Transcript
Page 1: Genetic characterization of Trypanosoma brucei gambiense and clinical evolution of human African trypanosomiasis in Côte d'Ivoire

Genetic characterization of Trypanosoma brucei gambiense

and clinical evolution of human African trypanosomiasis

in Cote d’Ivoire

V. Jamonneau1, A. Garcia2, S. Ravel3, G. Cuny3, B. Oury4, P. Solano1, P. N’Guessan1, L. N’Dri1, R. Sanon1,

J. L. Frezil3 and P. Truc5

1 Institut de Recherche pour le Developpement (IRD/UR 035), Centre Pierre Richet, Bouake, Cote d’Ivoire2 Institut de Recherche pour le Developpement (IRD/UR 010), Dakar, Senegal3 Institut de Recherche pour le Developpement (IRD/UR 035), Laboratoire de Recherche et de Coordination sur les Trypanosomoses,

Montpellier, France4 Institut de Recherche pour le Developpement (IRD/UR 062), Centre d’Etude sur le Polymorphisme des Microorganismes, Montpellier,

France5 Institut de Recherche pour le Developpement (IRD/UR 035), OCEAC, Yaounde, Cameroon

Summary Human African trypanosomiasis is a parasitic infection caused by protozoa belonging to Trypanosoma

brucei subspecies. The clinical evolution of this disease is complex and might be because of the

parasite itself, as genetic diversity has been observed in T. brucei ssp. We investigated the relationship

between the genetic diversity of trypanosomes and the diversity of clinical patterns in Cote d’Ivoire.

We studied clinical sleeping sickness cases, and genetically analysed the trypanosomes isolated from

these patients. An important genetic monomorphism among stocks isolated in Cote d’Ivoire was

observed by using various markers: isoenzymes electrophoresis, random amplified polymorphism DNA

and PCR of microsatellite sequences. At the same time, the diversity of clinical patterns and evolutions

was confirmed by clinical analysis. The existence of an individual susceptibility to disease (human

trypanotolerance) should be taken into account even if our genetic conclusions might be distorted

because the isolation success rates were particularly poor. In fact, we observed that the isolation success

rate varied significantly depending both on the focus of origin (P ¼ 0.0002) and on the ethnic group

(P ¼ 0.0317) of the patient. Further investigations are required in order to study a possible selective

impact of the use of the kit for in vitro isolation of trypanosomes as an isolation technique.

keywords Trypanosoma brucei gambiense, isoenzymes electrophoresis, RAPD, individual

susceptibility, Cote d’Ivoire

correspondence P. Solano, Institut de Recherche pour le Developpement (IRD), UR 035, Institut Pierre

Richet, 01 BP 1500 Bouake, Cote d’Ivoire. Fax: +225 31 63 27 38; E-mail: [email protected]

Introduction

Human African trypanosomiasis (HAT), or sleeping sick-

ness, is a major public health problem in sub-Saharan

Africa. Approximately 60 million people are daily exposed

to the risk of infection. It is estimated that there are about

500 000 infected but untreated persons (WHO 1998). The

pathogenic agent is the trypanosome Trypanosoma brucei.

Classically, T. brucei is subdivided into three subspecies on

the basis of extrinsic criteria: T. b. gambiense is responsible

for the chronic form in West and Central Africa, T. b.

rhodesiense is the agent of the acute form in East Africa,

and T. b. brucei, a parasite of cattle, is supposed to be non-

pathogenic to humans.

By definition, HAT evolves in two phases: a haemato-

lymphatic stage (first period), for which there are no

specific clinical signs (Jannin et al. 1993; Dumas &

Bouteille 1996), leading to a meningo-encephalitic stage,

usually characterized by neurological disorders (second

period). In the absence of treatment, the disease is

invariably fatal. In T. b. gambiense chronic form, the

duration of the first period may be several years: the

fluctuating parasitaemia remains low and tends to

decrease. The appearance of neurological disorders during

Tropical Medicine and International Health

volume 7 no 7 pp 610–621 july 2002

610 ª 2002 Blackwell Science Ltd

Page 2: Genetic characterization of Trypanosoma brucei gambiense and clinical evolution of human African trypanosomiasis in Côte d'Ivoire

the second period is often progressive. In acute HAT

caused by T. b. rhodesiense, the haemato-lymphatic stage

lasts a few weeks to a few months, passage to the meningo-

encephalitic stage is brutal and death can occur within a

few months of onset of this stage.

A diversity of clinical evolutions has been observed for

T. b. gambiense, from some chronic forms to asympto-

matic forms (Jamonneau et al. 2000a). At one extreme, a

patient from Togo carried trypanosomes (T. b. gamb-

iense) for 21 years with no clinical signs or neurological

disorders (Lapeyssonnie 1960). At the other extreme,

some patients detected in Cote d’Ivoire presented a

clinical evolution characteristic of acute HAT (Truc et al.

1997a). This clinical diversity does not correspond to the

traditional definition of the T. b. gambiense clinical forms

of HAT.

Genetic diversity within T. b. gambiense has been

demonstrated by several studies using molecular markers

(Gibson 1986; Paindavoine et al. 1986; Godfrey et al.

1990; Hide et al. 1990), but its influence on the clinical

evolution of the HAT remains unproven. We intended

to investigate the relationships between the genetic

diversity of the parasite and that of the clinical patterns.

With this aim, we launched a clinical study on patients

diagnosed in Cote d’Ivoire together with a genetic

analysis of the populations of trypanosomes isolated

from these patients.

Patients and methods

Study areas and patients

This study was conducted from 1996 to 1999 in two active

HAT foci of Cote d’Ivoire: the western-central part of the

country (Daloa, Vavoua, Bouafle, Sinfra, and Bonon) and

the south-east (Aboisso) (Figure 1). We included patients

detected (parasitological evidence) either actively (during

five medical surveys) or passively (patients presenting

themselves for treatment). All patients were treated in the

three centres specialized in HAT treatment: the Projet de

Recherches Cliniques sur les Trypanosomoses in Daloa,

and the local health centres in Bouafle and Aboisso.

