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PROJECT TOPIC AND MATERIAL ON A SURVEY FOR RUBELLA VIRUS AND ANTIBODIES AMONG PREGNANT WOMEN IN KADUNA STATE NIGERIA

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  • Name: A SURVEY FOR RUBELLA VIRUS AND ANTIBODIES AMONG PREGNANT WOMEN IN KADUNA STATE NIGERIA
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ABSTRACT

Rubella Virus (RV) causes a mild disease, but maternal infection early in pregnancy often leads to birth defects known as congenital rubella syndrome (CRS). Rubella remains poorly controlled in Africa despite being a vaccine preventable disease. The aim of this study were to determine the survey for rubella virus and antibodies among pregnant women in Kaduna State Nigeria. The study wascarried out on pregnant womenattending ante-natal clinic in three different senatorial district in Kaduna State from the year 2013-2014.Bloodserum samples from 900 pregnant women were screened for rubella IgM and IgG antibody usingenzyme linked immunosorbent assay (ELISA).The risk factors associated with rubella virus transmission among these pregnant women were identified. Reverse Transcription Polymerase Chain Reaction (RT-PCR), and gene sequencing were used to confirm the presence of rubella virus in the serum sample.Of the 900 pregnant women screened, 572(63.1%) were positive for rubella IgG and 39(4.3%) were positive for rubella IgM. The prevalence of rubella IgG was highest among the age group 41-45 years old (83.3%) and IgM was highest among the age group 21-25 years old (6.1%).The IgG test results shows that out of the 497 pregnant women enrolled in their first trimester,273 (54.9%) pregnant women tested positive,while the IgM positive results shows 24(4.8%) in their first trimester. Although the southern senatorial district had the highest sero-prevalence 14(35.9%) among the three centres, the differences were not statistically significant (p=0.05). Only three people claimed to have been vaccinated against rubella virus and these people were negative for rubella. Acquisition of secondary education and being a house wife were insignificantly associated (p=0.05).The serological evidence of rubella virus found in pregnant women in this study is an indication that rubella is prevalent in Kaduna State, Nigeria. It is however still necessary to immunize seronegative women against rubella before they get pregnant.

TABLE OF CONTENTS

DECLARATION ………………………………………………………………………………………………………………. i
CERTIFICATION ……………………………………………………………………………………………………………. ii
DEDICATION ………………………………………………………………………………………………………………. iii
ACKNOWLEDGEMENTS ………………………………………………………………………………………………… iv
ABSTRACT …………………………………………………………………………………………………………………… v
TABLE OF CONTENTS ……………………………………………………………………………………………………. vi
LIST OF TABLES …………………………………………………………………………………………………………… ix
LIST OF PLATES …………………………………………………………………………………………………………….. x
LIST OF FIGURES ………………………………………………………………………………………………………….. xi
2.1: Genome Cell Structure of Rubella Virus………………………………………… ………………………………… xi
LIST OF APPENDICES ……………………………………………………………………………………………………. xii
LIST OFABBREVIATIONS ……………………………………………………………………………………………… xiv
CHAPTER ONE ……………………………………………………………………………………………………………… 1
1.0 INTRODUCTION ………………………………………………………………………………………………………. 1
1.1Rubella Virus ……………………………………………………………………………………………………… 1
1.2 Statement of the Research Problem ……………………………………………………………………… 5
1.3 Justification ……………………………………………………………………………………………………… 6
1.4 Research Question ………………………………………………………………………………………………. 7
1.5 Aim …………………………………………………………………………………………………………………. 7
1.6 Specific Objectives ………………………………………………………………………………………………. 8
CHAPTER TWO …………………………………………………………………………………………………………….. 9
2.0 LITERATURE REVIEW ………………………………………………………………………………………………… 9
2.1 History of Rubella Virus …………………………………………………………………………………….. 9
2.2 Genome and Cell Structure of Rubella Virus ……………………………………………………….. 10
2.2.1 Genome Organization …………………………………………………………………………………………. 10
2.2.2The Capsid Protein ………………………………………………………………………………………………. 13
2.2.3 The E1 and E2 Glycoproteins …………………………………………………………………………………. 13
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2.3 Post-Natal and Congenital Rubella Viral Infections ………………………………………………. 15
2.3.1 Postnatally Acquired Rubella ……………………………………………………………………………….. 15
2.3.1.1 Clinical Features of Postnatally Acquired Rubella Virus …………………………………………… 16
