M Beerens

50 CHAPTER 3 TABLE 2. Group means of microbiologic data and outcome of independent samples t-tests with equal variances assumed. Group N Mean SD p-value (independent samples t-test) Total counts (CFU/surface) WSL 28 4.23E+07 4.58E+07 0.79 No WSL 9 3.78E+07 3.47E+07 % Aciduric flora WSL 28 40.43 25.27 0.21 No WSL 9 28.60 19.68 % S. mutans WSL 28 10.00 13.08 0.19 No WSL 9 4.02 3.62 % Lactobacilli spp. WSL 28 0.37 1.52 0.49 No WSL 9 0.01 0.02 % C. albicans WSL 28 0.03 0.10 0.36 No WSL 9 0.00 0.00 DISCUSSION In this study the automated band detection in DGGE gels proved ambiguous. The choice for minimum profiling settings either absolute or relative to the most intense band led to a very different outcome. It is concluded that DGGE has no predictive value with respect to caries risk in orthodontic patients undergoing treatment with fixed appliances. This finding is in contrast to existing literature, where DGGE was shown to have predictive value towards the development of early childhood caries (Li et al. , 2007; Yang et al. , 2010; Tao et al. , 2013; Tao et al. , 2015). However, these studies do not describe DGGE band lane detection settings in full. The choice of optimum band detection settings was not studied before. Minimum profiling settings relative to the most intense band are suggested when assessing DGGE patterns with different intensities, as is often the case when comparing clinical samples (‘Gelcompar II manual version 6.5,’ 2011). Nevertheless, in this study minimum profiling settings absolute to the most intense band had a better level of agreement with visually determined bands. Further, DGGE band patterns are best compared within one gel rather than among gels. For DGGE one is limited to a maximum of 20 specimens, including markers. The use of more samples, like in this study, implies the use of multiple gels. This issue can be overcome by aligning the gels using the lanes with mixture of reference species. The comparison of duplos from plaque samples from 4 subjects in this study, showed that a comparison among gels introduced little error.