Epidemiological aspects of the transmission of the parasites causing human African trypanosomiasis in Angola

Until the last few years, most cases of sleeping sickness or human African trypanosomiasis (HAT) occurred in Angola, the Democratic Republic of Congo and Sudan, with 17,673 cases detected in Angola alone among the 1,660,111 Angolans who were screened between 2001 and 2007 (T. Josenando, unpubl. obs.).

Angola is divided into 18 provinces (see Figure). The chronic form of HAT, caused by Trypanosoma brucei gambiense, occurs in seven of the 14 provinces where tsetse flies are present (in Bengo, Cuanza Norte, Uige, Cuanza Sul, Zaire, Malange, and the periphery of Luanda). The acute form of the disease, caused by T. b. rhodesiense transmitted by Glossina morsitans centralis, could once be found in the southern province of Cuando Cubango (where 30% of new Angolan HAT cases were diagnosed between 1964 and 1974; T. Josenando, unpubl. obs.). Most of the human population of this province moved to northern Angola during the civil war that ran from 1975 to 2002, however, probably leading to the disappearance of T. b. rhodesiense from Cuando Cubango and, therefore, from Angola as a whole (T. Josenando, unpubl. obs.).

Map of Angola, showing the provinces where human African trypanosomiasis (HAT) is endemic (hatched) and the location of the village of Maria Teresa, in and around which tsetse were caught in pyramidal traps. Note that HAT is only endemic on the periphery of the province of Luanda and not in Luanda city.

HAT in Angola is poorly documented, with no recent publications on the disease in the country. In 2007, the French Institut de Recherche pour le Développement (UMR 177) and the Angolan Instituto de Combate e Controlo das Tripanossomiases (ICCT) began collaborative research on the disease at Viana Hospital, which lies in Luanda province, 20 km to the east of Luanda city. The staff at the Viana Hospital only treat HAT cases, most of whom come from Bengo province. A year later, in an attempt to reinforce the tsetse-control capacity of the ICCT, an entomological study was conducted in and around the village of Maria Teresa (a community in Bengo province with particularly high incidences of HAT). The preliminary results of the collaborative research at Viana Hospital and the entomological survey in Maria Teresa are presented below.

SUBJECTS AND METHODS

The Province of Bengo

The province of Bengo, where most of the investigated cases resided, surrounds Luanda (Fig.). It is itself surrounded by other HAT-endemic provinces: Uige and Zaire to the north, Cuanza Norte to the east and Cuanza Sul to the south. The annual mean temperature is 25°C while annual rainfall varies from 475 to 724 mm. The sandy soils, which do not retain water, result in a low human density (10 inhabitants/km²) and no cattle production. However, the presence of Angola’s main rivers (such as the Kwanza River) in the province and the province’s proximity to the city of Luanda make Bengo a major source of food for the population of the Angolan capital. The main crops are cassava, potatoes and maize; there is also substantial charcoal production.

Definition of HAT Cases and Collection of Epidemiological Data

The HAT cases investigated, all of whom gave informed consent, were identified using the card-agglutination test (CATT) for T. b. gambiense (Magnus et al., 1978), mainly in Viana. Each suspected case was confirmed parasitologically by the microscopical detection of trypanosomes in (1) an aspirate from a swollen lymph node, (2) blood processed using capillary-tube (Woo, 1970) or mini-anion-exchange (Lumsden et al., 1979) centrifugation, and/or (3) a pellet produced by centrifuging a sample of cerebrospinal fluid (Miézan et al., 2000). Each confirmed case was admitted to Viana Hospital, for treatment.

At the time of his or her admission to Viana Hospital, a confidential file was completed for each case, by a clinician. This file included the case’s gender, age, place of birth, ethnic group, occupation, current place of residence, and place(s) of residence in the previous 4 years, and details of any journey made by the case, within Angola, in the previous 2 years, any previous treatment for HAT, and any previous HAT episode in the case and his/her family (with the date and location of each episode). Although each case was clinically examined on admission, the results of those examinations are not reported here.

The Entomological Survey

A tsetse survey was conducted in and around Maria Teresa in January 2008 (i.e. during the dry season). Besides training the ICCT staff in entomological techniques, the aim of the survey was to evaluate the risk of transmission in the study area, which lies close to Cuanza Norte province. Twenty pyramidal traps were set along a 40-km-long transect, with the tsetse flies caught in the traps collected once a day for 4 days. Each tsetse fly caught was identified to species and sexed. Any non-teneral fly was dissected so that its midgut could be checked, under a light microscope, for trypanosome infection. The salivary glands of any fly found midgut-positive for trypanosomes were also checked microscopically.

Every midgut was spread onto a piece of filter paper (Grade 4; Whatman, Maidstone, U.K.), which was allowed to dry and kept at room temperature until it could be tested in a PCR-based assay based on the internal-transcribed-spacer-1 sequences of Trypanosoma (Desquesnes and Dávila, 2002). DNA was extracted using QIAamp spin-columns (QIAGEN, Hilden, Germany). No attempt was made to identify any T. brucei s.l. encountered to subspecific level [e.g. by the use of iso-enzyme electrophoresis (Stevens et al., 1994)].

RESULTS AND DISCUSSION

HAT Case Profiles

The HAT in the 200 consenting cases (124 males and 76 females) included in the present study was confirmed between April 2007 and October 2008. Although most of the cases belonged to the Kimbundu (57·5%) or Umbundu (32·5%) ethnic groups (i.e. the two groups that predominate in Bengo province), the Kikongo (6·5%), Tchokoe (2·0%), Haneka Humbe (0·5%) and Mukubal (0·5%) ethnicities were also represented (and the ethnicity of one case could not be determined).

