able of ContentsIntroductionObjectives of Giemsa stainPrincipleReagents UsedProcedureStaining procedure 1: Thin Film stainingStaining Procedure 2: Thick Film StainingResultsInterpretation/ConclusionApplications Giemsa stainAdvantagesLimitationsReferencesFour Charged in Plot to Kidnap an Iranian Journalist in New YorkIntroductionGiemsa stain was a name adopted from a Germany Chemist scientist, for his application of a combination of reagents in demonstrating the presence of parasites in malaria.It belongs to a group of stains known as Romanowsky stains. These are neutral stains made up of a mixture of oxidized methylene blue, azure, and Eosin Y and they performed on an air-dried slide that is post-fixed with methanol. Romanowsky stains are applied in the differentiation of cells, pathological examinations of samples like blood and bone marrow films and demonstration of parasites e.g malaria. There are four types of Romanoswsky stains:Giemsa stainJenner StainWright stainMay-Grunwald StainLeishman stainObjectives of Giemsa stainTo accurately prepare the Giemsa stain stock solutionTo stain and identify blood cellsTo differentiate blood cells nuclei from the cytoplasmPrincipleGiemsa stain is a gold standard staining technique that is used for both thin and thick smears to examine blood for malaria parasites, a routine check-up for other blood parasites and to morphologically differentiate the nuclear and cytoplasm of Erythrocytes, leucocytes and Platelets and parasites.Like any type of Romanowsky stains, it composed of both the Acidic and Basic dyes, in relation to affinities of acidity and basicity for blood cells. Azure and methylene blue, a basic dye binds to the acid nucleus producing blue-purple color. Eosin is an acidic dye that is attracted to the cytoplasm and cytoplasmic granules which are alkaline-producing red coloration. The stain must be buffered with water to pH 6.8 or 7.2, to precipitate the dyes to bind simple materials.Classically, Giemsa stain is a differential stain which is made up of a combination of reagents (Azure, Methylene blue, and Eosin dye) used widely in cytogenetics and histopathology for the diagnosis of:Malaria, spirochetes and other blood parasitesChlamydia trachomatis inclusion bodiesBorrelia sppYersinia pestisHistoplasma sppPneumocystis jiroveci cystsReagents UsedMethanolGiemsa powderGlycerinWater (Buffer)ProcedurePreparation of the Giemsa Stain Stock solution (500ml)Into 250ml of methanol, add 3.8g of Giemsa powder and dissolve.Heat the solution up to ~60oCThen, add 250ml of glycerin to the solution, slowly.Filter the solution and leave it to stand for about 1-2 months before use.Preparation of Working solutionAdd 10ml of stock solution to 80ml of distilled water and 10ml of methanolStaining procedure 1: Thin Film stainingOn a clean dry microscopic glass slide, make a thin film of the specimen (blood) and leave to air dry.dip the smear (2-3 dips) into pure methanol for fixation of the

1. HCM 124: MEDICAL PARASITOLOGY AND
ENTOMOLOGY
JACKSON C. KORIR (PhD)
0721- 413 606
jcheruiyot@mmust.ac.ke
Masinde Muliro University
of Science and Technology
(MMUST)
University of Choice

2. MALARIA

3. Phylum Apicomplexa: Malaria
Taxonomy
Phylum: Apicomplexa
Class: Coccidia
Order: Haemosporida
Genus: Plasmodium

4. Background
 Malaria burden: -
 3 billion people at risk in 109 malaria prone countries
 250 million cases annually
 approximately 1 million deaths
 5 species of Plasmodium cause human malaria:
 P.falciparum,
 P.vivax,
 P.ovale
 P.malariae
 P. Knowlesi?
 Vector:- ♀ Anopheles mosquitoes (Anopheles gambie complex)
 Life cycle is complex, involving developmental stages in both
human and mosquito hosts

5. Life Cycle Overview
• The life cycle of Plasmodium that
infect humans includes 2 hosts:
•1) the human host and 2) the insect
vector, a female mosquito belonging to
the genus Anopheles
•Like other apicomlexa, a significant feature of the life cycle is the
alternation of sexual and asexual phases in the 2 hosts
• The asexual cycles, termed merogony, occur in the human
• The sexual cycle, termed gamogony occurs mainly in the mosquito
• Subsequent to the sexual stage, another asexual phase of
reproduction occurs in the mosquito, termed sporogony
• The infective form in humans is
the slender, elongated sporozoite
Anopheles sp.
Plasmodium sp. sporozoites

