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Plasmodium Species of Humans

Heinz Mehlhorn

Institut f€ur Zoomorphologie, Zellbiologie und

Parasitologie, D€usseldorf, Germany

Name

Greek, plasma = structured material. Latin,

mala = bad, aria = air, malaria = bad air,

vivax = permanent, ovale = ovoid,

falciparum = bended, knowlesi = honors,

Knowles = an English scientist.

Geographic Distributions/Epidemiology

Humid, warm countries between 40� North and

30� South. Worldwide, about 200 millions of

humans are infected and the yearly death rates

range between 700,000 and 1.3 million. The dis-

ease is called ▶malaria.

Morphology/Life Cycle

The parasites, which introduce malaria, belong to

the phylum Protozoa (single-celled organisms)

and are grouped into the so-called Apicomplexa

(formerly named Sporozoa). Their life cycle runs

between humans and blood-sucking vectors

belonging to mosquito species of the genus

▶Anopheles (Figs. 1, 2, and 3). The typical

human Plasmodium species have no reservoir

hosts except for some primate monkeys. The

course of the life cycle proceeds in all human

species as depicted in Fig. 1. There are five spe-

cies, which infect humans:

– Plasmodium vivax – agent of malaria

tertiana

– Plasmodium ovale – agent of malaria

tertiana

– Plasmodium falciparum – agent of the malign

malaria tertiana = malaria tropica

– Plasmodium malariae – agent of the malaria

quartana

– Plasmodium knowlesi – agent of a malaria

tertiana, which is mainly found in Southeast

Asian monkeys and in increasing numbers in

humans

The molecular biological relations between

the Plasmodium species are diagrammatically

depicted in Fig. 4.

The infection starts as soon as a female mos-

quito of the genus Anopheles has injected sporo-

zoites during its blood meal into a human (Fig. 5).

Mostly within less than 30 s, the injected sporo-

zoites (10–15 mm � 0.5–1.0 mm) enter a liver

cell, wherein they start the formation of a large

schizont. During their way from the biting site

into the liver cells, the sporozoites are protected

from defense reactions of the host by a surface

# Springer-Verlag Berlin Heidelberg 2015

H. Mehlhorn (ed.), Encyclopedia of Parasitology,DOI 10.1007/978-3-642-27769-6_4126-1

malaria

Anopheles

BITE

LIVER

SCHIZOGONY

SPOROGONY

GAMOGONY

RED BLOOD CELLS

BITE

PV

PV

E

PG

EE

PG

SG

IN

BM

NLP

1

22

21

20

19

18

17

16

15

14 1312

10

11

9.1 9.2

8

7

6

5

4

3

2

Plasmodium Species of Humans,Fig. 1 Diagrammatic representation of the life cycle of

malaria parasites. 1. Sporozoites are injected when the

mosquito bites. 2. Exoerythrocytic schizogony occurs in

the cells of the liver (see Table 1). In some species, some

of the sporozoites remain as dormozoites or hypnozoites

and cause relapses that occur at different intervals,

depending in species. 3, 4. Merozoites emerging from

hepatic schizonts normally penetrate erythrocytes. In the

case of P. vivax, they also appear to penetrate parenchy-

mal cells, though in smaller numbers, and cause subse-

quent blood parasitemias. 5–7. Erythrocytic schizogony.

Within the erythrocytes, merozoites are formed from

schizonts over a period of time that vary according to

species (see Table 1). This results in typical attacks of

fever. 8, 9. One portion of the erythrocytic stages is

transformed into gametocytes, which can survive inside

the mosquito. 10–13. Formation of gametes. Female

(macrogametes, 10) and male gametes (microgametes,

11–13) develop within a few minutes after the mosquito

feeds. 14. Syngamy, zygote formation. 15–17. A motile

kinete (= ookinete) grows out of the stationary zygote. 18.The ookinete passes through the peritrophic membrane

and penetrates the intestinal epithelial cell of the mos-

quito. 19–21. Formation of sporozoites within oocysts

(on the outside of the intestine). 22. Sporozoites in the

salivary gland of the mosquito form the “surface coat” (=become infectious). BM basal lamina, E erythrocyte, INintestine, LP liver parenchymal cells (hepatocytes),

N nucleus, PG pigment, PV parasitophorous vacuoles,

SG salivary gland

2 Plasmodium Species of Humans

coat (= circumsporozoite protein, CSP). The

exoerythrocytic schizonts (Fig. 6) in the liver

produce varying numbers of merozoites.

Depending on the species, 30,000 sporozoites

are developed in P. falciparum and 10,000 in

P. vivax but only 2,000 in P. malariae. The time

schedules from infection until formation of eryth-

rocytic merozoites and gamonts are depicted in

Table 1.