2.3.1.2 Complications from Postnatally Acquired Rubella Virus …………………………………………. 16
2.3.2 Congenital Rubella ……………………………………………………………………………………….. 17
2.3.2.1 Risk to the Fetus ……………………………………………………………………………………………… 17
2.3.2.2 Clinical Features of Rubella Virus ………………………………………………………………………… 18
2.4 Rubella Virus link to Teratogenicity ……………………………………………………………………… 19
2.4.1 Mitochondrial Changes ……………………………………………………………………………………….. 20
2.4.2 Cytoskeletal Changes ………………………………………………………………………………………….. 22
2.4.3 Apoptosis …………………………………………………………………………………………………………. 23
2.5 Life Cycle of Rubella Virus …………………………………………………………………………………. 26
2.5.1 Pathogenesis of Rubella Virus ………………………………………………………………………………. 26
2.6 Role of Cellular Proteins in RV Replication…………………………………………………………… 32
2.7 Replication Complexes ………………………………………………………………………………………. 34
2.8 Cellular Changes in RV-Infected Cells …………………………………………………………………. 35
2.9 Summary of Rubella Virus Life Cycle………………………………………………………………………….. 40
2.10 Rubella Re-Infection ………………………………………………………………………………………… 41
2.11 Epidemiology ………………………………………………………………………………………………….. 43
2.12 Laboratory Diagnosis ………………………………………………………………………………………. 44
2.13 Diagnosis of Rubella Congenital Acquired Infection ……………………………………………… 47
2.14 Prenatal Diagnosis of Congenital Rubella Infection ………………………………………………. 48
2.15 Prevention ……………………………………………………………………………………………………… 48
2.15.1 Vaccination Policies …………………………………………………………………………………………… 48
2.15.1.1 Two main policies were initially used …………………………………………………………………. 48
2.16 Immune Response ……………………………………………………………………………………………. 50
2.17 Reinfection ……………………………………………………………………………………………………… 52
2.18 Passive Immunization ………………………………………………………………………………………. 52
2.19 WHO recommendation …………………………………………………………………………………….. 52
CHAPTER THREE …………………………………………………………………………………………………………. 55
3.0 MATERIALS AND METHODS …………………………………………………………………………………….. 55
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3.1 Study Area……………………………………………………………………………………………………….. 55
3.2 Study Design …………………………………………………………………………………………………….. 57
3.3 Study Population ………………………………………………………………………………………………. 58
3.4 Inclusion Criteria ……………………………………………………………………………………………… 58
3.5 Exclusion Criteria……………………………………………………………………………………………… 58
3.6 EthicalApproval ……………………………………………………………………………………………….. 58
3.7 Sample Size Determination ………………………………………………………………………………… 59
3.8 Data Collection Using Questionnaire ……………………………………………………………………. 59
3.9 Collection of Samples…………………………………………………………………………………………. 60
3.10 Laboratory Analysis of Rubella Virus ……………………………………………………………….. 61
3.10.1 Procedure for Elisa Serum Analysis. ………………………………………………………………………. 61
3.11 Reverse Transcription Polymerase Chain Reaction (rtPCR). …………………………………. 62
3.12 RNA Extraction of Rubella Virus ………………………………………………………………………. 62
3.13 Reverse Transcription (RT) of Rubella Virus ……………………………………………………………… 63
3.14 Amplification and detection by RT-Polymerase Chain Reaction (RT-PCR) ………………………… 63
3.15 Sequencing of Rubella Virus …………………………………………………………………………………… 64
3.15.1 Nested-set RT-PCR for Sequencing. ………………………………………………………………………. 64
3.16 Statistical Analysis of the data …………………………………………………………………………… 65
CHAPTER FOUR ………………………………………………………………………………………………………….. 66
4.0 RESULTS …………………………………………………………………………………………………………….. 66
CHAPTER FIVE ……………………………………………………………………………………………………………. 86
4.0 DISCUSSION …………………………………………………………………………………………………….. 86
CHAPTER SIX ……………………………………………………………………………………………………………… 95
6.0 CONCLUSION AND RECOMMENDATIONS …………………………………………………………………… 95
6.1 CONCLUSION ………………………………………………………………………………………………… 95
6.2 RECOMMENDATIONS ……………………………………………………………………………………. 95
REFERENCES ……………………………………………………………………………………………………………… 97
APPENDIX I ……………………………………………………………………………………………………………… 111
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LIST OF TABLES
