Malaria

Detailed description of malarial parasites especially P. falciparum with regards to their Morphology, Life cycle, Pathogenesis, Epidemiology, Clinical manifestations and complications and Laboratory diagnosis including modern methods and treatment.

1. MALARIAL PARASITES Dr. Suprakash Das Assist. Prof. 1

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3. General Characteristics & Classification The Apical complex consists of  Polar rings, Rhoptries, Micronemes, Mitochondria, Microtubules and Micropores. It is visible only by EM. 3

4. Apicomplexa 4

5. Malarial Parasites of Medical Importance Plasmodium falciparum Plasmodium vivax Plasmodium ovale Plasmodium malariae Plasmodium knowlesi 5

6. Characteristics of the Family- PLASMODIIDEA Development of Micro- and Macro- gametes takes place independently Each microgamont produces eight microgametes Zygotes are motile Sporozoites are naked with a 3 layered wall Merogony and Sporogony takes place in Vertebrate hosts and Insect hosts respectively Parasites are transmitted by Insect hosts. 6

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8. Timeline of Malaria 8

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10. Timeline of Malaria 10

11. Timeline of Malaria 11

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13. Plasmodium falciparum - LIFE CYCLE Malarial parasites shows alteration of generation with alteration of hosts in Human ( INTERMEDIATE HOST ) and in Mosquitos ( DEFINITE HOST ). HUMAN CYCLE Human is the intermediate host. Parasite reproduce by ASEXUAL METHOD (SCHIZOGONY) 13

14. Plasmodium falciparum - LIFE CYCLE Human cycle begins with a bite of Infected Female Anopheles mosquito/ Transfusion of Infected blood (Except-> Transfusion malaria and Congenital malaria ) There are 2 stages in the Human cycle  1] Exo-erythrocytic (EE) schizogony in the Liver 2] Erythrocytic schizogony in the RBCs. 14

15. Exo-erythrocytic Schizogony Mosquito during biting inject SPOROZOITES along with saliva into the small blood vessels. Motile sporozoites are carried to the LIVER by the blood stream (30 mins ) The surface of the sporozoites is covered by a protein k/a Circumsporozoite protein (CSP) which has a ligand for receptor in the hepatocyte cell membrane. Within the hepatocytes , the sporozoites undergo a stage of ASEXUAL REPRODUCTION k/a- Primary EE schizogony (PE) 15

16. Exo-erythrocytic Schizogony During this cycle, the sporozoites are transformed into TROPHOZOITES. (Organelles of the Apical complex disappear and growing trophozoites feed on host cell cytoplasm) Mature trophozoites begin schizogony -> numerous daughter nuclei are first produced subsequently leading to the development of Multinucleate Liver Stage Schizonts ( EE Schizont ) Mature EE Schizonts are Spherical, 60µm in diameter, contain 2000- 50,000 uninuclate MEROZOITES 16

17. Exo-erythrocytic Schizogony Finally mature EE schizonts and enlarged liver cells ruptures releasing thousands of merozoites into the blood straem . In P. falciparum only a single cycle of Primary EE schizogony takes place. It is completed in 6 days . The secondary schizogony which occur in P. vivax and ovale is absent in falciparum and no HYPNOZOITES (the stage responsible for Relapse ) are produced in falciparum infection. 17

18. Exo-erythrocytic Schizogony Once the merozoites enter the erythrocytes , they never reinvade the liver. So EE forms are absent in Transfusion malaria . There may be recrudescence of fever even after remission due to persistence of small number of infected RBCs. 18

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22. erythrocytic Schizogony This begins with the attachment and invasion of erythrocytes with blood stream merozoites . The merozoites became attach to the GLYCOPHORINS ( the major surface glycoproteins of RBCs) and other Sialoproteins on the RBC membr . The Erythrocytes of Any Age & Reticulocytes are infected. 22

23. erythrocytic Schizogony During invasion the apical end of the merozoites first come in contact with the erythrocytes. Then the merozoite lies within an Intraerythrocytic Parasitophorus vacuole formed by erythrocyte plasma membrane. The nature of Haemoglobin and RBC enzymes influence the development of merozoites inside the RBC ….. Feotal Hb & HbS inhibit the development. 23

24. erythrocytic Schizogony Inside the RBCs, the Merozoite develop into Young TROPHOZOITES or, RING FORMS . These feed on Hb by ingesting the RBC cytoplasm. HAEMAZOIN ( Malarial pigment)  A compound of Haematin and Ferric acid , is produced as an end product of haemoglobin break down. The trophozoites multiply by division of nucleus by mitosis followed by division of cytoplasm , to become MATURE SCHIZONTS . 24

25. erythrocytic Schizogony A mature ERYTHROCYTIC SCHIZONT is asymmetrical and contains 8-32 MEROZOITES & Haemazoin . Rupture of schizont releases merozoites into circulation. These merozoites within seconds attach and penetrate new RBCs. The process of intracellular maturation leading to the development of Schizont and subsequent rupture is k/a- SCHIZOGONY . 25

26. erythrocytic Schizogony In P. falciparum , the erythrocytic schizogony is completed within 48 hrs . It always takes place in capillaries and vascular beds of internal organs. Therefore, in P. falciparum infection, SCHIZONTS & MEROZOITES are usually not demonstrated in the peripheral blood circulation. 26

