What is Plasmodium falciparum?

 Protozoa
 Malaria

http://www.traveldoctor.info/diseases/1.html
Taxonomy

Phylum Apicomplexa

Subphylum Sporozoa

Genus Plasmodium

Disease Malaria

Geographical Tropical &

distribution Subtropical countries
Structure
Malaria parasite (plasmodium)
 Pathogen of malaria
 P.vivax ; P.falciparum ;P.malariae ;
P.ovale
 P.vivax ; P.falciparum are more
common
 Plasmodium is a wide distribution
in many tropical or subtropical
regions of the world
 Morphology
Wright’s stain---reddish nuclei; bluish
cytoplasma and yellowish brown
malarial pigment
1. Morphological features of P. vivax
 Early trophozoite (ring form)
1 red nucleus on the ring-like light
blue cytoplasm ; single
infection in a cell.
infected RBC like
normal RBCs.
Late trophozoite
It is irregular shape like ameboid form
with pseudopodia; within
cytoplasm ,brown pigment granules
(malarial pigment---haemozoin) appear.
infected RBCs are
pale in color,and have
schuffner’s dots in it
(fine red granules) .
Immature schizont
oval in shape , nucleus
divided into 2-4 or more ,
malarial pigment begins to
concentrate in a mass.
Mature schizont
nucleus divided into
12-24 ;and cytoplasm also
divided , each nucleus
surrounded by a portion of cytoplasm to
form merozoites, malarial pigment clumped.
Male gametocyte
oval in shape; 1 loose
nucleus in centre of it ;
malarial pigments diffuse .

Female gametocyte
oval in shape ; 1 compact
nucleus not in centre of it .
2. Morphological features of P. falciparum
Early trophozoite (ring form)
1or 2 red nuclei on the ring-like light blue
cytoplasm ; multiple infection in a cell.
infected RBC like normal RBCs.

P. falciparum:
only can the early
trophozoites and gametocytes
be seen in the peripheral blood .
Male gametocyte
Sausage in shape; 1
loose nucleus in centre of it ;
malarial pigment diffuse .
Female gametocyte
Crescentic in shape ;
1 compact nucleus in
centre of it .
 Life cycle
In mosquito (final host)
Gametocytes(♀♂) gametes (♀♂)
(blood—stomach) (stomach of insect)
union of
zygote
rupture/release rounds up into
sporozoites oocyst motile ookinete
(Salivary glands) ( the body cavity side)
 Gametocyte development
 Sexual commitment
 Sequestered in bones
 Resistance to
quinine
 Longevity

“Female” gametocyte

http://www.bepast.org/dataman.pl?c=lib&dir=docs/photos/malaria
Gametocyte Infectivity
 Temp drop
 Red blood
cells lysed
 Xanthurenic
acid and
exflagellation
http://lozere.org/perso/malaria/parasit.htm  eye color
 Gametogenisis
 Rapid morphological changes

“male” gametocyte

http://www.ksu.edu/parasitology/625tutorials/Plasmodium01.html
Ookinets
 2 divisions
 Anatomy of a mosquito midgut

http://www.bc.edu/schools/cas/biology/news/encounter/
Oocysts
 Sporozoit
budding
 Number of
sporozoits per
oocyst

http://www.biosci.ohio-state.edu/~parasite/plasmodium.html
Sporozoits
 Migration

http://www.malarlife.dfl.org.za/malaria%20virus%20photos.htm
 In human body
1. Exoerythrocytic stage
bite/inject into
sporozoites exoerythrocytic schizonts
(mosquito blood) (hepatic cell)

rupture/release
exoerythrocytic sporozoites
( blood)
 There are two forms of sporozoites:
-----tachysporozoite and bradysporozoite
They are genetically distinct at the time of
maturation when they enter the hepatic cells
at the same time. tachysporozoite grow in
the hepatic cell and multiply to form
exoerythrocytic schizonts and then invade
RBCs to clinic malaria. Bradysporozoite is
the cause of relapse of malaria.
Bradysporozoite stay in the hepatic cells
and will multiply later.
2. Erythrocytic stage

