ABSTRACT
The study was conducted to evaluate the correlation of malaria parasite with ABO blood group and Rhesus factor in Nsukka and Enugu Metropolis. Three hundred blood samples were collected from Hansa Clinics, UNN Medical Center and UNTH, Enugu in which 169 were males and 131 were females between the ages of 1- 75 years. Full blood count was done using automated Homecue machine, Malaria parasite density was done using manual tally machine, blood grouping and genotyping was done using the tube method and cellulose acetate electrophoresis method respectively. The malaria positive samples were subjected to nested PCR for confirmation of P.falciparum. The result were analyzed using SPSS and P-value were used to determine significance. A total of 300 samples were included in this study out of which 19% were A+, 13.3% were B+,  0.3% were B-,  2% were AB+,  63.1% were O+, 2.3% were O-, Rhesus(+) were 97.3%, Rhesus(-) were 2.7%, 64.3% were AA and 35.7% were AS. Among the population sampled, 56% were males and 44% were females. Malaria affected all age groups and the age ranged from 1 to 75 years. The results show that age 6 -10 years has the highest mean parasite density, followed by 1-5years, 71-75 years and the least was recorded in age 36-40 years, 46-50 years and 40-45years. There were significant difference among the age groups (P<0.005).  The blood group AB has the highest mean PCV and RBC and lower mean parasite density ( 538±264.6) while the blood group B has the lowest PCV and RBC and highest mean parasite density (674.3±391.6), followed by blood group O (671.5±620.9). The blood group A has the lowest mean HB and highest platelet count.  There was no significant difference with the blood parameters and the parasite density across the various blood groups. Higher parasite density was observed among the females than the males, but there were no significant difference in the parasite density of male and female of various blood groups (P>0.005). No significant difference (P>0.005) were also seen regarding genotype and the blood parameters with AS having a higher parasite density (677±487) while AA has mean parasite density of 652±570. The Rhesus positive has mean parasite density of 668±551 and Rhesus negative has a mean parasite density of 463±326. There was no significant difference (P>0.005). There also was no correlation between malaria parasite, ABO and Rhesus factor while such relationship exists among the blood parameters and parasite density. There was a strong correlation where the HB, Platelets count and RBC decreases as parasite density increases, HB (P<0.005), RBC (P<0.005) and Platelets (P<0.005). The nested PCR carried out on positive blood samples revealed that out of 12 positive samples, OL1 – OL7 were positive to Malaria and PfEMP1. The blood groups are stated as A+, O+, O+ O+ O+ O+and B+. The study also supports that any of the ABO blood groups are at equal chances of malaria infection.
CHAPTER ONE
INTRODUCTION
1.0
1.1 Background of the study
Malaria is caused by an obligate, intercellular protozoan parasite of the genus Plasmodium. Of the four species that infect humans (Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae), P. falciparum is responsible for high mortality (Pathirana et al.,
2005).
Malaria is a very important disease in Sub-Saharan Africa with high morbidity and mortality rates (WHO, 1994), which consequently leads to a dwindle in the productivity of a nation (WHO, 1994). The most common individuals at high risk of malaria infection in endemic areas are people of low immunity, for instance, foreigners, pregnant women, children and perhaps HIV/AIDS patients (Migot et al., 1996).
Malaria is endemic throughout Nigeria (WHO, 2005) and the country is one of Africa’s hardest- hit, accounting for between 30 and 40 percent of malaria deaths on the continent. This magnitude of occurrence in this part of the world correlates with poverty, ignorance and social deprivations in the community (WHO, 2009).
In Nigeria, statistics show that malaria accounts for 25% of under-five mortality, 30% of childhood mortality and 11% of maternal mortality (Murphy et al., 2001). It also aggravates anemia and malnutrition in children and pregnant women (Murphy et al., 2001). All Nigerians are at risk of
malaria and the problem is compounded by the increasing resistance of malaria to hitherto cost- effective antimalaria drugs. It takes heavy tolls on pregnant women, in form of pre-term birth, intrauterine foetal death, miscarriages, maternal hypoglycaemia, cerebral oedema and maternal death (WHO, 2007). Prevalence of direct or indirect maternal death traceable to malaria in Nigeria has been reported as 10% in Calabar, Enugu 8%, 7.8% in Lagos and 8.2% in Kano (Gajida et al., 2010).
It has also been evidenced that the human plasmodium parasite can continue to exist in stored and frozen blood (Talib and Khurana, 1995), which makes malaria transmission by blood transfusion even more dangerous and of greater concern.
The WHO reports that, there are about 300-500 million incidences of malaria causing 2-3 million deaths each year in the tropical and subtropical regions of the world. About 90% of these deaths occur in Sub-Saharan Africa (WHO, 2003). This could be as a result of P. falciparum which is the most dangerous of the four parasites being the main cause of infections in this region and also the main malaria transmitting vector (Anopheles gambiae) being spread widely and very difficult to control (WHO, 2003).
