Triatominium search and diagnosis of trypanosoma cruzi in vectors captured in cane calk originating from sugarcane industry

The objective of the study was to survey triatomines and diagnose Trypanosoma cruzi in captured vectors and sugarcane juice from sugarcane mills. 100% (3/3) of the active sugar cane mills in the study area were surveyed. An active and passive search of the vectors and research of T. cruzi in the feces of engorged triatomines by compression of the abdomen and for the juice in natura by sedimentation with Giemsa staining technique were carried out, where five samples per mill were collected, totaling 15 researched. As a result, in 100% (3/3) of the mills, 22 hematophagous triatomines were captured, with 41% (9/22) by passive search and 59% (13/22) by active search. 81.8% (18/22) were found in the household environment and 18.2% (04/22) in the intra-household environment of the mills. Two species were Research, Society and Development, v. 10, n. 11, e96101119398, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i11.19398 2 identified being 68.18% (15/22) Triatoma brasiliensis and 9.09% (02/22) Triatoma pseudomaculata, with one adult and the four nymphs unable to be identified. T. cruzi diagnosis for triatomines was observed 4.54% (1/22) positive and 95.45% (21/22) negative, while broth, 100% (15/15) negative. However in 33.3% (5/15) broth samples the presence of soiling (microplastics) was observed. Demonstrating the importance of investigation and monitoring of milling sites with the possibility of oral transmission, which currently represent the majority of infection records in Brazil.


Introduction
Chagas Disease caused by the protozoan Trypanosoma cruzi can be considered one of the most important pathologies in America (Santana et al. 2019), with millions of people infected, mainly in Latin America (Briceño-León et al. 2007). In Brazil, when related to chronic cases the disease presents three million people affected, and increase in acute cases of pathology by ingestion of contaminated food (Brsil, 2019).
The oral infection route has configured the main form of transmission and a serious public health problem (Menezes, et al. 2019), due to the increase in the rates of the disease from 129 cases (2012) to 298 cases (2016) (Brasil, 2019). Among the foods, the contamination of the pulp or juice of açai (Euterpe oleracea) stands out, which during the period from 2007 to 2016 had an increase of 99.57%. Associated with the contamination of sugarcane juice, patuá (Oenocarpus bataua), buriti (Mauritia flexuosa) and bacaba (Oenocarpus bacaba) (Ferreira, et al. 2018).
In 2016, the Secretariat of Public Health of the State of Rio Grande do Norte (SESAP-RN) conducted an investigation in the municipalities of Alexandria, Marcelino Vieira, Pilões and Tenente Ananias, in order to confirm and notify cases associated with an outbreak of Chagas disease. According to SESAP-RN, the cases were mainly related to the ingestion of Research, Society andDevelopment, v. 10, n. 11, e96101119398, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i11.19398 3 sugarcane juice during the milling periods in the aforementioned region, making them risk areas (SESAP-RN, 2020). Thus, the objective of the study was to survey triatomines and diagnose T. cruzi in captured vectors and sugarcane juice from sugarcane mills.

Methodology
During a one-year period, a survey was conducted in the rural area of the municipality of Marcelino Vieira, Rio Grande do Norte (RN), in 100% (3/3) of the active sugarcane mills in the study area. This is considered one of the main local economic activities.
In the passive search, light traps were used and installed on the mills between 5:00 pm and 5:30 am for three consecutive days each month. The active search was carried out by means of a thorough inspection in all areas of the mills. In the intra-household, crevices or frechas, storage space, production and disposal of sugarcane bagasse were searched. In the peridomestic areas animal nests, bark and hollow tree trunks (Galvão et al, 2014). And when present inspection in piles of tiles and bricks, stone and wood fences (Jurber et al, 2014). (Lent Et Al. 1979). The investigation of T. cruzi positivity in the feces of engorged triatomines was performed by compression of the abdomen and fresh examination. Associated with the dissection of the beetle for definitive verification with analysis of the digestive tract, examining it under an optical microscope with the duodenum macerated with two drops of saline solution (Magallón-Gastélum et al, 1998).

The identification of hematophagous vectors was performed according to the classification of Lent & Wigodzinski
For the parasitological analyses of the sugarcane juice, five samples per mill were collected, totaling 15 broths surveyed. Two analyses were performed for each sample. The first was performed using the conventional spontaneous sedimentation method (Lutz, 1919) where 200 ml of the juice was homogenized with 50 ml of distilled water, sieved and placed in goblets for sedimentation. The second was performed according to Pinto et al. (1990) methodology where 2.0 ml of broth was measured and transferred to falcon tubes and then centrifuged (1500 rpm/3 minutes). The sediment was smeared and fixed in methanol for 3 minutes and stained with Giemsa for 30 minutes with subsequent visualization under a microscope (Pinto et al, 1990).

Discussion
Hematophagous triatomines were captured in all active mills, totaling 22 vectors. The diagnosis is related to the presence of the mills near the natural habitat of these insects. Where infestation can occur accidentally during the process of harvesting, storage, transport or processing of sugarcane (De Mattos et al., 2019).
In the search for triatomines, 18.2% (04/22) were found in the interior of the mill. The lack of maintenance in the structure of this environment can cause the presence of fissures in the walls, which makes the intra-household conducive to habitat similar to wild rock shelters (Lorenzo et al., 2000).
Among the species collected, T. brasiliensis stood out. It is a common vector in the Northeastern states, with a predominant characteristic, potentially invasive and difficult to control (Dias et al., 2000). They are found in the peridomicile of places near the forest and in the intradomicile attracted by light sources (Neves et al, 2016). While the other species identified was T. pseudomaculata, it stands out for its ease of adapting to high temperature environments, such as the semi-arid region of the Northeast (Argolo et al, 2008). In the mills, it may have been diagnosed by various factors such as the presence of hiding places between the storage sites of sugarcane, arranged on the ground in all the mills surveyed.
As for the positivity of the protozoan T. cruzi, it was diagnosed in 4.54% of the captured vectors. The vector presence in mills, production and distribution of broths causes a public health alert by the possibility of food contamination associated mainly with the hygienic-sanitary conditions of the environment (De Mattos et al, 2019). However, in the analyzed fresh broth, 100% of the samples were negative for T. cruzi parasite diagnosis.
In 33.3% of the sugarcane juice samples the presence of dirt (microplastics) was observed. Among the vulnerable situations found for food contamination, the hygienic-sanitary conditions of the production environments stood out, such as the absence of individual protection equipment and the manipulation of money and food at the same time. Research conducted with sugarcane juice produced by vendors, it was observed low hygienic-sanitary quality, where there was great precariousness of information and procedures, thus generating high rates of contaminants present (Prado et al, 2010).
With this, the National Health Surveillance Agency (ANVISA), constitutionalized the procedures for obtaining this broth, in order to avoid food contaminations in general, proposed and put into effect resolutions for food handling (Brasil, 2020).

Conclusion
The development of modern traps for vector research was relevant, with capture and positivity of hematophagous triatomines associated with the presence of dirt in the broth. This demonstrates the importance of investigating and monitoringsites with the possibility of oral transmission, which currently represent the majority of infection records in Brazil.