University of Ghana http://ugspace.ug.edu.gh SEROLOGICAL INDICATION OF ZIKA VIRUS INFECTION IN FEBRILE PATIENTS AT THE GREATER ACCRA REGIONAL HOSPITAL, ACCRA GHANA Godson Aryee Ankrah 10598763 This thesis/dissertation is submitted to the University of Ghana, Legon in partial fulfilment of the requirement for the award of MPHIL Microbiology degree July, 2018 University of Ghana http://ugspace.ug.edu.gh DECLARATION I Godson Aryee Ankrah declare that, this research was carried out by me at the Department of Virology, NMIMR. It was supervised by Professor Theophilus K. Adiku, Department of Medical Microbiology, SBAH and Dr. Joseph H. Kofi Bonney, Department of Virology, NMIMR. Work by other investigators which served as a source of information has been acknowledged in the form of referencing. …………………… …………………………. Godson Aryee Ankrah Date (Student) ……………………….. …………………………… Professor Theophilus K. Adiku Date (Supervisor) ………………………… ……………………………. Dr. JH Kofi Bonney Date (Co-supervisor) i University of Ghana http://ugspace.ug.edu.gh DEDICATION I dedicate this research work to God for his guidiance. ii University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENT I am thankful to God for the strength and grace given me to successfully complete this work. It was not always smooth, but the Lord has always been there to help. I want to sincerely acknowledge the support of my supervisor Professor Theophilus K. Adiku, a lecturer at the Department of Medical Microbiology, University of Ghana. I appreciate all his useful suggestions. I would like to also show my appretiation to Dr. Joseph H. Kofi Bonney, senior research fellow, NMIMR for his motivation and constructive criticisms. I am well pleased with the contributions of Ms Deborah Pratt and Ms Esinam Agbosu, research assistants, NMIMR. I appreciate their training and assistance in the laboratory investigations of my work. iii University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENT DECLARATION ..........................................................................................................i DEDICATION ………………………………………………………………………..ii ACKNOWLEDGEMENT …………………………………………………………...iii TABLE OF CONTENT ……………………………………………………………...iv LIST OF FIGURES …………………………………………………………………. x LIST OF TABLES ……………………………………………………………………xi LIST OF ABBRAVIATIONS ………………………………………………………..xii ABSTRACT …………………………………………………………………………..xv CHAPTER ONE ……………………………………………………………………...1 INTRODUCTION …………………………………………………………………1 1.1 Background ……………………………………………………………………..1 1.2 Problem Statement ……………………………………………………………...4 1.3 Justification ……………………………………………………………………..4 1.4 Main Objective ………………………………………………………………….5 1.4.1 Specific Objectives ………………………………………………………….5 iv University of Ghana http://ugspace.ug.edu.gh CHAPTER TWO ……………………………………………………………………..6 LITERATURE REVIEW …………………………………………………………...6 2.1 Structure, Genomic Organisation and Replication ………………………………6 2.1.1 Structural Morphology of Zika Virus ……………………………………......6 2.1.2 Replication Cycle of Zika Virus …………………………………………......8 2.2 Epidemiology of Zika Virus Infection ………………………………………......9 2.2.1 Zika Virus Infection in Africa ……………………………………………….14 2.2.2 Zika Virus Infection in Asia ………………………………………………....16 2.2.3 Zika Virus Infection in Oceania ……………………………………………..17 2.2.4 Zika Virus Infection in America ……………………………………………..19 2.3 Condition of Spread of Zika Virus Infection ……………………………………20 2.3.1 Climate Change and Variation ………………………………………………20 2.3.2 Social Change and Urbanization …………………………………………….21 2.4 Impact of Zika Virus Infection ………………………………………………….30 2.4.1 Public Health Impact ………………………………………………………...30 2.4.2 Socio-economic Impact ……………………………………………………...31 v University of Ghana http://ugspace.ug.edu.gh 2.5 Clinical Manifestation of Zika Virus Infection ………………………………….21 2.5.1 Adult ………………………………………………………………………....22 2.5.2 Children ……………………………………………………………………...22 2.6 Diagnosis of Zika Virus Infection …………………………………………….....24 2.6.1 Case Definition of Zika Virus Infection ……………………………………..24 2.6.2 Laboratory Diagnosis of Zika Virus Infection ………………………………25 2.6.2.1 Virus Isolation …………………………………………………………...26 2.6.2.2 Viral RNA Detection ……………………………………………………26 2.6.2.3 Serological Diagnosis ……………………………………………………27 2.6.2.3.1 Enzyme Linked Immunosorbant Assay ……………………………..27 2.6.2.3.2 Plague Reduction Neutralization Test ……………………………….28 2.6.2.3.3 Indirect Immunofluorescence …………………………………..……29 2.7 Treatment and Prevention of Zika Virus Infection ……………………………...32 2.7.1 Treatment and Therapeutic Approaches …………………………………….32 2.7.2 Prevention of Zika Virus Infection ………………………………………….33 CHAPTER THREE …………………………………………………………………..35 vi University of Ghana http://ugspace.ug.edu.gh Materials and Method ………………………………………………………………35 3.1 Study Design and Ethical Consideration .……………………………………..35 3.1.1 Study Design ……………………………………………………………....35 3.1.2 Ethical Consideration ………………………………………………………35 3.2 Study Site and Sample Size Determination ……………………………………36 3.2.1 Study Site ………………………………………………...………………..36 3.2.2 Sample Size Determination ………………………………………………..36 3.3 Eligibility Criteria ……………………………………………………………...37 3.5.1 Inclusion Criteria …………………………………………………………..37 3.5.2 Exclusion Criteria ……………………………………………………….....37 3.4.2 Sampling Procedure and Documentation ……………..……………………37 3.4.1 Sampling Procedure ………………….………..………………………....37 3.4.2 Documentation ………………………………………………….…..……38 3.5 Sample Processing and Strategy………………………………………………39 3.5.1 Sample Processing…………………………………..………………..…...39 3.5.2 Sampling Strategy…………………………………………………………39 vii University of Ghana http://ugspace.ug.edu.gh 3.6 Laboratory Testing of Zika Virus Infection …...................................................39 3.7 Calculation and Interpretation of Results (IgM and IgG)…………….…….....42 3.7.1 Calculation of Cut-off (IgM and IgG)…………………………………….42 3.7.2 Calculation of Results in Abcam Units (AU) (IgM and IgG) …………….42 3.7.3 Interpretation of Results (IgM and IgG) ……………………………….....42 3.8 Independent and Outcome Variable …………………………………………..43 3.8.1 Independent Variable ………………………………………………………43 3.8.2 Outcome Variable ……………………………………………………….....43 3.9 Statistical Analysis ……………………………………………………………..43 CHAPTER FOUR …………………………………………………………………….45 RESULTS …………………………………………………………………………….45 4.1.0 Demographic Characteristics of Febrile Patients …………………………….45 4.1.1 Seroprevalence of Zika Virus ………………………………………………...46 4.1.2 Characteristics of Zika Virus Seropositives ………………………………….46 4.1.3 Monthly Distribution of Zika Virus Antibodies ……………………………...48 4.1.4 Anti-Zika Virus IgM Monthly Distribution Stratified by Gender …………...48 4.1.5 Date of Anti-Zika virus IgM detection …………………………....................49 viii University of Ghana http://ugspace.ug.edu.gh 4.1.6 Town of Residence of Zika Virus Seropositive Patients …………………......50 4.1.7 Predictors of Zika virus Seropositivity ……………………………………….50 4.2.0 Symptoms Presented by Seropositive Febrile Patients ……………………….51 4.2.1 Bivariate Analysis of Zika Virus Seropositivity and Clinical Presentation.......52 CHAPTER FIVE ……………………………………………………………………...53 DISCUSSION, CONCLUSION, RECOMMENDATIONS AND LIMITATION.....53 5.1 Discussion ……………………………………………………………………….53 5.2 Conclusion ……………………………………………………………………….61 5.3 Recommendations ……………………………………………………………….61 5.4 Limitation ………………………………………………………………………..61 REFERENCES ……………………………………………………………………….62 APPENDIX …………………………………………………………………………..91 ix University of Ghana http://ugspace.ug.edu.gh LIST OF FIGURES Figure 2.1: Schematic diagram showing the structure of a Zika virus …………………8 Figure 2.2: Replication cycle of Zika virus …………………………………………….9 Figure 2.3: Severity of microcephaly ……….................................................................23 Figure 2.4: Major diagnostic markers for Zika virus infection ………………………...28 Figure 3.1: Samples …………………………………………………………………….42 Figure 3.2: Sample reading with automated microtitre plate reader …………………...42 Figure 4.1: Seroprevalence of Zika virus antibodies …………………………………...46 Figure 4.2: Monthly distribution of anti-Zika virus antibodies ………………………...48 Figure 4.3: Anti-Zika virus IgM monthly distribution stratified by gender ……………49 Figure 4.4: Date of Anti-Zika virus IgM detection ………………………….…………49 Figure 4.5: Symptoms presented by seropositive febrile patients …………………….. 51 x University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES Table 2.1: Prevalence of Zika virus infection in Africa since 2000 ……………………16 Table 2.2: Current Zika vaccine candidates at various stages of preclinical/clinical trials..32 Table 3.1: Arrangement of wells in the microplate (Zika plate map) ………………….41 Table 3.2: Criteria for Interpretation of results .………………………………………..43 Table 4.1: Characteristics of febrile patients …………………………………………...45 Table 4.2: Characteristics of Zika virus seropositives …………………………………47 Table 4.3: Bivariate logistic regression analysis showing predictors of Zika virus seropositivity …………………………………………………………………………...50 Table 4.4: Bivariate logistic regression analysis ……………………………………….52 xi University of Ghana http://ugspace.ug.edu.gh LIST OF ABBREVIATIONS CDC Centre for Disease Control and Prevention RNA Ribonucleic acid IgG Immunoglobulin G antibodies IgM Immunoglobulin M antibodies RT-PCR Real time polymerase chain reaction TBM 3, 3‘, 5, 5‘ tetramethylbenzidin ANOVA Analysis of Variance C Capsid Protein E Envelop Protein FL Fusion Loop HRPO Horseradish Peroxidase NIAID National Institute for Allergy and Infectious Disease NMIMR Noguchi Memorial Institute for Medical Research NS Non-Structural Protein OD Optical Density xii University of Ghana http://ugspace.ug.edu.gh PRNT Plaque Reduction Neutralization Test PrM Precursor Membrane SL Stem Loop UTR Untranslated Region M Membrane Protein IIFA Indirect Immunofluorescence Assays (IIFA) VNT Virus Neutralization Tests ELISA Enzyme Linked Immunosorbance Assay WHO World Health Organization USA United State of America MTPase N-terminal S-adenosyl Methionine Methyltransferase DC-SIGN Dendritic Cell-Specific Intercellular adhesion molecule -3- Grabbing Non- integrin ECDC European Centre for Disease Prevention and Control NIAID Nation Institute of Allergy and Infectious Disease IFRC International Federation of Red Cross xiii University of Ghana http://ugspace.ug.edu.gh UNDP United Nations Development Programme NMIMR Nugochi Memorial Institute for Medical Research xiv University of Ghana http://ugspace.ug.edu.gh ABSTRACT Background: Zika virus infection shares overlapping signs and symptoms with endemic diseases malaria and typhoid fever. These similarities have led to a high probability of false positive diagnosis leading to the over-diagnosis and over-emphasis of these endemic diseases to the neglect of other febrile illnesses of different aetiological agents. This study sought to serologically detect Zika virus infection in febrile patients in a selected health facility, the Greater Accra regional hospital in Ghana. Method: Archived human sera obtained from febrile patients and stored under ultra-low freezing conditions (-80oC) at Noguchi Memorial Institute for Medical Research were used. An Enzyme linked immunosorbent assay (ELISA) was used in the laboratory to detect anti- Zika virus antibodies (ZIKV IgM and IgG) in the sera. Results: Out of 160 sera evaluated, 119 were from females with an average age of 30 years whiles the males were of an average age of 29 years. In all, 30 were found to be positive for anti-Zika virus IgM and 3 for anti-Zika virus IgG. No patient had both anti-Zika virus IgG and IgM. Nineteen (63.3%) of the positives were from female patients and 14 (36.7%) from male patients. The highest proportion of seropositives were recorded among the 21-30 year age group with 8 (26.7%) anti-Zika virus IgM and 2 (66.7%) anti-Zika virus IgG. Monthly distribution of anti-Zika virus IgM peaked in March 7 (24.2%) and May 9 (30%) and decreased in September 1 (3.3%). Anti-Zika virus IgG were detected only in January 2 (66.7%) and March 1 (33.3%). Sera from patients who reside at Lapaz and Amasaman recorded the highest seropositivity of 12.1% each. All seropositive febrile patients developed at least one symptom consistent with Zika virus infection: 33 (100%) fever, 25 (76%) muscle pain, 24 (73%) joint pain, and conjunctivitis 2(1.1%). In addition to this, 28 (85%) loss of appetite, 14 (75%) rapid respiration and chest pain 15 (42%) were reported by seropositive febrile patients. Bivariate logistic regression analysis revealed an association between Zika virus seropositivity and chest pain, conjunctivitis, muscle pain and joint pain. A recent hearing loss was the complication detected. Conclusion: Data from the study indicates exposure to Zika virus which suggests the possible circulation of the virus among febrile patients in Ghana. This study therefore underscores the importance for sero-epidemiological surveillance for timely identification of Zika virus disease to prevent outbreaks. xv University of Ghana http://ugspace.ug.edu.gh CHAPTER ONE INTRODUCTION 1.1 Background Fever is a common medical symptom of many diseases (O’Neill, 1994). The sudden onset of fever may herald a serious life-threatening disease or a mild infection which could be of a virus, bacteria and/ or parasite (Wright et al., 1981). In Ghana, febrile illnesses account for about 30% and 75% (Farrar, 2011) of healthcare visit by children and adults (Kiekkas et al., 2013) respectively. However, these febrile illnesses, when presented at healthcare facilities are mostly treated for endemic diseases such as malaria and typhoid fever. This is because, case management of these endemic diseases