27. Gemetocytogenesis After 2-3 erythrocytic cycles, some of the merozoites invade the new erythrocytes and instead of developing into schizonts, they develop into  MALE MICROGAMETOCYTES AND FEMALE MACROGAMETOCYTES . These gametocytes develop in the RBC of the BONE MARROW AND SPLEEN . The early gametocytes are of irregular shape but finally they become CRSCENT- Shaped , a distinctive feature of P. falciparum . 27

28. Gemetocytogenesis The haemazoin granules of gametocytes of P. falciparum is found in central part of cytoplasm surrounding the nucleus of micro- & macrogamets . In P. vivax , heamazoin pigments are distributed throughout the cytoplasm. In PBS , only mature gametocytes are found . Gametocytogenesis completes in 96 hrs . 28

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33. Diagnostic forms of P. falciparum 33 STUDENTS ARE REQUESTED TO IDENTIFY THESE FORMS AS AN EXERCISE…!!

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38. Mosquito Cycle - SPOROGONY Sporogony , sexual cycle of parasite takes place in Female Anopheline Mosquito- Definitive Host . It begins with the ingestion of gametocytes by female mosquito during a blood meal. In the stomach of the mosquito the male gametocytes divides rapidly through a process of transformation k/a- Exflagellation . 38

39. Mosquito Cycle - SPOROGONY In exflagellation , the microgametocytes first become extracellular and Then within 10-12 mins, it’s nucleus dividing repeatedly to form 6-8 daughter nuclei. Each nucleus is surrounded by a developing Axoneme. Subsequently , the outer membrane of the microgamatocyte ruptures releasing 6-8 daughter nuclei, associated with Axoneme bud, each of which becomes Microgametes . 39

40. Mosquito Cycle - SPOROGONY The microgametes are sperm like organisms, highly motile with single flagellum. Female Macrogametocytes do not show any process of flagellation but mature by simple process of nuclear reduction and extension of Polar bodies. A single macrogamatocyte gives rise to only one Macrogamete . 40

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43. Mosquito Cycle - SPOROGONY The male gamate fertilizes the female gamate and the Zygote is produced within 20-120 minutes of blood meal. The zygote lengthens(slender) and develop into a motile Ookinete . It measures 11-13 × 2.5 µm and penetrates the gut wall of stomach where it secrets a thin wall and grows into a spherical structure- Oocyst (6- 12 µm) . 43

44. Mosquito Cycle - SPOROGONY Oocyst contains a number of haploid nucleated masses called SPOROBLASTS and the cytoplasm. The sporoblasts then divide repeatedly to form thousands of Sporpzoites , which are Raleased by rupture of Oocyst into the Heamocele , from there they migrate to the Salivary glands. Sporozoites are infectious for human and the process of formation of sporozoites are called SPOROGONY which completes in 9-10 days. 44

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46. Diagnostic forms in Human RING Form – this is the Young Trophozoite and found inside an RBC. it’s morphology resembles a Ring like structure The ring shaped parasite cytoplasm surrounding a central vacuole stain blue The nucleus ( Chromatin dot ) stain red. 46

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48. Diagnostic forms in Human TROPHOZOITES- they are vacuolated, more or less amoeboid, uninucleate . thin ring of cytoplasm and darkish stained nucleus. In heavy infection, the growing forms assume a compact form A single large mass of Pigment ( Heamazoin ) is present. In falciparum malaria both early and late trophozoites are rarely seen in peripheral blood . 48

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50. Diagnostic forms in Human ERYTHROCYTIC SCHIZONTS – they are asexual and dividing forms of the parasite. They occupy 2/3 of the infected RBCs. They contain 2-3 merozoites and dark stained pigments. Mature Schizonts contain 10-36 merozoites which are arranged in grape like clusters. Schizonts are very rarely seen and indicates severe infection. 50

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52. Diagnostic forms in Human GAMETOCYTES – They are sexual and erythrocytic stages of the parasite and Infectious for mosquitoes. they are crescent/ banana shaped with round or pointed ends. Mature gamatocytes are 1 and ½ times larger than the RBC Gamatocytes are 2 types- Microgamatocyte (Male) and Macrogamatocyte (female) 52

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57. Pathogenesis and Pathology P . falciparum is the most pathogenic malaria spp. It is protected from Immune system as most of it’s life cycle is inside Liver cells and RBCs ,invisible from immune surveillance. Infected RBCs are destroyed in the spleen. The clinical symptoms of malaria is caused by the asexual intra-erythrocytic stage of the parasite. 57

58. Pathogenesis and Pathology The disease process in malaria is due to- Local & systemic response to Host and Parasite antigens. Tissue hypoxia caused by sequestration of parasites. Anemia caused by destruction of large number of RBCs. 58

59. Virulence factors High level parasitaemia - Parasite density exceeds > 250,000 – 300,000/ml of blood. Nearly 30-40% of total body RBCs are parasitised . Erythrocytes of all ages are invaded. A level of 25% parasitaemia is usually fatal. 59

60. Virulence factors Sequestration of the parasite - It means a condition of holding back the mature parasites in the vital organs. This phenomenon is shown exclusively by P. falciparum and is due to it’s ability of cytoadherence . Inside RBCs , the P. falciparum merozoites produce a protein within the RBC surface membrane in the form of a deformation called KNOBS . These Knobs produce High molecular weight adhesive proteins resulting in RBCs to stick to walls of small blood vessels. 60