In the case of malaria parasites, with the

exception of P. vivax, only a single generation

of merozoites is formed in the liver (Fig. 6). Most

of these merozoites penetrate directly into the

erythrocytes. However, a small number of mero-

zoites remain in macrophages or stay in neighbor

liver cells, etc. Penetrating sporozoites (or even

schizonts) also may not develop immediately but

instead become so-called hypnozoites or

dormozoites (in P. vivax, P. ovale), which then,

after varying waiting periods, initiate the forma-

tion of merozoites, prompting a new invasion of

the erythrocytes. This process is known as a

relapse and differs from recrudescence. In the

case of the latter, residual erythrocytic

populations are reactivated and introduce new

attacks of fever. The merozoites emerging from

Plasmodium Speciesof Humans,Fig. 2 Macrophoto of a

female Anopheles stephensimosquito sucking sugar

solution

Plasmodium Speciesof Humans,Fig. 3 Scanning electron

micrograph of a female

mosquito of the species

Anopheles stephensi

Plasmodium Species of Humans 3

the schizonts in the liver penetrate the erythro-

cytes, merely denting their cellular membrane in

such a way so that the parasites become lodged

inside a tightly joined parasitophorous vacuole

where they can then begin the erythrocytic schi-

zogony (=merogony) process, which is likewise

asexual (Fig. 7). Individual Plasmodium species

prefers to penetrate at different life stages of the

erythrocyte. P. falciparum and P. vivax often

penetrate into the young stages (reticulocytes).

A double invasion of the erythrocytes is also

very common in the case of P. falciparum. Fol-lowing penetration (in less than 30 s), the mero-

zoites reach a stage named as the “signet ring”

because of its light microscopical appearance

(Fig. 8). Then the spherical parasite has a large

central vacuole with peripheral nucleus. Exami-

nation by help of an electron microscopy reveals

that these so-called trophozoites ingest erythro-

cyte plasma within a large cytostome

(▶micropore). Through growth and nuclear

divisions schizonts (meronts) are formed

containing a species-specific number of nuclei.

The metabolites of the ingested hemoglobin are

stored in crystalline form in vacuoles as so-called

pigment (Fig. 9; Table 2). The growth process of

the meronts ends with the production of erythro-

cytic merozoites, which are approximately

1.5 mm in size and are released with the pigment,

when the red cell membrane is ruptured. These

merozoites penetrate new erythrocytes. The time

Plasmodium Speciesof Humans,Fig. 4 Dendrogram

showing the relationship

(based on the mitochondrial

genome) of important

Plasmodium species. The

calibration mark indicates

the numbers of nucleotide

substitutions. The bird

blood parasite

Leucocytozoon sabrazesiwas used as out-group

(according to Carlton

et al. 2008)

Plasmodium Species of Humans, Fig. 5 Light (a) andscanning electron micrographs of sporozoites of Plasmo-dium falciparum without (b) or with (c) a surface coat


micropore

needed to form these merozoites varies among

the different malaria parasites (P. falciparum,P. vivax, P. ovale: 48 h; P. malariae: 72 h)

(Table 1). For reasons, which are not yet fully

understood, the formation of the merozoites

within the erythrocytes becomes almost

completely synchronized, so that large quantities

of pigment and parasites are released simulta-

neously accompanied by recurring attacks of

fever, which are known as tertian fever. Follow-

ing a species-specific period of time (Table 1),

some of the erythrocytic merozoites develop into

gamonts (gametocytes), while asexual schizog-

ony continues as a parallel process (Fig. 1). Pig-

ment is also stored as a metabolite in these

gametocytes, whose shape and coloring vary

according to species (see below). The further

development of the sexual stages does not take

place before the parasite has reached the intestine

of the mosquito, where the cooling down of the

blood acts as a stimulant as it has been proven by

in vitro studies. The erythrocytic stages of human

malaria parasites demonstrate the characteristics

shown in Fig. 8 and compiled in Tables 2 and 3.

They can be used for differential diagnosis

(Figs. 9–12).

In the case of Plasmodium falciparum, the

infected merozoites are changed in their struc-

ture. They appear with numerous protrusions at

their surface (Fig. 12), which are called “knobs”

and are produced by constriction processes of the

erythrocyte’s cytoskeleton (Fig. 13). These

infected erythrocytes lose their flexibility and

become closely attached at noninfected erythro-

cytes, thus forming clusters which block the fine

capillaries of the blood vessel system (Fig. 14),

Plasmodium Speciesof Humans, Fig. 6 Light

micrograph of a colored

section of a human liver

cell containing a large

developing schizont of

P. falciparum

Plasmodium Species of Humans, Table 1 Development of the Plasmodium species in humans

Species

Prepatent

period = minimum

duration of the

exoerythrocytic

merogony

Average

start for

erythrocytic

merogony

Duration of

erythrocytic

merogony

First

occurrence

of

gametocytes

in the blood

Common

incubation

times

Retention

in the

blood

(years)

P. vivax 8 d 13–17 d 48 h 11–13 d 8–31 d 4

P. ovale 8 d 13–17 d 48 h 20–22 d 11–16 d 4

P. falciparum 5 d 8–12 d 48 h 22–17 d 5–27 d 1–2

P. malariae 13–17 d 28–37 d 72 h 24–31 d 20–37 d 40

d days, h hours


introduce breakdown of the blood transportation,

and thus lead to a quickly occurring

insomneslescency (typical for malaria tropica)

and death of the patient.

