College of Medicine and Health Sciences

School Of Medical Laboratory

Lecture Note on Malaria,Leishmaniasis and

schistosomiasis on infectious Module For PCII
Medical students

1 Set by Z.K
 Introduction…Cont
What are protozoa? Proto = first, Zoa = animals
Single-celled eukaryotic organisms
Unknown until the invention of the microscope in 1675
Protozoan parasites are first recognized by Antony van leewenhoek
in 1676
He describe it as little animal or animacula

2 Set by Z.K
 Protozoan Diversity
=protozoa are extremely diverse organisms and found in a variety of
niches
=>200,000 named species
= Most species are free-living in
= Freshwater
= marine environments
= decaying organic matter and soil
=Some are beneficial to mankind by: being part of the
food chain , serving as experimental subjects.
3 Set by Z.K
=Few are adapted to a parasitic life but all plant and
animal species have at least one protozoan parasite
= ~10,000 are parasites in a wide range of hosts
= Vertebrate
= invertebrate
= Plants
= ~20 human pathogens
= Adapted to life in a wide range of sites within the host

4 Set by Z.K
 Ecological Niches in the Human Body:
 Skin: Leishmania

 Eye: Acanthamoeba

 Mouth: Amoebae and flagellates (usually non-pathogenic)

 Gut: Giardia, Entamoeba (and invasion to liver),

Cryptosporidium, Isospora, Balantidium

 G.U. tract: Trichomonas

5 Set by Z.K
 Ecological….

 Bloodstream: Plasmodium, Trypanosoma

 Spleen: Leishmania

 Liver: Leishmania, Entamoeba

 Muscle: Trypanosoma cruzi

 CNS: Trypanosoma, Naegleria, Toxoplasma, Plasmodium

6 Set by Z.K
 Importance of protozoa

• Medical importance
– Cause of more sickness and death, than any other
disease-causing organisms
– reduced working capacity
– Loss of productivity

7 Set by Z.K
 General Morphology
Protozoa exhibit a wide variety of morphologies
Size: vary in size, range from 1 to 150um
The smaller members, 1-10μm, include most of the
intracellular parasites (eg: plasmodium, Leishmania,
Toxoplasma)
The largest member is the ciliate Balantidium coli
Shape: No single shape that represent all
Ranges from amorphous shapeless amoeba to relatively
rigid forms
8 Set by Z.K
Morphology…cont
 Protozoa have relatively complex and specialized internal structure
which perform:
 The functions of locomotion, metabolism, and reproduction.
 The same physiological functions performed by many cells in a
more complex organism

9 Set by Z.K
 Taxonomic classification of protozoa
Sub Phylum Sub-phylum Genus- Species-
kingdom examples examples

Protozoa Sarcomastig- Sarcodina -- - Entamoeba E. histolytica

ophora move by pseudopodia
further divided into

Mastigophora Giardia G. lamblia

move by flagella

Apicomplexa Plasmodium P. falciparum,

no organelle of P. vivax,
locomotion
P. malariae,
P. ovale

Ciliophora Balantidium B. coli

move by cillia

Microspora Enterocyto- E. bienusi

Spore-forming zoa

10 Set by Z.K
 Flagellates

Luminal (intestinal,
Urogenital & oral) Haemo (blood & tissue)
flagellates flagellates

11 Set by Z.K
 General Characteristics blood & tissue flagellates
 Reproduces by simple longitudinal binary fission

 Transmission occurs through biological insect vectors as

intermediate hosts & human as definitive host

 The species are morphologically indistinguishable, but

they can be differentiated on the basis of on their

 Clinical features, geographical distribution, serologic

tests/immunological tests, etc

12 Set by Z.K
Introduction ...Cont
 Phylum Sarcomastigophora

 Order Kinetoplastida

 Family Trypanosomatidae

Medically important blood and tissue flagellates

belonging to the family trypomastidae
There are six genera under the family trypomastidae
but only two of them are responsible to cause disease to
man
Set by Z.K
 Genus Leishmania and Trypanosoma
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Learning objectives
After completing this session, students will be expected to;
– Describe the causative agents leishmaniasis
– describe the morphological stages of Leishmania parasites
– List the mode of transmission of Leishmania parasites
– Describe life cycle of Leishmania parasites
– List the clinical classifications of leishmaniasis
– Desribe the pathogenesis and clinical manifestations of
leishmaniasis
– Explain the laboratory dx

14 Set by Z.K
 Leishmania


 Causative agent of Leishmaniasis

 obligate intracellular protozoa of the genus Leishmania

 Named after Leishman, who first described it in London

in May 1903

 In the human host, Leishmania are intracellular parasites

that infect the mononuclear phagocytes

15 Set by Z.K
 Learning objectives

After completing this session, students will be expected to;

– Describe the causative agents leishmaniasis
– describe the morphological stages of Leishmania parasites
– List the mode of transmission of Leishmania parasites
– Describe life cycle of Leishmania parasites
– List the clinical classifications of leishmaniasis
– Desribe the pathogenesis and clinical manifestations of
leishmaniasis
– Explain the laboratory dx

16 Set by Z.K
  Human infection is caused by about 21 of 30 species that
infect mammals. These include:
 L. donovani complex with 3 species
L. donovani,
 L. infantum,
 L. chagasi;