early trophozoite later trophozoite

P.f/36-48hrs
P.v/48hrs
merozoite immature schizont

Mature schizont
*the process from trphozoite to merozoite is called
schizogony.
3. Gametgenesis
----After completing a few schizogonic
cycles, some merozoites develop into sexual
cells, the male and female gametocytes.
They continue their development in the
mosquito.
Life Cycle
 Pathogenicity
 Paroxysm (attack of malaria)
mechanism
----liberation of merozoites and malarial
pigment; RBC debris into the blood stream.
symptoms (in a typical case)
----p.v. attack occurs once every other day
(48 hours); P.f./36 to 48 hrs ;P.m./72 hrs
 Transmission & Life Cycle
 Intermediate host : human
 Final host : mosquito
 Infective stage : sporozoite
 Infective way : mosquito bite skin of human
 Parasitic position : liver and red blood cells
 Transmitted stage : gametocytes
 Schizogonic cycle in red cells : 48 hrs/P.v
 Sporozoite : tachysporozite and
bradysporozite
 Phases of Development in Man
2 phases of development
1. Inside the liver (tissue phase)
– Pre- erythrocytic schizogony – no clinical symptoms, no
pathological damage
– Exo- erythrocytic schizogony – cause of relapse

2. Inside the RBCs (erythrocytic phase)

– Erythrocytic schizogony – cause of malarial paroxsyms
– Gametogony – infects mosquito
 Morphological forms seen in Humans
 In liver:
1. Sporozoites
2. Pre erythrocytic schizonts
3. Merozoites – infect RBCs

 In RBCs :
1. Trophozoites – ring form
2. Schizonts
3. Merozoites – released by the rupture of schizonts –
infect other RBCs
4. Gametocytes – micro and macro gametocytes
 Morphological forms seen in Mosquito

 Further differentiation & development of gametocytes

take place in mosquito
1. Macro gametes (female gametes) – each macro
gametocyte develops in to one macro gamete in the mid
gut of mosquito

2. Micro gametes (male gametes) – one micro gametocyte

produces 6 to 8 micro gametes by exflagellation.

3. Zygote – Ookinete – Oocyst – rupture – release of

Sporozoites – predilection to salivary glands.
 Other modes of transmission
 Sporozoite- induced- malaria : injection of an
emulsion of salivary glands of mosquito containing
sporozoites

 Trophozoite- induced- malaria : injection of blood

from a malarial patient containing the asexual forms
of erythrocytic schizogony e.g.
1. Transfusion malaria – when persons with latent
infection are used as donors
2. Congenital malaria – transmission through some
placental defects (a healthy placenta acts as a
physiological barrier)
3. Drug addicts – by using same syringe
Incubation period
 P. vivax
 P. ovale 10 to 14 days
 P. falciparum

 P. malariae 18 days to 6
weeks
 Pathogenicity
 Infection causes intermittent fever – Malaria
 Each of the 4 species causes a characteristic fever:
P. vivax Benign tertian/ vivax malaria
P. falciparum Malignant tertian/ falciparum
malaria, black water fever
P. malariae Quartan malaria
P. ovale Tertian/ Ovale malaria
Clinical Features
 Series of febrile paroxysms – fever is caused by the
release of merozoites & toxins from ruptured
erythrocytic schizont which in turn causes the
release of cytokines.
Quartan malaria – every 72 hrs
Tertian malaria - every 48 hrs

* each paroxysm has 3 stages - cold stage (rigors),

hot stage (high temp., body & joint pains, vomiting
& diarrhoea) and perspiration stage (fall in temp.)
Clinical Features
 Anaemia – due to breakdown of RBCs,
particularly occurs in falciparum malaria