Table Title Page 4.1: Sero-Prevalence of Rubella Antibodies AmongPregnant Women Attending Ante-Natal Clinic in Kaduna State Based on Age…………………………………………………………………..68 4.2: Sero-Prevalence of Rubella Antibodies AmongPregnant Women Attending Ante-Natal Clinic in Kaduna State Based on Educational Status……………………………………………..69 4.3: Sero-Prevalence of Rubella Antibodies Among Pregnant Women Attending Ante-Natal Clinic in Kaduna State Based on Occupation………………………………………………………..70 4.4:Sero-Prevalence of Rubella Antibodies Among Pregnant Women Attending Ante-Natal clinic in Kaduna State Based on Marital Status………………………………………………………71 4.5: Sero-Prevalence of Rubella Antibodies Among Pregnant Women Attending Ante-Natal Clinic in Kaduna State Based on Vaccination Status……………………………………………..72 4.6: Sero-Prevalence of Rubella Antibodies Among Pregnant Women Attending Ante-Natal Clinic in Kaduna State Based on the three senatorial districts and their phcs. ………….74 4.7: Sero-Prevalence of Rubella Antibodies Among Pregnant Women Attending Ante-Natal Clinic in Kaduna State Based on Trimesters…………………………………………………………75 4.8: Follow up Cases of Rubella Virus in The Three Senatorial District in Kaduna State………………………………………………………………………………………………………………..79 4.9: Follow up Cases of Rubella Virus in Northern Senatorial District in Kaduna State………………………………………………………………………………………………………………..81 4.10: Follow up Cases of Rubella Virus in Southern Senatorial District in Kaduna State………………………………………………………………………………………………………………..82
4.11: Follow up Cases of Rubella Virus in Central Senatorial District in Kaduna State……………………………………………………………………………………………………………….84
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LIST OF PLATES
Plate Title Page
1: Molecular Detection of Rubella Virus Among Pregnant Women attending Ante-Na

CHAPTER ONE

1.0 INTRODUCTION
1.1Rubella Virus
Rubella virus (RV) causes a benign systemic rash illness when it infects humans as a result of acute infection and causes severe birth defects if acute maternal infection occurs during the first trimester of pregnancy(Willey et al., 2011). This virus is a positive-strand RNA virus that replicates in the cytoplasm of the infected cell. Rubella virus is taxonomically unique, being the sole member of the genus Rubivirus of the familyTogaviridae (Brooks et al., 2007). The rubella virus causes “German measles,” also known as “three-day measles”(Bukbuk et al., 2002), this is usually a milder disease than red measles. Infections occur most commonly in children and the resulting natural immunity is most probably lifelong (Matthews et al., 2011). However, in pregnant women the risk of intrauterine transmission is up to 90% if infection occurs in early pregnancy (8-10 weeks gestation). As the viraemia leads to placental infection and spread of the virus, it causes a chronic infection of the foetus leading to the development of congenital rubella syndrome (CRS) (Matthews et al., 2011).Red/Hard measles or just measles is caused by Rubeola virus.
A pregnant woman who is infected with rubella virus can transmit the virus to the foetus (Bukbuk et al., 2002), to cause congenital rubella infection (CRI). Several organ systems can be effected when CRI occurs and these includes the eyes, ears, heart, brain, and endocrine system (Chantler et al., 2001).A CRSencompasses all outcomes associated with intrauterine rubella infection, including miscarriage, stillbirth, abortion, combinations of birth defects, or asymptomatic infection in the infant (Reef and Cordero, 2000a).The pathogenesis of CRS is not understood and CRS patients are persistently infected with RV at birth and can suffer
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progressive squeal in addition to those apparent at birth(Brooks et al., 2007; Adewumi et al., 2013). The two clinical symptoms observed in rubella virus infection are CRI which is usually mild with most cases passing as subclinical or unrecognized events and CRS which is the outcome of infection of rubella virus from the mother to the foetus after transplacental transmission of rubella disease.The clinical manifestations of congenitally acquired rubella are usually severe (Chantler et al., 2001; Kimberlin, 2002). The classical sign of malformations in congenital rubella includes the eyes (cataract, microphthalmia, glaucoma and chorioretinitis), the heart (most often patent ductus arteriosus) and the ear (unilateral or bilateral sensoryneural deafness)(Haukenes, 2002). The virus is transmitted from person to person via respiratory aerosols (Lee and Bowden, 2000), and replicates in the nasopharynx and regional lymph nodes.Rubella virusinfects only humans and is spread from person to person through contact orfrom a cough or sneeze as the rubella virus lives in the mucus of an infected person (Agbede et al., 2011). Symptoms for postnatal Rubella virusinclude a rash spreading down from the face to the extremities and in some cases a runny nose, fever or joint pain. It is possible for these symptoms to be asymptomatic (Leeand Bowden,2000).