Seventy-one (35·5%) of the cases were farmers. The other cases were students, schoolchildren or other young people aged <14 years of age (20·5%), charcoal manufacturers (11%), household workers (7%), merchants (mainly coal merchants) (6%), fishermen (4%), bricklayers (3%), drivers (2·5%), unskilled workers (1·5%), mechanics (1·5%), teachers (1%), nurses (1%), locksmiths (0·5%), electricians (0·5%), soldiers (0·5%), policemen (0·5%) or hunters (0·5%) or had no occupation (2·5%) or an unknown occupation (0·5%).

The data collected on the cases were explored to see if certain occupations (e.g. those that could lead to close contact with infected tsetse), places of current or recent residence and/or places visited were associated with increased risk of HAT. A significant percentage of the cases had probably not been infected in their places of residence. Of the 159 cases identified in the HAT-endemic province of Bengo, for example, only 95 were residents of the province. The 64 non-residents had probably been infected as a result of their occupations, as most were farmers or charcoal manufacturers who had worked for short periods in areas where T. b. gambiense is transmitted or were merchants or drivers who regularly passed through such areas as they worked. In Cameroon, hunters (who regularly enter areas with high tsetse densities) are similarly known to be at relatively high risk of HAT (Grébaut et al., 2001).

Most (168) of the cases were detected in the provinces of Bengo or Luanda. Most of the cases who were resident in Bengo had apparently been infected in an area 60 km to the east of Viana, in a historic focus that includes the villages of Calomboloca (47 cases), Zenza do Gulungu (22), Catete Lalama (21) and Maria Teresa (11). Most of the cases from this focus had not been identified in a mass screening but, being very familiar with HAT, had presented at the Viana Hospital. Another nine cases came from Cuanza Norte province, which is also fairly close to Viana (about 6 h away by bus). Few of the cases came from the provinces of Zaire, Uige or Cuanza Sul but these provinces are relatively distant from Viana and each has its own designated centre for HAT diagnosis and treatment.

For six of the cases of HAT, the probable place of T. b. gambiense infection could not be determined. All six were in the late second stage of the disease, with severe neurological disorders, when they presented at the Viana Hospital. The family members who brought these cases claimed that the cases lived in the cities of Luanda or Viana, and had never visited any other place within Angola. The accuracy of these claims has to be questioned, however, as no transmission of T. b. gambiense within the cities of Luanda and Viana has ever been recorded. The possibility that some or all of these cases were infected when they were bitten by tsetse that had been infected elsewhere and then ‘imported’ into the cities (e.g. inside bags of charcoal) merits exploration.

Although many other of the cases lived in urban Luanda or Viana, they either had occupations that took them into areas where transmission is known to occur (farmers, charcoal manufacturers, merchants, bricklayers) or were students/young people who had each made at least one visit to such an area. Within Bengo province, farmers, charcoal manufacturers and fishermen, who probably work in close contact with tsetse flies in plantations, in forests and on riverbanks, appear to be at high risk of HAT. The data presented here are, however, preliminary and the observations and apparent associations made need to be confirmed in larger investigations, including entomological (Laveissière and Grébaut, 2003) and matched case–control studies. Similar observations have been made in the Central African Republic (Gouteux et al., 1993). The fact that many HAT cases in and around Bengo are not infected in their places of residence clearly means that T. b. gambiense is being dispersed as the cases travel and work away from their homes and return to their homes. This will complicate HAT surveillance and control activities, which tend to be focused on known foci, and leave some HAT cases undetected and untreated.

Profile of the Tsetse-fly Population in the Maria Teresa Area

All 319 tsetse flies that were captured (a mean of 2·7 flies/trap-day) were identified as G. palpalis palpalis. The traps sited in forest gallery caught more flies than traps sited in other habitats (data not shown). Only 1·9% of the tsetse flies caught were teneral and most of the rest (239) were dissected. Although, under the microscope, trypanosomes were only observed in 20 (8·4%) of the midguts that were investigated, 54 (22·6%) of the midguts were found PCR-positive for trypanosome DNA. Out of 20 midguts found positive by microscopy, however, only 11 were also found PCR-positive.

The amplicons produced in the PCR indicated that 32 (59·3%) of the 54 PCR-positive midguts contained the DNA of T. congolense of the savannah type, 20 (37%) the DNA of T. vivax, and only two (3·7%) the DNA of T. brucei s.l., with the PCR results also indicating that five flies (9·3% of those found PCR-positive) harboured mixed infections. No flagellates were found in any of the salivary glands that were examined.

The present results provide evidence of the predominance of G. p. palpalis among the tsetse flies in and around the HAT-endemic village of Maria Teresa and the presence of one or more subspecies of T. brucei s.l. in the local G. p. palpalis. Although they do not prove that T. b. gambiense is being transmitted in the area (most T. brucei s.l. in the area is likely to T. b. brucei), the number of HAT cases treated at the Viana Hospital demonstrates that there is active transmission of T. b. gambiense in the province of Bengo. Humans in the province who enter areas of forest gallery (where there are relatively high densities of G. p. palpalis) may be at particularly high risk of being infected with T. b. gambiense (or, if already infected, of passing the parasite to G. p. palpalis).