Malaria :protozoan disease transmitted by the bite of infected Anopheles mosquitoes.

1. Malaria

2. • The term malaria originates from Medieval Italian:
• – mala aria - bad air – swampy air was thought to
cause it .
• Tropical zone is endemic for all malaria.
• Each year 350–500 million cases of malaria occur
world wide,and over one million people die, most of
them young children in sub-Saharan Africa.

3. • Four Nobel prizes have been awarded for
work associated with malaria:
• Sir Ronald Ross (1902)
• Charles Louis Alphonse Laveran (1907)
• Julius Wagner-Jauregg (1927)
• Paul Hermann Muller (1948)
• Working in India in 1858, Dr. Ross watched through a
microscope the mechanism through which
mosquitoes spread malaria.

4. • Malaria is a protozoan disease transmitted by the
bite of infected Anopheles mosquitoes.
• Four species: P. Falciparum
P. Vivax
P. Ovale
P. Malariae
• Almost all deaths are caused by falciparum malaria.

5. Host
• In Man : parasite reside in liver &RBC (Asexual Cycle)
INTERMEDIATE HOST
• In Female Anopheline Mosquito: Gametocytes
formed in human host are transferred to insect
where they fuse and develop into sporozoites (sexual
cycle) DEFINITIVE HOST

6. Distribution of Malaria
6

8. • Plasmodium vivax, P. ovale, and P. malariae cause
low levels of parasitemia, mild anemia, and, in rare
instances, splenic rupture and nephrotic syndrome.
• P. falciparum causes high levels of parasitemia,
severe anemia, cerebral symptoms, renal failure,
pulmonary edema, and death

9. Features of P. falciparums greater
pathogenicity:
• Infect RBSs of any age high parasite burdens and
profound anemia.
• Infected red cells clump together (rosette) and stick to
endothelial cells lining small blood vessels
(sequestration), which blocks blood flow.
• Several proteins, including P. falciparum erythrocyte
membrane protein 1 (PfEMP1), form knobs on the
surface of red cells .
• PfEMP1 binds to ligands on endothelial cells, including
CD36, thrombospondin, VCAM-1, ICAM-1, and E-
selectin.

10. • Ischemia due to poor perfusion causes the
manifestations of cerebral malaria, which is the main
cause of death due to malaria in children.
• It stimulates production of high levels of cytokines,
including TNF, IFN-γ, and IL-1.
• GPI-linked proteins, including merozoite surface
antigens, are released from infected red cells and
induce cytokine production by host cells.
• These cytokines suppress production of RBC, increase
fever, stimulate nitric oxide production (leading to
tissue damage), and induce expression of endothelial
receptors for PfEMP1 (increasing sequestration).

11. Life Cycle
Human infection begins when a female anopheline mosquito
inoculates plasmodial sporozoites from its salivary gland during
a blood meal .
Intrahepatic or Preerythrocytic schizogony or
Merogony: sporozoites are carried rapidly via the
bloodstream to the liver, invade liver cells by binding to the
hepatocyte receptor for the serum proteins thrombospondin
and properdin and begin a period of asexual reproduction.
A single sporozoite eventually may produce from 10,000 to
>30,000 daughter merozoites.
The swollen infected liver cell eventually bursts, discharging
motile merozoites into the bloodstream.

13. • Plasmodium merozoites bind by a parasite lectin-like
molecule to sialic acid residues on glycophorin molecules on
the surface of red cells. Within the red cells the parasites
grow in a membrane-bound digestive vacuole, hydrolyzing
hemoglobin through secreted enzymes.

14. • Erythrocytic Scizogony: These merozoites then invade the
red blood cells (RBCs) and multiply six- to twenty fold every 48–
72 h.
• When the parasites reach densities of 50/L of blood (100
million parasites in the blood of an adult), the symptomatic
stage of the infection begins
• In P. vivax and P. ovale infections, a proportion of the
intrahepatic forms remain dormant from 3 weeks to a year or
longer before reproduction begins.
• These dormant forms, or hypnozoites, are the cause of the
relapses that characterize infection with these two species.

