unit 2-1 Blood & TISSUE coccidian PPT2 March 2023.ppt

Blood and tissue nematode

1. Unite-2
2.1.Blood and tissue sporozoa
(coccidian)

2. Outline
Blood and tissue sporozoa
Summary of taxonomic classification of protozoa
blood and tissue sporozoa
For each species:
Epidemiology , morphology, transmission
life cycle , clinical features, laboratory
diagnosis treatment, prevention& control

3. Learning objective
At the end of this unit the students will be able to:
Describe the epidemiological aspects of blood &
tissue sporozoa
Discuss the characteristics of each blood & tissue
sporozoa
Explain the life cycle of each blood & tissue sporozoa
Apply the necessary laboratory procedures for the
detection and identification of blood & tissue sporozoa

4. Blood and tissue sporozoa
Found inside blood , blood forming organs or
tissues
Blood and tissue sporozoa include
Plasmodium species
Babesia species
Toxoplasma gondii

5. Summary of taxonomic classification of
protozoa

6. PROTOZOA
PERCOLOZOA
PARABASALA
EUGLENOZOA KINETOPLASTIDEA TRYPANOSOMATIDAE
LEISHMANIA
TRYPANOSOMES
CILIOPHORA
APICOMPLEXA
COCCIDEA EIMERIIDA TOXOPLASMA
HEMATOZOEA
HAEMOSPORIDA PLASMODIUM
PIROPLASMIDA BABESIA
RHIZOPODA

7. Plasmodium species

8. Plasmodium species
Causative agent of Malaria: an acute and/or chronic
infection caused by protozoansof the genus
Plasmodium
Four plasmodium species causing human
malaria
Plasmodium falciparum(P. falciparum)
P. vivax
P.malariae
P.ovale

9. •Widespread species
•P. falciparum: most prevalent in the hotter and
more humidregions of the world.
•P. vivax: more common in temperateregion than
in the tropics
•Less widespread species
P. malariae: confined mainly to tropical Africa (25%)
P. Ovale: Low & restricted distribution
Occurs primarily in tropical west Africa (10%)

10. General feature of Plasmodium species
Intracellular obligate parasites. (liver cell & RBC)
Life cycle,
Alternation of generation ~alternation of hosts
Requires two hosts:
Man(IH)
Female Anophelesmosquitoes (DH)
Sexualand asexualreproduction
No animal reservoir hostexcept P.malariae

11. History of Malaria
Known since antiquity(oldest diseases to mankind )
Early medical writingsfrom India and China -
periodic fever malaria
Hippocratesdescribed symptoms (500 BC)
Italians –named mal aria(bad air) in 17th century
Laveranidentified parasite (1880)
Rossdemonstrated mosquito transmission (1898)

12. Garnhamdescribed liver stage 1940’s
WHO Launchworldwide malaria eradication in 1955
P. falciparumdescribed by Welch in 1897 and
Schaudinnin 1902
P.vivaxdescribed by Grassiandand Fellettiin 1890
and Labbein 1989

13. P. malariae described by Laveran in 1881 and
Grassiand and Felletti in 1890
P.ovale described by Stephens in 1922

14. Background …
World Malaria Report, 2020
Globally
27% of malaria cases decreased between 2000 and 2015.
Slow reduction in malaria cases (< 2%) between 2015 and 2019.
 241 mil241 million in 2020 cases Vs229 million case in 2019.
African Region in 2020
95% of malaria cases and deaths.
Ethiopia
Malaria prevalence 0.5-1.2 %.
(2015 malaria indicator survey repor)
< 5 % Annual malaria incidence. Figure 1:Map of malaria risk district in Ethiopia , 2017
14

15. Background …
Distribution malaria parasite varied by regions:
70% P. falciparum
30% P. vivax
i.e. P.falciparumis the main focus of this study
Plasmodium falciparum
Majority of malaria morbidity and mortality.
Rapid and accurate malaria diagnosis.
i.e. Important for the timely treatment of life threating malaria.
(FMOH., 2016, FMOH., 2017/18
(WHO., 2015a)
15

16. Plasmodium species
P.falciparum=60%
P.vivax= nearly 40%
P.malariae=1%cases ,focal distribution like
in Humera
P.ovale= less than 1%cases , found inSetit
Humera, Gambela& Arbaminch

17. Epidimologyof Malaria in Ethtiopia
The risk of malaria varies highly from season to
season and from place to place
Transmission-seasonal (Unstable)
Mainly depends on rain fall and Temp

18. Two major transmission periods
Major-September to December after main rainy
season
Minor-April to Junefollowing small showers of
rain in autumn.

