Malaria is an insect-transmitted parasitic disease characterized by recurrent episodes of fever and anaemia (loss of blood) in some cases caused by the Plasmodium species which is usually transmitted between mammals through the bite of the female Anopheles mosquito. It is a disease condition in human beings whereby there is an abnormally high body temperature and shivering (or cold) which generally affects the activeness of the affected individual. Malaria is one of the world’s deadliest vector-borne diseases that affect tropical regions of the world especially the sub-Saharan African continent where the disease burden is high. Vector-borne diseases are infectious diseases carried by other living organisms including mosquitoes, sand flies, ticks and water snails amongst others; and these diseases thrive predominantly in communities where environmental sanitation and living standards of the people are poor.
Vectors are living organisms that can transmit infectious diseases or infectious disease agents (parasites) between human beings and from animals to humans. A variety of vectors are blood-sucking in nature and this implies that they feed on human or animal blood during which they ingest pathogenic microorganism(s) that they transfer or inject into the body of susceptible human hosts when they feed on their blood. Typical example is the female Anopheles mosquito that feeds on human blood and ingests Plasmodium parasites (the causative agent of malaria) during the blood meal. The mosquito vector (in this case the female Anopheles mosquito) injects the ingested Plasmodium parasite into a new human host during their next blood meal, and this causes malaria in the later individual.
Malaria is by-far one of the world’s biggest killer infectious disease among the various vector-borne diseases in man. Other killer vector-borne diseases as shall be highlighted later in this Chapter includes Onchocerciasis, Schistosomiasis, Yellow fever, Dengue fever, Leishmaniasis and Chagas disease amongst others. Malaria to a great extent represents one of the world’s greatest public health problems, and it accounts for a high percentage of morbidity and mortality across the globe especially in subtropical and tropical countries as aforementioned. Historically, the disease is believed to have affected monkey and ape populations in Asia and Africa; from which it was transmitted to human populations. Malaria which was derived from an Italian word that means “Bad or spoiled Air” was first discovered by Charles Laveran (a French army surgeon) in 1880 from the blood of a soldier suffering from the disease.
Malaria is a disease condition caused by the bites from infected small flying, biting and sucking insects called mosquito. Particularly, the female Anopheles mosquito (a mosquito species responsible for biting and blood sucking) which feeds only on blood meal of animals (humans inclusive) is the vector that helps to transmit the parasite to humans especially after a successful blood meal. Ronald Ross was the first in 1877 to observe the parasitic forms of Plasmodium in the stomach cells of mosquito. This groundbreaking discovery in the malaria epidemic set the landmark for the definition of the different stages of the malaria disease episode (pre-erythrocytic, erythrocytic and sporogonic cycles) in both the mosquito vector and human host. The parasite responsible for the disease is known to live in the erythrocyte (red blood cells) of infected human hosts that has been previously bitten by the mosquito carrying the Plasmodium parasite.
Malaria is a disease that occurs predominantly in the tropical and subtropical regions of the world (Asia and Africa specifically), and it sometimes causes recurrent infections in millions of children and even adults in this part of the world. Despite global and national control strategies for malaria infection, coupled with the development of novel antimalarial drugs, the disease burden of Plasmodium malarial infection still persist. Plethora of programmes including the Roll back Malaria Initiative of the United Nations have all been geared towards finding a lasting solution to the malaria pandemic, yet total cure for the scourge is still far from reach. Though several effective treatment measures are available for malaria infection, the disease still remains a significant human illness worldwide.
Malaria is a major global public health problem, and it is adjudged to be by far one of the most important tropical diseases, causing great pain, suffering and even death to people in the tropic and subtropical regions of the world (Nigeria for example). Other parasitic diseases that are of great concern especially in Africa (which has had its fair share of the malady) according to the Tropical Disease Research (TDR) department of the World Health Organization (WHO) includes: Onchocerciasis, schistosomiasis, filariasis, trypanosomiasis and Leishmania. Together, these diseases (malaria inclusive) affect over 500 million people across the globe. Tropical Africa is endemic with malaria, and the disease according to the World Health Organization (WHO) affects about 350 million people worldwide. Each year over one million of these people die from the disease (according to WHO); and this has made the sickness one of the world’s leading cause of death.
