Choir singing as an activity to manage anxiety and temporomandibular disorders: Reports from a Brazilian sample

Abstract

The aim of this study was to evaluate anxiety, stress and symptoms of temporomandibular disorders (TMD) among a sample of participants that practice choir singing as a recreational activity. As the literature shows the positive effects of choir singing on mental and physical health, we hypothesized that anxiety, stress and TMD symptoms would be less frequent in the choir group when compared to participants from the general population. The choir group included 33 adults who participated weekly in a choral singing group as a recreational activity and the comparison group included 40 participants from the general population. Choir singers had significantly lower levels of anxiety and TMD. A significant positive correlation was observed between anxiety and TMD scores. The groups did not differ regarding the presence of stress symptoms. It seems that choir singing was associated with better mental and physical outcomes. This study reinforces the promising benefits of choir singing as a recreational activity. Although we only included amateur singers in our sample, the literature shows that singing as a professional may be an etiologic factor for developing TMD. Future studies should explore differences between professional and amateur choir singers regarding TMD symptoms.

Keywords

anxiety choir singing older adults quality of life temporomandibular disorders

Temporomandibular disorders (TMD) are pathological orofacial conditions characterized by a dysfunction at the temporomandibular joint, pre-auricular area, and masticatory and/or skull muscles. They are associated with acute and chronic pain in the temporomandibular joint and other bodily areas, such as the ears, neck, and head; limitations of jaw and joint functions; and the production of sounds during mastication (Araneda, Oyarzo, González, & Figueroa, 2013; Rhim, Han, & Yun, 2016). Temporomandibular disorders are a common group of disorders: approximately 75% of the population has one of the signs of TMD and 33% of the population has at least one TMD symptom, with a higher prevalence in women between 20 and 40 years (Cordeiro & Guimarães, 2012; De Leeuw & Klasser, 2008; Moraes, Sanches, Ribeiro, & Guimarães, 2013; Okeson, 1996; Schiffman & Fricton, 1988).

Temporomandibular disorders are multifactorial conditions caused by different etiologies, such as postural and proprioceptive changes; parafunctions (teeth-grinding and nail biting, for example); and psychosocial factors (anxiety, depression and stress; Etöz, Akcay, Neselioglu, Erel, & Alkan, 2012; Rhim et al., 2016; Sipila et al., 2001). Thus, TMD are associated with physiological, psychological and behavioral factors (Melchior, Mazzetto, & Felício, 2012; Suvinen, Reade, Kemppainen, Kononen, & Dworkin, 2005).

Some studies have shown that individuals with higher levels of anxiety and depression have an increased risk for developing TMD compared to individuals with lower levels of these symptoms (Giannakopoulos, Keller, Rammelsberg, Kronmüller, & Schmitter, 2010; Kindler et al., 2012; Torres, Pérez, & Molerio, 2013). The literature indicates a significant positive correlation between TMD, stress, anxiety and depression in non-clinical samples, such as undergraduate students (Minghelli, Kiselova, & Pereira, 2011). In clinical TMD samples it has been observed that more than 50% of the patients had a chronic and aversive stressor history prior to the establishment of the disorder (Leeuw, Bertoli, Schmidt, & Carlson, 2005). This may be explained by the fact that stressors and an increased level of anxiety might result in higher muscular tension, and these factors may lead to the development of TMD, which is endorsed by the fact that TMD frequently have a muscular origin (Kindler et al., 2012; Minghelli et al., 2011; Moraes et al., 2013). To summarize, Chisnoiu and colleagues (2015) suggest that anxiety and stress are part of the etiology of TMD and also play a significant role in maintaining the symptoms and worsening them.

Thus, as TMD are related to different etiologies, namely, psychosocial stressors, it has been hypothesized that therapeutic interventions that have the goal of directly or indirectly decreasing anxiety and stress levels may be useful to decrease the incidence of TMD or even prevent it. Among these differents activities, the literature has shown that music has been used to regulate emotional states as it has a positive effect on psychological indicators such as anxiety and depression symptoms, and neuroimmunological markers (Chanda & Levitin, 2013; Fancourt, Ockelford, & Belai, 2014; Kreutz, 2015).