L. mexicana complex with 3 main species

L. mexicana
 L. amazonensis
 L. venezuelensis;

17 Set by Z.K
  L. braziliensis complex
 L braziliensis

 L. Peruviana

 L. Guyanensis complex
 L. Guyanensis

 L. panamensis

 L. tropica; L. major & L. aethiopica

18 Set by Z.K
 Leishmaniasis can easily classified clinically as

• Visceral leishmaniasis
• Cutaneous leishmaniasis
• Mucocutaneous leishmaniasis
• Diffuse cutaneous leishmaniasis

 These different forms of the disease is caused by the

different species of Leishmania

19 Set by Z.K
  Cutaneous leishmaniasis(CL)
L. tropica
L. major
L. aethiopica
L. guyanensis
L. panamensis
L. peruviana
 Visceral leishmaniasis(VL)
L. donovani
L. infantum
 L. Chagasi

20 Set by Z.K
  Mucocutaneous leishmaniasis(MCL)
 L. panamensis
 L. guyanensis
 L. Brazilliensis

 Diffuse cutaneous leishmaniasis(DCL)

• L. amzonensis
• L. aethiopica

21 Set by Z.K
 Epidemiology

 more than 1 billion people are at risk of infection in 88
countries around the world

 72 of which are developing countries

 An estimated 30 000 new cases of VL and more than 1

million new cases of CL occur annually.

22 Set by Z.K
 Most of the affected countries are in tropics and sub
tropics
 90% of all VL cases occur in Bangladesh, Brazil,
India, Nepal and Sudan;

 90% of all MCL cases occurs in Bolivia, Brazil and

Peru

 90% of all CL cases occur in Afghanistan, Brazil,

Iran, Peru, Saudi Arabia and Syria
23 Set by Z.K
  Geographical distribution of leishmaniasis is limited by:
The distribution of the sandfly,

Its tendency to take blood from humans or

animals only, and

Its capacity to
support the internal
development of specific species of leishmania

24 Set by Z.K
  The incidence of leishmaniasis is increasing,
mainly because of:
Man-made environmental changes

Poverty and malnutrition

Movement of susceptible populations into

endemic areas

25 Set by Z.K
 Distribution in Ethiopia

 In Ethiopia
 Four species of Leishmania is found, namely,
 L. aethiopica,
 L.major

 L. tropica

 L. donovani

26 Set by Z.K
 Visceral leishmaniasis (VL)
 Occurs mainly in arid and semiarid lowlands below
1,300 m altitude

 Important endemic foci include

 Gelana focus at lake abaya,

 the segen valley (Aba- Roba focus) in Knoso

Wereda
 the Omo river plains and

 the Metema and Humera plains in north western

Ethiopia
27 Set by Z.K
 Cutaneous leishmaniasis
 Endemic at altitudes between 1400 and 2700 m in
most administrative regions

 Prevalence rates of 5.5 – 40% were reported from

villages in Shewa , Wello and G.Gofa with the highest
rate in Ocholo village in G. Gofa

 rock hyrax (Procavia habessinica) and tree

(Heterhyrax brucei ) hyraxes serving as reservoir host
for L. aethiopica

28 Set by Z.K
 Transmission and life cycle
 Common mode of transmission.
 Bite of sandfly
 Genera Phlebotomus in Old
world
 Lutzomyia in New world

 Uncommon modes of
transmission:
 Congenital transmission,
 Blood transfusion,
 Rarely, inoculation of cultures.
29 Set by Z.K
 Life cycle

Two Life-cycle stages
ProPromastigote
AmastigoAmastigote
-Elongated, with flagella

(10-20 µm long) -Round (3-7 µm diameter)
-Occur extracellularly in the -Occurs intracellularly,
-insect midgut & in artificial culture during mammalian stage
-Motile -Non-motile

30 Set by Z.K
 Mammalian R. Hosts
 Sloths
 Rodents  Primates
 Gerbils  Dogs
 Hyraxes  Foxes
 Bats  Anteaters
 Porcupines  .....
 Opossums

31 Set by Z.K


General life cycle of Leishmania species

 female sandflies inject the

infective stage, promastigotes,
during blood meals
 about 30 species of sand flies acts
a vector

32 Set by Z.K
 Promastigotes are phagocytized by macrophages &
transform into intracellular amastigotes form

 Amastigotes multiply by binary fission, rapture from

macrophages , and infect new cells
 In VL the amastigotes are carried through blood
circulation , then invade and multiply in the
macrophages of spleen, liver, Bone marrow, lymph
glands , etc.
 In Cl , MCL – the amasigote multiply in skin
macrophages (histocytes)
histocytes

33 Set by Z.K
 Sandflies become infected during blood meals when they
ingest macrophages infected with amastigotes

 the host cell break down and releasing the amasigotes which
is then transform to promastigotes

 multiply , fill the lumen of the gut and migrate to the

proboscis

34 Set by Z.K
35 Set by Z.K





Host-Parasite Interactions



 Entrance into the host and establishment of infection by
leishmanias is enhanced by saliva from the vector

 Two substances were involved

 maxadilin, or maximum dilation molecule: keeps the
capillary bed open
 SIP or salivary immunosuppressive protein :
restrains the immune system’s early efforts to
eliminate the parasites

36 Set by Z.K
 Infective promastigotes entering the blood of the
vertebrate are covered by two key molecules:
 the protein gp 63 and

 lipophosphoglycan (LPG)

 Both mediate the uptake of promastigotes by

macrophages

 The promastigotes are engulfed & form phagosome

37 Set by Z.K
 phagosome + lysosome to form a phagolysosome

 As the promastigotes transform into amastigotes,

which produce compounds that counter lysosomal
enzymes

 The gp 63 molecule inactivates proteolytic enzymes

 LPG protects against other enzymes

38 Set by Z.K
 Clinical features and pathology
Cutaneous leishmaniasis (CL)
 most common form,
 Relatively benign self-healing skin
lesions (localized or simple CL)