6. Life cycle of Plasmodium Sp
http://malariasite.com/MALARIA/lifecycle.jpg
Figure 1

7. Life Cycle (Detail)
• During feeding, the mosquito secretes sporozoite-bearing saliva
beneath the epidermis of the human victim, thus inoculating the
sporozoites into the blood stream
• About 24-48 hr
later, sporozoites
appear in the
parenchymal cells in
the liver, initiating
the exoerythrocytic
shizogonic cycle or
pre-erthrythrocytic
cycle

8. Exoerythrocytic Shizogonic Cycle
• Inside the liver cell, the sporozoite develops into a trophozoite,
feeding on host cytoplasm with its functional micropore
• After 1-2 weeks, the nucleus of the trophozoite undergoes
multiple fission, producing thousands of merozoites
• These rupture
from the host cell,
enter the blood
circulation, and
invade RBCs,
initiating the
erythrocytic
shizogonic cycle
• Some sporozoites
become dormant
hypnozoites

9. Note:
• Studies of P. vivax show that the membrane receptor
site for the engulfment phenomenon is determined by
the type of antigen present on the surface of the RBC -
e.g., merozoite penetration requires the presence of at
least one of two Duffy antigens (Fya+ or Fy b+ )
• People that lack the Duffy antigens (almost all West
Africans and about 70% of American blacks) are resistent
to vivax malaria
• However, P. ovale and P. falciparum malarias are not
influenced by Duffy antigens, thus accounting for their
prevalence in West Africa

10. Erythrocytic Shizogonic Cycle
• Electron microscopy has confirmed that merozoites interact with the RBC
plasma membrane and actively invade the cell
• During this process, rhoptries and micronemes are believed to secrete
surface active molecules that cause the host RBC membrane to expand and
invaginate to form a parasitophorous vacuole which envelops the parasite
Erythrocyctic trophozoite
Merozoite entering erythrocyte

11. Erythrocytic Shizogonic Cycle cont.
• Once in the RBC, the merozoite assumes an early trophozoite shape
consisting of a ring of cytoplasm and a dot-like nucleus - the signet ring
stage
• These early
trophozoites feed on
host hemoglobin,
grow to the mature
trophozoite stage,
and then undergo
multiple fission as
schizonts, producing
a characterisitc
number of
merozoites in each
infected RBC

12. Erythrocytic Shizogonic Cycle cont.
• Merozoites eventually rupture
RBCs and each merozoite is
capable of infecting a new RBC
Scanning electron micrograph of
Plasmodium-infected red blood cells

13. Erythrocytic Shizogonic Cycle cont.
One of 2 fates await
these merozoites:
1. Become signet ring
trophozoites and
begin shizogony
anew
2. Differentiate into
sexual stages,
becoming male
microgametocytes or
female
macrogametocytes

14. • Once the
surrounding RBC
material is lysed, the
gametocytes are
released into the
lumen of the
stomach
• The
microgametocytes
undergo a
maturation process
known as
exflagellation
Life Cycle cont.
• The sexual phase occurs in the female Anopheles mosquito and
begins when the mosquito takes a blood meal that contains
microgametocytes and macrogametocytes

15. Exflagellation
• The nucleus undergoes 3 mitotic
divisions, producing 6-8 nuclei that
migrate to the periphery of the
gametocyte
• Accompanying the nuclear divisions
are centriolar divisions, following
which one portion joins each nuclear
segment to become a basal body,
providing the center from which the
axoneme subsequently arises

16. • During this period
the macrogametocytes
have developed into
macrogametes which
become penetrated by
the microgamete
• The fusion of male
and female pronuclei
(syngamy) produces a
diploid zygote that
elongates into a motile
wormlike ookinete
Life Cycle cont.
• The nucleus with the axoneme and a small amount of cytoplasm
form a microgamete, which detaches from the mass and swims to
the macrogametocyte
Dr. JC korir 2015

17. Life Cycle cont.
•The ookinete penetrates the gut wall of the mosquito to the area
between the epithelium and the basal lamina, where it develops into
a rounded oocyst
• Growth of the
oocyst is, in part,
due to the
proliferation of
haploid cells called
sporoblasts, within
the oocyst

18. Life Cycle cont.
• Sporoblast nuclei undergo numerous divisions, producing
thousands of sporozoites enclosed within the sporoblast membranes
• As membranes rupture, sporozoites enter the cavity of the oocyst
• The sporozoite-
filled oocysts
themselves rupture,
releasing the
sporozites in the
hemocoel
• The sporozoites are
carried to the
salivary gland ducts
of the insect and are
ready to be injected
into the next victim
when another blood
meal is taken

19. Longitudinal section of mosquito
intestine showing numerous
oocysts
Sporozoites isolated from the
salivary glands of a mosquito

