Saliva samples were collected from participants in the morning (at least 2 h after brushing or consumption of meals) by the cotton swab method, as previously described
25)
using the saliva collection system SalivaBio
®Oral Swab and Swab Storage Tube (Salimetrics, State College, PA, USA) prior to oral examination. The samples were immediately frozen after collection and stored at −80℃ prior to DNA extraction.
All participants were subjected to a full-mouth clinical examination by certified dentists. Their periodontal condition was evaluated by measuring the periodontal pocket depth (PPD) and by bleeding on probing (BOP) at six sites for all teeth; the periodontal inflammation surface area (PISA), a periodontal parameter that quantifies the amount of inflamed periodontal tissue
26)
, was also calculated. A diagnosis of periodontitis was made following the recent diagnostic criteria for periodontal diseases based on the World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions
27)
.