1. II MBBS Dr Ekta Chourasia Microbiology
PLASMODIUM
II MBBS
Dr Ekta Chourasia
Microbiology

2. Dr Ekta Chourasia, Microbiology
Taxonomy
Phylum Apicomplexa
Subphylum Sporozoa
Genus Plasmodium
Disease Malaria
Geographical Tropical & distribution Subtropical countries
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Dr Ekta Chourasia, Microbiology

3. Dr Ekta Chourasia, Microbiology
Genus Plasmodium
Consists of 4 species:
P. vivax
P. falciparum
P. malariae
P. ovale
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Dr Ekta Chourasia, Microbiology

4. Landmarks in the evolution of Malaria
1880 – Laveran identified the malarial parasite in
an unstained smear
1885 – Golgi described the blood stage
(erythrocytic schizogony) of malarial
parasite – Golgi cycle
1898 – Amigo & Grassi described the life cycle
1891 – Romanowsky introduced the staining
method
1897 – Ronald Ross while in Calcutta, India,
demonstrated Anopheles sp. of mosquitoes as
vectors of malaria.
Got Nobel prize for his work in 1902
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Dr Ekta Chourasia, Microbiology

5. Transmission & Life Cycle
Definitive host Female Anopheles mosquito
Intermediate host Man
Infective form Sporozoites
Portal of entry Skin
Mode of transmission Bite of an infected mosquito
Site of localization First in liver cells & then in RBCs
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Dr Ekta Chourasia, Microbiology

6. Dr Ekta Chourasia, Microbiology
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Dr Ekta Chourasia, Microbiology

7. Phases of Development in Man
Inside the liver (tissue phase)
Pre- erythrocytic schizogony – no clinical symptoms, no pathological damage
Exo- erythrocytic schizogony – cause of relapse
Inside the RBCs (erythrocytic phase)
Erythrocytic schizogony – cause of malarial paroxsyms
Gametogony – infects mosquito
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Dr Ekta Chourasia, Microbiology

8. Morphological forms seen in Humans
In liver:
Sporozoites
Pre erythrocytic schizonts
Merozoites – infect RBCs
In RBCs :
Trophozoites – ring form
Schizonts
Merozoites – released by the rupture of schizonts – infect other RBCs
Gametocytes – micro and macro gametocytes
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Dr Ekta Chourasia, Microbiology

9. Morphological forms seen in Mosquito
Further differentiation & development of gametocytes take place in mosquito
Macro gametes (female gametes) – each macro gametocyte develops in to one macro gamete in the mid gut of mosquito
Micro gametes (male gametes) – one micro gametocyte produces 6 to 8 micro gametes by exflagellation.
Zygote – Ookinete – Oocyst – rupture – release of Sporozoites – predilection to salivary glands.
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Dr Ekta Chourasia, Microbiology

10. Other modes of transmission
Sporozoite- induced- malaria : injection of an emulsion of salivary glands of mosquito containing sporozoites
Trophozoite- induced- malaria : injection of blood from a malarial patient containing the asexual forms of erythrocytic schizogony e.g.
Transfusion malaria – when persons with latent infection are used as donors
Congenital malaria – transmission through some placental defects (a healthy placenta acts as a physiological barrier)
Drug addicts – by using same syringe
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Dr Ekta Chourasia, Microbiology

11. Dr Ekta Chourasia, Microbiology
Incubation period
P. vivax
P. ovale to 14 days
P. falciparum
P. malariae days to 6
weeks
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Dr Ekta Chourasia, Microbiology

12. Dr Ekta Chourasia, Microbiology
Pathogenicity
Infection causes intermittent fever – Malaria
Each of the 4 species causes a characteristic fever:
P. vivax Benign tertian/ vivax malaria
P. falciparum Malignant tertian/ falciparum
malaria, black water fever
P. malariae Quartan malaria
P. ovale Tertian/ Ovale malaria
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Dr Ekta Chourasia, Microbiology

13. Dr Ekta Chourasia, Microbiology
Clinical Features
Series of febrile paroxysms – fever is caused by the release of merozoites & toxins from ruptured erythrocytic schizont which in turn causes the release of cytokines.
Quartan malaria – every 72 hrs
Tertian malaria - every 48 hrs
* each paroxysm has 3 stages - cold stage (rigors), hot stage (high temp., body & joint pains, vomiting & diarrhoea) and perspiration stage (fall in temp.)
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Dr Ekta Chourasia, Microbiology

14. Dr Ekta Chourasia, Microbiology
Clinical Features
Anaemia – due to breakdown of RBCs, particularly occurs in falciparum malaria
Splenomegaly – all forms
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Dr Ekta Chourasia, Microbiology

15. Dr Ekta Chourasia, Microbiology
Falciparum Malaria
Most widespread
Accounts for 80% of malaria cases worldwide
Most pathogenic of human malaria species
Untreated infections - severe disease & even death, particularly in young children, pregnant woman & non immune adults.
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Dr Ekta Chourasia, Microbiology