HUMAN PLASMODIUM SPECIES
Malaria in humans is majorly caused by four (4) species of Plasmodium. Plasmodium species are in the Phylum Alveolata, Subphylum Apicomplexa, Class Haematozoa, Order Haemosporida, and Genus Plasmodium. Plasmodium parasite is naturally transmitted to susceptible human hosts from an insect vector called female Anopheles mosquito. The 4 major and well known infectious species of Plasmodium that cause malaria in humans are:
- Plasmodium falciparum
- Plasmodium vivax
- Plasmodium malariae
- Plasmodium ovale
Of these four species, P. falciparum is known to be extremely harmful and most aggressive of them all. P. falciparum it accounts for the main global burden of malaria. The genus Plasmodium is classified as a blood sporozoan. Species of parasites classified as sporozoa (singular: sporozoan) are known to undergo a complex form of life cycle with alternating asexual and sexual reproductive stages that usually occur in two different hosts (a human and an arthropod). Other examples of blood sporozoan includes: Toxoplasma gondii, Isospora belli, and Cryptosporidium.
Plasmodium species can also cause fever-like illnesses in rodents, but the species responsible for malaria in these animals are quite different from those that cause malaria in humans. The rodent malaria parasites are as follows:
- Plasmodium yoelii
- Plasmodium vinckei
- Plasmodium berghei
- Plasmodium chabaudi
Plasmodium knowlesi is parasitic in monkeys.
PATHOGENESIS AND LIFE CYCLE OF THE MALARIA PARASITE (Plasmodium)
Inside the human body, the Plasmodium parasite attacks the liver cells which are their first spot of call upon invasion. They penetrate into the liver cells and destroy the cells in which they reside (including the red blood cells). Anaemic conditions (i.e. shortage of blood in the human host) may arise since the Plasmodium parasite destroys some liver cells and many erythrocytes (red blood cells). This condition can lead to death following the drastic reduction in oxygen supply, hormonal circulation, and nutrient (food) circulation to cells of the body. The Plasmodium parasite is usually picked and ingested by the insect vector during a blood meal in which it ingests viable plasmodial gametocytes that develops in the insect’s gut (Figure 1).
This eventually transforms into the infective forms (sporozoites) that appears in the vector’s salivary gland and is passed on to uninfected human host during the insect’s next blood meal. After inoculation by the female anopheles mosquito, the Plasmodium parasites mature first in the liver of the human host for a period of about one week before it goes on to complete its multiplication in the red blood cell. This continues until it reaches levels that cause fever (a rise in body temperature) and a wide variety of clinical signs and symptoms in the infected host. The spleen is another vital organ which plays an important role in malaria disease because it is known to confer some level of immunity or protection against malaria to the host. The spleen usually enlarges during acute malaria as it helps to remove erythrocytes that have been invaded by Plasmodium parasites. Malaria disease causes a high mortality in asplenic individuals (those whose spleen has been surgically removed); and such individuals stand a high risk of P. falciparum infection because there spleen is no longer there to perform its usual flushing and cleansing action.
Figure 1: Life cycle of Plasmodium. 1. Malaria-infected female Anopheles mosquito inoculates sporozoites into the human host. 2. Sporozoites infect liver cells 3. And mature into schizonts 4. Schizonts rupture and releases merozoites 5. Merozoites infect red blood cells. 6. Ring stage trophozoites mature into schizonts, which rupture releasing merozoites. 7. Some parasites differentiate into gametocytes (sexual erythrocytic stage). 8. Gametocytes (microgametocytes and macrogametocytes) are ingested by a female Anopheles mosquito during a blood meal. 9. The microgametocytes (male gametes) penetrate the macrogametocytes (female gametes) to generate zygotes in the mosquito’s stomach (midgut). 10. The zygote transforms to ookinetes (motile and elongated zygotes). 11. Ookinetes invade the midgut wall of the female Anopheles mosquito and develop into oocysts. 12. Oocysts grow, rupture, and releases sporozoites that move to the mosquito’s salivary gland until the next blood meal. The life cycle of malaria is perpetuated when the sporozoites in the salivary gland of the female Anopheles mosquito is transferred to a new human host during a blood meal. CDC