61. Virulence factors This causes sequestration of parasites in general circulation and spleen. Attachment of these infected RBCs in endothelial venule in large amount causes blockage of vessels in the Brain, Kidneys, spleen and lungs . Gametocyte infected RBCs don’t have any knob-> don’t stick to vessels-> don’t sequestrate and seen in PBS. 61

62. Virulence factors Pf erythrocyte membrane protein 1 ( PfEMP1) are exposed to the immune system but don’t act as a good immune target due to it’s extreme diversity and switching of parasites between a broad repertoire of PfEMP1 surface proteins. CYTOKINES - Pf produces a number of cytokines such as IL-1, TNF, INF- γ . These cytokines act on various receptors on endothelial cells on small capillaries and post capillary venules-> End organ ds. of Kidney, Lungs and Brain. 62

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67. Pathological changes in various organs In malaria , typical pathological changes are seen primarily in the Spleen, Liver, Bone marrow, Lungs, Kidney and Brain . Effected organs shows the following features- Pigments are present in various organs- Characteristic Slate-grey/ Black appearance The cells of the RE system shows hyperplasia 67

68. Pathological changes in various organs Free pigments , free plasmodia, and infected erythrocytes are present within the capillaries of these organs. The capillaries also have Macrophages with infected RBCs and segmented plasmodia. Sometimes, thrombi caused by aggregation of pigments can be seen in capillaries. 68

69. Pathological changes in various organs SPLEEN - Markedly enlarged, In Acute infection- its soft, and moderately enlarged, and spleenic substance is congested / heamorrhagic . In chronic infection- Spleen is usually greyish or dark brown or even black and is k/a- Ague Cake . Histologically - (Acute) Marked congestion and hypertrophy and phagocytic activity of RE cells and Macrophages. 69

70. Pathological changes in various organs Capillaries are filled with Parasite infected RBCs Chronic – Spleen cells are filled with brown black pigments- Haemazoin Leucocyte and RBC debris are seen. 70

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72. Pathological changes in various organs At the top of this picture are the spleen (left) and liver (right) from an autopsy of a child. They show a normal spleen and liver appearance.  At the bottom of the picture are the spleen and liver from the autopsy of a child who died of malaria, which are a darker colour than the normal organs. The dark colour comes from extreme congestion and heavy deposition of haemozoin . 72

73. Genus:  Plasmodium  | Species:  falciparum  | Stage: Malaria pigment | Tissue: Human spleen | Stain:  Haematoxylin eosin | Imaging: Brightfield 73

74. Pathological changes in various organs LIVER – Acute - Moderately Enlarged, congested, Chronic - Enlarged, Pigmented and firm Hyperplasia of Kupffer cells and their cytoplasms filled with parasites, malarial pigment and cellular debris. Pigments are found in Parenchymal cells. 74

75. Pathological changes in various organs Bone marrow Acute - Red & Hyperplastic Chronic - Pale, RE Cells hyperplasia Capillaries are filled with infected RBCs KIDNEYS Enlarged & congested Glomeruli - Malarial pigments Tubules- Haemoglobin casts 75

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77. Pathological changes in various organs Histopathological changes of liver tissue  H– hepatocytes, A- hepatic artery, V- hepatic vein, B- bile duct, S- sinusoidal area, CV- central vein, Arrowheads- Kupffer cells, Star- fatty change, Asterisks- inflammatory cells, Arrows- PRBCs. 77

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79. Pathological changes in various organs Acute falciparum malaria: histopathology of the kidney This is a medium power view of a section of kidney from a child with falciparum malaria. It shows mild hyperplasia of the mesangium and small flecks of brown pigment ( haemozoin ) in mesangial cells. The adjacent tubules are normal. (H&E stain) 79

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81. Pathological changes in various organs This is a medium power microscopic view of a bone marrow specimen from a patient with falciparum malaria. It shows active haemopoietic tissue, with deposits of brown haemozoin . This marrow is hyperplastic, ie . the marrow occupies more than 50% of the total volume. ( H&E stain). 81

82. Pathological changes in various organs LUNGS The lungs are pigmented and show haemorrhagic patches . Pigments, pigmented leucocytes, phagocytes and infected RBCs are present in the capillaries. BRAIN Congested, capillaries are plugged with parasitised RBCs, each cell contains Malarial pigment. Small foci of heamorrhages are found in the parenchyma. Small foci of inflammatory granuloma - Durck’s Granuloma . 82

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84. Pathological changes in various organs Histopathology of the lung in a fatal case of adult falciparum malaria  There is expansion of alveolar capillaries by sequestered parasitized erythrocytes and host inflammatory leukocytes. Monocytes and neutrophils within alveolar septal capillaries contain phagocytosed hemozoin pigment (hematoxylin and eosin [H&E] staining, magnification 3 400, 84

85. Pathological changes in various organs HEAMATOLOGICAL CHANGES Anemia - 1] Enormous destruction of both Parasitised and non- parasitised RBCs - Antigenically different Parasitised RBCs bind with the non- parasitised and form Rossets and block vanules . Non parasitised RBCs are destroyed probably by auto-immune mechanism. 2] Decreased erythropoesis in Bone marrow - This is in part due to TNF Toxicity. In long term infection there may be Leucopenia & decreased Hb levels 85

