Stage determination and therapeutic decision in human African trypanosomiasis: value of polymerase chain reaction and immunoglobulin M quantification on the cerebrospinal fluid of sleeping sickness patients in Côte d'Ivoire.

In human African trypanosomiasis (HAT), two disease stages are defined: the first, or haemo-lymphatic stage, and the second, or meningo-encephalitic stage. Stage determination forms the basis of therapeutic decision and is of prime importance, as the drug used to cure second-stage patients has considerable side-effects. However, the tests currently used for stage determination have low sensitivity or specificity. Two new tests for stage determination in the cerebrospinal fluid (CSF) were evaluated on 73 patients diagnosed with HAT in Côte d'Ivoire. The polymerase chain reaction (PCR) detecting trypanosome DNA (PCR/CSF) is an indirect test for trypanosome detection whereas the latex agglutination test detecting immunoglobulin M (LATEX/IgM) is an indicator for neuro-inflammation. Both tests were compared with classically used tests, double centrifugation and white blood cell count of the CSF. PCR/CSF appeared to be the most sensitive test (96%), and may be of use to improve stage determination. However, its value for therapeutic decision appears limited, as patients whose CSF was positive with PCR were successfully treated with pentamidine. This result confirms those of previous works that showed that some patients with trypanosomes in the CSF could be treated successfully with pentamidine. LATEX/IgM, which depending on the cut-off, showed lower sensitivity of 76% and 88%, but higher specificity of 83% and 71% for LATEX/IgM 16 and LATEX/IgM 8 respectively, appears more appropriate for therapeutic decision making.

Comparison of different DNA preparation protocols for PCR diagnosis of Human African Trypanosomosis in Côte d'Ivoire.

During a medical survey the sleeping sickness focus in Bonon, Ivory Coast, PCR with Trypanosoma brucei specific primers (TBR 1-2 from Parasitology 99 (1989) 57) was tested on DNA derived from blood samples. DNA purification using a chelating resin was performed either on whole blood or on the buffy coat prepared in two different ways. The preparation based on whole blood performed better than those using the buffy-coat. Using this first method, the sensitivity was 100% on parasitologically confirmed patients, and the specificity was 92%. However, problems of reproducibility of the technique were pointed out, particularly on samples from serologically positive but apparently aparasitemic individuals. It is suggested that the PCR could help in the diagnosis of Human African Trypanosomosis, but the use of other primers should be investigated.

[Human African trypanosomiasis in Ivory Coast: biological characteristics after treatment. 812 cases treated in the Daloa focus (Ivory Coast)]. / Trypanosomose humaine africaine en Côte d'Ivoire: caractéristiques biologiques après traitement. A propos de 812 cas traités dans le foyer de Daloa (Côte d'Ivoire).

The treatment and post therapeutic follow up of patients diagnosed with HAT are important for HAT control. A longitudinal survey was implemented in the focus of Daloa (Côte d'Ivoire). A total of 812 patients infected with Trypanosoma brucei gambiense in meningoencephalitic stage and treated with melarsoprol were included, this study pointed out the biological characteristics of patients after treatment. The relapse occurs between 1 and 24 months after treatment. It is essentially neurological, and characterised by the presence in the CSF of antibodies, by the increase of cell count compared with value immediately after treatment, or by the presence of trypanosomes. The cure can be confirmed from 18 months after treatment, and is characterised by the absence of antibodies and trypanosomes in the CSF, by a normal cell count and a normal proteinorachy. Biological scares were recorded on some of the patients after 18 months of follow up, but no relapse occurred among them.

[Geographic distribution of trypanosomiasis treated in Ivory Coast from 1993 to 2000]. / Distribution géographique des trypanosomés pris en charge en Côte d'Ivoire de 1993 à 2000.

