1

Sleeping Sickness, human African trypanosomiasis

Alain Buguet1, M.D., Ph.D., Florian Chapotot

2, Ph.D., Raymond Cespuglio

1, Sylvie

Bisser3, M.D., Ph.D., Bernard Bouteille

4, Pharm.D., Ph.D.

1Neurobiologie des états de vigilance (EA 3734), Lyon, France;

2Centre de

recherches du service de santé des armées, La Tronche, France; 3Centre

international de recherche médicale, Franceville, Gabon; 4EA 3174 Institut de

neurologie tropicale, Limoges, France

Corresponding author: Prof. A. Buguet, EA 3734 Neurobiologie des états de

vigilance, Université Claude-Bernard Lyon 1, 8 avenue Rockefeller, 69373 Lyon

Cedex, France

Tel/Fax: +33 4 78 77 71 26 Cell Phone: +33 6 80 01 74 71

e-mail address: a.buguet@free.fr

Manuscript information: 7,275 words, 0 tables, 4 figures

Running title: Sleeping sickness

Keywords: Sleeping sickness – human African trypanosomiasis – treatment –

polysomnography – sleep-wake cycle – SOREMP

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Abstract

Human African trypanosomiasis (HAT), under control at the end of the colonial

era, is a re-emergent parasitic disease in intertropical Africa. Two trypanosome

groups, Trypanososma brucei, T. b., gambiense in Western and Central Africa and

T. b. rhodesiense in Eastern Africa, are transmitted to humans by tsetse flies.

Trypanosomes escape the host immune response due to antigenic variation related

to mantle variant surface glycoproteins. The chronic Gambian HAT evolves from the

hemolymphatic Stage I, treated with well-tolerated pentamidine, to the meningo-

encephalitic Stage II, treated with an arsenical derivative (melarsoprol), which may

provoke deadly arsenical encephalopathy. It is therefore crucial to diagnose

precisely the stages of HAT. The first 24-hour polysomnography (PSG) was

performed by our group in 1988. Since then, we recorded in endemic areas 106

PSG on 43 Stage II patients, 52 recordings on 35 Stage I patients, and 44 healthy

African controls. A PSG syndrome is invariably observed at Stage II, being

proportional to symptom severity and reversible after successful treatment: an

alteration of the 24-hour sleep and wake distribution, and an altered sleep structure

with the occurrence of frequent Sleep Onset REM Sleep Periods (SOREMPs).

These alterations were tested as potentially useful in diagnosing Stage I from Stage

II in patients at both stages, and in 15 healthy volunteers. Similarly to Stage II

patients, one third of Stage I patients showed abnormal PSG signs, especially

SOREMP occurrences. Healthy volunteers had normal sleep patterns. The

presence of SOREMP in HAT may therefore sign the impact of trypanosomes onto

the central nervous system.

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Epidemiological and clinical aspects of sleeping sickness

Introduction: present situation of sleeping sickness

Sleeping sickness, or human African trypanosomiasis (HAT), is an endemic

parasitic disease which is exclusively located in intertropical Africa, being rooted

between the 15th degrees of latitude North and South by the geographical

distribution of the tsetse fly (genus Glossina, G.). Although it has been known since

centuries, as shown by the description of the disease by the Arab historian Ibn

Khaldoun in 1406 [De Raadt, 2004], it has become epidemic at the end of the 19th

and beginning of the 20th

centuries. Colonization ended tribal skirmishes, installed a

rational administration, built roads and favored population exchanges. A devastating

epidemic developed, with an acme between 1915 and 1930, killing an estimated

million people. The colonial administration issued biomedical missions and, within a

few years, the medical services had found the cause of the disease, its

epidemiological and clinical characteristics, diagnostic criteria and had proposed

specific therapeutic approaches. The creation of mobile teams allowed the

progressive control of the disease. By the 1960s, its prevalence had dropped to

approximately 0.01 %. However, the large federal colonial entities were replaced by

several independent states, which soon lacked financial support, and the national

health services were not affected with the highest priority. Political turmoil, wars and

civil unrest contributed to this disorganization. Nowadays, the disease is considered

as a resurgent epidemic in the Sudan, Uganda, Democratic Republic of Congo and

Angola (Figure 1) and a public health preoccupation in several other countries of

Western and Central Africa. The World Health Organization [WHO, 1998] estimates

that among the 60 million people exposed to tsetse flies, no more than five million

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are benefiting from clinical and epidemiological surveillance. Therefore, only 40,000

new cases are reported each year. The WHO estimates the actual number of

patients between 300,000 and 500,000. Nowadays, sleeping sickness is considered

to rank among the most neglected diseases [Stich et al., 2003]. Another

characteristics is that resurgence always seems to originate from historical well-

identified foci, explaining some of WHO strategies.

