key: cord-1017932-qqoqlfzb
authors: Bahador, Mason; Alfirdous, Rayyan A.; Alquria, Theeb A.; Griffin, Ina L.; Tordik, Patricia A.; Martinho, Frederico C.
title: AEROSOLS GENERATED DURING ENDODONTIC TREATMENTS: A SPECIAL CONCERN DURING COVID-19 PANDEMIC
date: 2021-02-03
journal: J Endod
DOI: 10.1016/j.joen.2021.01.009
sha: 81a454b50a41deebca4ab51439a3693dad77b4bd
doc_id: 1017932
cord_uid: qqoqlfzb

AIMS: (1) To investigate aerosolized microorganisms generated during endodontic emergencies and non—surgical root canal therapy (NSRCT); (2) To assess how far airborne microbial spread; and (3) To verify the spatial distribution of airborne microbial spread. METHODS: A total of 45 endodontic procedures were sampled, including full pulpotomy (n=15), pulpectomy (n=15), and NSRCT (n=15). Samples were collected during room resting (s1) and after the treatment (s2). The passive air sampling technique using settle plates was applied. Agar plates were set at different locations in the operatory. The colony-forming unit (CFU) was counted in BHI blood agar plates. A set of agar plates containing selective chromogenic culture media was used for the isolation and presumptive identification of target microorganisms. Fungi were investigated using Sabouraud Dextrose Agar. RESULTS: Pulpotomy generated the lowest mean CFU count (p<0.05). There was no difference between the mean CFU counts found in pulpectomy and NSRCT (p>0.05). A higher mean CFU count was found close to the patient’s mouth (0.5m) than at a 2m distance in pulpectomy and NSRCT (P<0.05). There was no difference between the mean CFU count found in front of the patient’s mouth versus diagonal in pulpectomy and NSRCT (p>0.05). S. aureus (22/ 45, 48.8%) was the most frequent bacteria species. Longer treatment times were associated with higher CFU counts. CONCLUSION: Our findings indicated that pulpotomy generates less aerosolized microorganisms than pulpectomy and NSRCT. Moreover, the proximity to the patient’s mouth and the treatment duration implicated in the level of contamination.

Aerosols generated during dental procedures have recently taken the forefront of discussion in dentistry because of coronavirus disease 2019 disease (COVID-19) pandemic (1) (2) . As a result, there is a likely risk of transmission of Acute Respiratory Syndrome Coronavirus 2 virus (SARS-CoV-2) in dental practice (3). The primary mode of SARS-CoV-2 transmission is aerosol/droplet spread and contact with virus-contaminated surfaces acting as fomites (4). Due to the dual risk of high aerosol-generating in dentistry, plus saliva-borne SARS-CoV-2 in both symptomatic and asymptomatic individuals, dental associations, immediately implemented guidelines restricting aerosol-generation procedures at the early stage of the COVID-19 pandemic. However, dental associations' response to curb the clinic associated nosocomial transmission of SARS-CoV-2 varied at that time (5-6). Despite this guidance, the practitioners were still reluctant and fearful of disease transmission and crosscontamination within the dental clinic environment (7) .

Recommendation to avoid aerosol-generating procedures at the early stages of the pandemic posed significant challenges for managing dental emergencies, particularly to endodontists (3, [8] [9] . To avoid aerosol-generating procedures, palliative care with pharmacologic management of pain became the primary treatment rather than treating the endodontic emergencies with definitive root canal therapy, e.g., non-surgical root canal therapy (NSRCT) (3). The secondary management (3) for endodontic emergencies, in particular to symptomatic irreversible pulpitis (the most common endodontic emergency) (9) and symptomatic apical periodontitis, became full pulpotomy (10) (11) . The selection of full pulpotomy as secondary management was due to advantageously reduced treatment time, which could minimize endodontist's risk of being exposed to SARS-CoV-2 infection. At that time, the endodontists raised the question whether definitive root canal therapy or the full pulpotomy generates more aerosolized microorganisms.

Over the years, most studies in dentistry have investigated bacterial aerosols generated during restorative and periodontal procedures (1-2). However, current research has not assessed aerosolized microorganisms during root canal therapies. A systematic review and meta-analysis (1) suggested that studies are needed to measure aerosol contamination during dental procedures. The lack of studies evaluating aerosolized microorganisms in endodontic procedures and especially this meta-analysis raised concerns among endodontists.