The reasons for the limited success in efforts to eradicate malaria, a disease of poverty in Nigeria include lack of political will and commitment, low awareness of the magnitude of malaria problem, poor health practices by individuals and communities and resistance to drugs (Yusuf, 2007). Regardless of the soaring malaria incidence in endemic regions, a certain group of individuals seem to have more immunity to malaria than others. This could be accounted by several factors including haemoglobin variants, ABO blood group system, Rhesus factor and enzyme action, among others ( Chandramohan and Greenwood, 1998; Otajevwo, 2013). There is increasing evidence that P. falciparum malaria is influenced by ABO blood type of an individual but the
extent of association is not fully established (Ilozumba and Uzozie, 2009). Some investigators have expressed the opinion that genotype is a factor in susceptibility to Plasmodium species infection in humans (Hill et al., 1992; Ademowo et al., 1995; Omotade et al., 1999) but there is lack of consensus on possible association between ABO blood group genes and malaria parasitaemia (Hill et al., 1992; Ademowo et al., 1995; Fischer and Boone, 1998). A number of studies have been conducted to investigate the association between ABO blood group system and some disease conditions (Ndambaa et al., 1997; Blackwell et al., 2002 Tursen et al., 2005; Opera, 2007; Abdulazeeez et al., 2008). Some of these studies reported significant associations, suggesting that ABO blood groups have an impact on infection status of the individuals possessing a particular ABO blood type ( Ndambaa et al., 1997; Blackwell et al., 2002; Opera, 2007; Abdulazeez et al.,
2008). Variations in reports on the association of ABO blood groups and disease progression of P. falciparum malaria show the complexity of the interaction between the parasite and host immune responses (Miller et al., 1997).
Understanding the nature of the relationship between ABO blood groups and P. falciparum infections would lead to development of control strategies with a definite target group within the population hence reduce malaria transmission.
1.2 STATEMENT OF PROBLEM
There have been a very big problem trying to establish a relationship between malaria infection, Blood group and Rhesus factor following various results from different authors, this study will be helpful in elucidating any such relationship.
1.3 AIM
The aim of the study is to evaluate the co-relationship of malaria infection with ABO blood group and Rhesus factor.
1.4 OBJECTIVES OF THE RESEARCH
The specific objectives include;
ï‚· To determine the sex distribution as related to malarial infection.
ï‚· To determine if there is any significant association of the ABO blood types with malaria.
ï‚· To determine if there is any significant association of Rhesus blood grouping with malaria.
1.5 HYPOTHESES
HO – There is no relationship between malaria, ABO and RH blood group system. H1 – There is a relationship between malaria, ABO and RH blood group.
LITERATURE REVIEW
1.6
1.6.1 The ABO system
ABO and Rhesus factor are the most important blood group systems in medicine and transplantation immunology which differ by the presence or absence of antigens on red blood cells (RBCs) and antibodies in the blood plasma.
The most important human blood group system for transfusion or transplantation is the ABO system. Every human being is either of blood group O, A, B or AB, or of one of the minor variants of these four groups. In fact there are only two determinants in the ABO system: A and B. O is the absence of either A or B, and AB is the presence of both A and B on the red cells (Your blood,
2006). Blood groups are groups of antigens that are located on the red blood cell membranes and are coded by alleles at different loci on a chromosome. Although, about 400 blood grouping antigens have been reported, ABO and Rh are the most important. The ABO system derives its importance from the fact that A and B are strongly antigenic and anti A and anti B occur naturally in the serum of persons lacking the corresponding antigen, these antibodies being capable of producing hemolysis in vivo. Individuals are divided into four major blood groups: A, B, AB and O, depending on the antigens present on RBC (Pramanik and Pramanik, 2000; Conteras and Lubenko, 2001). An individual of type A blood group raises anti-B antibodies against B-blood group RBCs if transfused with blood from B group, with resultant lysis of the RBCs. This is due to the presence of isoantibodies against non-self blood group antigen. The same happens for B and O blood groups. AB does not have an anti-A and anti-B isoantibodies because A and B antigens are present on the RBC and are both self antigens.
The ABO blood groups are most important in transfusion because very early in life everybody who lacks either A or B develops antibodies against the A and B substances they lack. So, group O individuals develop anti-A and anti-B, group A develops anti-B, and group B develops anti-A. The very small proportion of group AB people has neither antibody. Thus, for example group A blood is dangerous if given to a group O individual because the anti-A in the plasma of the group O individual will rapidly destroy the transfused group A red cells. As well as on blood cells, ABO blood groups are also present on other tissues and, unless special precautions are taken, a group A kidney transplanted to a group O patient will be rapidly rejected.