in most healthcare facilities in Ghana are often by clinical judgement based on signs and symptoms (Chandler et al., 2008). However, clinical judgement based on signs and symptoms is of low specificity (52-61%) due to the overlapping clinical features of Zika virus infection with malaria and typhoid fever (Chandramohan et al., 2002). This, according to Chandramohan et al., (2002) and Nankabirwa et al., (2009) has led to a high probability of false positive diagnosis which inturn has contributed to the over- diagnosis and over-emphasis of these endemic diseases to the neglect of other febrile illnesses of different aetiological agents. A study conducted in Nigeria showed that 83% of children presented to the hospital with febrile illnesses were treated for malaria with artemisinin-based combination therapy (ACT) before microscopy results were shown to be negative (Oladosu and Oyibo, 2013). Similarly, in a study conducted in Tanzania, 870 hospital admissions showed that malaria was the clinical diagnosis given to 528 (61%) of the patients. However, only 14 (2%) were found to have malaria as the cause of the fever 1 University of Ghana http://ugspace.ug.edu.gh by laboratory confirmation (Crump et al., 2013). This is not different in Ghana; less than a third of febrile illnesses are diagnosed as malaria before confirmation with laboratory tests (John Snow Incorporated Research and Training Institute, 2013). Zika virus infection shares overlapping signs and symptoms with these endemic diseases and has been implicated as the cause of some febrile illnesses around the world (Center for Disease Control and Prevention, 2016). In Africa, the first serological evidence of Zika virus infection in febrile patients was reported in 1975 at the University College Hospital, Ibadan in Nigeria (Moore et al., 1975). Although Zika virus infection accounts for about 3 – 11% of febrile illnesses around the world, it remains under-recognized and under- reported in Ghana (Amarasinghe et al., 2011). Zika virus infection is arthropod borne (Speer & Pierson, 2016). The etiologic agent, Zika virus is among several neglected arboviruses with apparent pathogenicity in humans (Weissenböck et al., 2001). Zika virus was first isolated in Uganda in 1947 from a rhesus macaque monkey and Aedes africanus mosquito in 1948 (Duffy et al., 2009). Subsequently, Zika virus was isolated from other species of Aedes mosquitoes such as Aedes luteocephalus, Aedes aegypti, Aedes albopictus, Aedes furcifer and Aedes vittatus. Aedes spp. of mosquitoes reproduce in water and eggs are extremely hardy (Paixão et al., 2016). Viral reservoirs are monkeys and transmission is through the bite of Aedes spp. of mosquitoes, primarlly Aedes aegypti or Aedes albopictus which have acquired the virus from an infected monkey through a blood meal (Vasilakis & Weaver, 2016; Ferreira-de-Brito et al., 2016). Other means of transmission include sexual intercourse, vertical and blood transfusion (Oehler et al., 2014). 2 University of Ghana http://ugspace.ug.edu.gh In Africa, Zika virus infection was first documented in human in Uganda and Tanzania in 1952 (Haddow et al., 2012) and 1954 in Asia, notably India (Smithburn, 1954). Afterwards, Zika virus infection was found in Egypt, Nigeria, India, Pakistan, North Vietnam and Philippines, spreading from Africa to Southeast Asia (Calveta et al., 2016: Lanciotti et al., 2008). Zika virus infection in Asia and Africa never got the deserved attention due to the sporadic nature of the infection coupled with mild short-term fever. Other symptoms of Zika virus infection include pruritic rashes, non-purulent conjunctivitis and arthralgia (Brasil et al., 2016). As a result, no efforts were made to develop drugs and vaccine (Lanciotti et al., 2008). Since then, Zika virus infection never occurred in humans outside Africa and Asia until 2007, when the largest outbreak was reported in the Yap State of Micronesia (Duffy et al., 2009: Lanciotti et al., 2008). Outbreak was again documented in the French Polynesia with approximately 28,000 cases in 2013 (Musso et al., 2016: Klase et al., 2016). The devastating aspect of Zika virus infection reported during these outbreaks was the ability of the virus to infect the developing fetus causing birth defects such as microcephaly and intracranial calcifications, and causing Guillain-Barré Syndrome in adults (Coley, 2016). Subsequent spread and the congenital anomalies associated with Zika virus infection led the World Health Organization in 2016 to declare Zika virus infection as a worldwide public health emergency (Chan et al., 2016). Currently there are no treatment and vaccines available. Diagnosis is only possible in the 2 – 7 days of the infection and is based on detection of specific antibodies by IgG and IgM ELISA, indirect immunofluorescence assays (IIFA) and virus neutralization tests (VNT) (Waggoner and Pinsky, 2017). Virus isolation (Kostyuchenko et al., 2016) and detection of 3 University of Ghana http://ugspace.ug.edu.gh viral nucleic acid by RT-PCR (Dai et al., 2016) from cerebrospinal fluid, saliva, serum and urine are also used. 1.2 Problem Statement Currently, the geographical range of Zika virus infection and the number of susceptible individuals are predicted based on the distributions of Aedes aegypti and Aedes albopictus (Centers for Disease Control and Prevention, 2016). The presence of active vector population of Aedes spp. of mosquitoes in Ghana coupled with proof of contact with the virus in our neighbouring countries (Waddell and Greig, 2016), have necessitated the need for us to document the exposure levels in febrile patients (Plourde and Bloch, 2016). Additionally, in sub-Saharan Africa, febrile cases are mainly treated for endemic diseases like malaria and typhoid fever missing out on other possible aetiologies. Furthermore, the mild or asymptomatic nature and the overlapping clinical features with other endemic disease conditions make it probable for typical Zika virus infection to be missed out in diagnosis. This study therefore gives us the opportunity to document the exposure levels to Zika virus in febrile patients. 1.3 Justification The possibility that adverse clinical conditions can occur underscore the need to reinforce surveillance for Zika virus and in case of an outbreak, establish rigorous clinical monitoring to detect microcephaly, Guillain-Barré Syndrome and any other unusual clinical manifestations. In light of this, it is important to carry out a study to give an indication of 4 University of Ghana http://ugspace.ug.edu.gh the levels of exposure and possible infection due to the Zika virus. This study will determine whether suspected febrile cases presented to the Greater Accra regional hospital have had an exposure to Zika virus. Information that will be obtained on this infection such as prevalence and clinical manifestations will be helpful to healthcare policy makers, providers and health promotion activities. It will also justify the reasons to invest resources to carry out detailed studies to look for clinical cases of Zika viral disease at the Greater Accra regional hospital. This effort will enhance the work by health staff in the clinical determination of suspected Zika cases. This research will add to the body of knowledge in Ghana and the world at large. 1.4 Main Objective To determine Zika virus seroprevalence in febrile patients at the Greater Accra regional hospital, Ghana 1.4.1 Specific Objectives ➢ To determine the prevalence of anti-Zika virus antibodies in febrile patients ➢ To determine the clinical and demographic characteristics of sero-positive patients 5 University of Ghana http://ugspace.ug.edu.gh CHAPTER TWO LITERATURE REVIEW 2.1 Structure, Genomic Organisation and Replication 2.1.1 Structural Morphology of Zika Virus Zika virus is a single-stranded, enveloped icosahedral virus. Genome has a positive-sense RNA (Shi, 2012). Virion is 40–60 nm in diameter and contains 10,794 nucleotide encoding 3,419 amino acids (Kuno & Chang, 2007). Viral RNA has a distinct stretched open reading frame. This open reading frame encodes a polyprotein translated into three structural proteins (precursor membrane, capsid, and envelope) and seven non-structural proteins (5- C-prM-ENS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3) (Figure 2.1). Viral RNA is encapsulated in a C-protein with precursor membrane and envelope attached. The envelop protein facilitates attachment and entry of virus by membrane fusion (Rey et al., 1995; Nybakken et al., 2006). The envelope protein, capsid protein and precursor membrane play no part in viral replication and are only synthesized to package the matured virus (Pang et al., 2001; Alvarez et al., 2006). The non-structural proteins (5-C-prM-ENS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3) are responsible for viral replication and are often expressed in the cytoplasm of the infected cell (Pang et al., 2001; Alvarez et al., 2006). NS1 is a glycoprotein and possesses two glycosylation sites which are conserved among flaviviruses. NS1 functions in viral infection and elicits immune response (Avirutnan et al., 2010; Schlesinger et al., 1987). NS3 has three enzymatic activities such as 5’ RNA-triphosphatase (RTP), Nucleoside 6 University of Ghana http://ugspace.ug.edu.gh triphosphatase (NTPase) and a helicase. NS3 aids in viral replication and processing of polyproteins into structural and non-structural components (Bazan and Fletterick, 1989; Miller et al., 2010). NS5 is the largest protein measuring approximately 103KDa and has three enzymes namely: N-terminal S-adenosyl methionine methyltransferase (MTPase), nuclear localisation sequence and RNA-dependent RNA polymerase. It role includes transportation of protein to nucleus, guanine 7- N- and ribose 2’-O-methylation and synthesis of new vRNA genome (Miller et al., 2010). The 5’UTR contains 95-135 nucleotides and a type I cap (Yu et al., 2008). The cap structure is responsible for the cap dependent translation initiation of viral RNA by scanning 5’UTR for initiation codons. The 3’ UTR contains 114-650 nucleotides and folds into a stem loop (SL) but lacks a Poly (A) tail. A sequence that is conserved is found upstream of the SL (Men et al., 1996). This sequence contains a cyclization sequence (CS) which has a complementary sequence at the 5’ end of the genome (Hahn et al., 1987). Both untranslated regions are vital for replication and translation (Gamarnik, 2010; Padmanabhan and Strongin, 2010). Aside the cap structure at the 5’ UTR, there is also a large stem loop (SL) which act as a promoter for RNA polymerase-methyl transferase NS5 (Yu et al., 2008). The 5’ and 3’ UTR have complementary upstream AUG regions (UR) and cyclization sequence regions which hybridize during genome cyclization and RNA synthesis (Gamarnik, 2010). 7 University of Ghana http://ugspace.ug.edu.gh Figure 2.1: Schematic diagram showing the structure of a Zika virus Source: (Guzman et al., 2010) 2.1.2 Replication Cycle of Zika Virus Replication of Zika virus takes place in the salivary gland and midgut of the Aedes spp. of mosquitoes (Li et al., 2012; Wong et al., 2013). After viral inoculation by Aedes spp. of mosquitoes through a blood meal, the virus enters the skin fibroblasts, keratinocytes, undeveloped dendritic cells (Hamel et al., 2015), neurons and astroglial cells (Bell et al., 1971) using adhesion factors such as AXL receptor tyrosine, DC-SIGN (Dendritic Cell- Specific Intercellular adhesion molecule -3- Grabbing Non-integrin) and varied members of the phosphatidylserine receptor family (Hamel et al., 2015). Replication of Zika virus takes place near the inoculation site and later spreads to the blood and lymph nodes. After entry, viral particle is internalized in the cell and the viral genome is released inside the cytoplasm. This is achieved when the viral envelope merges with the membranes of the cellular endosomes of the host cell due to the acidic pH inside the cellular endosomes 8 University of Ghana http://ugspace.ug.edu.gh (Stiasny et al., 2011; Vazquez-Calvo et al., 2012). Viral RNA acts as mRNA and negative and positive strands of viral RNA are synthesized. Newly formed positive strand-RNA is packed into progeny virions and bud off into the endoplasmic reticulum to form enclosed immature virions. Immature virions are transported via the Golgi complex and the prM is cleaved (Hayes 2009). The virus replicates and spreads further to infect other parts of the body such as myocardium, central nervous system, skeletal muscles and to the fetus (Chan et al., 2016). The replication cycle of Zika virus is depicted in figure 2.2. Figure 2.2: Replication cycle of Zika virus Source: Chan et al., (2016) 2.2 Epidemiology of Zika Virus Infection Zika virus infection is among some of the fastest spreading arboviral infections with an estimated 30 percent growth in the number of reported cases since its inception in 1947 in Uganda (Dick, 1952). Most affected continents are Oceania and America with some parts 9 University of Ghana http://ugspace.ug.edu.gh of Africa and Asia showing high prevalence of the infection (Hill et al., 2017). Roughly two billion individuals live in Zika virus infection endemic areas with 390 million infected individuals showing symptoms of Zika virus infection annually (World Health Organization, 2009). An estimated 99 million individuals develop symptoms for the different levels of disease severity (Bhatt et al., 2013). Transmission of Zika virus infection exists as forest and urban cycles. Forest transmission cycle involves Rhesus monkeys and forest species of Aedes mosquitoes whereas urban transmission cycle involves human–mosquito–human. Zika virus is maintained in the forest cycle with cyclic epizootics in monkeys. Humans are accidently infected by the forest cycle (Martin- Acebes and Saiz, 2012). In the transmission cycle of Zika virus, Rhesus monkeys are the reserviors with intermittent involvement of humans (Haddow and Dick, 1948; Haddow et al., 1964). However, in areas without Rhesus monkeys, humans serve as the main reservior (Haddow et al., 2012). Zika virus is transmitted by mosquitoes of the Aedes (Stegomyia) genus. Zika virus is mainly transmitted by Aedes aegypti, Aedes africanus, Aedes hensilli and Aedes albopictus (Dick et al., 1952). Aedes aegypti is the main vector involved in the epidemic that occurred in Asia and the French Polynesia (Olson et al., 1981 and Oehler et al., 2014). Although Zika virus has not been detected in Aedes hensilli and Aedes polynesiensis, they have been reported to be the main vectors in the Yap outbreak (Musso et al., 2014). In Africa, the predominant vector is Aedes albopticus (Grard et al., 2007). In Africa and Asia, other vectors involved are Aedes africanus, Aedes luteocephalus, Aedes furcifer and Aedes Taylori. Aedes aegypti and Aedes albopictus are responsible for the epidemic in the Americas (Ledermann et al., 2014). 