Insect vectors of Chagas disease ( Trypanosoma cruzi ) in Northeastern Brazil

Introduction: Chagas disease remains a public health problem in the rural and urban areas of 19 countries in the Americas. Methods: The aim of the present study was to investigate the Trypanosoma cruzi infection rate of triatomines collected from both intraand peridomiciliary areas in eleven municipalities of Southeastern Ceará, Brazil, from 2009 to 2015. Results: A total of 32,364 triatomine specimens, including nymphs and adults, were collected, and 31,736 (98.06%) of these were examined. More nymphs were collected than adults, and the greatest number of triatomines (n = 8,548) was collected in 2010, for which the infection rate was 1.3%, with the highest rate of infections observed for specimens from Quixere. The species collected during the study were identified as Triatoma pseudomaculata, Triatoma brasiliensis, Panstrongylus megistus, Panstrongylus lutzi, and Rhodnius nasutus, with T. pseudomaculata being the most abundant (n = 19,962). Conclusions: These results verify the presence of triatomines in both intraand peridomiciliary areas, thereby ensuring persistence of the pathogen and consequently, the disease, as the presence of infected vectors in households is an important risk factor. According to these findings, the Chagas Disease Control Program should intensify its efforts in order to prevent the spread of the disease.


INTRODUCTION
Since its discovery by Carlos Chagas in 1909, Chagas disease, which is caused by the flagellated protozoan Trypanosoma cruzi and transmitted by hematophagous insect or triatomines, has been a public health problem in rural and urban areas of the 19 American countries colonized by Iberian empires, owing to the migration of its vectors 1 .It is estimated that 8 to 14 million people are infected worldwide 2 , especially in Latin America, and more than 100 million are at risk of contracting the disease.Previously, the disease was only found in the American continent.However, due to the intense migratory flows of Latin American populations, Chagas disease is now considered a global problem 3 .
Chagas disease is typically transmitted by vectors (80% of cases) when triatomines, while feeding on a host's blood, deposit feces that contain the metacyclic trypomastigote form of T. cruzi, which then penetrates the host's skin or mucous membranes.The second transmission route of epidemiological importance is through blood transfusion (5-20% of cases), which, along with international migration, has introduced Chagas disease to non-endemic countries, including Canada, the United States, European countries, and some Eastern Pacific countries 4 .With advances in vector control and transfusion monitoring, alternative routes, such as oral and transplacental transmission, laboratory accidents, animal handling, and organ transplants, have increased in relevance 5 .In recent years, the oral route transmission has increased in northern Brazil, owing to the ingestion of T. cruzi-infected foods, such as açai berry and sugarcane 6 .For example, 41 cases of Chagas disease were confirmed in the City of Belém, State of Pará, in 2011 (January to October, with 24 cases in October alone) 7 .
In Brazil, vector transmission occurs in all states of the northeast region and primarily depends on the presence of vectors in households 8 .The northeastern region of Brazil is considered endemic for Chagas disease and is second among the top regions infested by triatomines 9,10 , with the State of Bahia harboring the greatest diversity of triatomine species in the country 11,12 .
The rural area of the State of Ceará has caatinga-type vegetation, and precarious human dwellings, which facilitate the presence of several triatomine species that are important in the transmission of Chagas disease in Ceará: Triatoma brasiliensis, Triatoma pseudomaculata, Panstrongylus megistus, Panstrongylus lutzi, and Rhodnius nasutus 13 .These species are the most important in the Brazilian caatinga region, with a high invasive potential, and occupy a place in the domestic, peridomiciliary, and wild chain of Chagas disease 11,14 .
Since the first studies carried out by Alencar, Chagas disease has been highly prevalent in the State of Ceará (14.8%), especially in the municipality of Limoeiro do Norte 15 .Because of the high number of triatomines captured in Limoeiro do Norte by Vasconcelos in 2013 and the difficulty of controlling T. brasiliensis and T. pseudomaculata in this municipality 16 , we decided to assess the prevalence of Chagas disease (i.e., rates of infestation and triatomine infection) in Southeastern Ceará, where Limoeiro do Norte is located, as well as to identify host species (domiciliary, peridomiciliary, or wild) that contribute to the maintenance and dispersion of T. cruzi so that preventive action can be taken to control the disease.

