High prevalence of Trypanosoma brucei gambiense group 1 in pigs from the Fontem sleeping sickness focus in Cameroon.

To understand the importance of domestic pigs in the epidemiology of human trypanosomiasis, PCR was used to identify trypanosome populations in 133 pigs from the Fontem sleeping sickness focus of Cameroon. The results from this study show that 73.7% (98/133) of pigs from the Fontem area carry at least one trypanosome species. Trypanosoma vivax, T. brucei s.l. and T. congolense forest were found in 34.6% (46/133), 40.0% (53/133) and 46.0% (61/133) of the pigs respectively. T. simiae and T. congolense savannah were not identified in these animals. The use of repeated DNA sequences detected T. b. gambiense group 1 in 14.8% (15/101) of the pigs. Such pigs can be possible reservoir hosts for T. b. gambiense group 1 and contribute to the maintenance of the disease in the area. Mixed infections were revealed in 35.3% (47/133) of the pigs. Furthermore, we observed that under natural conditions, 52.4% (11/21) of the pigs from the Fontem focus carry mixed infections with T. b. gambiense group 1. No significant difference was observed between the percentage of T. b. gambiense group 1 single and mixed infections, and between the prevalence of this trypanosome in pigs from villages with and without sleeping sickness patients.

Wild fauna as a probable animal reservoir for Trypanosoma brucei gambiense in Cameroon.

In order to study the existence of a wild animal reservoir for Trypanosoma brucei gambiense in South Cameroon, blood was collected from wild animals in three human African trypanosomiasis foci and from a nonendemic control area. The 1142 wild animals sampled belonged to 36 different species pertaining to eight orders (407 primates, 347 artiodactyls, 265 rodents, 54 pangolins, 53 carnivores, 11 saurians and crocodilians, and five hyraxes). QBC and KIVI tests detected trypanosomes on 1.7% (13/762) and 18.4% (43/234) of animals examined, respectively. Using specific primers, T. brucei non-gambiense group 1 DNA was detected on 56 animals (4.9%). This infection rate was 5.3% in the endemic zone and 3.8% in the control zone. Of the 832 animals of the endemic zone, PCR revealed T. b. gambiense group 1 DNA in 18 (2.2%). These hosts included two rodents, two artiodactyls, two carnivores and two primates. T. b. gambiense group 1 was absent from animals from the nonendemic zone. A decrease in the prevalence of T. b. gambiense group 1 was observed in wild animals from the Bipindi sleeping sickness focus after a medical survey and vector control in this area. The epidemiological implications of these findings remain to be determined with further investigations.

Infection rate of Trypanosoma brucei s.l., T. vivax, T. congolense "forest type", and T. simiae in small wild vertebrates in south Cameroon.

In order to identify the infection rate of trypanosome species infecting wild animals in four localities (Bipindi, Campo, Fontem and Nditam) of southern Cameroon, 1,141 wild animals were sampled. These animals belonged to 36 species grouped in 8 orders including 407 primates, 347 artiodactyls, 264 rodents, 54 pangolins, 53 small carnivores, 11 saurians and crocodilians and 5 hyraxes. PCR using specific primers for Trypanosoma vivax, T. brucei s.l., T. congolense "forest type", and T. simiae showed that 18.7% of the animals were infected by at least one of these trypanosome species. A positive PCR result may not indicate absolutely an active infection because PCR can detect also transient infections. T. vivax (Duttonella) had the highest infection rate (9.5%) and was found in almost all the host orders studied. T. brucei s.l. mostly infected primates, rodents and some duikers (Cephalophus dorsalis and C. monticola). Trypanosomes of the subgenus Nannomonas had a lower infection rate of 5.5% (2.4% for T. simiae and 3.1% for T. congolense "forest type"). They were harboured mainly by primates, ungulates and rodents. Trypanosome infection rates were highest in Nditam (24.5%) and Bipindi (21%). T. brucei s.l. (Trypanozoon) had its maximum infection rate of 10.4% in Bipindi. The "Quantitative Buffy Coat" (QBC) and Kit for in vitro isolation techniques were used to identify 48 (6.1%) infected animals. 13 were positive using QBC, and 42 were positive by KIVI. However, PCR was negative on 16 of these infected animals, probably due to infections with other trypanosome species. This study showed that trypanosomes of the subgenera Duttonella, Nannomonas and Trypanozoon could infect small wild vertebrates as has been shown for large ungulates and carnivores. The presence of T. brucei s.l. in a large range of wild animals strengthens the hypothesis of the existence of a wild animal reservoir of T. b. gambiense in Cameroon.