19. degazone(> 2,500 m) mean annual temperature
of 10-15
0
C,is malaria free
weynadegazone( 1,500 -2,500 m) mean annual
temperatures range from 15-20o c
malaria most often occurs below 2,000
meters, with short-lived transmission following
the rains
kollazone (< 1,500 m), mean annual
temperatures are 20-25oc, malaria transmission is
endemic

20. Endemicity : defined in terms of parasitemia rates
or palpable spleen rates in children 2 to 9 years of
age as
hypoendemic (<10%)
mesoendemic (11 to 50%)
hyperendemic (51 to 75%)
holoendemic(>75%)
Hyper and Holoendemic malaria are found in
areas of stable malaria transmission

21. Hypo and Mesoendemic: are found in areas of
unstable malaria transmission
Characteristics of stable malaria:
~ Constant incidence over several years
Includes seasonal transmission
Immunity and disease tolerance developed by adult
Usually affects children

22. Characteristicsofunstablemalaria:
Malariaincidencevariesfromweekto
week,monthtomonth,yeartoyear,daytoday.
Communalimmunityofthepopulationlow.
Makestheregionpronetomalariaepidemics.
Highmorbidityandmortality

23. 23
Morphological stages
Sporozoite: develops in the mosquito salivary gland
Hepatic schizontactively dividing, multinucleated,
parasite form in hepatocytes
Trophozoite: metabolically active form living within
the RBC
Sometimes called the ring form

24. 24
Erythrocytic schizont: multinucleated stage in a RBC
resulting from asexual multiplication of trophozoite
Each schizont contains a species determined
number of meroziotes
Merozoite: infective schizont components that break
out of hepatocyte or RBC

25. Gametocyte: morphologically distinctive sexual
(male or female) form which develops from
some trophozoitesin RBCs

26. •Sequential developmental stages
•Distinct morphological features helps for
species identification

27. 27
Terms in malaria
Prepatent period
Incubation period:
Recurrence:
Relapse:
Recrudescence:
Inadequate treatment
Drug resistance
Unusual pharmacokinetics
Incomplete dosage
Reinfection:

28. Transmission and life cycle of Malaria
Principal modeTransmission
bites of female anopheles
mosquito
60 speciesof
mosquito
sucks the gametocytes
during blood meal
bites between 5 PM and
7 AM, with maximum
intensity at midnight.
Anopheles

29. Mosquito transmission depends
•susceptibility of anopheline species
More than 200 known species of Anopheles
, 60 of them are considered to be vectors of
malaria
•feeding habits
•Density
•Longevity
•climatic factors
•temperature, humidity, rainfall, wind, etc

30. In Ethiopia : A.gambiae, A.funestus, A.nili,
A.arebiansis& A.pharonensisare
main vectors
A. arabiensisis responsible for most
epidemics in the country

31. Other modes of transmission
1. Blood transfusion (Transfusion malaria):
This is fairly common in endemic areas
Following an attack of malaria, the donor may
remain infective for:
1-3 years in P. falciparum,
3-4 years in P. vivax, and
15-50 years in P. malariae.

32. Most infections occur:
in blood stored for less than 5 days and
rare in blood stored for more than 2 weeks
Frozen plasma is not known to transmit malaria
blood transfusions malaria
Infective stage-trophozoites/merozoites
shorter incubation period,becauseno exo-
erythrocyticshizogony

33. no relapses possible (vivax/ovale)
clinical features & management of cases are
the same as naturally acquired infection
Donor blood should be screened

34. 2. Mother to the growing fetus (congenital
malaria)
occurs in 5 % of new borne whose mothers are
infected
relatively rare although placenta is heavily
infected
Congenital malaria is more common in first
pregnancy, among non -immune populations
3. Needle stick injury:
Accidental transmission can occur among drug
addicts who share syringes and needles

35. Life cycle
Require two host
Man:-
intermediate host
Asexual reproduction
Liver cell
RBC
Mosquitoes:-
Definite host
Sexual
reproduction

36. 36
Feeding posture of different types of adult mosquito. (a) Culicine, (b) anopheline.

37. Exo-
erythrocytic
(hepatic) cycle
Sporozoites
Mosquito Salivary
Gland
Malaria Life
Cycle
Life Cycle
Gametocytes
Oocyst
Erythrocytic
Cycle
Zygote
Schizogony
Sporogony
Hypnozoites
(for P. vivax
and P. ovale)

38. When the female mosquito bites a person, she pierces the skin and injects
saliva (which contains anticoagulants to stop the blood clotting whilst she
takes her meal

40. P. falciparum
mature and released simultaneously
from liver, no relapse
P. malariae
Pvivax
may remain latent in the liver and
relapse
Povale

41. Comparison of malarial parasites
Pf Pv Po Pm
Tissue schizogony
8 -25 days
8 -27
days
9 -17
days
15 -30 days
Erythrocytic phase
48 hours 48 hours 48 hours 72 hours
Red cells affected
All
Reticulocy
tes
Reticuloc
ytes
Mature RBC's
Merozoites per
schizont 8 -32 12 -24 4 -16 6 -12
Relapse from
Hypanozoites
No Yes Yes
No, but blood
forms can
persist up to
30 years

42. Clinical Features & pathology
Characterized by acute febrile attacks(malaria
paroxysms)
•caused by the release of toxins (when erythrocyticschizonts
rupture) stimulate the secretion of cytokines from leucocytes
and other cells
Manifestations and severity depend on parasite
species, parasitemiaand host status, i,eimmunity,
general health, nutritional state, genetics
Without treatment, P.vivax,P. ovale, P. malaria
ultimately may result in spontaneous cure
P. falciparumcan develop severe complications

43. Prodromal Symptoms
Malaria paroxysm preceded by Prodromal
period
2-3 days before 1st paroxysm
includes: malaise, fatigue, headache, muscle pain,
nausea, anorexia(i.e., flu-like symptoms)
can range from none to mild to severe