86. Immunity Against Malaria Nature of Hemoglobin - Sickle- cell trait ( HbS ) Thalassemia trait Foetal Hb G6PD Deficiency Humoral - Circulating Abs against asexual forms may protect against the malarial parasite by- Inhabiting RBC invasion Inhibiting grown inside RBC Sequestration of RBC These antibodies results in decreased susceptibility to malaria and reduce transmission. 86

87. Immunity Against Malaria CMI Activated Macrophages may phagocytose and induce extracellular killing of the target cells. It may be enhanced by Antibodies bound to the target cell surface. This natural immunity is supressed in Pregnant women, children and patients on immuno-supressed drugs. 87

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90. Clinical manifestations The clinical symptoms can be divided into – A] Prodromal period B] Malarial paroxysm C] Anemia D] Hepatospleenomegaly . Prodromal period - Malarial paroxysm is preceded by a Prodromal period which varies from a few to several days. Non-specific symptoms such as  Malaise, Myalgia, Headache, and Fatigue are seen. Local symptoms like Chest pain, Abdominal pain and Arthralgia are also found. 90

91. Clinical manifestations Malarial paroxysm - It is the classic manifestation of Acute malaria characterised by – Fever, Chill & Rigor . Malignant Tertian Fever - Key manifestation that occurs every 48 hours in falciparum malaria. Fever is irregular and doesn’t show any periodicity pattern. Ruture of RBCs-> erythrocytic debris-> activation of Macrophages-> IL-1 & TNFs. -> Fever/ Chill. 91

92. Clinical manifestations Anemia - It is haemolytic Normocytic & Normochromic anemia. Hepatosplenomegaly - In uncomplicated malaria there is moderate splenomegaly (4-8 cms ) and tender hepatomegaly . Lymphadenopathy don’t occur. 92

93. Clinical manifestations The first symptoms of malaria are nonspecific; the lack of a sense of well-being, headache, fatigue, abdominal discomfort, and muscle aches. Followed by fever are all similar to the symptoms of a minor viral illness. In some instances, a prominence of headache, chest pain, abdominal pain, arthralgia , myalgia , or diarrhea may suggest another diagnosis. Although headache may be severe in malaria, there is no neck stiffness or photophobia resembling that in meningitis . While myalgia may be prominent, it is not usually as severe as in dengue fever , and the muscles are not tender as in leptospirosis or typhus. 93

94. Clinical manifestations Nausea, vomiting, and orthostatic hypotension are common. The classic malarial paroxysms, in which fever spikes, chills, and rigors occur at regular intervals, are relatively unusual and suggest infection with P. vivax or P. ovale . The fever is irregular at first (that of falciparum malaria may never become regular) The temperature of non-immune individuals and children often rises above 40°C in conjunction with tachycardia and sometimes delirium. 94

95. Clinical manifestations Although childhood febrile convulsions may occur with any of the malarias, Generalized seizures are specifically associated with falciparum malaria and may herald the development of cerebral disease. Uncomplicated infections have few abnormal physical findings other than fever, malaise, mild anemia, and (in some cases) a palpable spleen . Anemia is common among young children living in areas with stable transmission, particularly where resistance has compromised the efficacy of antimalarial drugs. 95

96. Clinical manifestations In nonimmune individuals with acute malaria, the spleen takes several days to become palpable, Splenic enlargement is found in a high proportion of otherwise healthy individuals in malaria-endemic areas and reflects repeated infections . Slight enlargement of the liver is also common, particularly among young children. Mild jaundice is common among adults; It may develop in patients with otherwise uncomplicated falciparum malaria and usually resolves over 1–3 weeks. 96

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99. Complications of Severe Falciparum malaria Appropriately and promptly treated, uncomplicated falciparum malaria (i.e., the patient can swallow medicines and food) carries a mortality rate of ~0.1%. However, once vital-organ dysfunction occurs or the total proportion of erythrocytes infected increases to >2% (a level corresponding to >10 12 parasites in an adult), mortality risk rises steeply. The condition occurs more frequently in – Non-immune persons Immuno -suppressed persons Pregnant women Splenectomy patients. 99

100. Complications of Severe Falciparum malaria- BLACKWATER FEVER The syndrome is manifestation of repeated infection from falciparum malaria which was inadequeately treated with quinine. The condition is characterized by Rapid & Massive Intravascular heamolysis of both infected and non-infected RBCs. It is associated with high levels of Hb and Hb breakdown products in blood and urine. 100

101. Complications of Severe Falciparum malaria- BLACKWATER FEVER Clinical features - High fever, Vomiting, Pain in loin, Jaundice, Heamoglobinaemia , Heamoglobinuria , Circulatory collapse and renal failure. 1-2 haemolytic episodes are usually present. Cold, abnormal physical activity and Alcohol abuse are precipitating factors. The urine is dark red to brown black in appearance due to presence of metheamoglobin or oxyheamoglobin . Protein, epithelial cells and casts are present in the urine. 101

102. Complications of Severe Falciparum malaria- BLACKWATER FEVER Acute Renal Failure is the immediate cause of death. Mortality rate- 20- 50% An autoimmune mechanism has been suggested in the pathogenesis of Black water fever. Erythrocytic autoantibodies produced in previous P. falciparum infections probably combine with the autoantigens occuring in the newer infections of the erythrocytes with the same P. falciparum , resulting in heamolysis . The quininised and parasitised RBCs act as autoantigens against which autoantibodies are produced. 102

