AFRICAN SLEEPING SICKNESS
history of North Africa. The next report came from Guinea in 1734.
INTRODUCTION: The Disease and Importance
Human African trypanosomiasis (HAT), also called
sleeping sickness, is an illness endemic to sub-Saharan Africa. It is
caused by the flagellate protozoan Trypanosoma brucei, which exists
in 2 morphologically identical subspecies: Trypanosoma brucei
rhodesiense (East African or Rhodesian African trypanosomiasis) and
Trypanosoma brucei gambiense (West African or Gambian African
trypanosomiasis). Both of these parasites are transmitted to human
hosts by bites of infected tsetse flies (Glossina palpalis transmits T
brucei gambiense and Glossina morsitans transmits T brucei
rhodesiense), which are found only in Africa.
The reservoirs of infection for these vectors are exclusively
human in West African trypanosomiasis. However, East African
trypanosomiasis is a zoonotic infection with animal vectors. African
trypanosomiasis is distinct from American trypanosomiasis, which is
caused by Trypanosoma cruzi and has different vectors, clinical
manifestations, and therapies.
The major epidemiology factor in African trypanosomiasis
is contact between humans and tsetse flies. An increasing tsetse fly
density, changing feeding habits, expanding human development into
tsetse fly–infested areas, and an increasing number of
immunologically naïve persons in previously endemic areas,
influences this interaction. Major outbreaks from 1920-1950 led to
extensive treatment and, apparently, immunity for 50 years. Now,
infection is occurring again as the same populations lose their
immunity.
Trypanosomes are parasites with a 2-host life cycle:
mammalian and arthropod. The life cycle starts when the
trypanosomes are ingested during a blood meal by the tsetse fly from
a human reservoir in West African trypanosomiasis or an animal
reservoir in the East African form. The trypanosomes multiply over a
period of 2-3 weeks in the fly midgut; then, the trypanosomes migrate
to the salivary gland, where they develop into epimastigotes. The
metacyclic trypomastigotes infect humans.
Although human sleeping sickness may not seem as
important on the world stage as diseases such as malaria and AIDS, it
is nevertheless an important disease in Sub-Saharan Africa and is
responsible for a considerable degree of suffering and mortality in
countries where it is endemic. Some 55 million people in 37 countries
are at risk, with an estimated 50,000 new cases reported annually.
Left untreated, the outcome of the disease for the individual is death,
but equally insidious is the effect on communities and quality of life
resulting from the debilitating symptoms. In public health terms, the
effects of the disease on the community life and, in particular, the
contribution of individuals to food production and community
support can be measured in terms of disability-adjusted life years lost
(DALYS). Human sleeping sickness is responsible for 1.78 million
DALYS, and the magnitude of this effect can be seen when compared
with the related disease leishmaniasis, which has a value of 2.06
million DALYS despite there being 350 million people at risk in 88
countries.
Early History
The earliest recorded account of sleeping sickness comes
from upper Niger during the 14th century in the historical writings of
Ibn Khaldoun, who wrote about the disease in his account of the
In 1803, the diseases that caused visible swollen lymph glands in
West Africa came to be known as Winterbottom's sign, after the
description of the disease by Cris Winterbottom. Slave traders who
avoided trading and buying slaves who displayed those symptoms
readily recognized such signs.
The earliest detection of trypanosomes in human blood was
in 1902, when R.M. Forde discovered what was then thought to be
filiaria in the blood of a steamboat captain who had traveled
extensively along the River Gambia. J.H. Cook in East Africa made
similar discoveries of filiaria-like organisms in the blood, but
confusion arose as to how filiaria worms could cause such varying
clinical symptoms. J.E. Dutton first correctly identified the parasite as
a trypanosome and subsequently named it Trypanosoma gambiense.
In 1902, A. Castellani observed the presence of trypanosomes in
cerebrospinal fluid taken from a sleeping sickness patient, but it
wasn't until 1903 that D. Bruce correctly recognized that
trypanosomes were the causative agents of sleeping sickness
transmitted to humans by tsetse flies, and that “trypanosome fever”
and “sleeping sickness” - both thought to be different diseases at the
time - were in fact the same.
Morphologically indistinguishable from the West African
species as well as the animal infecting species Trypanosoma brucei
brucei, Trypanosoma brucei rhodensiense was first discovered in
Zambia by J.W.W. Stephens and H.B. Fantham in 1910. By 1926,
T.b. rhodensiense could be found along the fly-belt between Tabora
and Kigoma, Tanzania. The difficulties in identifying this virulent
form of sleeping sickness lead to uncertainties today regarding the
evolution and progression of T.b. rhodensiense through the continent,
although it is generally agreed upon that it originated from the West
African form.
The earliest recorded major epidemics of sleeping sickness
took place in Uganda and Congo between 1896 and 1908, where
roughly 500,000 people were estimated to have died in the Congo
Basin and approximately 300,000 died in Busoga, Uganda. With the
Rift Valley transecting the country, Uganda is in the precarious
position of having foci of both forms of diseases which resulted in
two other major epidemics of sleeping sickness - one in the late
1940's and another in 1980. Throughout West Africa, smaller
epidemics of sleeping sickness rapidly spread from Senegal to
Cameroon during the 1920's, and died down by the late 1940's.
ETIOLOGY
There are two clinical forms of African trypanosomiasis
(also called sleeping sickness); each is named for the region of Africa
in which they were found historically.
East African sleeping sickness is caused by the parasite
Trypanosoma brucei rhodesiense, commonly found in Uganda,
Kenya, Tanzania, Malawi, Ethiopia, Zaire, Zimbabwe and Botswana.
East African sleeping sickness is an acute or rapidly
progressing disease that typically leads to death within weeks or
months if not treated. The initial bite leaves a distinctive sore spot
called a chancre. Symptoms, which appear one to four weeks after
infection, may include swollen lymph nodes, irritability, fever, severe
headache, fatigue, muscle and joint pain, and a skin rash. During the
second stage of the disease, the parasite crosses the blood-brain
barrier and attacks the central nervous system. Neurological
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complications include slurred speech, confusion, and difficulty with
walking.
West African Sleeping Sickness also called Gambian
sleeping sickness is caused by a parasite called Trypanosoma brucei
gambiense commonly found in Democratic Republic of Congo,
Angola, Sudan, Central African Republic, Republic of Congo, Chad
and Northern Uganda.
Like East African sleeping sickness, West African
(Gambian) sleeping sickness is a serious slow developing disease that
is fatal if not treated. However, symptoms may not appear for months
to years after the initial infection in a chronic disease. Initial
symptoms include swollen lymph nodes, swelling of face and hands,
skin rash, fever, severe headaches, muscle and joint pain, and fatigue.
Weight loss is common as the disease progresses. Neurological
impairment occurs during the second stage in which the victim can
experience personality changes, slurred speech, changes in sleep
patterns, progressive confusion, difficulty with walking, and seizures.
BACTERIOLOGY
The Trypanosome
Trypanosomes have been around for more than 300 million
years. They are microscopic spindle-shaped unicellular protozoan of
genus Trypanosoma, which are ubiquitous parasite of insects, plants,
birds, bats, fish, amphibians and mammals. Because they have been
around for so long, they and their natural hosts have evolved together
to ensure their mutual survival. Trypanosomes can be found from
Nairobi to New York, from Sydney to San Francisco, and from
Birmingham to Buenos Aires. Fortunately, few species of
trypanosomes are pathogenic. Trypanosomes, and other parasites,
mainly cause disease when they spread to new hosts, like humans and
their domestic animals, especially recent imports into endemic areas
of species that diverged since continent separated.
African trypanosomes are extracellular organisms, both in
the mammalian and insect host. T. b. gambiense and T. b.
rhodesiense are morphologically indistinguishable, measuring 25-40
µ m in length. Infection in the human host begins when the infective
stage, known as the metacyclic stage, is injected intradermally by the
tsetse fly. The organisms rapidly transform into blood-stage
trypomastigotes (long, slender forms), and divide by binary fission in
the interstitial spaces at the site of the bite wound. The buildup of
metabolic wastes and cell debris leads to the formation of a chancre.
Trypanosomes have a single specialized mitochondrion
called a kinetoplast mitochondrion. One of its unusual features is
that the entire DNA of the mitochondrion, which can be up to 25% of
the total cell DNA, is localized in the kinetoplast, adjacent to the
flagellar pocket. Kinetoplast DNA or kDNA exists in two forms:
mini-circles and maxi-circles. Mini-circle DNA encodes guide RNAs
that direct extensive editing of RNA transcripts post-transcriptionally.
Maxi-circle DNA contains sequences that, when edited, direct
translation of typically mitochondrially encoded proteins.
In the vertebrate host, trypanosomes depend entirely upon
glucose for energy and are highly aerobic, despite the fact that the
kinetoplast-mitochondrion completely lacks cytochromes. Instead,
mitochondrial oxygen consumption is based on an alternative oxidase
that does not produce ATP. When in the insect vector, the parasite
develops a conventional cytochrome chain and TCA cycle.
The surface of the trypanosome has numerous membrane-
associated transport proteins for obtaining nucleic acid bases,
glucose, and other small molecular weight nutrients. None of these
proteins reacts well with antibodies, because although they lie in
exposed regions of membrane, they are shielded by allosteric
interference provided by the variant surface glycoprotein (VSG) coat
proteins. This flagellated stage enters the bloodstream through the
lymphatics and divides further, producing a patent parasitemia. The
number of parasites in the blood is generally so low that diagnosis by
microscopic examination is often negative. At some point,
trypanosomes enter the central nervous system, with serious
pathological consequences for humans. Some parasites transform into
the non-dividing short, stumpy form, which has biochemistry similar
to those of the long, slender form and the form found in the insect
vector.
The tsetse fly becomes infected by ingesting a blood meal
from an infected host. These short, stumpy forms are pre-adapted to
the vector, having a well-developed mitochondrion with a partial
TCA cycle. In the insect vector, the trypanosomes develop into
procyclic trypomastigotes in the midgut of the fly, and continue to
divide for approximately 10 days. Here they gain a fully functional
cytochrome system and TCA cycle. When the division cycles are
completed, the organisms migrate to the salivary glands, and
transform into epimastigotes. These forms, in turn, divide and
transform further into metacyclic trypanosomes, the infective stage
for humans and reservoir hosts. The cycle in the insect takes 25-50
days, depending upon the species of the fly, the strain of the
trypanosome, and the ambient temperature. If tsetse flies ingest more
than one strain of trypanosome, there is the possibility of genetic
exchange between the two strains, generating an increase in genetic
diversity in an organism that may not have a sexual cycle.
Flies can remain infected for life (2-3 months). Tsetse flies
inject over 40,000 metacyclic trypanosomes when they take a blood
meal. The minimum infective dose for most hosts is 300-500
organisms, although experimental animals have been infected with a
single organism. Infection can also be acquired by eating raw meat
from an infected animal. In East Africa, this mode of transmission
may be important in maintaining the cycle in some reservoir hosts.
We generally associate trypanosomes with disease in
Africa and South America. African trypanosomiasis is commonly
known as Sleeping Sickness in humans and Nagana (meaning “loss
of spirit” in Zulu language) in cattle.
Many trypanosomes do not appear to harm their hosts, but
a number of species cause serious disease in humans or domestic
animals. Trypanosoma brucei gambiense and Trypanosoma brucei
rhodesiense causes African sleeping sickness and is transmitted
Trypanosomes in the blood of vertebrates have been
observed to have three body types; a short, broad form often without
a flagellum, called the promastigote; a long and narrow form, the
trypomastigote, and an intermediate between these two, the
epimastigote. In arthropod hosts, all developmental stages have been
reported to derive from an amastigote stage, which lacks a flagellum
and has a circular shape. Trypomastigote form may be active and
proliferative, at this stage they are referred to as metacyclic, which is
presumed to be the infective stage for vertebrates.
• Amastigote - Basal body anterior of nucleus, with a short,
essentially non-functional, flagellum.
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 • Promastigote - Basal body anterior of nucleus, with a long
detached flagellum.
• Epimastigote - Basal body anterior of nucleus, with a long
flagellum attached along the cell body.
• Trypomastigote - Basal body posterior of nucleus, with a
long flagellum attached along the cell body.
These names are derived from the Greek “mastig”-
meaning whip, referring to the trypanosome's whip-like flagellum.
T. brucei is found as a trypomastigote in the slender,
stumpy, procyclic and metacyclic forms. The procylic form
differentiates to the proliferitive epimastigote form in the salivary
glands of the insect.
Subs-species of Trypanosome brucei
Trypanosoma brucei spp. is an extracellular gram-negative
parasitic protist species that causes African trypanosomiasis (or
sleeping sickness) in humans and nagana in animals in Africa. There
are 3 sub-species of T. brucei; T. b. brucei, T. b. gambiense and T. b.
rhodesiense.
MORPHOLOGY
A sound knowledge of the basic features of the various trypanosomes enables the identification of each species and so the exact cause of
the disease. Once the basic features possessed by all trypanosomes are appreciated, the diagnostic differences can be recognized and the species
identified.
Basic morphology of trypanosomes
Diagram of a trypanosome Fig. 1
The parasite consists of a single cell varying in size from 8
to over 50 μm. All the activities associated with a living organism
• T. brucei gambiense - Causes slow onset chronic
trypanosomiasis in humans. Most common in central and
western Africa, where humans are thought to be the
primary reservoir.
• T. brucei rhodesiense - Causes fast onset acute
trypanosomiasis in humans. Most common in southern and
eastern Africa, where game animals and livestock are
thought to be the primary reservoir.
• T. brucei brucei - Causes animal African trypanosomiasis,
along with several other species of trypanosoma. T. b.
brucei is not human infective due to its susceptibility to
lysis by human apolipoprotein L1. However, as it shares
many features with T. b. gambiense and T. b. rhodesiense
(such as antigenic variation) it is used as a model for
human infections in laboratory and animal studies.
These obligate parasites have two hosts - an insect vector
and mammalian host. Because of the large difference between these
hosts, the trypanosome undergoes complex changes during its life
cycle to facilitate its survival in the insect gut and the mammalian
bloodstream. It also features a unique and notable variable surface
glycoprotein (VSG) coat in order to avoid the host's immune system.
There is an urgent need for the development of new drug therapies, as
current treatments can prove fatal to the patient as well as the
trypanosomes
take place within this unicellular organism — nutrition, respiration,
excretion, reproduction. The substance of which all living cells
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consist, the protoplasm, comprises three parts, an outer protective and
retaining layer, the pellicle = cell envelope = cell membrane, within
which the cytoplasm forms the bulk of the contents. Suspended in the
cytoplasm are various structures, the most prominent being the
nucleus, which may be regarded as the command centre of the cell
and which also plays a major part in reproduction. It contains DNA
(deoxyribonucleic acid), which is arranged in the form of genes and
chromosomes; it represents the genetic information and is responsible
for the manufacture of enzymes and other proteins of the cell.
Small granules (formerly called “volutin granules”) can
sometimes be seen in the cytoplasm; they may have various origins,
they may be food or nuclear reserves, or result from a reaction
between the trypanosome and the host's immune system.
Trypanosomes are thoroughly adapted to living and moving
in the blood plasma or tissue fluid of the host. They are elongated and
streamlined, and tapered at both ends. The pellicle, the outer layer of
the cytoplasm, is flexible enough to permit a degree of body
movement, while retaining a definite shape. As shown in Figure 1 a
flagellum arises near to the posterior end from a parabasal body, and
runs the length of the trypanosome; it may be continued beyond the
anterior end of the body as a whip-like free flagellum. Along the
length of the body, the pellicle and cytoplasm are pinched up into a
thin sheet of tissue called the undulating membrane, through the outer
margin of which runs the flagellum, as shown in Figure 1.
Among other basic morphological features, a distinct well-
defined body, the kinetoplast, is seen near to the posterior end of the
trypanosome and differs in size and position according to the species.
It is adjacent to the parabasal body (from which the flagellum arises),
and so close to it that it cannot easily be seen separately with the light
microscope. The kinetoplast has important functions in reproduction
and metabolism and is probably essential for cyclical transmission by
tsetse flies. (It is sometimes absent in a proportion of. Trypanosomes,
especially of some strains of T. evansi, a species which has lost its
ability of being cyclically transmitted.) The extent of the undulating
membrane and the absence or presences of the free flagellum are also
precious in specific identification of trypanosomes. Other
morphological characters are the average length and the shape of the
body.
Differential morphology
There are distinct differences in appearance, shape and size
between the various species of trypanosomes, allowing specific
identification. It must be remembered, however, that in any biological
material there is some variability. In addition, trypanosomes are not
rigid and continuously change their shape slightly; the individual
parasite seen in the stained preparation presents the shape it had now
of dying. It has also been subjected to the unnatural stresses of drying
out and being fixed and stained. Many variations in appearance are
therefore seen. It is thus necessary to observe carefully and
systematically all the features in a sufficiently large number of
individual trypanosomes: only after such an examination is it possible
to arrive at a reasonably accurate diagnosis. There will be examples
where trypanosomes are so few, or the staining so inadequate, that
identification may not be possible or only after a prolonged search. It
is also essential to examine several individual trypanosomes, because
even if one specimen is perfect to establish its identity beyond any
doubt, further search may reveal another species and thus a mixed
infection.
For specific identification, a number of trypanosomes
should be examined systematically for the presence or absence, size
and position of a number of features:
• Presence or absence of trypanosomes of different
appearance. If all individual trypanosomes are alike, the
infection is called monomorphic (of one form); if there are
distinctly different types it can be either a polymorphic (=
pleiomorphic) species, or a mixed infection of different
species.
• Presence or absence of a free flagellum. In certain species,
there may be some trypanosomes with, and some without, a
free flagellum.
• Size of the trypanosome (expressed in μm).
• The size and position of the kinetoplast. The position is
related to proximity to the posterior extremity (rear end) of
the organism.
• The degree of development of the undulating membrane. It
may be conspicuous or inconspicuous.
• The shape of the parasite, particularly the shape of its
posterior part. The posterior extremity may vary from blunt
to pointed.
Specific morphology
The sub genus Trypanozoon (the brucei group). This group
comprises five members: T. brucei brucei, T. brucei gambiense, T.
brucei rhodesiense, T. evansi and T. equiperdum. The three
subspecies of T. brucei are normally transmitted by tsetse flies (in
contrast to T. evansi and T. equiperdum) and are exactly similar in
morphology, but only T. brucei gambiense and T. brucei rhodesiense
are the cause of human sleeping sickness, the former mainly in West
and Central Africa and the latter in eastern and southern Africa. T.
brucei brucei is not infective to humans.
T. brucei (see Figure 2). T. brucei is polymorphic, with
three main forms, all of which have a small kinetoplast and a
conspicuous undulating membrane:
• Long slender forms (23–30 μm in length) with a free
flagellum, which may be up to one half of the length of the
organism. The posterior end is pointed and the nucleus is
central. The kinetoplast is placed up to 4 μm in front of the
posterior extremity.
• Short stumpy forms (17–22 μm in length) normally without
a free flagellum, but in which there may occasionally be
individuals with a short free flagellum. The kinetoplast is
usually sub terminal. The position of the nucleus varies
greatly and it is in some cases in the posterior part of the
cell, sometimes so far posterior that the kinetoplast is
anterior to it (so-called postero-nuclear forms). There is
considerable variation in appearance between short stumpy
forms, from broad, squat types (which include the postero-
nuclear forms) to a form similar to T. congolense, although
longer. In stained specimens, blue volutin granules are
often present in the cytoplasm, often arranged in a line
along the margin of the cell.
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 • Intermediate forms, varying in length between the two
previously mentioned types. A free flagellum, of varying
length, is always present. The nucleus is centrally placed.
The posterior end is somewhat variable in shape, but
usually bluntly pointed. The kinetoplast is close to the
posterior extremity. Volutin granules are occasionally
present but neither as common nor as plentiful as in the
short, stumpy forms.

