Malaria is an infectious disease caused by Plasmodium parasites that are transmitted via mosquito bites. The document discusses the history and life cycle of malaria parasites, describing how they were first observed by Hippocrates and eventually their full life cycle was discovered in the late 19th/early 20th century. It also summarizes methods for diagnosing malaria, including examining blood smears under microscopy, rapid diagnostic tests, fluorescent microscopy using stains, and PCR to detect parasite genetic material.

2. Laboratory diagnosis of
Malaria
MODERATOR - DR. A. PANCHONIA

3. Malaria and Man

4. Ancient History of Malaria
• Malaria parasites have been with us since the
dawn of time. They probably originated in
Africa (along with mankind), and fossils of
mosquitoes up to 30 million years old, show
that the malaria vector, the malaria mosquito,
was present well before the earliest history.

5. Hippocrates and Malaria
• Hippocrates, a physician
born in ancient Greece,
today regarded as the
"Father of Medicine", was
the first to describe the
manifestations of the
disease, and relate them to
the time of year and to
where the patients lived.

6. History – Events on Malaria
• 1753- name of malaria was given.
• 1847-Meckel observed presence of pigment in
organs.
• 1880 - Charles Louis Alphose Lavern discovered
malarial parasite in wet mount. wins Nobel Prize in
1907
• 1883 - Methylene blue stain - Marchafava
• 1891 - Polychrome stain- Romanowsky
• 1898 - Roland Ross - Life cycle of parasite
transmission, wins Nobel Prize in 1902
• 1948 - Site of Exoerythrocytic development in Liver
by Shortt and Garnham

7. Major Developments in 20th Century
• 1955 - WHO starts world wide malaria
eradication programme using DDT
• 1970 – Mosquitos develop resistance to DDT
Programme fails
• 1976 – Trager and Jensen in vitro cultivation
of parasite

8. Lavern and Ronald Ross
Pioneered the Events on Malaria

9. Introduction
• Malaria is probably one of the oldest diseases known to
mankind that has had profound impact on our history.
• It is a huge social, economical and health problem,
particularly in the tropical countries.
• Malaria is a vector-borne infectious disease caused by single-
celled protozoan parasites of the genus Plasmodium.
• Malaria is transmitted from person to person by the bite of
female mosquitoes.

10. Global problem

12. What causes Malaria
• Malaria is caused by a parasite called
Plasmodium, which is transmitted via the bites
of infected mosquitoes. In the human body,
the parasites multiply in the liver, and then
infect red blood cells.
• Transmission of Malaria do not occur <160c
and >330c
• Do not occur > 2000 meters altitude.

13. Etiology of Malaria
• Five Species known to infect Man
1 Plasmodium vivax – Benign Tertian, Tertian
Malaria(Grassi and Feletti)
2 P.ovale - Ovale tertian Malaria(Stephens)
3 P.malariae – Quartan malaria (Laveran)
4 P.falciparum – Falciparum malaria or
Malignant Tertian malaria(Welch)
5 P. knowlesi (Sinton and Muller)

14. LIFE CYCLE OF MALARIA
Oocyst
Sporozoites
Zygote Stomach Wall
Salivary Gland
Gametocytes Pre-erythrocytic
(hepatic) cycle
Exo-erythrocytic
(hepatic) cycle
Erythrocytic Hypnozoites
Cycle
Adapted from:

15. LIFE CYCLE OF MALARISA PARASITE
IN MAN IN MOSQUITO
• PRE ERYTHROCYTIC • GAMETOCYTE
SCHIZOGONY- • ZYGOTE
CRYPTOZOITES. • OOKINETE
• ERYTHROCYTIC CYCLE- • OOCYST
MEROZOITES.
• SPOROZOITE
• GAMETOGONY-
GAMETOCYTES
• EXOERYTHROCYTIC CYCLE-
PHANEROZOITES.