19. P.Vivax P.Falciparum P.Malariae P.ovale
Schizogony 48 hr 48 hr /less 72 hr 48 hr
Forms in PBF Trophoz.,schizo
nt,gametocyte
Rings&crescent
mainly
Trophoz.,schizo
nt,gametocyte
Trophoz.,schizo
nt,gametocyte
Trophozoites Size 2.5µm 1.25-1.5µm 2.5µm 2.5µm
Ring Form Cytoplasm opp.
The nucleus is
thicker
Cytoplasm –
fine,regular
,ACCOLE
Cytoplasm opp.
The nucleus is
thicker
Cytoplasm opp.
The nucleus is
thicker
Growing Form Irregular,vacuol
e,ameboid
Compact
form,early
pigment
Band like
slightly ameboid
No ribbon
shape.
Schizont 9-10µm
completely fill
enlarged RBC
4.5-5µm fills 2/3
of RBC
6.5-7µm fill
normal RBC
6.2µm fill ¾ of
slightly enlarged
RBC
Merozoites 12-24,Grape
like clusters
18-24,Grape
like clusters
6-12,
rosette,daisy
6-12 irregularly
arranged
Infected RBC Enlarged pale
Schuffners dots
Unaltered
Maurers dots
Not enlarged,no
dots(Ziemans )
Slightly
enlarged,oval,
James dots

20. Pre Erythrocytic Scizogony
Length of pre-
erythrocytic schizogony
Size of mature
scizont
No.of merozoites
P Vivax 8 days 42 µm 12,000
P. Falciparum 6 days 60 by 30µm 40,000
P.Ovale 9 days 80 by 50µm 15,000
P.Malariae 7-12 days 22 µm 2,000

21. Host Resistance to Plasmodium
• Two general mechanisms
• 1.Inherited alterations in red cells make people
resistant to Plasmodium.
• 2.Repeated or prolonged exposure to Plasmodium
species stimulates an immune response that reduces
the severity of the illness caused by malaria.

22. • Plasmodial antigen (asexual phase schizont &
merozoite) are Labile , Resistant & Stable : produce
antibodies Ig G , Ig M.
• Antibodies to La are all in IgG fraction of gamma
globulin , it crosses placenta , give immunity to
foetus for 6 months.
• PABA necessary for malarial parasites metabolism.
Mothers milk is deficient in PABA so, infants are
protected.

23. • People heterozygous for the sickle cell trait (HbS)
less likely to die from infection(parasites to grow
poorly or die because of the low oxygen
concentrations).
• The geographic distribution of the HbS trait is similar
to that of P. falciparum, suggesting evolutionary
selection of the HbS trait in people by the parasite.
• HbC, hemoglobin mutation, also protects against
severe malaria by reducing parasite proliferation.

24. • P. vivax enters red cells by binding to the Duffy blood
group antigen. Many Africans, including most
Gambians, are not susceptible to infection by P. vivax
because they do not have the Duffy antigen.
• Parasite needs G6PD for metabolism, in patients
with G6PD deficiency confers protection.

25. Modes of transmission
• Transfusion Malaria
• Congenital Malaria
• Malaria in Drug Addicts
• Therapeutic Malaria :- Artificially induced or
treatment of neuro- syphilis, P. Vivax is used.

26. Clinical Features
• Febrile Paroxysms
• Anemia
• Splenic Enlargment
• Incubation Period :- time b/w sporozoites entry into
blood and development of fever
• For P. Vivax, Ovale & Falciparum :10-14 days
• For P Malariae: 18 days to 6 weeks.

27. • 1 ) Febrile Paroxysm :
a) Cold Stage : 20 min -1 hr.
b) Hot Stage :1-4 hr.
c) Sweating Stage – 2-3 hr.
Types of Fever :-
48 hr cycle : Tertian Fever
72 hr cycle : Quartan Fever
24 hr cycle : Quotidian

28. 2 ) Anemia : microcytic or normocytic hypochromic
type.
3 ) Spleenomegaly : after few paroxysms by 2nd
wek
spleen is palpable.

