A. actinomycetemcomitans profile and red complex bacterial species of an Afro-Brazilian community: A comparative study

archives of oral biology 60 (2015) 753–759

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A. actinomycetemcomitans profile and red complex bacterial species of an Afro-Brazilian community: A comparative study Murilo de Araujo Neris a, Sheila Cavalca Cortelli b, Davi Romeiro Aquino b, Taı´s Browne de Miranda c, Fernando de Oliveira Costa d, Jose´ Roberto Cortelli b,* a

Dental School, Department of Periodontology, University of Taubate´, Rua Expediciona´rio Ernesto Pereira, 110 – Centro, Taubate´, SP, Brazil b Dental School, Nucleus of Periodontal Research, University of Taubate´, Rua Expediciona´rio Ernesto Pereira, 110 – Centro, Taubate´, SP, Brazil c Department of Biology, University of Taubate´, Rua Expediciona´rio Ernesto Pereira, 110 – Centro, Taubate´, SP, Brazil d Periodontal Department, Dental School, Federal University of Minas Gerais, Av. Antonio Carlos, 6.627 – Pampulha, CEP: 31270-901 Belo Horizonte, Minas Gerais, Brazil

article info

abstract

Article history:

Purposes: The primary aim of this cross-sectional study was to compare the levels of red

Accepted 31 January 2015

complex bacteria between Afro-Brazilian and non Afro-Brazilian cohort. The secondary aim was to compare the distribution of both Aggregatibacter actinomycetemcomitans serotype b and

Keywords:

its JP2 strains among participants who harboured this bacterial species.

Periodontitis

Methods: A total of 84 individuals were included in this study: 42 Afro-descendants (mean

Ethnic groups

age 35.9  13.1 years) and 42 non-Afro-descendants (mean age 36.2  13.1 years) matched

Serotype

(1:1) by periodontal diagnosis, age and gender. All participants received clinical examina-

Leucotoxin

tions of periodontal pocket depth, clinical attachment level, and plaque and gingival indices.

Bacteria

Subgingival samples were taken for microbial analysis. First, genomic DNA (gDNA) was extracted and purified and the quantification of total number of bacterial cells, A. actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola was carried out by qPCR. Then, A. actinomycetemcomitans strains were classified according to serotype b and JP2 profiles by conventional PCR. Results: Clinically, mean PD, mean CAL and percentage of CAL  3 mm differed between groups (Student’s t-test p < 0.05). The levels of red complex bacteria between Afro-Brazilian and non-Afro-Brazilian populations were similar. The exception was verified to A. actinomycetemcomitans showing significantly higher levels among Afro-Brazilian descendants in comparison to non-Afro-Brazilian descendants. Afro-Brazilian descendants were clearly infected by more virulent serotype b and JP2 strains. Conclusions: Despite no statistically significant differences related to the red complex species, Afro-Brazilian descendants harboured higher levels of A. actinomycetemcomitans.

* Corresponding author at: Rua Expediciona´rio Ernesto Pereira, 110 – Centro, 12020-330 Taubate´, SP, Brazil. Tel.: +55 12 30250058; fax: +55 12 30250058. E-mail address: [email protected] (J.R. Cortelli). http://dx.doi.org/10.1016/j.archoralbio.2015.01.013 0003–9969/# 2015 Elsevier Ltd. All rights reserved.

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Also, our findings confirm that Afro-descendant populations are preferably colonised by A. actinomycetemcomitans serotype b as well as JP2 strains. # 2015 Elsevier Ltd. All rights reserved.

1.