Choir or choral singing is a type of music activity which is very common in different countries and practiced by thousands of people. Being part of a choir singing community involves creating a goal as a group and working in a collaborative way to overcome a new challenge (Tonneijck, Kinébanian, & Josephsson, 2008). A recent systematic review (Clements-Cortes, 2015) assessed the effects of singing on health measures in older adults and the author observed different main effects of singing, namely, psychological, social and physiological benefits.

Regarding the psychological benefits, it seems that choral singing has two main general effects related to this dimension: (a) increase of positive feelings; and (b) reduction of negative emotional states (Clements-Cortes, 2013, 2015; Coulton, Clift, Skingley, & Rodriguez, 2015; Sanal & Gorsev, 2014). Increase of positive feelings promoted by choral singing is associated with an improvement in quality of life, vitality, positive affect, focused attention, mental health, and cognitive stimulation; and reduction of negative emotional states is associated with a decrease in stress, anxiety and depressive symptoms (Clift & Hancox, 2010; Clift et al., 2010; Dingle, Brander, Ballantyne, & Baker, 2012; Gale, Enright, Reagon, Judd, & Pooley, 2012).

Related to the social benefits of choral actitivy, choir singers commonly report feelings such as: happiness about creating and working to achieve a group goal; connection; belonging; sense of identification to a group; and wholeness, which indicates that choir singing may increase social functioning (Bailey & Davidson, 2005; Clements-Cortes, 2015; Coulton, Clift, Skingley, & Rodriguez, 2015; Durrant, 2005; Stewart & Lonsdale, 2016).

There is some evidence that choir singing promotes physiological benefits as well. Some studies have indicated that this activity increases the level of immunoglobulin A (an antibody that reinforces immune system activity), and decreases cortisol and salivary amylase (considered stress markers). It has been hypothesized that this recreational activity may strengthen the immune system and help participants to reduce the stress responses (Kreutz, Bongard, Rohrmann, Hodapp, & Grebe, 2004; Sanal & Gorsev, 2014). Changes in oxytocin levels have also been related to choir singing. This hormone is associated with sociobiological bonding and the creation and endurance of intimate relationships, and is considered a physiological indicator of social affiliation among human beings (Gordon, Zagoory-Sharon, Leckman, & Feldman, 2010). Kreutz (2014) found an increase of oxytocin concentration in choral singers after rehearsal, but not after chatting about positive events in the group, indicating that this increase was specific to the activity of singing with others.

To summarize, the literature shows that choir singing may promote social, physical and mental health (Clements-Cortes, 2013; Dingle et al., 2012; Sanal & Gorsev, 2014). However, the results are inconclusive, and additional research using different methodological approaches should evaluate the putative therapeutic function of this activity (Gick, 2011).

So far, and to the best of our knowledge, this study is the first to investigate the relationships among TMD, anxiety and stress levels, and choir singing practice. The primary aim of this study was to evaluate anxiety, stress and TMD scores in a sample of choir singing participants, and then compare the results with members of the general population. Consistent with the literature data about the putative therapeutic effects of choral or choir singing practice on different physical and mental health markers, we hypothesized that anxiety, stress and TMD would be less frequent for the choir singing sample compared to participants from the general population who do not practice this activity. We also hypothesized that these symptoms would be correlated, as the literature shows that TMD may be caused/affected by psychosocial factors.

Methods

Participants

This was a cross-sectional study that evaluated the prevalence of temporomandibular disorders, anxiety, and stress symptoms in a sample of adults in a southeastern area of Brazil. This study consisted of 73 participants (62 women, 11 men) ranging in age from 18 to 75 years (M = 54.84, SD = 13.61). They formed two different groups: the choir singing group and the comparison group.

The choir singing group included 33 adults (27 women and 6 men) ranging in age from 18 to 75 years (M = 55.29, SD = 17.34) who participated weekly in a choral singing group as a recreational activity. These participants were recruited from three different choral singing groups that offer this activity for free to members of the general community. This group had a varied background in choral singing activity. The years of choral singing practice varied between 0.5 to 56 years (M = 13.66, SD = 13.97). Regarding the weekly frequency of choral singing rehearsal, 60.6% of participants rehearsed once a week, 30.3% twice a week, 6.06% three times, and 3.04% four times a week. These participants evaluated choral singing activity as a source of pleasure in life and as a recreational activity. They joined choir singing rehearsals of their own volition and did not receive any financial support for taking part in this activity.

