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

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

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.
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 positive samples
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 of 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 Gunbe metropolis. Ejim and mbanugo in their study reported of that out of 400 children living in Awka were facliparum more found. 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 disquinshing 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 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. The 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 the technicians 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.

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