PLASMODIUM

 belongs to the class Sporozoa and includes the species of malarial parasites that infect
humans, namely:
1. Plasmodium falciparum
2. Plasmodium vivax
3. Plasmodium malariae
4. Plasmodium ovale
5. Plasmodium knowlesi

MALARIA WORLDWIDE

 3.2 billion people live in areas at risk of malaria transmission in 106 countries and
territories
 The World Health Organization estimates that in 2016, malaria caused 216 million
clinical episodes and 445,000 deaths

MALARIA PARASITES

 first noticed in the blood of a patient suffering from malaria on November 6, 1880 by
Charles Louis Alphonse Laveran, a French army surgeon stationed in Constantine,
Algeria (he was awarded the Nobel Prize in 1907 for the discovery of the parasite)

1. Plasmodium falciparum
 discovered by William Welch in 1897
 most common in the Philippines (around 70% of cases)
 causes severe or complicated malaria and death if not treated promptly and
appropriately
*cerebral malaria – parasite alters the red cell surface, making it “sticky”, so it sticks to
the blood vessel walls, causing further anemia and blockage of capillaries in the brain

2. Plasmodium vivax
 named by Giovanni Batista Grassi and Raimondo Filetti in 1890
 around 30% of cases in the country
 very rarely causes severe disease
 relapse is common if not treated adequately; can develop dormant liver stages
(hypnozoites) that can reactivate after being asymptomatic for up to 2 years

3. Plasmodium malariae
 first identified species of Plasmodium described by Charles Louis Alphonse Laveran in
1880; Laveran had believed that there was only one species, and he originally named it
Oscillaria malariae
 less than 1% of cases in the country
 infection is usually not severe but may last up to 50 years if not treated

4. Plasmodium ovale
 very rare; last documented 2 cases in the Philippines during the 1950s
 relapse may occur if not treated adequately; can develop dormant liver stages
(hypnozoites) that can reactivate after being asymptomatic for up to 4 years

5. Plasmodium knowlesi
 first described by Robert Knowles and Biraj Gupta in 1931 in a long-tailed monkey
 simian (primate) malaria parasite commonly found in Southeast Asia
 found normally among long-tailed monkeys (reservoir host) but can be transmitted to
man by blood passage
 the first documented human infection with P. knowlesi was in 1965
 with the increasing popularity of deforestation and development efforts in Southeast
Asia, many long tailed-monkeys (macaques) are now coming in close contact with
humans; hence more people who live in the semi-urban areas are being found to be
infected with P. knowlesi
 all stages of P. knowlesi are microscopically indistinguishable from P. malariae and the
early trophozoites are identical to those of P. falciparum; differentiation can only be
made through molecular detection assays such as PCR which are employed to confirm
malaria infection and to determine definitively which species are involved

VECTOR
 the male Anopheles feeds on nectar and fruit juices
 female Anopheles feeds primarily upon blood; need blood for egg development
  most Anopheles species feed on warm-blooded animals, with some preferences for
humans or certain animal hosts
 longevity – 30 days

A. Primary Vector
Anopheles flavirostris
 breeds in slow-flowing, partly shaded clear streams
 indoor and outdoor biter
 bites both man and animals
 flight range of 1.5 to 2.0 kms
 peak biting time – 10:00 pm to 2:00 am

B. Secondary Vectors
1. Anopheles litoralis – vector in coastal areas; brackish water
2. Anopheles maculatus – semi-stagnant streams along sides of shallow rivers with mat
of algae
3. Anopheles mangyanus – slow flowing streams in wooded areas
4. Anopheles balabacensis – stagnant water in forest areas, hoofprints, animal wallows

LIFE CYCLE OF THE MALARIA PARASITE

The malaria parasite life cycle involves two hosts. During a blood meal, a malaria-
infected female Anopheles mosquito inoculates sporozoites into the human host (1).
Sporozoites infect liver cells (2) and mature into schizonts, which rupture and release
merozoites (4). (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 (A)), the parasites undergo
asexual multiplication in the erythrocytes (erythrocytic schizogony (B)). Merozoites infect red
blood cells (5). The ring stage trophozoites mature into schizonts, which rupture releasing
merozoites (5). Some parasites differentiate into sexual erythrocytic stages (gametocytes) (7).
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 (8). The parasites’ multiplication in the mosquito is
known as the sporogonic cycle (C). While in the mosquito’s stomach, the microgametes
penetrate the macrogametes generating zygotes (9). The zygotes in turn become motile and
elongated ookinetes (10) which invade the midgut wall of the mosquito where they develop into
oocyst (11). The oocysts grow, rupture, and release sporozoites (12), which make their way to
the mosquito’s salivary glands. Inoculation of the sporozoites (1) into a new human host
perpetuates the malarial life cycle.