10 University of Ghana http://ugspace.ug.edu.gh Aedes aegypti and Aedes albopictus bite during the day and are commonly found in the tropical and subtropical regions. Aedes aegypti and Aedes albopictus have intrinsic ability to transmit the Asian genotype of Zika virus strain. The ability of Aedes aegpti to transmit Zika virus in a given location is because they live in close association with human, feed mostly on humans, bite multiple humans in a single blood meal and have an almost imperceptible bite (Dick et al., 1952; Marchette et al., 1969). Aedes albopictus is found in more temperate regions compared to Aedes aegypti, thus extending the possible range where epidemics can occur. Aedes aegypti is distributed throughout America, Asia, Oceania, Europe and Africa (Kraemer et al., 2015) and can be shielded in local settings. Meanwhile Aedes albopictus, the most aggressive species of Aedes mosquitoes (Medlock et al., 2012), have extended it territory to North, Central and South America, Africa, southeastern Asia, China, Japan, northern Australia, and southern Europe in the last 30–40 years (Paupy et al., 2009). This success is as a result of the ability of Aedes albopictus to adjust to different climates through the production of cold-resistant eggs. Preference of Aedes albopictus to containers in domestic settings has also increased it contact with humans (Medlock et al., 2012). Zika virus has uncommonly been isolated from Aedes unilineatus, Anopheles coustani and Mansonia uniformis mosquitoes (Dick et al., 1952). Zika virus infection can be transferred from mother to fetus. Viral RNA and antigens have been isolated from amniotic fluids, placentas and brain tissues of mothers whose children were diagnosed with cerebral abnormalities and microcephaly (Besnard et al., 2014; Musso et al., 2015). Peripartum transmission of Zika virus infection involving mother and infant has also been reported (Besnard et al., 2014; Musso et al., 2015). Although transmission of 11 University of Ghana http://ugspace.ug.edu.gh Zika virus infection through sexual intercourse has been documented, risk factors and duration have not been determined.Viral particles and RNA often in high quantities have been identified in sperms up to 62 days after the onset of symptoms (Besnard et al., 2014; Musso et al., 2015). Given the transmission routes of other arboviral infections, transmission of Zika virus through blood transfusion is likely to occur even though it has not been reported. An individual became infected with Zika virus after been biten by a monkey in Indonesia (Besnard et al., 2014; Musso et al., 2015). Two infections in laboratories have been reported. Zika viral RNA has been found in the breast milk of a symptomatic woman on the day of delivery (Besnard et al., 2014; Musso et al., 2015). In a study carried out at the Colombia-Venezuela border, 29 (18.47%) of the 157 sera sampled from febrile patients were positive for Zika virus by real-time PCR. Prevalence of Zika virus co-existing with dengue and chikenguya was 6.37% and 5.10% respectively. Zika virus was also detected in a 27-year-old man who sought treatment at the Jambi city hospital 2 days after a sudden onset of fever in Indonesia (CDC, 2016). Zika virus was diagnosed as the cause of fever in a 52-year-old woman after a visit to El Salvador (Teale et al., 2016). Anti-Zika virus IgM 17 (8.4%) and IgG 120 (59.1%) were detected from 203 sera sampled in 2000 in Senegal. Fourteen out of the 17 anti-Zika virus IgM positives were confirmed by seroneutralization test (Fatim et al., 2000). 12 University of Ghana http://ugspace.ug.edu.gh In 2014 in Puerto Rico, out of the 29,345 Zika virus infected individuals reported, the highest incidence occurred among persons aged between 20 and 29 years. Subsequently in 2015–2016, of the 28,341 Zika virus infected individuals documented, the median age was 32 years. Incidence increased in persons aged 20 –29 years and 10–19 years when pregnant women were included (Matthew et al., 2016). Outbreak in the French Polynesia recorded 383 serologically confirmed Zika virus infected individuals of which prevalence was higher in 28 years old individuals (Paixão et al., 2016). According to Matthew et al., (2016), reasons for the higher number of individuals infected with Zika virus aged 20 years and above are unknown. In Bahia state and El Salvador, age group 20–49 years were the most affected (Dos Santos et al., 2016). During the 2007 epidemic in the Yap State, the attack rate among confirmed cases of Zika virus infection peaked among persons aged 30–39 years (Duffy et al., 2009). Zika virus infection is more prevalent in females than males (Paixão et al., 2016). In Puerto Rico, among the 28,219 patients confirmed with Zika virus infection, frequency was greater among women compared to men for all age groups. Even though prevalence of Zika virus infection in females age group 10–19 years was similar the observation in males of the same age group, prevalence in females persistently escalated with age and peaked among women aged 3 to 39 years (Matthew et al., 2016). Similarly, in Bahia state and El Salvador, total number of individuals infected with Zika virus was 75% higher in females than in males (Dos Santos et al., 2016). Outbreak of Zika virus infection in the Yap State of Micronesia recorded 61% of females’ infected (Duffy et al., 2009). 13 University of Ghana http://ugspace.ug.edu.gh Rate of infections has increased at different epochs. In Southern Cone and Andean, transmission of Zika virus infection was intensified in January, peaked in February and decreased (Ikejezie et al., 2016). Again, in the Central America, rates escalated in January and June (Ikejezie et al., 2016). Non-Latin Caribbean sub-regions recorded high rates of Zika virus infection in the months of February and June respectively (Ikejezie et al., 2016). In the Latin Caribbean sub-region, where prevalence of Zika virus infection is at its peak, rates increased in 2016, and continued at high intensity throughout the month of July (Ikejezie et al., 2016). These differences are associated with temperature, rainfall and humidity (Brisbois et al., 2010). In Indonesia a maximum rainfall of 2463 mm and minimum of 16.8 mm with an average rainfall of 352.2mm are appropriate for Zika virus transmission (Gharbi et al., 2011). A link between the incidence of Zika virus infection and rainfall has also been established (Ariati et al., 2012). 2.2.1 Zika Virus Infection in Africa Despite the presence of Zika virus infection in Africa since 1947 (Dick, 1952), limited data exist on the prevalence especially in febrile patients. This has been attributed to the sporadic nature of the infection with overlapping signs and symptoms resembling other infectious diseases, misdiagnosis and limited availability of tools for testing (Foy et al., 2011). Zika virus infection was first documented in 1947 in a rhesus macaque monkey and 1948 in an Aedes africanus mosquito in Uganda (Dick, 1952). Subsequently in 1952, Zika virus infection was found in humans in Tanzania and Uganda (Haddow et al., 2012). 14 University of Ghana http://ugspace.ug.edu.gh In 1954, a 10 years old female who was reported to a hospital in Nigeria was diagnosed with Zika virus infection after several laboratory tests (Macnamara, 1954). Other two cases reported in 1954 in Nigeria confirmed the possible circulation of Zika virus (Macnamara, 1954). In 1964 and 1975, Nigeria became endemic especially in Ibadan (Moore et al., 1975). Although, Zika virus infection was found in all age groups, children below 4 years were the most affected (Moore et al., 1975). Later, the infection was found among people in Kenya (Geser et al., 1970), Gabon (Jan et al., 1978; Grard et al., 2014), Central African Republic (Saluzzo et al., 1981), Sierra Leone (Robin and Mouchet, 1975), Senegal (Monlun et al., 1993; Diallo et al., 2014; Althouse et al., 2015), Uganda (McCrae and Kirya, 1982) and Ivory Coast (Akoua-Koffi et al., 2001), with a prevalence level ranging between 1.3–52%. Higher number of individuals with Zika virus infection was recorded in 1969 as compared to 1965 and 1967. Prevalence of Zika virus infection in 1970 in Kenya was attributed to the higher housing density (Geser et al., 1970). Growing age, farming and grass roof were the associated risk factors for the occurrence of Zika virus infection in Zambia. However, indoor residual spraying and having an iron roofing sheet over the house reduced the risk for Zika virus infection (Babaniyi et al., 2015). From 2008 to 2013, three people were diagnosed with Zika virus infection in Senegal (Heang et al., 2012; Heang et al., 2011). In 2016, Zika virus infection was transmitted locally in Guinea Bissau and Angola (World Health Organization, 2016 and 2017). The table below gives a brief description of the prevalence of Zika virus infection in Africa since 2000 (Waddell and Greig, 2016). 15 University of Ghana http://ugspace.ug.edu.gh Table 2.1: Prevalence of Zika virus infection in Africa since 2000 Country Date Population sampled IgM and IgG Prevalence Gabon 2007-2010 All ages-hospital submissions 0.1% Senegal 2009-2013 Adult-clinic submission 0.1% Cameroon 2010 All ages-hospital submissions 38% Zambia 2010 General population 6% Cape Verde 2010-2016 General population Outbreak with 19,083 cases Ethiopia 2013 Hospital submissions 10% Kenya 2013 General population 0.0% Source: Waddell and Greig, (2016) 2.2.2 Zika Virus Infection in Asia First reported cases of Zika virus infection in Asia was in India (Smithburn, 1954). Afterwards, clusters of Zika virus infection were found between 1977 and 1980 in Malaysia, Indonesia and Pakistan toward the end of the raining season (Olson et al., 1981; (Darwish et al., 1983). Zika virus infection was subsequently detected in Sabah, Malaysia in 144 orangutans (Kilbourn et al., 2003). In 2010, few individuals were sporadically infected with Zika virus infection in Cambodia (Heang et al., 2012). Because of the outbreak in the Americas, alertness and observation for 16 University of Ghana http://ugspace.ug.edu.gh Zika virus infection in Asia was improved and this resulted in better recognition and reporting. Rigorous analysis of sera from Cambodia, Bangladesh and Lao People’s Democratic Republic identified patients who were initially diagnosed as negative for Zika virus infection (WHO, 2017 and Duong et al., 2017). Implementation of improved surveillance for Zika virus infection in Thailand in 2016 increased the total number of Zika virus infections reported (WHO, 2016). A sero- epidemiological study found proof of Zika virus infection in some parts of Thailand (Wikan et al., 2016). Zika virus infection was also reported in tourists returning from Thailand (Ellison et al., 2016). Evidence of possible circulation of Zika virus was provided in Singapore in 2016 and 2017 (Leo and Chow, 2016). Zika virus infection was also found in South Korea in 2016 (Leo and Chow, 2016). 2.2.3 Zika Virus Infection in Oceania After the emergence of Zika virus infection in 1947, Zika virus infection showed a geographical restriction (Duffy et al., 2009). Zika virus infection was confined to Africa and Asia until 2007 when evidence of exposure was found on the island of Yap in the Federated States of Micronesia with about 5000 infected individuals. The occurrence of Zika virus infection in the Yap State of Micronesia was initially misdiagnosed as Dengue, Chikungunya or Ross River disease. However, further analyses revealed Zika virus RNA in the sera of these patients. About 49 individuals were confirmed with Zika virus infection with no hospitalizations or deaths (Duffy et al., 2009). The infection was predominant in children aged 3 years (Duffy et al., 2009). 