 Splenomegaly – all forms

 •Splenomegaly and anemia
----Rupture of the infected RBCs and
destruction of normal RBCs enhance
phagocytosis stimulate phagocytes to
grow in number and enhance in function.
Finally, lead to anemia and enlargement of
the spleen.
Question: Which reasons are there in the
anemia of malaria?
 •Relapse
----a specific attack that it is up to months or
even years after the primary attacks.
----The bradysporozoites in the liver spend a
rest and sleeping times of months or even
years , then they start develop in
exoerythrocytic stage and erythrocytic stage.
at this time, the patient occurs paroxysm ,
showing as periodic fever like the primary
attacks, it is called relapse.
----Relapse only occurs in P.v.
 •Malignant malaria
Malaria caused by P.f. is more severe
than that caused by other plasmodia.
----The serious complication of P.f. involves
cerebral malaria (involving the brain);
massive haemoglobinuria (blackwater fever)
in which the urine becomes dark incolor,
because of acute hemolysis of RBC; acute
respiratory distress syndrome; severe
gastrointestinal symptoms; shock and renal
failure which may cause death.
 Falciparum Malaria
 Most widespread
 Accounts for 80% of malaria cases
worldwide
 Most pathogenic of human malaria species
 Untreated infections - severe disease &
even death, particularly in young children,
pregnant woman & non immune adults.
Falciparum malaria
 Severe falciparum malaria is associated
with
1. Pernicious malaria /cerebral malaria
2. Blackwater fever
3. Anaemia
4. Hypoglycaemia
5. Hypotension
6. Complications in pregnancy
Pernicious Malaria
 Def: refers to a series of phenomenon occurring
during infection with P. falciparum which, if not
effectively treated, threatens the life of the patient
with in 1 to 3 days

 In children & non immune adults, can cause coma

& death – Cerebral malaria.

 Occurs as a result of capillary blockage.

Black Water Fever
 Occurs in previously infected subjects
 Can also occur in non immune adults with
severe falciparum malaria, and also as a
complication of quinine therapy.
 A rare but acute condition characterised by
sudden & massive hemolysis of parasitised &
non parasitised RBCs followed by fever and
haemoglobinuria.
 Often fatal due to renal failure
Black Water Fever
 Difficult to find the parasites
in the blood following a
hemolytic attack.
 Urine appears dark red to
brown black due to the
presence of free Hb.
 Clinical features – fever, rigor, aching pains in the
loin, icterus, bilious vomiting, circulatory collapse,
haemoglobinuria & acute renal failure.
 Treatment – Chloroquine, blood transfusion,
peritoneal dialysis in ARF.
 Anaemia
 Can be severe & occur rapidly, particularly
in young children

 Occurs due to destruction of parasitised

RBCs – phagocytosis & destruction in the
spleen

 Decreased production of RBCs in the bone

marrow.
Falciparum malaria in Pregnancy
Can result in:
 Severe anemia
 Low birth weight babies
 Greatest risk in 1st pregnancy
 Malaria caused by P.vivax,
P.ovale & P.malariae
 Rarely life threatening
 Relapses/ recurrences are a feature

Recurrences in Malaria
 May result from – reinfection or
- due to certain events related
to the parasite’s life cycle
 Recurrence of Malaria
 Two types of recurrences known in malaria:
1. Recrudescence –
– seen in P. falciparum & P. malariae
– due to persistence of blood infection (some erythrocytic
forms evade host immunity) even after clinical illness
has subsided.
– The numbers may increase later, leading to
reappearance of clinical symptoms
– Occur mostly up to one year or so but in P. malariae, it
can occur even after decades
Recurrence of Malaria
2. Relapse
– Occurs due to a special form of parasites –
hypnozoites.

– Hypnozoites are the sporozoites that remain

dormant after infecting liver

– Activated from time to time to initiate pre

erythrocytic schizogony - Exoerythrocytic
schizogony
Genetic factors protecting
against Malaria
 Sickle cell anaemia – sickle celled RBCs are
removed by the spleen before the development of
schizonts

 Ovalocytosis – RBCs are rigid and they resist

parasitic invasion

 Duffy blood group negative individuals – duffy

blood group Ag is the receptor for the attachment
of merozoites of P.vivax
 Genetic factors protecting
against Malaria

 Newborn infants – natural protection for 1st

few months of life due to high conc. of HbF
in their RBCs.