MALARIAL PAROXYSM

 cold stage
• feeling of intense cold
• vigorous shivering
• lasts 15-60 minutes
 hot stage
• intense heat
• dry burning skin
• throbbing headache
• lasts 2-6 hours
  sweating stage
• profuse sweating
• declining temperature
• exhausted and weak → sleep
• lasts 2-4 hours

CHARACTERISTICS OF PLASMODIUM SPECIES INFECTING HUMANS

Species P. vivax P. malariae P. falciparum P. ovale P. knowlesi

Benign Benign Malignant Mild benign

Other names Simian
tertian quartan tertian tertian
Erythrocytic
48 72 48 48 24
cycle
Hypnozoite
Present None None Present None
stage
Erythrocyte
Young RBCs Old RBCs All RBCs Young RBCs All RBCs
preference
Average
50,000 –
parasitemia 20,000 6,000 9,000 600 – 10,000
500,000
(mm3)

EXAMINATION OF BLOOD SMEARS FOR MALARIA

1. Thick smear
 Layer of red blood cells 10 to 20 times thicker than in a thin film
 Parasites are within red cell ghosts (staining unfixed red cells causes hemolysis)
 Used to detect parasites and estimate parasite density
 Gives sensitivity to diagnosis – parasite detection
 If parasites are found, extend more time to examine the whole smear before confirming
identification of species; this allows for possible detection of a mixed infection
 A parasite count may be established

2. Thin smear
 Single layer of red blood cells
 Used as label to identify patient
 Used to identify parasite species, after they have been seen in the thick film
 Gives specificity to diagnosis – confirmation of species identification
 Examine the distal 1/3 of the smear where cells are likely to be evenly distributed, in a
single layer, and have minimum distortion

MORPHOLOGY OF MALARIA PARASITES

Developmental Stages Seen in Blood Films:

1. Trophozoite – this stage is the most commonly seen; often called the ring stage; growing
stage

2. Schizont – the malaria parasite starts to reproduce by asexual reproduction (because the
parasite is neither male nor female but reproduces itself by simple division

3. Gametocyte – sexual stage (the parasites become either male or female in preparation for
the next stage which takes place in the stomach of the female Anopheles mosquito; can be
rounded or banana-shaped

a. Macrogametocyte (female) – deep blue cytoplasm and clearly stained bright red
chromatin
b. Microgametocytes (male) – reddish cytoplasm with indistinct chromatin
Features of Plasmodium:

Chromatin – part of the parasite nucleus;usually round in shape and stains a deep red

Cytoplasm – occurs in a number of forms, from ring shape to irregular shape; it always stains
blue, although the shade of blue may vary between the malaria species

Pigment – by-product of the growth or metabolism of the parasite; it does not stain, but has its
own color, which may range from pale yellow to dark brown or black

Red Cell Morphology

1. Size of infected RBCs
a. enlarged – P.vivax or P. ovale
b. not enlarged – P. falciparum or P. malariae (occasionally smaller than normal)
2. Infection of RBCs
a. single – usually suggestive of P. vivax
b. multiple – common to P. falciparum
3. Presence of stipplings or clefts
a. stippling clearly visible (Schuffner’s) – P. vivax or P. ovale
b. stipplings not clearly visible – P. falciparum or P. malariae

Plasmodium falciparum (Trophozoite Stage)

Diagnostic Points:
 small, regular, fine to fleshy cytoplasm
 infected RBCs are not enlarged
 numerous, multiple infection is common
 ring, comma, marginal or accole forms are seen; often have double chromatin dots
 Maurer’s dots are not clearly visible

Plasmodium falciparum (Schizont Stage)

Diagnostic Points:
 small, rarely fill the RBC
 rare in peripheral blood (indicates heavy infection if present)
 16-32 or more merozoites in compact cluster
 single dark pigment
 usually associated with many young ring forms

Plasmodium falciparum (Gametocyte Stage)

Diagnostic Points:
 banana-shaped or rounded
 macrogametocyte (female):
o small, compact, and has a central chromatin dot
o pigments closely adhere to the chromatin
 microgametocyte (male):
o broader, shorter and more sausage shaped
o loosely scattered chromatin
o golden brown pigments scatters at the central half

Plasmodium vivax (Trophozoite Stage)

Diagnostic Points:
 infected RBCs are usually enlarged
 irregular or fragmented cytoplasm (ameboid)
 mature ring forms tend to be large and coarse
 Schuffner’s dots (stippling) are frequently visible

Plasmodium vivax (Schizont Stage)

Diagnostic Points:
 large, covering almost or the entire enlarged RBC
  few to moderate
 12-24 merozoites in irregular cluster
 yellowish-brown loose pigments

Plasmodium vivax (Gametocyte Stage)

Diagnostic Points:
 round and large
 usually compact nucleus at the periphery
 chromatin is deep red or magenta

Plasmodium malariae (Trophozoite Stage)

Diagnostic Points:
 small and few
 ring to rounded, compact, vacuolated or non-vacuolated
 band forms seen
 dark, scattered pigments
 Ziemann’s stipplings are not clearly visible
 infected red cell is not enlarged

Plasmodium malaria (Schizont Stage)

Diagnostic Points:
 small, compact, dark
 usually few
 6-12 merozoites in “rosette” formation, but more often in irregular cluster
 concentrated pigments