17 University of Ghana http://ugspace.ug.edu.gh Epidemic of Zika virus infection in the French Polynesia was in 2013. Cases were reported from Marquesas and Tuamotu Islands in Mo’orea, Tahiti, Tahaa, Raiatea, Bora Bora, Arutua, Nuku and Hiva. Initially, it was assumed as Dengue with approximately 19,000 cases. By 2014, cases had increased to 29,000 with 8,503 suspected as Zika virus infection. Outbreak declined in March 2014, abating in October 2014 with approximately 30,000 Zika virus infected individuals (Derraik and Staney, 2015). Outbreak lasted for 21 weeks with an estimated 3,500 individuals complaining of fever. Actual number of infections in the French Polynesia was not known because patients did not seek medical care (Aubry et al., 2015). Zika virus infection also occurred in New Caledonia with 64 cases reported from Dumbea, Noumea and Ouvea of which thirty were introduced from the French Polynesia. By 2014, 140 individuals were infected with Zika virus with thirty two imported. Outbreak increased in April and by September, number of individuals with Zika virus infection had increased to 1,400 with thirty five imported. Zika virus infection circulated in the country until 2015 with 137 confirmed cases. Outbreak lasted for 29 weeks (European Centre for Disease Prevention and Control, 2015). In Cook Island, outbreak occurred in February 2014 with eighteen confirmed and two imported cases (European Centre for Disease Prevention and Control 2014). Outbreak ended in May with fifty cases confirmed as Zika virus infection and 932 suspected as Zika virus infection (WHO, 2014). Forty individuals were suspected to have Zika virus infection in 2014 in the Easter Island. Fifty individuals were infected with Zika virus in Chile (Tognarelli et al., 2016). These individuals were suspected to have been infected in the French Polynesia during the annual Tapati festival (Musso, 2015). Zika virus infection was 18 University of Ghana http://ugspace.ug.edu.gh also reported in Vanuatu weeks after the tropical cyclone in 2015 (European Centre for Disease Prevention and Control, 2015). 2.2.4 Zika Virus Infection in America About 175,063 individuals have been infected with Zika virus in the Americas since the epidemic in 2015 (Brazil Ministry of Health, 2016). Epidemic of Zika virus infection in America first occurred in Brazil and Colombia. Brazil is the most affected country with approximately 500,000 to 1,500,000 individuals (Bogoch et al., 2016). Case counts for Zika virus infection was not available in Brazil until 2016 when Zika virus infection became a national notifiable condition (Brazil Ministry of Health, 2016). Most of the individuals infected with Zika virus were native of Natal. Afterwards, Zika virus infection was found in individuals of Brazil, Guatemala, Colombia, Mexico, El Salvador, Paraguay, Bonaire, Samoa, Aruba, Sint Maarten, Trinidad and Tobago, Argentina, Venezuela and USA (Corsica, 2015). 2.3 Conditions for Spread of Zika Virus Infection 2.3.1 Climate Change and Variation The geographical distribution of Aedes spp. of mosquitoes has been exaggerated by climate variation and change. Climate change and variation have enlarged temperatures to a level that favour transmission and lengthen the transmission season in the tropical and subtropical regions. Temperature affects incubation and fecundity rates, survival, development and biting of Aedes aegypti and Aedes albopictus (Mordecai et al., 2016). 19 University of Ghana http://ugspace.ug.edu.gh The occurrence of Zika virus infection in the Americas has been ascribed to temperatures appropriate for Aedes aegypti (Hales et al., 2002). Warmer winter temperatures have enlarged the number of Aedes aegypti breeding sites in Brazil (Stewart et al., 2013). Climate variation and change have become a great worry in the temperate regions where temperatures were not always suitable for Aedes aegypti. Warmer temperatures recorded in the winter have increased the survival rate of the Aedes aegypti’s egg that was formally restricted to 10°C, thereby expanding the geographical range of Aedes aegypti’s (Foote et al., 1961; WHO, 2009). Warmer temperatures have also expanded the extent of transmission in the temperate regions especially in areas where Aedes aegypti or Aedes albopictus were locally established (Mordecai et al., 2016). 2.3.2 Social Change and Urbanization Aedes aegypti and Aedes albopictus are spreading to new countries due to human behaviors such as economic enterprises and expansion, trade and economic globalization, urbanization, population growth and poor sanitation (Simmons et al., 2012). Social changes such as natural disasters increase human contact with Zika virus-infected Aedes spp. of mosquitoes. In Ecuador, increased local transmission was linked with a tremor that hit the Manabi province in 2016, contaminating drinking water, destroying infrastructure and forcing people to live outdoors (Mis, 2016). 20 University of Ghana http://ugspace.ug.edu.gh Disasters also interfere with educational programs, health delivery and vector control programs (Watson et al., 2007). Apart from this, socioeconomic and political changes also expose people to poor infrastructure, water access and sanitation. Fluctuating vector control efforts, human movement and public sanitation policy further exacerbate present differences in these services (Watson et al., 2007). 2.4. Impact of Zika Virus Infection 2.4.1 Public Health Impact Because the infection has been found outside Africa and Asia, the virus is considered as an evolving pathogen. Zika virus infection up to date has been mild and benign until 2007 when severe forms of the infection were observed (Petersen & Hayes, 2004). It discovery in Micronesia and the neurological abnormalities such as Guillian Barre Syndrome and microcephaly that were associated with the virus proves that the virus has evolved and can spread across large geographical areas. Diagnosis is difficult due to the cross-reactive antibodies produced by these flaviviruses and the overlapping symptoms and signs that resemble dengue and other flaviviral infections. Given its overlapping signs and symptoms, prevention and control strategies include the use of insect repellent and implementation of interventions that will reduce the number of potential mosquito vectors. The rapid spread of Zika virus infection and its impact on health requires the cooperation of medical professionals, public health officers and first- class reference laboratories (Duffy et al., 2009). 21 University of Ghana http://ugspace.ug.edu.gh 2.4.2 Socio-economic Impact Socioeconomic impact has been anticipated to be long-term, most likely to affect the poorest and helpless countries, and also widen inequalities in the world. The cost of the recent spread to Latin America and the Caribbean has been projected at 7-18 billion US dollars, impact that is more than what was recorded in South America. Apart from this, more than 80 percent of revenues annually earned from tourism were reduced, with the possibility of reaching a total of US$9 billion. Haiti and Belize are estimated to loss about 1.13% and 1.19% of GDP annually (UNDP and IFRC, 2017). Aside revenue loss, the magnitude of stress that is likely to be placed on health care facilities can retard social development. Severest impact on development will be felt by the poorest economies in the world. In these regions, income that is estimated to be lost is projected at a half a billion dollars. Although several intensive efforts by most affected nations have been put in place to control the spread of Zika virus infection, these efforts have been faced with several challenges (UNDP and IFRC, 2017). These include allocation of resources and coordination, prevention, surveillance and diagnosis. Social inequalities and inadequate health service coverage is a challenge for national response teams to reach the most helpless groups (UNDP and IFRC, 2017). Furthermore, huge investment is required in durable control measures such as vaccine development, production of mosquito repellants, waste management, epidermiologic and entomologic researches to define the range of Zika virus vectors and identify new areas where autochthonous transmission could take place to enable early intervention. Cost of diagnosis is expected to high particularly in countries where resources for diagnosis are 22 University of Ghana http://ugspace.ug.edu.gh lacking and potential intervention measures (e.g., contraception or termination of pregnancy) discouraged or illegal (Fauci et al., 2016). 2.5 Clinical Manifestation of Zika Virus Infection Gestation period of Zika virus infection is three to twelve days and indications are usually mild and asymptomatic (Majumder et al., 2016). Symptoms are relieved within two to seven days but virus can still be detected in the sera of the infected person after one week of cure. Severe clinical forms of the infection and mortality rates are very low. Persons develop immunity after exposure (Ahmad et al., 2016). Key complications include microcephaly and Guillain–Barré syndrome 2.5.1 Adults About 20–25 % of individuals infected with Zika virus develop Zika virus disease (Duffy et al., 2009). Symptoms include mild fever with a tempereature greater than 37 °C, puritic rashes, headache, non-purulent conjunctivitis, myalgia, arthralgia (Brasil et al., 2016), hematospermia (Musso et al., 2015), asthenia, transient hearing loss (Tappe et al., 2015), retro-orbital pain and subcutaneous bleeding (Karimi et al., 2016). Other symptoms include mucous membrane ulceration, lymphopenia, diarrhea, neutropenia, thrombocytopenia, nausea, monocytosis, increased erythrocyte sedimentation rate, thrombocytopenia (Zammarchi et al., 2015), raised serum levels of gamma-glutamyl transpeptidase, lactate dehydrogenase, aspartate aminotransferase, abdominal pain, pruritus, 23 University of Ghana http://ugspace.ug.edu.gh ferritin, fibrinogen, transient and mild leucopenia and C-reactive protein during the course of the viremic phase (Dupont et al., 2014). Outburst of Zika virus infection in the Americas was characterized by an unusual increase in Guillain–Barré syndrome. However, the casual link was unknown (Mlakar et al., 2016). During the outbreak of Zika virus infection in the French Polynesia, Zika virus infection was associated with Guillain–Barré syndrome. Anti-Zika virus IgM was positive in 93 % of Guillain–Barré syndrome cases (Cao-Lormeau et al., 2016). 2.5.2 Children Children are infected with Zika virus through the intrauterine, intrapartum and postnatal routes (Hennessey, 2016). Intrauterine consequences of Zika virus infection and its determinants are not fully known. Although the virus has been isolated from several cases of microcephaly, actual number of cases caused by Zika virus is unknown (Mlakar et al., 2016). Symptoms include low birth weight, polyhydramnios, redundant scalp skin, arthrogryposis and anasarca. Neurological deformities include cerebral lesions, brainstem dysfunction and polymalformative syndromes (WHO, 2015). Intracranial calcifications are mostly seen over the white matter of the lentostriatal vessels, frontal lobes, cerebellum, vermis and thalami, caudate, around the ventricles and asymmetrical cerebral hemispheres (Schuler-Faccini, 2015). Symptoms in the later stages of pregnancy include cerebral atrophy, enlarged cisterna magna, dysgenesis of corpus callosum, severe unilateral ventriculomegaly and thinning of 24 University of Ghana http://ugspace.ug.edu.gh the parenchyma on the dilated side, pons and brainstem, sensorineural deafness, mental retardation and ophthalmological lesions (Schuler-Faccini, 2015). During the Brazil epidemic, there were 35 microcephaly cases (Schuler-Faccini, 2015). Heads have circumferences ≥2 standard deviations (Schuler-Faccini, 2015). Figure 2.3: Range of microcephaly severity Source: Lyle et al., (2016) 2.6 Diagnosis of Zika Virus Infection 2.6.1 Case Definition Zika virus infection is a self-limiting infection with patients recovering after a non-severe clinical course (Tripathi et al., 2011). However, if Zika virus infection is not properly managed it can lead to severe forms of the infection such as Microcephaly and Guillian Barre syndrome. The WHO Zika virus infection case definition was therefore introduced for the timely identification and management of Zika virus infection by clinicians. This saves resources and leads to a reduction in Zika complications (Horstick et al., 2014). Definitions according to the WHO, (2016) include; 25 University of Ghana http://ugspace.ug.edu.gh Suspected case: a person who has fever and/or rashes with either non-purulent conjunctivitis or arthralgia Probable case: a person who has anti-Zika virus IgM with no proof of any other aboviral infection, and has not had any interaction with an infected person or resided in or travelled to an area with active transmission of Zika virus infection two weeks before onset of symptoms. Confirmed case: is a person who has antigen or Zika virus RNA and a plaque reduction neutralization test titre value of ≥20 for Zika virus and titre ratio ≥ 4 compared to other flaviviruses 2.6.2 Laboratory Investigation Investigation of Zika virus infection is often based on defined criteria for case definition and travel history (Rigau-Perez et al., 1994). Clinical evaluation of Zika virus infection is undertaken concurrently with CHIKV and DENV for patients diagnosed with fever, myalgia, rash or arthralgia. Laboratory investigation of Zika virus infection can be made by one of the following methods; ➢ Cell culture to isolate virus (Vordam and Kuno, 1997) ➢ Real time PCR to identify viral nucleic acid ➢ Serology to detection of virus specific antibody Viral detection (virus isolation, genome detection and antigen detection) is done at the acute phase (0-5) of the infection in plasma, serum, whole blood and tissues following the 26 University of Ghana http://ugspace.ug.edu.gh onset of symptoms. However, detection of the virus and its components from samples are not routinely employed in laboratories due to the few commercial kits available. As a result, commonly diagnostic tools routinely employed for the diagnosis of Zika virus infection are serological tests (Peeling et al., 2010). 