Human African Trypanosomosis (HAT, or sleeping sickness) caused by Trypanosoma brucei gambiense develops chronically in Côte d'Ivoire. From 1993 to 2000, a total of 1616 patients were taken in charge in the three treatment centres of the country, which means an average of 202 patients a year. The patients came from two main areas in the Centre West of the country in the Marahoué region: the districts of Sinfra, South of Bouaflé, and Bonon, West of Bouaflé. In the Centre West and in the South East of the country (Aboisso-Ayamé), patients are still struck by the disease, although these foci are less active. The remaining foci seem to be controlled, although no active survey has been carried out. The areas where the greatest number of patients were recorded are the ones where rental crops are located (cocoa and coffee mainly) and where rural activities tend to bring humans and tsetse flies in contact. In this study, are figured the number of treated patients, the endemic and risk areas. It will help to design control strategies and decision makers to know where priority control programs should be implemented.

Discrepancy in plasma melarsoprol concentrations between HPLC and bioassay methods in patients with T. gambiense sleeping sickness indicates that melarsoprol is metabolized.

OBJECTIVE: With the use of a specific high-performance liquid chromatography (HPLC) method and a bioassay which determines trypanocidal activity, concentrations of melarsoprol were assessed in plasma, urine and cerebrospinal fluid (CSF) from 8 patients with late-stage Trypanosoma gambiense sleeping sickness. The aim was to unravel to what extent the bioassay codetermines biologically active metabolites of melarsoprol. METHODS: Subjects were given one dose of melarsoprol i.v. per day for 4 days (1.2, 2.4, 3.0-3.6, 3.0-3.6 mg per kg b.w., respectively). Plasma samples were obtained before the first melarsoprol injection, immediately after and at 1 h, 24 h and 5 days after the 4th injection. Urine was collected before melarsoprol therapy and at 24 h after the 4th injection. CSF samples were taken once before treatment and at 24 h after the 4th injection. RESULTS: HPLC analyses showed that plasma concentrations immediately after the 4th injection varied from 2200 to 15,900 nmol/l; dropping to 0-1800 nmol/l at 1 h; and to undetectable levels at 24 h. In urine small amounts of melarsoprol were recovered. Melarsoprol could not be detected in CSF by HPLC. Immediately after injection, bioassay analyses showed plasma concentrations of the same magnitude as HPLC assays but at 1 h they were 4-65-fold higher than the levels assessed by HPLC. Even 24 h and 5 days after the 4th injection there was significant but decreasing activity. Urine levels were 40-260-fold higher than the measured HPLC concentrations, whereas there was low but detectable activity in CSF. CONCLUSION: Results indicate that melarsoprol is rapidly eliminated from plasma. The significant trypanocidal activity determined by bioassay and simultaneous low or undetectable levels of melarsoprol assayed by HPLC indicate that the compound is transformed into metabolites with parasiticidal activity.

Assessment of central nervous system involvement in gambiense trypanosomiasis: value of the cerebro-spinal white cell count.

OBJECTIVE: To assess, in a clinical setting, the comparative values of conventional criteria used in the diagnosis of central nervous system (CNS) involvement in Trypanosoma brucei gambiense sleeping sickness: white cell count (WCC) in cerebrospinal fluid (CSF) > 5 x 10(6) cells/l; total protein concentration in CSF > 40 mg/100 ml); evidence of trypanosomes in CSF following double centrifugation (DC). METHOD: In vitro culture of CSF was used as the gold standard. RESULTS: The study showed that WCC is, by itself, as sensitive for the diagnosis of the CNS involvement as the usually recommended combination of three conventional criteria. The specificity of WCC is improved while the sensitivity is reduced when the cut-off point is set at a higher value (WCC > 10 X 10(6)/l). CONCLUSION: In poorly equipped laboratories, the diagnosis of CNS involvement in patients with confirmed systemic infection should be based only on the WCC. However, a pilot study is needed to assess the feasibility and reliability of the WCC handled by 'front line' personnel, for different cut-off values.

In vivo evaluation of sixteen plant extracts on mice inoculated with Trypanosoma brucei gambiense.