Our study focuses on investigating aerosolized microorganisms generated during different root canal therapies due to this lack of evidence. First, we successfully investigated J o u r n a l P r e -p r o o f the aerosolized microorganisms generated during endodontic emergency procedures (pulpotomy and pulpectomy) and non-surgical root canal therapy (NSRCT), describing the microbial load and composition; second, we assessed how far airborne microbial spread during endodontic procedures and the level of contamination; third, we verified spatial distribution of airborne microbial spread during endodontic procedures and the level of contamination.

This study was approved by the local Institutional Review Board (IRB) at the University of Maryland, Baltimore (#HP-00092103).

The passive air sampling technique using "settle plates" was applied to investigate microbial fallout during pulpotomy, pulpectomy, and NSRCT. This sampling technique has been widely used in different fields (12) (13) (14) . Microbial fallout samples were collected from a total of 45 endodontic procedures, including full pulpotomy (n=15) (10), pulpectomy (n=15), and non-surgical root canal therapy (NSRCT) (n=15). The sampling was performed in maxillary and mandibular teeth with primary root canal infection and symptomatic apical periodontitis undergoing the aforementioned treatment. Non-surgical retreatment and periapical surgery were excluded from this study. The root canals were irrigated with 2.5% sodium hypochlorite (2.5% NaOCl).

All samples were collected in the Endodontic resident's operatory in a 4 x 4 m 2 room with closed doors. Samples were obtained first in the morning after overnight room resting.

High-efficiency particulate air filters (HEPA filter), OSO Pure ADP-70 Air Disinfecting Purifier (Skaare Enterprises Inc. of Glendale, AZ, USA) was left on overnight and throughout the procedure.

For the first sample (s1), room resting sampling, a set of agar plates (Table 1) was exposed to air for 30 min in the operatory before the treatment. The plates were then closed and incubated accordingly (Table 1 ). This s1 sample was used to determine the CFU count. This s2 sample was used to determine the microbial contamination levels (CFU/plate) and the composition of target bacterial species.

To measure the fallout microorganisms, standard Petri dish plates 9 cm in a diameter containing Brain Heart Infusion agar (BHI) agar + 5% sheep-blood were used. After the incubation period (Table 1) , the number of CFU was count in the plate using a Stereomicroscope with 1000 x magnification (VWR Radnor, PA, USA). The mean number of CFU were calculated.

For the investigation of microbial composition, plates containing selective chromogenic culture media (CHROMagar) were used for the isolation and presumptive identification of target Staphylococcus spp., Streptococcus spp., and Pseudomonas spp. (Table 1) . Additionally, Sabouraud Dextrose Agar + 0.05g Chloramphenicol was used for fungi investigation (Table 1 ). All culture media were prepared according to the manufacturer's instructions. A set of standard Petri dish plate 9 cm in diameter containing the aforementioned culture media were left open throughout the treatment spatially distributed as described above. The plates were closed and incubated accordingly (Table 1) . After the incubation period, the colony's presence was verified in the agar plate, and the presumptive identification was performed according to the typical colony appearance described by the manufacturer (Table 1 ). ATCC strains were tested as a positive control before presumptive identification of the samples. 

The time required to complete the endodontic procedures was measured by stopwatch in minutes (min), beginning when first drilling with the highspeed for access cavity until the completion of the procedure with the placement of a temporary restoration.

Data were expressed as mean ± standard deviation (SD). After the Shapiro-Wilk test, data were analyzed by One Way Analysis of Variance to assess differences between groups, followed by a Tukey test for multiple comparisons. Two-way ANOVA was applied to determine differences within groups (position and distance factors). Kruskal-Wallis One Way Analysis of Variance on Ranks was used to evaluate the treatments' duration (time in minutes). To explore the possible association between time and bacteria levels, Pearson correlation analyses were performed. Correlation analyses were performed for the overall study sample. For all tests, a significance level of 5% was used.

level of contamination) -At s1, bacteria were detected in only 3/45 room resting samples with a low mean CFU value of 0.177 ± 0.386. No difference was found among the treatment modalities at s1 (p>0.05). There was a significant difference between the mean CFU values found in s1 versus s2 for all treatments (p<0.05). At s2, pulpotomy generated the lowest mean CFU count (p<0.05) ( Table 2 ). There was no significant difference between the mean CFU count found in NSRCT and pulpectomy at s2 (p>0.05) ( Table 2) .

