Synanthropic triatomines ( Hemiptera , Reduviidae ) in the State of Pernambuco , Brazil : geographical distribution and natural Trypanosoma infection rates between 2006 and 2007

Introduction: The present study shows a descriptive analysis of triatomine occurrence and its natural Trypanosoma infection rates in the state of Pernambuco, Brazil, between 2006 and 2007. Methods: Entomological data for the species, such as specimens captured in both intra and peridomiciles and natural infection index, were obtained via domiciliary capture in 147 municipalities from 11 Regional Managements of Health. The database was obtained from a sample of insects (100% infected and 20% non-infected) sent to the Central Laboratory of Pernambuco. Results: A total of 18,029 triatomines were analyzed from 138 municipalities of the state. Triatoma pseudomaculata (35%), Triatoma brasiliensis (34%), and Panstrongylus lutzi (25%) were the most captured species. These species also showed a widespread geographical distribution in the state. Panstrongylus megistus, Triatoma petrocchiae, Triatoma melanocephala, Triatoma sordida, Rhodnius nasutus, Rhodnius neglectus, and Triatoma infestans showed more limited geographical distribution and lower relative abundance. The parasitological research showed that 8.8% of the triatomines were naturally infected with flagellates morphologically similar to Trypanosoma cruzi and 91.3% of them were captured inside houses in 113 municipalities. P. lutzi showed the highest rates of natural infection. Conclusions: After the control of T. infestans, synanthropic species, such as T. brasiliensis, T. pseudomaculata, and P. lutzi, maintain the risk of T. cruzi transmission to humans in the state of Pernambuco. These species are widely distributed, and infected specimens have been found inside houses. Thus, an enhanced surveillance and vector control of Chagas disease is recommended in Pernambuco.

Today, Chagas disease or American Trypanosomiasis continues to be a chronic and potentially fatal illness in Latin America 1 .According to estimates by the Pan American Health Organization, 20% of Latin American population was at risk (109 million individuals), and nearly 7.7 million individuals were infected in 2005 (1.9 million in Brazil) [2][3] .The etiological agent, Trypanosoma cruzi (Chagas, 1909), is mainly transmitted by hematophagous insects of the subfamily Triatominae (Hemiptera, Reduviidae) adapted to colonize human dwellings 4 .
The estimated prevalence of human Chagas disease in the State of Pernambuco, Northeastern Brazil, in the 1980s was 2.8%, and seropositive people were detected in 144 of 163 municipalities 5 .In the 1990s, serological surveys showed that infections with T. cruzi still occur in the state [6][7] .According to the last national survey of Chagas disease seroprevalence carried out in Brazil between 2001 and 2008, four positive cases were detected in Pernambuco.One case involved a child below 5 years whose mother was also seropositive 8 .
Currently, the subfamily Triatominae contains 142 species, grouped into 18 genera and five tribes [9][10][11][12] .In Brazil, there are 62 recognized species of Triatominae (Galvão and Gurgel-Gonçalves unpublished data).Pioneering studies on the vectors of Chagas disease in Pernambuco reported the occurrence of Triatoma infestans (Klug 1834) and other eight species [13][14][15]  The occurrence of native triatomine species that sporadically invade or reinvade human dwellings is a major difficulty for the consolidation of vector control [18][19] .Knowledge of geographical distribution and natural infection of these bugs is fundamental to