The comparison group included 40 participants (35 women and 5 men) ranging in age from 21 to 73 years (M = 53.62, SD = 11.06). These participants were selected to be paired by age, gender and education level with the choir singing group. They were members of the general population who did not participate in the singing group. These participants had never belonged to a choir group as adults.

Procedures

After contacting the choir leaders and scheduling an appointment with the participants, the researchers explained to the singers the study and its goals. Participants interested in enrolling completed the consent form, after which they completed a demographic questionnaire and self-report measures. The participants answered the questionnaires before the choir singing rehearsal, and the assessment occurred as a group. The only exclusion criterion to this group was being younger than 18 years old.

After the assessment of the choir singing group, the researchers determined the age range, education level, and proportion of female and male participants to be recruited from the general population to compose the comparison group. Thus, these participants were selected to be paired by age, gender and education level with the choir singing group. Participants were recruited from the community via advertisements that were posted in public areas. Those participants interested in enrolling completed the consent form, a demographic questionnaire and self-report measures. The participants in the comparison group were individually tested. We used two exclusion criteria to this group: being younger than 18 years old, and having previously been part of a choir.

Measures

Demographic questionnaire

Participants answered a demographic questionnaire about age, gender, and educational level. The choir singing group answered questions regarding time and frequency of choral singing practice, and whether this activity was evaluated as being a source of stress or pleasure in life. As TMD, anxiety, and stress scores may be affected by the practice of physical activity and other activities, such as dancing or painting, all participants answered questions about these other activities.

Lipp’s Stress Symptoms Inventory for Adults (LSSI)

This inventory was created by Lipp and Guevava in 1994, and revised in 2000 (Lipp, 2000). It evaluates the stress response in two different domains: psychological and physical distress. It evaluates whether the responder has a significant level of stress, and discriminates between participants with and without stress symptoms. If the participants have a significant level of stress, the inventory classifies which stage of stress they are in: alarm, this being the initial phase of stress response; resistance, the intermediary stage; near exhaustion, the third phase; and exhaustion, the last and most damaging and chronic stage.

Beck Anxiety Inventory (BAI)

The BAI is a 21-item self-report inventory designed to evaluate the presence and severity of anxiety in adults and adolescents. It was created by Beck and colleagues, and it is a well-accepted measure of anxiety (Beck, Epstein, Brown, & Steer, 1988). This instrument discriminates between the cognitive, emotional, and physiological symptoms of anxiety. Responses are rated on a four-point Likert scale and range from 0 (not at all) to 3 (severely) and they range along a continuum of symptom severity. The total score ranges from 0 to 63 and it is possible to classify the responders according to the severity of anxiety. Scores from 0 to 9 represent normal or no anxiety; scores of 10 to 18 represent mild to moderate anxiety; scores of 19 to 29 represent moderate to severe anxiety; and scores of 30 to 63 represent severe anxiety (Julian, 2011). The BAI has good statistical properties, such as a high internal consistency (Cronbachs α = .92) and a test-retest reliability of .75 over one week.

Fonseca Anamnesis Questionnaire (FAQ)

This is a ten-item questionnaire commonly used by dentists and researchers in Brazil to evaluate the symptom severity of TMD. The items are questions related to the presence of pain or discomfort at the temporomandibular joint. The participant may choose among three different responses (yes/ten points; no/0 points; and sometimes/five points). At the end each participant has a different score. Scores between 0 and 15 points indicate absence of TMD; 20–45 points classify the participants with mild TMD; 50–65 points indicate moderate TMD; and upwards to 70 points indicates severe TMD (Chaves, Oliveira, & Grossi, 2008; Fonseca, Bonfate, Valle, & Freitas, 1994).

Data analysis

Since none of the quantitative data followed a normal distribution, the Mann–Whitney U test was used to evaluate differences between the two groups in age, anxiety and TMD symptoms, and educational level. Chi-square analysis was used to evaluate differences in gender, practice of physical and recreational activity, and presence or absence of stress. Spearman’s correlation was used to test the correlation between anxiety and TMD symptoms. The significance level was set at p < .05. We used the software Statistical Package for Social Sciences (SPSS) for Windows, 16.0 version (Nie, Hull, & Bent, 2003) to analyze the data.