 Diffuse cutaneous leishmaniasis (DCL)

 Rare cutaneous infection with non- ulcerating
Nodules resembling lepromatous leprosy

39 Set by Z.K
  Mucocutaneous Leishmaniasis (MCL)
- simple skin lesions that metastasize to
mucosae especially nose and mouth
region

 Visceral Leishmaniasis (VL)

 generalized infection of the reticuloendothelial
system, high mortality response

40 Set by Z.K
 Cutaneous Leishmaniasis
Causative agents

Leishmania tropica
Leishmania major
Leishmania aethiopica
Leishmania mexicana
Leishmania peruriana
Leishmania panamensis
Leishmania guyanensis

41 Set by Z.K
 Cutaneous
Leishmaniasis
• incubation period: 2 weeks
to several months
• Initially, the lesion is a small,
red papule up to 2 cm in
diameter
• change in size and
appearance over time

Chiclero Ulcer (L.

42 Set by Z.K
mexicana)
 Cutaneous
Leishmaniasis
• chronic ulcerated, papular, or
nodular lesion
• lesion is painless, non-tender,
non-pruritic and usually
clean

Chiclero Ulcer (L.

mexicana)
43 Set by Z.K
 Cutaneous
Leishmaniasis
• chronic ulcerated, popular,
or nodular lesion
• lesion is painless, non-
tender, non-pruritic and
usually clean

44 Set by Z.K
 Cutaneous
Leishmaniasis

• self-healing, months to
years
• Sores can leave significant
scars and be disfiguring if
they occur on the face

45 Set by Z.K
 • metastasis via blood or
lymphatic systems
• especially L. braziliensis

46 Set by Z.K
 Cutaneous
Leishmaniasis
Often described as looking
somewhat like a volcano with a
raised edge and central crater
• occasionally palpable lymph
nodes

The clinical forms of CL vary

according to
-The species of parasite
-Region and
-Response of patient
47 Set by Z.K
 Soldier in Afghanistan
with Leishmania
Officer holding Iraqi child tropica on hand
with Leishmania tropica on
face
48 Set by Z.K
 Diffuse Cutaneous
Leishmaniasis
• Lesion develop over large
areas of the body
• Scaly, not ulcerated,
nodules
• Chronic and painless
• Numerous parasites in
lesions
• Seldom heal despite
treatment
L. aethiopica
49 Set by Z.K
50 Set by Z.K
 Mucocutaneous Leishmaniasis
• primarily L. braziliensis
• two stages
• simple skin lesion
• 2o mucosal involvement
• metastasis via blood or lymphatic
systems
• can occur long after primary
lesion (up to 16 years)
• frequently in naso-pharyngeal
mucosae
• junction of skin and mucosa

51 Set by Z.K
 Mucocutaneous
Leishmaniasis
L. braziliensis
• variable types and sizes of
lesions
• chronic and painless
• ulcerative type
• rapid and extensive mutilation
• non-ulcerative type
• local edema (upper lip)
• 'tapir' nose
52 Set by Z.K
53 Set by Z.K
 Visceral Leishmaniasis
 caused by the Leishmania donovani complex,
 Leishmania donovani
L. infantum
L. chagasi
• reticuloendothelial system affected
• spleen, liver, bone marrow, lymph nodes

• onset is generally insidious

• progressive disease
• 75-95% mortality if untreated
• death generally within 2 years
54 Set by Z.K
 Death usually occurs because of severe secondary bacterial
infections in advanced disease

 Pneumonia is the common complication

55 Set by Z.K
 Clinical Presentation
• incubation period
• generally 2-6 months
• can range 10 days to years
• fever, malaise, weakness
• wasting despite good appetite
• spleno- and hepatomegaly,
enlarged lymph nodes
• depressed hematopoiesis
• severe anemia
• leucopenia
• thrombopenia  petechial
hemorrhages in mucosa
56 Set by Z.K
57 Set by Z.K
Profile view of a
teenage boy suffering
from visceral
leishmaniasis. The boy
exhibits splenomegaly,
distended abdomen and
severe muscle wasting.

Set by Z.K 58
A 12-year-old boy
suffering from visceral
leishmaniasis. The boy
exhibits splenomegaly
and severe muscle
wasting

Set by Z.K 59
Jaundiced hands of
a visceral
leishmaniasis
patient

Set by Z.K 60
Enlarged spleen and
liver in an autopsy of an
infant dying of visceral
leishmaniasis.

Set by Z.K 61





Post Kala Azar Dermal leishmaniasis (PKDL)
 characterized by hypo pigmented and raised erythematous
patches on the face, trunk of the body and limbs

 may develop in to nodules and resembles those of

lepromatous leprosy, fungi infections or other skin disorders

 Occasionally there is ulceration of lips and tongue

62 Set by Z.K
  occurs in 1-3% of Indian and 50% of Sudanese VL
patients

 It require expensive and prolonged treatment

 easily cured with treatment (in contrast to DCL)

63 Set by Z.K
64 Set by Z.K
 Leishmaniasis and HIV Infection
 Coexistence of leishmaniasis with HIV infection is a serious

concern
 Leishmaniasis is spreading in several areas of the world because

of the rapidly spreading epidemic AIDS

 In 2021, Leishmania-HIV coinfection has been reported from 45

countries. High coinfection rates are reported from Brazil,

Ethiopia and India.
 The immune deficiency has lead to increased susceptibility to

infections, including leishmaniasis

65 Set by Z.K
 Co-infection with HIV has lead to the spread of
leishmaniasis, typically a rural disease, into urban areas