The development of malaria parasites in the

intestine of mosquitoes

The female mosquito ingests all blood

stages during a blood meal (Fig. 1). However,

while the asexual stages are being digested,

the gametocytes continue to develop. The for-

mation of gametes begins a few minutes fol-

lowing the blood meal in the midgut, where

the ingested blood is surrounded by a

▶ peritrophic membrane. The male

gametocyte divides into 4–8 flagellated

microgametes, the formation of which has

been described in light microscopy studies as

exflagellation (Fig. 1). However, the oocyte

(= macrogamete) stays behind without

undergoing nuclear division and becomes

somewhat more spherical. Following fertiliza-

tion, the zygote elongates to form a motile

ookinete (Fig. 15), which penetrates the

peritrophic membrane, passes through the

cytoplasma of the intestinal cells, and settles

outside between the intestinal epithelium and

basal lamina. Here, the ookinete continues to

grow and is more correctly termed a

zygotokinete, until it becomes an oocyst and

Plasmodium Species of Humans, Fig. 7 (a–d) Dia-grammatic demonstration of the penetration process of a

Plasmodium merozoite into a red blood cell. (a) After

adhesion of a merozoite, the active penetration process is

started (anterior pole forward). (b, c) During the penetra-

tion process, the contents of the organelles of the anterior

pole are discharged leading to the formation of a depres-

sion of the outer surface of the erythrocyte. During this

penetration process, the parasite becomes constricted by a

so-called moving junction, which is formed at the

penetration site. (d) At the end of the penetration process,the parasite is completely surrounded by the host cell

membrane and has a position in a so-called

▶ parasitophorous vacuole. DB dense bodies, DVapicoplast (containing an own genome), E erythrocyte,

EP developing parasitophorous vacuole, G Golgi appara-

tus, MI mitochondrion, MJ moving junction, MNmicronemes, N nucleus, PV parasitophorous vacuole, RHrhoptries, SC surface coat on the outer membrane of the

merozoites, Z cell membrane of the erythrocyte


peritrophic

parasitophorous

Plasmodium Species of Humans, Fig. 8 Light micro-

graphs of the intraerythrocytic stages of the four typical

Plasmodium species of humans (Giemsa-stained version).

(a–d) Plasmodium falciparum. (a) Young trophozoites,

(b) older trophozoites = growing young schizonts, (c)macrogamont, (d) microgamont, (e–h) Plasmodium


seems to form a protective wall around itself

(Fig. 16). Asexual multiplication takes place

within this oocyst (sporogony), producing

thousands of long and slender sporozoites,

which are released into the body cavity when

the oocyst bursts and these find their way to

the salivary glands of the mosquito. Here, the

surface coat is formed that will protect against

the human immune system.

The duration of development in the mos-

quito vector varies depending on the species

and is controlled by the ambient temperature.

��

Plasmodium Species of Humans, Fig. 8 (continued)

vivax. The infected erythrocyte is considerably enlarged

and shows so-called ▶ Sch€uffner’s dots. (e) Young tro-

phozoites, (f) young schizonts, (g) older schizonts, (h)macrogamont. (i–l) Plasmodium malariae. (i) Young tro-

phozoites (arrow), (j) growing schizonts, (k) mature

schizonts, (l) microgamont. (m–p) Plasmodium ovale.

The infected erythrocytes appear with star-like protru-

sions and are filled with Sch€uffner’s dots. (m) Trophozo-

ites, (n, o) growing schizonts, (p) macrogamont.

E erythrocyte, ES growing schizonts, G gamont,

N nucleus, SH adult schizonts, T Sch€uffner’s dots

Plasmodium Species of Humans, Table 2 Characteristics of asexual blood stages of human malaria parasites

Species

Parasite stages

in the peripheral

blood

Size of the

trophozoites

Number of

nuclei in

schizonts

Appearance of

pigment

Changes in the cells

of the host

P. vivax All 2/5 RBC 12–24 Yellowish brown Greatly enlarged,

with Sch€uffner’sdots

P. ovale All 2/5 RBC 6–12 Light brown Slightly enlarged,

ragged surface,

Sch€uffner’s dots

P. falciparum Signet ring only

(trophozoites)

1/5 RBC 8–32 Scattered, light

brown; clumped,

blackish brown

Usually none, but

sometimes Maurer’s

clefts

P. malariae All 2/3 RBC 6–12

(8 often

arranged in

shape of a

rosette)

Dark brown Usually none

RBC red blood cell, the trophozoites are also known as signet ring stages (because of the peripherally located nucleus)

Plasmodium Species of Humans, Table 3 Characteristics of the sexual blood stages of human malaria parasites

Species Shape Microgamont Macrogamont

P. vivax Spherical/

ovoid

10 mm; red, eccentric nucleus; light blue/

pink plasma; pigment in the form of grains

11 mm; small, dark red nucleus; blue

plasma; lots of pigment

P. ovale Spherical/

ovoid

9 mm; similar to P. vivax 9 mm; similar to P. vivax

P. falciparum Sickle or

banana

shaped

9–11 mm; large and diffuse nucleus; pink

plasma; diffusely scattered pigment

12–14 mm; central and red nucleus;

blue-to-purple plasma; pigment

centered around the nucleus

P. malariae Spherical/

ovoid

7 mm similar to P. vivax 7 mm; similar to P. vivax


Schüffner’s

Schüffner’s

A lower limit of 15 �C was determined for

development in P. vivax and P. malariae; forP. ovale it was 16 �C and for P. falciparum a

temperature of at least 18–21 �C is required.

