in Internal Laboratory for all their help in the laboratory.
7.
Lastly, I would like to thank my family, my husband, Omer, and my daughters, Nawsha and Nyar, for their support and encouragement. Shawnm A. Aziz
Abstract
To determine the percentage rates of anti-hepatitis A and E (HAV and HEV) IgG and IgM seropositivity and RNA among chemical bombarded survivors in different villages and cities in Iraqi Kurdistan Region including Halabja, Goptapa, Sewsenan, Shanakhse, Sheikh Wasan and Balisan , 137 blood samples were collected randomly, 92 from chemical bombarded exposures and 45 from non-exposures - controls- from July 2013 to November 2013. Males and females were included. ELISA, conventional and nested PCR techniques were used to detect anti-hepatitis A and E IgG, IgM and RNA respectively. Some blood parameters ( Complete blood count ), liver enzymes ( ALT , AST and ALP) and total serum billirubin (TSB) also were measured in chemical weapons exposures and controls using auto analyzers. It was observed that the percentage rates of hepatitis A seropositivity was higher than hepatitis E. All exposures100% were seropositive for antihepatitis A IgG, whereas 61.79% was positive for anti-hepatitis E IgG. Similarly, anti-hepatitis A IgM seropositivity was higher than that of antihepatitis E 9.89% and 1.089% respectively. It was concluded that there were significant differences between exposures and controls regarding anti IgG for both HAV and HEV (p= 0.0001 and 0.0002), unlike anti-IgM for both viruses which demonstrated no significant differences (p= 0.621 and 0.56). Moreover, noticeable differences were observed among anti-hepatitis A and E IgG and IgM seropositivity among exposures (p= 0.000 and 0.0055) respectively. The percentage rate of hepatitis A RNA positivity was 15.68%, whereas no positive results were seen for HEV. Significant differences were seen among exposures and controls in regard to HAV RNA (p = 0.000). As well I
as the percentage rates of seropositivity for both HAV and HEV regarding anti-IgG, anti-IgM and PCR results were varied among different areas of the study. Morever, the geographical distribution of exposures had a significant effect on the results (p ‹ 0.05). It was noticed that only lymphocytes were significantly different between HAV seropositive and seronegative exposures from studied areas (p ‹ 0.05), whereas no significant differences were observed among all other obtained hematologic and liver function test results from HAV seropositive and seronegative exposures (p › 0.05). The effect of the geographical areas revealed to be significant on liver function test results (p=0.038). It was noticed that a relatively high percentage rate of exposures with HAV and HEV seropositivity as well as those who showed PCR positive results, were suffering from lymphopenia. The highest percentage of lymphocyte abnormalities was among exposures with antiHAV IgM seropositive results followed by anti-HAV IgG and then exposures with anti-HEV IgG seropositive results.
II
______________________________________________________________Contents
CONTENTS Abstract ………………………………………………………….….. Contents ……………….……………………………………………. List of Tables………………………………………………………… List of Figures …………………………………..…………………… List of abbreviations…………………………………………………...
I III V VI VII
Chapter One: Introduction
1.1
Introduction ……………………………………….…….…..
1
Chapter Two: Literature review 2.1 Hepatitis ……………………………………………….…… 2.2 Etiology of hepatitis ……………………………………..…… 2.2.1 Drugs and toxins …………………………………………...... 2.2.2 Metabolic causes ………………………………………..…… 2.2.3 Autoimmune …………………………………………...…… 2.2.4 Bacteria ……………………………………………….……. 2.2.5 Fungi ……………………………………………….….. ….. 2.2.6 Viruses ……………………………………………….…….. 2.2.6.1 Viral Hepatitis produced due to non hepatotropic viruses………….. 2.2.6.2 Hepatitis B Virus (HBV) …………………………………..…. 2.2.6.3 Hepatitis C Virus (HCV) ………………………………….…. 2.2.6.4 Hepatitis D Virus (HDV) …………………………………….. 2.2.6.5 Hepatitis A Virus (HAV) ……………………………………. 2.2.6.5.1 Morphology and physiochemical properties of HAV……………. 2.2.6.5.2 Genome and proteins of Hepatitis A virus…………………..….. 2.2.6.5.3 HAV life cycle……………………………………….….….. 2.2.6.5.4 HAV replication cycle …………………………………...….. 2.2.6.5.5 Epidemiology of hepatitis A virus …………………………..… 2.2.6.5.6 Antigenicity and Serotype……………………………………. 2.2.6.5.7 HAV Genetic diversity…………………………………...….. 2.2.6.5.8 HAV detection……………………………………………… 2.2.6.5.9 HAV Transmission …………………………………..…….. 2.2.6.5.10 Immune response to hepatitis A ……………………………... 2.2.6.5.11 Clinical features of hepatitis A ……………………………… 2.2.6.5.12 HAV Prevention ……………………………………….….. 2.2.6.6 Hepatitis E Virus ……………………………………….…... 2.2.6.6.1 HEV Viral particle structure ………………………….……… 2.2.6.6.2 Classification and genotypes ………………………………… 2.2.6.6.3 Characteristics and genome organization of HEV ……………… 2.2.6.6.4 Genome replication and life cycle ………………………….… 2.2.6.6.5 HEV Transmission ……………………………………….… III
4 5 5 5 6 6 7 7 7 8 8 9 9 10 12 13 14 15 20 20 21 22 23 24 26 28 29 29 30 31 32
______________________________________________________________Contents 2.2.6.6.6 Epidemiology of hepatitis E Virus ……………………………. 2.2.6.6.7 Clinical symptoms and course of infection ………………….…. 2.2.6.6.8 Immunity response to HEV ………………………………….. 2.2.6.6.9 Diagnosis of HEV infection ……………………………….…. 2.2.6.6.10 Prevention and control …………………………………….… 2.2.6.6.11 Vaccination …………………………………………………
33 34 35 36 36 37
Chapter Three: Materials and Methods 3.1 3.1.1 3.1.2 3.1.3 3.1.4 3.1.5 3.1.6 3.1.7 3.2 3.2.1 3.2.2 3.2.3 3.2.3.1 3.2.3.2 3.2.3.3 3.2.3.4 3.2.3.4.1 3.2.3.4.2
4.1
Materials …………………………………………………… Materials and instruments ……………………………………. Chemicals and biological materials ……………….…………... Enzyme immune assay reagents and solutions ………………….. Nucleic acid extraction kit, buffers and dyes ………………….... HAV 430 kit ……………………..……………………….… Agarose gel ………………………………………………… Primers used for HEV detection ………………………………. Methods ……………………………………………………. Samples ……………………………………………………. Detection of anti-HAV and HEV IgG and IgM by ELISA………... Detection of HAV and HEV RNA by PCR …………………….. Viral nucleic acid extraction ………………………………….. Reverse transcription of HAV extracted RNA ………………..... Amplification of HAV cDNA by PCR …………………..…….. Molecular detection of HEV by nested RT- PCR ……………….. Reverse transcription of HEV extracted RNA…………………... Preparation of agarose gel and Electrophoresis…………………..
38 38 39 40 45 45 46 46 47 47 48 54 55 56 57 58 59 60
Chapter Four: Results Results…………………………………………………..…
61
Chapter Five: Discussions 5.1 5. 2 5. 3
ELISA results (anti- Hepatitis A and E IgG and IgM) ………....... Molecular detection of HAV and HEV ……………………...… Hematological and liver function tests results ………………..…
75 79 82
Chapter Six: Conclusions and Recommendations 6. 1 6. 2
Conclusions ……………………………………………….. Recommendations…………………………………………..
85 86
References ………………………………………………….
87
IV
_________________________________________________________List of Tables
List of Tables Table No. 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9
Table Title
Page No.
Percentage of HAV and HEV nucleic acid detection among exposures and controls……………………………………………………
64
Anti-IgG, IgM and nucleic acid detection for HAV and HEV for all tested exposures in different chemical bombed areas in Kurdistan……
66
Hematological and liver function tests of HAV seropositive exposures in different areas……………………………………………….
68
Hematological and liver function tests of HAV seronegative exposures in different areas ……………………………………………….
69
Hematological and liver function tests of HEV seropositive exposures in different areas ………………………………………………….
70
Hematological and liver function tests of HEV seronegative exposures in different areas ……………………………………………….
71
Hematological and liver function tests of HAV-PCR positive exposures in different areas ……………………………………………….
72
Hematological and liver function tests of HAV-PCR negative exposures in different areas ………………………………………………..
72
Hematological and liver function tests of HEV-PCR negative exposures in different areas ………………………….……………………..
73
V
_________________________________________________________List of figures
List of Figures Figure No.
Figure Title
Page No
2.1
Electron micrograph of HAV…………………….……
11
2.2
Hepatitis A virus genomic organization and expression…..
13
2.3
Possible "enterohepatic" cycling of HAV………….……
13
2.4
The HAV replication cycle……………………………
15
2.5
HAV global distribution………………………………
17
2.6
Age-specific prevalence of antibodies to HAV……….…
17
2.7
Distribution of HAV in EMRO Countries in adult group…
19
2.8
Natural history of hepatitis A……………………….…
26
2.9
Structural domains of the HEV capsid protein…………..
29
2.10
Genome organization and proteins of HEV…………..…
31
2.11
Proposed replication cycle of HEV………………….…
32
2.12
Global prevalence of HEV……………………………
34
4.1
Percentage rates of Anti-IgG, anti-IgM and PCR for both HAV and HEV ……………………..……………….
62
Agarose gel electrophoresis showing the RT- PCR amplified products genes of HAV……………………..
62
4.3
Agarose gel electrophoresis of first round nested RT-PCR..
63
4.4
Agarose gel electrophoresis of second round nested RTPCR ……………………………………………….
63
Seropositivity and RNA detection for HAV among different groups of chemical exposures and controls……
65
Seropositivity and RNA detection for HEV among different groups of chemical exposures and controls……
66
Interactions of seropositivity and PCR results for HAV and HEV among exposures………….……………………
68
Lymphocyte abnormalities among HAV and HEV PCR and seropositive exposures …………………..……….
74
Liver function test result abnormalities among HAV, HEV PCR and seropositive exposures ………………………
74
4.2
4.5 4.6 4.7 4.8 4.9
VI
_________
Abbreviations
List of abbreviations Ag : AIDS : ALF : ALP : ALT : AST : bDNA : BLK : CD : cDNA : CMV : ddH2O : DEPC-D.W: EBV : EIA : EDTA : ELISA : EMRO : ET-NANBH FHF : FHF-A : HAV : HBV : HBcAg : HBsAg : HCC : HCV : HD-Ag : HEV : HGV : HLA : HRP : HSV : ICTV : ICU : IEM : IFN : IgG : IgM : MAbs : m RNA : NANBH : NC : NCR : NK : ORFs : PAGE :
Antigen Acquired immune deficiency syndrome Acute liver failure Alkaline phosphatase Alanine aminotransferase Aspartate aminotransferase Branched deoxyribonucleic acid Blank Cluster of differentiation Complementary DNA Cytomegalovirus deionized distilled water Diethyl pyro carbonate deionized water Epstein-Barr virus Enzyme immunoassay Ethylene di amine tetra acetic acid Enzyme-linked immunosorbent assay East Mediterranean Region Organization Enterically transmitted non-A, non-B hepatitis fulminant hepatic failure Fulminant hepatic failure associated with hepatitis A Hepatitis A virus Hepatitis B virus Hepatitis B core antigen Hepatitis B surface antigen Hepatocellular carcinoma Hepatitis C virus Hepatitis D antigen Hepatitis E virus Hepatitis G virus Human leukocyte antigen Horseradish peroxidase Herpes simplex virus International committee on taxonomy of viruses Intensive care unit Immune electron microscopy Interferon Immunoglobulin G Immunoglobulin M monoclonal antibodies messenger RNA Non-A, non-B hepatitis Negative control Non-coding region Natural killer cells Open reading frames Polyacrylamide gel electrophoresis
VII
_________ PBS : PC : PCR : PT : RC qRT-PCR : RdRp : RFLP : RIA : RIBA : RT-PCR : SDS : SGOT : SGPT : TNF : TSB : UTR : VLP :
Abbreviations phosphate buffered saline Positive control Polymerase chain reaction Prothrombin time Replication complex Quantitative real time- polymerase chain reaction RNA-dependent RNA-polymerase Restriction fragment length polymorphism Radio- immuno-assay Recombinant immunoblot- assay Reverse transcriptase- polymerase chain reaction Sodium dodecyl sulfate Serum glutamic pyruvic transaminase Serum glutamic oxaloacetic transaminase Tumor necrosis factor Total serum bilirubin Un-translated region Virus-like particle
Units Da : µl : Hr : nm : °C : ml : Gm : KD : Min : M: U : Mm : V: µg : Ng : w/v : MW : Yrs : Nt : Bp :
Dalton Microliter Hour Nanomole Degree Celsius Milliliter Gram Kilodalton Minute Molar Enzyme Unit Activity Millimolar Volt Microgram Nanogram Weight/Volume Molecular weight Years Nucleotide(s) Base pair
VIII
Chapter One
Introduction
Chapter One
Introduction
Introduction Kurdistan Region of Iraq was exposed to the chemical bombardment in the 1980s during the Iran-Iraq war and in 1988, in particular, several cities and villages were the target of the bombardment (Dickman, 1988). As a result different health complaints were found among survivors who were exposed to chemical gases, included respiratory, eye, dermatological, immunologic complaints (Hama et al., 2008). Extensive exposure to chemical weapons such as masterd gas, nerve gas and cyanide causes high mortality , morbidity, injuries, and chronic side effects on vital organs, especially the respiratory tract. Chemical weapons were heavily used by Iraq against the Iraqi kurds in Sheikh Wasan and Balisan valley, during April 1987 and in Halabjah on 16 th march 1988. Reports suggest that as many as 2.9% of the Kurdish population have been exposed to chemical weapons at some level ( Dizaye, 2012) . Immunologic defects among survivors of the chemical attack make the possibility of infections higher than the normal, including opportunistic and viral infections, as it is known that defects in cell-mediated immunity increase the risk of viral infections. Among viral infections are those which can be transmitted easily through common routes like fecal-oral route, both share the same routes and mechanisms of spread, and they can cause similarly severe epidemics (Traore´et al., 2012). The two most common infectious viral hepatitis are hepatitis A and E. Hepatitis A virus (HAV) belongs to the genus Hepatovirus and is a member of the Picornaviridae family (Roque-Afonso, 2006). It is non-enveloped, positive single-stranded RNA, non-segmented. While the hepatitis E virus (HEV) belongs to the genus Hepevirus, a member of the family Hepeviridae (Menget al., 2011). It is the etiological agent of an acute self-limiting hepatitis
-1-
Chapter One
Introduction
and is a major cause of enterically transmitted hepatitis worldwide (Khuroo, 2011). In humans, the symptoms are hepatomegaly, jaundice, fever, anorexia, nausea and abdominal pain. Generally, HAV is an asymptomatic disease in children, but it can cause severe symptomatology in young people and mainly in older adults, and it may lead to fulminant hepatitis at a rate of 1.8% in individuals over 50 years old. Among immunocompromised patient and pregnant women, the mortality rates are relatively higher (Ferreira et al., 2008). Although there are seven viral genotypes (I–VII) and viral strains of the same genotype share greater than 85% nucleotide identity ( Costa-Mattioli et al., 2003), most infections of HAV strains belong to a single serotype. It is believed that most of the waterborne outbreaks of hepatitis in endemic regions were caused by HEV (Khudyakov and Kamili, 2011). The World Health Organization (WHO) estimated that one-third of the world’s population have been infected with HEV. The primary transmission routes of HAV and HEV are fecal-oral, mostly by contaminated drinking water; low sanitary standards contribute to the frequent spread of Hepatitis A and E (Ashbolt, 2004). Unfortunately, some of these risk factors can be present in chemically bombarded areas, which may contribute to a high rate of infection by these viruses. Moreover, defects in the immune system of most of the survivors may be another risk factor, which contributes to the high percentage of infections by these viruses in comparison with normal conditions. The risk groups for HAV and HEV infection include those with liver disease, the elderly and the immunocompromised individuals (Kamar et al., 2012). The diagnosis of hepatitis is made by the serology investigation through enzyme immunoassay (ELISA) technique as well as biochemical assessment -2-
Chapter One
Introduction
of liver function tests, including urine bilirubin, total and direct serum bilirubin, alanine aminotransferase (ALT) and /or aspartate aminotransferase (AST), alkaline phosphatase, total protein, serum albumin,etc. Nowadays, reverse transcription-polymerase chain reaction (RT-PCR) assays represent the most commonly used molecular investigation for HEV genome detection. The usage of RT-PCR as a diagnostic tool has become feasible
since
the
development
of
the
real-time
PCR
platforms.
Real-time PCR is a closed system that minimizes the risk of contamination by the amplified target (Pelosi and Clarke, 2008).
