Blood and Tissue Protoza

Plasmodium
Prof Shahina Yasmin
 Plasmodium- LOs

• Enlist the medically important four species of

Plasmodium and diseases caused by each
• • Compare the life cycles of the Plasmodium
species
• • Compare the pathogeneses of the diseases
caused by different Plasmodium species
• • Describe the lab diagnosis of malaria
 Classification of Protozoa- Depending on Motility
Flagellates (Super class – • Sporozoa (Sub phylum –
Mastigophora) Sporozoa)(Restricted Motility)
(moving by flagella) • Blood/Tissue – Plasmodium
• Intestinal – Giardia lamblia • Tissue – Toxoplasma
• Genitourinary • Intestinal – Cryprosporidium,
• Trichomonas vaginalis Isospora
• Tissue – Leishmania, Ciliates (Sub phylum – Ciliophora)
Trypanosoma (Move by Cilia)
Amoebae (Super Class – Sarcodina) • Intestinal & Tissue –
(moving by Pseudopodia) Balantidium coli
• Intestinal – Entamoeba
• Free living/Tissue – Naeglaria,
Acanthamoeba
 Plasmodium
• Sporozoa (Sub phylum – Sporozoa) (Restricted Motility)
– Blood/Tissue – Plasmodium
– Tissue – Toxoplasma
– Intestinal – Cryprosporidium, Isospora
• Species causing Malaria - Malaria (Mal– bad; aria – air)
– Plasmodium falciparum (tertian)
– Plasmodium vivax (tertian)
– Plasmodium ovale (tertian)
– Plasmodium malariae (quartian)
 Life Cycle
Sexual & asexual life cycles
Two phases in life cycle:
• Sporogony
– Sexual cycle
– Sporozoites are produced
– Occurs in mosquitoes
• Schizogony
– Asexual cycle
– Schizonts are produced
– Occurs in humans
• Vector: Female Anopheles mosquito and definitive host for
plasmodia
• Intermediate hosts: Humans
Sexual life Cycle - In Mosquito [7-21 days]
• Female Anopheles mosquito feeds on an infected person.
Male malaria parasites (microgametocytes) sucked into
mosquito’s stomach, produce four to eight flagellated
ganetocytes, each separates from parent body. when it
finds female malaria parasite (macrogametocyte), it
enters and fertilizes it.
• A zygote is formed, which squeezes between the cells of
stomach wall, settles under the outer lining and encysts.
• Parasites multiply in oocyst until the oocyst contains many
thousands of new parasites.
• Eventually, oocyst ruptures and releases the spindle-
shaped sporozoites, which make their way to mosquito’s
salivary glands.
 Asexual life Cycle (in Man)
Exo-Erythrocytic
• Mosquito bites man, sporozoites are introduced.
• Sporozoites move to liver and infect liver cells, single
parasite divides and generates many thousands of new
parasites over 7–21 days.
• The enlarged liver cell (liver schizont), finally bursts,
releases thousands of merozoites into bloodstream, each
one enter a new red blood cell.
• Parasite starts to grow in the cell, using the contents of
the cell as food, and becomes a trophozoite.(P. falciparum
and P. malariae).
• P. vivax and P. ovale, when originally enter liver cells do
not immediately become liver schizonts but remain
dormant as hypnozoites, responsible for the relapses
 Asexual life Cycle (In Man)
Erythrocytic
• Released merozoites enter RBCs and developed to
trophozoites, Immature Trophozoites (Ring Form)
• Mature Trophozoites either converted to gametocytes or
multiply within the RBC and become Schizont (Red cell
Schizont)
• Schizont rupture and releases merozoites to enter in new
RBCs
• Gametocytes wait to be ingested by the Mosquitoe to
complete the cycle
Schizogenic periodicity and fever patterns
• Schizogenic periodicity is length of asexual
erythrocytic phase
– 48 hours in P.f., P.v., and P.o. (tertian)
– 72 hours in P.m. (quartian)
• Initially may not see characteristic fever pattern if
schizogeny is not synchronous
• With synchrony, periods of fever or febrile
paroxsyms assume a more definite 3 (tertian)- or
4 (quartian) day pattern
 Immunity
• Influenced by
– Genetics, Age, Health condition, Pregnancy status
– Intensity of transmission in region,
– Length of exposure, Maintenance of exposure
• Innate
– Red cell polymorphisms associated with some
protection. Hemoglobin S sickle cell trait or disease,
Hemoglobin C and hemoglobin E, Thalessemia – α and
β, Glucose – 6 – phosphate dehydrogenase deficiency
(G6PD)
• Red cell membrane changes: Absence of certain
Duffy coat antigens improves resistance to P.v.
 Immunity
Acquired
• Transferred from mother to child, 3-6 months protection.
• Increased susceptibility during early childhood
– Hyper- and holo-endemic areas: By age 5 attacks usually < frequent
and severe. Can have > parasite densities with fewer symptoms
– Meso- or hypoendemic areas: Less transmission and repeated
attacks. May acquire partial immunity and be at higher risk for
symptomatic disease as adults.
• No complete immunity: Can be parasitemic without clinical disease
• Need long period of exposure for induction. May need continued
exposure for maintenance
• Immunity can be unstable: Can wane as one spends time outside
endemic area. Can change with movement to area with different
endemicity. Decreases during pregnancy, risk improves with increasing
gravidity
• Phenomenon of Premunition: Partial immunity based on humoral
antibodies that block merozoites from invading RBCs. Low level of
 PARASITE DEVELOPMENT IN RBC
(P.falciparum)
SURFACE CHANGES IN RBC METABOLISM OF HB LYSIS OF INFECTED CELLS

