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 gambienseand clinical evolution of human African trypanosomiasisin Cote dIvoireV. Jamonneau1, A. Garcia2, S. Ravel3, G. Cuny3, B. Oury4, P. Solano1, P. NGuessan1, L. NDri1, R. Sanon1,J. L. Frezil3 and P. Truc51 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, CameroonSummary Human African trypanosomiasis is a parasitic infection caused by protozoa belonging to Trypanosomabrucei subspecies. The clinical evolution of this disease is complex and might be because of theparasite itself, as genetic diversity has been observed in T. brucei ssp. We investigated the relationshipbetween 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 fromthese patients. An important genetic monomorphism among stocks isolated in Cote dIvoire wasobserved by using various markers: isoenzymes electrophoresis, random amplified polymorphism DNAand PCR of microsatellite sequences. At the same time, the diversity of clinical patterns and evolutionswas confirmed by clinical analysis. The existence of an individual susceptibility to disease (humantrypanotolerance) should be taken into account even if our genetic conclusions might be distortedbecause the isolation success rates were particularly poor. In fact, we observed that the isolation successrate 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, individualsusceptibility, Cote dIvoirecorrespondence P. Solano, Institut de Recherche pour le Developpement (IRD), UR 035, Institut PierreRichet, 01 BP 1500 Bouake, Cote dIvoire. Fax: +225 31 63 27 38; E-mail: solano@ird.ciIntroductionHuman African trypanosomiasis (HAT), or sleeping sick-ness, is a major public health problem in sub-SaharanAfrica. Approximately 60 million people are daily exposedto the risk of infection. It is estimated that there are about500 000 infected but untreated persons (WHO 1998). Thepathogenic agent is the trypanosome Trypanosoma brucei.Classically, T. brucei is subdivided into three subspecies onthe basis of extrinsic criteria: T. b. gambiense is responsiblefor 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 nospecific clinical signs (Jannin et al. 1993; Dumas &Bouteille 1996), leading to a meningo-encephalitic stage,usually characterized by neurological disorders (secondperiod). In the absence of treatment, the disease isinvariably fatal. In T. b. gambiense chronic form, theduration of the first period may be several years: thefluctuating parasitaemia remains low and tends todecrease. The appearance of neurological disorders duringTropical Medicine and International Healthvolume 7 no 7 pp 610621 july 2002610 2002 Blackwell Science Ltdthe second period is often progressive. In acute HATcaused by T. b. rhodesiense, the haemato-lymphatic stagelasts a few weeks to a few months, passage to the meningo-encephalitic stage is brutal and death can occur within afew months of onset of this stage.A diversity of clinical evolutions has been observed forT. b. gambiense, from some chronic forms to asympto-matic forms (Jamonneau et al. 2000a). At one extreme, apatient from Togo carried trypanosomes (T. b. gamb-iense) for 21 years with no clinical signs or neurologicaldisorders (Lapeyssonnie 1960). At the other extreme,some patients detected in Cote dIvoire presented aclinical evolution characteristic of acute HAT (Truc et al.1997a). This clinical diversity does not correspond to thetraditional definition of the T. b. gambiense clinical formsof HAT.Genetic diversity within T. b. gambiense has beendemonstrated 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 clinicalevolution of the HAT remains unproven. We intendedto investigate the relationships between the geneticdiversity of the parasite and that of the clinical patterns.With this aim, we launched a clinical study on patientsdiagnosed in Cote dIvoire together with a geneticanalysis of the populations of trypanosomes isolatedfrom these patients.Patients and methodsStudy areas and patientsThis study was conducted from 1996 to 1999 in two activeHAT foci of Cote dIvoire: the western-central part of thecountry (Daloa, Vavoua, Bouafle, Sinfra, and Bonon) andthe south-east (Aboisso) (Figure 1). We included patientsdetected (parasitological evidence) either actively (duringfive medical surveys) or passively (patients presentingthemselves for treatment). All patients were treated in thethree centres specialized in HAT treatment: the Projet deRecherches Cliniques sur les Trypanosomoses in Daloa,and the local health centres in Bouafle and Aboisso.Patients were told of the objectives and protocol of thestudy, and only those who gave their consent were includedin the study (patients younger than 10 years were notincluded).Epidemiological data collectionFor 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, existenceof family history of HAT, time and mode of diagnosis(passive or active), and