16. Period of Pre erythrocytic cycle
• 1 P.vivax 8 days
• 2 P.falciparum – 6 days
• 3 P.malariae - 13 – 16 days,
• 4 P.ovale 9 days
On maturation Liver cells ruputure
Liberate Merozoites into blood stream

17. Affinity of Parasite to Erythrocytes
• P.vivax Young red blood cells
• P.malariae Old red blood cells
• P.ovale Young red blood cells
• P.falciparum Infects all age groups
Also adhere to the endothelial lining of Blood
vessesl
Causes the obstruction, Thrombosis and Local
Ischemias

18. Erythrocytic Schizogony
• Liberated Merozoites
penetrate RBC
• Three stages occur
1 Trophozoites
2 Schizont
3 Merozoite

19. Trophozoites
• After invasion grow and
feed on hemoglobin
• Blue cytoplasm and red
nucleus, Called as
Signet ring appearance
• Hence called ring form

20. Schizont
• When the Trophozoite is fully developed
becomes compact.
• Malarial pigments are scattered through the
cytoplasm
• The Nucleus is large and lies at the periphery
starts dividing.
• Becomes Schizont

21. Exo Erythrocytic Schizogony
• Some Sprozoites do not undergo sporogony in
the first instance
• But go into resting stage called as
Hypnozoites,( hibernation )
• Within 2 years reactivate to form Schizonts
release Merozoites and attack red cell and
produce relapses
• Absent in P falciparum

22. Gametogony
• Merozoites differentiate into Male and female
gametocytes
• Macrogametocytes also called female gametocytes
• Microgametocyte also called as male gametocytes
• They develop in the red cells
• Found in the peripheral blood smears
• Microgametocyte of all species are similar in size
• Macro gametocytes are larger in size.

24. Mosquito cycle
A definitive Host – Mosquito

25. Clinical Features of
Malaria

27. Malaria Diagnosis
Clinical Diagnosis
Malaria Blood Smear
Fluorescent microscopy
Antigen Detection
Serology
Polymerase Chain Reaction

28. Malaria Blood Smear
• Prepare smears as soon as possible after
collecting venous blood to avoid
• Changes in parasite morphology
• Staining characteristics
• Take care to avoid fixing the thick smear
• Risk of fixing thick when thin is fixed with
methanol if both smears on same slide
• Let alcohol on finger dry to avoid fixing
thick
• Be careful if drying with heat

29. Collection of Blood Smears
1. 4.
The second or third Slide must always be
finger is usually grasped by its edges.
selected and
cleaned.
2. 5.
Puncture at the side Touch the drop of
of the ball of the blood to the slide
finger. from below.
3.
Gently squeeze
toward the puncture
site.

30. Preparing thick and thin films
1. 4.
Touch one drop of Carry the drop of blood
blood to a clean to the first slide and hold
slide. at 45 degree angle.
2. 5.
Spread the first Pull the drop of blood
drop to make a 1 across the first slide in
cm circle. one motion.
3. 6.
Touch a fresh drop Wait for both to
of blood to the edge dry before fixing
of another slide. and staining.

31. Recognizing Malaria Parasites
Blue cytoplasm
Inside a red blood cell
One or more red chromatin dots

32. Recognizing Erythrocytic Stages:
Schematic Morphology
Blue
Cytoplasm
RING Red TROPHOZOITE
Chromatin
Brown
Pigment
SCHIZONT GAMETOCYTE

33. Malaria Parasite Erythrocytic Stages
Ring form
Trophozoite
Schizont
Gametocytes

34. Plasmodium falciparum
Infected erythrocytes: normal size
M I
Gametocytes: mature (M)and
immature (I) forms (I is rarely
Rings: double chromatin dots; accole forms;
seen in peripheral blood)
multiple infections in same red cell
Schizonts: 18-32 merozoites
(rarely seen in peripheral blood)
Trophozoites: compact
(rarely seen in
peripheral blood)

35. Plasmodium vivax
Infected erythrocytes: enlarged up to 2X; deformed; (Schüffner’s dots)
Rings Trophozoites: ameboid; deforms the erythrocyte
Schizonts: 12-24 merozoites Gametocytes: round-oval

