SCHOOL OF PUBLIC HEALTH COLLEGE OF HEALTH SCIENCES UNIVERSITY OF GHANA FACTORS ASSOCIATED WITH ASYMPTOMATIC MALARIA AMONG PREGNANT WOMEN ATTENDING ANTENATAL CLINIC AT RIDGE REGIONAL HOSPITAL ACCRA, GHANA DANIEL KIPTOO (10584999) A DISSERTATION SUBMITTED TO THE SCHOOL OF PUBLIC HEALTH, UNIVERSITY OF GHANA IN PARTIAL FULFILLMENT FOR THE AWARD OF THE MASTER OF PUBLIC HEALTH (MPH) DEGREE JULY, 2016 University of Ghana http://ugspace.ug.edu.gh II DECLARATION This work was the result of my research, except for references to other people’s work which I have duly acknowledged. It has not been submitted for the award of any other degree apart from this. I am responsible for the views expressed and the factual accuracy of its contents. …………………………… date …………………………. Daniel Kiptoo (Student) …………………………… date ……………………………….. Dr. Reuben K Esena (Supervisor) University of Ghana http://ugspace.ug.edu.gh III DEDICATION This work is dedicated to my wife Jerusha Nyabiage, my mother Esther Kaitany, my late father William Chemase and my family members for their unflinching support in prayers, cash and in kind throughout my education. University of Ghana http://ugspace.ug.edu.gh IV ACKNOWLEDGEMENT I wish to express my profound gratitude to the Almighty God for his unfailing love, care and grace to me throughout this programme. I am very grateful to my academic supervisor, Dr Reuben K. Esena for his guidance, insightful suggestions, support and time that led to the production of this useful work. I also extend my sincere gratitude to all lecturers of the School of Public Health who in diverse ways made my stay in the School productive and memorable. Finally, I want to express my sincere appreciation to my research assistants, study participants, administration and staff of Ridge Regional hospital and my fellow TDR colleagues for being part of this memorable journey University of Ghana http://ugspace.ug.edu.gh V LIST OF ABBREVIATIONS/ACRONYMS ANC Antenatal clinic BDS Bachelor of Dental Surgery DL Deciliter DOT Direct Observed Therapy DHDS Ghana Health Demographic survey EDTA Ethamine Diaminetetraaceticacid GHSREC Ghana Health Service Ethical Review Committee IRS Indoor residual spraying ITN Insecticide Treated Nets IPT Intermittent Preventive Treatment K C S E Kenya Certificate of Secondary Education LLINs Long Lasting Insecticide Nets MPH Master of Public Health MDGs Millennium Development Goals NMCP National Malaria Control Programme OPD Out-patient department PMI Presidents malaria initiative PVC Packed Cell Volume RBM Roll Back Malaria SP Sulphadoxine-Pyrimethamine SDGs Sustained development goals USA United States Of America TDR Training and Research in Tropical Diseases WHO World Health Organization WBC White Blood Cells University of Ghana http://ugspace.ug.edu.gh ABSTRACT Background: Asymptomatic malaria increases the risk of anaemia, stillbirths, spontaneous abortion, premature delivery and low birth weight. The asymptomatic nature of individuals with malaria has led to difficulties in diagnosing, inconsistencies in defining and a general lack of urgency to investigate this particular disease outcome and is a major hurdle for malaria elimination, as infected hosts serve as silent reservoirs for transmission of malaria. This study was aimed at identifying factors associated with asymptomatic malaria infection in pregnant women in Ridge Regional Hospital Accra, Ghana. Methods: A total of 400 asymptomatic pregnant women were enrolled using simple random approach. Demographic information and malaria prevention practices were obtained using a structured questionnaire. About 2ml of blood was obtained from a peripheral blood vessel for Microscopy to determine parasitaemia, species and parasite density. Chi square test and logistic regression analysis were used to compare factors associated with malaria in the pregnant women. Results: The prevalence of malaria using peripheral blood from the 400 pregnant women who participated in the study by microscopy was 5.5%. Plasmodium falciparum parasite were seen in 95.5% of the cases while Plasmodium malariae accounted for the remaining 4.5%. Age, gravidity, education level, gestation, IPT-sp use and ITN use were not significantly associated with malaria infection (p≥0.05). Conclusion: Malaria prevalence was low among the pregnant women studied. Age, gravidity, education level, occupation, gestation, IPT-sp use and ITN use were not significantly associated with malaria infection (p≥0.05). Key words: Asymptomatic malaria, Pregnancy, risk factors, Malaria in pregnancy University of Ghana http://ugspace.ug.edu.gh VII TABLE OF CONTENTS DECLARATION ............................................................................................................................ II DEDICATION .............................................................................................................................. III LIST OF ABBREVIATIONS/ACRONYMS ................................................................................ V ABSTRACT .................................................................................................................................. VI TABLE OF CONTENTS ............................................................................................................. VII LIST OF TABLES ......................................................................................................................... X LIST OF FIGURES ...................................................................................................................... XI CHAPTER ONE ............................................................................................................................. 1 INTRODUCTION .......................................................................................................................... 1 1.1 Background ...................................................................................................................... 1 1.2 Problem statement ............................................................................................................ 3 1.3 Justification of the study .................................................................................................. 5 1.4 Conceptual framework ..................................................................................................... 6 1.5 Narrative of the conceptual framework ............................................................................ 6 1.6 Research Questions .......................................................................................................... 7 1.7 Objectives of the study ..................................................................................................... 7 1.7.1 General objective ...................................................................................................... 7 1.7.2 Specific objectives .................................................................................................... 7 CHAPTER TWO ............................................................................................................................ 9 LITERATURE REVIEW ............................................................................................................... 9 CHAPTER THREE ...................................................................................................................... 16 METHODOLOGY ....................................................................................................................... 16 3.1 Study Design .................................................................................................................. 16 3.2 Study Area ...................................................................................................................... 16 3.3 Study Variables .............................................................................................................. 17 3.4 Sampling......................................................................................................................... 18 3.4.1 Study population ..................................................................................................... 19 3.4.2 Sample size ............................................................................................................. 19 3.4.3 Sampling method/procedure ................................................................................... 19 University of Ghana http://ugspace.ug.edu.gh VIII 3.4.4 Laboratory test and diagnosis of asymptomatic malaria......................................... 20 3.4.5 Staining technique for thick blood film .................................................................. 20 3.4.6 Staining technique for thin film .............................................................................. 21 3.5 Data collection techniques and tools/instruments .......................................................... 22 3.5.1 Inclusion criteria ..................................................................................................... 22 3.5.2 Exclusion criteria .................................................................................................... 22 3.5.3 Training of interviewers .......................................................................................... 22 3.5.4 Pre-testing and review of instruments..................................................................... 22 3.5.5 Data collection ........................................................................................................ 23 3.5.6 Quality control ........................................................................................................ 23 3.5.7 Data processing and analysis .................................................................................. 24 3.5.8 Ethical clearance ..................................................................................................... 