On October 7th

1994, the WHO appealed to international solidarity [Louis et

al., 2004]. The international organization obtained support from the French and

Belgian governments, OCEAC (Organisation pour la lutte contre les endémies en

Afrique Centrale, Yaoundé, Cameroon), and non governmental organizations such

as “Médecins sans frontières”. In 2000, the WHO used HAT as a model for the

concept of “orphan medication”. Since then, industrials have issued a collaboration

with the WHO: funds are being raised and medications made freely available to

health services. The WHO is able to organize, homogenize and coordinate medical

actions. Concomitantly in 2001, African governments decided to develop and

coordinate their actions for the control of the disease by creating the Pan African

Tsetse and Trypanosomiasis Eradication Campaign (PATTEC), under the auspices

of the African Unity Organization. Indeed, trypanosomiasis is not only a human

disease, but also an animal pathology, which induces terrible economical losses

depriving African developing countries of meat.

Tsetse flies and trypanosomes

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The bloodthirsty tsetse flies infect their hosts with trypanosomes of the

Trypanosoma brucei (T. b.) species, which is itself divided into several subspecies of

which only two are infective to humans. Infected by T. b. brucei, wild and

domesticated animals develop the nagana. Humans are resistant to T. b. brucei.

Exposure to normal human serum triggers trypanolysis within a few minutes [Lorenz

et al., 1995]. The trypanolytic factor was identified as a haptoglobin-related protein

[Raper et al., 1996; Vanhamme and Pays, 2004]. The human disease is

schematically divided into two separate geographical entities. In Western and

Central Africa, T. b. gambiense is mainly transmitted by G. palpalis, a forest tsetse

fly. The protozoa reservoir is most exclusively human. In Eastern Africa, T. b.

rhodesiense has almost solely an animal reservoir, including wild big game and

domestic animals. Humans are accidentally infected. T. b. rhodesiense is most

likely transmitted by G. morsitans in the savannah, G. pallidipes at the forest edge or

G.fuscipes in riverine and swamplands. The Rhodesian parasites are mainly

differentiated from T. b. brucei by the human serum resistance test.

African trypanosomes are extra cellular parasites characterized by their ability

to alter the composition and organization of their plasma membrane to escape

immune defences of the host, the latter being elicited within a few days after

infection [Pays, 1999]. Antibodies from the host bind to the parasite surface,

activate complement and destroy the trypanosome. The parasites are mantled with

variant surface glycoproteins (VSG). Although only one variable antigen type (VAT)

is expressed at a given time, it can be replaced by another type of VSG. The

sequential expression of VSG genes realizes the so-called antigenic variation.

Therefore, the trypanosome glycoprotein shell is subject to constant variation of

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exposed epitopes. Antibodies against one VAT provoke trypanolysis. However,

some trypanosomes expressing another VSG escape the host reaction. They are

able to proliferate and initiate a new wave of parasitemia, until the host produces an

adapted antibody response. Once again, antigenic variation will allow some other

trypanosomes to express a new VSG, and so on. Trypanosomes possess about

1,000 VSG genes, representing about 10 % of their genome.

Immunopathology

Major immunological changes are observed in HAT, being marked by

lymphadenopathy, splenomegaly and hypergammaglobulinemia, as well as

autoimmune reactions and immunodepression. At the hemolymphatic stage of the

disease (Stage I), trypanosomes (via the VSGs) express VATs, leading to a specific

antibody response in the lymph and plasma [Barry and Emergy, 1984]. With each

new wave of trypanosomes, a corresponding wave of immunoglobulins induces a

massive hypergammaglobulinemia (mainly IgMs), which are not all VSG-specific. A

marked polyclonal B-cell activation generates specific and non specific antibodies,

autoantibodies and immune complexes [Greenwood and Whittle, 1980].

Autoantibodies are associated with the stage of meningo-encephalitis (Stage II).

Their detection in the serum and cerebrospinal fluid (CSF) could be a marker of

central nervous system (CNS) involvement.

Macrophages play a key role in specific immunity, as antigen presenting cells in

synergy with antibodies and cytokines, and are involved in immunosuppressive and

immunopathological phenomena. Macrophages are activated by interferon- (IFN-),

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itself produced by CD8+ T cells stimulated by the trypanosome lymphocyte triggering

factor [Bakhiet et al., 1996], and also by a lipopolysaccharide-like product released

by trypanosomes. Activated macrophages produce tumor necrosis factor- (TNF-),

which, in conjunction with the latter two substances, should lead to the production

and release of nitric oxide (NO), a trypanostatic and trypanocidal molecule.

