Sleeping sickness transmission in Campo (Cameroon)

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Introduction. Human African Trypanosomiasis (HAT) is considered by W.H.O. (2010) as a neglected disease being eliminated in the next decades. In 2012, the ...
Sleeping sickness transmission in Campo (Cameroon): interest of xenomonitoring. Grébaut P., Njitchouang G.R., Melachio T., Nyangmang S., Ebo’o E. V, Ofon E., G. Simo.

Introduction Human African Trypanosomiasis (HAT) is considered by W.H.O. (2010) as a neglected disease being eliminated in the next decades. In 2012, the number of diagnosed cases has fallen below ten thousands, reaching the historical level of the independencies period in the sixties (ref). One could think that this disease is on the way to disappear, but several reasons are still making this elimination a real challenge (Grébaut, 2012). First, HAT is considered as a neglected disease, which means that in most countries where the endemic level is low, little efforts are made to support active screening and vector control in the historical foci. Secondly, diagnosis and treatment must be supplied by a specialized medical team; in most countries, most of the staff, which was formed in the 1970s and 1980s, begins to retire. At last, the maintenance of an active reservoir, for more than a century in some foci, is the key for the disease to re-emerge. The sleeping sickness focus of Campo, located in the South-West of Cameroon is characterizing this situation. The historical of this sleeping sickness focus was described by Penchenier et al. (1998). In the beginning of the XXth century, German colonizers already considered the region of Campo as a dangerous one (Zyemann, 1910). After the First World War, the French colonizers continue to describe an endemic situation even after the departure of Dr. Jamot: prevalence is low and three villages are principally concerned, such as Ipono, Mabiogo and Campo Beach (Ledentu, 1934). In 1974, Eouzan et al. (unpublished data) realized an entomological survey in Campo in order to prepare vector control actions. But one has to wait for the yearly reports of the “Organisation de Coordination contre les Endémies en Afrique Centrale” (OCEAC), between 1977 and 1997, to know that 146 sleeping sickness patients were diagnosed through a passive way in the Campo area. In 1980, Eteme and Chauvet proposed a vector control campaign and insecticide spraying was realized twice a year between 1981- and 1986. In 1998, OCEAC realized a mass screening in the focus, including the Guinea-Equatorial population living on the other side of the river Ntem, a natural boarder between both countries. At that time, among 5660 persons that were screened, 16 cases were diagnosed. Still in 1998, Morlais et al. studied the situation of the vector and found 15.6% of infected flies with trypanosomes in the area. One year after, in 1999, Nkinin et al. using isoenzymes analysis, found that 13 domestic mammals were serological suspects, among them one was diagnosed positive to T. b. gambiense. During the last decade, from 2000 to 2010, Cameroonian scientists achieved several studies in this HAT focus, trying to understand the maintenance of an active reservoir. Njiokou et al. (2004), working with wild mammals, observed a 25.3% infection rate with trypanosomes, using molecular markers; but 11.7% of the mammals had unidentified trypanosomes. The year after, Simo et al. (2005), using the analysis of the Mobile Genetic Element (MGE-PCR), suggested a flow of genotypes of trypanosomes between Man and Pig in this focus. Anyway, one animal was diagnosed positive to T. b. gambiense defining 0.6 % prevalence in wild mammals, while 2.6% were positives to T. b. gambiense Non Group 1. Studying the blood meals of Glossina palpalis, Simo et al. (2008) found that 61.4 % of the blood meals in the area were taken on human that gave evidence for an important