Pre-erythrocytic stage: Pre-erythrocytic stage which can also be called the exo-erythrocytic cycle is the first stage of Plasmodium parasite development in humans, and it occurs inside the cells of the liver (hepatocytes). It is the stage of the Plasmodium parasite development that occurs in the liver cells of the human host (outside the red blood cells) immediately after the introduction or inoculation of sporozoites (infective stage of Plasmodium parasite) into the human blood stream after the bite of an infected female Anopheles mosquito. In this stage, the Plasmodium parasite only multiplies in the hepatocytes of the human liver. After a successful blood meal (or bite from a female Anopheles mosquito), sporozoites leaves the salivary gland of the mosquito and find their way to the bloodstream of the human host where they circulate for a little time period before attacking or entering the hepatocytes. The infection of the liver cells (hepatic infection) in humans is usually asymptomatic (i.e. without any fever or cold), and this may last for several days. The sporozoites remain in the hepatocytes for about 10 days for replication and, eventually mature into schizonts. Upon the rupturing of the schizonts in the hepatocytes, thousands of merozoites are released into the human bloodstream. This marks the beginning of the erythrocytic stage (i.e. the invasion of the red blood cells). It is noteworthy that relapsing fever (or recurrent malaria infection) can also occur in humans after successful treatment, when the dormant stage (hypnozoites) of some Plasmodium species (in particular: sporozoites of P. vivax and P. ovale) persist in the liver; and in turn invade the erythrocytes to cause relapsing fever weeks or years later. The merozoites do not return again to the liver cells from the bloodstream after its departure or release from hepatocytes. This implies that malaria infection (P. falciparum infection) can naturally terminate without any therapy in a year except the disease ends in the death of the sufferer.
Erythrocytic stage: Erythrocytic stage is the phase of Plasmodium parasite invasion into the red blood cells (RBCs), and which usually marks the clinical manifestation of the malaria disease. It is the stage where the Plasmodium manifests itself in the bloodstream with several nuclear division and replication. The erythrocytic stage usually has two phases: the asexual stage (where schizogony are formed) and sexual stage (where gametocytes are formed). The asexual stage is marked by the invasion of the erythrocyte by merozoites after their release from the liver cells. The Plasmodium parasite undergo asexual multiplication in the erythrocytes (this is known as erythrocytic schizogony), and the merozoites mature inside the RBCs from a ring to a mature trophozoite. The ring stage trophozoites then undergo asexual division (schizogony) to form schizonts that ruptures to release more merozoites. The rupturing of schizonts marks the start of malaria symptoms in the human host following the action of the Plasmodium parasite on the host’s cells to release cytokines. This cycle is repeated for approximately 48 hours (for P. falciparum, P. vivax, and P. ovale) or 72 hours (for P. malariae) and parasitaemia continues depending on the infecting Plasmodium parasite. Sexual stage in the human host is usually characterized by the entering of some parasite merozoites into the erythrocytes. These merozoites later differentiate into male gametocytes (microgametocytes) and female gametocytes (macrogametocytes). This marks the beginning of the sexual phase in the human host, but this sexual stage of the Plasmodium development is not completed in humans but in the insect vector. This occurs when the gametocytes (male and female) are picked up and ingested by female Anopheles mosquitoes during a blood meal; and this ingestion allows the insect vector to continue the transmission of the Plasmodium parasite to susceptible human hosts as it sucks and feed on their blood. It is noteworthy that not all merozoites released from RBCs can go on to infect other erythrocytes. The gametocytes (which are offshoots of merozoites) cannot infect the erythrocytes but rather infects only mosquitoes.
Sporogonic stage: The sporogonic phase occurs solely in the female Anopheles mosquito, and it is characterized by the multiplication of the Plasmodium parasite in the midgut (stomach) of the insect vector. This stage in the Plasmodium parasite life cycle begins immediately after the ingestion of the gametocytes by the female anopheles mosquito during a blood meal. Inside the stomach of the mosquito, the male and female gametocytes undergo fertilization (usually meiosis and fusion of both cells) to form a zygote which later transforms into a motile and elongated form called ookinete. The ookinete enters the midgut wall of the mosquito’s stomach where they later develop into oocysts. The Plasmodium parasite usually takes about 10-35 days to develop within the insect vector (and this stage is called sporogony). The oocysts mature and rupture to release plenty of sporozoites which migrate to the salivary glands of the female Anopheles mosquito where they wait to be transmitted to a susceptible human host during a blood meal. The life cycle of the Plasmodium parasite infection (malaria episode) becomes completed when the sporozoites successfully gain entry into the vascular system of a human host during a blood meal.