The distribution of the ABO groups in England, Wales and Northern Ireland is about 40% A, 46% O, 11% B and 4% AB, though this will vary slightly in different parts of the country (Bakare et al., 2006).
The distribution, however, differs significantly around the world, with higher levels of B in Asia and more O in Africa and in Native Americans and Australians. In addition, in Nigeria, among
7653 individuals in Ogbomoso, Oyo State, 50% had type O, type A, 22.9%; type B, 21.3% and type AB, 5.9% (Bakare et al., 2006).
Also, in Benin, Niger-Delta region, Nigeria, blood group distribution among 160,431 individuals showed phenotypes A, B, AB and O as 23.72, 20.09, 2.97 and 53.22%, respectively (Enosolease and Bazuaye, 2008).
How the ABO blood groups evolved is not known. There is an argument that blood group A came before O in evolution, yet O is more common than A in many populations of the world. One possible explanation for this, which is supported by substantial evidence, is that group O people
are marginally more resistant to malaria than group A people, giving group O a slight advantage in area where malaria is endemic. In fact, there is a geographical trend for group O to be more common than A in those countries where malaria is common.
1.6.2 RHESUS BLOOD GROUP (Rh)
The Rhesus factor is proteins found on the covering of red blood cells. If the Rhesus factor protein is present on the cells, the person is Rhesus positive. If there is no Rhesus factor protein, the person is Rhesus negative. Rhesus factors are genetically determined.
Rhesus factor are the second most important blood group system in transfusion, though of little significance in transplantation; It is called Rhesus, because it was originally believed to be similar to blood groups in rhesus monkeys, but now simply called Rh. In the UK about 83% of people are Rh-positive and 17% are Rh-negative (NHS Blood and Transplant Active Donor Base 2013/14). However, the latest statistics of blood issued to hospitals across England and Wales is as follows; positive 77%, Rh-negative 23% to the nearest %.
The human RBCs that contain antigen D are known as rhesus positive (Rh+), while those without antigen D in their RBCs are rhesus negative (Rh¯) (Conteras and Lubenko, 2001; Knowles and Poole, 2002). The D-antigen is immunogenic and induces an immune response in 80% of D- negative individuals when transfused with 200 ml of D-positive blood (Mollison et al.,
1997). This also occurs in pregnancy resulting in hemolytic disease of the newborn, HDN (Knowles and Poole, 2002). Rhesus incompatibility is a condition that occurs during pregnancy, if the woman has Rh-negative blood and her baby has Rh-positive blood. Rhesus is particularly important in pregnancy because when an Rh-negative woman gives birth to an Rh-positive baby
she may develop antibodies that can harm Rh-positive babies in future pregnancies (Avert and Reid, 2000). Paradoxically, injecting these mothers with antibodies during and after the pregnancy prevents them from making the potentially harmful antibodies. In the Far East, Rh-negative is rare and so there are very few problems associated with pregnancy and the Rh blood group.
The Rhesus blood group system has around 50 different red blood cell antigens. D is the most important antigen of the Rhesus system. It is also known as Rhesus (D) or Rhesus factor or RH1. The percentage of Rhesus negative people varies in different countries, e.g. less than 5% of India’s population is Rhesus negative. Moreover, Rh-positive is documented as 95% in African- Americans, 97% in Africans whereas Rhesus negative is 5.5% in South India, 5% in Nairobi, 7.3% in Lahore, 4.8% in Nigeria (Mwangi, 1999; Omotade et al., 1999)
1.6.3 PREVALENCE OF MALARIA
Malaria is a disease that is associated with poverty due to poor sanitary and environmental conditions. The rich and powerful live in sanitary surroundings with easy access to medical facilities, while the poor live in crowded urban slums and remote rural areas which favor transmission. In 2006, the World Health Organization (WHO) estimated that 3.3 billion persons were at risk of acquiring malaria (WHO, 2006).
Of these, 247 million were infected (86% in Africa) and nearly 1 million (mostly African children) die of the infection. In 2008, malaria was still endemic in 109 countries worldwide, and 45 of the countries are in Africa. WHO estimated that approximately 1.1 million persons were still dying of malaria (WHO, 2008). Some 11 percent of all child deaths worldwide are estimated to occur in Nigeria.
Malaria is the leading cause of child death in the country and around 250,000 Nigerian children die every year from the disease. While children under the age of five and pregnant women are particularly vulnerable, almost the entire population of Nigeria is at risk of contracting malaria (WHO, 2008). The disease is strongly resurgent owing to the effects of climate change, war, large scale population movements, increased breeding opportunities for the vector mosquitoes, rapidly spreading drug and insecticides resistance as well as neglect of public health infrastructure (Martens, 2000; Wellems and Miller, 2003). Martens, 2000; Wellems and Miller, (2003) further stated that malaria is currently endemic in over 100 countries which are visited by over 125 million international travelers each year. WHO (2011) showed that malaria is endemic in 106 countries where it leads to an estimated 216 million cases per year and 655,000 deaths, majority of which are in Sub-Saharan Africa.
