The document provides an extensive overview of malaria, a protozoal disease caused by Plasmodium species primarily transmitted by female Anopheles mosquitoes. It outlines the historical context, life cycle, morphology, pathogenicity, and clinical manifestations of the disease, particularly focusing on different species such as Plasmodium vivax, Plasmodium falciparum, Plasmodium malariae, and Plasmodium ovale. Additionally, it discusses laboratory diagnosis methods for malaria and highlights the global impact of malaria, with significant morbidity and mortality rates.

1. MALARIAL PARASITE
By
Sarita Rizal r.n. : 6
& Shimraun Baidya r.n. : 7
B.Sc. MLT 1st
year

2. INTRODUCTION
 Malaria : Italian word mal- bad & aria- air.
 Malaria was commonly found in swampy areas and was thought to be
contracted by breathing in "bad air" (= mal aria) in the swamps.
 Malaria is a protozoal disease caused by Plasmodium species which
infects RBC of humans.
 Transmitted by Female Anopheles mosquitoes(vector) from humans to
humans.
 Besides humans, parasite can cause the disease in higher apes,
monkeys ,birds, rodents, bats and cold blooded animals e.g lizard.

3.  40% of the world’s population are at risk
 300 million illnesses per year
 1.2 million deaths per year
 90% deaths in sub-Saharan Africa

5. HISTORY
 Disease has been known since antiquity - one of first reports described
fevers in 1550 BC.
 In early 17th
century, bark of cinchona was successively used to treat the disease
 In 1880, Charles Loius Laveran discovered the parasite (exflagellated gametocytes)
in an unstained preparation of fresh blood.
 Methylene blue was used by Marcheafava for staining malaria parasites in 1883.
 Golgi demonstrated the multiplication of asexual blood forms of benign tertian
malaria in 1885.
 Romanowsky’s polychrome stain was introduced for staining malaria parasites in
1891.

6.  In 1898, Ross demonstrated developing plasmodia in the stomach of
mosquito. Later he demonstrated the life cycle of malaria parasite in
mosquito for which he was awarded Nobel prize in 1902.
 In 1900 observation of mosquito transmission by Ross was supported
by Patrick Manson.
 1948 Shortt and Garnham demonstrated the site of the exo-
erythrocytic development in the liver.
 In the next year, demonstrated pre-erythrocytic cycle.
 In 1955, WHO launched a worldwide malaria eradication program
which continued till 1976 when it was officially declared failure
 In 1976, a continuous P. falciparum culture was established in RPMI
1640 medium containing a thin layer of stationary human RBCs and
overlay of medium containing human serum

7. CLASSIFICATION
Phylum: Apicocomplexa
Class: Sporozoa
Sub class: Coccidia
Order: Eucoccidia
Sub order: Hemosporina
Genus: Plasmodium
Species: Plasmodium vivax
Plasmodium falciparum
Plasmodium malariae
Plasmodium ovale

8. SUBDIVISION OF Plasmodium
The genus Plasmodium is subdivided into the subgenus:
1. Laverania: P. falciparum
2. Plasmodium: P. vivax, P. ovale & P. malariae

9. Species parasite of human
Four recognized & distinct species:
I. Plasmodium vivax
II. Plasmodium falciparum
III. Plasmodium malariae
IV. Plasmodium ovale
Other species:
 Plasmodium knowlesi: Human infections with P. knowlesi, a species that
naturally infects monkeys (macaques), have been reported from Malaysia
and Thailand.
 Morphologically P.knowlesi resembles P. malariae but unlike P. malariae,
parasite numbers have been reported as high and clinical symptoms more
severe.

10. Generic character
 Parasites belonging to phylum apicomplexa.
1. Lacks motile organs.
2. Shows slight amoeboid form change.
3. Asexual reproduction (schizogony) followed by sexual (syngamy) i.e.
alternation of generation.
 Sporozoa infecting human divided into 2 groups:
1. Intestinal parasite
2. Blood inhabiting parasite

11.  In malaria, alternation of generation with alternation of host.
 Asexual cycle (schizogony cycle) : occurs in RBC of the vertebrate host
forming Schizonts
 Sexual cycle (sporogony cycle) : occurs in invertebrate host forming
Sporozoites
 Gametogony (gametocytes formation) : starts in host RBC & completes in
mosquitos producing sporozoites (infective form)

12. Geographical distribution
 Found in all countries.
1. P. malariae: subtropical zone
2. P.vivax: temperate zone
3. P.ovale: tropical Africa, west Africa, Asia (Philippines) ,Endemic in Ethiopia
4. P. falciparum: tropical and sub- tropical zone

13. Habitat
 In human- liver parenchymal cells & inside RBC
 carried to all organs.

14. Method of transmission
 Mode of infection: by biting of female anopheles mosquito through
proboscis, puncturing human skin & releasing large no. of sporozoites into
blood stream.
 Transmitting agent: female anopheles- 10 species.
 Infective forms: sporozoites.
 Portal of entry: skin.
 Localization site: 1st
liver cells, 2nd
RBCs.