103. Complications of Severe Falciparum malaria- CEREBRAL MALARIA Cerebral malaria is defined as any abnormality of mental status in a person with malaria. It is a diffuse symmetric encephalopathy and is believe to represent metabolic encephalopathy . Coma is a characteristic and ominous feature of falciparum malaria and, despite treatment, is associated with death rates of ~20% among adults and 15% among children 103

104. Complications of Severe Falciparum malaria- CEREBRAL MALARIA Cerebral malaria manifests as diffuse symmetric encephalopathy; Focal neurologic signs are unusual . Although some passive resistance to head flexion may be detected, signs of meningeal irritation are lacking. The eyes may be divergent and a pout reflex is common, but other primitive reflexes are usually absent. 104

105. Complications of Severe Falciparum malaria- CEREBRAL MALARIA The corneal reflexes are preserved, except in deep coma. Muscle tone may be either increased or decreased. The tendon reflexes are variable, and the plantar reflexes may be flexor or extensor; The abdominal and cremasteric reflexes are absent. Flexor or extensor posturing may be seen. 105

106. Complications of Severe Falciparum malaria Approximately 15% of patients have retinal hemorrhages; with pupillary dilatation and indirect ophthalmoscopy , this figure increases to 30–40%. Other funduscopic abnormalities include discrete spots of retinal opacification (30–60%), Papilledema (8% among children, rare among adults), Cotton wool spots (<5%), and decolorization of a retinal vessel or segment of vessel Below Image- The eye in cerebral malaria: perimacular whitening and palecentered retinal hemorrhages 106

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108. Complications of Severe Falciparum malaria Convulsions, usually generalized and often repeated, occur in up to 50% of children with cerebral malaria. More covert seizure activity is also common, particularly among children, and may manifest as repetitive tonic- clonic eye movements or even hypersalivation . ~15% of children surviving cerebral malaria—especially those with hypoglycemia, severe anemia, repeated seizures, and deep coma —have some residual neurologic deficit when they regain consciousness; hemiplegia, cerebral palsy, cortical blindness, deafness, and impaired cognition and learning (all of varying duration) have been reported. Approximately 10% of children surviving cerebral malaria have a persistent language deficit. 108

109. Complications of Severe Falciparum malaria The plugging of capillaries by Rossets of sequestered parasitised RBCs in the cerebral microvasculature -> Circulatory stasis and hypoxia. The below images shows the autopsy of Brain from patient died with Cerebral malaria. 109

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112. Complications of Severe Falciparum malaria- HYPOGLYCEMIA Hypoglycemia, an important and common complication of severe malaria, is associated with a poor prognosis and is particularly problematic in children and pregnant women. Hypoglycemia in malaria results from a failure of hepatic gluconeogenesis and an increase in the consumption of glucose by both host and, to a much lesser extent, the malaria parasites. To compound the situation, quinine and quinidine—drugs used for the treatment of severe chloroquine-resistant malaria—are powerful stimulants of pancreatic insulin secretion . Hyperinsulinemic hypoglycemia is especially troublesome in pregnant women receiving quinine treatment. 112

113. Complications of Severe Falciparum malaria- HYPOGLYCEMIA - Diagnosis of hypoglycemia is difficult: The usual physical signs (sweating, gooseflesh, tachycardia) are absent , and the Neurologic impairment caused by hypoglycemia cannot be distinguished from that caused by malaria. 113

114. Complications of Severe Falciparum malaria- ACIDOSIS Acidosis, an important cause of death from severe malaria, results from accumulation of organic acids . Hyperlactatemia commonly coexists with hypoglycemia. In adults, coexisting renal impairment often compounds the acidosis; in children, ketoacidosis may also contribute. Other still-unidentified organic acids are major contributors to acidosis. Acidotic breathing , sometimes called respiratory distress, is a sign of poor prognosis . It is often followed by circulatory failure refractory to volume expansion or inotropic drugs and ultimately by respiratory arrest. 114

115. Complications of Severe Falciparum malaria- ACIDOSIS The plasma concentrations of bicarbonate or lactate are the best biochemical prognosticators in severe malaria . Lactic acidosis is caused by the combination of anaerobic glycolysis in tissues where sequestered parasites interfere with  microcirculatory flow, hypovolemia, lactate production by the parasites, and a failure of hepatic and renal lactate clearance. The prognosis of severe acidosis is poor. 115

116. Complications of Severe Falciparum malaria- Noncardiogenic Pulmonary Edema Adults with severe falciparum malaria may develop noncardiogenic pulmonary edema even after several days of antimalarial therapy. The pathogenesis of this variant of the adult respiratory distress syndrome is unclear. The mortality rate is >80%-> Most serious complication. This condition can be aggravated by overly vigorous administration of IV fluid. Noncardiogenic pulmonary edema can also develop in otherwise uncomplicated vivax malaria, where recovery is usual. Most commonly occur in Pregnant women. 116

117. Complications of Severe Falciparum malaria- Renal Impairment Renal impairment is common among adults with severe falciparum malaria but rare among children. The pathogenesis of renal failure is unclear but may be related to erythrocyte sequestration interfering with renal microcirculatory flow and metabolism. Clinically and pathologically, this syndrome manifests as acute tubular necrosis , although renal cortical necrosis never develops. Acute renal failure may occur simultaneously with other vital-organ dysfunction (in which case the mortality risk is high) or may progress as other disease manifestations resolve. 117

