CHAPTER ONE
1.0 INTRODUCTION
Malaria is a life-threatening illness, that has continued to pose public health challenges. It affects millions of people all around the globe especially, in Africa, Asia and South America. Malaria is currently endemic in over 100 countries with 3 billion people at risk of infection and around 225 million cases in 2009, leading to approximately 781,000 deaths (WHO, 2010). Malaria has remained a major public health problem in Nigeria, and is responsible for 30% childhood and 11% maternal mortality (FMoH, 2005). It accounts for 300,000 deaths each year and about 60% of outpatient visits (President’s Malaria Iniative, 2011). Together Nigeria, and the Democratic Republic of Congo account for over 40% the estimated total malaria burden and deaths globally (WHO, 2012). It is caused by the asexual form of the parasitic protozoan know as Plasmodium. The species incriminated are Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale which is found humans and Plasmodium knowlesi which found in non-humans. Among these parasites, Plasmodium falciparum and Plasmodium vivax are the most widespread and common causes of mixed-species malaria, which is defined as co-infection with more than one species or genotype of Plasmodium (Mayxay et al., 2004).
Most cases of malaria are uncomplicated, commonly presenting with fever and sometimes with other non-specific symptoms including headache, and aches and pains elsewhere in the body (Gilles, 1991; WHO, 2003). Mtoni and Senosi (2007) noted that early diagnosis and treatment are key to addressing morbidity and mortality due to malaria. Proper management of malaria cases within the first 24 hours of onset is considered to be the best way to reduce its morbidity and mortality (Singh et al., 2013). This would be adequately achieved if most of the patients have access to laboratory facilities (Kamugisha et al., 2008). Most victims of malaria still die, because the disease is not diagnosed in time by health workers (Uzochukwu et al., 2009). Microscopy is the gold standard for laboratory diagnosis of malaria in many developing countries, though expertise may be lacking in both endemic and non-endemic settings (Moody, 2002), especially in Nigeria. However, in situations lacking reliable microscopic diagnosis, rapid diagnostic tests (RDTs) may offer a useful alternative to microscopy (Nour et al., 2009).
In general, RDTs are fast, easy to perform and relatively cheap (Lubell et al., 2007). A lot of research and development has been going on to develop alternative methods for laboratory diagnosis of malaria. Rapid diagnostic tests have been developed, validated and field tested. It was introduced in the nineties, but has now undergone many improvements (Martha et al., 2010). Malaria rapid diagnostic test plays a key role in malaria control and elimination programmes in order to avoid unnecessary anti-malarial therapy, to prevent drug resistance and to enhance case finding (Eibach et al., 2013). The RDTs are based on the principle of immunochromatography, which require finger prick blood and detect malaria specific antigen. There are three different RDTs that are available commercially; one of them is specific for detecting P. falcipraum antigens, while the other two detects one or more of the three human malaria species. The RDTs provide quick results, are reliable, and require less skilled persons as compared to microscopic diagnosis. They do not require electricity or any equipment. It promotes patient’s confidence as well as health services.
More than 60 RDT brands and over 200 different products have been developed. Of these, the WHO and Foundation for Innovative New Diagnostics (FIND) evaluated 70 from 26 manufacturers (WHO, 2008; 2009). Of these products, 39 are three-band tests that detect and differentiate P. falciparum from non falciparum species (Martha et al., 2010). The CareStart™ Malaria HRP-2/ pLDH (Pf/pan) Combo Test and the SD Bioline Ag pf/pan, HRP-2 and pan-pLDH are both a three-band RDT detecting HRP-2 and pan-pLDH. This present study is focused on evaluating the efficacy of two of the many RDTs; SD Bioline and CareStart™ Malaria kits using it microscopy test as the gold standard for the diagnosis of malaria.