16. Dr Ekta Chourasia, Microbiology
Falciparum malaria
Severe falciparum malaria is associated with
Pernicious malaria /cerebral malaria
Blackwater fever
Anaemia
Hypoglycaemia
Hypotension
Complications in pregnancy
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Dr Ekta Chourasia, Microbiology

17. Dr Ekta Chourasia, Microbiology
Pernicious Malaria
Def: refers to a series of phenomenon occurring during infection with P. falciparum which, if not effectively treated, threatens the life of the patient with in 1 to 3 days
In children & non immune adults, can cause coma & death – Cerebral malaria.
Occurs as a result of capillary blockage.
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Dr Ekta Chourasia, Microbiology

18. Dr Ekta Chourasia, Microbiology
Black Water Fever
Occurs in previously infected subjects
Can also occur in non immune adults with severe falciparum malaria, and also as a complication of quinine therapy.
A rare but acute condition characterised by sudden & massive hemolysis of parasitised & non parasitised RBCs followed by fever and haemoglobinuria.
Often fatal due to renal failure
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Dr Ekta Chourasia, Microbiology

19. Dr Ekta Chourasia, Microbiology
Black Water Fever
Difficult to find the parasites
in the blood following a
hemolytic attack.
Urine appears dark red to
brown black due to the
presence of free Hb.
Clinical features – fever, rigor, aching pains in the loin, icterus, bilious vomiting, circulatory collapse, haemoglobinuria & acute renal failure.
Treatment – Chloroquine, blood transfusion, peritoneal dialysis in ARF.
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Dr Ekta Chourasia, Microbiology

20. Dr Ekta Chourasia, Microbiology
Anaemia
Can be severe & occur rapidly, particularly in young children
Occurs due to destruction of parasitised RBCs – phagocytosis & destruction in the spleen
Decreased production of RBCs in the bone marrow.
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Dr Ekta Chourasia, Microbiology

21. Falciparum malaria in Pregnancy
Can result in:
Severe anemia
Low birth weight babies
Greatest risk in 1st pregnancy
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Dr Ekta Chourasia, Microbiology

22. Malaria caused by P.vivax, P.ovale & P.malariae
Rarely life threatening
Relapses/ recurrences are a feature
Recurrences in Malaria
May result from – reinfection or
- due to certain events related
to the parasite’s life cycle
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Dr Ekta Chourasia, Microbiology

23. Dr Ekta Chourasia, Microbiology
Recurrence of Malaria
Two types of recurrences known in malaria:
Recrudescence –
seen in P. falciparum & P. malariae
due to persistence of blood infection (some erythrocytic forms evade host immunity) even after clinical illness has subsided.
The numbers may increase later, leading to reappearance of clinical symptoms
Occur mostly up to one year or so but in P. malariae, it can occur even after decades
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Dr Ekta Chourasia, Microbiology

24. Dr Ekta Chourasia, Microbiology
Recurrence of Malaria
Relapse
Occurs due to a special form of parasites – hypnozoites.
Hypnozoites are the sporozoites that remain dormant after infecting liver
Activated from time to time to initiate pre erythrocytic schizogony - Exoerythrocytic schizogony
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Dr Ekta Chourasia, Microbiology

25. Genetic factors protecting against Malaria
Sickle cell anaemia – sickle celled RBCs are removed by the spleen before the development of schizonts
Ovalocytosis – RBCs are rigid and they resist parasitic invasion
Duffy blood group negative individuals – duffy blood group Ag is the receptor for the attachment of merozoites of P.vivax
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Dr Ekta Chourasia, Microbiology

26. Genetic factors protecting against Malaria
Newborn infants – natural protection for 1st few months of life due to high conc. of HbF in their RBCs.
Beta thalassaemia – protects against severe falciparum infection
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Dr Ekta Chourasia, Microbiology

27. Dr Ekta Chourasia, Microbiology
Laboratory Diagnosis
Microscopy – detecting & identifying malarial parasites in peripheral blood films.
Concentrating parasites in venous blood by centrifugation when they can not be found in blood films
Using a rapid malaria Ag or enzyme detection test
Other tests – Hb, PCV, Blood glucose, total WBC & platelet count.
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Dr Ekta Chourasia, Microbiology

28. Examination of Blood film
Collection of blood
- best prepared directly from capillary blood
- in EDTA bulb (used within 30 mins)
Time of collection
- as soon as possible if malaria is suspected
- before administering antimalarials
- during pyrexial phase
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Dr Ekta Chourasia, Microbiology

29. Dr Ekta Chourasia, Microbiology
Types of Blood film
Two types:
Thick films :
- 30 to 40 times more sensitive than thin films
- more suitable for detection of malarial parasite when they are few in number
- blood is not fixed, RBCs are lysed during staining (only parasitic forms will be seen)
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Dr Ekta Chourasia, Microbiology

30. Dr Ekta Chourasia, Microbiology
Types of Blood film
2. Thin films :
- to confirm the Plasmodium species
- assists in the identification of mixed infections
- blood is fixed, parasites are seen within the RBCs
- also helps in assessing the response to treatment especially in areas where drug resistance is suspected (by counting the number of parasitised RBCs before & after the treatment)
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Dr Ekta Chourasia, Microbiology

