Single nucleotide polymorphisms (SNPs) resolve the smallest degree of genetic difference between individuals and in the post-genomic era are increasingly being used to identify genetic markers for complex disease traits (Cargill M et al). SNPs at the TNF can be identified to be associated with the pathogenesis of Plasmodium falciparum.
A large body of clinical and epidemiological evidence has also been accumulated which clearly demonstrates that host iron deficiency is protective against falciparum malaria and host iron supplementation may increase the risk of malaria (Tsytsykova et al 2003). Furthermore, the wisdom of universal iron supplementation campaigns in malaria-endemic regions has recently been questioned due to clinical evidence that suggests iron deficiency protects against malaria and iron supplementation of women and children may increase the incidence of malaria when given without malaria prophylaxis or access to adequate health care (Zlotkin et al 2013). Thus, it is important to understand the significance of host iron level in regard to the severity of falciparum malaria.
This study seeks to:
Explore the genetic diversity and SNPs that occur in the TNF of Plasmodium falciparum patients.
Epidemiological data that describes the relationship between host iron status and malaria infection will also be analyzed.
The serum iron level in the blood will be measured by complete blood count (CBC) after 12 hours fast, in falciparum malaria patients and controls. DNA is extracted from patients, the TNF gene is detected and primers are designated using primer3 software.
It is expected that there will be some SNPs in the TNF in patients with falciparum malaria which may be associated with the severity of the disease. It is also expected that there will be low iron levels in the blood serum of patients with the disease as compared to controls. Lower iron levels even further expected in patients who have been infected for a longer time.
Introduction
Background Information
P. falciparum, is found in tropical and subtropical areas, and especially in Africa where this species predominates. P. falciparum can cause severe malaria because it multiples rapidly in the blood, and can thus cause severe blood loss (anemia). although falciparum is the most severe strain of malaria, the other species that cause malaria include; p.vivax, p.ovale and p.malariae in humans. The WHO estimates that in 2015 there were 214 million new cases of malaria resulting in 438,000 deaths. Others have estimated the number of cases at between 350 and 550 million for falciparum malaria. The majority of cases (65%) occur in children under 15 years old. About 125 million pregnant women are at risk of infection each year; in sub-Saharan Africa. Maternal malaria is associated with up to 200,000 estimated infant deaths yearly. (google scholar). There has been a lot of success in the diagnosis of malaria but this has been neutralized with the development of resistance in the available medicines hence the need for the study.
TNF is a cell signaling protein like cytokines involved in systemic inflammation and is one of the cytokines that make up the acute phase reactions. The human TNF gene is located on chromosome number 6 in a region of a major histocompatibility complex between the class 1 HLA-B and class 2 HLA-DR loci (Wilson AG et al). The TNF gene provides a unique opportunity to study the distribution of SNPs in a genetic regulatory region where critical regions involved in the transcriptional regulation of genes have been well characterized (tsytsykova AV et al). several of the extended haplotypes that contain TNF SNPs specifically -856, -862, and -307 have themselves been associated with differential susceptibility to a variety of autoimmune diseases. (Alper CA et al).
On the other hand, Iron is an essential nutrient for nearly every living organism including humans and the malaria parasite. Iron impacts a broad range of Iron is an essential nutrient for nearly every living organism including humans and the malaria parasite. Iron impacts a broad range of biological processes; including host and parasite cellular function, erythropoiesis and immune function (Martha A Clark et al, 2014). A general consensus was generated that iron deficiency is protective against malaria and iron supplementation increases malaria risk in absence of access to adequate health care(Spottiswood et al, 2012). Iron is essential for the survival of malaria parasites. It multiples 8-32 times in the course of a single intra-erythrocytic proliferation (Rubin et al, 1993).
Problem statement
In Kenya, malaria remains a major cause of morbidity and mortality with more than 70% of the population at risk the disease (MOH 201in Kenya, malaria remains a major cause of morbidity and mortality with more than 70% of the population at risk the disease (MOH 2014). Malaria was predominantly in the lake basin and at the coast of Kenya but it has re-emerged in areas with little or no transmission (Oloo AJ et al; 1996). Although intravenous artesunate for severe malaria and oral arteminism-based therapies are the first-line treatment for all plasmodium species, malaria has continued to cause death. Falciparum malaria has become resistant to artemisinin and partner drugs which increasingly causes treatment failure. Therefore, there is a need to analyze the SNPs of falciparum patients to come up with better ways of eliminating malaria.