However, in animals and humans, our group has shown that blood NO decreases

due to the inhibitory effect of interleukine-10 (IL-10) on TNF- and the activation of

arginase, which deprives the macrophagic NO synthase of its substrate, L-arginine

[see Buguet et al, 2002]. A profound deregulation of the cytokine network is

therefore involved in the pathogenic mechanisms of HAT. On the contrary, in the

brain, NO is increased due to the infiltration of macrophages [Buguet et al, 2002].

TNF- is also produced by astrocytes and microglial cells in the CNS [Chao et al,

1995]. TNF- and other cytokines contribute to the generation of somnogenic

molecules such as IL-1 [Pentreath, 1989].

Precisely how the trypanosomes enter the CNS is still unknown. The parasites

occupy the meninges soon after the infection, inducing meningitis. The crossing of

the blood-brain barrier by trypanosomes is not yet elucidated, whether it is directly

due to the parasite or to substances released locally at the choroids plexus level.

Then, the trypanosomes reach the Virchow-Robin spaces, and penetrate the cortex.

They may also exfiltrate from damaged or permeable meningeal and parenchymal

vessels, especially at the level of meso-diencephalic regions. The mechanism by

which trypanosomes damage the CNS remains unanswered. Toxin production by

trypanosomes, leading to inflammation and vascular permeability, may contribute to

it. It may be related to elevated cytokine concentrations found in both patients and

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experimental models. However, the most important mechanism may be related to

autoantibody production against CNS components, a consistent feature of HAT

[Okomo-Assoumou et al., 1995].

Clinical aspects

Just after the bite by the tsetse fly, a chancre appears at the point of the

inoculation. The illness then evolves in two stages, the hemolymphatic Stage I

followed by the meningo-encephalitic Stage II, ending with demyelinisation, altered

consciousness, cachexia, and death if untreated [Dumas and Bisser, 1999]. The

acute Rhodesian form of HAT evolves towards death in weeks, while the chronic

Gambian form will do so in several months or years [Dumas et al., 1999].

The trypanosomes reach the lymphatic and blood circulations. Clinical

symptoms are diverse, non specific and the diagnosis is difficult. One of the

characteristic signs is the classical cervical adenopathies (firm, mobile, painless).

Fever is irregular, recurrent, oscillating in three- to five-day cycles. Cardiac

symptoms with arrhythmia or even pancarditis may be observed, especially in the

Rhodesian form, persistent tachycardia being seen in the Gambian form. Other

symptoms are identified such as: cutaneous eruptions, pruritus with skin lesions

being common, facial edema, rare hepatosplenic disorders. Stage I is considered to

end when neurological symptoms begin and/or when trypanosomes and/or

mononuclear inflammatory cells appear in the CSF, marking the beginning of CNS

invasion.

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Among several neurological and psychiatric symptoms developing insidiously,

excessive daytime sleepiness is one of the most reported signs at Stage II.

However, apart from the signs already described for Stage I, any kind of neurological

or psychiatric symptoms can be observed, especially in the Gambian disease:

headaches, sensory disturbances with uncomfortable diffuse superficial or deep

painful sensations (hyperpathia), sensory deficit, presence of primitive reflexes

(palmo-mental reflex, sucking reflex), exaggerated deep tendon reflexes, tremor (fine

and diffuse without any myoclonic jerk at rest or during movement), abnormal

movements (upper members, choreic gesticulations), pyramidal alterations with

Babinski sign, alterations in muscle tone, numbness, cerebellar signs (ataxia), and

psychiatric disorders (indifference, absent gaze, mutism, confusion, mood swings,

euphoria, agitation, aggressive behavior).

Diagnosis

An accurate identification of the evolutionary stage of HAT is crucial, as

treatment for Stage II remains toxic. As yet, there are no specific clinical signs nor

blood tests for stage determination [Bisser et al., 2002; Lejon et al., 2003; Lejon et

al., 2004]. The finding of trypanosomes in the blood, lymph, or CSF is the only direct

means to positively diagnose HAT. Several ways of improving the direct microscopic

examination have been proposed: blood sample centrifugation, centrifugation of

blood in a capillary tube, use of a Mini Anion Exchange Centrifugation Technique,

double CSF centrifugation, etc.

