Occurrence of the off-host life stages of Tunga penetrans (Siphonaptera) in various environments in Brazil

Transcription

1 Annals of Tropical Medicine & Parasitology, Vol. 104, No. 4, (2010) Occurrence of the off-host life stages of Tunga penetrans (Siphonaptera) in various environments in Brazil P. M. LINARDI *, C. M. L. CALHEIROS {, E. B. CAMPELO-JUNIOR {, E. M. DUARTE {, J. HEUKELBACH { and H. FELDMEIER 1 * Department of Parasitology, Federal University of Minas Gerais, Avenida Antônio Carlos 6627, Belo Horizonte, CEP , MG, Brazil { Institute of Biological Sciences and Health, Federal University of Alagoas, Praça Afrânio Jorge s/n, Trapiche, Maceió, CEP , AL, Brazil, and Alagoas State University of Health Sciences and School of Biological Sciences and Health, Avenida Siqueira Campos 2095, Maceio, CEP , AL, Brazil { Department of Community Health, Federal University of Ceará and Mandacaru Foundation, Rua Vilar de Andrade 257, Fortaleza, CEP , CE, Brazil 1 Institute of Microbiology and Hygiene, Charité Universitätsmedizin, Campus Benjamin Franklin, Hindenburgdamm 130, Berlin, Germany Received 15 February 2010, Revised 16 April 2010, Accepted 20 April 2010 To explore the local transmission dynamics of Tunga penetrans in Brazil, 134 soil samples from various environments were collected in three different endemic regions of the country and checked for the presence of the flea s larvae, pupae and adults. The samples, which came from an urban slum in the north east, a village of Xavante Indians in the central west and a community of Yanomami Indians living in traditional longhouses (malocas) in the north, were categorized as indoor, outdoor or indoor outdoor (the latter representing samples collected in the malocas). The proportion of samples found positive for T. penetrans was lowest in the slum (9.3%) and highest in the Yanomami village (32.0%; P50.01). Soil samples collected below bedsteads or hammocks or from the indoor resting places of dogs were significantly more likely to be positive than the indoor samples collected at other sites (65.0% v. 35.0%; P50.02). There was no evidence indicating that the presence of T. penetrans in a soil sample was markedly affected by soil temperature, air temperature or air humidity. As no life stages of T. penetrans were found in any outdoor sample, it seems likely that, in resource-poor settings in Brazil, most transmission of T. penetrans occurs indoors. Control measures against the off-host life stages of T. penetrans should therefore be targeted at particular indoor micro-environments. Although human infection with Tunga penetrans (L., 1758) (i.e. tungiasis) has been known for 500 years and remains common in resource-poor communities in South America, the Caribbean and sub-saharan Africa, the reproductive biology of this flea Reprint requests to: C. M. L. Calheiros, Travessa Menino Marcelo, Quadra B, Lote 12, Nu 213, Residencial Jardim Europa, Antares, Maceió, CEP , AL, Brazil. claudiamcz@ig.com.br. remains enigmatic (Heukelbach, 2005). For example, it is still not known whether, in an endemic area, humans become infested predominantly while outdoors or whether considerable intradomiciliary transmission also occurs (Heukelbach, 2007; Witt et al., 2007). The data available on the off-host life stages of T. penetrans and the environments where these developmental stages thrive are generally outdated or were collected in studies that were each limited to a single setting (Hicks, 1930; Nagy et al., 2007). # W. S. Maney & Son Ltd 2010 DOI: / X

2 338 LINARDI ET AL. FIG. 1. The locations of the study sites at Alto Alegre (&), Fortaleza (N) and Barra do Garças (m), in the Brazilian states of Roraima (RR), Mato Grosso (MT) and Ceará (CE). In the present study, in a first step to describe, comprehensively, the epidemiology and ecology of the off-host stages of T. penetrans in Brazil, soil samples collected from three endemic areas, with different climatic, soil and socio cultural characteristics, were checked for the presence of the flea s larvae, pupae and adults. MATERIALS AND METHODS The study was performed in three geographical regions of Brazil with different environmental and socio cultural characteristics (Fig. 1): an urban slum (favela) on the outskirts of the north eastern city of Fortaleza (Ceará state), a central western village of Xavante Indians in the municipality of Barra do Garças (Mato Grosso state), and a community of Yanomami Indians living in four traditional longhouses (malocas) in the Alto Alegre municipality, in the north of the country (Roraima state). The houses in these study sites were constructed from bricks and/or recycled materials (the slum) or from wooden poles covered with palm leaves (the Xavante village and Yanomami malocas). Between November 2005 and July 2006, soil samples, each of g, were collected in and (in the urban slum and Xavante village) around each household in which at least one family member had been found