Justification
Malaria is a disease that has been there for centuries. Intravenous quinine, usually formulated as a dihydrochloride salt, is currently the most widely used agent in the treatment of severe falciparum malaria.
Objectives
The main objective of the study is to analyze SNPs that occur at TNF and the iron status in falciparum malaria patients.
The specific objectives of this study are:
To explore the genomic diversity and haplotype frequency of SNPs at the TNF and extrapolate possible associations with markers of severity between malaria-infected and healthy controls.
To determine how the level of iron in the blood serum affects the epidemiology of malaria in patients and controls.
To determine the severity of malaria in patients according to their iron level.
Literature review
Malaria.
Plasmodium falciparum is a unicellular protozoan parasite of humans and the deadliest species of plasmodium that cause malaria in humans. It is transmitted to humans through the bite of a female anopheles mosquito. After a bite from the female anopheles mosquito, there is the time that goes by before the first symptoms appear; the incubation period. The incubation period in a most case varies from 7-30 days but is shorter in falciparum malaria. Malaria can be complicated or severe uncomplicated.
Uncomplicated Malaria
The classical but rarely observed malaria attack and lasts 6–10 hours. Infrequently observed, the attacks occur every second day. The patient has the following symptoms: fever, chills, sweats, headaches, nausea and vomiting, and body aches.
In Kenya, malaria is frequent, people often recognize the symptoms as malaria and treat themselves without seeking diagnostic confirmation (“presumptive treatment”).
Severe (complicated)
Severe malaria occurs when infections are complicated by serious organ failures or abnormalities in the patient’s blood or metabolism. The manifestations of severe malaria include the following: first, cerebral malaria, with abnormal behavior, impairment of consciousness, seizures, coma, or other neurologic abnormalities. Second, severe anemia due to hemolysis (destruction of the red blood cells). Third, abnormalities in blood coagulation. Fourth, low blood pressure is caused by cardiovascular collapse. Fifth, acute kidney injury. Sixth is hyperparasitemia, where more than 5% of the red blood cells are infected by malaria parasites. Seventh, metabolic acidosis (excessive acidity in the blood and tissue fluids), often in association with hypoglycemia. Lastly, hypoglycemia (low blood glucose). Hypoglycemia may also occur in pregnant women with uncomplicated malaria, or after treatment with quinine.
Severe malaria is a medical emergency and should be treated urgently and aggressively.
The life cycle of plasmodium falciparum
The life cycle of P. falciparum involves two hosts: the female anopheles’ mosquito and humans. The life cycle of the parasite P. falciparum starts in humans with the inoculation of parasite through mosquitoes. The female anopheles’ mosquito inoculates the sporozoites through a bite. The sporozoites travel through the bloodstream to the liver and then the RBCs.
Exoerythrocytic schizogony
This cycle takes place in the liver. The sporozoites migrate to the liver and develop into schizonts. This period has little or no pathology and it takes approximately 5-6 days for Plasmodium falciparum. The schizonts then burst and each one of them releases approximately 10,000 merozoites (Meaden,2013). The sporozoites invade hepatocytes using a membrane-associated actin-myosin motor with the resulting formation of a Parasitophorous vacuole membrane (PVM) (Aly et al., 2010). The merozoites are contained and transported out of the liver inside merosomes (host-derived vesicles) thus protecting them from phagocytosis by kupffer cells. They are deposited into the blood and the blood stage initiates (Janse et al., 2004).
Erythrocycycticschizogony
The merozoites invade red blood cells and form the ring stage. The mature schizonts then burst to release approximately 16-24 merozoites per Erythrocytic schizont(Bannister and Mitchell, 2003).The merozoites are then released and some invade fresh red blood cells while others differentiate into male or female gametocytes. This is because, during the ring stage, the parasites are committed to either asexual or sexual reproduction (Josling and Llinás, 2015a). This stage occurs in approximately 48 hours for Plasmodium falciparum(Trager and Jensen, 1976).
Figure 1: Illustration of the life cycle of plasmodium falciparum.
