Screening for Genetic Diversity of Malaria Positive Samples Gotten From Mater Misericordae Hospital, Afikpo Ebonyi State

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Malaria is a mosquito borne parasite disease caused by the unicellular, eukaryotic and protozoan parasites of the genus plasmodium. Infective Anopheles Mosquitoes are the sole vector of the transmission from human to human. Malaria still remains an important public health disease in the tropical parts of the world especially in the African continent.

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In Nigeria, the disease is responsible for 60% outpatient visits to health facilities, 30% deaths of under-five children’s, 11% maternal mortality and 300,000 deaths per year. There are five causative plasmodium parasites which have been recognized by the world health organization which are able to infect humans.

These plasmodium species include Plasmodium falciparum (P. falciparum), Plasmodium Vivax (P. vivax), Plasmodium Ovale (P. ovale), Plasmodium malariae (P. malariae) and Plasmodium knowlesi (P. knowlesi). Malaria affects 300 to 500 million deaths mainly in African children. (Bender et al., 2001). Malaria has been associated to gene present in the human genome and it has been associated as an evolutionary force of some genetic disease such as sickle cell disease (SCD), thalassemia, glucose-6-phosphate dehydrogenase (G6PD) deficiency, and other red blood cell (RBC) genetic anemia with mendelian inheritance. Reports associating several genetic disorders with malaria susceptibility or resistance are on the risk and studies of heritability indicate that approximately 25% of the risk for severe malaria progression is determined through human genetic factors (Mwagi et al., 2005). Genetic epidemiology may help in pointing out major molecular pathways of some infectious disease such as malaria which involve a robust immune and inflammatory response and the participation of erythrocytes and other blood cells in its pathogenesis.

The immune response is critical for controlling plasmodium infection and the balance between proinflamatory anti-inflammatory cytokines has been implicated in both the control of parasite multiplication and the development of symptoms.
The genetic background of the affected individual might also influence the cytokine expression and disease outcomes. Notably, the frequency of genetic alterations differs depending on the population origin and structure and some mutations might differently influence the disease outcome in different patterns. Understanding the genetic alterations involving RBC disorder and the immune response might provide insight into the development of new strategies for host genotype treatment and the prevention of malaria.
Genetic diversity is the variety and frequency of different genotypes or combination of different genotypes within a population. A population is a geographically, socially or culturally linked group whose reproductive decision affects those within the group. Genetic diversity is measured by genetic variability, which diminishes in a population when the number of different phenotypes or the number of different combinations of genotypes decreases. Since populations are composed of individuals that carry genotypes, individual reproductive outcomes affect the genetic variability within specific populations (Resnik, 2000). Genetic diversity provides the resource for phenotypic variation that is integral in determining the rate of evolutionary change in an environment. A population that lacks genetic diversity will be poorly equipped to meet environmental changes and demands. The importance of genetic diversity is undeniable; the survivability of a population is directly related to genetic diversity. Humans place positive value in genetic diversity as it promotes the extrinsic value of survivability.

There is an ethical duty to prevent decreases in the genetic diversity of populations because of those populations. Decreases in genetic diversity in population are ethically undesirable because actions that reduce the survivability of the population are unethical.
There are many emerging technologies that could potentially affect genetic diversity. These include genetic testing and screening, selective breeding, population control, sterilization, selective abortion, embryo testing and selection, sperm donation, egg donation, embryo donation, surrogate pregnancies, fertility drugs, contraception, cloning embryos and germ line or somatic cell manipulation. Each of these reproductive technologies affects the composition of the human gene pool by increasing or decreasing the frequency of different genotypes or combinations of genotypes. Genetic testing and screening examines the genetic information contained in a person’s cells to determine whether that person has or will develop a certain disease is more susceptible to certain environmental risk or could pass a disease onto his or her offspring. Parents could subject themselves to testing to determine whether or not to produce based on the likelihood of their potential children inheriting their genetic maladies. Also, embryos can be subjected to testing and screening to determine the likelihood that the future individual will develop a genetic disease.
Plasmodium falciparum is the most virulent of the four parasites which cause malaria in humans.

These malaria parasites are genetically diverse at all levels of endemicity. The study of genetic diversity in malaria parasites is expected to provide new insight for the development of control measures. The genetic complexity of P.falciparum and in particular its ability to generate mutant variant, makes it a successful pathogen. Although information on the frequency of genes conferring, for example, resistance to a certain drug or a vaccine in a given area has obvious relevance to the implementation of control measure using such agent, a critical first step is to obtain information on the genetic polymorphism of the parasites in the human host. Single nucleotide polymorphism contribute largely to the variability of Plasmodium falciparum and provide multiple effective evasion and drug resistance mechanisms for the parasite necessitating the use of molecular technique to differentiate alleles responsible for recrudescence’s and re-infections after treatment. Polymorphic genes within a parasite species are used as genetic markers thereby providing a means to assess the composition of the parasite population. Several strategies have been used for plasmodium parasite genotyping. At present, there are various techniques based on DNA fingerprinting for microorganism such as PCR-restriction fragment length polymorphism (PCR-RFLP).
Other methods for typing Plasmodium falciparum are based on microsatellites analysis, reverse transcriptase, sequence analysis, mini satellite variant repeat (MVR) mapping and ligase detection reaction-florescent microsphere assay (LDR-FMA). The most widely used methods are PCR based (Farnet et al., 2001), since they require small amount of blood. Polymerase chain reaction techniques are also being used for the detection of drug resistance marker and differentiation of recrudescence’s and re-infection after treatment but a recent technique using microsatellites and capillary electrophoresis showed higher sensitivity and specificity in differentiating recrudescence’s from new infection than conventional PCR methods. Nevertheless, the analysis of parasites genotypes provides measures that can be used to characterize the malariological picture in human populations and to extend our understanding of the epidemiological effects of natural immunity to Plasmodium falcifalrum. The importance of human genetic background on malaria morbidity and infections has become increasingly recognized. Haemoglobin (Hb), Hemoglobin cell, and a-thalassemia confer protection from malaria. Accordingly, these traits may influence mode of infection of Plasmodium falcifarum and the presence of distinct genotypes. Of the three traits, HbS in the heterozygous form, sickle-cell traits (HbAS) has been studied most. In areas endemic for falcifarum malaria, HbAS is widespread due to its advantage against fatal malaria. In such areas, individuals with HbAS have lower parasite rates and densities than individual with normal hemoglobin (HbAA). Sickle cell traits protects against severe falcifarum malariae and reduces susceptibility to mild malaria but does not prevent malaria infection therefore the protection is not absolute. The protective effect of the traits is highest during first 12 months after the loss of passively acquired immunity and before specific protection mechanisms are developed.
However, microscopy is the common method used to detect malaria parasites. Meanwhile, as microscopy detection and identification of P. falciparum in Giema- stained, thick and thin blood smears continues to be used in the laboratory diagnosis of malaria, Polymerase Chain Reaction is also an attractive and additional technique to microscopy for the confirmatory, identification and genotyping of Plasmodium Species. Polymerase Chain Reaction has been adopted to detect the DNA of the parasites. It also distinguishes between P.vivax, and P.ovale. meanwhile RADP markers have been used to study the genetic diversity of individuals who are attack by malaria parasites, showing common genetic characteristics, variability among individuals who are infected with malaria parasites, either according to age, gender etc

AIM/OBJECTIVES OF THE STUDY
AIM
To determine the genetic diversity of plasmodium species of malaria positive samples gotten from patients attending mater misericordiae hospital, Afikpo

OBJECTIVES OF THE STUDY
a) To extract DNA form Malaria positive samples.
b) To study the genetic diversity of malaria parasites.
c) To study the prevalence of malaria among genotypes.