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Following the WHO recommendations [1998], the diagnosis of Stage II is

based on the CSF examination to search for: i) the presence of trypanosomes; ii)

elevated white blood cell counts; the cut-off proposed by the WHO is <5/L for Stage

I; and iii) determination of total protein concentration; however, according to the

technique used, different cut-offs have been proposed and vary from 250 mg/L to

450 mg/L.

Immunologically-based techniques are not yet totally sure, although they have

greatly improved field diagnosis of HAT. This is the case of the Card Agglutination

Trypanosomiasis Test (CATT) for T. b. gambiense and the Procyclic Agglutination

Trypanosomiasis Test (PATT) for T. b. rhodesiense. Indirect immunofluorescence

tests, Enzyme Linked Immunosorbent Assay (ELISA), Polymerase Chain Reaction

(PCR), and the most recent surface-enhanced laser desorption-ionisation time-of-

flight mass spectrometry [Papadopoulos et al., 2004] are sensitive but cannot be

used as field diagnosis techniques. There is hope that a new latex technique using

given VSGs or recombinant antigens or synthetic peptides will be available for use in

the field [Büscher et al., 1991; Lejon and Büscher, 2001].

Treatment

Although there are few available active medications to treat HAT [Bouteille et

al., 2003], the WHO [Bauquerez and Jannin, 2004] has obtained commitment from

pharmaceutical companies to constitute stocks of all trypanocides for at least five

years. Trypanocides can therefore be obtained via the WHO in Geneva. However,

what makes the therapeutics of HAT difficult is that the operational treatment of

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Stage II is based on the use of melarsoprol, an arsenical derivative issued in 1949

[Friedheim, 1949]. Melarsoprol may cause dramatic side effects, which are

potentially lethal, emphasizing the imperious need for accurate stage diagnosis. On

the contrary, treatment of Stage I uses relatively well-tolerated medications such as

pentamidine or suramin.

Pentamidine (pentamidine isethionate BP, Pentacarinat®), introduced in 1936,

is the treatment of choice for Gambian infections at Stage I [Apted, 1980]. The

commonly recommended dosage is 4 mg/kg per day by intramuscular injection either

every day or every other day, for a series of 7 to 10 injections. Pentamidine is

generally well tolerated, although various adverse effects can be observed, such as

reversible renal toxicity [Coulaud et al, 1975].

Suramin (Bayer 205, Germanin®), derived from trypan red and commercially

available since 1916, is preferred for treating Rhodesian infections at Stage I. The

usual dosage is 20 mg/kg by a series of five intravenous injections at 5- to 7-day

intervals. Suramin has few side effects.

Melarsoprol (Mel B, Arsobal®) remains the principal medication for the

treatment of Stage II HAT, although concentrations in the CSF were found to be 50

to 100 times lower than that in plasma [Burri et al., 1993]. The commonly accepted

protocol consists of daily slow intravenous injections, realized during three to four

consecutive days weekly, throughout a three-week period, with a maximum of 3.6-

mg/kg injected daily. The number of series of injections is often based on the CSF

white blood cell count [Neujean, 1950]. A continuous treatment schedule at 2.2

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mg/kg per day for 10 days has been recently shown to be effective [Burri et al.,

2000]. Melarsoprol is primarily neurotoxic, with a risk of fatal encephalopathy

[Apted, 1980; Pépin et al., 1995], but other undesirable side effects may occur.

Overall fatality ranges from 2% [Dutertre and Labusquière, 1966] to 9.8% [Bertrand

et al., 1973] for T. b. gambiense-infected patients and from 3.4% [Buyst, 1975] to

12% [Apted, 1957] in Rhodesian infections. Furthermore, 10 to 30% of patients may

be resistant to melarsoprol, especially in Uganda and Angola [Legros et al., 1999;

Stanghellini and Josenando, 2001]. In such a case, a second melarsoprol

administration may be effective, but an alternative treatment (eflornithine or

nifurtimox) is recommended.

Eflornithine (difluoromethyl-ornithine, DFMO, Ornidyl®), used against Stage II

HAT since 1992, is an irreversible specific inhibitor of the trypanosome ornithine

decarboxylase [Bitonti et al., 1986], efficient in both stages of the T. b. gambiense

infection [Milord et al., 1992], although results are disappointing in Rhodesian forms

[Iten et al, 1995]. The standard intravenous treatment schedule is hard to set up in

field conditions and consists of 100 mg/kg of DFMO given in slow infusion every six

hours during 14 consecutive days. This costly and complicated treatment should be

reserved for patients refractory to melarsoprol. Furthermore, DFMO is not devoid of

side effects, which are similar to those of anti-cancer drugs.

