Effects of chlorhexidine preprocedural rinse on bacteremia in periodontal patients: a randomized clinical trial

Abstract Objective: Single dose of systemic antibiotics and short-term use of mouthwashes reduce bacteremia. However, the effects of a single dose of preprocedural rinse are still controversial. This study evaluated, in periodontally diseased patients, the effects of a pre-procedural mouth rinse on induced bacteremia. Material and Methods: Systemically healthy individuals with gingivitis (n=27) or periodontitis (n = 27) were randomly allocated through a sealed envelope system to: 0.12% chlorhexidine pre-procedural rinse (13 gingivitis and 13 periodontitis patients) or no rinse before dental scaling (14 gingivitis and 15 periodontitis patients). Periodontal probing depth, clinical attachment level, plaque, and gingival indices were measured and subgingival samples were collected. Blood samples were collected before dental scaling, 2 and 6 minutes after scaling. Total bacterial load and levels of P. gingivalis were determined in oral and blood samples by real-time polymerase chain reaction, while aerobic and anaerobic counts were determined by culture in blood samples. The primary outcome was the antimicrobial effect of the pre-procedural rinse. Data was compared by Mann-Whitney and Signal tests (p<0.05). Results: In all sampling times, polymerase chain reaction revealed higher blood bacterial levels than culture (p<0.0001), while gingivitis patients presented lower bacterial levels in blood than periodontitis patients (p<0.0001). Individuals who experienced bacteremia showed worse mean clinical attachment level (3.4 mm vs. 1.1 mm) and more subgingival bacteria (p<0.005). The pre-procedural rinse did not reduce induced bacteremia. Conclusions: Bacteremia was influenced by periodontal parameters. In periodontally diseased patients, pre-procedural rinsing showed a discrete effect on bacteremia control.


Introduction
Periodontal diseases contribute to systemic bloodstream and the migration of microorganisms and their products throughout the body 14,15 . Bacteremia can be induced by simple daily habits such as oral hygiene 14,16 and mastication 6,9 or by more invasive procedures such as dental scaling 13,14,20,30 . The intensity and local infection affect bacteremia 27  We hypothesised that susceptible individuals undergoing manual dental scaling develop bacteremia, which could be reduced employing a pre-procedural rinse. Therefore, this study evaluated whether a single dose of pre-procedural mouthrinse in periodontally diseased patients reduces bacteremia stimulated by manual dental scaling. We also analyzed the occurrence and magnitude of bacteremia based on the results from culture and real-time polymerase chain reaction (PCR) techniques.  The sample size calculation was determined based on a previous study 6 and adjusted after the pilot study (n=2 per group) that included the processing of realtime PCR. Volunteers from the pilot study did not participate in the main study. The minimum number of 12 individuals per group was designed to provide According to periodontal condition, two blocks of patients were randomly allocated to one of two groups by opaque closed and numbered envelopes containing non-rinse groups (Figure 1). One hour prior to the induction of bacteremia, subgingival samples were collected 5 and stored at -80°C in empty minitubes. Blood samples were collected 6 at 3 sequential times: before dental scaling (T0), 2 minutes (T1), and 6 minutes (T2) after dental scaling had started. At each time, 2 mL of peripheral blood was collected into citrated vacuum tubes, which were slightly agitated to prevent the blood from coagulating. One milliliter was pipetted from the tube and poured into a second tube containing 1 mL of a 1% sterile solution of sodium polyanethol sulfonate (Sigma Chemical Co., St. Louis, MO, USA) to deactivate complement proteins. The additional 1 mL was stored at -80°C until PCR processing.

Real-time PCR procedures
First, DNA was extracted from the samples.
To quantify the total bacterial load and the Bacterial culture procedures Immediately after collection, blood samples were spread out onto agar plates in duplicate 6,9 . Tryptic Soy Agar (TSA), incubated for 2 days at 35°C, was used to determine total aerobic counts, while Schaedler Blood Agar (SBA), incubated for 5-7 days at 35°C in an anaerobic chamber, was used to determine total anaerobic counts. Bacterial colony-forming units (CFU) were counted by an automated colony-counting system by a single researcher.

Statistical analysis
Bacteremia occurrence (presence and/or increase of bacterial levels in the blood) was determined according to dental scaling. In addition, bacteremia data was compared between the groups (gingivitis vs. periodontitis) and among the times of sampling (preand post-scaling). The association between oral and blood bacterial levels and between PD and CAL and bacteremia were statistically analyzed. The bacterial levels determined by both laboratorial techniques were compared.
The primary outcome was the antimicrobial effect of a single mouth rinse use. To check this effect on bacteremia, the levels of viable anaerobic and aerobic bacterial cells were compared between 0 (T0) and 2 (T1) minutes, 0 (T0) and 6 (T2) minutes, and 2 (T1) and 6 (T2) minutes. Bacterial levels at 2 and 6 minutes were compared among the volunteers who performed the pre-procedural rinse and those who did not. As a secondary analysis, similar comparisons were performed considering the qPCR results.
Mann-Whitney and Signal tests were used in the above-mentioned comparisons (p<0.05). Data from the intention-to-treat analysis did not differ from the per protocol analysis.    Health care procedures, such as periodontal t h e ra p e u t i c s c a l i n g , c a n i n d u c e t ra n s i t o r y bacteremia 13,14 . In dentistry, the use of antimicrobial mouthrinses has different aims, however, the number of studies evaluating the effectiveness of preprocedural rinses on bacteremia is scarce, especially when considering RCT design in periodontal research.

Differences of mean bacterial levels (T1,T2) -T0
Due to its well-known antimicrobial properties, 0.12% chlorhexidine is one of the most recommend pre-procedural mouthrinse 29 . Therefore, this study evaluated blood bacterial levels after dental scaling, days reduced bacteremia after mastication in gingivitis individuals 6,9 . However, in periodontitis patients, subgingival irrigation with essential-oils combined with one-week rinsing was not enough to decrease bacteremia following quadrant scaling 20 . Therefore, it could be speculated that to reduce bacteremia, antimicrobials would be prescribed 15 days before dental scaling and possibly continued for the duration should be done aiming to control aerosol contamination and intra-oral infection.
Dental professionals should be able to appropriately manage periodontal patients and recognize bacteremia.
In periodontally compromised individuals, high rates of bacteremia before scaling indicated the occurrence of bacteremia associated with daily activities. Preprocedural rinse did not reduce, effectively, levels of bacteria in the blood. Other strategies should be adopted to reduce bacteremia. The prescription of mouthrinse as a preventive measure against bacteremia should be further investigated. An Effects of chlorhexidine pre-procedural rinse on bacteremia in periodontal patients: a randomized clinical trial appropriate management of bacterial biofilm in the subgingival area could contribute to prevent bacteremia in periodontally diseased individuals.
Pre-procedural rinse did not reduce, effectively, levels of bacteria in the blood. Other strategies should be adopted to reduce bacteremia in periodontally diseased individuals.

Conclusions
Dental scaling induced bacteremia in both gingivitis and periodontitis. However, bacteremia increased as periodontal compromising increased. The magnitude of bacteremia was greater among periodontitis patients.
In periodontally diseased patients, pre-procedural rinsing showed a discrete effect on bacteremia control.