After examination of the drugs used by traditional practitioners in Côte d'lvoire, nine formulas prescribed in the treatment of African human trypanosomiasis (AHT) were selected for investigation. These formulas made use of 40 plants, 16 of which were studied because of their properties, as described in the literature, and their frequent use by practitioners. The plant extracts were administered, after maceration or decoction, either orally or intraperitoneally to Swiss mice that had previously been inoculated with Trypanosoma brucei gambiense (Tbg), strain MHOM/Cl/81/Dal 083. The parasitaemia in each mouse was followed for three consecutive days and compared with that in control mice, which had been given either a saline solution (SS: negative control) or well-known drugs (melarsoprol, difluoromethylornithine, and pentamidine: positive control). Our investigations led to the following conclusions. (a) None of the plant extracts revealed trypanocidal or trypanostatic activity relative to SS controls (P > 0.05). In fact, the mice that received the extracts died on the third day after inoculation, with 0% survival and an average parasitaemia of 10.8 +/- 2 x 10(7) trypanosomes/ml. (b) The treated positive controls, relative to SS, showed 100% survival and no parasitaemia (P < 0.05). Melarsoprol appeared to be active when given orally at a dose of 3.6 mg/kg body weight twice a day for 3 days. This method of testing the sensitivity of trypanosomes to plant extracts is easy and inexpensive, and could be applied to other areas of research on tropical diseases.

The efficacy of pentamidine in the treatment of early-late stage Trypanosoma brucei gambiense trypanosomiasis.

Fifty-eight patients in the early-late stage (early central nervous system involvement) of Trypanosoma brucei gambiense trypanosomiasis were treated with pentamidine and divided into four groups (G1, G2, G3, and G4) according to cerebrospinal fluid (CSF) indicators: white blood cell (WBC) count, protein level (CSF protein), and the presence or absence of trypanosomes. Group G1 consisted of eight patients with normal CSF WBC counts and CSF protein levels and trypanosomes in the CSF. Group G2 consisted of nine patients with elevated CSF WBC counts, normal level of CSF protein, and trypanosomes in the CSF. Group G3 consisted of 31 patients with high CSF WBC counts, normal CSF protein levels, but no trypanosomes in the CSF. Group G4 consisted of 10 patients with normal CSF WBC counts and CSF protein levels and trypanosomes demonstrated by CSF culture. Post-treatment follow-up of all patients for at least one year revealed three relapses. There were two deaths from diseases unrelated to trypanosomiasis or to the treatment protocol. Of these patients, 52 were followed for more than two years, the time necessary to confirm a complete cure, indicating a cure rate of 94%. Pentamidine is therefore effective in treating the early-late stage of T. b. gambiense trypanosomiasis, and is comparable with melarsoprol or eflornithine in terms of its tolerance and availability.

Human immunodeficiency virus infection and human African trypanosomiasis: a case-control study in Côte d'Ivoire.

To assess the association between human immunodeficiency virus (HIV) infection and human African trypanosomiasis (HAT) in Côte d'Ivoire, West Africa, a cross-sectional case-control study was conducted on 301 HAT patients recruited in the main foci of the country. For each HAT patient, 3 controls, matched for sex, age and residence, were selected. Data relating to socio-demographic factors and potential risk factors for Trypanosoma brucei gambiense and HIV infections were obtained, and serum samples were collected for HIV-1 and HIV-2 tests. A positive test consisted of enzyme immunoassay reactive to HIV-1, HIV-2 or both and confirmed by a synthetic peptide test or Western blot. Data were analyzed using conditional logistic regression with EGRET software. No statistically significant difference was found between the prevalence of HIV infection in HAT patients and controls (4.3% and 3.5% respectively; crude odds ratio (OR) 1.28, 95% confidence interval (CI) 0.65-2.50). In multivariate analysis, allowance for 5 covariates did not change the association between the 2 infections (adjusted OR 1.27, 95% CI 0.64-2.52). Although this study had limited statistical power, no significant association was found between HIV infection and T.b. gambiense infection in rural Côte d'Ivoire. Studies are needed to determine whether HIV infection influences the clinical course of HAT, a question not addressed in the present study.

Pharmacokinetics and adverse reactions after a single dose of pentamidine in patients with Trypanosoma gambiense sleeping sickness.