Study design and sampling
This is a retrospective descriptive study that was performed using records from the Vector Control Center of Ceará [Núcleo de Controle de Vetores do Ceará (NUVET)], of the State Health Secretariat of Ceará State [Secretaria da Saúde do Estado do Ceará (SESA).The data were collected from 2009 to 2015 and included all the municipalities (11 towns) belonging to the 13 th Regional Health Coordination (CRES) in Southeastern Ceará: Alto Santo, Ererê, Iracema, Jaguaribara, Jaguaribe, Limoeiro do Norte, Pereiro, Potiretama, Quixeré, São João do Jaguaribe, and Tabuleiro do Norte (Figure 1A and Figure 1B).
During the study period, both nymph and adult triatomines were collected from intra-and peridomiciliary (chicken coops, barns, pigpens, cattle sheds) areas, and dead triatomines were excluded, as it is not possible to detect T. cruzi in dead vectors using the abdominal compression method.In this method, the material obtained from triatomine compression is placed on a microscope slide, along with a drop of 0.9% saline, covered with a coverslip, and then analyzed under an optical microscope.

Data analysis
In order to identify statistical associations between collection site and triatomine species, the data were analyzed using chisquare tests (p < 0.05) in GraphPadPrism 5.0.which allowed us to identify statistical associations between collection site and triatomine species.Proportions, infection rates, and infestation rates were estimated using simple percentage calculations.
In the present study, more nymphs were collected than adults, from both intra-and peridomiciliary areas, with 1,806 (8.2%) and 20,346 (91.9%) of the nymphs collected from intra-and peridomiciliary areas.More specimens were also collected from peridomiciliary areas (n = 29,209; 90.3%) than intradomiciliary areas.
There was a statistical association between the occurrence of T. pseudomaculata and peridomiciliary areas, whereby the proportions of T. pseudomaculata were higher than those of the other species in peridomiciliary areas, and a similar association was noted for T. brasiliensis in intradomiciliary areas (p < 0.001; Table 3 and Table 4).

Infection rate
A total of 432 triatomines tested positive for T. cruzi infection, indicating an infection rate of 1.4% (Table 2).The greatest number of infected triatomines (n = 144; 2.7%) was observed in 2009, with more adults being infected than nymphs, and during the same year (i.e., 2009), the greatest infection rate was observed in specimens from Limoeiro do Norte (7.6%), followed by the specimens from Quixeré (3.5%) and Tabuleiro do Norte (3.2%).Specimens from Limoeiro do Norte also yielded the greatest number of infected specimens throughout the entire study period (n = 205), indicating an infection rate of 5.3% (Table 2).

Triatomine collection
From 1975 to 1980, a national investigation was carried out, during which 15,342 triatomine specimens were collected from northeastern Brazil, and in 1996, the Chagas Disease     16 .
These findings indicate, according to WHO entomological indicators, that triatomine colonization is still occurring and reinforce statements by Coura, in 2008, and Coura and Viñas, in 2010, that man, through burning and other changes to natural triatomine habitat, has forced these insects to adapt to manmade environments.Coura and Borges-Pereira, in 2012, stated that the adaptation of triatomines to household environments might be the most concerning matter of Chagas disease transmission 4,18,19 .
In the present study, T. pseudomaculata was the most frequently collected species in all the municipalities, except Quixeré, in which T. brasiliensis predominated.These results differ from those reported by Sarquis et al. 17 , who collected 3,082 triatomines in Jaguaruana, Ceará, from 2000 to 2002, with 2,307 (74.9%) identified as T. brasiliensis and 229 (7.4%) identified as T. pseudomaculata specimens, but corroborate the results of Vasconcelos et al., who also collected a higher proportion of T. pseudomaculata specimens, Limoeiro do Norte from 2006 to 2009 16 .A notable aspect of T. pseudomaculata is that is a low-efficacy vector, in that only 10% of T. cruzi within the species are able to develop into the infectious metacyclic form 19 .Therefore, the risk that these specimens contribute to the transmission of Chagas disease is relatively low.Nevertheless, T. pseudomaculata have been reported to colonize dwellings in Berilo, Minas Gerais 20 , and in the urban area of Sobral, Ceará 21 .Because the species is well-adapted to high temperatures, it is usually found in roofs and other parts of buildings that receive generous amount of sun 22 , thereby making such sites more significant in terms of T. cruzi transmission 23 .Chagas disease has been endemic to Ceará since the 1940s, as reported by Alencar et al. 24 .Lima et al. 25 collected 829 R. nasutus specimens from palm trees in the rural and peri-urban areas of Jaguaruana, Ceará, and Dias et al. 26 reported that palm trees are the natural habitat of Rhodnius spp.and that microclimate plays an important role in the interaction of Rhodnius spp.and palm trees.However, in the present study, 417 R. nasutus specimens were collected from homes, rather than from palm trees.
Freitas et al. 27 reported that R. nasutus specimens occur in households more frequently during the period when carnauba trees are pruned and suggested that anthropogenic environmental degradation contributes to the domiciliation of triatomines and facilitates vector transmission of Chagas disease in the municipality of Limoeiro do Norte.
A study carried out in the municipality of Russas, Ceará, from 2008 to 2009, reported a high (28.6%)prevalence of T. cruzi infection among 761 triatomine specimens, mostly Triatoma brasiliensis 22 , and Silva et al. 28 reported an infection rate of 8.8% for triatomines collected from 113 municipalities in the State of Pernambuco, most inside households, in 2006 and 2007, with P. lutzi exhibiting the highest rates of natural infection.Therefore, we recommend that Chagas disease surveillance and vector control should be improved in the State of Pernambuco.
The gradual reduction in specimens collected by NUVET from 2009 to 2015 may have been influenced by control program success, improvement of housing, and improvement of the sanitary habits of the population.Otherwise, it may be the result of worsening triatomine control programs, owing to reduced funding, as investments in control programs for other emerging diseases, such as dengue fever and Zika virus, have increased.Together, these results indicate that efforts to educate populations in recognizing triatomines and notifying health authorities should be extended and reinforced in order to ensure that the disease is controlled in places where it is still found.