118. Complications of Severe Falciparum malaria- Renal Impairment In survivors, urine flow resumes in a median of 4 days, and serum creatinine levels return to normal in a mean of 17 days . Early dialysis or hemofiltration considerably enhances the likelihood of a patient’s survival, particularly in acute hypercatabolic renal failure 118

119. Complications of Severe Falciparum malaria- Liver Dysfunction Mild hemolytic jaundice is common in malaria. Severe jaundice is associated with P. falciparum infections ; is more common among adults than among children; and Results from hemolysis, hepatocyte injury, and cholestasis. When accompanied by other vital-organ dysfunction (often renal impairment), Liver dysfunction carries a poor prognosis. Hepatic dysfunction contributes to hypoglycemia, lactic acidosis,and impaired drug metabolism. Occasional patients with falciparum malaria may develop deep jaundice (with hemolytic, hepatitic , and cholestatic components) without evidence of other vital-organ dysfunction. 119

120. Complications of Severe Falciparum malaria- Hematologic Abnormalities Anemia results from  Accelerated RBC removal by the spleen, Obligatory RBC destruction at parasite schizogony, and Ineffective erythropoiesis. In severe malaria, both infected and uninfected RBCs show reduced deformability, which correlates with prognosis and development of anemia. Splenic clearance of all RBCs is increased. In nonimmune individuals and in areas with unstable transmission, anemia can develop rapidly and transfusion is often required. As a consequence of repeated malarial infections, children in many areas of Africa may develop severe anemia resulting from both shortened RBC survival and marked dyserythropoiesis . 120

121. Hematologic Abnormalities Slight coagulation abnormalities are common in falciparum malaria, and mild thrombocytopenia is usual. Of patients with severe malaria, <5% have significant bleeding with evidence of disseminated intravascular coagulation. Hematemesis from stress ulceration or acute gastric erosions may also occur 121

122. CHRONIC COMPLICATIONS OF MALARIA- TROPICAL SPLENOMEGALY (HYPERREACTIVE MALARIAL SPLENOMEGALY) Chronic or repeated malarial infections produce hypergammaglobulinemia; normochromic, normocytic anemia; and, in certain situations, splenomegaly. Some residents of malaria-endemic areas in tropical Africa and Asia exhibit an abnormal immunologic response to repeated infections characterized by Massive splenomegaly, Hepatomegaly, Marked elevations in serum titers of IgM and malarial antibody, Hepatic sinusoidal lymphocytosis, and (in Africa) Peripheral B cell lymphocytosis. 122

123. CHRONIC COMPLICATIONS OF MALARIA- TROPICAL SPLENOMEGALY (HYPERREACTIVE MALARIAL SPLENOMEGALY) This syndrome has been associated with the production of  cytotoxic IgM antibodies to CD8+ T lymphocytes, antibodies to CD5+ T lymphocytes , and an increase in the ratio of CD4+ T cells to CD8+ T cells. These events may lead to uninhibited B cell production of IgM and the formation of cryoglobulins ( IgM aggregates and immune complexes ) 123

124. CHRONIC COMPLICATIONS OF MALARIA- TROPICAL SPLENOMEGALY (HYPERREACTIVE MALARIAL SPLENOMEGALY) This immunologic process stimulates reticuloendothelial hyperplasia and clearance activity and eventually produces splenomegaly . Patients with hyperreactive malarial splenomegaly (HMS ) present with an abdominal mass or a dragging sensation in the abdomen and occasional sharp abdominal pains suggesting perisplenitis . Anemia and some degree of pancytopenia are usually evident, and in some cases malarial parasites cannot be found in peripheral-blood smears 124

125. CHRONIC COMPLICATIONS OF MALARIA- TROPICAL SPLENOMEGALY (HYPERREACTIVE MALARIAL SPLENOMEGALY) Vulnerability to respiratory and skin infections is increased ; many patients die of overwhelming sepsis. Persons with HMS who are living in endemic areas should receive antimalarial chemoprophylaxis; In nonendemic areas, antimalarial treatment is advised. 125

126. MALARIA IN PREGNANCY In heavily endemic (hyper- and holoendemic ) areas, falciparum malaria in primi - and secundigravid women is associated with low birth weight (average reduction, ~170 g) consequently increased infant and childhood mortality. In general, infected mothers in areas of stable transmission remain asymptomatic despite intense accumulation of parasitized erythrocytes in the placental microcirculation. 126

127. MALARIA IN PREGNANCY Maternal HIV infection predisposes pregnant women to malaria, predisposes their newborns to congenital malarial infection, and exacerbates the reduction in birth weight associated with malaria. In areas with unstable transmission of malaria , pregnant women are prone to severe infections and are particularly vulnerable to high-level parasitemia with anemia, hypoglycemia, and acute pulmonary edema. Fetal distress, premature labor, and stillbirth or low birth weight are common results. 127

128. MALARIA IN PREGNANCY Fetal death is usual in severe malaria. Congenital malaria occurs in <5% of newborns whose mothers are infected Its frequency and the level of parasitemia are related directly to the parasite density in maternal blood and in the placenta. P. vivax malaria in pregnancy is also associated with a reduction in birth weight (average, 110 g) 128