36. Plasmodium ovale
Infected erythrocytes: moderately enlarged (11/4 X); fimbriated; oval; (Schüffner’s dots)
Trophozoites: compact
Rings
Schizonts: 6-14 merozoites; Gametocytes: round-oval
dark pigment;

37. Plasmodium malariae
Infected erythrocytes: size normal to decreased (3/4X)
Trophozoite: Trophozoite: Schizont:
compact typical 6-12 merozoites(rosette like);
band form coarse, dark pigment

38. Species Differentiation on Thin Films
Feature P. falciparum P. vivax P. ovale P. malariae
Enlarged infected RBC + +
Infected RBC shape round round, oval, round
distorted fimbriated
Stippling infected RBC Maurer Schuffner Schuffner Ziemann
dots dots dots dots
Trophozoite shape Small ring, large ring, large ring, small ring,
accoleform amoeboid compact compact
Chromatin dot often double single large single
Mature schizont rare, 18-32 12-24 8-12 6-12
merozoites merozoites merozoites merzoites
Gametocyte crescent shape large, large, compact,
round round round

39. Species Differentiation on Thin Films
P. falciparum P. vivax P. ovale P. malariae
Rings
Trophozoites
Schizonts
Gametocytes

40. Species Differentiation on Thick Films
Feature P. falciparum P. vivax P. ovale P. malariae
Uniform trophozoites +
Fragmented trophozoites ++ +
Compact trophozoites + +
Pigmented trophozoites +
Irregular cytoplasm + +
Stippling (“RBC ghosts”) + +
Schizonts visible very rarely often often often
Gametocytes visible occasionally usually usually usually

41. Calculating Parasite Density - 1
• Using 100X oil immersion lens, select area
with 10-20 WBCs/field on Thick smear
• Count the number of asexual parasites
and white blood cells in the same fields on
thick smear
• Count ≥ 200 WBCs
• Assume WBC is 8000/µl (or count it)
parasites/µl = parasites counted X WBC count/µl
WBC counted

42. Calculating Parasite Density - 2
• Count the number of parasitized and
nonparasitized red blood cells (RBCs) in
the same fields on thin smear
• Count 1000 RBCs (fewer RBCs if
parasitemia is high)
Number of parasite in 1 µl
Of blood = RBC IN million/cmm X Parasite %

43. Estimating Parasite Density
Alternate Method
• Count the number of asexual parasites
per high-power field (HPF) on a thick
blood film
+ 1-10 parasites per 100 HPF
++ 11-100 parasites per 100 HPF
+++ 1-10 parasites per each HPF
++++ > 10 parasites per each HPF

44. Differentiating Babesia
from Malaria

45. High Power
• Ring shaped trophozites • White eccentric “food
• The intraerythrocytic vacuole” in a ring form.
trophozoites multiply by binary • Very transient stage in Malaria.
fission or schizogony, forming Very rarely seen.
two to four separate
merozoites. .

46. the famous Maltese Cross
• Presence of 4 daughter merozoites in a tetrad is
pathomnemonic.
• However, rarely seen.
• Never seen in malaria.

47. Fluorescent Microscopy
• Modification of light microscopy
• Fluorescent dyes detect RNA and DNA that is
contained in parasites
• Nucleic material not normally in mature RBCs
• Kawamoto technique
– Stain thin film with acridine orange (AO)
– Requires special equipment – fluorescent
microscope
– Nuclei of malaria parasites floresce bright
green and cytoplasm red.
– Staining itself is cheap
– Sensitivities around 90%

49. Quantitative Buffy Coat (QBC ®)
• Useful for screening large numbers of samples
• Quick, saves time
• Requires centrifuge, special stains
• Malaria parasite floresce green yellow against
dark red –black background.
• 3 main disadvantages
– Species identification and quantification difficult
– High cost of capillaries and equipment
– Can’t store capillaries for later reference

51. Principle of QBC System

52. Malaria Serology – antibody detection
• Immunologic assays to detect host
response
• Antibodies to asexual parasites appear
some days after invasion of RBCs and may
persist for months
• Positive test indicates past infection
• Not useful for treatment decisions