This study aims to: -
determine the servo-positivity of both hepatitis A and E viral infections among survivors of the chemical attack in different cities and villages in the Iraqi Kurdistan region using ELISA technique.
-
study the presence or absence of hepatitis A and E nucleic acid (RNA) among survivors depending on PCR technique.
-
study changes in liver function test and some hematologic parameters of survivors.
-3-
Chapter Two
Literature Review
Chapter Two
Literature review
2.1 Hepatitis Hepatitis is a clinical and pathological condition that results from the damage of liver cells, either by viral, bacterial, pharmacological or immunemediated damage (Bass et al., 1990). Clinically, the liver may be enlarged and become tender with or without jaundice, and laboratory evidence of hepatocellular also may be found which can be represented in the form of elevated transaminase levels. The clinical course of hepatitis may range from mild or inapparent to a dramatic illness with severe hepatocellular dysfunction, marked jaundice, impairment of coagulation and disturbance of neurological function (Alter and Mast, 1994). Hepatitis is further divided into acute and chronic types on the basis of clinical and pathological criteria. Acute hepatitis implies a condition lasting less than six months, culminating in complete resolution of the liver damage with return to normal liver function and structure, or resulting in rapid progression of the acute injury toward extensive necrosis and a fatal outcome (Dusheiko, 1992),while the latter is defined as a sustained inflammatory process in the liver lasting more than six months (Sherlock and Dooley, 1997). The term viral hepatitis generally refers to inflammatory disease of the liver resulting from one of the hepatotropic viruses, which includes A, B, C, D, and E (Rehermann, 1996 and O'Connor, 2000), or may be resulted from other non-hepatotropic viruses, as acute hepatitis was believed to be caused by a variety of bacterial and parasitic organisms, and was described as catarrhal jaundice caused by occlusion of the bile by a mucus plug (Chironna et al.,2003). There are different ways of grouping or classifying the hepatitis viruses, among these is by their routes of transmission, fecal-oral route,which is the characteristic for the hepatitis A and E virus, whereas parenterally transmitted viruses include hepatitis B, C, and D viruses (Rehermann, 1996 and Lammert et al, 2000). -4-
Chapter Two
Literature review
Acute hepatitis due to these viruses may be asymptomatic, symptomatic but icteric, or it may take the form of classical icteric hepatitis, and rarely become very severe with a prolonged or fulminant course which has high mortality rates (McIntyre, 1990 and Ryder et al., 2001).
2.2 Etiology of hepatitis 2.2.1 Drugs and toxins The liver is the main site of drug metabolism, and thus it is particularly susceptible to structural and functional damage after xposure to chemicals, drugs or toxins. Hepatotoxic agents may damage the hepatocytes directly through the alteration of membrane lipids or through denaturation of proteins or indirectly through injury, which may occur by interference with metabolic pathways essential for cell integrity, or through distortion of cellular constituents by covalent binding of a reactive metabolite. The clinical spectrum of these damages might vary from asymptomatic liver biochemical abnormality to fulminant hepatic failure (Mannucci et al., 1994).
2.2.2 Metabolic causes These causes are common in infants and children, and many metabolic causes of hepatitis were known, including galactosemia, which is a rare autosomal recessive disorder that might be presented by hypoglycemia, jaundice, hepatomegaly and cataract (Balistreir, 1992, Hyams et al., 1992), alpha-1-antitrypsin deficiency, which is an autosomal recessive disorder with low incidence accompanied by cholestasis, failure to thrive, hepatomegaly or coagulopathy (Mannucci et al., 1994). Moreover, other metabolic cases are included such as tyrosinemia type 1, anunusual autosomal recessive disorder that may present with acute liver failure or with chronic liver disease (Mannucci et al., 1994), and chromosomal disorders such as trisomy 18 and
-5-
Chapter Two
Literature review
12, which were found to be presented with neonatal hepatitis (Kelly, 1994 and Mannucci et al., 1994). 2.2.3 Autoimmune Autoimmune hepatitis is a syndrome of unknown cause. A loss of tolerance to autologous liver tissue is regarded as the principal mechanism. These problems usually run a chronic progressive course, one-third of patients, however, may present with an episode of acute hepatitis (Desmet et al., 1994). It is diagnosed after ruling out viral, metabolic or drug-induced etiologies (Raghuveera et al., 1996), rather than the absence of circulating auto antibodies (Manns and Obermyer, 1997). 2.2.4 Bacteria Many bacteria can infect hepatocytes and cause acute or chronic hepatitis that needs to be differentiated from other causes of hepatitis.The liver may be infected by Mycobacterium tuberculosis, which usually produces a chronic form of hepatitis and rarely causes fulminant liver failure (Hussain et al., 1995). Mycobacteria other than tuberculosis can produce a granulomatous hepatitis, particularly as part of acquired immune deficiency syndrome AIDS (Patel, 1981). The spirochetes can affect liver cells as a part of congenital, secondary or tertiary syphilis (Schlossberg, 1987), also Borrelia recurrentis invades liver cells and causes focal necrosis (Felsefeld and Wolf, 1969). In addition, Borrelia burgdorferi is known to cause hepatitis (Rahn and Malawista, 1991). Moreover, Coxiella burntii occasionally causes hepatitis, which clinically may mimic anicteric viral hepatitis (Tissot-Dupont et al., 1992).
-6-
Chapter Two
Literature review
2.2.5 Fungi Fungal infections of the liver can be caused by different fungi, including Cryptococcus
neoformans,
Candida
albicans.
Histoplasmosis
and
coccidiodomycosis which are usually part of disseminated diseases and usually infect immunocompromised individuals, acute leukemia patients and those having malignant diseases (Maxwell and Mamtora, 1988), in addition to infections that occur following liver transplantation (Thaler et al., 1988).
2.2.6 Viruses 2.2.6.1 Viral Hepatitis produced due to non hepatotropic viruses and virus-like agents: Many viruses could infect hepatocytes, and, sometimes, can cause hepatitis rather than hepatotropic viruses including Herpes simplex viruses (HSV), Cytomegalovirus (CMV), Epstein-Barr virus (EBV), Yellow fever virus, Coxsackie A and B virus, Mumps, Rubella and Influenza virus, although the infection differs from those included in the term of viral hepatitis, in the fact that they do not primarily cause liver damage but nevertheless, occasionally display increased hepatotropism resulting sometimes in jaundice (Green et al., 1989). Epstein-Barr virus was reported to cause hepatitis, particularly in children (Duffy et al., 1986). Both HSV and CMV also can infect hepatocytes as part of disseminated disease (Fishman et al., 1996), whereas Rubella and entero viruses were isolated from children with acute hepatitis (Brunel, 1992).
Although many viruses could infect hepatocytes, only some of them are termed hepatitis viruses or hepatotropic viruses, six different hepatitis viruses designated by the letters A, B, C, D, E and G have been identified,which can cause acute or chronic hepatitis in human, although each of them belongs to a distinct viral group, but they share the features of hepatotropism (Jameel et al.,1994). -7-
Chapter Two
Literature review
2.2.6.2 Hepatitis B Virus (HBV): Hepatitis B virus is a member of hepadnaviridae family, and the complete veroin (Dane particle) is a 42 nm, spherical particle consisting of HBsAg (the outer lipoprotein surface envelope), HBcAg (the internal core, which surrounds the viral DNA genome) and HBeAg (a subunit of HBcAg). The transmission of HBV occurs via parenteral routes through exposure to contaminated blood or blood products, primarily by transfusion of infected blood or sharing of infected needles during drug abuse (Balayan et al., 1983). Sexual transmission is also a major factor in the spread of hepatitis B and from 60 to 80% of babies born to mothers infected with hepatitis B virus become carriers who can spread the virus for the rest of their lives. It can become chronic in a certain number of cases and can lead to chronic active hepatitis, cirrhosis and hepatocellular carcinoma (Hyams et al., 1992). Chronic HBV infection is inversely proportional to age of acquisition, and persistent infection developed in 90% of neonates compared with 20% to 50% of young children and 5% of adults, although the majority of pediatric HBV infections were found to be asymptomatic, most of the pediatric cases became chronic (Wong et al., 2000).
2.2.6.3 Hepatitis C Virus (HCV): It is a single stranded RNA virus, its infection predominantly spreads between individuals by the parenteral routes through exposure to contaminated blood or one of its products, an accidental needle stick, illicit self infection, hospital exposure or tattooing.The majority of cases becomes chronic; at least 50% of all HCV infections and 70% of post transfusion HCV infections persist of more than 6 months (Arora et al., 1996).
-8-
Chapter Two
Literature review
HCV infection may progress to cirrhosis and/ or Hepatocellular carcinoma. It was noticed in certain areas of the world that Hepatoceluller carcinoma was associated more frequently with HCV than with HBV (Arora et al., 1996).
2.2.6.4 Hepatitis D Virus (HDV): It is a circular single standard RNA virus,which encodes a single protein called delta antigen, and is associated with the viral genome to form an amorphous structure (Arora et al., 1996). HDV infection may be acquired by simultaneous HBV coinfection or by super-infection of chronic HBV carriers. Its coinfection with HBV was reported to cause more severe acute disease and carry a higher risk of fulminant hepatitis than HBV infection alone; furthermore, the HDV super-infection was found to become chronic in 90% of cases (Behrens and Doherty, 1993).
2.2.6.5 Hepatitis A Virus (HAV) : Hepatitis A virus is an enveloped virus, with about 27 nm indiameter that belongs to the Picornaviridae family. It contains a single-stranded positive RNA genome with 7500 nucleotides in length, which codes for four structural capsid proteins as well as contains a viral polymerase and proteases (Saffar et al., 2009).The incubation period of HAV is around 28 days, and its transmission is predominantly fecal oral. Parenteral transmission could occur because of transient viremia in the prodromal stage. In cases of post transfusion, hepatitis A was reported in neonatal nurseries and in hemophilias (Shamsizadeh et al., 2009). It does not cause chronic infections and no chronic viral carriers exit, although infection with HAV occurs sporadically or in epidemics (Okuda and Hai, 2001). Hepatitis A virus occurs worldwide and humans are thought to be its principal host (Cuthbert, 2001). Transmission occurs via fecal-oral route by either person-to-person contact or consumption of contaminated food or -9-
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water. The incidence of infection is highly related to the prevailing level of hygiene and sanitation, and the disease is most endemic in the less-developed parts of the world, where poor socioeconomic conditions facilitate transmission of the virus, while in the developed world and in some developing countries, the seroprevalence of HAV infection has declined, presumably because of improvements in hygiene associated with rising socioeconomic conditions (Jacobsen and Koopman, 2004). However, the introduction of effective hepatitis A vaccine in the US, Israel, selected regions of Italy, Spain and Australia in the mid-1990s provided the first specific tool for preventing HAV infection (Wasley et al., 2006). More than 70% of cases of HAV infection occurring in children less than 6 years old are asymptomatic, or if illness occurs, it is not accompanied by jaundice (Hadler et al., 1980), however, in 70% of older children and adults, HAV infection causes more-severe clinical illness, including jaundice malaise, fever and dark urine (Lednar et al., 1985). 2.2.6.5.1 Morphology and physiochemical properties of Hepatitis A virus Hepatitis A virus was formerly classified as Enterovirus (serotype 72) within the Picornaviridae family. However, due to its unique structural composition, stability, tissue tropism and genetic distance from members of other picornaviruses genera, it is now classified as a separate genus, Hepatovirus (Rueckert and Wimmer, 1984; Melnick, 1992). Hepatitis A virus was first identified in 1973 by electron microscope and is one of the smallest and structurally simplest RNA animal viruses (Fig. 2.1). The viral particle is non-lipid enveloped; therefore, it is resistant to ether, chloroform and alcohol. Morphologically HAV is an isometric particle with a diameter of 27-32 nm and composed entirely of 70% viral protein and 30% ribonucleic acid (Lemon, 1994; Stapleton and Lemon, 1994; Koff, 1998).
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The buoyant density of the full viral particles is 1.32-1.34g/cm3 in CsCl and a sedimentation coefficient of 156-160 S in neutral sucrose solutions. During early infection, empty capsids collected in feces, band at 1.20 and 1.29-1.31g/cm3, with a sedimentation coefficient ranging from 50 S to 90 S, predominantly 70 S (Koff, 1998). HAV, unlike other members of the Picornaviridae family, is stable at (pH 1), resistant to heat (56°C for 30 minutes) (Hollinger et al., 1996), can remain infectious in the environment for weeks, but the virus is inactivated by heating to more than 85°C for at least one minute or by exposure to bleach (Wasley et al., 2006). It is acid-stable and able to retain infectivity below pH 3, can remain infectious after refrigeration and freezing; besides, the chlorine is partially effective in removing the virus; however, it is readily inactivated by ionizing radiation, phenol and formaldehyde (Siegl et al., 1984).
Figure-2.1-Electron micrograph of HAV (Hepatitis A-E slide set) From: Centers for Disease Control and Prevention (CDC, 2000), Atlanta, USA.
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2.2.6.5.2 Genome and proteins of Hepatitis A virus The genome of the virion consists of positive-stranded RNA, which is approximately 7.5 kb in length, there are non-translated regions (NTR) at the both ends of the viral genome. The highly conserved 5’end NTR extending over 10% of the total genome and is covalently attached to the viral protein, VPg (2.5kD) (Brown et al., 1991; Melnick, 1992). The 3’end terminates with a poly (A) tail of 40-80 nucleotides, a single open reading frame (ORF) encoding a single poly protein of 2227 aminoacids, is divided into three functional regions termed P1, P2 and P3. P1 encodes for the polypeptides, which are post-translationally processed into the capsid, whereas P2 and P3 encode the non-structural polypeptides, which are associated with replication (Fig. 2.2) (Totsuka and Moritsugu, 1999). HAV differs from other Picornaviridae by having a smaller VP4, which has not yet been isolated in mature virus particles (Probst et al. 1999). Both the virion capsid proteins VP1, VP4 and non-structural proteins are generated from the poly protein by a series of viral protease (3Cpro) cleavage (Toyoda et al., 1986; Sommergruber et al., 1989). There are naturally-occurring strains that infect non human primates (three genotypes) as well as four genotypes that comprise the human-infectious viruses (Lemon et al., 1992).
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Figure-2.2- Hepatitis A virus genomic organization and expression (Pintó et al., 2012)
2.2.6.5.3 HAV life cycle: After oral intake of viral particles, the viral antigens can be detected in the stomach, small intestine, and large intestine not only after the initial oral inoculation, but also later throughout
the disease, which suggests viral
replication in the intestine (Asher et al., 1995).
Figure-2.3- Possible "enterohepatic" cycling of HAV (Cuthbert, 2001) - 13 -
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The virion presumably reaches the liver in the portal blood (or after systemic circulation) and is taken up by hepatocytes. Once HAV has replicated in the liver and been released into the bile, the enterohepatic cycle of gastrointestinal uptake and transfer to the liver could continue until neutralizing or other antibodies interrupted the cycle (Cuthbert, 2001) (Fig.2-3). 2.2.6.5.4 HAV replication cycle: In the first step of the viral life cycle (Fig. 2.4), HAV binds to its cellsurface receptor called HAVcr (kaplan et al.,1996), which is a mucin like integral membrane glycoprotein whose exact role in viral entry is not clear. This protein is not selectively expressed in the liver and hence liver tropism does not appear to be defined by the cell receptor. Interestingly, an alternate binding and entry mechanism mediated by IgA molecules has been proposed for HAV (Dotzauer et al.,2000). Upon binding and probably receptor-mediated endocytosis, it is assumed that the HAV particle structure is modified in such a way that the RNA genome is uncoated and released into the cytoplasm (Bishop and Anderson, 2000). The incoming viral RNA genome initially serves as the template of translation. The primary translation product, the poly protein, is cleaved into structural proteins and non-structural proteins that presumably assemble into an RC together with host proteins. Viral RNA synthesis proceeds through a negative strand intermediate. The subsequently formed positive RNA strands are either used as a translation or a replication template or packaged by the structural proteins to form infectious viral capsids (Gauss-Müller et al.,2002). HAV replicates persistently without inducing gross changes in cellular functions and morphology (Gauss-Müller et al.,1984; Racaniello, 2001). The cell exit strategy appears to be tightly
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connected with capsid maturation and the removal of the assembly signal by a host proteinase (Rachow et al., 2003).
