Forty children and young people participated in this study: 19 males and 21 females, ged 2 to 22 years old (mean age 8.6 years). All had neurologiacal impairment (cerebral palsy and genetic syndrome) and neurogenic oropharyngeal dysphagia confirmed in their medical records. Participants were divided into two groups: Group I (GI, n = 20), composed of children and young people using gastrostomy, and Group II (GII, n = 20), composed of children and young people with oral feeding. Exclusion criteria were : children and young people with physical conditions that prevented the collection of clinical specimens, with an unstable general clinical picture, with the absence or incomplete eruption of dental elements number 11, 31, 16, 26, 36, and 46 (permanent dentition) or 51,71, 55, 65, 75, and 85 (primary dentition) necessary for evaluation and continuous antibiotic use.
The research project was approved by the Research Ethics Committee of the institution under the number 4.391.196. All those responsible for the individuals included in the study protocol gave informed consent for participation. The selection of children and young people occurred according to their presence in outpatient care and previous analysis of medical records confirming the diagnosis of oropharyngeal dysphagia. They were invited to participate in the research and received explanations about the objectives and the method of collection. Such children and young people are followed up at the Specialties Clinic of Hospital XXX.
Assessment of oral hygiene and saliva collection were always performed by the same two professionals, one from the dentistry area and the other from the nursing area, both trained and experienced in the field. The child or young person was positioned in the Kavo dental chair, model Unik, or even in their own wheelchair, according to the positioning need. During evaluation and collection, sterile gloves, dental oral clinical mirror, dental explorer probe, disposable plastic sucker, triple syringe attached to the dental chair with an air and water jet, reflector light from the dental chair, and an oral swab were used.
Simplified Oral Hygiene Index (SOHI)
In order to qualify oral hygiene, quantification of plaque deposit and dental calculus was performed in the entire sample population involved. Evaluation was carried out by the same professional specializing in the field of pediatric dentistry, with seven years of experience in serving this population. For this, the Simplified Oral Hygiene Index (SOHI) proposed by Greene and Vermillion (1964) 13 was applied, in which the existence of a plaque or calculus was verified on the buccal surface of the number 11 dental element (upper right central incisor), 31 (lower left central incisor), 16 (upper right first molar) or 26 (upper left first molar), or on the lingual surface of element 36 (lower left first molar) or 46 (lower right first molar). In the absence of one element, we replaced it with another from the same group; that is, we used the same dental arch and the same quadrant. Only fully erupted elements were considered. In deciduous or mixed dentition, the buccal surface of dental elements number 51 (upper right central incisor), 71 (lower left central incisor), 55 (upper right first molar), 65 (upper left first molar) was evaluated, as well as the lingual surface of elements 75 (lower left first molar) and 85 (lower right first molar).
Saliva collection
After cleaning the mouth with 100 mL of water, saliva was collected by scraping the inside of the cheeks with sterile swabs, making circular movements approximately 30 times. These swabs were cut and placed in 2mL microtubes with gel inside. The collected samples were stored in a refrigerator at 4°C for a period of up to seven days before the extraction of genomic DNA.
Bacterial analysis
The saliva collected by oral swab allowed the investigation of two bacteria, Streptococcus mutans and Streptococcus sobrinus, through the expression of mRNA as measured by polymerase chain reaction (PCR). The analysis process is described below:
DNA was extracted with the commercial DNA isolation kit (Puregene, Gentra Systems, Minneapolis/EUA). In the microtubes containing the swabs, 300 µL of lysis solution was added. Then, 1.5 µL of proteinase K (20 mg/mL) and 100 µL of precipitation solution were added. Then, 300 µL of 100% isopropanol and 0.5 µL pf glycogen (20 mg/mL) were added, and the tubes were centrifuged at 1500 rpm for 3 min. The supernatant was discarded, and the tube was inverted onto absorbent paper. Then, 300 µL of 70% ethanol was used to wash the DNA. The tubes remained open 15 min for evaporation of residual ethanol, and the DNA was dissolved in 20 µL of DNA elution solution.
Extracted DNA samples were subjected to electrophoresis in 1.5% agarose gel in TBE (tris, boric acid and EDTA 0.001 M, pH 8.0) containing ethidium bromide at a concentration of 0.5 ug/mL of gel and observed in a Hoefer transluminator (model Macro-Vue UV-20) to check its integrity. The concentration of the DNA samples obtained were measured in a spectrophotometer (Ultrospec III, Pharmacia LKB Biochrom Ltd, Cambridge, England), at 260 nm. The 260/280 ratio equal to 1.8 was used to characterize the purity of the material. The samples were stored at 4 ºC until use.
In PCR, amplification of the constitutively expressed gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was carried out as an internal control for the reactions to confirm whether the DNA extraction process was successful. The Invitrogen protocol was adopted: in a sterile tube was added 2.5 µL of 10× PCR buffer, 0.75 µL of MgCl2 (50 mM), 0.5 µL of dNTP mix (10 mM), 1.0 µL of oligonucleotide primer forward (2 mM), 1.0 µl of oligonucleotide primer reverse (2 mM), 0.5 µL of DNA sample, 0.2 µL of Taq DNA polymerase (5 units/µL) and distilled and autoclaved water to complete the final volume of 25 µL. The reaction was performed on the Perkin Elmer GeneAmp PCR System 2400 thermal cycler, under the conditions of 94°C for 45 s (denaturation), 60°C for 45 s (annealing), 70°C for 1 minute (extension), and 70°C for 15 min (final extension), for 25 cycles.
The FIREpol protocol was adopted for the amplification of S. mutans and S. sobrinus by PCR. In a sterile tube, 4 µL of 5× Master Mix, 0.6 µL of forward primer oligonucleotide (10 µM), 0.6 µL for reverse primer oligonucleotide (10 µM), 1.0 µL of the DNA sample and distilled, autoclaved water was added to give the final volume of 20 µL. This reaction was also performed on the Perkin Elmer GeneAmp PCR System 2400 thermal cycler, under conditions of 95°C for 30 s (denaturation), 59°C for 30 s (annealing), 72°C for 1 min (extension) and 72°C for 5 min (final extension).
The sequences of the oligonucleotide primers for the amplification of GAPDH and specific bacteria are show in Table 1.
Table 1. Sequences of oligonucleotide primers used for the amplification of GAPDH and bacteria Streptococcus mutans and Streptococcus sobrinus.
The amplified DNA samples were subjected to electrophoresis on 1.5% agarose gel in TBE containing ethidium bromide at a concentration of 1.0 µg/mL of gel and observed in a Hoefer transilluminator model Macro-Vue UV-20 to verify the expression of the analyzed genes.
Statistical analysis
The data were previously analyzed for normality using the Kolmogorov-Smirnov test at 5% probability. Once the presence of the normal distribution of the analyzed variables was not confirmed, it was decided to use non-parametric statistics. Thus, the Mann-Whitney test was applied to compare groups in relation to oral hygiene, with the median and interquartile range (IQR) values being presented together for each group. For the analysis of bacteria, the chi-square test was used. For both tests, the level of 5% probability was used. All statistical analyses were conducted using the R software package.