Results

This study consisted of 73 participants. The majority of the sample had a college degree (n = 41). Regarding the regular practice of physical and recreational activities, such as painting, dancing, or playing an instrument, 52% of participants (n = 38) did physical activity, and 23% (n = 17) practiced recreational activity. There were no differences across the groups in age, U = 487, p = .16; gender, χ²(1) = 0.456, p = .49; education level, U = 617, p = .74; or practice of other recreational activity (with exception of choir singing), χ²(1) = 1.51, p = .22. Although there is a tendency for choir singers to practice more physical activity than the comparison group, this difference did not reach statistical significance, χ²(1) = 2.88, p = .09. Table 1 shows the sample characteristics of the groups.

Table 1.

Participant characteristics.

Variable	Choir singing group(n = 33)	Comparison group(n = 40)
Gender
Women, n (%)	27 (81.8)	35 (87.5)
Age
Mean (SD)	55.29 (17.34)	53.62 (11.06)
Range	18–75	21–73
Education
Fundamental, n	7	4
High school, n	4	9
Incomplete Bachelor’s degree, n	3	5
Bachelor’s degree, n	19	22
Physical activity
Yes, n (%)	21 (63.6)	17 (42.5)
Recreational activity*
Yes, n (%)	10 (30.3)	7 (17.5)

Painting, dancing, or playing an instrument.

Regarding the presence of stress symptoms, 47.5% of comparison group (19 participants) had a significant level of stress, while only 30.3% of the choir singing group (10 participants) had a high level of this symptom. However, this difference did not reach statistical significance, χ²(1) = 2.23, p = .135.

The choir singing group had a mean anxiety score evaluated through BAI of 4.88 points (MDN = 3; SD = 4.98), which indicates an absence of anxiety symptoms. The members of the comparison group had a mean anxiety score of 11.08 points (MDN = 8.5; SD = 10.42), which represents mild to moderate anxiety. Anxiety symptoms were significantly more common among participants from the comparison group compared to the participants in the choir group, U = 424.000, p = 0.009. Figure 1 shows the mean anxiety scores through the groups. The data are reported as mean ± SEM (standard error of the mean).

Figure 1.

Mean anxiety and temporomandibular disorders (TMD) scores measured by Beck Anxiety Inventory and Fonseca Anamnesis Questionnaire in the choir singing group (n = 33) and comparison group (n = 40).

The FAQ showed that the comparison group had a mean of 26.88 points in TMD symptoms (MED = 22.5; SD = 22.09), which indicates a mild level of symptoms. The choir singers had a mean of 10.66 points (MED = 5; SD = 16.33), which indicates that they do not present TMD symptoms. Statistical analysis revealed a significant difference between these groups (U = 335, p < .001). Symptoms of temporomandibular disorders were significantly more common in the comparison group. Figure 1 shows the mean TMD scores through the groups. The data are reported as mean ± SEM.

To evaluate possible correlations between the anxiety level and TMD symptoms we performed a Spearman’s correlation analysis. A positive and strong correlation between anxiety and TMD scores was found, r_s = .72, p < .001. This indicates that the more anxiety symptoms the participants of this study had, the more TMD levels they presented. Figure 2 shows the relation between anxiety and TMD scores.

Figure 2.

Linear relationship between anxiety symptoms (measured by Beck Anxiety Inventory) and temporomandibular disorders (TMD) scores (measured by Fonseca Anamnesis Questionnaire).

Conclusion

The present study sought to evaluate anxiety, stress and TMD symptoms in a sample of participants who practice choir singing as a recreational activity, and to compare their scores with members of the general population who do not practice this musical activity. Consistent with expectations, results of this study indicate that choir singers had lower levels of anxiety and TMD symptoms than the comparison group. Quantitative analysis confirmed statistically significant differences between the groups, which suggests that singing in a choir was associated with better mental (anxiety) and physical (TMD) outcomes. This study aligns with previous research about the effects of choir singing on anxiety levels (Clements-Cortes, 2015; Clift et al., 2010; Coulton et al., 2015; Gale, Enright, Reagon, Lewis, & van Deursen, 2012).

In addition, we found a significant positive correlation between anxiety and TMD symptoms. Regardless of the group, it was observed that, the greater the number of symptoms of anxiety participants had, the higher their TMD scores. This association is consistent with other studies that show a relationship between psychosocial factors, namely, anxiety and TMD, and reinforces previous findings indicating that these factors are related to the etiology of temporomandibular disorders (Giannakopoulos et al., 2010; Kindler et al., 2012; Torres et al., 2013).