25 CHAPTER FOUR ......................................................................................................................... 26 RESULTS ..................................................................................................................................... 26 4.1 Demographic characteristics of the study population .................................................... 26 4.2 Obstetric and prevalence characteristics of the pregnant women .................................. 28 4.3 Malaria prevention methods adopted by pregnant women in the study population....... 30 4.4 Results of Sociodemographic factors associated with malaria parasitaemia from chi square test .................................................................................................................................. 31 4.5 Obstetric characteristics and preventive methods associated with malaria parasitaemia from chi square test ................................................................................................................... 33 4.6 Sociodemographic characteristics of participants from logistic regression models ...... 34 4.7 Obstetric characteristics and preventive methods associated with parasitaemia from logistic regression models ......................................................................................................... 35 CHAPTER FIVE .......................................................................................................................... 37 DISCUSSION ............................................................................................................................... 37 5.1 Introduction .................................................................................................................... 37 5.2 Prevalence of malaria parasitaemia ................................................................................ 37 5.3 Risk factors associated with Parasitaemia ...................................................................... 38 5.4 Limitations ..................................................................................................................... 40 CHAPTER SIX ............................................................................................................................. 41 CONCLUSIONS AND RECOMMENDATIONS ....................................................................... 41 University of Ghana http://ugspace.ug.edu.gh IX 6.1. Conclusion ...................................................................................................................... 41 6.2. Recommendations .......................................................................................................... 41 REFERENCES ............................................................................................................................. 43 APPENDICES .............................................................................................................................. 48 Appendix1: Consent form ............................................................................................................. 48 Appendix2: Questionnaire ............................................................................................................ 43 University of Ghana http://ugspace.ug.edu.gh X LIST OF TABLES Table 1: Variables of the study ..................................................................................................... 17 Table 2: Demographic characteristics of the study participants ................................................... 27 Table 3: obstetric characteristics, prevalence and parasite density of the study participants ....... 29 Table 4: Malaria preventive methods adopted by the study participants...................................... 30 Table 5: Sociodemographic characteristics of the participants associated with malaria prevalence ....................................................................................................................................................... 32 Table 6: Obstetric characteristics and preventive methods associated with malaria parasitaemia 33 Table 7: Sociodemographic characteristics of participants from logistic regression models ....... 35 Table 8: Obstetric characteristics and preventive methods associated with parasitaemia from logistic regression models ............................................................................................................. 36 University of Ghana http://ugspace.ug.edu.gh XI LIST OF FIGURES Figure 1: Conceptual framework of factors associated with asymptomatic malaria compiled by the researcher .................................................................................................................................. 6 Figure 2: Map of Ridge Hospital, the study location .................................................................... 17 University of Ghana http://ugspace.ug.edu.gh 1 CHAPTER ONE INTRODUCTION 1.1 Background Malaria occurs worldwide, affecting 3.3 billion people of all ages with children and pregnant women being at a higher risk. Of these number, about 1.2 billion are at high risk of malaria and an estimated 198 million cases of malaria occurred globally in 2013 of which an estimated 584,000 annual deaths occurred (WHO, 2014). The heaviest burden being in the African region where about 90% of all malaria deaths occur with deaths in children under five accounting for about 78% of all malaria-related deaths in Africa (Snow et al., 2005). Malaria infection during pregnancy causes an enormous risk to the mother, fetus and neonates (Gajida, Iliyasu, & Zoakah, 2010). It increases the risk of stillbirths, spontaneous abortion, premature delivery and low birth weight (WHO, 2010). In the African continent, where the greatest burden of malaria occurs, about 30 million women living in malaria endemic regions become expectant each year. Malaria is a threat to these women and their babies, with up to 200,000 new born deaths occurring each year as a result of malaria in pregnancy (WHO, 2010). There are five species of Plasmodium that infect humans: falciparum, ovale, malariae, and vivax. Of these, Plasmodium falciparum is the most deadly. The infection is transmitted by the female anopheles mosquito. It is transmitted when an infected mosquito feeds on human blood and the Plasmodium sporozoites are transferred from the saliva into the capillary bed of the host. The Plasmodium parasites then migrate to the liver, where they undergo further cycling and replication before being released back into the host’s bloodstream. University of Ghana http://ugspace.ug.edu.gh 2 The phenomenon of asymptomatic malaria is recognized in malaria endemic areas due to partial immunity to malaria with low levels of plasmodium parasites, being detectable in blood without the individual presenting with any clinical signs of malaria (Nkoghe, Akue, Gonzalez, & Leroy, 2011). Gametocytes that persist post-treatment are always asymptomatic as they are non- pathogenic (Schneider et al., 2006). Asymptomatic malaria parasitaemia poses a diagnostic challenge as unlike patients who present with malaria symptoms, these patients don’t have any classical signs and symptoms of malaria. Asymptomatic malaria parasitaemia varies greatly in known endemic areas. Malaria in Ghana is hyper-endemic and accounts for a considerable disease burden and is the single most important cause of morbidity and mortality among children under five and pregnant women. Ghana committed itself to a number of malaria control programs including the Roll Back Malaria (RBM) (Roll Back Malaria, 2000) Initiative and the Abuja Declaration on Roll Back Malaria in Africa which builds on the Global Malaria Strategy with a focus on Africa. The main goal of the Roll Back Malaria Initiative was to halve the world's malaria burden by 2010. Unfortunately, the various control measures undertaken over the years met with limited success mainly due to factors including focus on single strategies, lack of funding, poor human resource capacity and non-involvement of other stakeholders. The government of Ghana developed and adopted a new national strategic malaria control plan in 2001 based on multiple interventions, involvement of all the stakeholders and evidence-based interventions. These interventions included use of Insecticide Treated Nets (ITN)/Long Lasting Insecticide Nets (LLIN), promoting home-based care, improving case management in health facilities and use of appropriate chemoprophylaxis in pregnancy (Malaria & Programme, 2013). Despite all the efforts put by the Government of Ghana, malaria remains a public health problem. Recent data on malaria showed University of Ghana http://ugspace.ug.edu.gh 3 that it is endemic in all parts of the country with over 25 million people at risk of malaria infection every year (WHO, 2014). Factors associated with asymptomatic malaria infections are still poorly understood and therefore remain a challenge to malaria control programs as it significantly influences transmission dynamics. This study therefore sought to determine the factors associated with asymptomatic malaria among pregnant women attending antenatal clinic at Ridge Regional Hospital Accra, Ghana. Health care workers, policy makers and other stakeholders will use the outcome of the study to inform decisions in the control of the burden of malaria among pregnant women and by extension the adverse effects on the fetus and the neonate in the study area. 1.2 Problem statement In sub-Saharan Africa, the burden of malaria is mainly in children and pregnant women (Menéndez, Alessandro, Kuile, & Leopold, 2007). Malaria in these regions of Africa are predominantly asymptomatic in pregnancy and yet it causes severe maternal anemia and low birth weight babies. Because of this strong