Rubella occurs worldwidewith a seasonal distribution. The peak incidence ofinfection is in late winter or early spring (Adesina et al., 2008). The mechanism by which rubella virus causesfoetal damage is not well understood till now. The possible mechanism is direct viral damage of infected cells (Margaret, 2011).Worldwide, Rubella is known to affect children,
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andbeing a childhood diseaseit is predominantly endemic throughout the world (Kimberlin, 2002; Haukenes, 2002; Brooks et al., 2007; Willey et al., 2011). In 1998, standard case definitions for surveillance of CRS and rubella were developed byW.H.O. In 2001, 123 countries/territories reported a total of about eight hundred and thirty five thousand rubella cases. More countries are expected to report on rubella as a global measles/rubella laboratory network is further developed under the coordination of W.H.O (Frey, 2008). Rubella epidemic and pandemics are controlled in the United States (Brookset al., 2007;Centers for Disease Control and prevention, 2008; NewYork Times, 2015). During the 1962–1965 global pandemic, an estimated 12.5 million rubella cases occurred in the United States, resulting in 2000 cases of encephalitis, 11,250 spontaneous abortions, 2100 neonatal deaths, and 20,000 infants born with congenital rubella syndrome(Mayo Clinic, 2011).The rubella-induced congenital anomalies were observed and collectively referred to as the expanded congenital rubella syndrome (Best and Banatvala, 2000; Kimberlin, 2002). The economic impact of this epidemic in the United States was estimated to be $1.5 billion (Kimberlin, 2002).
In a study in Karachi, the prevalence for rubella IgM and IgG rubella were 13% and 29% respectively among 355 pregnant women screened. Out of this number, total of 212 pregnant women had abortion(Bamboyeet al., 2004). It was recommended that to reduce morbidity and mortality related to rubella virus, premarital screening and vaccination of sero-negative girls should be done(Bamboyeet al., 2004). There is at least a 20% chance of damage to the foetus if a woman is infected with rubella early in her pregnancy (Adesina et al., 2008).Immunizing children on schedule is recommended to protect children and others,
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including pregnant women and their unborn babies from rubella infection (Bukbuk et al., 2002). In Africa there is general lack of awareness of Rubella and it is not often diagnosed clinically, which may be due to the dark skin colour (Christelleet al., 2010). Sero-epidemiological survey conducted throughout Africa has shown the presence of the virus throughout the continent(Christelleet al., 2010). World Health Organization (2015) hasreported 466 rubella cases in 2007 in Nigeria. Studies in Ghana, Togo, Ivory Coast, BurkinaFaso and Mali all showed a high prevalence of rubella antibody in the adult population (Christelleet al., 2010).In Lagos State Nigeria, a study on rubella-IgG antibody in women of childbearing age was conducted and 77% of all the female subjects were positive for Rubella-IgG antibody. This means that the remaining 23% did not have immunity to this rubella virus(Dontigny et al., 2008).
The prevalence of IgG antibody specific to the rubella virus was assessed in 207 consenting pregnant women in Maiduguri Borno State (Bukbuk et al., 2002). The pregnancy outcome was normal in 27% of the women studied (Bukbuk et al., 2002). Miscarriage, premature delivery and stillbirth occurred with the others. There was no clinically detectable malformations in 25 (44.6%) of all the deliveries from the pregnant women (Bukbuk et al., 2002).A study conducted at the Ahmadu Bello University Teaching Hospital, Zaria, showed a rubella IgG sero-prevalence of 97.9% among pregnant women and 2.1% sero negative for rubella IgG antibody this was out of 430 serum samples tested (Muhammad et al., 2010). Differences in socio demographic factors were of little significance between the groups but awareness of the infection was low (Bamboyeet al.,2004). Another study on rubella conducted at the Ahmadu Bello University Zaria showed a prevalence of 1.9% for rubella
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IgM antibodies(Pennap et al., 2009). This was determined from 160 pregnant women(Olajide et al.,2012).A study in Benue state showed that 4.2% of the pregnant women tested were seropositive to rubella IgM (Pennap et al., 2009; WHO, 2015).
The advent of molecular techniques such as the polymerase chain reaction and sequencing has made it possible to examine the genetic makeup of the virus itself and perform molecular epidemiology studies (Lee and Bowden, 2000). Genotypes 1A, 1E, 1F, 2A and 2B have been isolated in China. Genotype 1j has only been isolated from Japan and the Philippines. Genotype 1E is found in Africa, the Americas, Asia and Europe. Genotype 1G has been isolated in Belarus, Cote d’Ivoire and Uganda. Genotype 1C is endemic only in Central and South America. Genotype 2B has been isolated in South Africa. Genotype 2C has been isolated in Russia (Dominguez et al.,1990).