2.6.2.1 Virus Isolation Common methods used in the isolation of Zika virus is the inoculation of serum into Aedes albopictus cell lines (C6-36) and Aedes pseudoscutellaris (AP61) (Guzman and Kouri, 1996; Race et al., 1979), Vero cells and LLC-MK2 and inoculation into intra-cerebra of suckling mice (Peeling et al., 2010). Isolation of Zika virus from larval or adults of Toxorchunchitis spp mosquitoes (T. ambionesis, T. splendens) (Gubler and Rosen, 1976; Rosen and Shroyer, 1985) has a higher viral isolation rate of 80% as compared to other methods (Vordam and Kuno, 1997). Immunofluorescent assay is performed to identify the virus, either seven to ten days after inoculation in cell lines or fourteen days after inoculation in mosquitoes (Guzman and Kouri, 1996; Henchal et al., 1983; Vordam and Kuno, 1997). Culturing of Zika virus has a lot of limitations. It requires specimen from patients within two to seven days of infection (with sufficient viral loads) and isolating the virus immediately as the period for successful isolation of the virus is short. Culturing of Zika virus is time consuming, labour intensive and requires training (World Health Organization, 1997). 27 University of Ghana http://ugspace.ug.edu.gh 2.6.2.2 Viral RNA Detection Viral RNA can be isolated from tissues and sera using molecular amplification methods. Molecular amplification (e.g., RT-PCR) is the preferred method for diagnosing Zika virus infection due to its high sensitivity and specificity. Currently, there are seven real-time RT- PCR assays for Zika virus RNA detection (Waggoner and Pinsky, 2016). Protocols developed for the isolation of Zika viral RNA employ the use of primers that target specific regions in the genome such as membrane (M), envelop (E) and the nonstructural proteins (NS) 1, NS2, NS5 and NS3 (Lanciotti et al., 1992; Johnson et al., 2005; Wu et al., 2001). 2.6.2.3 Serological Diagnosis Serological methods used for diagnosing Zika virus infection include ELISA, indirect immunofluorescence assays (IIFA) and virus neutralization tests (VNT) (Guzman and Kouri, 1996). Furthermore, these assays can only detect antibodies five days after the onset of disease, making them unsuitable for rapid diagnosis (Guzman and Kouri, 1996). 2.6.2.3.1 Enzyme-Linked Immunosorbent Assay (ELISA) ELISA is the best tool for detecting anti- Zika virus IgG and IgM antibodies during the acute phase of Zika virus infection. They include the IgM based assays, IgG based assays or rapid immunochromatographic test (Guzman and Kouri, 1996; Vordam and Kuno, 1997; Innis et al., 1989). Anti-Zika virus IgM is detectable four to five days after the onset of symptoms and can remain detectable 2-3 months. Positive result for anti-Zika virus IgM can be suggestive of a current infection. However, in an endemic area, demonstration of a fourfold increase in antibody titres of paired samples is required to diagnose a current infection (World Health Organization, 2009). Anti-Zika virus IgG ELISA kits are employed to establish a previous 28 University of Ghana http://ugspace.ug.edu.gh exposure to Zika virus (Peeling et al., 2010). During a primary infection, anti-Zika virus IgG levels are often low but increases over time (Halstead and Papaevangelou, 1980). Anti-Zika virus ELISAs has microplates treated with anti-human IgM or IgG antibodies to bind the equivalent antibodies in the sample. After washing the well, horseradish peroxidase (HRP) labeled Zika virus antigen is added. This antigen-conjugate binds to the captured anti-Zika virus IgG or IgM antibodies. In a second washing step unbound conjugate is removed and Tetramethylbenzidine (TMB) substrate is added which gives a blue reaction product. The resultant colour change after adding stop reagent is quantified by a spectrophotometric reading of optical density (OD) which is directly proportional to the amount of antigen-specific IgM or IgG present in the sample. The samples OD reading is compared to a reference cut-off value to determine results (Peeling et al., 2010). Figure 2.4: Major diagnostic markers for Zika virus infection Source: Peeling et al., (2010). 29 University of Ghana http://ugspace.ug.edu.gh 2.6.2.3.2 Plaque Reduction Neutralization Test It is recommended that presumptive positives, equivocal or unconvincing IgM test results be confirmed by this method. Sera or solution of antibody is diluted and mixed with a constant volume of Zika viral suspension. This is incubated and poured over a confluent monolayer of host cells. The surface of the cell layer is covered in a layer of agar or carboxymethyl cellulose to prevent the virus from spreading indiscriminately. The concentration of plaque forming units is estimated by the number of plaques (regions of infected cells) formed after a few days (Schmidt et al., 1976). Vital dye such as neutral red is then added for visualization of the plaques and the percentage neutralization estimated by dividing the quantity of plaques formed in individual plate by the original number of virions. The plaque forming units are measured by microscopic observation, fluorescent antibodies or specific dyes that react with infected cells. The concentration of serum to reduce the number of plaques by 70% compared to the serum free Zika virus gives the measure of how much antibody is present or how effective it is (Schmidt et al., 1976). Currently it is considered to be the "gold standard" for detecting and measuring antibodies that can neutralize Zika virus (Thomas et al., 2009-11; Ratnam et al., 1995). It has a higher sensitivity and specificity than Enzyme immunoassay. However, the test is relatively cumbersome and time intensive (few days) relative to ELISA kits that give quick results (usually several minutes to a few hours) (Schmidt et al., 1976). 30 University of Ghana http://ugspace.ug.edu.gh 2.6.2.3.3 Indirect Immunofluorescence In indirect immunofluorescence assay, two antibodies are used in the diagnosis of Zika virus infection. A primary antibody which has not been labeled binds specifically to the antigen. A secondary antibody which has been labeled with a fluorophore recognizes the primary antibody and binds to it. This provides signal amplification by increasing the number of fluorophore molecules per antigen (Fritschy and Hartig, 2001). 2.7 Treatment and Prevention of Zika Virus Infection 2.7.1 Treatment and Therapeutic Approaches Treatment of Zika virus infection is directed to symptoms relief with fluids, rest, anti- pyretics and analgesics (Engle and Diamond, 2003). Acetaminophen is used to relief patients of headache, myalgia and fever. Pruritus is managed with antihistamines whiles fluid loss is fixed with adequate rehydration (Chen and Hamer, 2016). For effective treatment of latent neurological complications particularly Guillian Bare Syndrome, it is advised that diagnosis be done quickly. Treatment involves the use of immunoglobulins and plasmapheresis (Chen et al., 2016). Infected children and family are supposed to be counseled by pharmacists, medical geneticists, neurologists and other health specialists (Staples et al., 2016). Currently, thirty-eight vaccines have been reported to the World Health Organisanition. Thirty two are in the pre-clinical stage, five in phase one trial and one in phase two trail. These vaccines include DNA, mRNA, inactivated and live attenuated vaccines, proteins 31 University of Ghana http://ugspace.ug.edu.gh and peptides (Lagunas-Raangel et al., 2017). The table below (Table 2.2) shows some of the vaccines that have been developed and their trial stage. Table 2.2: Current Zika vaccine candidates at various stages of preclinical/clinical trials Vaccine Approach Developer Status DNA-based vaccine NIAID Phase 2 Purified inactivated Zika WRAIR Phase 1 vaccine A live-attenuated vaccine NIAID Phase 1 mRNA vaccines NIAID and GSK Phase 1 AGS-v, SEEK Phase 1 Source: NIAID, (2017) 2.7.2 Prevention of Zika Virus Infection Control and prevention of Zika virus infection is mostly focused on preventing the bites of Aedes spp. of mosquitoes. Methods include putting on long-sleeved shirts and pants, using insect repellent, minimization of outdoor activities, using screened windows and doors, elimination of standing water and enactment of mosquito control programs (Martin-Acebes and Saiz, 2012). The European Center for Disease Prevention and Control and the Center for Disease Control and Prevention recommend that, pregnant women and those who want to get 32 University of Ghana http://ugspace.ug.edu.gh pregnant should not to travel to areas with active Zika virus transmission. Again, areas where Zika virus infection has not reported, but has potential mosquito vectors, it is recommended that a rapid response system be adapted by the public health institutions to prevent local transmission when a case is detected. The World Health Organisation on the other hand calls for collaboration between the health authorities and the transport sectors in various countries in order to disinfect any air craft travelling from affected areas (Musso et al., 2015). The Center for Disease Control and Prevention also endorses the use of condom when having sexual intercourse while traveling or if partner has just come from an area with active viral transmission. Furthermore, pregnant women are advised to abstain from sexual intercourse. Likewise, since Zika virus infection can be spread through blood transfusion, countries with or without active transmission of Zika virus infection are advised to take measures to prevent transmission through blood transfusion (Musso et al., 2014). 33 University of Ghana http://ugspace.ug.edu.gh CHAPTER THREE MATERIALS AND METHODS 3.1 Study Design and Ethical Consideration 3.1.1 Study Design The study was a cross sectional study involving archived serum samples from febrile patients at the Greater Accra regional hospital between December 2016 and November 2017. These clinical specimens were obtained as part of an ongoing project with the aim of using serological and molecular tools to detect Dengue and Chikungunya in febrile patients within selected health facilities in Ghana. A structured case investigation form was used to collect information about demographic features and clinical symptoms of the febrile patients (Case Investigation Form, Appendix 1). 3.1.2 Ethical Consideration This study used archived sera from an on-going project at Noguchi Memorial Institute for Medical Research, thus ethical clearance obtained for this ongoing project from the Noguchi Memorial Institute for Medical Research Institutional Review Board covered this work. Besides this, ethical clearance was also solicited from the College of Health Sciences Ethical and Protocol Review Committee, University of Ghana. 34 University of Ghana http://ugspace.ug.edu.gh 3.2 Study Site and Sample Size Determination 3.2.1 Study Site Greater Accra regional hospital serves as a secondary referral center as well as the main hospital for the Greater Accra Region. Catchment area of Greater Accra regional hospital is the whole Greater Accra region with a population of about 4,283,322 inhabitants. The hospital was selected for this study based on the high number of febrile cases recorded at the hospital and the presence of well-established clinical and laboratory facilities. The hospital has a 600-bed capacity and provides a wide range of services including out- patients, in-patient, specialist services and administration and support services. 3.2.1 Sample Size Determination Sample size was determined using hospital admissions prevalence rate obtained from a study conducted by Tsegaye (2014) in Ethiopia in 2013. This is the only recent seroprevalence study conducted in Africa I found during my literature review, as a result it selection. With 95% confidence level and a corresponding Z- value of 1.96; Error margin of 0.05, Prevalence (P) of Zika virus infection of 10% (0.10) Sample size required was determined using the formula; N=Z2P(1-P) d2 35 University of Ghana http://ugspace.ug.edu.gh Sample size = (1.962x 0.10x 0.88)/0.052 =135 samples However, one hundred sixty (160) serum samples were used for this study because of the design of anti-Zika virus ELISA test kits used. 3.3 Eligibility Criteria 3.3.1 Inclusion Criteria The study recruited all persons in all ages including pregnant women and children. For a consented participant to meet the inclusion criteria he/she should have fever (body temperature ≥ 38oC) and at least two of the following signs or symptoms: conjunctivitis, diarrhea, nausea, vomiting, muscle pain, joint pain, jaundice, loss of appetite, chest pain, rapid respiration and recent loss of hearing. 