Adherence of Haemazoin Formation Stroma Hb Antigen

parasitized RBC
to endothelium
Haemoglobinemia Fever

Splenomegaly

Fe Store depletion +Antibody

Auto Ab (P.falciparum)
(P.malariae)
Increased Haemoglobinuria
DIC Destruction (Black WaterFever)
Normal RBC Nephritis
Anemia
Localized decreased
microcirculation Tissue anoxia

Adrenal Bacterial Cerebral Gastrointestinal Hepatic Pulmonary Renal

Sepsis

Shock Hyperpyrexia Coma Dysentry Jaundice Oedema Anuria

 Clinical presentation
Prodromal / Early symptoms: Headache, Malaise, Fatigue,
Nausea, Muscular pains, nausea, anorexia, Slight diarrhea,
Slight fever, usually not intermittent. Can be mistaken for
influenza or gastrointestinal infection
• Acute febrile illness, may have periodic febrile paroxysms
every 48 – 72 hours, rigors, 40-41o C, Lasts 8-12 hours,
start between midnight and midday. Days 1 and 3 for P.v.,
P.o., (and P.f.) tertian. persistent fever or daily paroxyms
for P.f.. Days 1 and 4 for P.m. - quartian
• Sweating stage, afebrile asymptomatic intervals
• Tendency to recrudesce or relapse over months to years
• Anemia, thrombocytopenia, hepatosplenomegaly,
jaundice, respiratory distress syndrome, renal dysfunction,
hypoglycemia, mental status changes.
 Clinical presentation
Varies in severity and course
• Parasite factors
– Species and strain of parasite
– Geographic origin of parasite
– Size of inoculum of parasite
• Host factors
– Age, Immune status
– General health condition and nutritional status
– Chemoprophylaxis or chemotherapy use
• Mode of transmission
– Mosquito
– Bloodborne, no hepatic phase (transplacental,
needlestick, transfusion, organ donation/transplant)
 Complications of Malaria
• Fever& hyperpyrexia: Cytokines, Ischaemia of heat
regulating centre.
• Cerebral Malaria: Headache, Drowsiness, Confusion,
Coma, Cerebral Ataxia, No edema
• Anaemia: Heavy Parasitic Load, Haemolytic, Autoimmune
– type II hypersensitivity, Hypersplenism, DIC
• Renal disease: Nephrotic Syndrome – Immune complex
disease, Acute Renal Failure – Tubular necrosis
• Black water fever: Haemoglobinuria due to increased
RBC destruction, Splenomegally, Autoimmune
• Algid Malaria: Vascular collapse and shock, gram
negative septicemia, Pulmonary oedema, massive GIT
bleeding, Splenic rupture, Uncorrected dehydration
 Complications of Malaria
• Pulmonary oedema: Parentral fluid therapy, Cardiac
decompensation due to DIC or anoxia
• Tropical Splenomegaly: Enlarged Spleen, Hypersplenism,
Anaemia, Increased Immunoglobin (Antibodies to
P.falciparum), Reduction in T-Lymphocytes
• Hyperparasitaemia: 10-20%, High Mortality
• Hypoglycemia: Hyperinsulinaemia due to Antimalarials,
Impaired Hepatogluconeogenesis
• Suppression of cell mediated immune responses: EB
virus under influence of Chronic falciparum malaria leads
to Burkitt’s lymphoma
 Congenital malaria
• Transplacental infection
– Can be all 4 species
– Commonly P.v. and P.f. in endemic areas
– P.m. infections in nonendemic areas due to long