There was a significant difference between the level of contamination encountered close to the patient's mouth (0.5 m) than at a 2 m distance both in the pulpectomy and NSRCT (p<0.05) ( Table 2) . Furthermore, there was no significant difference between the CFU count set directly in front of the patient's mouth versus diagonal (p>0.05) irrespective of the distance (0.5 or 2 m distant) both in the pulpectomy and NSRCT (Table 2) . Treatment duration (min) and level of contamination -The mean time required to complete the endodontic procedures was 34.8 ± 3.3 min for the pulpotomy, 73.7 ± 13.7 min for the pulpectomy, and 108 ± 16.8 min for the NSRCT, respectively. There was a positive correlation between the procedure duration and the level of contamination (CFU count) ( Table 3) . Longer treatment times were associated with higher CFU counts. Dispersion graphs show the correlation between the duration of the procedure and the level of contamination.

Data obtained in the present study revealed that pulpotomy generated the lowest mean CFU count when compared to pulpectomy and NSRCT (p<0.05). No difference between the mean CFU counts found in pulpectomy and NSRCT (p>0.05). We found a higher level of contamination close to the patient's mouth (0.5m) than at a 2m distance both in pulpectomy and NSRCT (P<0.05). Additionally, there was no difference between the mean CFU count found in front of the patient's mouth versus diagonal in pulpectomy and NSRCT (p>0.05).

Furthermore, longer treatment times were associated with higher CFU counts.

In this study, to achieve our results, we investigated aerosolized microorganisms using the passive air sampling technique with 'settle plate.' This sampling technique has been widely used in dentistry (12) (13) (14) . This method quantifies the viable microorganisms that can settle, grow, and multiply in a plate (15) . Some authors have listed several advantages of passive air sampling (15) . It uses Petri dishes containing culture media exposed to the air for a given time to collect biological particles (15) . These biological particles 'sediment' out and are subsequently incubated, and results are expressed in CFU. Besides collecting microbial fallout onto agar plates, it provides a valid risk assessment to measure the airborne population's harmful part (16). One of the disadvantages of this passive air sampling technique is the lack of standardization across the studies, limiting the comparison of results.

For example, Petri dishes of different diameters, exposure times, nutrient media, incubation temperatures, and times make it difficult to compare data. Here, we followed the most common parameters described in the literature for the CFU count with Petri dishes 9 cm in diameter, Brain Heart Infusion Agar (BHI-agar) supplemented with 5% Sheep-blood and incubation at 37 °C for 48h. (1), in a systematic review (SR), revealed a lack of clinical evidence on the ability of rubber dam isolation in reducing the spread during aerosol-generating procedures. It is worth pointing out that most dental aerosols studies reported in the literature are in restorative procedures and periodontal treatments with a lack of evidence in endodontic procedures. To the best of our knowledge, this is the first study to compare aerosols generated during pulpotomy, pulpectomy, and NSRCT.

The high-volume evacuation system (HVE) used here to prevent contaminated aerosols from scaping the mouth can draw a large air volume within a short period (18, 28). How far the aerosols spread and what level of contamination is of concern (13-14).

Here, for pulpectomy and NSRCT, the contamination level (CFU count) was significantly higher at 0.5m than 2m. In agreement, Monteiro et al. (14) reported a higher mean bacteria CFU count at 0.5m [21.5 (± 12.1)] at 2m [17.8 (± 9)] distance from the patient's head position during endodontic treatment. Besides investigating how far the aerosols can spread during endodontic procedures, we also investigated the spatial distribution of airborne microbial spread during endodontic procedures and contamination level. Our data indicated no difference in the contamination level found in front and diagonal of the patient's mouth.

According to Bentley et al. (32), there is an extremely variable distribution of bacterially contaminated aerosols and spatter. However, it seems to be of a consensus that the highest contamination is found close to the patient's mouth and at the patient's chest area (13) (14) 32) .

Studies on dental aerobiology reveal that, depending on airborne particles' size, they can remain suspended as aerosols or fall rapidly and splatter on objects in their trajectory (33) .

Airborne particles larger than 50 to 100 µm in diameter have initial forces greater than the frictional air forces and are ballistic in nature. True aerosol particles are usually less than 50 µm in diameter, invisible, and airborne for longer periods (33) . Of interest as infective agents are bacterial aerosol particles in the 0.5-10 µm diameter range, which can be inhaled and impinged in the terminal bronchioli and alveoli of the human lung (33) .