 Beta thalassaemia – protects against severe

falciparum infection
 Laboratory Diagnosis
 Microscopy – detecting & identifying malarial
parasites in peripheral blood films.
 Concentrating parasites in venous blood by
centrifugation when they can not be found in blood
films
 Using a rapid malaria Ag or enzyme detection test
 Other tests – Hb, PCV, Blood glucose, total WBC &
platelet count.
Examination of Blood film
 Collection of blood
- best prepared directly from capillary blood
- in EDTA bulb (used within 30 mins)

 Time of collection
- as soon as possible if malaria is suspected
- before administering antimalarials
- during pyrexial phase
Types of Blood film
 Two types:
1. Thick films :
- 30 to 40 times more sensitive than thin films
- more suitable for detection of malarial
parasite when they are few in number
- blood is not fixed, RBCs are lysed during
staining (only parasitic forms will be seen)
Types of Blood film
2. Thin films :
- to confirm the Plasmodium species
- assists in the identification of mixed
infections
- blood is fixed, parasites are seen within
the RBCs
- also helps in assessing the response to
treatment especially in areas where drug
resistance is suspected (by counting the
number of parasitised RBCs before & after the
treatment)
Making of Thin & Thick films
Fixation & Staining
 Fixation – thin films are fixed with absolute
alcohol for 1 to 2 mins.

 Staining – films are stained with

Romanowsky stain: giemsa,
field’s, wright’s

 Giemsa – 10% solution for 10 mins

Reporting of Blood film
 Look for the different morphological
forms of parasite in blood smear:
1. Trophozoites / ring forms
2. Schizont
3. Gametocytes

 Identify species – differences in the

characteristics of morphological forms in
different species
 Trophozoites / Ring forms
Character P. vivax P. falciparum
Size 2.5µ (1/3rd of RBC) 1.25 to 1.5 µ
Cytoplasm Thick opposite to Uniform thickness
nucleus
Nucleus One/ ring Can have >1
Number of rings One ring/ RBC >1/ RBC
Location in RBCs Always inside RBCs Inside as well as on the
surface (accole’ forms)
Type of RBC Preferentially young All types
infected RBCs &
reticulocytes
Thin Blood Film Thick Blood Film

Ring Forms /
Trophozoites
 Schizont
Character P. vivax P. falciparum

Size of RBC Increases to Does not change

twice its size
No of 16 8 to 32
merozoites
Arrangement of Symmetric in Asymmetrical
merozoites form of rosette
Presence in Present Absent
peripheral blood
P. vivax P.falciparum
 Gametocytes (male & female)
Character P. vivax P. falciparum
Shape – Male Spherical Cresentic
Female Spherical Sausage shaped
Nucleus- M Central, diffuse Central, diffuse
F Peripheral,small Central,compact
Infected RBC Enlarged Deformed, with its
membrane
stretched.
P.vivax P. falciparum
Counting the % age of
parasitised RBCs
 On thin blood films
 When falciparum malaria parasitemia is high
 Method of counting:
1. Select an area where no of RBCs is roughly 250.
2. Count the no of parasitised RBCs in 4 such fields i.e.
approximately 1000 RBCs.
3. Divide by 10 to obtain the percentage.

