Methods. Results. Variable. Strain. Fluoride (Test). Standard (Reference). Strain. Caries (ICDAS 1–6). Caries-free (ICDAS 0)

Methods

This cross-sectional study was carried out as a part of a cluster-randomized controlled field trial aiming to evaluate the effectiveness of fluoride varnish applications on caries development in toddlers living in multicultural areas in greater Stockholm, Sweden [10]. The original trial was designed with two parallel arms that compared children receiving a standard oral health program (reference group) with children who had the same standard oral health program supplemented with semi-annual fluoride varnish applications (test group). The study comprised 23 public dental clinics involving 3403 toddlers and the intervention was carried out between 1- and 3-years of age. From the main study, 7 public dental clinics representing both programs were selected by convenience and invited to the present investigation. 507 children (263 from the test group and 244 from the reference group) were consecutively enrolled in connection with the scheduled 36-month examination. All parents gave their written informed consent after verbal and written information and an interpreter was used when necessary. The project was approved by the Regional Ethic Committee (EPN; no 2013/143–32).

Intervention

The children in the test group received topical applications of fluoride varnish (Duraphat®, 22. 5 mg of fluoride per ml, Colgate-Palmolive) every 6th month from 1 year of age (totally 5 applications) as a supplement to a standard oral health program. The standard program (reference group) consisted of yearly dental examinations with tooth-brushing instructions, fluoride toothpaste enforcement and dietary counselling. The children of the reference group did not receive any fluoride varnish applications. At the end of each dental visit, all participating children were given a free tube of fluoride toothpaste (1100 ppm) and a soft toothbrush.

Sample collection

The samples were collected with aid of a sterile swab that was rotated inside the lips to capture saliva and supra-gingival plaque. The swab was then transferred to a test tube coded with a unique number and immediately sent by surface mail to the laboratory. The samples were stored frozen at − 20 °C until further processing. The samplings were performed between March and November 2013 and the samples were analysed within 3–15 months (median 12 months) after collection.

Microbial analyses

The samples were processed with the checkerboard DNA-DNA hybridization technology as described by Wall-Manning et al. (2002) [11]. DNA was extracted with mutanolysin and lysozyme as previously described [12] and the DNA quality was evaluated from the UV extinction at 260 nm using NanoDrop 2000 (Thermo Scientific, Fisher Scientific, Gothenburg, Sweden). Whole genomic DNA probes were prepared from a panel of 12 bacterial species with strain designation according to the Culture Collection, University of Gothenburg (CCUG), or Oral Microbiology, Gothenburg, Sweden (OMGS): Actinomyces odontolyticus OMGS G67; Bifidobacterium dentium OMGS G174; Capnocytophaga ochracea OMGS 1233; Haemophilus parainfluenzae CCUG 12836; Lactobacillus casei OMGS 3184; Lactobacillus salivarius OMGS 3830; Neisseria subflava CCUG 23930; Streptococcus intermedius CCUG 17827; Streptococcus oralis OMGS 2470; Streptococcus mutans OMGS 2482; Streptococcus salivarius OMGS 2473; Veillonella parvula OMGS G186. The detection level was > 10⁴ cells per mL sample.

Caries data

Caries data was extracted from the clinical examination at 3 years of age performed by the child’s regular dental team as previously described [10]. The International Caries Detection and Assessment System (ICDAS II) was used according to Ismail and co-workers [13]. Furthermore, data from a structured parental interview concerning socioeconomic conditions, the child’s general health and oral health related habits were registered.

Dropouts

Data from seven children were lost due to technical errors and these children were excluded from the final material.

Statistical analysis

All data was processed with the IBM SPSS software (version 22. 0, Chicago, IL USA). Differences in percentage distribution of bacterial growth between the groups were calculated with chi-square tests. We considered a p-value less than 0. 05 as statistically significant. A power estimation based on the prevalence of S. mutans was made since systematic reviews have highlighted this bacterium as a strong biomarker for caries development in early childhood [14, 15]. We anticipated that a 50% difference in the prevalence of high counts (≥ 10⁵ cells) would be of “clinical importance”. With α and β set at 0. 05 and 0. 2 respectively, it was calculated that 170 participants in each group should be recruited to get sufficient power to limit the risk of Type I and Type II errors.

Results

The background characteristics and caries data of the participating children is summarized in Table 1. The proportion of parents with immigrant background (other language than Swedish at home) was significantly higher in the test group (p < 0. 05). Likewise, one or both parents were more often smoking on a daily basis in the test group. The children of the test group displayed also a higher prevalence of caries than the reference group.

Variable	Fluoride	Standard	P
	(Test)	(Reference)
	n = 263	n = 237
Girls/boys	55/45%	53/47%	NS
Chronic disease (yes)	11%	10%	NS
Immigrant background^a (yes)	86%	76%	p < 0. 05
Mother’s education (< 9 years)	22%	21%	NS
Family income (< 20, 000 SEK/month)	46%	37%	NS
Mother and/or father smoking (yes)	42%	34%	p < 0. 05
Candy (> 1 week)	37%	32%	NS
Sweet drinks (> 2 times per day)	6%	2%	p < 0. 05
Daily tooth brushing (yes)	91%	94%	NS
ICDAS 1–6 (all lesions)	29%	17%	p < 0. 05
ICDAS 3–6 (moderate/extensive lesions)	17%	8%	p < 0. 05

The microbial data is presented in Table 2. Gram positive streptococci dominated the samples in both groups; there were no significant differences in the prevalence of S. intermedius, S salivarius and S. mutans between the groups while S. oralis seemed to occur less frequently in the reference group (p < 0. 05). Among the non-streptococci, V. parvula, L. salivarius, B dentium and H. parainfluenze were frequently harboured in both groups albeit in low counts. High counts were commonly displayed for S. salivarius and N. subflava. The results from the checkerboard hybridisation in relation to caries prevalence are shown in Table 3. The prevalence (ICDAS 1–6) for the entire study group (n = 500) was 23. 4% and we found a more frequent occurrence of V. parvula among children with caries compared with those that were caries free (ICDAS 0). On the other hand, the B. dentium, L. casei, L. salivarius and N. subflava strains were more frequently detected from the caries-fee children (p < 0. 05). No differences were obtained concerning A. odontolyticus or any of the streptococci strains.

Strain	Fluoride (Test)		Standard (Reference)
	n = 263		n = 237
	≥ 10⁴	> 10⁵	≥ 10⁴	> 10⁵
Actinomyces odontolyticus
Bifidobacterium dentium
Capnocytophaga ochracea
Haemophilus parainfluenzae
Lactobacillus casei
Lactobacillus salivarius
Neisseria subflava
Streptococcus intermedius
Streptococcus mutans
Streptococcus oralis			72^a
Streptococcus salivarius
Veillonella parvula

Strain	Caries (ICDAS 1–6)		Caries-free (ICDAS 0)
	n = 117		n = 383
	≥ 10⁴	> 10⁵	≥ 10⁴	> 10⁵
Actinomyces odontolyticus
Bifidobacterium dentium			40^a
Capnocytophaga ochracea
Haemophilus parainfluenzae
Lactobacillus casei			4^a
Lactobacillus salivarius			44^a
Neisseria subflava			30^a	9^a
Streptococcus intermedius
Streptococcus mutans
Streptococcus oralis
Streptococcus salivarius
Veillonella parvula	56^b

1. ^aSignificantly higher compared with children with caries (p < 0. 05; chi-square test)

2. ^bSignificantly higher compared with caries-free children (p < 0. 05; chi-square test)

3. Values in table denote percentage

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