Patients were told of the objectives and protocol of the

study, and only those who gave their consent were included

in the study (patients younger than 10 years were not

included).

Epidemiological data collection

For each patient, the following data were recorded: sex,

age, nationality, ethnic group, geographical origin

(south-east or mid-west), HAT focus of provenance (Sinfra,

Bonon, Daloa, Bouafle or Aboisso), occupation, existence

of family history of HAT, time and mode of diagnosis

(passive or active), and treatment schedule. We distin-

guished two ethnic grouping groups: natives (including

patients born in the study areas) and migrants (including

subjects from northern Cote d’Ivoire, Burkina Faso and

Mali).

Clinical assessment before treatment

Objective clinical signs that were looked for were:

• palpation (hepatomegaly, splenomegaly, swollen lateral

cervical lymph nodes);

• cardiovascular investigation (dysrhythmia, heart

murmurs and low blood pressure);

• dermatological examination (search for initial lesion of

inoculation, trypanids);

• assessing possible endocrinological disorders (impotence,

facial oedema, amenorrhea, abortion);

• neurological examination (alteration of mental state,

abnormal reflexes, tone disorders, sensory disorders,

coordination disorders).

A questionnaire presented to patients covered subjective

clinical signs such as asthenia, anorexia, cachexia, fevers,

repeated headache, nausea, pruritus, cutaneous rash, sleep

disturbances (alteration of circadian rhythm). Questions

about the approximate date of appearance of the initial

symptoms and the mode of evolution of the disease were

also included.

On the basis of clinical signs, patients were classified

according to the existence and the importance of the

following: infectious or inflammatory syndrome, cardio-

vascular syndrome, digestive syndrome, dermatological

syndrome and neuro-psychiatric syndrome (Table 1).

Serological and parasitological examinations,

and stage determination

Each patient underwent serological and parasitological

investigations. For serology, the Card Agglutination Test

for Trypanosomiasis (T. b. gambiense) was performed

using whole blood and plasma (Magnus et al. 1978).

Trypanosomes were detected using the mini Anion

Exchange Centrifugation Technique (Lumsden et al. 1979)

and by direct microscopic examination of the lymphatic

fluid if lymph nodes were swollen.

For stage determination, the tests used on the cereb-

rospinal fluid (CSF) were: trypanosome detection by

double centrifugation (Cattand et al. 1988) and leukocyte

counting using a Nageotte counting chamber. A low

leukocyte count (£ 5 cells/ll) combined with absence of

Tropical Medicine and International Health volume 7 no 7 pp 610–621 july 2002

V. Jamonneau et al. Human African trypanosomiasis in Cote d’Ivoire (Ivory Coast)

ª 2002 Blackwell Science Ltd 611

Page 3: Genetic characterization of Trypanosoma brucei gambiense and clinical evolution of human African trypanosomiasis in Côte d'Ivoire

trypanosomes in CSF is defined as the first stage of the

disease. An elevated leukocyte count (>5 cells/ll), with or

without trypanosomes in CSF, forms the basis for second-

stage diagnosis (WHO 1998).

Isolation of trypanosomes

The stocks were isolated by using the KIVI (Kit for In

vitro Isolation of trypanosomes, Aerts et al. 1992) and

were then multiplied using semidefined culture medium

(Cunningham 1977) according to the protocol described

by Truc et al. (1992). For each stock, two pellets of

trypanosomes were obtained by centrifugation and stored

in liquid nitrogen. Reference stocks (Tables 2 and 3) had

been isolated with the KIVI method during or after

1991, the others having been isolated by rodent inocu-

lation.

Multilocus enzyme electrophoresis

Proteins were extracted from one of the two pellets (Truc

et al. 1991). Stocks were characterized by the technique of

multilocus enzyme electrophoresis (MLEE) on cellulose

acetate plates and 11 enzymatic systems were revealed:

ALAT (EC.2.6.1.2), GOT (EC.2.6.1.1), Nhi (EC.3.2.2.1),

Nhd (EC.3.2.2.1), ME (EC.1.1.1.40), PEP-2 (EC.3.4.11),

MALI

GUINEA

BURKINA FASO

Bouake

Vavoua

BononDaloa

Sinfra

Bouafle Yamoussoukro

LIBERIA

GHANA

Aboisso

Abidjan

ATLANTIC OCEAN

LEGEND:RiverLake

Towns of the study areaOther towns

Scale0 25 50 75 100 km

N

Figure 1 Geographic location of the studyarea.

Tropical Medicine and International Health volume 7 no 7 pp 610–621 july 2002

V. Jamonneau et al. Human African trypanosomiasis in Cote d’Ivoire (Ivory Coast)

612 ª 2002 Blackwell Science Ltd

Page 4: Genetic characterization of Trypanosoma brucei gambiense and clinical evolution of human African trypanosomiasis in Côte d'Ivoire

MDH (EC.1.1.1.37), IDH (1.1.1.42), TDH (1.1.1.103),

PGM (EC.2.7.5.1) according to Truc et al. (1991) and

Truc and Tibayrenc (1993), and SOD (EC.1.15.1.1)

according to Stevens et al. (1989).

Random amplified polymorphism DNA

The DNA was extracted from the second pellet according to

the protocol described by Oury et al. (1998). Stocks were

characterized by the RAPD technique (random amplified

polymorphism DNA, Welsh & McClelland 1990; Williams

et al. 1990; Tibayrenc et al. 1993). Seven primers were used:

A2 (5¢-TGCCGAGCTG-3¢), A4 (5¢-AATCGGGCTG-3¢),A7 (5¢-GAAACGGGTG-3¢), A8 (5¢-GTGACGTAGG-3¢),A10 (5¢-GTGATCGCAG-3¢), A11 (5¢-CAATCGCCGT-3¢)and A18 (5¢-AGGTGACCGT-3¢).