SIGNS AND SYMPTOMS OF MALARIA
Malaria is usually accompanied with some specific symptoms, and these include: fever that reaches 40oC or more, chills or cold, headache, nausea, fatigue, vomiting, diarrhea, occasional cough, abdominal pain, splenomegaly (enlargement of the spleen), hepatomegaly (enlargement of the liver), jaundice, chest pain, and myalgia (muscle pain). Loss of appetite and bitterness of the mouth can also be experienced by some patients. Malaria symptoms are usually manifested in a human host following the rupturing of the red blood cells (erythrocytes) to release erythrocyte schizonts. This action triggers immune response in the host which leads to the formation of cytokines and other immune system products that spark up an inflammatory action that usually results to the chills and fever experienced by the individual. It is noteworthy that the clinical manifestation of malaria is ushered in following the invasion of the erythrocyte by the Plasmodium parasite.
FACTORS THAT AFFECT THE RESULT OF MALARIA DIAGNOSIS
The clinical and laboratory diagnosis of malaria is challenging to health practitioners, physicians and scientists alike owing to some diagnostic complexity associated with the disease. Malaria is a febrile infection that usually presents clinically with signs and symptoms that are significantly related to other feverish infections (of bacterial or viral origin) common in most tropical regions where the disease is widespread. To detect the parasite and administer therapy to the affected patient appropriately, it is vital that scientist take into consideration some of the prevailing factors that affect the reliability of malaria test results. Some of these factors which commonly promote the indiscriminate use of antimalarial drugs (a cause for emergence and spread of resistant Plasmodium strains) and hold back the specificity of identifying and correctly interpreting malaria parasitaemia diagnostic test results include:
- Administration of antimalarial drugs based only on clinical diagnosis.
- Presence of persisting viable or non-viable Plasmodium parasites in blood.
- Drug resistance of some Plasmodium
- Sequestration of Plasmodium parasites in host tissues.
- Movement of people from malaria-endemic regions to non-endemic areas and vice-versa.
- Endemicity of some Plasmodium species in some regions ( falciparum in sub-Saharan Africa).
- Signs and symptoms, and diagnosis of malaria infection.
- Issues arising from reporting non-malarial febrile infections as malaria in endemic regions (Africa and Asia).
LABORATORY DIAGNOSIS OF MALARIA
Aside, laboratory diagnosis, clinical diagnosis of malaria is often practiced in some quarters to diagnose the disease but this practice is not versatile (though traditional amongst most physicians) due to its non-specificity and the possibility of missing out other febrile non-malarial infections in the process. The diagnosis of malaria in the hospital or laboratory is usually suspected when patients present with febrile conditions. Usually venous blood or capillary blood specimens are obtained from affected patients, and these are examined microscopically in the laboratory. The diagnosis of malaria infection normally begins with the identification of Plasmodium parasites in the blood smears of patients. Thick and thin blood smears are usually made using the Giemsa staining technique. The ring forms of Plasmodium species as well as their trophozoites and gametocytes are often among the key features of the Plasmodium parasite looked for when performing microscopy on blood smears for detection of malaria parasitaemia. The ring forms of Plasmodium species (particularly P. falciparum) are morphologically endowed with cytoplasm and one or two small chromatin dots; and P. falciparum gametocytes usually assumes a crescent or sausage shape when observed under the microscope (Figure 2).
The trophozoites of P. falciparum assumes an amoeboid shape (Figure 3), and they are rarely seen in peripheral blood smears. While the thin blood smear helps to differentiate the different species of Plasmodium parasites, the thick blood film preparation helps to concentrate the parasite and help to detect mild cases of malaria parasitaemia. Serological techniques which involve the use of rapid diagnostic tests (containing monoclonal antibodies) to detect specific Plasmodium parasite antigens from blood specimens have also been introduced in malaria laboratory diagnosis. Most recently in the diagnosis of malaria parasitaemia is the use of molecular detection techniques including gene amplification methods, polymerase chain reaction (PCR) and nucleic acid probes to detect malaria parasites from blood specimens of patients. However, the Giemsa staining technique involving thick and thin blood films is still being used in most part of the developing nations as the primary diagnostic protocol for malaria parasitaemia. Abnormal liver function tests and an elevated lactate dehydrogenase tests coupled with low haemoglobin levels should also raise suspicion of malaria parasitaemia.