*WHO – if it is >5%, then the parasitemia is

heavy & prognosis is poor.
Buffy Coat preparation
 To concentrate malarial parasite
 Centrifuge EDTA anticoagulated venous
blood in a thin bore capillary tube
 Buffy coat layer is formed between the
RBCs & the plasma.
 Break the tube & transfer buffy coat &
RBCs to a slide - make a thin smear – air
dry – fix with ethanol – stain with Giemsa.
Quantitative Buffy Coat
 Capillary tube is coated with an
anticoagulant & Acridine orange
fluorescent dye
 After centrifugation, the tube can be used
for two purpose:
1. Complete blood count
2. Identification of malarial parasite using a
fluorescence microscope.
Quantitative Buffy Coat
Rapid Diagnostic tests
 Developed to diagnose falciparum malaria rapidly &
without a microscope.
 Can also detect vivax malaria
 Three tests are available commercially
 Detects either HRP2 Ag (Histidine rich protein) or
specific pLDH (parasite lactate dehydrogenase)
 Both HRP2 & pLDH are produced by the parasites
during their growth & differentiation in RBCs.
 Rapid Diagnostic tests
HRP2 tests
 detection of P.falciparum
 Two types of test – ParaSight F
- ICT Malaria Pf

pLDH test e.g. OptiMAL test

 Detection of P.falciparum & P.vivax
 Produced by all human malarial parasites
 Differentiation of species is based on antigenic
differences between pLDH isoforms.
ParaSightF test

Optimal test

ICT Malaria Pf / Pv
 Treatment
Chlorquine and quinine----anti-
erythrocytic stage drugs. (question: Which
stage of plasmodium can these drugs kill?)
Primaquine and pyrimethamine
----anti-exoerythrocytic stage drugs.
 Prevention
Chemoprophylaxis
-----Chloroquine / pyrimethamine
used for
prophylaxis of malaria
-----Chemotherapy: 1 week before entry into
the endemic area ; for 4 weeks after
returning from the endemic area.
 Stage specificity of antimalarial drugs
Stage of malarial Antimalarial drug
parasite
Sporozoite Proguanil, Pyrimethamine

Hypnozoite Primaquine

Pre- erythrocytic Proguanil, Pyrimethamine

schizont
Blood schizont Chloroquine, Quinine, Artemisinin

Gametocyte Primaquine
 Pathogenicity

http://www.nmm.ac.uk/server/show/conMediaFile.5757/outputRegister/lowhtml
Toxins
 Resistance
 Hemozin
 TNF-alpha IL-1
and IFN-gamma

http://www.wordsources.info/words-mod-malaria.html
Tumor Necrosis factor

http://www.farm.kuleuven.ac.be/anafar/lab/protein/tnf.htm
Toxicity and pathogenicity
Proteins and cell membranes
Rosetting
 Five receptors
 Immune
system evasion?
 Antigenic Variation
 Opportunities abound

http://www.lovetoparty.co.nz/images/SURPRISE.jpg
Crossover

http://www.stanford.edu/group/Urchin/GIFS/crossover.jpg
Var Family

http://www.niaid.nih.gov/dir/labs/lmvr/mgs.htm
Research About Malaria
 PO phosphoriboprotein

http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=15322057
 Vaccines
 New treatment
 Acquired
immunity
 challenges

http://web.uct.ac.za/depts/mmi/jmoodie/vacc2.html
Prevention of Liver stage infection

http://www.med.nyu.edu/parasitology/faculty/ufrevert.html
Vaccinating mosquitoes

http://www.bigthings.ca/manitoba/pictures/1mos1.jpg
 Paratransgenesis

http://www.mbl.edu/Astrobiology/Riley/image/E.co
li.gif
http://www.invivo.fiocruz.br/celula/imagens/bacillus.jpg
Engineering mosquitoes
 3 stages
AgAper promoter
vitellogenin
promoter
salvitory promoters

http://jeb.biologists.org/cgi/content/full/206/21/3809/FIG2
 Other ways to reduce infection

http://www.wmconnolley.org.uk/bees/

http://k12education.uams.edu/scvlab/history.htm
Future challenges
 Funding
 Research
 Distribution
 Funding
Conclusion
 "I have not failed. I've just found 10,000
ways that won't work." Thomas Alva
Edison
Sources
 See notes section for a list of references
arranged by slide number.