RESULTS
Silva MBA et al -Triatomines in the State of Pernambuco the understanding of epidemiological aspects related to the T. cruzi transmission and should be considered to guide the actions of control and surveillance of Chagas disease.Thus, the aim of the present work was to analyze the geographical distribution of triatomines and their natural Trypanosoma infection in the State of Pernambuco, according to the Regional Managements of Health between 2006 and 2007.
The state of Pernambuco is geographically situated in the northeast region of Brazil.According to the IBGE (Census 2010), this state is divided into 185 municipalities, covering an area of 98,146.315km 2 , with a population of 8,796,448 people.Eighty percent of the population is in urban areas (http://www.ibge.gov.br/estadosat/).Municipalities are distributed in five geographical regions: the metropolitan area of Recife (coastal region with mangrove and Atlantic rainforest), Zona da Mata (Atlantic rainforest), Agreste (area of transition between the Atlantic rainforest and the Caatinga), Sertão, and Sertão do São Francisco (areas of semi-arid Caatinga).All of them are operationally subordinated to eleven Regional Managements of Health (Gerência Regional de Saúde -GERES), Figure 1.Entomological data were obtained via domiciliary capture in 147 municipalities from 11 GERES of the State of Pernambuco between 2006 and 2007.During the conduct of this research, Afogados da Ingazeira and Serra Talhada were part of the same GERES, but in 2007, this GERES was divided (Figure 1).

FIGURE 1 -Location of the State of Pernambuco in Brazil and its 11 Regional Managements of Health (GERES). The numbers indicate the municipality administrative center of each GERES.
Triatomines were collected manually by the agents of endemic diseases in the municipalities and sent to the GERES.The materials used for triatomine collection were metal tweezers and flashlights to inspect cracks and sites deprived of light, and, when necessary, dislodging liquid (Pirisa 2%) was applied.
The database used in this study considered a sample of insects sent by the 11 GERES of the state to the Laboratory of Endemic Diseases, Central Laboratory of Pernambuco (LABEND/LACEN-PE).This laboratory was responsible for quality control (taxonomic identification and parasitological exams of triatomines) and helped in the surveillance activities.The database included all infected bugs and 20% of the negative bugs after laboratory examination in the GERES.Those insects were taken for a reexamination between January 2006 and December 2007.For the 2006 database, we considered the following entomological indicators: number of insects captured in the municipality (males, females, and nymphs) in both intra and peridomiciles and the number of infected bugs by flagellates morphologically similar to T. cruzi.
Triatomines were identified to the species level according to Lent and Wygodzinsky 4 .The parasitological research was conducted through abdominal compression of triatomines and subsequent examination of fresh feces.The phenotypic identification of the parasites was made by observation under a microscope of Giemsastained insect feces.The natural infection rate was obtained from the ratio between the number of infected insects and the number of insects examined ×100 20  Triatoma pseudomaculata (35%), T. brasiliensis (34%), and P. lutzi (25%) were the most captured species considering the sample of insects examined.These species also showed a widespread geographical distribution in the state.The other species showed more limited geographical distribution and lower relative abundance (Figure 2).The parasitological research showed that 8.8% of the triatomines were naturally infected with flagellates morphologically similar to Trypanosoma cruzi, and 91.3% of them were captured inside houses in 113 municipalities.T. melanocephala and P. lutzi showed the highest rates of natural infection (Table 1).However, T. melanocephala had a little representation in the sample analyzed.
The geographical distribution and relative abundance of triatomine species captured at 11 GERES are shown in Figure 2. Details about the occurrence and natural infection of these species in each region are listed below.

GERES I -Recife
This GERES includes the Metropolitan Region of Recife and other 18 municipalities.Between 2007 and 2007, 16 municipalities did not send bugs to control quality in LABEND.Chagas disease vectors were found in two municipalities (Vitória and Pombos).By the time of this research, a total of 215 specimens of T. pseudomaculata and P. lutzi were examined with predominance of the first (Figure 2).In relation to the place of capture, 92.2% were found inside houses.Adults and nymphs of T. pseudomaculata were recorded, but only adult specimens of P. lutzi were captured.No triatomine was found infected in this GERES.

GERES II -Limoeiro
This GERES is composed of 31 municipalities located in the northern Zona da Mata, and 22 (71%) of them were positive for four species: Panstrongylus lutzi, T. pseudomaculata, T. melanocephala, and P. megistus.A total of 2.667 triatomines were examined.T. pseudomaculata was predominant in this GERES (Figure 2).The majority of the insects (96.7%) were found inside houses.Nymphs of all species were captured in this GERES, except T. melanocephala.Infected triatomines were found in 19 municipalities.