3 Tunga penetrans LIFE STAGES 339 infested with T. penetrans (Tables 1 and 2). All samples were collected in the morning, between and hours. The peridomestic or outdoor samples from the slum and Xavante village were collected in front of the entrance to the house, along the outsides of the walls of the house, in the backyard, and from the outdoor resting place(s) of any dog(s). The intradomiciliary or indoor samples from the same two study sites were collected from earthen floors in sleeping and living rooms, from crevices in concrete floors and between solid floors and walls, and from the indoor resting places of the dog(s). (The resting places of dogs in the slum were exclusively outdoors but the Xavante Indians allowed their dogs to rest indoors.) In the Yanomami malocas (open shelters covered by palm leaves and encircled by forest), so-called indoor outdoor samples were taken from soil beneath the hammocks and from the resting places of any dogs. Each sample was collected with a small shovel or a brush (used to collect samples from crevices in concrete floors) and stored in a plastic bag. Care was taken to ensure that each sample came from the top of the soil (to a maximum depth of 1 cm). Bags were coded and kept at ambient temperature until their microscopical analysis (see below), 2 22 days after the samples were collected. Each sample was classified, according to colour and structure, as sand, red-brown laterite or yellow laterite (Anon., 1999). After collection of the soil samples, the physical characteristics of the surrounding micro-environment were recorded. Soil temperature was measured using an infrared thermometer (HI testo; Conrad, Hirschau, Germany) while air temperature and relative humidity were measured with a hygro thermometer (HT 100 Voltcraft; Conrad). For the microscopical analysis, each sample was checked, under a dissecting microscope (Zoom 2000; Leica, Wetzlar, Germany), for the off-host life stages of T. penetrans, which were identified using the guidelines provided by Hicks (1930). Larvae and pupae were mounted on a slide in Berlese solution and measured using a calibrated eye-piece micrometer. The gender of each adult was recorded (Linardi and Guimarães, 2000). A soil sample was defined as positive if was found to contain at least one larval, pupal or adult T. penetrans. Relative frequencies of positivity were compared in x 2 tests, with a P- value of,0.05 being considered indicative of a statistically significant difference. RESULTS Of the 134 samples collected, 28 (20.8%) contained at least one larval (Fig. 2), pupal TABLE 1. The numbers of soil samples collected, stratified according to micro-environment (N5134) Indoor samples from: * Outdoor samples from: Indoor outdoor samples from: Location Sandy Resting place floor Crevices { of dog Compound Resting place of dog Beneath hammock Resting place of dog Urban slum Xavante village Yanomami 19 6 malocas All three sites * Collected in sleeping and living rooms. { In concrete floor or between floor and walls.

4 340 LINARDI ET AL. FIG. 2. A photomicrograph of a second-stage larva of Tunga penetrans that was found in a soil sample from a crevice in a concrete floor. The larva measured 3.1 mm in length and 0.42 mm in width. or adult T. penetrans. The percentage of samples found positive varied considerably not only with study site being lowest in the slum (9.3%) and highest in the Yanomami malocas (32.0%; P50.02; Table 3) but also with micro-environment, ranging from 0% for the outdoor samples to 57.1% for the indoor samples from the Xavante village (P,0.001). All 28 positive soil samples were from microenvironments that were not directly exposed to sunlight. Although the frequency of positivity appeared to be unaffected by the delay between sample collection and the microscopical analysis, only larvae and pupae were found in the samples analysed within 14 days whereas the samples examined after a longer delay only contained pupae and adults. Thus, all the adults identified microscopically had probably developed during sample storage, from immature life stages that were present in the soil samples when they were collected. When the indoor samples (all of which came from the slum or Xavante village) were stratified according to micro-environment, those collected beneath hammocks and bedsteads were more likely to be positive than the other samples (65.0% v. 35.0%; TABLE 2. Regions, areas and sites of the soil collection No. of samples: Region State Location Month Season Indoor Outdoor Indoor outdoor North east Ceará Urban slum, Vicente Pizon, Fortaleza Central Mato Xavante village, west Grosso Barra do Garças North Roraima Yanomami malocas, Alto Alegre municipality November 2005 End of dry season May 2006 Dry Season July 2006 Rainy season 25