relationship between Man and the vector. Working on the infection rates of tsetse with trypanosomes, Farikou et al. (2009) observed that these rates were about the same with T. brucei in all the villages, and that T. congolense infection rate was significantly superior in the villages closed to the forest by the east of the area. Continuing their studies on animals, Njiokou et al. (2009) diagnosed 4.83 % infected domestic mammals with T. b. gambiense, thus demonstrating the reality of an animal reservoir. At last, Mélachio et al. (2010) confirmed that tsetse population was structured in panmictic subpopulations; their sizes were estimated between 20 and 300 individuals and assumed densities between 120 and 2000 flies per square kilometer, with a dispersion of adults between 60 and 300 meters. Today, xenomonitoring is more efficient, due to the use of molecular tools that allow the identification of trypanosomes and of the origin of the tsetse blood. These monitoring was successfully used in Kinshasa (Simo et al., 2009; Grébaut et al. 2009) and in Cameroon (Labou et al., 2013). In the present study, we elaborated a projection of the epidemiological data to get a spatial vision of HAT in this focus, including human cases and entomological information. An entomological study was conducted in the Campo focus in dry season 2012 that brought an acute vision of the distribution of the actors of the pathogen complex which is human Trypanosomiasis. This information can bring a precious help to eliminate the disease in the area, showing the real interest of xenomonitoring.

Methods Study zone Campo is a small town of about 2000 inhabitants, located in the extreme south-west of Cameroon (2°22N, 9°49E) along the Atlantic and the mouth of the Ntem River that separates Cameroon from Equatorial-Guinea. The environment is composed of a coastal plain along the ocean, mangrove swamp along the Ntem River by the south of the area, then evergreen forest everywhere else. Our study zone includes Campo and four villages located at the east of the city. In the area, 3 rivers feed the watershed Ntem: Bitandé, Nyamelandé and Bibabimvoto rivers. Climate is equatorial of the Guinean type, with four seasons a year. The annual changes of temperature are low and the medium temperature is 25.7 °C, with an average relative humidity of 87 % and an annual rainfall superior to 2700 mm3. Population density is low and inferior to one inhabitant per square kilometer. Most of the people belong to four ethnical groups (Yassa, Mvae, Mabea and pigmies). Habitat is a linear and water points are for public use. Fishing is both maritime and inland, and excepted shrimp fishery, is exclusively practiced by men. Hunting was one of the most practiced activities but since the creation of the Campo Ma’An national park in the 2000s, hunting is highly restricted in the area. Epidemiological context The National Control Program (NCP) for sleeping sickness was created in 1998 in Cameroon, but we had to wait 2002 for its first medical survey in Campo. Excepted in 2005, where there were no medical surveys, the NCP came regularly and diagnosed cases every year up to 2011. In 2012, two cases were diagnosed passively in the Campo Hospital.







15 10 10 5


7 2

6 3

0 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011

Figure1. Number of HAT cases diagnosed in Campo from 1998 to 2011. Between 2001 and 2012, 61 HAT cases were diagnosed in Ipono, small village of about 300 inhabitants, which constitutes the epicenter of the HAT focus in the subdivision of Campo. All other cases were from Mabiogo (16 cases), Campo-Beach (15 cases) and Campo (3 cases). Five other cases were diagnosed in Mvasse (besides Ipono), Bouandjo on the road to Kribi and in Doumassi, a small village at the entrance of Campo. Taking into account these results, we observed the distribution of entomological results. Entomological study An entomological study was conducted in dry season 2012 in the five villages most affected by the disease: Campo, Campo-beach, Ipono, Mabiogo and Akak. One hundred and sixty-one capture points were spread over the 5 sites upon 295 km², using pyramidal traps during three days of capture. All these points were georeferenced. Tsetse flies were collected twice a day and after counting for ADT (Apparent Density per day and per Trap) have undergone a determination at the species level, if at the teneral state or not, then were dissected to isolate the midgut for further molecular analysis; midgut were stocked in 70 % ethanol at ambient temperature. Molecular analysis All samples isolated in the field were incubated at 80°C for ethanol evaporation. DNA was extracted using Chelex method (Walsh et al., 1991). After extraction, DNA was stored at -20°C until Polymerase Chain reaction (PCR) analyses. To detect trypanosomes in the midgut, PCR was performed using TBR1 and TBR2 primers, specific for Trypanozoon (); TCF1 and TCF2 for T. Congolense Forest (); TCS1 and TCS2 for T. Congolense savannah (); TVW1 and TVW2 for T. vivax () and TS1 and TS2 for T. simiae (). All the samples positive to TBR were amplified using TRPA primers (), in order to identify T. b. gambiense at the subspecific level of brucei. To identify the feeding hosts of tsetse flies we used heteroduplex PCR-based assay (Njiokou et al., 2005). The cytochrome B gene was amplified with primers of the vertebrate cytochrome B gene. Then, amplified fragments were hybridized with a driver, in this case with the Crycetomys gambianus (Giant rat) Cytochrome B DNA. After a migration in a 5% acrylamide/urea gel, profiles were compared to references such as Man or domestic mammals. Transmission Risk Index (TRI) This index was elaborated in the 1990’s (Laveissière et al., 1994) then simplified (Laveissière & Grébaut, 2003). In the formula r = 104(t+1)1.23*n²*(C0.46/TD3.69), are taken into account the number of