Figure 2: Gametocytes of P. falciparum (arrows) in thin (A) and thick (B) blood smear. CDC
Figure 3: Ring forms or trophozoites (arrows) of P. falciparum in thick (A) and thin (B) blood smear. CDC
OVERVIEW OF PROTOCOLS USED FOR THE DIAGNOSIS OF MALARIA INFECTION
- Microscopy: Microscopy as earlier explained is this textbook is still the gold standard for the laboratory diagnosis of malaria infection in many parts of the world where the disease occurs. It usually involves the microscopical investigation of peripheral blood smear (PBS) – which can either be a thick smear (for parasite detection) or thin smear (for parasite-species identification). All the four species of Plasmodium e. P. falciparum (Figure 4), P. malariae (Figure 5), P. ovale (Figure 6) and P. vivax (Figure 7) that causes malaria in humans appear differently under the microscope when a thick blood smear is made and examined. And these morphological appearances of the Plasmodium species aids in their identification in the laboratory. Microscopic technique though very relevant in the laboratory diagnosis of malaria is laborious, time-consuming and parasite-specie identification at low levels of parasitaemia is very challenging. This method requires well trained personnel for optimum result. Briefly, two types of blood specimens may be required for laboratory detection of malaria parasites using the microscopy technique: capillary blood obtained by fingerstick and venous blood obtained by venipuncture. To obtain capillary blood, clean the middle or ring finger of the patient with 70 % alcohol; allow to dry. Puncture the ball of the finger and wipe away the first drop with a clean cotton wool. For infants, the heel is usually punctured to obtain venous blood. Touch the next drop of blood with the center of a clean glass labeled slide. Venous blood is usually obtained in an EDTA bottle after drawing blood from the patient using a sterile string. For thin blood smear, place a drop of the blood at one end of a clean glass slide, and use another slide to push and spread the blood forward in a smooth and rapid fashion. For thick blood smear, place a drop of blood at the center of a clean glass slide, and use the edge of another slide to spread the blood in a circular fashion while making sure not to make it too thick. Allow the thin and thick blood smears to dry properly before staining. Thin blood smear is fixed with methanol prior to staining but thick blood smear is not fixed before staining.
Figure 4: Illustration of the morphological features of P. falciparum in a thick smear. 1. Small trophozoites. 2. Gametocytes (normal). 3. Slightly distorted gametocyte. 4. Rounded-up gametocyte. 5. Disintegrated gametocyte. 6. Nucleus of leukocyte. 7. Blood platelets. 8. Cellular remains of young erythrocyte. CDC.
Figure 5: Illustration of the morphological features of P. malariae in a thick smear. 1. Small trophozoites. 2. Growing trophozoites. 3. Mature trophozoites. 4, 5, 6. Immature schizonts with varying numbers of divisions of the chromatin. 7. Mature schizonts. 8. Nucleus of leukocyte. 9. Blood platelets. 10. Cellular remains of young erythrocytes. CDC
Figure 6: Illustration of the morphological features of P. ovale in a thick smear. 1. Small trophozoites. 2. Growing trophozoites. 3. Mature trophozoites. 4. Schizonts. 5. Gametocytes. 6. Nucleus of leukocyte. 7. Blood platelets. CDC
Figure 7: Illustration of the morphological features of P. vivax in a thick smear. 1. Amoeboid trophozoites. 2. Schizont- 2 divisions of chromatin. 3. Mature schizonts. 4. Microgametocyte. 5. Blood platelets. 6. Nucleus of neutrophil. 7. Eosinophil. 8. Blood platelets associated with cellular remains of young erythrocytes. CDC
Note: Allow the fixed thin film to dry before staining. Giemsa stain is usually used for staining blood smears required for detection of Plasmodium parasites, and stained smears are viewed under oil immersion objective lens. In thin smear preparations, the morphological appearances of the components of the Plasmodium species i.e. P. falciparum (Figure 8), P. malariae (Figure 9), P. ovale (Figure 10) and P. vivax (Figure 11) also differ slightly from their appearances in a thick smear preparation.
Figure 8: Illustration of the morphological appearance of P. falciparum in a thin smear preparation. 1. Normal red cell. 2-18. Trophozoites (Note: image 2-10 shows the ring-stage trophozoites). 19-26. Schizonts (Note: image 26 is a ruptured schizont). 27, 28. Mature macrogametocytes (female). 29, 30. Mature microgametocytes (male). CDC.