The optimal ambient temperature for all

malaria parasites has been shown to be

25 �C. But then, differing time periods are

required for each species to form infectious

sporozoites. In the case of P. vivax, it takes

9–10 days (for P. falciparum 10–12 days, for

P. ovale 12–16 days, and for P. malariae

15–21 days), until the process of sporogony

is completed within the mosquito. With an

ambient temperature of only 20 �C, these

processes can take significantly longer, for

example, 16–17 days (P. vivax), 22–28 days

(P. falciparum), or 20–25 days (P. malariae).

This also lengthens the span of the infection

risk in any region following the first occur-

rences of people falling ill.

The amount of malaria infection within a

region is measured by various degrees of

so-called ▶ endemicity. This depends on the

so-called ▶ entomological inoculation rate

(EIR), which results from the number of bites

by infectious mosquitoes per person per year

within a region. In Africa, the EIR can encom-

pass several thousand bites, but even in highly

endemic regions it is often only 40–400.

Plasmodium Species of Humans,Fig. 10 Transmission electron micrograph of

P. falciparum: an erythrocyte is bursted and released a

schizont during the phase of protruding merozoites.

MA = merozoite anlage; MI = mitochondrion; N =nucleus; P = pigment; R = rhoptry; RE, RP = remnants

of the bursted erythrocyte; RL = remnants of the limiting

membrane of the red blood cell

Plasmodium Species of Humans, Fig. 9 TEM of an

erythrocyte infected with two young schizonts

(▶ trophozoites) of Plasmodium falciparum (agent of

human malaria tropica). The stages are situated in a very

narrow parasitophorous vacuole (PV). Note the typical

knobs (KN) at the erythrocyte surface. The larger stage

represents a so-called signet ring stage including a large

vacuole (V). CL cleft in the red blood cell plasm,

E erythrocyte, EM outer membrane of the red blood cell,

KN knoblike structure, MI mitochondrion, N nucleus, PGcrystalline pigment, V vacuole


endemicity

entomological inoculation rate

trophozoites

Symptoms of Disease

Clinical Symptoms of Nontropical Malaria

At the beginning, malarias start with non-defined

fever, since the intraerythrocytic reproduction is

not yet synchronized. These fever phases are

called ▶ Sch€uffner’s introductory fever, which

may even persist as a continua type for many

hours. This is followed at the end of the first

infection week by a palpable enlargement of the

spleen. Since mostly headache and muscle pain

are accompanying symptoms, malaria is not

recognized and influenza is diagnosed. The con-

tinua fever phases do not fluctuate more than 1�

between highest and lowest peaks, while

remittent fevers reach differences of at least

1.5 �C during 24� hours and intermittent fever is

interrupted by fever-free phases.

The nontropical malaria occurs either as

malaria tertiana (P. ovale, P. vivax, P. knowlesi)

or as malaria quartana (P. malariae). The typical

fever curves are diagrammatically depicted in

Figs. 17 and 18.

(a) Plasmodium vivax (Fig. 17)After an incubation period of about

9–18 days, fever starts, which runs synchron-

ically only after a few days. The fever phases

start every 48 h, with shivering of about 1 h

followed by temperatures of 40–41 �C, whichpersist for several hours before the final phase

with 2–3 h sweat finishes the cycle. Without

treatment, about 10–15 of such phases may

follow each other. Finally, the fevers are

reduced in intensity. However, recidives

(= new fever phases) may occur within the

next 5–7 years. In cases, when fever occurs

all 24 h, a double infection is present and,

thus, the symptoms are called malaria

duplicate. Similar symptomology occurs in

Plasmodium Species of Humans, Fig. 11 TEM of a

human red blood cell infected with two mature schizonts

of Plasmodium falciparum both being situated in large

parasitophorous ▶ vacuoles (PV). The merozoites are

built up leaving a large residual body (RB) containing

crystalline pigment (PG). L lipid, ME merozoites,

N nucleus, PE pellicle, PG pigment, PV parasitophorous

vacuole, RE remnants of the host cell

Plasmodium Species of Humans, Fig. 12 Scanning

electron micrograph of an erythrocyte which is infected

with two protruding schizonts (blue). Note the typical,

here whitish appearing “knobs” at the surface of the

erythrocyte


Schüffner’s

Schüffner’s

vacuoles

infections with P. knowlesi – a current para-

site of monkeys in Asia.

(b) Plasmodium ovaleAfter an incubation period of 10–17 days,

fevers occur with a 48 h periodicity. Without

treatment, in general, about 4–8 fever phases

follow each other before fever stops.

However, recidives may occur mostly within

the next 2 years.

(c) Plasmodium malariae (Fig. 18.)

After an incubation period of up to

6 weeks, synchronized fever (40–41 �C)occurs repeatedly all 72 h after an initial

phase of shivering. In non-treated cases,

these fever phases may be repeated about

20 times. Recrudescences may occur up to

30 years (or longer).

Clinical Symptoms of Malaria Tropica

Plasmodium falciparum (Fig. 19)

After an incubation period of about 14 days

(ranging from 8 to 24 days), irregular fevers

start reaching temperatures up to 39 �C(occasionally also more than 40 �C). The

fever phases may appear permanently, remain

high, or may be even absent (algid malaria,

afebrile malaria). These fevers as leading

symptoms are not very significant, while

patients feel weakened and suffer from severe

headache.