persistence of species
• Neonate can be diagnosed with parasitemia within 7
days of birth or longer if no other risk factors for malaria
(mosquito exposure, blood transfusion)
• Fever, irritability, feeding problems, anemia,
hepatosplenomegaly, and jaundice
• Be mindful of this problem even if mother has not been
in malarious area for years before delivery
 Types of Infections
• Recrudescence
– exacerbation of persistent undetectable parasitemia,
due to survival of erythrocytic forms, no exo-
erythrocytic cycle (P.f., P.m.)
• Relapse
– Reactivation of hypnozoites forms of parasite in liver,
separate from previous infection with same species
(P.v. and P.o.)
• Recurrence or reinfection
– exo-erythrocytic forms infect erythrocytes, separate
from previous infection (all species)
• Can not always differentiate recrudescence from re-
infection
 Lab Diagnosis

• Blood peripheral smear

• Bone marrow smear
• Immunochromatographic (ICT Malaria)
• Immunofluoresence
• Blood PCR
• Identification/diagnosis of complications
 Preparation of blood films
• Thick films
– Place a drop of blood in the middle
of a clean microscope slide
– With the corner of a second slide
spread the drop until it is about 10-
15mm in diameter.
– The thickness should be such that it
is just possible to see news print
through it.
• Thin films
– These are made in standard manner.
– Allow the films to dry, do not leave
on the bench in a laboratory which is
not fly proofed otherwise the film
will be eaten.
 P. falciparum
Red Cells not enlarged.
Trophozoites Rings Asexual growing form. having
chromatin dot and thin blue ring of cytoplasm.
Fine, delicate, may be several in one cell. Some
rings may have two chromatin dots. unusual to
see developing forms in peripheral blood films.
Presence of marginal or appliqué forms.
Red mauve staining Maurer's dots may be seen in
cell containing trophozoites
Gametocytes have crescent (banana) shape. do
not usually appear in blood for first four weeks of
infection. Oval forms may be seen
Schizont: have merozoites and dark colored
pigment. Rarely seen. Indicate severe infection
1. Red cells containing
parasites are usually
enlarged.
2. Schuffner's dots are
frequently present in the
red cells as shown
above.
3. The mature ring forms
tend to be large and
coarse.
4. Developing forms are
frequently present.
Trophozoites: thick compact
densely staining. Yellow
brown pigment may be seen
in late stages. Ring forms
may have a squarish
appearance. Characteristic
Band forms may be seen.
Mature schizonts may have a
typical daisy head
appearance with up to ten
merozoites.
Red cells are not enlarged.
Chromatin dot may be on the
inner surface of the ring.
1. Red cells enlarged.
2. Comet forms common
3. Rings large and coarse.
4. Schuffner's dots, when
present, may be
prominent.
5. Trophozoites compact,
small, little pigment.
6. Mature schizonts
similar to those of P.
malariae but larger and
more coarse
 Infected RBCs