Figure-2.4-The HAV replication cycle HAV binds to the cellular receptor (1).The binding-induced conformational change of the viral particle allows RNA genome uncoating and release into the cytoplasm (2). The messenger-sense (+) RNA associates with ribosomes and translation is initiated. A polyprotein is generated that is proteolytically cleaved by the viral proteinase 3C into the structural proteins (SP) that assemble to form the viral capsid (C, 5). The non-structural proteins associate with host proteins to form the replication complex (RC) that catalyzes viral genome replication. Viral proteins 2B and 2C induce the formation of membrane vesicles that are the site of viral RNA synthesis. The (+) strand RNA is copied through a (-) strand intermediate (dashed line, 4a) into multiple new (+) strands (solid line,4b). Some (+) strand RNA molecules are again translated, others are encapsidated by structural proteins to initially form immature particles (hatched hexagon, 6). Before or concomitant with maturation the virion exits the cell (7). (Gauss-Müller,2007).
2.2.6.5.5 Epidemiology of hepatitis A virus Approximately 1.5 million clinical cases of hepatitis A occur worldwide annually but the rate of infection is probably as much as ten times higher. The incidence rate is strongly related to socioeconomic indicators and access to safe drinking water (WHO, 2010). Hepatitis A virus (HAV) has emerged as an important public health problem in many countries from the Middle East region, and Iraq is no exception (Turkey, 2011).
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Tens of millions of
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individuals worldwide are estimated to become
infected with hepatitis A virus (HAV) each year (Wasley et al., 2006). HAV infection occurs with distinct patterns of geographic distribution and transmission (Fig.2.5) (Jacobsen KH and Wiersma ST. 2010). Socioeconomic conditions, standards of hygiene and sanitation, household crowding, and access to clean drinking water are factors strongly associated with the incidence of acute hepatitis A disease and endemicity (Murphy et al.,2012). In highly endemic areas (i.e., parts of Africa and Asia), almost all infections occur in children, and this results in high rates of population immunity and a low burden of disease. In areas with intermediate endemicity (i.e., Central and South America, Eastern Europe, and parts of Asia), childhood transmission is less frequent; more adolescents and adults are susceptible to infection, and outbreaks are common. In areas with low and very low endemicity, most disease occurs among adolescents and adults in defined high-risk groups (e.g., injection drug users and international travelers), during community or cyclic outbreaks facilitated by transmission among children, or through exposure to contaminated food (Wasley et al,. 2006; Murphy TV et al., 2012 and Franco E et al., 2012).
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Figure-2.5-HAV global distribution (Jacobsen and Wiersma, 2010).
Figure-2.6- Age-specific prevalence of anti-hepatitis A antibodies. Diamonds, high endemicity; X's, intermediate endemicity; circles, low endemicity; squares, very low endemicity (Bell, 2002). - 17 -
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Adults in these areas are at a high risk of infection and disease, especially who are susceptible; but overall disease rates are generally low and outbreaks rare due to the past acquisition of immunity in the population (Wasley et al., 2006). Great differences exist among the countries and also among various parts of the same country in some regions due to their sanitary and socioeconomic levels. Residents of the Nile delta were shown 100% seropositive to anti-HAV in 1996. (Darwish et al.,,1996). Moreover, in another study on Egyptian blood donors in 1986, it was noticed that 91-94.5% were anti-HAV seropositive (Ramia,1986). Other researchers found that 99% were seropositive with anti-HAV in Kabul district visiting the French military field hospital (Carmoi et al.,2009). In 2010, a study on Iranian general population showed positive results for anti-HAV among 86% of the studied population aging 18-65 years (Merat et al., 2010). Also other investigators in Tehran showed that 97.63% of army draftees in Tehranwere anti-HAV seropositive (Ghorbani et al., 2007). Another study in 2008 from Isfahan province showed that 8.33% of the population had positive results for anti-HAV (Ataei et al., 2008), although in a previous study, it was reported that 30.9% of the studied population in Mazandaran province were positive for anti-HAV (Nassrolahei et al.,2004). In a study done in Riyadh in 2006, 67% were seropositive (Almuneef et al.,2006), whereas 86% of seropositivity was shown by others among a number of healthy populations of Eastern Saudi (Fathalla SE et al., 2000 (Fig.2.7). Moreover, in previous studies, it was reported that 76.3% and 61.3% of the studied population were shown to be anti-HAV seropositive in Giza and Riyadh respectively (Arif,1996), others found 96-95.1% positive results among males and females (el-Hazmi,1989). In a study done in 2005 from Lebanon, it was shown that 78% of the population older than 21 years had positive findings for anti-HAV IgG (Sacy et al., 2005), while a seropositivity rate of 97.7% was reported in previous studies among adults in - 18 -
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1982 (Shamma'a et al.,1982). 89% of Syrians showed to be anti-HAV seropositive (Antaki and Kebbewar, 2000), while only 28.8% were shown to have positive results among studied groups in Kuwait (Alkhalidi et al.,2009). A study done in Iraq (Ataallah M. Turky, 2011) showed that the prevalence of anti HAV IgG is 96.4% and of anti HEV IgG is 20.3%. In areas of moderate endemicity, HAV is not transmitted as readily because of better sanitary and living conditions, and the average age of infection is higher in these areas than in areas of high endemicity (Cianciara, 2000).The prevalence of anti-HAV increases gradually with age, primarily reflecting the declining incidence, changing endemicity, and resultant lower childhood infection rates over time. Some regions have very low endemicity, with most cases occurring in defined risk groups, such as travelers returning from endemic areas and injection drug users (Bottiger et al., 1997).
Figure-2.7- Distribution of HAV in EMRO countries, among adult groups (Mahboobi N et al., 2014).
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2.2.6.5.6 Antigenicity and Serotype Only a single serotype of HAV exists, despite genetic heterogeneity at the nucleotide level. Individuals infected with HAV in one area of the world are protected from reinfection by HAV from other parts of the world. The vaccine prepared from virus isolates originating in Australia or Costa Rica can cause protection from infection worldwide (Innis et al., 1994).The antigenic structure of the virus is relatively simple, with a restricted number of overlapping epitopes combining to forma single dominant antigenic site that interacts with virus-neutralizing antibodies, and these epitopes are highly conformational and are formed by amino acid residues located on more than one capsid protein (Ping and Lemon,1992).
2.2.6.5.7 HAV Genetic Diversity RNA viruses exploit all known mechanisms of genetic variation to ensure their survival (Domingo and Holland, 1997), including mutation and genetic recombination.Their high rates of mutation and replication allow them to move through sequence space at a rate that often makes their DNA-based host’s evolution look glacial by comparison (Worobey et al., 1999). HAV displays a high degree of antigenic and genetic conservation throughout the genome (Lemon et al., 1992; Sanchez et al., 2003). However, enough genetic diversity exists to define several HAV genotypes and sub-genotypes (Robertson et al., 1992). The entire nucleic acid sequences of several HAV strains have been determined by molecular cloning (Graff et al.,1994), and a large number of HAV isolates have been characterized by sequencing of short genome segments. The genomic regions most commonly used to define HAV genotypes include the C terminus of the VP3 region (Jansen et al., 1990), the N terminus of the VP1 region, the 168 bp junction of the VP1/P2A regions (Robertson et al., 2000), the 390 bp region of the VP1-P2B regions (Nainan, 2005), and the entire VP1 region (Nainan et al., 1991). - 20 -
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Seven HAV genotypes have been identified; four genotypes (I, II, III, and VII) are of human origin, thegenotypes I and III are most prevalent, and three (IV, V, VI) are of simian origin. Genotypes II and VII were initially defined based upon a single isolate for each (Robertson et al., 1992). However, further investigations have reclassified genotype VII as a sub-genotype of genotype II (Costa-Mattioli et al., 2002).
2.2.6.5.8 HAV detection: HAV was first visualized after the aggregation of fecal material with serum containing specific homologous antibodies (Feinstone et al., 1973). The technique of immune electro-microscopy of stool was used to assay for specific anti-HAVantibodies in convalescent phase sera after episodes of naturally-occurring hepatitis and to investigate the transmission of the virus (Dienstag et al., 1975). HAV replicates in cell culture without cytopathic signs of infection and without apparent host cell damage. Because of the lack of a cytopathic effect in cell culture, immunological assays are required to detect HAV antigen (Siegl et al., 1984). Previous
(resolved) HAV
infection is diagnosed by detection of IgG anti-HAV. However, commercially available assays detect total anti-HAV (both IgG and IgM antibodies). The presence of total anti-HAV and the absence of IgM anti-HAV can be used to differentiate between past and current infections (Nainan et al., 2006). HAV has been detected with techniques such as restriction fragment length polymorphism (Goswami et al., 1997), single-strand conformational polymorphism (Fujiwara et al.,2000), Southern blotting (Sanchez et al.,2004), nucleic acid sequencing-based amplification (Jean et al., 2004), nucleic acid hybridization (Zhou et al., 1991), and reverse transcription-PCR (RT-PCR) and antigen capture RT-PCR (Cromeans et al., 2001). Amplification of viral RNA by RT-PCR is the most sensitive and widely used method for the detection of HAV RNA. (Nainan et al., 2006). - 21 -
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Nowadays, real-time PCR becomes an essential diagnostic technique. The main advantage of this technique is that with real-time PCR, the starting template copy number can be determined with accuracy and high sensitivity over a wide dynamic range. It can even detect a single copy of the target gene. In addition, real-time PCR assays can reliably detect gene copies with lower coefficients of variation than other detection methods. Another advantage of real-time PCR is that data can be evaluated without gel electrophoresis, resulting in reduced experiment time. Finally, the real-time PCR assay is performed, and data are evaluated in a closed-tube system, eliminating the need for post-amplification manipulation and, thus, reducing opportunities for contamination (Feng Qiu et al.,2014).
2.2.6.5.9 HAV transmission: HAV is most commonly transmitted through close person-to person contact in households and extended family settings. Young children have the highest infection rates, and in most communities with sustained transmission, asymptomatic young children are the primary source of infection (Bell et al., 1998). Serologic studies conducted within households of hepatitis A patients have shown that asymptomatic HAV-infected children are often present and presumably were the source of infection (Staes et al., 2000). Additionally, food borne or waterborne transmission can occur when fecal material from HAV-infected persons contaminates foodor water. In the developed world, food borne transmission is most commonly identified when HAV-infected food service workers contaminate food that is served to others without being cooked (Fiore, 2004).
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2.2.6.5.9.1 Parenteral hepatitis A transmission: Parenteral transmission of hepatitis A complicating transfusion of blood and blood products were
reported many times (Noble et al., 1984).
Transmission associated with platelet and plasma donation processing, and anti cancer immunotherapy reagents had also been documented (Weisfuse et al., 1990). Identical HAV sequences were detected in clotting factor concentrates and hemophiliac recipients in Italy (Mannucci et al., 1994). The other group at risk for HAV infection by parenteral transmission is the injection drug-using population (Grinde et al.,1997). Hepatitis A can also be potentially spread within this group by contamination from rectally carried drugs as well as by unsanitary living conditions, crowding, and the lack of the necessary personal hygiene to prevent infection. Approximately 40 to 50% of injection drug users in northern Europe are anti-HAV positive (Krook et al.,1997). 2.2.6.5.10 Immune response to hepatitis A: The immunology of hepatitis A is important for two reasons; first, specific diagnostic tests for the confirmation of HAV as the etiologic agent depend upon the production of antibody by the humoral immune response, which leads to the development of circulating immune complexes with associated symptoms and signs in some patients. Second, clearance of viral infection and the disease manifestations associated with this process is most likely produced by the cellular immune response(Margolis et al., 1990). The symptoms of acute hepatitis A are similar to those of other viral hepatitis, and serologic testing for the detection of Immunoglobulin M (IgM) antibodies to HAV (anti-HAV) is required to confirm the diagnosis. IgM anti-HAV is usually noticeable when symptoms appear, and concentrations decline to undetectable levels within six months ofinfection in most patients (Stapleton, 1995). While Immunoglobulin G anti-HAV appears early during - 23 -
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infection and remains noticeable throughout the person's lifetime.Total antiHAV tests are often used in epidemiologic investigationsor in determining susceptibility to HAV infection but do not identify acute infection (Wasley et al., 2006).
2.2.6.5.11 Clinical features of hepatitis A: The clinical presentation of viral hepatitis is similar, regardless of the etiology, and the diagnosis must be made on the basis of serologic tests. However, each type of viral hepatitis has clinical features and a natural history that is more characteristic of that agent than of the other hepatitis viruses (Poovorawan et al., 2005). Infection with HAV may result in a wide spectrum of clinical outcomes, ranging from a completely unapparent infection detected serologically by evidence of a rising titer of anti-HAV, to subclinical disease characterized by limited symptoms and biochemical abnormalities of liver function tests to classical icteric hepatitis, to potentially fatal fulminant hepatitis with hepatic failure and coma. Adults develop icteric clinical illness, regardless of the strain of the infecting virus and fulminant hepatitis A occurs most commonly in patients over 50 years of age and death from hepatitis A is also more common in this age group (Kyrlagkitsis et al., 2002). Depending on experimental infections and analyses of common-source epidemics, the average incubation period of hepatitis A is approximately four weeks (range 2-7 weeks), (Brown and Persley, 2002).Variations in the incubation period may be related to the infectious dose, differences in strain virulence or host factors. Patients developing symptomatic disease usually experience aprodromal period beginning several days before the onset of illness and characterized by malaise, flu-like symptoms, anorexia and fever (Purcell et al., 2002). These symptoms may continue into the acute phase followed by darkening of the urine and lightening of the faeces, but they - 24 -
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usually decrease as jaundice, manifest by icterus ofthe sclera, skin and mucous membranes, develops (Bower et al.,2000). Hepatitis A viremia and faecal shedding, detected initially by volunteer studies, but now by Enzyme-Linked Immunosorbent Assay (ELISA) or Reverse Transcriptase Polymerase Chain Reaction (RTPCR), appears approximately 2-3 weeks after infection, during the incubation period, and peaks before the onset of disease (Fig.2.8). With the increasing use of hepatitis A vaccination, cases of modified HAV infection may become more common (Chitambar et al., 2001). Biochemical evidence of acute hepatitis is indistinguishable from that observed in other forms of viral hepatitis. Serum aminotransferases [Alanine aminotransferase (ALT) and Aspartateaminotransferase (AST)] formerly known as (SGPT) and (SGOT) respectively are sensitive indicators of liver cell amage. They rise rapidly during the late incubation period, reaching peak levels (500-2000 U/L) generally within one week of the onset of symptoms and fall relatively rapidly after the onset of jaundice inuncomplicated cases. Early changes in the hepatocyte reflect altered permeability of the plasma membrane, resulting in the loss of ALT from the cell, found in the cytosol, before the loss of AST, which is found in the mitochondria. Thus, serum ALT levels are generally higher than serum AST levels in acute uncomplicated hepatitis A. High levels of AST indicate severe tissue necrosis. Generally, bilirubin reaches peak levels of 170-200 μmol/L shortly after the transferase peak (Kabrane-Lazizi et al., 2001).
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Figure-2.8-Natural history of hepatitis A (Martin and Lemon,2006).
2.2.6.5.12 HAV Prevention: 1. Gamma globulin (passive immune-prophylaxis): Preliminary data indicated that persons under age 20 were uniformly susceptible, whereas the majority of older individuals were predicted to be immune. Gamma globulin can be used for protection from HAV as a passive prophylactic mechanism (Winokur et al., 1992).
2. Vaccination (active immune-prophylaxis): Recommendations for the use of hepatitis A vaccine varies considerably among countries. Guidance from the World Health Organization on hepatitis A vaccines emphasizes the need to consider the cost-benefit and sustainability of various prevention strategies within the context of the epidemiologic characteristics of the setting where vaccination is being considered (Wasley et al., 2006). Hepatitis A vaccination has few indications in the developing world, where hepatitis A is highly endemic and where most of the population is already - 26 -
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immune as a result of HAV infection during early childhood (Bell, 2002). In more developed countries, hepatitis A vaccine is primarily being used to protect persons with increased risk of hepatitis A or its consequences, such as travelers to areas where hepatitis A is endemic (Crowcroft et al., 2001). However, in some of these countries, the epidemiology of hepatitis A is heterogeneous, with much of the disease burden being focused in certain regions where large community-wide epidemics occur (Germinarioet al., 2000). These regions have been the focus of programs using routine vaccination of children to reduce HAV transmission (Lopalco et al., 2001). In the United States, hepatitis A vaccination has been recommended since 1996 for groups who are at increased risk of hepatitis A or its consequences (Wasley
et
al.,
2006),
many
developed
countries
have
similar
recommendations as well. Hepatitis A vaccine is recommended for persons who travel to countries where hepatitis A is of high or intermediate endemicity (Franco et al., 2003). In the few past years, it was noticed that the majority and the highest rates of HAV infection were in areas in which hepatitis A vaccination of children was not recommended (Samandari et al., 2004). The analysis of the National Immunization Survey data found that in addition to living under a state where routine vaccination was recommended, other factors associated with having received hepatitis A vaccine included living in an urban area, having a mother with less than a high school education (Amon et al., 2006).