3.3.2 Exclusion Criteria 1. Patients positive for Malaria by blood film test 3.4 Sampling Procedure and Documentation 3.4.1 Sampling Procedure Frozen sera were used for the study. Sampling procedure used for the collection of the sera is as follows; Human whole blood samples were taken by nurses that have been trained on the case definition at the Greater Accra regional hospital. From an eligible patient who had consented to be part of the study, a good-sized vein was identified, usually in the antecubital fossae or on the dorsum (back) of the hand. A 36 University of Ghana http://ugspace.ug.edu.gh tourniquet was then applied proximal to the site of venipuncture to ensure engorgement of vein with blood. The site of the venipuncture was cleaned with an alcohol swab and approximately 5 milliliters of blood is collected into a BD Vacutainer with SST II Advance Semi-separator gel (BD, Belliver Industrial Estate, Plymouth, PL6 7BP, United Kingdom), mixed by turning it 5 times upside-down. Once enough blood was withdrawn, the tourniquet was removed, with the needle still in place, a cotton swab was taken and placed over site of needle insertion and the needle was gently removed. Direct pressure was applied with the cotton swab over the puncture site to stop any bleeding (about 2 minutes), after which the swab was removed to ensure bleeding has stopped. If not, the swab was affixed with gauze tape. 3.4.2 Documentation Participant who consented and their clinical samples taken also provided demographic information which were captured on a structured case investigation form (Appendix 1) as well as their clinical information. All the serum collecting tubes were labeled with unique study number that also corresponded with their identification number on the case investigation form. 3.5 Sample Processing and strategy 3.5.1 Sample Processing Samples were processed into sera and cryogenized as follows; The blood samples (approximately 5ml) collected were processed into serum at the virology department, Noguchi Memorial Institute for Medical Research. The processing involved the blood in the serum separator tube been centrifuged at 1000 × g for 10 minutes 37 University of Ghana http://ugspace.ug.edu.gh to separate the serum which usually will be about 3.5mls in volume. The serum was carefully removed and transferred aseptically using a fine-bore pipette into two separate sterile labeled 1.8ml-sized cryogenic vials. Aliquots of the serum were used in the laboratory investigation and the rest stored permanently under ultra-low (-80°C) freezing conditions at NMIMR. It was from these frozen sera, samples were taken to run for anti- Zika virus IgG and IgM. 3.5.2 Sampling Strategy Every respondent who met the eligibility criteria and consented to the study was sampled. Recently collected será (between December 2016 and November 2017) were purposively collected from the banked será. 3.6 Laboratory Testing of Zika Virus Infection All reagents and sera were allowed to thaw and mixed well before used according to Abcam anti-Zika virus IgM and IgG capture ELISA kits (Abcam, Cambridge, United Kingdom) protocols (Figure 3.1). Abcam anti-Zika IgM and IgG capture ELISA kits contain Zika virus IgG and IgM microplate, IgG and IgM sample diluent, stop solution, 20X wash buffer solution, horseradish peroxidase (HRP) Zika virus conjugate, 3, 3’, 5, 5’-tetramethylbenzidine (TMB) substrate solution, Zika virus IgG and IgM positive control, Zika virus IgG and IgM cut–off control, Zika virus IgG and IgM negative control and cover foil. Working washing solution was prepared by diluting 1 part (eg. 10ml) of the Washing solution with 19 parts (190ml) of fresh distilled water. Subjects’ sera were diluted with the 38 University of Ghana http://ugspace.ug.edu.gh dilution buffer specially prepared for testing for IgM and IgG antibodies to Zika as provided by the manufacturer. One part (10 μl) of serum was diluted with 100 parts (1ml) of the dilution buffer. Microtitre strip wells were arranged according to the number of samples. One hundred microlitres of the negative control were aliquoted into wells C1 and D1 while well A1 and B1 were left blank according to the Abcam anti-Zika virus IgM and IgG capture ELISA kits protocols. One hundred microlitres of the cut-off and positive control were also aliquoted into wells E1 and F1 and G1 and H1. One hundred microlitres of diluted patients’ serum were then aliquoted into each of the remaining microplate wells (80 of them). The wells were covered with a foil and incubated at 37ºC for an hour. After incubation, the foil was removed, content of wells aspirated and washed with 300μl of washing buffer. The wells were blotted on an adsorbent paper until completely dried. One hundred microlitres of HRP Zika virus conjugate (peroxidase labeled Zika virus antigen) were added to each well except for the substrate blank wells and incubated for 30 minutes at 37ºC. The washing procedure was repeated after which 100μl of substrate solution (3,3´,5,5´- tetramethylbenzidine (TMB) hydrogen peroxide) was added to each well and incubated in the dark at room temperature 20º C - 25º C for 15 minutes. One hundred microlitres of the stop solution was added to each well. The ELISA microtitre plate reader (Human Diagnostic Worldwide, Germany) (Figure 3.2) was set to zero using the substrate blank in wells A1 and B1 as shown in Table 3.1. The absorbance was measured at 450nm within 30 minutes after terminating the reaction using a reference wavelength of 620nm. Reagents and serum samples were dispensed into the wells using a micropipette. 39 University of Ghana http://ugspace.ug.edu.gh Table 3.1: Arrangement of wells in the microplate 1 2 3 4 5 6 7 8 9 10 11 12 A Blank B Blank C Negative Control D Negative Control E Cut-off Control F Cut-off Control. G Positive Control. H Positive Control. Figure 3.1: Thawing of Samples Figure 3.2: Sample reading with automated microtitre plate reader 40 University of Ghana http://ugspace.ug.edu.gh 3.7 Calculation and Interpretation of Results (IgM and IgG) 3.7.1 Calculation of Cut-off (IgM and IgG) The Cut-off was calculated as the mean absorbance value of the Cut-off Controls determined (Table 3.1). Cut-off = absorbance value cut-off control + absorbance value cut-off control 2 3.7.2 Calculation of Results in Abcam Units (AU) (IgM and IgG) Results in Abcam Units (AU) = Sample (mean) absorbance value ×10 Cut-off 3.7.3 Interpretation of Results (IgM and IgG) Tests results were interpretated according to Abcam anti-Zika virus Igm and IgG protocols. Interpretation was done according to the table below; Table 3.2: Criteria for Interpretation of Results Result Value Cut-off 10 AU Positive >11 AU Equivocal 9-11 AU Negative <9 AU 41 University of Ghana http://ugspace.ug.edu.gh 3.8 Independent and Outcome variables 3.8.1 Independent variable The independent variables were: age, yellow fever vaccination and sex. 3.8.2 Outcome variable The outcome variable were seropositivity, muscle joint pains, conjunctivitis, diarrhoea, nausea, vomiting, muscle pain, joint pain, jaundice, loss of appetite, chest pain, rapid respiration and recent loss of hearing. 3.9 Statistical Analysis Clinical and laboratory data were collected and recorded manually. Data were double entered, cleaned and stored in Microsoft Excel. Analyses were done using IBM SPSS version 21 (IBM Corp., Armonk, United States). Bivariate logistic regression model was used to investigate association between Zika virus seropositivity and age, yellow fever vaccination and sex. It was also used to investigate the association between Zika virus seropositivity and clinical characteristics such as muscle joint pains, conjunctivitis, diarrhoea, nausea etc. In the bivariate analysis, variables that were associated with Zika virus seropositivity at a significance level of p-value < 0.05 were considered to be statistically significant. One way-Anova was also used to test the association within groups at a significance level of p-value < 0.05. 42 University of Ghana http://ugspace.ug.edu.gh CHAPTER FOUR RESULTS 4.1.0 Demographic Characteristics of Febrile Patients A total of 160 febrile patients were enrolled, 119 (74.4%) were females while 41 (25.6%) were males. Eighty seven (54.4%) were within the reproductive age group but four (12.1%) were children. Sixty one (38.1%) were vaccinated against yellow fever whereas 99 (61.9%) were unvaccinated. Demographic characteristics of febrile patients involved in the study are summarized in Table 4.1 below. Table 4.1: Demographic characteristics of febrile patients Variable Number Percentage (%) Age (years) Children 16 10 Reproductive age 87 54.4 > (above) 57 35.6 Gender Male 41 25.6 Female 119 74.4 Yellow fever vaccination Vaccinated 61 38.1 Unvaccinated 99 61.9 43 University of Ghana http://ugspace.ug.edu.gh 4.1.1 Seroprevalence of Zika Virus Seroprevalence of Zika virus from the study was 20.6%. Of the 33 anti-Zika virus antibodies detected, 3 were anti-Zika virus IgG while 30 were anti-Zika virus IgM. No patient had both anti-Zika virus IgG and IgM antibodies (Figure 4.1). Serological test for ZIKA 200 150 100 50 0 0 5 10 15 20 25 IgG (Standard Units) Figure 4.1: Seroprevalence of Zika virus 4.1.2 Characteristics of Zika Virus Seropositives Among the age groups identified, 14 (42.5%) of the anti-Zika virus IgM were recorded in the reproductive age group but 4 (12.1%) were recorded in children. Anti-Zika virus IgG was detected in two individuals (66.7%) within the reproductive age group and a child 1 (33.4%). From the total Zika virus seropositives, 11 (33.3%) were males and 22 (66.7%) females. As a result, Zika virus infection was estimated to affect males and females in a ratio of 1:2. Number of anti-Zika virus IgM males was 11 (36.7%) and that of females was 19 (63.3%). Anti-Zika virus IgG was detected only in females representing 100%. No significant 44 IgM (Standard Units) University of Ghana http://ugspace.ug.edu.gh difference was observed between anti-Zika virus IgM males and females (P=0.016) and IgG males and females (P=0.500). Twenty (66.7%) and ten (33.3%) of the anti-Zika virus IgM were detected in yellow fever unvaccinated and vaccinated febrile patients respectively with a significant difference of P=0.000. Again, 2 (66.7%) of the anti-Zika virus IgG were detected in unvaccinated febrile patients. Vaccinated febrile patients recorded only one (33.3%) as shown in Table 4.2. Table 4.2: Characteristics of Zika virus seropositives Variables Zika virus antibodies n= 33 IgM (30) P-value IgG (3) P-value Age groups Children 4(12.1%) 0 Reproductive age 14(42.5%) 0.08 2(66.7%) - > (above) 12(36.4%) 1 Gender Male 11(36.7%) 0 Female 19(63.3%) 0.016 3(100%) - Yellow fever vaccination Vaccinated 10(33.3%) 1(33.3%) Unvaccinated 20(66.7%) 0.000* 2(66.7%) - 45 University of Ghana http://ugspace.ug.edu.gh One –way Anova was used to estimate the difference between groups. “n” represents the number of Zika virus seropositives. * denotes statistically significant difference between groups. P is significant at 0.05 4.1.3 Monthly Distribution of Anti-Zika Virus Antibodies Anti-Zika virus IgM detection peaked in March 7 (23.3%) and May 9 (30%) and decreased in September 1 (3.3%). However, anti-Zika virus IgG were detected only in January and March, 2 (66.7%) and 1 (33.3%) respectively. Anti-Zika virus IgM monthly distribution showed no significant difference (P = 0.06). Again, no significant difference was observed between anti-Zika virus IgG monthly distribution (P=0.205) (Figure 4.2). Monthly distribution of Anti-Zika virus antibodies 10 9 8 7 6 IgM 4 IgG 3 3 3 2 2 2 1 0 Dec. 2016 Jan. March April May Sept. Oct Nov. Figure 4.2: Monthly distribution of anti-Zika virus antibodies 4.1.4 Anti-Zika Virus IgM Monthly Distribution Stratified by Gender Anti-Zika virus IgM females were recorded in the month of May 8 (24.2%) whereas, males were recorded in the month of March 3 (9%) (Figure 4.3) 46 Number of occurances University of Ghana http://ugspace.ug.edu.gh 8 7 6 5 4 8 3 2 4 3 3 1 2 2 2 2 2 1 1 1 1 1 0 Dec. Jan March April May Sept Oct. Nov. Anti-Zika virus IgM monthly distribution stratified by gender Figure 4.3: Anti-Zika virus IgM monthly distribution stratified by gender 4.1.5 Date of Anti-Zika virus IgM detection The study recorded 8 (24%) of the anti-Zika virus IgM on the 2nd day after the onset of illness. One (3%) of the anti-Zika virus IgM was detected on the 7th and 8th days respectively (Figure 4.4). 8th day 1 7th day 1 6th day 3 5th day 5 4th day 6 3rd day 4 2nd day 8 1st day 2 0 1 2 3 4 5 6 7 8 Number of occurances Figure 4.4: Date of Anti-Zika virus IgM detection 47 Date of Anti-Zika virus IgM frequency detection Female Male Female Male Female Male Female Male Female Male Male Female Male Female University of Ghana http://ugspace.ug.edu.gh 4.1.6 Town of Residence of Zika Virus Seropositive Patients Proportion of Zika virus seropositives residing in Lapaz and Amasaman accounted for 4 (12.1%) each. Ashaley Botwe, Adabraka, Adenta and Kosoa recorded 2 (6.1%) each. One (3.0%) each was recorded in 37, Alajo, Asylum down, Ordorna, Dome kwabenya, Teshie, Airport, Darkuman, Nima, Weija, Pokuase, Kokomlemle, Winneba, Achimota, Circle, Awodome and Asafo. 4.1.7 Predictors of Zika Virus Seropositivity Bivariate logistic regression analysis revealed that gender, age and yellow fever vaccination were not associated with Zika virus seropositivity (Table 4.3). Table 4.3: Bivariate logistic regression analysis showing predictors of Zika virus seropositivity Variables OR (95% Cl) P-value Age groups 3-20 1 21-30 0.293 (0.048-1801) 0.185 31-40 0.480 (0.094-2.458) 0.378 41-50 0.778 (0.141-4.304) 0.773 51-60 0.283 (0.52-1.548) 0.145 61 and above 0.533 (0.076-3.725) 0.526 Sex 48 University of Ghana http://ugspace.ug.edu.gh Male 1 Female 0.696 (0.306-1.587) 0.389 Vaccination 0.935 (0.426-2.049) 0.866 4.2 Symptoms Presented by Seropositive Febrile patients From the figure below, 33 (100%) of the seropositive febrile patients were presented with fever. The least 1 (0.8%) symptom reported was Jaundice. Conjunctivitis was not reported by any seropositive Zika virus female. All other symptoms were reported in both males and females. 