 In patients infected with HIV, leishmaniasis accelerates

the onset of AIDS
 by cumulative immunosuppression and
 by stimulating the replication of the virus

 It may also change asymptomatic Leishmania infections

into symptomatic infections

66 Set by Z.K
 Diagnosis of CL, MCL, DCL
• suspected because of:
• geographical presence of parasite
• history of sandfly bite
• + skin lesion:
• chronic, painless, ‘clean’ ulcer
• nasopharyngeal lesions
• nodular lesions
1. Demonstration of parasite amastigotes (scrapings, biopsy,
aspirates)
2. culture from ulcer material
3. Leishmainin test
4. serology?
67 Set by Z.K


1.Collection and examination of slit skin
smears for amastigotes

 should be taken from the inflamed raised swollen
edge of an ulcer or nodule not from its base or centre
which usually contains only necrotic tissue

 if bacterial infection is present, examination for

Leishmania amastigotes is best delayed until
antimicrobial treatment has been completed and the
bacterial infection has cleared.

68 Set by Z.K
 make incision in active part of lesion

69 Set by Z.K
 scrape cells from incision

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 prepare Giemsa-stained smear

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72 Set by Z.K
 Amastigote

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2.Culture of ulcer material

 when cutaneous leishmaniasis is suspected and parasites

cannot be found in smears

 Material for culture is best obtained by injecting and then

aspirating a small quantity of sterile physiological saline in
and out of the hardened margin of the ulcer

 A few drops of the final aspirate is used to inoculate the

culture medium

74 Set by Z.K
75 Set by Z.K aspiration and culture
 promastigotes following in vitro culture
76 Set by Z.K
 3.Leishmainin or Montenegro test

 The antigen is prepared from killed promastigotes of L.

braziliensis , L.mexicana or L.tropica with a concentration
of 10x106 parasites per ml

 Positive reaction: when the area of indurations is 5mm in

diameter or more

77 Set by Z.K
 Delayed Hypersensitivity Skin Test
• leishmanin skin test, Montenegro reaction
• intradermal inoculation of leishmanin
• suspension of whole or
disrupted promastigotes
• preferably from local area
• include negative control
• induration ± erythema in 48-72 hours

78 Set by Z.K
 4.Serology
 Because of the poor antibody response in CL, serological
tests are of little value in diagnosis

79 Set by Z.K
 DIAGNOSIS Visceral leishmaniasis

(i) Demonstration of parasite in tissues by
 light microscopic examination of the stained
specimen,
 culture
 animal inoculation
(ii) Detection of parasite DNA in tissue samples

(iii) immunodiagnosis by detection

 Antigen detection

 antibody detection
80 Set by Z.K


1.Demonstration of the parasite


 Finding of amasigote from aspirate

Aspirate %positive
Spleen …………………………………….95-98%
Bone marrow …………………………….64-86%
Enlarged lymph node …………………About 64%
Buffy coat (India) ………………………….67-99%
Buffy coat (Africa)……………….........About 50%

81 Set by Z.K


Examination of aspirates for amastigotes

Splenic aspiration
 must not be performed without training and
experience

 Before performing a splenic aspiration the following

tests must be performed:
 Platelet count and prothrombin time

 Hemoglobin

82 Set by Z.K


Examination of Buffy coat preparations for
amastigotes

 VL parasites can be detected in stained Buffy coat
smears prepared from EDTA (sequestrene)
anticoagulated venous blood

83 Set by Z.K
Amastigotes (*) of
Leishmania
donovani in the
cells of a spleen.
The individual
amastigotes
measure
approximately 1
µm in diameter.

Set by Z.K 84
Amastigotes of
Leishmania in a
macrophage from a
lymph node of a dog.

Set by Z.K 85
A macrophage
filled with
Leishmania
amastigotes.

Set by Z.K 86
 Culture
 cultures are required for

(i) Obtaining a sufficient number of organisms to use an antigen

for immunologic diagnosis and speciation,
(ii) Obtaining parasites to be used in inoculating susceptible
experimental animals,
(iii) In vitro screening of drugs, and
(iv) as a supplement to other methods or to provide a diagnosis
when routine methods have failed

87 Set by Z.K
  Leishmania strains can be maintained as
promastigotes in artificial culture medium

 Some of the culture media used are :Novy –Nicolle-

MacNeal(NNN), M199, or Grace’s and Tobies
medium

88 Set by Z.K
 Animal inoculation
 inoculation of laboratory animals (such as hamsters, mice or

guinea pigs) with infected specimen

 not usually employed as a diagnostic test, since several

months may be required to obtain a positive result.

 can be infected via many routes, including across mucous

membranes, intraperitoneal and intrasplenic routes

89 Set by Z.K
 Both amastigotes and promastigotes can infect the animal

 the animal is examined weekly for signs of infection

 In the absence of signs of obvious infection, the animal is

generally sacrificed after 4 months, at which point liver and
spleen samples are examined for the presence of the parasite

90 Set by Z.K
(ii) Detection of parasite DNA in tissue
samples
 by using molecular techniques like PCR

 highly sensitive and specific tool for diagnosis of both VL and

PKDL

 useful method for species identification

 used to distinguish between relapse and reinvention in treated

VL patients

91 Set by Z.K
 (iii) Immunodiagnosis

A) Antigen detection
 more specific than Ab –based tests

 Ag detected quite early

 the amount of detectable Ag tends to decline rapidly following

chemotherapy

 useful in the diagnosis of disease in cases where there is

deficient antibody production (as in AIDS patients)