CHAPTER TWO
LITERATURE REVIEW
HISTORY OF MALARIA
Early theories reported that malaria was caused by bad air (“mala aria” in Italian) (CDC, 2012). The parasites in the blood were first seen in 1880 by French army surgeon Alphonse Laveran, who was looking for a bacterial cause of malaria. He immediately realized that parasites were responsible for the disease other than bacteria (Ridley, 2012).
Malaria parasites are micro-organisms that belong to the genus Plasmodium. There are more than 100 species of Plasmodium, which can infect many animal species such as reptiles, birds and various mammals. Four species of Plasmodium have long been recognized to infect humans in nature. In addition, there is one species that naturally infects macaques which has recently been recognized to be a cause of zoonotic malaria in Humans (CDC, 2012). The species infecting humans are: P. falciparum, which is found worldwide in tropical and subtropical areas.
It is estimated that every year approximately one million people are killed by Plasmodium falciparum, especially in Africa where this species predominates. Plasmodium vivax is usually found in Africa. Plasmodium ovale is found mostly in Africa (especially West Africa). It is biologically and morphologically very similar to Plasmodium vivax. However, differently from P. vivax, it can infect individuals who are negative for the duffy blood group, which is the case for many residents of sub-Saharan Africa. This explains the greater prevalence of P. ovale (rather than P. vivax) in most of Africa. Plasmodium malariae, found worldwide, is the only human malaria parasite species that has a quartan cycle (three-day cycle). Plasmodium knowlesi is found throughout Southeast Asia as a natural pathogen of long-tailed and pig-tailed macaques. It has recently been shown to be a significant cause of zoonotic malaria in that region, particularly in Malaysia. Plasmodium knowlesi has a 24-hour replication cycle and so can rapidly progress from an uncomplicated to a severe infection; fatal cases have been reported (CDC-b, 2012).

 

W.H.O estimated that in 2010 there were 219 million cases of malaria resulting in 660,000 deaths. Others have estimated the number of cases at between 350 and 550 million for Plasmodium falciparum malaria and deaths in 2010 at 1.24 million up from 1.0 million deaths in 1990.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. There are about 10,000 malaria cases per year in Western Europe, and 1300–1500 in the United States. About 900 people died from the disease in Europe between 1993 and 2003. Both the global incidence of disease and resulting mortality has declined in recent years. According to the WHO, deaths attributable to malaria in 2010 were reduced by over a third from a 2000 estimate of 985,000, largely due to the widespread use of insecticide-treated nets and artemisinin-based combination therapies. In 2012, there were 207 million cases of malaria. That year, the disease is estimated to have killed between 473,000 and 789,000 people, many of whom were children in Africa.
A 2010 estimate indicated the deadliest countries per population were Burkina Faso, Mozambique and Mali. The Malaria Atlas Project aims to map global endemic levels of malaria, providing a means with which to determine the global spatial limits of the disease and to assess disease burden. Malaria is prevalent in tropical and subtropical regions because of rainfall, consistent high temperatures and high humidity, along with stagnant waters in which mosquito larvae readily mature, providing them with the environment they need for continuous breeding. In drier areas, outbreaks of malaria have been predicted with reasonable accuracy by mapping rainfall.

TRANSMISSION OF MALARIA
The malaria parasite typically is transmitted to people by mosquitoes belonging to the genus Anopheles (NIH-b, 2009). The female anopheles mosquito is the chief vector and the most common means for transmitting malaria to humans. Over 60 species of this mosquito have been identified as vector for malaria. The infection is transmitted by the bite of an infected female anopheles mosquito as reported by Ridley et al., The mosquito most frequently bites at dawn and at dusk, as this is the most active feeding times for mosquitoes. The mosquito is infected by biting a patient infected with malaria, where it aspirates the sexual forms of the parasite, the gametocyte continue the sexual phase of the cycle and the sporozoites fill the salivary glands of the infested mosquito (Ridley, 2012). In rare cases, a person may contract malaria through contaminated blood. Malaria also may be transmitted from a mother to her fetus before or during delivery (i.e. congenital malaria). Because the malaria parasite is found in red blood cells, malaria can also be transmitted through blood transfusion, organ transplant, or the shared use of needles or syringes contaminated with blood (NIH-b, 2009). Transmission of malaria also depends on climate conditions that may affect the number and survival of mosquitoes such as rainfall pattern, temperatures and humidity. In many places, transmission is seasonal with the peak during and just after the rainy season. Malaria epidemics can occur when climate and other conditions suddenly favor transmission in areas where people have little or no immunity to malaria. They can also occur when people with low immunity move into areas with intense malaria transmission.

 

In the life cycle of Plasmodium, a female Anopheles mosquito (the definitive host) transmits a motile infective form (called the sporozoite) to a vertebrate host such as a human (the secondary host), thus acting as a transmission vector. A sporozoite travels through the blood vessels to liver cells (hepatocytes), where it reproduces asexually (tissue schizogony), producing thousands of merozoites. These infect new red blood cells and initiate a series of asexual multiplication cycles (blood schizogony) that produce 8 to 24 new infective merozoites, at which point the cells burst and the infective cycle begins anew. Other merozoites develop into immature gametocytes, which are the precursors of male and female gametes. When a fertilized mosquito bites an infected person, gametocytes are taken up with the blood and mature in the mosquito gut. The male and female gametocytes fuse and form an ookinetea fertilized, motile zygote. Ookinetes develop into new sporozoites that migrate to the insect’s salivary glands, ready to infect a new vertebrate host. The sporozoites are injected into the skin, in the saliva, when the mosquito takes a subsequent blood meal.
Only female mosquitoes feed on blood, male mosquitoes feed on plant nectar, and thus do not transmit the disease. The females of the Anopheles genus of mosquito prefer to feed at night. They usually start searching for a meal at dusk, and will continue throughout the night until taking a meal. Malaria parasites can also be transmitted by blood transfusions, although this is rare.

PATHOLOGY OF MALARIA
A research made by John and perti in 2006 indicated that pathogenic effect of malaria infection have been considered to be directly related to the hemolysis of infected red cell and uninfected cell, liberation of the metabolites of the parasites and the immunologic response of the host to this antigenic material and the formation of malaria pigment. In falciparum malaria, the phenomenon of cytoaldherence is basic to the locally diminished tissue perfusion seen in it is more severe complication. In persons subjected to repeat attack of malaria anemia is disproportional to the number of red blood cells infected and indicates that non infected red blood cells may become sensitized and destroyed. The clinical symptoms of malaria are primarily due to schizont rupture and destruction of erythrocytes (Trampuz et al., 2003). Malaria typically produces a string of recurrent attacks, or paroxysms, each of which has three stages-chills, followed by fever and then sweating. Along with chills, the person is likely to have headache, malaise, fatigue, muscular pains, occasional nausea, vomiting and diarrhea. Within an hour or two, the body temperature rises, and the skin feels hot and dry. Then, as the body temperature falls, a drenching sweat begins. The person, feeling tired and weak, is likely to fall asleep. The symptoms first appear some 10 to 16 days after the infectious mosquito bite and coincide with the bursting of infected red blood cells (RBCs). When many red blood cells are infected and break at the same time, malaria attacks can recur at regular time periods-every two days for P. vivax malaria and P. ovale and every three days for P. malariae (NIH-c, 2009).