35 33 30 28 25 24 25 20 18 16 15 14 15 12 10 5 2 1 0 Figure 4.5: Symptoms presented by seropositive febrile patients 49 University of Ghana http://ugspace.ug.edu.gh 4.2.1 Bivariate Analysis of Zika Virus Seropositive and Clinical Presentation Zika virus seroprevalence was associated with muscle pain, joint pain, conjunctivitis and chest pain as shown in the bivariate logistic regression analysis table below (Table 4.5). Table 4.4: Bivariate logistic regression analysis Variables OR (95% Cl) P-value n=33 Chest pain - 0.000* Fever 0.686(0.256-1.838) 0.453 Diarrhoea 0.000(0.000) 0.999 Nausea 0.476(0.178-1.277) 0.140 Loss of appetite 1 (0.367-2.727) 1.000 Muscle pain 0.102(0.31-0.334) 0.000* Joint pain 0.183(0.063-0.531) 0.002* Conjunctivitis 0.214(0.075-0.609) 0.004* Rapid Respiration 9.143(1.855-45.056) 0.007 Vomiting 0.607(0.227-1.625) 0.320 “n” represents the number of Zika virus seropositive .* denotes statistically significant difference. P is significant at 0.05 50 University of Ghana http://ugspace.ug.edu.gh CHAPTER FIVE DISCUSSION, CONCLUSION, RECOMMENDATION AND LIMITATION 5.1 Discussion Data from the study indicates exposure to Zika virus which suggests the possible circulation of the virus in Ghana. The recent rapid expansion, association with severe neurological disorders and the overlapping clinical features of Zika virus infection with other endemic disease conditions have made prediction of exposed population a necessity. Until recently, identifying exposed population has been of a low priority because of the mild symptoms presented by Zika virus infection (Petersen et al., 2016). This study therefore sought to bring some degree of attention to Zika virus infection in Ghana where little is been done on surveillance. It was conducted to serologically determine and establish Zika virus infection in febrile patients in a selected health facility, the Greater Accra regional hospital in Accra, Ghana. Cross-reactive antibodies produced by flaviviruses have made diagnosis of Zika virus infection difficult especially, when dengue has been found in the sub-region. A study conducted by Safronetz et al., (2017) to evaluate the performances of 5 commonly used Zika virus immunoassays against an ELISA (MAC ELISA) developed by the Center for Disease Control and Prevention and cross-PRNTs showed that, the Abcam ELISA kits used in this study has a specificity of 100% and a sensitivity of 57% (Safronetz et al., 2017). Zika virus infection (33 seropositive febrile patients) was not widespread among the febrile patients. The prevalence of Zika virus antibodies recorded from the study, 20.6% was in line with findings from other African countries, 1.3-52% prevalence level (Akoua-Koffi et 51 University of Ghana http://ugspace.ug.edu.gh al., 2001). However, prevalence recorded was greater than seroprevalence in hospital admissions in Zambia 6%, Senegal 0.1%, Gabon 0.1%, Ethiopia 10% but less than Cameroon 38%. Prevalence was also higher than seroprevalence in the French Polynesia 0.8% before the epidemic based on screening banked blood from 593 blood donores (Aubry et al., 2015). These differences could be due to inconsistency in the study participants’ inclusion criteria or diagnostic test used (Rupert et al., 2014). This study recruited febrile patients whereas those studies were conducted in the general population. Despite the low seroprevalence of Zika virus recorded, real-time reverse transcription polymerase chain reaction (RT-PCR) and plaque reduction neutralization test (PRNT) which could eliminate the potential for cross-reactivity and increase sensitivity were not used in this study. Of the 160 febrile patients, 3 (1.9%) tested positive for anti-Zika virus IgG, suggestive of previous exposure to Zika virus (Vaughn et al., 2000). This indicates susceptibility level of 98.1% among the study population which confirms studies carried out in other sub-Saharan African countries (Cannon et al., 2010). Due to the high susceptibility levels (98.1%), appropriate preventive measures should be taken to curb the occurrence of congenital infection and devastating consequences of Zika virus infection. These preventive measures as advised by Matthew et al., (2016) include practicing mosquito abatement, employing mosquito bite avoidance behaviors and taking precautions to reduce the risk for sexual transmission. Thirty (18.8%) patients had anti-Zika virus IgM. The observed IgM prevalence could be due to recent exposure to Zika virus or a wave of Zika virus infection. No patient had both anti-Zika virus IgG and IgM. The absence of 52 University of Ghana http://ugspace.ug.edu.gh patients with both anti-Zika virus IgM and IgG show no possible recurrent Zika virus infection (Lazzarotto et al., 2008). Among the age groups identified, 14 (42.5%) of the anti-Zika virus IgM were recorded in the reproductive age group but 4 (12.1%) were recorded in children, with no significant difference of P = 0.08. This may be due to the pattern of sexual activities and exposure among the age groups. Active sexual involvement and exposure to Zika virus has been proven to begin in the reproductive age group, increases gradually and then declines as one ages (Ayensu et al., 2014). Taking into account the possible adverse effects associated with Zika virus infection, it is important to focus on prevention efforts in this age group and surveillance. Anti-Zika virus IgG was detected in two individuals (66.7%) within the reproductive age group and a child 1 (33.4%). This data suggests that, Zika virus infection is not endemic in Ghana, rather the virus had been introduced to a non-exposed population as endemicity is attained when the adult infection decreases and only the new entrants into the population are affected more (Kumar et al., 2015). This is different from what has been observed around the world in feberile patients (Fatim et al., 2000). High prevalence of anti-Zika virus IgM or IgG in the reproductive age group compared to children supports data from earlier studies by Matthew et al., (2016) and Kumar et al., (2015). Possible explanation to this observation could be the activeness of the reproductive age group during the early hours of the day which exposes them to the bites of Zika virus- carrying Aedes spp. of mosquitoes more frequently than children (World Health Organization, 2009). 53 University of Ghana http://ugspace.ug.edu.gh Zika virus infection was estimated to affect females and males in a ratio of 2:1 which correlate well with several studies (Mathew et al., 2016; Plourde and Bloch, 2016). Number of anti-Zika virus IgM males was 11 (36.7%) and females, 19 (63.3%). Female accounting for greater number of anti-Zika virus IgM is consistent with a study conducted in Puerto Rico in 2016 which showed females to be more predisposed to Zika virus infection (Matthew et al., 2016). Considering the findings of Matthew et al., (2016) and that of this study, there may be gender-related differences in Zika virus infection incidence, which might be due to exposure differences (Kaplan et al., 1983). Activeness of females during the day time exposes greater proportion of females to Zika virus-carrying Aedes spp. of mosquitoes either at work or while travelling to and from work (Matthew et al., 2016). This may also be attributed to possible differences in who sought medical care following symptomatic infection (Matthew et al., 2016). Yew et al. found males to be significantly more likely to have past Zika virus infection in a multivariate regression analysis (Yew et al., 2009). In contrast, this study demonstrated females to have past Zika virus infection, anti-Zika virus IgG of 100% (3). But no significant difference was observed between anti-Zika virus IgM males and females (P=0.166) and IgG males and females (P=0.500). Absence of anti-Zika virus IgG in males could mean that males are highly susceptible to Zika virus infection. Analysis of data based on yellow fever vaccination revealed high prevalence 20 (66.7%) of anti-Zika virus IgM in yellow fever unvaccinated compared to yellow fever vaccinated individuals 10 (33.3%) with a significant difference of P=0.000. Further, one (33.3%) anti- 54 University of Ghana http://ugspace.ug.edu.gh Zika virus IgG was recorded in yellow fever vaccinated and two (66.7%) in unvaccinated. Finding agrees with Rupert et al., (2014) and Stoler et al., (2015), and may suggests that yellow fever vaccination could provide immunity against Zika virus infection probably due to the cross-reactive antibodies produced by yellow fever vaccines (Cavalcanti et al., 2016). This draws parallel with a research in Brazil by Cavalcanti et al., (2016). However, in a bivariate logistic regression analysis, yellow fever vaccination was not associated with Zika virus seropositivity (P>0.05). This was in contrast to the results of Bouba et al., (2017); in a multivariate analysis, yellow fever vaccination was significantly associated with Zika virus seropositivity. Bivariate logistic regression analysis revealed that gender and age were not associated with Zika virus seropositivity. This observation is in accordance with a prevalence study of symptomatic Zika virus infection by age and gender in Puerto Rico, 2016 (Matthew et al., 2016). Zika virus antibodies detection has been found to vary by season. Detection peaks in the raining season and decreases in the dry season (Heang et al., 2012; Heang et al., 2011). Similar to Mark et al., (2009), monthly trends indicate that anti-Zika virus IgM detection peaked in March 7 (24.2%) and May 9 (30%) and decreased in September 1 (3.3%). Interestingly, this was different for anti-Zika virus IgG. Anti-Zika virus IgG peaked in January and decreased in March representing 2 (66.7%) and 1 (33.3%) respectively. Anti- Zika virus IgM monthly detection showed no significant difference (P = 0.006). No significant difference was observed between anti-Zika virus IgG monthly detection (P=0.205). 55 University of Ghana http://ugspace.ug.edu.gh Monthly distribution of Zika virus infection with higher prevelence in the raining season as seen in this study synchronizes with reported pattern of Zika virus transmission (Gupta et al., 2006). The high frequency of anti-Zika virus IgM in the months of March and May could be due to the high amount of rainfall which provided temperatures suitable for virus survival, incubation, development and biting of Aedes aegypti and Aedes albopictus (Mordecai et al., 2016). The compact clusters of cases in March and May, and the high prevalence of IgM against Zika virus are consistent with an acute outbreak of Zika virus infection in the population without previous immunity to Zika virus (Mark et al., 2009). In the absence of data on the estimated persistence of anti-Zika virus IgM and reports of outbreak of Zika virus infection, but anti-Zika virus IgM can be detected after 90 days of infection (Nogueira et al., 1992); outbreak might have resulted from a recent contact with Zika virus. Monthly distribution of Zika virus infection is very important at the local level for effective control measures. Preventive measures to curb the occurrence of any congenital anomalies associated with Zika virus infection should come into full action during water stagnation periods after the initial bouts of rainfall (Ukey et al., 2010 and Kumar et al., 2015). Relatively, majority (6) of the anti-Zika virus IgM females were recorded in the month of May while 3 of anti-Zika virus IgM males were recorded in the month of March. Yew et al. suggested that, dissimilarities in the type of work might account for the differences in number of female to male in the monthly distribution of Zika virus antibodies (Yew et al., 2009). Again, reporting biases by health care workers, severity of symptoms among gender 56 University of Ghana http://ugspace.ug.edu.gh (male and female), sexual transmission and differences in health care seeking behavior may also account for the differences in the distribution of Zika virus antibodies (Dos Santos et al., 2016). Sera from patients who reside at Lapaz and Amasaman recorded the highest seropositivity of 12.1% each. Ashaley Botwe, Adabraka, Adenta and Kosoa recorded 2 (6.1%) each. One (3.0%) was recorded in 37, Alajo, Asylum down, Ordorna, Dome kwabenya, Teshie, Airport, Darkuman, Nima, Weija, Pokuase, Kokomlemle, Winneba, Achimota, Circle, Awodome and Asafo. The high number of Zika virus seropositive patients residing in Abeka Lapaz and Amasaman could be due to environmental factors present in these localities which favour the breeding of the active vector Aedes spp. of mosquitoes. These environmental factors according to Thomson et al., (2006) include precipitation and temperature. Additionally, housing type and lifestyle present in these localities may also contribute to populations’ contact with the active vector Aedes spp. of mosquitoes (Moreno-Madrin˜´an and Turell, 2017). The study recorded 8 (24%) of the anti-Zika virus IgM on the 2nd day after the onset of illness. One (3%) of the anti-Zika virus IgM was detected on the 7th and 8th days respectively. This indicates anti-Zika virus IgM is detectable in high numbers on the first day of infection to the fifth day but persists in low numbers after the fifth day. This observation is consistent with what has been observed in other countries (Sant´e, 2015). This further suggests the seropositive febrile patients to be recently infected since anti-Zika virus IgM are detectable within two to seven days after infection (Vaughn et al., 2000). 