Introduction

Malaria the most important parasitic disease

transmitted by a vector mosquito

One of the oldest diseases known to mankind

influenced human history as well

Human malaria likely originated in Africa, associated

with the origin of mankind
 malaria on going transmission reported in 91 countries

half of the worlds population are at risk

The burden of malaria;

Direct burden mortality an morbidity

In year 2015
212 million clinical cases (148 - 304 million)

429,00 deaths (235000 639000) 99% due to P.

falciparum, rest due to P. vivax

303,000 deaths were of children aged under year 5;

mainly in Africa

Taking the life of child in every 2 minutes

Indirect burden

Human development:
loss school attendance

productivity at work (greatest risk men at 15 50 y)

impaired intellectual development,

developmental abnormalities (sp. following

cerebral malaria),
 Classification:

Sub-kingdom - Protozoa

Phylum - Apicomplexa -
(apical organelle complex for attachment)
Class - Sporozoa
(reproductive cells called spores)
Sub-class - Coccidia

Order - Coccidiida

Sub-order - Haemosporina

Genus - Plasmodium
 >100 species of Plasmodium - variety of
hosts;
rodents, birds, reptiles and non-human
Five species of obligate protozoans that naturally
primates
infect and cause malaria in humans:

P. falciparum P. vivax P. malariae P. ovale

Fifth species of human malaria ?????

P. knowlesi - monkey malaria
species
P. falciparum highest numbers of
severe complicated malaria and
mortality

P. vivax most wide spread human malaria

parasite outside Africa
rarely cause severe complicated malaria

P. ovale mainly confined to Africa

P. malariae least severe infection

 Transmission of malaria

Primarily transmitted by the bite of an infected

female Anopheles mosquito (subcutaneous inoculation

of sporozoites)

Congenital

Blood transfusion

Through contaminated
syringes of drug addicts
 Malaria in Sri Lanka
History
Reported from early days
The most devastating epidemic in recent history was in
1936-37 ;
1.5 million cases with 80,000 deaths

P. vivax Vectors
Anopheles culicifacies
P. falciparum
principal vector
Anopheles subpictus
Anopheles annularis
P. vivax was the predominant species
P falciparum was the second most predominant

Since October 2012 no indigenous malaria cases were

reported from Sri Lanka

2013 95
2014 49
2015 - 36 cases of malaria were reported, all in
individuals that had contracted the disease overseas
Imported malaria

In September 2016 WHO certified Sri Lanka as

malaria free country
Presently in Sri Lanka;

Malaria is a notifiable disease

No indigenous malaria cases

Problems?????

Vector is still prevalent

Parasite source available (imported cases)

Lack of awareness

Can reemerge
Morphology and Life cycle of
Malaria Parasites
 Life cycle of Plasmodium

Human / vertebrate Mosquitoes

(intermediate host) (definitive host)
Asexual cycle Sexual cycle
(schizogony) (sporogony)

Exo-erythrocytic cycle Erythrocytic cycle

(pre-erythrocytic schizogony) (erythrocytic schizogony)
Inside man ..
Infective stage
sporozoites

Exo-erythrocytic
stages

Erythrocytic c
ycle

Sexual multiplication
of Plasmodium
in the man -
Sporogonic cycle
 Exo-erythrocytic cycle
Infective stage - Sporozoite
Sporozoite concentrated in salivary glands of Anopheles
species mosquitoes.

Elongated & spindle shaped.

12-14 m in length.

Large number of sporozoites

are injected into the bite wound with the mosquito saliva

Sporozoites circulate in the blood stream for about 30

minutes.
 Exo-erythrocytic cycle

Sporozoites invade the paranchymal cells of the liver.

Increase in size.

Asexual multiplication begins by division of nucleus large

number of daughter nuclei.

Parasite cytoplasm condenses around daughter nuclei and

begins the production of merozoites Liver Schizont

Mature schizont occupies entre liver cell cytoplasm and then

rupture - merozoites released
 Exo-erythrocytic cycle

Time to complete liver schizogony and the numbers of

merozoites released different according to the
Plasmodium species

Length of Number of
exo-erythrocytic merozoites released
schizogony (days)
P. falciparum 5-6 30,000
P. vivax 8 10,000-12,000
P. ovale 9 15,000
P. malariae 15 2,000
 Exo-erythrocytic cycle