1.2 Statement of the Research Problem
The research Problem of this study include: 1. The lack of routine immunization against the infection 2. Cases of misdiagnosis which include a rash spreading down from the face to the extremities and in some cases a runny nose, fever or joint pain. 3. The sequalae of outcome which includes CRS and the irreversibility of the outcome 4. Lack of awareness of the disease and control measures
Worldwide, it is estimated that there are more than 100.000 infants born withCRS each year(Frey, 2008). Cases of rubella infection still occur in Nigeria among pregnant women (Bamboye et al., 2004, Olajide et al., 2012). In 2006, a case of confirmed CRS was reported in Port Harcourt in a three month old male with heart failure (Otaigbe et al., 2009). Other
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studies conducted in Maiduguri, Ibadan, Lagos and Zaria have shown the presence of rubella virus in pregnant women with prevalence of rubella IgG antibodies of76%, 54%, 68.5%, 95.9% and 97% respectively (Onyenekwe et al., 2000;Bukbuk et al., 2002; Bamboye et al.,2004;Muhammad et al., 2010; Olajide et al., 2012).
1.3 Justification
Rubella virus only has a large risk of infection if the disease is contracted by the mother in the first trimester, after which birth defects are less likely(Lee and Bowden, 2000; Sallamet al., 2003). Even if a woman who develops rubella has no rubella symptoms, her baby can still develop serious congenital rubella syndrome symptoms. Therefore, it is important to determine the rubella immunity status for all women, either before conception or very early in the first trimester of pregnancy. Worldwide, it is estimated that there are more than 100.000 infants born withCRS each year (Frey, 2008). Cases of rubella infection still occur in Nigeria among pregnant women (Bamboye et al., 2004, Olajide et al., 2012). In 2006, a case of confirmed CRS was reported in Port Harcourt in a three month old male with heart failure (Otaigbe et al., 2009). Other studies conducted in Maiduguri, Ibadan, Lagos and Zaria have shown the presence of rubella virus in pregnant women with prevalence of rubella IgG antibodies of 76%, 54%, 68.5%, 95.9% and 97% respectively (Onyenekwe et al., 2000; Bukbuk et al., 2002; Bamboye et al.,2004;Muhammad et al., 2010; Olajide et al., 2012). In Markurdi, Benue state, a serological survey of specific rubella IgM in the sera of pregnant women showed that only 4.2% of the women were susceptible (Pennap et al., 2009).
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A study in Kaduna State by Muhammad et al. (2010) reported a rubella IgG prevalence of 97.9% among pregnant women attending ante natal clinic in Ahmadu Bello University Teaching Hospital Shika. Olajide et al. (2012) also conducted a study between the months of June and August 2012 on rubella IgM among pregnant women and reported a prevalence of 38.8%. This makes it necessary to conduct a survey across the senatorial districts of Kaduna State. This research will give information on the current prevalence of rubella virus infection in pregnant women in Kaduna State. The result would provide data that the Ministry of Health could usethis information toguide its routine immunization programs in health facilities in Nigeria. Prenatal diagnosis of infectious diseases has been shown to be indispensable to confirm or exclude due to rubella.Consequently, the central challenge and the most important public health priorities at this time are to determine if there are other infected persons and if necessary, implement measures to prevent additional human infections (WHO, 2015).
1.4Research Question
1. What is the prevalence of rubella virus among pregnant women in Kaduna State Nigeria?
2. What are the socio-demographic characteristics of women with rubella IgM or IgG antibodies?
3. What are the risk factors for rubella virus among pregnant women in Kaduna State?
1.5 Aim
―Aimed at monitoring the disease by way of surveillance study in Kaduna Nigeria‖
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1.6Specific Objectives
The specific objectives of this studywere to:
1. Identify the risk factors associated with rubella virus infection among pregnant women in Kaduna State and Follow up the babies of these pregnant women whose serum were positive for IgM by checking their case note, calling or tracing their homes.
2. Determinerubella viruse IgG and IgM antibodies among pregnant women in Kaduna State Nigeria using Enzyme Linked Immuno- sorbent Assays.
3. Confirmrubella virusin the serum of pregnant women attending ante-natal clinic in Kaduna State using conventional Reverse Transcription Polymerase Chain Reaction (RT-PCR).
4. Sequence the Rubella virus isolated from these pregnant women attending ante-natal clinic in Kaduna State.

 

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