association between low birth weight and child survival, successful control of malaria in pregnancy could prevent 75,000 – 200,000 infant deaths every year (Back, 2010). In the past years, Ghana has adopted a number of national and global initiatives aimed at reducing the burden of malaria targeting especially the vulnerable groups; pregnant women and children. These programs include the Roll Back Malaria (RBM) partnership, the Presidents Malaria Initiative (PMI), the African summit on RBM, Millennium Development Goals (MDGs) and recently sustainable Development Goals (SDGs). Despite all these efforts, Malaria remains a significant public health problem in Ghana and many African countries. University of Ghana http://ugspace.ug.edu.gh 4 Recent Malaria data showed that it is endemic in all parts of the country with over 25 million people at risk of malaria infection every year (WHO, 2014). In 2013, 11.3 million cases of out- patient department (OPD) records was due to malaria (Ghanaweb, 2014). Of these number, over 8 million were presumed and confirmed malaria cases (WHO, 2014). Diagnosis of asymptomatic malaria in pregnancy is very difficult yet such information is very crucial to preventing child and maternal mortality in Africa due to malaria. While some studies have been done on the asymptomatic malaria in other countries (Agomo, Oyibo, Anorlu, & Agomo, 2013; Baliraine et al., 2009; Kern et al., 2011; Nkoghe et al., 2011; Otupiri, Yar, & Hindin, 2012; Zoghi et al., 2012), there is paucity of data on factors associated with asymptomatic malaria in pregnancy in Ghana. Malaria infection in pregnancy is a significant public health problem causing a substantial risk to the pregnant woman, the fetus and the newborn child. This study therefore sought to determine the factors associated with asymptomatic malaria among pregnant women attending antenatal clinic (ANC) at Ridge Regional Hospital Accra, Ghana. It is expected that the findings from this study will assist in policy decisions in the implementation of focused and effective interventions in child and maternal morbidity and mortality in Ghana. University of Ghana http://ugspace.ug.edu.gh 5 1.3 Justification of the study Malaria is an enormous global health problem, affecting over 3.3 billion people. In Ghana, malaria among pregnant women accounts for 13.8% of OPD cases, 10.6% of hospital admissions and 9.4% of deaths. If it occurs in pregnancy, malaria poses a substantial risk to the mother, her fetus and the neonate because pregnant women have a lowered immunity to malaria infection (Desai et al., 2007). Despite the adverse effects caused by malaria during pregnancy, malaria control in pregnancy has not received adequate program support. This is largely due to the fact that most malaria infections in pregnancy remain asymptomatic in hyper-endemic countries such as Ghana. This study therefore sought to determine the factors associated with asymptomatic malaria in pregnant women attending ANC at Ridge Regional Hospital Accra, Ghana. The findings of this study will be useful in instigating active surveys to identify asymptomatic carriers and treatment of the infectious parasite reservoirs. This will provide a positive impact in the elimination of malaria transmission and hence the adverse effects on the pregnant women, fetus and the newborn. These efforts will also have a direct impact on reducing maternal and neonatal mortality in Ghana. University of Ghana http://ugspace.ug.edu.gh 6 1.4 Conceptual framework Figure 1: Conceptual framework of factors associated with asymptomatic malaria compiled by the researcher 1.5 Narrative of the conceptual framework Factors associated with asymptomatic malaria include maternal age, trimester of pregnancy, socio- economic status, education level, the use of ITNs, IRS and IPT-sp. Pregnant women of younger ASYMTOMATIC MALARIA Key: ITN Insecticide Treated Nets IRS Indoor Residual Spray IPT-sp Intermittent Preventive Treatment with sulphadoxine-pyrimethamine Utilization of ANC services IPT-SP use Knowledge of malaria in pregnancy Ownership and use of ITNs, insecticide spray Socio- demographic characteristics Socio- cultural beliefs Trimester of pregnancy University of Ghana http://ugspace.ug.edu.gh 7 maternal age are at a greatest risk of asymptomatic malaria infection. It has been demonstrated that infection rates are higher in women in their first and second pregnancies, with lower rates in later pregnancies. This is understandable as pregnancy is naturally accompanied by general immune suppression that may cause loss of acquired immunity to malaria especially among primigravidae. There is an association between education level and malaria infection. Prevalence of asymptomatic malaria among pregnant women decrease with the increase in education level. This is due to the fact that women with higher education level are more likely to use malaria preventive methods during pregnancy due to their increased knowledge on malaria. The use of ITNs, IRS,IPT-sp decreases both the number of malaria cases and malaria deaths in pregnant women. 1.6 Research Questions  What is the prevalence of asymptomatic malaria among pregnant women attending antenatal clinic (ANC) at Ridge Regional Hospital Accra, Ghana?  What are the factors associated with asymptomatic malaria in pregnancy? 1.7 Objectives of the study 1.7.1 General objective The general objective of this study was to determine the factors associated with asymptomatic malaria among pregnant women attending ANC clinic at Ridge Regional Hospital Accra, Ghana. 1.7.2 Specific objectives The specific objectives included: University of Ghana http://ugspace.ug.edu.gh 8  To determine the prevalence of asymptomatic malaria among pregnant women with parasitaemia.  To determine the risk factors associated with asymptomatic malaria among pregnant women attending ANC at Ridge Regional Hospital Accra, Ghana. University of Ghana http://ugspace.ug.edu.gh 9 CHAPTER TWO LITERATURE REVIEW Malaria is the most deadly tropical infectious disease affecting disproportionately pregnant women, children under five and the poor. Malaria in pregnancy is a major public health problem in both tropic and sub-tropical regions of the world. Every year, millions of women become pregnant in malaria endemic regions of Africa and a significant proportion of individuals are asymptomatic though with plasmodium species (Baliraine et al., 2009; Nkoghe et al., 2011). Plasmodium falciparum is the prevalent species in these regions and is usually asymptomatic in pregnancy. Despite being asymptomatic, parasites may be present in the placenta and is responsible for anaemia and parasitaemia in pregnant women which causes adverse effects to the fetus and newborn, contributing to neonatal mortality. The sequestration of plasmodium species in the placenta is associated with low birth weight, pre-term delivery, miscarriages and stillbirths(Sullivan et al., 2001). The symptoms and complications of malaria in pregnancy vary according to malaria transmission intensity in the given geographical area and individuals acquired immunity. With high acquired immunity, malaria remains asymptomatic in pregnancy. The major risk factors associated with malaria in pregnancy include gestational age in pregnancy, young maternal age and a lower number of previous pregnancies (Agomo et al., 2013). There are four species of plasmodium with falciparum being the cause of the highest morbidity and mortality. Pregnant women with compromised immune system and young children with low immune system are more vulnerable to this disease. Plasmodium falciparum disease severity ranges from mild uncomplicated, severe complicated to asymptomatic. Researchers have developed diagnostic criteria based on clinical manifestations upon onset to help in the treatment and management of malaria. Individuals with mind malaria typically present with fever and University of Ghana http://ugspace.ug.edu.gh 10 perhaps one or more of the following; chills, sweat, headache, vomiting, anemia and splenomegaly. If such individuals are properly treated, the recovery process is high for the patient, Such uncomplicated malaria usually occurs in endemic areas and is likely due to the development of immunity from previous exposure (Grobusch, 2005). Unlike severe and mild malaria, the diagnosis asymptomatic malaria is not straight forward due to lack of clinical manifestation and often sub-patent levels of malaria parasites (Mosha et al., 2013). Researchers have investigated for decades the development of 2 types of immunity which may result in asymptomatic malaria: An anti-disease immunity that allows one to carry the parasite with no symptoms and an anti-parasite that may allow the suppression of parasite loads after a certain age. The prevalence of asymptomatic malaria in endemic regions present a serious challenge in the sustained efforts to curtail, control and eliminate malaria. This is due to the fact that, they act as silent reservoir of gametocytes for transmission by mosquito vector (Rodrigues, Martha, & Ladeia- andrade, 2006). A study in Nigeria found that the prevalence of asymptomatic malaria was 77.6% (Igwe et al., 2014). Previous studies in several parts of Nigeria reported a prevalence of asymptomatic malaria among pregnant women to be between 16-95.4% (Agomo et al., 2013; Gajida, Iliyasu, & Zoakah, 2010; Ifeanyi, Onyebuchi, Ukamaka, & Ugochukwu, 2009; Igwe et al., 2014; Ogbu, Aimakhu, Anzaku, Ngwan, & Ogbu, 2015). Asymptomatic malaria is not only limited to regions of high transmission where exposure-related immunity is expected to develop but has also been reported in low transmission areas. Amazonian regions of Brazil, Peru and Columbia has reported cases of asymptomatic malaria (Branch et al., 2005; Rodrigues et al., 2006; Vallejo et al., 2015). In these regions of low transmission, immunity is developed much earlier due to