Life cycle Plasmodium

The malaria parasite life cycle involves two hosts. During a blood meal, a malariainfected female Anopheles mosquito inoculates sporozoites into the human host.
Sporozoites infect liver cells and mature into schizonts, which rupture and release
merozoites . (Of note, in P. vivax and P. ovale a dormant stage [hypnozoites] can persist
in the liver and cause relapses by invading the bloodstream weeks, or even years
later.) After this initial replication in the liver (exo-erythrocytic schizogony ), the
parasites undergo asexual multiplication in the erythrocytes (erythrocytic schizogony ).
Merozoites infect red blood cells. The ring stage trophozoites mature into schizonts,
which rupture releasing merozoites . Some parasites differentiate into sexual
erythrocytic stages (gametocytes). Blood stage parasites are responsible for the clinical
manifestations of the disease. The gametocytes, male (microgametocytes) and female
(macrogametocytes), are ingested by an Anopheles mosquito during a blood meal. The
parasites multiplication in the mosquito is known as the sporogonic cycle. While in the
mosquito's stomach, the microgametes penetrate the macrogametes generating
zygotes. The zygotes in turn become motile and elongated (ookinetes) which invade
the midgut wall of the mosquito where they develop into oocysts. The oocysts grow,
rupture, and release 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.

1. Discuss the pathogenesis, pathology, clinical

laboratory diagnosis of the malarial parasites.

manifestations,

and

a. PATHOGENESIS
When the parasite develops in the erythrocyte, numerous known and unknown
waste substances such as hemozoin pigment and other toxic factors accumulate in
the infected red blood cell. These are dumped into the bloodstream when the
infected cells lyse and release invasive merozoites. The hemozoin and other toxic
factors such as glucose phosphate isomerase (GPI) stimulate macrophages and
other cells to produce cytokines and other soluble factors which act to produce
fever and rigors and probably influence other severe pathophysiology associated
with malaria.
Plasmodium falciparum-infected erythrocytes, particularly those with mature
trophozoites, adhere to the vascular endothelium of venular blood vessel walls and
do not freely circulate in the blood. When this sequestration of infected erythrocytes
occurs in the vessels of the brain it is believed to be a factor in causing the severe
disease syndrome known as cerebral malaria, which is associated with high
mortality.
Malaria Relapses
In P. vivax and P. ovale infections, patients having recovered from the first episode
of illness may suffer several additional attacks ("relapses") after months or even
years without symptoms. Relapses occur because P. vivax and P. ovale have
dormant liver stage parasites ("hypnozoites") that may reactivate.
b. CLINICAL MANIFESTATIONS
All the clinical symptoms associated with malaria are caused by the asexual
erythrocytic or blood stage parasites.
Uncomplicated Malaria
The classical (but rarely observed) malaria attack lasts 6-10 hours. It consists of
a cold stage (sensation of cold, shivering)
a hot stage (fever, headaches, vomiting; seizures in young children)
and finally a sweating stage (sweats, return to normal temperature, tiredness).
Classically (but infrequently observed) the attacks occur every second day with the
"tertian" parasites (P. falciparum, P. vivax, and P. ovale) and every third day with the
"quartan" parasite (P. malariae).
More commonly, the patient presents with a combination of the following
symptoms:
Fever
Nausea and vomiting
Chills
Body aches
Sweats
General malaise
Headaches

In countries where cases of malaria are infrequent, these symptoms may be

attributed to influenza, a cold, or other common infections, especially if
malaria is not suspected. Conversely, in countries where malaria is frequent,
residents often recognize the symptoms as malaria and treat themselves
without seeking diagnostic confirmation ("presumptive treatment").
Physical Findings may include:
Elevated temperatures
Perspiration
Weakness
Enlarged spleen

Mild jaundice
Enlargement of the liver
Increased respiratory rate

i.