5 Tunga penetrans LIFE STAGES 341 P50.001; Table 3). In addition, in both the Xavante village and in the Yanomami malocas, the soil samples collected below hammocks were more likely to be positive than the samples collected from the resting places of dogs, with frequencies of 68.7% v. 6.2% (P50.02), and 62.5% v. 37.5% (P50.02), respectively. Although the cat flea, Ctenocephalides felis felis, was detected in many of the soil samples, as larvae, pupae or adults, its distribution among the samples differed considerably from that of T. penetrans, even within the same study site (Table 4; P50.001). Only in 11 samples were the two flea species found together. Of these 11 samples, four (36%,), three (28%) and four (36%) were collected from the resting places of dogs, beneath hammocks and bedsteads, and from other micro-environments (such as the fissures between concrete floors and walls), respectively. At six locations, all of which were the resting places of dogs, C. felis felis but no T. penetrans were found. In 15 (53%) of the soil samples positive for T. penetrans, adult arthropods other than C. felis felis (Acari, Hymenoptera, Collembola and Coleoptera) were identified. As expected, air temperature and relative humidity differed between indoor and outdoor micro-environments, with medians of 27.7uC and 70.4% relative humidity indoors compared with 26.8uC and 56.9% indoors (the corresponding P-values were 0.02 for air temperature and 0.05 for relative humidity). The presence of T. penetrans life stages in soil close to the ground surface did not, however, appear to be significantly affected by soil temperature, air temperature or relative humidity (data not shown). DISCUSSION This appears to be the first study to assess, systematically, the occurrence of the off-host stages of T. penetrans in varied environments and socio cultural settings. The results indicate that, in resource-poor communities in urban and rural Brazil, most, and perhaps all, of the development of T. penetrans, from eggs to adults, occurs either indoors or in the indoor outdoor habitats of the traditional maloca. Hitherto, only anecdotal reports have indicated that human infestation may take place inside dwellings. For instance, in the travel literature of the 18th and 19th TABLE 3. Detection of the larvae (L), pupae (P) and adults (A) of Tunga penetrans in the soil samples, split by location and micro-environment Study site Urban slum (sandy soil) Xavante village (red brown laterite) Yanomami malocas (yellow laterite) NO. OF SAMPLES POSITIVE/INVESTIGATED AND (% OF SAMPLES POSITIVE) Indoor 4 (L)/18 (22.2) 16 (9L, 2P (57.1) 0/0 and 5A)/28 Outdoor 0/25 (0.0) 0/38 (0.0) 0/0 Indoor 0/0 0/0 8 (2L, 3P (32.0) outdoor and 3A)/25 From any area 4/43 (9.3) 16/66 (24.2) 8/25 (32.0) NO. AND [%] OF POSITIVE SAMPLES FROM SITE RECOVERED: Under the resting 0 [0.0] 1 (L) [6.3] 3 (1L, 1P [37.5] places of dogs and 1A) Beneath hammocks 2 (L) [50.0] 11 (6L, 2P [68.8] 5 (1L, 2P [62.5] or bedsteads and 3A) and 2A) In other areas 2 (L) [50.0] 4 (2L and 2A) [25.0] 0 [0.0]