Malaria is a public health problem in Nigeria where it accounts for more cases and death than any other country in the world. It is risk for 97% of Nigeria’s population. The remaining 3% of the population live in the malaria free highlands. There are estimated 100 million cases with over 300,000 deaths per year in Nigeria. This compares with 215,000 deaths per year in Nigeria from HIV/AIDS. Malaria contributes up to 11% of maternal mortality. It accounts for 60% of outpatient’s visits and 30% of hospitalizations among children under five years of age in Nigeria. Malaria has greatest prevalence close to 50% in children age 6 – 59 months in the South West, North Central and North West regions. Malaria has the least prevalence, 27.6% in children 6 – 59 months in the South East region (Malaria Fact Sheet, 2011). Malaria contributes greatly to the increase in hospital attendance across the six geo political zones of Nigeria. World malaria report indicated that Nigeria accounted for a quarter of all malaria cases in the 45 malaria endemic countries in Africa, showing clearly the challenges of malaria in Nigeria (WHO, 2008).
Studies carried out to determine the prevalence of malaria parasite infection among 19 infants and children (0 – 12 years) in South west Nigeria shows an overall prevalence rate of 89.5%. Age group (0 – 5years) had the highest frequency rate of 84.7% with mean parasite density of 900. Children of illiterates from suburb villages had the highest mean parasite density of 850 with 78.1% prevalence rate (Olasehinde et al., 2010). Onyido et al. (2011) obtained a prevalence of 70.8% for malaria parasite in Anambra State, Nigeria. Abdullahi et al. (2009) recorded prevalence of 27.29% for malaria parasite in Sokoto State, Nigeria.
In a study to determine the prevalence of malaria infections among children aged six months to eleven months in a tertiary institution in Benin City, Okafor and Oko-Ose (2012) recorded the highest prevalence of 58.57% between ages 6 months and 2 years when compared with the other age groups with 41.43%. Olasehinde et al. (2010) recorded a prevalence rate of 80.5% among infants and children (0-12 years) in south western Nigeria. Houmsou et al. (2011) recorded a malaria prevalence of 39.5% among children attending hospital in a semi urban hospital in central Nigeria.
1.6.3.1 LIFE CYCLE OF P. falciparum
The life cycle of the malaria parasite is complex. Figure 1.1 is a flow chart showing the life cycle of P. falciparum. Understanding the complete life cycle is important in knowing the pathogenicity of P. falciparum infections (Sherman and Irwin, 1998). Human and other vertebrates are the secondary hosts while female Anopheles mosquitoes are the definitive hosts for the malaria parasites and therefore act as transmission vectors. The mosquitoes first ingest the malaria parasite by feeding on an infected human carrier and the infected Anopheles mosquitoes carry Plasmodium
sporozoites in their salivary glands. A mosquito becomes infected when it takes a blood meal from an infected human.
Once ingested, the parasite gametocytes taken up in the blood further differentiate into male or female gametes and then fuse in the mosquito’s gut (Billker et al., 1998). This produces an ookinete that penetrates the gut lining and produces an oocyst in the gut wall. When the oocyst ruptures, it releases sporozoites that migrate to the salivary glands, where they are ready to infect a new human host (Bledsoe, 2005).
The sporozoites are injected into the skin of a new host, alongside saliva, when the mosquito takes a subsequent blood meal.
1.6.3.2. PATHOGENESIS OF P.FALCIPARUM MALARIA
In Plasmodium falciparum infections, the interaction between the erythrocyte, the host’s immune system and the parasite is central to the pathogenesis of severe malaria and results in mechanical and rheological changes to the infected erythrocyte (Hommel, 1993). The membrane of infected cell becomes rigid and results in knob protrusions, cytoadherence and rosette formation. The release of malaria antigens, pigment and toxins gives rise to a cascade of pathological events such as production of cytokines, particularly tumor necrosis factor (TNF), induced by the release of parasite products during schizont rupture. TNF or cachexin has been implicated as the cause of malarial fever. Merozoites are released into blood circulation at the time of schizont rupture, thus beginning the erythrocytic stage of the life cycle (Miller et al., 1994; Pasvol and Hogh, 1995). Within the red blood cells, the parasites multiply further asexually, periodically breaking out of their hosts to invade fresh red blood cells. Parasitaemia levels are greatest in the brain, heart, liver, lung, kidney and blood (Warrell, 1993).
This material content is developed to serve as a GUIDE for students to conduct academic research
EVALUATION OF CO-RELATIONSHIP OF MALARIA INFECTION WITH ABO BLOOD GROUP AND RHESUS FACTOR IN SOME LOCATIONS IN ENUGU AND NSUKKA>
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