Infection rate
Even though the infection rate of T. brasiliensis was relatively low (0.8%) in the present study, Sarquis et al. 17 reported an infection rate of 15.3% for the species, and many other studies have reported T. brasiliensis as the most frequently collected and infected species 29,30 .Therefore, T. brasiliensis likely plays an important role in the transmission of Chagas disease in the northeast region and, especially, in the State of Ceará.Meanwhile, the infection rate of R. nasutus (24.3%) in the present study was similar to the rate reported by Vasconcelos et al. 16  (19.4%).

Food eclecticism
In the present study, P. megistus was the least frequently collected species, with only 50 specimens collected in total.This is in contrast to the findings of Villela et al. who reported that P. megistus was the most frequently collected species in the State of Minas Gerais, with a total of 1,380 specimens (99.3% of all triatomines) collected from 2003 to 2007.However, this finding was expected as the species predominates triatomine communities in midwestern, eastern, and southeastern Brazil 31,32 .In addition, some authors have reported that P. megistus exhibits food eclecticism, feeding on the blood of birds, humans, cats, and other animals, with human blood being the second most frequently observed in their digestive tracts 33,34 .
Moreover, 60-70% of T. cruzi in P. megistus are able to transform into the form that infects humans and other vertebrates 19 .Therefore, according to Perlowagora-Szumlewicz 34 , who reported that metacyclogenesis is one of the most important factors in disease transmission, this species may pose a risk of Chagas disease transmission, even though it was relatively infrequent.
Similarly, P. lutzi was also relatively infrequent but is still a concern for Chagas disease transmission as the species food eclecticism 32,35 , a factor in the transmission of Chagas disease, and because it is considered one of the most important secondary species for Chagas disease maintenance, owing to its high rates of natural infection and great capacity for household invasion, as it can fly into dwellings.This species also exhibited the highest rates of infection in the present study.
Considering all the observations and limitations of the present study, it can be concluded that the municipalities of Southeastern Ceará need to intensify their surveillance efforts, in order to prevent the colonization of intra-and peridomiciliary areas by these species, and because nymphs were more prominent than adults, suggesting domiciliation and a higher risk of infection for the exposed population, this is especially important for households.
Although the infection rates observed in the present study were relatively low, when compared to previous studies, the infestation rate of each municipality was quite high, which confirms that Chagas disease is still a matter of concern.
Therefore, considering that the presence of infected vectors in the household is an important risk factor for the transmission of Chagas disease, the Chagas Disease Control Program should intensify its efforts in order to prevent the spread of the disease.
The involvement of the populace is also crucial for establishing and maintaining permanent epidemiological surveillance.

1 :
Frequency of triatomines collected in Southeastern Ceará, Brazil, from 2009 to 2015.data was not collected.Source: Vector Control Center of Ceará.

TABLE 3 :
: number of vectors examined.No. infect.(%): Number of positive results (infection rate).NA: data was not collected.Source: Vector Control Center of Ceará.Frequency of triatomines collected in southeastern Ceará, Brazil, from 2009 to 2011.

TABLE 4 :
Frequency of triatomines collected in southeastern Ceará, Brazil, from 2012 to 2015.Number of adults (% total number of individuals).N (%): Number of nymphs (% total number of individuals).Source: Vector Control Center of Ceará.

TABLE 2 :
Frequency of examined and infected triatomines collected in southeastern Ceará, Brazil, from 2009 to 2015.