6 342 LINARDI ET AL. Centuries, it is repeatedly reported that foreign travellers became heavily infested in the first night that they spent in a house after arriving in an endemic community (Bonnet, 1867; Hoeppli, 1963). More recently, the results of attempts to identify the main risk factors for tungiasis, in community-based studies, have also indicated that human infestation with T. penetrans may occur indoors, at least in Brazil (Muehlen et al., 2006) and Nigeria (Ugbomoiko et al., 2007). In the West African study by Ugbomoiko et al. (2007), a sandy floor inside a house was identified as an important independent risk factor for the presence of tungiasis in the house, although people who rested outside also frequently became infested. There is no doubt that, in some settings, humans become infested with T. penetrans while outdoors, in peridomestic areas (Nagy et al., 2007; Witt et al., 2007). In the present study, larval, pupal and adult T. penetrans were found in soil samples that had been collected in houses with and without a solid, concrete floor. In houses with concrete floors, the sand/dust that had accumulated in crevices in the floors or in the fissures between the concrete floors and the walls was sometimes found to contain T. penetrans. Under the physical characteristics prevailing inside simple dwellings in tropical Brazil (in the present study, these included air temperatures between 22.0uC and 31.2uC, and relative humidities between 51.4% and 95.1%), larval T. penetrans can survive and develop in the rather small amounts of organic material present in crevices and fissures in concrete floors. In Mexico, curiously, Faust and Maxwell (1930) found that the larvae of T. penetrans could live in skin lesions on humans, where they fed on small amounts of human blood. Among the soil samples collected indoors, those taken from beneath bedsteads or hammocks were particularly likely to contain T. penetrans larvae or pupae (Table 3). This makes sense, since in tropical Brazil people do not cover their body with a blanket during sleep, and eggs expelled from embedded female T. penetrans may therefore easily reach the ground below a sleeping case of tungiasis (Eisele et al., 2003). Such contamination of the soil is, perhaps, particularly likely to occur under a case who is sleeping in a hammock, with his or her feet left exposed and dangling over the floor. If TABLE 4. The numbers of the life stages of Tunga penetrans and Ctenocephalides felis felis isolated from soil samples collected from 28 indoor areas No. and (%) of the recovered: Larvae Pupae Adults Sample site Micro-environment T. penetrans C. felis felis T. penetrans C. felis felis T. penetrans C. felis felis Sleeping room Living room Resting place of dog Beneath bedstead/ hammock 41 (45.6) 6 (26.2) 13 (68.4) 1 (20.0) 36 (37.9) 12 (38.7) Sandy soil 10 (11.1) 4 (4.2) Gap between floor and walls 9 (10.0) 2 (10.5) Crevices in 12 (13.3) 3 (13.1) concrete floor Gap between floor 7 (7.8) 3 (3.2) and walls Sandy soil 11 (12.2) 14 (60.2) 4 (21.1) 4 (80.0) 52 (54.7) 19 (61.3) All

7 Tunga penetrans LIFE STAGES 343 the local environmental characteristics are suitable, the shed eggs of T. penetrans can develop into adults within 18 days (Hicks, 1930). Tungiasis is one of only a few parasitic diseases in which the whole lifecycle, including off-host and on-host stages, can be completed in one bedroom. In the present study, the indoor outdoor samples of soil collected in the traditional Yanomami community, where several families (sometimes more than 100 people) and their pets may live closely together in one maloca, were more likely to be found positive than the indoor or outdoor samples from elsewhere. Malocas are rather simple round shelters with open entrances, outer walls but no inner walls, and palm-leaf roofs. All cooking, eating and sleeping occurs inside the maloca, and pets and rodents can roam freely in and around the shelter. In many ways, the resultant mixed indoor outdoor micro-environment resembles that of the open kitchen stalls frequently found in the compounds of those who live in other impoverished rural communities in Brazil. Interestingly, such stalls have been identified as sites where infestation with T. penetrans frequently occurs (Witt et al., 2007). In the Xavante village and in the Yanomami malocas, T. penetrans life stages were recovered from several resting places of dogs. In these settings, dogs probably act as important reservoir hosts (Rieschel, 1989; Heukelbach et al., 2003). In an epidemiological study in north eastern Brazil, between 30.9% and 67.1% of the dogs in impoverished rural and urban communities were found infested with T. penetrans, some of the animals harbouring more than 100 embedded fleas each (Heukelbach et al., 2003). Dogs could clearly maintain the transmission cycle, in indoor or peridomestic sites, provided that the type of soil where the dogs rest is suitable for the development of the off-host stages of the fleas. As rodents can also act as reservoir hosts for T. penetrans (Heukelbach et al., 2003; Ugbomoiko et al., 2008), it is (theoretically) possible that adult but unembedded fleas could have been carried inside the dwellings of the Xavante and Yanomami Indians by various species of rodent. In the present study, however, it seems unlikely that any adult fleas were present in the soil samples at the time the samples were collected, and the life stages that were collected mostly came from the resting places of dogs and below bedsteads and hammocks. If fleas were frequently being carried into homes and malocas by rodents, the indoor distribution of the fleas might be expected to be more random. The simultaneous presence of different life stages at the same sampling sites, and the co-occurrence of T. penetrans and C. felis felis life stages in the resting places of dogs, are indicators that the life-cycles of both flea species are being maintained in quite restricted patches of soil or detritus. One of the most surprising results of the present study was the failure to detect any larval, pupal or adult T. penetrans in any of the outdoor samples of soil from peridomestic sites in the slum or Xavante village, even though these samples were collected during the dry season, when the prevalence of human tungiasis peaks in Brazil (Heukelbach et al., 2005). In peridomestic environments, the immature life stages of T. penetrans may be much more highly dispersed than the stages to be found indoors. In consequence, the 63 outdoor samples examined in the present study may simply have been too few to pick up any T. penetrans in the peridomestic areas. In addition, such areas are often exposed to intense sunshine, with relatively high ground temperatures and relatively low relative humidities, and these characteristics may impede the development and survival of any off-host stages of T. penetrans (Bonnet, 1867). During the rainy season, any heavy precipitation could further disperse the off-host life stages that are outdoors and if the site is inclined wash the superficial soil layers, together with any ectoparasites that they contain, far away. The predation of the developing fleas, by Acari, Coleoptera and Hymenoptera, is also