SD Bioline (Ag pf/pan, Cassette, RDT, kit) is a one step differential diagnosis by detecting HRP-II antigen from Plasmodium falciparum and pLDH antigen from other species (P. vivax, P. malariae, P. ovale) in human whole blood. The CareStart (Combo, dev., RDT) is a test designed for the differential diagnosis between Plasmodium falciparum and other Plasmodium species such as Plasmodium vivax, Plasmodium ovale and Plasmodium malariae. Though, the gold standard for malaria testing remains microscopy, but the limitations associated with this technique could affect the speed of delivery of quality services to the patients (Ameh et al., 2012).
1.1 Statement of the Problem
Microscopy has been in use for over 100 years and is inexpensive, rapid and relatively sensitive when used appropriately (Laveran, 1891). Microscopy is regarded as the ‘gold standard’ for malaria diagnosis (WHO, 1999). However, the lack of skilled scientists in medical facilities in affected areas often leads to poor interpretation of data. In addition, microscopy is time consuming, labour intensive, and cannot detect sequestered P. falciparum parasites (Leke et al., 1999). It is less reliable at low-density parasitaemia that is, 50 parasites (ml blood) (Kilian et al., 2000; Bell et al., 2005). Even though microscopy is cheap, reliable and available on an instant base, it has limitations. For instance, in resource-limited centres, there are problems of equipment, training manpower, and workload, whereas in non-endemic countries, laboratory staff may lack sufficient exposure to malaria positive samples resulting in low expertise (Moody, 2002; Hanscheid, 2003).
In Nigeria, RDTs are still new to the people, and they are unsure of the efficacy, accuracy and authenticity. It has been 7 years since the launching of malaria RDTs in Nigeria but the populace know little or nothing about Malaria RDTs due to poor promoting from the part of manufacturers. In addition, the implementation of RDTs also faces many difficulties such as logistics; transport and continuous supply, limited shelf life and the need of proper storage rooms. RDTs are quickly affected by humidity and extreme temperatures (Wongsrichanalai et al., 2007). They are not able to quantify parasitaemia and may give false positive results owing to the persistence of antigens that can remain in the circulation of a patient after treatment (Wongsrichanalai et al., 2007).
1.2 Significance of the Study
The essence of continuous research and development is to find a way to improve the lives of people around the globe. Thus, finding an alternatively cheap, fast, convenient and effective way to diagnosis malaria is a key to control malaria. This study is therefore significant in many ways:
1.3 Research Questions
1.4 Research Hypothesis
HA: RDTs are more efficient in the detecting of malaria cases than microscopy
HO: Microscopy is more efficient in defecting malaria than RDTs
The aims and objectives of this study were to:
CHAPTER TWO
LITERATURE REVIEW
2.1 Taxonomy of Malarial parasite:
Phylum - Apicomplexa (Sporozoa)
Class - Haemosporidea (Sporozoea)
Order - Haemosporidia
Genus - Plasmodium
Sub-genus: Plasmodium, Laverania, Vinckeia
Species: There are about 120 species, of which 4 infect man. These are P.falciparum, P.vivax, P.ovale, P.malariae (Sinden and Gilles, 2002) and P. Knowlesi (which found in non-human species).
2.2 Morphological forms of Malaria parasites
Plasmodium falciparum exists in the tropics and sub-tropics, and is responsible for approximately 50% of all malaria cases. The incubation period of P. falciparum malaria is the shortest, between 8 and 11 days and has a periodicity of 36–48 hours. It can be differentiated from the other species by the morphology of the different stages found in the peripheral blood. In infections with Plasmodium falciparum usually only young trophozoites and gametocytes are seen in peripheral blood smears, the schizonts are usually found in capillaries sinuses of internal organs and in the bone marrow. The disease it produces runs an acute course and often terminating fatally. It is a significant cause of abortions and stillborns and even death of non- immune pregnant women (Mejbah, 2007; Ahiable, 2009).