92 Set by Z.K
(B) Antibody detection.
 Direct agglutination test (DAT)
 is a rapid and reliable screening test for VL

 sensitivity of 91-100% and specificity of 72-100%

 some cross reaction with leprosy, Chagas disease,

malaria and schisotomiasis may occur

 may also yield positive result for long time after

complete cure

93 Set by Z.K
New K39 antigen strip test to diagnose
visceral leishmaniasis
 has high sensitivity and specificity for active kala-azar

 The nitrocellulose strips used in the test are impregnated

with recombinant K39 antigen

 can be used to test peripheral (finger prick) blood samples

94 Set by Z.K
 Rapid latex agglutination test
 quicker and easier to perform and interpretable than
the DAT

 Equal volumes of test serum and sensitized dyed

latex particles are mixed on a cavity microscope slide
and rotated for up to 2 minutes. A positive test is
shown by agglutination.

95 Set by Z.K
 Shown an estimated sensitivity 100% and specificity 98%.

 yield positive result for long time after complete cure

 some cross reaction with malaria ,Tuberculosis or typhoid fever

may also occur.

96 Set by Z.K
 Formol gel (aldehyde) test

 non-specific test which detects marked increases in IgG

 Simple, low cost and widely used to assist in the diagnosis of

VL

 positive in 94% of patients with VL which becomes positive

about 3 months after infection and negative about 6 months

after successful treatment

97 Set by Z.K
Other tests

 Haematological investigations including:

Measurement of the hemoglobin anaemic

Total and differential white cell (leucocytes) count:

leucopenic

Platelet (thrombocyte) count: thrombocytopenic

98 Set by Z.K
 a raised ESR

Plasma albumin levels are greatly reduced

 total protein raised in patients with VL

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 Treatment
Treatment

• Sodium stibogluconate (Pentostam)

• Pentamidine isethionate

• amphotericin B

• cryotherapy and thermotherapy

100 Set by Z.K

 prevent and control

1. Early detection by serological diagnosis (VL) and

treatment of infected persons

1. Personal protection from sandfly bites by:

 Using insect replants
 Avoiding endemic areas especially at times when sandifies are
most active
 Use of pyrethroid impregnated bed nets and curtains

3. Vector control by the use of light traps, sticky paper

traps, or residual insecticide spraying of houses
101 Set by Z.K
 4. Destruction of stray dogs and infected domestic dogs

5. Elimination and control of rodents

6. sitting human dwellings away from the habitats of animal

reservoir hosts where sandifies are known to breed

102 Set by Z.K

 Summary

 Discuss the general charcterstics of blood and tissue

flagelates

 List the laboratory diagnosis approaches for Cl DCL, MCL

and VL

 List the general control and prevention methods used for

leishmaniansis

103 Set by Z.K



Learning Objectives
 After completing this session, students will be expected to;

 – Describe the causative agents malaria

 – describe the morphological stages of malaria

 – List the mode of transmission of malaria parasites

 – Describe life cycle of malaria parasites

 – describe the pathogenesis and clinical manifestations of malaria

 – Explain the laboratory dx of malaria

104 Set by Z.K



Malaria
 Malaria: an acute and/or chronic infection caused by protozoans of

the genus Plasmodium

 Causitive agents: Five plasmodium species causing human malaria

 Plasmodium falciparum (P.falciparum)

 P.vivax

 P.malariae

 P.ovale

 P.knowlesi

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Epidemiology
 Nearly half of the world's population is at risk of malaria

 According to WHO, malaria report there were 247 million cases of

malaria in 2021
 The WHO African Region continues to carry a disproportionately

high share of the global malaria burden

 In 2021 the Region was home to about 95% of all malaria cases

and 96% of deaths

 Children under 5 years of age accounted for about 80% of all

malaria deaths in the Region

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 Burden of Malaria
 An estimated 881 000 malaria deaths ww

— of which 91% were in Africa and 85% were of < 5children

 One child dies of malaria in Africa every 20 sec

 One malarial death every 12 sec in the world

 Kills in 1 year what AIDS killed in 15 years

 Every year ~ 30000 visitors to endemic areas develop malaria

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 Burden of Malaria CONT...
 In Ethiopia,

 ¾landmass malarious

 68% of the population at risk

 Annual clinical cases estimated 4-5 million

– 10-40% of all outpatient consultations

– 13-26% of all inpatient admissions

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Burden of malaria in Ethiopia cont..
P.falciparum =60%

P.vivax = nearly 40%

P.malariae =1% cases ,focal distribution like in Humera

P.ovale = less than 1% cases , found in Setit Humera ,

Gambela & Arbaminch

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 Widespread species
• P. falciparum
most prevalent in the hotter and more humid
regions of the world
• P. vivax
more common in temperate region 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%)
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 Epidimology of 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

 Two major transmission periods

Major - September to December after main rainy

season
Minor- April to June following small showers of rain in

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 Dega zone(> 2,500 m) mean annual temperature of 10

-150C , is malaria free

 Weyna dega zone( 1,500 - 2,500 m) mean annual

temperatures range from 15-20oc

malaria most often occurs below 2,000 meters, with

short-lived transmission following the rains

 Kolla zone (< 1,500 m), mean annual temperatures are

20-25oc, malaria transmission is endemic

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Endemicity : defined in terms of parasitemia rates

hypoendemic (<10%)

 mesoendemic (11 to 50%)

 hyperendemic (51 to 75%)

 holoendemic(>75%)