Polymorphism of chain reaction of microsatellite DNA

sequences

From the same DNA extraction, microsatellite sequences

(PCR/microsatellite) were amplified with four primer pairs:

M6C8-CAF/R, MT30-33F/R (Biteau et al. 2000),

TRBPA1/2 (Simo et al. 2000) and TBDAC1/2 (Truc et al.

2002). Amplifications followed the protocol described by

Truc et al. (2002).

Analysis procedures

Whenever possible, a comparison of two qualitative

variables was performed by means of Pearson chi-square

tests. Alternatively, we performed Fisher’s exact test. The

level of significance retained for the tests was 5%. These

Table 1 Clinical signs and corresponding syndromes

Syndrome Clinical signs Expected answers

Inflammatory/infectious Temperature at inclusion Quantitativesyndrome Asthenia Absence/presence

Anorexia Absence/presenceWeight loss Absence/presence(feeling) Fever Absence/presenceRecurrent headache Absence/presenceCervical lymph nodes Absence/presence

Digestive syndrome Nausea Absence/presenceVomiting Absence/presenceHepatomegaly Absence/presenceSplenomegaly Absence/presence

Cardiovascular syndrome Arrhythmia (heart rhythm) Normal/abnormalHeart rate Normal/abnormalBlood pressure Normal/abnormalSubjective heart troubles Absence/presence

Dermatological syndrome Chancre Absence/presenceTrypanids Absence/presenceOedema Absence/presencePruritus Absence/presenceSkin rash Absence/presence

Neuro-psychiatric syndrome Sleep disorders Absence/diurnal drowsiness/night insomniaEating disorders Absence/lack of appetite/compulsive eatingThirst disorders Absence/polydipsiaSexual disorders Absence/lower libido/lost libido/sexual impotenceSensibility disorders Absence/hyperpathyConsciousness disorders Absence/lower consciousness/mental confusion/comaBehavioural disorders Absence/agitation/indifferenceEmotional disorders Absence/euphoria/sadness/aggressivenessMotor disorders Absence/lower motility/no motilityCoordination disorders Absence/impaired coordinationMuscular tonus disorders Absence/hypertonicity/hypotonicityArchaic reflexes Absence/presenceOsteo-tendinous reflexes Normal/excessive/abolitionPlantar-skin reflexes Normal/excessive/abolitionObjective sensitivity Normal/insensitivity

Tropical Medicine and International Health volume 7 no 7 pp 610–621 july 2002

V. Jamonneau et al. Human African trypanosomiasis in Cote d’Ivoire (Ivory Coast)

ª 2002 Blackwell Science Ltd 613

Page 5: Genetic characterization of Trypanosoma brucei gambiense and clinical evolution of human African trypanosomiasis in Côte d'Ivoire

analyses were done using BMDP software (BMDP statis-

tical Software, University of California, Los Angeles, CA,

USA).

Unweighted Pair Group Method Analysis (UPGMA)

dendrograms were built, starting with the Jacquard genetic

distances (Jacquard 1973) calculated from MLEE and

RAPD results for visualizing the relationships between

stocks (Sneath & Sokal 1973). Reference stocks of T. b.

rhodesiense, T. b. brucei and T. b. gambiense groups 1 and

bouafle were included, as well as stocks of T. congolense-

like groups for the UPGMA comparison (Tables 2 and 3).

For the PCR/microsatellite DNA, the four primers

were specific to T. b. gambiense group 1; thus, only

two reference stocks, Jua and Peya (Table 3), were

used. Each band obtained, defined by its molecular

weight (in base pairs or bp), corresponds to an allele.

For each stock, two bands per primer were revealed

(X bp/Y bp).

Results

Patients

A total of 139 patients (harbouring trypanosomes) partici-

pated in the study; nine came from Aboisso in the South-

East, and of the 130 remaining patients from the midwest, 82

Table 2 Reference stocks used for MLEE characterization

Stock Host Year Country Focus Zymodeme Species Reference

DAL072 Human 1978 CI Vavoua 1 T. b. g 1 1Trazie Human 1991 CI Sinfra 2 T. b. g 1 2Sique Human 1991 CI Sinfra 3 T. b. g 1 2SH017 Human 1989 CI Aboisso 6 T. b. bfl 2SH196 Human 1990 CI Daloa 7 T. b. bfl 2SH276 Human 1992 CI Daloa 10 T. b. bfl 2SINF1 Human 1992 CI Sinfra 11 T. b. g 1 2SINF5 Human 1992 CI Sinfra 12 T. b. g 1 2TH2 Human 1978 CI Daloa 14 T. b. bfl 3TSW53 Pig 1982 CI Bouafle 15 T. b. bfl 1TSW103 Pig 1977 Liberia Sanniquelle 27 T. congo 4132 Kob 1993 CI Comoe 30 T. b. bfl 5KK39 Kob 1980 CI Comoe 33 T. b. bfl 6AB14 Hartebeest 1980 CI Comoe 37 T. b. bfl 11972 Human 1993 CI Sinfra 38 T. b. g 1 2Bub6 Hartebeest 1994 CI Marahoue 40 T. b. g 1 2

CI, Cote d’Ivoire; T. b. g 1, Trypanosoma brucei gambiense group 1; T. b. bfl, Trypanosoma brucei bouafle group; T. congo, Trypanosomacongolense.References: 1. Stevens et al. (1992); 2. Truc et al. (1997a); 3. Mehlitz et al. 1982; 4. Gashumba et al. (1988); 5. Truc et al. (1997b);6. Young and Godfrey (1983).

Stock Host Year Country Specie Reference

Jua* Human 1979 Cameroon T. b. g 1 1Peya* Human 1980 Congo T. b. g 1 1KP465 Pig 1991 Cote d’Ivoire T. b. bfl 2TSW65 Pig 1982 Cote d’Ivoire T. b. bfl 3058clA3 Human 1974 Zambia T. b. rh 4Eatro 1125 w.m. 1966 Uganda T. b. b 5TRPZ105 Dog 1981 Zambia T. congo 6

w.m., Wild mammal; T. b. g 1, Trypanosoma brucei gambiense group 1; T. b. bfl,Trypanosoma brucei bouafle group; T. b. rh, Trypanosoma brucei rhodesiense; T. b. b,Trypanosoma brucei brucei; T. congo., Trypanosoma congolense.* For PCR of microsatellites sequences, only Jua and Peya were used as reference stocks.References: 1. Truc et al. (1991); 2. Truc et al. (1997a); 3. Stevens et al. (1992);4. Gibson et al. (1980); 5. Hide et al. (1990); 6. Gashumba et al. (1988).

