Genetic characterization of Trypanosoma brucei gambiense and clinical evolution of human African trypanosomiasis in Côte 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

    Tropical Medicine and International Health

    volume 7 no 7 pp 610621 july 2002

    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

    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)

    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.

    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)

    612 2002 Blackwell Science Ltd

  • 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

    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)

    2002 Blackwell Science Ltd 613

  • 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 Harte...

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