44. Febrile Attack (Malaria
Paroxysm), 4-8 hr
periodic febrile episodesalternating with
symptom-free periods
initially fever may be irregular before developing
periodicity
may be accompanied by splenomegaly,
hepatomegaly (slight jaundice), anemia
P. falciparum can be lethal in non-immune
paroxysms comprises of three successive
stage: cold stage, hot stage and sweating stage

45. cold stage
•feeling of intense cold
•vigorous shivering, rigor
•lasts 15-60 min

46. hot stage
•intense heat
•dry burning skin
•throbbing headache
•lasts 2-6 hours

47. sweating stage
•profuse sweating
•declining temperature
•exhausted, weak sleep
•lasts 2-4 hours

48. •paroxysmsassociated with
synchrony of merozoite
release
•between paroxysms
temperature is normal and
patient feels well
•falciparum may not exhibit
classic paroxysms
•continuous fever
•24 hr periodicity
Malaria Paroxysm
tertian malaria
quartan malaria

49. Complications of acute
malaria

50. Malaria caused by P. falciparum
Falciparum/subtertian/malignant malaria
Most pathogenicof all species
Almost all deathsare due to falciparum malaria

51. Factors for Malignance of P.falciparum
Rapid multiplication
Infected red blood cells become "stick"
Infects all age group of red blood cells
A single red blood cell can be infected by more
than one parasites
Erythrocytic schizogonic reproduction takes place
in the deep capillaries of organs such as brain,
lung, heart, spleen, bone-marrow, placenta,
intestine, etc.

52. 1.Higher Parasitemiain
FalciparumMalaria
•all erythrocytes
invaded
•up to 36 merozoites
•Pv/Po = reticulocytes
•Pm = senescent RBC
P.falciparum.
-Up to 30-40%of RBC
-sever if > 5%RBC are
infected.
P.vivax& P.ovalerarely
exceeds 2%
P.malariae.Usually < 1%
Pathogenecity of P. falciparum

53. •avoidance of
spleen
•low oxygen
tensions
•better invasion
2. Cytoadherence of infected
erythrocytes
-trophozoite and schizont
stages
-primarily in brain, heart,
lungs, and gut
complications
-immune evasion (spleen
avoidance

54. Predisposing factors for complications of P.
falciparummalaria
(1.) Extremes of age.
(2.) Pregnancy,especially in primigravidaeand in
2nd half of pregnancy.
(3.) Immunosuppressed-patients on steroids, anti-
cancer drugs, immunosuppressant drugs
(4.) Splenectomy.
(5.) Lack of previous exposure to malaria (non-
immune) or lapsed immunity
(6.) Pre-existing organ failure.

55. Blackwater fever

56. Malaria caused by P. vivax, P. ovale P. malariae
Plasmodium vivaxis referred to as vivax
malaria , benign tertian(BT) malaria
Plasmodium ovaleis referred to as ovale
malaria, ovale tertianmalaria
Plasmodium malariae is referred to as malariae
malaria, quartan malaria

57. Infections caused by P. vivax, P. ovaleorP.
malariaeare rarely life threatening
no Cytoadherenceof parasitized cells
parasitic densities are lower
Relapsesare a feature of vivaxand ovalemalaria
Recrudescencesare a feature of P. falciparum
P. malariaeis nephritic syndrome which may
progress to renal failure.

58. Hyper-reactive malaria splenomegaly

59. Genetic factors That Provide Protection
Against Malaria
1.Nature of hemoglobine
HgbS (Sickle cell anemia trait) –p.f
ThalassemiaHgb-P.f
Fetal Hgb–all sps
HgbE –P.v
2. Enzyme content of erythrocyte
Glucose-6-phosphate dehydrogenasedeficiency ,-P.f
3. Presence or absence of certain factor
Ovalocytosis-P.f& P.v
Duffy blood group antigens (i.e., Fyaand Fyb)
negative RBCs-P.v

60. Laboratory Diagnosis

61. Laboratory diagnosis
Clinical Diagnosis
Microscopic
•Thin film
•Thick film
•QBC
Immunological
Ag /enzyme
•RDT.ICT Malaria Pf
ParaSightF
OptiMAL
Ab-ELISA
Molecular
PCR
etc.
Malaria Diagnosis
MALALRIA Diagnostics approaches

62. Clinical diagnosis
Based on clinical signs and symptom:
Fever,Chills, perspiration, anorexia, headaches,
vomiting, and malaise
It is inexpensiveto perform and requires no
special equipment or supplies.
Are non-specificand symptoms overlap with
those of other febrile illnesses.
A Dxof MAL based on Clinical grounds alone is
therefore unreliable-Overdiagnosis

63. Laboratory diagnosis Techniques
I. Microscopy examination
Peripheral smear study
Detecting and identifying malaria parasite in direct blood films
CONCENTRATING MALARIA PARASITES
Concentrating parasite in venous blood by centrifugation when
note found in blood films
1.Buffy Coat preparation
2. Quantitative buffycoat( QBC) system
II. Immunologic/Biochemical techniques
detection of malaria antigen, antibody & parasite products
III. Molecular diagnosis techniques

64. I. Microscopy examination

65. Microscopic examination of blood film
Established method for
confirmation of malaria-the
gold standard.
Requirements:
Carefully collected
blood specimen/sample
Well prepared blood
films
Well stained smears(
Romanowskystains)
Careful examination of
the smears