29. Relapse in Malaria
• Relapse: In P. vivax and P. ovale infections, a
proportion of the intrahepatic forms remain dormant
from 3 weeks to a year or longer before reproduction
begins.
• Recrudescence : surviving population of erythrocytic
forms are increased
• seen in P. Falciparum

30. Pernicious Malaria
• Acute Falciparum malaria
• Death in 1-3 days if not treated
• Decreased effective circulating volume due to capillary
blockage
• Recession of asexual parasite from peripheral circulation
to capillaries of internal organs .
• Stickiness of infected RBC.
• Vasoconstriction.
• Hypovolemia , Increased permiability , increased blood
viscosity (mature schizont).

31. • Pernicious menifestation is anticipated when > 5 %
RBCs are parasitised.
• Clinical Types :
• Cerebral Malaria : hyperpyrexia,coma,paralysis
• Algid Malaria : vascular collapse cold clammy skin ,
vomiting, diarrhoea, blood in faeces.
• Septicaemic Malaria : High fever, pneumonia,cardiac
syncope.

32. Pathology of Malaria
• Destruction of cells.
• Release of Haematin pigment granules .
• Malaria Toxin (inorganic phosphate & lactic acid):
depress respiration , interfere with mitochondrial
activity .
• Shock in plasmodium infection is related to kinin
release.

33. Main Features
• Pigmentation of various organs slate grey or black.
• Hyperplasia of RE cells.
• Parasitised RBCs fill the lumen of capillaries.
• Congestion & dilatation of sinusoidal vessels.
• Parenchymal degeneration.
• Mesenchymal reaction : fibrosis mild.
• Immunosuppression.

34. Changes in Blood
Anemia : Destruction of infected RBCs.
• AI haemolysis also seen.
• TLC : raised initiallynduring rising temprature
(20,000/cmm)normal
• With recurring paroxysm leucopenia occurs(3000-5000/cmm.
•Monocytes increased(10-20%)
• A:G ratio reversed.
•Increase in Potassium levels.
•ESR raised.
•Raised PH (Inc. lactate & pyruvate)
•Increased indirect bilirubin.

35. MORPHOLOGY
• SPLEEN :-
• Congestion and enlargement of the spleen, > 1000 gm in
weight.
• Parasites are present within red cells, which is the basis of the
diagnostic test, and there is increased phagocytic activity of the
macrophages in the spleen.
• In chronic malaria infection, the spleen becomes increasingly
fibrotic and brittle, with a thick capsule and fibrous trabeculae.
• Gray or black parenchyma because of phagocytic cells
containing granular, brown-black, faintly birefringent hemozoin
pigment.

36. LIVER
• Liver becomes progressively enlarged and pigmented.
• Kupffer cells are heavily laden with malarial pigment,
parasites, and cellular debris
• some pigment is also present in the parenchymal
cells.
• Pigmented phagocytic cells may be found dispersed
throughout the bone marrow, lymph nodes,
subcutaneous tissues, and lungs.

37. • KIDNEY :-
The kidneys are often enlarged and congested
with a dusting of pigment in the glomeruli
and hemoglobin casts in the tubules.

38. Bone Marrow
• Yellow fatty marrow is replaced by vascular
cellular tissue
• Erythroblastic reaction of normoblastic type ,
increase in nucleated RBC number.
• Hyperplasia of RE cells which are laden with
malarial pigments.

39. • HEART:-
• Nonspecific focal hypoxic lesions in the heart
may be induced by the progressive anemia
and circulatory stasis in chronically infected
people.
• Myocardium : focal interstitial infiltrates.
• Nonimmune patient: pulmonary edema or
shock with DIC may cause death.

40. Cerebral Malaria
• Pial & cerebral vessels are markedly congested.
• C/S : multiple punctiform haemorrhages.
• M/E :
• a) Dilation & congestion of cerebral capillaries
filled with parasitised RBCs.
• b) perivascular Ring haemorrhage around
plugged vessel
• c) scattered areas of softening (degeneration of
nerve tissue)invaded by glial cells  Malaria
Granuloma

41. Retina
Malarial retinopathy consists of four main components: retinal
whitening, vessel changes, retinal hemorrhages, and
papilloedema

42. 42
Pregnancy
• Serious complication in pregnancy
• maternal deaths, foetal death (x10) & foetal
retardation
• Placental sequestration & clumping
• accumulation of intervillus macrophages & fibrin
deposits

43. Foetal Malaria
PMVF:Pigmented
monocytes in
foetal vessel

44. Pathology of Algid Malaria
• GIT : mucus membranes show slate grey
pigmentation,punctiform haemorrhage.
• M/E : mucosal & submucosal capillaries are congested
with parasitised RBCs.
• Adrenal : necrosis and haemorrhage of zona fasciculata
and zona reticulata.
• Peripheral Blood Vessels : Generalised vascular collaps.