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2.2.6.6 Hepatitis E Virus (HEV): Hepatitis E virus is an enveloped, single stranded RNA that occurs in both epidemic and sporadic form and is generally associated with drinking water contaminated with sewage. Infection among household contacts indicates person to person spread. The outbreaks were mainly found among young and middle aged adults with very high mortality in infected pregnant woman (Khuroo et al.,2004). Hepatitis E virus (HEV) is responsible for endemic hepatitis as well as sporadic epidemics of acute, enterically transmitted hepatitis in the developing world, including parts of Asia, the Middle East, Africa, and Mexico (Emerson and Purcel, 2007; Chandra et al., 2008). Hepatitis E is an infectious viral disease with clinical and morphological features of acute hepatitis observed in young, middle-aged adults and among pregnant women, and also in the few past years reported in kids (Thapa, 2009). Hepatitis E is typically a self limited, acute viral hepatitis lasting 1-4 weeks; it does not progress to chronic disease. In rare cases, some patients have severe disease, which progresses to fulminant liver failure; the overall case fatality rate for the general population in disease-endemic countries range from 0.1 to 4%. Case fatality rates are much higher (up to 25%) among pregnant women infected with HEV during the third trimester (Krawczynski, 2007). Large outbreaks of HEV are linked to contaminated waterborne sources and are also a zoonosis, as anti-HEV antibodies have been detected in rodent, swine, sheep, bovine and poultry (Meng, 2009 and FitzSimons et al., 2010). The first epidemiological study about hepatitis E came from India in the early fifties, and the infectious acute hepatitis outbreak in Delhi was extensively described (Viswanathan, 1957).
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2.2.6.6.1 HEV viral particle structure The HEV capsid subunits are formed by two identical molecules, which represent the main structure responsible for the virion shell (Xing et al 1999). The capsid protein comprises about 660 amino acids with a molecular size of approximately 70 KD and can be divided into three different domains: S (shell), M (middle) and P (protruding) (Fig.2.9) (Xing et al., 2010). The S domain forms the internal skeleton of the particle, forming a continuous capsid shell. (Guu et al., 2009; Yamashita et al., 2009). The M domain is tightly associated with the S domain and linked to the P domain by a long proline-rich hinge (Yamashita et al., 2009). The association of these two domains makes it possible for the capsid protein dimer to change its conformation, allowing a unique topology (Mori and Matsuura, 2011). The P domain is a single individual domain forming a twisted antiparallel ß-sheet structure, and forms dimeric spikes stabilizing protein interactions across the two-fold like spikes (Guu et al., 2009; Yamashita et al., 2009; Mori and Matsuura, 2011).
Figure -2.9- Structural domains of the HEV capsid protein ( Xing et al., 2010)
2.2.6.6.2 Classification and genotypes The International Committee for Taxonomy of Viruses has classified HEV as a Hepevirus in the family Hepeviridae.The HEV genomes of several geographically distinct isolates showed a high degree of sequence - 29 -
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conservation (Arankalle et al.,1999). At least four phylogenetically distinct genotypes have been defined, which distribute by geographic regions, although all HEV genotypes show different intra-genome diversity (Okamoto, 2007). Genotype 1 includes Asian and African HEV strains; genotype 2 includes the single Mexican HEV strain, and few variants identified from endemic cases in African countries; genotype 3 includes human and swine HEV strains from industrialized countries, and genotype 4 includes human and swine HEV strains from Asia, particularly China, Taiwan and Japan. The avian HEV was proposed to belong to a new genotype 5 (Haqshenas et al., 2001; Huang et al., 2004); however, this has not yet been confirmed. 2.2.6.6.3 Characteristics and genome organization of Hepatitis E Virus Hepatitis E virus was first described in 1983 as a spherical, non-enveloped particle 27-34 nm in diameter, containing a polyadenylated, positive strand RNA genome (Fig. 2.10) (Jothikumar et al, 1993; Purcell, 1996; Aggarwal et al, 1999). The HEV viral genome consists of a single positive stranded RNA genome of approximately 7.2 Kb in length (Tam et al., 1991; Fauquet et al., 2005). Sequence analysis revealed that the HEV genome contained two large potential open reading frames (ORF's) (Fig. 2.10) within the full-length genomic transcript, and a third small positive-polarity ORF was identified by the immunoreactive epitope that it encodes (Zafrullah et al., 1997; Aggarwal et al., 1999; Aggarwal and Krawczynski, 2000; Tyagi et al, 2001).
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Figure-2.10-Genome organization and proteins of HEV (Chandraet al.,2008).
2.2.6.6.4 Genome replication and life cycle Due to the lack of an efficient cell culture system or animal model, the mechanisms of HEV replication are not well known. A replication model has been proposed based on analogy to other single stranded RNA viruses and some knowledge of HEV (Fig. 2.11) (Ahmad et al., 2011). It is believed that HEV particle uptake occurs by receptor-mediated endocytosis using a not yet identified receptor on the cell surface. After uncoating, RNA is translated into the non-structural poly protein by host ribosomes; it is assumed that the papain-like protease cleaves the ORF one encoded poly protein. The RdRp replicates (alone or with the aid of cellular proteins) the positive RNA into negative RNA strands (Agrawal et al., 2001), which will serve as a template for synthesis of the positive sense RNA strand by the viral RNA polymerase. In parallel, the sub-genomic RNA is translated by the structural proteins in the ORF 2 and ORF 3, and the capsid protein packages the genome probably with the aid of the cytoskeleton phosphoprotein (ORF 3) - 31 -
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and the virions are assembled and released by a mechanism not yet identified (Mori and Matsuura, 2011).
Figure-2.11- Proposed replication cycle of HEV(Chandraet al.,2008).
2.2.6.6.5 HEV transmission The main route of human HEV transmission is fecal-oral as the first report on the outbreak pointed to water, and food contaminated related to HEV infections (Wong et al., 1980; Sreenivasan et al., 1984b; Aye et al., 1992; Huang et al., 1992; Skovgaard, 2007). Other fewer common routes are vertical transmission (transplacental) as well as horizontal via blood transfusion or organ transplantation (Kumar et al., 2001; Tamura et al., 2007a; Panda et al., 2007; Khuroo and Kamili, 2009; Halac et al., 2011; Hosseini Moghaddam, 2011; Rostamzadeh Khameneh et al., 2011). However, there are studies that suggest other routes - 32 -
Chapter Two
Literature review
of transmission in Europe, which are related to consumption of offal, wild boar or food contaminated during preparation (Wichmann et al., 2008). As food borne pathogens, HEV particles can actually be ingested via water, undercooked meat from swine or wild animals such as deer, crops, as well as ingestion of mollusks from contaminated water or sewage (Li et al., 2007; Meng, 2011). Other evidence also showed transmission through blood transfusion, as the first molecular evidence for transfusion-transmitted HEV came in 2004 from a 67- year-old Japanese patient, the HEV sequence was highly similar to that of one donor sample (Matsubayashi et al., 2004). 2.2.6.6.6 Epidemiology of hepatitis E virus HEV seropositivity is globally present and endemic in large parts of Africa, Latin-America and Asia (Fig. 2.15) (Teo, 2011). Regions of the world can be considered as hepatitis E disease endemic or non-endemic, based on the periodic occurrence of disease outbreaks. Several large epidemics of hepatitis E have been observed in the Indian subcontinent, and in developing countries of Southeast and central Asia (He et al., 2006). Outbreaks of hepatitis E have been observed in the Middle East, and northern and western parts of Africa. In North America (Mexico), two small outbreaks were reported in the years 1986-1987 (Valazquez et al., 1990). In non-endemic regions, where outbreaks have not been reported, the disease
accounts
for < 1% of reported cases of acute viral hepatitis,
although hepatitis E virus is restricted to tropical and subtropical countries, where it causes epidemics of viral hepatitis, often involving large numbers of patients (Hunter, 1997; Jameel, 1999; Schlauder and Mushahwar, 2001). Iraq is a country with few suspected outbreaks of HEV. Starting in May 2004, an outbreak of acute hepatitis was identified in several cities of Baghdad, including Al-Sader, AL-Ubidy and AL-Kamalya, in Al- Rusafa district, Al-Mahmodya and Abu Graib Cities, in Al-Karkh district - 33 -
Chapter Two
Literature review
(Mministry of health, 2004, 2005, 2006). Hepatitis is also reported from Basrah
Governorate, south of Iraq
(Turky, 2011). The disease was
recognized among young adults, pregnant women with fewer deaths among them and was therefore, clinically diagnosed as HEV. Hepatitis E is prevalent in most developing countries, and common in any country with a hot climate (Uchida, 1992) .
Figure-2.12- Global prevalence of HEV (Teo, 2011) .
2.2.6.6.7 Clinical symptoms and course of infection Based on the symptoms, acute HEV infection cannot be distinguished from hepatitis A (Purcell and Emerson, 2008). HEV infection can cause acute liver disease, which is mild and self-limited in the majority of cases. However, in some cases it can induce the so-called “Fulminant Hepatic Failure” (FHF), which is a severe acute hepatic disease with low chances of recovery. The - 34 -
Chapter Two
Literature review
non-specificity and diversity of the clinical symptoms may lead to misdiagnosed cases; it can be misdiagnosed in drug induced acute liver injury cases (Davern et al., 2011). HEV infection often manifests as subclinical disease. Usually, the patients show typical signs and symptoms of acute liver disease, very similar to HAV infection. Unlike Hepatitis Virus infections, the clinical response to HEV is dosedependent and the incubation period can range from 15 to 60 days (Balayan et al., 1983, Chauhan et al., 1993).The classical symptomatic infection can be divided into pre-icteric from (1-10 days), icteric from (12-15 days up to one month) and post-icteric, which is characterized by normalization of liver enzyme levels (Panda et al., 2007; Aggarwal, 2011).
2.2.6.6.8 Immunity response to HEV In healthy individuals, HEV infection leads to innate and adaptive immune responses. The number of natural killer (NK) and T-cells and their activation state increases during acute hepatitis infection (Srivastava et al., 2007; Srivastava et al., 2008). Moreover, the numbers of
both CD8+ and
CD4+high, CD8+low T-cells increased in acute hepatitis E virus-infected patients (Srivastava et al., 2007; Husain et al., 2011; Prabhu et al., 2011). A new study suggests the association of a specific T-cell response with clearance of infection (Suneetha et al., 2012). In parallel, an adaptive immune response leads to the production of antiHEV IgM and IgG. Although detectable IgG anti-HEV antibody levels rise quickly and peak at around four weeks after infection, subsequently they drop until they are undetectable (Corwin et al., 1995). It is believed that IgG antibody presence persists over many years, and several reports indicate a gradual decrease of anti-HEV IgG seropositivity in an infected population (Khuroo et al., 1993; Corwin et al., 1995; Khuroo, - 35 -
Chapter Two
Literature review
2011). Specific anti-HEV IgM antibodies can be detected quickly upon infection by the hepatitis E virus, and decrease after HEV infection has been cleared (3-5 months after clearance). Therefore, these antibodies provide an accurate marker for present or recent HEV infection. 2.2.6.6.9 Diagnosis of HEV infection: Different serological and molecular assays (RT-PCR and qRT-PCR) were developed (Jothikumar et al., 2006; Li et al., 2006 b). In general, the diagnosis includes the detection of IgG and IgM antibodies against HEV as well as HEV RNA in serum and feces (Teshale and Hu, 2011). ELISA based on clonal recombinant HEV antigen has been developed to detect HEV-IgM and HEV-IgG antibodies, so ELISA system is regarded as a convenient method in the diagnosis of acute or past HEV infection (Zhou et al., 2004). Alternatively, the presence of the viral genome in patients’ serum or faeces as the most reliable marker for ongoing HEV infections can be demonstrated by reverse transcriptase polymerase chain reaction (RT-PCR) (Erker et al., 1999; Schlauder et al., 1999; Mizuo et al., 2002). Quantitative RT-PCR is also widely used (Enouf et al., 2006; Jothikumar et al., 2006). A few years ago, several different PCR-based methods have been developed to test for HEV RNA presence, including conventional reverse transcriptase RT-PCR, real-time RT-PCR and reverse transcription loopmediated isothermal amplification (RT-LAMP) (Lan et al., 2009).
2.2.6.6.10 Prevention and control In developing countries, good sanitation conditions such as access to clean water and sewage systems are fundamental in the control of hepatitis E outbreaks. For instance, the use of chlorination reduces the amount of fecal coliforms and contributes to the control of hepatitis E (Naik et al., 1992).
- 36 -
Chapter Two
Literature review
In developed countries, the consumption of raw or undercooked meat and meat products from swine, wild boar and deer should be avoided. Few measures can be applied in order to prevent vertical transmission of HEV (Kumar et al., 2001; Chibber et al., 2004).
2.2.6.6.11 Vaccination At least two distinct recombinant HEV vaccines went into clinical trials (Li et al., 2005a ; Shrestha et al., 2007; Zhu et al., 2010). One vaccine is based on a recombinant capsid protein expressed via the baculovirus system using Spodoptera frugiperda (Fall armyworm) cells. The vaccine seems to be efficient in preventing hepatitis E infection (Shrestha et al., 2007).
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Chapter Three Materials and Methods
Chapter Three
Materials and Methods
3 Materials and Methods 3.1 Materials The following materials and instruments were used during conducting the current study: 3.1.1 Materials and Instruments Instrument
Manufacturer, Origin
Absorbent paper tissues
Selpak,Turkey
Alcohol Pad
Falcon, UAE
Autoclave Automated clinical chemistry analyze
Yamato, Japan ELITech, USA
Balance
Metller, China
Camera
Polaroid Co. USA
Centrifuge
Biofugeheraeus, Germany
Cool box
Tank, Iran
Coulter counter analyzer
Beckman
disposable plastic tips
Citotest, China
Distillator
Exelo, UK
EDTA Tubes
Vacutest, Italy
Electrophoresis
Major Science, USA
Eyela Freeze Dryer
Tokyo Riakikai Co. Japan
Gloves
Sempercare, nitrile, UK
Hot Plate Incubator Medical freezer- 40 microcentrifuge tubes (DNase and RNase free) Microelisaplate Microelisaplate Reader Microelisaplate Shaker Micropipette(0-50)μl,(100-200)μl,(100-1000)μl Microscope Microwave oven Oven PH-Meter Refrigerator -20, -30 Shaker-Incubator Syringe Thermocycler UV- Transilluminator Vacuum tubes Vortex Water Bath
Medax, UK Memmert, Germany SANYO CitoTest, CHINA
- 38 -
Teco diagnostics, USA Slamed, Germany Olympus, Japan Arcelik, Turkey Gallenkamp, Australia Metrohm AG, Switzerland VESTEL, Turkey Memmert, Germany Greetmed, China Techne TC-512, UK Major Science, USA Vacutest, Italy Grant –bio, Latvia Memmert, Germany
Chapter Three
Materials and Methods
3.1.2 Chemicals and biological materials 3.1.2.1 Solutions, buffers and reagents Chemical
Company
10X TBE (Tris-Borate-EDTA) PBS (phosphate-buffered saline) Ethidium bromide 100 bp DNA Ladder Absolute Ethanol DEPC distilled water
Biotech Biotech BIO BASIC INC GeneDirex Scharlau–spain BIONEER
3.1.2.2 Auto analyzer biochemical liver enzyme tests reagents 1. AST/GOT 4+1 SL: LotNo 12-1326; ELITech Clinical Systems SelectraProM, USA; contains: Kinetic: AST Reagents: R1 contains Tris buffer PH (7.8), L-Aspartate: Lactate dehydrogenase (LDH) (microorganisms), Malate dehydrogenase (MDH) (bacterial) and sodium azide. R2 contains α-Ketoglutarate, NADH. 2. ALT/GPT 4+1 SL: LotNo 12-1634; ELITech Clinical Systems SelectraProM, USA; ALT Reagents: R1 contains Tris buffer PH(7.5), Reagents, L-Alanine: Lactate dehydrogenase (LDH), R2 contains α-Ketoglutarate, NADH. 3. Total Bilirubin Reagent: R1 contains sulfanilic acid, Hydrochloric acid and cetrimide. R2 contains sodium nitrite. 4. Alkaline phosphatase: Lot No 12-1154; ELITech Clinical Systems SelectraProM, USA; It consists of 2 reagents; R1 contains 2-Amino-2methyl-1-propanol (AMP) buffer (pH 10.45), Magnesium ions (2.4 mM), and Zinc ions (1.2 mM). R2 contains phosphate (p-NPP; 80 mM) and sodium azide (<0.1%).