An isoenzyme survey of Trypanosoma brucei s.l. from the Central African subregion: population structure, taxonomic and epidemiological considerations.

In order to improve our knowledge about the taxonomic status and the population structure of the causative agent of Human African Trypanosomiasis in the Central African subregion, 169 newly isolated stocks, of which 16 came from pigs, and 5 reference stocks, were characterized by multilocus enzyme electrophoresis, for 17 genetic loci. We identified 22 different isoenzyme profiles or zymodemes, many of which showed limited differences between them. These zymodemes were equated to multilocus genotypes. UPGMA dendrograms revealed one main group: Trypanosoma brucei gambiense group I and 3 T. brucei 'non-gambiense' stocks. T. b. gambiense group I zymodemes were very homogenous, grouping all the human stocks and 31% of the pig stocks. Two main zymodemes (Z1 and Z3) grouping 74% of the stocks were found in different remote countries. The genetic distances were relatively high in T. brucei 'non-gambiense' zymodemes, regrouping 69% of pig stocks. The analysis of linkage disequilibrium was in favour of a predominantly clonal population structure. This was supported by the ubiquitous occurrence of the main zymodemes, suggesting genetic stability in time and space of this parasite's natural clones. However, in some cases an epidemic population structure could not be ruled out. Our study also suggested that the domestic pig was a probable reservoir host for T. b. gambiense group I in Cameroon.

Characterization of Trypanosoma brucei s.l. subspecies by isoenzymes in domestic pigs from the Fontem sleeping sickness focus of Cameroon.

Though it has been established that domestic animals (especially the pig) are potential reservoir hosts for Trypanosoma brucei gambiense in West Africa, there is little data to this effect concerning Central Africa. Instead, some previous authors report the absence of Trypanozoon type trypanosomes in domestic animals in Cameroon. Thirty-two domestic pigs were sampled by KIVI (kit for in vitro isolation) of trypanosomes in the northern region (Bechati) of the Fontem sleeping sickness focus of Cameroon. Twenty-one of these were found positive, from 15 of which 17 isolates were successfully obtained. Isoenzyme characterization revealed that isolates from 4 of the 15 pigs belonged to zymodemes associated with T. brucei gambiense group 1. The prevalence of this disease in the local human population is, however, very low. It is evident from this study that the domestic pig may be a potential reservoir host for T. brucei gambiense in the Fontem focus. There is, however, need for an extensive study on domestic animals in Cameroon and other neighbouring countries for a better comprehension of the epidemiology of sleeping sickness within the Central African region.

Identification of trypanosomes in wild animals from southern Cameroon using the polymerase chain reaction (PCR).

One possible explanation of the maintenance of many historical foci of sleeping sickness in Central Africa could be the existence of a wild animal reservoir. In this study, PCR was used to detect the different trypanosome species present in wild animal captured by hunters in the southern forest belt of Cameroon (Bipindi). Trypanosomes were also detected by a parasitological method (Quantitative buffy coat: QBC). Parasite could not be isolated in culture medium (Kit for in vitro isolation: KIVI). Specific primers of T. brucei s.l., T. congolense forest type, T. congolense savannah type, T. vivax, T. simiae and T. b. gambiense group 1 were used to identify parasites in the blood of 164 animals belonging to 24 different species including ungulates, rodents, pangolins, carnivores, reptiles and primates. Of the 24 studied species, eight were carrying T. b. gambiense group 1. Those parasites pathogenic to man were found in monkeys (Cercocebus torquatus and Cercopithecus nictitans), in ungulates (Cephalophus dorsalis and C. monticola), in carnivores (Nandinia binotata and Genetta servalina) and in rodents (Cricetomys gambianus and Atherurus africanus). 13 species (54%) were carrying T. brucei s.l. identified as non-gambiense group 1.

