This prospective controlled study is a part of long-term research project supported by the Ministry of Health of the Czech Republic and performed at the Department of Dentistry and Neonatology Faculty of Medicine and University Hospital in Pilsen and Hradec Kralove. Ethical approval for the investigation was obtained from the Research Ethic Committees of both institutions. The study was conducted in accordance with the Helsinki Declaration of children´s rights 1975 (revised 1983). The study group infants were selected from the Departments of Neonatology, University Hospital in Pilsen and Hradec Kralove. The control group infants were selected similarly from the Department of Paediatric Dentistry of both institutions. The laboratory tests were performed at the Department of Microbiology and Immunology, University Hospital in Hradec Kralove and the Research Institute of Clinical and Experimental Dentistry, University Hospital in Prague. The written informed consent was obtained from all patents of all infants before the study.
Study group inclusion criteria were: corrected age one year, birth weight below 1500 grams and gestation age shorter than 37 weeks. All consecutive children coming for the paediatric examination at the time of corrected age one year were recruited during the period of one year.
The total number of pre-term, VLBW infants born during the recruitment period was 157. The reasons for dropout from evaluation were especially child's death, serious health complications, remote residence and parental disapproval of participation in the study. Finally 102 VLBW were evaluated.
As pre-term born infants are not fully mature at the time of birth, their chronological age does not correspond to their true biological age. Hence, a comparison with full term infants can only be made if the age of pre-term infants is corrected. The corrected age was calculated by subtracting the number of weeks born before 40 weeks of gestation from the chronological age.
Control group including criteria were chronological age (a measure of an individual's age based on the calendar date on which he or she was born) one year, birth weight above 2500 grams, gestation age longer, then 37 weeks and no systemic disease. These children were selected over the period of one year as they came for their first dental examination at one year of age.
Altogether 189 of one year old infants (102 VLBW and 87 NBW) were included in the study. In two cases there were twins in the study group. Information on their gestational age, mode of delivery, Apgar score (Apgar is a quick test performed on a baby at 1 and 5 minutes after birth. The 1-minute score determines how well the baby tolerated the birthing process. The 5-minute score tells the health care provider how well the baby is doing outside the mother's womb), general health status (congenital heart defects, renal and respiratory disorders, neurological disorders, vision impairments), birth weight and antibiotic use were obtained from hospital records. Feeding practice, bed-time sugar drinks and oral hygiene onset were obtained from questionnaires. All infants participated in the study were residents of communities with low natural water fluoride content (< 0.3 ppm). Children were examined clinically at the age of one year (corrected in VLBW, chronological in NBW) by two trained calibrated examiners using the dental mirror and headlight. Examiners were trained to provide treatment using the protocol approved for this study. Inter- and intra-examiner reliability was evaluated by using kappa coefficients before the start of the study. The standard methodology recommended by WHO has been used (https://apps.who.int/iris/handle/10665/66783). The dental examiners were blinded to the group of infants. The number of erupted teeth (if any part of its crown had penetrated the oral mucosa), developmental defects of enamel (DDE) based on DEE criteria and hard palate deformities were evaluated [8] . The morphology assessment of the infant palate was visual using the non-standardized visual and metrical measurements, because the adequate three-dimensional shape methodology has not been developed [9].
Samples for microbial analysis
Samples for microbial evaluation were taken from both children (91 VLBW and 86 NBW) and mothers (89 VLBW and 86 NBW). In two cases of the VLBW study group there were twins. Some samples were collected in insufficient quantities for microbial and serotype analysis, therefore there were less samples then primary included individuals. Unstimulated saliva samples from the dorsum of the tongue of children and their mothers were collected with a sterile cotton swab. Each swab was transferred into a sterile tube for the detection and identification of the main known cariogenic pathogens Streptococcus mutans, Streptococcus sanguinis, Streptococcus salivariu and Streptococcus viridans. Samples for the detection of cariogenic bacteria were inoculated on the selective diagnostic media Mitis Salivarius Agar (OXOID, UK) and incubated 48 hours in 37 degree of Celsius at atmosphere with 10% CO2. The main characteristics of the cariogenic bacteria were identified (STREPTOtest 24, ERBA-LACHEMA). Serotypes of the isolated Streptococcus mutans were detected using the latex agglutination test Prolex Streptococcal Grouping Latex Kit (BioVendor-Laboratorní medicína a.s, Czech Republic) according the manufacturer instructions. 74 isolates from children and 90 isolates from mothers (both VLBW and control group) were used for the serotyping.
Statistical analyses
Data were described by absolute and relative frequencies of categorical variables and median of quantitative variables. For comparison of categorical variables in groups chi-square test was used, in case of quantitative variables the Mann Whitney U test was adopted, kappa statistics was used for correlation. Analysis was performed in the Stat Graphics Software (Stat Point Technologies, Inc. Warrenton, Virginia, USA), MedCalc v. 9.5.2.0 (MedCalc Software, Belgium) and Microsoft Excel 2016 (Microsoft, USA). Statistical significance was set at p<0.05 two sided.