8 344 LINARDI ET AL. likely to be heavier outdoors than indoors (Fox and Bayona, 1968; Fox, 1969). Although, at least at the sites investigated in the present study, the transmission of T. penetrans may be more intense indoors than outdoors, the marked seasonality seen in the incidence of tungiasis in Brazil (Heukelbach et al., 2005) indicates that the life-cycle of T. penetrans is not exclusively maintained indoors. Although soil and air temperatures and relative humidity had no apparent effect on the detection of T. penetrans life stages in the present study, the values recorded for these variables were not that wide-ranging. The off-host development of T. penetrans, like that of several other flea species (Silverman et al., 1981; Krasnov et al., 2001, 2002), is probably possible over a considerable range of temperatures and humidities. It seems likely that, in Brazil at least, transmission of T. penetrans to humans occurs indoors and in the maloca-type indoor outdoor environments the whole year round, whereas peridomestic transmission is limited to those seasons in which the air humidity is neither too low nor too high. As indoor transmission depends on the existence of suitable soil/detritus in those places where the flea s eggs are expelled and fall to the ground, the importance of such transmission in impoverished communities has probably diminished over the last few decades, when many earthen floors were covered in tiles or concrete (Feldmeier et al., 2003; Muehlen et al., 2003). The present results have important implications for tungiasis control, at least in slum and indigenous communities in Brazil. Residual spraying with insecticides could and probably should be targeted at indoor sites where the development of off-host stages is very likely (such as the resting places of pets and below hammocks and bedsteads), thereby diminishing the amount of insecticide required. Solid floors, made of a smooth layer of concrete, would also probably help diminish the indoor propagation of T. penetrans. In conclusion, in Brazil, the life-cycle of T. penetrans may be completed indoors or in the mixed indoor outdoor habitats provided by the traditional dwellings of indigenous populations, especially below hammocks, bedsteads and the resting places of dogs. ACKNOWLEDGEMENTS. The study described formed part of the Ph.D. thesis of C.M.L.C., within the Programa de Pós-graduação em Parasitologia/Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais. The study was partially supported by the Fundação de Educação em Saúde Mandacarú, the Fundação de Amparo à Pesquisa de Alagoas, and the Ärztekomitee für die Dritte Welt, Frankfurt, Germany. The authors thank R. Cruz and W. Uieda of the Department of Indigenous Health (National Health Foundation) for technical assistance, and M. Feldmeier for her secretarial assistance. Two of the authors (J.H. and P.M.L.) are research fellows of the Brazilian Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). REFERENCES Anon. (1999). Sistema Brasileiro de Classificação de Solos. Rio de Janeiro, Brazil: Empresa Brasileira de Pesquisa Agropecuária. Bonnet, G. (1867). Memoire sur la puce penétrante ou chique (Pulex penetrans). Archives de Médecine Navale, 8, 19 53, , Eisele, M., Heukelbach, J., Van Marck, E., Mehlhorn, H., Meckes, O., Franck, S. & Feldmeier, H. (2003). Investigations on the biology, epidemiology, pathology and control of Tunga penetrans in Brazil. I. Natural history of tungiasis in man. Parasitology Research, 90, Faust, E. C. & Maxwell, T. A. (1930). The finding of the larva of the chigo, Tunga penetrans, in scrapings from human skin. Archives of Dermatology and Syphology, 22, Feldmeier, H., Eisele, M. & Saboia Moura, R. C. (2003). Severe tungiasis in underprivileged communities: case series from Brazil. Emerging Infectious Diseases, 9,

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