Table 3 Reference stocks used for RAPDand PCR/microsatellite characterization*

Tropical Medicine and International Health volume 7 no 7 pp 610–621 july 2002

V. Jamonneau et al. Human African trypanosomiasis in Cote d’Ivoire (Ivory Coast)

614 ª 2002 Blackwell Science Ltd

Page 6: Genetic characterization of Trypanosoma brucei gambiense and clinical evolution of human African trypanosomiasis in Côte d'Ivoire

came from Sinfra, 27 from Bonon, 13 from Bouafle and 8

from Daloa. Sixty-eight per cent of patients were detected

passively and 71 actively through five medical surveys.

Thirty-nine patients were natives (28%) and 100 migrants

(72%). This unequal distribution is traditional in Cote

d’Ivoire as most of the migrants work and live close to the

cocoa and coffee plantations, which constitute areas of

greater HAT transmission risk. One-hundred and nine

patients (78.4%) were farmers and 30 (21.6%) declared a

family history of HAT.

Isolation of trypanosomes

Of the 139 KIVIs performed before treatment, only

62 (44%) gave a positive result allowing the in vitro

culture and multiplication of procyclics. To isolate a

maximum of stocks, another KIVI was inoculated for

46 patients (some of whom had already given positive

KIVI in the first round). Only 20 of these 46 KIVI

(43%) were positive. In total, 64 stocks (46%) were

isolated and cultured for genetic identification.

These isolation success rates were particularly poor.

Thus, we checked whether some of the host characteristics

could have affected the success of isolation. We compared

the epidemiological parameters of the human population

from whom the stocks were isolated (population ‘KIVI

positive’) with the population from whom the isolation

failed (population ‘KIVI negative’). The HAT focus of

origin and the ethnic group had a significant influence on a

positive in vitro isolation (P ¼ 0.0002 and P ¼ 0.0317,

respectively). More precisely, the isolation rate ranged

from 33.7% in the Sinfra focus to 85.2% in the Bonon one.

The isolation rate was particularly poor with the Baoule

group, natives of the area (7.7%) and was most elevated

for the Senoufo migrant group (66.7%).

Isozyme characterization

Of 64 stocks, 61 were zymodeme 3 (Z3, Truc et al. 1997a).

The three other stocks were zymodeme 38 (Z38, Truc et al.

1997a). The dendrogram (Figure 2) shows the relationships

between these two zymodemes, which differ only by the ME

locus: Z3 showed heterozygotes at this locus (three bands),

while Z38 was a homozygote (one band). These two

zymodemes are genetically closely related (d < 0.1). They

both belong to group 1 of T. b. gambiense (Gibson 1986).

Z27/TSW103

Z11/SINF1

Z40/Bub6

Z1/DAL072

Z12/SINF5

Z38/1972

Z2/TRAZIE

Z3/SIQUE

Z30/132

Z14/TH2

Z37/AB14

Z7/SH196

Z33/KK39

Z15/TSW53

Z6/SH017

Z10/SH276

T.b. bouafle

T.b. gam

biense group 1

0.1

T. congolense

Figure 2 UPGMA dendrogram based onthe matrix of Jacquard genetic distancescalculated on isoenzymatic results of 15known zymodemes of the species Trypa-nosoma brucei. T. congolense was chosenas the outgroup and is identified byzymodeme 27. Zymodemes 3 and 38underlined on the tree are the only zymo-demes that have been sampled in this study.

Tropical Medicine and International Health volume 7 no 7 pp 610–621 july 2002

V. Jamonneau et al. Human African trypanosomiasis in Cote d’Ivoire (Ivory Coast)

ª 2002 Blackwell Science Ltd 615

Page 7: Genetic characterization of Trypanosoma brucei gambiense and clinical evolution of human African trypanosomiasis in Côte d'Ivoire

Thus, MLEE results showed very low genetic variability in

our sample.

RAPD characterization

Random amplified polymorphism DNA analysis allows

a very clear distinction between T. congolense and

T. brucei ssp. and a clear individualization of group 1

of T. b. gambiense within T. brucei (Waitumbi &

Murphy 1993). Owing to logistic constraints, only 50

stocks in our study were characterized. They all

belonged to T. b. gambiense group 1. These results

were concordant with those of MLEE, as a low genetic

polymorphism was observed. Of the seven primers

used, only one (A2) showed a microvariability with

three profiles differing between them by only one band

(data not shown). The dendrogram is presented in

Figure 3.

Characterization by PCR/microsatellite DNA

Because of logistic constraints, only 16 stocks were charac-

terized by PCR/microsatellite (Table 4). Using MT30-33

F/R, the 16 stocks showed two bands identical to Jua and

TRPZ105058Cl.A 3Eatro1125KP465TSW656346142582257425602508250724972498659PeyaJuaB120/9806/96706696686666646626616606576556546486386366226126106066002604260326022601260025982597259525882587258425702569256225612557254924992548

0.1

Figure 3 UPGMA dendrogram based onthe Jacquard genetic distances calculated onthe RAPD results of the 50 stocks of ourstudy. Trypanosoma congolense, repre-sented by stock TRPZ 105, was chosen asthe outgroup. Stocks 058CL.A3, Eatro1125, KP465, TSW65, Jua and Peya werechosen as reference stocks for T. brucei.