teneral flies (t), the number of human blood meals (r), the number of flies caught (C) by (T) traps during (D) days. Gradient of Risk Grebaut et al. (2009) used this gradient of risk during an entomological study about transmission in Kinshasa (RDC) suburbs. This gradient is defined by 5 levels of risk:      

Risk 0: no tsetse fly; Risk1: at least one tsetse fly; Risk2: at least one teneral fly or one human blood meal; Risk3: Both one teneral and one human blood meal; Risk4: presence of T. b. gambiense in the midgut; Risk5: risk4 and Risk2

Geographical Information System Mapping support of this region of Cameroon was provided by National Geographic ®. Projections of all epidemiological results and entomological results were realized using ArcGis®.

Results Nine hundred and ninety-one tsetse flies were caught in traps, among them 96.6% (957) were Glossina palpalis palpalis; the other 3.4% were Glossina pallicera (29), Glossina caliginea (1), Glossina tabaniformis (3) and Glossina nigrofusca (1). 5% of the flies were at the teneral stage. Distribution of the vector Campo Beach, which is located at the west of the study area, offers the lowest ADT (0.2 fly per day and per trap) that could be observed during this study (figure 2). The ADT around Campo, which is the most populous place in the area, is 1.7 tsetse flies per trap a day. In forest places, such as Mabiogo and all the villages along the eastern axis the ADTs are far more important, reaching 9.2 in Akak, which constitutes the most important density we observed in the area. The average ADT is 1.5 glossina.

Figure 2. Map of the distribution of the apparent density of tsetse flies per day and per trap (ADT) in dry season 2012 in Campo. Blood meals analysis Among 991 glossinas captured, 962 (97%) were dissected and 143 blood meals were analyzed (figure 3) with the heteroduplexe technics (). 60.1% of the blood meals stayed unidentified, but the main host in the area is man, with 32.2% of the whole isolated blood meals. With 4.2%, pigs are the second feeding hosts of tsetse flies then we found Sitatunga (1.4%), sheep (1.4%) and goat (0.7%).





RSHOM 32,2




Figure 3. Determination of the origin of isolated blood meals from tsetse flies, with RS = blood meal from, HOM = human, ND = undetermined, SUI = Suidae, CAP = goat, OV = sheep and SIT = Sitatunga.

Trypanosomes infections Twenty-eight midguts were diagnosed infected using PCR (Figure 4), that made a 19.6% infection rate. Among these infected midguts, 5 were mixed infections (TBB/TCF, TBB/TCS). T. congolense Forest type was the most current infection (17/28), followed by 7 flies infected by T. c. Savannah type. One fly was found infected with T. simiae besides a pigsty in Campo. Among the 3 samples infected with T. brucei sl., 1 was a T. b. gambiense one.







