Study on Chagas disease occurrence in the municipality of Monte Negro , State of Rondônia , Brazilian Amazon

Introduction: Studies on Chagas disease deal with the perspective of its occurrence in the Amazon region, which is directly correlated to the population growth and the spread of the bug biotope. The state of Rondônia has an immense source of vectors (Triatomine) and reservoirs of Trypanosoma cruzi. Environmental changes brought forth by the deforestation in the region may cause vector behavior changes and bring these vectors to a closer contact with humans, increasing the probability of vector infection. Methods: This study was carried out to check the occurrence of Chagas disease in the municipality of Monte Negro, Rondônia, Brazil, based on a random sampling of the farms and people wherein blood collection from the population and capturing triatomines were done. The blood samples were submitted to serologic tests to detect antibodies of the IgG class against T. cruzi. The triatomines that were collected had their digestive tract checked for the presence of trypanosomatidae with morphology resembling that of the T. cruzi. Results: The population examined was mostly from other states. From the 322 bugs examined on the microscope, 50% showed parasites with morphology compatible with T. cruzi. From the serology of 344 random samples of human blood, 1.2% was found positive, 6% showed inconclusive results, and 92.8% were negative. Conclusions: Monte Negro shows low prevalence of human infection by T. cruzi and none active vector transmission; however, preventive and surveying measures, which are not performed until now, shall be taken due to the abundance of vectors infected by trypanosomatidae.

Chagas disease is endemic in the American continent, and it is more prevalent in South America.In Brazil, this parasitosis has contaminated about 5 million people, while 20 million are at risk of being infected [1][2][3] .Typically, Chagas disease occurs in people who live in rural areas and in poor quality houses where the vector insects can easily lodge and remain out of sight.The infection in humans may be very severe.It may cause a significant mortality rate among children in its acute phase and seriously compromise the heart and/or the digestive tract in adults with chronic disease 4 .
Triatomines are hematophagous insects that transmit Trypanosoma cruzi by the time they have their blood meal when they excrete fecal matter and urine containing parasites over the mucosa or in small lesions in the skin or by contaminating food with their feces.Other mechanisms of transmission are vertical, accidental transmission, by blood transfusion, and organ transplantation [5][6] .Most of these insects are sylvatic and are associated with a broad variety of habitats and vertebrate hosts.Some of them may be adapted to peridomestic and domestic habitats [5][6] .
The epidemiology of Chagas disease in the Amazon region is still being understood in a superficial and fragmental way.Despite the broad Amazon biodiversity, with the presence of diverse triatomines and many mammalian classes that may host T. cruzi, this region has been spared from the disease.Today, some works have considered the disease as emerging around Brazilian Amazon, with some autochthonous cases and some triatomines being reported around the dwellings 2,7-10 .
Up to now, there are no ongoing preventive programs and/or specific disease control measures being implemented in Amazon.A recent research carried out in Monte Negro by the investigators of the V Biomedical Science Institute of the University of São Paulo (ICB5/USP) in the past years showed a large occurrence of infected triatomines (on palms) around the rural area of the municipality and an indication of none vector transmission 9 in a small area of the municipality.
The study was carried out in the municipality of Monte Negro (S 10º 15'35" e W 63º 18'06") in the State of Rondônia, which has a population of about 14,010 inhabitants, stretches over an area of 1,413.4km 2 , and is located 250km away from the state capital, the city of Porto Velho 11 .The weather is equatorial and super humid, with an annual rain precipitation of 2.020mm (1,800-2,200mm), an average temperature of 25.8°C ranging from 12°C to 37°C, and air humidity ranging between 70% to 80% over most part of the year 9,12 .There are considerable anthropogenic activities along the BR421 road, which crosses the municipality and its vicinal districts.The original forest was replaced mainly by coffee plantations and pastures.
In this study, 100 sampling farms were randomly chosen out of the 1,093 existing farms located along the vicinal roads of the municipality (Figure 1), and 344 people out of a total of 398 inhabitants in these randomly sampled farms were included.Two visits were done in each farm with the aim of catching bugs inside the dwelling and around them, and dissection of two specimens of babassus (Orbginya speciosa) for each visit, besides the collection of blood samples from the inhabitants, was carried out.These visits were done during the rainy season and during the dry season to determine the influence of the rain precipitation on the diversity of the triatomine species (Figure 1).

Capture of triatomines
With the owners' authorization, domestic and peridomestic inspections were carried out for manually catching the bugs without using any dislodging chemical product.In addition to the manual search, samples of triatomines and collecting kits (gloves, tweezers, and collecting bottle) were given to the general population and to every farm owner to catch and keep any suspicious specimen in the absence of the researchers for future identification.
Four babassus samples were dissected at each farm, two for each visit with the aim of finding bugs.The distance of each babassu being dissected from the dwelling house was measured by a GPS (Global Positioning System) device.
The existing fauna around the dwellings, peridomestic areas and facilities, and babassus was described, along with the information on rain precipitation.The insects captured were placed in collecting bottles, labeled, and taken to the laboratory where they were examined and randomly split into two portions -one to be checked for infection by trypanosomatidae specimens and another to be forwarded to the Biologic Sciences Department of the Pharmaceutical Sciences College of Araraquara for morphological identification of the species based on the key elaborated by Lent & Wygodzinsky 13 .The nymph stage was identified based on the works of Correa et al. 14

