Confirmation of the use of Latex IgM on cerebrospinal fluid for improving stage determination of Human African Trypanosomiasis

The clinical evolution of the chronic form of Human African Trypanosomiasis starts with the haematolymphatic or first stage (P1). The meningoencephalitic or second stage (P2) begins when trypanosomes reach the cerebrospinal fluid (CSF). The classical stage determination method is based on CSF cell count, CSF protein concentration and/or the presence of trypanosomes detected in CSF. However their cutoff values and the sensitivity of detection of trypanosomes in CSF remains doubtful while the appropriate treatment depends on this determination of disease stage. Thus, the classical stage determination is reconsidered using new serological tests, and results were compared to the clinical data. Thirty-eight patients were classified into 4 clinical groups according to the observed degree of severity of neuropsychiatric signs. Based on multivariate analysis to evaluate the relevance of the new serological tests as compared with clinical groups, we confirm that Latex IgM CSF, cheap and easy to perform under field conditions, may improve stage determination of the disease

In vitro activity of commercial formulation and active principle of trypanocidal drugs against blooststreams forms of Trypanosoma brucei gambiense

The in vitro trypanocidal activities of 4 commercial formulations Ornidyl®, Pentamidine isethionate®, Germanin® and Lampit® and their corresponding active principles (Dl-difluoromethylornithine, pentamidine isethionate, suramine and 5-nitrofuran) were compared against Trypanosoma brucei gambiense. Differences of minimum inhibitory concentration (MIC) were observed between Ornidyl® and Dl-difluoromethylornithine and between Lampit® and 5-nitrofuran. For RO 15 strain and the comparison of Ornidyl®/ DFMO, the MIC when using the commercial drug was more than twice the MIC value obtained with the active principle. For all 3 trypanosome strains, MICs were identical for Lampit® and 5-nitrofuran but the MIC with the commercial formulation was twice the MIC obtained with the active principle. The active principles, rather than commercial formulations, should be used for standardization of in vitro assay protocols. Key words: In vitro activity, trypanocidal drugs, commercial formulations, Trypanosoma brucei gambiense. African Journal of Biotechnology Vol.2(11) 2003: 474-47

Characterization of Trypanosoma brucei gambiense stocks isolated from humans by RAPD fingerprinting in Côte d'Ivoire: another evidence for multiple infections

Trypanosoma brucei gambiense was isolated twice from each of 23 patients in Côte d'Ivoire. Genetic characterization using RAPD (Random Primed Amplified Polymorphic DNA) showed additional variability within a given isoenzyme profile (zymodeme), confirming that this fingerprinting method has a higher discriminative power (faster molecular clock) than isoenzymes. RAPD confirmed also the evidence of multiple infections by different genotypes in the same patient despite a low genetic variability among Trypanosoma brucei gambiense stocks. The involvement of this phenomenon in treatment failure is discussed. Key Words: Human African Trypanosomiasis, Trypanosoma brucei gambiense, RAPD, multiple infections. African Journal of Biotechnology Vol.3(1) 2004: 94-9

The separation of trypanosomes from blood by anion exchange chromatography: From Sheila Lanham's discovery 50 years ago to a gold standard for sleeping sickness diagnosis

Human African trypanosomiasis (HAT), or sleeping sickness, is a neglected tropical disease that is fatal if untreated, caused by Trypanosoma brucei gambiense and T. brucei rhodesiense. In its 2012 roadmap, WHO targeted HAT for elimination as a public health problem in 2020 and for zero transmission in 2030. Diagnosis of HAT is a multistep procedure comprising of clinical suspicion, confirmation, and stage determination. Suspects are identified on clinical signs and/or on screening for specific antibodies. Parasitological confirmation of suspects remains mandatory to avoid unnecessary toxic drug administration. The positive predictive value of the antibody detection tests is low. Simple parasite detection techniques, microscopic examination of lymph node aspirate, or stained thick blood films lack sensitivity, whereas in T. brucei gambiense patients, the number of blood trypanosomes may be very low. Parasite concentration techniques are therefore indispensable. Half a century ago, Sheila Lanham discovered a technique to separate trypanosomes from the blood of infected rodents, based on anion exchange chromatography with diethyl amino ethyl (DEAE) cellulose, a weak anion exchanger. Between pH 6−9, trypanosome surface is less negatively charged than that of blood cells. When blood is poured on top of a DEAE cellulose column, blood cells are retained, whereas parasites pass the column together with the elution buffer. The result is a pure suspension of trypanosomes that retain their morphology and infectivity. Because cell surface charges vary among trypanosome and mammal species, the optimal buffer pH and ionic strength conditions for different combinations of host and trypanosome species were established. Lanham's technique revolutionized the diagnosis of HAT. It is indispensable in the production of the Card Agglutination Test for Trypanosomiasis (CATT), the most used field test for screening in T. brucei gambiense HAT foci and essential to confirm the diagnosis in suspected people. Lumsden and colleagues developed the mini anion exchange centrifugation technique (mAECT). After adaptation for field conditions, its superior diagnostic and analytical sensitivity compared to another concentration technique was demonstrated. It was recommended as the most sensitive test for demonstrating trypanosomes in human blood. At the beginning of the 21st century, the mAECT was redesigned, allowing examination of a larger volume of blood, up to 0.35 ml with whole blood and up to 10 ml with buffy coat. The plastic collector tube in the new kit is also used for detection of trypanosomes in the cerebrospinal fluid. Unfortunately, mAECT also has some disadvantages, including its price, the need to centrifuge the collector tube, and the fact that it is manufactured on a noncommercial basis at only two research institutes. In conclusion, 50 years after Sheila Lanham's discovery, CATT and mAECT have become essential elements in the elimination of HAT

Confirmation of antibodies against L-tryptophan-like epitope in human African trypanosomosis serological diagnostic

Antibodies directed against L-tryptophan epitope (WE - W for tryptophan, E for epitope), a constant epitope borne by variant surface glycoproteins (VSG), have been detected in sera of all 152 Human African Trypanosomosis (HAT) patients from Angola. The WE is present in VSG hydrophobic regions of the C terminal domains. In the assay, L-tryptophan was linked to bovine serum albumin (BSA) with glutaraldehyde to synthesize W-G-BSA conjugate which was used in an enzyme-linked immunosorbent assay (ELISA) to detect the antibodies. A significant difference was found between HAT patients and controls confirming previous results obtained with a lower number of patients in Congo. A diagnostic test based on this synthetic epitope, especially in combination with other tests, might improve the HAT diagnostic test in field conditions.Key words: Tryptophan, enzyme-linked immunosorbent assay (ELISA), human African trypanosomosis, serological diagnostic

Etude de la prévalence des infections à trypanosomes chez différentes espèces d'animaux sauvages du parc national de la Comoé en Côte d'Ivoire : résultats préliminaires sur la comparaison de trois méthodes de diagnostic

Si de nombreuses études portent sur la trypanosomoses chez les animaux domestiques, peu de rcherches ont été effectuées sur la faune sauvage d'Afrique occidentale. Les résultats préliminaires sur la comparaison de trois méthodes de dépistages des trypanosomes : le frottis de sang, la détection des antigènes circulants par le technique ELISA et l'isolement in vitro des trypanosomes par le "kit for in vitro isolation" (KIVI), chez les animaux sauvages du parc national de la Comoé en Côte d'ivoire, ont permis de confirmer l'existence de cette infection chez ces animaux sans toutefois identifier de façon précise les espèces de trypanosomes en cause. Des investigations ultérieures permettront d'affiner l'identification des souches de parasites isolées par KIVI. (Résumé d'auteur