66. Collection of Blood Specimen
I. Collect sufficient quantity of blood
1.Capillary bloodfrom finger prick , toes, or
ear lobes –best
2.Venous blood.(EDTA anticoagulant)
3.In obstetric practice, cord blood and
placental impressionsmears can be used
II. Time of collection
When the patients feels febrile
Before anti-malaria drugsare given to the
patients

67. Note:-
A negative testDOES NOT rule out
malaria.
Repeated testsmay have to be done in all
doubtful cases
1.Preparation of blood film
I.thick blood film
II.thin blood film

68. I. Preparation of thick films
I.slides must be clean, free from
dirt, grease and fingerprints
II.Mix the blood
III.Using an applicator stick, apply
4 drops of blood on to a
microscope slide
IV.Spread the blood without
excessive stirring to form a
smear approximately a cm
2
,
through which newspaper print
can be red.
V.This should be approximately
5 red blood cells thick.
VI.Allow to air dry horizontally
(without using heat).
VII.Label slides.

69. II. Preparation of Thin Films
1.The microscope slides
must be clean as for thick
films otherwise the blood
will not adhere and the
smear will be irregular.
2.Apply 1 drop of blood to
the end of the slide, pace
another slide at an angle
of 45
o
and bring it towards
the drop of blood.
3.As soon as it touches it,
the blood will disperse
along the width of the
slide.

70. 5.Before it reaches the edges, pull the drop
along the length of the slide.
6. the correct amount of blood in the drop the
film will form a good tail before the end of
the slide. The film here should be 1 RBC
thick. Make 2 slides for each test.
7. Air dry, Label slides

71. Preparation and processing the blood film
1 2 3 4
5 6 7 8
9 10 11

72. Thick blood film
•Good for rapid detection malaria parasites ,
particularly when they are few
In P.malariaeparasitaemiais normallylow
•About 30 times more sensitive (detecting about
20 parasites/µl)
•In a thick film the blood is not fixed

73. Thin blood film
required to confirm the plasmodiumspecies
enabling the parasites to be seen in the red cells
greatly assists in the identification of mixed infection
value in assessing whether a patient with falciparum
malaria is responding to treatment
gives the opportunity to investigate anaemiaand
white cell abnormalities

74. Good blood film macroscopic appearance
both thin and thick on the same slide
thick film 10mm diameter
news print read under thick film before staining
10mm from frosted end & thick film and b/n thick
& thin film
estimated blood volume 6µl for thick & 2µl for thin
film
tongue shape & feathery end thin film

75. characteristics Thin Thick
Thickness Single layer No. of layers
FewP/F More/ F
1/field 20-30/field
Time for DX 20-25min /200 3-5min
/100
RBC morphology Fixed Hemolized
Parasite morphology Framed in RBC Distorted

76. Common Mistakes in Making Blood Films
1. Too much blood
On thick film
After staining the background will be
too blue.
Result too many WBCs per thick film
field, and these could obscure malaria
parasites .
On thin film
RBCs will be on top of one another
impossible to examine them properly
after fixation.
76

77. Common mistakes
2.Too little blood
Not able to examine
enough blood in the
standard examination.
False negative result is
likely.
77

78. Common mistakes…
3.Blood films spread on a
greasy slide
The blood films will spread
unevenly .
Some of the thick film will
probably come off the slide
during the staining
78

79. Common mistakes…
4. Edge of spreader slide
chipped
the thin film spreads unevenly,
is streaky and has many “tails”.
The spreading of the thick film
may also be affected.
79

80. Common Mistakes…
5.Badly positioned blood films
Blood films should be correctly sited
on the slide.
If not, it may be difficult to examine
the thick film.
80

81. Common mistakes…
6. Thin film too big, thick film in the wrong
place
The thick film will be out of place and may be so near the edge
of the slide that it can’t be seen through the microscope.
During staining or drying, portions of the thick film will scraped
off by the edges of the staining trough or drying rack.
Very difficult to position the thick film on the microscope stage.
81

82. 3. STAINING MALARIA PARASITE
The stains most frequently used
1.Giemsa-alcohol based Romanowsky
Proved to be the best for routine diagnosis
It gives best consistence quality of staining malaria parasites
Allow differentiation of species in thin films
Good at low conc.
Not satisfactory when conc. Is increased to reduce time
Requires dilution with buffered water( pH 7.1-7.2) before use.
Usually bought as a solution but can be prepared carefully
SOP for preparation of Stock solutions & buffer -and Standard lab.
Manual very important

83. Staining With Giemsa
Immediately before use dilute the Giemsaas required
3% for 30 min
10% for 10 min
Thin films must be fixed in absolute methanol for at
least 30 seconds(1-2min)
Stain
Wash
Drain & dry vertically/ at an angle.