PREVALENCE OF MALARIA
Malaria is a major public health problem and cause of suffering and premature death in tropical countries. This preventable disease has reached epidemic proportion in many regions of the world and continues to spread unchecked (WHO, 2010). African children under five years and pregnant women are more at risk of malaria. Fatally afflicted children often die less than 72 hours after developing symptoms. Malaria drain vital, nutrients from children suffering from malaria thereby impairing their physical and intellectual development.
Fawole and Onadeko estimates that more than one million children living in Africa die yearly, from direct and indirect effect of malaria infection. Malaria parasites (plasmodium species) are spread from one person to another through the bites of hemategenous female adult mosquitoes belonging to the insect genus Anopheles. These adult female Anopheles Mosquitoes are hence said to be carriers of malaria inhibit the tropical and subtropical part of the world (Epidi et al., 2008).
The major vectors of human malaria are Anopheles gambiae, Anopheles funertus Anopheles arabiensis, and Anopheles melas. Transmission of malaria is intense and stable in Nigeria because the intensity of attack remains constant throughout the year. In Nigeria, malaria is holendemic in the urban areas. In the southern part of the country the transmission rate is approximately uniform throughout the year north, there is a marked differences between the high transmission rate in the short wet season and low transmission rate in the long dry season. Man and malaria seem to have evolved together. It is believed that most, if not all, of populations of human malaria may have had their origin in West Africa (Plasmodium falcifarum) and central Africa (Plasmodium vivax) on the basis of the presence of hormozygous alleles for hemoglobin C and red blood cell duffy negativity that confer production against P. falciparum and P. vivax respectively. Recent molecular studies have found evidences that human malaria parasites probably jumped onto human through the bites of vector mosquitoes (Krief et al., 2010). W.H.O (1998), reported a prevalence of 58% malaria parasite in children living in Gambia. Umar (2006) reported 94% prevalence of malaria parasites among children living in Gombe metropolis. Ejim and mbanugo in their study reported of that out of 400 children living in Awka were affected by P. facliparum. Krogstad et al., reported also that plasmodium infection was more prevalent in young children because of their relatively less developed body structure.

DIAGNOSIS OF MALARIA
There is need for prompt and accurate diagnosis to the effective management of malaria. Malaria diagnosis involves identifying malaria parasites or antigens in patient’s blood. Although these may seem simple, the diagnostic efficacy is subject to many factors. The different forms of the five malaria species, the different stages of erythrocytic schzogony, the endemicity of different species, the interrelation between levels of transmission, population movement, parasitemia, immunity and signs and symptoms, drug resistance, the problems of recurrent malaria, persisting viable or non-viable parasitemia, the sequestration of the parasites in the deeper tissues and the use of chemoprophylaxis can all influence the identification and interpretation of malaria parasitemia in a diagnostic test. Delay in diagnosis and treatment are the leading cause of death. (CDC, 2008). Diagnosis can be difficult when malaria is no longer endemic for health care providers unfamiliar with the disease. Clinicians tend to forget to consider malaria as a potential diagnosis for some patients and not order the necessary diagnostic tests. In many malaria endemic countries, the lack of resources is a major barrier to reliable and timely diagnosis.

CLINICAL DIAGNOSIS OF MALARIA
A clinical diagnosis is least expensive and most widely practiced. Clinical diagnosis is based on the patient’s signs and symptoms and on physical findings at examination. The earliest sign of malaria are very nonspecific and variable and include fever, headache, weakness, chills, dizziness, abdominal pain, diarrhea, nausea, vomiting and anorexia. A clinical diagnosis of malaria is still challenging because of the non-specific nature of the signs and symptoms which considerably overlap with other common life threatening diseases impairs diagnostic specificity which can promote the indiscriminate use of anti-malaria and compromise the quality of care for patients with non malaria fevers in endemic areas .(Mwangi et al.,2005).
According to the integrated management of Children illness (IMCI), clinical algorithm for managing and diagnosing common illness by children by health care provider in the developing world is due to inappropriate equipment for laboratory diagnosis. A widely utilized clinical algorithm for malaria diagnosis compared with a fully trained pediatrician with access to laboratory support showed a very low specificity (0-9%) but 100% sensitivity. This lack of specificity reveals the perils of distinguishing malaria from other cases of fever in children on clinical grounds alone. Another recent study showed that the use of integrated management of children illness clinical algorithm resulted in 30% over diagnosis of malaria ( Tarimo et al., 2001),the accuracy of malaria diagnosis can be greatly enhanced by combining clinical and parasitic –based findings (Odong et al., 2008).

LABORATORY DIAGNOSIS OF MALARIA
Rapid and effective malaria diagnosis has not only alleviated suffering but has also reduced community transmission. The nonspecific nature of the clinical signs and symptoms of malaria has resulted in over-treatment of malaria or non-treatment of disease in malaria-endemic areas and misdiagnosis in non-endemic areas (Bhandari et al., 2008). In the laboratory, malaria is diagnosed by different techniques, e.g Conventional microscopic diagnosis by staining thin and thick peripheral blood smears ( Mubi et al., 2008), other concentration techiques, e.g quantitative buffy caot (QBC) method (Odong et al., 2008), rapid diagnostic tests e.g OptiMAL (Tagbor and Gavidia, 2008), ICT (Ratsimbasoa, 2008), ParaHTT-f(McMorrow and Masanja,2007), paraScreen(Endeshaw,2008), SD Bioline(Lee et al, 2009), Paracheck(Harvey and Jennings, 2006), and molecular diagnostic method such as polymerase Chain Reaction (PCR), but some of the shortcomings of these methods is related to specificity, accuracy, precision, sensitivity, time consuming, cost effectiveness, labor intensive, the need for skilled microscopic and the problem of inexperienced technicians.

Microscopic diagnosis using staining thin and thick peripheral blood smears (PBS)
Malaria is diagnosed by microscopic examination of stained blood films using Giesma, Wrights or Fields stains. More than a century, microscopic detection and identification of plasmodium species in Giesma-stained thick blood films and thin blood films remain the gold standard for laboratory diagnosis. Malaria is diagnosed microscopically by staining thick and thin blood films on a glass slide, to visualize malaria parasites. Briefly the patient’s finger is cleaned with 70% ethyl alcohol, allowed to dry and then the slide of fingertip is picked with a sharp sterile lancet and two drops of blood are placed on a glass slide. To prepare a thick blood film, a blood spot is stirred in a circular motion with the corner of the slide, taking care to make sure the preparation is not too thick and also allowed to dry without fixative. After drying, the spot is stained with diluted Giesma(1:20,vol/vol) for 20mins and washed by placing the film in a buffered water for 3 minutes. The slide is dried by placing it in a vertical position and examined using a light microscope. The wide acceptance of this technique by laboratories can be attributed to its low cost, simplicity, ability to identify the presence of parasites, the infecting species and assess parasite density.
A recent study showed that conventional malaria microscope diagnosis at malaria healthcare facilities could reduce the prescription of anti-malaria drugs and also improve the appropriate management of non-malarial fevers. However, the staining and interpretation processes are time consuming, laborious and require considerable expertise and trained personnel especially in the area of identifying species accurately at low parasitemia. The shortcoming of microscopic examination is its low specificity, particularly at low parasite levels. Microscopy is laborious and ill-suited for high-through-put use and species determination at low parasite density is challenging.

Quantitative buffer coat technique (QBC)
The QBC technique is a technique that was designed to enhance microscopic detection of parasites and simplify malaria diagnosis. Quantitative buffer coat method involves staining parasite deoxyribonucleic acid (DNA) in micro-hematocrit tubes with flurosence dyes such as acridine orange and its subsequent detection by epi-flouresnt microscopy. In a brief state, finger-prick blood is collected in a hematocrit tube containing acridine orange and anticoagulant. The tube is centriguge at 12,000g for 5minutes and immediately examined using an epi-fluoresent microscope .Studies shows that quantitative buffer coat technique is rapid and sensitive for diagnosing malaria in numerous laboratories settings. While it enhances sensitivity for Plasmodium falciparum species, it reduces sensitivity for non falciparum species and decreases specificity due to staining of leukocyte DNA. Recent studies shows that acridine orange is the preferred diagnostic method (over light microscopy and immunochromatographic tests) in the context of epidemiologic studies in endemic areas probably because of increased sensitivity at low parasitemia. Although the QBC method is simple, reliable and user friendly, it also requires specialized instrumentation, is more costly than conventional light microscopy and is poor at determining species and numbers of parasites.