Nifurtimox (Bayer 2505, Lampit®), used for the treatment of American

trypanosomiasis, Chagas’ disease, is active per os (three daily doses of 5 mg/kg

each, during two to three weeks) in both stages of T. b. gambiense infection. Its

efficacy against T. b. rhodesiense is unknown. Since it has not been approved in

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HAT, initial trials have involved patients refractory to melarsoprol with no other

treatment alternatives. The incidence and severity of neurological side effects

increase with the duration of treatment.

No specific remedies for HAT have been developed since melarsoprol first

appeared in 1949. Trypanocides are not considered by pharmaceutical companies

as being a priority, particularly because of the small and unpredictable market. The

only trypanocidal drug currently under clinical trial, DB-289, an orally administered

pentamidine substitute, is reserved for Stage I [Donkor et al., 2001].

Sleep in sleeping sickness

Clinical descriptions of sleep-wake disturbances

Sleep alterations in sleeping sickness have been described by physicians since

more than one century. Most reports describe somnolence, but insomnia is not rare.

Mackensie [1890] had ruled out hypersomnia having observed that the patients slept

often, in short sleep bouts, during the day as well as at night. However, the most

widely accepted description was that of patients being sleepy by day and restless by

night [Manson-Bahr, 1942]. Although Mackensie’s description evoked a major

disturbance in the 24-hour alternation of the sleep-wake cycle, this aspect was not

specifically studied. In 1910, Lhermitte, and concomitantly Van Campenhout,

described the suddenness with which the patients fall asleep and established a

parallel with the sleep crises of narcolepsy.

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Polysomnographic approach of sleep-wake alterations: nighttime or daytime

recordings

Polysomnographic (PSG: electroencephalogram, EEG; electro-oculogram,

EOG; electromyogram, EMG) recordings were conducted early after the

development of this new technique. Polysomnography represents the only objective

means to distinguish between wakefulness, REM (rapid eye movement) sleep, and

non-REM sleep and its four stages, sleep stages 3 and 4 representing slow-wave

sleep [Rechtschaffen and Kales, 1968]. However, PSG recordings were only taken

at night [Bert et al., 1965] or during diurnal naps [Schwartz and Escande, 1970].

The largest study of nocturnal sleep conducted by Bert et al. [1965] in Dakar

(Senegal) revealed abnormalities in 16 out of 17 Stage II patients, the only patient at

Stage I and one patient clinically ranked as being at early Stage II showing a normal

sleep structure. In their meningo-encephalitic patients, the authors had difficulties in

scoring intermediary stages. However, stage 4 and REM sleep were normal in most

patients. They noted the scarcity of vertex sharp waves, spindles and K complexes.

The authors stressed the parallel between anomalies of the sleep traces and

severity of the clinical state. They also discussed the similarity of sleep structure

with that of narcoleptic patients, showing one hypnogram revealing sleep onset REM

periods (SOREMP), but they did not attribute a special importance to the

phenomenon.

In Paris, Schwartz and Escande [1970] examined a Lebanese patient from

Dakar, who had contracted HAT in Senegal and was at an advanced stage of

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meningo-encephalitis. The patient was treated with melarsoprol. During the

treatment procedure, he underwent three standard wake recordings in the morning

and one PSG in the afternoon (between 14:00 h and 16:00 h). During the four

following months, PSG was performed at three-week intervals: one nocturnal PSG,

performed two months after treatment, was preceded and followed by 2 afternoon

PSG recordings. Overall, sleep episodes occurred by night and by day. The authors

noted three SOREMPs in the afternoon tests and a short REM sleep latency in the

night recording. They stressed the resemblance with narcoleptic sleep organization.

They also experienced sleep stage scoring difficulties and noted the paucity of

vertex spikes, K complexes and spindles. Sleep patterns improved with time.

To our knowledge, besides our published recordings, the literature only reports

on three other patients recorded by PSG. Sanner et al. [2000] reported on a German

patient, who presented nocturnal insomnia and daytime hypersomnia, starting 12

days after returning from a 20-day trip to Zambia, Zimbabwe and Tanzania. The

patient rapidly developed multiorgan failure attributed to T. b. rhodesiense infection.

She recovered in a few days after suramin treatment. Nocturnal PSG recordings

were performed on days 7, 9 and 15, and 6 months after the onset of the disease.