GERES III -Palmares
It is composed of 22 municipalities located in the southern Zona da Mata region.However, 18 municipalities did not send bugs to LABEND.Panstrongylus megistus was the main species among the 180 triatomines captured (Figure 2) in four municipalities (Quipapá, São Benedito do Sul, Belém de Maria e Lagoa dos Gatos) especially inside domiciles (87.3%)where adults and nymphs were found.No triatomine was found infected in this GERES.

GERES IV -Caruaru
This GERES is located in the northern Agreste region, and 30 out of 32 municipalities surveyed were positive for triatomines.Six triatomine species were detected, mainly P. lutzi (Figure 2).A total of 1,578 specimens were captured (92.4% of them inside houses).Nymphs, males, and females of P. lutzi, T. pseudomaculata, and T. brasiliensis were collected.For other species, only adult specimens were collected.By the time of the study, 293 triatomines were infected in 16 municipalities.

GERES V -Garanhuns
This GERES is composed of 21 municipalities located in the southern Agreste region, and 20 of them registered the presence of vectors.Four species were detected (Figure 2).A total of 1,171 specimens were captured (92.4% of them inside houses).Nymphs of T. pseudomaculata and P. lutzi were not registered.Infected triatomines were found in 18 municipalities.

GERES VI -Arcoverde
Triatoma pseudomaculata, T. brasiliensis, and P. lutzi were the most captured species in all the 13 municipalities that compose this GERES, located in the Sertão region.A total of 2,531 specimens were captured; 80.9% of them were found inside houses with predominance of T. brasiliensis (Figure 2).Nymphs of T. brasiliensis and T. pseudomaculata were captured.Infected bugs were detected in 10 municipalities.

GERES VII -Salgueiro
Triatomines were captured in all 7 municipalities that compose this GERES, which is located in the Sertão region.Triatoma brasiliensis predominated among the four species detected (Figure 2).Exceptionally, the presence of Rhodnius neglectus was detected (two non-infected males inside domicile).Most of the 992 (94.8%) triatomines captured were found inside domiciles.Infected bugs were detected in 6 municipalities.

GERES VIII -Petrolina
Triatomines were captured in 6 out of the 7 municipalities of this GERES located in the Sertão do São Francisco region.This region presented a different profile in relation to the site of capture; 65% of all triatomines were found in the peridomiciles, totaling 1,974 specimens with predominance of Triatoma brasiliensis (Figure 2).Another peculiarity of this region was the presence of two species not detected in other GERES during the development of this work (Rhodnius nasutus and Triatoma infestans).Infected bugs were detected in 3 municipalities.Triatoma infestans specimens were captured inside houses in one municipality (Lagoa Grande), but they were not infected.

GERES IX -Ouricuri
Triatomines were captured in all the 11 municipalities that compose this GERES, located in the Sertão region.Similar to GERES VIII, there was a predominance of triatomines in peridomiciles (79.2% of the 1,967 captured triatomines).Triatoma pseudomaculata (Figure 2) was the most captured species.As observed in GERES VIII, Triatoma sordida occurred rarely.Infected bugs were detected in 10 municipalities.

GERES X and GERES XI -Afogados da Ingazeira and Serra Talhada
Triatomines were captured in all the 22 municipalities that compose these GERESes, located in the Sertão region.Seven species were detected (Figure 2), and a total of 1,730 specimens were captured from both GERESes in 2006.In 2007, after the separation of these two GERESes, 1,929 triatomines were captured in Afogados da Ingazeira and 1,071 in Serra Talhada.T. brasiliensis predominated among the seven species found, and co-occurrence of R. neglectus and R. nasutus was detected in Afogados da Ingazeira (Figure 2).Most of the triatomines (82.9%) captured were found inside domiciles.Infected bugs were detected in 20 municipalities.