Introduction

Periodontal disease is one of the most common infectious diseases affecting humans. The microbial aetiology of periodontal disease has been the focus of researchers for a long time. So far, more than 700 species of bacteria have been detected in the oral microflora, either by cultivation or by DNA-based procedures.1 On the other hand, it is clear that not all subjects harbour the same supra and subgingival bacteria, and that a limited number of subgingival species, including Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola, have been recognised as indicators for the progression of periodontal destruction.2,3 A. actinomycetemcomitans is a nonmotile, gram negative, facultatively anaerobic rod that has been implicated in the aetiology and pathogenesis of aggressive4 and chronic periodontitis.5 In addition, a second group of bacteria, P. gingivalis, T. forsythia, and T. denticola, categorised as the red complex bacterial taxa by Socransky et al.6 has been mainly associated with severe forms of periodontal disease. Traditionally, epidemiological data regarding levels of oral diseases are based on national samples. Due to the limited number of Afro-descended populations in most of these studies, analysis regarding blacks is often restricted to overall descriptions of major oral diseases and conditions and oral hygiene practices, rather than descriptions of the diversity within Afro-descendent populations.7 Several studies have examined the prevalence of periodontal pathogens in populations in developed and developing countries (as detailed in Rylev and Kilian8). Our group has previously reported two studies including Afro-descendent populations from Brazil. In the first study, conducted in Bahia,9 a state in the northeast of the country, we investigated the presence of P. gingivalis, A. actinomnycetemcomitans, E. corrodens and F. nucleatum in urban young adult subjects. The main results of Vitor’s study9 were a high percentage (96.96%) of subjects harbouring at least one selected periodontal pathogen, despite the fact that most of them showed a healthy periodontal status. In the second study, conducted by Bonifacio et al.,10 we examined the prevalence of periodontopathogens in a secluded black Brazilian community compared to a black urban population. In this particular study, the results showed that except for Campylobacter rectus, all pathogens were present in both groups with no statistically significant difference. C. rectus was more prevalent only in gingivitis patients from the black urban area. We also verified a high frequency of periodontopathogens related to the severity of periodontal disease. Taking into consideration these previous results, it was assumed that African ethnic ancestry would not affect bacterial profile regarding key periodontal pathogens. To

address this question, in the present study a microbial comparative analysis was conducted between Afro-Brazilian and non-Afro-Brazilian descendants considering oral levels of A. actinomycetemcomitans, P. gingivalis, T. forsythia and T. denticola. In addition, occurrence of more virulent A. actinomycetemcomitans strains was also compared.

2.

Objectives

The first aim of this cross-sectional study was to compare the levels of selected periodontal pathogens between an AfroBrazilian and a non-Afro-Brazilian cohort. The second aim was to compare the distribution of both A. actinomycetemcomitans serotype b and JP2 strains among participants who harboured this bacterial species.

3.

Material and methods

3.1.

Subjects and clinical exams

Participants included in the present study were recruited in Brazil’s Bahia state (Afro-Brazilian descendants-AD) and Sa˜o Paulo state (non-Afro-Brazilian descendants-NAD) from March 2012 to February 2014. The study population was defined as a convenience sample, because all the selected patients were individuals who sought general dental treatment at a Public Health Center (Bahia) or at the University of Taubate’s Department of Dentistry in Taubate, Sa˜o Paulo, Brazil. First, the AD study population was allocated; then we matched with the NAD study population (1:1) by periodontal diagnosis (chronic periodontitis), age and gender. Data and personal information on the medical and dental histories were obtained by interview. All subjects signed an informed consent form, which was previously approved by the Institutional Committee on Research Involving Human Subjects of the University of Taubate (protocols 304-532/13 and 521/10). Clinical measurements and microbial sample collection in Bahia were performed by one trained and calibrated examiner (MAN), while the clinical measurements and microbial sample collection in Sa˜o Paulo were performed by another examiner (JRC), both of whom were first trained and calibrated at the Department of Periodontology-Nuper at the University of Taubate. The agreement between examiners was high (kappa = 0.83 for PD and 0.81 for CAL). Clinical measurements were used to establish the periodontal condition. Periodontal probing depth (PD), clinical attachment level (CAL), plaque index11 (PI) and gingival index12 (GI) were taken from all teeth, except for third molars, with the help of a manual periodontal probe (PCPUNC 15

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Hu-friedy Mfg Co. Inc. Chicago, IL, USA). A diagnosis of periodontal conditions followed the criteria defined by the American Academy of Periodontology.13

3.2.

Included and excluded criteria

To be included in the study the subjects had to be in good general medical health and had received chronic periodontitis diagnosed, both genders and at least 18 years of age. Individuals were excluded if they had the following conditions: (I) any condition requiring antibiotic prophylaxis for dental exam, (II) uncontrolled systemic diseases, (III) immunological compromise, (IV) pregnancy or women currently breast-feeding, (V) periodontal treatment 12 months before the beginning of the study, (VI) antibiotic treatment 3 months prior to the clinical and microbial examination and (VII) diagnosis of aggressive periodontitis or other type of periodontal diseases than chronic periodontitis.13

3.3.