 Hyper and Holoendemic malaria are found in areas of stable

malaria transmission
 Hypo and Mesoendemic : are found in areas of unstable

malaria transmission
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Characteristics of stable
Characteristics of unstable malaria
malaria
 Malaria incidence varies from week to
 Constant incidence over week, month to month, year to year, day to
several years day.
Communal immunity of the population
 Includes seasonal


low.
transmission
 Makes the region prone to malaria
 Immunity and disease epidemics.
tolerance developed by adult  High morbidity and mortality

 Usually affects children

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General feature of Plasmodium species
 obligate Intracellular parasites. (liver cell & RBC))

 Life cycle

Alternation of generation ~ alternation of hosts

Requires two hosts

 Man (IH)

Female Anopheles mosquitoes (DH)

 Sexual and asexual reproduction

 No animal reservoir host except P.malariae

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 Life cycle
 Require two host Mosquitoes:-
Man:- Definite host
intermediate host Sexual
Asexual reproduction reproduction
 Liver cell
 RBC

Mosquitoes cycle
A- Sporogony
Human cycle
Two phases
B- exo-erythrocytic schizogony in liver
C- Erythrocytic schizogony & gametocytogenesis in RBC
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 Life Cycle:

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IN THE MOSQUITO
 During a blood meal on man, female Anopheles mosquito picks
up mature gametocytes
 In the mosquito's mid gut, a micro gamete(male) penetrates a
macro gamete(female) and form a zygote
 The zygotes in turn become motile and elongated form called
ookinetes
 invade the midgut wall of the mosquito where they develop into
oocysts
 The oocysts expanding by asexual multiplication, grow, rupture,
and release motile sporozoites , which make their way to the
mosquito's salivary glands
 Inoculation of the sporozoites into a new human host
perpetuates the malaria life cycle

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IN MAN
 During a blood meal, malaria-infected female Anopheles
mosquito inoculates sporozoites and salivary fluid
 The sporozoites remains in the circulating blood for only 30
minutes
 The kupfer cells of the liver kill and clear many sporozoites
from blood stream
 Fraction sporozoites escape destruction are carried rapidly via
the blood stream and invade hepatic parenchymal cells of the
liver
 begin their initial asexual replication : Exo- erythrocytic/
Intrahepatic / Pre-erythrocytic schizogony
 within 5-15 days mature into pre-erytrocytice(PE)
schizonts containing 10,000-30,000 merozoites
 rupture the swollen liver cells & release merozoites in to blood
stream
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 P. falciparum
mature and released simultaneously
from liver, no relapse
 P. malariae

 P vivax
may remain latent in the liver and
relapse
 P ovale

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 A proportion of the merozoites are phagocytosed & destroyed
 The remaining Enter in to red cells starts erythrocytic
schizogony which to complete takes 36-48 hours (P.
falciparum),48 hours (P. ovale/ vivax) &72 hours (P. malariae).
 At this time the intracellular merozoites develop in to
trophozoites (‘ring form’)
 When the trophozoites fully developed ,then schizogony
takes place resulting in the formation of schizont
containing 8-32 merozoites

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 Development to erytrocytic schizont in P. falciparum takes place in

the capillaries of deep tissue

 The mature schizont rapture from red blood cells

releasing merozoites , malaria pigment and toxins in to

plasma
Merozoites ,which are not destroyed by host immune

system infect new red blood cells, initiates further cycle of

erythrocytic schizogony with more red blood cells begin
destroyed.
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 After several erythrocytic schizogony cycle , some of the

trophozoites in the red blood cells develop in to male female

gametocytes
 P. vivax , P. ovale and P. malariae at least two cycle
eryhrocytic schizgony
 P. falciparum ,the asexual parasites in the circulation for ten
days

 The gametocytes are now ready to be ingested by an

Anopheles mosquito during a blood meal

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Morphological stages
 Sporozoite: develops in the mosquito salivary gland
 Hepatic schizont actively dividing, multinucleated, parasite form in
hepatocytes
 Trophozoite: metabolically active form living within the RBC
 Sometimes called the ring form
 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
 Gametocyte: morphologically distinctive sexual (male or female)
form which develops from some trophozoites in RBCs

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 Transmission of Malaria
Principal mode Transmission
Bites of female anopheles
mosquito
60 species of mosquito
Sucks the gametocytes
during blood meal
 Bites between 5 PM and 7
AM, with maximum intensity
at midnight.

Anopheles

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  Anopheles mosquito In Ethiopia :
A.gambiae, A.funestus, A.nili, A.arebiansis &
A.pharonensis are
main vectors
A. arabiensis is responsible for most epidemics in the
country

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 Other modes of transmission
1. Blood transfusion (Transfusion malaria):
 Frozen plasma is not known to transmit malaria
Infective stage-trophozoites/merozoites
Sho r t e r i nc uba t i o n pe r i o d, be c a us e no e xo -
erythrocytic shizogony
No relapses possible (vivax/ovale)

 Clinical features & management of cases are the same

as naturally acquired infection
 Donor blood should be screened
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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

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Clinical Features & pathology
 Characterized by acute febrile attacks (malaria paroxysms)

 caused by the release of toxins (when erythrocytic schizonts

rupture) stimulate the secretion of cytokines from leucocytes

and other cells
 Manifestations and severity depend on parasite species, parasitemia

and host status, i,e immunity, general health, nutritional state,

genetics
 Without treatment, P.vivax, P. ovale, P. malaria ultimately may result
in spontaneous cure
 P. falciparum can develop severe complications
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 P. falciparum can develop severe complications and most

likely to result in death if left untreated

 Most pathogenic of all species.