Sexual reproduction in the mosquito
The female anopheles mosquito during a blood meal takes up the gametocytes and in its mid-gut the microgamete (male gametocyte) ex-flagellates under the influence of xanthurenic acid to form 8 microgametes. These microgametes fertilize the macrogamete (female) to form a zygote(Janse et al., 1986). The zygotes mature into ookinete which crosses the mid-gut of the mosquito. The ookinete transverses the mid-gut encysts to form oocytes which undergo sporogony to form sporozoites. On bursting the oocyte releases approximately 10,000 sporozoites which invade the salivary glands of mosquitoes ready for injection into the human skin during the next blood meal(Baton and Ranford-cartwright, 2005).
The tumor necrosis factor
The tumor necrosis factor is a cell signaling protein (cytokine) involved in systemic inflammation and is one of the cytokines that make up the acute phase reactions. It is produced chiefly by activated macrophages, although it can be produced by many other cell types such as CD4 lymphocytes, mast cells, natural killer cells, and neurons.
Tumor necrosis factor α (TNFα) is a potent immunomodulatory and proinflammatory cytokine that has been implicated in the pathogenesis of autoimmune and infectious diseases. For example, plasma levels of TNFα are positively correlated with severity and mortality in malaria (Anthony G Wilson, J A Symons, et al, 1997). Homozygosity for TNF2 carries a sevenfold increased risk of death from cerebral malaria. TNF is involved in multiple inflammatory and immune responses and plays an important role in the pathogenesis of many infectious diseases including P. falciparum malaria.
Single Nucleotide Polymorphism gene
SNP is a variation that occurs when one nucleotide of the Adenine, Guanine, Cytosine, or Thymine in a genome is altered. The variations differ between members of the same species. The variations can affect how humans develop diseases and also how they respond to drugs and pathogens (Oeveren and Janssen, 2009).
The association of the severity of malaria with several human genetic factors is well documented (Kwiatkoski D,2005) and the disease has been the selective pressure behind several erythrocytic defects such as sickle cell disease, G6PD deficiency and thalassemia (Tishkoff SA, Varkoryi R, et al,2001). Malaria susceptibility/resistance has been correlated with polymorphisms in more than 30 other genes, some of which have exhibited differential associations in distinct populations of the world (Kwiatkoski D, 2005). Plasmodium falciparum blood infection levels and fever episodes have been linked to chr 5q31–33 (Garcia A, Marquet S, et al, 1998) and chr10 (Timmann C, Evans JA, 2007). While most human gene polymorphism-disease association studies for malaria susceptibility have been carried out on populations from Africa and south-east Asia, there is limited information on malaria-associated gene polymorphisms in Indian populations (Sukumar S et al 2004).
SNPs in the TNF of P. falciparum malaria
SNPs in the 5' regulatory region of TNF and coding region of FCGR2A have been associated with P. falciparum malaria (McGuire W, Hill AVS, et al, (1994) Ubalee R, Suzuki F et al (2002). The transcription of TNF is complex and tightly regulated (Tsai EY et al, 2000). SNPs in the 5' regulatory region of the gene have been shown to correlate with many infectious and inflammatory diseases (Heel DAV, et al, 2001) with conflicting reports regarding their functional significance. SNPs at positions -1031, -857, -376 (rs1800750, G>A), -308 and -238 in the proximal enhancer of the TNF gene exhibit differential associations to malaria and TNF production in different populations (Ubalee R, et al, 2001) suggesting that individual TNF responses may be genetically determined.
Correlation of TNF levels with severity of P. falciparum malaria
Elevated TNF levels in malaria patients have been correlated with severe disease manifestation in other world populations (Clark IA et al, 2004) although significant differences between plasma TNF levels were not observed between uncomplicated, severe anemia, and cerebral malaria patients in a recent report on Malian children (Cabantous et al, 2005) suggesting the possibility of population-specific differences. An earlier study from India (Manish R et al, 2003) reported higher TNF levels in patients with multiple organ dysfunction and those who died.