Trypanosoma brucei blood stream forms Figure 2

The cell structure

The structure of the cell is typical

of eukaryotes, see eukaryotic cell. All major
organelles are seen, including the nucleus,
mitochondria, endoplasmic reticulum, Golgi
apparatus etc. Unusual features include the
single large mitochondria with a condensed
mitochondrial DNA structure, and its
association with the basal body of the
flagellum, unusually the cytoskeleton
organization mechanism of the cell. The cell
surface of the bloodstream form features a
dense coat of variable surface glycoproteins
(VSGs) which is replaced by an equally
dense coat of procyclins when the parasite
differentiates into the procylic in the tsetse
fly midgut.

The genome • 11 pairs of large chromosomes of 1 to 6 megabase pairs.

• 3-5 intermediate chromosomes of 200 to 500 kilobase
The genome of T. brucei is made up of: pairs.

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 • Around 100 mini chromosomes of around 50 to 100
kilobase pairs. These may be present in multiple copies per
haploid genome.
The large chromosomes contain most genes, while the
small chromosomes tend to carry genes involved in antigenic
variation, including the VSG genes. The genome has been sequenced
and is available online.
The mitochondrial genome is found condensed into the
kinetoplast, an unusual feature unique to the kinetoplastea class. It
and the basal body of the flagellum are strongly associated via a
cytoskeleton structure.
The cytoskeleton
The cytoskeleton is predominantly made up of
microtubules, forming a sub-pellicular corset. The microtubules lie
parallel to each other along the long axis of the cell, with the number
of microtubules at any point roughly proportional to the
circumference of the cell at that point. As the cell grows (including
for mitosis) additional microtubules grow between the existing
tubules, leading to semi conservative inheritance of the cytoskeleton.
The microtubules are orientated + at the posterior and - at the
anterior. Microfilament and intermediate filaments also play an
important role in the cytoskeleton, but these are generally
overlooked.
Flagellar structure
The trypanosome flagellum has two main structures. It is
made up of a typical flagellar axoneme, which lies parallel to the
paraflagellar rod, a lattice structure of proteins unique to the
kinetoplastida, euglenoids and dinoflagellates.
The microtubules of the flagellar axoneme lie in the normal
9+2 arrangement, orientated with the + at the anterior end and the - in
the basal body. The cytoskeletal structure extends from the basal
body to the kinetoplast. The flagellum is bound to the cytoskeleton of
the main cell body by four specialised microtubules, which run
parallel and in the same direction to the flagellar tubulin.
The flagellar function is twofold - locomotion via
oscilations along the attached flagellum and cell body, and
attachment to the fly gut during the procyclic phase.
Variant Surface Glycoprotein (VSGs)
Trypanosomes have a specialized mechanism to overcome
the obstacles of the mammalian immune system. Days after
infection, host antibodies recognize surface glycoprotein that coat the
protozoa—the antigenic determinants of the organism—and kill the
organisms by labeling them destruction. However a few protozoa
escape destruction via a programmed system that changes their
glycoprotein composition and thus enables them to evade immune
recognition. Again, a day later the immune system recognizes this
change and mounts an immune response against the new
glycoprotein. This cycle is repeated with a pattern of high parasite
load followed by a period of low parasite load. For this reason,
patients exhibit an irregular pattern of high-grade fevers followed by
a febrile period throughout the course of a systemic infection. For
this reason, vaccine development has been thwarted.
The VSG coat
The surface of the trypanosome is covered by a dense coat
of ~1x107 molecules of Variable Surface Glycoprotein (VSG). This
coat enables an infecting T. brucei population to persistently evade
the host's immune system, allowing chronic infection. The two
properties of the VSG coat that allow immune evasion are:
• Shielding - the dense nature of the VSG coat prevents the
immune system of the mammalian host from accessing the
plasma membrane or any other invariant surface epitopes
(such as ion channels, transporters, receptors etc.) of the
parasite. The coat is uniform, made up of millions of copies
of the same molecule; therefore, the only parts of the
trypanosome the immune system can 'see' are the N-
terminal loops of the VSG that make up the coat.
• Periodic antigenic variation - the VSG coat undergoes
frequent stochastic genetic modification - 'switching' -
allowing variants expressing a new VSG coat to escape the
specific immune response raised against the previous coat.
Antigenic variation
Sequencing of the T. brucei genome has revealed a huge
VSG gene archive, made up of thousands of different VSG genes. All
but one of these is 'silent' VSGs, as each trypanosome expresses only
one VSG gene at a time. VSG is highly immunogenic, and an
immune response raised against a specific VSG will rapidly kill
trypanosomes expressing this VSG. This can also be observed in
vitro by a complement-mediated lysis assay. However, with each cell
division there is a possibility that one or both of the progeny will
switch expression to a silent VSG from the archive. The frequency of
such a switch has been measured to be approximately 1:100. This
new VSG will likely not be recognized by the specific immune
responses raised against previously expressed VSGs. It takes several
days for an immune response against a specific to develop, giving
trypanosomes, which have undergone VSG coat switching some time
to reproduce (and undergo further VSG coat switching events)
unhindered. Repetition of this process prevents extinction of the
infecting trypanosome population, allowing chronic persistence of
parasites in the host. The clinical effect of this cycle is successive
'waves' of parasitaemia (trypanosomes in the blood).
VSG structure
VSG genes are hugely variable at the sequence level.
However, for them to fulfill their shielding function, different VSGs
have strongly conserved structural features. VSGs are made up of a
highly variable N terminal domain of around 300 to 350 amino acids,
and a more conserved C terminal domain of around 100 amino acids.
The C terminal domain forms a structural bundle of 4 alpha helices,
while the N terminal domain forms a 'halo' around the helices. The
tertiary structure of this halo is well conserved between different
VSGs (in spite of wide variation in amino acid sequence) allowing
different VSGs to form the physical barrier required to shield the
trypanosome's surface. VSG is anchored to the cell membrane via a
glycophosphatidylinositol (GPI) anchor - a covalent linkage from the
C terminus, to approximately 4 sugars, to a phosphatidylinositol
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phospholipid acid, which lies in the cell membrane. VSGs form
homodimers.
VSG archive structure
The VSG gene archive is the collection of silent VSGs in
the T. brucei genome. Some of these are full-length, intact genes;
others are pseudogenes) typically with omitted sections or premature
stop codons. Expression of an antigenically novel VSG can occur by
simply switching to a different full-length VSG gene. However, only
5% of the archive is made up of such complete silent VSGs. To
utilize the rest of the silent VSG archive, ‘mosaic’ VSGs can be
formed by replacing part of the expressed VSG with a structurally
homologous region from the archive. The combinatorial nature of
mosaic formation in conjunction with the huge silent VSG archive
gives the parasite a theoretically limitless VSG library, and is the
major barrier to vaccine development.
VSG expression
One major focus in trypanosome research is how the
majority of VSG genes are kept silent, and how these genes are
switched. The expressed VSG is always located in an Expression Site
- found at the telomeres of the large and intermediate chromosomes.
Each is a polycistronic unit, containing a number of Expressions Site-
Associated Genes (ESAGs) all expressed along with the active VSG.
While there are at least 20 known expression sites, only a single one
is ever active at one time. A number of mechanisms appear to be
involved in this process, but the exact nature of the silencing is still
unclear
The VSG can be switched either by changing the active
expression (from the active to a previously silent site) or by changing
the VSG gene in the active site. The genome contains many copies of
possible VSG genes, both on minichromosomes and in repeated
sections in the interior of the chromosomes. These are generally
silent, typically with omitted sections or premature stop codons, but
are important in the evolution of new VSG genes. It is estimated up
to 10% of the T.brucei genome may be made up of VSG genes or
pseudogenes. Any of these genes can be moved into the active site by
recombination for expression. Again, the exact mechanisms that
control this are still only partially known.
The Trypanosome cell cycle (procyclic form)
The mitotic division of T.brucei is unusual in terms of the
cytoskeletal process. The basal body, unlike a centrosome of most
eukaryotic cells, plays an important role in the organization of the
spindle.

Stages of mitosis:

1. The basal body replicates, both remaining associated with the kinetoplast.
2. The kinetoplast undergoes replication, and the daughter kinetoplasts are separated by the basal bodies.
3. The second flagellum grows while the nucleus undergoes replication.
4. The mitochondrion divides, and cytokinesis progresses from the anterior to posterior end.
5. The division resolves. The daughter cells may stay connected for a significant length of time after cytokinesis is complete

7
The process of endocytosis in trypanosomes
Trypanosomes, like all organisms, need nutrients to live
and grow. The parasites obtain these nutrients from their environment
by taking up molecules and particles in a process known as
endocytosis. Past studies using the electron microscope have shown
that trypanosomes draw in molecules by way of a structure named the
flagellar pocket. Molecules binding to the surface of the
trypanosome, including antibodies, have been found also to be
engulfed in this way. Therefore, in addition to changing its surface
coat of proteins to avoid antibody binding, the parasite seems able to
take up antibodies, and possibly in this way to neutralize their effect.
It was demonstrated during the year that clearance of antibodies from
the surface of the trypanosome in this way renders parasites in vitro
resistant to the effects of “complement”, the collective name for a
group of proteins in mammalian blood so named because they
complement and amplify the action of antibodies to cause the lysis
(rupture) of parasites.
One approach to learning more about the physical
organization of the endocytic pathway is to determine the functional
activity of defined fractions and subcomponents of trypanosomes. In
1990, in collaboration with scientists from the European Molecular
Biology Laboratory (Heidelberg), a free-flow electrophoresis
technique was used to separate microsomal trypanosome fractions.
The fractions obtained were characterized to some extent based on
demonstrations of enzymatic activity.
Progress was also made during the year in purifying and
characterizing a receptor molecule of T. b. brucei that recognizes and
binds to transferring circulating in the blood of the mammalian host.
The receptor appears to be much smaller than its mammalian
equivalent. The possibility that trypanosomes have, and make use of,
receptors to mammalian growth factors will be examined in the
coming year.
Parasite enzymes that break down proteins, called
proteases, appear to be involved in the endocytic process. A
lysosomal thiol protease of T. congolense was purified in 1990.
Antibodies to the enzyme have proved to be valuable markers of the
lysosome. An important discovery made during the year was that this
enzyme is better recognized by antibodies in the sera of
trpyanotolerant N'Dama cattle recovering naturally from infections
than in Zebu cattle that required treatment to recover from infection.
Furthermore, the enzyme is detectable in the sera of cattle early in an
infection and so is a possible cause of the immune and physiological
dysfunctions that characterize trypanosomiasis in animals. The
degree of protection afforded livestock by immunization with the
purified molecule, and the possibility that it is involved in the
pathogenesis of the disease, are subjects of current studies.
Locomotion
Trypanosomes move actively and progress by movement of
the undulating membrane and the free flagellum (when present),
which acts as a kind of propeller, thus drawing themselves through
the blood plasma or tissue fluid. (The free flagellum, when present,
arises from the anterior [front] end of the parasite.)
Reproduction
This is by a process of division to produce two daughter
cells. However, as stated above, it has been shown that exchange and
recombination of genetic material may take place in the tsetse fly
between two trypanosomes, but it is unknown how frequently this
occurs.
The division into two daughter cells (binary fission)
follows the sequence of events illustrated in Figure 3. The kinetoplast
divides first. A second parabasal body develops, from which a second
flagellum develops. The nucleus divides next, followed by the rest of
the trypanosome body duplicating all the structures present in the
cytoplasm. The body then divides into two daughter cells, beginning
at the anterior end. The process is rapid, and may result in a vast
population in the host within a short period.
8
Division of a trypanosome Fig. 3

Habitat differentiate into several forms, culminating the metacyclic form,

which is able to infect mammalian hosts. When the infected fly next
feeds, these metacyclic trypanosomes are injected into the skin along
• Blood with tsetse saliva. In the animal, the parasites differentiate into a
• Lymph channel throughout the body bloodstream form specially adapted to live in mammalian blood. The
• CSF bloodstream parasites multiply by binary fission and enter the
animal's lymphatic and blood circulation. As flies feed on animals
• Connective Tissue infected with the parasite, they take up blood containing
• Intracellular spaces trypanosomes, which then completes the life cycle.
• Brain
Insects are usually involved in the natural transmission of
Life Cycle the African pathogenic trypanosomes with which we are concerned in
this field guide. When this is the case, the life cycle has two phases,
one in the insect vector and one in the mammalian host. Transmission
The life cycle of the single-celled trypanosome parasite is
by insects may be cyclical by tsetse flies, Glossina species, or
complex. In both the tsetse fly vector and the mammalian host, mechanical by other biting flies (but apart from transmitting
trypanosomes undergo a series of transformations into different
trypanosomes cyclically, tsetse flies can also act as mechanical
forms. The tsetse fly ingests trypanosomes when it feeds on an vectors).
animal infected by the parasite. In the fly, the trypanosomes