15. MORPHOLOGY
 Plasmodium is a Eukaryote, an organism whose cells have a nucleus, but with
unusual features

17. Life cycle of Plasmodium species
 Passes its life cycle in 2 hosts.
 Human : liver parenchymal cell and RBCs,
 Reproduces asexually(schizogony) ,
 Represents intermediate host.
 Female anopheles mosquito: gametocytes developed in human further
develops in mosquito forming sporozoites(infective form)
 Sexual reproduction.
 Represents definitive host.

20. Pre-erythrocytic Schizogony
 Occurs inside liver parenchymal cells.
 Sporozoites do not directly enter into RBCs but undergoes for development
inside liver parenchyma.
 After development liberated as merozoites. Smaller:
micromerozoites Larger: macromerozoites
 No any clinical manifestation & pathological damage are seen.
 Parasites not found in peripheral blood.

21. Erythrocytic Schizogony
 Parasites reside inside RBCs.
 Passes 3 stages:
 Trophozoite Schizont Merozoite.
 During this phase clinical manifestations occurs.
 May be continued for considerable time & finally infection reside due to
decreased reproduction capacity or destruction.

22. Gametogony
 Occurs after erythrocytic stage with gametocyte production.
 Host is called as ‘carriers’
 Some merozoites instead of developing into trophozoites & schizonts, forms
gametocytes(sexual function) after leaving human host.
 Develop in RBCs of internal organs capillaries(spleen & bone marrow).
 Mature forms found in peripheral blood.
 No ferbrile reaction, produced for continuity of generation.

23. Latent Stage(Hepatic)
 Formation of resting phase form k/a hypnozoites in liver cells after infection
establishment.
 Some of the sporozoites of P. vivax and P. ovale after invading liver cells
delay their development into Pre-erythrocytic(PE) schizonts.
 Becomes active and develop into PE schizonts later, causing relapses.
 Varies according to the species; absent in P. falciparum & present in P.
vivax, P.ovale.

24. Mosquito Life Cycle: Sexual Cycle
 Starts after gametocyte production in human which is then ingested by mosquito
during biting/blood meal.
 Mosquito ingests both sexual and asexual forms but mature are capable of
development & rest die off.
 Development occurs only in female anopheline mosquito.
 Human carrier need at least 12 gametocytes/mm³ of blood with fe
gametocyte>male gametocyte.
 1st
phase development occurs in stomach.

25.  Microgamete- exflagellation with 4-8 filamentous structure.
 Macrogamete – doesn’t show any flagellation.
 Fertilization : chemotaxis action & fusion forming zygote(20min - 2hrs)
 After 24hrs zygote lengthens and mature into ookinete(vermicule)
 Ookinetes develop into oocyst: spherical mass with capsule size 6-12µm in
diameter.

26.  Oocyst(upto 100 in stomach wall) matures to form haploid sporozoites(100-
1000).
 Oocysts rupture after 10 days releasing sporozoites in the body cavity &
circulated all over the body except ovary.
 Concentration is maximum on salivary gland duct.
 This stage is capable of transmitting infection to man with a single bite.
 Different spp. Can develop in same mosquito leading to mixed infection.

27. Life cycle of different plasmodium
species in human
I. Plasmodium vivax
II. Plasmodium falciparum
III. Plasmodium malariae
IV. Plasmodium ovale

28. 1.Plasmodium vivax
• These species have the widest geographic area. This particular species is
found mostly in the tropics and throughout Asia.
• They cause “Benign tertian malaria” with low deaths.
• Trophozoites, schizonts and gametocytes can be seen in the peripheral
blood.