Role of TNF and SNPs polymorphism in P. falciparum malaria
The role of TNF during P. falciparum malaria infection has been described as both protective and pathogenic (Gimenez F et al,2003). At low levels, TNF is believed to augment parasite killing by macrophage activation and subsequent release of cytokines, whereas high TNF level has been associated with severe manifestations like acute respiratory distress and cerebral malaria. A recent study on lethal malaria in mice has implicated high levels of TNF in the impairment of dendritic cell function thus contributing to immunosuppression associated with malaria (WykesMN, et al, 2007). Individual variation in TNF production mainly by macrophages and NK cells is likely to influence severe disease manifestation. Although the effect of TNF enhancer SNPs on transcription levels of the cytokine remains controversial (Abraham LJ, et al, 1999), several studies have implicated their role in determining TNF levels in individuals and consequently influencing their response to a gamut of autoimmune and infectious diseases including P. falciparum malaria (Yangs SK et al, 2006) While the association of the -863A substitution with reduced TNF levels was described in the Swedish population (Skoog, et al, 1999), a study from Japan (Hitachi T, et al, 1998) reported association of the -1031, -863 and -857 polymorphisms with increased reporter gene expression and increased concanavalin A-stimulated TNF production from peripheral blood mononuclear cells. Additionally, the ubiquitous transcription factor OCT-1 has been reported to exhibit allele-specific binding to the variant allele -863A or -857T (Hohjoh H et al,2001). The two SNPs may play a significant role in modulating the immune response and influencing the outcome of several infectious diseases
Iron in the human body iron is stored in an intracellularuniversal protein ferritin which releases it in a controlled fashion. The protein is produced by almost all living organisms, including algae, bacteria, higher plants, and animals. In humans, it acts as a buffer against iron deficiency and iron overload (Rachel Cassidey et al, 2002). Ferritin is found in most tissues as a cytosolic protein, but small amounts are secreted into the serum where it functions as an iron carrier. Plasma ferritin is also an indirect marker of the total amount of iron stored in the body, hence serum ferritin is used as a diagnostic test for iron deficiency anemia.
Figure 2: Structure of the murine ferritin complex (Granier T, et al, 2003)
Iron status in P. falciparum malaria patients
Understanding the influence of iron status on the risk of malaria is necessary for planning and implementing iron deficiency (ID) control programs in sub-Saharan Africa. Both ID and malaria are common in this region and are major causes of anemia. ID anemia is associated with impaired cognitive and motor development, reduced growth velocity, and anorexia in children (Sheard NF,1994). International guidelines recommend iron and folic acid supplementation in children under 2 years of age in areas with a high prevalence of anemia
Although the detrimental effects of ID argue for aggressive intervention, the safety of universal routine iron supplementation remains unclear. Children in a malaria-endemic region of Tanzania who were randomized to receive iron supplements suffered from a 15% increased all-cause mortality (Sazawal S, et al, 2006). Whereas results from several earlier studies also identified increased malarial risk in individuals treated with iron (Smith AW, et al, 1989)
Therefore, ID protects children from malaria infection, malaria morbidity, and mortality (Moses Gwamaka, et al, 2012).
Iron deficiency protection against P. falciparum malaria
Malaria and iron deficiency anemia (IDA) impact the same geographic and demographic groups and the pathophysiological relationship between the two is complex. Acute malaria can cause severe anemia due to hemolysis of infected and uninfected RBCs, and chronic or subclinical malaria can induce anemia of inflammation (Clark et al, 2014a). There is clear epidemiological evidence in both children (Gwamaka et al, 2012, Jonker et al 2012) and pregnant women (Senga et al, 2011) that, once established, IDA is protective against malaria infection. In fact, in pregnant women, iron deficiency has been shown to reduce the risk of placental malaria to a greater extent than multiparity (Kabyemela et al, 2008).
Evaluation in vitro parasite growth in RBCs from anemic children at baseline was found lower in parasite growth rates than in RBCs from iron-replete individuals. (M.M Goheen et al, 2016).
Iron deficient RBCs have a shorter circulation lifetime (90 vs 120 days, on average) and exhibit physiological differences such as microcytosis, decreased deformability, and increased oxidative membrane stress, among other effects – similar to changes in aged RBCs (Brandao et al, 2009). The parasites preferentially infect young RBCs and reticulocytes (Clark et al, 2014a). Studies show that RBCs from anemic African children were resistant to invasion with both laboratory and clinical P. falciparum strains and that iron supplementation increased invasion susceptibility(M.M Goheen et al, 2016).
Parasite growth and invasion in RBCs from anemic children (Hgb < 11 g/dl) at baseline.
A) P. falciparum (strain FCR3-FMG) growth rates are proportional to hemoglobin concentration. Growth assays were performed in RBCs drawn from anemic children at baseline (Day 0) and values are presented relative to growth in RBCs from non-anemic donors. Each dot represents the mean result of triplicate growth assays from each donor and the error bars represent 95% CI. One-way ANOVA indicates the means are significantly different between Days (p< 0.05); specifically, post-hoc analysis with Tukey's test indicates significant differences between Hgb levels 7–9 g/dl and 10.1–11 g/dl (*p< 0.05). (M.M Goheen et al, 2016).