Normal amounts of REM sleep were observed, but slow-wave sleep almost

disappeared in the early days to be restored to normal 6 months later. In a review

paper on waking disorders, Billiard and Ondzé [2001] presented the 24-hour

hypnogram of a 26-year-old Cameroon patient showing one SOREMP and altered

sleep-wake cycle. The same hypnogram was presented in Billiard’s book [Billiard

and Carlander, 2003]. The third patient, a 39 year-old Zaïre man (Democratic

Republic of the Congo), was examined in Paris [Monge-Strauss et al., 1997]. He

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had a complicated 6-year long history with several psychiatric hospitalizations and

was even sent to jail. The diagnosis of trypanosomiasis was finally made and the

patient was treated with eflornithine. All clinical symptoms (meningo-encephalitis,

confusion, and epileptic seizures) disappeared rapidly. He underwent two 48-hour

PSG recordings, 15 days after completion of the treatment procedure and two

months later. The patient presented disrupted distribution of sleep and wake

episodes and a shortened REM latency indicating the presence of SOREMPs.

The 24-hour polysomnographic methods used in our investigations in Africa

The first 24-hour sleep recording in sleeping sickness was performed in 1988 in

Niamey (Niger) on a migrant worker who had contracted the disease in Côte d’Ivoire

[Buguet et al., 1989]. Although the EEG traces were loaded with slow waves,

sleeping, waking, and REM sleep were identifiable. The 24-hour recording revealed

the disappearance of the normal distribution of sleeping and waking, which occurred

indifferently throughout the nychthemeron.

Our team then used PSG to analyze sleep and wake modifications, firstly in

Stage II patients, and more recently in patients at Stage I. Altogether, in 8 field trials,

106 PSG recordings were taken on 43 Stage II patients, 52 on 35 patients at Stage I,

and 44 on 44 healthy villagers who had volunteered as controls. All patients had

been infected by T. b. gambiense. In addition, two Caucasian Stage II patients, who

had contracted Rhodesian HAT in Rwanda, were also followed-up after melarsoprol

treatment in France over a period of 11 months. Informed consent to participate in

the study was always obtained from either the patients or their families, as was the

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agreement from national health authorities (Angola, Congo, Côte d’Ivoire, Niger). In

all investigations, the diagnosis of sleeping sickness was confirmed by finding

trypanosomes in the blood, lymph gland fluid and/or CSF.

The techniques used have evolved over this 16-year period. A total of fifty-

three 24-hour PSG paper recordings were taken on 8- or 10-channel polygraphs

following a previously published procedure [Buguet et al., 1987]. Basically, two

channels were devoted to the EEG, while the EMG and EOG were taken on

separate channels. Other channels served to record the electrocardiogram and the

respiratory cycle (nasal and buccal airflow with Cu-Ct thermocouples; chest

movements with a strain gauge). Paper speed was 15 mm.s-1

, thus determining 20 s

scoring periods. In most patients, classical scoring of sleep states and wakefulness

could be performed [Rechtschaffen and Kales, 1968]. In a few patients at a very

advanced Stage II, the scoring technique had to be adapted following Schwartz and

Escande [1970] and Buguet et al. [1989], because of the invasion by ubiquitous EEG

slow waves. In these patients, non-REM sleep was thus divided into light sleep and

SWS. In all patients, wakefulness and REM sleep were easily identified.

The remaining PSG recordings used portable Holter-type equipment (Figure 2).

As none of the patients recorded on paper had shown any respiratory disturbance,

most of the recordings included only EEGs, EOG and EMG traces. Recently,

analogical portable recorders were replaced by ambulatory digital recording systems,

which allow for quantitative EEG analyses.

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Eighteen of our meningo-encephalitic patients participated in a study on

hormonal secretory rhythms. They were catheterized in the median basilic vein, and

10-mL blood samples were removed each hour over a 24-hour period in 8 of them.

Blood was continuously withdrawn through peristaltic pumps in the other 10 patients.

The overall-24-hour blood withdrawal was always kept below 300 mL. A similar

investigation with continuous blood withdrawal was conducted in Abidjan (Yopougon

University Hospital) on 6 healthy subjects. In these three investigations, designated

to study hormonal secretion, blood sampling was terminated either after 24 hours or

upon request from the patients or if the patient’s hemoglobin dropped below 10 g.dL-

1.

Sleep patterns in the healthy subjects

In villages from Côte d’Ivoire and Angola, 44 healthy subjects volunteered to

serve as controls for the HAT villagers and were recorded by 24-hour ambulatory

PSG. All healthy volunteers had a major sleep episode at night and sometimes a

small nap in the afternoon or late morning [Buguet et al., 2002]. They did not show

any disturbance in their sleep-wake cycle nor any SOREMP (Figure 3).