treatment schedule. We distin-guished two ethnic grouping groups: natives (includingpatients born in the study areas) and migrants (includingsubjects from northern Cote dIvoire, Burkina Faso andMali).Clinical assessment before treatmentObjective 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 subjectiveclinical signs such as asthenia, anorexia, cachexia, fevers,repeated headache, nausea, pruritus, cutaneous rash, sleepdisturbances (alteration of circadian rhythm). Questionsabout the approximate date of appearance of the initialsymptoms and the mode of evolution of the disease werealso included.On the basis of clinical signs, patients were classifiedaccording to the existence and the importance of thefollowing: infectious or inflammatory syndrome, cardio-vascular syndrome, digestive syndrome, dermatologicalsyndrome and neuro-psychiatric syndrome (Table 1).Serological and parasitological examinations,and stage determinationEach patient underwent serological and parasitologicalinvestigations. For serology, the Card Agglutination Testfor Trypanosomiasis (T. b. gambiense) was performedusing whole blood and plasma (Magnus et al. 1978).Trypanosomes were detected using the mini AnionExchange Centrifugation Technique (Lumsden et al. 1979)and by direct microscopic examination of the lymphaticfluid if lymph nodes were swollen.For stage determination, the tests used on the cereb-rospinal fluid (CSF) were: trypanosome detection bydouble centrifugation (Cattand et al. 1988) and leukocytecounting using a Nageotte counting chamber. A lowleukocyte count ( 5 cells/ll) combined with absence ofTropical Medicine and International Health volume 7 no 7 pp 610621 july 2002V. Jamonneau et al. Human African trypanosomiasis in Cote dIvoire (Ivory Coast) 2002 Blackwell Science Ltd 611trypanosomes in CSF is defined as the first stage of thedisease. An elevated leukocyte count (>5 cells/ll), with orwithout trypanosomes in CSF, forms the basis for second-stage diagnosis (WHO 1998).Isolation of trypanosomesThe stocks were isolated by using the KIVI (Kit for Invitro Isolation of trypanosomes, Aerts et al. 1992) andwere then multiplied using semidefined culture medium(Cunningham 1977) according to the protocol describedby Truc et al. (1992). For each stock, two pellets oftrypanosomes were obtained by centrifugation and storedin liquid nitrogen. Reference stocks (Tables 2 and 3) hadbeen isolated with the KIVI method during or after1991, the others having been isolated by rodent inocu-lation.Multilocus enzyme electrophoresisProteins were extracted from one of the two pellets (Trucet al. 1991). Stocks were characterized by the technique ofmultilocus enzyme electrophoresis (MLEE) on celluloseacetate plates and 11 enzymatic systems were revealed:ALAT (EC., GOT (EC., Nhi (EC.,Nhd (EC., ME (EC., PEP-2 (EC.3.4.11),MALIGUINEABURKINA FASOBouakeVavouaBononDaloaSinfraBouafle YamoussoukroLIBERIAGHANAAboissoAbidjanATLANTIC OCEANLEGEND:RiverLakeTowns of the study areaOther townsScale0 25 50 75 100 kmNFigure 1 Geographic location of the studyarea.Tropical Medicine and International Health volume 7 no 7 pp 610621 july 2002V. Jamonneau et al. Human African trypanosomiasis in Cote dIvoire (Ivory Coast)612 2002 Blackwell Science LtdMDH (EC., IDH (, TDH (,PGM (EC. according to Truc et al. (1991) andTruc and Tibayrenc (1993), and SOD (EC. to Stevens et al. (1989).Random amplified polymorphism DNAThe DNA was extracted from the second pellet according tothe protocol described by Oury et al. (1998). Stocks werecharacterized by the RAPD technique (random amplifiedpolymorphism DNA, Welsh & McClelland 1990; Williamset 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 DNAsequencesFrom 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 byTruc et al. (2002).Analysis proceduresWhenever possible, a comparison of two qualitativevariables was performed by means of Pearson chi-squaretests. Alternatively, we performed Fishers exact test. Thelevel of significance retained for the tests was 5%. TheseTable 1 Clinical signs and corresponding syndromesSyndrome Clinical signs Expected answersInflammatory/infectious Temperature at inclusion Quantitativesyndrome Asthenia Absence/presenceAnorexia Absence/presenceWeight loss Absence/presence(feeling) Fever Absence/presenceRecurrent headache Absence/presenceCervical lymph nodes Absence/presenceDigestive syndrome Nausea Absence/presenceVomiting Absence/presenceHepatomegaly Absence/presenceSplenomegaly Absence/presenceCardiovascular syndrome Arrhythmia (heart rhythm) Normal/abnormalHeart rate Normal/abnormalBlood pressure Normal/abnormalSubjective heart troubles Absence/presenceDermatological syndrome Chancre Absence/presenceTrypanids Absence/presenceOedema Absence/presencePruritus Absence/presenceSkin rash Absence/presenceNeuro-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/insensitivityTropical