RESULTS
The triatomines selected for infection analysis were anaesthetized with ethyl acetate, rinsed with a 70% ethanol solution, and had their terminal digestive tube dissected, from which the smears were prepared on slides diluted in saline solution.The checkout of the slides for trypanosomatidae presence was carried out by optical microscope examination (400X).
The approach of human beings After signing the written consent form, the inhabitants of the visited farms were subjected to medical examinations and blood collection where a physician filled out the epidemiologic clinical cards of the patients.
Serologic exams A 10mL blood sample was collected from each inhabitant, which was then centrifuged for 8min at 2,500rpm.The blood serum was separated into 3 portions of 1mL each and then frozen at -20°C.A portion of each person's sample was forwarded to the laboratory of the Federal University of Goiás for checking of anti-T.cruzi antibodies of the IgG class by the ELISA method and the Indirect Immunofluorescence method.For the ELISA test, a Chagatest ELISA Kit (Wiener®) and a PaGIA DiaMed Kit (DiaMed®) were used.For the immunofluorescence, an anti-IgG Human globulin was used, which was conjugated with fluorescein (Biomerieux®).Evans Blue (Bioshop®) and T. cruzi microscope slides (Y strain prepared from LIT cultures in house) were used.For ELISA, the cutoff test optical density ratio was considered as negative when it was below 0.9, undetermined when it was between 0.9 and 1.1, and positive for values above 1.1.For the immunofluorescence, the positive values were considered when the titles were above 1/20.

Statistical analysis and data storage
The information found was added to a databank in Microsoft Access 2.0.The statistical analysis of the epidemiologic data was carried out by using Epilnfo 6.04 b and Excel 2007 software.Kolmogorov-Smirnov test with Lilliefors correction, Chi-square test, Fisher's exact test, and Wilcoxon-Mann-Whitney U test were performed.In all cases, a significance level of 5% was considered (α=0.05).

Property, dwelling, and population profiles
The properties visited had an average colonization time of 16.3 years, ranging from 2 months to 38 years.The areas of the properties ranged from 33 to 231ha, and the inhabitants were engaged mostly in the cattle farming business.Most of the dwellings had a uniform shape, while the walls were made of wood and bricks, and the roofs were covered by ceramic tiles or cement asbestos roofs.The peridomestic improvements (cattle-shed, granary, hennery, and pigsty) were usually made of wood and covered with cement asbestos roofs or ceramic tiles (95%), with very few exceptions, covered with wood of babassus tree feathery leaves.
The population studied was mostly people who were from other states (57%).The average age was 34.2 years, ranging between 2 and 78 years.

Triatomine quest
Eight hundred fifty-three triatomines were collected; 5 were found inside the dwellings and 848 on the babassu (around 1 triatomine/babassu).No bug colony was found inside the dwellings and around their peridomestic area, which indicated that there were no bug lodging occurrences in the region researched.
Most of the triatomine specimens collected in this study were found on babassus (Orbignya speciosa) (99.4%), and the others were not lodged in the dwellings.All the dissected babassus had an average distance from the dwelling of 234.7m.The shortest distance was 76.6m, while the longest was 392.8m.
The relation between the quantity of triatomines collected and the distance the babassus were found from the dwelling was confirmed by the Chi-square statistical test, where χ2=15.3 and p=0.0321, that is, the closer to the dwelling, the higher is the amount of bugs found on each babassu.Also noticed was the clear relationship between the presence of mammalians and/or birds found on babassus and the presence of collected bugs, as the mammalians and birds are different feeding sources for triatomines and live on the babassu.The Fisher's exact test confirmed such a relation, where p=0.0000000000.
By using the Kolmogorov-Smirnov test with Lilliefors correction, it was found that the distribution of a variable quantity of triatomines for the samples collected over the dry and rainy seasons was not regularly distributed, p<0.01.To compare the two populations, the Wilcoxon-Mann-Whitney U test was used.It was found that there was no statistically significant difference between the rainy season and the dry season, U=4,799.5 and p=0.851.
Out of the 853 bugs collected, 652 specimens were selected for the analysis, and the others were discarded.Others were discarded due to the death of the insects, which impaired their morphology.
The triatomines analyzed showed the predominance of specimens in nymph stage (63.2%), and 36.8% were adult insects, out of which 46.6% were male.
The triatomine identification showed a uniform outcome in which all the specimens were identified as Rhodnius robustus.
Three hundred twenty-two triatomines were microscopically analyzed for trypanosomatidae specimens with resembling T. cruzi morphology in their digestive tract, and 161 (50%) triatomines were found to be positive.Among all locations studied, the one found with the highest average number of triatomines positive for trypanosomatidae was the LC-40 locality, where 87.5% of the insects analyzed were found positive.There were four locations where no bug was found to be positive in localities LC-0, LC-52, LC-12,5, and TB-24.
and Rosa et al.15
This study has been submitted and approved by the Research Ethical Commission on Human Beings of the College Sao Lucas (Letter AP/CEP/173/08) (Originally, Comitê de Ética em Pesquisa em Seres Humanos da Faculdade São Lucas -Carta AP/CEP/173/08 in Portuguese).The cutting down of palms was authorized by the State Department of Environmental Development (Originally, Secretaria de Estado do Desenvolvimento Ambiental -SEDAM), with Special Authorization nr.195/2007, and by the Renewable Resources -IBAMA (Licence nr.05/2007, process nr.02024.001826/2006-47).