PROJECT TOPICS ON PREVALENCE OF PLASMODIUM SPECIES AND CO-INFECTION WITH EPSTEIN-BARR VIRUS AMONG SELECTED PATIENTS ATTENDING ABUTH, ZARIA-NIGERIA

ABSTRACT

Coinfection between Plasmodium species and Epstein-Barr virus (EBV) has been linked to many EBV-related cancers, especially in regions where malaria is endemic. The aim of this study was to determine the prevalence of Plasmodiumspeciesand co-infection with EBV among selected patients attending Ahmadu Bello University Teaching Hospital, Zaria-Nigeria. While microscopy was used in the detection and quantification of Plasmodium species, the enzyme-linked immunosorbent assay was used for the detection of IgG antibodies to EBVNA-1 and quantification of the selected patients‘ immune status ratio (ISR) to EBVNA-1. A total of 279 patients were enrolled in this study, out of which 44.8% (125/279) were male and 55.2% (154/279) were female. Based on their clinical presentations, the selected patients, were separated into three study groups-Febrile (58.8%:164/279), Cancer(27.2%:76/279) and the Sickle-cell group(14.0%:39/279). The only Plasmodium species found among the study population was P. falciparum with a prevalence of 63.4%. The highest prevalence of falciparum malaria was found among the cancer group(89.5%: 68/76) while the least was found among the febrile group(48.2%: 79/164). The female patients had higher falciparum parasitaemia (67.5%: 104/154) compared to their male counterparts (58.4%: 73/125). Malaria infection was highest among the 61-70age-group (100%; 4/4) and least was among the 21-30 age-group (52.8%; 28/53) (P<0.05). Also, a higher parasitaemia was found among the divorced (83.3%: 5/6) compared to the married (54.6%: 89/163) (P>0.05). Those who had tertiary education had lowerfalciparum malaria (41.2%: 39/94) compared to those who have had only primary education (87.5%: 56/64)(P<0.05). All the patients had IgG antibodies to EBVNA-1. However, the highest ISRwas found among cancer patientswho also had malaria(1.34; 68/76) while the least ISR was found among malaria-negative febrile patients (0.59; 85/164) (P<0.05; OR=7.08). Sixty-three point four percent (63.4%) of the study population were co-infected with EBV and P. falciparum. Thefactorsfound in this study to predispose to EBV-related cancers wereexposure to X-rays (59.86%: 167/279)(P<0.05) and a family history of cancer (15.4%; 43/279)(P<0.05). There was no significant statistical association between genotype and blood group of study patientsand infection withP. falciparum malaria (P>0.05). Malaria is still endemic in Zariaand P. falciparumis the most prevalent species (63.4%). More research still needs to be done in the ongoing fight to roll back malaria, especially asfalciparummalaria in co-infection with EBV has been shown to increase ISR levels, a risk factor for EBV-related cancers.

CHAPTER ONE

     INTRODUCTION

 

Infectious agents are important contributors to the global cancer burden particularly in Africa. Of the 12.7 million new cancer cases diagnosed worldwide in 2008, about two million were attributable to infectious agents, of which 1.6 million occurred in less developed regions of the world (Odutola, et al., 2016). In its 2009 review of the carcinogenicity of infectious agents, the International Agency for Research on Cancer (IARC) found sufficient evidence to conclude that seven viruses: human papillomavirus (HPV), Epstein–Barr virus (EBV), hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus type-1 (HIV-1), Kaposi‘s sarcoma herpes virus (KSHV), and human T-cell leukemia virus type-1 (HTLV-1), were carcinogenic to humans (Odutola, et al., 2016).

In March, 1964 the Lancet Medical Journal published a remarkable piece of research from three scientists called Anthony Epstein, Yvonne Barr, and Burt Achong. They had discovered the first human virus that is the etiology of cancer, and the virus was named after them: Epstein-Barr virus, EBV (Smith, 2014). Owing to Burkitt‘s intrepid studies in Africa and Epstein‘s persistence, we now know that EBV infection plays a role in diverse types of cancer, including lymphomas, nasopharyngeal cancers and some stomach cancers (Smith, 2014).