9
 The tsetse fly is large, brown and stealthy. While taking
blood from a mammalian host, an infected tsetse fly (genus Glossina)
injects metacyclic trypomastigotes into skin tissue. The parasites
enter the lymphatic system and pass into the bloodstream
1. Inside the host, they transform into bloodstream
trypomastigotes
2. are carried to other sites throughout the body, reach other
blood fluids (e.g., lymph, spinal fluid), and continue the
replication by binary fission
3. The entire life cycle of African Trypanosomes is
represented by extracellular stages. A tsetse fly becomes
infected with bloodstream trypomastigotes when taking a
blood meal on an infected mammalian host
4. In the fly's midgut, the parasites transform into procyclic
trypomastigotes,
5. multiply by binary fission,
6. leave the midgut, and
7. transform into epimastigotes
8. The epimastigotes reach the fly's salivary glands and
continue multiplication by binary fission.
MODE OF TRANSMISSION
Cyclical transmission
When a tsetse fly hatches from its pupal case, it is free from
trypanosomes. Until its first bloodmeal, it is called a teneral fly. It
acquires a trypanosomal infection when feeding on a parasitaemic (=
having parasites in the circulating blood) mammalian host. The
trypanosomes undergo a cycle of development and multiplication in
the digestive tract of the fly until the infective metacyclic
trypanosomes (metatrypanosomes) are produced. Different
trypanosome species develop in different regions of the digestive
tract of the fly, and the metatrypanosomes occur either in the biting
mouthparts or in the salivary glands. The period from ingesting
infected blood to the appearance of these infective forms varies from
one to three weeks; once infective metatrypanosomes are present, the
fly remains infective for the remainder of its life. During the act of
feeding, the fly penetrates the skin with its proboscis. The rupture of
small blood vessels forms a pool of blood in the tissues and the fly
injects saliva to prevent coagulation. Infection of the host takes place
at this stage, with infective metacyclic trypanosomes in the saliva.
Although no classical sexual processes in the life cycle of
trypanosomes have been described, it has been shown that exchange
of genomic material (DNA) between trypanosomes sometimes occurs
in the tsetse fly, although it is not clear how significant this is.
Life cycle in the mammalian host. The infective metatrypanosomes
undergo development and multiplication at the site of infection where
a swelling or chancre may be detected in the skin, and finally the
mature blood trypanosomes (or trypomastigotes) are released via
lymph vessels and lymph nodes into the blood circulation.
Reproduction in the mammalian host occurs through a
process of binary division.
Trypanosomes feed by absorbing nutrients, through their
outer membrane, from the body fluids of the host. The proteins,
carbohydrates and fats are digested by enzyme systems within their
protoplasm. Oxygen dissolved in the tissue fluids or blood plasma of
their host is absorbed in a similar manner, to generate the energy
necessary for the vital processes.
10
 Waste products are disposed of by a reverse process,
through the outer membrane, into the body fluids of the host. They
include carbon dioxide formed during respiration, as well as more
complex metabolic products.
Life cycle in the tsetse fly. Blood stream forms (trypomastigotes)
ingested by the fly undergo considerable changes, in morphology as
well as in their metabolism. They change into long slender forms
called epimastigotes, which multiply and finally give rise to the
infective meta-trypanosomes.
Mechanical transmission
By biting insects. The process is purely mechanical. A biting insect
passes the blood forms from an infected animal to another in the
course of interrupted feeding. The time between the two feeds is
crucial for effective transmission because the trypanosomes die when
the blood dries. The importance of this mode of transmission is
variable from place to place, depending on the numbers of hosts and
biting insects present, and on the species of trypanosome. Large
biting insects such as tabanids carry more blood and are more likely
to act as mechanical vectors than for example mosquitoes. (Tsetse
flies themselves can of course also act as mechanical vectors.) This
mode of transmission has proved to be sufficiently effective to
maintain Trypanosoma vivax and Trypanosoma evansi in South and
Central America, and the latter species in North Africa and Asia as
well. No tsetse flies occur outside tropical Africa, apart from small
tsetse pockets in the southwest of the Arabian peninsula.
High Risk Group
• Hunters
• Rural villagers living in areas with a lack of infrastructure
 Like running water, electricity
• Workers in game reserves or park
• Tourist who spend an extended time in rural areas or game reserves
By iatrogenic means. This can occur when using the same needle or
surgical instrument on more than one animal, at sufficiently short
intervals that the blood on the needle or instrument does not dry. It is
not an uncommon occurrence when animals are vaccinated or treated
by injection, or when blood is collected from several animals in a
row, without changing or disinfecting needles or pins. It may also
occur when several animals are subjected at short intervals to a
surgical intervention (dehorning, castration, etc.) without properly
disinfecting the instruments.
Transmission by other means
• It is well known that carnivores may be infected with T.
evansi and T. brucei by ingesting meat or organs from
infected animals, as long as these are still sufficiently fresh
to contain live trypanosomes. Infection occurs probably
through the mucosa of the mouth (in which moreover bone
splinters make wounds through which the parasites
penetrate even more easily).
• All trypanosome species are occasionally transmitted
congenitally, or vertical transmission from the mother to
the offspring, through the placenta either while the fetus is
still in the uterus, or when bleeding occurs during birth.
Congenital transmission of T. vivax, for example, has been
observed in Latin America as well as in Africa, but its real
importance is not well known.
11
Disease Course
Infection with African trypanosomes can result in disease
manifestations ranging from asymptomatic or mild to a severe
fulminating disease. T. rhodesiense is more likely to cause a rapidly
progressing and fulminating disease than T. gambiense. T. gambiense
tends to cause either a slow progressing, which may be self-limiting,
or the central nervous system (CNS).
The infection is initiated when metacyclic trypomastigotes
are introduced from the saliva of the tsetse into the bite wound.
Generally, there is an asymptomatic incubation period of 1-2 weeks
in which the trypomastigotes are replicating within the tissue near the
site of the bite. Occasionally, a local inflammatory nodule known as a
‘trypanosomal chancre’ is observed during this period. Chancres are
usually tender and painful and ulceration may occur.
The trypomastigotes will invade the capillaries, enter the
circulatory system during this incubation period, and continue to
replicate within the blood of the human host. The establishment of
this acute blood stage infection is characterized by irregular episodes
of fever and headache. In the case of T. gambiense the number of
parasites in the blood tends to be very low and often the infected
person exhibits no symptoms, whereas most persons infected with T.
rhodesiense will exhibit much higher parasitemias and a more
pronounced fever sometimes associated with rigor.
Disease progression is often characterized by invasion of
the lymphatics in T. gambiense infections. Symptoms during the
lymphatic stage include enlarged lymph nodes (particularly post-
cervical group), weight loss, weakness, rash, itching, and edema as
well as the continued intermittent febrile attacks. Higher parasitemias
are often associated with the symptomatic periods. The infection can
spontaneously resolve during either the blood stage or the lymphatic
stage. There is usually little evidence of lymphatic involvement in T.
rhodesiense infections. In general, the symptoms during the earlier
stages of the infection tend to be non-specific (fever, malaise,
headache, weakness) and may infect multiple organs.
A hallmark feature of African trypanosomiasis is the
invasion of the CNS and nervous system impairment. Trypanosomes
crossing the blood-brain barrier result in a generalized
meningocephalitis characterized by progressively worsening
symptoms. Indications of nervous impairment include apathy,
fatigue, confusion, somnolence, and motor changes (such as tics,
slurred speech, and in coordination). The changes in sleep patterns
are often characterized by extreme fatigue during the day and
extreme agitation at night. Generally it is 6-12 months (or even years)
after the infection before the neurological symptoms start to become
apparent in the case the CNS stage of the disease will almost always
progress to include convulsions or coma by death in both T.
gambiense and T. rhodesiense infections.
Clinical Presentation
Incubation Period: The first clinical manifestation of African
trypanosomiasis occurs a few days after infection as a chancre at the
site of tsetse fly inoculation. This is due to the localized proliferation
of the pathogens within the subcutaneous tissue. Incubation period
for T.b. rhodesiense may be two to three weeks, while the incubation
period for the Gambian species may last several weeks to months.
Dissemination: With the conclusion of the incubation period, the
organisms have already disseminated into the bloodstream, leading to
the emergence of a characteristic intermittent fever pattern that
correlates directly with high versus low levels of parasitemia. The
reason for the oscillating levels of parasite load is linked to the ability
of the organisms to change their variable surface glycoproteins
(VSGs) and evade the host’s immune system. Lymphadenopathy, the
swelling of lymph nodes, especially in the posterior cervical nodes
(on the back of the neck) is characteristic sign of African sleeping
sickness and is termed Winterbottom’s sign.
Invasion of the Central Nervous System: Invasion of the central
nervous system (CNS) occurs within several weeks in the Rhodesian
species and months to even years in the Gambian species of
trypanosomiasis. Symptoms include headache, stiff neck, sleep
disturbance, and depression, followed by progressive mental
deterioration, focal seizures, tremors, and palsies. This progresses to
coma and the ultimate death of the patient often secondary to
pneumonia or sepsis. Without treatment, African trypanosomiasis is
a universally fatal illness.
PATHOGENESIS
During the bite of an infected tsetse fly, the protozoan
enters the bite wound in the fly saliva. The organism multiplies at the
skin site and within a few weeks enters the lymphatics and blood
circulation. The patient responds with fever and production of IgM
antibody against the protozoa, and symptoms improve. Within about
a week and at roughly weekly intervals thereafter, however, there are
recurrent increases in the number of parasites in the blood. Each of
these bursts of increased parasitemia, meaning parasite in the
circulating blood, coincides with the appearance of a new
glycoprotein on the surface of the trypanosomes. More than a
thousand genes, each coding for a different surface glycoprotein, are
present in the protozoan chromosome. Only one of these genes is
activated at a time, and the patient’s immune system must respond to
each gene product with production of a new antibody. The recurrent
cycles of parasitemia and antibody production continue until the
patient is treated or dies.
In Trypanosoma brucei rhodesiense infections, the disease
tends to progress rapidly, with the heart and brain invaded within 6
weeks of infection. Irritability, personality changes and mental
dullness results from involvement, but the patient usually dies from
heart failure within 6 months. With Trypanosoma brucei gambiense,
progression of infection is much slower, and years may pass before
death occurs, often from secondary infection. Much of the damage to
the host is due to immune complexes formed when antibody reacts
with complement and high levels of protozoan antigen.
PATHOLOGY
T. brucei effectively evade the human immune system by a
fascinating genetically programmed system of antigenic variation.
When even a single parasite crosses the epithelial layer via the bite of
an infected tsetse fly, local pain and inflammation result as the innate
immune system responds to the invader. Nevertheless, when innate,
non-specific mechanisms of destruction are not sufficient to clear the
pathogen (as is often the case in humans), the parasite grows and
multiples in the bloodstream, while a primary adaptive immune
response is built. Indeed, the adaptive response is evident by the
appearance of redness and swelling at the site of infection 1-2 weeks
after the tsetse fly bite. VSGs on the surface of the parasite are
recognized as foreign antigens by specific BCRs and TCRs.
Generally, a highly specific B-cell mediated antibody response is
generated against to certain VSG epitopes. The adaptive immune
response eventually kills all of the clones of the original parasite via
12
antibody-mediated adaptive immune response. However, some
parasites spontaneously change the VSG coating by altering which
VSG gen is expression. This process is known as gene conversion. In
fact, 806 different genes encode for VSGs. Each VSG gene encodes
for structurally similar, but extremely unique VSGs. Because
different VSGs are distinct enough that they are each seen as
representative of ‘new’ pathogens by the immune system, a separate,
primary adaptive immune response must be generated to clear the
parasites with new VSGs. The time required to mount a sufficient
primary adaptive immune response allows the parasite to divide,
multiply, and occasionally change its VSG surface again. Thus,
although theoretically all T. brucei can eventually be cleared by the
human immune system, spontaneous changes in which VSG is
expressed allow the parasite to stay one step ahead of the adoptive
immune system. Indeed, patients with African trypanosomiasis suffer
from chronic cycles of infection. The disease progresses as these
cycles lead to chronic inflammation, fever, and the build-up of
immune complexes. Furthermore, the severe and often irreversible
neurological damage occurs as parasites reaches deeper and into less
protected areas of the body, such as brain. Fatigue is probably a result
of the body constantly funneling of resources to fight a never-ending
infection.
PATHOPHYSIOLOGY
Humans are infected with T brucei following a fly bite,
which occasionally causes a skin chancre at the site. These injected
trypomastigotes further mature and divide in the blood and lymphatic
system, causing malaise, intermittent fever, rash, and wasting.
Eventually, the parasitic invasion reaches the central nervous system
(CNS), causing behavioral and neurologic changes such as
encephalitis and coma. Death may occur.
The parasites escape the initial host defense mechanisms by
extensive antigenic variation of parasite surface glycoproteins known
as major variant surface glycoprotein (VSG). This evasion of the
humoral immune responses contributes to parasite virulence. During
the parasitemia, most pathologic changes occur in the hematologic,
lymphatic, cardiac, and central nervous systems. This may be the
result of immune-mediated reactions against antigens on red blood
cells, cardiac tissue, and brain tissue, resulting in hemolysis, anemia,
pancarditis, and meningoencephalitis.
A hypersensitivity reaction causes skin problems, including
persistent urticaria, pruritus, and facial edema. Increased lymphocyte
levels in the spleen and lymph nodes infested with the parasite leads
to fibrosis but rarely hepatosplenomegaly. Monocytes, macrophages,
and plasma cells infiltrate blood vessels, causing endarteritis and
increased vascular permeability.
The gastrointestinal system is also affected. Kupffer cell
hyperplasia occurs in the liver, along with portal infiltration and fatty
degeneration. Hepatomegaly is rare. More commonly in East African
trypanosomiasis, a pancarditis affecting all heart tissue layers
develops secondary to extensive cellular infiltration and fibrosis.
Arrhythmia or cardiac failure can cause death prior to the
development of CNS manifestations. CNS problems include
perivascular infiltration into the interstitium in the brain and spinal
cord, leading to meningoencephalitis with edema, bleeding, and
granulomatous lesions.
FREQUENCY
United States
All cases of African trypanosomiasis are imported from
Africa by travelers to endemic areas. Infections among travelers are
rare, with less than 1 case per year reported among US travelers.
Most of these infections are caused by T brucei rhodesiense and are
acquired in East African game parks.
International
African trypanosomiasis is confined to tropical Africa
between latitudes 15°N and 20°S, or from north of South Africa to
south of Algeria, Libya, and Egypt.
The prevalence of African trypanosomiasis varies by
country and region. In 2005, major outbreaks were observed in
Angola, the Democratic Republic of Congo, and Sudan.
In Central African Republic, Chad, Congo, Côte d'Ivoire,
Guinea, Malawi, Uganda, and United Republic of Tanzania, sleeping
sickness remains an important public health problem.
Fewer than 50 new cases per year are reported in countries
such as Burkina Faso, Cameroon, Equatorial Guinea, Gabon, Kenya,
Mozambique, Nigeria, Rwanda, Zambia, and Zimbabwe.
T. brucei transmission seems to have stopped and no new
cases of African trypanosomiasis have been reported for several
decades in countries such as Benin, Botswana, Burundi, Ethiopia,
Gambia, Ghana, Guinea Bissau, Liberia, Mali, Namibia, Niger,
Senegal, Sierra Leone, Swaziland, and Togo.
Sleeping sickness threatens millions of people in 36
countries of sub-Saharan Africa. The current situation is difficult to
assess in numerous endemic countries because of a lack of
surveillance and diagnostic expertise.
In 1986, a panel of experts convened by the World Health
Organization (WHO) estimated that 70 million people lived in areas
where transmission of African trypanosomiasis is possible. In 1998,
almost 40,000 cases of the disease were reported, but this number did
not reflect the true situation given the remoteness of affected regions
and the focal nature of the disease. Between 300,000 and 500,000,
more cases were estimated as remaining undiagnosed and therefore
untreated.
During recent epidemic periods, the prevalence of sleeping
sickness has reached 50% in several villages in the Democratic
Republic of Congo, Angola, and Southern Sudan. Sleeping sickness
was considered the first or second greatest cause of mortality in those
communities, even ahead of HIV infection and AIDS. By 2005,
surveillance had been reinforced and the number of new cases
reported throughout the continent had substantially reduced; between
1998 and 2004, the figures for both forms of African trypanosomiasis
together fell from 37,991 to 17,616.
The estimated number of cases is currently between 50,000
and 70,000. The current epidemic, which began in 1970, is thought to
have been facilitated by factors such as the halting of screening
programs, population migration, civil war, economic decline, and
reduced health care financing.
13
Mortality/Morbidity
• The symptoms of East African trypanosomiasis develop
more quickly (starting 1 month after bite) than the
symptoms of West African trypanosomiasis, which can
begin months to a year after the first bite.
• Both types of African trypanosomiasis cause the same
generalized symptoms, including intermittent fevers, rash,
and lymphadenopathy. Notably, individuals with the East
African form are more likely to experience cardiac
complications and develop CNS disease more quickly,
within weeks to a month. The CNS manifestations of
behavioral changes, daytime somnolence, nighttime
insomnia, stupor, and coma result in death if untreated.
• In West African trypanosomiasis, the asymptomatic phase
may precede onset of fevers, rash, and cervical
lymphadenopathy. If unrecognized, the symptoms then
progress to weight loss, asthenia, pruritus, and CNS disease
with a more insidious onset. Meningismus is rare. Death at
this point is usually due to aspiration or seizures caused by
CNS damage.
Race
African trypanosomiasis has no racial predilection.
Sex
African trypanosomiasis has no sexual predilection.
Age
Exposure can occur at any time. Congenital African
trypanosomiasis occurs in children, causing psychomotor retardation
and seizure disorders.
CLINICAL SYMPTOMATOLOGY OF HUMAN AFRICAN
TRYPANOSOMIASIS
The symptoms of two diseases are more pronounced in
Caucasian than in the local African population.
After a painful tsetse bite, the chancre represents the initial
lesion at the bite site, characterized by local erythema, edema, heat,
tenderness and a lack of any suppuration. Trypanosomes are present
in the inflammatory tissues. The chancre disappears within 2 or 3
weeks. The disease evolves in two distinct successive phases
determining its two pathological stages. Within a few days after the
tsetse bite, the patient enters the haemolymphatic stage I of the
illness.
Stages 1, The Haemolymphatic Stage
Clinical signs appear very early. Intermittent fever develops
because of the successive waves of invasion of the blood by the
trypanosomes. Adenopathies, splenomegaly, or even hepatological
signs mark the invasion of the reticulo-endothelial system. Skin
eruptions or trypanides are commonly observed. Severe pruritus with
scratching skin lesions becomes unsupportable for the patient.
Cardiovascular alterations are less prominent, especially in
the Gambian form. Irregular febrile episodes are accompanied by
headaches, malaise, exhaustion, anorexia, extreme thirst, muscle and
joint pains, pruritus, anaemia, rash and often-deep hyperesthesia (the
sign of the key of Kerandel). The lymph nodes are generally rubbery
and mobile, painful at the beginning. Palpation of the subclavicular
region (Winterbottom sign) is an important part of the diagnostic
procedure in the Gambian form. Any adenopathy accompanied by
fever should evoke the diagnosis of sleeping sickness in patients from
endemic areas. Later, pruritus generalizes. Edema of the face and
extremities appears early.
Few minor neurological and endocrine disorders may
reveal the precocity of central nervous system (CNS) involvement,
long before any detectable changes occur in the cerebrospinal fluid
(CSF). Daytime somnolence or nighttime insomnia may already be
reported and electroencephalographic (EEG) tracings may reveal
abnormalities. Psychiatric signs, with the alternation of irritability,
changes in personality or mood affecting the daily and professional
life of the patients, constitute often the first manifestation of the
disease. A permanent feeling of coldness marks the endocrine