- 39 -
p-Nitrophenyl
Chapter Three
Materials and Methods
3.1.2.3 Different materials Names, kits
Supplied Company
HAV Ab ELISA (EIA- 4233) Kit Anti-HAV IgM ELISA (EIA-3889) kit HEV Ab ELISA (EIA-4416) kit HEV IgM ELISA (EIA-4417) kit Viral Nucleic Acid Extraction kit II VR100 HAV 430 Reverta and amplification Kit AccuPowerR RT/ PCR PreMix kit AccuPowerR PCR PreMix kit AGAROSE M
DRG Diagnostics DRG Diagnostics DRG Diagnostics DRG Diagnostics Geneaid, Taiwan Sacace,(Italy) Bioneer, KOREA Bioneer, KOREA BIO BASIC INC
(Germany) (Germany) (Germany) (Germany)
3.1.2.4 Sodium hydroxide solution 1N: It was used for adjusting the pH of the solutions. 3.1.3 Enzyme immune assay reagents and solutions: 1. HAV Ab ELISA(EIA- 4233) kit Component (Each kit contains sufficient reagents to perform 96 tests). A. Microtiter strips: 8x12 microwell strips coated with purified and inactivated HAV, sealed into a bag with desiccant. Allow the microplate to reach room temperature before opening. Reseal unused strips in the bag with desiccant and store at 2-8°C°. B. Negative Control: 1 x 2. 0 ml/vial . Ready to use. Contains bovine serum proteins, 10 mM phosphate buffer pH 7.4+/-0.1, 0.02% gentamicine
sulphate
and
0.1%
Kathon
GC as
preservatives. The negative control is color coded pale yellow. C. Positive Control:1 x 2.0 ml/vial. Ready to use. Contains bovine serum proteins, anti HAV antibodies at a concentration higher than 100 WHO mI U/ml, 10 mM phosphate buffer pH 7.4+/-0.1, 0.02% gentamicine
sulphate
and
0.1%
Kathon
preservatives. The positive control is colour coded green.
- 40 -
GC as
Chapter Three
Materials and Methods
D. Calibrator: n° 1 vial. Lyophilized.To be dissolved with EIA grade water as reported in the label. Contains bovine serum proteins, anti HAV antibodies at a concentration of about 10 WHO mI U/ml, 10 mM phosphate buffer pH 7.4+/-0.1, 0.02% gentamicine sulphate and 0.1% Kathon GC as preservatives. E. Wash buffer concentrate: WASHBUF 20X: 1 x 60 ml/bottle. 20x concentrated solution. to be diluted up to 1200 ml with distilled water before use. Once diluted, the wash solution contains 10 mM phosphate buffer pH 7.0+/-0.2, 0.05% Tween 20 and 0.05% Kathon GC. F. Enzyme conjugate: 1 x 16 ml/vial. Ready-to-use solution. Contains
Horseradish
peroxidase
conjugated
antibody,
specific to HAV, in presence of 10 mMTris buffer ph 6.8+/-0.1, 2% BSA, 0.1% Kathon GC and 0.02% gentamicine sulphate as preservatives. The reagent is colored with a red dye. G. Chromogen/Substrate: SUBS TMB: 1 x 16ml/vial. Contains a 50 mM citrate-phosphate buffered solution at pH 3.5-3.8, 0.03% tetra-methyl-benzidine or TMB and 0.02% hydrogen peroxide of H2O2. H. Specimen Diluent: DILSPE: 1 x 8ml. Buffered solution suggested to be used in the follow up of vaccination. It contains 0.09% sodium azide and 0.1% Kathon GC as preservatives.The reagent is color coded dark green. I. Sulphuric Acid: H2SO4 O.3 M: 1 x 15 ml/vial. Contains 0.3 M H2SO4 solution. J. Plate sealing foils and Package insert
- 41 -
Chapter Three
Materials and Methods
2.Anti-HAV IgM ELISA (EIA-3889) kit component: 1. Anti-μ-chain Coated Wells 2. Enzyme conjugate 3. Positive Control Serum 4. Negative Control Serum 5. Wash Buffer (1:20 dilution prior to use) 6. Substrate (TMB) A 7. Substrate (TMB) B 8. Stop Solution 9. HAV Ag 10. Seal Paper
1 block (96wells) 1 bottle (6ml) 1 vial (0.5ml) 1 vial (0.5ml) 1 bottle (50ml) 1 bottle (6ml) 1 bottle (6ml) 1 bottle (6ml) 1 bottle (6ml) 2 pieces
3. HEV Ab ELISA(EIA-4416) kit component (96 tests). A. Microplate, n°1 microplate: 12 strips of 8 microwells coated with HEV specific synthetic antigens derived from ORF2 and ORF3 regions. Plates are sealed into a bag with desiccant. B. Negative Control 1 x 2.0 mL/vial. Ready to use control. It contains 1%
goat serum proteins, 10 mM Na-citrate buffer
pH 6.0 ± 0.1, 0.5% Tween 20, 0.09% Na-azide and 0.1% Kathon GC as preservatives. The negative control is olive green colour coded. C. Positive Control 1 x 2.0 mL/ vial. Ready to use control. It contains 1% goat serum proteins, human antibodies positive to HEV, 10 mM Na-citrate buffer pH6.0 ± 0.1, 0.5% Tween 20, 0.09% Naazide and 0.1% Kathon GC as preservatives. The Positive Control is blue colour coded. D. Calibrator n°1 vial. Lyophilized calibrator.To be dissolved with the volume of EIA grade water reported on the label. It contains foetal bovine serum proteins, human antibodies to HEV whose content is calibrated on 1st WHO reference reagent for HEV antibody, NIBSC code 95/584 at 1IU /ml ± 10 % ,10 mM Na-citrate buffer pH 6.0 ± 0.1, 0.3 mg/ml gentamicine sulphate and 0.1% Kathon GC as preservatives.
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Chapter Three
Materials and Methods
E. Wash buffer concentrate (WASHBUF 20X)
1 x 60 mL/ bottle.
20x concentrated solution. Once diluted, the wash solution contains 10 mM phosphate buffer pH 7.0 ± 0.2, 0.05% Tween 20 and 0.1% Kathon GC. F. Enzyme Conjugate 1 x 16 mL/ vial. Ready to use and red colour coded reagent. It contains Horseradish Peroxidase conjugated goat polyclonal antibodies to human antibodies, 5% BSA, 10 mM Tris buffer pH 6.8 ± 0.1, 0.1% Kathon GC and 0.02% gentamicine sulphate as preservatives. G. Chromogen/Substrate SUBS TMB1 x 16 mL/vial. Ready-to-use component. It contains 50 mM citrate-phosphate buffer pH 3.5-3.8, 4% dimethyl sulphoxide, 0.03% tetra-methyl benzidineor TMB and 0.02% hydrogen peroxide or H2O2. H. Assay Diluent (DILAS) 1 x 8 mL/ vial. 10 mM tris buffered solution pH 8.0 ± 0.1 containing 0.1% Kathon GC for the pre-treatment of samples and controls in the plate, blocking interference. I. Stop solution (SulphuricAcid) H2SO4 0.3 M1 x 15 mL/vial. It contains 0.3 M H2SO4 solution. J. Sample Diluent: DILSPE1 x 50 mL/vial. It contains 10 mM Na-citrate buffer pH 6.0 ± 0.1, 0.5% Tween 20, 0.09% Na-azide and 0.1% Kathon GC as preservatives. K. Plate sealing foils n°2 L. Package insert n°1
4.HEV IgM ELISA (EIA-4417) kitcomponent (96 tests). A. Microplate: 12 strips of 8 microwells: coated with HEV specific synthetic antigens derived from ORF2 and ORF3 regions of all the for subtypes. Plates are sealed into a bag with desiccant. B. Negative Control 1 x 2.0 ml/ vial; Ready to use control. - 43 -
Chapter Three
Materials and Methods
It contains 1% goat serum proteins, 10 mM Na-citrate buffer pH 6.0 +/0.1, 0.5% Tween 20,0.09% Na-azide and 0.1% Kathon GC as preservatives. The negative control is yellow colour coded. C. Positive Control
1 x 2.0ml/ vial; ready to use control.
It contains 1% goat serum proteins, human anti HEV IgM, 10 mM Nacitrate buffer pH 6.0 +/-0.1, 0.5% Tween 20,0.09% Na-azide and 0.1% Kathon GC as preservatives.The positive control is dark green colour coded. D. Wash buffer concentrate WASHBUF 20X ; 1 x 60ml/ bottle. 20x concentrated solution. Once diluted, the wash solution contains 10 mM phosphate buffer pH 7.0+/-0.2, 0.05% Tween 20 and 0.1% Kathon GC. E. Enzyme Conjugate:1 x 16ml/ vial. Ready to use and red colour coded reagent. It contains Horseradish Peroxidase conjugated goat polyclonal antibodies to human IgM, 5% BSA, 10 mM Tris buffer pH 6.8+/-0.1, 0.1% Kathon GC and 0.02% gentamicine sulphate as preservatives. F. Chromogen/Substrate: SUBS TMB; 1 x 16ml/ vial. Ready-to-use component. It contains 50 mM citrate-phosphate buffer pH 3.5-3.8, 4% dimethyl sulphoxide, 0.03% tetra-methyl-benzidine or TMB and 0.02% hydrogen peroxide or H2O2. G. Sulphuric Acid H2SO4 O.3 M; 1 x 15ml/vial. It contains 0.3 M H2SO4 solution. H. Specimen Diluent: DILSPE; 2 x 60ml/ vial. It contains 2% casein, 10 mM Na-citrate buffer pH 6.0 +/-0.1, 0.1% Tween 20, 0.09% Na-azide and 0.1% Kathon GC as preservatives. To be used to dilute the sample. I. Neutralizing Reagent: SOLN NTR; 1x 8ml/ vial. Ready-to-use Reagent. It contains goat anti hIgG, 2% casein, 10 mM Na-citrate buffer pH 6.0 +/-0.1, 0.1% Tween 20, 0.09% Na-azide and 0.1% Kathon GC as preservatives. J. Plate sealing foils 2 x and Package insert 1 x - 44 -
Chapter Three
Materials and Methods
3.1.4 Nucleic acid extraction kit, buffers and dyes Geneaid viral nucleic acid extraction kit II VR100 Kit Contents
50 T
VB Lysis Buffer AD Buffer1 W1 Buffer Wash Buffer2 RNase- Free Water VB-Column 2 ml- Collection Tube
60 ml 8 ml 50 ml 25 ml 6 ml 100 pcs 100 pcs
3.1.5 HAV 430 kit: The kit HAV 430
is intended for the qualitative
detection of HAV RNA by reverse transcription (RT) and nucleic acid amplification. 1. Part N° 1 – “Reverta-L”: reverse transcription of the RNA Kit Contents RT-G-mix-1 RT-mix Reverse transcriptase (M-MLV) TE-buffer
60T 5 x 0,01 ml 5 x 0,125 ml 0,03 ml 1,2 ml
2. Part N° 2 – “HAV 430”: amplification kit; Kit Contents PCR-mix-1 PCR-mix-2 HAV cDNA (C+) DNA-buffer (C-) Negative Control C-* HAV RNA Rec (C+)* HAV IC (Internal Control)**
55T 55 ready-to-use single-dose test 0,6 mL 0,1 mL 0,5 mL 1,2 mL 5 x 0,03 mL 5 x 0,06 mL
* Must be used in the isolation procedure as Negative and Positive Controls (add 10 μl) of Extraction. ** Ten μl of internal control was added during RNA extraction procedure directly to the sample/lysis mixture. - 45 -
Chapter Three
Materials and Methods
3.1.6 Agarose gel 2%: It was prepared by dissolving 2gm of agarose in 100 ml Tris-borate buffer, boiled and dissolved in microwave, cooled to 45-50ºC, then poured into electrophoresis tank.
Position(nt)
HEV1 (F*)
5'-AATTATGCC(T)CAGTAC(T)CGG(A)GTTG-3' 3156 N
HEV2 (R**) HEV3 (F*) HEV4 (R**)
5'-CCCTTA(G)TCC(T)TGCTGA(C)GCATTCTC3' 5'-GTT(A)ATGCTT(C)TGCATA(T)CATGGCT-3'
Housekeeping gene β-actin: (S***)
5’-GTCGTACCACTGGCATTGTG-3’
Housekeeping gene β-actin (AS****)
5'-AGCCGACGAAATCAATTCTGTC-3'
5’-CCATCTCTTGCTCGAAGTCC-3
(5711-5732) 3157 N (6419-6441) 3158 N (5996-6017) 3159 N (6322-6343)
* [forward primer], ** [reverse primer], *** [sense primer], **** [antisense primer]
- 46 -
OLIGO MACROGEN
First and second round
Company
Name
(Yijia Yan et al.,2008); (Liu Min et al., 2013)
Primer sequence 5´ - 3´
References
3.1.7 Primers used for HEV detection extracted from the literature
Chapter Three
Materials and Methods
3.2 Methods 3.2.1 Samples Between July - September 2013, 137 blood samples were collected 92 from chemical bombed exposures survivors and 45 from non chemical exposures - controls-
including both sexes from different areas of Iraqi
Kurdistan Region, which were exposed to chemical bombardment by Sadam’s regime.The cities and villages included were: Goptapa, Sewsenan, Shanakhse, Sheikh Wasan, Balisan and Halabja. To each case subjected to the study, the following was done: 1) Patient’s medical history: Through the preparation of a questionnaire form, the following information was collected from each patient: their age, gender, time of exposure to the chemical warfare; past medical history of jaundice, past surgical operations, blood transfusions, medications and recent travel abroad. 2) Laboratory investigations: Ι. Sample collection: Serum separator tubes (SST) and EDTA tubes were used for collecting 7-10 ml of venous blood from each patient under aseptic conditions. The clotted blood specimen was centrifuged at 3000 r/minute for 15 minutes to collect the serum. The sera were separated from each sample within three hours, and divided into three aliquots of 2.0 ml screw capped tubes. The first aliquot was used for routine liver function tests, the second and the third aliquot were kept frozen after adequate labeling at -40°C until the time of assays. The sera samples of the second aliquot were screened for the detection of antibodies against Hepatitis A and E virus (both IgG and IgM) using ELISA kit techniques (DRG Diagnostics,Germany).
- 47 -
Chapter Three
Materials and Methods
The sera samples of the third aliquot were tested for HAV and HEVRNA by RT-PCR kit. II. Complete blood counts (C.B.C). Complete blood counts was made from tubes with EDTA by the automated Coulter counter analyzer. III.Liver function enzymes (ALT, AST, ALP and TSB) All blood samples were tested for routine biochemical liver function tests (LFTs) such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP).The total serum bilirubin (TSB) were assayed using an automated clinical chemistry system and colorimetric method.
3.2.2 Detection of anti-HAV and anti-HEV IgG and IgM by ELISA 3.2.2.1 Detection of anti-HAV IgG by ELISA (Willner I. R. et al .,1998) 1. Test Principle The assay was based on the principle of competition where the antibodies in the sample compete with an anti-HAV specific antibody, labeled with HRP, for a fixed amount of antigen on the solid phase. A purified and inactivated HAV was coated to the micro-wells. The patient’s serum was added to the micro-well and antibodies to HAV were captured by the solid phase. After washing, the enzyme conjugate was added and binds to the free HAV antigen, if still present. The plate was washed to remove unbound conjugate and then the chromogenic substrate was added. In the presence of peroxidase, the colorless substrate was hydrolyzed to a colored end-product, whose optical density detected and is inversely proportional to the amount of antibodies to HAV present in the sample.
- 48 -
Chapter Three
Materials and Methods
An additive was added to the sample directly into the well to block interferences able to mask the presence of antibodies, mostly appearing in the follow-up of vaccination. 2. Assay procedure: The assay had to be carried out according to what was reported before. 1. The required numbers of strips were placed in the micro-plate holder and the A1 well was left empty for the operation of blanking, the other strips were stored into the bag in the presence of the desiccant at 2°- 8°C and sealed. 2. Fifty μL specimen diluent was dispensed in all the wells used for identifying samples and controls/ calibrator, except for A1. Later 100 μl of Negative Control was pipetted in triplicate,100 μl of Calibrator in duplicate,100 μl Positive Control in single, after that 100 μL of samples was added according to the company instructions. The plate was incubated at 37°C for 60 min. 3. The micro-plate was washed. 4. For all the wells, except A1, 100 μl enzyme conjugate was pipetted and incubated at 37°C for 60 minutes. 5. The micro-plate was washed as described in step 3. 6. The amount of 100 μl TMB/ H2O2 mixture was pipetted into each well, including the blank wells and incubated at room temperature for 20 minutes. 7. The amount of 100 μl sulphuric acid was pipetted into each well to stop the enzymatic reaction using the same pipetting sequence as in step 6. Then the color intensity was measured with a micro-plate reader at 450nm (reading) and possibly at 620-630nm (blanking), blanking the instrument on A1 well.
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Chapter Three
Materials and Methods
3.2.2.2 Detection of anti-HAV IgM by ELISA (Liaw Y. F. et al .,1986; Stapleton J. T. 1995). 1.
Principle of the assay The purified Anti-μ-chain was coated on the solid phase of multi-wells.