Only in P. vivax and P. ovale, some sporozoites remain

dormant for varying periods - Hypnozoites

Hypnozites later reactivate and starts liver schizogony

and results relapse infections
 Erythrocytic
stage
Important in clinical practice:

only stages causing the clinical symptoms in humans

the recognition of parasites in blood cells allows the

diagnosis

Starts when merozoites from liver schizonts released to

the circulation
Morphology is described as parasites appear in smears
stained with Romanowsky stains

Malaria pigment - Haemozoin :

by product of Heam
In erythrocyte;

Merozoite appears as a rounded body with vacuole in the

middle, cytoplasm pushed to the periphery and nucleus at
one pole

When stained with Romanowsky stains

nucleus deep red
cytoplasm blue
vacuole unstained

Parasite feeds on haemoglobin of the RBC and leaves

residues of haemozoin pigments - malaria pigments

These iron containing pigments accumulate in the body of

the parasite as dark granules
 Erythrocytic cycle of P. vivax

P. vivax merozoites enters young RBCs.

Usually one merozoite invades a red cell.

Need 48 hours to complete the cycle.

Entire cycle takes place in peripheral circulation.

 Erythrocytic cycle of P. vivax

Ring stage
Vacuole appear in the parasite.

Single nucleus.

Thick cytoplasm

RBC becomes lager and paler.

Parasite remains as ring stage for 6
Vacuole to 8 hours
Appears numbers of pink stained
particles Schuffners dots
 Erythrocytic cycle of P. vivax

Amoeboid stage Parasite produce pseudopodia

after 10 to 12 hours of
invasion.

One or more vacuoles in the

cytoplasm.

Malaria pigments (Haemozoin)

brown colour
 Erythrocytic cycle of P. vivax
Schizogony Starts about 36th hours.

Early or immature schizont

Occupies almost all of the RBC.

Cytoplasm condenses round each of 14

to 24 nuclei.

Late or mature schizont

14-24 erythrocytic merozoite

Malaria pigment found the centre.

Schuffners dots clearly visible.

Starting of the sexual cycle

After 4 6 days - new stage of
parasites are formed and
gametocytes.
No vacuoles.
vacuoles Scattered Schuffners dots
are found.
Gametocytes
Nucleus stains pink
Spread over a wide area.
Malaria pigments scattered in
the cytoplasm.
Gametocytes
Concentrated nucleus.
Malaria pigments scattered
around the nucleus.
 Erythrocytic cycle of P. falciparum

P. falciparum prefers RBCs of all ages.

Need 48 hours to complete the cycle.

Usually one or more merozoites invades a red cell multiple

invasions.

One or two chromatin dots can be found in one merozoite

Applique form marginal parasites

 Erythrocytic cycle of P. falciparum

Ring stage
Single or double nuclei
Applique form
Multiple infection (a characteristic
feature)
Thin cytoplasm
No changes in size of RBC.
Schuffners dots are absent.
Vacuole
Maurers cleftsirregular clefts
staining deep redappears.
 Erythrocytic cycle of P. falciparum
Amoeboid stage is not found with P.falciparum
Visceral scizogony-
By about 24th hour, infected RBCs disappear from the
peripheral circulation and are sequestrated in the
capillaries and veins of deep organs
Early or immature schizont occupies all of the RBC
The schizont ruptures at 48 hours release 8-24
erythrocytic merozoites
Erythrocytic merozoites enter new RBCs in capillaries
of deep organs
Beginning of the sexual cycle of P. falciparum

After 10 days gametocytes produced

Appears in the peripheral blood

Sausage / banana shaped

Devoid of vacuoles

Gametocytes blunt ends

Nucleus stains pink and spread
over a wide area
Malaria pigments scattered in the
cytoplasm

Gametocytespointed ends
Concentrated nucleus
Pigments scattered around the nucleus
Erythrocytic cycle of Plasmodium malariae
-prolonged - takes around 72 hours to complete
-infected RBCs do not enlarged and normal shape
-multiple infections are rare
-all parasitic stages are present in peripheral blood
-ring stage having thicker cytoplasm than in other species
-Schuffners dots rare; coarse and dark pigments present
-Trophozoite has a characteristic band form
-Schizonts have characteristic daisy head pattern
-Pattern of the life cycle is similar to that of P. vivax with
some exceptions
Erythrocytic cycle of Plasmodium ovale

-development is similar to that of P. vivax

-infected RBCs are enlarged as for P. vivax

-stippling / Schuffners dots is intense

-infected RBCs wall become weakened and may have

irregular shapes

-Oval RBC with fimbriated edges is characteristic, but

not always present
 P. f. P. v.