syndrome, lack of appetite or in contrast hyperphagia, polydipsia and
impotence, amenorrhea or infertility, indicative of vegetative and
sexual disturbances.
Stage 2, The Meningoencephalitic Stage
The meningoencephalitic stage appears slowly and
insidiously over a period of months or years depending on the
trypanosome. However, the clinical signs remain reversible for a long
time with treatment attesting to the predominance of potentially
reversible inflammatory lesions over irreversible demyelinating
lesions. The general signs of the haemolymphatic stage do not
completely disappear: spikes of fever (but sometimes hypothermia),
adenopathies and splenomegaly, cardiovascular manifestations
endocrine disturbances and typical pruritus.
The development of the neurological symptoms is
progressive. As neurological signs occur already in stage I, biological
criteria are the only means to confirm CNS invasion. The threshold
criteria, which are commonly used, are based on CSF examination:
more than 5 cells/µL and/or the presence of trypanosomes.
A wide variety of symptoms is encountered. The main
symptoms from which sleeping sickness was named are daytime
somnolence and nocturnal insomnia, the patients being "sleepy by
day and restless by night". The sleep-wake cycle disturbances are
accompanied with either or many of the following symptoms:
headaches, sensory disturbances with diffuse superficial or deep
sensations (muscle and bone hyperesthesia, either spontaneous or
provoked; hyperpathia), presence of primitive reflexes (palm-mental
reflex, sucking reflex), exaggerated deep tendon reflexes, psychiatric
disorders (confusion, mood swings, agitation, aggressive behaviour,
euphoria, absent gaze, mutism, indifference), and tremor (fine and
diffuse without any myoclonic jerk at rest or during movement).
Pyramidal alterations revealed by a Babinski sign can also be
observed along with alterations in muscle tone, numbness or sensory
deficit.
An abnormal number of monocytic cells is observed in the
CSF. The early neurological symptoms correlate with the widespread
meningeal inflammation, which occurs in both forms of HAT. The
selective CNS locations explain in part the principal clinical
neurological signs. Sleep-wake disturbances may result from invasion
14
of the median eminence by a parasite, which also accounts for neuro-
endocrine dysfunctions with the involvement of the suprachiasmatic
nuclei. Disorders of the sleep-wake cycle are accompanied by a state
of apathy in the patient, the loss of muscle tone especially in the neck
muscles and a drooping of the eyelids. Extrapyramidal symptoms
signal the involvement of the striatum. Deep sensory disturbances
with hyperpathia may result from the involvement of the thalamus
and the early invasion of posterior spinal roots.
Apart from the disruptions of the circadian rhythm of the
sleep-wake cycle, other biological rhythms are disturbed, such as
body temperature, cortisol and prolactin or growth hormone
secretion. The invasion of the subthalamic and hypophyseal regions
account for the persistence of ad-endocrine disturbances such as
impotence, amenorrhea or infertility and the development of
disturbed sensations of hunger and thirst, with often hyperphagia and
polydipsia in contrast to the poor general state of malnutrition of the
patients. At the terminal phase of the disease, CNS demyelination and
atrophy are accompanied with disturbances in consciousness and the
development of dementia with incoherence, incontinence and
epileptic fits. The patient dies in a state of cachexia and physiological
misery.
History
• Stage 1 (early, or hemolymphatic, stage)
o Painless skin chancre that appears about 5-15
days after the bite, resolving spontaneously after
several weeks (seen less commonly in T brucei
gambiense infection)
o Intermittent fever (refractory to antimalarials),
general malaise, myalgia, arthralgias, and
headache, usually 3 weeks after bite
o Generalized or regional lymphadenopathy
(Posterior cervical lymphadenopathy
[Winterbottom sign] is characteristic of T brucei
gambiense African trypanosomiasis [sleeping
sickness].)
o Facial edema (minority of patients)
o Transient urticarial, erythematous, or macular
rashes 6-8 weeks after onset
o Trypanids (ill-defined, centrally pale, evanescent,
annular or blotchy edematous erythematous
macules on trunk)
• Stage 2 (late, or CNS, stage)
o Persistent headaches (refractory to analgesics)
o Daytime somnolence followed by nighttime
insomnia
o Behavioral changes, mood swings, and, in some
patients, depression
o Loss of appetite, wasting syndrome, and weight
loss
o Seizures in children (rarely in adults)
Physical
• Stage 1 (early, or hemolymphatic, stage)
o Indurated chancre at bite site
o Skin lesions (trypanids) in light-skinned patients
o Lymphadenopathy: Axillary and inguinal
lymphadenopathy are more common in patients
with East African trypanosomiasis. Cervical
lymphadenopathy is more common in patients
with West African trypanosomiasis. The classic
Winterbottom sign is clearly visible (ie, enlarged,
nontender, mobile posterior cervical lymph
node).
o Fevers, tachycardia, irregular rash, edema, and
weight loss
o Organomegaly, particularly splenomegaly (T
brucei gambiense African trypanosomiasis)
• Stage 2 (late, or CNS, stage)
o CNS symptoms: The CNS symptoms of West
African trypanosomiasis have a slower onset of,
ie, months to a year. Symptoms include
irritability, tremors, increased muscle rigidity and
tonicity, occasional ataxia, and hemiparesis, but
rarely overt meningeal signs. East African
trypanosomiasis usually has a faster onset, ie,
weeks to a month, and does not exhibit a clear
distinction between the two stages.
o Kerandel sign, including delayed pain on
compression of patient's soft tissue
o Behavioral changes consistent with mania or
psychosis, speech disorders, and seizures
o Stupor and coma (giving rise to the name
sleeping sickness)
o Psychosis
o Sensory disorders, tremor, and ataxia
Disease Management
Disease management is performed in three steps:
• Screening for potential infection. This involves the use of
serological test and/or checking for clinical signs –
generally swollen cervical glands.
• Diagnosing shows whether the parasite is present.
• Staging to determine the state of progression of the disease
entails examination of cerebro-spinal fluid obtained by
lumbar puncture and is used to determine the course of
treatment.
Diagnosis must be made as early as possible and before the
neurological stage in order to avoid complicated, difficult and risky
treatment procedure.
DIAGNOSIS
Laboratory Studies
• General
o In African trypanosomiasis (sleeping sickness),
the most significant laboratory abnormalities
include anemia, hypergammaglobulinemia, low
complement levels, elevated erythrocyte
sedimentation rate (ESR), thrombocytopenia, and
hypoalbuminemia, but not eosinophilia or
abnormal liver function.
o In West African trypanosomiasis, the total
immunoglobulin M (IgM) level is notably higher
in blood and CSF (along with high CSF protein).
o A definitive diagnosis of infection requires actual
detection of trypanosomes in blood, lymph nodes,
CSF, skin chancre aspirates, or bone marrow.
However, symptomatic improvement after
15
 empiric treatment is the usual confirmatory test in
areas where diagnostic studies are not readily
available.
• Lymph node aspiration at a high dry magnification (X400)
is commonly used as a rapid test for trypanosomes. It
requires immediate search for parasites because they are
mobile for only 15-20 minutes. This test has more utility in
T brucei gambiense trypanosomiasis.
• Blood smear
o A wet smear of unstained blood or Giemsa-
stained thick smear (more sensitive) is used to
evaluate for mobile trypanosomes, again for 15-
20 minutes. Wright and Leishman stains are
inadequate. This technique is most sensitive in
early stages of disease, when the number of
circulating parasites is highest (≥5000/mL),
particularly in T brucei rhodesiense
trypanosomiasis.
o Better assays are now available, including the
hematocrit centrifugation technique for buffy
coat examination and the miniature anion-
exchange centrifugation technique (mAECT),
which filters out the red cells but not the
trypanosomes. This test can be used to detect
parasitemia levels as low as 5 parasites/mL; the
test can be repeated on subsequent days to
increase the yield when results are negative.
• Chancre aspirate can be used as a wet preparation,
especially in East African trypanosomiasis, but a blood
smear is more sensitive.
• Bone marrow aspiration results may be positive in some
patients.
• CSF assay
o Lumbar puncture should be performed whenever
trypanosomiasis is suspected. CSF examination
helps to diagnose and stage the disease. However,
a negative result does not necessarily rule out the
diagnosis.
o The double centrifugation technique is the most
sensitive method to detect the trypanosomes.
o Other CSF findings include elevated WBC count,
elevated IgM levels, elevated total protein levels,
and raised intracranial pressure. An uncommon
characteristic finding is Mott cells, which are
thought to be large eosinophilic plasma cells
containing IgM that have failed to secrete their
antibodies.
o Increased intrathecal synthesis of IgM has been
found to be the most sensitive indicator of CNS
involvement in African trypanosomiasis.
Imaging Studies
• CT scanning and MRI of the head: Both head CT scanning
and MRI reveal cerebral edema and white matter
enhancement, respectively, in patients with late-stage
African trypanosomiasis.
• EEG in neurologic involvement usually shows slow wave
oscillations (delta waves), a nonspecific finding.
Other Tests
• General: Field serology-based diagnosis of African
trypanosomiasis has been slow to progress over the past
decades. Although many research tools are available for
diagnosis, few are used clinically in endemic areas.
• Serologic antibody detection
o The standard serologic assay to diagnose West
African trypanosomiasis is the card agglutination
test for trypanosomiasis (CATT).
o The CATT can be conducted in the field without
electricity, and results are available in only 10
minutes. It is highly sensitive (96%) but less
specific because of cross-reactivity with animal
trypanosomes.
o Commercial antibody tests for Eastern African
trypanosomiasis are not available.
• Antigen detection tests based on enzyme-linked
immunosorbent assay (ELISA) technology have been
developed. They have shown inconsistent results and are
not yet commercially available.
• Culture of CSF, blood, bone marrow aspirate, or tissue
specimens can be performed in liquid media.
• Other tests developed but not frequently used clinically
include antibody detection in the CSF and intrathecal space
(low sensitivity), polymerase chain reaction (PCR), and
serum proteomic tests.
• Research tools such as isoenzyme analysis and restriction
fragment length polymorphism (RFLP) are used for
definitive subspecies identification.
Procedures
• Lumbar puncture: CSF fluid is used to detect trypanosomes
and to measure WBC counts, protein, and IgM in patients
with parasitemia or positive serologies or symptoms.
Importantly, CNS disease can manifest early in East
African trypanosomiasis.
Differential Diagnoses
Other Problems to Be Considered
• Stage 1 (early) African trypanosomiasis (sleeping sickness)
Symptoms
Differential diagnoses of recurrent fever include malaria,
HIV infection, borreliosis, and brucellosis, typhoid fever, and other
enteric fevers.
Differential diagnoses of lymphadenopathy include
tuberculosis (TB) lymphadenitis, HIV infection, and cancer.
• Stage 2 (late) African trypanosomiasis symptoms
Differential diagnoses of mental status changes include TB,
meningitis, and HIV-related opportunistic infections, including
cryptococcal meningitis.
TREATMENT
Medical Care
16
 • Prehospital care of African trypanosomiasis (sleeping
sickness) centers on management of the acute symptoms of
fever and malaise while closely monitoring the patient’s
neurologic status.
• In the emergency department, if CNS symptoms are severe,
then airway management to prevent aspiration becomes
important, along with an immediate blood smear, CBC
count, and lumbar puncture for trypanosome detection.
Medications
Stage 1
Type of Trypanosomiasis
(Hemolymphatic Stages)
East African trypanosomiasis
Suramin 100-200 mg IV test dose, then
(caused by T brucei rhodesiense)
1 g IV on days 1,3,7,14,21
West African trypanosomiasis
Pentamidine isethionate 4 mg/kg/d IM
(caused by T brucei gambiense)
for 10 d or
Suramin 100-200 mg IV test dose, then
1 g IV on days 1,3,7,14,21
Suramin (Metaret)
Antiparasitic agent used IV in early-stage African
trypanosomiasis and onchocerciasis. Suramin is a polysulfonated
naphthylamine derivative of urea. Suramin is trypanocidal and works
by inhibiting parasitic enzymes and growth factors. Highly bound to
serum proteins and, thus, crosses the blood-brain barrier poorly.
Serum levels are approximately 100 mcg/mL. Suramin is more
effective and less toxic than pentamidine. Excreted in the urine at a
slow rate.
Suramin was introduced in 1920 to treat the first stage of
the disease. By 1922, Suramin was generally combined with
Tryparsamide (another pentavalent organo-arsenic drug) in the
treatment of the second stage of the gambiense form. It was used
Medication
The type of drug treatment used depends on the type and
stage of African trypanosomiasis (sleeping sickness).The drugs used
in the first stage of the disease are less toxic, easier to administer and
more affective. Treatment success in the second stage defends on a
drug that can cross the blood –brain barrier to reach the parasite.
Such drugs are quite toxic and complicated to administer. Four drugs
are registered for the treatment of sleeping sickness and provided free
of charge to endemic countries through a WHO private partnership
with anofi-aventis (pentamidine, melarsoprol, eflornithine) and Bayer
AG (suramin).
Medications
Stage 2
(Neurologic [CNS] Stages)
Melarsoprol 2-3.6 mg/kg/d IV for 3d;
after 1 week, 3.6 mg/kg/d for 3days; after
10-21 d, repeat the cycle
Melarsoprol 2-3.6 mg/kg/d IV for 3d;
after 1 week, 3.6 mg/kg/d for 3 days;
after 10-21 d, repeat the cycle or
Eflornithine 400 mg/kg/d IV in 4 divided
doses for 14 days
during the grand epidemic in West and Central Africa in millions of
people and was the mainstay of therapy until 1969.
Adult
100-200 mg test dose, then 1 g IV on days 1, 3, 7, 14, 21
Pediatric
1-2 mg test dose, then 20 mg/kg IV on days 1, 3, 7, 14, 21
Melarsoprol (Melarsen Oxide-BAL, Mel B, RP 3854)
Trivalent arsenical used in the late or CNS stage of African
trypanosomiasis. Trypanocidal, inhibiting parasitic glycolysis. Water
17
insoluble and has a half-life of 35 h. Serum levels range from 2-5
mcg/mL, but CSF levels are 50-fold lower. The kidneys primarily
excrete the drug. Clinical improvement is usually observed within 4 d
after starting the drug. Therapy is as high as 90-95% successful in
clearing the parasitemia. However, it can be toxic and even fatal in 4-
6% of cases. Studies have now demonstrated the effectiveness of 10-
day melarsoprol treatments for late-stage African trypanosomiasis. In
addition, melarsoprol resistance has become a concern in the Congo
and Uganda; up to 30% of cases do not respond to the drug.
The organo-arsenical melarsoprol (Arsobal) was developed
in the 1940s, and is effective for patients with second stage sleeping
sickness. However, 3 - 10% of those injected have reactive
encephalopathy (convulsions, progressive coma, or psychotic
reactions), and 10 - 70% of such cases result in death; it can cause
brain damage in those who survive the encephalopathy. However,
due to its effectiveness, melarsoprol is still used today. Resistance to
melarsoprol is increasing, and combination therapy with nifurtimox is
currently under research.
Adult
2-3.6 mg/kg/d IV for 3 d; after 1 wk, 3.6 mg/kg/d IV for 3 d; after 10-
21 d, repeat the cycle; always administer each dose slowly on an
empty stomach
Pediatric
0.36 mg/kg IV initially; increase gradually to a maximum 3.6 mg/kg
at intervals of 1-5 d for a total of 9-10 doses; average dosing is 18-25
mg/kg over 1 month
Eflornithine (Ornidyl)
Recommended for treatment of patients with West African
trypanosomiasis, especially late (or CNS) disease. Selective and
irreversible inhibitor of ornithine decarboxylase, which is a critical
enzyme for DNA and RNA synthesis. Generally tolerated better and
is less toxic than arsenic drugs. Available via World Health
Organization. Initial response time is 1-2 wk, used for patients in
whom melarsoprol fails.
Eflornithine (difluoromethylornithine or DFMO), the most
modern treatment, was developed in the 1970s by Albert
Sjoerdsmanot and underwent clinical trials in the 1980s. The drug
was approved by the United States Food and Drug Administration in
1990, but Aventis, the company responsible for its manufacture,
halted production in 1999. In 2001, however, Aventis, in association
with Médecins Sans Frontières and the World Health Organization,
signed a long-term agreement to manufacture and donate the drug.
Adult
100 mg/kg IV q6h for 14 d
Pediatric (Not established)
Pentamidine isethionate (Pentam 300, Pentacarinat, NebuPent)
Antiprotozoal agent usually used for early (or stage 1)
African trypanosomiasis as well as Pneumocystis carinii pneumonia
and leishmaniasis. Works by inhibiting dihydrofolate reductase
enzyme, thereby interfering with parasite aerobic glycolysis. Because
of poor GI absorption, the drug is administered IV/IM and is strongly
bound to tissues, including spleen, liver, and kidney. Clinical
improvement usually noted within 24 h of injection. Reported to have
a >90% cure rate. Pentamidine does not penetrate the blood-brain
barrier effectively and, therefore, does not treat CNS infection.
Pentamidine, a highly effective drug for the first stage of
the disease, has been used since 1939. During the fifties, it was
widely used as a prophylactic agent in Western Africa, leading to a
sharp decline in infection rates. At the time, it was thought that
eradication of the disease was at hand.
Adult
4 mg/kg/d IM/IV for 10 d
Pediatric
Administer as in adults
During the first half of the 20th century, there were very few
efficacious medications. The only one, which was known and used at
first, was an arsenical by-product named Atoxyl it had to be
administered over a several week period of time through course of
subcutaneous injection. As it was a swift killer of trypanosomes
roaming the blood stream, its use during the first stage prevented the
risk of transmission with new fly bites. However, as this medication
does not pass through the meningeal barrier it is not efficacious
against trypanosomes which are already present in the cerebro-spinal
fluid so that the disease can continue to progress in patients who are
experiencing the second stage of this condition.
In 1926, a new arsenical by-product named Tryparsamide
became available. It is given intravenously once a week over a few
weeks. This medication has a great advantage over Atoxyl as it
penetrates into the cerebro-spinal fluid. It is therefore a medication
for the second stage of the disease. Nevertheless about 20% of all
patients are not going to recover and their outcome will be fatal one.
When mass therapy campaigns against Tyrpanosomiasis
got started, only these two medications were available. They are
arsenical by-products with deleterious side effects for the optic nerve,
as a result, most of the time patients are cured but some complain of
ocular side effects and may even wind up fully blind. The need of
repeated injections is another shortcoming as it is difficult to have
patients stick to the program unless they are inpatient over two or
three month period. Then incomplete treatment course give rise to
strains which are resistant to these medication. These first generation
arsenical drugs are obsolete now and not used any longer with the
availability of new molecules, which were developed through
research and development.
An international research team working in the Democratic
Republic of the Congo, New Sudan and Angola involving Immtech
International and University of North Carolina Hill has completed a
Phase IIb clinical trial and commenced a Phase III trial in 2005
testing the efficacy of the first oral treatment for Sleeping Sickness,
known at this point as “DB289”.
18
Combination therapy
Combination therapy may be more effective than
monotherapy for the treatment of late-stage T brucei gambiense
trypanosomiasis. An open randomized trial involving 278 patients
compared melarsoprol monotherapy (two different dosing regimens),
nifurtimox monotherapy, and melarsoprol-nifurtimox combination
therapy. The 48 relapses reported in the study were limited to patients
receiving one of the 3-monotherapy regimens. The trial concluded
that a consecutive 10-day low-dose melarsoprol-nifurtimox
combination is more effective than the standard melarsoprol regimen.
A trial that compared the efficacy of eflornithine
monotherapy to nifurtimox-eflornithine combination therapy in
patients with late-stage T brucei gambiense infection found that cure
rates were similar. However, adverse effects were more common in
patients who received eflornithine monotherapy (25.5% vs 9.6%).
Thus, the nifurtimox-eflornithine combination appears to be a
promising regimen for use in late-stage T brucei gambiense
trypanosomiasis.
Anthelmintics
Parasite biochemical pathways are sufficiently different
from the human host to allow selective interference by
chemotherapeutic agents in relatively small doses.
Follow-up
Further Inpatient Care
• If late or CNS disease complications and coma occur,
intensive care unit (ICU) staff are needed while treatment is
administered (i.e., melarsoprol for East African
trypanosomiasis or eflornithine for West African
trypanosomiasis). Monitor potential adverse effects from
such drugs, including hematologic, renal, and hepatic
function.
Further Outpatient Care
• In both early- and late-stage trypanosomiasis, treatment
usually resolves symptoms and clears the parasitemia on
repeat blood smears.
• Patients who have recovered from late-stage East African
trypanosomiasis should undergo lumbar punctures every 3
months for the first year. Patients who have recovered from
West African trypanosomiasis should undergo lumbar
punctures every 6 months for 2 years. A relapse is
suggested if symptoms return, the CSF WBC count is
above 20 cells/µL, CSF pleocytosis occurs, or if
trypanosomes are still present in blood or CSF. A
persistently elevated CSF WBC count can be observed in
recovering patients, however, so the change (increase or
decrease) in WBC count is more helpful diagnostically. If a
relapse is noted, consider repeat treatment with melarsoprol
or eflornithine.
Prognosis
• In early, or stage 1, trypanosomiasis, most patients
experience full recovery following treatment.
• In late, or stage 2, trypanosomiasis, the CNS manifestations
are ultimately fatal if untreated. The cure rate approaches
95% with drugs that work inside the CNS (eg,
melarsoprol).