Medicine and International Health volume 7 no 7 pp 610621 july 2002V. Jamonneau et al. Human African trypanosomiasis in Cote dIvoire (Ivory Coast) 2002 Blackwell Science Ltd 613analyses 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 geneticdistances (Jacquard 1973) calculated from MLEE andRAPD results for visualizing the relationships betweenstocks (Sneath & Sokal 1973). Reference stocks of T. b.rhodesiense, T. b. brucei and T. b. gambiense groups 1 andbouafle 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 primerswere specific to T. b. gambiense group 1; thus, onlytwo reference stocks, Jua and Peya (Table 3), wereused. Each band obtained, defined by its molecularweight (in base pairs or bp), corresponds to an allele.For each stock, two bands per primer were revealed(X bp/Y bp).ResultsPatientsA 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, 82Table 2 Reference stocks used for MLEE characterizationStock Host Year Country Focus Zymodeme Species ReferenceDAL072 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 2CI, 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 ReferenceJua* 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 6w.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 610621 july 2002V. Jamonneau et al. Human African trypanosomiasis in Cote dIvoire (Ivory Coast)614 2002 Blackwell Science Ltdcame from Sinfra, 27 from Bonon, 13 from Bouafle and 8from Daloa. Sixty-eight per cent of patients were detectedpassively and 71 actively through five medical surveys.Thirty-nine patients were natives (28%) and 100 migrants(72%). This unequal distribution is traditional in CotedIvoire as most of the migrants work and live close to thecocoa and coffee plantations, which constitute areas ofgreater HAT transmission risk. One-hundred and ninepatients (78.4%) were farmers and 30 (21.6%) declared afamily history of HAT.Isolation of trypanosomesOf the 139 KIVIs performed before treatment, only62 (44%) gave a positive result allowing the in vitroculture and multiplication of procyclics. To isolate amaximum of stocks, another KIVI was inoculated for46 patients (some of whom had already given positiveKIVI in the first round). Only 20 of these 46 KIVI(43%) were positive. In total, 64 stocks (46%) wereisolated and cultured for genetic identification.These isolation success rates were particularly poor.Thus, we checked whether some of the host characteristicscould have affected the success of isolation. We comparedthe epidemiological parameters of the human populationfrom whom the stocks were isolated (population KIVIpositive) with the population from whom the isolationfailed (population KIVI negative). The HAT focus oforigin and the ethnic group had a significant influence on apositive in vitro isolation (P 0.0002 and P 0.0317,respectively). More precisely, the isolation rate rangedfrom 33.7% in the Sinfra focus to 85.2% in the Bonon one.The isolation rate was particularly poor with the Baoulegroup, natives of the area (7.7%) and was most elevatedfor the Senoufo migrant group (66.7%).Isozyme characterizationOf 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 relationshipsbetween these two zymodemes, which differ only by the MElocus: Z3 showed heterozygotes at this locus (three bands),while Z38 was a homozygote (one band). These twozymodemes are genetically closely related (d < 0.1). Theyboth belong to group 1 of T. b. gambiense (Gibson 1986).Z27/TSW103Z11/SINF1Z40/Bub6Z1/DAL072Z12/SINF5Z38/1972Z2/TRAZIEZ3/SIQUEZ30/132Z14/TH2Z37/AB14Z7/SH196Z33/KK39Z15/TSW53Z6/SH017Z10/SH276T.b. bouafleT.b. gambiense group 10.1T. congolenseFigure 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 610621 july 2002V. Jamonneau et al. Human African trypanosomiasis in Cote dIvoire (Ivory Coast) 2002 Blackwell Science Ltd 615Thus, MLEE results showed very low genetic variability inour sample.RAPD characterizationRandom amplified polymorphism DNA analysis allowsa very clear distinction between T. congolense andT. brucei ssp. and a clear individualization of group 1of T. b. gambiense within T. brucei (Waitumbi &Murphy 1993). Owing to logistic constraints, only 50stocks in our study were characterized. They allbelonged to T. b. gambiense group 1. These resultswere concordant with those of MLEE, as a low geneticpolymorphism was observed. Of the seven primersused, only one (A2) showed a microvariability withthree profiles differing between them by only one band(data not shown). The dendrogram is presented inFigure 3.Characterization by PCR/microsatellite DNABecause of logistic constraints, only 16 stocks were charac-terized by PCR/microsatellite (Table 4). Using MT30-33F/R, the 16 stocks showed two bands identical to Jua andTRPZ105058Cl.A 3Eatro1125KP465TSW656346142582257425602508250724972498659PeyaJuaB120/9806/967066966866666466266166065765565464863863662261261060660026042603260226012600259825972595258825872584257025692562256125572549249925480.1Figure 