Tissue
5-6 dys 8 dys
schizogony

Erythrocytic
48 hrs 48 hrs
phase

Red cells Reticulocytes

All
affected Young red cells

Normal size
Enlarge,

Infected RBC Maurers dots

Schuffners dots

Basophilic stipplings
 P. f. P. v.
Rings Rings

Stages found in Gametocytes Trophozoites

peripheral blood
(Occasionally late Schizonts
Schizonts)
Gametocytes

Delicate, small,
1/3 of RBC Large
Ring Double chromatin Single,
Multiple rings Prominent Chromatin
Applique form
 P. f. P. v.
Merozoites per
8 - 32 12 - 24
red cell schizont
Sausage / banana Spherical
shaped,
Microgamete Nucleus spread
(male) Large diffuse nucleus wide area

Blue / pink cytoplasm, Pale blue cytoplasm

Crescent
Spherical
Macrogamete
Deep blue cytoplasm
(female)
Large,
Compact nucleus
Compact nucleus
 Inside mosquito - Sporogonic cycle

Male and female gametocytes enter the mid gut with the
blood meal
Male gametocyte Exflagellation male gametes
Female gamete gets fertilized by male gamete in the mid
gut of the mosquito.
Zygote motile Ookinete
Ookinete cross mosquito mid gut wall and produce Oocysts
Oocysts attached to the outside mid gut wall
Inside Oocysts sporozoites are produced
Sexual multiplication of Plasmodium in the vectorSporogonic cycle

GAMETOGENESIS microgametes

megagamete Fertilization

Development
inside mosquito
Ookinate
Mosquito salivary
gland
SPOROGONY

Oocyst

Formation of sporozoite
Sexual multiplication of Plasmodium in the
vectorSporogonic cycle
 Clinical features of malaria

Asymptomatic (clinical
immunity)
Acute, uncomplicated
Sever complicated

The primary symptom of all types

of malaria is the "malaria fever"
(chills and fever).
The Characteristic text-book picture of
malarial fever is not commonly seen.
It includes,
1. Cold stage

2. Hot stage

3. Sweating stage
The interval between paroxysms is determined by
the length of the erythrocytic cycle of the
parasite species involved:

In vivax - tertian malaria (every 48h)

In ovale - tertian malaria

In falciparum - tertian /sub tertian malaria

In malariae - quartan malaria (every 72h)

 Treatment of malaria - Class of anti
malaria drugs Clinical
Target Type of drug
application
Acute
Trophozoite in
Blood
blood
schizontocide
Site A
malaria
Active schizont in
Primary tissue
liver None
Schizontocide
Site B
Dormant Prevention of
Hypnozoitocide
hypnozoite relapse

Gametocyte in
Gametocytocid Prevention of
blood
e transmission
Site C

Forms in mosquito
 Site B

Site D
Site A

Site C
 Malaria control

Drugs - parasite

Insecticides - vector

Drug resistant
In Sri Lanka CQ resistant

Insecticide resistant
Malaria vaccines are a

viable option
Transmission blocking
vaccines Preerythrocytic
vaccines
The immune
mediators
implicated at the
different stages
of the life cycle
are shown.

Vaccines are
being developed
based on 4
strategies.

Anti-disease
Good, M.F. & Doolan, D.L. vaccines
(2010). Immunity,33, 555-566
Asexual blood stage
vaccines
DIAGNOSIS OF
MALARIA

54
 Prompt and accurate diagnosis -
key to effective disease management

Diagnosis 1. Parasitological (Laboratory Ix)

Demonstration of the parasite
Direct evidence of the parasite presence

Indirect presence of malaria parasite

antigens or antibodies (immunodiagnosis)

2. Clinico-Epidemiological (Hx, Ex)

 Clinical diagnosis
The most important element in the clinical
diagnosis of malaria, in both endemic and non-
endemic areas, is to have a high index of
suspicion

Visit to a Malaria endemic area?