20. Dr. JC korir 2015

21. Plasmodium vivax (benign tertian malaria)
• Less than 1% of the
total RBC population is
parasitized
• Predilection for
immature RBCs
(reticulocytes)
• Schuffner’s dots
usually stains pink to
red when subjected to
stains
• Hemozoin granules,
by-products of
hemoglobin
degradation by the
parasite, are prominent
•The cytoplasm of the
trophozoite stages is
very irregular and
displays an active
ameboid movement
Dr. JC korir 2015

22. P. ovale (mild tertian malaria)
•Less than 1% of the
total RBC population
is parasitized
• Predilection for
immature RBCs
(reticulocytes)
• Schuffner’s dots
usually stains pink to
red when subjected to
stains
• Hemozoin granules,
by-products of
hemoglobin
degradation by the
parasite, are
prominent
•The cytoplasm of the
trophozoite stages is
very irregular and
displays an active
ameboid movement
Dr. JC korir 2015

23. Plasmodium malariae (quartan malaria)
• Parasitizes about
0.2% of older RBCs
• Trophozoites
accumulate pink
staining Ziemann’s
dots
• Hemozoin granules
appear in the center
or periphery of the
shizont
• Trophozoite often
appear as a band
across the cell
• Mature trophozoites
resemble
macrogametocytes
• Recrudescensces as
long as 52 years after
initial infection
Dr. JC korir 2015

24. • Only ring trophozoites and
gametocytes seen in
peripheral circulation; later
stages trapped in capillaries of
muscle and visceral organs
• Plasma membranes of
infected RBCs undergo
alteration causing them to
adhere to the walls of
capillaries
• Infects RBCs of any age;
about 10% of the total RBCs
• Multiple infections of single
RBCs are common
• Gametocytes are crescent
shaped cells
• Hemozoin as well as
Maurer’s dots (precipitates
in the cytoplasm of RBCs
infected to P. falciparum),
tend to aggregate around the
nuclear region of gametocytes
Plasmodium falciparum (Malignant tertian malaria)
Dr. JC korir 2015

25. Diagnosis
Asexual stages seen in Both thin and
thick blood smears stained with:
Giemsa (preferred)
Field's
Wright's
Leishman's stain

26. RDTs
• Rapid, simple, sensitive, and specific antibody-based
diagnostic stick or card tests that detect P. falciparum–
specific, in finger-prick blood samples are now being
used widely in control programs
• RDTs are replacing microscopy in many areas because of
their simplicity and speed, but they are relatively
expensive and do not quantify parasitemia.

27. PCR
PCR tests is increasingly used for genotyping and
speciation in mixed infections

28. Pathogenesis of malaria
 Pathology and clinical manifestations almost exclusively due
to the asexual erythrocytic stage parasites.
 Plasmodium infection causes an acute febrile illness
 Notable for its periodic fever paroxysms at either 48 or 72
hour intervals depending on the species
 Severe disease forms associated with P.falciparum:
• Cerebral malaria
• metabolic acidosis
• Hypoglycemia
• Severe anemia

29. Cerebral malaria
- Characterized by impaired consciousness.
- In malaria endemic areas affects older children >5yrs
- May be due to:
 microvascular obstruction by parasites and
platelets
 Rosettes
 Microparticles
 Effects of pro-inflammatory cytokines

30. Metabolic acidosis
- Predominantly but not exclusively a lactic acidosis
- Commonly manifested as organic acidosis
- increased production and impaired metabolism of lactate and
ketoacids
Hypoglycaemia
- Glucose levels are lower than normal(<40 mg/dL)
-Usually associated with severe anemia, jaundice,
hyperparasitemia and there may be lactic acidosis
- In children it is independently associated with poor outcome
and an increased mortality

31. Severe malarial anemia
 SMA commonly occurs predominantly in children who are <3
years
 Primagravidae and secundagravidae women also at risk in
holoendemic areas
 defined as having hemoglobin (Hb) concentration of 5 g/dL
and a P. f parasitemia of 10,000 parasites/mL or more
 Development of SMA is multifactorial, involving both the
destruction of RBC’s and their decreased production.

32. Postulated
mechanisms of
malarial anaemia
Parasitology today (Mackintosh
et al. 2004)
Figure 2
Dr. JC korir 2015

33. Epidemiology
• Endemicity of human malaria is usually determined by the geographic
distribution of its anophelene mosquito; areas where the vector is not present are
free of the disease
• Local environmental factors determine which particular species of mosquito
transmits malaria in a given area; local epidemiological surveys can be used to
assay the prevalent vectors
• Precipitin tests of ingested blood from infected mosquitoes reveal whether the
vectors have zoophilic or anthrophilic feeding preferences
• Water dependency for breeding varies greatly
• The control of malaria depends on a variety of factors, such as availability of
antimalarial drugs, use of screens on houses to keep out mosquitoes, proper use of
insecticides, elimination of mosquito breeding sites, etc.