Serum sample, HAVAg and Horseradish peroxidase labeled with Anti-HAV (conjugated) were added to coated wells. After incubation, if HAV-IgM was present in the sample, a complex of Anti-μ-chain-HAV-IgM-HAVAg-AntiHAV labeled with HRP will form. After washing the wells and removing other unbounded serum components, the plate was incubated with the substrate (TMB) to form a colored product the absorbance at 450nm was measured to indicate the presence or absence of HAV-IgM in the sample.
2.Test procedure 1. The samples were diluted with physiological saline solution to 1:1000. 2. One blank, two positive and two negative controls were set for each test, the amount of 100μl serum sample, positive and negative control was added into the coated wells, the wells were sealed with seal paper, and incubated for 30 minutes at 37°C. 3. The liquid in the coated wells was discarded, then the wells were left for a while to dry. The wells were filled with wash solution, and laid aside for 30 seconds, the liquid was discarded, and were left for a while to dry (the process was repeated five times). 4. One drop (approximately 0.05 ml) of enzyme conjugant and HAV-Ag was added into the same coated wells (The blank well is omitted), and mixed thoroughly, the wells were sealed with seal paper, and incubated for 30 minutes at 37ºC. 5. The wells were washed.
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Chapter Three
Materials and Methods
6. One drop (approximately 0.05 ml) of substrate A and B respectively was added to each well, mixed thoroughly, and incubated for 10 minutes at 37ºC. 7. One drop (approximately 0.05 ml) of stop solution was added into each well, mixed thoroughly, and measured the absorbance at 450 nm against the blank.
3.2.2.3 Detection of HEV-Ab (IgG) by ELISA (Ellner P. D. and Neu H. C., 1992) . 1.
Test Principle: Micro-plates were coated with HEV-specific synthetic antigens encoding
for conservative and immune-dominant determinants derived from ORF2 and ORF3 of all the viral subtypes (1, 2, 3 and 4). The solid phase was first treated with the diluted sample and anti-HEV IgG was captured, if present, by the antigens. After washing out all the other components of the sample, in the 2nd incubation bound, anti-HEV IgG was detected by the addition of polyclonal specific anti-human IgG antibodies, labeled with peroxidase (HRP). The enzyme was captured on the solid phase, acting on the substrate/chromogen mixture, generated an optical signal that was proportional to the amount of anti- HEV IgG present in the sample. A cutoff value let optical densities be interpreted into anti-HEV IgG negative and positive results.
2. Assay procedure 1. The required number of Micro-wells was placed in the micro-well holder, the first well was left empty for the operation of blanking. 2. The amount of 200 μL of negative control was dispensed in triplicate, 200 μL Calibrator in duplicate and 200 μL positive controls in single in proper wells. Controls and calibrator were ready to use. - 51 -
Chapter Three
Materials and Methods
3. The amount of 200 μL of sample diluent (DILSPE) was added to all the sample wells; then 10 μL samples was dispensed in each properly identified well. The wells were mixed gently in order to fully disperse the sample into its diluent. 4. The amount of 50 μL assay diluent (DILAS) was dispensed into all the controls/ calibrator and sample wells. 5. The micro-plate was incubated for 45 min at 37°C. 6. The micro-plate was washed with an automatic washer by delivering and aspirating 300 μL/well of diluted washing solution. 7. The amount of 100 μL enzyme conjugate was pipetted into each well, except the first blanking well and covered with the sealer and the plate was incubated for 45 min at 37°C. 8. The plate (micro-wells) was washed. 9. The amount of 100 μL chromogen/substrate mixture was pipette into each well, including the blank well, then the micro-plate was incubated at room temperature (18- 24°C) for 15 minutes. 10. The amount of 100 μL sulphuric acid was pipetted into all the wells using the same pipetting sequence as in step 10 to stop the enzymatic reaction. 11.The color intensity of the solution was measured in each well at 450 nm filter (reading) and possibly at 620-630 nm, blanking the instrument on A1.
3.2.2.4
Detection of anti-HEV IgM by ELISA (Hollinger F B and
Dreesman G R ,1992) . 1.
Test Principle: Micro plates were coated with HEV-specific synthetic antigens encoding
for conservative and immune-dominant determinants derived ORF2 and ORF3 of all the 4 subtypes . The solid phase was first treated with the diluted - 52 -
Chapter Three
sample
Materials and Methods
and anti-HEV IgM were captured, if present, by the antigens
adsorbed on wells. After washing out all the other components of the sample, in the 2nd incubation bound, anti HEV IgM antibodies were detected by the addition of polyclonal specific anti-human IgM antibodies, labeled with peroxidase (HRP). The enzyme was captured on the solid phase, acting on the substrate/chromogen mixture, generated an optical signal that is proportional to the amount of anti HEV antibodies present in the sample. A cutoff value let optical densities be interpreted into anti-HEV IgM negative and positive results.
2.
Assay procedure
The assay had to be carried out according to what follows, taking care to maintain the same incubation time for all the samples in testing. 1. Samples were diluted by 1:101 into a properly defined dilution tube; the calibration set was ready to use. All the liquid components on vortex were mixed carefully and then preceeded.The required numbers of micro-wells were placed in the micro-well holder. A1 well was left empty for the operation of blanking. 2. The amount of 50 μl of the neutralizing reagent (SOLN NTR) was dispensed in all the wells of the samples. 3. The amount of 100 μl of negative control was dispensed in duplicate and 100 μl of positive control in single, then 100 μl of diluted samples was dispensed in each properly identified well, and the micro-plate was incubated for 60 min at 37°C. 4. The micro-plate was washed with an automatic washer by delivering and aspirating 300 μl/well of diluted washing solution. 5. The amount of 100 μl enzyme conjugate was pipetted into each well, except the A1 well, and covered with the sealer, and the red-colored
- 53 -
Chapter Three
Materials and Methods
component was checked to ensure that it had been dispensed in all the wells, except A1. 6. The micro-plate was incubated for 60 min at 37°C. 7. The micro-wells were washed. 8. The amount of 100 μl chromogen/substrate mixture was pipetted into each well, including the blank well, and the micro-plate was incubated at room temperature (18-24°C) for 20 minutes. 9. The amount of 100 μl sulphuric acid was pipetted into all the wells using the same pipetting sequence (addition of acid was turned the positive Calibrators, the control serum and the positive samples from blue to yellow). 10. The color intensity of the solution was measured in each well at 450nm filter (reading) and possibly at 620-630 nm, blanking the instrument on A1.
3.2.3 Detection of HAV and HEV RNA by PCR 3.2.3.1 Viral nucleic acid extraction (Vogelstein and Gillespie, 1979). 1. Assay Principle: The viral nucleic acid extraction ( Kit II VR 100) was designed specifically for high-thorough purification of viral DNA/RNA from cell-free samples such as serum, plasma, body fluids and the supernatant of viral infected cell cultures. DNA/RNA viruses were lysed quickly and efficiently using the lytic buffer which is a highly concentrated solution of chaotropic salt. When combined with ethanol, the AD Buffer created optimum conditions for Nucleic Acid binding to the glass-fiber matrix of the column. Contaminants such as salts, metabolites and soluble macromolecular cellular components were removed in the wash step. Nucleic Acid was eluted in RNase-free water and was then ready for use in subsequent reactions, including Real-time PCR/RT-PCR, Automated Fluorescent DNA Sequencing, PCR, and other enzymatic - 54 -
Chapter Three
Materials and Methods
reactions. The detection limit for certain viruses depended on the sensitivity of individual PCR or RT-PCR assays.
2. Viral Nucleic Acid Extraction Protocol: According to the instructions supplied by the company, the following steps were followed: Sixty (60) ml absolute ethanol was added to the AD buffer prior to initial use, according to the bottled label for volume. The amount of 100 ml absolute ethanol was added to the wash buffer prior to initial use, according to the bottle labeled for volume.
Step1: Lysis
The amount of 200 μl of the sample (serum) was transferred into a 1.5 ml micro-centrifuge tube. The amount of 400 μl of VB lytic buffer was added to the sample and mixed by vortex. The mixture was incubated at room temperature for 10 minutes.
Step 2: Nucleic AcidBinding
The table below indicates extraction steps:
The amount of 450 μl of AD buffer (ethanol added) was added to the sample lysate and shacken vigorously. VB Column was placed in a 2 ml Collection Tube. The amount of 600 μl of the lysate mixture was transferred to the VB column. The mixture was centrifuged at 14-16,000 x g for 1 minute. The flow-through was discarded and the VB Column was placed back in the 2 ml collection tube. The remaining lysate mixture was transferred to the VB column, and centrifuged at 14-16,000 x g for 1 minute. The 2 ml collection tube containing the flow-through discarded and transferred the VB column to a new 2 ml collection tube.
- 55 -
Step 3: Wash
Materials and Methods
The amount of 400 μl of W1 buffer was added to the VB column, and centrifuged at 14-16,000 x g for 30 seconds. The flow-through was discarded and the VB column was placed back in the 2 ml collection tube. The amount of 600 μl of washing buffer was added to the VB column, and centrifuged at 14-16,000 x g for 30 seconds. The flow-through was discarded and the VB column was placed back in the 2 ml collection tube, then centrifuged at 14-16,000 x g for 3 minutes to dry the column matrix.
Step 4: Nucleic AcidElution
Chapter Three
The dried VB column was placed in a clean 1.5 ml microcentrifuge tube. Fifty μl of RNase-free water was added to the center of the VB column matrix, and left standing for 3 minutes and centrifuged at 14-16,000 x g for 1 minute to elude the purified nucleic acid.
3.2.3.2 Reverse transcription of HAV extracted RNA (cDNA synthesis): 1.
Assay Principle: The HAV Kit was intended for the qualitative detection of HAV RNA by
reverse transcription (RT) and nucleic acid amplification. The Kit was based on four major processes: Isolation of HAV RNA from specimens, reverse transcription of the RNA, nucleic acid amplification and detection of the amplified products on agarose gel. Hepatitis A virus detection by the polymerase chain reaction (PCR) was based on the amplification of a pathogen cDNA specific region using specific HAV primers
after PCR,
the amplified products could be detected in
agarose gel. The analysis of PCR results was based on the presence or absence of specific bands of amplified DNA in Agarose gel (2%). The length of specific amplified
DNA fragments
was
( HAV - 290 bp). The sample was - 56 -
Chapter Three
Materials and Methods
considered positive for hepatitis A virus RNA if the band of 290 bp was present in agarose gel and the sample was considered to be negative for hepatitis A virus RNA if the band of 290 bp was absent. Besides specific bands, the appearance of the
indistinct washed-out bands
indicates the
presence of primer-dimers.The indistinct washed-out bands of primer-dimers were situated lower than level of 100 bp of nucleotide pairs. Target region: 5’UTR. 2. Assay procedure According to the instructions supplied by the company, the following steps were followed: 1) For preparing 12 reactions,5 µl RT-G-mix-1 was added to the tube containing RT-mix and vortexed for at least 5-10 seconds, then centrifuged, after that 6 µl M-MLV ( Revertase) was added to a tube with Reagent Mix, and mixed by pipetting, vortexed for 3 sec, and centrifuged for 5-7 sec. 2) Ten µl of the reaction mix was added to each sample tube. 3) Ten µl RNA sample was pipetted to the appropriate tube and mixed by pipetting. 4) The tubes were placed in the thermo-cycler and incubated at 37ͦ C for 30 minutes. 5) Each obtained cDNA sample was diluted with TE- buffer by adding 20 µl TE-buffer to each tube.
3.2.3.3 Amplification of HAV cDNA by PCR 1) Required quantities of PCR-mix-1tubes, including two additional tubes (one for the negative other for positive control) were prepared. 2) To the each tube, 10 µl PCR- mix-2 were added.
- 57 -
Chapter Three
Materials and Methods
3) Ten µl of obtained cDNA after reverse transcription step was added to the appropriate tube. 4) Ten µl of DNA buffer was added to the tube for negative control of amplification. 5) Ten µl HAV cDNA was added to the tube for positive control of amplification. 6) PCR -mix-1 tubes were closed and transferred to the thermo-cycler only when temperature reached 95ͦ C and the following program were started:
Thermocyclers with active tempreture adjustment Step
T ͦC
1
95ͦ C
2
95ͦ C
5 min
95ͦ C
30 min
Time Pause
ͦ
67 C
30 min
72 ͦ C
30 min
4
72 ͦ C
1 min
5
10 ͦ C
3
Cycles 1
42 1 Storage
3.2.3.4 Molecular detection of HEV by nested RT- PCR (Yijia Yan et al., 2008) ; (Liu Min et al. 2013). Reverse transcriptase PCR (RT- PCR) was performed using the set of nested RT-PCR primers and the conserved region located in ORF2 was selected . This was designed to produce a 348- nt segment of open reading frame (ORF) 2, and were capable of detecting all four known HEV genotypes, also housekeeping gene β-actin was used which served as replication control.
- 58 -
Chapter Three
Materials and Methods
3.2.3.4.1 Reverse transcription of HEV synthesis)andamplification: Experimental Protocol
extracted
RNA (cDNA
1. Template RNA and the HEV2 reverse primer were mixed in a sterile tube as indicated in the table below:
Reaction volume RNA Template Total RNA Primer Primer
20 µl reaction 0.5-1.0µg 10-30 pmole
2. The mixture was incubated at 70 °C for 5 min and placed on ice. 3. The incubated mixture and the HEV1 forward primer were transferred to AccuPower® RT/PCR PreMix tube, then filled up the reaction volume with DEPC-DW. 4. The lyophilized blue pellet was dissolved by vortexing, and briefly spun down. 5. The thermal cycler with top heating, so the mineral oil was not added to each tube. 6. cDNA synthesis reaction was performed as follows: 42° C for 60 minute (cDNA synthesis) 94° C for 5 minute (RTase inactivation) 7. First round of nested RT-PCR cycles performed included a 35 cycles of denaturation for 45 s at 94°C, annealing for 30 s at 55°C, and extension for 1 min at 72°C, and a final incubation for 10 min at 72°C. 8. 10 µl of the products from the first round was used as the templates for the second round of nested PCR and 1 µl of each primer (HEV3 forward primer and HEV4 reverse primer) with housekeeping gene primers, β-actin (served as replication control)
were
added to
AccuPower® PCR tube. 9. AccuPower® PCR tubes completed to a total volume of 50 µl by adding DEPC- distilled water. - 59 -
Chapter Three
Materials and Methods
10. The lyophilized blue pellet was Dissolved by vortexing, and briefly spun down. 11. Second round of nested PCR of samples was performed in the same way as first amplification including: 35 cycles of denaturation for 45 s at 94°C, annealing for 30 s at 55°C, and extension for 1 min at 72°C, and a final incubation for 10 min at 72°C. 12. Samples were loaded on agarose gel, and electrophoresis were performed. 3.2.3.4.2 Preparation of agarose gel and Electrophoresis:Sambrook et al. (1989) method was used for HAV electrophoresis by dissolving 2 gm agar in 100 ml Tris-borate buffer, then the mixture was heated to boiling and cooled to 45°C then 5 µl of Ethidium bromide at final concentrations 0.5 µg /ml was added.The mixture was poured on to a glass plate surrounded by a gel former, the comb was inserted and the gel was allowed to set. The comb and surrounded cover were removed and the gel was soaked in a gel tank containing Tris- borate buffer. Five µl of the cDNA sample was added to the wells, and then the gel tank was covered by a lid. The gel was run at 90 Volt/cm for an hour; but for HEV electrophoresis 5 µl of the amplified products samples and DNA marker were loaded on 1.5% agarose gel without adding a loading-dye mixture because each AccuPower® PCR tube contained a tracking dye and precipitant, and electrophoresis was performed for 45 minutes at 100 V. Gel fluorescence was photo-documented through a UV light transilluminator.
- 60 -
Chapter Four
Results
Chapter Four
Results
4.1 Results : For the current study, both anti-IgG and IgM seropositivity of hepatitis A and E were studied among chemical bombardment survivors in different areas of Iraqi Kurdistan Region and studying the presence or absence of HAV and HEV RNA using conventional and nested PCR technique respectively. Moreover, 45 persons from distinctive areas also were submitted to the same tests and examinations performed for chemical survivors. These cases were regarded as the control group. Generally, it was noticed that the percentage of anti-IgG seropositivity was higher for both viruses than anti IgM seropositivity (Fig. -4.1-). Statistical analysis revealed that there were significant differences between exposures and controls regarding anti IgG and for both HAV and HEV (p= 0.0001 and 0.0002) respectively, whereas no noticeable differences were observed regarding anti-IgM for both HAV and HEV (p= 0.621 and 0.56) respectively. (Fig. -4.1-). Moreover, it was noticed that there were significant differences between anti-HAV IgG and anti-HEV IgG seropositivity among exposures (p= 0.000), similarly considerable differences were observed between anti-HAV IgM and anti-HEV IgM among exposures (p= 0.0055) ( Fig-4.1-). The percentage rates of RNA detection for HAV and HEV among both exposures and controls varied. The percentage rate of HAV RNA positivity was 15.68% , whereas no positive results were seen for HEV as well as among controls (Figs. -4.2; 4.3; and 4.4-).