predictably low parasite genetic diversity and few overlapping University of Ghana http://ugspace.ug.edu.gh 11 infections. Unlike plasmodium falciparum, few reports are available on asymptomatic malaria caused by other species of malaria parasite. Plasmodium vivax malaria has been reported in low transmission settings of Solomon Islands, Temotu province, (Harris et al., 2010) and highly endemic areas of Rio Negro in the Amazon state, Brazil. The greatest challenge posed by asymptomatic malaria is due to its lack of standard diagnostic criteria. Infected individuals who are in pre-symptomatic period present with symptoms later and may be missed (Rodrigues et al., 2006). Alternatively, without thorough clinical history, patients who had experienced brief periods of malaria symptoms and taken medication suppresses symptoms and parasitaemia. The widely used method of diagnosing asymptomatic malaria is the use of thick blood smear to detect presence of parasites and the absence of malaria related symptoms (Leoratti et al., 2008; Mast et al., 2010; Port et al., 2008). There has been no universal standard parasite threshold for classifying an infection as asymptomatic because different studies use variable cut off levels for parasite density. Asymptomatic malaria usually goes untreated and undetected therefore becoming a major source of gametocyte for local mosquito vector (Alves, Gil, Marrelli, Ribolla, Camargo, 2005). P.falciparum has been shown to persist asymptomatically in people with immunity for more than 18 months (Babiker, Abdel-Muhsin, Ranford-Cartwright, Satti, 1998). In areas with annual malaria transmission asymptomatic falciparum parasitaemia is common and a large proportion of individuals harbor malaria without any clinical symptoms of malaria. Asymptomatic carriers of submicroscopic gametocytes densities have been shown to be infectious to the mosquito host as found in high transmission regions (Paul RE, Bonnet S, Boudin C, Tchuinkam T, 2007; Schneider P, Bousema JT, Gouagna LC, Otieno S & Omar SA, 2007). Studies have shown that antimalarial drugs impact negatively on the gametocyte quality by reducing its University of Ghana http://ugspace.ug.edu.gh 12 infectivity upon a blood meal by the vector (Hallett et al., 2006). Evidence suggest that individuals with severe anaemia have higher levels of gametocytes (Meerman, Ord , Bousema , Niekerk, Osman & Pinder, Walraven, 2005) Further studies provided evidence of this important development of falciparum gametocytaemia and transmission potential (Price et al., 1999). It suggests that the sexual stages are not pyrogens and are commonly seen in people without fever. A study in Thailand reported that febrile individuals were more infective than asymptomatic individuals to mosquito host (Pethleart, Prajakwong, Suwonkerd, Corthong, Webber, 2004), In contrast to a study done in Kenya that reported that asymptomatic individuals were more infective to mosquitoes than parasites from symptomatic individuals with their conclusion based on gametocyte abundance in asymptomatic individuals (Baliraine et al., 2009). In the past years, a number of national and global efforts aimed at reducing the burden of malaria have been initiated targeting especially the vulnerable groups, pregnant women and children. This programs include the roll back malaria (RBM) partnership, the African summit on RBM, president’s malaria initiative (PMI), millennium development goals (MDGs) and recently sustained development goals. The roll back malaria partnership was launched in 1998 and its aims were to half the 1990 morbidity and mortality by 2010. The strategy laid down by the roll back malaria partnership included, early case detection, rapid treatment, multiple prevention, well-coordinated action and focused research. The World Health Organization (WHO) recommends a three- pronged approach for reducing malaria in pregnancy, the use of chemoprophylaxis, ITNs and rapid case management. In an attempt to achieve the RBM, African heads of states resolved in 2000 to strengthen national health systems to ensure that by the year 2015, three out of every five patients (60%) malaria University of Ghana http://ugspace.ug.edu.gh 13 patients will access treatment within 24hrs of onset of malaria symptoms, 60% of pregnant women and children use insecticide-treated nets and 60% of pregnant women will have access to chemoprophylaxis (RBM, 2000). The impact of malaria prevention in pregnancy using presumptive treatment with intermittent preventive treatment (IPT) with sulphadoxine-pyrimethamine (sp) and routine antimalarial drugs has been documented. IPT with SP has been shown to reduce malaria episodes, maternal parasitaemia, anaemia and incidence of low birth weight (Guyatt, Noor, Ochola, 2004; Schultz, Steketee, Macheso, Kazembe., 1994; Steketee, Nahlen, Parise, 2001). A study done in Kenya showed that IPT with SP could reduce severe anemia among primigravidae by 39% (Shulman, Dorman, Cutts, Kawuondo, Bulmer, 1999).Despite the beneficial effects of IPT-sp, poor accessibility and use has been shown during its implementation. A study done in Malawi showed that although 90% of pregnant women knew about IPT-sp, only 365 received the recommended 2 doses (Holtz, Kachur, Roberts, Marum, Mkandala, 2004). In Kenya, 96% of the healthcare providers were aware of IPT-sp despite this, only 5% of pregnant women received the recommended doses, cost and constraints in commodity supply were cited as the causes for the discrepancy. The cause of low use and adherence of IPT-sp has been attributed to late booking and presentation of pregnant women to antenatal clinic (van Eijk, Ayisi, ter Kuile Slutsker, Otieno, 2004). In Ghana, malaria prevention policies during pregnancy are well articulated. Intermittent preventive treatment (IPTsp) of malaria during pregnancy is administered free under direct observed therapy (DOT) at all public hospitals and provided as part of the antenatal care (ANC) package with sulphadoxine-pyrimethamine (sp) being the recommended drug. According to Ghana Health Demographic Survey (DHDS, 2014), 83% of women with live births, reported to University of Ghana http://ugspace.ug.edu.gh 14 have used at least one dose of IPT-sp during ANC visit with 63% reporting to have taken 3 or more doses. This proportion was higher in urban than in rural areas (42 – 36%) respectively. According to the survey, there was an increased chance of taking sp with increased education level and wealth. The problems associated with using IPT-sp include, rapidly increasing cases of resistance and shortage of sp in healthcare facilities in the country (Ghana statistical service, 2014). The use of long-lasting insecticide-treated nets (LLINs) is an effective form of protection that reduces mosquito bites therefore reducing the risk of malaria illness, severe disease and death in malaria endemic regions. World Health Organization and roll back malaria partners promote the use of LLINs as a sustainable and cost effective form of protection against malaria in malaria endemic regions in the world. The Ghana ministry of health and the Ghana health service in collaboration with other stakeholders provide free mosquito nets. The Government made the importation of LLINs tax free in 2002 in the efforts to ensure universal coverage of LLINs through the availability of mosquito nets in the country. About 70% of households in Ghana own at least one mosquito net with disparities in ownership of ITN between urban and rural dwellers (60% -78%) respectively (GHDS, 2014). Households in Greater Accra region are the least likely to own an ITN (53%) and least likely to sleep under a mosquito net (16%) according to the survey. In spite of this high ownership of nets, only (37%) of household population slept under a mosquito net the night before the survey with Wealth increase having a negative correlation with the possibility of sleeping under a mosquito net. Indoor residual spraying (IRS) is an essential University of Ghana http://ugspace.ug.edu.gh 15 component of the integrated vector management strategy in Ghana in the prevention of malaria and involves spraying the interior wall of households with the aim of killing the vector. The combination of IRS and ITN confers the greatest protection to pregnant women and children against malaria (71%) of households are protected by owning at least ITN and or by having received IRS in the past one year (Ghana statistical service, 2014) Asymptomatic malaria increases the risk of stillbirths, spontaneous abortion, premature delivery and low birth weight (WHO, 2010).Yet it has rarely been a major research focus. The asymptomatic nature of individuals with malaria has led to difficulties in diagnosing, inconsistencies in defining and a general lack of urgency to investigate this particular disease outcome. In some sense, asymptomatic malaria has become the “Neglected” malaria, although recently asymptomatic malaria has become accepted as a major hurdle for malaria elimination, as infected hosts serve as silent reservoirs. Routine treatment of asymptomatic carriers of malaria parasite as part of surveillance strategies has the potential to make a significant contribution in the fight against malaria in endemic regions. University of Ghana http://ugspace.ug.edu.gh 16 CHAPTER THREE METHODOLOGY 3.1 Study Design This was a cross-sectional study aimed at determining the factors associated with asymptomatic malaria among pregnant women attending antenatal clinic at Ridge Regional Hospital Accra, Ghana in June 2016. 