57 University of Ghana http://ugspace.ug.edu.gh Eighty percent of Zika virus infections are asymptomatic (Shan et al., 2016). Symptomatic cases account for about 18-20%, manifest with nonspecific clinical symptoms and resemble dengue or chikungunya confounding diagnosis (Duffy et al., 2009). In this study, commonly reported symptoms of Zika virus infection documented were fever, muscle pain, joint pain and conjunctivitis. This is similar to a Zika virus infection clinical profile documented in Martinique in asymptomatic blood donors (Gallian et al., 2016). Strikingly, conjunctivitis often observed in Zika virus infection (Heukelbach et al., 2016), was not reported by any seropositive female. This agrees with a study conducted in Brazil by Cassia et al., (2016). Digestive symptoms noted in Zika virus infected patients but rarely observed, however, found in this study include nausea, vomiting and diarrhea (Oehler et al., 2013). A bivariate logistic regression analysis demonstrated an association between Zika virus seropositivity and chest pain, conjunctivitis, muscle pain and joint pain (P<0.05). 5.2 Conclusion Results from this study indicate the possible exposure of these febrile patients to Zika virus with a prevalence of 20.6%. This may be the tip of the iceberg since our population size is relatively small compared to the total number of febrile patients in Ghana. Characteristics such as age, gender and yellow fever vaccination were found not to be the predictors for Zika virus infection. 58 University of Ghana http://ugspace.ug.edu.gh 5.3 Recommendations The current study revealed possible exposure of these febrile patients to Zika virus and therefore this study should be replicated in other parts of Ghana to determine the overall susceptibility levels in the entire country. Again, a critical study in newborns would be necessary to estimate the number of newborns who suffer abnormalities because of maternal primary infection among the few susceptible individuals. This is due to overlapping symptoms of most of the childhood illnesses. 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J Clin Virol off Publ Pan Am Soc Clin Virol. 63:32–5. 88 University of Ghana http://ugspace.ug.edu.gh APPENDIX Appendix 1: Case Investigation Form 89 University of Ghana http://ugspace.ug.edu.gh 90 University of Ghana http://ugspace.ug.edu.gh 91 University of Ghana http://ugspace.ug.edu.gh Appendix 2: Characteristics of Patients Town of Ag Se Date Date Dia Na Vo La M Jp Co Cp Rr Residen e x of of last r. u. m. p. p nj. ce onset YF vacci natio n Ablekum 66 M 16/09 No N Y Y Y Y Y N Y Y a /2017 Weija 36 F 14/09 No Y Y N Y Y Y N N N /2017 Kwashie 26 F 15/09 2011 Y Y Y N Y Y N N man /2017 Pokuase 26 F 16/09 2011 N N N Y Y Y N N N / 2017 Lapaz 64 F No N N N N Y Y N Y N Gbawe 29 F No Y N N Y N N Y N N Ashiama 31 F 19/09 No N N N N N Y N Y N n / 2017 Abeka 43 F No N N N N Y N N N N Lapaz Sakumo 79 F No N N N N Y Y N Y N no Awoshie 30 F 24/09 No N N N Y Y Y N Y /2017 Kokomle 52 M 23/09 No N N N Y Y Y N Y N mle /2017 Kasoa 32 F 23/09 2011 Y Y N Y Y Y N Y Y /2017 Kokomle 20 F 22/09 No N N N Y Y Y N Y Y mle /2017 Lapaz 27 F 21/09 2011 N Y Y Y Y Y N Y N /2017 Nima 24 M 23/09 No Y Y N Y Y Y N N N /2017 Winneba 47 M 25/09 No N N N Y Y Y N N Y /2017 Achimot 32 F 22/09 2011 Y Y N Y Y Y N N N a /2017 Awoshie 28 F 29/09 No N Y Y Y Y Y N N N /2017 25 No Kasoa 14 f 08/10 No Y N Y Y Y N N N Y /2017 Achimot 52 M 09/10 No Y Y N Y Y Y N Y N 92 University of Ghana http://ugspace.ug.edu.gh a /2017 Kaneshie F No N Y N Y Y Y N Y Y Circle 58 M 04/10 No N Y Y Y Y Y N N Y /2017 Adabrak 67 F 06/10 No Y Y Y Y Y Y N Y Y a /2017 Ridge- 44 F 08/10 No Y Y Y Y Y Y N Y N Accra /2017 Mallam 30 M 19/10 No Y N N N Y N N N N /2017 Teshie F 20/10 No N N N N Y N N Y N /2017 Amasam 38 M 19/10 No N N N N N N N N N an /2017 West 46 F 21/10 No N N N Y Y N N N N Legon /2017 Nsawam 32 F 20/10 No N Y N N N N N N N /2017 McCarth 48 F 22/10 No N N N Y N N N N N y Hill /2017 Lapaz 37 F 18/10 No N N N N N N N N N Nii Boye /2017 Town Amasam 62 F 29/10 No N N N N Y N N N N an /2017 Madina 31 F 26/10 No N N N Y N N N N N /2017 Asheley 39 F 23/10 No N N N N N N N N N Botwe /2017 Akwetey 49 F 27/10 No N Y N N N N N N N man /2017 Kokomle 58 F 25/10 No N N N Y Y N N N N mle /2017 Adenta 31 M 27/10 No Y N N Y N N N N N /2017 Amasam 49 F 28/10 No N N N Y N N N N N an /2017 Osu 71 F 28/10 No N Y Y N Y N N N N /2017 Kotobabi 35 F 02/11 No Y N N N N N N Y Y /2017 Bubuash 39 F 03/11 No N N N N N N N N N ie /2017 Ridge 53 M 05/11 No N N N Y N N N N N /2017 93 University of Ghana http://ugspace.ug.edu.gh Achimot 80 F 05/11 No N N N N Y Y N N N a /2017 Osu 61 F 09/11 No N N N Y Y N N N N /2017 Amasam 35 F 09/11 No N N N N N N N N N an /2017 Madina 45 M 10/11 No N Y N N N N N N N /2017 Kanda 35 M 08/11 No N N N N N N N N N /2017 Dome 60 M 16/11 No N N N N Y N N N N /2017 Awodom 3 F 18/11 No N N N N N N N N N e /2017 Amasam 40 F 17/11 No N N N Y N N N N N an /2017 Botwe 23 F 18/11 No Y N N N N N N N N /2017 Asofa 9 M 20/11 No N N N N N N N N N /2017 Pokuase 32 F 17/12 2011 Y Y Y Y Y Y N Y Y /2016 Agirigan 34 F 17/12 2011 N N N Y Y Y N Y o /2016 Kaneshie 23 F 16/12 2011 /2016 Anyaa 59 F 16/12 No N Y Y Y Y Y N N N /2016 Abeka 39 F 21/12 No N N N Y Y Y N Y N / 2016 Osu 15 M 2011 Y N N Y Y Y N N N 37 29 F 24/12 2011 Y N N Y Y Y N Y Y /2016 24 F 24/12 2011 Y Y Y Y N N N Y Y /2016 Adabrak 61 F 20/12 2012 Y Y Y Y N N N N Y a /2016 Spintex 31 F 21/12 No Y N N Y Y Y N Y Y /2016 Mesie 42 F 09/01 No Y Y Y Y Y Y N Y Y /2017 Kokomle 44 F 09/01 No Y Y Y Y Y Y N Y Y mle /2017 East 22 F 08/01 No Y N N Y Y Y N Y Y Legon /2017 Asylum 22 F 09/01 No Y N N Y Y Y N Y Y 94 University of Ghana http://ugspace.ug.edu.gh Down /2017 Nungua 27 F 10/01 No Y Y Y Y N Y N Y Y /2017 Spintex 38 F 07/01 2011 Y Y Y Y Y Y N N Y /2017 Achimot 9 M 10/01 2008 Y Y Y Y Y Y N N N a /2017 Ridge 30 M 13/1// 2010 N N N N Y Y N Y Y 2017 Botwe 60 F 10/01 No N Y Y Y Y Y N N Y /2017 Spintex 44 F 14/01 No N N N N N Y N N N /2017 Madina 49 M 09/01 No Y N N Y N N N N N /2017 Adabrak 24 M 11/01 No N Y Y Y Y Y N Y Y a /2017 Sowutuo 48 F 08/01 No Y Y Y Y Y Y N N N m /2017 Ordorna 21 F 10/01 2010 N N N Y Y Y N Y Y /2017 Accra 27 M 11/01 2010 Y Y Y Y Y Y N Y Y Newtow /2017 n Nungua 11 F 14/01 2009 Y Y Y Y Y Y N N N /2017 Achimot 45 M 12/01 2010 N Y Y Y Y Y N Y Y a /2017 Lapaz 25 F 12/01 No Y Y Y Y Y Y N Y N /2017 Kasoa 16 M 20/01 No /2017 Nsawam 63 M 16/03 No N N N Y Y Y N Y N /2017 Nima 54 F 19/03 No N N N Y Y Y N N Y /2017 Madina 34 F 20/03 2010 Y Y N Y Y Y N Y Y /2017 Adabrak 20 F 14/03 2010 Y N N Y Y Y N Y Y a /2017 Weija 29 M 18/03 2010 Y Y Y N Y Y N N N /2017 Kasoa 87 M 15/03 No Y Y Y Y Y Y N N Y /2017 Pokuase 25 F 18/03 2010 Y Y Y Y Y Y N Y Y /2017 95 University of Ghana http://ugspace.ug.edu.gh Lapaz 29 F 18/03 2010 Y Y Y Y N N N Y Y /2017 Tema 30 F 18/03 2010 N Y Y Y Y Y N Y Y / 2017 Lapaz 28 F 2010 Y N N Y Y Y N Y Y Korle- 22 F 20/03 2010 Y N N Y Y Y N N N Bu /2017 Accra 32 F 20/03 2010 Y Y N Y Y Y N Y Y Newtow /2017 n Lapaz 78 F 19/03 No N Y Y Y Y Y N N N /2017 Kokomle 21 F 20/03 No N N N Y Y Y N Y Y mle /2017 Achimot 34 F 16/03 No Y N N Y N N N Y Y a /2017 Osu 25 F 21/03 2010 N N N Y Y Y N N N /2017 Teshie 44 F 26/03 No Y Y Y Y Y Y N Y Y /2016 Newtow 17 F 19/03 2010 N N N Y Y Y N N N n /2017 Kasoa 37 M 20/03 2010 Y Y N Y Y Y N N Y /2017 Nima 67 M 2010 N N N Y Y Y N N N Amasam 32 M 23/03 2010 N Y Y Y Y Y N Y Y an /2017 Madina 32 F 24/03 No Y Y Y Y Y Y N Y N /2017 Madina 40 F 25/03 No N N N Y Y Y N Y N /2017 Weija 26 F 25/03 2010 Y Y N Y Y Y N N Y /2017 Madina 33 F 2010 N Y N Y Y Y N Y N Sapeima 34 F 20/03 No N Y N Y Y Y N Y Y n /2017 Adabrak 51 M 21/03 No N N N Y Y Y Y N Y a /2017 Kanda 29 F 22/03 No N N N Y Y Y N Y Y /2017 Kasoa 44 F 21/03 No Y Y N Y Y Y N Y Y /2017 Kiseima 20 F 20/03 2010 N N N Y Y Y N N Y n /2017 Dome 24 F 19/04 2010 N N N Y Y Y N Y Y 96 University of Ghana http://ugspace.ug.edu.gh /2017 North 37 M 19/04 2010 Y Y N Y Y Y N N Y Kaneshie /2017 Sakaman 23 F 21/04 No N N N Y Y Y N N N /2017 Darkuma 23 F 23/04 2010 Y N N Y Y Y N Y Y n /2017 Madina 29 F 22/03 2010 N N N Y Y Y N Y Y /2017 Kokomle 33 M 22/04 2010 Y N N Y Y Y N N N mle /2017 Dome 37 F 23/04 No N N N Y Y Y N Y N Kwaben /2017 ya Madina 70 F 20/04 No N Y Y Y Y Y N Y N /2017 Kanda 68 F 22/04 No Y N N Y Y Y N N N /2017 Adenta 17 M 24/04 2010 Y Y Y Y N N N Y N /2017 Alajo 29 F 25/04 2010 Y Y Y Y Y Y N Y Y /2017 Pokuase 25 F 27/04 2010 Y Y Y Y Y Y N N Y /2017 Alajo 42 F 30/04 No N Y N Y Y Y N N N /2017 Asylum 38 F 02/05 2010 Y N N Y Y Y N N Y Down /2017 Kasoa 49 F 01/05 No Y Y N Y Y Y N Y Y /2017 West 31 F 27/04 2010 N Y N Y Y Y N Y Y Legon /2017 Osu 55 F 04/05 No Y N N Y Y Y N Y N /2017 Darkuma 42 F 02/05 No Y N N Y Y Y N N N n /2017 Adabrak 42 F 04/05 2011 Y N N Y Y Y Y Y Y a /2017 Achimot 33 F 06/05 2011 Y N N Y Y Y N Y Y a 2017 Madina 51 F 06/05 No Y Y N Y Y Y N Y Y /2017 Weija 26 M 02/05 No Y Y Y Y Y Y N N N /2017 Amasam 30 F 06/05 2011 Y N N Y Y Y N N N an /2017 97 University of Ghana http://ugspace.ug.edu.gh Lapaz 53 M 02/05 No N Y Y Y Y Y N N N /2017 Circle 23 M 06/05 2011 Y Y Y Y Y Y Y N Y /2017 Alajo 13 M 01/05 2005 Y Y Y N Y Y N Y N /2017 Achimot 45 F 20/05 2011 Y Y Y Y Y Y N Y Y a /2017 Mampro 63 F 19/05 No Y Y Y Y Y Y N Y Y bi /2017 Odorkor 30 F 17/05 No Y N N Y Y Y N Y Y /2017 Osu 27 F 20/05 2011 N Y N Y Y Y N N N /2017 Airport 20 F 20/05 2010 Y N N Y Y Y N Y N /2017 Pig Farm 37 F 20/05 No N Y Y Y Y Y N N N /2017 Dome 37 F 19/05 No N Y Y Y Y Y N Y N /2017 Lapaz 59 F 18/05 No Y Y Y Y Y Y N Y Y /2017 Maamob 54 F 18/05 No Y N N Y Y Y N Y Y i /2017 Adenta 72 F 20/05 No N N N Y Y Y N Y Y /2017 Adenta 21 F 17/05 2011 N Y N Y Y Y Y N N /2017 Amasam 44 F 27/05 No Y Y Y N Y Y N N an /2017 Asofa 47 F 22/05 No Y Y Y Y Y Y N N Y /2017 Amasam 26 F 26/05 2011 Y N N Y Y Y N Y N an /2017 Pokuase 26 F 22/05 2011 Y N N Y Y Y N Y N /2017 Nima 34 M 27/05 No Y Y Y Y Y Y N Y Y /2017 Adenta 35 F 27/05 No N N N Y Y Y N Y Y /2017 Mampro 49 M 26/05 2011 N Y N Y Y Y N Y Y bi /2017 Nima 42 F 24/05 No N N N Y N N N Y N /2017 Adenta 28 F 26/05 2011 Y N N Y Y Y N Y N /2017 98 University of Ghana http://ugspace.ug.edu.gh Ofankor 40 M 26/05 2011 Y Y N Y Y Y N Y Y /2017 Akwetey 35 M 15/09 2011 N Y N Y Y Y N Y Y man /2017 Appendix 3: Zika virus ELISA Results (Raw results) Plate 1: Zika IgG 1 to 80 Plate 2: Zika IgG 81 to 160 99 University of Ghana http://ugspace.ug.edu.gh Plate 1: Zika IgM 1 to 88 Plate 2: IgM 89 to 160 100 University of Ghana http://ugspace.ug.edu.gh Appendix 4: Calculated Zika Virus Elisa Results Plate 1: IgG 1 to 80 Blan 0.0 3.500 3.091 6.100 3.091 3.336 4.749 2.538 2.395 2.538 2.702 k 56 512 095 307 095 745 232 383 087 383 149 Nega 0.1 3.111 3.091 3.172 10.29 7.082 2.415 5.301 3.050 2.763 2.517 tive 62 566 095 979 683 907 558 945 154 562 912 Cut- 0.4 3.009 2.804 2.804 2.886 3.070 2.313 2.681 2.354 2.456 3.132 off 885 212 504 504 387 624 204 679 145 499 037 Posti 1.1 2.906 3.909 2.947 3.009 3.787 2.599 4.257 2.558 3.111 2.968 ve 685 858 928 799 212 103 795 932 854 566 27 3.009 2.763 2.845 3.050 3.418 3.418 2.845 2.476 2.620 20.85 212 562 445 154 628 628 445 97 266 977 3.541 2.968 5.445 2.845 2.988 2.415 2.415 3.357 2.579 2.497 453 27 241 445 741 558 558 216 324 441 2.784 2.763 2.804 2.579 4.135 2.415 2.640 5.568 2.251 2.743 033 562 504 324 107 558 737 066 791 091 3.193 3.234 7.430 7.246 3.029 3.009 2.865 2.722 2.865 3.213 449 391 911 673 683 212 916 62 916 92 Plate 2: IgG 81 to 160 Blan 0.02 12.59 2.762 2.642 2.342 2.182 3.003 2.242 6.586 2.262 5.405 k 6 259 763 643 342 182 003 242 587 262 405 Neg 0.13 2.782 2.162 2.322 3.483 2.162 2.762 3.803 2.122 3.943 2.402 783 162 322 483 162 763 804 122 944 402 Cut 0.49 2.302 2.142 14.87 2.442 2.202 4.124 2.082 2.302 - 4.064 95 302 142 487 442 202 124 082 302 3.603 064 6 Post 1.09 2.402 2.682 2.342 2.182 2.562 2.462 2.662 3.003 2.202 2.502 101 University of Ghana http://ugspace.ug.edu.gh 75 402 683 342 182 563 462 663 003 202 503 2.862 2.302 2.302 2.362 2.502 2.342 2.182 2.322 2.662 4.004 863 302 302 362 503 342 182 322 663 004 2.262 2.302 2.442 3.483 2.262 2.282 4.364 2.282 2.422 3.863 262 302 442 483 262 282 364 282 422 864 2.442 2.642 2.222 2.822 2.322 2.362 3.363 6.166 1.921 2.262 442 643 222 823 322 362 363 166 922 262 3.003 2.702 2.422 2.882 2.762 3.603 4.044 2.342 2.762 2.302 003 703 422 883 763 604 044 342 763 302 Plate 3: IgM 1 to 88 Bla 0.0 2.28 4.32 3.61 7.17 10.2 1.75 7.07 2.80 3.37 3.32 5.98 nk 115 0285 304 0451 3397 1378 772 8385 285 2922 5416 5748 Neg 0.0 3.37 5.27 7.64 11.2 2.42 2.85 28.6 24.4 4.70 2.80 3.70 225 2922 3159 8456 5891 2803 0356 4608 6556 3088 285 5463 Cut 0.2 11.8 2.18 5.41 6.03 5.41 3.27 2.13 1.90 3.46 89.4 8.88 105 2898 5273 5677 3254 5677 791 7767 0238 7933 5368 361 Pos 0.5 4.94 2.56 2.75 24.6 3.32 2.32 29.9 12.2 1.66 2.61 4.94 1 0618 5321 5344 0808 5416 7791 2874 5653 2708 2827 0618 4.22 5.93 2.56 1.66 14.7 2.09 27.1 8.07 10.1 4.03 5.08 8029 8242 5321 2708 7435 0261 734 601 6627 8005 3135 4.65 2.28 5.89 4.84 2.18 7.41 2.56 6.69 14.2 1.94 4.51 5582 0285 0736 5606 5273 0926 5321 8337 5178 7743 3064 4.60 5.17 11.8 6.08 19.1 2.28 1.61 2.18 1.28 2.89 2.66 8076 8147 2898 076 4489 0285 5202 5273 266 7862 0333 3.94 13.6 10.2 7.88 3.13 3.84 2.85 10.5 24.6 2.99 39.0 2993 342 6128 5986 5392 7981 0356 9382 0808 2874 0238 102 University of Ghana http://ugspace.ug.edu.gh Plate 4: IgM 89 to 160 Blan 0.03 5.285 2.663 2.832 3.509 4.228 8.118 24.35 8.160 42.74 k 15 412 848 981 514 33 393 518 677 841 Neg 0.04 38.30 6.807 46.34 7.399 20.42 40.67 7.610 3.044 4.270 55 867 611 249 577 283 653 994 397 613 Cut 0.23 3.974 3.044 3.720 2.367 9.556 2.536 7.230 8.202 3.551 65 63 397 93 865 025 998 444 96 797 Pos 0.54 3.044 - 4.143 8.160 7.272 3.847 6.257 6.131 11.96 6 397 11.07 763 677 727 78 928 078 617 82 3.086 11.83 27.31 4.016 15.43 6.427 8.118 5.116 163.3 681 932 501 913 34 061 393 279 827 7.019 6.257 3.002 2.579 14.29 8.118 3.890 62.24 6.807 027 928 114 281 175 393 063 101 611 42.07 23.00 24.69 32.43 3.213 9.302 16.65 3.932 9.640 188 211 345 129 531 326 962 347 592 2.832 10.27 4.904 5.665 6.342 2.917 4.651 22.28 2.621 981 484 863 962 495 548 163 33 564 103