Regarding the lower levels of TMD observed within the choir singing group, it must be highlighted that one of the first treatment choices for TMD is the prescription of specific exercises. These have the goal of reducing pain, tension of the muscles fibers, and muscle spasm; promoting coordination and strength of orofacial muscles; and increasing local circulation of this area. The most common exercises involve opening and closing of the mouth slowly; and stretching, contracting and relaxing the masticatory and elevator jaw muscles. Postural exercises are also prescribed in some cases with the goal of promoting the alignment of the craniomandibular system (Moraes et al., 2013; Yuasa et al., 2013). Singing involves the use of orofacial areas, such as exercising the masticatory and elevator jaw muscles. It has different impacts on bodily posture, breathing, and speaking voice, with trained singers having a higher fundamental frequency of the speaking voice and better vocal capability than non-singers (Siupsinskiene & Lycke, 2011). Thus, the lower level of TMD observed in this study among the choir singing group may be related to the effect of singing exercises on orofacial muscles. As the choir singing sample evaluated has a medium to high level of experience in choir singing (M = 13.66 years), it is possible that the long period practicing an activity that recruits the orofacial muscles might have had a positive effect on TMD levels, preventing the ocurrence of these disorders within the choir singing group.

Nevertheless, a body of literature consistently indicates the effects of choir singing on psychosocial outcomes, as a reduction of anxiety, stress and depressive symptoms and an increase in quality of life, positive feelings and social functioning (Clements-Cortes, 2015; Clift et al., 2010; Coulton et al., 2015; Gale et al., 2012; Judd & Pooley, 2014; Stewart & Londsdale, 2016). As mentioned before, in this study a significantly lower level of anxiety was observed in the choir singing group. Taken together with the significant positive correlation between anxiety and TMD, and the knowledge that anxiety is one of the etiologic factors in TMD, it is possible that anxiety may mediate the lower levels of TMD that were found among choir singers. Additional research is needed to test this hypothesis: whether the effect of choir singing on TMD symptoms occurs primarily through an effect on anxiety; or whether the exercise of orofacial muscles promoted through choir singing is the factor that is related to a decrease in TMD levels (Melchior et al., 2012; Torres et al., 2013).

There are specific etiologic factors which contribute to the development of TMD among musicians, especially violin, viola, trumpet, trombone and tuba players. The repetitive and excessive upward and backward force against the mandible and the temporomandibular joint might predispose those professionals to develop TMD (Taddey, 1992). Singing may involve the overuse and misuse of orofacial muscles as well, as professional singers tend to present a heavy vocal load and an increased risk for the development of voice disorders (Phyland et al., 2013), as well as the presence of body pain near to the larynx area (Rocha, Moraes, & Behlau, 2012). Thus, it is important to stress that singing may be an etiologic or maintaining factor for TMD, which might predispone singers to develop that condition.

However, Gick (2011) highlights the differences between professional and amateur singers’ responses, with professional singers presenting a higher cortisol response (a stress marker) than amateurs (Grape, Sandgren, Hansson, Ericson, & Theorel, 2003). Additionaly, amateur singers describe singing as a meaningful activity that promotes self-expression, while professional singers tend to focus on the technichal aspect of singing (Grape et al., 2003). Thus, singing may have negative impacts on different measures when it involves excessive muscle tension in orofacial and laryngeal muscles, and an overuse of the voice, which are commonly observed in professional singers, but not in amateurs. In our study we only included individuals who sing in a choir group for recreational purposes, and none of the participants received financial support for being part of a choir. Thus, the choir singing group was formed only of amateurs who evaluate choral singing activity to be a source of pleasure. Moreover, there was not an overuse of the voice in our sample, as 60% of participants rehearsed only once a week. Taken together, those sample characteristics may rule out the etiologic impact of singing on TMD in our study.