Morphology of Trophozoites
Red blood cells in Plasmodium falciparum infections are not enlarged and they may have a spiky outline which is common in cells which have dried slowly. The typical arrangement of the cytoplasm in young trophozoites is the well-known ring formation which thickens and invariably contains several vacuoles as the trophozoite develops. Chromatin is characteristically found as a single bead, but double beads and small curved rod forms frequently occur. Maurer’s dots are slow to appear and are first seen as minute purplish dots, 6 or less in number as described in Table 2.1. The points become spots, still few in number and are now characteristic enough to be recognized. Maurer describes them as fine ringlets, loops or streaks (Arora and Arora, 2009). They are seldom absent from the red blood cells containing large rings but the staining of the spots is very sensitive to pH and are seldom seen if the pH falls below 6.8. Trophozoites of P. falciparum can be found on the edge of the red blood cells. These are known as accole forms and are found as three distinct types:
1. Common: The single chromatin bead lies on the edge of the cell with most of the cytoplasm extended along the edge on both sides of the bead.
2. Rim: The complete parasite lies in a thickened line along the edge of the cell with no evidence of ring formation.
3. Displaced: The parasites are displaced beyond the edge of the host cell. All degrees of displacement may occur, from partial to marked displacement with most of the parasite lying beyond the cell margin. Pigment is not a characteristic finding in the early stages of P. falciparum infections.
Morphology of Gametocytes
Gametocytes are the sexual stage of the malaria parasite. Plasmodium falciparum gametocytes appear in the peripheral circulation after 7-12 days of patent parasitemia and by then, they have assumed their typical crescent shapes. They soon reach their peak density, and then decline in numbers, disappearing in about three months as a rule. The ring thickens and invariably contains several vacuoles as the trophozoite develops. Maurer’s dots are slow to appear and are first seen as minute purplish dots. The female form, or macrogametocyte, is usually more slender and somewhat longer than the male, and the cytoplasm takes up a deeper blue color with Giemsa stain. The nucleus is small and compact, staining dark red, while the pigment granules are closely aggregated around it. The male form, or microgametocyte, is broader than the female and is more inclined to be sausage shaped. The cytoplasm is either pale blue or tinted with pink and the nucleus, which stains dark pink, is large and less compact than in the female, while the pigment granules are scattered in the cytoplasm around it.
Morphology of Schizonts
Schizonts are rarely seen in the peripheral blood and their presence may indicate a potentially serious parasitemia. Schizonts have 8-36 merozoites and a large mass of golden brown pigment (hemozoin) is seen in the pre-schizont and schizont stage as described in Table 2.1. They are small, about 4.5-5.0 µm in diameter, fills two thirds of normal-sized red blood cell (Arora and Arora, 2009)
Fig 2.1: Plasmodium falciparum schizont. Rarely seen in the peripheral blood, a good indicator of a potentially serious parasitemia. They have 8 – 36 merozoites and a large golden brown pigment. (Giemsa stain) (SOURCE: CDC/Steven Glenn, Laboratory & Consultation Division, 2014)
Plasmodium vivax is found almost everywhere malaria is endemic and is the most predominant of the malaria parasites. Causing 43% of all cases of malaria in the world, it also has the widest geographical distribution. Although the disease itself is not usually life threatening, it can cause severe acute illness. Plasmodium vivax does not infect West Africans due to the fact that West Africans do not possess the Duffy Antigen on the red blood cells which the parasite requires to enter the red blood cell. It has an incubation period of between 10 and 17 days which is sometimes prolonged to months or years due to the formation of hypnozoites. It has a periodicity of 48 hours (Arora and Arora, 2009). Plasmodium vivax infections are usually characterized by the presence of more than one developmental stage in the peripheral blood film. The parasites parasitize young enlarged erythrocytes and Schüffner’s dots develop on the erythrocyte membrane.
Morphology of Trophozoites
Most trophozoites of P. vivax are already several hours old when they appear in peripheral blood and by that time the Schüffner’s dots are already visible. The trophozoites are actively amoeboid and contain single or sometimes double chromatin dots that are either circular or ovoid. As the trophozoites mature, the Schüffner’s dots increase in number and size and the parasite changes from large irregular rings to rounded or ovoid forms in mature trophozoites. Trophozoites of Plasmodium vivax are already several hours old when they appear in the peripheral blood and therefore, you can already see the Schüffner's dots as shown in Table 2.1. They contain single or sometimes double chromatin dots.