129. MALARIA IN CHILDREN Most of the estimated 1–3 million persons who die of falciparum malaria each year are young African children. Convulsions, coma, hypoglycemia, metabolic acidosis, and severe anemia are relatively common among children with severe malaria, whereas deep jaundice, acute renal failure, and acute pulmonary edema are unusual. Severely anemic children may present with labored deep breathing, which in the past has been attributed incorrectly to “anemic congestive cardiac failure ” but in fact is usually caused by metabolic acidosis, often compounded by hypovolemia . Evidence is accruing that severe malaria can result in longterm neurocognitive and developmental deficits . In general, children tolerate antimalarial drugs well and respond rapidly to treatment. 129

130. TRANSFUSION MALARIA Malaria can be transmitted by blood transfusion, needlestick injury, sharing of needles by infected injection drug users, or organ transplantation. The incubation period in these settings is often short because there is no pre-erythrocytic stage of development. The clinical features and management of these cases are the same as for naturally acquired infections. Radical chemotherapy with primaquine is unnecessary for transfusion-transmitted P. vivax and P. ovale infections. 130

131. Mosquito borne Vs. Transfusion Malaria Feature Mosquito borne Transfusion Infective stage Sporozoite Trophozoite Incubation period Long Short PE Schizogony Present Absent EE Schizogony May be present Absent Relapse May occur Don’t T/t Radical cure not possible Radical cure possible 131

132. Recurrence of Clinical malaria Recurrence of clinical malaria after treatment may occur due to 3 reasons- 1] True relapse : It is caused by Hypnozoites in P. vivax & P. ovale . It is due to re- emrgence of blood stage parasites from latent parasites(Hypnozoites) in liver. Since no Hypnozoites in P. falciparum  No true relapse. 2] Recrudescence - It is seen falciparum malaria d/t inadequate t/t and seen in – Drug resistance, Immuno -suppression & pregnancy. 132

133. Recurrence of Clinical malaria 3] Latent malaria - This condition refers to a state of asymptomatic malaria harbouring plasmodia gametocytes in the peripheral blood. These persons are infectious to mosquitoes and act as Reservoirs . 133

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135. EPIDEMIOLOGY Malaria occurs throughout most of the tropical regions of the world . P. falciparum predominates in Africa, New Guinea, and Haiti; P. vivax is more common in Central America. The prevalence of these two species is approximately equal in South America, the Indian subcontinent, eastern Asia, and Oceania. P. malariae is found in most endemic areas, especially throughout sub-Saharan Africa, but is much less common. P. ovale is relatively unusual outside of Africa and, where it is found, comprises <1% of isolates. 135

136. EPIDEMIOLOGY Endemicity traditionally has been defined in terms of parasitemia rates or palpable-spleen rates in children 2–9 years of age as hypoendemic (<10%), mesoendemic (11–50%), hyperendemic (51–75%), and holoendemic (>75%) In holo - and hyperendemic areas (e.g., certain regions of tropical Africa or coastal New Guinea) where there is intense P. falciparum transmission, people may sustain more than one infectious mosquito bite per day and are infected repeatedly throughout their lives. In such settings, rates of morbidity and mortality due to malaria are considerable during childhood. 136

137. EPIDEMIOLOGY Constant, frequent, year-round infection is termed stable transmission . In areas where transmission is low, erratic, or focal, full protective immunity is not acquired, and symptomatic disease may occur at all ages. This situation usually exists in hypoendemic areas and is termed unstable transmission . Malaria behaves like an epidemic disease in some areas, particularly those with unstable malaria, such as northern India, Sri Lanka, Southeast Asia, Ethiopia, Eritrea,Rwanda , Burundi, Southern Africa, and Madagascar 137

138. EPIDEMIOLOGY An epidemic can develop when there are changes in environmental, economic, or social conditions, such as heavy rains following drought or migrations (usually of refugees or workers) from a non- malarious region to an area of high transmission; a breakdown in malaria control and prevention services can intensify epidemic conditions. This situation usually results in considerable mortality among all age groups. 138

139. EPIDEMIOLOGY The principal determinants of the epidemiology of malaria are the number (density), the human-biting habits, and the longevity of the anopheline mosquito vectors. More specifically, the transmission of malaria is directly proportional to the density of the vector, the square of the number of human bites per day per mosquito, and the tenth power of the probability of the mosquito’s surviving for 1 day. Mosquito longevity is particularly important, because the portion of the parasite’s life cycle that takes place within the mosquito— from gametocyte ingestion to subsequent inoculation ( sporogony )—lasts 8–30 days, depending on ambient temperature; thus, to transmit malaria, the mosquito must survive for >7 days. 139

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142. DIAGNOSIS- Demonstration of Parasites - Light Microscopy The diagnosis of malaria rests on the demonstration of asexual forms of the parasite in stained peripheral-blood smears. It is the Gold standard for confirmation of malaria. After a negative blood smear, repeat smears should be made if there is a high degree of suspicion. Of the Romanowsky stains, Giemsa at pH 7.2 is preferred ; Wright’s, Field’s, or Leishman’s stain or, JSB stain can also be used. Both thin and and thick blood smears should be examined. The thin blood smear should be rapidly air-dried, fixed in anhydrous methanol, and stained; the RBCs in the tail of the film should then be examined under oil immersion (×1000 magnification). The level of parasitemia is expressed as the number of parasitized erythrocytes per 1000 RBCs. 142

