qPCR assay optimisation for a clinical study comparing oral health risk in Rett syndrome.

PURPOSE: This study aimed to validate qPCR assays for specific microbiota, for use on dental plaque samples stored on Whatman FTA cards to compare relative oral health risk in Rett syndrome. METHODS: Supragingival dental plaque samples were collected, using a sterile swab, (COPAN FLOQswab™) swabbed onto Whatman FTA™ cards. DNA extraction was performed using a modified Powersoil™ protocol. Where published assays were unsuitable, species-specific qPCR assays for caries-associated, gingivitis-associated and oral-health-associated bacteria were designed using multiple sequence alignment, Primer3Plus and PrimerQuest. Assays were run using absolute quantification. Limit of detection (LOD) and limit of quantification (LOQ) were calculated, and PCR products verified by Sanger sequencing. RESULTS: Most assays allowed detection using real-time qPCR with high specificity on samples collected on FTA cards. Several assays showed low or even single gene copy numbers on the test samples. CONCLUSION: Assays were optimised for detection and evaluation of oral health risk in dental plaque samples stored on FTA cards when cold storage is not feasible, except for F. nucleatum. Several assays showed gene copy numbers less than the LOQ or outside the range of the standard curve, so there is merit in optimising these assays using digital droplet PCR.

Oral health care and service utilisation in individuals with Rett syndrome: an international cross-sectional study.

BACKGROUND: There is a dearth of literature available on the comparative oral health status of those with Rett syndrome (RTT) despite diurnal bruxism being a supportive diagnostic criterion for the disorder. This study was designed to investigate the dental experiences of individuals with RTT in terms of perceived at-home and professional dental care. METHODS: Using data in the InterRett database, provided by English-speaking families of individuals with a confirmed MECP2 genetic mutation, the study investigated relationships between dental problems, oral care, child factors including bruxism and use of gastrostomy, and socioeconomic indicators. The study also explored relationships between dental presentations and socioeconomic, child, and family-related factors. RESULTS: Individuals with RTT exhibiting bruxism were more likely to access dental treatment. Those who had full oral feeding had a higher incidence rate of dental treatment than those with full tube feeding. A conservative (under) estimation of the overall dental caries progression rate revealed that this may be similar to that of the normal population. CONCLUSIONS: Drivers for dental treatment in RTT include bruxism as well as dental caries. Those who have full oral feeding experience more dental treatment than those with full tube feeding. A higher maternal education level may confer a protective effect for oral health outcomes in those with RTT. Nevertheless, families generally tended to value the importance of oral health despite reported difficulties in day-to-day mouth care.

Oral health experiences of individuals with Rett syndrome: a retrospective study.

BACKGROUND: There is relatively little literature on the oral health experiences of individuals with Rett syndrome. This study described the incidence of dental extractions and restorations in a population-based cohort, according to a range of demographic and clinical factors. The association between bruxism and age was also investigated. METHODS: Existing questionnaire data in the population-based Australian Rett Syndrome Database for the years 2004, 2006, 2009 or 2011 on genetically confirmed female cases (n = 242) were analysed. RESULTS: The incidence rate of restorations and extractions were 6.8 per 100 person years (py) and 9.3 per 100 py respectively. The incidence of extractions decreased with increasing levels of income. Compared to those with a C-terminal mutation, the incidence rate of extraction was higher for those with large deletions (Incidence Rate Ratio (IRR) 4.93; 95% CI 1.46-16.7, p = 0.01). There was a 5% decrease in the risk of frequent bruxism for every one-year increase in age (Risk Ratio 0.95; 95% CI 0.94-0.97). CONCLUSIONS: Social advantage may provide some protection for dental health in individuals with Rett syndrome. Those with more severe genotypes seemed to have poorer oral health outcomes.