Table 1 – Primer and probe sets used in the qPCR reactions to bacterial analysis. Bacterial speciesa

Primers/probe – sequence (50 –30 )

A. actinomycetemcomitans

F-CAA GTC TGA TTA GGT AGT TGG TGG G R-TTC ATT CAC GCG GCA TGG C P-6FAMATC GCT AGC TGG TCT GAG AGG ATG GCCTAMRA F-CCG AAT GTG CTC ATT TAC ATA AAG GT R-GAT ACC CAT CGT TGC CTT GGT P-6FAMATG GGC CCG CGT CCC ATT AGC TAMRA F-ACC TTA CCC GGG ATT GAA ATG R-CAA CCA TGC AGC ACC TAC ATA GAA P-VICATG ACT GAT GGT GAA AAC CGT CTT CCC TTC TAMRA F-AGC GAT GGT AGC AAT ACC TGT C R-TTC GCC GGG TTA TCC CTC P-6FAMCAC GGG TGA GTA ACGTAMRA F-TGG AGC ATG TGG TTT AAT TCG A R-TGC GGG ACT TAA CCC AAC A P-VICCAC GAG CTG ACG ACA AGC CAT GCATAMRA

T. denticola

P. gingivalis

T. forsythia

Sampling for microbial analysis Universal

Subgingival samples were collected from mesio-buccal aspect of the four teeth showing more evidence of periodontal disease (preferably showing higher values of probing depth, clinical attachment loss and gingival inflammation) using sterile paper points inserted into the depth of the pocket after the removal of supragingival plaque using sterile curettes. Sixty seconds after placement in the pocket, paper points were immediately inserted in a microtube and kept on ice. The bacterial cells in the microtubes were dispersed using a vortex mixer at maximum speed for 1 min, and the resulting bacterial suspension was saved in a freezer at 80 8C until laboratory processing.

3.4.

Polymerase chain reaction (PCR)

3.4.1.

A. actinomycetemcomitans (JP2 strain)

To identify JP2 strains, conventional PCR was performed on the extracted DNA using two oligonucleotide primers directed toward the A. actinomycetemcomitans leucotoxin promoter region (see details in Cortelli et al.14).

3.4.2.

A. actinomycetemcomitans (serotype b)

Serotyping was carried out by conventional PCR using a primer specific for serotype b (see details in Cortelli et al.15).

3.5.

Real-time quantitative PCR – qPCR

Genomic DNA (gDNA) was extracted and purified from the pellet using a PureLink1 Genomic DNA Mini Kit (Life Technologies, Carlsbad, CA, USA) according to manufacturer’s specifications. The quantification of the total number of bacterial cells, A. actinomycetemcomitans, P. gingivalis, T. forsythia and T. denticola was determined by qPCR using TaqMan assay (TaqMan1 Universal PCR Master Mix II, Life Technologies) with a specific set of primers/probes (Table 1) in an ABI 7500 Fast Real Time PCR System1 (Life Technologies) following manufacturer’s instructions in 20 ml reactions. The qPCR conditions were: 50 8C for 2 min, 95 8C for 10 min, 40 cycles of 95 8C for 15 s and 60 8C for 1 min.

F, forward; R, reverse; P, probe. Considering reference bacterial strains, and Escherichia coli for Universal, the specificity was checked using NCBI/Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast). a

The absolute quantification of the target organisms was determined by the plotting of the cycle threshold (Ct) value obtained from each clinical sample against a standard curve generated with known concentration of gDNA of reference bacterial strains (Table 1) in 10-fold serial dilutions (102–107 cells). Negative control (purified PCR-grade water instead of the DNA template) was included in all PCR reactions.

3.6.

Statistical analysis

Demographic and clinical periodontal parameters between Afro-descendants and non-Afro-descendants were compared using the Student’s t-test, chi-square test and Fisher’s exact test. Microbial analysis was performed using Mann–Whitney and Student’s t-tests. Statistical significance was taken as a p value less than 0.05.

4.

