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.

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%).

Monitoring the developmental status of Trypanosoma brucei gambiense in the tsetse fly by means of PCR analysis of anal and saliva drops.

Teneral Glossina palpalis gambiensis (Diptera: Glossinidae) were infected with a culture of procyclic forms of Trypanosoma brucei gambiense using a single-bloodmeal membrane feeding technique. The infection was monitored by analysing the saliva (mature infection) and anal drop (midgut infection) of each fly at different post-infection times both by microscopic observation and polymerase chain reaction (PCR). Amplification revealed many more positive anal drops than microscopy. The monitoring showed that the installation of T. b. gambiense in Glossina took place at least 11 days after the infection and that maturation occurred after 29 days. It also reflected precisely the parasitic status of each tsetse fly as determined by the dissection, microscopic examination and PCR amplification of the midguts and salivary glands 47 days post-infection. Twice as many tsetse flies with mature salivary glands infection were revealed by PCR than by microscopic examination, but the two techniques gave exactly the same results regarding the proportion of flies with midgut infection. This study also demonstrated the ability of natural non-infective procyclic forms of T. b. gambiense, to colonise the midgut and subsequently establish in the salivary glands of G. p. gambiensis.

Evaluation of LATEX/T.b.gambiense for mass screening of Trypanosoma brucei gambiense sleeping sickness in Central Africa.

We compared the Card Agglutination Test for Trypanosomiasis (CATT), which consists of lyophilized bloodstream form trypomastigotes of Trypanosoma brucei gambiense (T.b.g.) variable antigen type LiTat 1.3, with LATEX/T.b.g., which consists of a lyophilized suspension of latex particles coated with variable surface glycoproteins of T.b.g. variable antigen types LiTat 1.3, 1.5 and 1.6. This study was carried out during two mass screening surveys in 1998 in Campo, a sleeping sickness focus in Cameroon, with a low prevalence (0.3%) and in 1999 in Batangafo which belongs to the Central African focus of Ouham which has a higher prevalence (3%). In Campo, we compared the CATT performed on whole blood with the LATEX/T.b.g. on diluted blood. In Batangafo, both tests were performed on diluted blood. In all circumstances, the specificity of the LATEX/T.b.g. was higher than of CATT. The use of LATEX/T.b.g. on diluted blood instead of CATT results in an important decrease of workload and as a consequence, of costs related to parasitological examinations. In the case of Campo the workload was up to 12 times less than when using CATT 1.3 on whole blood and the cost divided by 3. In Batangafo the workload was decreased by nearly 20% with the LATEX/T.b.g. Finally, it should be noted that in Batangafo, one of the parasitologically confirmed sleeping sickness patients was negative in CATT and positive in LATEX/T.b.g. and that the reading of the test result in LATEX/T.b.g. is easier than in CATT.

New molecular marker for Trypanosoma (Duttonella) vivax identification.

Trypanosoma vivax is a widespread hemoparasite in tropical areas and is pathogenic to ruminant domestic livestock as well as wild ruminants. The accurate identification of parasites in both hosts and vectors is crucial for epidemiological studies and disease control programs. We describe here the development of molecular markers specific for T. vivax identification. These markers were used to identify mouthpart infections in field-collected tsetse flies from Cameroon. The markers target the genomic sequence of a species-specific antigen from the bloodstream stages. No cross amplification with other trypanosome species was observed, which makes the markers a reliable tool to detect T. vivax infections, both in hosts and vectors. The PCR-amplified sequence contains a (CA)(n) microsatellite repeat for which 11 different alleles were identified. This microsatellite, which showed high polymorphism, provides a suitable marker for population genetic studies.

Detection and identification of trypanosomes by polymerase chain reaction in wild tsetse flies in Cameroon.

The prevalence of various species and subgroups of trypanosomes in infected flies from three sleeping sickness foci in Cameroon was determined by the use of polymerase chain reaction (PCR). The predominant tsetse species found were Glossina palpalis palpalis. Microscopical examination of 943 non-teneral tsetse flies revealed an average infection rate of 10.4%. A total of 90 flies were analyzed for trypanosome identification with primer sets specific for Trypanosoma (Trypanozoon) brucei s.l., T. (Duttonella) vitax, T. (Nannomonas) simiae, and forest type T. (Nannomonas) congolense. PCR succeeded in identifying 52 of the 90 infected flies. Other primers were also tested on microscope positive/PCR-negative infections, and trypanosome subgroups were detected (Kilifi type and savannah type T. congolense). PCR amplification allowed identification of immature infections and revealed mixed-infections. The PCR technique failed to identify 42.2% (38/90) of the parasitologically positive flies and the reasons for this failure are discussed.

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.

Spatial and temporal patterns of human African trypanosomosis (HAT) transmission risk in the Bipindi focus, in the forest zone of southern Cameroon.