Rapid Diagnostic Tests (RDTs)
The world health organization (WHO) has recognized the urgent need for new, simple , quick, accurate and cost effective diagnostic tests for determining the presence of malaria parasites to overcome the deficiencies of light microscopy, and also numerous new malaria diagnostic techniques methods has been developed. These has lead to an increase in the use of rapid diagnostic tests for malaria, which are fast and easy to perform and do not require electricity. Unlike conventional microscopic diagnosis by staining thin and thin blood smears and QBC methods, RDTs are all based on the same principle to detect malaria antigen in blood flowing along a membrane containing specific anti-malaria antibodies, they do not require laboratory equipment. Although most RDTs products are suitable for P.falciparum malaria diagnosis, some also claim that they can effectively and rapidly diagnose P.vivax malaria. Recently a new RDT method has been developed for detecting P. knowlesi. Rapid diagnostic tests appears to be a valuable, rapid malaria diagnostic tool for healthcare workers, however, it must be used in conjunction with other method to confirm the results, characterize infection and monitor treatment. In malaria endemic areas where no light microscopy facility exists that may benefit from RDTs, improvements are required for ease of use, sensitivity for non falciparum infection, stability and affordability. Due to the simplicity and reliability of rapid diagnostic test (RDTs) has been improved for use in rural endemic areas, RDT diagnosis in non-endemic areas is becoming more feasible which may reduce time for the treatment of cases of imported malaria.

Serological Tests
Diagnosis of malaria using serological methods is usually based on the detection of antibodies against asexual blood malaria parasites. Immunofluorescence antibody testing (IFA) has been a reliable serologic test for malaria. Although immunofluorescnce antibody testing is time consuming and subjective, it is highly sensitive and specific. This literature clearly illustrates the reliability of immunofluorosence antibody testing so that it was usually regarded as the gold standard for malaria serology testing.
Immunofluorescent antibody testing is useful in epidemiological surveys for screening potential blood donors and occasionally for providing evidence of recent infection in non-immunes. Recent studies shows that it is a validated method for detecting Plasmodium-specific antibodies in various blood bank units, which was useful for screening prospective blood donors, so avoiding transmitted malaria . The principle of IFA is that following infection with any Plasmodium species specific antibodies are produced within two weeks of initial infection and persist for 3-6 months after parasite clearance. IFA uses specific antigen or crude antigen prepared on a slide, coated and kept at -30C until used and quantifies both IgG and IgM antibodies in patient’s serum samples. IFA is simple and sensitive but time consuming .it can’t be automated which limits the number of serum that can be studied daily. It also requires fluorescent microscopy and trained technicians; readings can be influenced by the level of training of thechnicians particularly for serum samples with low antibodies titer. The lack of IFA reagent standardization makes it impractical for routine use in blood transfusion centers and for harmonizing inter-laboratory results.

MOLECULAR DIAGNOSTIC METHODS
Traditional malaria diagnostic methods remain problematic. New laboratory diagnostic techniques that display high sensitivity and high specificity without subjective variation are needed in various laboratories. Recent developments in molecular biological technologies such as polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), mass spectrometry (MS), and flow cytometric assay (FCM) techniques have permitted extensive characterization of malaria parasites and are generating new strategies for malaria diagnosis.

Polymerase Chain Reaction Technique
Polymerase chain reaction (PCR) is a powerful method for amplifying particular segments of DNA, distinct from cloning and propagation within the host cell. Polymerase chain reaction is also an efficient and cost effective tool to copy or amplify small segments of DNA or RNA. Polymerase chain reaction combines the principles of complementary nucleic acid hybridization with those of nucleic acid replication that are applied repeatedly through numerous cycles. This in vitro amplification technique can amplify a single copy of nucleic acid target by using two synthetic oligonucleotide primers that bind to the target genomic sequence which are extended by a Taq polymerase (a thermostable DNA polymerase). An automated process of repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94C), annealing of primers to their complementary sequences and primer extension (70C) is employed for the amplification of target sequence. Polymerase chain reaction was invented by Kary Mullus in 1983.
Polymerase chain reaction based techniques are a recent development in the molecular diagnosis of malaria and have proven to be the one of the most sensitive and specific diagnostic methods particularly for malaria with low parasitemia or mixed infection. The PCR technique continues to be used extensively to confirm malaria infection, follow-up therapeutic response and identify drug resistance. It was found to be more sensitive than quantitative buffer coat technique and some rapid diagnostic test. With global standard of malaria diagnosis, polymerase chain reaction has shown higher sensitivity and specificity than conventional microscopic examination of stained peripheral blood smears and seems the best method for malaria diagnosis. Polymerase chain reaction can detect as few as 1-5parassites of blood compared with 50-100 parasites of blood by microscopy. Polymerase chain reaction can detect drug resistant parasites, mixed infection and may be automated to process large numbers of samples.

Principles of Polymerase Chain Reaction
Polymerase chain reaction uses the enzyme DNA polymerase that directs the synthesis of DNA from deoxynucleotide substrate on a single stranded DNA template. DNA polymerase adds nucleotides to the 3 end of a custom designed oligonucleotide when it is annealed to a longer template DNA. If a synthetic oligonucleotide is annealed to a single stranded template that contains a region complementary to the oligonucleotide, DNA polymerase can use the oligonucleotide as a primer and elongate its 3 end to generate an extended region of double stranded DNA.

Steps of Polymerase Chain Reaction
Polymerase chain reaction consist the following steps.

a. Initialization step: – This step consists of heating the reaction to a temperature of 94-960C which held’s for 1-9.

b. Denaturation step: – This step is the first regular cycling event and consist of heating the reaction to 94-980C for 20-30 seconds. It causes DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases yielding single-stranded DNA molecules.

c. Annealing step: The reaction temperature is lowered to 50-650C for 20-40 seconds allowing annealing of the primers to the single-stranded DNA template. This temperature must be low enough to allow for hybridization of the primer to the strand, but high enough for the hybridization to be specific.

d. Extension step: The temperature at this step depends on the DNA polymerase used. In this step, the DNA polymerase synthesizes a new strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5 to 3 direction, the extension time depends both on the DNA polymerase used and on the length of the DNA fragment to amplify.

 

NESTED POLYMERASE CHAIN REACTION
This polymerase chain reaction increases the sensitivity due to small amounts of the target are detected by using two sets of primers, involving a double process of amplification (Jann-Yuan et al., 2004). The first set of primers allows a first amplification. The product of this PCR is subjected to a second set of primers. These primers used in the second PCR are specific to an internal amplified sequence in the first PCR. Therefore, specificity of the first PCR product is verified with the second one. The disadvantage of this technique is the possibility of contamination during transfer from the first amplified product into the tube which the second amplification will be performed. Contamination can be controlled using primers designed to anneal at different temperatures. Contamination can also be controlled by adding ultra pure oil to make a physical separation of two mixtures of amplification (Kitagawa et al., 1996)

SYMPTOMS OF MALARIA
The clinical symptoms of malaria are due to the rupture of the schizont and destruction of erythrocytes as reported by Trampuz et al. Malaria typically produces a string of recurrent attacks each of which has three stage-chills, followed by fever and then sweating. Along with chills, the person is likely to have headache, fatique, muscular pain, occasional nausea, vomiting and diarrhea. Within an hour or two, the body temperature rises and the skin feels hot and dry. Then as the body temperature falls, a drenching sweat begins. The person feeling tire and weak is likely to fall asleep. The symptoms first appear some 10 to 16 days after the infectious mosquito bite and coincide with the bursting of infected red blood cells. When many red blood cells are infected and break at the same time, malaria attacks can recur at regular time periods every two days for Plasmodium vivax malaria and plasmodium ovale and every three days for Plasmodium malariae.