Severe Malaria

Severe malaria occurs when infections are complicated by serious

organ failures or abnormalities in the patient's blood or metabolism. The
manifestations of severe malaria include:
Cerebral malaria, with abnormal behavior, impairment of consciousness,
seizures, coma, or other neurologic abnormalities
Severe anemia due to hemolysis (destruction of the red blood cells)
Hemoglobinuria (hemoglobin in the urine) due to hemolysis
Acute respiratory distress syndrome (ARDS), an inflammatory reaction in
the lungs that inhibits oxygen exchange, which may occur even after the
parasite counts have decreased in response to treatment
Abnormalities in blood coagulation
Low blood pressure caused by cardiovascular collapse
Acute kidney failure
Hyperparasitemia, where more than 5% of the red blood cells are infected
by malaria parasites
Metabolic acidosis (excessive acidity in the blood and tissue fluids), often
in association with hypoglycemia
Hypoglycemia (low blood glucose). Hypoglycemia may also occur in
pregnant women with uncomplicated malaria, or after treatment with
quinine.

c. DIAGNOSIS
Microscopic examination remains the "gold standard" for laboratory
confirmation of malaria
A blood specimen collected from the patient is spread as a thick or thin
blood smear, stained with a Romanovsky stain (most often Giemsa),
and examined with a 100X oil immersion objective. Visual criteria are used
to detect malaria parasites and to differentiate (when possible) the
various species,
Wrights stain can be used if Giemsa stain is not available. However,
species determination might be more difficult.

ii.

Rapid Diagnostic Test (RDT) is an alternate way of quickly establishing

the diagnosis of malaria infection by detecting specific malaria antigens in
a person's blood

A blood specimen collected from the patient is applied to the

sample pad on the test card along with certain reagents. After 15 minutes,
the presence of specific bands in the test card window indicate whether
the patient is infected with Plasmodium falciparum or one of the other 3
species of human malaria. It is recommended that the laboratory maintain
a supply of blood containing P. falciparum for use as a positive control.

iii.

Indirect Fluorescent Antibody Test

Blood stage Plasmodium species schizonts (meronts) are used as
antigen. The patient's serum is exposed to the organisms; homologous

antibody, if present, attaches to the antigen, forming an antigen-antibody

(Ag-Ab) complex. Fluorescein-labeled anti-human antibody is then added,
which attaches to the patient's malaria-specific antibodies. When the slide
is examined with a fluorescence microscope, if parasites fluoresce an
apple green color, a positive reaction has occurred.

iv.

Simian Malaria Species Confirmation Service (SMSCS)

In addition to the four classic human species of malaria, there are more
than 20 species of malaria parasites that naturally infect non-human
primates.
Molecular techniques such as polymerase chain reaction (PCR) and
microsatellite testing can assist in definitive species determination

2. What species of malarial parasites will you consider as the most

probable diagnosis of all forms found in the blood smear are ring
forms? Why so?

P. falciparum
o P. falciparum rings have delicate cytoplasm and one or two small
chromatin dots. RBCs that are infected are not enlarged; multiple
infection of RBCs is more common in P. falciparum than in other
species. Occasional appliqu forms (rings appearing on the
periphery of the RBC) can be present.
o It affects all ages of RBC
o It causes severe malaria because it multiplies rapidly in the blood
thus causing severe blood loss. It can also clog small blood
vessels when it occurs in the brain, cerebral malaria occurs and it
is fatal.

3. What is the old method of sampling for malarial smear preparation?

What is the modified method of sampling and the reason for its
modification?

The old method of sampling was the Classical thick-smear method.

Modifications of this method brought about the new Fast thick-smear method.

Inexpensive modifications of sampling for malarial smear preparation

from the old method of Classical thick-smear method involve rapid drying, an
isotonic fixative and a haemolysing solution containing saponin. The drying,
haemolysing, fixing, and staining steps, together called the fast-thick-smear
method (FTS), can be completed in less than 10 minutes.

Results from researches indicated that there was no statistically

significant differences between the two methods in terms of their sensitivity,
specificity or predictive values for parasite detection. However, estimates of
the intensities of the Plasmodium falciparum infections observed, based on
counts of trophozoites against 200 leucocytes, were markedly higher with the
FTS than with the CTS.