Vector control is an effective and cost-efficient way to disrupt the transmission of human African trypanosomosis (HAT); it has nonetheless been little used to date in the disease's foci. With the aim to target trapping more precisely and to develop an optimized vector control system, a transmission risk index was used in the HAT focus of Bipindi, in the forest zone of southern Cameroon. The authors used a simplified version of the index originally developed by Laveissière et al. in 1994. The calculation of this new index only requires knowledge of the proportion of teneral flies and the proportion of flies with human blood meals in samples caught in different biotopes. This makes it possible to identify the biotopes displaying permanent risk, such as riverbanks, as well as biotopes displaying seasonal risk, such as marshy hollows and encampmemts. In the villages, the domestic pig, with 49% of the identified blood meals, is the favorite host of the tsetse flies during the short rainy season. The proportion of blood meals taken on human beings does not significantly increase when domestic pigs are absent. Game animals, contributing to 46% and 64% of the blood meals during the short rainy season and the long dry season, respectively, are also favored as feeding hosts in this particular HAT focus.

Improvement of blood and fly gut processing for PCR diagnosis of trypanosomosis.

We have adapted a simple and efficient technique to detect trypanosomes in human blood, without DNA purification, and increased the sensitivity threshold to 1 parasite in 1 ml. We have then applied it for detection of parasites in midguts of tsetse flies, negative by microscopy. This technique has been developed for field conditions and could greatly facilitate epidemiological studies.

Inhibition of the DNA amplification of trypanosomes present in tsetse flies midguts: implications for the identification of trypanosome species in wild tsetse flies.

The present study was carried out in order to investigate if there was really a failure of PCR in identifying parasitologically positive tsetse flies in the field. Tsetse flies (Glossina palpalis gambiensis and Glossina morsitans morsitans) were therefore experimentally infected with two different species of Trypanosoma (Trypanosoma brucei gambiense or Trypanosoma congolense). A total of 152 tsetse flies were dissected, and organs of each fly (midgut, proboscis or salivary glands) were examined. The positive organs were then analysed using PCR. Results showed that, regardless of the trypanosome species, PCR failed to amplify 40% of the parasitologically positive midguts. This failure, which does not occur with diluted samples, is likely to be caused by an inhibition of the amplification reaction. This finding has important implications for the detection and the identification of trypanosome species in wild tsetse flies.

[The focus of human trypanosomiasis in Campo (Cameroon). History and endemic situation in 1998]. / Le foyer de trypanosomose humaine de Campo (Cameroun). Historique et situation de l'endémie en 1998.

For the first time in the last thirteen years, the human sleeping sickness focus at Campo, spanning the Cameroon-Equatorial Guinea border areas, has been prospected. The screening was carried out simultaneously on both sides of the border. This focus has been known since the beginning of the century but, contrary to what took place in other well-known foci in bordering countries south of Cameroon, either in the 1920s or the 1980s--there has never been an epidemic outbreak in that area. Such an epidemiological situation makes this focus particularly interesting. Though still active, trypanosomiasis is not very manifest. According to passive screening carried out in recent years, the estimated prevalence ranges between 0.2 and 0.5%. For this screening, 5,255 persons were examined on the Cameroonian side of the focus (90.6% of the census population). The serological screenings were carried out with the CATT 1.3, which is the CATT generally used in screening, and with the latex CATT which associates LiTat 1.3, 1.5 and 1.6. The search for trypanosomes was made by testing the lymph nod juice in presence of adenopathy and in the blood by Quantitative Buffy Coat (QBC), the mini anion exchange centrifugation (mAEC), as well as the in vitro culture using the kit for in vitro isolation of trypanosomes (KIVI) for individuals suspected to be serologically positive. 16 patients were identified in Cameroon but none in Equatorial Guinea. The results show that the Campo focus is active only on the Cameroonian side, centred on the village of Ipono with a limited prevalence (0.3%). The persisting epidemic is most likely to be associated with the presence of pigs carrying the Trypanosoma brucei gambiense which was identified during the study in Ipono. The strain that we isolated was studied by isoenzyme electrophoresis on cellulose acetate. Its zymodeme is the same as that of the human strain isolated in Campo. With the collected epidemiological data, a concerted medical and entomological action could be planned within the limits of the village of Ipono to eradicate the disease. This action may be organised by the existing local health structures. During this study, the latex CATT proved to be more cost-effective than the CATT 1.3 since a similar result was reached requiring eight times less work at a lower cost. This remains to be confirmed in a hyperendemic focus.

[Risk factors for human African trypanosomiasis in the Bipindi region of Cameroon]. / Recherche des facteurs de risque de la trypanosomose humaine africaine dans le foyer de Bipindi au Cameroun.

In the course of two surveys carried out at the end of 1998 and beginning of 1999, sleeping sickness was diagnosed in a total of 43 people in the Bipindi region of Cameroon. This observation led us to investigate the mechanisms of transmission of human African trypanosomiasis in the epicentrer of the outbreak. A case-control study showed a particularly high risk of infection associated with hunting activities (Odds-Ratio: 2.87; CI 95%: 0.96-9.52). Interpretation of this finding in the light of local geographical features and current entomological data suggests that the higher risk in hunters is linked to the presence of a perennial vector population and absence of domestic pigs.