In the case of the most severe form of

malaria (malaria tropica due to

P. falciparum, which is often fatal – if left

untreated), the following symptoms (usually

in combinations) are the most common symp-

toms among infected persons:

1. Severe fever (99–100 %)

2. Headache (84 %)

3. Chills (81 %)

4. Splenomegaly (69 %)

5. Anemia (68 %)

6. Outbreaks of sweating (67 %)

7. Nausea (30 %)

8. Vomiting (30 %)

9. Arthralgia (39 %)

10. Diarrhea (18 %)

11. Coughing (16 %)

12. Abdominal pain (16 %)

Plasmodium Species of Humans,Fig. 13 Diagrammatic representation of the peripheral

regions of an uninfected (above) and an infected erythro-

cyte (according to Folly and Tilley, USA). The knobs

(seen in the infected erythrocytes) are formed by actions

of adhesion proteins (AD) and surface structure proteins,

which in general reduce or even stop flexibility. A actin;

AD adhesion protein (e.g., sequestrin); AN ancyrin; EMmembrane of the erythrocyte; H HRP-1 protein (= knob

protein); MP3 modified protein 3; MS MESA (= mature

parasite-infected erythrocyte surface antigen); P 3, P 4.1,

P 4.9 proteins; SP dimers of spectrin


In laboratory tests, serious deviations

were found for the following parameters:

1. Thrombocytopenia (standard:

>150,000 ml)110 % of

patients

2. LDH (standard: >240 U/l) 74 % of

patients

3. Leukocytopenia (standard: 5,000/

ml)68 % of

patients

4. Anemia (standard: Hb f: >12 g/dl,

m: 14 g/dl)

68 % of

patients

5. GPT (standard: f:>19 U/l, m:>23

U/l)

48 % of

patients

6. Gamma-GT (standard: f: >18 U/l,

m: >28 U/l)

48 % of

patients

7. AP (standard: >190 U/l) 48 % of

patients

There is a risk of multiple organ failure,

which is ultimately the cause of death in

most cases of malaria tropica. In addition to

these measurable symptoms, more compli-

cated cases of cerebral malaria tropica may

also include numerous other symptoms, such

as strabismus, miosis, hallucinations, disorien-

tation, nystagmus, ataxia, psychoses, spasms,

tremors, hemiparesis, weakness of the facial

muscles, hemoglobinuria, jaundice, acidosis,

etc. With parasitemia levels at or above 40 %

of red blood cells, the prognosis for surviving

malaria tropica is extremely poor, despite

treatment.

In the case of the primary, non-immediate

life-threatening species of malaria

(M. tertiana, M. quartana), direct symptoms

include fever every 48 or 72 h, respectively.

However, in cases of double infections, the

bouts of fever can overlap and thus appear

atypical. Seldom are more than 2–3 % of red

blood cells infected with these species. The

incubation times for both malaria species can

last a very long time (weeks to months), such

that a possible link with a previous trip to the

tropics is often overlooked.

Plasmodium Speciesof Humans, Fig. 14 Light

micrographs of sections

through blood vessels

(capillaries) of patients

being blocked by infected

erythrocytes. P pigment


Pathogenicity factors for severe = complicated

malaria

Severe symptoms and cerebral infection

may occur, especially in the case of

P. falciparum infections and recently in the

case of Plasmodium vivax infections as well

(Anstey et al. 2009). The degree of severity of

certain factors ranges from the known spec-

trum of asymptomatic progression to compli-

cations resulting in death. Thus, the following

factors have an influence on the progression of

the disease:

– Reduced blood flow due to the adhesion of

infected erythrocytes to vascular walls or

through the formation of thrombi in narrow

blood vessels

– Upregulation in the development of adhe-

sion molecules due to cytokines

– Increased cytokine induction as a result of

the release of toxic by-products from the

parasites

– Secretion of nitric oxide (NO) from endo-

thelial cells and various other sources

Plasmodium species of humans

Clinical symptoms See Table 4.

Diagnosis

The demonstration of malarial parasites inside

the erythrocytes by the help of the thick droplet

method and blood films is the most significant

diagnostic method before starting treatment of

malaria infections (see below). The thick droplet

method (Fig. 20) reaches a 20–40-fold enrich-

ment of parasitic stages compared to the results

of the thin film method. Thus, the thick film

technique is able to detect parasitemia rates of

about 0.1 %. Since the preservation of the para-

sitic structures is rather crude, species determina-

tion is mostly not possible but can be done by the

help of thin films (Fig. 8). Such▶Giemsa-stained

blood films show the following main species-

specific criteria:

Plasmodium Species of Humans, Fig. 15 TEM of an

ookinete (motile zygote). Note the blue-stained nucleus at

the posterior pole Plasmodium Species of Humans, Fig. 16 TEM of an

ookinete which starts the production of sporozoites. This

stage protrudes into the body cavity of the mosquito


Giemsa

Plasmodium Species of Humans, Fig. 17 P. vivax.Diagrammatic representation of the relations between

the development of the parasites in the blood of a patient

and the development of fever in the case of malariatertiana (being similar in P. ovale, however, the parasit-ized erythrocytes appear with ray-like protrusions). The

numbers of the so-called Sch€uffner’s dots inside the eryth-rocytes are constantly increasing. The pikes of the fever