MEDICAL ECOLOGY OF SLEEPING SICKNESS: THE
VECTOR
Distribution
Tsetses (pronounced se’tse’, tse’tse’), sometimes-spelled
tzetze, are large biting flies inhabiting much of mid-continental
Africa between the Sahara and Kalahari deserts. They live by feeding
on the blood of vertebrate animals and are the primary biological
vectors of trypanosomes, which causes human sleeping sickness and
animal trypanosomiasis, called nagana.
Tsetse flies are 6-14 mm long, excluding the proboscis. The
broad head bears three ocelli and two large compound eyes, which
are widely spaced in both sexes. The pair of short antennae has three
segment and each an arista with its characteristically branched hairs.
Hygro-, thermo-, chemo- and mechanoreceptors are concentrated on
the antennae. The mouthparts consist of three unpaired components,
the piercing labium, the labrum enclosing the food channel, and the
hypopahrynx with the salivary channel. All are sheathed by two
modified maxillarypalps. On the back, the thorax ends in a caudal,
triangular scutellum. The eight abdominal segments can distend
greatly after feeding. Genital organs, males possessing a button-like
hypopygium under the dorsal end of the body, which covers the
clasper, can only distinguish males and females. The white larvae are
elongated, without eyes and legs, at the posterior end possessing a
pair of black polypneustic lobes.
There are 31 species and subspecies of tsetse flies under the
genus Glossina, family Glossinidae, and order Diptera. Tsetse flies
are largely classified into three subgenera based on morphological
differences in the structure of the genitalia: Morsitans (Glossina),
Palpalis (Nemorhina), and Fusca (Austenina) groups. Although the
tsetse flies can be found over some 9 million squared kilometers of
the African continent, presence of glossina populations throughout
the continent are far from continuous. In general, the Sahara and
Somali Deserts limit the populations in the north, extending across
the entire continent from Senegal in the west to southern Somalia in
the east. Tsetse populations are denser in West and Central Africa,
and are found more sporadically to the East and down to the borders
of the Kalahari and Namibian Deserts in Southern Africa. Although
tsetse fly habitats may vary considerably, climate and altitude -
through their direct effects on vegetation, rainfall, and temperature -
are still the primary determinants for proliferation. Unlike other
insects, there are no seasonal interruptions in the life cycles of tsetse
flies. However, both adult longevity and puparial duration are related
to temperature, and a significant seasonal decline in tsetse
populations is normal, particularly in savannah habitats during the
dry season.
Behavioral Ecology of the Vector: Mating
The mating behaviors of tsetse flies have received much
attention because of the development of the Sterile Insect Technique
(SIT) for tsetse fly control. The existences of tsetse flies at low
densities in certain areas suggest highly specific mating mechanisms
involving visual and olfactory responses. Female tsetse flies only
need to mate once in their life time, but multiple mating have been
19
known occur occasionally. Cross mating is possible in areas where
habitats of different species overlap, however male hybrids are
infertile. Mating of female is mostly confines to early life with mean
duration of mating declining by age. Most female flies are
successfully inseminated even at very low population densities;
usually during their first blood meal right after emerge from the pupal
stage.
Behavioral Ecology of Vector: Host Feeding
Tsetse flies use visual and olfactory characteristics to
recognize potential hosts before initiating host-oriented responses.
There are series of behavioral responses involved in the process of
obtaining a blood meal. Host-seeking behaviors are influenced by
endogenous and exogenous factors. Endogenous factors include
circadian rhythm of activity level of starvation, age, sex, and
pregnancy status of the fly. Exogenous factors include temperature,
vapor pressure, visual and olfactory stimuli, and mechanical
stimulation.
Four Stages of Host-locating Behavior
• Ranging
Flying in search of a host in the absence of an
external cue.
• Activation
Change in behavior caused by perception of
external stimulus
• Orientation
Upwind anemotaxis in response to complex
chemical and visual directing the insect to the
host
• Landing
Generally, the tsetse fly will detect an odor plume
upwind until it visually recognizes the host. After
landing on the host, heat stimulation cause a
probing and feeding response.
Life Cycle of the Vector
Unlike most insect species (r-strategists) that produce large
quantities of eggs, fertilized female tsetse flies (k-strategies) “give
birth” to one larva. A typical female tsetse fly will produce one full
grown larva approximately every 9-10 days depending on the
temperature and humidity. A single egg will hatch and develop to a
third-stage larva in the uterus of the female fly, where it is nurtured
and supplied with nutrients. This reproductive process is known as
adenotrophic viviparity.
MEDICAL ECOLOGY OF SLEEPING SICKNESS: THE
HUMAN
Although epidemics as large as the ones in Uganda at the
turn of the century have not been repeated, there is much concern
over the re-emergence and increase in the number of sleeping
sickness cases being reported every year in Africa. In 1994, there
were an estimated 150,000 cases in Congo, with prevalence as high
as 70% in some villages. Despite the WHO projection of 60 million
people at risk in Africa, only a fraction of the population at risk is
currently under surveillance, and relatively few cases are accurately
diagnosed annually. Although sleeping sickness was largely under
control during the 1960s, recent epidemics have been strongly
associated with political and civil unrest in West and Central Africa
resulting in mass movement of populations into areas formerly
uninhabited by humans.
In 2005, major outbreaks have been observed in Angola,
the Democratic Republic of Congo and Sudan. In Central African
Republic, Chad, Congo, Côte d’Ivoire, Guinea, Malawi, Uganda and
United Republic of Tanzania sleeping sickness remains an important
public health problem. Countries such as Burkina Faso, Cameroon,
Equatorial Guinea, Gabon, Kenya, Mozambique, Nigeria, Rwanda,
Zambia and Zimbabwe are reporting fewer than 50 new cases per
year. In countries such as Benin, Botswana, Burundi, Ethiopia,
Gambia, Ghana, Guinea Bissau, Liberia, Mali, Namibia, Niger,
Senegal, Sierra Leone Swaziland and Togo transmission seems to
have stopped and no new cases have been reported for several
decades. Nonetheless, it is difficult to assess the current situation in a
number of endemic countries because of a lack of surveillance and
diagnostic expertise.
Epidemiology of West African Sleeping Sickness
Both protozoa species are morphologically
indistinguishable but have drastically different epidemiological
features
West African sleeping sickness is typically a chronic
disease, making it a difficult disease to diagnose in the field. Low
levels of trypanosomes in circulating blood make it difficult to detect
the presence of parasites in blood smears, requiring more
sophisticated means of detecting trypanosomes such as with the use
of miniature anion-exchange/centrifugation (mAEC) technique. In
comparison to the East African form, T. b. gambiense has a longer
evolutionary history with humans, having successfully adapted to
establishing infections in human hosts without manifesting severe
symptoms. Astonishingly, infection rates of T. b. gambiense in wild
glossina populations are as low 0.1%, even in areas with an epidemic
of sleeping sickness.
Vectors of West African sleeping sickness are species of
the palpalis group, most of which are in close contact with humans.
Several different reservoirs for T. b.gambiense have been identified,
strongly suggesting that other animals, such as the African domestic
pig, may maintain the persistence of sleeping sickness in human
populations. However, T.b.gambiense has not been observed or
proven experimentally to reach significantly infectious levels of
parasetemia in other reservoir host. Although it is widely accepted
that the human-to-fly contact is the main route of transmission, some
suggest a minor cycle involving an animal reservoir may help explain
the re-emerge and persistence of the disease in West Africa.
The epidemiology of T.b. gambiense sleeping sickness is
far from being fully understood. Despite the low levels of parasitemia
in humans, the disease has successfully established endemicity in
many regions of West Africa. It has also long been observed that the
incidence of disease is not related to the density of the glossina
populations, and that epidemics often occur in areas where the
density of the vector is low. In Nigeria, sleeping sickness occurred in
the north where the distribution of G.p. palpalis and G.tachinoides
were scare and restricted to vegetation close to watercourses during
the dry season. In Southern Nigeria, the same species of tsetse flies
are found in abundance due to favorable climatic conditions, yet
20
cases of sleeping sickness have never been observed. It is thought
that the nature of the human-fly contact is of particular importance in
the transmission of T. b. gambiense and the distribution of the
disease, and that human-fly contacts can be classified as “personal”
or “impersonal” depending on the ecological circumstances of the
interaction.”Personal” contact refer to situations where fly
movements are restricted to areas where exposure to humans are
frequent, such as a watering hole or a stream, and single tsetse fly can
have multiple opportunities to feed on humans. “Impersonal” contact
occurs when fly movements are less restricted, and where repeated
contacts are not likely. In general, ecological isolation of tsetse flies
in the vicinity human populations leads to increased “personal”
contact. Climatic stress, lack of natural host where humans have
destroyed wild animals close to villages, or clearing vegetation for
cultivation are all examples of restrict movements of palpalis group
vectors.
Epidemiology of East African Sleeping Sickness
East African sleeping sickness differs from West African
sleeping sickness in both its epidemiology as well as its clinical
manifestations in mammalian host. The clinical symptoms of East
African sleeping sickness are more severe, and the onset of disease is
rapid. In contrast to T. b. gambiense, T.b. rhodesiense occurs with
higher levels of parasitemia in ungulates, and humans are the
adventitious hosts. The vectors of T.b. rhodesiense and the
G.morsitans subspecies, G.pallidipes and G.swnnertoni species from
the morsitans group, and on lesser occasions the peridomestic vectors
from the palpalis group, G.fuscipes and G.tachinoides. Sporadic cases
usually arise from among those in the population whose activities
bring them into contact with the savannah woodland habitats of the
morsitans group. Although the vectors normally feed on game
animals, under extreme situations where “personal” contact is
increased due to social and environmental factors, a human-fly-
human transmission cycle may ensue resulting in an outbreak.
Droughts and political turmoil are known to increase the number of
cases when entire communities relocate to hitherto unoccupied areas
in search of safety or fertile lands and water.
PREVENTION AND CONTROL
Prevention
There is no available vaccine and Chemoprophylaxis in
unavailable. Trypanosomes are able to switch at random to over
1000, different antigens making it difficult for the body to create the
appropriate antibodies
Preventive measures can help:
1. Wear protective clothing, including long-sleeved shirts and
pants. The tsetse fly can bite through thin fabrics, so
clothing should be made of medium-weight material.
2. Wear neutral-colored clothing. The tsetse fly is attracted to
bright colors and very dark colors.
3. Inspect vehicles for tsetse flies before entering. The flies
are attracted to moving vehicles.
4. Avoid bushes. The tsetse fly is less active during the hottest
period of the day. It rests in bushes but will bite if
disturbed.
5. Use insect repellant. Though insect repellants have not
proven effective in preventing tsetse fly bites, they are
effective in preventing other insects from biting and
causing illness.
Control
In the absence of vaccine for trypanosomiasis and the
looming threat of further trypanocidal drug resistance, the most
theoretically desirable means of controlling the disease is through the
vector population. Although complete eradication of the vector is
impossible, the most successful attempts at controlling tsetse flies are
likely to be at the extreme limits for survival of the fly, where both
the density of the fly is low and “personal” contact with humans may
be highest.
Chemical Control
There are several different control techniques available
today, but the use of chemicals in controlling tsetse populations is
still the most common method. In brief, whether aerial or from the
ground, residual insecticides such as organochlorines (DDT, Dieldrin,
Endosulfan), pyrethroids (deltamethrin, pyrethoids (deltamethrin,
permethrin, and alphamethrin), and avermectins (ivermectin) are used
as a target areas where human-to-fly contact are likely. Pyrethroids
are preferred because they are rapidly degraded in soil and are
environmentally safe, unlike organochlorines, carbamates and
organophospates that a bioaccumulate in the food chain and are
highly toxic to mammals and other vertebrates. Despite being
effective, the use of organochlorines and organophosphates are now
banned for widespread outdoor spraying. Susceptibility to
insecticides varies from one species to another, and between the
different classes of species.
Targets and Traps
Traps and target are mechanical devices used to kill or
weaken tsetse flies through insecticides or various trapping methods.
The use of traps and targets to control tsetse populations have been
successful primarily because tsetse flies are k-strategies with a low
rate of reproduction, and require very little sustained mortality
pressure to bring about a reduction in population or even eradication
from an area. Hargrove estimated that an additional mortality of 4%
per day imposed on female flies was enough to cause extinction, in
the absence of immigration. The traps and targets attract tsetse flies
by taking advantage of their host-seeking behaviors, visual and
olfactory stimulation. The developments of potent attractants in the
last 20 yrs as well as the production of second-generation synthetic
pyrethroid insecticides are making this form of control technique
highly successful.
Bush Clearing
Exploiting the knowledge that tsetse flies concentrated in
certain areas lead to numerous bush-clearing projects all over West
and East Africa to drastically alter and maintain the area unsuitable
for tsetse fly habitation. Discriminative bush clearing was used in
Uganda to control G.m. centralis is by clearing taller Acacia trees in
the Ankole district. In Tanzania, between 1923 and 1930, bush-
clearing methods were also widely employed to stop the spread of
sleeping sickness epidemic in Maswa district, where G. swynnertoni
was prevalent. Similar tactics were used in Ghana to control sleeping
sickness around villages were human-fly contacts were high. Despite
the apparent success of these methods, it is widely accepted that
bush-clearing is unsuitable as a long term control measure due to the
expense and speed of reinvasion, as well as the environmental
21
damage it causes through soil erosion, decreased soil fertility, and its
adverse effects its adverse effects on water supplies
Sterile Insect Techniques
One of the modern methods of non-insecticidal control is
the Sterile Insect Technique (SIT), which was first considered as a
means to control tsetse by Simpson in 1958. This technique relies on
the mating of wild females with sterile male flies. Physiologically,
female tsetse flies only required to mate once to store sperm in its
spermathecae in sufficient quantity such that fertilization can occur
over its entire reproductive life. Mating with a sterile male would
thus result in no offspring.
Sterilization of male Tsetse flies can be carried out by:
 Irradiation
Gamma rays, Beta rays
 Chemo sterilization
Bisazir, Metepa, Tepa, Apholates, Phytosterols
 Physiologic sterilization
Pyriproxyfen, Sulphaqunizaline, Chlordimeform
The Future of Sleeping Sickness Control
After the publication of works such as “Silent Spring”,
public awareness of the dangers associated with insecticides are
increasingly changing the way we treat our environment, and the way
we institute environmental controls. Consequently, efforts to
introduce more environmentally friendly methods of vector control,
such as the use of traps without insecticides, challenges us to
understand more about the vectors that transmit the disease, as well
as the ecological balance that we - as humans - strike with them.
We live in a world where various technical means of
control are available to address the spread of the disease. However,
sleeping sickness is a disease of the developing world, where despite
the multitude of control strategies, the issues have widely been
neglected and abandoned. One of the key components required to
bring about effective change is to consider the sustainability of the
control strategy, and to encourage local communities to take
ownership over the process, thereby empowering people to take an
active role in an environmentally conscious solution. Increasing
knowledge through culturally sensitive education, providing technical
support, and a long-term commitment of basic resources to
beneficiary communities is essential for large-scale tsetse control.
Alongside efforts to reduce the spread of disease through
environmental controls, there is also an urgent need to improve
current surveillance and diagnostic procedures. Mortality can be
drastically reduced when cases can be diagnosed early enough to
prevent the progression of late-stage sleeping sickness. Training and
resources are desperately needed in endemic areas for proper
diagnostics and sero-surveillance.
Perhaps the most mysterious aspect of this disease relates
to the issue of treatment options, and the availability of drugs in
Africa. Drug and vaccine development for diseases in developing
countries have always been lagging, and unfortunately, trypanocidal
drugs are no exception. An estimated 300,000 – 500,000 people are
currently infected and suffering from the disease with no hope for
treatment. In 2000, the USFDA approved the use of eflornithine by
the Bristol-Myers Squibb Company and The Gillette Company in a
product called Vaniqa TM, a topical eflornithine HCl cream to
remove facial hair. Perhaps some of the profits generated from the
sale of this form of the drug will be used to underwrite the free use of
the drug in Africa; similar to what has already happened at Merck,
who donates invermectin for the treatment of river blindness, and at
Pfizer Inc for their azithormycin give away program for the treatment
of trachoma.
SURVEILLANCE
Sleeping sickness is one of the few communicable diseases
where systematic population screening is necessary, particularly for
gambiense sleeping sickness which has a very long almost
asympotomatic period. There are several reasons for this including
the difficulty of diagnosis, which cannot normally be made in remote
primary health care facilities (4), the difficulty and high risk of
treatment for the late stage, for which special skills are required, and
the near impossibility of vector control. Therefore, the control
measure most often used for gambiense sleeping sickness is
systematic screening of the population to detect all cases, including
those in both the first and second stage of disease, and then curing
them. Guidelines for sleeping sickness surveillance have been
developed by WHO in collaboration with sleeping sickness endemic
countries (5).
______________________________________________________
(4) Serological detection with the CATT test (Card Agglutination
Trypanosomiasis Test) is commonly used in screening. However, this
test has insufficient specificity (too many false positives) to be used
as a definitive diagnosis. Parasitological examinations are sufficiently
specific, but are not sensitive enough unless done over a period of
several successive days, because the level of parasites in the blood
oscillates rapidly. If the blood is taken during the part of the cycle
when few parasites are circulating, then a parasitological examination
is likely to be negative, even though the disease is present.
Appropriate treatment depends on whether or not the parasite has
passed the blood/brain barrier. A spinal tap is needed for determining
this.
(5) Trypanosomiase Humaine Africaine: Surveillance
epidemiologique et systeme d'information geographique (S.I.G),
Geneva, World Health Organization, 1996.
Surveillance Strategies
The five alternative surveillance strategies. These were the
classic mobile teams, fixed-post surveillance and the less widely used
innovative techniques of filter paper sampling by trained community
animal health workers, either visiting the community or based at rural
health centers.
• Fixed-post or passive surveillance, where patients
presenting with symptoms that are difficult to diagnose or
don't respond to treatments, say for malaria, are eventually
referred to a treatment centre and tested for a variety of
disease, including trypanosomiasis, and those with the
disease are eventually diagnosed. The initial screening test
is performed on wet blood.
• Filter paper sampling at rural health centers, where
community health workers based at rural health centers
receive some training in collecting samples on filter paper
22
 and then routinely test any new patients presenting © 2007 by the Infectious Diseases Society of America. All rights
themselves at the health centre for whatever reason. reserved.
• Filter paper samplings by community health workers, 1058-4838/2007/4511-0006$15.00
who have been trained in collecting filter paper samples DOI: 10.1086/522982
and then spend 20% of their time collecting samples and CSE THEME ARTICLE MAJOR ARTICLE
following up seropositive individuals. This was based on
experience in Uganda and Côte d’Ivoire. Nifurtimox‐Eflornithine Combination Therapy for Second‐Stage
• Monovalent mobile teams, the classic surveillance teams; Trypanosoma brucei gambiense Sleeping Sickness: A Randomized
in the scenario the CATT is performed on whole blood and Clinical Trial in Congo
all the parasitological tests except for lumbar punctures are
done by the team in the field. The monovalent teams work Gerardo Priotto, 1 Serena Kasparian,1 Daniel Ngouama,2
only on trypanosomiasis. Sara Ghorashian,3 Ute Arnold,3
• Polyvalent mobile teams, which operate in the same way
as monovalent teams, except that only a third of their work
Salah Ghabri, 1 and Unni Karunakara3
consists of screening for trypanosomiasis.