[The trapping of tsetse flies (Diptera: Glossinidae). Improvement of a model: the Vavoua trap]. / Recherches sur les pièges à glossines (Diptera: Glossinidae). Mise au point d'un modèle économique: le piège "Vavoua".

The control of tsetse flies with traps needs a decrease of their cost/efficiency. In the forest belt of Côte d'Ivoire, the research on Glossina palpalis palpalis behaviour allows to propose a new model of trap, the "Vavoua" trap, issued from the biconical and the pyramidal traps, with a similar efficiency but a twice lower cost (1139 F CFA without manpower, i.e. 3.55 US $, respectively 6.68 and 6.98 US $ for the biconical and the pyramidal). This trap has an upper cone (polyamide mosquito net) overcoming three screens (length 45 cm), sewed at 120 degrees, composed of a blue external part (cotton/polyester) and a black internal part (polyamide) with a blue/black ratio equal to 2. Its low cost and the possibility for the farmer to soak themselves the trap with insecticide allow to consider its use for large-scale control of tsetse flies in the forest zones by rural communities.

Characterization of trypanosome infections by polymerase chain reaction (PCR) amplification in wild tsetse flies in Cameroon.

The polymerase chain reaction (PCR) method was used to characterize trypanosome infections in tsetse flies from 3 sleeping sickness foci in Cameroon. The predominant tsetse species found was Glossina palpalis palpalis. An average infection rate of 12.1% was revealed by microscopical examination of 888 non-teneral tsets flies. PCR amplification analyses for trypanosome identification were carried out on 467 flies, with primer sets specific for Trypanosoma (Trypanozoon) brucei s.1., T. (Duttonella) vivax, T. (Nannomonas) simiae and forest type T. (Nannomonas) congolense. Of 467 flies 93 were positive by microscopical analysis while PCR succeeded in identifying 89 positive flies. Of the PCR-positive flies 34 (38.2%) were negative by microscopical examination. PCR amplification, when compared to the parasitological technique, gave a higher estimate of infection rate of trypanosomes in natural tsetse populations. The PCR technique did, however, fail to identify 40.9% (38/93) of the parasitologically positive flies. The reasons for this failure are discussed. The overall prevalence of mixed infections, assessed by PCR, was 37.1%; the majority (72.7%) involved T. brucei and forest type T. congolense.

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.

Monitoring the susceptibility of Glossina palpalis gambiensis and G. morsitans morsitans to experimental infection with savannah-type Trypanosoma congolense, using the polymerase chain reaction.

Teneral Glossina palpalis gambiensis and G. morsitans morsitans (Diptera: Glossinidae) were fed on mice infected with savannah-type Trypanosoma (Nannomonas) congolense. The infection was monitored by checking the post-feeding diuresis fluid (midgut infection) and saliva (mature infection) of individual flies for parasites, at different times post-infection, using microscopical examination and a PCR-based assay. The results indicated that both tsetse species supported established midgut infections by 10 days post-infection and that maturation occurred after 24 days in G. m. morsitans. Although, for both diuresis fluid and saliva, the results of the microscopy showed good concordance with those of the PCR, the PCR identified more positive samples. Monitoring allowed determination of the status of the infection in individual flies, which was confirmed, 48 days post-infection, by the microscopical examination of the midguts and probosces dissected out of the flies and by the PCR-based amplification of any trypanosome DNA in these organs. Again, in terms of the detection of trypanosomes in the dissected organs, there was good concordance between the results of the PCR and those of the microscopy, although PCR revealed many more mature infections than did microscopical examination, particularly in the G. p. gambiensis investigated. There was a higher prevalence of immature infection in G. p. gambiensis than in G. m. morsitans (P<0.05) but the inter-specific differences seen in the prevalences of any infection and of mature infection were not statistically significant. The intrinsic vectorial capacity for T. congolense of both tsetse species therefore appeared quite similar, although the true vectorial competence of G. p. gambiensis remains to be determined.

Characterization of Trypanosoma brucei s.l. infecting asymptomatic sleeping-sickness patients in Côte d'Ivoire: a new genetic group?

Six villagers in the Sinfra focus of sleeping sickness in Côte d'Ivoire who in 1995 were asymptomatic and refusing treatment, despite then being serologically and parasitologically positive for trypanosomes, were followed-up, while still refusing treatment, until 2002. In 2002, five of the six cases remained serologically positive but no trypanosomes could be found in any of them by use of the classical parasitological methods. A PCR-based assay, however, revealed that all six had the DNA of Trypanosoma brucei s.l. in their blood, so confirming the low sensitivity of the classical parasitological tests. The analysis of satellite, minisatellite and microsatellite markers indicated that, in 2002, all six cases were infected with a 'new' distinct genetic group of T. brucei s.l. and four were co-infected with T. b. gambiense group 1. The epidemiological consequences of such co-infections are discussed. The 'new' group of T. brucei had a molecular pattern that differed from those of the classical T. b. gambiense group 1 and the 'bouaflé' group.