TNF SNPs haplotypes associated with iron deficiency
Plasma levels of tumor necrosis factor-α (TNF-α) are significantly raised in malaria infection and TNF-α is thought to inhibit intestinal iron absorption and macrophage iron release. There is compelling evidence that plasma levels of tumor necrosis factor-α (TNF-α) are significantly raised in malaria infection compared with other illnesses, (Kwiatkoski D et al, 1990) even in children with asymptomatic infection. (Kurtzhals JA et al, 1999)
Studies show that TNF, mapped on 6p21.3 in the major histocompatibility complex (MHC), is involved in the regulation of iron metabolism. TNF-α induces hypoferremia by inhibiting iron release from macrophage storage compartments (Alvarez-Hernandez X et al, 1989) and increasing the transcriptional induction of ferritin (Torti SV, 1988). TNF-α also inhibits iron uptake in erythroid precursors, (Moldawer LL et al,1989) blocks the differentiation and proliferation of erythroid progenitor cells (Bird A et al,1987) and causes erythrophagocytosis and dyserythropoietic (Ulich TR et al, 1990) Recent in vitro and animal studies have demonstrated that TNF-α also directly inhibits intestinal iron absorption independently of hepcidin production (Sharma N,2005)
The TNF−308 and the TNF−238 promoter polymorphisms were found to alter the phenotypic expression of hereditary hemochromatosis. (Kyenbuehl PA et al, 2006). The TNF−308 polymorphism was associated with an increased risk of severe anemia in low-birth-weight children in Kenya, (Aidoo M et al, 2001)although not severe malaria anemia. The TNF−238 polymorphism was associated with an increased risk of severe malarial anemia in Gambian children(McGuire W et al,1999).
Diagnosis of P. falciparum malaria
Malaria diagnosis involves identifying malaria parasites or antigens/products in the patient blood. The diagnostic efficacy is subject to many factors which include; the different forms of the 5 malaria species, the different stages of erythrocytic schizogony, parasitemia, the interrelation between levels of transmission, population movement, sequestration of the parasites in deeper tissues and the use of chemoprophylaxis or even presumptive treatment on basis of clinical diagnosis which can influence the identification and interpretation of malaria parasitemia a in the diagnostic test.( Noppadon T et al, 2009).
In the laboratory, malaria is diagnosed using different techniques. This includes convectional microscopic diagnostic by staining thin and thick peripheral blood smears (Ngasala B, et al, 2008) then stained with Giemsa stain, and then screening the presenting parasite under a microscope.
Other concentration techniques include quantitative buffer coat (QBC) method (Bhandari PL, et al, 2008), rapid diagnostic tests e.g. OptiMAL(Zerpa N et al, 2008) ICT (Ratsimbasoa A et al, 2008) para-HIT-f (McMorrow ML et al, 2008) and molecular diagnostic methods, such as polymerase chain reaction (Vo TK et al, 2007)
Most Rapid diagnostic tests (RDTs), target Plasmodium Falciparum- specific proteins such as histidine-rich protein (HRP-II). RTDs are easy to use, fast and do not require electricity or specific equipment hence useful in remote areas.
Malaria treatment
Uncomplicated P. falciparum malaria can be treated orally with quinine, atovaquone plus proguanil (Malarone®), or co-artemether (Riamet®); quinine is highly effective but poorly tolerated in prolonged dosage and is always supplemented by additional treatment, usually with oral doxycycline. ALL patients treated for P. falciparum malaria should be admitted to the hospital for at least 24 h, since patients can deteriorate suddenly, especially early in the course of treatment. (David G. Lalloo et al, 2007).
Severe falciparum malaria, or infections complicated by a relatively high parasite count (more than 2% of red blood cells parasitized), should be treated with intravenous therapy until the patient is well enough to continue with oral treatment. This may exacerbate hypoglycemia that can occur in malaria; patients treated with intravenous quinine, therefore, require careful monitoring. Intravenous artesunate reduces high parasite loads more rapidly than quinine and is more effective in treating severe malaria in selected situations (David G. Lalloo et al, 2007).