Alanine aminotransferase, bilirubin, and creatinine levels were

In order to standardize the results, these were calculated for
measured by a colorimetric method (Randox) in a subgroup of
an area with a human population of 100,000 people, containing ten
patients. The reagents for alanine aminotransferase were different
rural health centers, and where 20 community animal health workers
(Human) for the last 39 patients because of logistical constraints.
were operating.
Abnormal values were defined as follows: bilirubin, >17 μmol/L,
with a >1.5‐fold increase; alanine aminotransferase, >12 IU/L (as
REFERENCES determined for 43 patients using the Randox reagents) or >32 IU/L
(for female subjects) and >42 IU/L (for male subjects, as determined
for 39 patients using the Human reagents), with a >2.5‐fold increase;
www.who.int and creatinine, >80 μmol/L (for female subjects) and >97 1Epicentre,
Paris, France; 2Programme National de Lutte contre la Trypanomose
www.cdc.gov Humaine Africaine, Ministry of Health, Republic of Congo; and
3
Médecins Sans Frontières, Amsterdam, Holland
www.new-medical.net

www.thefreedictionary.com Background. Human African trypanosomiasis caused by

Trypanosoma brucei gambiense is a fatal disease. Current treatment
www.microbiologybyte.com options for patients with second‐stage disease are either highly toxic
or impracticable in field conditions. We compared the efficacy and
www.wikipedia.org safety of the nifurtimox‐eflornithine drug combination with the
standard eflornithine regimen for the treatment of second‐stage
www.pathmicro.med disease.

www.iaea.org.at Methods. A randomized, open‐label, active‐control, phase III

clinical trial comparing 2 arms was conducted at the Sleeping
www.nhm.ac.uk Sickness Treatment Center, which was run by Médecins Sans
Frontières, in Nkayi, Bouenza Province, Republic of Congo. Patients
www.medicalecology.org were screened for inclusion and randomly assigned to receive
eflornithine alone (400 mg/kg per day given intravenously every 6 h
www.medicalhelthcare.info for 14 days) or eflornithine (400 mg/kg per day given intravenously
every 12 h for 7 days) plus nifurtimox (15 mg/kg per day given orally
www.nature.com
every 8 h for 10 days). Patients were observed for 18 months. The
study's outcomes were cure and adverse events attributable to
www.virtualcentre.org
treatment.
www.dictionary.com
Results. A total of 103 patients with second‐stage disease were
www.wrongdiagnosis.com enrolled. Cure rates were 94.1% for the eflornithine group and 96.2%
for the nifurtimox‐eflornithine group. Drug reactions were frequent in
www.healthsystem.virginia.edu both arms, and severe reactions affected 25.5% of patients in the
eflornithine group and 9.6% of those in the nifurtimox‐eflornithine
group, resulting in 2 and 1 treatment suspensions, respectively. There
was 1 death in the eflornithine arm and no deaths in the nifurtimox‐
eflornithine arm.
Journal
Conclusions. The nifurtimox‐eflornithine combination appears to
1 December 2007 be a promising first‐line therapy for second‐stage sleeping sickness.
If our findings are corroborated by ongoing findings from additional
Volume 45, Number 11 sites (a multicenter extension of this study), the new nifurtimox‐
Clinical Infectious Diseases 2007; 45:1435–1442 eflornithine combination therapy will mark a major and multifaceted
advance over current therapies.
23
Received 30 March 2007; accepted 23 May 2007; electronically
published 22 October 2007.
• (See the editorial commentary by Chappuis on pages 1443–
5)
Reprints or correspondence: Dr. Gerardo Priotto, Epicentre, 8 rue
Saint‐Sabin, 75011 Paris, France (gpriotto@epicentre.msf.org).
Human African trypanosomiasis (HAT), or sleeping sickness,
remains a public health challenge in sub-Saharan Africa, with an
estimated 50,000–70,000 new cases per year, of which 20,000 are
detected and reported [1]. The disease is caused by the protozoan
parasite Trypanosoma brucei gambiense, which is transmitted by the
tsetse fly (Glossina species), and it progresses from the
hemolymphatic first stage to the meningoencephalitic second stage. It
is invariably fatal without appropriate treatment. Since 1949,
melarsoprol is the most commonly used treatment for second‐stage
HAT. This arsenical derivative is associated with severe toxic effects
—in particular, reactive encephalopathy, which is fatal in 10%–70%
of cases and affects 5%–10% of treated patients [2, 3]. Moreover,
increasing melarsoprol failure rates (up to 30%) have been reported
in several countries [4–6].
Eflornithine (diethylfluoromethylornitihine), the only new drug
registered in 58 years for the treatment of second‐stage HAT, is a
trypanostatic that inhibits ornithine decarboxylase, an enzyme
essential for cell multiplication and differentiation [7–9]. It is better
tolerated than melarsoprol, and its toxic effects—mainly seizures,
gastrointestinal disorders, and myelosuppression—are reversible if
well managed. Its efficacy is comparable to that of melarsoprol.
However, a major disadvantage of eflornithine is the mode of
administration, requiring 1 slow infusion every 6 h for 14 days (56
infusions in total), a regimen imposed by its short half‐life of 1.5–5 h
[10, 11]. The difficulty in administering eflornithine in resource‐poor
settings explains why melarsoprol continues to be the first‐line
treatment.
Nifurtimox is an orally administered drug used in the treatment of
Chagas disease (American trypanosomiasis) at dosages of 8–20
mg/kg per day for 90–120 days. Although it has not been approved
for treatment of HAT, nifurtimox is used for compassionate treatment
of relapsed disease. Its toxicity, which has been poorly documented,
includes mainly neurological (headache, sleep disorders, agitation,
and confusion) and gastrointestinal (anorexia, nausea/vomiting, and
dyspepsia) dysfunctions [12, 13], some of which increase with the
duration of intake [14, 15]. In the 1970s and 1980s, nifurtimox was
tested empirically in several HAT case series, and the results were
conflicting [14–17]. These studies, which applied different treatment
regimens and evaluation criteria, are difficult to compare.
Drug combinations can potentially avert or delay the emergence of
drug‐resistant organisms. Dosage reductions of each drug combined
may reduce the overall toxicity while maintaining good efficacy.
Combinations may also allow for a simpler administration of
treatment, improving the feasibility of therapy in remote areas with
logistic and staffing limitations.
A first attempt to assess various combinations for the treatment of
second‐stage HAT in Uganda was interrupted because of excess
fatality in the melarsoprol‐nifurtimox arm. Although the results were
inconclusive, the trial showed promising safety and efficacy results
with the nifurtimox‐eflornithine combination [18]. Assessment of this
combination was therefore extended to a case study of 31 patients
that yielded similar results [19].
In 2003, Médecins Sans Frontières and Epicentre, in collaboration
with the Ministry of Health, initiated a randomized, open‐label
clinical trial in Nkayi, Republic of Congo, to evaluate the efficacy
and toxicity of the nifurtimox‐eflornithine combination with doses
equal to those in the 2 previous studies, with additional simplification
of the administration schedule. In 2005, the study was extended to the
Democratic Republic of Congo and Uganda, in partnership with the
Drugs for Neglected Diseases initiative, the World Health
Organization Special Program for Training and Research in Tropical
Diseases, the Swiss Tropical Institute, and national HAT programs.
Methods
To facilitate external comparability, the study methodology was
similar to the methodologies in previous clinical trials involving
second‐stage HAT [18–22].
Participants. Study participants were identified among persons
with cases diagnosed at the treatment center or during active
screening. Inclusion criteria were as follows: confirmed second‐stage
infection, with trypanosomes detected in specimens of blood, lymph,
or CSF, with >20 leukocytes/μL in CSF specimens. Exclusion criteria
were as follows: age, <15 years; pregnancy; history of second‐stage
HAT treated during the preceding 36 months; severe comorbidities
likely to lead to early death during the follow‐up period; hemoglobin
concentration, <5 g/dL; or inability to complete 18 months of follow‐
up for other reasons.
Three ethics committees approved the study protocol and protocol
amendments: (1) the Médecins Sans Frontières Ethical Review
Board, (2) Comité Consultatif de Protection des Personnes dans la
Recherche Biomédicale (Saint‐Germain‐en‐Laye, France), and (3)
the World Health Organization Research Ethics Review Committee
(Geneva, Switzerland). The Congolese Ministry of Health issued
authorization. All participants provided written informed consent.
A data safety monitoring board consisting of 4 independent experts
was formed before study initiation. The data safety monitoring board
received regular reports and issued recommendations for the study
continuation.
Interventions. Participants were randomized into 1 of 2 arms: the
eflornithine arm or the nifurtimox‐eflornithine arm. A scientific
committee established the dosages on the basis of published and
unpublished evidence. The investigational arm received eflornithine
(400 mg/kg per day given intravenously every 12 h for 7 days) plus
nifurtimox (15 mg/kg per day given in oral tablets every 8 h for 10
days), and the active comparator arm received eflornithine (400
mg/kg per day given intravenously every 6 h for 14 days).
Eflornithine was infused over a 2‐h period and was diluted in 250 mL
of normal saline. Nifurtimox doses were repeated if vomiting
occurred within 30 min. All doses were directly administered and
observed by the medical staff. Before commencement of treatment,
all patients with malaria (as determined by microscopic evaluation
and rapid diagnostic testing) received arthemeter‐lumefantrine for 3
days; study treatment was started at least 1.5 days after the last dose
of antimalarial therapy. The administration of drugs for all other
concomitant conditions was postponed until the end of the
hospitalization, unless the clinical need warranted immediate
treatment. Patients and attendants received a food ration of at least
2100 kcal/day each.
24
All patients were examined daily and hospitalized for 7 days after the
end of treatment (or longer, if deemed necessary). A lumbar puncture
was performed on the day after the patient received his or her last
dose, and CSF specimens were examined for trypanosomes.
Laboratory studies, including lumbar puncture examinations and
examinations of blood and lymph specimens, were performed at 3, 6,
12, and 18 months. At each follow‐up visit, the CSF specimen was
examined for parasites after double‐centrifugation, a parallel CSF
leukocyte count was determined, and IgM titers in CSF were
determined using the Latex/IgM reagent (Institut de Médecine
Tropicale) [23], which aimed at increasing the sensitivity of detection
of relapses. Blood samples were examined by capillary tube
centrifugation and quantitative buffy coat [24]. Lymph was aspirated
from any palpable posterior cervical lymph node.
The study follow‐up period was set at 18 months, as recommended
by the Informal Consultation on the Conduct of Clinical Trials in
HAT (World Health Organization, 2004) on the basis of data that
suggested that 70%–90% of relapses occur 18 months after the
completion of treatment. Although the study end point was
determined at 18 months, the national protocol required a last follow‐
up visit at 24 months for all patients.
The definition of relapse is not currently standardized. Relapse was
diagnosed if trypanosomes were seen in body fluid specimens or if
CSF leukocyte counts increased twice consecutively by at least 20
cells/μL any time after the completion of treatment. Patients who
presented with a single increase were reexamined 1 month later. At
the month 18 examination, relapse was diagnosed if the CSF
leukocyte count was 20 cells/μL, regardless of previous counts.
Distinction between relapse and reinfection was not made. The
probability of reinfection was considered to be minimal, because
disease transmission had been substantially reduced after 3 years of
intensive disease‐control activities by Médecins Sans Frontières.
Safety was assessed with the international Common Toxicity Criteria
[25], which grade adverse events by intensity from 1 to 4 (for mild,
moderate, severe, and very severe, respectively), drug‐event
relationship (unrelated, unlikely, possible, probable, and definite),
and outcome (complete recovery, still present, sequelae, and death).
All patients had a blood sample taken before and after treatment; the
sample was examined for hemoglobin concentration, total and
differential leukocyte counts, total bilirubin level, creatinine level,
and alanine aminotransferase level.
Anemia was defined as a hemoglobin concentration <13 g/dL for
male patients and <11 g/dL for female patients that had decreased by
>20%. Leukopenia was defined as a leukocyte count <4000 cells/μL
that had decreased by >30%. Neutropenia as a neutrophil count
<2000 cells/μL that had decreased by >30%.
μmol/L (for male subjects), with a >1.5‐fold increase.
Pharmacokinetic analysis of plasma and CSF drug concentrations
was performed in an ancillary study related to the clinical trial. The
methodology and results will be reported elsewhere.
Outcomes. The primary outcome was cure. The following end
points were regarded as therapeutic failures: (1) death in temporal
relation to treatment (i.e., 30 days after the commencement of
treatment), and (2) relapse of HAT or death compatible with HAT
within the 18 months of follow‐up. Deaths due to disease without
clearly established alternative causality were regarded as compatible
with HAT. Secondary outcomes were the adverse events in temporal
relation to treatment, particularly the major adverse events graded as
severe (grade 3) and very severe (grade 4).
Sample size. The sample size to test non inferiority in cure rates
for the complete multicenter study was set at 280 subjects. This
report analyzes the data of the 103 patients enrolled in Nkayi.
The randomization list (in blocks of 10) was electronically generated.
The list and the block size were concealed from the field team.
Participants were enrolled in the same order in which they received
diagnoses. Sealed and numbered opaque envelopes containing the
treatment allocation were opened in strict numeric sequence.
Blinding was unfeasible owing to the different drug administration
modes.
Data management. Data were collected in purposely designed
patient charts. Trial‐specific data were extracted onto case report
forms. These data were double‐entered electronically with EpiData,
version 3.0 (The EpiData Association), and were analyzed with Stata,
version 9.0 (Stata). No statistical comparisons were performed,
because interim analysis would alter the statistical power of the
overall trial.
Result
Participants. Of 630 patients who had HAT diagnosed during the
trial period, 261 had cases that were in the second stage, of which
103 met the entry criteria and were enrolled in the study (figure 1).
The main reasons for ineligibility were relapsed status, young age,
and/or low CSF leukocytes count. All enrolled patients were treated:
51 were treated with eflornithine, and 52 were treated with
nifurtimox‐eflornithine.
25
Figure 1. Trial profile. E, eflornithine; HAT, human African trypanosomiasis; N+E, nifurtimox‐eflornithine. aRelapses were detected at 12
months in one patient and 18 months in the other. bRelapses were detected both at 18 months. cCondition was controlled at 12 months, with favorable
evolution; patient moved away later. dOne patient had controlled disease at 3 and 6 months, with favorable evolution; the patient later died of cerebral
malaria. The other patient had controlled disease at 3, 6, and 12 months, with favorable evolution; the patient later died of ovarian cancer.