(FS) appear all 48 h. 1 = signet ring stage, 2 = polymor-

phous trophozoites, 3 = immature schizonts, 4 = mature

schizonts just before merozoites formation, H hours, FSfever pike

Plasmodium Species of Humans, Fig. 18 P. malariae. Diagrammatic representation of the relations between fever

and parasite development inside the erythrocytes during malaria quartana (for definition of the stages, see Fig. 17)


Plasmodium falciparum

(a) In general, the blood smears show mainly

uninuclear trophozoites (signet ring stages),

which are rather tiny reaching only 1/5 of the

diameters of the erythrocyte. Rarely, also two

nucleated ring forms occur. In other rare

cases, trophozoites are closely attached at

the inner side of the erythrocytic membrane

Plasmodium Species of Humans, Fig. 19 Diagrammatic representation of the fever curve and the intraerythrocytic

events in infections with Plasmodium falciparum

Plasmodium Species of Humans, Table 4 Comparison of clinical symptoms due to the different human Plasmodiumspecies

Criteria P. falciparum P. vivax P. ovale P. malariae

Common incubation

periods

8–24 d 9–18 d 10–17 d 18–40 d

Prodromal symptoms Influenza-like Influenza-like Influenza-

like

Influenza-like

Initial fevers Daily, remittent, or

continuous

Irregular until daily Irregular

until daily

Regular all 72 h

Periodicity of

established fever

No fever continuous fever

all 36–48 h

48 h 48 h 72 h

Initial paroxysmus Severe, for 16–36 h Slight until severe,

for 10 h

Slight, for

10 h

Slight until severe,

for 11 h

Length of untreated

disease

2–3 weeks 3–8 and more

weeks

2–3 weeks 3–24 weeks

Duration of detectable

parasites

6–8 months 5–7 years up to 2 years 30 years and more

Anemia ++++ ++ + ++

ZNS syndrome ++++ +/� +/� +/�Kidney syndrome +++ +/� � +++

Blackwater fever ++++ + + +

d days, h hours, + existing, � not occurring


(being named accolle stages). The erythro-

cytes show so-called knobs at their surface.

(b) Schizonts containing 8–32 merozoite anla-

gen occur only in rare cases – mostly only

at extremely high infection rates (often prior

to death).

(c) The infected erythrocytes retain their normal

size, are not deformed, appear slightly red-

dish, and do not contain ▶ Sch€uffner’s dots.

Only in erythrocytes with late schizonts

so-called ▶Maurer’s clefts appear as tiny

bluish structures.

(d) The large, banana-shaped gamonts (♀, ♂)

are characteristic for P. falciparum. They

occur about 7–8 days after the occurrence of

the first fever period. The gamonts persist for

months in the blood even after a successful

treatment stopping the schizogonic reproduc-

tion. Thus, they remain infectious for blood-

sucking mosquitoes.

(e) In contrast to the other human Plasmodium

species, the infected erythrocytes of

P. falciparum become attached to

noninfected erythrocytes and to the walls of

tiny blood vessels, where they may block

completely the bloodstream. Therefore,

repeated blood investigations are needed

(e.g., at the end of a fever phase) especially

in order to detect low-graded infections.

Plasmodium vivax

(a) Depending on the timing of an infection (e.g.,

on the relevant fever period), the erythrocytes

show uninuclear (= signet ring stages),

young, and old schizonts (the latter with

12–24 merozoites). The signet ring stages of

P. vivax are larger than those of P. falciparum

reaching diameters of about 2/5 of an eryth-

rocyte. In rare cases, also two nucleated

stages and so-called ▶ accolle forms are

seen in blood smears.

(b) The parasitized erythrocytes (reticulocytes)

appear enlarged and hypochromatic. They

contain eosinophilic granula (▶Sch€uffner’sdots), which, however, often lack in freshly

infected erythrocytes.

Plasmodium Speciesof Humans,Fig. 20 Diagrammatic

representation of

procedures of the thick film

(thick droplet) method

(below) respectively(above) the thin film (blood

smear) technique to

diagnose Plasmodiumstages (for explanations,

see Boxes 1 and 2)


Schüffner’s

Schüffner’s

Maurer’s

accollé forms

Schüffner’s

Schüffner’s

(c) Male and female gamonts possess a cen-

trally (♂) or peripherally (♀) situated

nucleus and may be diagnosed often already

3 days after the starting of the blood

schizogony.

Box 1 Description of the preparation

procedures to obtain thin and thick films

for detection of malaria parasites and other

blood organisms

1. Blood smear – thin film preparation

A small drop of blood is spread by the help

of a slide or a coverslip on a glass slide

(Fig. 20). After drying the following,

procedures are done:

(a) Fixation for 3 min with absolute

methanol

(b) Drying in the air

(c) Staining according to Giemsa (see

below) for 30 min

(d) Removement of the color solution

by dipping the slide into water bath

(e) Drying of the preparation on air

(f) Covering of the preparation by, e.g.,

Eukitt and a cover slide

(g) Examination in light microscope

2. Thick film (= thick droplet method)

(a) A larger drop of blood (diameter

ca. 1.5 cm) is placed on a glass slide

(Fig. 20) and stirred in order to elimi-

nate the fibrin and to avoid

coagulation.