Results

A total of 84 individuals were included in this study, 42 AD (mean age 35.9  13.1 years) and 42 NAD (mean age 36.2  13.1 years). Table 2 shows the distribution of both populations according to age, smoking habits, gender and systemic conditions. The periodontal clinical variables analysed between AD and NAD groups were: probing depth (PD), clinical attachment level (CAL), plaque index (PI), gingival index (GI), percentage of PD sites  4 mm, percentage of CAL sites  3 mm and percentage of PD  4 mm and CAL  3 mm. Statistically significant

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Table 2 – Demographic characteristics of AD and NAD populations according to age, smoking habits, gender and systemic conditions. Population

Table 3 – Periodontal clinical variables between AD (Afrodescendants) and NAD (non-afro-descendants) groups. Periodontal clinical variables

p

Afro-Brazilian Non Afro-Brazilian (AD) N = 42 (NAD) N = 42

PD

Age

35.9  13.1 Md = 31.5

36.2  13.1 Md = 33.0

0.921 A AD = NAD

CAL

Smoking

4.8%

12.2%

0.265 C AD = NAD

IP

Gender Female Male

73.8% 26.2%

73.8% 26.2%

1.000 B AD = NAD

% PD sites  4 mm

Diabetes

11.9%

7.3%

0.713 C AD = NAD

% CAL sites  3 mm

Cardiopathy

11.9%

2.4%

0.202 C AD = NAD

% PD  4 mm and CAL  3 mm

IG

AD

NAD

2.5  0.6 Md = 2.4 2.9  1.2 Md = 2.4 46.1  31.7 Md = 43.6 42.5  27.1 Md = 38.0 13.4  16.7 Md = 5.1 49.5  26.9 Md = 43.9 11.6  16.3 Md = 3.6

2.2  0.5 Md = 2.1 2.2  1.1 Md = 2.0 37.5  15.6 Md = 35.3 31.5  26.1 Md = 29.9 9.5  8.8 Md = 6.8 36.4  24.6 Md = 28.9 8.1  8.6 Md = 6.0

p

0.014 AD > NAD 0.019 AD > NAD 0.120 AD = NAD 0.061 AD = NAD 0.194 AD = NAD 0.023 AD > NAD 0.228 AD = NAD

Probability of significance: Student’s t-test. PD, probing depth; CAL, clinical attachment level; PI, plaque index; GI, gingival index.

Probability of significance: Student’s t-test (A), chi-square test (B), Fisher exact test (C).

differences between groups were detected related to PD, CAL and percentage of CAL  3 mm (Table 3). Total bacterial load and levels of the periodontopathogens A. actinomycetemcomitans, P. gingivalis, T. forsythia and T. denticola were determined. As shown in Table 4, all bacteria investigated were present in both groups. We also observed that there was no statistically significant difference between AD and NAD groups considering P. gingivalis, T. forsythia and

Study population

T. denticola bacteria species. Only A. actinomycetemcomitans showed higher levels in the AD group ( p < 0.001). After quantification, distribution of both A. actinomycetemcomitans serotype b and JP2 strains were determined. Out of 84 individuals examined in this study, these A. actinomycetemcomitans subspecies were identified in 14 (16.66%) subjects. More importantly, a higher number of participants (around 20%) in

Table 4 – Comparative analysis of mean microbial levels (T103) and percentage of positive bacterial samples between AD (Afro-descendants) and NAD (non-Afro-descendants) groups. Bacteria

Study population AD mean  standard deviation median (% of positive samples)

Total bacterial load

Student’s t-test p value Aggregatibacter actinomycetemcomitans

Student’s t-test p value Porphyromonas gingivalis

Student’s t-test p value Tannerela forsythia

Student’s t-test p value Treponema denticola

Student’s t-test p value

Mann–Whitney test p value

NAD mean  standard deviation median (% of positive samples)

3303.8  3236.8 Md = 2308 (100%)

2746.2  3477.6 Md = 1415.6 (100%)

0.111 AD = NAD

104.5  299.5 Md = 10.7 (92.85%)

<0.001 AD > NAD

1276.9  3284.2 Md = 3.1 (54.76%)

0.364 AD = NAD

1066.7  2106 Md = 228.1 (76.19%)

0.111 AD = NAD

14028.2  23230.5 Md = 1968.6 (83.33%)

0.128 AD = NAD

1.00 136.4  179.5 Md = 60.8 (85.71%) 0.43 163.3  314.1 Md = 2.4 (52.38%) 0.23 167.7  279.7 Md = 52.7 (78.57%) 0.56 3674.2  7126.9 Md = 758.8 (92.85%) 0.54

Significant differences between AD and NAD mean bacterial levels (Mann–Whitney test; p < 0.05); significant differences between AD and NAD bacterial percentages (Student’s t-test; p < 0.05).