84. Quality control of Giemsa
The stain dilutions and the buffered water (pH 7.1-2)
must be accurately prepared and the stock stain must
be of good quality.
Filter Giemsabefore use
The staining procedure should be followed very
carefully
The methanol for fixing must be free from water
The bottles of stock Giemsastain must be kept
tightly closed , free from contamination and out of
sun light

85. Each batch should be labelled & tested with
reference slide of P. falciparumor P. vivax

86. Microscopic examination of blood films
Focus on the film with the X10 objective.
Examining the film first with 40x objective to select a
well stained area
Apply a drop of immersion oil on the slide and switch to
the oil-immersion objective(100).
Examine at least 100 fields(100Xobjective)
*P.malariaeexam approximately 200 fields

87. Microscopic differentiation
Microscopic differentiation of species depend on
Host celland Parasite characteristics
1.Feature of infected red cell and ghosts
change in size,shapeand colour
Presence of dots, maurer'sclefts(not on ghost
cell) on infected red cell
Single or multipleinfection of each cell

88. 2.Parasite morphology at specific stages
Number and sizeof chromatin beads
Shape and sizeof cytoplasm
Degree of pigmentationwithin cytoplasm
Stages of parasiteseen together

89. Appearance of different species of
Plasmodium in a thick blood film
89

90. Appearance of different Plasmodium
Species in Thin blood film
90

91. Microscopic features of three blood stages
-trophozoites, schizonts, gametocytes
All these three stages have:
Rednuclear chromatin, bluecytoplasm and
pigment(except young troph)
What are the key features of :
trophozoites?
single (Sometimes double) chromatin bead
Cytoplasm as a ring uniform or fragmented mass
Pigment absent from young (ring) form

92. Schizonts?
2-32 chromatin beads
Cytoplasm-typically irregular, amoeboid
Pigment in early and late form
Gametocyte?
Single chromatin bead (diffuse in male)
Round or crescent shaped
Solid cytoplasm
Pigment in early and late stage

93. Notes: the mature gametocytes of Pv, Po and Pm
are hard to distinguish from mature trophof these
species.
Artefacts
Potential source:
•Vegetable spores, yeast, pollen, algae and
bacteria in the stain or on the slide
•Platelets
•Howell-jolly bodies in anaemic patients
•Ghosts of immature red cells mimicking
schuffner'sstippling

94. Examining Microscopic feild

97. Examination
P.falciparem
Young Trophozoite (Ring
forms)
Stage frequently found in
blood film
Size:Very delicate, 0.15-0.5
diameters of RBCs which is
unaltered in size.
Shape:small fine pale blue
ring
Chromatin: 1 or 2 small red
dots.
Often with double chromatin
dot
May lie on red cell membrane
(accole forms)
Pigment: absent:

98. The Trophozoit
/Ring/ stage
Most commonly seen
As it is a growing stage, the
parasite within the RBC
may vary in size.
Malaria pigment is a by-
product of the growth or
metabolism of the parasite.
It does not stain, but has a
color of its own, which may
range from pale yellow to
dark brown or black.
98
Red blood cell
Chromatin dot (red stain)
Cytoplasm (blue
stain)
Chromatin
dot (red
stain)
Cytoplasm (blue stain)
Red blood cell

99. Schizontstage
Not usually seen in
peripheral blood
RBC unaltered in size ,
sometimes stippled, pale.
Size: parasite about 0.6-
fill RBC
Shape: compct
Chromatin: numerous
irregular masses
Merozoites:8-32;
average 24
Pigment: clumped black

100. Mature Trophozoite
Stage rarely seen in
peripheral blood
RBC unaltered in size,
sometimes stippled, pale
Size: small
Shape: compact thin blue
ring , comma or
exclamation mark
Chromatin: 1 or 2 red
dots
Pigment: black or dense
brown mass
StipplingMaurer's cleft

101. Plasmodium vivax
Young Trophozoites
Stage frequently seen
Size : 0.3-0.5 diameter of
RBC which is unaltered in
size.
Cytoplasm-irregular blue
quite thick ring
Chromatin : one rather
large red dottes/ some
times two
Pigment:absent.
RBCs are enlarged and
distorted.
Schüffner's dotsmay be
seen

102. Mature Trophozoite
Not frequently seen
Size: large ( RBC
enlarged,stippled)
Parasite-large blue
irregular/ amoeboid
Chromatin:Dots or
threads of red colour
Pigment:Golden
brown and scattered

103. Gametocytes
RBC is distorted.
Fairly frequently found
Size:larger than red cell
Shape: crescent or banana
Male:-Bluntly round ends
Female:-Rounded/pointed ends
Cytoplasm:Reddish
blue(Male)/Dark blue(female)
Chromatin:
Male:-fine granules scattered
Female:-compact masses near center
Pigment: black granules
Rounded forms may be seen if film
dries slowly

104. Shizonts stage…
104

105. Schizonts
Quite frequently
seen
RBC much enlarged
Size:Almost fills red
blood cells
Shape: amoeboid or
segmented, parasite
large, filling enlaged
RBC
Cytoplasm: pale blue
Merozoites: 14-24;
average 16
Pigment: Golden
brown central loose
mass

106. Gametocytes
Are round to ovalwith blue
stain
Scattered brown pigment
Dense red triangular nucleus
often at one end
May almost fill the red blood
cell (RBC).
Rbcs are enlarged 1 1/2 to 2 ×
and may be distorted.
Under optimal conditions,
schüffner's dots may appear
more fine than those seen in p.
Ovale.
Difficult to differentiate from
late trophozoites

