## 2 Materials and Methods  
### Study Sample  
This study evaluated the effectiveness of laser therapy combined with two different types of protocols. Thirty patients aged between 18 and 65 (18 men and 12 women) were recruited. Patients were treated between February 2019 and January 2020 at a private practice in Bergamo, Italy, and all of them signed the appropriate informed consent. The present pilot study was approved by the ethics committee of the School of Medicine and Surgery at Milano Bicocca University (protocol n. 11/17), derived from the approval of the Italian National Institute of Health (ISS, Rome, Italy) prot. 30/07/2007-0040488, and it was conducted in observance of the Declaration of Helsinki. All patients met the inclusion criteria described below. The study population was randomly divided into 3 groups of 10 subjects each:  
* GROUP A: patients treated following the LANAP protocol;
* GROUP B: patients treated following the OHLLT protocol;
* GROUP C: patients treated exclusively with scaling and root planing (control group). From each patient with periodontal disease, a triple sampling of subgingival plaque was performed in 2 distinct sites of the oral cavity. The first sampling was carried out before the application of the protocols, the second happened 1 week after the end of the treatments, and the third was 9 months after the end of the treatments. The operative procedure for subgingival plaque removal was carried out through the following steps: removal of supragingival plaque, isolation of the operative field, sampling with a sterile paper cone, insertion in the appropriate tubes, fast and careful shipping by a courier and delivery of the sample to the laboratory in good time. Each plaque sample arrived in the laboratory accompanied by a patient card containing information about the patient's age, sex, date of recruitment, tooth number and sampling site where the sample was taken, along with the depth of the periodontal pocket (PPD) and any presence of bleeding on probing or plus at the time of sampling. The primers and probe oligonucleotides were designed based on 16S rRNA gene sequences of the Human Oral Microbiome Database (HOMD 16S rRNA RefSeq Version 10.1), totaling 845 entries. All the sequences were aligned in order to find either a consensus sequence or less conserved spots. Three real-time polymerase chain reaction (RT-PCR) runs were performed for each sample. The first reaction quantified the total amount of bacteria using two degenerate primers and a single probe matching a highly conserved sequence of the 16S ribosomal RNA gene. The second reaction detected and quantified the three red complex bacteria (i.e., _P. gingivalis, T. forysthia_ and _T. denticola_) in a multiplex RT-PCR. The third reaction detected and quantified Aggregatibacter actinocyetemomi-tans, Fusobacterium nucleatum, Echinococcus corrodens and Campylobacter rectus. The oligonucleotide concentrations and RT-PCR conditions were optimized to ensure sensitivity, specificity and no inhibitions in case of unbalanced target amounts. Absolute quantification assayswere performed using the Applied Biosystems 7500 Sequence Detection System. The amplification profile was initiated by a 10 min incubation period at 95 \({}^{\circ}\)C to activate the polymerase, followed by a two-step amplification of 15 s at 95 \({}^{\circ}\)C and 60 s at 57 \({}^{\circ}\)C for 40 cycles. All these experiments were performed including nontemplate controls to exclude reagent contamination. Plasmids containing synthetic DNA target sequences (Eurofin MWG Operon, Ebersberg, Germany) were used as a standard for the quantitative analysis. Standard curves for each target were constructed in a triplex reaction by using a mix of the same number of plasmids in serial dilutions ranging from 101 to 107 copies. There was a linear relationship between the threshold cycle values plotted against the log of the copy number over the entire range of dilutions (data not shown).