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 610621 july 2002V. Jamonneau et al. Human African trypanosomiasis in Cote dIvoire (Ivory Coast)616 2002 Blackwell Science LtdPeya bands (154 bp/162 bp). Using M6C8-CA F/R, 16stocks 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 sevenstocks). Finally, when using TBDAC1/2, a higher micro-variability was highlighted showing four differentpatterns.Clinical studyForty-four patients were in the first stage of the disease (P1),while 95 were in the second stage (P2). The degree of severityfor the five syndromes is given in Table 5. Syndromes werenot significantly linked to one another, and only the neuro-psychiatric syndrome was significantly linked to stage ofdisease (P < 0.0001), indicating a high diversity of clinicalsymptoms among these patients before treatment.The time of appearance of symptoms was significantlylonger (P 0.0002) for the patients in P2 than for thepatients in P1 (Table 6). Among the 62 patients feelingunwell for more than 12 months, 50 (81%) were diag-nosed in P2. However, of 30 patients whose symptomsappeared within 6 months, 22 (73%) were already in P2.Thus, though the appearance of symptoms for more than12 months seemed to confirm a diagnosis of second stage,a recent appearance of clinical signs was not necessarily acriterion for a first-phase diagnosis. This result suggests adiversity of clinical evolutions.Correlation between clinical and genetic diversityWe compared the clinical patterns of the patients Z3with those of the patients Z38. Whatever the clinicalvariable, there was no significant difference between theTable 4 Results of the PCR/microsatellitetechnique carried out using four primerpairs, on 16 stocks of our studyStock TRBPA1/2 TBDAC1/2 M6C8-CAF/R MT30-33F/RJua* 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 TotalInfectious 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 139The 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.Tropical Medicine and International Health volume 7 no 7 pp 610621 july 2002V. Jamonneau et al. Human African trypanosomiasis in Cote dIvoire (Ivory Coast) 2002 Blackwell Science Ltd 617two populations by means of Fisher exact test. The lowgenetic variability detected by MLEE did not seem to becorrelated to the clinical variability observed. The sameresult and conclusion were obtained when comparing thegenetic groups identified by both RAPD and PCR/micro-satellite analysis. Neither was there any significant differ-ence between KIVI positive and negative populations interms of clinical variables. Thus, genetic variability did notseem to be related to the observed clinical variability.DiscussionThe isolation rates observed were particularly low com-pared with previous works where success rates were up to90% in Cote dIvoire and R.P. Congo (Aerts et al. 1992;Truc et al. 1992). This may be because of the quality ofKIVI batches. To check this hypothesis, two differentbatches were used during the whole study but the rates ofisolation 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 alsoexplain the low rate of isolation. In fact, we observed thatthe isolation rate varied significantly depending on thefocus of the origin of the patient. As the composition of thehuman populations were similar between one focus andanother, a factor related to the parasite might have aninfluence on the isolation success. Further investigationsare required in order to study a possible selective effect ofthe use of the KIVI. We also observed that the isolation ratevaried significantly according to the ethnic group. Thus, afactor related to the host might also have an influence onthe success of in vitro isolation of trypanosomes.According to MacNamara et al. (1995), the failure orthe success of KIVI may depend first on the proportion ofshort (stumpy) forms and long (slender) forms in the bloodof the patient at the moment of KIVI isolation. A highnumber of stumpy forms in blood increases the success ofisolating trypanosomes using KIVI because, as within thetsetse fly, only these stumpy forms are able to betransformed into procyclic forms and multiply. Furtherinvestigations are required to study the proportions ofstumpy and slender forms in patients blood when per-forming KIVI isolation in various foci while taking intoaccount the ethnic origin of patient.The genetic characterization by MLEE and RAPDrevealed a very low genetic polymorphism within thestocks under study. Some genetic homogeneity amongT. 