Tropical Medicine and International Health volume 7 no 7 pp 610–621 july 2002

V. Jamonneau et al. Human African trypanosomiasis in Cote d’Ivoire (Ivory Coast)

616 ª 2002 Blackwell Science Ltd

Page 8: Genetic characterization of Trypanosoma brucei gambiense and clinical evolution of human African trypanosomiasis in Côte d'Ivoire

Peya bands (154 bp/162 bp). Using M6C8-CA F/R, 16

stocks showed two bands identical to those of Peya (85 bp/

89 bp). Two profiles were observed with TRBPA1/2

(149 bp/203 bp for nine stocks and 149 bp/185 bp for seven

stocks). Finally, when using TBDAC1/2, a higher micro-

variability was highlighted showing four different

patterns.

Clinical study

Forty-four patients were in the first stage of the disease (P1),

while 95 were in the second stage (P2). The degree of severity

for the five syndromes is given in Table 5. Syndromes were

not significantly linked to one another, and only the neuro-

psychiatric syndrome was significantly linked to stage of

disease (P < 0.0001), indicating a high diversity of clinical

symptoms among these patients before treatment.

The time of appearance of symptoms was significantly

longer (P ¼ 0.0002) for the patients in P2 than for the

patients in P1 (Table 6). Among the 62 patients feeling

unwell for more than 12 months, 50 (81%) were diag-

nosed in P2. However, of 30 patients whose symptoms

appeared within 6 months, 22 (73%) were already in P2.

Thus, though the appearance of symptoms for more than

12 months seemed to confirm a diagnosis of second stage,

a recent appearance of clinical signs was not necessarily a

criterion for a first-phase diagnosis. This result suggests a

diversity of clinical evolutions.

Correlation between clinical and genetic diversity

We compared the clinical patterns of the patients ‘Z3’

with those of the patients ‘Z38’. Whatever the clinical

variable, there was no significant difference between the

Table 4 Results of the PCR/microsatellitetechnique carried out using four primerpairs, on 16 stocks of our study

Stock TRBPA1/2 TBDAC1/2 M6C8-CAF/R MT30-33F/R

Jua* 149/203 152/160 83/87 154/162Peya* 149/149 154/164 85/89 154/1622499 149/203 154/162 85/89 154/1622508 149/185 150/156 85/89 154/1622562 149/203 154/162 85/89 154/162611 149/203 154/162 85/89 154/162614 149/203 154/162 85/89 154/162622 149/203 154/162 85/89 154/162634 149/203 154/162 85/89 154/162654 149/185 150/156 85/89 154/162659 149/203 152/160 85/89 154/162662 149/185 150/156 85/89 154/162664 149/203 156/164 85/89 154/162666 149/203 156/164 85/89 154/162668 149/185 150/156 85/89 154/162669 149/185 150/156 85/89 154/162384 149/185 152/156 85/89 154/162387 149/185 152/156 85/89 154/162

* Reference stocks (see Table 3).

Table 5 Occurrence of syndromes among139 patients Syndrome Absence Presence Heavy Very heavy Total

Infectious or inflammatory 4 62 73 0 139Cardiovascular 120 16 3 0 139Digestive 89 47 3 0 139Dermatological 50 53 36 0 139Neuro-psychiatric 32 66 29 12 139

The degree of importance of the different syndromes was determined as follows:Infectious or inflammatory, cardiovascular, digestive and dermatological syndrome:absence ¼ no clinical sign, presence ¼ 1–2 clinical signs, and heavy ¼ more than twoclinical signs.Neuro-psychiatric syndrome: absence ¼ no clinical sign, presence ¼ 1–2 clinical signs,heavy ¼ 3–8 clinical signs, and very heavy ¼ more than 8 clinical signs.

Tropical Medicine and International Health volume 7 no 7 pp 610–621 july 2002

V. Jamonneau et al. Human African trypanosomiasis in Cote d’Ivoire (Ivory Coast)

ª 2002 Blackwell Science Ltd 617

Page 9: Genetic characterization of Trypanosoma brucei gambiense and clinical evolution of human African trypanosomiasis in Côte d'Ivoire

two populations by means of Fisher exact test. The low

genetic variability detected by MLEE did not seem to be

correlated to the clinical variability observed. The same

result and conclusion were obtained when comparing the

genetic groups identified by both RAPD and PCR/micro-

satellite analysis. Neither was there any significant differ-

ence between KIVI positive and negative populations in

terms of clinical variables. Thus, genetic variability did not

seem to be related to the observed clinical variability.

Discussion

The isolation rates observed were particularly low com-

pared with previous works where success rates were up to

90% in Cote d’Ivoire and R.P. Congo (Aerts et al. 1992;

Truc et al. 1992). This may be because of the quality of

KIVI batches. To check this hypothesis, two different

batches were used during the whole study but the rates of

isolation remained low whatever the batch used. Circula-

tion of particular stocks in HAT foci of Cote d’Ivoire,

which could be difficult to isolate with KIVI, could also

explain the low rate of isolation. In fact, we observed that

the isolation rate varied significantly depending on the

focus of the origin of the patient. As the composition of the

human populations were similar between one focus and

another, a factor related to the parasite might have an

influence on the isolation success. Further investigations

are required in order to study a possible selective effect of

the use of the KIVI. We also observed that the isolation rate

varied significantly according to the ethnic group. Thus, a

factor related to the host might also have an influence on

the success of in vitro isolation of trypanosomes.

According to MacNamara et al. (1995), the failure or

the success of KIVI may depend first on the proportion of

short (stumpy) forms and long (slender) forms in the blood

of the patient at the moment of KIVI isolation. A high

number of stumpy forms in blood increases the success of

isolating trypanosomes using KIVI because, as within the

tsetse fly, only these stumpy forms are able to be

transformed into procyclic forms and multiply. Further

investigations are required to study the proportions of

stumpy and slender forms in patient’s blood when per-

forming KIVI isolation in various foci while taking into

account the ethnic origin of patient.

The genetic characterization by MLEE and RAPD

revealed a very low genetic polymorphism within the

stocks under study. Some genetic homogeneity among

T. b. gambiense stocks was already known (Gibson 1986;

Paindavoine et al. 1986; Godfrey et al. 1990; Hide et al.