53. Malaria Serology – antibody detection
• Valuable epidemiologic tool in some settings
• Useful for
– Identifying infective donor in transfusion-
transmitted malaria
– Investigating congenital malaria, esp. if mom’s
smear is negative
– Diagnosing, or ruling out, tropical splenomegaly
syndrome
– Retrospective confirmation of empirically-treated
non-immunes

54. Target antigens for malaria RDT
pLDH HRP2
Pf-only
Pf and pan-specific bands Persists after parasite death
Closely reflects parasite
viability Aldolase
Pan-specific
Asexual and sexual stages
Closely reflects parasite viability
? Potential for monitoring
treatment efficacy
Pv, Po, Pm-specific Mabs
developed

55. RDTs : test formats

56. Current RDT formats
• Card / cassette / dipstick
• HRP2
• HRP2 & aldolase
• pLDH Pf & pan
• pLDH Pf & Pv "COMBO" tests
• HRP2, pLDH pan
• HRP2, pLDH pan & pLDH Pv
• aldolase

58. RDT procedure

66. Plastic cassette format of
RDT

67. Card format of RDT

68. Dipstick format
of RDT

70. Potential applications for RDTs.
Diagnosis in Confirmation of
remote areas dubious
microscopy
diagnosis
Rapid outbreak
investigation
and surveillance
Laboratory-based
screening / diagnosis

71. Polymerase Chain Reaction (PCR)
• Molecular technique to identify parasite
genetic material
• Uses whole blood collected in
anticoagulated tube or directly onto filter
paper

72. Polymerase Chain Reaction (PCR)
• Threshold of detection
– 5 parasites/µl
• Definitive species-specific diagnosis now
possible
• Can identify mutations – try to correlate to drug
resistance
• Parasitemia not quantifiable
• May have use in epidemiologic studies
• Requires specialized equipment, reagents, and
training

73. PCR: identification of malaria species
Lane S: Molecular base pair
standard (50-bp ladder). Black
arrows :size of standard bands.
Lane 1: P. vivax (size: 120 bp).
Lane 2: P. malariae (size: 144 bp).
Lane 3: P. falciparum (size: 205 bp).
Lane 4: P. ovale (size: 800 bp).

74. Comparison of methods for diagnosing Plasmodium
infection in blood
PARAMETER MICROSCOPY PCR FLUORESCENCE Dipstick HRP-2 Dipstick pLDH, ICT-Pf/Pv
Sensitivity
50 5 50 >100 >100
(parasites/micol)
P.f good, others P. falciparum and P.vivax good P.o
Specificity All species All species P. falciparum
difficult and P.m only Pldh
prarasite density crude
Yes No No crude estimation
or parasitemia estimation
time for result 30-60 min 24 hr 30-60 min 20 min 20 min
skill level High High Moderate Low Low
QBC apparatus
PCR or direct
equipment Microcsope Kit only Kit only
appratus fluorescence
microscope
cost /test Low High moderate/low Moderate Moderate

75. Hame jindgi apni kamiyo ko door karne ke bajay.
Bhagwan ne jo hame khubiya di hai unka upyog
karne me gujarni chahiye.
SPEAKER-DR. NARMADA PRASAD
TIWARI

76. a consequence was natural selection for
sickle-cell disease
, thalassaemias, glucose-6-phosphate
dehydrogenase
deficiency, ovalocytosis, elliptocytosis and
loss of the Gerbich antigen (glycophorin C)
and the Duffy antigen on
the erythrocytes because such blood
disorders confer a selective advantage
against malaria infection (balancing
selection).[7] The three major types of
inherited genetic resistance (sickle-cell
disease, thalassaemias, and glucose-6-
phosphate dehydrogenase deficiency)
were present in the Mediterranean world by
the time of the Roman Empire, about 2000
years ago.[8]

77. The term 'miasma' was coined
by Hippocrates of Kos who used it to
describe dangerous fumes from the ground
that are transported by winds and can
cause serious illnesses.[13] The name
malaria, derived from ‘mal’aria’ (bad air
in Medieval Italian). This idea came from
the Ancient Romans who thought that this
disease came from the horrible fumes from
the swamps