The sample evaluated in this study was predominantly older adults. It is well known that aging is related to impairments in sensory, perceptual, and cognitive areas, and an increased likelihood of developing diseases. In addition, there are important impacts on mental health, as older adults frequently report depression and anxiety symptoms and feelings of loneliness and loss of life meaning, which makes this age range more vulnerable to developing physical diseases, such as TMD (Yinger, 2014). With the increase in the lifespan of the general population, it has become an area of interest for medical and healthcare research to understand the mechanisms and putative interventions that promote quality of life and health in older people. This research may have a therapeutic function on mental and physical health. Studies with choir singing have shown that this activity may combat the negative impairments of aging, and promote positive psychophysiological and social effects in older adults (Clements-Cortes, 2015; Hillman, 2002; Tomaino, 2013). Choir singing has been associated with reduction in pain perception and an increase in positive mood and energy in older adults with dementia and their family and caregivers (Clements-Cortes, 2015); and fewer symptoms of depression and higher quality of life and satisfaction with health in this aging group (Johnson et al., 2013). In a randomized controlled trial, Coulton and colleagues (2015) evaluated the effects of choir singing on quality of life, anxiety and depression in older adults at three- and six-month follow-up. The authors found a better quality of life at the six-month follow-up, and lower levels of anxiety and depression three months after the random allocation within the choir singing group, compared to a group randomized to usual activities (Coulton et al., 2015).

Thus, choir singing may be an activity adopted by public health programs in order to enhance quality of life and promote mental and physical health of vulnerable populations. Adults with a history of chronic hospitalization or suffering from different health problems have high levels of anxiety and stress, which worsen the disease. Choir singing may be a relatively new therapeutic avenue and a health promotion strategy to be considered in public health systems, since the literature shows that this acitivity is related to a good cost-effectiveness and different health benefits.

The present study is not without limitations. First, this study was a cross-sectional study and not an experimental one, which precludes causal conclusions among choir singing, anxiety, and TMD. Even though we found statistically significant differences between the groups, we cannot affirm that the results obtained are related specifically to the practice of this musical activity. However, it is important to highlight that the groups did not differ in any of the demographic variables evaluated, which raises the hypothesis of the positive effects of choir singing on the variables measured.

Another limitation is related to the higher incidence of TMD symptoms in the comparison group than in the choir singing group, which could indicate the positive effects of singing in a choir on TMD symptoms. However, TMD are characterized by a dysfunction at temporomandibular joint, pre-auricular area, and masticatory and/or skull muscles, which causes acute and chronic pain, and affects mouth opening and jaw movements (Araneda et al., 2013; Rhim et al., 2016; Voog, Alstergren, Leibur, Kallikorm, & Kopp, 2003). As singing involves the use of orofacial areas, such as jaw muscles (Siupsinskiene & Lycke, 2011), the presence of TMD may prevent individuals from singing in a choir, as it limits the jaw and joint functions required during singing activity. Thus, the presence of a lower level of TMD symptoms in the choir singing group and a greater incidence in the comparison group may be related to a lower probablitily of individuals with TMD joining choral groups, rather than being due to a putative therapeutic effect of this activity.

A third limitation of this study is the sample size used. Even though the choir singing group had a lower prevalence of stress symptoms (30.3%) compared to the comparison group (47.5%) for example, the difference was marginally significant (p = .13), which may have been due to our small sample size. As there is no other study in the literature that has used the LSSI inventory to evaluate stress symptoms in a choir singing sample, it is not possible to compare the stress results obtained in this study with other research.

Another limitation relates to the fact that different studies show that being part of a recreational group or doing some leisure activity such as dancing, painting or even practicing physical activity have positive and well-established effects on mental and physical health, such as decreasing anxiety and stress responses. Therefore, choir singing may be just one additional form of leisure activity and we cannot affirm with this study that it is has more of an effect than other activities on anxiety and TMD. Further research is necessary to evaluate whether choir singing is more benefical and cost-effective on mental and health measures than other activities. Future studies should explore, either through the use of randomized controlled trials or longitudinal studies, the effects of choir singing on TMD and anxiety, manipulating the variables among different groups.

Despite these limitations, the present study provides useful information about choir singing as a potential activity that may be used to promote mental and physical health. It is consistent with the literature regarding the benefits of choir participation for choir members.

Footnotes

Acknowledgements

We thank Sharon Eldar, PhD, for comments that greatly improved the manuscript.

Ethical approval

Ethical approval for this project was given by the Institutional Review Board at the School of Dentistry of Ribeirão Preto [ref number 50415315.8.0000.5419], University of São Paulo. The research was undertaken in accordance with the Declaration of Helsinki.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article. However, the principal investigator (Kátia Alessandra de Souza Caetano) received a scholarship as a PhD student during the execution of this study from the São Paulo Research Foundation/FAPESP [FAPESP process number: 2013/ 19263-1].

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