3.2 Study Area The study was conducted at Ridge Regional Hospital in the Greater Accra Region of Ghana (Figure 1). Ridge Hospital is the Regional Hospital of Greater Accra Region located in the Ridge residential area of the Osu Klottey Sub-Metro. It was established in 1919 and was formerly referred to as ‘European Hospital’, built exclusively for the European colonial community during the Gold Coast era. It shares boundaries with West Africa Examinations Council to the north, to the West, Kanda Highways; East, Sixth Avenue, and to the South, Osu Castle Road. The hospital is a 348- bed regional teaching and referral Centre in the Greater Accra Region providing general and specialized services for the population in the city and surrounding areas. In 2011, the hospital had about 1,064 admissions, and 2,382 outpatient visits. A patient’s average length of stay on admission was eight (8) days. The hospital occupancy rate was about 80% at any given time. It has staff strength of about 872 comprising doctors, nurses, laboratory technicians, pharmacists and other healthcare workers. The hospital acts as a regional referral point for other health facilities within the region. University of Ghana http://ugspace.ug.edu.gh 17 Figure 2: Map of Ridge Hospital, the study location 3.3 Study Variables Table 1: Variables of the study Variable name Variable definition Variable measurement Dependent variable Parasitaemia Presence of malaria parasites under microscopy Dichotomous University of Ghana http://ugspace.ug.edu.gh 18 Variable name Variable definition Variable measurement Independent variables Age Age in completed years Discrete. Completed years Educational level Educational level reached. Nominal: Marital status Current marital status Nominal: Single Married Divorced Knowledge of malaria prevention Measures of malaria prevention Nominal: ITN, IRS ,IPT-sp ITN Ownership and sleeping under it. Dichotomous: Yes No IPT-sp Use of IPT-sp Nominal Occupation Professional activity Nominal Indoor residual spray(IRS) Use of indoor residual spray Nominal Age of the pregnancy Current Trimester of the pregnancy. Discrete Number of children All children born alive or dead by the pregnant woman Discrete 3.4 Sampling All asymptomatic pregnant women attending antenatal clinic at Ridge Regional Hospital Accra, Ghana during the study period were sampled using consecutive sampling method. University of Ghana http://ugspace.ug.edu.gh 19 3.4.1 Study population The study population included all pregnant women with no symptoms of malaria as per a clinician’s assessment attending ANC at Ridge Regional Hospital Accra, Ghana All pregnant women who did not consent to the study were excluded before testing. Also those who were symptomatic for malaria were excluded from the study. 3.4.2 Sample size Sample size was determined using the formula n=, (z 2 pq)/d 2 where n is the sample size. A prevalence of 47% reported from a study in Northern Ghana (Clerk, Bruce, Greenwood, & Chandramohan, 2009) was used at 95% confidence interval. P= estimated proportion with condition of interest. n = sample size = (z 2 pq)/d 2 Z=1.96 at 95% confidence interval (CI) d= Desired difference between observed proportion and true proportion= 5% (95% CI) n = sample size = 382 n= 400 after factoring for non-completeness 3.4.3 Sampling method/procedure Informed consent was obtained from the women who attended antenatal care within the month of June 2016 during the study period. After providing a brief description of the study, willing and consenting pregnant women were recruited into the study. Women who were symptomatic of malaria and those who did not consent to the study terms were excluded from the study. A Pre- University of Ghana http://ugspace.ug.edu.gh 20 tested structured questionnaire was administered and completed for each participant. Information obtained included age of respondents, educational status, occupation and ITN ownership, Insecticide spray use as well as the use of malaria chemoprophylaxis. Obstetrics information included, gestational age, parity and the number of children alive. 3.4.4 Laboratory test and diagnosis of asymptomatic malaria About 2ml of blood was obtained by a Phlebotomist from a peripheral vein into an ethamine diaminetetraaceticacid (EDTA) bottle for preparation of thick and thin blood film. Two glass slides were labeled for each participant. A drop of blood was then placed on the clean, grease free glass slide and allowed to dry. Precaution was taken to maintain a constant volume of the blood as much as possible. The thin smear were made to spread on the glass slide so that newsprint could be read through it. This was immediately fixed in absolute methanol for 5 seconds and allowed to air dry completely before staining. The dried slides were then placed on a rack in preparation for staining. 3.4.5 Staining technique for thick blood film Thick blood film was used to determine the presence of and quantification of malaria parasites. The thick blood smear was allowed to dry completely under a drier before staining. Giemsa staining technique was used for staining the slides. A staining time of 30 minutes in a 2% volume/volume dilution was used. The air–dried thick blood film was stained in a trough containing the 2% giemsa stain for 30 minutes. The slides were then removed, rinsed in buffer water and the back wiped clean with dry wool. The slides were then placed vertically on the staining rack to air–dry before examination. University of Ghana http://ugspace.ug.edu.gh 21 3.4.6 Staining technique for thin film The film staining detects the species of plasmodium. The giemsa staining technique was also used. The thin film already fixed in absolute methanol for 5 seconds was allowed to air dry completely on the staining rack. The slides were then immersed in a trough containing 2% giemsa for 30 minutes. The stained slides removed and rinsed in buffer water (PH 7.2). The back side of the slide were wiped with dry cotton wool, kept vertically on the rack to air- dry before examination. 3.4.7 Reading of slides and counting of parasites When the slides were completely dry, a drop of oil immersion was placed on each slide and examined using a compound microscope with a x100 objective magnification. Properly stained areas were selected and read by two competent microscopists for malaria parasites. Thick blood film was used and, the method of parasite enumeration was based on WHO approved method. The number of parasites in a film per 200 white blood cells was counted. At least 200 white blood cells were counted. The number of parasites was then divided by the number of leucocytes and multiplied by a factor of 8000. This gave the number of parasites per deciliter. The average number of white blood cells (WBC) in blacks per deciliter is 8000 cells. Parasites count (per dl) = number of parasite X 8000/Number of leucocyte. Any discrepant (discordance) result in terms of the presence of parasites is re-read by a more experienced microscopist (Reader 3).The discrepant microscopy result was resolved by calculating the percentage discrepancy. The percentage discrepancy was determined using the formula: % Discrepancy = [count 1] - [count 2] × 100 [Mean of counts 1 & 2] For parasite counts with % discrepancy less than 20%, the count is accepted and the mean parasite count was taken as the parasite density. For parasite counts with % discrepancy ≥ 20%, the films University of Ghana http://ugspace.ug.edu.gh 22 were examined by Reader 3. The count by Reader 3 and the closest from either Reader 1 or Reader 2 was used to calculate the % discrepancy, and their mean count was taken as parasite density provided the % discrepancy is < 20%. For the purpose of this study, the following quantification was used to describe the densities. Mild parasitaemia =<1000 per dl, Moderate parasitaemia 1000- 2999, Severe parasitaemia>3000 per dl 3.5 Data collection techniques and tools/instruments 3.5.1 Inclusion criteria All consenting asymptomatic pregnant women who attended antenatal clinic (ANC) at Ridge Regional Hospital Accra, who were 16 weeks and above of pregnancy were included in this study. 3.5.2 Exclusion criteria Exclusion criteria included women with gestational age <16 weeks, those who had taken antimalarial drugs in the last 2 weeks, presentation with clinical symptoms of malaria (fever, chills, rigor, nausea, vomiting, headache anorexia and joint/muscle pains) and women who did not consent to the study. 3.5.3 Training of interviewers Research assistants recruited were trained to administer the study questionnaires in a friendly and professional manner. They were particularly coached on ways of conducting the interviews efficiently for quality data collection. 3.5.4 Pre-testing and review of instruments The questionnaire pre-testing was done at Ridge Regional Hospital antenatal clinic Accra, Ghana. A random selection of pregnant women attending ANC were interviewed and questionnaires filled. University of Ghana http://ugspace.ug.edu.gh 23 The format and women’s comprehension of the questionnaires were assessed and revised accordingly. 3.5.5 Data collection The trained data collectors visited the antenatal clinic (ANC) and recruited subjects meeting the eligibility criteria and provided a brief overview of the study. Individuals who could be recruited as per the healthcare worker’s assessment were then referred for an informed consent session. A clinician administered the informed consent, subjects who consented were re-assessed by the study clinician and data on age, sex, number of children, education, knowledge of malaria prevention, ownership of ITNs, sleeping under the ITN, use of intermittent preventive treatment(IPT), and use of insecticide spray were obtained through a closed ended personal interviews with structured questionnaire. About 2ml of blood was obtained by a phlebotomist from the participants from a peripheral vein into an ethamine diaminetetraaceticacid (EDTA) bottle for preparation of thick and thin blood film by a phlebotomist. 