Epstein-Barr virus was the first human virus to be directly implicated in carcinogenesis (Thompson and Kurzrock, 2004). It infects >90% of the world‘s population (Thompson and Kurzrock, 2004). Although most humans coexist with the virus without serious sequelae, a small proportion progress to develop tumors (Thompson and Kurzrock, 2004). Normal host populations can have vastly different susceptibility to EBV-related tumors as demonstrated by geographical and immunological variations in the prevalence of these cancers (Thompson and

Kurzrock, 2004). The presence of this virus has also been associated with epithelial malignancies arising in the gastric region and the breast, although some of this work remains in dispute(Thompson and Kurzrock, 2004). Epstein-Barr virus uses its viral proteins, the actions of which mimic several growth factors, transcription factors, and anti- apoptotic factors, to usurp control of the cellular pathways that regulate diverse homeostatic cellular functions (Thompson and Kurzrock, 2004).

The Epstein-Barr virus (EBV) is classically associated with malignancies such as Burkitt’s lymphoma, B-cell lymphoproliferative syndromes, nasopharyngeal carcinoma, Hodgkin’s disease, T-cell lymphomas, and gastric carcinoma, as well as being the causal agent for infectious mononucleosis (Pagano, 1999). The relation of the virus to the malignancies varies from primary etiologic agent to necessary or contributory cofactor (Pagano, 1999). Most of the malignancies occur after years of viral dormancy and are accompanied or triggered by viral reactivation, in contrast to infectious mononucleosis, which results from primary infection with EBV (Pagano, 1999).

The Epstein–Barr virus also called human herpesvirus 4 (HHV-4), is one of eight viruses in the herpesviridae family, and is one of the most common viruses in humans. It is best known as the cause of infectious mononucleosis (glandular fever)(Cherry-Peppers et al., 2003; Maeda et al., 2009). It is also associated with particular forms of cancer, such as Hodgkin’s lymphoma and conditions associated with human immunodeficiency virus (HIV), such as hairy leukoplakia and central nervous systemlymphomas (Cherry-Peppers et al., 2003; Maeda et al., 2009).

The EBV is approximately 122 nmto 180 nm in diameter and is composed of a double helix DNA of about 172,000 base pairs coding for about 85 genes (Amon, 2004). The DNA is surrounded by a protein nucleocapsid. This nucleocapsid is surrounded by a tegument made of protein, which in turn is surrounded by an envelope containing both lipids and surface projections of glycoproteins which are essential forinfection of the host cell (Odumade and Hogquist, 2011).

Burkitt‘s lymphoma, a childhood cancer common in parts of sub-Saharan Africa, has been associated with EBV and malaria (Mutalima et al., 2008). A protein produced by the malaria parasite can trigger an aggressive and torturous form of cancer called Burkitt’s lymphoma(Khamsi, 2007).It was found that the cysteine-rich inter-domain region 1α (CIDR1α) of thePlasmodium falciparum membrane protein 1,is a polyclonal B cell activator that preferentially activates the memory compartment where EBV is known to persist (Chêne et al., 2007).

It has been shown that CIDR1α binds to the EBV-positive B cell line and increases the number of cells switching to the viral lytic cycle as measured by green fluorescent protein (GFP) expression driven by a lytic promoter (Chêne et al., 2007). The virus replication in CIDR1α-exposed cultures was directly proportional to the number of GFP-positive cells (lytic EBV) and to the increased expression of the EBV lytic promoter BZLF1 (Chêne et al., 2007).

Furthermore, CIDR1α stimulated the replication of EBV in peripheral blood mononuclear cells derived from healthy donors and children with Burkitt‘s lymphoma (Chêne et al., 2007). Also,P. falciparum antigens such as CIDR1α can directly induce EBV reactivation during malaria infection, increasing the risk of Burkitt‘s lymphoma development for children living in malaria-endemic areas (Chêne et al., 2007).The Burkitt‘s lymphoma illness causes horrendous suffering. The immune cells turn cancerous and tumours develop in the lymph nodes, often making the tongue

swell and cheeks bulge (Khamsi, 2007). The tumour is characterized by chromosome translocations (Manolov and Manolova, 1972). Usually, the terminal portion of chromosome 8 containing the c-myc gene is translocated to chromosome 14 (Taub, 1982), near the enhancer of the immunoglobulin heavy-chain locus (Hayday, 1984).

It has been discovered that attack byPlasmodium falciparum malaria results in alteration of the T-cell subpopulations so that, in vitro, B lymphocytes infected with EBV proliferate abnormally to secrete large amounts of immunoglobulin and antibody. This phenomenon offers some explanation for the increased incidence of Burkitt’s tumor and the high levels of immunoglobulin found in people living in areas where P. falciparum malaria is common (Greenwood, 1974).