-61-
Chapter Four
Results
100 90
% of positive results
80 70 60 50 40 30 20 10 0
Exposures Non-Exposures
HAV HEV Anti- IgG 100 61.79 86 36
HAV HEV Anti-IgM 9.89 1.08 8 2
HAV
HEV PCR
15.68 0
0 0
Figure-4.1- Percentage rates of Anti-IgG, anti-IgM and PCR for both HAV and HEV
Figure 4.2: Agarose gel electrophoresis showing the RT- PCR amplified products genes of HAV (Lane M and 21 : M = DNA marker, PC: positive control( 290bps) , lane 4 , lane 6, lane 7, lane10, lane11, lane13, lane16, and lane 18 were positive samples which were 290 bps.)
-62-
Chapter Four
Results
Figure -4.3- Agarose gel electrophoresis of first round nested RT- PCR showing no HEV amplified products genes (M = marker, 1-22 tested samples).
Figure -4.4- Agarose gel electrophoresis of second round nested RT- PCR using second set of primers and β- actin housekeeping gene 313 bps , showing no HEV amplified products genes.
-63-
Chapter Four
Results
When the results were analyzed statistically, it was noticed that there were significant differences between HAV RNA for both exposures and controls, unlike HEV RNA, which was negative for both tested groups (Table, 4.1) (Figs. -4.5 and -4.6-).
Table-4.1- Percentage of HAV and HEV nucleic acid detection among exposures and controls
% HAV Positive Tested groups
Tested No. PCR
Exposed cases
% HEV Positive
51
P-Value
15.68
PCR 0
p=0.000 Controls
45
P-Value
0
p= 1 0
The percentage rates of seropositivity for both HAV and HEV regarding anti-IgG, anti-IgM and PCR results also were different among distinctively chosen areas, although the rates, in general, were higher among total exposures in comparison with controls. All tested exposures in all areas were seropositive for anti HAV IgG, whereas the percentage was lower generally for anti-HEV IgG. The highest percentage was among exposures in Goptapa 92% while the lowest was in Sheikh Wasan 22.22% (Table, 4.2) (Fig.-4.5-, -4.6-). Regarding anti-IgM for both HAV and HEV, the percentage rate of seropositivity was higher for anti-HAV IgM than that of anti-HEV IgM, when the data were statistically analyzed, it was noticed that there were significant differences (p= 0.000) (Fig-4.5-). -64-
Chapter Four
Results
Moreover, when the obtained results were analyzed statistically, it appeared that anti-IgM seropositivity for HAV was meaningfully different among groups (p= 0.000), whereas the vast majority of anti-HEV IgM was negative, and no significant differences were recorded among the groups (p≥0. 5) except for exposures from Sheikh Wasan, which was statistically different from other areas (p = 0.000) (Table, 4.2) (Fig-4.5-).
Percentage of positve results
100 90 80 70 60 50 40 30 20 10 0
Halabja
Goptapa
Sewswnan
Balisan
Shex
Shanaxse
Controls
wasanan Anti-HAV IgG
100
100
100
100
100
100
86
anti HAV IgM
13.15
14.28
7.69
7.14
0
0
8
PCR
8.69
20
25
20
0
0
0
Figure-4.5- Seropositivity and RNAdetection for HAV among different groups of chemical exposures and controls
The result of PCR also was distinct among exposures in the studied areas, including both HAV and HEV. Statistical differences were observed among the obtained results from both viruses in all areas (p = 0.000), although all results for HEV was negative. Moreover, significant differences were found among exposures themselves in different areas regarding HAV depending on PCR results (p = 0.000) (Table -4.2- and Fig-4.6-). -65-
Chapter Four
Results
Table -4.2- Anti-IgG, IgM and nucleic acid detection for HAV and HEV for all tested exposures in different chemical bombed areas in Kurdistan.
Tested Areas No.
% HAV serpositivity and RNA
% HEV serpositivity and RNA
IgG
IgM
PCR
IgG
IgM
PCR
Halabja
38
100
13.15
8.69
63.15
0
0
Goptapa
14
100
14.28
20
92.85
0
0
Sewsenan
13
100
7.69
25
76.92
0
0
Balisan
14
100
7.14
20
35.71
0
0
Sheikh Wasan
9
100
0
0
22.22
11.11
0
Shanakhse
2
100
0
0
50
0
0
Controls
45
86
8
0
36
2
0
Percentage of positive results
100 90 80 70 60 50 40 30
20 10 0
anti HEV IgG anti- HEV IgM PCR
Halabja 63.15 0 0
Goptapa Sewswna n 92.85 76.92 0 0 0 0
Balisan 35.71 0 0
Shex Shanaxse Controls wasanan 22.22 50 36 11.11 0 2 0 0 0
Figure-4.6- Seropositivity and RNA detection for HEV among different groups of chemical exposures and controls -66-
Chapter Four
Results
When statistical analysis was performed, it was noticed that only lymphocytes were significantly different between HAV seropositive and seronegative exposures from studied areas (p ‹ 0.05), whereas no significant differences were observed among all other obtained hematologic and liver function test results from HAV seropositive and seronegative exposures (p › 0.05) (Tables, 4.3 and 4.4). Moreover, it was noticed that there were no significant effects of the studied areas on the hematological parameters for HAV seropositive exposures (p=0. 99). At the same time, it was appeared that some exposures were positive for anti-HAV IgG and PCR 8.9%, moreover, some exposure cases were positive for anti-HEV IgG and HAV PCR at the same time 10.9% of those who were anti-HEV IgG) (Fig.-4.7-). In the current study, the effect of the geographical location (areas) was also significant on liver function tests (p=0.038), the liver function test results among HAV seropositive exposures were significantly characteristic regarding
distinguishing
selected
areas
of
chemical
bombardment
(Table, 4.3). Among HAV seronegative exposures no significant effects of geographic locations (areas) were observed using Chi-square test (p › 0.05), although as in the case of hematologic parameters, it was found that the liver function test results were significantly unusual among HAV seronegative exposures in distinctive studied areas (p= 0.0002) (Table, 4.4)
-67-
Chapter Four
Results
100 90 80 Positive percentages %
70 60 50 40 30 20
10 0
anti-HAV
anti
antiHAV
anti-HEV
anti-HEV
anti-HEV
antiHEV
IgG
HAVIgG=
IgM=PCR
IgG
IgG+PCR
IgM+PCR
IgG+HAV
PCR % Positive
100
PCR
8.9
0
61.79
0
0
10.9
Figure-4.7- Interactions of seropositivity and PCR results for HAV and HEV among exposures
Table- 4.3- Hematological and liver function tests of HAV seropositive
AST
TSB
7.39
2.2
4.5
214
181.58
17.2
16.6
0.86
Goptapa
14
5.0
14.9
7.04
2.2
4.3
204
196.85
22.2
10.1
0.74
Sewsenan
13
5.2
13.3
8.17
2.8
4.9
214
85.230
16.3
16.6
0.48
Balisan
14
5.0
14.4
10.5
2.4
7.4
248
169.3
25.7
10.0
0.77
SheikWasan
9
4.9
13.7
8.37
2.2
5.6
259
87.4
25.8
13.6
0.82
Shanakhse
2
5.3
14.4
8.75
2.0
6.3
156.
146.45
27.1
29
0.78
-68-
ALP
13.3
PLT
4.6
GRA
38
LYM
Halabja
RBC
ALT
Liver function tests
WBC
Mean Hematologic parameters HGB
Areas
Tested No
exposures in different areas
Chapter Four
Results
Table- 4.4- Hematological and liver function tests of HAV seronegative
4.9
13.4
7.6
2.2
4.7
257
183.7
12.5
15.8
0.6
Goptapa
3
5.1
15.5
8.2
2.01
5.8
251
190
26
21.6
0.76
Sewsenan
3
5.0
13.4
6.5
2.3
3.9
235
85
24.3
18.6
0.2
Balisan
2
5.0
14.4
6.85
1.95
4.35
219
92
22.5
7
0.7
SheikhWasan
1
4.4
12.3
7
1.9
4.6
215
170
18
8
0.8
TSB
70
ALP
Halabja
PLT
AST
Liver function tests
ALT
GRA
LYM
HGB
WBC
Mean Hematologic parameters
RBC
Areas
Tested No
exposures in different areas
When the obtained parameters from HEV seropositive and seronegative exposures were statistically analyzed, it appeared that there were no significant differences between hematological and liver function test results from both HEV seropositive and seronegative exposures (p › 0.05) for each parameter; also no significant effects of geographic areas (studied areas) were seen on the haematological results (p = 0.499), whereas it was noticed that liver function tests from seropositive exposures from unusual studied areas were significantly distinctive (p = 0.0005) (Tables, 4.5). Similarly, significant differences were observed between liver function test results from HEV seronegative exposures when the Chi-square test was depended for the analysis (p = 0.00021) (Tables, 4.6).
-69-
Chapter Four
Results
Table- 4.5- Hematological and liver function tests of HEV seropositive exposures in different areas
Areas
RBC
HGB
WBC
LYM
GRA
PLT
ALP
ALT
AST
TSB
Liver function tests
Tested No
Mean Hematologic parameters
Halabja
38
4.5
13.2
7.44
2.3
4.5
215
191.55
17.2
15.3
0.84
Goptapa
14
4.9
14.8
7
2.2
4.3
205
183
22.4
10.2
0.7
Sewsenan
13
5
13.2
8.5
2.9
5.1
223
83.7
13.1
16.4
0.5
Balisan
14
5.1
14.9
8.4
2.3
5.4
254
174.56
32.2
11.2
0.84
Sheikh Wasan
9
5.2
14.8
13.7
2.4
10.5
390
101.75
30.5
17
1.1
Shanakhse
2
4.9
15.8
10.8
2.7
7.86
138
146.45
34
26
0.78
Comparisons of the obtained hematologic and liver function tests also were performed for HAV PCR positive and negative exposures. It was concluded that there were no noticeable differences between obtained hematologic and liver function test results from HAV PCR positive and HAV PCR negative exposures (p › 0.05) (Tables, 4.7 and 4.8). Moreover, it was concluded that there were no significant effects of the selected studied areas on the hematological parameters from HAV PCR exposures (p= 0.984), whereas significant differences were seen among HAV PCR positive exposures with regard to liver function test results (p= 0.0186) (Table, 4.7).
-70-
Chapter Four
Results
Table- 4.6- Hematological and liver function tests of HEV seronegative exposures in different areas
Areas
RBC
HGB
WBC
LYM
GRA
PLT
ALP
ALT
AST
TSB
Liver function tests
Tested No
Mean Hematologic parameters
Halabja
38
4.81
13.4
7.14
2.0
4.45
214
164
17.2
20.3
0.9
Goptapa
14
5.20
15.8
7.98
2.2
5.46
234
235
24.5
18.5
0.9
Sewsenan
13
5.45
13.6
6.81
2.3
4.16
209
87.6
25.8
18
0.3
Balisan
14
5.06
14.1
10.8
2.4
7.75
239
152
22.1
9
0.7
SheikhWasan
9
4.77
13.2
6.65
2.1
4.05
215
95.1
23.4
11.8
0.7
Shanakhse
2
5.86
13
6.7
1.4
4.8
175
206
20.3
32
1.2
Similarly, no statistically significant differences were seen in hematological parameters for HAV-PCR positives exposures in different studied areas (p= 0.998), while as among PCR positive cases, significant differences were observed in liver function test results from different studied areas (p= 0.0108) (Table, 4.8). Regarding HEV PCR results, although there were no positive results, it was noticed that studied areas had no significant effect on the hematological parameters among HEV PCR negative exposures (p › 0.05). However, the results of
liver function tests showed significant differences among
exposures from different studied areas (p= 0.0038) (Table, 4.9). -71-
Chapter Four
Results
Table- 4.7- Hematological and liver function tests of HAV-PCR positive exposures in different areas
Areas
RBC
HGB
WBC
LYM
GRA
PLT
ALP
ALT
AST
TSB
Liver function tests
Tested No
Mean Hematologic parameters
Halabja
38
4.62
14
7.6
3.05
3.75
244
185
18
14.5
0.7
Goptapa
14
4.92
15.2
6.42
1.76
4.28
148
184
11
7
0.9
Sewsenan
13
5.19
12.7
7.15
2.39
4.4
214
88.6
12.6
12.3
0.2
Balisan
14
5.44
13.5
6.8
1.8
4.4
262
85.2
18
5
0.6
Table-4.8 - Hematological and liver function tests of HAV-PCR negative exposures in different areas
Areas
RBC
HGB
WBC
LYM
GRA
PLT
ALP
ALT
AST
TSB
Liver function tests
Tested No
Mean Hematologic parameters
Halabja
38
4.70
13.5
7.18
2.27
4.38
221
155
11.6
13.9
0.76
Goptapa
14
5.0
15.4
7.09
2.39
4.36
214
202.
25.5
11.2
0.72
Sewsenan
13
5.13
12.9
7.81
2.76
4.70
212
80.3
13.5
15.3
0.54
Balisan
14
4.96
14.4
12.7
2.55
9.52
230
177
21
15.2
0.82
Sheikh Wasan
9
5.11
14.2
7.88
2.2
5.18
198
110
26.2
16.6
0.96
Shanakhse
2
5.39
14.4
8.75
2.09
6.33
156
146
27.1
29
0.78
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Chapter Four
Results
Table-4.9 - Hematological and liver function tests of HEV-PCR negative exposures in different areas
WBC
LYM
GRA
PLT
ALP
AST
TSB
Halabja
38
4.70
13.2
7.26
2.23
4.46
224
178
16
16.1
0.8
Goptapa
14
5.06
15.0
7.17
2.34
4.41
201
197
22.9
10.4
0.7
Sewsenan
13
5.30
13.3
8.08
2.82
4.87
213
87.5
17.2
16.2
0.5
Balisan
14
5.0
14.4
10.1
2.42
7.04
244
159
25.3
9.68
0.8
Sheikh Wasan
9
4.88
13.5
8.22
2.23
5.48
254
96.5
25
13
0.8
Shanakhse
2
5.39
14.4
8.75
2.09
6.33
175
146
27.1
29
0.8
ALT
HGB
Liver function tests
RBC
Areas
Tested No
Mean Hematologic parameters
It was noticed that a relatively high percentage rates of exposures with HAV and HEV seropositivity, as well as those who showed PCR positive results, were suffering from lymphocyte number (count) abnormalities (Either higher or lower than the normal range. The higher percentage of lymphocyte abnormalities were among exposures with anti-HAV IgM seropositive results followed by those who were anti-HAV IgG seropositive and then exposures with anti-HEV IgG positive results (Fig. -4.8-). Generally, liver function test result abnormalities also were seen among chemical exposures with PCR and seropositive results for both HAV and HEV. The highest percentage of abnormalities were within the ALP 34.5% followed by ALT and TSB 19.97% and 18.18% respectively (Fig. -4.9-).
