Genetic characterization of Trypanosoma brucei gambiense and clinical evolution of human African trypanosomiasis in Cte d'Ivoire

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  • Genetic characterization of Trypanosoma brucei gambiense

    and clinical evolution of human African trypanosomiasis

    in Cote dIvoire

    V. Jamonneau1, A. Garcia2, S. Ravel3, G. Cuny3, B. Oury4, P. Solano1, P. NGuessan1, L. NDri1, R. Sanon1,

    J. L. Frezil3 and P. Truc5

    1 Institut de Recherche pour le Developpement (IRD/UR 035), Centre Pierre Richet, Bouake, Cote dIvoire2 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 dEtude 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 dIvoire.

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

    these patients. An important genetic monomorphism among stocks isolated in Cote dIvoire 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 0.0002) and on the ethnic group(P 0.0317) of the patient. Further investigations are required in order to study a possible selectiveimpact 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 dIvoire

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

    Richet, 01 BP 1500 Bouake, Cote dIvoire. Fax: +225 31 63 27 38; E-mail: solano@ird.ci

    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

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    610 2002 Blackwell Science Ltd

  • 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 dIvoire 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 dIvoire 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 dIvoire: 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 dIvoire, Burkina Faso and

    Mali).

    Clinical assessment before treatment

    Objective clinical signs that were looked for were:

    palpation (hepatomegaly, splenomegaly, swollen lateralcervical lymph nodes);

    cardiovascular investigation (dysrhythmia, heartmurmurs and low blood pressure);

    dermatological examination (search for initial lesion ofinoculation, 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

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    V. Jamonneau et al. Human African trypanosomiasis in Cote dIvoire (Ivory Coast)

    2002 Blackwell Science Ltd 611

  • trypanosomes in CSF is defined as the first stage of the

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

    SinfraBouafle 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.

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  • 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 Fishers 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

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  • 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 dIvoire; 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 dIvoire T. b. bfl 2TSW65 Pig 1982 Cote dIvoire 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*

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  • 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

    dIvoire 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 0.0002 and P 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. bouafleT.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.

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  • 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.

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  • 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 0.0002) for the patients in P2 than for thepatients 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 no clinical sign, presence 12 clinical signs, and heavy more than twoclinical signs.Neuro-psychiatric syndrome: absence no clinical sign, presence 12 clinical signs,heavy 38 clinical signs, and very heavy more than 8 clinical signs.

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  • 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 dIvoire 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 dIvoire,

    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 patients 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

    dIvoire 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 dIvoire 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 dIvoire. 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 06 months 612 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 610621 july 2002

    V. Jamonneau et al. Human African trypanosomiasis in Cote dIvoire (Ivory Coast)

    618 2002 Blackwell Science Ltd

  • (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 dIvoire. This work was supported by a grant from

    the Fonds dAide a` la Cooperation du Ministe`re des

    Affaires Etrange`res, 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.

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