ABSTRACT
Malaria is a major global public health concern especially in countries where transmission occur regularly. A larger proportion of the malaria cases that resulted in mortality occurred among children less than five years old. This cross sectional study was aimed at determining the genetic diversity of Plasmodium falciparum and antibody responses to erythrocyte surface antigens among children in Minna, Nigeria. Blood samples were collected from children with no signs of fever (asymptomatic cases) within the residential communities in Minna, and children with symptoms of fever (symptomatic cases) who attended selected healthcare facilities within the period of the study. Thick and thin films of blood samples were prepared using Giemsa staining technique to determine the parasite density and the infective stage. Subsequently, a complete blood count to determine the haematological indices of the blood samples was carried out. Blood samples were exposed to enzyme linked immunosorbent assay (ELISA) for the measurement of IgG antibody levels against P. falciparum. Samples were further analyzed for the molecular characterization and gene type using polymerase chain reaction (PCR). A total population of 316 children both symptomatic and asymptomatic were sampled for this study from children between 6 months and 17 years between the months of February to July, 2018. The sampled population comprises 181 (57 %) males and 135 (43 %) females. Of the 316 blood samples screened for P. falciparum infection, 238 (75.37 %) were infected with P. falciparum which comprises of 39 (16.39 %) from asymptomatic children from the communities and 199 (83.61 %) symptomatic children attending the six healthcare facilities. The male and female categories recorded a P. falciparum prevalence of 126 (39.61 %) and 112 (35.44 %), respectively, and a non-significant difference (P>0.05) was observed between sex and P. falciparum infection. P. falciparum infected cases with anaemia recorded a high prevalence rates as compared to the non-infected cases. However, the multinomial logistic regression model demonstrated that P. falciparum was not a significant predictor to anaemia. In addition, the results of ELISA showed P. falciparum infected cases had higher specific antibody IgG responses of 64 (68.81 %) as compared to the negative P. falciparum samples, though a non-significant difference (P>0.05) between the negative and positive P. falciparum infected cases and the production of IgG antibody was observed (χ2=0.896, P=0.979). Determination of gene diversity revealed MAD20 and FC27 as the predominant allele for MSP1 and MSP2, respectively. However, K1 and FC27 were the predominant allele from asymptomatic children and on the other hand, FC27 and MAD20 were predominant for symptomatic cases. A high malaria transmission intensity as a result of mixed infection was observed in Minna with an overall multiplicity of infection (MOI) of 2.22. The MOI for symptomatic cases was lower 2.21 than what was observed from asymptomatic cases with 2.41. Finally, findings from this study revealed a high level of gene diversity within the antigenic markers of MSP1 and MSP2 (0.714 and 0.830, respectively). This study has demonstrated the potential of malarial antibody and gene diversity, as important markers for assessing differences in malaria parasite transmission intensity. Continuous genetic surveillance in P. falciparum will therefore serve as an important tool in monitoring changes in gene types and for intervention programmes effectiveness.
CHAPTER ONE
1.0 INTRODUCTION
1.1 Background to the Study
Malaria is a mosquito-borne infectious disease that affects humans which is caused by parasitic protozoans that belong to the genus Plasmodium (WHO, 2014). It causes symptoms that normally include fever, fatigue, vomiting, and headaches. In severe cases, it can cause seizures, yellow skin, coma, or death (Caraballo, 2014). There are however, five species of Plasmodium that can infect humans and spread by mosquitoes (Caraballo, 2014). These species are Plasmodium falciparum, which causes most deaths, while P. vivax, P. ovale, and P. malariae cause a milder form of malaria (Caraballo, 2014; WHO, 2014). Meanwhile, the species P. knowlesi rarely causes disease in humans (WHO, 2014). Plasmodium falciparum is the most prevalent malaria parasite in sub-Saharan Africa and it accounts for 99 % of the estimated malaria cases in 2016. Outside of Africa, P. vivax is the predominant parasite in America, which represent 64 % of malaria cases, and is above 30 % in South- East Asia and 40 % in the Eastern Mediterranean regions (WHO, 2017).