45. • Malaria Granuloma : collection of
proliferated glial cells radialy arranged
around occluded blood vessel.

46. Erythrocytic forms (signet)
Young ring form trophozoites

47. Gametocytes
Micro
Macro
P.falciparum

48. 48
Tertian Malaria - P. vivax & P. ovale
• Rarely fatal- relapses common
• Prodrome
• myalgia, headache, chills, low grade irregular
fever
• paroxysms on alternate days
• Bengn tertian fever
• Spleen palpable 10-14 days
• P. ovale milder with shorter initial attacks
• RBC: Enlarged, Distoted,Washed out,Cytoplasmic
Stippling

49. 49
Qartan Malaria - P. malariae
• Paroxysms every third day
• Mildest and most chronic of the 4
• Immune complex nephropathy
• Seasonal variation (wet season)

50. 50
Falciparum Malaria
• Cause of virtually all malaria deaths
• asynchronous cycle
• onset insidious - fever variable
• Rapid onset of splenomegaly
• Severe anaemia, jaundice, hyperventilation, CNS
dysfunction (delirium, stupor, coma)

51. 51
Blackwater Fever
• Massive intra vascular haemolysis
1)Haemoglobinuria
2)Acute renal failure
3}Tubule necrosis
• parasitemia may be absent
• Mortality 20-30%

52. 52
Diagnosis
1. Clinical Diagnosis
2. Microscopic examination of blood Smear
3. Rapid Diagnostic Tests( antigen or enzyme)
4. Fluorescence Microscopy
5. Serological Technique
6. PCR : nucleic acid detection of parasite.

53. Microscopic Examination
• Gold Standard for diagnosis, sensitive can detect
about 50 parasite/µl in experienced hands
• Smear obtained at the onset of fever and chills.
• Before starting antimalarials
• Skin Puncture smear made immediately is used
• If necessary use EDTA
• Thick Smear : Detection of parasite
• Thin Smear : Identification of species.

54. Examination of Thin Blood Film
•Alkaline buffer solution is necessary to bring out schuffeners dots:
•Sodium phosphate 2g
•Potassium dihydrogen phosphate 1mg
•Thymol 1mg
•D.W 1000ml
• Important to see 200 oil immersion fields before reporting negative.

55. Degree Of Parasitemia
• Number of parasite amongst 1000 RBC counted &
reported as %.
• No. Of parasite in 1 µl is calculated
=RBC Count in million/µl X Parasite%
red cell count in million/µl is known(5 million/µl)
• Parasitemia>10% in P falciparum is indication for
Exchange Transfusion.

56. Examination of Thick Smear
• Big drop of blood taken on slide , spread with needle
to form ½ inch square or
• 4 small drops are joined by a needle
• In moist temprature about ½ hr is taken for thick
smear to dry at room temprature
• Thick smear is dehaemoglobinised before staining by
• Glacial acetic acid 2% 4 parts , Tarteric acid 2 % 1
part mixture
• Atleast 100 oil immersion fields seen in thick smear.

57. Fields Stain
• Two solutions A & B used
• Thick film placed in sol. A for 1-2 sec till Hb is removed
• Rinsed in clean water
• Placed in sol. B for 1 sec
• Rinsed in clean water for 2-3 sec
Solution A Solution B
Methylene blue 8 g Eosine 1g
Azure I 0.5g Disodium hydrogen phosphate 5g
Disodium hydrogen phosphate 5g Potassium dihydrogen phosphate 6.25g
Potassium dihydrogen phosphate 6.25g D.W 500ml
D.W 500ml

58. Combined Thick & Thin Film
• Two drops of blood taken one ½ inch & other 1 Inch
from Lt & Rt edge of the slide.
• Former is made into thick & later into thin film.
• Thick is dehaemoglobinised & then stained with thin
film
• The undilluted leishman is poured over thin & after
dilution the stain is poured over thick film Or
• Thin film is fixed with methanol & then whole slide is
flooded with Geimsas Stain for1/2 to 2 hr.