DISCUSSION
Pioneering studies on the epidemiology of Chagas disease in Pernambuco carried out in the 1950s 13 reported ecological aspects of seven species of triatomines: P. megistus, P. lutzi, Triatoma rubrofasciata (De Geer, 1773), T. pseudomaculata, T. sordida, T. brasiliensis, and T. melanocephala.Later, Psammolestes tertius Lent and Jurberg, 1965  and Triatoma infestans were recorded 14,21 .Triatomine surveys carried out in the 1970s and 1980s recorded three more species in the state: Triatoma tibiamaculata (Pinto, 1926), R. nasutus, and R. neglectus [15][16] .Dias et al. 7 included T. petrocchiae in the list of 13 recognized species of triatomines in the State of Pernambuco.Differently from what was observed by Dias et al. 7 , our study did not detect these 3 species: Psammolestes tertius, T. tibiamaculata, and T. rubrofasciata.The first species occurs frequently in bird nests of the family Furnariidae 22 with a wide distribution in the State of Pernambuco 23 .However, records of this species in domestic environments are rare, and although the species has already been experimentally infected with T. cruzi, natural infection is not frequent, due to a marked ornithophily.To our knowledge, T. tibiamaculata was recorded in Bonito (GERES IV) 16 , and the occurrence of T. rubrofasciata has been recorded since 1950s in Recife 13 .
As observed by Dias et al. 7 , T. brasiliensis and T. pseudomaculata remain as the main species caught in the State of Pernambuco.In the present study, these insects were often found inside houses, both in semi-arid Caatinga and in the Zona da Mata.Triatoma brasiliensis is considered the main vector of T. cruzi in northeastern Brazil 17 .In the wild environment, this species is often found under rocks, developing colonies associated with rodents (Kerodon rupestris) with high rates of infection with T. cruzi 24 .Our results showed that T. brasiliensis has a wide geographical distribution in the State of Pernambuco and it was one of the main species caught between 2006 and 2007.According to Costa et al. 25 , the State of Pernambuco includes hybrid zones of members of T. brasiliensis complex, and biological, morphological, ecological and genetic studies of populations of this complex should be developed in these areas.Unlike T. brasiliensis, specimens of T. pseudomaculata live under bark of dead trees and bird nests 26 .The species is most frequent in the peridomiciles, usually feeds on birds 27 and has lower rates of infestation, colonization, and infection 7 .However, Assis et al. 28 suggest that the increasing presence of T. pseudomaculata in household environments is related to climate change, deforestation, and expansion of agricultural areas.Moreover, the known ability of dispersal by flight guided by artificial light of T. pseudomaculata and T. brasiliensis 29 favors the invasion of households in the northeastern region of Brazil.
The epidemiological importance of P. lutzi is increasing in the State of Pernambuco.According to Lucena 13 , only seven adult specimens of P. lutzi were recorded among the 15,653 captured triatomines in the state in a survey conducted in the 1950s.The same author indicated a low synanthropic potential of this species, considering its rare presence in the households and even the absence of nymphs.According to Costa et al. 17 , P. lutzi was the fifth most common species by analyzing the number of triatomine captures in 12 Brazilian states in 1998 (2,900 specimens captured).Moreover, Silveira and Dias 30 indicated an increase in the number of P. lutzi specimens captured in Brazil, especially in the State of Pernambuco over the past 30 years.Our results showed that P. lutzi was the third most abundant species in Pernambuco, with a wide geographical . A total of 18,029 triatomines were analyzed from 138 municipalities of the state between 2006 and 2007.The 10 identified species were: Triatoma pseudomaculata, Triatoma brasiliensis, Panstrongylus lutzi (Neiva and Pinto, 1923), Panstrongylus megistus (Burmeister, 1835), Triatoma petrocchiae Pinto & Barreto, 1925, Triatoma melanocephala Neiva and Pinto, 1923, Triatoma sordida (Stål, 1859), Triatoma infestans, Rhodnius nasutus Stål, 1859 and Rhodnius neglectusLent, 1954.