29. Morphological structures
 Trophozoite :
i. Early(Ring stage):
 Large about 2.5 µm in diameter.
 Usually presence of 1 nucleus, sometimes 2.
 Development of central large non-contractile vacuole which pushes the
nucleus to one side of peripheral cytoplasm.
ii. Late:
 Non-contractile vacuole disappears.
 Develops pseudopodial processes into cytoplasm forming amoeboid stages.

30.  Shinzont :
 Large about 9-10 µm in diameter.
 Almost completely fill an enlarged RBC.
 Nucleus undergoes multiple fission to form nuclei (12-24).
 The nuclei are arranged at the periphery of the parasite.
 Mature schizonts contains 12-14 uni-nucleated oval merozoites.
 Merozoites are arranged in grape-like cluster.

31.  Gametocytes :
i. Microgametocytes:
Spherical in shape and is about 9 -10 µm .
Cytoplasm stain light blue.
Nucleus is large and lies centrally.
ii. Macrogametocytes:
Oval in shape and is about 10 -12 µm .
Cytoplasm stain deep blue.
Nucleus is compact and lies peripherally.

33. 2.Plasmodium falciparum
 These species accounts for the majority of the inflections and is the most
lethal.
 They cause “Malignant malaria”.
 Only the early trophozoites and gametocytes can be seen in the peripheral
blood.

34. Morphological structures
 Trophozoite :
i. Early:
 Small about 12.5 – 1.5 µm in diameter.
 Often with 2 rings in one RBC.
 Sometimes parasite lie along the red cell membrane known as accole form.
ii. Late:
 Assumes a compacted and rounded form.
 Vacuole inconspicuous.

35.  Shinzont :
 Large about 4.5 - 5 µm in diameter.
 Fills two-third of a normal sized RBC.
 Mature schizonts contains 18-24 merozoites.
 Merozoites are arranged in grape-like cluster.

36.  Gametocytes :
i. Microgametocytes:
 Crescent-shaped and is about 8-10µm by 2–3 µm.
 Cytoplasm stain light blue.
 Nucleus is scattered throughout the cytoplasm.
 Pigment scattered throughout the cytoplasm.

37.  Gametocytes :
ii. Macrogametocytes :
 Crescent-shaped and is about 10-12µm by 2–3µm.
 Cytoplasm stain deep blue.
 Nucleus is condensed into a small compact mass at the center.
 Pigment surrounds the nucleus.

39. 3.Plasmodium ovale
 It is the rarest of the top four species of Plasmodium, and is found in West Africa.
 They cause “Mild tertian malaria”.
 Trophozoites, schizonts and gametocytes can be seen in the peripheral blood.

40. Morphological structures
 Trophozoite :
i. Early(Ring stage):
 Large about 2.5 µm in diameter.
 Usually presence of 1 nucleus, sometimes 2.
 Development of central large non-contractile vacuole which pushes the
nucleus to one side of peripheral cytoplasm.
ii. Late:
 Non-contractile vacuole disappears.
 Small, compact and rounded.
 Not amoeboid.

41.  Shinzont :
 Large about 6.2 µm in diameter.
 Fill about three quarters of slightly enlarged RBC.
 Nucleus undergoes multiple fission to form nuclei.
 Mature schizonts contains 6-12 merozoites usually 8.
 Merozoites are arranged irregularly.

42.  Gametocytes :
i. Microgametocytes:
 Spherical in shape.
 Cytoplasm stain light blue.
 Nucleus is large and lies laterally.
ii. Macrogametocytes:
 Spherical in shape.
 Cytoplasm stain deep blue.
 Nucleus is compact and lies peripherally.

44. 4.Plasmodium malariae
 It is the form of malaria that has been shown to be the cause of zoonotic malaria, or
malaria that is transmitted from animals to human.
 They cause “Quartan malaria”.
 Trophozoites, schizonts and gametocytes can be seen in the peripheral blood.

45. Morphological structures
 Trophozoite :
i. Early(Ring stage):
 Large about 2.5 µm in diameter.
 Usually presence of 1 nucleus, sometimes 2.
 Development of central large non-contractile vacuole which pushes the
nucleus to one side of peripheral cytoplasm.
ii. Late:
 Non-contractile vacuole disappears.
 Elongation of the parasite occurs leading to formation of band form.
 Not amoeboid.

46.  Shinzont :
 Large about 6.5-7 µm in diameter.
 Almost completely fill an normal sized RBC.
 Nucleus undergoes multiple fission to form nuclei.
 Mature schizonts contains 6-12 merozoites usually 8.
 Merozoites are arranged around a central mass of pigment like a “daisy”.