Morphology of Gametocytes
Mature female gametocytes are large rounded parasites which fill or nearly fill the host cell. The cytoplasm is blue and fairly homogenous. The nuclear chromatin is a single, well-defined purplish mass, varied in form and usually peripheral in distribution. Mature male gametocytes can be distinguished from females by the large, loose and ill-defined mass of chromatin and by their paler color and smaller mass. Mature female Plasmodium vivax gametocytes are large rounded parasites which fill or nearly fill the host cell. The cytoplasm is blue and fairly homogenous. The nuclear chromatin is a single, well-defined purplish mass, varied in form and usually peripheral in distribution.
Morphology of Schizonts
The parasitized red cells are much enlarged containing Schüffner’s dots. The parasites are large, filling the enlarged red cell. There are between 14-24 merozoites in the schizonts (Arora and Arora, 2009). The pigment is a golden brown central loose mass.
Fig 2.2: Trophozoites of Plasmodium vivax are already several hours old when they appear in the peripheral blood and therefore, you can already see the Schüffner's dots. They contain single or sometimes double chromatin dots. (Giemsa stain) (SOURCE: CDC/ Steven Glenn, Laboratory & Consultation Division, 2014)
Plasmodium ovale is widely distributed in tropical Africa especially the west coast, but it is not often encountered. It has also been reported in South America and Asia. It has an incubation period of 10–17 days which is sometimes prolonged to months or years due to the formation of hypnozoites. It has a periodicity of 48 hours; the fever it produces is milder than that caused by the benign tertian P. falciparum as shown in Table 2.1.
Morphology of Trophozoites
Parasites of P. ovale are usually found in enlarged and stippled red blood cells (James’s dots), similar to those found in P. vivax infections. Host cells show an oval shape, particularly those containing younger stages of the parasites and the host cell may also show “spiking” or fimbriation.
Young trophozoites are found as compact rings in cells containing Schüffner’s dots. The trophozoite rings remain compact as they develop and show little of the amoeboid features common in P. vivax Small, scattered pigment granules can be seen in developing trophozoites which disperse as the trophozoite matures. Late trophozoites are round and consolidated with an increase in cytoplasm, they are very similar to P. vivax at this stage.
Morphology of Gametocytes
The mature gametocytes are round, filling two thirds of the red cell. The red blood cell is slightly enlarged and stippled and contains pigment which has a distinct arrangement of concentric rods, mostly at the periphery.
Morphology of Schizonts
The parasite is smaller than red blood cells and contains 6-12 merozoites, usually 8 in a single ring (Arora and Arora, 2009). The pigment is a dark brown (Arora and Arora, 2009). The red cell slightly enlarged, stippled, frequently oval and fimbriated.
4. Plasmodium malariae
Plasmodium malariae occurs mainly in the subtropical and temperate areas where P. falciparum and P. vivax occur. It is responsible for approximately 7% of all malaria in the world. It has an incubation period of 18–40 days and a periodicity of 72 hours (Arora and Arora, 2009).
Morphology of Trophozoites
Parasites of P. malariae are typically compact heavily pigmented parasites which are usually smaller and more deeply stained than normal. They tend to parasitize small, old red blood cells, they do not contain any inclusion dots and the parasitaemia is usually low.
Trophozoites are found as fairy large fleshy rings with a single chromatin dot. These can be very distorted and can often take the form of bands across the cell. All trophozoites have a single chromatin dot and contain pigment.
Plasmodium malariae gametocyte.
They contain large amounts of black pigment, with chromatin present as a compact mass in females (macrogametocyte) and diffuse in males (microgametocyte).
Morphology of Schizonts
Schizonts are usually few in numbers with 6-12 large merozoites in a single ring (Arora and Arora, 2009). Pigment is usually present as a central black mass. The parasites present are generally only found at one stage of schizogony development.
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