Its pathogenicity is mainly due to:

 1. cytoadherence

 Infected RBCs adhere to the cells lining the wall of

capillaries

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 This leads to sequestration(holding back) of infected RBCs

in capillaries of internal organs, with subsequent

 congestion, hypoxia, blockage and sometimes rupture of

small blood vessels

 Example: parasitized RBCs blocking capillaries and venules

in the brain in Cerebral malaria

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 Sequestration Hypothesis
cytoadherence

cerebral ischemia

hypoxia, metabolic effects

coma

death

Cerebral malaria
 severe complication of
falciparum malaria
 mortality of 30-50%
 associated with sequestration in
micro-vasculature of brain
 a diffuse encephalopathy with
loss of consciousness
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2. High level of parasitaemia:

 A single red blood cell can be infected by more than one

parasites

 All age groups of RBCs can be infected, and

 Rapid multiplication of parasites occur

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Complications of Severe P.falciparum
 cerebral malaria
 blackwater fever
 Severe anemia
 hypoglycemia
 pulmonary edema
 Renal failure
 Generalized convulsions
 Circulatory collapse and shock
 Acidosis / hyperlactatemia
 jaundice or other liver malfunctions
 hyperparasitemia
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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.
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Predisposing factors for complications of P. falciparum malaria
(1.) Extremes of age.
(2.) Pregnancy
 especially in primigravidae and 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

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P. vivax
 In 50% of patients, relapses occur after weeks, months, or

up to 5 years(or more)
 Severe complication are rare in P. vivax infections

P. ovale
 Although P. ovale and P. vivax infections are clinically similar,

P. ovale malaria is usually less severe, tend to relapse less

frequently, and usually ends with spontaneous recovery

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Malaria caused by Plasmodium spp
 Plasmodium falciparum: malignant tertian malaria
 Plasmodium vivax is referred to as vivax malaria , benign tertian
(BT) malaria
 Plasmodium ovale is referred to as ovale malaria, ovale tertian
malaria
 Plasmodium malariae is referred to as malariae malaria , quartan
malaria
 Infections caused by P. vivax, P. ovale or P. malariae are rarely life
threatening
 no Cytoadherence of parasitized cells
 parasitic densities are lower
 P. malariae is nephritic syndrome which may progress to renal
failure

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Prodromal Symptoms of malaria
 Malaria paroxysm preceded by Prodromal symptoms

2-3 days before 1st paroxysm

 includes: malaise, fatigue, headache, muscle pain, nausea,

anorexia
 In all four species, prodromal symptoms may mimic

symptoms seen with influenza

 Can range from mild to severe (usually mild to moderate)

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 Paroxysms Comprises of three Successive Stage
Cold stage Hot stage
• feeling of intense cold • intense heat
• vigorous shivering, • dry burning skin
rigor and headache • severe headache
• lasts 15-60 min • pains in the back & joints
• lasts 2-6 hours

Sweating stage
• profuse sweating
• declining temperature
• -headache & other pain relieved
• exhausted, weak  sleep
• lasts 2-4 hours
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 cold stage
• feeling of intense cold
• vigorous shivering, rigor
• lasts 15-60 min

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

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

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 Malaria paroxysm may be accompanied by splenomegaly,

hepatomegaly (slight jaundice), anemia

 Acute febrile attacks (malaria paroxysms) is caused by

— the release of toxic materials & malaria pigments

(when erythrocytic schizonts rupture) into the circulation

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 Clinical characteristics of the four plasmodium
species

Infection parameter P.vivax P.ovale P.malariae P.falciparum

Incubation period 8 -17day 10 8 -17 daY s8 -11 days

s -17days
Initial Paroxysm Severity moderate moderate severe
to severe mild to severe

Symptom periodicity 48 hrs 48 hrs 72 hrs 36-48 hrs

Maximum Infection 5-7 years 12 20+ years 6-17

Duration (untreated) months months
Anemia Mild to mild Mild to Severe
moderate moderate
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Complications renal cerebral
 Relapse
 is a feature of vivax and ovale malaria due to the delayed
development of PE schizonts from hypnozoites in liver
cells. (the reappearance of parasitmia in a sporozoite-
induced infection following adequate blood schizonticidal
therapy )
Recrudescence :- is a feature of P. malariae and also P.
falciparum but this is not relapse caused by dormant
forms called hypnozoites
• Recrudescence caused by small numbers of erythrocytic
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the parasite persisting in the host after a previous


Hyper-reactive malaria splenomegaly (Tropical
splenomegaly syndrome)


 massive and chronic splenomegaly with

 high levels of IgM ,

 malaria antibody,

 circulating immune complexes

 a moderately enlarged liver with hepatic sinusoidal

lymphocytosis
 The patient is usually anaemic (normocytic)
 low white cell and platelet counts
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Hyper-reactive malaria splenomegaly
Genetic factors that provide protection against Malaria
Nature of hemoglobine
 Hgb S (Sickle cell anemia trait) –p.f

 Thalassemia Hgb-P.f

 Fetal Hgb – all sps

 Hgb E – P.v

Enzyme content of erythrocyte

 Glucose-6-phosphate dehydrogenase deficiency ,P.f

Presence or absence of certain factor

 Ovalocytosis -P.f & P.v
 Duffy blood group antigens (i.e., Fya and Fyb) negative RBCs, P.v