59. JSB Stain
• Jaswant Singh & Bhattacharji Stain
• Rapid Romanowsky Stain with 2 solutions.
• Sol. 1 : Methylene blue 0.5 g, K.Dichromate 0.5g,1% H2SO4
3ml,1%KOH 10ml , Water 500ml
• Sol 2 :1g Eosin in 500ml tap water.
Steps : fix slide in alcohol
Immerse slide in Sol. 1 : 30 sec
Wash in a jar III having tap water (PH 6.2-6.6)
Stain with Sol.2 :1 sec
Wash in Jar III : 4 sec.
Immerse slide in Sol. 1 :30 sec
Wash in a jar III: 10 sec
Dry & examine.

60. Degree of Parasitemia in Thick Film
• No. Of parasite counted till 200 WBC
No.of Parasite/µl =TLC/µl X No. Of Parasite
200
TLC is taken generally as 8000/µl
= No. Of Parasite X 40.

61. Rapid Immunochromatographic Test
• Antigens against which commercial test kits are
available are :
1) Histidine rich protein-2(HRP-2)
• Senstivity & Specificity > 90%(P.D>100µl)
• Synthesised by asexual blood stages & young
gametocyte of P. Falciparum
• Expressed on RBC surface.
2) Parasite lactate dehydrogenase (pLDH)
• Glycolytic pathway enz. Of all 4 species.
• Levels correlate with parasite density

62. pLDH

64. Advantage of commercial test kit
• Confirmation of P.falciparum in doubtful identity
• Confirmation of P.falciparum in mixed infection
• When facilities of microscopic diagnosis are not
present
• Rapid self diagnosis by tourists travelling to endemic
countries is possible.
• Disadvantage : Test remain positive following
several days of treatment
• Distinction B/W gametocyte and other pathogenic
form is not possible

65. Quantitative Buffy Coat (QBC)
• Useful for screening large number of samples
• Quick, saves time.
• Requires centrifuge, special stains.
• Disadvantage:-
• Species identification & Quantification difficult
• Cant store capillaries for later reference.

66. • Blood centrifuged in a spl. Capillary tube
containing a float coated with acridine orrange and
an anticoagulant.
• parasite is concentrated in upper layer of red cell
just below buffy coat & stained with fluorescent
dye.
• When capillary is viewed using spl.
Objective(paralens) attached to fluorescent
microscope,malaria fluoresce yellow against red
black backgroud.
• Howell Jolly Bodies gives false positives reaction.
• Scizont & gametocytes are localised in buffy layer
so, missed

67. Fluorescent Microscopy
• Modification of light microscopy
• Fluorescent dyes detect RNA & DNA that is contained
in parasites.
• Kawamoto Technique
• Stain thin film with acridine orange (AO)
• Requires Fluorescent Microscope
• Nuclei of malaria parasite fluoresce bright green &
cytoplasm red.
• Sensitive around 90 %

68. Polymerase Chain Reaction(PCR)
• Molecular technique to identify parasite genetic
material
• Uses whole blood directly collected in anticoagulated
tube or directly onto filter paper
• Threshold for detection 5 parasites/µl
• Definitive species diagnosis possible
• High sensitivity & specificity
• Screening of blood donors,& epidemiological surveys

69. Serology
• Serology detects antibodies against malaria parasites
• Using : Indirect immunofluorescence (IFA)
Enzyme-linked immunosorbent assay (ELISA).
• Serology does not detect current infection but rather
measures past exposure.
• Helps in retrospective epidemiological surveys
• Screening of donor for asymptomatic infection
• Investigating congenital malaria
• Retrospective diagnosis.

70. Treatment
• 1)Therapeutic: 4-aminoquinolines i.e
• Quinine , chloroquine , Artemisinin, &mefloquine
( erythrocytic phase)
• Primaquine for radical cure .
• 2) Prophylaxis : Proguanil, pyrimethamine &
trimethoprim.
• Destuction of adult mosquito.
• Antilarval measures.
• use of repellants , bed nets.

71. Thank
You