35. Relapse of Infection
• Victims of vivax or ovale malaria may suffer a relapse
• Originally, the relapse was thought only to be due to populations
of cryptozoites (pre-erythrocyte shizont) being maintained in the
exoerythrocytic cycle
•A more recent view also recognizes the existence of 2 different
populations of sporozoites
• Short prepatent sporozoites - upon entering the human host,
undergo the usual exoerythrocytic and erythrocytic phases of
development and cause malaria
• Long prepatent sporozoites or hypnozoites - remain dormant in
the hepatocytes for an indefinite period
• Some kind of physiological fluctuation activates them into
exoerythrocytic and erythrocytic cycles and a relapse occurs

36. Recrudesence
• Recurrence of malaria among victims infected by P. malariae
many years after apparent cure fostered the idea that this species
produced relapses like those produced by P. vivax and P. ovale
• But, it has been shown that the periodic increase in numbers of
parasites results from a residual population persisting at very low
levels in the blood after inadequate or incomplete treatment of
the initial infection
• The situation may persist for as long as 53 years before
something triggers a parasite population explosion with
accompanying disease manifestations -
•This phenomenon is referred to as recrudesence

37. Symptomatology and Diagnosis
• Pathology in human malaria (P. falciparum) is generally manifested in 2 basic
forms: host inflammatory reactions and anemia
• Host inflammatory reactions are initiated by the periodic rupture of infected
RBCs, which release malarial pigment such as hemozoin and parasite metabolic
wastes
• These ruptures are accompanied by fever paroxysms that are usually
synchronous except during the primary attack (correlated with the merozoites
rupturing from RBCs)
• During cell rupturing, toxins are released which in turn cause macrophage
cells to release tumor necrosis factor (TNF); it’s TNF that actually induces the
fever
• During the primary attack synchrony may not be evident, since the infection
may arise from several populations of liver merozoites at different stages of
development

38. Black Water Fever
• A condition known as black water fever often accompanies
falciprum malaria infections
• It is characterized by massive lysis of RBCs and it produces
abnormally high levels of hemoglobin in urine and blood
• Fever, vomiting with blood, and jaundice also occur
• There is between 20-50% mortality rate, usually due to renal
failure; probably due to renal anoxia
• The exact cause of this condition is uncertain
• It may be a reaction to quinine, or it may result from an
autoimmune phenomenon in which hemolytic antibodies are
produced in response to chemotherapy

39. Chemotherapy
• Malaria control requires effective treatment of the disease in
humans and continuous efforts to control mosquito populations
• The first known antimalarial drug was quinine
• The drug primarily destroys the schizogonic stages of malaria
•Since WWII several synthetic drugs have been used: chloroquine,
amodiaquin, and primaquine
• Chloroquine is a weak base and it increases the pH of the food
vacuole which in turn prevents the digestion of hemoglobin
•Pyrimethamine used in combination with sulfadoxine have been
effective in inhibiting the folic acid cycle of malarial parasites
•Current first line therapy is a combination of Artemesinin and
lumefantrin

40. Immunity
• In addition to chemotherapy research, development of a protective vaccine
against malaria is being pursued
• Interestingly, the surface coat of the sporozoite acts as a renewable “decoy” to
the vertebrate host’s immune system, stimulating the production of antibodies
• When the sporozoite is attacked and its “decoy” coat sloughs off, a replacement
coat is synthesized and its “decoy” effect continues
• This system provides ideal protection for the sporozoite which only spends a
brief amount of time in the blood before it penetrates a liver cell as is protected
from circulating antibodies
• In endemic areas, premunition is the basis for protective immunity as long as
low-level infection persists; however, with complete cure, the victim regains
susceptibility
• Also, while nursing infants in endemic areas are protected through antibodies in
their mother’s milk, they are at risk at the time of weaning
• Also, P. falciparum can cross the placenta and cause infection on the fetus

41. Genetics and Malaria Infections
Several genetic conditions are known to affect the malarial
organism:
• Susceptibility is conferred by the presence of Duffy antigens
e.g., vivax merozoite penetration of RBCs requires 1 of 2 Duffy
antigens
•Genetic deficiency in G6PDH activity in RBCs (favism) creates and
inhospitable environment for the parasites
• Humans heterozygous for sickle cell anemia possess a selective
advantage over individuals with normal hemoglobin in regions
where P. falciparum is endemic