47.  Gametocytes :
i. Microgametocytes:
 Spherical in shape.
 Cytoplasm stain light blue.
 Nucleus is large and lies laterally.
ii. Macrogametocytes:
 Oval in shape.
 Cytoplasm stain deep blue.
 Nucleus is compact and lies peripherally.

49. Pathogenicity:
1) Febrile paroxysm: begins in early afternoon
-consists of 3 stages:
i) cold stage: for 15-60 minutes
-patients feel intense cold and shivering
ii) hot stage- for 2-6 hours
-develops fever (40.0-40.6o
C), severe headache, nausea and vomiting
iii) sweating stage- fever ends by profuse sweating

50. Periodicity of attack:
-Benign tertian: P. vivax and P. ovale
-Quartan: P. malariae
2) Anaemia:- destruction of parasitized RBCs
-reduced erythropoiesis
-lysis and phagocytosis of infected RBCs
-autoimmune destruction of RBCs in malignant malaria
3) Spleenomegaly: due to massive proliferation of macrophages

51. 4) Pernicious malaria: life threatening complications in acute falciparum malaria due to
heavy parasitization
a. Cerebral malaria:
-characterized by hyperpyrexia, coma and paralysis
-capillaries of brain are plugged with parasitized RBCs
b. Algid malaria:
-cold skin, peripheral circulatory failure and shock
-may cause vomiting and diarrhea or dysentery
c. Septicaemic malaria:
-high degree of prostration
-high continuous fever

52. 5) Blackwater fever: due to repeated infections with P. falciparum
-quinine therapy for new attack causing massive destruction of RBCs
-fever, haemoglobinuria and renal failure

53. LABORATORY DIAGNOSIS
 Lab diagnosis contains:
1. Blood films
2. Antigen tests
3. Molecular method
4. Subjective diagnosis
5. Differential

54. Blood Films
 A microscopical examination of blood films forms one of the most important
diagnostic procedure in malaria.
 It contains:
1. Thin films
2. Thick films
A malarial parasites can be detected in the thick films and then the thin films are
examined for identifying the species.
At least 200-300 oil-immersion field should be examined before the smears are
considered negative.

55.  The parasites are most abundantly found in peripheral blood late in the febrile
paroxysm. So, collection should be done during this period.
 Schuffner’s, Maurer’s, ziemann’s and jame’s dots can be seen in RBC infected with P.
vivax, P. falciparum, P. malariae and P. ovale respectively.

57. Species Differentiation on Thin Films
Feature P. falciparum P. vivax P. ovale P. malariae
Enlarged infected RBC + +
Infected RBC shape round round,
distorted
oval,
fimbriated
round
Stippling infected RBC Mauer clefts Schuffner
spots
Schuffner
spots
none
Trophozoite shape small ring,
appliqu
large ring,
amoeboid
large ring,
compact
small ring,
compact
Chromatin dot often double single large single
Mature schizont rare, 12-30
merozoites
12-24
merozoites
4-12
merozoites
6-12
merzoites
Gametocyte crescent shape large,
round
large,
round
compact,
round

58. Species Differentiation on Thick Films

59. Antigen test
 For areas where microscopy is not available, or where laboratory staff are
not experienced at malaria diagnosis, there are commercial antigen
detection tests that require only a drop of blood.
 Immunochromatographic tests (also called: Malaria Rapid Diagnostic Tests
, Antigen-Capture Assay or "Dipsticks") have been developed, distributed
and fieldtested.

60.  Most frequently a dipstick or test strip bearing monoclonal antibodies is
used, which is directed against the target parasite antigens.
 Several commercial test kits are currently available.
 The test can be performed in about 15 minutes.
 Some antigens targeted by currently available RDTs are:- HRP-II(detect p.
falciparum only) and pLDH(detect all four plasmodium sps).

61.  Test strip consist of a sample pad, 3-detection lines(contains capture
antibodies specific for P. falciparum and all plasmodium sps and control
antibody respectively) and an absorbent pad.
 Depending on the kit, 2-50micro-L of finger prick blood specimen is
collected.
 The blood specimen is mixed on a sample pad with buffer solution that
contains hemolysing compound as well as specific antibody that is labelled
with detectable marker, such as: colloidal gold.