Blood transfusions
History of Malaria

Severe malaria can mimic many other diseases

56
 Differential
Diagnosis
The most important of these
All types of Meningitis
Typhoid fever
Septicaemia
Other differential diagnoses include
Influenza
Dengue and other arboviral infections
Hepatitis
Leptospirosis
The relapsing fevers
Haemorrhagic fevers
Scrub typhus
All types of viral encephalitis (including rabies)
Gastroenteritis
African trypanosomiasis
In pregnant women, malaria must be distinguished
from sepsis arising in the uterus, urinary tract or breast
In children, convulsions due to malaria must be
differentiated from febrile convulsions
57
 Parasitological Diagnosis
DIRECT METHODS
Demonstration of Malaria parasite in blood or tissue

Eg:- Thin or Thick Blood Film

Intradermal smears &
Bone marrow aspirate (not done routinely)

INDIRECT METHODS
Demonstration of Malaria Antigen, Antibody or DNA material
Eg:- 1. Histidine Rich Protein II, (ParaSight-F, ICT)

2. Parasite Lactate Dehydrogenase (OptiMAL)

3. Nucleic acid sequences (PCR - DNA Probe)

58
 Blood film
Blood film examination GOLD standard

based on its
ability to allow speciation,
quantification of parasitaemia,
assessment of the distribution of parasite
forms

Two types of blood film for malaria parasites

Thick blood smear
use to determine if parasite is present
Thin blood smear
use to confirm the different Plasmodium species
Blood obtained by pricking a finger or earlobe
is the ideal sample

density of developed trophozoite or

schizonts is greater in blood from this
capillary-rich area

Venous blood
Cord blood
Blood from donor pack

Ideally few hours after the fever attack ????

60
 Thick Blood Film

Thick film about 20-30 layers of RBC on a small area

Unfixed film is stained

red cells are haemolysed &
only leucocytes & parasites are found

The thick blood film ;

provides enhanced sensitivity of the blood film
(in lab about 5-20 parasites/l of blood;
in field about 50-100 parasites/l )

is much better than the thin film for detection of

low levels of parasitaemia and reappearance of
circulating parasites during infection recrudescence
or relapse.
 Thin Blood film

Morphological identification of the parasite to the

species level is much easier

Provides greater specificity than thick-film

examination

Stages are identifiable more easily

Can use to monitor the treatment response and

detect drug resistance

Estimation of parasitaemia possible

In low parasitaemia - miss the diagnosis

 Blood film examination
Advantages
Available in countries where malaria is endemic
Cost for the test is minimal / Recurrent expenditure less
Species identification possible
Provides a permanent record of results

Disadvantages
Needs good microscope & well trained staff
Only one sample can examined at a time
High false negative rates
False positives (rate is low depending on the quality of film)
Labour intensive process
Repeated examinations are necessary to exclude malaria
(Pf infections easily be missed due to sequestration)
Plasmodium falciparum Infected erythrocytes: normal size

Rings: double chromatin dots; accole forms;

multiple infections in same red cell

M I

Schizonts: 18-32 merozoites Gametocytes: mature (M) and

(rarely in peripheral blood) immature (I) forms (I is rarely
seen in peripheral blood)
Plasmodium vivax
Infected erythrocytes: enlarged up to 2X; deformed; (Schffners dots)

Rings Trophozoites: ameboid; deforms the erythrocyte

Schizonts: 12-24 merozoites Gametocytes: round-oval

Plasmodium ovale
Infected erythrocytes: moderately enlarged (11/4 X); oval; (Schffners dots)

Trophozoites: compact
Rings

Schizonts: 6-14 merozoites; Gametocytes: round-oval

dark pigment;
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
69
Quantitative Buffy Coat technique

Test based on the ability of acridine orange (AO) to stain

nucleic acid containing cells

Briefly,
A microhaematocrit tube containing acridine orange stain
and anticoagulant is filled with patients blood (55-65 l)

After centrifuged immediately placed the vial under an

ultraviolet light source microscope for observation
The blood cellular components separated in the tube
platelets
lymphomonocytes
granulocytes
erythrocytes
and forced against the tube wall by the internal float

The parasites are seen as fluorescent bodies standing

at different levels of the sedimentation column
depending on the stage and species of the parasite
Advantages
Short time per test (5-10 minutes)
Useful for screening large numbers of samples
Higher sensitivity than thick films (3-4 parasites/l blood)
Ease of interpretation
Technically easy to perform

Disadvantages
Species identification and quantification difficult
High cost equipment and high recurrent expenditure
Detection of Malarial Antigen

Highly specific
Diagnostic
No false positives - minimal cross
reactions
RAPID DIAGNOSTIC TESTS / RDTs

Based on the detection of plasmodium antigens (Ag)

derived from malaria parasites in lysed blood

Malaria Ags currently used as diagnostic targets are

either specific to a one Plasmodium species or
conserved across the human malaria spp (pan
malaria antigens)

Using immunochromatographic lateral flow-strip

technology
 Mode of action of common RDTs

Mixing the patients blood with a lysing agent and ruptures the red blood cells,
releasing more parasite protein

