This study was designed as a cross-sectional observational study and it is reported following the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines . The protocol of this investigation was approved by the local ethical committees in the respective countries (references 18/128-E in Spain and 197 in Albania) and considers all aspects of the Helsinki Declaration regarding experimentation involving human.
Subjects seeking dental attending the Faculty of Dentistry in Tirana (Albanian University, Albania) and the Faculty of Dentistry in Madrid [University Complutense of Madrid (UCM), Spain] were screened between April–May 2018 and March 2020 and registered the socio-demographic characteristics of the patients, such as age, gender, origin, smoking, systemic health, medications, and conditions. Patients included in this study met the following criteria: (1) age between ≥ 30 and ≤ 60. The following exclusion study criteria were also evaluated: (1) having less than 16 teeth; (2) patient with a periodontal abscess or necrotizing periodontal diseases; (3) use of systemic antibiotics in the previous month; (4) patient with relevant systemic diseases (diabetes, polymorphonuclear neutrophil defects, other immune system disorders); (5) pregnant or lactating patient; and (6) patient with current anti-inflammatories, anticonvulsant, calcium channel blockers, or immunosuppressant treatments, or 6 months prior to the sample.
When subjects fulfilled these criteria, they were verbally informed about the study and were asked to participate by signing an informed consent. Upon acceptance, each patient was appointed for the study visits.
Clinical and radiological examination
The patients received a complete periodontal and radiographic examination, including gingival recession, probing depth (PD), clinical attachment loss (CAL) and bleeding on probing (BoP)  using a UNC-15 periodontal probe (HuFriedy, Leinmen, Germany). Plaque index (PlI)  was evaluated after rinsing the patient with a plaque disclosing solution containing erythrosine (Plac-Control®, Dentaid, Barcelona, Spain).
After this visit, the included subjects were segmented by their periodontal status in three categories using the following criteria [19, 20]: (1) periodontal health and gingivitis: no CAL, no radiographic bone loss (RBL) and PD ≤ 3 mm, assuming no pseudo-pockets; (2) stages I and II periodontitis: PD 4–5 mm, mostly horizontal RBL and no tooth loss due to periodontal reasons. CAL will be 1–2 mm (stage I) or 3–4 mm (stage II), while RBL affects only the coronal third (< 15% for stage I and 15–33% for stage II); and (3) stages III and IV periodontitis: At least two non-adjacent sites with CAL ≥ 5 mm or reaching the middle third of the root, with PD ≥ 6 mm. Evidence of tooth loss due to periodontal reasons.
Samples were taken with two consecutive standardized 30# sterile paper points (Maillefer, Ballaigues, Switzerland). Paper points were inserted into the crevice or pocket and left in place for 10 s. Prior to sampling, four sites per patient were selected, one in each quadrant. The selected sites were isolated from saliva and supragingival plaque contamination with the use of cotton rolls and compressed air. In periodontal health/gingivitis subjects, subgingival samples were taken from the mesio-buccal sites of the first molars and, when absent, from the adjacent second molars (the next alternative was the second premolars and from there, any teeth present mesially). In subjects with periodontitis, subgingival samples were taken from the most accessible site with the deepest PD and BoP, per quadrant. The eight paper points were transferred into a screw-capped vial, containing 1.5 ml of reduced transport fluid (RTF) , so an individual pooled sample was obtained from each patient. Samples were sent directly (Spanish samples) or via courier (Albanian samples) to the Laboratory of Research at UCM, Spain, where they were processed within 24–36 h. RTF was the ideal transport medium, as it has been shown to maintain a good viability of anaerobes up to four days after sample collection .
Direct anaerobic culture
At the Laboratory, samples were homogenized by vortexing for 30 s, and serially diluted in phosphate buffer saline (PBS) (dilutions 10–1, 10–2, 10–3 and 10–4). For each sample, 100 µl of at least two of the dilutions were plated on non-selective blood agar medium (Blood Agar Base II, Oxoid, Basingstoke, England), supplemented with haemin (5 mg/l), menadione (1 mg/l) and 5%, sterile horse blood. Plates were incubated for up to 14 days in anaerobic conditions (80% N2, 10% CO2 and 10% H2) at 37 °C. After 7–14 days of anaerobic incubation, suspected colonies were further identified by microscopy, gram-staining and enzyme activity (see Additional file 1: Table S1). The counts of representative colonies (those with colony morphologies compatible with target pathogen morphology) were carried out.
For isolation and quantification of A. actinomycetemcomitans, another 100 µl of the 10–1 dilution of each sample and 100 µl without dilution were plated onto the selective medium Dentaid-1 , that was incubated for 3 days in air with 5% CO2 at 37 °C.
Sample size calculation
The outcome variable “proportion of the anaerobic cultivable bacteria of P. gingivalis” was selected to calculate the sample size. With a proportion of P. gingivalis in Spain of 22.21%  and in order to detect a difference in proportions of 16.72% between Albania and Spain, with a 90% of power and a significance of 95%, at least 88 patients per country were necessary. Besides, and to narrow differences between different age groups in different conditions, the overall sample was a uniformed stratified sampling, in which the same size for all the defined categories were assigned. The desired sampling distribution was 30 patients in each category (periodontal health/gingivitis, stages I–II periodontitis, stages III–IV periodontitis), and 10 patients per age cohort, 30–40 years, 41–50 years, 51–60 years, within each periodontal status category.
The statistical unit of the study was the patient. For continuous data, Kolmogorov–Smirnov test and distribution of data were used to assess normality. Data were expressed as means and standard deviations (SD), and as median and interquartile ranges (IQR) for non-parametric data. Categorical data were expressed as percentages.
Demographic data and clinical variables were analysed by Student t test, ANOVA test and chi-square test with probability values adjusted with the Bonferroni correction. For microbiological outcome variables, total anaerobic counts were calculated on blood-agar plates and expressed in total colony-forming units/ml (CFU/ml). Counts for each specific bacterial species, as well as their percentage of total cultivable bacteria, were also calculated for each patient. Counts and proportions were calculated considering all samples. The logarithmic transformation of CFU of bacterial counts was designed to normalise the data distribution. Microbiological variables were compared by t test and ANOVA test, for parametric data, and U Mann–Whitney test or Kruskal–Wallis test with Dunn–Bonferroni post hoc tests, for non-parametric data. Differences between the two countries were further explored by analysis of covariance (ANCOVA), with country as the factor, and PlI and PD were entered into the model as co-variates. In this case, ANCOVA model adjusted means and confidence intervals (CI) were calculated. Proportions of target pathogens were log-transformed to achieve homogeneity of variances.
For categorical data, chi-square test was used, with Bonferroni correction for multiplicity when was necessary.
All statistical analyses were performed using SPSS 20 program package (SPSS Inc, Chicago, IL, USA) and the level of significance was set in 0.05.