**2. Methods**

*Gingival Disease - A Professional Approach for Treatment and Prevention*

with house dust mite allergy.

**1. Introduction**

literacy [6].

periodontal pathogen, *Porphyromonas gingivalis*

Immunoglobulin-E and Immunoglobulin-G4 in periodontally healthy children

Given the complexity of community-based health care systems in the era of national insurance and high rate of poverty in Indonesia, it is not surprising that limited health literacy is common especially in rural areas [1]. Numerous studies show a link between limited parental health literacy with poor oral health among their child [2–4]. Parents' health literacy is often positively correlated with the frequency of dental visits and their knowledge/understanding of preventive measures in terms of oral or gingival diseases [5]. Thus, problems in the mouth range from bacterial biofilm, dental caries, chronic gingivitis, and locally aggressive gingivitis often reported in children whose parents have limited health

The presence of bacterial biofilm has an association with the presence of periodontal pathogen, which may lead to a periodontal disease when left untreated or undertreated [6]. Bacterial biofilm is often found among children with limited health literacy [7]. When left untreated or undertreated, these periodontal pathogens can spread below the gum line and may develop into gingivitis and periodontitis in the future [8]. Gingivitis is a reversible dental plaque-induced inflammation limited to the gingiva [9], while periodontitis is usually accompanied by gingivitis

While many people believe that periodontal pathogen is an adult issue, nowa-

Evidence suggests that the composition of the oral microbiome differs between children with and without allergy [18], and disruption of the bacterial biofilm in children leads to allergic responses following allergen challenge in subjects not previously sensitized to the allergen [19]. It is likely that the greatest concentration of *Porphyromonas gingivalis* lipopolysaccharides can be found in saliva, since it is a prerequisite for their transmission [20]. Hygiene hypothesis explained a protective role of microbiome (including oral microbiome) in the development of asthma and allergy [21]; on the other hand, in periodontally healthy and diseased mouths, *Porphyromonas gingivalis* in subgingival plaque elicit both local and systemic

immune responses [22]. To our knowledge, however, studies investigating the direct

days this kind of pathogen is also a pediatric issue, since its presence is often reported in children aged 6–16 years [11, 12]. Even though the origin and transmission of these pathogens are not fully understood, few researches have linked them with salivary transmission. Saliva is the most probable vehicle for person-to-person transmission of periodontal pathogens, such as *Porphyromonas gingivalis* [13]. *Porphyromonas gingivalis*, a black-pigmented gram-negative anaerobic rod, is one of the most crucial periodontal pathogen, not only found in adults but also common in children [14]. It can be cultured occasionally from periodontally healthy mouths [14]. Their lipopolysaccharide and whole cell were detected in the gum and oral cavities of approximately 37% of people and at similar frequencies across ages and genders [15]. This fact highlights that *Porphyromonas gingivalis* may be acquired in the first days of life [16]. These results are intriguing, while some researchers argued that children may acquire these periodontal pathogens from their parents,

but involves irreversible destruction of the supporting tissues [10].

especially if the parent has periodontitis [17].

**Keywords:** Immunoglobulin-E, Immunoglobulin-G4, lipopolysaccharide,

**32**

#### **2.1 Ethical considerations**

This is the grant number for ethics approval (20/Panke.KKE/I/2017). A written informed consent was obtained from every parent to be included in the study. This study was approved by Institutional Review Boards of Dr. Soetomo General Hospital Ethics Committee for Health Research at Surabaya, January 20, 2017. Results will be disseminated through peer-reviewed publications within 1 year after experiment has been finished.

#### **2.2 Research design and setting**

This is an analytic observational study with cross-sectional study design to assess the correlation between salivary lipopolysaccharide of *Porphyromonas gingivalis* with the circulatory Ig-E and Ig-G4. The study has been conducted by the support from Faculty of Dental Medicine Universitas Airlangga (Indonesia). Overall study started on January 1, 2017, and finished on December 31, 2017, at pediatric allergy-immunology clinic at Dr. Soetomo General Hospital (Indonesia) for data collection and Airlangga Oral and Dental Laboratory for IgE and IgG4 measurement.

#### **2.3 Sample size estimation**

The sample size is based on a substantial meaningful change in IgE level observed in the group [23]. Korn et al. [23] stated that ELISA is able to assay for free IgE in a concentration range of 1–2000 pg/ml from peripheral blood samples with a substantial meaningful change of 0.5 pg/ml and the expected standard deviation of IgE concentration is assumed to be 0.01 pg/ml based on findings by Korn [23]. For this randomized controlled trial design, the formula is:

$$N = \text{--}\, 2 \times \left(\frac{\mathbb{Z}\_{1-\frac{n}{2}} + \mathbb{Z}\_{1-\emptyset}}{8}\right)^2 \times s^{\frac{n}{2}}$$

All parameters were assumed as follows: mean change of IgE in intervention group = 10 pg/ml; mean change of IgE in control group = 0 pg/ml; α = 0.05; β = 0.20; δ = 10 pg/ml; s = 7; and s2 = 49.

We calculated and found the value of N = 17.71. This estimate requires 18 patients, to obtain 80% statistical power with 5% significance level for an independent samples and paired t-test. Due to the nature of this study being a pilot study, we aimed to recruit a minimal of 22 subjects in order to compensate for an estimate of 20% drop outs.