Enrollment started in August 2003 and was closed in December 2004 because of a decrease in the disease prevalence in the area, resulting in a low
enrollment rate. Patient characteristics were similar in the 2 groups (table 1). Four patients (2 in each arm) had CSF leukocyte counts of 5–20
cells/μL and were wrongly enrolled. Because these 4 patients had trypanosomes in the CSF, they were kept in the study. All patients received
complete treatment in accordance with the protocol.

Table 1. Baseline characteristics of trial participants, by treatment arm.

Characteristic Eflornithine arm Nifurtimox‐

(n = 51) eflornithine arm
(n = 52)

Demographic characteristics

Female sex 23 (45.1) 26 (50.0)

Age, mean years (range) 36.1 (15–70) 33.1 (15–69)

Weight, mean kg ± SD 53.1 ± 7.2 51.7 ± 7.4

Mean body mass index a ± SD 19.7 ± 2.2 19.1 ± 2.0

Body mass index a <18.5 15 (29.4) 22 (42.3)

Parasitologic findings

Case detected by active screening 29 (56.9) 31 (59.6)

Presence of trypanosomes

In lymph nodes 40 (78.4) 36 (69.2)

26
 In blood 47 (92.2) 42 (80.8)

In CSF 37 (72.6) 30 (57.7)

Leukocyte count in CSF

5–20 cells/μL 2 (3.9) 2 (3.9)

21–99 cells/μL 10 (19.6) 19 (36.5)

100 cells/μL 39 (76.5) 31 (59.6)

IgM titer in CSF >1:128 b 28 (57.1) 20 (40.8)

Clinical characteristics

Mean Karnofsky score ± SD 84.6 ± 9.6 80.9 ± 15.9

Mean Glasgow coma score ± SD 15.0 ± 0.1 14.8 ± 0.8

Hemoglobin concentration, mean g/dL ± SD 12.7 ± 1.9 13.0 ± 1.8

Presence of malaria parasites 14 (27.5) 11 (21.2)

Lymphadenopathy 40 (78.4) 36 (69.2)

Hepatomegaly 2 (3.9) 5 (9.6)

Splenomegaly 14 (27.5) 11 (21.2)

Headache 35 (68.6) 38 (73.1)

Fever c 13 (25.5) 2 (3.8)

Pruritus 33 (64.7) 33 (63.5)

Daytime somnolence 35 (68.6) 32 (61.5)

Insomnia 18 (35.3) 16 (30.8)

History of seizures 5 (9.8) 4 (7.7)

Psychiatric signs 25 (49.0) 21 (40.4)

Speech disorder 8 (15.7) 9 (17.3)

Impotence or amenorrhea 15 (29.4) 19 (36.5)

Tremors 20 (39.2) 18 (34.6)

Anorexia 6 (11.8) 12 (23.1)

Duration of symptoms, mean months (range) 9.8 (1–72) 8.7 (0–24)

Table 1. Baseline characteristics of trial participants, by treatment arm.

Outcomes and estimation. One patient died in temporal relation to the treatment regimen and was considered to have experienced treatment
failure. The large majority of patients (98 [95.1%] of 103) completed the 18‐month follow‐up period or reached the study end point earlier, and the
rest (5 [4.9%]) underwent partial follow‐up. Of the 5 patients who underwent partial follow‐up, all had demonstrated decreasing CSF leukocyte
counts and IgM titers at their last follow‐up visit. One died of cerebral malaria (last follow‐up visit was at month 6), another died after ovarian cancer
surgery (last follow‐up visit was at month 12), and 3 moved away after the month 12 follow‐up visits. There were 4 relapses in total (2 per arm). All
4 patients who experienced relapse had increasing CSF leukocyte counts without detected trypanosomes; 1 patient had relapse at 12 months, and 3

27
had relapse at 18 months. Cure rates were 94.1% (48 of 51 patients) in the eflornithine arm and 96.2% (50 of 52) in the nifurtimox‐eflornithine arm.
If patients with partial follow‐up are excluded from analysis, the cure rates are 93.3% (46 of 49 patients) in the eflornithine arm and 95.9% (45 of 49)
in the nifurtimox‐eflornithine arm.

Paired before‐after treatment CSF IgM titers were available for 88 patients. Blood contamination of CSF (6% of specimens) and other constraints
(1% of specimens) explain the missing measurements. Lumbar puncture was performed 12 h after the last dose of eflornithine and revealed that the
titers decreased in 40 (46%) of 88 patients, stayed unchanged in 38 patients (43%), and increased in 10 patients (11%). The evolution of the IgM titer
values during follow‐up ( ) showed a consistent decrease over time. Only in patients who experienced relapse did increases in IgM titers
accompany increases in CSF leukocyte counts.

Drug reactions. Only 1 patient, who was in the eflornithine group, died within 30 days after the start of treatment; this death was attributed to
septic shock following severe neutropenia. Of a total of 261 adverse events, 14 were classified as unrelated to treatment, and 247 were regarded as
drug reactions (table 2). Noteworthy differences in overall toxicity included fever, hypertension, and diarrhea, all of which were less common in the
nifurtimox‐eflornithine arm. On the other hand, the combination more frequently provoked nausea and vomiting, which tended to appear 1 h after
the simultaneous administration of both drugs (for the first daily dose) and less frequently when the drugs were administered at different times.

Table 2. Clinical and biological drug reactions during hospitalization.

Event No. (%) of events

Eflornithine arm Nifurtimox‐

(n = 51) eflornithine arm
(n = 52)

All Major All Major

Treatment‐related death 1 (2.0) … 0 (0.0) …

Neurologic reactions

Seizures 2 (3.9) 2 4 (7.7) 4

Confusion 0 (0.0) … 1 (1.9) …

Anxiety and/or agitation 4 (7.8) … 2 (3.8) …

Dizziness 1 (2.0) … 1 (1.9) …

Insomnia 1 (2.0) … 2 (3.8) …

Gastrointestinal reactions

Anorexia 0 (0.0) … 1 (1.9) …

Abdominal pain 11 (21.6) … 10 (19.2) …

Diarrhea 4 (7.8) … 0 (0.0) …

Constipation 0 (0.0) … 2 (3.8) …

Nausea and/or vomiting 8 (15.7) … 26 (50.0) …

Cardiovascular reactions

Arrythmia 3 (5.9) … 1 (1.9) …

Hypertension 8 (15.7) 1 1 (1.9) …

Edema 4 (7.8) … 0 (0.0) …

Infection

Tissue infection 1 (2.0) … 1 (1.9) …

28
 Septic shock (neutropenic) 1 (2.0) 1 0 (0.0) …

Other infection 2 (3.9) 1 3 (5.8) …

Other clinical events

Fever a 13 (25.5) 2 5 (9.6) 1

Headache 15 (29.4) … 13 (25.0) …

Cough 3 (5.9) … 0 (0.0) …

Pruritus 7 (13.7) … 3 (5.8) …

Skin rash 3 (5.9) … 0 (0.0) …

Chest pain 3 (5.9) … 0 (0.0) …

Myalgia and/or arthralgia 3 (5.9) … 3 (5.8) …

Other 2 (3.9) … 4 (7.7) …

Biological reactions

Anemia 11 (21.6) 1 4 (7.7) 0

Leukopenia 1 (2.0) 0 2 (3.8) 0

Neutropenia 29 (56.9) 6 11 (21.2) 1

Abnormal bilirubin levelb 1 (2.0) … 2 (3.8) …

Abnormal alanine aminotransferase levelc 0 (0.0) … 0 (0.0) …

Abnormal creatinine levelc 0 (0.0) … 1 (1.9) …

Weight loss 5% 0 (0.0) … 3 (5.8) …

Total no. of eventsd 141 14 106 6

No. (%) of patients who experienced major events … 13 (25.5) … 5 (9.6)

Treatment interruption 2 (3.9) … 1 (1.9) …

Treatment suspension 2 (3.9) … 1 (1.9) …

Treatment termination 0 (0.0) … 0 (0.0) …

Table 2. Clinical and biological drug reactions during hospitalization.

There were 14 major drug reactions (i.e., those of grades 3 and 4) in the eflornithine arm and 6 such reactions in the nifurtimox‐eflornithine arm, and
the proportions of patients who experienced major reactions were 25.5% and 9.6% per arm, respectively (table 2). Treatment was suspended because
of severe complications for 2 patients in the eflornithine arm and 1 patient in the nifurtimox‐eflornithine arm. These interruptions were made as a
result of seizures (1 patient in each arm) and arrhythmia (1 in the eflornithine arm). The 6 major adverse events observed in the nifurtimox‐
eflornithine arm were seizures (4 patients), fever (1 patient), and neutropenia (1 patient), all of which resolved favorably.

Before‐and‐after treatment hematologic data were available for all patients. Neutropenia and anemia were the most common biological adverse
events, appearing 3 times more frequently in the eflornithine arm. Grade 3 neutropenia (i.e., a neutrophil count <1000 cells/μL) developed in 6
patients in the eflornithine arm, compared with only 1 patient in the nifurtimox‐eflornithine arm. Abnormal biochemical values were rare and mild.