Malaria is a major global public health concern especially in countries where transmission occur regularly. In 2016, an estimated 216 million cases of malaria occurred worldwide, compared with 237 million cases in 2010 and 211 million cases in 2015 (WHO, 2017). About 90 % of most malaria cases in 2016 were found in African, followed by South-East Asia region with 7 % and the Eastern Mediterranean region with 2 %. Out of the 91 countries that reported malaria cases in 2016, 15 countries – all in sub-Saharan Africa – carried 80 % of the global malaria burden (WHO, 2017). In sub-Saharan Africa, malaria is one of the leading causes of morbidity and mortality, with 191 million cases and over 390 thousand deaths reported in 2016. Nevertheless, with the scaling up of malaria prevention, diagnosis and treatment, the prevalence of Plasmodium falciparum infection in many parts of sub-Saharan Africa declined by half, and the frequency of clinical disease fell by 40 % between the year 2000 and 2015 (Bhatt et al., 2016). Malaria incidence among populations at risk dropped globally by 21 % between 2010 and 2015. In that same period, malaria mortality rates among populations at risk globally dropped by 29 % among all age groups, and by 35 % among children under the age of five (WHO, 2016). In addition, Sub-Saharan Africa carries an excessively high proportion of the global malaria burden. In 2015, the region recorded 90 % of malaria cases and 92 % of malaria deaths (WHO, 2016). There were an estimated 33,000 malaria cases per 100,000 people in Nigeria, with 110,000 deaths (WHO, 2015a). Malaria prevalence among children in Nigeria has decreased steadily over the years (from 42 % in 2010 to 27 % in 2015), but malaria remains a leading cause of death among them (WHO, 2015a).
A larger proportion of the malaria cases that resulted in mortality probably occurred among children less than 5 years of age. Due to the endemic nature of malaria in Nigeria, partial immunity to malaria is acquired among older children. However, severe forms of anaemia can be seen in children less than 5 years of age who have not yet acquired immunity. According to WHO, in 2015, 69 % of the malaria deaths that occurred worldwide were among children below 5 years (WHO, 2015b).
In 2016, there were an estimated 445,000 deaths from malaria globally, compared to 446,000 estimated deaths in 2015 with a decline of 1000. Meanwhile, the African region accounted for 91 % of all malaria deaths in 2016, followed by South-East Asia region with 6 %. Fifteen countries from the sub-Saharan Africa recorded 80 % of global malaria deaths in 2016. In 2016, all regions recorded mortality reductions compared to 2010, with the exception of the Eastern Mediterranean region, where mortality rates remained virtually unchanged over the period. The largest decline occurred in the regions of South-East Asia (44 %), Africa (37 %) and the America (27 %) (WHO, 2017).
In Africa, malaria was estimated to result in economic loss of US$12 billion a year due to increased healthcare costs, lost ability to work, and negative effects on tourism (Greenwood et al., 2005), and by 2016, an estimated US$ 2.7 billion was invested in malaria control and elimination efforts globally by governments of malaria endemic countries and international partners.
In areas of stable malaria transmission, children and pregnant women are the population groups of highest risk for malaria morbidity and mortality (Bourdy et al., 2008) and most children experience their first malaria infections during the first year or two of their life, when they have not yet acquired adequate clinical immunity – which makes these early years particularly dangerous. This rises the burden of deaths due to malaria in young children to about Ninety percent in Africa. Adult women in areas of stable transmission have a high level of immunity, but this is impaired especially in the first pregnancy, with the result that risk of infection increases (WHO, 2003).
During the blood stage of Plasmodium falciparum infection, anaemia may be induced as a result of destruction of red blood cells by the parasite and soluble derivatives released by the parasite induce bone marrow dysfunction at the period of malaria infection. As such, populations most affected by malaria often are at high risk for anaemia (McCuskee et al., 2014). Severe malarial anaemia caused by P. falciparum is responsible for approximately a third of the deaths associated with the disease (Haldar and Mohandas, 2009). Anaemia is an important contributor to malaria attributable deaths in hospitals, with severe anaemia accounting for between 17 % and 54 % of malaria attributed deaths in children under 5 years of age (Biemba et al., 2000).
Plasmodium parasites, expresses many variant antigens on cell surfaces. Variant surface antigens (VSAs) are typically organized into large subtelomeric gene families that play critical roles in virulence and immune evasion (Frech and Chen, 2013). Antigenic variation at the Plasmodium-infected erythrocyte surface plays a critical role in malaria disease severity and host immune evasion (Noelle et al., 2010). Severe cases of malaria can occur when parasite invades and then proliferates within red blood cell erythrocytes. The parasite produces many variant antigenic proteins, encoded by multigene families, which are present on the surface of the infected erythrocyte and play important roles in virulence (Chen et al., 2000). The major surface proteins are the Merozoite Surface Protein 1 and 2 (MSP 1 and 2).