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Chapter Four
Results
Percentage op positive %
35 30 25 20 15 10 5 0 anti-HAV IgG
anti-HAV IgM
HAV RNA
anti-HEV IgG
Lymphocytes % High
3.37
0
0
5.45
Lymphocytes % Low
24.7
33.3
0
21.81
Lymphocytes % Total abnormal
28.07
33.3
0
27.26
Figure -4.8- Lymphocyte abnormalities among HAV and HEV PCR and seropositive exposures
% of positive results
70 60 50 40 30 20 10 0
anti-HAV IgG +ve anti-HAV IgM +VE HAV RNA +VE anti-HEV IgG +VE
ALT 22.2 22 0 19.97
AST ALP Liver function tests 11 37.9 11.1 66.5 0 12.5 0 34.5
TSB 14.5 0 37.5 18.18
Figure -4.9- Liver function test result abnormalities among HAV, HEV PCR and seropositive exposures
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Chapter Five
Discussions
Chapter Five
Discussions
5.1 ELISA Results (anti- Hepatitis A and E IgG and IgM detection): The most commonly used procedures in the diagnosis of HAV and HEV infections are enzyme immunoassays for detecting IgG and IgM in serum samples,
although,
immune
assays
often
have
limited
sensitivity
(Wang et al., 2001; Innis et al., 2002), as these techniques cannot be used for genotype determination and, therefore, provide limited information for epidemiological studies. The diagnosis of hepatitis A and E is usually made serologically by commercial ELISA kits; however, limitations for serodiagnosis of these viruses may exist due to various envelope antigens in diagnostic kits of various origins; especially different HEV genotypes result in different antibody response to corresponding antigens, which make results from different ELISA kits liable to be different (Chen et al., 2005). In this study, all tested exposures were seropositive for anti-HAV IgG, whereas the percentage rates of anti-IgM seropositivity were very low 9.89% in comparison with the anti-IgG. The higher seropositivity rates of hepatitis A virus, especially anti-HAV IgG, indicates past infection by this virus due to several factors, including water sanitation and supply as HAV transmits through oral-fecal routes. The areas under study unfortunately had no perfect water supply with international standards, which made the possibility of infectious viral hepatitis (HAV and HEV) more. Other factors behind higher percentage rates may be due to another health complaints such as defects in their immune system (Hama et al., 2008) and psychological problems as all inhabitants of chemical exposed areas have instability in their psychological conditions due to
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Chapter Five
Discussions
long-term effects of chemicals. The vast majority of the Iraqi Kurdistan Region was exposed to massive destruction in 1980s due to Iraq-Iran war and the attacks of Sadam’s regime on the Kurdish villages, towns and cities. All these resulted in destruction of all service projects, including water supply projects
in
these
areas;
this
led
to
unhygienic
conditions
and
contamination of drinking water, which may be behind the higher percentage rates of anti-HAV and anti- HEV IgG seropositivity among tested chemical exposures. There are two important facts that can explain the obtained results; firstly, all tested exposures were from different areas that exposed to destruction and massacres; and secondly, there were no considerable differences between tested exposures in different areas, i.e., there were no important effects of the study areas on the rates of seropositivity (p ‹ 0.05). In countries with poor sanitary conditions, HAV infection is highly endemic. Household crowding, poor levels of sanitation, and inadequate water supplies contribute to the propagation of infection within communities in countries such as Africa, Asia, central and south America. Most individuals become infected within the first few years of life and are asymptomatic; therefore, reported rates of infection are low (Shaprio and Margolis, 1993). The similarities of the current results may be due to the mentioned risk factors which are common in Kurdistan Region, and especially in the studied areas. IgM indicates recent or acute infections, the lower rates of anti-HAV and anti-HEV IgM seropositivity indicated that the vast majority of examined exposures had no acute hepatitis A and E infection. This fact also may indicate the slight positive changes in the hygienic education levels of most inhabitants, rather than improvements in the water-supply projects. The
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Chapter Five
Discussions
principles of hygiene are observed in projects of drinking water supply but in their minimum level. Studies done in Iraq indicate that the percentage rates of anti-HAV IgG seropositivity were comparatively higher than that of Hepatitis E. Among patients with a clinical diagnosis of acute viral hepatitis, two fifths had serologic evidence of type A and another one fifth had type E viral hepatitis (Turkey et al., 2011). This means that the rates of HAV and HEV infection are reasonably high in Iraq. The results of the current study are similar to these observations. It was reported that anti-HAV IgG seropositivity was 96.4%, while that of Hepatitis E-IgG antibodies was 20.3 % (Turkey, 2011). The conclusions of the present study are in agreement with those of pervious investigations. In the current study, it was concluded that the percentage rates of anti-HAV IgM was higher than that of HEV, which was similar to results reported in previous studies done by Turkey and his colleagues in 2011. Another study among patients with suspected acute viral hepatitis in Baghdad-Iraq reported that more than two-fifth 44.8% of cases were positive for anti HAV IgM antibodies and 1.6 % showed positive anti-HEV IgM antibodies (Al-Naaimi et al., 2012), which is similar to the results obtained by this study but the current study among chemical
weapon
exposure which is due to many risk factors and immunologic defects among survivors of the chemical weapon exposure make the possibility of infections higher than the normal as it is known that defects in cell-mediated immunity increase the risk of viral infections.
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Chapter Five
Discussions
In a study done in India, it was noticed that about 18.8% showed anti-HEV IgM seropositivity (Mishet al., 2003). The study of HAV prevalence in adults of EMRO Countries show that 100% of residents of the Nile delta were shown to be anti-HAV seropositive in 1996 (Darwish , 1996); moreover, in a relatively new published study on samples all over Iranian, 86% of the studied population aging 18- 65 years were discovered to be anti-HAV seropositive (Merat et al., 2010), similarities can be observed between the results obtained by the present study and the conclusions recorded by the above investigators. Another study conducted in 2008 from Isfahan province indicate that about 10% of the population had positive results for anti-HAV (Ataei et al., 2008), while the rates were higher for about three times among studies, population in Mazandaran province in regard to anti-HAV seropositivity (Nassrolahei and Khalilian, 2004). These observations are different from those of the current study. Other investigators in Saudi Arabia report higher percentage rates of antiHAV seropositivity incomparable to the earlier Iranian studies. It was reported that about 70% were seropositive (Almuneef et al., 2006); whereas, about 90% anti-HAV seropositivity were observed among a group of healthy populations of Eastern Saudi in a proceeding study (Fathalla et al., 2000). Moreover, in other studies in the Middle East, various results were recorded. In a study done in 2005 in Lebanon, about 80% of the population older than 21 years showed positive findings for anti-HAV IgG (Sacyet al., 2005), whereas about 90% of Syrians showed to be anti-HAV seropositive in 2000 (Antaki et al., 2000).
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Chapter Five
Discussions
Unlike these finding, lower rates of anti-HAV seropositivity are recorded in Kuwait, only about 30% appear to be seropositive (Alkhalidi et al., 2009). In the same year and in a study done in Turkey, it is reported that the Northern Cyprus appeared to be with an intermediate endemicity of HAV infection. It is clarified that the seropositivity of anti-HAV was related to the age of the studied groups, the rate increases with increasing age (Zafer-Kurugol et al., 2009). The conclusions of the present study are in agreement with these observations, although there are differences in the percentage of seropositivity results. Studies in experimentally infected macaques reported that serum anti-HEV immunoglobulin G (IgG) appeared around 3-4 weeks post-inoculation at the peak of ALT elevation. A human volunteer study shows anti-HEV IgM to peak in the symptomatic period and then decline to baseline within three to six months of illness. Serum anti-HEV IgG levels continued to rise during the symptomatic phase and became detectable in the convalescent phase for two years (Zhu et al., 2008; Meng, 2010). These conclusions are generally similar to observations recorded in the current study.
5.2 Molecular detection of HAV and HEV Polymerase chain reaction is known to be a sensitive and specific technique for the detection of active viremic stage of all hepatitis patients. All the HAV and HEV seropositive samples were further subjected to total RNA extraction and RT-PCR. Positive results among cases showing seropositivity for HAV, although the percentage was low. This means that most of the exposures had no HAV active RNA in their blood (no viremic), which indicates either
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Chapter Five
Discussions
negative results or the infection did not reach the viremic stage, otherwise, sometime, production of anti-RNA antibodies may impair the detection of virus nucleic acid. However, no positive PCR results were recorded for HEV, which means that the percentage rates of active HEV infections (indicated by viremic stage) did not exist or at least was not detected, as in the case of serology test results, which were lower than that of HAV results, no HEV RNA was detected. RNA was normally detected in both serum and stool in late latent period and early acute infection of patients with HAV and HEV. In some cases with negative results of anti-IgM , the RNA was positive, as the RNA was generally detected in early acute infection of patients with infection, and tended to decline to an undetectable level during the course of the disease progressing. On the other hand, since anti-IgM might not reach the detectable level in early infection stage, depending mainly on serological tests, it could misdiagnose cases with early viremia before seroconversion. Instead, positive results of nucleic acid would be more helpful for early diagnosis (Liu et al., 2013). The molecular test results clarified that it is not necessarily seropositive samples that can be also positive for PCR or vice versa. This fact confirms that it is not true to confirm seropositive results by PCR and sometimes serologic tests may be more beneficial, especially for HEV infections. Moreover, performing of real time PCR may be the gold standard here for avoiding false positive or false negative results, especially these viral infections are infectious and can be easily transmitted by fecal-oral routes, as
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Chapter Five
Discussions
real time -PCR can detect the minimum amount of the viral nucleic acid and minimize contamination (Pelosi and Clarke, 2008); it also can clarify the life cycle state of the virus. This test helps early diagnosis and as a result it helps in controlling of infection spreading. Although in the current study for the detection of HEV RNA nested PCR was used, but no positive results were recorded. This method is a modification of reverse transcription PCR, which aims to eliminate unspecific amplification in the first round RT-PCR due to the unexpected primer binding site, while RT-PCR is a process where RNA is reverse transcribed into DNA and subsequent exponential amplification of the resultant DNA (Yong and Son, 2009). It was concluded that the detection of HEV RNA in serum or stool using nested or real time PCR is the most sensitive and definitive diagnostic test; however, the viremic period is short (10 to 30 days after the onset of symptoms) and the detection of HEV RNA within the proper time of diagnosis in the clinical setting was not easy, while fecal shedding of virus may last longer with a high viral titer compared with viremia in the blood (Inoue et al., 2006). Some difficulties are also recorded by other investigators who were trying to study and detect HEV using molecular techniques including conventional amplification of nucleic acid by PCR. They report that Hepatitis E viremia can be detected before the onset of liver abnormality, which is accompanied by humoral immune reaction. Moreover, since there is no robust system to grow HEV in culture, there are some limitations in the field of HEV study (Chandra et al., 2008).
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Chapter Five
Discussions
Other factors may be behind the molecular results of the current study as HEV of different genotypes were genetically very heterogeneous on the nucleotide level, some universal primers are designed, which can detect HEV strains from all four known genotypes (Cooper et al., 2005; Inoue et al., 2006). This set of RT-PCR primers used here belongs to one of them and have been used in many previous studies. Finally, HEV RNA was usually detected in early acute infection of patients with hepatitis E infection, and tended to decline to an undetectable level during the course of the disease progressing, and since anti-HEV, IgM might not reach the detectable level in early infection stage, false-negative records may occur.
5.3 Hematological and liver function tests results. Clinical investigation and medical reports reveal that all survivors suffered from different health complaints due to the long-lasting effects of chemicals used in bombing the studied areas by Sadam’s regime. It was noticed in previous studies that respiratory, ophthalmological and dermatological complaints were common among exposures (Hama et al., 2008.), and most of the survivors were suffering from an interaction of the three mentioned complaints. Survivors in studied areas, until now, are suffering from a variety of health complaints from mild to severe and fatal ones. It was recorded by the current study through their medical reports that exposures to have respiratory complain including shorten breath, cough, expectorates due to chronic bronchitis, lung diseases, and asthma. The increasing incidence of chronic
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Chapter Five
Discussions
respiratory tract problems among survivors can be strongly related to Sulfur Mustard (SM) attack, which was used against innocent inhabitants of those cities, while dermatological problems including chronic eczema, and other allergic diseases were due to the direct contact of SM and other chemicals. Some of the hematologic results reported in the current study are similar to observations recorded by other investigators (Davidsohn and Nelson, 1974) who observed polycythemia, lymphocytopenia and thrombocytopenia. The wide range of changes seen in the total leukocyte count of survivors might be due to the reduction in neutrophils or lymphocytes that comprise a large ratio of total leukocytes. As a result, exposed cases with a low total leukocyte count were suffering from neutropenia, sub-normal lymphocytes, or both. Several factors may cause leukopenia including a reduced flow of leukocytes (neutrophils) from the bone marrow into the peripheral blood, due to the lack of production or ineffective production, the increased removal of leukocytes from the blood, a change in distribution between the circulating granulocyte pool and the marginal granulocyte pool and, sometimes, a combination of these factors (Davidsohn and Nelson, 1974). Moreover, some other hematologic changes among exposures are the same as those reported by Hama et al. (2008), especially changes in lymphocyte counts, these because of the long-lasting effects of mustard compounds, which may be the main factor behind the changes in leukocyte levels. Alexander, in 1947, reported the effect of SM on the leukocytes in the circulating blood of humans and found severe toxic effects causing leukopenia. He also noted that lymphocytes were the first to disappear, followed by granulocytes, which were severely affected.
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Chapter Five
Discussions
In 1991, Dean and Murray reported that mustard was a leukocytic toxin acting on the bone marrow with myelotoxicity causing leukopenia, pancytopenia, anemia and plastic or hypoplastic bone marrow in experimental animals. The reduction in the total leukocyte, neutrophil and lymphocyte counts among chemical survivors in bombarded areas may, therefore, be due to the severe effects of long-lasting effects of mustard compounds. The results of liver function tests indicated that most studied cases were within elevated values rather than the normal range, which were similar to observations recorded by other investigators (Becker and Stauffer 1982). As with the clinical symptoms and signs, there were no pathognomonic findings in the laboratory investigations that distinguish HAV from other hepatotropic viruses, although the elevation of aminotransferase serum levels roughly correlated to the severity of the acute hepatitis A or E among asymptomatic cases or some other factors may be responsible. The overall severity of the infection, however, is demonstrated by the bilirubin level (Cuthbert, 2001).
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Chapter Six Conclusions and Recomendations
Chapter Six
Conclusions and Recommendations
6.1 Conclusions From the current study, the following points are concluded: 1. The percentage of anti-HAV and HEV IgG seropositivity is higher than that of anti-HAV and HEV IgM. 2. The percentage rate of HAV RNA positivity is 15.68%, whereas no positive results are seen for HEV as well as among controls. Significant differences are found between exposures themselves in different areas regarding HAV depending on PCR results. 3. It is noticed that only lymphocytes are significantly different between HAV seropositive and seronegative exposures from studying areas. Exposures with HAV and HEV seropositivity as well as those who showed PCR positive results, suffer from low lymphocyte numbers (Lymphopenia). 4. Some exposures are positive for anti-HAV IgG and PCR (8.9%), moreover, some exposure cases are positive for anti-HEV IgG and HAV PCR at the same time (10.9% of those who are anti-HEV IgG). 5. Liver function test result abnormalities are seen among exposures with PCR and seropositive results for both HAV and HEV, and the highest percentage of abnormalities are within ALP followed by ALT and TSB respectively. 6. Both molecular and serological methods are important for HAV and HEV diagnosis, so they could not be replaced by each other for detection.
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Chapter Six
Conclusions and recommendations
6. 2 Recommendations In the current study, the following recommendations are made: 1. Using molecular diagnostic detection in the health centers, especially in chemical exposed areas and supplying newly available diagnostic equipment like ELISA and PCR machines to enable them early and correct diagnosis of different infections, including HAV and HEV infections, 2. Hepatitis E virus should be considered during dealing with patients suffering from hepatitis, 3. Controlling drinking water and water supplying projects through collaboration between related sectors to work together to supply safe water and safe sewage disposal, especially in chemical exposed cities and villages, 4. Hygienic education and vaccination will be the gold standard to minimize and controlling infectious hepatitis (A and E), which can be transmitted through fecal-oral routes.
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References
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IgG , IgM E A
1995
7 5
5
RNAIgMIgG E A Conventional and Nested PCR ELISA Auto analyzer A E EAIgG AIgM E p=E A IgG p= IgM p= IgM IgG
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ر ﺎﻟﺔ ﻓﺎ ﻲ ا ﻮم و ﺮ ﺔ ا ﻮم ﺪ ﺔاﻰ ﺎ ﺔ ﻮل ا ﻮم ﻓﻲ ﺎ ﺔ ا ﺷﮭﺎدة ﻄ ﺎت ﺰء ﺮ ﻓﻲ ﻮم ا ﺎة ﺎ ) ا ﺎء ا ﮭﺮ ﺔ(
ﻮ ا ﺪ ﺰﺰ ﺎ ﻮر ﻮس ﻮم ا ﺎة ) ،(1995ﺎ ﺔ ﺻ ح ا ﺪ د ﻮم ا ﺎ ﻲ ﻓﻲ ا
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ا ﻮع Eﺎ
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ﺮو ﺎت
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.(P< ,
ا ﺎ
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ا
ﺮا ﻲ
ا
ﺮا ﻲ
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اﺬ
ا ﻮ ﺔ
RNAﺎ ﺮس ﻮع A ھﺬه ا
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ا ﻮ ﺔ
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ا
اظﮭﺮوا ا ﺎ
اظﮭﺮوا ا ﺎ
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ﻰ ا ﺰ ﺎت ا ﺪ ﺔ ) اظﮭﺮوا ا ﺎ
ا ﺎ
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ﺪاد IgGﺎ ﺮس ﻮع ) A
ﻲ درا ﺔ ﺎ ﺮ ا ﻮز ﻮز
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ا ﻮ ﺔ
ا
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. ( P= ,ﺬ
ﻮ ﻮح ا ﺎ ﺮ ا ﻮ ﻮح ﺎن ا
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ﺪاد IgMا ﺎص ﺎ ﺮس ﻮع Aﮭﺎ ا ﻮ ﺔ
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ﺪاد IgGا ﺎص ﺎ ﺮس ﻮع Eﻲ ا ﺮ ﺔ ا ﺎ ﺔ .