1990) but a polymorphism as low as that described by

Jamonneau et al. (2000b) using 222 stocks isolated in Cote

d’Ivoire from 1992 to 1999 remains unusual. Moreover, a

recent study on the characterization of trypanosome stocks

from various geographical origins using PCR/microsatellite

also highlighted the absence of genetic variability among

nine stocks isolated in Cote d’Ivoire since 1993 belonging

to zymodeme Z3 (Biteau et al. 2000). In the present work,

stocks characterized by PCR/microsatellite analysis were

monomorphic for two primers, whereas a microvariability

was found when using the two other primers. We can

notice that the evaluation of the discriminatory potential of

the primers actually used for PCR/microsatellite is still in

progress. Some patterns obtained with these primers could

be specific to T. b. gambiense group 1 (Biteau et al. 2000;

Truc et al. in press).

Thus, whatever the technique used (MLEE, RAPD, PCR/

microsatellite), a low genetic polymorphism was revealed

within the stocks currently isolated in Cote d’Ivoire. Only

Z3 seems to be able to spread within the whole country,

confirming previous observations (Jamonneau et al.

2000b). However, according to the assumption of a

selection by KIVI, the isolated stocks might not be

representative of the natural populations (KIVI positive

stocks could be then genetically different from KIVI

negative stocks). This hypothesis confirms the need of

studying selectivity in isolation techniques. This could be

done by trying to directly identify trypanosomes within the

biological fluids of man (blood, CSF and lymph juice)

without isolation and culture, and using specific molecular

markers. The technique of PCR/microsatellite seems to be a

promising tool for this purpose, as two primers used in this

study highlighted a microvariability within stocks belong-

ing to the same zymodeme (Z3).

The results of the clinical study indicate a significant

diversity of clinical pictures. This is in accordance with a

previous longitudinal follow-up of patients refusing the

treatment in the Sinfra area between 1996 and 1999, which

revealed the existence of a diversity of clinical evolutions

Date of appearance of the first symptoms

Stage 0–6 months 6–12 months >12 months No response Total

1 8 20 12 4 442 22 15 50 8 95Total 30 35 62 12 139

Table 6 Stage (period) of the disease anddate of appearance of the first symptoms

Tropical Medicine and International Health volume 7 no 7 pp 610–621 july 2002

V. Jamonneau et al. Human African trypanosomiasis in Cote d’Ivoire (Ivory Coast)

618 ª 2002 Blackwell Science Ltd

Page 10: Genetic characterization of Trypanosoma brucei gambiense and clinical evolution of human African trypanosomiasis in Côte d'Ivoire

(Jamonneau et al. 2000a). In this latter work various

clinical patterns were observed, from the chronic form to

an acute form, and self-cure cases were suspected. What-

ever the parasitic disease, diversity of clinical evolution can

be explained either by the virulence of the parasite or by

the host susceptibility to disease. In the particular case of

Chagas disease, Andrade et al. (1992) have suggested that

the various genotypes described within the species

Trypanosoma cruzi (Miles et al. 1981) could be partly

responsible for the diversity of clinical evolutions. Evidence

of a correlation between genotypes and clinical evolutions

was shown through experimental pathology (Laurent et al.

1997), as well as in humans (Montamat et al. 1996). For

HAT caused by T. b. rhodesiense in the Southeast of

Uganda, Smith and Bailey (1997) showed that stocks of the

Busoga group were more pathogenic than stocks of the

Zambezi group using isoenzymatic characterization. In our

study, no correlation between clinical diversity in human

and genetic diversity of parasites (observed or not) could be

shown.

The role of the host in clinical variability could be

considered. The existence of an individual susceptibility to

disease has been shown, for example, in malaria (Garcia

et al. 1998) and leishmaniasis (Mary et al. 1999). It has

been suspected for HAT (Ginoux & Frezil 1981), being

called trypanotolerance. This phenomenon has been des-

cribed for animal trypanosomiasis (Murray et al. 1990;

Authie 1994). There is no experimental data on individual

susceptibility to HAT and its genetic explanation, only

indirect evidence (Authie et al. 1991; Garcia et al. 2000;

Jamonneau et al. 2000a). In our study, the individual

susceptibility to disease seems to better explain the

observed data than the genotype of the infecting

T. b. gambiense strains. The hypothesis of an individual

susceptibility for HAT and its genetic mechanism must be

investigated. The fast evolution of part of the patients to

the second stage of the disease, together with the suspected

existence of trypanotolerant patients, who do not see a

physician and/or refuse treatment because they do not feel

sick, is a new useful plea again for early diagnosis and

treatment and for regular supervision of HAT foci by

National Control Programmes.

Acknowledgements

We thank the HAT team of the Institut Pierre Richet in

Bouake and the National Control Program for HAT of

Cote d’Ivoire. This work was supported by a grant from

the Fonds d’Aide a la Cooperation du Ministere des

Affaires Etrangeres, Direction du Developpement et de la

Cooperation and by the Agence Francaise de la Fran-

cophonie (AUF). We thank two anonymous referees for

their great help in the improvement of the manuscript, and

we thank G. Manners for editorial advice.

References

Aerts D, Truc P, Penchenier L, Claes Y & Le Ray D (1992) A kit

for in vitro isolation of trypanosomes in the field: first trial

with sleeping sickness patients in the Congo. Transactions of

the Royal Society of Tropical Medicine and Hygiene 86,

394–395.

Andrade SG, Rassi A, Magalhaes JB, Ferriolli Filho F & Luquetti

AO (1992) Specific chemotherapy of Chagas disease. A com-

parison between the response in patients and experimental

animals inoculated with the same strains. Transactions of the

Royal Society of Tropical Medicine and Hygiene 86, 624–626.

Authie E (1994) Bovine trypanosomosis and trypanotolerance: a

role for ‘congopain’? Parasitology Today 10, 360–364.