For the investigation of microbial composition, we used agar plates containing selective chromogenic culture media (CHROMagar) for presumptive identification of Staphylococcus spp., Streptococcus spp., and Pseudomonas spp. CHROMagar media is widely used for presumptive identification of pathogens (34) (35) . This chromogenic culture media technology is based on soluble colorless molecules (called chromogens), composed of a substrate (targeting a specific enzymatic activity) and a chromophore for the microbial investigation (36) . When the target organism's enzyme cleaves the colorless chromogenic conjugate, the chromophore is released. This method has several advantages, including easy to read at a glance, once it is a color-based differentiation method, and distinguishable with the naked eye under normal light conditions. Moreover, it allows for easy differentiation of microorganisms (36) . We examined Staphylococcus spp., Streptococcus spp., and Pseudomonas spp. was based on previous investigations reporting their occurrence in aerosols generated during dental procedures (13, (37) (38) (39) (40) (41) . Pseudomonas was investigated because it is an opportunistic pathogen present in biofilm in dental unit waterlines (DUWL) and may also be aerosolized during dental procedures (39) . Additionally, we investigated fungi' presence using Sabouraud Dextrose Agar supplemented with chloramphenicol, previously reported in dental aerosols (13) .

The most frequent bacteria species identified here were S. aureus, followed by S.

epidermidis. Previous studies have demonstrated that Staphylococcus spp. are frequently identified in dental aerosol studies (13-14, 37-38,40) . We detected oral streptococcus only close to the patient's mouth (0.5m distant) but not at 2 m. The presumptive identification of oral streptococcus found here is a relevant indicator of salivary contamination of air. Such finding in consonance with Bennett et al. (41) supporting the concept that saliva is one of the sources of pathogens in dental aerosols. Despite plausible evidence suggesting that dental unit waterlines (DUWL) might contribute to a large fraction of the microbial load in dental aerosols, mainly Pseudomonas spp. (39), we verified no colony growth in the CHROMagar Pseudomonas media. Furthermore, we recovered no fungi with sabouraud dextrose agar supplemented with chloramphenicol. It is worth pointing out that it is expected to find a certain microbial heterogeneity across clinical studies.

Up to now, there is no direct evidence indicating that the spread of microorganisms during dental treatment is a major cause of infectious disease in dentists and patients.

However, the possibility cannot be ignored, especially during the current COVID-19 pandemic, where symptomatic and asymptomatic patients carrying SARS-CoV-2 can be a source of infection in dental practices.

In light of the current COVID-19 pandemic situation, our data showed that fewer aerosolized microorganisms are generated during pulpotomy than in pulpectomy or NSCRT.

Additionally, our results indicated a higher level of contamination closes the patient's mouth with no difference in spatial distribution directly in front or diagonal of the patient's mouth.

However, it is important to highlight that our study evaluated bacteria contamination. Viruses are much smaller, and it can be speculated that viruses suspended in the air in small airborne particles can reach greater distances from the patient's mouth than was found here.

One of the limitations of this study is that because of the current COVID-19 pandemic, we could not assess individual interventions adopted here to verify their single effectiveness in reducing dental aerosols during endodontic treatment. However, while waiting for more researchers to share their preventive measures adopted to reduce aerosolized microorganisms generated during endodontic procedures during the current COVID-19 pandemic, our study shows that the preventive measures adopted here resulted overall in a low number of CFU counts for all treatment modalities.

In conclusion, our findings indicated that pulpotomy generates less aerosolized microorganisms than pulpectomy and NSRCT. Moreover, the proximity to the patient's mouth and the treatment duration implicated in the level of contamination. J o u r n a l P r e -p r o o f Table 3 . Correlation between the overall duration of the procedure (time in min) and the level of contamination (CFU count). 

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Data curation; Formal analysis; Investigation; Methodology. Rayyan Alfirdous: Data curation; Formal analysis. Theeb Alquria: Data curation; Formal analysis. Ina Griffin: Data curation; Investigation; Supervision. Patricia Tordik: Investigation

Data curation; Formal analysis; Investigation; Methodology; Project administration; Supervision; Validation

Roles/Writing -original draft; Writing -review & editing