Size Shape Schüffner’s

Dots

<N, N:PM Crescent: PF PV,PO

N: PF (gametocytes)
>N: PO Ameboid: PV
> >N: PV Fimbriation:PO
Elongated:PM
 Parasites Found In Circulating Blood

Rings Trophozoites Schizonts Gametocytes

(mature)
Accessory Compact: 6-12 nuclei:PM Crescent: PF
chromatin PO, PM 6-14 nuclei:PO Round: PV
dots: PF PF (rarely seen) 12-24: PV PO
Delicate: PF Band form:PM 8-24: PF PM
(rarely seen)
Rosettes: PM
 Techniques for Parasite Density
• Percentage of Red Cells infected (For prognosis)
– 10-20% infected – severe
– 20-30% infected – very severe
– >30% infected – grave
• Number of parasite present in l of blood
• (Calculation with counting of WBC)
• Parasite density in (+) sign (thick film)
1-10/100 fields +
11-100/100 fields ++
1-10/every field +++
>10/every field ++++
 Malarial Index
• Number of infected Red cells in relation to 100
WBCs
• Exact number of parasites per l of blood
calculated as follow:
TLC x Parasites in relation to 100 WBCs
100
 In a blood smear, diagnostic stages of falciparum are
cigar shaped gametocytes (top row) and the small ring
(Trophozoite) stages (bottom row). No other stages are
seen.
 Plasmodium vivax Plasmodium
Plasmodium vivax
trophozoites on a thin vivax double
gametocyte
film. the red blood cells ring on a thin
(female) on a thin
are enlarged and contain film. The red
film. the red cell
Schuffner's dots and the blood cell is
being enlarged
growing Trophozoite enlarged with
with Schuffner's
appears motile with a Schuffner's dots.
dots.
spreading appearance.
 Plasmodium malariae
Plasmodium malariae schizont
trophozoite on a thin film.
on thin film. It has 8
Trophozoite is basket
merozoites in a rosette shape.
shaped. Red blood cells
red blood cells are normal size
small and no Schuffner's
and no Schuffner's dots.
dots.
 Biological Resistance Against Malaria
1. Hemoglobin ‘S’ Gene (Heterozygous)
a. Affected RBC Sickle morequickly due to lower pH
b. The parasite is phagocytosed and destroyed.
c. (In homozygous phagocytic action is inadequate)
2. Thalassemia Gene (Beta Thalassemia Trait)
a. Affected RBCs membrane show increased sensitivity to Peroxidase
damage
3. Ovalucytosis
a. Morphological abnormal,
b. Changes in surface antigenic composition
4. G6PD Deficiency Gene (SEX LINKED)
a. Heterozygous have double population of normal and deficient RBCs
b. G6PD Deficient invites more parasites than normal. This genetic
mosaicism interfrere with adaptation of Parasite.
5. Duffy Negative
a. Glycophorin receptors which P.vivax needs to attach and invade RBCs
are missing
 Drug Resistance
• R- I: Following treatment, parasitaemia clears but a
recrudescence occurs
• R- II: Following treatment, there is a reduction but not a
clearance of parasitaemia
• R- III: Following treatment, there is no reduction in
parasitaemia
 Prevention
• Communal measures are directed against reducing mosquito
population. Drainage of stagnant water in swamps, ditches
reduces breeding areas. Many insecticide sprays are ineffective
due to development of resistant strains
• Protection is particularly important during the night
• Use of mosquito netting, Window screens,
• Protective clothing, Insect repellents
• Chemoprophylaxis for travelers going to endemic areas for
Chloroquine resistant P falciparum by Mefloquine. Chloroquine
used in areas where P falciparum is sensitive. In areas where
other plasmodia are found, should take Chloroquine starting 2
wks before arrival and continuing for 6 wks after departure.
Should be followed by 2 week course of primaquine if exposure
was high
Rabbi zidni ilmaa