1. Plasma concentrations of pentamidine were measured up to 1-8 months after a single 2 h i.v. infusion of 3.0 to 4.8 mg kg-1 pentamidine isethionate in 11 patients with late stage Trypanosoma gambiense sleeping sickness. 2. Maximum plasma drug concentrations varied between 713 and 2461 nmol 1-1. After termination of infusion, a rapid distribution phase over 10 min was followed by a slower distribution phase and an elimination phase prolonged over weeks to months. 3. The 'terminal' elimination rate constant could be determined in six patients and subsequent kinetic calculations showed a three to fourfold variation in plasma clearance and 'terminal' half-life (median 1126 (range 553-2036) ml min-1 and 265 (107-446) h, respectively). The median apparent volume of distribution (Vss) was 11,850 1. Renal clearance accounted for a median of 11% of total plasma clearance, indicating that metabolism is a major route of pentamidine elimination in man. 4. Side effects were few and mild and a slight or moderate decrease in blood pressure was the most common registered adverse reaction observed in four subjects. 5. The prolonged elimination of pentamidine seems inconsistent with the present recommended dosage regimen of pentamidine for treatment of trypanosomiasis of 7 to 10 parenteral doses given once daily or every second day.

Long-term exposure of Trypanosoma brucei gambiense to pentamidine in vitro.

In order to study the sensitivity in vitro of Trypanosoma brucei gambiense to pentamidine, 5 x 10(4) parasites were exposed to 0, 0.1, 1.0, 2.0, 10, 100, 1000 and 10,000 micrograms/L of pentamidine isethionate for up to 10 d. The viability of parasites was determined each day by microscopy. Multiplication was retarded during continuous exposure to 2 micrograms/L. After 4 d no further multiplication took place, although the trypanosomes remained alive for another 3 d. The parasiticidal effect was more pronounced when higher concentrations were used; when exposed to 10 and 100 micrograms/L, all parasites were dead after 4 and 3 d, respectively. Despite exposure to 1000 micrograms/L, 74% of the parasites were still alive the next day. 10,000 micrograms/L killed all parasites within 24 h of exposure. Our results show that the time period of exposure to pentamidine plays a major role in determining the sensitivity in vitro of T. b. gambiense, and we suggest that prolonged exposure in vivo may be more important than attaining high but brief peak concentrations.

[Evaluation of the parasitologic technics used in the diagnosis of human Trypanosoma gambiense trypanosomiasis in the Ivory Coast]. / Evaluation des techniques parasitologiques utilisées dans le diagnostic de la trypanosomose humaine a Trypanosoma gambiense en Côte-d'Ivoire.

The investigators carried out a comparative evaluation of twelve or parasitological techniques used nowadays in the diagnosis of human trypanosomiasis and parasite isolation in the lymph fluid, blood and cerebro-spinal fluid (CSF). The tests were performed on 64 seropositive suspects selected with TESTRYP-CATT among 661 attendants examined at the Projet de Recherches Cliniques sur la Trypanosomiase (PRCT), Daloa, Côte-d'Ivoire. The study showed that the sensitivity of the different techniques varies between 17.2% (for CSF inoculation to Mastomys) and 84.5% (for the anion exchange centrifugation technique-mAECT). The classical techniques, says lymph fluid examination, direct blood examination and thick blood have a sensitivity of 58.6, 22.4 and 34.5% respectively. The most sensitive methods are lymph fluid examination, mAECT and double centrifugation of CSF (69%). The sensitivity increases up to 98.3% with the combination of two or three techniques. The combination of lymph fluid examination/mAECT/double centrifugation of CSF is either the most sensitive and the most suitable one for use in the field. The combination of lymph fluid examination and mAECT which detects 91.4% of the infected subjects is the most efficient. The authors discussed the results and recommended that similar study be done in field conditions to assess methods which either demonstrated better sensitivity and are more suitable for field use.

Pentamidine concentrations in plasma, whole blood and cerebrospinal fluid during treatment of Trypanosoma gambiense infection in Côte d'Ivoire.

Pentamidine concentrations in plasma, whole blood and cerebrospinal fluid (CSF) were determined in 11 patients with Trypanosoma gambiense infection without involvement of the central nervous system in Côte d'Ivoire. Blood samples were drawn during a 48 h period after the first and last dose of pentamidine dimesylate given as 10 intramuscular injections on alternate days. Maximum plasma concentrations were generally attained within one hour after injection but varied extensively (420-13420 nmol/litre). The median plasma concentration 48 h after the last dose was approximately 5 times higher than that after the first dose. The ratio between whole blood and plasma concentration was approximately 2. Small amounts of the drug were found in the CSF after the last dose. The findings showed inter-individual differences in the pharmacokinetics of pentamidine. The currently recommended daily dose regimen could be questioned, as drug accumulation was pronounced. All patients were cured and the concentrations attained should be considered as parasiticidal. Further studies on the kinetics and distribution after single and multiple doses of pentamidine as well as studies on the possible relationship between adverse effects and plasma concentrations are, however, needed.