29
Discussion
The results obtained with the nifurtimox‐eflornithine combination are
similar to those obtained with standard eflornithine treatment, even
showing a trend of superiority in the safety parameters. These data
confirm the observations made in our earlier studies from Uganda
[18, 19]. With nearly complete follow‐up data for patients (100%
reviewed at least 2 times and 95% with complete follow‐up), the high
cure rates are reassuring.
The fact that 3 of the 4 relapses were detected at the month 18 visit,
having been controlled at 12 months, suggests that the follow‐up in
clinical studies should be no shorter than 18 months. The lower
occurrence of major drug reactions in the nifurtimox‐eflornithine arm
suggests a better safety profile of the combination.
Neutropenia occurred more frequently among recipients of
eflornithine alone than among recipients of combination treatment,
which contains half as much eflornithine. Patients with grade 3
neutropenia are vulnerable to bacterial infection, which explains the
frequent infectious complications emerging during or after treatment,
as well as the only death in our cohort. The combination clearly
offers improved safety over melarsoprol, which causes reactive
encephalopathy in 5%–10% of recipients, with a high fatality rate.
There were no clinical data to support theoretical concerns about
toxicity related to drug interactions with the combination therapy.
Nkayi presented unusual advantages in comparison to most other
HAT foci, including sociopolitical stability, the supply of electricity,
the availability of internet communication, daily flights to the capital,
good hospital infrastructure, and qualified medical staff. The study
did not achieve the planned sample size of 280 subjects in Nkayi
because of bureaucratic delays that resulted in the bulk of patients
being detected and treated by Médecins Sans Frontières before
enrollment could begin. There were, however, some advantages to
this situation: the smaller caseload facilitated good medical
supervision and eased laboratory work. With a decreased prevalence
of disease, the probability of reinfection during follow‐up was also
minimized.
Limitations. As a result of the insufficient number of patients
recruited at this site, we have not yet performed the noninferiority
analysis designed in the protocol; this will only be possible when the
studies from additional sites are completed. Therefore, these data
should not be regarded as definitive proof.
Overall evidence. In the face of unsatisfactory therapeutic options
for second‐stage sleeping sickness, the need to identify new, safe,
feasible, and effective therapies is urgent [3]. Research is hampered
by the fact that foci with high caseloads that can sustain good
enrollment rates are often affected by conflict and located in isolated
and impoverished parts of Africa. Gathering the minimal elements for
quality research projects (e.g., qualified personnel, infrastructure,
communications, and stable and functional logistics) at such sites
represents a challenge that very few researchers are willing to
assume.
Even if, at this first study site, we did not attain the complete sample
size, we believe that our data are of crucial interest because of the
promising efficacy and safety results of the nifurtimox‐eflornithine
combination, which was tested here for the first time with a simpler
(twice‐daily) eflornithine administration schedule.
The short half‐life of eflornithine theoretically requires shorter
administration intervals for effective therapy, and this was one of the
key issues addressed in this study. We hypothesize that this adverse
pharmacokinetic profile may be balanced by the long‐lasting
pharmacodynamic effect on trypanosomes, explained by the long
time (18–19 h) needed by T. brucei gambiense to replenish their
ornithine decarboxylase after inhibition by eflornithine [26]. This
would give sufficient opportunity for trypanocydal nifurtimox to
eliminate the parasites. Our data seem to confirm that the 12‐h
intervals are possible if the agent is combined with nifurtimox.
The simplified regimen in this nifurtimox‐eflornithine schedule,
which involves 4‐fold fewer eflornithine infusions (i.e., 14 infusions
instead of 56), represents an important advance in terms of access to
safer and effective treatment. Most treatment centers are located near
disease transmission foci, in remote areas where logistical means and
trained staff are scarce, and most patients need treatment for second‐
stage disease. The twice‐per‐day infusions are key because they fit
well in the routine of rural health facilities. Another important
advantage is the cost reduction coming from the shorter
hospitalization durations and from the 4‐fold reduction in the number
of intravenous infusions, requiring fewer infusion materials and
logistics and less staffing.
A degree of protection against the development of drug resistance can
also be expected from combination versus monotherapy, as is the
case for drug combinations in use for other infectious diseases. In the
context of increasing parasite resistance to melarsoprol, and because
eflornithine is the only alternative agent for second‐stage HAT, the
availability of a combination treatment regimen is urgent, to avert the
development of eflornithine resistance as well.
Because these new data reinforce the evidence in favor of the
nifurtimox‐eflornithine combination, timely completion of the
additional sites and ensuring good follow‐up of patients are essential.
It will also be necessary to organize field studies with simpler—but
sound—methodology to assess the administration of treatment in
more‐realistic situations. Studies of children will also be needed. For
all this to be possible, the continued production and availability of
nifurtimox must be ensured. If our findings are corroborated by
ongoing studies from additional sites (i.e., from a multicenter
extension of this study), the new nifurtimox‐eflornithine combination
therapy will mark a major and multifaceted advance over current
therapies.
Acknowledgments
We thank the Congolese Ministry of Health—in particular, Claude
Manthelot and Damase Bodzongo, whose cooperation in setting up
the study was invaluable. We acknowledge the clinical and laboratory
field team members, international and local, whose hard work
permitted this research to happen. The following individuals
contributed to the protocol development in the scientific committee:
Christian Burri, Cyrus Bacchi, Dominique Legros, François
Chappuis, Marc Gastellu‐Etchegorry, Philippe Büscher, and Simon
von Nieuwenhove. The following provided laboratory technical
advice and training: Olivier Denis, Christian Kibonge, Barrie
Rooney, Marina Pozzoli, Laurence Bonte, Philippe Büscher, and
Veerle Lejon. Loretxu Pinoges kindly provided advice on statistical
matters. We are indebted to the members of the Data and Safety
Monitoring Board: Dr. Pieter De Raadt, Dr. Jean Dupouy‐Camet, Dr.
Marleen Boelaert, Dr. Jacques Chandenier and Joris Menten. The
30
Drugs for Neglected Diseases initiative and the Swiss Tropical
Institute provided advice on the Good Clinical Practice guidelines.
Financial support. This study was funded by the Dutch section of
Médecins Sans Frontières. The Médecins Sans Frontières
International Nobel Prize Fund supported the original protocol
development.
Potential conflicts of interest. All authors: no conflicts.
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CSE Global Theme Issue on Poverty and Human Development
Cited by
D. Steverding, X. Wang. (2009) Evaluation of anti-sleeping-sickness
drugs and topoisomerase inhibitors in combination on Trypanosoma
brucei. Journal of Antimicrobial Chemotherapy 63:6, 1293-1295
Online publication date: 1-Jul-2009.
CrossRef
D. Mumba Ngoyi, V. Lejon, F.X. N’Siesi, M. Boelaert, P.
Büscher. (2009) Comparison of operational criteria for treatment
outcome I n gambiense human African trypanosomiasis. Tropical
Medicine & International Health 14:4, 438-444
Online publication date: 1-May-2009.
CrossRef
Peter G. E. Kennedy. (2008) The continuing problem of human
African trypanosomiasis (sleeping sickness). Annals of Neurology
64:2, 116-126
Online publication date: 1-Sep-2008.
CrossRef
Veerle Lejon, Isabelle Roger, Dieudonné Mumba Ngoyi,
Joris Menten, Jo Robays, Francois X. N’Siesi, Sylvie Bisser,
Marleen Boelaert, and Philippe Büscher. (2008) Novel Markers for
Treatment Outcome in Late‐Stage Trypanosoma brucei gambiense
Trypanosomiasis. Clinical Infectious Diseases 47:1, 15-22
Online publication date: 1-Jul-2008.
Abstract-Full Text-PDF Version (402 kB)
François Chappuis
CSE Poverty and Human Development. (2007) Editorial
Commentary: Melarsoprol‐Free Drug Combinations for Second‐
Stage Gambian Sleeping Sickness: The Way to Go. Clinical
Infectious Diseases 45:11, 1443-1445
Online publication date: 1-Dec-2007.
Citation-Full Text-PDF Version (162 kB)
Medical Research
Submitted to: Dr.Mazhar Qayyum
Submitted by: Farthat Yasmeen
Date: 23/o6/2008
Trypanosoma brucei
32
Scientific Classification
Kingdom: Protista
Phylum: Euglenozoa
Subphylum: Mastigophora
Class: Kinetoplastoa
Order: Trypanosomastida
Genus: Trypanosoma
Species: Trypanosoma brucei
Description and Significance:
The genus Trypanosoma contains a large number of
parasitic species, which infect wild and domesticated animals and
humans in Africa. Commonly known as African sleeping sickness,
human trypanosomiasis is caused by the species Trypanosoma brucei
and is transmitted to human through either a vector or the blood of
ingested animal. The most common vector of Trypanosoma brucei is
the tsetse fly, which may spread the parasite to humans and animals
through bites. Through the process of antigenic variation, some
trypanosomes are able to evade the host’s immune system by
modifying their surface membrane, essentially multiplying with every
surface change. As the disease progress, Trypanosoma brucei
gradually infiltrates the host’s central nervous system. Symptoms
include headache, weakness, and joint pain in the initial stages;
anemia, cardiovascular problems, and kidney disorders as the disease
progresses; in its final stages, the disease may lead to extreme
exhaustion and fatigue during the day, insomnia at night, coma, and
ultimately death. Human trypanosomiasis affects as many as 66
million people in Sub-Saharan Africa. Trypanosomes are also found
in the America in the form of Trypanosoma cruzi, which causes
American human trypanosomiasis, or Chagas’ disease. This disease is
found in two forms: as an amastigote in the cells, and as a
trymastigote in the blood. The vectors for Trypanosome cruzi include
members of the order Hemiptera, such as assassin flies, whichingest
the amastigote or trymastigote and carry them to animals or humans.
The parasites enter the human host through mucus membranes in the
nose, eye, or mouth upon release from the insect vectors. Left
untreated, Chagas’ disease may cause dementia, megacolon and
megaesophagus and damage to the heart muscle and may result in
death.
The infection: Trypanosomiasis
The insect vector for T. brucei is the tsetse fly. The parasite
lives in the midgut of the fly (procyclic form), whereupon it migrates
to the salivary glands for injection to the mammalian host on biting.
The parasite lives within the bloodstream (bloodstream form) where
it can reinfect the fly vector after biting. Later during a T. brucei
infection the parasite may migrate to other areas of the host. A T.
brucei infection may be transferred human to human via bodily fluid
exchange, primarily blood transfer.
There are three different sub-species of T. brucei, which cause
different variants of trypanosomiasis.
• T. brucei gambiense - Causes slow onset chronic
trypanosomiasis in humans. Most common in central and
western Africa, where humans are thought to be the
primary reservoir.
• T. brucei rhodesiense - Causes fast onset acute
trypanosomiasis in humans. Most common in southern and
eastern Africa, where game animals and livestock are
thought to be the primary reservoir.
• T. brucei brucei - Causes animal African trypanosomiasis,
along with several other species of trypanosoma. T. b.
brucei is not human infective due to its susceptibility to
lysis by human apolipoprotein L1. However, as it shares
many features with T. b. gambiense and T. b. rhodesiense
(such as antigenic variation) it is used as a model for
human infections in laboratory and animal studies.
The cell structure
The structure of the cell is typical of eukaryotes, see
eukaryotic cell. All major organelles are seen, including the nucleus,
mitochondria, endoplasmic reticulum, Golgi apparatus etc. Unusual
features include the single large mitochondria with a condensed
mitochondrial DNA structure, and its association with the basal body
of the flagellum, unusually the cytoskeleton organization mechanism
of the cell. The cell surface of the bloodstream form features a dense
coat of variable surface glycoproteins (VSGs) which is replaced by
an equally dense coat of procyclins when the parasite differentiates
into the procylic in the tsetse fly midgut. Trypanosomatid cellular
forms.
Trypanosomatids show specific cellular forms:
• Amastigote - Basal body anterior of nucleus, with a short,
essentially non-functional, flagellum.
• Promastigote - Basal body anterior of nucleus, with a long
detached flagellum.
• Epimastigote - Basal body anterior of nucleus, with a long
flagellum attached along the cell body.
• Trypomastigote - Basal body posterior of nucleus, with a
long flagellum attached along the cell body.
These names are derived from the Greek mastig- meaning whip,
referring to the trypanosome's whip-like flagellum.
T. brucei is found as a trypomastigote in the slender, stumpy,
procyclic and metacyclic forms. The procylic form differentiates to
the proliferitive epimastigote form in the salivary glands of the insect.
Unlike Leishmania, the promastigote and the amastigote form does
not form part of the T.brucei life cycle.
The genome
The genome of T. brucei is made up of:
• 11 pairs of large chromosomes of 1 to 6 megabase pairs.
• 3-5 intermediate chromosomes of 200 to 500 kilobase
pairs.
• Around 100 mini chromosomes of around 50 to 100
kilobase pairs. These may be present in multiple copies per
haploid genome.
33
The large chromosomes contain most genes, while the small
chromosomes tend to carry genes involved in antigenic variation,
including the VSG genes. The genome has been sequenced and is
available online.
The mitochondrial genome is found condensed into the kinetoplast,
an unusual feature unique to the kinetoplastea class. It and the basal
body of the flagellum are strongly associated via a cytoskeleton
structure.
The cytoskeleton
The cytoskeleton is predominantly made up of
microtubules, forming a subpellicular corset. The microtubules lie
parallel to each other along the long axis of the cell, with the number
of microtubules at any point roughly proportional to the
circumference of the cell at that point. As the cell grows (including
for mitosis) additional microtubules grow between the existing
tubules, leading to semiconservative inheritance of the cytoskeleton.
The microtubules are orientated + at the posterior and - at the
anterior. Microfilament and intermediate filaments also play an
important role in the cytoskeleton, but these are generally
overlooked.
Flagellar structure
The trypanosome flagellum has two main structures. It is
made up of a typical flagellar axoneme, which lies parallel to the
paraflagellar rod, a lattice structure of proteins unique to the
kinetoplastida, euglenoids and dinoflagellates.
The microtubules of the flagellar axoneme lie in the normal 9+2
arrangement, orientated with the + at the anterior end and the - in the
basal body. The cytoskeletal structure extends from the basal body to
the kinetoplast. The flagellum is bound to the cytoskeleton of the
main cell body by four specialised microtubules, which run parallel
and in the same direction to the flagellar tubulin.
The flagellar function is twofold - locomotion via oscilations along
the attached flagellum and cell body, and attachment to the fly gut
during the procyclic phase.
The VSG coat
The surface of the trypanosome is covered by a dense coat of
~1x107 molecules of Variable Surface Glycoprotein (VSG).[4] This
coat enables an infecting T. brucei population to persistently evade
the host's immune system, allowing chronic infection. The two
properties of the VSG coat that allow immune evasion are:
• Shielding - the dense nature of the VSG coat prevents the
immune system of the mammalian host from accessing the
plasma membrane or any other invariant surface epitopes
(such as ion channels, transporters, receptors etc.) of the
parasite. The coat is uniform, made up of millions of copies
of the same molecule; therefore the only parts of the
trypanosome the immune system can 'see' are the N-
terminal loops of the VSG that make up the coat.
• Periodic antigenic variation - the VSG coat undergoes
frequent stochastic genetic modification - 'switching' -
allowing variants expressing a new VSG coat to escape the
specific immune response raised against the previous coat.
Antigenic variation
Sequencing of the T. brucei genome has revealed a huge
VSG gene archive, made up of thousands of different VSG genes. All
but one of these is 'silent' VSGs, as each trypanosome expresses only
one VSG gene at a time. VSG is highly immunogenic, and an
immune response raised against a specific VSG will rapidly kill
trypanosomes expressing this VSG. This can also be observed in
vitro by a complement-mediated lysis assay. However, with each cell
division there is a possibility that one or both of the progeny will
switch expression to a silent VSG from the archive (see below). The
frequency of such a switch has been measured to be approximately
1:100. This new VSG will likely not be recognised by the specific
immune responses raised against previously expressed VSGs. It takes
several days for an immune response against a specific to develop,
giving trypanosomes, which have undergone VSG coat switching
some time to reproduce (and undergo further VSG coat switching
events) unhindered. Repetition of this process prevents extinction of
the infecting trypanosome population, allowing chronic persistence of
parasites in the host. The clinical effect of this cycle is successive
'waves' of parasitaemia (trypanosomes in the blood).
Trypanosome cell cycle
The mitotic division of T.brucei is unusual in terms of the
cytoskeletal process. The basal body, unlike a centrosome of most
eukaryotic cells, plays an important role in the organisation of the
spindle.
Stages of mitosis:
6. The basal body replicates, both remaining associated with
the kinetoplast.
7. The kinetoplast undergoes replication, and the daughter
kinetoplasts are separated by the basal bodies.
8. The second flagellum grows while the nucleus undergoes
replication.
9. The mitochondrion divides, and cytokinesis progresses
from the anterior to posterior end.
10. The division resolves. The daughter cells may stay
connected for a significant length of time after cytokinesis
is complete.
Prevention and Control
Infection by trypanosome species is acquired from the bite
of an infected tsetse fly. Thus, preventing flies from biting with
repellants or insect nets will reduce the transmission of the parasite.
Control of the flies through insecticides and habitat alteration
(removal of plant cover near the water sources) is possible, but has
shown to be very difficult.
Case management and Treatment
There are three stages to case management:
• Screening is the initial sorting of people who might be
infected. This involves checking for clinical signs or the
use of serological test.
• Diagnosis shows whether the parasite is present. The only
sign, one that has been known for centuries, is swollen
cervical glands (Winterbottom sign)
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 • Phase diagnosis shows the state of progression of the
disease. It entail examination of CSF obtained by lumbar
puncture and is used to determine the course of treatment
The long, asymptomatic first phase of T.b. gambiense sleeping
sickness is one of the factors that make treatment difficult. Diagnosis
must be made as early as possible in order to preclude the onset of
irreversible neurological disorders and prevent transmission. Case
detection is difficult and requires major human, technical and
material sources. Since the disease is rife in rural areas among poor
people with little access to health facilities, this problem is all the
more difficult.
Treatment
If the disease is diagnosed early, the chances of cure are
high. The type of treatment depends on the phase of disease; initial or
neurological. Success in the latter phase depends on having a drug
that can cross the blood-brain barrier to reach the parasite. Four drug
have been used until now.
• Suramine
• Pentamidine
• Melarsoprol
• Eflornithine
Drug Target and Drug Discovery
The genome of the parasite has been decoded and several
proteins have been identified as potential targets for drug treatment.
The decoded DNA also revealed the reason why generating a vaccine
for this disease has been so difficult. Trypanosoma brucei has over
800 genes that manufacture proteins that the disease mixes and
matches to evade immune system detection.
Recent findings indicate that the parasite is unable to
survive in the bloodstream without its flagellum. This insight gives
researchers a new angle with which to attack the parasite.
A new treatment based on a truncated version of
apolipoprotein L-1 of high-density lipoprotein and a nanobody has
recently been found to work in mice, but has not been tested to
human.
New Scientist, 25 Aug.2007, pp. 35.7 ref
The cover story of the August 25,2006 issue of Cell journal
describe an advance; Dr. Lee Soo Hee and colleagues, working at
John Hopkins, have investigated the pathway by which the organism
make myristate, a 14-carbon length fatty acid. Myristate is a
component of the VSG, the molecule that makes trypanosome’s outer
layer. This outer surface coat of VSG is vital to the trypanosome’s
avoidance of immunological capture, Dr. Lee and Colleague
discovered trypanosomes use a novel fatty acid synthesis pathway
involving fatty acid elongases to make myristate and other fatty
acids.
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