In the six healthy subjects, who slept with an indwelling catheter as controls for

endocrine studies, sleep efficiency was impaired due to frequent awakenings,

resulting in a reduction in total sleep time. REM sleep was also reduced. These

data obtained in Africa were concordant with the observation of Adam [1982] in 7

healthy Scots also sleeping with a catheter.

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The polysomnographic syndrome in Stage II patients (Figure 4)

The meningo-encephalitic patients showed a major disturbance in the 24-hour

distribution of sleep and wakefulness episodes, the extension of which increased

with the severity of the disease. Severely ill patients experienced sleep episodes

throughout the nychthemeron. As there was no increase in the 24-hour total sleep

time, i.e., no hypersomnia, the average duration of wakefulness and sleep episodes

was inversely related to the severity of the disease. Therefore, the sleep-wake

alternation occurred in shorter cycles. The occurrence of REM sleep episodes

throughout the nychthemeron is the best example of such a disturbance in 24-hour

rhythms.

The deregulation of sleep and wakefulness 24-hour distribution was completed

by an alteration of the structure of sleep episodes. The latter started often with REM

sleep phases (SOREMPs). The number of SOREMPs increased in relation to the

severity of clinical symptomatology.

The sleep disturbances were not aggravated in the patients examined in the

Daloa clinic (Côte d’Ivoire) whether they had a catheter or not [Buguet et al., 1993].

It seems that these hyperpathic patients had a lowered sensitivity to environmental

stimuli.

Contrary to our expectations, the numerous alterations of the EEG were not

specific of HAT [Tapie et al., 1996]. During sleep, normal features were seen.

However, transient activation phases were decreased in the patients. Four patients

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presented monophasic frontal delta bursts predominant during slow-wave sleep

along with paroxysmal hypnopompic hypersynchronic events during slow-wave

sleep. It was therefore concluded that in sleeping sickness patients, although

dampened, the waking process remains responsive, and slows down only at a late

stage of meningo-encephalitis.

The polysomnographic syndrome as a diagnostic mean

The PSG alterations may be determinant in diagnosing the passage from

Stage I to Sage II. We explored this hypothesis in two investigations conducted in

Angola, during which we examined 11 patients at Stage II and 24 at Stage I, as well

as 15 healthy volunteers. All Stage II patients exhibited the complete sleep-wake

syndrome. Among Stage I patients, 13 showed abnormal PSG signs, especially

sleep episodes with SOREMPs (Figure 5). Healthy volunteers had absolutely

normal sleep patterns and distribution. The presence of SOREMPs in patients with

HAT may therefore sign the passage of trypanosomes into the CNS or the induction

of CNS reactions through the action of trypanosome-induced chemokines across the

blood brain barrier.

Treatment effect on the polysomnographic syndrome

In the investigation conducted in Brazzaville [Buguet et al., 1999] on 10

meningo-encephalitic patients, 24-hour PSG was recorded before treatment with

melarsoprol, and then weekly during the three-week treatment procedure. In all

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patients, the disturbed sleep-wake alternation improved after treatment. The

number of SOREMPs also normalized, especially in the less severely sick patients.

The treatment-related improvement of the sleep-wake 24-hour alternation was

also observed in two Caucasian patients with severe meningo-encephalitis. These

had been infected by T. b. rhodesiense in Rwanda. They were treated in Marseille

(France) with melarsoprol and had PSG recorded 4, 6 and 11 months later

[Montmayeur et al., 1994]. Although their sleep disturbances were improved by

treatment, normalization was only obtained 11 months later in one of the two

patients. Reversibility of the PSG syndrome may therefore take several months to

be completed.

In summary

The major findings of PSG in Stage II patients with African trypanosomiasis can

be summarised as follows. The 24-hour alternation of sleep and wakefulness is

altered proportionally to the severity of the disease. It is reversed by melarsoprol

treatment. Sleep structure alterations appear early in Stage II, with frequent

SOREMPs. SOREMPs recede or disappear after melarsoprol treatment.

The occurrence of SOREMP-like episodes has also been shown in a rat model

of African trypanosomiasis [Darsaud et al., 2004]. The animals were infected by T.

b. brucei and followed with PSG until death, which occurred on an average three

weeks after the inoculation of the parasites. SOREMPs appeared immediately after

22

the decline of food intake and body weight (12 to 14 days after infection), which also

corresponded to the presence of trypanosomes in the CNS [Darsaud et al., 2003].