Figure 4. Trypanosomes infections in the midguts of captured tsetse flies, with TBB = T. brucei brucei, TBG = T. b. gambiense, TSIM = T. Simiae, TCF = T. Congolense Forest type and TCS = T. congolense Savannah type. Transmission Risk index When applying the TRI, one can notice that this index goes from 0 to 700 according to the village where applied (figure 5). It is particularly high in some villages of the eastern axis (700 in Akak) and no really important in the villages of the epicenter of the focus. In Campo, this risk translates a close contact between the human population and the glossinas. Gradient of Risk The gradient of risk was applied to each capture point and we can propose a map of distribution of this gradient (figure 6). No risk5 and risk6 levels were observed. As with the TRI, the eastern road cumulates places at higher risk (risk3) that could favor an endemic situation if a reservoir was introduced in this area. We also notice that several places around Campo city were at the risk3 level. In Mvasse, which is a village of the epicenter we can note one level 4 capture point, with evidence of circulation of the human pathogen in the vector. Xenomonitoring using entomological observations brings us essential information about tsetse flies in this HAT focus. In the discussion chapter we will try to understand how this focus can still be active

despite the regular screening that is conducted by the Cameroonian NCP.

Figure 6. Gradient of risk of transmission of HAT in the Campo Focus.

Discussion This entomological study allows making an inventory of the vector and trypanosomes situation in the Campo area, results are focusing on the apparent density of the fly population and their infections with trypanosomes. Combined with the identification of the host blood, we can point places at risk in this area and make recommendation for vector control and epidemiological surveillance showing all the interest of this xenomonitoring approach. The absence of geo-referencing of the traps during past studies impels us to be cautious about our conclusions. With 96.6% of individuals Glossina palpalis palpalis is the predominant Glossina species in the Campo area. Nevertheless, and for the first time since Eouzan’s study (1974), Tabaniformis were captured and identified in the area. This re-apparition of tabaniformis mainly occurred in the traps located at the eastern side of the study area, at a few kilometers of the Campo Ma’an National Park. During our study we also captured 29 Glossina pallicera (2.9% of captured flies) when Mbida Mbida described a 25.1% of the captured effectives at the same period of the year. In 1998, Morlais noticed that the Glossina palpalis population was 81 % of the captured flies, and Mbida Mbida (2005) estimates this proportion to 61.8 % at the same season we realized our study. Our estimation could indicate that, except the case of tabaniformis, the other tsetse flies species are decreasing in the Campo area.

When observing the ADT’s distribution, we can notice, as described in most of all the entomological studies that were realized in the area, that closer you get to the coast most glossina densities fall and are up to 30 times greater on the eastern axis where there are almost no cases. One more time, this confirms the fact that transmission of HAT does not depend on the vector’s density. We were able to identify trypanosomes using molecular markers. In order to isolate the midgut in optimistic conditions for laboratory analyses, we limited the number of traps to 50 by circuit during 3 days of captures to get enough flies and allow 2 collects per day. After dissection, midgut was isolated in a 70% ethanol solution to preserve DNA from degradation. This strategy allowed us to dissect 97% of caught flies and diagnosed a 19.6% infection rate with trypanosomes. This rate is similar to the 16.16% infection rate determined by Morlais (1998) in the Campo area.

Acknowledgements References

Dans la région de Campo, 11% (14/127) des repas de glossines ont une origine multiple. Les repas de sang mixtes correspondent probablement à des repas interrompus, la glossine se gorgeant sur différents hôtes (Gouteux et al., 1982~). Ceci assurerait une circulation non cyclique du parasite d’hôte à hôte, les glossines devenant alors des vecteurs mécaniques ’ (Gingrich, 1982). Les taux d’infection étant particulièrement élevés à Campo, ce mode de piqûre pourrait jouer un rôle non négligeable dans l’amplification de la transmission. Les types Forest et Savannah de T. congolense sont les plus fréquents. Les potamochères, abondants sur cette zone, comme le confirme l’analyse des résidus de repas sanguins, pourraient constituer le réservoir naturel du type Forest de T. congolense. Les suidés sauvages sont en effet présumés être d’importants hôtes réservoir (Aschrofi, 1959 ; Gruvel, 1975). Les ruminants sauvages hébergeraient plutôt le type Savannah. Ce dernier est héquemment identifié chez les bovins (de La Rocque, 1997 ; Reifenberg et al., 1997) et les travaux de Truc et al. (1994) sur la faune sauvage d’un parc animalier ivoirien confi’ient

la forte prévalence de T. congolense chez ces animaux.

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