Diagnosis of human trypanosomiasis, due to Trypanosoma brucei gambiense in central Africa, by the polymerase chain reaction.

During a mass screening of sleeping sickness conducted in 1998 and 1999, and involving 27,932 persons in Cameroon and the Central African Republic, we tested the polymerase chain reaction (PCR) on whole blood for the diagnosis of human African trypanosomiasis due to Trypanosoma brucei gambiense. The 1858 samples obtained were from 4 groups: 155 infected patients, 1432 serological suspects detected by the card agglutination test for trypanosomiasis (CATT), 222 negative controls living in the prospected area (negative with the CATT and parasitological methods), and 49 negative controls (CATT and parasitological methods) and unexposed to the disease (Europeans). The technique of DNA extraction used made it possible to preserve the blood samples in the field. The primers used were specific for T. brucei s.l. Only 1 patient was PCR negative, and 3 of the negative controls, exposed to the disease, were PCR positive. Among the 1432 serological suspects, only 50 were PCR positive. During the 6-month follow-up after the surveys, the 3 negative controls, who were initially positive by PCR, were found to be negative. These initial positive PCR results are unlikely to have been due to a cross-reaction with T. brucei brucei, which is non-pathogenic for man, but are more likely to have resulted from a mislabelling of sample tubes. All control individuals, exposed or not to the disease, were negative by PCR. The PCR-negative patient was possibly a registration error. Among 50 PCR positive serological suspects, 39 of them were re-examined. Five were found to be positive by the kit for in-vitro isolation of trypanosomes, representing an increase in patients of almost 13%. At the end of the study, 160 patients were diagnosed, and the PCR was positive for 159 of them (99.4%). Moreover, the PCR made it possible to reduce the number of suspects to be re-examined (50 instead of 1432; a reduction of 96.5%).

[Release of Loa loa antigens after treatment with ivermectin]. / Libération d'antigènes de Loa loa après traitement par l'ivermectine.

This study concerns 112 patients of whom 104 were followed up. Microfilaricidal treatment of loaiasis is sometimes followed by severe adverse reactions. Using an immunodiffusion technique and the measure of microfilaraemia by calibrated thick smear, the authors show that the intensity of adverse reactions is proportional to the quantity of microfilariae eliminated by the treatment. The appearance of Loa loa antigens within three days following treatment was evident in 83% of the subjects. Five patients presenting the serious adverse reactions described belonged to this group. However, the precise cause of these adverse reactions, allergic or toxic, has not been demonstrated.

[Genetic origin of venom variability: impact on the preparation of antivenin serums]. / L'origine génétique de la variabilité des venins: impact sur la préparation des sérums antivenimeux.

One of the main possible origin of the biochemical variations of venoms could be genetic. We studied the venom of members of litters born in a snake farm (12 Crotalus atrox and 21 Naja haje). We first used the electrophoresis in cellulose acetate (AE). Then, variations were confirmed by immunoelectrophoresis (AIE) using an antivenom (IPSER Africa, Pasteur Mérieux Sérums & Vaccines) and immunsera prepared on rabbit from i) venom presenting the maximum of bands in electrophoresis (complete venom) and ii) pure toxins (neurotoxin-alpha and cardiotoxin-gamma). At last, the toxicity of some samples was measured and the ability of SAV to neutralise the corresponding sample was measured. The AE of C. atrox venoms showed a good homogeneity, probably due to a good genetic stability of the investigated group. On the other hand, N. haje venoms have revealed a great heterogeneity. The 13 samples were allocated to five groups according to the absence of some fractions compared to the complete venom. The AIE showed that the neurotoxin-alpha is present in every sample, but variable in quantity, even when it did not appear on AE. We suggest that these pattern variations are due either to relative variations of protein fractions in samples or to modifications of the chemical composition of the neurotoxin-alpha. However, the variation of toxicity between the different samples questioned the neutralisation ability of antivenoms. We propose that venom sample choice for SAV production should be based on biochemical criteria and toxicity of samples rather than random pooling.