143. DIAGNOSIS- Demonstration of Parasites - Light Microscopy The thick blood film should be of uneven thickness. The smear should be dried thoroughly and stained without fixing. As many layers of erythrocytes overlie one another and are lysed during the staining procedure, The thick film has the advantage of concentrating the parasites (by 40- to 100-fold compared with a thin blood film) and thus increasing diagnostic sensitivity. Both parasites and white blood cells (WBCs) are counted, and the number of parasites per unit volume is calculated from the total leukocyte count. Alternatively, a WBC count of 8000/ μL is assumed. A minimum of 200 WBCs should be counted under oil immersion. This figure is converted to the number of parasitized erythrocytes per microliter -> Quantitative test-> Prognostic value . 143

144. DIAGNOSIS- Demonstration of Parasites - Light Microscopy Before a thick smear is judged to be negative, 100–200 fields should be examined under oil immersion. The presence of only malarial pigments in the absence of Malarial parasites suggests recent P. falciparum i nfection. Disadvange of thick smear is that Plasmodial spp. cant be identified. 144

145. DIAGNOSIS- Preparation of thick blood smears Prepare at least 2 smears per patient. Place a small drop of blood in the center of the pre-cleaned, labeled slide. Using the corner of another slide or an applicator stick, spread the drop in a circular pattern until it is the size of a dime (1.5 cm2). A thick smear of proper density is one which, if placed (wet) over newsprint, allows you to barely read the words. Lay the slides flat and allow the smears to dry thoroughly (protect from dust and insects!). Insufficiently dried smears (and/or smears that are too thick) can detach from the slides during staining. 145

146. DIAGNOSIS- Preparation of thick blood smears The risk is increased in smears made with anticoagulated blood. At room temperature, drying can take several hours; 30 minutes is the minimum; in the latter case, handle the smear very delicately during staining. Drying can be accelarated by using a fan or hair dryer (use cool setting). Protect thick smears from hot environments to prevent heat-fixing the smear. Do not fix thick smears with methanol or heat. If there will be a delay in staining smears, dip the thick smear briefly in water to hemolyse the RBCs. 146

147. DIAGNOSIS- Preparation of thin blood smears Thin smears consist of blood spread in a layer such that the thickness decreases progressively toward the feathered edge. In the feathered edge, the cells should be in a monolayer, not touching one another. Prepare at least 2 smears per patient! A thin smear being prepared. Place a small drop of blood on the pre-cleaned, labeled slide, near its frosted end. Bring another slide at a 30-45° angle up to the drop, allowing the drop to spread along the contact line of the 2 slides. Quickly push the upper (spreader) slide toward the unfrosted end of the lower slide. Make sure that the smears have a good feathered edge. This is achieved by using the correct amount of blood and spreading technique. Allow the thin smears to dry. (They dry much faster than the thick smears, and are less subject to detachment because they will be fixed.) Fix the smears by dipping them in absolute methanol. https://www.youtube.com/watch?v=WPP7AjmStBg 147

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155. DIAGNOSIS- Quantitative Buffy Coat (QBC) This is a sensitive microscopic test based on the ability of Acridin Orange to stain the nucleic acid contained parasites . In this method, blood is collected in a capillary tube coated with fluorescence dye and subjected to microheamatocrit centrifugation in a special centrifugation apparatus. After centrifugation, the buffy coat in the centrifused capillary tube is examined directly under a fluorescence microscope. Dye stained malarial parasites appear bright green. It can detect parasite count as low as 3-4 parasites/ µL. The disadvantage is inability to identify parasite spp. 155

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160. DIAGNOSIS- Fluorescence Microscopy Kawamoto technique is a fluorescent-staining method for demonstrating the malarial parasite. the blood smears are prepared in a slide and is stained with Acrydine organge . this results in differential staining of Malarial parasite- Nuclear DNA- Stained GREEN, Cytoplasmic RNA- Stained RED 160

161. DIAGNOSIS- SERODIAGNOSIS Serological tests are used for  Identify infected donors in case of Transfusion malaria Confirm past malaria in patients. Epidemiological survey Indirect heamaglutination (IHA), Indirect Fluorescence antibody (IFA) & ELISA are most frequently used test to detect malarial antibody in serum. ELISA –Inhibition Test is a recent method to detect malarial antigen in serum. 161

162. DIAGNOSIS- Immunochromatographic tests- They are used for Malarial Antigen detection from blood or urine aka- Malarial rapid diagnostic tests, Antigen capture assay or Dipstics . The ICTs are monoclonal antibody based assays to detect  Plasmodium falciparum histidine-rich protein-2, Parasite lactose dehydrogenase or Plasmodium aldolase. PfHRP-2 persists in blood for few months so can’t be used to predict t/t failure. 100 parasites/ µL density is required for the test . 162

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165. DIAGNOSIS- Molecular diagnosis DNA and RNA probes are highly sensitive and specific molecular methods for d/g of falciparum malaria. DNA probes can detect <10 parasites/ µL of blood. PCR including real time assay like QT-NASBA (quantitative nucleic acid sequence-based amplification) can detect parasite as low as 1 parasite in 20 µL of blood using PBRK 1 primer> 100 times more sensitive than Thick smear. PCR can detect parasites in dry blood spots and drug resistance malaria. 165

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170. Treatment of Malaria 170

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172. THANKS FOR YOUR ATTENTION 172