General conclusion

As stated recently by Peter De Raadt [2004], who worked for the WHO for over

30 years, history repeats itself. Nowadays, sleeping sickness has come back. It is

considered to be a re-emergent disease, menacing 60 million people living in the

tsetse belt of Africa, one of the poorest areas of the world [Cox, 2004]. It is also an

orphan disease with a limited therapeutic arsenal. However, despite a past

disinterest by government and international institutions and little funding, research on

HAT has developed considerably [Dumas et al., 1999]. Furthermore, African

trypanosomiasis is not only deadly for humans, it represents also a major

economical plague infecting and killing domestic cattle in an economically sensitive

continent [Kristianson et al., 1999]. The recent Congress on the tsetse fly and

trypanosomiasis held in Brazzaville in March 2004 [Milleliri et al., 2004], insisted on

the fact that both animal and human African trypanosomiases represent major

obstacles for the economic development of the African continent. There is however

hope due to the recent promotion of cooperation between pharmaceutical groups

and governments from Africa and the rest of the world, under the auspices of the

WHO [Bauquerez and Jannin, 2004]. Sleeping sickness has found its place within

the concept of neglected diseases and patients can now have access to free

medications provided by Aventis (pentamidine, melarsoprol, eflornithine) and Bayer

(suramin). At Lomé (Togo) in 2001, the African governments committed themselves

to eliminate the disease. The objective is not only to develop new medications to

23

treat the patients, but also to obtain new means to improve the stage diagnosis of

HAT in order to optimise treatment strategies.

Research on sleep in sleeping sickness belongs to this logic. It has provided

with new knowledge, which may be used for the determination of the evolutionary

stages of HAT, and also for the follow-up of treatment efficacy.

24

Acknowledgements

This work was supported by the World Health Organization (grant n° TDR/ID

910048), the French Ministry of Cooperation, the Société Elis, companies UTA and

Air Afrique and Région Rhône-Alpes grant under the Priority Thematic Program

“Emerging and transmissible diseases, parasitic diseases” (grants n° 00816028 and

00816053). Institutions which co-participated in this work were as follows: Defence

Research and Development Toronto, Canada; EA 3734 Neurobiologie des états de

vigilance, Université Claude-Bernard, Lyon, France; EA 3174 Institut de neurologie

tropicale, Université de Limoges, France; Laboratoire de physiologie, Faculté de

médecine, Abidjan, Côte d'Ivoire; Laboratoire de physiologie et de psychologie

environnementales, CNRS, Strasbourg, France; Projet de recherches cliniques sur

la trypanosomiase, Daloa, Côte d'Ivoire; Service des grandes endémies, Brazzaville,

Congo; Service de neurologie, CHU de Brazzaville, Congo. The software used for

ambulatory studies was provided by the PhiTools company (Strasbourg, France).

25

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Figure Captions

Figure 1: Map of Africa showing the geographical distribution of human African

trypanosomiasis adapted from WHO African trypanosomiasis web pages

(http://www.who.int/health-topics/afrtryps.htm).

Figure 2: Voluntary subject from an Angolan village being recorded with an

ambulatory digital acquisition system.

Figure 3: Young patient with meningo-encephalitis being recorded with an

ambulatory digital acquisition system under the hospital tent set up in an Angolan

village.

Figure 4: Hypnogram showing a 24-hour distribution of sleep states (SP, REM

sleep; 1-4, stages 1 to 4 of non-REM sleep) and wakefulness (W) in a healthy

subject in Angola. As in the rest of the world, the subject sleeps at night, with a

well-structure sleep.

Figure 5: Hypnograms of two meningo-encephalitic Stage II patients. The upper

graph represents 24-hour sleep and wakefulness distribution with nocturnal

episodes of destructured sleep, the alterations being essentially shown by restless

sleep and the presence of Sleep Onset REM Periods (SOREMP), especially in the

second sleep episode. The lower graph is that of an advanced meningo-

encephalitic patient, demonstrating the complete polysomnographic syndrome:

34

disturbed distribution of sleep and wakefulness throughout the nycthemeron;

presence of SOREMPS in several sleep episodes.

Figure 6: Hypnograms of three patients diagnosed clinically and biologically as

being at Stage I of human African trypanosomiasis. The upper graph shows a

hypnogram demonstrating a normal sleep pattern. The middle graph represents a

slightly disturbed nocturnal sleep pattern, with the occurrence of a phase of REM

sleep within a long wakefulness episode. The lower graph demonstrates a

completely disturbed sleep pattern showing an obvious alteration of sleep-

wakefulness function.