COMPLICATIONS IN PATIENTS WITH MALARIA

CEREBRAL MALARIA
This has a high case fatality and is a pathological condition resulting from infection with P. falciparum. A number of hypotheses have been proposed to explain the phenomenon of cerebral malaria, but in general it is thought to stem from immune responses against sequestered infected RBCs (Lamb, 2012). The onset may be dramatic with a generalized convulsion, or gradual with initial drowsiness and confusion, followed by coma lasting from several hours to several days (Trampuz et al., 2003). Sections of brain tissue from fatal P. falciparum infections reveal micro vascular obstruction in the brain due to the accumulation of sequestered infected RBCs, auto agglutinates (where by infected RBC adhere to each other) and rosettes of infected RBCs, as well as infiltrates of lymphocytes. Brain-resident macrophages, or macrophage/monocyte population that migrate to the brain tissue as a result of inflammatory immune responses against sequestered infected RBCs, directly contribute to the pathogenesis of cerebral malaria (Lamb, 2012).

 

PULMONARY COMPLICATIONS
Acute lung injury usually occurs a few days into the disease course. It may develop rapidly, even after initial response to anti malarial treatment and clearance of parasitemia. The first indications of impending pulmonary edema include tachypnea and dyspnea, followed by hypoxemia and respiratory failure requiring intubation. Pulmonary edema is usually non cardiogenic and may progress to acute respiratory distress syndrome (ARDS) with an increased pulmonary capillary permeability. Acute lung injury is defined as the acute onset of bilateral pulmonary infiltrates with an arterial oxygen tension/fractional inspired oxygen ratio of 300 mmHg or less, a pulmonary artery wedge pressure of 18 mmHg or less, and no evidence of left aerial hypertension. ARDS is defined as acute lung injury and an arterial oxygen tension/fractional inspired oxygen ratio of 200 mmHg or less. Volume overload and hypoalbuminemia may aggravate pulmonary capillary leakage. Chest radiograph abnormalities range from confluent nodules to basilar and/or diffuse bilateral pulmonary infiltrates. Non cardiogenic pulmonary edema rarely occurs with P. vivax and P. ovale malaria (Trampuz et al., 2003).

METABOLIC ACIDOSIS
The development of metabolic acidosis, whereby the pH of the blood lowers due to increased production of hydrogen in the body or defective removal of bicarbonate from the body by the kidneys, is often accompanied by respiratory distress and is strongly correlated with fatal malaria infection. Metabolic acidosis is exacerbated by the lack of
Circulating RBCs in patients with SMA, due to a reduction in the amount of oxygen delivered to the tissues and anaerobic metabolism. Hypovolaemia, whereby the volume of circulating blood decreases (presumably volume loss is partially due to lost RBC mass), is associated with severe anemia, and this also exacerbates metabolic acidosis (Lamb, 2012).

Hypoglycemia
Hypoglycemia is a common feature in patients with severe malaria. It may be overlooked because all clinical features of hypoglycemia (anxiety, dyspnea, tachycardia, sweating, coma, abnormal posturing, and generalized convulsions) are also typical of severe malaria itself. Hypoglycemia may be caused by quinine- or quinidine-induced hyperinsulinemia, but it may be found also in patients with normal insulin levels (Trampuz et al., 2003).

TREATMENT OF MALARIA
Malaria is treated with anti malarial medications; the ones used depend on the type and severity of the disease. While medications against fever are commonly used, their effects on outcomes are not clear. Uncomplicated malaria may be treated with oral medications. The most effective treatment for P. falciparum infection is the use of artemisinins in combination with other anti malaria’s (known as artemisinin-combination therapy, or ACT), which decreases resistance to any single drug component. These additional antimalarials include: amodiaquine, lumefantrine, mefloquine or sulfadoxine/pyrimethamine. Another recommended combination is dihydroartemisinin and piperaquine. To treat malaria during pregnancy, the WHO recommends the use of quinine plus clindamycin early in the pregnancy (1st trimester), and ACT in later stages (2nd and 3rd trimesters). In the 2000s (decade), malaria with partial resistance to artemisins emerged in Southeast Asia.
Infection with P. vivax, P. ovale or P. malariae is usually treated without the need for hospitalization. Treatment of P. vivax requires both treatment of blood stages (with chloroquine or ACT) and clearance of liver forms with primaquine. Recommended treatment for severe malaria is the intravenous use of ant malarial drugs. For severe malaria, artesunate is superior to quinine in both children and adults. Treatment of severe malaria involves supportive measures that are best done in a critical care unit. This includes the management of high fevers and the seizures that may result from it. It also includes monitoring for poor breathing effort, low blood sugar, and low blood potassium..

PREVENTION AND CONTROL OF MALARIA
Vector control is the main way to reduce malaria transmission. It is the only intervention that can reduce malaria transmission from very high levels to close to zero. For individuals, personal protection against mosquito bites represents the first line of defense for malaria prevention. Two forms of vector control are effective in a wide range of circumstances: insecticide-treated mosquito nets (ITNs): long-lasting insecticidal nets (LLINs) are the preferred form of ITNs for public health distribution programmes. WHO recommends coverage for all at-risk persons; and in most settings. The most cost effective way to achieve this is through provision of free LLINs, so that everyone sleeps under a LLIN every night. Indoor spraying with residual insecticides: indoor residual spraying (IRS) with insecticides is a powerful way to rapidly reduce malaria transmission. Its full potential is realized when at least 80% of houses in targeted areas are sprayed. Indoor spraying is effective for 3-6 months, depending on the insecticide used and the type of surface on which it is sprayed. Dicholoro Diphenyl Trichloroethane (DDT) can be effective for 9-12 months in some cases. Longer-lasting forms of existing IRS insecticides, as well as new classes of insecticides for use in IRS programmes, are under development. Ant malarial medicines can also be used to prevent malaria (WHO, 2015).

 

GENETIC DIVERSITY OF MALARIA PARASITES
Genetic diversity is the total number of genetic characteristics in the genetic makeup of specie. It is distinguished from genetic variability which describes the tendency of genetic characteristics to vary. Genetic diversity serves as a way for populations to adapt to changing environments. With more variation, it is more likely that some individuals in a population will possess variation of alleles that are suited for the environment. Those individuals are more likely to survive to produce offspring bearing that allele. The population will continue for more generation because of the success of these individuals.
The academic field of population genetics includes several hypotheses and theories regarding genetic diversity. The neutral theory of evolution proposes that diversity is the result of accumulation of neutral substitutions diversifying selection is the hypothesis that two subpopulations of a species live in different environments that selects for different alleles at a particular locus. This may occur, for instance, if specie has a large range relative to the mobility of individuals within it. Frequency- dependent selection is the hypothesis that as alleles become more common, they become more vulnerable. This occurs in host pathogen interaction where a high frequency of a defensive allele among the host means that it is more likely that a pathogen will spread if it is able to overcome that allele.

 

MEASURES OF GENETIC DIVERSITY
Genetic diversity of a population can be assessed by some simple measures which include:

Heterozygozity: The term heterozygous refers to a pair of genes where one is dominant and the other is recessive i.e they are different. Heterozygosity refers to the fraction of individuals in a population that are heterozygous for a particular locus.

Gene Diversity: This is the total number of characteristics in the genetic makeup of a species. It is distinguished from genetic variability which describes the tendency of genetic characteristics to vary. Gene diversity is the proportion of polymorphic loci across the genome.