Figure 9: Illustration of the morphological appearance of P. malariae in a thin smear preparation. 1. Normal red cell. 2-5. Young trophozoites (rings). 6-13. Trophozoites. 14-22. Schizonts. 23. Developing gametocyte. 24. Macrogametocyte (female). 25. Microgametocyte (male). CDC.
Figure 10: Illustration of the morphological appearance of P. vivax in a thin smear preparation. 1. Normal red cell. 2-6. Young trophozoites (ring stage parasites). 7-18. Trophozoites. 19-27. Schizonts. 28, 29. Macrogametocytes (female). 30. Microgametocyte (male). CDC.
Figure 11: Illustration of the morphological appearance of P. ovale in a thin smear preparation. 1. Normal red cell. 2-5. Young trophozoites (ring). 6-15. Trophozoites. 16-23. Schizonts. 24. Macrogametocytes (female). 25. Microgametocytes (male). CDC.
- Rapid diagnostic tests (RDTs): RDTs are immunochromatographic techniques that employ specialized malaria detection test kits for the laboratory diagnosis of malaria infection. They are easy to use and very reliable in the prompt detection of malaria parasites from patient’s specimens. Unlike the microscopic technique, RDTs does not require any specialized training as they can be used based on the manufacturers instruction. RDTs are cost-effective and they were developed to take care of some of the lapses associated with microscopy such as time wastage and inability to detect parasites at low levels of parasitaemia. Some of the commercially available RDTs include ParaSight F, ICT Malaria Pf, and OptiMAL amongst others.
- Quantitative Buffy Coat (QBC): The QBC technique is a centrifugation method used for the laboratory diagnosis of malaria infection. In this method, blood specimens are centrifuged in specialized tubes (e.g. haematocrit tubes) containing anticoagulant such as EDTA and acridine orange. After centrifugation, the tubes are examined under a fluorescent microscope to detect Plasmodium nuclei or DNA and other parasite components. The acridine orange stains the DNA of the parasite during centrifugation, and it appears as a fluorescing bright-green matter under the fluorescent microscope. The cytoplasm appears as a yellow-orange matter. QBC technique is not versatile in the laboratory detection of malaria infection due to its high cost and inability or poor performance to detect parasite species and numbers.
- Serology: Serological tests have also been employed in the laboratory diagnosis of malaria infection. They are mainly based on the detection of specific antibodies produced against the malaria parasite in the blood (serum) of infected individuals. Immunoflourescence antibody testing (IFA) which employs antigen-antibody reactions is one of the versatile methods used in most serological tests.
- Polymerase Chain Reaction (PCR): PCR is the most sensitive and specific technique for the diagnosis of malaria infection as it can differentiate between mixed infection and actual malaria infection. Unlike other techniques, PCR can detect malarial parasites at very low levels of parasitaemia at the molecular level. However, its usage for the routine diagnosis of malaria in most malaria-endemic regions has been limited by unavailability of specialized trained personnel, high cost of purchase and maintenance, and its mode of operation is more complex than the microscopic technique and other methods used for the laboratory diagnosis of malaria infection.
- Other tests: Most traditional methods of diagnosing malaria though versatile and inexpensive to perform are usually problematic and challenging. Some of these methods (for example clinical diagnosis) are unreliable while others require a well trained health personnel and equipments which may be lacking in some local communities where the incidence of malaria infection is high. Prompt detection and diagnosis of malaria infection in places where Plasmodium parasite transmission is widespread is vital to the reduction of the morbidity and mortality due to the disease. The challenges associated with some of the traditional methods of diagnosis and detecting malaria parasites in the laboratory and clinical settings have stepped up the need for newer and better detection protocols with high specificity and sensitivity for parasite detection. These newer techniques used for the diagnosis of malaria infection detect malaria parasites from patient’s specimens at the molecular level; and they include: real-time PCR technique, reverse transcription PCR, nested PCR, and DNA microarrays. Though very reliable, specific and sensitive in detecting malaria parasites from patient’s specimen; some of these techniques are not versatile and available for the routine diagnosis of malaria infections in most regions of developing countries where the disease is prevalent. They require specialized trained personnel and high cost of maintenance. Thus, some of these techniques are only practiced or used in reference laboratories for the detection of malaria parasites. ELISA, parasite culture techniques, enzyme immunoassay, flow cytometry, LAMP technique and mass spectrophotometry are other non-molecular techniques which are currently been employed for the prompt detection and diagnosis of malaria infection.
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