3.5.6 Quality control Completed questionnaires were checked for consistency and completeness by the principal investigator before the data were entered. Species discrepancies while reading the microscope were resolved by a third external microscopist. Parasite density with discrepancies ≥20% were resolved by averaging the third microscopist’s results and the closer of the two first results. Double entry of all data was done to reduce the chances of data entry errors. Eligibility criteria was assessed and confirmed by the clinician administering the informed consent. University of Ghana http://ugspace.ug.edu.gh 24 3.5.7 Data processing and analysis The data were cleaned, coded and entered into stata version 13 (STATA, College Station, TX, USA) for analysis. Descriptive univariate analyses were performed to assess the frequency distribution of the various factors. Bivariate analysis was employed to examine the associations of individual factors with parasitaemia. The chi square statistic with its corresponding probability level, odds ratio (OR), and 95% confidence interval (CI) were computed to examine the magnitude and significance of the bivariate associations between pairs of dichotomous variables. In the associated factors model, a likelihood ratio with a P–value of <0.05 was considered as statistically significant while those p-values ≥0.05 were not considered significant. University of Ghana http://ugspace.ug.edu.gh 25 3.5.8 Ethical clearance Ethical clearance: Ethical approval for the study was obtained from the Ghana Health Service Ethical Review Committee (GHSREC) before commencement of the study. Approval from study area Permission was also sought from Health management team at Ridge Regional Hospital. Voluntary consent All study procedures were clearly explained to participants while obtaining informed consent. Hospital staff and study participants were assured of the confidentiality, data safety and appropriate data usage. University of Ghana http://ugspace.ug.edu.gh 26 CHAPTER FOUR RESULTS 4.1 Demographic characteristics of the study population A total of 400 pregnant women who visited the antenatal clinic at Ridge Regional hospital between 4th─30th June 2016 participated in the study. The demographic characteristics of the pregnant women that participated in this study are summarized in Table 2. Majority of the women were in the age group (25-29) representing 35.75% of the total participants while the least number were those of the age group (15-19) with 2.5%. Almost half of the pregnant women (48.12%) had basic education while only (8.52%) had no formal education. Most of the study participants were traders, (41.75%); full-time housewives, (22.75%) and artisans, (21%). 338(84.50%) of the pregnant women were married and over 80% were Christians University of Ghana http://ugspace.ug.edu.gh 27 Table 2: Demographic characteristics of the study participants Variable (n, %) Age (years) 15-19 10(2.5) 20-24 29 (7.3) 25-29 143(35.8) 30-34 127(31.8) ≥35 91(22.8) Education level No education 34(8.5) Basic 192(48.1) Secondary 105(26.3) Tertiary 68(17.0) Marital status Married 338(84.5) Single 60(15.0) Divorced 2(0.5) Occupation Trader 167(41.8) Artisan 84(21) Housewife 91(22.8) Civil servant 58(14.5) Religion Christian 329(82.3) Muslim 69(17.8) University of Ghana http://ugspace.ug.edu.gh 28 4.2 Obstetric and prevalence characteristics of the pregnant women The obstetric characteristics of the pregnant women that participated in this study are summarized in Table 3 .The prevalence of malaria in our study was 5.5% with the Plasmodium species detected in the positive malaria slide of pregnant women whose peripheral blood smear was tested being: P. falciparum 21 (95.50) P. malariae 1 (4.5%). Majority of the study participants had mild parasitaemia 18 (81.8%) while 3(13.6%) had severe parasitaemia. Primigravidae formed the lowest percentage of our study participants 61(15.25%) while multigravidae constituted over half of the study participants 214(53.5%). Most of the study participants were in their third trimester of pregnancy 258 (64.5%). University of Ghana http://ugspace.ug.edu.gh 29 Table 3: obstetric characteristics, prevalence and parasite density of the study participants Variable (N, %) Gravidity Primigravidae 61(15.25) Secundigravidae 125(31.25) Multigravidae 214(53.50) Gestation Second trimester 142 (35.50) Third trimester 258 (64.50) Parasitaemia Yes 22 (5.50) No 378 (94.50) Parasite Density Mild 18 (81.80) Moderate 1 (4.60) Severe 3 (13.60) Parasite species Plasmodium falciparum 21(95.50) Plasmodium malariae 1(4.50) University of Ghana http://ugspace.ug.edu.gh 30 4.3 Malaria prevention methods adopted by pregnant women in the study population The prevention methods adopted by the pregnant women that participated in this study are summarized in Table 4. In this study, pregnant women adopted several methods of malaria prevention: use of ITN, Insecticide spray and IPT-sp. About 243 (60.75%) owned at least a mosquito bed net while only 188 (47%) actually used the mosquito nets in the prevention of malaria. Over seventy percent of the study participants used insecticide spray 283 (70.75%). Majority of the pregnant women had taken at least one dose of antimalarial prophylaxis (IPT-sp) 327 (81.75%) while less than thirty percent had not taken any prophylaxis. Almost all the study participants knew the importance of the preventive methods 397 (99.25%). Table 4: Malaria preventive methods adopted by the study participants VARIABLE (N, %) Ownership of ITN Yes 243(60.75) No 157(39.25) ITN use Yes 188(47.00) No 212(53.00) Insecticide spray use Yes 283(70.75) No 117(29.25) IPT-sp use Yes 327(81.75) No 73(18.25) Knowledge on malaria prevention Yes 397(99.25) No 3(0.75) University of Ghana http://ugspace.ug.edu.gh 31 4.4 Results of Sociodemographic factors associated with malaria parasitaemia from chi square test The epidemiological characteristics of malaria and the sociodemographic characteristics are reported in table 5. The study participants in the age group (30-34) had the highest number of parasite positives 8 compared to 1 in the age group (15-19). The percentage prevalence of parasite positives among the different age groups were: 15-19 (10%), 20-24 (6.9%), 25-29 (4.9%), 30-34 (6.9%), ≥35 (4.6%). Participants who had basic education had the highest number of malaria parasite positives while those who had tertiary and no education had the least number. Women who were single had the highest percentage of malaria parasite 5 (8.3%) compared to those who were married 17 (5%). Participants who were traders had the highest percentage of malaria compared to other cadres while civil servants had the least percentage of malaria parasite. The chi square or fisher’s exact test showed that the prevalence of malaria among participants had no significant differences (p≥0.05) with the demographic variables. University of Ghana http://ugspace.ug.edu.gh 32 Table 5: Sociodemographic characteristics of the participants associated with malaria prevalence Variable Parasitaemia no parasitaemia p -Value Age (years) (n, %) 0.915 15-19 1(10.00) 9(90.00) 20-24 2(6.90) 27(93.10) 25-29 7(4.90) 136(95.10) 30-34 8(6.30) 119(93.70) ≥35 4(4.60) 87(95.40) Education level 0.116 No education 2(5.90) 32(94.10) Basic 11(6.0) 182(94.00) Secondary 7(6.70) 98(93.30) Tertiary 2(2.90) 66(97.10) Marital status 0.756 Married 17(5.00) 321(95.0) Single 5(8.30) 55(9.70) Divorced 0(0.00) 2(0.50) Occupation 0.395 Trader 11(6.70) 156(93.30) Artisan 4(5.70) 80(94.50) Housewife 5(5.40) 86(94.60) Civil servant 2(3.40) 56(96.60) University of Ghana http://ugspace.ug.edu.gh 33 4.5 Obstetric characteristics and preventive methods associated with malaria parasitaemia from chi square test Primigravidae had the highest percentage of malaria parasite positives in this study (8.2%) while multigravidae had the least percentage (4.7%). The characteristic distribution is shown in table 6. Participants in the third trimester had a slightly lower percentage prevalence of malaria parasite compared to those in their second trimester. The percentage prevalence of malaria parasite in those who used ITN was (5.3%) while those who did not use had a prevalence of (5.7%). Those who used IPT-sp had (20.8%) malaria prevalence while those who did not take had 6.8%). The chi square or fisher’s exact test showed that all the factors were not significant (p≥0.05). Table 6: Obstetric characteristics and preventive methods associated with malaria parasitaemia VARIABLE Parasitaemia No Parasitaemia P-Value Gravidity 0.251 Primigravidae 5(8.20) 56(91.80) Secundigravidae 7(5.60) 118(94.40) Multigravidae 10(4.70) 204(95.3) Gestation 0.931 Second trimester 8(5.60) 134(94.40) Third trimester 14(5.40) 244(94.6) ITN use 0.881 Yes 10(5.30) 178(94.70) No 12(5.70) 200(94.30) Insecticide spray use 0.785 Yes 15(5.30) 268(94.70) No 7(6.00) 110(94.00) IPT-sp use 0.576 Yes 17(20.8) 310(79.2) No 5(6.80) 68(93.20) University of Ghana http://ugspace.ug.edu.gh 34 4.6 Sociodemographic characteristics of participants from logistic regression models The logistic regression analysis and the results are presented in Table 7. It showed that participants who were in the age group above 35 were about 60% less likely to get malaria than those who were in the age group (15-19) (OR= 0.41, 95% CI .04─4.11).It showed that as age increases, the odds of malaria reduces. Traders had about 19% higher chance of getting malaria compared to artisans while civil servants had the least likely chance of getting malaria. Those participants who had basic education were about 33% more likely to get malaria that those who had no formal education while those who had tertiary education were about 50% less likely to get malaria (OR= 0.48, 95% CI.07─3.60). Participants who were single were about 70% more likely to get malaria than married ones (OR= 1.72, 95% CI .61─4.84) .All the factors were not significantly associated with malaria (p≥0.05). University of Ghana http://ugspace.ug.edu.gh 35 Table 7: Sociodemographic characteristics of participants from logistic regression models VARIABLE OR C (95% CI) P-Value Age (years) 15-19 reference 20-24 0.67 (.05─8.25) 0.752 25-29 0 .46 (.05─4.19) 0.493 30-34 0.61 (.07─5.39) 0.652 ≥35 0.41 (.04─4.11) 0.451 Education level no education reference basic 1.33 (.21─8.50) 0.761 secondary 0.59 (.11─3.19) 0.543 tertiary 0.48 (.07─3.60) 0.479 Marital status married reference single 1.72 (.61─4.84) 0.307 Occupation artisan reference trader 1.19 (.36-3.86) 0.777 housewife 0.96 (.39─2.35) civil servant 0.59 (.10-3.34) 0.550 4.7 Obstetric characteristics and preventive methods associated with parasitaemia from logistic regression models The logistic regression analysis and the results are presented in Table 8. Pregnant women in their third trimester had about 5% reduced chance of getting malaria compared to those in their second trimester. Women who did not use ITN had 7% increased chance of getting malaria (OR= 1.07, 95% CI 0.45─2.53).Participants who used insecticide spray were less likely to get malaria compared to those who did not. Women who have had more than four pregnancies were about University of Ghana http://ugspace.ug.edu.gh 36 50% less likely to get malaria compared to those who were having their first pregnancy (OR= 0.55 95% CI 18-1.67). All the factors were not significantly associated with malaria (p≥0.05). Table 8: Obstetric characteristics and preventive methods associated with parasitaemia from logistic regression models VARIABLE OR C (95% CI) P Gravidity Primigravidae reference Secundigravidae 0.66 (.20-2.19) 0.501 Multigravidae 0.55 (.18-1.67) 0.291 Gestation Second trimester reference Third trimester 0.96 (.39─2.35) 0.931 ITN use Yes reference No 1.07 (.45─2.53) 0.881 Insecticide spray use Yes reference No 1.14 (.45─2.86) 0.785 IPT-sp use Yes reference No 1.34 (.48─3.76) 0.577 University of Ghana http://ugspace.ug.edu.gh 37 CHAPTER FIVE DISCUSSION 5.1 Introduction This study examined factors associated with asymptomatic malaria among pregnant women attending antenatal clinic in Ridge Regional Hospital Accra, Ghana. This chapter therefore discusses the findings of the study. In some instances the findings are also compared and contrasted with previous studies. 5.2 Prevalence of malaria parasitaemia This study aimed to determine the prevalence of asymptomatic malaria infection and possible associated risk factors in pregnant women attending ANC in Ridge regional hospital Accra, Ghana. The prevalence of maternal peripheral malaria infection by microscopy was 5.5% with P.falciparum being the predominant species (99.5%) confirming the national prevalent species of falciparum with over 99% prevalence (WHO, 2015). Steffen et al. (Steffen & Ban, 2003) reported that 80-95% of malaria infections in tropical Africa are caused by P. falciparum which is in agreement with the findings of this study. The prevalence of 5.5% is comparable to findings of studies done in coastal Ghana which found a prevalence of 5% (Stephens, Ofori, Quakyi, Wilson, & Akanmori, 2014) but in contrast to those reported in various parts of the country (Clerk et al., 2009; Douamba et al., 2012; Stephens et al., 2014) of 47%, 35.1% and 19.7% respectively. The figure is also much lower than the 58% reported in urban pregnant women as reported by Nwagha and co-workers (Ifeanyi et al., 2009) and the 59.9% reported by Ogbodo et al. (2009) for some University of Ghana http://ugspace.ug.edu.gh 38 rural women in southeastern Nigeria. Similarly, another study conducted in southeastern Nigeria by Nwonwu and his colleague reported a prevalence of 29% among a group of urban, asymptomatic pregnant women (Nwonwu et al, 2009). There is, therefore, a very wide variation in reported prevalence. The low prevalence of peripheral blood malaria parasites in our study could be explained by the improved education on malaria during pregnancy in Ghana. Indeed, over 99% of the pregnant women had knowledge on malaria preventive methods. Another plausible explanation to the wide difference in prevalence is the fact that we used microscopy which has less sensitivity compared to PCR, it could be that if we had used PCR, the prevalence would have been higher (Rogerson, Hviid, Duff, Leke, & Taylor, 2007). The large differences in the reported prevalence rates of malaria may also be attributed to skill and experience of laboratory personnel involved in blood film preparation, staining and reading of the slides. In our study, strict adherence to procedures for slide preparation and staining ensured the production of clear, well-stained slides, thereby reducing chances of errors. In this study, very strict inclusion criteria were used to recruit women who were asymptomatic and study participants were assessed twice by experienced clinicians before being included in the study. This strict adherence to inclusion and exclusion criteria ensured that only eligible women participated in the study. Another important factor that could explain the low prevalence is the urban setting of our study with participants having high knowledge on malaria. Over 80% of the study participants had taken at least one dose of IPT-sp at the time of the study way above the national prevalence of 54.1% (NMCP, 2015). 5.3 Risk factors associated with Parasitaemia Studies have shown that age, gravidity, gestation, use of ITN, insecticide spray, education level and the use of IPTsp are associated with malaria in pregnancy. These studies have shown that University of Ghana http://ugspace.ug.edu.gh 39 pregnant women of young maternal age are at the greatest risk of malaria infection, as well as having the highest parasite densities (Adam, Khamis, & Elbashir, 2005; Agomo & Oyibo, 2013). Multigravidas have been noted to have lower effects of malaria in pregnancy than in other gravidities. This is as a result of acquisition of specific immunity to placental malaria due to previous exposure. Acquired specific immunity accumulates with subsequent infection and subsequent pregnancies (Staalsoe et al., 2004). In our study, older women and multigravidas had reduced chance of getting malaria 59%, 45% respectively compared to younger and primigravidas. However, this reduced chance was not significant. This could be because in a malaria endemic area such as Ghana, it is possible that the women could have had a number of encounters with malaria infection prior to visiting the antenatal clinic used in this study. There could also have been no difference in the level of specific immunity of the study participants based on gravidity. Reports indicate that the use of ITN substantially reduces the risk of malaria in pregnancy. Indeed, WHO has advocated for a three pronged approach to tackling malaria and part of the strategy is the use of ITN (WHO, 2014). In our study, those who did not use ITN had 7% increased risk of getting malaria. Marchant and co-workers (Marchant, Schellenberg, Edgar, Nathan, & Abdulla, 2002) also reported a non-significant but modest impact of ITN on malaria in pregnancy .The use of bed nets, and insecticide spray, did not have a significant impact on malaria infection in this study Education and the gestation ages of the women were not significantly associated with malaria infection. Compared to participants who had no formal education, participants who had tertiary level of education had 52% reduced chance of getting malaria but this reduced prevalence was not statistically significant. This lack of significant association could be explained by the fact that majority of the study participants had very good knowledge of malaria preventive methods. This University of Ghana http://ugspace.ug.edu.gh 40 is attributed to radio and television campaigns on malaria prevention strategies and appropriate treatment options available in Ghana. Moreover, pregnant women attending antenatal clinic at Ridge regional hospital are usually given a health talk on malaria and other conditions affecting them before being attended to. These campaigns have bridged the gap between those who had formal education and those who did not have. The use of IPT-sp has been shown to reduce malaria prevalence in pregnancy significantly (Ofori, Ansah, Agyepong, Hviid, & Akanmori, 2009; Stephens et al., 2014).In our study, the coverage of IPT-sp was over 80% way above the national coverage of 54.1% (NMCP, 2015).In this study, the use of IPT-sp was not significantly associated with malaria infection in pregnancy. Similar finding was also observed in a study in Lagos, Nigeria (Agomo & Oyibo, 2013). Occupation was not significantly associated with malaria infection in this study. 5.4 Limitations The use of microscopy instead of the more sensitive PCR to determine the prevalence of parasitaemia could have underestimated the real prevalence of parasitaemia. Results of our study cannot be generalized to rural areas because it was conducted in an urban setting. Study may have been underpowered and thus undermined statistical significance of test of associations. Responses and study findings may have been compromised by the study design (i.e. cross sectional study) which allows for only temporality and point assessment, as well as the choice of study site (regional hospital) may have. University of Ghana http://ugspace.ug.edu.gh 41 CHAPTER SIX CONCLUSIONS AND RECOMMENDATIONS 6.1. Conclusion The prevalence of malaria parasitemia among asymptomatic pregnant women attending ANC in Ridge Regional hospital was low. This low prevalence could be attributed to the urban setting of our study site. It could also be due to the scaling-up of malaria interventions that has seen the level of knowledge, the use of ITN and IPT-sp increase tremendously in Ghana. These effective interventions have had a positive impact on malaria infection among pregnant women. Strict inclusion criteria and the use of Microscopy instead of a more sensitive PCR could have also contributed to the low prevalence. Gravidity, maternal age, usage of insecticide spray, ITN use, occupation, gestation and the use of IPT-sp had modest but non-significant impact on malaria infection in pregnancy. This lack of significant association could have been attributed to the low prevalence of malaria positives. 