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 Laboratory Diagnosis
Clinical Diagnosis

Malaria Diagnosis

Laboratory diagnosis

Molecular
Microscopic Immunological
•Thin film PCR
•Thick film RDT.ICT
ICT etc.
• QBC  Ag /enzyme
 Ab- ELISA
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Malaria Diagnostic Cont..
 Clinical Diagnosis

 The first essential steps to disease management

 The only approach where lab support doesn’t exist

 Based on clinical signs and symptom

 Most prominent symptom is fever

 Symptoms are non-specific and overlap with other febrile

illnesses
 Alone is unreliable, and when possible should be confirmed by
laboratory tests
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Laboratory diagnosis Techniques
I. Microscopic 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 buffy coat( QBC) system
II. Immunologic/Biochemical techniques
detection of malaria antigen, antibody & parasite products
III. Molecular diagnosis techniques
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 Microscopic examination
 Microscopic examination of blood film

 Blood collection

—When the patients feels febrile

—Before anti-malaria drugs are given to the patients
 Blood film preapration

—Thin BF: to identify Plasmodium species

—Thick BF: to screen/detect Plasmodiumparasite
 Staining

 Microscopic examination

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 I. Microscopy examination

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 Microscopic examination of blood film
 Established method for confirmation of malaria-
the gold standard.
 Requirements:
 Carefully collected blood specimen/sample

 When the patients feels febrile

 Before anti-malaria drugs are given to the

patients

 Well prepared blood films

 Thin BF: to identify Plasmodium species

 Thick BF: to screen/detect Plasmodiumparasite

 Well stained smears( Romanowsky stains)

 Careful examination of the smears

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 Examination

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Reporting the result of Blood film
 Species,stage & approximate No of parasites are included
 Dx stages to be reported
 Trophozoite
 Schizont
 gametocyte
 – Pf – if schizonts seen report immediately
 Using thin blood film report the percentage
 Using thin/thick blood film ----number of parasite / µLof
blood

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Reporting blood films for malaria parasites

A. REPORTING THICK BLOOD FILMS
 plus sign scheme :
Parasites
1 – 10 per 100 high power fields ………………… +
11 -100 per 100 high power field…………………++
1 – 10 in every high power field…………………+++
More than 10 in every high power field ………. ++++

 Example: P. falciparum trophozoites +++, gametocytes +, with

malaria pigment in white cells

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  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)

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B. REPORTING THIN BLOOD FILMS




COUNTING PARASITE NUMBERS
The following two methods are commonly used:

1. Estimating parasite numbers/µl of blood from the

thick film.
count a total of at least 500 red cells making a note of the
number that contain parasites excluding gametocytes and
at the end report in percentage

Note:-A serious P.falciparum infection is indicated

 when 5% of more of the red cells are infected.
 E.g. When 10-20% of the cells are parasitized the
prognosis is serious, with 20-30% it is grave and over
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Z.K it is exceptionally grave.
2. Estimating parasite numbers/µl of blood by counting
parasites against white cells

No of P/Per l of blood = WBC counts X parasites

counted against 100WBC
100
Example.
Patients WBC count =8000 /l
Parasites count against 100 WBC =680
Parasite count  8000 X 680 5440/l
100
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 antigens

Based on detection of parasite antigen

 Includes:

 Histidine-rich protein 2 of P. falciparum (PfHRP2)

 Parasite lactate dehydrogenase

 Plasmodium aldolase

Ways of reporting antigen test

 Postive :if detected

 Negative: if not

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Polymerase Chain Reaction (PCR)

 highly sensitive and specif ic for detecting all species

of malaria and permits genotyping

 particularly in cases of low level parasitemia and

mixed infections

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Treatment
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

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164
 •Currently Artemisinin-based combination therapy (ACT)

drugs:- Artemether-Lumefantrine,
 Artesunate-Mefloquine, Artesunate-Sulfadoxine-

pyrimethamine and
 Dihydroartemisinin-Piperaquine are potent drugs used for the

treatment of uncomplicated P. falciparum malaria

 Artemether/Lumefantrine (AL), a fixed dose in a 1:6 ratio

(20mg/120mg) approved as Coartem®, is used as oral

medication in endemic countries for P. falciparum including
Ethiopia
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 Severe falciparum malaria is treated with parenteral

Quinine or Artesunate (IV route or injection) and other supportive

therapies towards all kinds of organ and system complications
 Treat adults and children with severe malaria (including infants,
pregnant women in all trimesters and lactating women) with IVor IM
artesunate for at least 24 hr and until they can tolerate oral
medication
 Once a patient has received at least 24 hr of parenteral therapy and
can tolerate oral therapy, complete treatment with 3 days of an ACT
 Plasmodium vivax malaria is treated with oral Chloroquine for 3days

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Prevention and Control
Avoid mosquito bites by
 Using impregnated bed nets
 Wearing protective clothes
 Using mosquito repellents
 screens, house spraying

Destroy adult mosquitoes by

 Indoor residual spraying :-DDT, Pyrethrum, Pyrethrins, Pyrethroides, Malathaion

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Preventing breeding of mosquitoes by
 Environmental modification
 Spraying breeding places with effective larvicides
 Biological control( bacteria (Bacillus sphaericus & B.
thuringiensis), growing plants that inhibit mosquito breeding,
introducing fishs that feed on mosquito larvae into breeding
water )
Treatment
 Active infection
 Chemoprophylaxis

Health education
Blood screening for malaria
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The End!