Plasmodium malariae (Gametocyte Stage)

Diagnostic Points:
 round, compact, smaller than those of the P. vivax
 scattered, coarse, pigments
 eroded forms with only chromatin and pigment present
 not much difference in the size of both sexes, as well as in the staining quality

Plasmodium ovale (Trophozoite Stage)

Diagnostic Points:
 James dots present at early ring forms; dots may be larger and darker than P. vivax
 Ring is larger than P. vivax
 ring shape maintained until late development

Plasmodium ovale (Schizont Stage)

Diagnostic Points:
 stippled
 ¾ of cell occupied by 8-12 merozoites in rosettes or irregular
 pigment clustered (as compared to P. malariae’s concentrated pigments)

Plasmodium ovale (Gametocyte Stage)

Diagnostic Stage:
 fimbriated edges of cell
 smaller than P. vivax

MIXED INFECTIONS

 infection of an individual by more than one species of Plasmodium

 common in endemic areas in the Philippines
 P. falciparum and P.vivax (most common)
 can easily be overlooked ( blood films must be studied carefully to rule out mixed
infections)
P. falciparum
P. vivax
P. malariae
P. ovale
COCCIDIAN PARASITES

 Coccidian parasites are known to infect a wide variety of animals, including humans,
birds and livestock. They are usually species-specific, but the well-known exceptions are
toxoplasmosis caused by Toxoplasma gondii and cryptosporidiosis caused by
Cryptosporidium parvum. The zoonotic coccidian parasites known to cause disease in
humans belong to the genus Cystisospora, Cryptosporidium, Toxoplasma and
Sarcocystis respectively. 

 Cystisospora belli is the only species of Cystisospora that infects man and is frequently
responsible for “traveller’s diarrhea”. C. belli is found throughout the world but is more
common in tropical and subtropical regions. This disease is typically mild in healthy
individuals but can be life threatening in people who are young or immunodepressed.

 Cystisosporosis was largely ignored until its recent emergence as one of the
opportunistic infections affecting AIDS patients.

 Toxoplasmosis is an opportunistic infection in humans caused by protozoan parasite

Toxoplasma gondii, widespread globally and responsible for serious complications in
individuals with impaired immune defences as well as congenitally infected infants. The
high prevalence rate in some parts of the world coupled with the current drug treatments
that trigger hypersensitivity reactions makes the development of immunotherapeutic
interventions a highly important research priority. Immunotherapeutic strategies could
either be a vaccine which would confer a pre-emptive immunity to infection, or passive
immunization in case of recurrent clinical diseases. 

 Cryptosporidium is increasingly gaining attention as a human and an animal pathogen

mainly due to its dominant involvement in worldwide waterborne outbreaks. Ingestion of
oocysts can cause gastrointestinal disease in immunocompetent and
immunosuppressed human patients and those working with animals, including farmers
and veterinarians, are considered to be at increased risk. 

 Sarcocystis spp. have indirect life cycle with an intestinal infections occur in the definitive
host, and tissue invasion is seen in the intermediate host. Three species viz.,
Sarcocystis hominis, S. heydorni (intermediate hosts: cattle) and S. suihominis
(intermediate hosts: pig) have been identified where humans serve as definitive hosts
and get infected by ingesting raw or undercooked beef and pork respectively. Although
others may exist, till now only S. nesbitti has been identified in humans serving as
intermediate hosts based on 18S ribosomal DNA (rDNA) sequence analysis. Though the
life cycle of S. nesbitti remains unknown, this zoonosis is linked to ingestion of food and
water contaminated with the sporocysts of this species.
CYCLOSPORA

 Cyclospora cayetanensis is a coccidian protozoan. It appears that all human cases are
caused by this species; no animal reservoirs for C. cayetanensis have been identified.
TOXOPLASMA GONDII

 Toxoplasma gondii is a protozoan parasite that infects most species of warm-blooded

animals, including humans, and causes the disease toxoplasmosis.

CRYPTOSPORIDIUM
 Many species and genotypes of the apicomplexan protozoan Cryptosporidium can infect
humans and have a wide range of host animals. Zoonotic species and genotypes
of Cryptosporidium are those transmitted from animal hosts to humans, and non-
zoonotic species and genotypes are host-adapted without evidence of transmission from
animals to humans. Cryptosporidium parvum (formerly known as C. parvum genotype II)
and C. hominis (formerly known as C. parvum genotype I) are the leading causes of
human cryptosporidiosis. C. meleagridis, C. felis, C. canis, C. ubiquitum, C. cuniculus,
C. viatorum, Chipmunk genotype I, Cryptosporidium mink genotype, and C. muris can
also infect humans.
CYPTOISOSPORIASIS

 Cystoisosporiasis (formerly known as isosporiasis) is an intestinal disease of humans

caused by the coccidian parasite Cystoisospora belli (formerly known as Isospora belli).
Cystoisosporiasis is most common in tropical and subtropical areas of the world. The
parasite can be spread by ingesting contaminated food or water. The most common
symptom is watery diarrhea. The infection is treatable and preventable.