Authie E, Cuisance D, Force-Barge P et al. (1991) Some new

prospects in epidemiology and fight against human African

trypanosomiasis. Research Revue of Parasitology 51, 29–46.

Biteau N, Bringaud F, Gibson WC, Truc P & Baltz T (2000)

Characterisation of Trypanozoon isolates using micro- and

minisatellite markers. Molecular and Biochemical Parasitology

105, 185–201.

Cattand P, Miezan TW & de Raadt P (1988) Human African

trypanosomiasis: use of double centrifugation of cerebrospinal

fluid to detect trypanosomes. Bulletin of the World Health

Organization 66, 83–86.

Cunningham I (1977) New culture medium for maintenance of

Tsetse tissues and growth of Trypanosomatids. Journal of

Protozoology 21, 325–329.

Dumas M & Bouteille B (1996) Human African trypanosomiasis.

Compte-Rendu de la Societe de Biologie 190, 395–408.

Garcia A, Cot M, Chippaux JP et al. (1998) Genetic control of

blood infection levels in human malaria: evidence for a complex

genetic model. American Journal of Tropical Medicine and

Hygiene 58, 480–488.

Garcia A, Jamonneau V, Magnus E et al. (2000) Longitudinal

survey of positive Card Agglutination Trypanosomiasis

Test (CATT) but apparently aparasitemic individuals in

Cote d’Ivoire: evidence for complex and heterogeneous popu-

lation. Tropical Medicine and International Health 5,

786–793.

Gashumba JK, Baker RD & Godfrey DG (1988) Trypanosoma

congolense: the distribution of enzymic variants in East and

West Africa. Parasitology 96, 475–486.

Gibson WC (1986) Will the real Trypanosoma brucei gambiense

please stand up. Parasitology Today 2, 255–257.

Gibson WC, Marshall TF & Godfrey DG (1980) Numerical

analysis of enzyme polymorphism: a new approach to the

epidemiology and taxonomy of trypanosomes of the subgenus

Trypanozoon. Advances in Parasitology 18, 175–246.

Ginoux PY & Frezil JL (1981) Recherches sur la latence clinique et

la trypanotolerance humaine dans le foyer du couloir du fleuve

Congo. Cahier ORSTOM, Serie Entomologie Medicale et

Parasitologie 1, 33–40.

Tropical Medicine and International Health volume 7 no 7 pp 610–621 july 2002

V. Jamonneau et al. Human African trypanosomiasis in Cote d’Ivoire (Ivory Coast)

ª 2002 Blackwell Science Ltd 619

Page 11: Genetic characterization of Trypanosoma brucei gambiense and clinical evolution of human African trypanosomiasis in Côte d'Ivoire

Godfrey DG, Baker RD, Rickman LR & Mehlitz D (1990) The

distribution, relationships and identification of enzymatic var-

iants within the subgenus Trypanozoon. Advances in Parasit-

ology 29, 1–39.

Hide G, Cattand P, Le Ray D, Barry JD & Tait A (1990) The

identification of T. brucei subspecies using repetitive DNA

sequences. Molecular and Biochemical Parasitology 39, 213–226.

Jacquard A (1973) Distances genealogiques et distances genetiques.

Cahiers d’Anthropologie et d’Ecologie Humaine 1, 11–124.

Jamonneau V, Garcia A, N’Guessan P et al. (2000a) Clinical and

biological evolution of Human African Trypanosomiasis in Cote

d’Ivoire. Annals of Tropical Medicine and Parasitology 94, 831–

835.

Jamonneau V, N’Guessan P, N’Dri L, Simaro P & Truc P (2000b)

Exploration of the distribution of Trypanosoma brucei ssp. in

West Africa, by multilocus enzyme electrophoresis. Annals of

Tropical Medicine and Parasitology 94, 643–649.

Jannin J, Moulia-Pelat JP, Chanfreau B et al. (1993) Trypanos-

omiase Humaine Africaine: etude d’un score de presomption de

diagnostic au Congo. Bulletin of the World Health Organization

71, 215–222.

Lapeyssonnie L (1960) Deuxieme note concernant un cas exep-

tionnel de trypanosomiase, parasitemie observee pendant 21 ans

sans signes cliniques appreciables chez une malade traitee

inefficacement pendant les 10 premieres annees. Bulletin de la

Societe de Pathologie Exotique 57, 28–32.

Laurent JP, Barnabe C, Quesney V, Noel S & Tibayrenc M (1997)

Impact of clonal evolution on the biological diversity of

Trypanosoma cruzi. Parasitology 114, 213–218.

Lumsden WHR, Kimber CD, Evans DA & Doig SJ (1979)

Trypanosoma brucei: miniature anion-exchange centrifugation

technique for detection of low parasitemia; adapted for field use.

Transactions of the Royal Society of Tropical Medicine and

Hygiene 73, 312–317.

MacNamara JJ, Bailey J, Smith DH, Wakhooli S & Godfrey DG

(1995) Isolation of Trypanosoma brucei gambiense from Nor-

thern Uganda: evaluation of the Kit for in vitro isolation (KIVI)

in an epidemic focus. Transactions of the Royal Society of

Tropical Medicine and Hygiene 89, 388–389.

Magnus E, Vervoort T & Van Meirvenne N (1978) A card-

agglutination test with stained trypanosomes (CATT) for the

serological diagnosis of T. gambiense trypanosomiasis. Annales

de la Societe Belge de Medecine Tropicale 59, 169–176.

Mary C, Auriault V, Faugere B & Dessein AJ (1999) Control of

Leishmania infantum infection is associated with CD8(+) and

gamma interferon- and interleukin-5-producing CD4(+) antigen-

specific T cells. Infection and Immunity 67, 5559–5566.

Mehlitz D, Zillmann U, Scott CM & Godfrey DG (1982)

Epidemiological studies on the animal reservoir of gambiense

sleeping sickness. Part III. Characterisation of Trypanozoon

stocks by isoenzymes and sensitivity to human serum. Tropen-

medizin und Parasitologie 33, 113–118.