107. Plasmodium malariae
Mature Trophozoite
RBC unaltered
Parasite-compact often band or rounded s
Chromatin: round do or red band
Pigment: dark brown or black pigment, often
concentrated along one edge of the band
Band formscan be seen in thin films
Occasionally “birds-eye”ring form may be seen 8-10
merozoites in mature schizont
•‘rosette’
Young Trophozoite
Size: Up to 1/3 Red
blood cell.
Cytoplasm–
Thicker & dense
(compact) blue ring
Chromatin: one
large red dot
Pigment: absent

108. Schizont
RBC unaltered
Size: small compact nearly fills red cells
Merozoites 6-12; average 8,
sometimes forming rosette
Pigment: Brown aggregated

109. Gametocytes
Shape-large, oval/round
Nucleus-1 round red chromatin at
one edge
Pigment-black brown course
RBC unaltered, parasite small round
filling RBC
Infected Red Blood Cells
Oldest erythrocytes are infected
Stippling-None (Ziemanns dots often
after prolonged leishmania staining
Parasite Density: low density,
Rarely more than 1% of cells infected
(easily missed in Laboratory
diagnosis

110. P.ovale
•Red cells enlarged.
•Comet forms common (top right
•Schüffner’s dots
•Rings large and coarse.
•similar to P. vivax
•subtle differences
•‘compact’ trophozoite
•fewer merozoites
•elongated erythrocyte
Mature schizonts similar
to those of P. malariae
but larger and more coarse

111. features are:
Developing form of plasmodium
"Comet-like" red cells
Enlarged red cell
This is a typical Plasmodium ovale
presentation.

112. features are:
Broad band form of plasmodium
Red cells not enlarged
This is a typical Plasmodium malariae
presentation.

113. Estimating Parasitaemia
Important for clinical purposes
To monitor the progress of the disease and
The efficacy of therapy.
Methods
1.Number of parasites/µL of blood (thick film)
2.Number of parasites/µL of blood (thin film)
3.Proportion of parasitized erythrocytes (thin film)
4.Semi quantitative count (thick film):
113

114. 1. Number of parasites/µL of blood (thick
film):
•Requires observation of at least 100 fields
while you count 200 WBCs.
•Number of asexual parasites and WBCs
should be counted in each field until the
number of WBCs reaches 200.
•If number of WBCs is unknown, it can be
assumed to be 8000/µL

115. Example:
Patient WBC = 8000 ul
Parasite count against 200 WBCs = 650
Parasite count/ul= 650x 8000/ul
200
= 26 000 parasites/ul
115

116. 2. Number of parasites/µL of blood (thin
film)
Requires the preliminary determination of RBCs number present in
the average microscopic field.
The number of asexual parasites is counted in at least 25
microscopic fields.
The number of RBCs in the average microscopic field is about 200,
so total RBCs counted in 25 fields is about 200 x 25 = 5000.
RBCs/µl is assumed to be 5 millions/µlfor malesand 4.5
millions/µl for females.
116

117. Example:
Parasite counted against 5000 RBCs/25 fields/= 50
# of RBCs in 25 fields= 5000
RBC count = 5 million/Male patient/
50 x 5,000,000
5000
50,000/µl of blood.
117

118. 3. Proportion of parasitized erythrocytes
(thin film):
Indicate the percentage of erythrocytes that are
infected by malaria parasites.
The number of parasitized erythrocytes (asexual
forms) present in 25 microscopic fields is
counted divided by the total number of
erythrocytes present in these fields (about
5000), and multiplied by 100.
118

119. Example
Average # of RBCs/25 fields=5000
# Parasitized RBCs/25 fields=100
% of parasitized RBCS= 100 X 100
5000
2% of RBCs are infected with asexual for of malaria
parasite.
119

120. 4. Semi quantitative count (thick film):
Very quick but less accurate.
Used only when not possible to perform more
accurate methods.
Reporting:
+ 1-10 asexual parasites / 100 thick film fields
++ 11-100 asexual parasites / 100 thick film fields
+++ 1-10 asexual parasites / single thick film field
++++ > 10 asexual parasites / single thick film field
120

121. Reporting of BF results
If positive :
Check the presence of
Different stages ( Throphozoits, schizontsand gametocytes).
Mixed infection
Report the species, stage and density of parasites and if present
malaria pigments .
If negative after examination of a minimum of 100x
thick film fields.
Report No parasite or hemoparasitefound.

121

122. Diagnostic quality Control: Depends
upon
Compliance with standards
Availability of supplies/equipments/infrastructure
Condition of microscope
Training of laboratory personnel
Regular supervision
quality of reagents & stains
cleanliness
Work load
Technical ability & type of techniques used
122

123. If no parasites are found after examining 100
fields (or if indicated 200 fields),report the film
as:
Malaria thick film: NPF (No parasites found)

124. Features are:
No parasites seen in this field
This film would need to be examined for 10
minutes before being called negative.
Repeat films should be prepared and
examined on at least 2 further occasions,
ideally as the temperature peaks, before
the presence of Malaria can be excluded.

