Malaria

MALARIAL PARASITES
Dr. Suprakash Das
Assist. Prof.
1

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

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

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

Timeline of Malaria
First
Reference
• Early Hindu and Chinese writings
Early 17th
• The bark of Cinchona was successfully used to treat the
disease
1880
• Charles Louise Alphonse Leveran , a French army
surgeon- Discovered malarial parasite( Exoflagellated
gametocytes) in an unstained preparation of fresh blood.
8

Timeline of Malaria
1883
• Methylene Blue was used by Marchiafava
for staining malarial parasites
1885
• Golgi demonstated the multiplication of
asexual blood forms
1891
• Romanowsky Polychrome stain was
introduced for staining malarial parasites.
9

Timeline of Malaria
1898
• Ross demonstrated developing Plasmodia in the
stomach of mosquito. Later he discovered the life cycle
of malarial parasites in mosquito. Noble- 1902
1900
• Patrick Manson- supports the Ross observations
1948
• Shortt & Granham- Exo-erythrocytic development in
Liver
10

Timeline of Malaria
1955
• WHO launched worldwide Malaria
Eradication Programme
1976
• Continuous P. falciparum culture was
establishes using RPMI 1640 medium.
11

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

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

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

 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

 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

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

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

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

 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

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

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

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

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

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

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

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

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

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

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

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

 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

 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

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

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

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

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

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

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

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

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

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

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

Capillaries are filled with Parasite infected
RBCs
Chronic – Spleen cells are filled with brown
black pigments- Haemazoin
 Leucocyte and RBC debris are seen.
70

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

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

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

 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

• 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

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

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

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

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

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

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

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

 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

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

 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

 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

 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

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

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 >1012 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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

 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

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

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

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

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

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

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

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

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

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

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

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

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

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

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 infection.
Disadvange of thick smear is that Plasmodial
spp. cant be identified.
144

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

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

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

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

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

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

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

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 PBRK1 primer> 100
times more sensitive than Thick smear.
 PCR can detect parasites in dry blood spots and drug resistance
malaria.
165

Malaria

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