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 byJamonneau et al. (2000b) using 222 stocks isolated in CotedIvoire from 1992 to 1999 remains unusual. Moreover, arecent study on the characterization of trypanosome stocksfrom various geographical origins using PCR/microsatellitealso highlighted the absence of genetic variability amongnine stocks isolated in Cote dIvoire since 1993 belongingto zymodeme Z3 (Biteau et al. 2000). In the present work,stocks characterized by PCR/microsatellite analysis weremonomorphic for two primers, whereas a microvariabilitywas found when using the two other primers. We cannotice that the evaluation of the discriminatory potential ofthe primers actually used for PCR/microsatellite is still inprogress. Some patterns obtained with these primers couldbe 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 revealedwithin the stocks currently isolated in Cote dIvoire. OnlyZ3 seems to be able to spread within the whole country,confirming previous observations (Jamonneau et al.2000b). However, according to the assumption of aselection by KIVI, the isolated stocks might not berepresentative of the natural populations (KIVI positivestocks could be then genetically different from KIVInegative stocks). This hypothesis confirms the need ofstudying selectivity in isolation techniques. This could bedone by trying to directly identify trypanosomes within thebiological fluids of man (blood, CSF and lymph juice)without isolation and culture, and using specific molecularmarkers. The technique of PCR/microsatellite seems to be apromising tool for this purpose, as two primers used in thisstudy highlighted a microvariability within stocks belong-ing to the same zymodeme (Z3).The results of the clinical study indicate a significantdiversity of clinical pictures. This is in accordance with aprevious longitudinal follow-up of patients refusing thetreatment in the Sinfra area between 1996 and 1999, whichrevealed the existence of a diversity of clinical evolutionsDate of appearance of the first symptomsStage 06 months 612 months >12 months No response Total1 8 20 12 4 442 22 15 50 8 95Total 30 35 62 12 139Table 6 Stage (period) of the disease anddate of appearance of the first symptomsTropical Medicine and International Health volume 7 no 7 pp 610621 july 2002V. Jamonneau et al. Human African trypanosomiasis in Cote dIvoire (Ivory Coast)618 2002 Blackwell Science Ltd(Jamonneau et al. 2000a). In this latter work variousclinical patterns were observed, from the chronic form toan acute form, and self-cure cases were suspected. What-ever the parasitic disease, diversity of clinical evolution canbe explained either by the virulence of the parasite or bythe host susceptibility to disease. In the particular case ofChagas disease, Andrade et al. (1992) have suggested thatthe various genotypes described within the speciesTrypanosoma cruzi (Miles et al. 1981) could be partlyresponsible for the diversity of clinical evolutions. Evidenceof a correlation between genotypes and clinical evolutionswas shown through experimental pathology (Laurent et al.1997), as well as in humans (Montamat et al. 1996). ForHAT caused by T. b. rhodesiense in the Southeast ofUganda, Smith and Bailey (1997) showed that stocks of theBusoga group were more pathogenic than stocks of theZambezi group using isoenzymatic characterization. In ourstudy, no correlation between clinical diversity in humanand genetic diversity of parasites (observed or not) could beshown.The role of the host in clinical variability could beconsidered. The existence of an individual susceptibility todisease has been shown, for example, in malaria (Garciaet al. 1998) and leishmaniasis (Mary et al. 1999). It hasbeen suspected for HAT (Ginoux & Frezil 1981), beingcalled trypanotolerance. This phenomenon has been des-cribed for animal trypanosomiasis (Murray et al. 1990;Authie 1994). There is no experimental data on individualsusceptibility to HAT and its genetic explanation, onlyindirect evidence (Authie et al. 1991; Garcia et al. 2000;Jamonneau et al. 2000a). In our study, the individualsusceptibility to disease seems to better explain theobserved data than the genotype of the infectingT. b. gambiense strains. The hypothesis of an individualsusceptibility for HAT and its genetic mechanism must beinvestigated. The fast evolution of part of the patients tothe second stage of the disease, together with the suspectedexistence of trypanotolerant patients, who do not see aphysician and/or refuse treatment because they do not feelsick, is a new useful plea again for early diagnosis andtreatment and for regular supervision of HAT foci byNational Control Programmes.AcknowledgementsWe thank the HAT team of the Institut Pierre Richet inBouake and the National Control Program for HAT ofCote dIvoire. This work was supported by a grant fromthe Fonds dAide a` la Cooperation du Ministe`re desAffaires Etrange`res, Direction du Developpement et de laCooperation and by the Agence Francaise de la Fran-cophonie (AUF). We thank two anonymous referees fortheir great help in the improvement of the manuscript, andwe thank G. 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