[Evaluation of Testryp CATT applied to blood samples on filter paper and on diluted blood in a focus of trypanosomiasis due to Trypanosoma brucei gambiense in the Ivory Coast]. / Evaluation du Testryp CATT appliqué au sang prélevé sur papier filtre et au sang dilué, dans le foyer de trypanosomiase à Trypanosoma brucei gambiense en Côte d'Ivoire.

The Testryp CATT was performed on dried blood samples on filter-paper and on diluted blood using a microtechnique. This method was applied to both sample collection techniques and was evaluated in parallel with the classical Testryp CATT on whole blood, as described in the instructions provided with the reagents by the manufacturer. A total of 2087 people were tested; 453 samples were tested in the laboratory and 1634 during a field survey in 5 villages of a trypanosomiasis focus in Daloa, Côte d'Ivoire. This study has demonstrated that the Testryp CATT micromethod on either type of sample collection gives results comparable to the Testryp CATT on whole blood. The collection of dried blood samples on filter-paper can be performed by non-specialized staff in trypanosomiasis control programmes of the national health services. In addition, a flask of CATT reagent will allow testing of 6 times more people by the micromethod than by the classical whole-blood method. The micromethod is suitable in the implementation of programmes for the serological surveillance of populations at risk.

Treatment of human late stage gambiense trypanosomiasis with alpha-difluoromethylornithine (eflornithine): efficacy and tolerance in 14 cases in Côte d'Ivoire.

alpha-Difluoromethylornithine (DFMO; eflornithine), an inhibitor of polyamine biosynthesis, was used to treat 14 patients with late stage gambiense sleeping sickness, 12 cases having been previously treated with and considered refractory to melarsoprol. alpha-Difluoromethylornithine was administered intravenously at a dose of 400 mg/kg/day for 14 days followed by oral treatment, 300 mg/kg/day, for 21-28 days. In all patients treatment was associated with rapid disappearance of trypanosomes from body fluids (in several cases within 24 hr) and decreased cerebrospinal fluid white blood cell counts. In all but one patient, who died of a pulmonary infection during treatment, alpha-difluoromethylornithine produced a dramatic reversal of clinical signs and symptoms of the disease. Determination of drug concentrations in serum and cerebrospinal fluid of 5 patients demonstrated that alpha-difluoromethylornithine diffuses into the central nervous system with cerebrospinal fluid levels representing up to 51% of corresponding serum concentrations. Diarrhea, abdominal pain, and anemia were the most frequent side effects associated with therapy, but were reversible and did not necessitate discontinuation of treatment. Four patients have been followed for more than 2 years post-treatment without evidence of relapse.

Integration de la surveillance de la maladie du sommeil aux soins de sante primaires dans le foyer de Sinfra (Cote d'Ivoire); rapport provisoire

Dans le foyer de Sinfra en Cote d'Ivoire; 102 agents ont recu une formation specialisee sur les soins de sante primaires et un entrainement au prelevement de sang sur confettis en decembre 1994. 60.043 personnes sont recensees et apres un mois; sur 24.344 confettis analyses; 402 sont seropositifs; 34 malades sont confirmes. L'assainissement du reservoir humain qui doit etre associe a l'elimination du vecteur sera fait selon deux protocoles : - confection de confettis en zones de basse prevalence; - intervention d'equipes mobiles (PRCT; IPR BSR) dans les zones de forte prevalenve avec l'appui des agents de sante pour la mobilisation de la population et l'apport inestimable de leurs registres de recensement. Le cout (provisoire) de l'installation d'un couple d'agents de sante pour la lutte contre la maladie du sommeil est d'environ 114.000F CFA (soit approximativement 213 US$). L'installation d'un laboratoire revient a 2.500.000F CFA (4700 US$)