62.  If malaria antigen is present, the antigen-antibody complex is formed.
 The labelled antigen-antibody complex migrates up the test strips by
capillary action towards the detection line.
 The washing buffer is then added to remove the hemoglobin and permit
visualization of any coloured line on the strip.
 If the blood contain malaria antigen, the labelled antibody-antigen
complex will be immobilized from the pre-diposited line of captured
antibody and will be visually detectable.
 The complete test run takes 5-15 minutes.

63. Rapid diagnostic tests detect malaria antigens

65. Molecular methods
 Molecular methods are available in some clinical laboratories and rapid
real-time assays (for example, QT-NASBA based on the
polymerase chain reaction) are being developed with the hope of being
able to deploy them in endemic areas.

66. PCR as a Confirmatory Technique for Laboratory
Diagnosis of Malaria
 PCR was more sensitive compared to microscopy, allowing the detection of
Plasmodium in cases with low parasitemias, as well as mixed infections of
malaria.
 PCR was also able to detect one mixed infection with P. falciparum and P.
ovale that was missed by microscopy, as well as detect three from
submitted specimens from telediagnosis cases.
 PCR also better distinguished between P. vivax and P. ovale. We found that
2.2% of samples were incorrectly identified as P. vivax or P. ovale by
microscopy.

67.  The nested PCR used here allowed reliable detection of all four species of
Plasmodium.
 We conclude that this nested PCR is valuable as a confirmatory test and
implementation should be considered by reference laboratories and
worldwide with adequate laboratory infrastructure to perform molecular
procedures.
 Molecular techniques are a costly procedure, including the cost of labor
and access to reagents, compared to the examination of blood smears.

68. Subjective Diagnosis
 Areas that cannot afford laboratory diagnostic tests often use only a history
of subjective fever as the indication to treat for malaria.
 Using Giemsa-stained blood smears from children in Malawi, one study
showed that when clinical predictors (rectal temperature, nailbed pallor,
and splenomegaly) were used as treatment indications, rather than using
only a history of subjective fevers, a correct diagnosis increased from 2% to
41% of cases, and unnecessary treatment for malaria was significantly
decreased.

69. Quantitative Buffy Coat(QBC) test:-
 It is a new method for identifying the malarial parasite in the peripheral
blood.
 It involves staining of the centrifused and compressed red cell layer with
acridine orange and its examination under UV- light source.
 It is fast, easy and claimed to be more sensitive then the traditional thick
smear examination.

70.  The QBC tube is a high precision glass hematocrit tube, pre-coated
internally with acrydine orange stain and potassium oxalate.
 It is filled with 60micro-L of blood from finger, ear or heel puncture.

71.  A clear plastic closure is then attached.
 A precisely made cylindrical float designed to be suspended in packed
red cell is inserted.
 The tube is then centrifuged and the component of the Buffy coat separate
according to their densities forming discrete bands.
 Because of the presence of the leucocyte and thrombocyte cell band
width, the topmost area of the red cell is enlarged to 10-times normal.
 Then the QBC tube is placed on the tube holder and examined using
standard white light microscope.

72.  Fluorescing parasites are then observed at RBC/WBC interface.

73. Culture
 Trager and jensen successfully cultivated and maintained P. falciparum in
vitro, in human red blood cells.
 They used medium RPMI in a continuous flow system in which human
erythrocytes were in a shallow stationary layer covered by a shallow layer
of medium.
 The medium was made to flow slowly and continuously over the layer of
settled red cells under an atmosphere with 7% CO2 and 1-5% oxygen.
 This culture system is now widely used for the production of antigens.

74. Treatment:-
 Chloroquine was the standard treatment for acute malaria for many years
 But, some plasmodium are chloroquine resistant.
 So, Quinine is the most reliable alternative to chloroquine for the treatment
of malaria.
 Tetracycline and Clindamycin are used as an adjunct to quinine therapy.
 Chloroquine and quinine do not eliminate exo-erythrocytic parasites in the
liver. So, for this primaquine should be used.

75. Prophylaxis
 Malaria control depends on:
Spraying residual insecticides such as: DDT or melathion.
Spraying the breeding sites with petroleum oils as larvicides.
Using larvivorous fish, Gambusa affinis.
Flooding and flushing of breeding places.
Avoiding exposure to mosquito bites by
Wearing long sleeve clothing and trousers after sunset when insects are
most active
Using bed nets impregnated with pyrethroids
Using electric mat to vaporize synthetic pyrethroids or burning mosquito
coils
Application of mosquito repellents containing diethyltoluamide to exposed
skin