Merozoite Surface Protein 1 and 2 of P. falciparum are major targets for bloodstage malaria vaccine (Chaitarra et al., 1999) and are also appropriate markers for the identification of genetically distinct P. falciparum parasite sub- populations. MSP1 is a major surface protein of approximately 190-kDa size. It plays a major role in erythrocyte invasion (Holder et al., 1992) and is a major target of immune responses (Apio et al., 2000). MSP1 contains 17 blocks of sequence flanked by conserved regions (Takala et al., 2002) Block 2, which is the most polymorphic part of MSP1, is grouped into three allelic families namely K1, MAD20, and RO33 type (Takala et al., 2006). MSP2 is glycoprotein consisting five blocks where the central block is the most polymorphic (Ferreira and Hart, 2007), and is grouped into two allelic families, FC27 and 3D7/IC1.
Furthermore, individuals living in malaria endemic areas develop naturally acquired immunity to Plasmodium spp. infections as they grow, but only slowly and only after repeated exposure (Marsh and Kinyanjui, 2006; Doolan et al., 2009). Anti-disease immunity production mechanisms and factors controlling effective protection are still largely unknown (Reddy et al., 2012). However, it was well established that antibodies contribute to protection against clinical malaria due to P. falciparum. Antibodies directed against cell surface proteins of either the merozoite form of the parasite or of infected red blood cells have been shown to be important components of acquired protective immunity against malaria (Richards and Beeson, 2009).
Naturally acquired antibodies to sporozoites can protect against liver-stage infection and antibodies targeting blood-stage antigens can repress high parasite densities and progression to symptomatic disease (Doolan et al., 2009). The magnitude of antibody responses are important, with responses to a repertoire of antigens associated with increased disease protection (Richards et al., 2013; Rono et al., 2013), while also acting as biomarkers of past exposure (Elliott et al., 2014).
1.2 Statements of the Research Problem
In malaria-endemic regions, the most vulnerable populations that suffered severe and complicated malaria, including malarial anaemia, are children less than five years of age and pregnant women (Quintero et al., 2011). The vast majority of deaths due to malaria parasite is caused by Plasmodium falciparum which is the most virulent species of the malaria parasites (Abdel Hamid et al., 2013).The special pathology of P. falciparum as compared to other species of Plasmodium that infect humans has been attributed to the ability of P. falciparum-infected erythrocytes to cytoadhere to receptors expressed on the surface of capillary endothelial cells (Pongponratn et al., 1991; Berendt et al., 1994; Turner et al., 1994; MacPherson et al., 1995), and the parasite mediates adherence of infected erythrocytes to uninfected erythrocytes, which is responsible for the most severe clinical complications of P. falciparum malaria. These parasites also have the ability to undergo antigenic switching to evade a developing antibody response (Bull et al., 2005). Many important aspects of these variant surface antigen (VSA) function and evolution remain obscure, impeding the understanding of virulence mechanisms and vaccine development (Frech and Chen, 2013).
Severe malaria is one of the most feared complications that is associated with an increased mortality, especially in children and is one of the factors that contributes to the anaemia burden in children. Hence, immunological factors and mechanisms appear to have great relevance in the anaemia pathogenesis during a malaria infection (Quintero et al., 2011). At the period of malaria infection soluble derivatives released by the parasite induce bone marrow dysfunction. These derivatives are therefore implicated in the pathogenesis of malarial anaemia (Silverman et al., 1987; Miller et al., 1989; Jootar et al., 1993).
In addition, Plasmodium is known to induce a strong inflammatory response with a robust production of immune effectors, including cytokines and antibodies. Therefore, it is possible that the extent of the immune response not only may facilitate the parasite killing but also may provoke severe illness, including anaemia (Castro-Gomes et al., 2014). However, despite enormous health implication of this parasite, the immunological mechanisms involved in malaria- induced anaemia remain incompletely or poorly understood (Castro-Gomes et al., 2014.)
Genetic diversity of malaria parasite creates a great hindrance to the development of vaccine and enhances antimalarial drug resistance (Duah et al., 2016). On the other hand, vaccine designed for Plasmodium falciparum was hindered by polymorphisms in certain vaccine candidate loci (Hughes and Verra, 2002; Tongren, 2004). Highly polymorphic regions have been observed in P. falciparum antigenic surface proteins, such as the merozoite surface protein 1 (MSP1), circumsporozoite protein (CSP), the apical membrane antigen 1 (AMA-1), the liver stage antigen (LSA-1) and the thrombospondin-related anonymous protein (TRAP) (Escalante et al., 1998).
Therefore, knowledge of the distribution of polymorphic sites on malaria antigens is necessary to obtain a detailed understanding of their significance for vaccine development. Moreover, this study also includes infections occurring in both symptomatic and asymptomatic parasitemia, its relation with anaemia and immune response to the antigen.
1.3 Justification for the Study
Nigeria is an endemic country for malaria, its large population, various weather conditions and beliefs make it a bit difficult implementing the same malaria control measures throughout the country. Genotyping malaria parasites to assess their diversity in different geographic settings have become necessary for the selection of antigenic epitopes for vaccine development and for further implementation of regional intervention programs.