125. II. Immunologic/Biochemical techniques
Immunochromatographic
tests for malaria antigens
Antimalarialantibody

126. Immunochromatographic tests for malaria
antigens
Are based on the capture of the parasite
antigensfrom the peripheral blood
Uses either monoclonal or polyclonal
antibodiesagainst the parasite antigen targets.
RDTs do not require a laboratory, electricity, or
any special equipment.
Targets
1. Histidine-rich protein2 of P. Falciparum,
2. Pan-malarial plasmodium aldolase, and
3. Parasite specific lactate
dehydrogenase(pldh)

127. Histidine-rich protein 2 of P. falciparum
(PfHRP2)
Proteinproduced by the asexual stages and
gametocytes of P. falciparum,
Remain in the blood for at least 28 days after the
initiation of antimalarialtherapy
Several RDTs targeting PfHRP2 have been
developed.

128. Parasite lactate dehydrogenase(pLDH)
is a soluble glycolyticenzymeproduced by the
asexual and sexual stages of the live parasites
present in and released from the parasite infected
erythrocytes
found in all 4 human malaria species, and different
isomers of pLDHfor each of the 4 species exist
With pLDHas the target, a quantitative
immunocaptureassay,

129. Plasmodium aldolase
Is an enzymeof the parasite glycolyticpathway
Expressed by the blood stages of P. Falciparumas
well as the non-falciparummalariaparasites
Monoclonal antibodies against plasmodium
aldolaseare pan-specific in their reaction
Have been used in a combined 'p.F/P.V' ICT test
that targets the pan malarial antigen (PMA) along
with pfhrp2

130. RDTS procedures
1.50 µl of blood from finger prick
A blood specimen is mixed in a buffer
The labeled Ag –Ab complex migrates up the test
strip
Sample origin
Detection lineAbsorbent pad
anti-Pf Ab
Ant-MAL Abs
(all species)
Control
Ab

132. RDTs General procedures
Labeled antibodies pre deposited
Finger prick blood is mixed with buffer solution with
haemolysingcompound & specific antibody
If the target Antigen is present, antigen /antibody is formed
Antigen –antibody complex migrates up the test strip by
capillary action towards test specific reagents, which have
been predepositedduring manufacture
Buffer added to wash hemoglobin and permit visualization of
any colored line on the strip
Performance of the test
Assessed in divers clinical situation
Some RTD detect two parasites

133. Currently available RTDs:
advantagesover microscopy
•Simpler to perform & interpret
•Do not require electricity, special equipment & training in
microscopy
•Community volunteers can be taught the procedure in hrs.
•RDTs detect circulating antigen may detect Pf antigens even
when the parasites are sequestered
disadvantagesover microscopy
•Highly sensitive, false positive result
•Can not detect density of the parasite
•Expensive

134. •+ve circulating antigens
•Not quantitative
•Do not differentiate P.vivax , P.ovale P.malariae
•Specific to malaria parasite only, can not detect other
haemo-parasites

135. III. Molecular diagnosis

136. Polymerase Chain Reaction (PCR)
Using labeled probe following PCR amplification
highly sensitive and specific for detecting all 4
species of malaria and permits genotyping
particularly in cases of low level parasitemia and
mixed infections
allows the detection of drug resistant

137. The PCR test is reportedly 10-fold more
sensitive than microscopy
with one study reporting
a sensitivity to detect 1.35 to 0.38
parasites/µL for P. falciparum and 0.12
parasites/µL for P. vivax
expensive and requires a sophisticated
laboratory with well-trained staff.

138. Other tests
Measurement of haemoglobin or packed cell
volume
Measurment of blood glucose to detect
hypoglycaemia
Total white cell count and platelet count (severe
falciparum malaria)

139. Testing urine for free haemoglobin if black water
fever is suspected
Blood urea or serum creatinine to monitor renal
failure
urine protein if nephritic syndrome is suspected

140. 140
Antimalarial drugs like
Chloroquine
Widespread resistance has now rendered it
virtually useless against P. falciparum
Artemisinin-active against all Plasmodium species
Pyrimethamine in combination with a sulfonamide
Effective against all four human malarias
And other can be used
Treatment

141. Prevention and Control
1. Avoid mosquito bites by
Using impregnated bed nets
Wearing protective clothes
Using mosquito repellents
screens, house spraying
2. Destroy adult mosquitoes by
Indoor residual regular effective spraying

142. 3.Preventing breeding of mosquitoes by
environmental modification
Spraying breeding places with effective
larvicides
Biological control
4. Treatment
Active infection
Chemoprophylaxis
5)Health education
6) Blood screening for malaria

143. Babesia species

144. Babesia species
Causative agent of Babesiosis , piroplasmosis,
thick fever, or red fever
More than 100 species have been reported
known as parasites of domestic and wild animals
Humans are accidental host

145. Four species known to infect humans
Name Normal parasite
B. bovis Ox
B. divergens Ox
B. equi Horse
B. microti microtus Rodents

146. Transmission and life cycle
Transmission
By bite of infected ixodid or hard –bodied tick
(definitive host )
blood transfusion
Life cycle

148. During a blood meal, a Babesia-infected tick
introduces sporozoites into the mouse host
Sporozoites enter to erythrocytes and undergo
asexual reproduction (budding)
In the blood, some parasites differentiate into
male and female gametes
The gametes ingested by appropriate tick

149. gametes unite and undergo a sporogoniccycle
resulting in sporozoites
Babesia-infected tick introduces sporozoitesinto
the human hostduring a blood meal
Sporozoitesenter erythrocytes and undergo
asexual replication (budding)
Humans are, for all practical purposes,
dead-endhosts