31. Making of Thin & Thick films
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Dr Ekta Chourasia, Microbiology

32. Dr Ekta Chourasia, Microbiology
Fixation & Staining
Fixation – thin films are fixed with absolute alcohol for 1 to 2 mins.
Staining – films are stained with Romanowsky stain: giemsa, field’s, wright’s
Giemsa – 10% solution for 10 mins
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Dr Ekta Chourasia, Microbiology

33. Reporting of Blood film
Look for the different morphological forms of parasite in blood smear:
Trophozoites / ring forms
Schizont
Gametocytes
Identify species – differences in the characteristics of morphological forms in different species
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Dr Ekta Chourasia, Microbiology

34. Trophozoites / Ring forms
Character
P. vivax
P. falciparum
Size
2.5µ (1/3rd of RBC)
1.25 to 1.5 µ
Cytoplasm
Thick opposite to nucleus
Uniform thickness
Nucleus
One/ ring
Can have >1
Number of rings
One ring/ RBC
>1/ RBC
Location in RBCs
Always inside RBCs
Inside as well as on the surface (accole’ forms)
Type of RBC infected
Preferentially young RBCs & reticulocytes
All types
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Dr Ekta Chourasia, Microbiology

35. Dr Ekta Chourasia, Microbiology
Thin Blood Film
Thick Blood Film
Ring Forms /
Trophozoites
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Dr Ekta Chourasia, Microbiology

36. Dr Ekta Chourasia, Microbiology
Schizont
Character
P. vivax
P. falciparum
Size of RBC
Increases to twice its size
Does not change
No of merozoites 16
8 to 32
Arrangement of merozoites
Symmetric in form of rosette
Asymmetrical
Presence in peripheral blood
Present
Absent
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Dr Ekta Chourasia, Microbiology

37. Dr Ekta Chourasia, Microbiology
P. vivax
P.falciparum
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Dr Ekta Chourasia, Microbiology

38. Gametocytes (male & female)
Character
P. vivax
P. falciparum
Shape – Male
Female
Spherical
Cresentic
Sausage shaped
Nucleus- M F
Central, diffuse
Peripheral,small
Central,compact
Infected RBC
Enlarged
Deformed, with its membrane stretched.
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Dr Ekta Chourasia, Microbiology

39. Dr Ekta Chourasia, Microbiology
P.vivax
P. falciparum
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Dr Ekta Chourasia, Microbiology

40. Dr Ekta Chourasia, Microbiology
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Dr Ekta Chourasia, Microbiology

41. Counting the % age of parasitised RBCs
On thin blood films
When falciparum malaria parasitemia is high
Method of counting:
Select an area where no of RBCs is roughly 250.
Count the no of parasitised RBCs in 4 such fields i.e. approximately 1000 RBCs.
Divide by 10 to obtain the percentage.
*WHO – if it is >5%, then the parasitemia is heavy & prognosis is poor.
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Dr Ekta Chourasia, Microbiology

42. Buffy Coat preparation
To concentrate malarial parasite
Centrifuge EDTA anticoagulated venous blood in a thin bore capillary tube
Buffy coat layer is formed between the RBCs & the plasma.
Break the tube & transfer buffy coat & RBCs to a slide - make a thin smear – air dry – fix with ethanol – stain with Giemsa.
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Dr Ekta Chourasia, Microbiology

43. Quantitative Buffy Coat
Capillary tube is coated with an anticoagulant & Acridine orange fluorescent dye
After centrifugation, the tube can be used for two purpose:
Complete blood count
Identification of malarial parasite using a fluorescence microscope.
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Dr Ekta Chourasia, Microbiology

44. Quantitative Buffy Coat
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Dr Ekta Chourasia, Microbiology

45. Rapid Diagnostic tests
Developed to diagnose falciparum malaria rapidly & without a microscope.
Can also detect vivax malaria
Three tests are available commercially
Detects either HRP2 Ag (Histidine rich protein) or specific pLDH (parasite lactate dehydrogenase)
Both HRP2 & pLDH are produced by the parasites during their growth & differentiation in RBCs.
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Dr Ekta Chourasia, Microbiology

46. Rapid Diagnostic tests
HRP2 tests
detection of P.falciparum
Two types of test – ParaSight F
- ICT Malaria Pf
pLDH test e.g. OptiMAL test
Detection of P.falciparum & P.vivax
Produced by all human malarial parasites
Differentiation of species is based on antigenic differences between pLDH isoforms.
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Dr Ekta Chourasia, Microbiology

47. Dr Ekta Chourasia, Microbiology
ParaSightF test
Optimal test
ICT Malaria Pf / Pv
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Dr Ekta Chourasia, Microbiology

48. Dr Ekta Chourasia, Microbiology
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Dr Ekta Chourasia, Microbiology

49. Stage specificity of antimalarial drugs
Stage of malarial parasite
Antimalarial drug
Sporozoite
Proguanil, Pyrimethamine
Hypnozoite
Primaquine
Pre- erythrocytic schizont
Blood schizont
Chloroquine, Quinine, Artemisinin
Gametocyte
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Dr Ekta Chourasia, Microbiology

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Dr Ekta Chourasia, Microbiology