The main advantages of the new FTS method allows a greater volume
of blood to be examined, it is not time-consuming, and it improves the
appearance of parasites for easier detection.

4. Name the other modes of transmission besides mosquito bites.

Other modes of transmission of malarial parasites aside from mosquito

bites can be any of the following:
Transfusion of blood from infected persons
Use of contaminated needles and syringes
Congenital transmission
Organ transplant

5. Tabulate the advantages and disadvantages between different

diagnostic tests for malaria

Test

Advantages

DIRECT FECAL SMEAR

1. Thick smear
Rapid detection
Examine large amount
of blood increase
sensitivity
Requires examination
of lesser oil immersion
fields (100 OIF)
2. Thin smear
Used for species
identification
Examine intact
plasmodium species
stages increased
specificity
3. QBC
Uses fluorochromes
Microhematocrit
(Acridine Orange,
Centrifugation
Benzothiocarboxypurin
Method
e) to stain Nucleic Acid
of plasmodium species
increased sensitivity
o Acridine Orange
DNA - Bright
apple greenyellow color
RNA Yellow or
Orange in color
Rapid detection
IMMUNODIAG Rapid diagnostic test
for malaria detection
NOSIS

Disadvantage
s

Not specific Failure to

identify the stage and
species of plasmodium
RBCs are lysed leading
to remnant stages of
plasmodium species
Requires examination
of larger oil immersion
fields (200-300 OIF)
Decreased sensitivity
depending on parasitic
load on the blood
Expensive
Not exclusive for
malarial parasites
Non-specific Not
used for identification

Expensive
Not widely available

of presence of antigen
& antibody for malarial
identification
Malarial Antigens
4. HRP-2
Specific for
(Histidine-Rich
Plasmodium
falciparum
Protein 2)
5. PLDH
Increased sensitivity
(Parasite Lactate
due to antigen being
produced by all
Dehydrogenase)
plasmodium species
6. Plasmodium
Increased sensitivity
aldolase
due to antigen being
(Pan Specific
produced by all
plasmodium species
Malarial Antigen)
a. Parasight F Test Detects presence of
HRP-2 specific for
Plasmodium
falciparum
b. optimal test
Detects HRP-2 & pLDH
Detects plasmodium
falciparum and
Plasmodium vivax
o (+): 2 lines P.
vivax; 3 lines P.
falciparum
c. ICT malaria
Detects HRP-2 and
P.f/P.v
Plasmodium aldolase
o (+) 3 Lines
1st line: Control
2nd line: Detects
HRP-2 (P.f)
Detects
Plasmodium
aldolase
MOLECULAR DIAGNOSIS
7. PCR
Specific detects DNA
by gene amplification

Diagnosis based only

by asexual stage

Non-specific Produced by all

malarial parasite

Does not detect other

malarial parasite

Does not distinguish

mix infection with
other species

Does not distinguish

mix infection with
other species

Expensive
Last line of diagnostic
test to be used

6. Differentiate morphologically Plasmodium falciparum from

Plasmodium knowlesi

It is not possible to accurately identify P. knowlesi by microscopy, since the

morphological features of the early trophozoites of P. knowlesi are identical to
those of P. falciparum, with double-chromatin dots, multiple infections per

erythrocyte, and no enlargement of infected erythrocytes. The rest of the

blood stages of P. knowlesi resemble those of P. malariae, including bandform trophozoites
o Careful examination of well-stained slides shows minor differences in
morphology between P. knowlesi and P. malariae, such as a certain
proportion of early trophozoites of P. knowlesi with double-chromatin
dots and schizonts of P. knowlesi having up to 16 merozoites,
compared to 6 to 12 for P. malariae

Reference/s:
http://www.cdc.gov/dpdx/diagnosticprocedures/blood/specimenproc.html
Thellier, M., et al.. (2002). Diagnosis of Malaria Using Thick Smears. ATMP.
96:2, 115-124
https://www.cdc.gov/malaria/about/biology/parasites.html