(b) Drying at air or by help of a heater.

(c) Introduction of the dried slide into tap

water, which leads to hemolysis of the

erythrocytes.

(d) The now pale glass slide has to be

dried on air.

(e) Without fixation, the probe is colored

by help of the ▶Giemsa stain for

30 min.

(f) Drying and covering the glass by a

cover slide.

(g) Microscopical examination.

Box 2 Materials/procedures needed for thin

and thick film preparations

A. Avoiding blood coagulation

1. 0.1 ml heparin (100 units/ml) for

5–10 ml blood or

2. 0.1 ml K3 EDTA (0,38 M/ml) for

10 ml blood or

3. Citrate anticoagulants: 7.3 g citric

acid, 22 g Na-citrate; 24.5 g glucose;

for 1 l aqua bidest. 15 ml of these

anticoagulants is sufficient for

100 ml blood

B. Coloration according to ▶Giemsa

0.3 ml Azur-Eosin methylene blue

solution (Merck No. 9204) for 10 ml

Weise buffer (pH 7.2). Weise buffer:

0.49 g KH 2 PO 4 + 1.14 g Na 2 HPO 4

for 1 l aqua bidest.

C. Eukitt incubation

One droplet of Eukitt or of a similar

cover solution is placed on the dry sur-

face of the probe and covered by a

coverslip.

D. Inspection of the preparation

Microscopical inspection can be

done immediately. It must not be waited

until the Eukitt has been dried.

Plasmodium ovale

The blood stages look similar to those of P. vivax;

however, parasitized erythrocytes have changed

their shape and appear ovoid, respectively, poly-

morphous and/or with ray-like protrusions. Inside

the erythrocytes Sch€uffner’s dots are present,

which appear mostly more intensive than those

in P. vivax-infected erythrocytes. The schizonts

produce mostly only 8–10 merozoites, and the

gamonts do not fill the whole interior of the

erythrocyte.


Giemsa

Giemsa

HONG KONG

MACAOBRUNEI

DARUSSALAM

SINGAPORE

MALDIVES

VANUATU

B

C

C

A

A

c

C

A

A

a

b

COMOROS

MAURITIUS

CAPE VERDE

Plasmodium Species of Humans, Fig. 21 Diagrammatic representations (a, b) of world maps showing occurrences

of chloroquine-resistant strains of ▶P. falciparum with increasing severity (a-c) according to WHO (2012)


P. falciparum

Plasmodium malariae

Tape-like young schizonts are especially charac-

teristic of P. malariae, while the mature schizonts

mostly produce only eight merozoites, which are

often ringlike arranged around the centrally situ-

ated pigment. The infected erythrocytes are nei-

ther enlarged or deformed nor contain

▶ Sch€uffner’s dots. Simultaneous infections

with P. falciparum or P. vivax are common,

may produce unusual fever periods, and must be

considered in treatment activities. The gamonts

remain rather small.

Besides the microscopic investigation of thin

and thick film preparations, other methods such

as the ▶QBC (quantitative buffy coat) analysis

method, investigation of circulating antigenic

material, PCR, or DNA in situ hybridization

may be used to clarify finally the diagnosis.

Plasmodium Speciesof Humans, Fig. 22 Life

cycle stages of Plasmodiumfalciparum, the agent ofmalaria tropica. 1 The

female Anopheles injectssporozoites which enter

liver cells via the

bloodstream. 2, 3 Schizontsdevelop numerous

merozoites (3) which after

rupture of the host cell

leave the liver and enter

erythrocytes. 4 Merozoite

directly after penetration

(so-called signet ring

stage) – this stage is very

small (one-fifth of the red

blood cell’s diameter) and

is the only stage found in

blood cell smears of

patients. 5–8 Schizonts,

which are blocked within

capillaries (e.g., of the

brain), give rise to several

merozoites (6) whichinvade other red blood cells

and again become schizonts

(5) or develop into male or

female banana-shaped

gamonts (7 a, b) which are

taken up by another

engorging mosquito (8).E erythrocyte, H skin

surface, N nucleus, NHnucleus of host cell, PVparasitophorous vacuole,

R remnants of the

erythrocyte


Schüffner’s

Schüffner’s

QBC

Infection

Main infections occur during bites of females of

about 70 ▶Anopheles species, which inject

saliva containing the slender sporozoites. Infec-

tions are also possible during blood transfusion,

since schizonts stop division during cool storage

but start activity after warming up. Furthermore,

blood-licking leeches may also transmit infected

erythrocytes, if they had previously sucked at an

infected human.

Prophylaxis

(a) Avoidance of mosquito bites by using repel-

lents (e.g., ▶ Icaridin = Saltidin, ▶DEET).

(b) Use of insecticide-impregnated mosquito

nets during sleep.

(c) Spraying of insecticides inside of houses.

(d) Chemoprophylaxis: oral uptake of

chemotherapeuticals (see Table 5).

(e) Vaccination: Several products are under

development, but none is already officially

registered. One product (RTS,

S) – developed by GlaxoSmithKline and

sponsored by the Bill & Melinda Gates

Foundation – is now used for children in

Africa and protects from cerebral malaria.