150. Clinical feature
Multiplication of the blood stage parasites is
responsible for the clinical features
chills, sweating, fatigue, hemolytic anemia,
jaundice, fever and hepatomegaly, usually 1-2
weeks after infection
it is self-limiting disease infection in human

151. Laboratory Diagnosis
1. Examination of stained smear
identifying poleomorphicring like intra-
erythrocyticparasite in Giemsa-stained blood
films
The small parasites appearing much like
P.falciparum
canbe differentiated from malaria parasite by
the absence of pigment in the infected
erythrocytes

152. 2. Serologic test:IFA
3. Molecular technique: PCR
Treatment
Clindamycin and quinine or Atovaquone plus
Azithromycin

153. Prevention and control
1.Tick control through acarcidaltreatment (
animal host )
2. Chemotherapy to infected animal and host
3. Avoidance of exposure to tick

154. Toxoplasma gondii
Causative agent of toxoplasmosis
Tissue or extraintesinalcoccida
infects most species of warm blooded animals ,
including humans
Epidemilogoy
World wide

155. About 47% of African , more than 50% adult
population in north America and west Europe
shows antibodies to T. gondii
Only about 1% of persons showing antibody to
T. gondiihave signs and symptoms of diseases
In Ethiopia: few studies indicated the disease
found in Ethiopia

156. Habitat
In the skeletal muscle, heart, lymph nodes,
lungs, spleen, bone marrow, mononuclear
leukocytes, brain, CSF, Spleen, etc. of man,
domestic and wild animals
Morphology
has five main developmental forms
only trophozoiteand cyststages are
found in man
but all occur in the feline (cats family)

157. Toxoplasm(trophozoite):-has two forms
I. Tachyzoite/endozoite
occurs in the early acute stage of infection
Size: 3m by 7 m
Shape: crescent or oval in shaped, one end
is rounded and the other end is pointed
Content: In Giemsastain, paranuclearbody-
stains red, nucleus stains dark red and
cytoplasm stains blue

158. Bradyzoites/cryptozoites
Occurs in the chronic stage of infection,
develops slowly and multiplies in the tissues to
form a true cyst
Cyst:-10-100m & may contain about 3,000
trophozoites

159. Transmission and life cycle
Human infection may be acquired by
A) ingestion of undercooked infected meat
containing Toxoplasma cysts
B) ingestion of the oocyst from fecally
contaminated hands or food
C) organ transplantation or blood transfusion;
D) transplacental transmission;
E) accidental inoculation of tachyzoites

160. life cycle

161. Intermediate host : Humans , rodents , chicken
, pigs and other animals
Definitive host: cat family (Felidae)
Cats become infected with T. gondii by
ingesting tissue cysts or oocysts
viable organisms are released and invade
epithelial cells of the small intestine where they
undergo an asexual followed by a sexual cycle
Form oocysts which is excreted in faeces

162. Human ingested cyst and oocyst
Following ingestion of infective oocyst or tissue
cyst, the organism excyst in the intestine, but
leave it and develop in other tissues like lymph
node, brain, eye and etc.
develop quickly and produce tachyzoites then
bradyzoites which form tissue cyst
These cysts may remain throughout the life of
the host

163. Clinical Features
adults acquired infection are often asymptomatic
, they can cause
fever, rash
enlargement of lymph gland with
lymphocytosis
occasionally inflammation of the eye
(ocular toxoplasmosis) , myocardities,
menengoencephlitiesand atypical
pneumonia.

164. HIV associated toxoplasmosis
serious & often fatal opportunistic toxoplama
infections
infections are due to the reactivation of cysts
cause encephalitis with fever , headache,
mental deterioration and seizures

165. Congenital infections
occur in about 1-5 per 1000 pregnancies of
which 5-10% result in miscarriage and 8-10%
result in serious brain and eye damage to the
fetus
10-13% of the babies will have visual
handicaps
Infection can be acquired due to active
parasitaemiaduring pregnancy or reactivated in
immunocompromised mothers

166. Laboratory Diagnosis
1.Identifying toxoplasmain Giemsastained
histological sections, aspirates of lymphnode,
bone-marrow, CSF, pleural fluid, peritoneal
fluids and sputum
2. Serologic tests such as , ELISA, IFAT, CFT.

167. Treatment
Pyrimethamine-sulfadiazine (Fansider) or
Spiramycin
Prevention and Control
1.Avoid contamination of hand, food and water
with the faecesof cat
2.Not eating raw or under cooked meat such as
pork, beef

168. 3. Screening of blood and organ of individuals for
the parasites
4. Treatment and health education
5. Ciliates

169. summary
Discuss about the epidemiology of malarial in
Ethiopia
List all laboratory diagnosis technique for malaria
examination
What characters help to differentiate one
plasmodium parasite species?
What are the significance of thin and thick blood in
the laboratory diagnosis of malaria parasites?
Which plasmodium species is more malignant
that others? Why?

170. Explain methods used to estimate parasitemia
load of malaria parasites
What characters help to differentiate plasmodium
parasite species from babesia species ?
List prevention and control methods used for
prevention malaria
Discuss about modes of transmission , laboratory
diagnosis , prevention and control of T. gondii