Nucleotide Diversity: This is a concept in molecular genetics which is used to measure the degree of polymorphism within a population. One commonly used measure of nucleotide diversity was introduced by Nei and Li in 1979. This measure is defined as the average numbers of nucleotide differences chosen randomly from the sample population. Nucleotide diversity is a measure of genetic variation. It is usually associated with other statistical measure of population diversity and is similar to expected heterozygosity. This statistic may be used to monitor diversity within or between ecological populations to examine the genetic variation in crops and related species or to determine evolutionary relationships. Nucleotide diversity can be calculated by examining the DNA sequences directly or may be estimated form molecular marker data such as Random amplified Polymorphic DNA (RADP) data and Amplified Fragment Length Polymorphism.

Alleles per Locus: This is also used to demonstrate variability.

GENETIC SCREENING OF MALARIA PARASITES
The principle of genetic screening is based on the binding of a probe to the DNA molecule of the patient or the person to e screened. Complementary DNA nucleotide sequences bind to each other. The probe used is usually single stranded DNA which binds to the test sample. The DNA sample can be analyzed by a technique called restriction fragment polymorphism (RFLP). In this technique, the DNA sample is cut up with a mixture of restriction enzyme which cut the DNA at a specific sequence. A radioactive probe is added to the fragments that can bind to certain sequences in the DNA. The fragments are then separated by gel electrophoresis. The DNA fragments move through the gel at varying rates depending on their molecular weight. The pattern of size distribution is quite individual and the disease causing or linked fragment can e found. Currently the number of disorder that are screen able is measured in hundreds in hundreds and is rapidly increasing, genetic disease that have DNA probe available include: cystic fibrosis, thalassemia, Hemophilia, Huntington’s chorea, Sickle cell anaemia and many more.
One of the problems in genetic screening is the genetic heterogeneity that is the disease causing mutation may occur anywhere in the gene and there are also many mutations that do not cause disease. It is possible to use different probes to cover different mutations, but it is impossible to be sure of a negative result of a disease. Another problem of genetic screening is the difference in the expressivity. One person may express the disease to a different extent or at different age to another and this could be due to multiple gene action or variations in the environmental factors which can affect the expression of a certain gene, an example of such differences in expressivity can be found in a disease like Wilms tumor or Glucose-6-phosphate dehydrogenase deficiency.

TYPES OF GENETIC SCREENING
Generally there are two types of genetic screening: screening of children and adults and screening of unborn children. Genetic screening of children and adults has two purposes: first it can confirm whether the person has mutated genes of certain disease or characteristics. The second purpose is to test adults to see if their children will be at risk of certain disease. Knowing that one or both parents carries a dominant allele for genetic disease might affects the decision parents make about having children, sometimes this kind of genetic screening is used for approval of marriage licenses in some countries.
The second type of genetic screening involving screening of unborn children is called prenatal screening. The purpose of this kind of screening is to detect genetic disorders during early pregnancy. An example of this type of screening is testing for Down’s syndrome when the mother is over 40. Prenatal screening has been used for several decades using different techniques. The older technique used is aminocentesis, where cells from the amniotic fluid are removed and cultured. No harm is done to the fetus as these cells are no longer needed for the growth. The fetal sample can be taken at 12-16 weeks and provide sufficient material for analysis. Preimplantation screening has been used since 1989. The first study involved embryos that were not implanted after screening. Evidence suggested that embryos are not harmed by this procedure. One ethical objection, once it is confirmed that this procedure is safe, is the interference with nature by discarding of disease embryos at this stage. But given the economic factors that often influence health care for the people suffering from severe disease, his kind of screening may be encouraged by governments. On the other hand, religion has a problem with this kind of screening before the birth of the child; because the fact that carrying a defective gene does not mean that the child will express this gene.

BENEFITS OF GENETIC SCREENING
Genetic screening could have great value if used properly to help people take preventive measures to avoid suffering from some diseases. The first type of genetic screening used was screening of newborn children for PKU deficiency.
Genetic screening also works out economically, because it is cheaper to treat patients before serious damage is done to them by a genetic disease, than to keep sufferers in institutions. There is screening for genetic susceptibility to environmentally induced disease like elevated blood cholesterol. If screening shows the person is at risk, advice can be given on how to lower the risk. It has been found that many common cancers such as lung, breast and colon develop by step-wise accumulation of mutations affecting many genes. The disease includes the loss of some suppressor’s genes which inhibit cell growth. Genetic screening may help predicting a cancer and can be used to warn people to avoid agents that cause more mutations like smoking.

MOLECULAR MARKERS IN GENETIC DIVERSITY
Molecular markers or genetic markers are any stable and inheritable variation at DNA sequences level that can be measured or detected by a suitable method and can be used subsequently to identity a specific genotype. Molecular markers are considered as constant landmarks in the genome. They are also identifiable DNA sequences found at specific locations of the genome and transmitted by the standard law of inheritance from one generation to another. Molecular markers are tools used to study diversity at DNA level (polymorphism) and help breeders to identify specific chromosome segments that contain genes of interest. Molecular markers are one of the most recent development advances in DNA technologies and it has made it possible to reveal large number of genetic variation within a population. Consequently they are used for evaluation of the genetic basis of the observed phenotypic variability. Molecular markers are not functional genes, but they are less or more genes with no function and no impact on organism performance.

However, they are located either near or within major or functional genes such that their presence indicates to a large extent, the presence of the functional gene.
DNA markers are useful in both basic (e.g phylogenetic analysis and search for useful genes) and applied research (e.g. marker assisted selection, paternity testing and food traceability). Diversity among organism is as a result of variation in DNA sequences and of environmental effects. Genetic variation is substantial and each individual of a species, with the exception of monozygotic twins, possess a unique DNA sequence. DNA variations are mutations resulting from substation of single nucleotides, insertion or deletion of various length or duplication or inversion of DNA fragment.

THE ROLE OF MOLECULAR TECHNOLOGIES IN GENETIC DIVERSITY
Information on genetic diversity is essential in optimizing both conservation and utilization strategies. As resources for conservation are limited, prioritization is often necessary. New molecular tools hold the promise of allowing the identification of genes involved in a number of traits including adaptive traits and polymorphism causing functional genetic variation. In the absence of reliable phenotype and quantitative trait nucleotide data to complement the existing data, the most rapid and cost effective measures of genetic diversity are obtained from the assay of polymorphisms using anonymous molecular genetic markers. Anonymous markers are likely to provide indirect information on functional genes for important traits assuming that unique populations that have had a particular evolutionary history at the neutral markers are likely to carry unique variants of functional variations. Molecular techniques have also proved useful in the investigation of the origin and domestication of livestock species and their subsequent migration as well as providing information on evolutionary relationships and identifying geographical areas of admixture among populations of different genetic origins.

 

TECHNIQUES USING DNA MARKERS TO ASSESS GENETIC DIVERSITY

NUCLEAR DNA MARKERS
A number of markers are now available to detect polymorphism in nuclear DNA. In genetic diversity studies, the most frequently used markers are microsatellites.

Microsatellites
Currently, microsatellites are the most popular markers in genetic characterization studies (Sunnucks, 2001). Their high mutation rate and co dominant nature permit the estimation of within and between breed genetic diversity and genetic admixture among breeds even if they are closely related. The mean number of alleles per population, and observed and expected heterozygosity are the most common parameters for assessing diversity among breeds within-breed diversity. The simplest parameter for assessing diversity among breeds is the genetic differentiation indices. Some estimations have been proposed and the most widely used being FST (Weir and Basten, 1990) which measures the degree of genetic differentiation through calculation of the standardized variances in allele frequencies among population. Microsatellites data are also used to commonly assess genetic relationships between individuals through the estimation of genetic distances. The most commonly used genetic measure is the Nei’s standard genetic distance (Nei, 1972).