6.2.Recommendations Based on the findings, there is need to:  Introduce a systematic test and diagnosis of malaria at various stages of pregnancy as part of antenatal care package.  The use of intermittent preventive treatment, insecticide spray and ITNs should be strengthened among all pregnant women in all parts of Ghana. University of Ghana http://ugspace.ug.edu.gh 42  Future research should be conducted in different transmission settings to provide current data on the national prevalence of asymptomatic malaria and risk factors in the context of scaled-up malaria control efforts. University of Ghana http://ugspace.ug.edu.gh 43 REFERENCES Adam, I., Khamis, A. H., & Elbashir, M. I. (2005). 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Intermittent sulfadoxine—pyrimethamine to prevent severe anemia secondary to malaria in pregnancy: a randomised placebo controlled trial. Lancet, 353., 632—6. Snow, R. W., Guerra, C. A., Noor, A. M., Myint, H. Y., & Hay, S. I. (2005). The global distribution of clinical episodes of Plasmodium falciparum malaria. Nature, 434(7030), 214-217. Staalsoe, T., Shulman, C. E., Bulmer, J. N., Kawuondo, K., Marsh, K., & Hviid, L. (2004). Mechanisms of disease Variant surface antigen-specific IgG and protection against clinical consequences of pregnancy-associated Plasmodium falciparum malaria, 363. Steffen, R., & Ban, A. (2003). Tra v el epidemiology * a global perspecti v e, 21, 89–95. Steketee RW, Nahlen BL, Parise ME, M. C. (2001). The burden of malaria in pregnancy in malaria-endemic areas. Am J Trop Med Hyg, 64, 28–35. Stephens, J. K., Ofori, M. F., Quakyi, I. A., Wilson, M. L., & Akanmori, B. D. (2014). Prevalence of peripheral blood parasitaemia , anaemia and low birthweight among pregnant women in a suburban area in coastal Ghana, 17(Supp 1), 1–4. http://doi.org/10.11694/pamj.supp.2014.17.1.3541 Sullivan, A. D., Nyirenda, T., Cullinan, T., Taylor, T., Harlow, S. D., James, S. A., & Meshnick, S. R. (2001). Malaria Infection during Pregnancy : Intrauterine Growth Retardation and Preterm Delivery in Malawi, 1580–1583. Vallejo, A. F., Chaparro, P. E., Benavides, Y., Álvarez, Á., Quintero, J. P., & Padilla, J. (2015). High prevalence of sub-microscopic infections in Colombia. ???, 1–7. http://doi.org/10.1186/s12936-015-0711-6 van Eijk AM, Ayisi JG, ter Kuile FO, Slutsker L, Otieno JA, O., & JO, et al. (2004). Implementation of intermittent preventive treatment with sulphadoxine—pyrimethamine for control of malaria in pregnancy in Kisumu, western Kenya. Trop Med Int Health, 9(630-7). WHO. (2014). World malaria report. Geneva. WHO. (2015). African Region, 2015, 2015. Zoghi, S., Mehrizi, A. A., Raeisi, A., Haghdoost, A. A., Turki, H., & Safari, R. (2012). Survey for asymptomatic malaria cases in low transmission settings of Iran under elimination programme, 1–10. University of Ghana http://ugspace.ug.edu.gh 48 APPENDICES Appendix1: Consent form Introduction We are asking you to take part in a research study on factors associated with asymptomatic malaria among pregnant women at Ridge Regional Hospital. We want to be sure that you understand the purpose and your responsibilities in the research before you decide if you want to be part of the study. Please ask us to explain any words or information that you may not understand. Information about the Research This is a research study that would involve collection of about 2ml of blood from a peripheral vein for the detection of plasmodium parasitaemia. Possible Risks (explain risks –blood collection) It is very unlikely that participation in this research will expose you to any physical, social or psychological risks. Possible Benefits Participation in this research may not benefit you directly. But results from this study will be used to inform decisions in implementation and strengthening of programs aimed at controlling maternal and child mortality from malaria in Ghana. If You Decide Not to Be in the Research You are free to decide if you want to be part of this research or not. University of Ghana http://ugspace.ug.edu.gh 49 Confidentiality We will protect information about you taking part in this research to the best of our ability. We will neither use your name in any reports nor discuss your participation with anyone outside the research team. Payment No payments will be made for participation. However, there will be reimbursement of transport equivalent to five Ghana cedes only (GH¢ 5). Leaving the Research You may end your participation at any time with no negative consequence to you. If You Have Questions about the Study If you have any questions about the research, call 0553017342 Your rights as a Participant This research has been reviewed and approved by the Ghana Health Service ethical review board and permission from the administration of Regional Ridge Hospital. If you have any questions about how you are being treated by the study or your rights as a participant you may contact Dr Hannah Firmpong (Hannah.Frimpong@hru-ghs.org) Telephone number: 233 021 681 109 University of Ghana http://ugspace.ug.edu.gh 43 Appendix2: Questionnaire Factors associated with asymptomatic malaria among pregnant women attending antenatal clinic (ANC) at ridge regional hospital Accra, Ghana. Introduction Hello thanks so much for your permission. I am Daniel Kiprono Kiptoo, a student pursuing a Master of Public Health (MPH) Program at the University of Ghana School of Public Health Accra, Ghana. This interview is being conducted as part of a research into the factors associated with asymptomatic malaria for Malaria control in pregnancy. I would be very much grateful if you would kindly find some time to answer these questions. Your views, opinions and contributions are very valuable and important and would go a long way to help me determine the factors associated with asymptomatic malaria in pregnancy. This study is strictly for academic purposes and I can assure you of the confidentiality on any information that you would provide. Thanks for your cooperation. University of Ghana http://ugspace.ug.edu.gh 44 Please tick [√] where appropriate and give the appropriate response to each item as presented. Section A Socio-demographic information 1 1. Sex 2. Male 3. Female |___| 2 What is your age? 1. 15-19 2. 20- 24 3. 25-29 4. 30-34 5. 35-39 6. 40- 44 7. 45- 49 8. 50 and above |___| 3 What is the highest level of school you attended? 1. No education 2. Primary 3. Middle 4. JSS/JHS 5. Secondary/Vocational 6. SSS/SHS 7. Higher |___| 4 What is your current marital Status 1. Married 2. Single 3. Divorced 4. Separated 5. Widowed |___| 5 What is your nationality? ………………………… 6 What is your ethnicity? 1. Akan 2. Ga/Adangbe 3. Ewe 4. Hausa 5. Other |___| 7 How many of children Do you have? 1. No child 2. 1-3 children 3. 4-6 children 4. 7-9 children |___| University of Ghana http://ugspace.ug.edu.gh 45 5. more than 9 children 8 If employed, what is your occupation, that is, what kind of work do you mainly do? …………………………. 9 Which religion do you belong to? 1. Christian 2. Islam 3. Traditionalist 4. Others (Please specify).............. |___| Section B Obstetric characteristics 10 How many times have you gotten pregnant? 1. Once 2. Twice 3. Thrice 4. > Four |___| 11 How many times have you delivered? 1. Once 2. Twice 3. Thrice 4. Four or more |___| 12 What is the gestational age of your current pregnancy? 1. First trimester 2. Second trimester 3. Third trimester |___| Section C Intermittent preventive treatment use (IPT-sp) 13 Have you ever heard or been told that pregnant women are supposed to take malafan/sp in pregnancy to prevent malaria? 1. Yes 2. No 3. Don’t know |___| 14 Have you taken malafan/sp during your current pregnancy? 1. Yes 2. No If no, go to question 16 |___| 15 If the answer to the above question is yes, how many times? 1. Once 2. Twice 3. Thrice 4. Four or more times |___| Section D Possession/ownership of ITN. 16 Do you have a mosquito bed net? 1. Yes 2. No |___| University of Ghana http://ugspace.ug.edu.gh 46 If no, go to question 18 17 If yes, how many mosquito bed nets do you have? 1. One 2. Two 3. Three 4. Four or more 5. Five or more |___| 18 Does any member of your household have any ITN? 1. Yes 2. No |___| Section E Use of ITN 19 Do you hang the net on your bed? 1. Yes 2. No If yes, go to question 21 |___| 20 If no, why? 1. Causes heat 2. no space to hang 3. looks like burial shroud 4. allergic reasons 5. Others (Please specify)............................... |___| 21 What time do you go to bed? 1. 7:00 pm – 8:00pm 2. 8:00- 9:00pm 3. 9:00pm – 10:00pm |___| 22 Did you sleep under a mosquito net (ITN) last night? 1. Yes 2. No If yes, go to question 24 |___| 23 If no, why? 1. Feels hot inside . Looks like a burial shroud 3. No space to hang 4. Others (Please specify)……………. |___| 24 Which periods do you use the net? 1. All year round 2. During rainy season 3. During dry season 4. Others (Please specify)................................ |___| 25 Why do you use the net during that period? 1. To prevent malaria 2. For warmth 3. As a partition in the room 4. Others ( please specify)…………. |___| 26 What are the benefits of ITN use? 1. To prevent malaria University of Ghana http://ugspace.ug.edu.gh 47 2. To sleep soundly 3. To provide warmth 4. To prevent insects bites 5. Other (Please specify)............................... |___| Section F. Use of indoor residual spray. 27 Have you ever heard about IRS? 1. Yes 2. No |___| 28 Do you use indoor residual spray? 1. Yes 2. No |___| 29 If yes to the above question, how often do you spray? 1. All year round 2. During rainy season 3. During dry season 4. Others (specify)........................................ |___| University of Ghana http://ugspace.ug.edu.gh