Malaria is caused by parasites that are spread by mosquitoes. The Anopheles mosquitoes transmit the malaria parasites that cause malaria in humans(Jimoh et al., 2007). In adults, its common symptoms are headache, weakness, fever, aches and pains, high body temperatures and bitterness of the mouth (loss of appetite) while in children, in addition to the already mentioned symptoms, it may also manifest in more than normal sleeping, nausea and vomiting(Jimoh et al., 2007). It is a serious disease affecting children and adults but its consequences are graver among children and pregnant women (Jimoh et al., 2007).

Malaria is endemic in Nigeria and is a leading cause of morbidity and mortality in the country. The disease accounts for 25% infant mortality and 30% of childhood mortality in Nigeria alone(Jimoh et al., 2007). Nigeria can be divided into three major malaria epidemiological zones, namely, forest, savannah and the grass-land zones(Jimoh et al., 2007). The forest zone consists of coastal areas stretching from Lagos in the South-Western Nigeria to the forest areas in the Eastern Nigeria, up to the Northern portion of the forest zone of Oyo state. The Savannah zone

consist of areas north of Oyo state to the central areas of Kogi and Benue states, while the Grass-land zone consists of the Nothern parts of Nigeria (Jimoh et al., 2007).

The most dominanat species of Anopheles mosquito in Nigeria are Anopheles funestus, A. gambiae, A. complex, A. arabiensis, and A. melas. The dominant vector in the forest zone is the A. melas while the dominant vectors in the Savannah zone are a combination of A. melas and A. arabiensis (Jimoh et al., 2007). The dominant vector in the Grass-land zone is A. arabiensis. The most deadly strain of malaria, Plasmodium falciparum has been reported in all zones and other parts of Africa (Jimoh et al., 2007; WHO, 2016).

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It is widely accepted that the sickle cell trait protects the carrier from malaria because only a small number of P. falciparum are able to infect sickled cells due to their shapes and increased rates of phagocytosis of infected sickled cells (Lamonte et al., 2012). It can therefore be implied that an inverse association of Burkitt‘s lymphoma with the sickle-cell trait (AS haemoglobin) would provide strong evidence for the role of intense falciparum malariain the increased incidence of EBV-related tumors (Morrow, 1985).

PROJECT TOPICS ON PREVALENCE OF PLASMODIUM SPECIES AND CO-INFECTION WITH EPSTEIN-BARR VIRUS AMONG SELECTED PATIENTS ATTENDING ABUTH, ZARIA-NIGERIA

1.1         Statement of the Research Problem

Epstein-Barr virus, a ubiquitous B-lymphotrophic herpesvirus (Hsu and Glaser, 2000) is an accepted carcinogen, since experimental studies have clearly established its presence in tumour cells of several malignancies (Gulfaraz and Muhammad, 2014). Some of these malignancies include Burkitt‘s lymphoma, lymphomas associated with immunosuppression, Hodgkin‘s disease, other non-Hodgkin‘s lymphomas, nasopharyngeal carcinoma, gastric adenocarcinoma, lymphoepithelioma-like carcinomas and immunodeficiency-related leiomyosarcoma (Hsu and Glaser, 2000).Cancer is one of the leading causes of death worldwide and research focused on

understanding itsaetiology and pathogenesis is a major challenge. Over a period of 20 years, global mortality from cancer has increased from 5.779 million in 1990 to 7.978 million in 2010. This represents an increase of approximately 2% per year (Gulfaraz and Muhammad, 2014). It has also been estimated that oncogenic viruses play an etiological role in the development of 12% of all human malignancies (Gulfaraz and Muhammad, 2014).

Epstein-Barr virus has continued to attract considerable attention, primarily due to its oncogenic properties and its association with a number of malignancies including post-transplant lymphoproliferative disease (PTLD) (Gulfaraz and Muhammad, 2014).About 80% of PTLD cases have been attributed to EBV (Gulfaraz and Muhammad, 2014).Some published researches have estimated that approximately 1.5% of transplant recipients develop PTLD while 50% of these die within the first year (Gulfaraz and Muhammad, 2014). It has been estimated that the risk of developing PTLD and dying from it was the same for both sexes (Gulfaraz and Muhammad, 2014).For the age-group of 0-14 years, it was estimated that mortality of 90.5%, 33.3% and 15.2% were found in regions where Burkitt‘s lymphoma is endemic, intermediate or sporadic respectively (Gulfaraz and Muhammad, 2014). Burkitt‘s lymphoma is the fastest growing human tumour (Robbiani et al., 2015). The annual incidence has been estimated at 40-50 per million children younger than 18 years (Robbiani et al., 2015).A vast majority of the world‘s population exhibits antibodies to EBV and the infection usually occurs early in childhood (Thompson and Kurzrock, 2004).