– Artemether/lumefantrine (Riamet®) and

piperaquintetraphosphat/dihydroartemisinin

(Eurartesim®) are not recommended/registered

for prophylactic use, but can be used upon med-

ical device in cases of patent malaria infections.

– Qinghaosu – a Chinese antimalarial product

made originally from extracts of the plant

Artemisia – is now named in Western regions

▶Artemisinin.

Incubation period

– P. falciparum 8–24 days

– P. vivax 12–18 days

– P. ovale 10–17 days

– P. malariae 18–42 days

Prepatent period

19–21 days.

This period is defined in the present case as the

minimal period of the exoerythrocytic schizog-

ony in the liver (= first appearance of first mero-

zoites in blood/inside erythrocytes).

– P. falciparum 5 days (8–12 days)

– P. vivax 8 days (13–17 days)

– P. ovale 8 days (13–17 days)

– P. malariae 13–17 days (13–37 days)

Plasmodium Species of Humans, Table 5 List of compounds/products registered for malaria prophylaxis

Compounds (products) Start of uptake End of uptake Further informations

Atovaquone/proguanil

(Malarone®)

1–2 days before entering

endemic regions

7 days after leaving

endemic regions

Limitation of uptake: 28 days

Chloroquine

(Resochin®,

Weimerquin®,

Quensyl®)

Entering regions without

chloroquine-resistant strains

6 weeks after leaving

endemic regions

Prophylactic doses:

300 mg base per week

Chloroquine plus

proguanil

(Paludrine®)

See proguanil See proguanil -

Doxycycline 1–2 days before entering

endemic regions

Up to 4 weeks after

leaving endemic

regions

In many countries not

registered for prophylaxis

Mefloquine (Lariam®) 1–2 weeks before start in

mefloquine resistance-free

countries

2–3 weeks after

leaving endemic

regions

Attention: There is a warningfor psychic problems during

uptake


Anopheles

Icaridin

DEET

Artemisinin

Patency

– P. falciparum 4–6 weeks during treatment, but

without max. 18 months

– P. vivax 5–7 years

– P. ovale up to 2 years

– P. malariae 30 years and even more

Treatment

Figures 1, 2, and 3 in the keyword “hemoglobin

interaction” show activity of several antimalarial

drugs. The following doses are recommended by

the producers for therapy of persisting malaria

fevers.

1 Artemether/lumefantrine(Riamet®)

Adults80 mg/480 mg (=four pills) initially,

after 8 h further four

pills, then 2 x daily

each 4 pills on day

2 and 3 starting at a

body weight of

35 kg

ChildrenSee packages

2 Atovaquone/proguanil(Malarone®)

Adults1,000 mg/400 mg

(= four pills) 1 x on

3 consecutive days

starting at a body

weight of at least

40 kg


3 Chloroquine (Resochin®,Quensyl®)

Adults600 mg base

(= four pills

Resochin), 6 h after

start of therapy and

24 and 48 h

afterwards always

300 mg

Children10 mg/kg

bodyweight first,

later 3 � 5 mg/kg

bodyweight

4 Mefloquine (Lariam®) AdultsInitially 750 mg

(= three pills), after

(continued)

6–8 h another

500 mg plus after

another 6 h 250 mg

(= 1 pill)


5 Piperaquintetraphosphat/dihydroartemisinin(Eurartesim®)

Adults120 mg/960 mg

(= three pills) 1 x

on 3 consecutive

days for 36–75 kg

bodyweight


This list of medications is internationally incomplete.

Recently (2015) Mrs Tu Youyou (China) received the

Nobel Prize for Medicine and Physiology for the chemical

characterization of Artemisine.

Further Readings

Allison AC (2008) Genetic control of resistance to human

disease. Curr Opinion Immunol 21:499–505

Anstee DJ (2010) Relationship between blood groups and

disease. Blood 115:4635–4643

Anstey NM, Russell B, Yeo TW, Price RN (2009) The

pathophysiology of vivax malaria. Trends Parasitol

25:220–227

Beutler E (2008) Glucose-6-phosphate dehydrogenase

deficiency: a historical perspective. Blood 111:16–24

Carlton JM, Escalante AA, Neafsey D, Volkman SK

(2008) Comparative evolutionary genomics of human

malaria parasites. Trends Parasitol 24:545–550

Culleton RL et al (2008) Failure to detect Plasmodiumvivax in West and Central Africa by PCR species

typing. Malaria J 7:174–182

Elliot DA et al (2008) Four distinct pathways of hemoglo-

bin uptake in the malaria parasite P. falciparum. ProcNatl Acad Sci U S A 105:2463–2468

Lopez C et al (2010) Mechanisms of genetically based

resistance to malaria. Gene 467:1–12

May J et al (2007) Hemoglobin variants and disease man-

ifestations in severe falciparum malaria. JAMA

297:2220–2226

Piel FB et al (2010) Global distribution of sickle cell gene

and geographical confirmation of the malaria hypoth-

esis. Nat Commun 104:1–7

Santos-Ciminera PD et al (2007) Malaria diagnosis and

hospitalization trends. Brazil Emerg Infect Dis

13:1597–1600

WHOMalaria Policy Advisory Committee and Secretariat

(2012) Malaria Policy Advisory Committee to the

WHO: conclusions and recommendations of Septem-

ber 2012 meeting. Malar J 2012 11:424


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