However, for closely related populations where genetic drift is the main factor of genetic differentiation particularly in the developing world, the modified cavallisforza distance is recommended (Nei et al., 1983).
Genetic relationship between breeds is often visualized through the reconstruction of a phlogeny, most often using the neighbor-joining (N-J) method (Saitou and Nei, 1987). However, a major drawback of phylogenetic tree reconstruction is that the evolution of lineages is assumed to be non-reticulate, i.e. lineages can diverge, but can never result from crosses between lineages. Molecular genetic data, in conjunction with, and complemented by, other sources such as archeological evidence and written records provide useful information on the origin and subsequent movement and development of genetic diversity. Mapping the origin of current genetic diversity potentially allows inferences to be made about where functional genetic variation might be found within a species for which only limited data on phenotypic variation exist.

Single Nucleotide Polymorphism (SNPs)
SNPs are used as an alternative to microsatellites in genetic diversity studies. Several technologies are available to detect and type SNP markers. Being a balletic marker, SNPs have rather low information content and larger numbers to have reached the level of information obtained. SNPs seem to be the appealing markers to apply in the future for genetic diversity studies because they can be easily used to asses functional or neutral variation. SNPs can be generated through various experimental protocols such as sequencing, single-stranded conformational polymorphism and comparing multiple sequences of the same region form public genome and expressed sequence databases. A frequent example is when SNPs initially identified in a small sample of individual and then typed in a large sample of chromosomes.

MITOCHONDRIAL DNA MARKERS
Mitochondrial DNA (mtDNA) polymorphisms have been extensively used in phylogenetic and genetic diversity analyses. The haploid mtDNA carried by the mitochondrial in the cell cytoplasm has a maternal role of inheritance and high mutation rate, it does not recombine. These characteristics enable biologist to reconstruct evolutionary relationship between and within species by assessing the patterns of mutations in mtDNA. MtDNA markers may also provide a rapid way of detecting hybridization between species (Nijman et al., 2003)
On the basis of techniques used for detection, molecular markers are classified into two major categories;
• Hybridization based markers
• PCR based markers

HYBRIDIZATION BASED MARKERS- These refers to the traditional or first generation restriction fragment length polymorphism which requires the use of appropriately labeled DNA probe to select the gene of interest from digested DNA sample by hybridization. The generated DNA fragment are separated by gel electrophoresis and polymorphism or genetic variation among genotypes are visualized as hybridization bands, wherein identical genotypes produce similar banding patterns while different genotypes have different banding pattern.

PCR BASED MARKERS- These markers do not require the probe hybridization step and it has led to the discoveries of several useful and easy to screen new generation markers. Being PCR based, these categories of markers require the use of primer pairs to select specific regions of the DNA for which genetic variation is measured. Primers are short sequences or nucleotides designed to be complementary to specific regions of DNA for amplification by PCR and sequence analysis. They therefore initiate the amplification of a particular DNA segment. The amplification products of DNA from different genotypes are separated on a gel to observe size dependent variation in banding pattern and may be further sequenced to identify sequence variations. PCR based molecular markers analysis involves DNA extraction from the Source material, determination of quantity and quality of the isolated DNA, amplification of the isolated DNA using PCR and gel electrophoresis. If sequencing is required, the amplification products are usually purified, subjected to sequencing PCR and further purified before sequencing. Some of the PCR based markers include, Random amplified Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP), Microsatellites or Inter specific simple Repeats and Single Nucleotide Polymorphism (SNP).

Random Amplified Polymorphic DNA (RADP) Marker
Random amplified polymorphic DNA were the first PCR-based molecular markers to be employed in genetic variation analysis as introduced by Williams et al., 1990. Random amplified polymorphic DNA markers are generated through the random amplification of genomic DNA using short primers, sepa4ration of the obtained fragment on agarose gel in the presence of ethidium bromide and finally visualization under ultraviolet light. The RAPD technique based on the polymerase chain reaction has been of the most commonly used molecular technique to develop DNA markers (Kumar, 2011). The RAPD technology provides quick and efficient screening for DNA sequence based polymorphism at a very large number of loci. The RAPD analysis has been extensively used for various purposes which include identification and classification of accession (Fukoka et al., 1992), identification of breeds (Qian et al., 1996) and genetic diversity analysis (Cao et al., 1997). The principle of RAPD includes a single short oligonucleotide primer which b nds to many different loci is used to amplify random sequences form a complex DNA template. This means that the amplified fragment generated by PCR depends on the length and size of both the primer and the target genome (Nandani, 2014). Random amplified polymorphic DNA is decamers (10 base pairs) in size, randomly generated, dominant markers and PCR –based marker system.
The success of their PCR is dependent on short arbitrary oliginucleotides that hybridizes onto the complimentary DNA fragments. These short oligonucleotide function in pairs with forward and reverse primers and they are used to amplify a set of DNA fragment. These markers are simple, inexpensive, and rapid and use arbitrary primers without any previous knowledge of individual sequences.

They may be used to detect DNA variations at different levels, ranging from single use changes to insertion and deletions. They are predominantly dominant and cannot distinguish heterozygous individuals. This system yields high polymorphism and it is easy and rapid because of its simplicity and requirement of minimal amounts of genomic DNA. The RADP can enhance the resolution of the PCR products and its repeatability. Several factors affect the reproducibility of RAPD reactions. These include quality and quantity of template DNA, PCR buffer, concentration of magnesium chloride, Taq DNA Polymerase source, annealing temperature, thermo cycler brand. The major advantage of RAPD include that it does not require pre-sequencing of DNA. Since the advantages of RAPD are the technical simplicity and the independence of any prior DNA sequence information (Qian et al., 1996). It is viewed as having several advantages compared to resolution fragment length polymorphism and finger printing. A disadvantage of RAPD marker is the fact that the polymorphism are detected only as the presence or absence of band of a certain molecular weight with no information on heterozygosity besides being dominantly inherited and also show some problems with reproducibility of data (Brumlop, 2010).

APPLICATIONS OF RAPD ANALYSIS
Because of the simplicity and low cost of the RAPD technique, it has found a wide range of applications in many areas of biology. Some of the areas where the technique is used are described below:

Genetic Mapping
Restriction fragment length polymorphisms (RFLPs) have been commonly used to map genes (Botstein et al., 2010). This approach involves hybridization of a probe (usually a cloned fragment) to Southern blotted genomic DNA digested with restriction endonucleases. A useful probe will detect differences in restriction fragment lengths arising from loss or gain of recognition sites or from deletions or insertions of stretches of DNA between sites. Because the Southern blot approach of RFLP analysis is time consuming and the identification and isolation of clones is often tedious, there is increasing interest in PCR technology, which allows amplification of any sequence of interest from nanogram amounts of DNA, and direct visualization of the amplified product. Although PCR seems promising in the amplification of mini satellites, macro satellites and sequence tagged site (STS) loci to make genetic mapping easy, the prior sequence information needed to design specific primers is a limiting factor in developing large numbers of genetic markers for any organisms (Goodier et al., 1993).

The necessity for sequence information for PCR was circumvented using short primers of arbitrary sequences to amplify DNA segments, namely RAPD. The speed and efficiency of RAPD analysis encouraged scientists to perform high-density genetic mapping in many plant species such as alfalfa, faba bean and apple in a relatively short time. Therefore, when mapping with dominant markers, it is necessary to use backcross or recombinant inbred populations, haploid or gametophytic tissue, or alternatively an F2 population where only RAPD markers amplified from a single parent are mapped (Williams et al., 1993). Alternatively, the RAPD technique could provide a ready source of hybridization probes for standard Southern blot analysis simply by isolating bands from gels to detect RFLPs. However, some polymorphic RAPD bands are not suitable as RFLP probes because of hybridization to repetitive DNA sequences (Williams et al., 1990).