Malaria is the number one killer of all parasitic diseases and more than 80% of the deaths worldwide occur in sub-Saharan Africa (Brooks et al., 2010). According to the World Health Organization, in 2015, there were about 214 million cases of malaria. The disease caused around 438,000 deaths of which 306,000 were of children under 5 years (WHO, 2015)and about 3.2

billion people, almost half of the world‘s population are at risk of malaria (WHO, 2016). The Centre for Disease Control further reported malaria to be the number one cause of death in Nigeria (CDC Nigeria, 2015). The majority of these deaths are due to Plasmodium falciparum malaria (Targett and Greenwood, 2008).Understandably, the focus of enhanced research and malaria control has been primarily on Plasmodium falciparum malaria, given the mortality and severity of disease associated with this species (Targett and Greenwood, 2008).

A co-infection with Plasmodiumfalciparummalaria and EBV is a major risk factor for endemic Burkitt lymphoma (eBL), one of the most prevalent paediatric cancers in Equatorial Africa (Chattopadhyayet al., 2012). A coinfection with malaria parasite and EBV can increase the mortality of malaria patients. Children in sub-Saharan Africa become infected with EBV in infancy and it is quite difficult to distinguish from other mild childhood illnesses. Almost all African children are infected with EBV by the age of 6-12 months(FNIL, 2016). Since both of these infections occur around the same time in these children, many are at high risk for co-infection before the age of 1 (FNIL, 2016).

Finally, the chance of any individual developing an EBV-related lymphoma is modulated by ethnic, geographic (areas with holoendemic malaria), genetic, immunologic and other infectious cofactors (Kanakry and Ambinder, 2014).Furthermore, individuals said to carry the sickle cell trait, were in fact highly protected against malaria, thus explaining the high prevalence of this mutation in geographical areas where malaria is endemic (Instituto Gulbenkian de Ciencia, 2011). These findings lead to the widespread belief in the medical community that understanding the mechanism whereby sickle cell trait protects against malaria would provide critical insight into developing treatment or a possible cure for this devastating disease, responsible for over a million premature deaths in sub-Saharan Africa (Instituto Gulbenkian de Ciencia, 2011).

1.2         Justification of the study

This study will determine the prevalence of Plasmodium species, as well as the parasite density in the study area. Current information on the prevalence of EBV and the ISR of patientsto EBVNA-1in the study area will be provided. Since EBVNA-1 is an established tumor marker (a substance that is produced by cancer or by other cells of the body in response to cancer or certain benign conditions) the presence of measureable antibodies to it can provide data on possible risk for carcinogenesis. A corresponding data on the complications associated with a co-infection of EBV and Plasmodium falciparum,especially that predisposing to cancer will be brought to fore. If other Plasmodium species asideP. falciparumare present in the study area, an association between them and EBV will be determined with the hope of providing current information. Furthermore, the study will determine the protective role of the sickle-cell trait against malaria which has been a subject of much speculation and debate among researchers. More so, the likely risk factors associated with EBV-related cancers as well as EBV and malariaco-infection will be determined among patients in the study area. Finally data generated from this research will be useful in both preventive and control measures as well as in the treatment of EBV, malaria and EBV-related cancers.

1.3          Aim of the Study

The aim ofthe study was to determine the prevalence of Plasmodiumspecies and co-infection with Epstein-Barr virusamong selected patients attending Ahmadu Bello University Teaching Hospital, Shika-Zaria, Nigeria.

1.4          Objectives of the Study

The objectives of the study were to:

1. Detect the presence of Plasmodium species and their density among the selected patients using light microscope and the atlas of Medical Parasitology.
2. Screenfor anti-EBVNA-1 (IgG) and measure the Immune Status Ratio (ISR) of the selectedpatientsto EBVNA-1usingELISA.
3. Determine co-infection of Epstein-Barr virus and Plasmodium species among the selected patients.
4. Determine likely risk factors for EBV-related cancersand co-infection with EBVand Plasmodium species.

See Also :PREVALENCE OF SCHISTOSOMIASIS AMONG PRIMARY SCHOOL CHILDREN IN DAKACE DISTRICT, ZARIA LOCAL GOVERNMENT AREA, KADUNA STATE, NIGERIA