Miles MA, Cedillos RA, Povoa MM, Prata A, Sousa AA &

Macedo V (1981) Do radically dissimilar Trypanosoma cruzi

strains (zymodemes) cause Venezuelan and Brazilian forms of

Chagas’s disease? Lancet 20, 1338–1340.

Montamat EE, De Luca GM, Gallerano RH, Sosa R & Blanco A

(1996) Characterisation of Trypanosoma cruzi populations by

zymodemes: correlation with clinical pictures. American Journal

of Tropical Medicine and Hygiene 55, 625–628.

Murray M, Trail JCM & d’Ieteren GDM (1990) Trypanotolerance

in cattle and prospects for the control of trypanosomiasis by

selective breeding. Revue Scientifique et Technique de l’Office

Internationnal Des Epizooties 9, 369–386.

Oury B, Dutrait N, Bastrenta B & Tibayrenc M (1998) Trypan-

osoma cruzi: use of a synapomorphic RAPD fragment as a

species-specific DNA probe. Journal of Parasitology 83,

52–57.

Paindavoine P, Pays E, Laurent M et al. (1986) The use of DNA

hybridisation and taxonomy in determining the relationship

between Trypanosoma brucei stocks and subspecies. Parasitol-

ogy 92, 31–50.

Simo G, Njiokou F, Nkinin SW, Mgbedie M, Laveissiere C &

Herder S (2000) Etude de la prevalence des infections a

trypanosomes chez les animaux sauvages du foyer de la maladie

du sommeil de Bipindi, Cameroun. Bulletin de Liaison de

L’oceac 33, 8–15.

Smith DH & Bailey JW (1997) Human African trypanosomiasis in

South-eastern Uganda: clinical diversity and isoenzyme profiles.

Annals of Tropical Medicine and Parasitology 7, 851–856.

Sneath PHA & Sokal RR (1973) Numerical Taxonomy. The

Principles and Practice of Numerical Classification. W.H.

Freeman and Co, San Francisco, 573 pp.

Stevens JR, Nunes VLB, Lanham SM & Oshiro ET (1989)

Isoenzyme characterisation of Trypanosoma evansi isolated

from capybaras and dogs in Brazil. Acta Tropica 46, 213–222.

Stevens JR, Lanham SM, Allingham R & Gashumba JK (1992) A

simplified method for identifying subspecies and strain groups in

Trypanozoon by isoenzymes. Annals of Tropical Medicine and

Parasitology 86, 9–28.

Tibayrenc M, Neubauer K, Barnabe C, Guerrini F, Skarecky D &

Ayala FJ (1993) Genetic characterisation of six parasitic

protozoa: parity between random-primer DNA typing and

multilocus enzyme electrophoresis. Proceedings of the National

Academy of Sciences of the USA 90, 1335–1339.

Truc P, Mathieu Daude F & Tibayrenc M (1991) Multilocus

isoenzyme identification of Trypanosoma brucei stocks isolated

in Central Africa: evidence for an animal reservoir of sleeping

sickness in Congo. Acta Tropica 49, 127–135.

Truc P, Aerts D, McNamara JJ et al. (1992) The direct in vitro

isolation of Trypanosoma brucei from man and animals, and its

potential value for the diagnosis of Gambian trypanosomiasis.

Transactions of the Royal Society of Tropical Medicine and

Hygiene 86, 627–629.

Truc P, Formenty P, Diallo PB, Komoin-Oka C & Lauginie F

(1997a) Confirmation of two distincts classes of zymodemes of

Trypanosoma brucei infecting patients and wild mammals in

Cote d’Ivoire: suspected difference in pathogenicity. Annals of

Tropical Medicine and Parasitology 91, 951–956.

Truc P, Formenty P, Duvallet G, Komoin-Oka C, Diallo PB &

Lauginie F (1997b) Identification of trypanosomes isolated by

KIVI from wild mammals in Cote d’Ivoire: diagnostic, taxo-

Tropical Medicine and International Health volume 7 no 7 pp 610–621 july 2002

V. Jamonneau et al. Human African trypanosomiasis in Cote d’Ivoire (Ivory Coast)

620 ª 2002 Blackwell Science Ltd

Page 12: Genetic characterization of Trypanosoma brucei gambiense and clinical evolution of human African trypanosomiasis in Côte d'Ivoire

nomic and epidemiological considerations. Acta Tropica 67,

187–196.

Truc P, Ravel S, Jamonneau V, N’Guessan P & Cuny G (2002)

Genetic variability within Trypanosoma brucei gambiense:

evidence of the circulation of different genotypes in Human

African Trypanosomiasis patients in Cote d’Ivoire. Transactions

of the Royal Society of Tropical Medicine and Hygiene 96,

1–4.

Truc P & Tibayrenc M (1993) Population genetics of Trypano-

soma brucei in Central Africa: taxonomic and epidemiological

significance. Parasitology 106, 137–149.

Waitumbi JN & Murphy NB (1993) Inter- and intra-species

differentiation of trypanosomes by genomic fingerprinting with

arbitrary primers. Molecular and Biochemical Parasitology 58,

181–186.

Welsh J & McClelland M (1990) Fingerprinting genomes using PCR

with arbitrary primers. Nucleic Acids Research 18, 7213–7218.

WHO (1998) Control and Surveillance of African Trypanosomi-

asis. WHO Technical Report Series 881, WHO, Geneva.

Williams JGK, Kubelik AR, Livak KJ, Rafalski JA & Tingley SV

(1990) DNA polymorphisms amplified by arbitrary primers are

useful as genetic markers. Nucleic Acids Research 18,

6531–6535.

Young CJ & Godfrey DG (1983) Enzyme polymorphism and the

distribution of Trypanosoma congolense isolates. Annals of

Tropical Medicine and Parasitology 77, 467–481.

Tropical Medicine and International Health volume 7 no 7 pp 610–621 july 2002

V. Jamonneau et al. Human African trypanosomiasis in Cote d’Ivoire (Ivory Coast)

ª 2002 Blackwell Science Ltd 621


Recommended