Furthermore, the level of antigenic diversity varies from one malaria endemic region to another and even between countries, in such a way that the variant forms of the parasite exist at different frequencies in different geographic areas presenting different complexities of infection (Raj et al., 2004). Merozoite Surface Protein 1 and 2 of P. falciparum are one of the variant forms of the parasite which are the most abundant and a major blood-stage malaria vaccine targets (Chaitarra et al., 1999) and are also suitable markers for the identification of genetically distinct P. falciparum parasite sub-populations. Antibodies against several of these merozoite antigens have been found to be associated with protective immunity (Fowkes et al., 2010) and Immunoglobulin IgG which is a type of antibody found in all body fluids was used. The lack of in vitro functional assays that correlate with protective immunity in vivo has hampered the development of effective blood stage vaccines (Crompton et al., 2010). There have been inconsistencies in the correlations of antibody responses to recombinant antigens and protection from malaria using enzyme-linked immunosorbent assays (ELISAs) (McCallum et al., 2008). P. falciparum Parasite genetic diversities has been implicated in evolutionary fitness and consequently populations with high diversity have the ability to survive against most intervention efforts in the control of malaria thereby frustrating control efforts (Barry et al., 2013).
Therefore, to control and eventually eradicate malaria, an effective vaccine is considered to be needed in addition to the existing strategies such as insecticidetreated bed nets (ITNs) and chemotherapy (Kilama and Ntoumi, 2009). However, the development of an effective vaccine is being hampered by genetic diversity (Healer et al., 2004) even though extensive research on the diversity in the parasite field isolates have been conducted in several studies (Babiker et al., 2000 and Zakeri et al., 2005). This diversity has the potential to alter the conformation of antimalarial drug targets and render the parasites drug resistant (Blasco et al., 2017) which will hinder malaria treatment outcome (Felger et al., 1999 and Raj et al., 2004). Genetic diversity and multiplicity of P. falciparum infections are therefore, essential parasite indices that could determine the impact of malaria intervention programs as well as the endemicity of parasite infections in varying transmission settings (Barry et al., 2013, Nabet et al., 2016 and Razak et al., 2016).
Malarial anaemia is acknowledged as an important cause of morbidity and mortality in endemic regions (Quintero et al., 2011). Its importance as a cause of death may well be underestimated because of difficulty in diagnosis, especially where parasitemia may be low and the clinical picture may be confused with other causes of anaemia (Phillips and Pasvol, 1992). Complications of severe anaemia and cerebral malaria were assumed to be the major cause of morbidity and mortality, however, recent evidence indicates that the immunological response of the host could also contribute to the pathophysiology of the disease in human beings (Malaguarnera and Musumeci, 2002). However, giving that specific haematological changes associated with malaria infection may vary with the level of malaria endemicity (Idro et al., 2006), nutritional status (Friedman et al., 2005), demographic factors (Barcus et al., 2007), malaria immunity (Langhorne et al., 2008) and the parasite species. It is therefore, essential to study and characterize the indices involved in malarial anaemia in endemic regions like Minna metropolis. Furthermore, to elucidate the molecular mechanisms involved in the pathogenesis of malaria- induced anaemia, this study addresses the malarial anaemia immune pathogenesis process.
In view of this, haematological, immunological and molecular studies are essential for better understanding of the parasite diversity, and to generate information on the immunological mechanisms involved in malaria induced anaemia as well as the nature and extent of antibody response to infected P. falciparum erythrocyte which is essential in understanding the mechanism underlying the pathology of malaria, the acquisition of immunity, and also the development of vaccine candidate against the parasite. This research, therefore, focuses on hematological indices, genetic diversity of parasite antigens and immune responses to the parasite antigens in vitro.
1.4 Aim and Objectives
1.4.1 Aim of the study
This study evaluated the genetic diversity of P. falciparum and antibody responses to erythrocyte surface antigens among children in Minna, Nigeria.
1.4.2 Objectives of the study
The objectives of this study are to determine the:
i. prevalence of P. falciparum among children in Minna ii. haematological indices for malaria induced anaemia
iii. specific immunoglobulin IgG levels among P. falciparum infected children in Minna
iv. immunological indices involved in malaria- induced anaemia
v. molecular characterization of P. falciparum field isolates from Minna vi. estimate of multiplicity of infection of the alleles of P. falciparum
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GENETIC DIVERSITY OF PLASMODIUM FALCIPARUM AND ANTIBODY RESPONSES TO ERYTHROCYTE SURFACE ANTIGENS AMONG CHILDREN IN MINNA, NIGERIA
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