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in untreated caries and severe periodontitis in younger age groups in regions less advanced in the demographic and epidemiologic transition may lead to high levels of tooth loss in these areas in the future. Disparities in oral health as well as in the prevalence of periodontitis by race/ethnicity and socioeconomic status have been documented by several authors.17–19 As mentioned by these authors, both African Americans and individuals with low income or education have a higher prevalence of oral diseases, including periodontal disease, dental caries and tooth loss. These disparities in oral health have also been observed in studies conducted outside the United States.8,20 Considering that there were reciprocal interests between the Afro-Brazilian communities and the researchers examining these populations, we had the opportunity to identify periodontal conditions in the Tijuac¸u community and to conduct preventive periodontal treatment. To consider the clinical and microbial profile of Tijuac¸u population, we matched them with a non-African descendant population from Sa˜o Paulo state, Brazil. The community of Tijuac¸u was formed by people of African descent, is estimated that the population of the community today is around 3000. In our study we compared AD and NAD populations matched by gender, age and clinical periodontal diagnosis. According to our results, statistically significant differences between groups were detected related to mean values of PD and CAL as well as percentage of CAL  3 mm, revealing a worse clinical status among those of African ancestry (Table 3). These data confirm that the AD population showed a more severe pattern of periodontal diseases than NAD subjects. It is possible to speculate that the poorer social and educational conditions observed in the AD population (data not shown) could have had a negative impact on their periodontal condition. In our evaluation of bacterial levels, we used the population of both groups as the basis for analysis. There were no differences among the pathogens studied, with the exception of A. actinomycetemcomitans. As shown in Table 4 levels, of the red complex species – P. gingivalis, T. forsythia and T. denticola – were not influenced by ethnicity. In fact, based on their worse periodontal status, our expectation was to find higher levels of these bacteria in the AD population. Our study slightly disagrees with what has been postulated about red complex species, i.e., increased levels and proportions among subjects showing greater severity of periodontal disease. Usually, these bacteria isolate and as a bacterial consortium tend to be at higher levels in the presence of greater attachment loss, deepest or more inflamed periodontal sites.21,22 However, it

Graph 1 – Distribution (%) of two most virulent strains of A. actinomycetemcomitans (Aa), JP2 and serotype b in AD and NAD groups.

the AD group were infected by more virulent strains in comparison to the NAD group (14%), as shown by Graph 1. Table 5 shows the association between JP2 strains and clinical parameters. Separately, the analysis of serotype-specific strain (serotypes b) showed that out of the 14 individuals positive for one of the searched A. actinomycetemcomitans subspecies, 6 (42.8%) individuals were positive for serotype b. This serotype was predominately found in the AD population (83.3%). Similar findings were observed when occurrence of JP2 strains were compared between AD and NAD groups.

5.

Discussion

Knowledge of the disease burden in populations is essential for health authorities to plan the use of resources in programmes for prevention and care. As pointed out by Marcenes et al.16 in a recent publication, the global burden of oral conditions is shifting from severe tooth loss towards severe periodontitis and untreated caries. Tooth loss is a final common finding when preventive or conservative treatments fail or are unavailable. Thus, a dramatic reduction in the prevalence of caries and periodontitis in regions with advanced demographic and epidemiologic transition may underlie some of the reduced prevalence of severe tooth loss. Conversely, it is quite conceivable that the increases observed

Table 5 – Periodontal clinical comparative profiles between individuals from the AD and NAD groups harbouring JP2 Aggregatibacter actinomycetemcomitans strains. Study group Afro-Brazilian (AD) Non-Afro-Brazilian (NAD) p value

Probing depth (mean  SD)

Clinical attachment level (mean  SD)

Plaque index (mean  SD)

Gingival index (mean  SD)

3.42  1.05 3.33  1.32 0.2143

2.88  1.34 2.99  1.37 0.2341

0.60  0.31 0.55  0.29 0.1011

0.47  0.31 0.41  0.28 0.0990

SD, standard deviation. Comparisons between AD and NAD groups by Student’s t-test ( p < 0.05).