Developing Genetic Markers Linked to a Trait in Question
One of the most widely used applications of the RAPD technique is the identification of Markers linked to traits of interest without the necessity for mapping the entire genome. Martin et al., have described an efficient method based on the RAPD technique to DNA segments linked to certain traits. This approach based on near-isogenic lines (NILs) is accomplished by repeatedly backcrossing a line carrying a gene of interest (donor parent) to a cultivated line having otherwise desirable characteristics (recurrent parent). The introgression of the target gene produces a line with a small segment of donor parent genome in a genetic back round, which is almost exclusively from the recurrent parent. Thus, markers that show polymorphisms between these 2 lines are likely to be linked to the gene of interest. Another RAPD-based approach is bulked segregant analysis (BSA) described by Michelmore et al., Arnheim et al., previously outlined a genome pooling strategy for RFLP markers that are not in linkage equilibrium to the targeted region of the genome. Bulk Segregate Analysis involves bulking DNA samples of individuals segregating in a single population. Bulked DNA samples from individuals that have the target trait or gene are compared to bulked DNA samples of individuals lacking the trait. RAPD markers that are linked to a chromosome or specific region of a chromosome were developed using the pooled DNA method. RAPD analysis of pooled DNA samples has further increased the efficiency of gene tracking (Barua et al., 1993).

 

Population and Evolutionary Genetics
The advances in DNA techniques have had a great impact in addressing problems in many aspects of biology. Application of DNA-based approaches to population genetic studies has been limited, probably due to the need for large samples of individuals from each population to provide an accurate estimate of allele and genotype frequencies. The relatively high cost, the requirement for sophisticated equipment and well-trained personnel, and low speed are other limiting factors in population genetic studies. The RAPD technique has received a great deal of attention from population geneticists (Hedrick, 1992) because of its simplicity and rapidity in revealing DNA-level genetic variation, and therefore has been praised as the DNA equivalent of allozyme electrophoresis (skibinki, 1992). A major drawback of RAPD markers in population genetic studies of out breeding organisms is that they are dominant. Thus gene frequency estimates for such loci are necessarily less accurate than those obtained with co dominant markers such as allozymes and RFLPs. Lynch and Milligan suggested that 2 to 10 times more individuals need to be sampled for dominant markers to achieve the same degree of statistical power as co dominant markers such as allozymes and RFLPs. The assumption of homology between bands of apparently the same molecular weight from the same primer is potentially another problem for RAPD surveys. Homology between co migrating bands in different individuals is a good assumption when individuals are from the same population. This may not be true when individuals belong to different species or widely divergent populations (Smith et al., 1994). Because the chance of co migrating bands being homologous becomes less as populations diverge, it was suggested that
RAPD analysis gives more accurate estimates between closely related populations and less accurate estimates for distantly related populations. RAPD data has been used for polygenetic studies and generally supported existing taxonomies based on morphology, isozymes and RFLPs. Some contradictions were found, especially among more distantly related plant species. The technique has also been used to study genetic variation in several fish species. RAPD markers are more suitable for clonal organisms than sexually reproducing organisms. As they breed asexually, a polymorphic fragment among individuals can be used to determine clonal identity. Species-specific markers were developed in species and strains of microorganism (Chalmers et al., 1992). Clone-specific markers have been identified in hydroids. Although the value of RAPD markers in taxonomic and phylogenetic studies is not very clear, there is no doubt that these markers can be used for diagnostic purposes. RAPD markers unique to individuals from 1 species within a genus will be species-specific (inter-specific).

Random Amplified Polymorphic DNA (RAPD) Markers Similarly, genus-specific markers can be generated if the fragment is a unique polymorphism to individuals belonging to a certain genus. Species-specific markers can be used in inter-specific gene flow and hybrid identification. Similarly, population-specific markers will be useful in identification of hybrid populations (Hadrys et al., 1992). RAPD polymorphism detected among individuals within a given species has been used to determine paternity and kinship relationships. To date, RFLP markers have been used as genetic markers to monitor the transmission of useful QTL alleles from generation to generation in the course of breeding programs (Beckmann, 1993).
The RAPD technique enabled the development of large numbers of genetic markers more efficiently than RFLP based methods that have been used to construct saturated mapping of complex genomes. These markers can be used in monitoring these loci during introgression and selection programs. Markers linked to simple commercially important genetic traits such as disease resistance genes can also be identified from natural resources and introgressed into domestic strains or varieties. The ability of the RAPD technique to reveal intra-specific variation can be used in screening for the degree of inbreeding in commercial plant and animal species to prevent an increase in the frequency of deleterious recessive alleles in populations. Polymorphic RAPD markers transformed to SCAR markers can be more advantageous in commercial breeding programs if a quick assay can be developed to detect the presence or absence of the product (Paran, 1993).

REPRODUCIBILITY OF RAPD MARKER
Although the RAPD method is relatively fast, cheap and easy to perform in comparison with other methods that have been used as DNA markers, the issue of reproducibility has been of much concern since the publication of the technique. In fact, ordinary PCR is also sensitive to changes in reaction conditions, but the RAPD reaction is far more sensitive than conventional PCR because of the length of a single and arbitrary primer used to amplify anonymous regions of a given genome. This reproducibility problem is usually the case for bands with lower intensity. The reason for bands with high or lower intensity is still not known. Perhaps some primers do not perfectly match the priming sequence, amplification in some cycles might not occur, and therefore bands remain fainter. The chance of these kinds of bands being sensitive to reaction conditions of course would be higher than those with higher intensity amplified with primers perfectly matching the priming sites. The most important factor for reproducibility of the RAPD profile has been found to be the result of inadequately prepared template DNA. Differences between the template DNA concentration of 2 individuals DNA samples result in the loss or gain of some bands. Since RAPD amplification is directed with a single, arbitrary and short oligonucleotide primer, DNA from virtually from all sources is amenable to amplification. Therefore, DNA from the genome in question may include contaminant DNA from infections and parasites in the material from which the DNA has been isolated. Special care is needed for keeping out the DNA to be amplified from other sources of DNA.

INTER-SIMPLE SEQUENCE REPEAT (ISSR) MARKER
Inter simple sequence repeat (ISSR)-PCR is a technique which involves the use of microsatellite sequence as primers in a polymerase chain reaction to generate multilocus markers. It is a simple and quick method that combines most of the advantages of microsatellites and amplified fragment length polymorphism to the universality of random amplified polymorphic DNA. Inter simple sequence repeat markers are highly polymorphic and are very useful in studies on genetic diversity, phylogeny, gene tagging, genome mapping and evolutionary biology. Among the PCR based marker technique, ISSR is one of the simplest and widely used techniques which involve amplification of DNA segment present at an amplifiable distance in between two identical microsatellites repeat region oriented in opposite direction.
Though ISSR markers are dominant, they are more stable and reproducible because of their properties which are: ISSR markers have recently been found using extensively for finger printing, phylogenetic analysis, population structure analysis, and genetic mapping and marker assisted selection. Inter simple sequence repeat are usually 16-25 base pairs in a single primer PCR reaction in multiple genomic loci for the amplification of different sizes of ISSR sequence. The microsatellite can be either di-nucleotide or tirnucleotide. The marker uses PCR technique which overcomes limitations like high cost of AFLP, low reproducibility of RAPD and the flanking sequences to develop species-specific primers for single sequence repeat (SSR) polymorphism. Inter-simple sequence repeat markers segregate mostly as dominant markers following mendelian law of inheritance. These markers enable distinction between homozygote and heterozygote. They are simple, quick, highly reproducible and polymorphic. There is also no use of radioactivity and no prior information about the genomic sequence.

 

APPLICATIONS OF ISSR MARKERS
Because of the multilocus fingerprinting profiles obtained, inter simple sequence repeat analysis can be applied in studies involving genetic identity, parentage, clone and strain identification and taxonomic studies of closely related species. In addition, ISSRs are considered useful in gene mapping studies.

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