Dye-labeled antibody, specific for target antigen, is present on the lower end of
nitrocellulose strip

Same antibody also bound to the strip in a test line

Different antibody specific for the dye-labeled antibody, or another antigen, is

bound at the control line
Mode of action of common RDTs

Blood and buffer placed on strip are mixed with dye-labeled antibody and are
drawn up the strip across the lines of bound antibody.
Mode of action of common RDTs

If antigen is present,
some labeled Ab-Ag complex trapped on the test line;
excess-labeled antibody is trapped and accumulates on the control line
(A visible control line indicates that labeled antibody has traversed the full length of
the strip, past the test line, and that at least some free antibody remains conjugated
to the dye and that some of the capturing properties of the antibodies remain intact)

The intensity of the test band will vary with the amount of antigen present, at least
at low parasite densities (antigen concentration)
Parasite antigens currently used in commercial RDTs

1) Plasmodium Histidine-rich protein 2 (HRP-2)

Unique to Plasmodium falciparum or P. vivax

Commercial kits are currently available detect HRP-2 only from P.

falciparum

Found in parasite cytoplasm and on the IRBC surface; produced

mainly by trophozoites and young gametocytes

Also found as a soluble protein in plasma

HRP-2 concentration is increased as parasite develops from ring

to late trophozoites

HRP-2 can remain in the blood at least 28days after the

initiation of antimalarial therapy
2) Plasmodium lactase dehydragenase (pLDH) -

Produced by asexual and sexual stages of all 4 human

malaria parasites

pLDH is remove from plasma within 4 days after initiation

of the antimalarial treatment

Monoclonal antibodies have been developed that can target

a conserved element of pLDH on all human malaria species
(panmalarial) or
specific regions unique to P. falciparum or P. vivax.

They can distinguish Pf from non Pf species but can not

distinguish between Pv, Pm and Po
 3) Plasmodium aldolase
Plasmodiumaldolaseis an enzyme of the parasite
glycolytic pathway expressed by the blood stages ofall
four malaria parasites.

Reaction against monoclonal antibodies forthis enzyme

have been used in a combined immunochromatographic test
that targets the pan malarial antigen (PMA) along with
PfHRP2.
 Commercially available RDTs
By combining detection of these 3 antigens on an
immunochromatographic strip (ICS) assay, RDTs have been
developed that can be used to detect malaria species:
P. falciparumalone,P. vivaxalone, or any combination
thereof.

HRP-2 (Plasmodium falciparumspecific)

HRP-2 (P. falciparumspecific) and aldolase (panspecific)
HRP-2 (P. falciparumspecific) andpLDH (panspecific)
pLDH (P. falciparumspecific) andpLDH (panspecific)
pLDH (P. falciparumspecific) andpLDH (P. vivaxspecific)
HRP-2 (P. falciparumspecific),pLDH (panspecific), andpLDH
(P.vivaxspecific)
Aldolase (panspecific)
Several commercial test kits are currently
available developed in different test formats
mainly like the dipstick, card, cassette

HRP-2 detection based RDTs

Immunochromatographic Test

ParaSight-F tests
Anti Malaria Campaign (AMC) Sri Lanka currently
using
Malaria Combo Test Kit

P. falciparum and / or
P. vivax, P. malariae, P. ovale

HRP-2 (P. falciparumspecific) and aldolase

(panspecific)

Results with in 20 minutes

Negative should be confirmed

after 30 min.
 Detection of Malaria Antibodies
Antibodies to malaria can be detected using
enzymatic immunoassays - ELISA
or
immunofluorescence techniques

Antibodies to the asexual blood stages appear days to weeks

after the infection and may persist for months

Can useful in survey work and screening of blood donors

But are little value to confirm "acute" malaria

Cross reactions are high- false positives ++++

 Florescence Microscope Technique

Malaria parasites

88
 Anti-body detection by ELISA
Expensive not freely available in endemic counties

Need highly skilled labour

expensive equipments necessary
eg: ELISA reader
ELISA plates
Reagents

89
 Anti-body detection by ELISA

Coloured product
Substrate

Enzyme labeled
Anti globulin
(Anti human Ab)
Non-Specific Ab Specific Antibody
Human serum

Well Coated Ag
(MALARIA)90
 Molecular Diagnostics methods - PCR

Detection of parasite genetic materials

The major advantages

the ability to detect malaria parasites in patients with
low levels of parasitaemia and
identify them to the species level.
high specificity and sensitivity
(infection with five parasites or less per l can be
detected with 100% sensitivity and equal specificity)