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should be remembered that this is the first periodontal microbial description of Tijuac¸us? population. The highest level ( p < 0.001) of A. actinomycetemcomitans in the AD group displayed a more conventional pattern. These data were somewhat unsurprising considering the worse clinical periodontal condition and the ethnicity of this specific group.23 However, there are some studies that questioned the role of A. actinomycetemcomitans in periodontitis aetiology and pathogenesis.24,25 Faveri et al.24 when studying generalised aggressive periodontitis failed to detect this bacterium. This year in a nice written review25 authors widely talked about A. actinomycetemcomitans and periodontal statuses. They mentioned that individuals without periodontal disease also harbour this bacterium and that not always aggressive periodontitis patients are infected. But interestingly, they mentioned that this relation is questionable only in non-black populations. This controversial scenario could be under influenced of many aspects, which among them are technical procedures. Although, paper points and curettes sampling methods did not impact red complex bacterial profiles A. actinomycetemcomitans was more consistently detected by means of paper point sampling.26 As mean levels of A. actinomycetemcomitans differed between groups, we performed a specific microbial analysis regarding serotype b and JP2 strains. Our data showed higher frequencies of A. actinomycetemcomitans serotype b and JP2 in the AD group. Different serotypes of A. actinomycetemcomitans have been shown to be associated with a healthy or a diseased periodontium worldwide. However, researchers have failed to determine a unique serotype-disease pattern.4,27 Bandhaya et al.28 stated that genotype distribution of A. actinomycetemcomitans varies between ethnic groups. However, no clear relationship between a specific genotype and periodontal conditions was observed. In a Brazilian population15 it was found that serotype c was the most prevalent in both diseased and healthy subjects. Aggressive periodontitis subjects were not exclusively associated with A. actinomycetemcomitans serotype b, and serotypes d and f were not detected in this examined Brazilian population. Among serotype b there are JP2 and non-JP2 genotypes being the first more virulent.29 Previous studies have reported that the presence of the JP2 strain of A. actinomycetemcomitans was implicated in the aetiology and severity of periodontal diseases (preferably in aggressive periodontitis) in different geographic regions in the world (for details see Haubek and Johansson23). For example, Haubek et al.30 examined 326 subjects from five continents and observed that 38 individuals were infected by JP2. In 2005, our group investigated the prevalence of A. actinomycetemcomitans in Brazilians with aggressive and chronic periodontitis. We verified that from out of 203 individuals, 13 were also ˚ berg et al.31 reported that the infected by JP2. Recently, A presence of both JP2 and non-JP2 genotypes of A. actinomycetemcomitans were strongly associated with the onset and progression of attachment loss in the whole group of Ghanaian adolescents. In part, the greater severity of periodontal disease in the AD group could be understood by the presence of JP2 strains, which were 6 times more prevalent than in NAD group. Colonisation by serotype b in this same group AD group could also contribute to their worse clinical condition.

6.

Conclusions

In comparison to non-Afro-Brazilian descendants, Afrodescendants showed a poorer periodontal status accompanied by higher levels of A. actinomycetemcomitans and similar levels of red complex species. Therefore, our findings confirm that Afro-Brazilian populations are preferably colonised by A. actinomycetemcomitans serotype b as well as JP2 strains.

Funding Governmental grants – the Sa˜o Paulo Foundation for Research (FAPESP-2010/19079-8) and the National Council for Scientific and Technological Development (CNPq-552264/2011-3).

Competing interests The authors declare that they have no conflict of interests related to this study.

Ethical approval The present study was approved by the Institutional Committee on Research of the University of Taubate (protocols # 304532/13 and 521/10), Sa˜o Paulo, Brazil, in accordance to the Helsinki Declaration of 1975, as revised in 2000. Prior to selection, oral and written explanations regarding the research protocol were given to the eligible participants. All subjects provided written informed consent before enrolling in the present study.

Acknowledgments This study was supported by a grant from the Sa˜o Paulo Foundation for Research (FAPESP-2010/19079-8) and also by The National Council for Scientific and Technological Development (CNPq-552264/2011-3).

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