DOI: 10.31038/JCRM.2021414

Abstract

Background: We describe the results of the application of Incobotulinumtoxin A reconstituted in a solution composed of zinc gluconate 0.02% and we evaluated whether the presence of zinc would increase or not the duration of treatment.

Methods: The sample consisted of 48 female participants, over 50 years old, with no history of chronic gastrointestinal disease or diabetes, and who had not undergone treatment with botulinum toxin less than 6 months ago. All patients answered the questionnaire with socio-demographic and health questions in which the patient’s perception of her quality of life was assessed through validated WHOQOL-Bref questionnaire before and after application.

Results: The results obtained showed that there is no relationship between race, alcoholism, smoking and use of sunscreen with the effect of the toxin in the two study groups. Concerning the variables age and amount of weekly zinc intake, the result of the Manny-Whitney test indicated that there is no significant difference between the groups, p-value>0.05. All patients were evaluated with the frontalis muscle at rest and in movement. In the group of patients who received solubilized incobotulinumtoxine A in sodium chloride, within 14 weeks of application there was a 66% reduction in the effects in patients with frontal wrinkles at rest. In the group of patients who received incobotuliniumtoxin A solubilized in zinc gluconate, within 14 weeks of application, there was a 100% reduction in effects in patients with frontal wrinkles at rest.

Conclusions: The data identified that the patients’ lifestyle habits do not influence the final result of the procedure, and that the duration of the effect is not linked to the dilution of the product with a substance different from the classic dilution with physiological saline solution.

Keywords

Zinc, Botulinum toxins type A, Facial muscles, Efficiency, Side effects, Adverse drug-related reactions, Record of clinical trial: ISRCTN27486491

Introduction

Botulinumtoxin type A is a zinc-dependent endoprotease that acts on vulnerable cells to separate the polypeptides, which are essential for the cation. When the exogenous Zinc (Zn²⁺) cation is added to the toxin that was removed by soluble chelators, the molecule coats the cation and recovers the activity of catalytic and neuromuscular block. Exogenous Zn²⁺ can restore activity of the toxin, either when the toxin is free in solution outside the cell or when internalized in cytosol [1]. Because they are zinc-dependent proteases, the question arose whether the intracellular zinc concentration could influence the action of the toxin [2].

Materials and Methods

This is a prospective, double-blind study. The sample size was composed of 48 individuals and was calculated by use of the G-Power program to verify the association between variables. For this purpose, an effect of 0.5 was used, a test power of 80% and a confidence level of 95%. The sample was composed of female participants, over 50 years of age, without a history of chronic gastrointestinal disease or diabetes, and who had not undergone treatment with botulinum toxin less than 6 months ago. Every patient answered the questionnaire with socio-demographic and health questions, in which the patient’s perception of her quality of life was assessed through the validated WHOQOL-bref¹¹ questionnaire before and after application. The study assessed the effectiveness and duration of the effect of botulinumtoxin type A (Incobotulinium) reconstituted in a 0.02% zinc gluconate solution versus dilution in 0.9% saline. The participants were photographed in similar lighting conditions before treatment in weeks 0, 2, 4 and 14 after the procedure.

Discussion

The results obtained showed that there is no relationship between race, alcoholism, smoking and use of sunscreen with the effect of the toxin in the two study groups. Concerning the variables age and amount of weekly zinc intake, the result of the Manny-Whitney test showed that there is no significant difference between the groups, p-value> 0.05. Before checking the duration of the relaxing effect of the frontalis muscle in the application of botulinum toxin solubilized with 0.9% sodium chloride and with zinc gluconate 0.02%, the individualized views of patients and the examining physician regarding the degree of front line wrinkles [3] that each participant presented before the treatment. The result indicated that there was agreement between the patients and the doctor, that is, when the participant looked at herself in the mirror and compared it to the scale of the lines the doctor also found similar results. Another study [4] which also used the same scale of front lines to check the duration of three types of botulinum toxin type A in 180 patients (incobotulinumtoxin A, onabotulinumtoxin A, and abobotulinumtoxin A) considered only the opinion of subjects and did not compare the doctor’s point of view. The beginning of the treatment was defined as the fourteenth day (or second week) after execution of the application, since the effect of botulinum toxin, according to the literature [5] is complete in two weeks, enough time for the observer and patient to find a decrease in frontal muscle activity compared to photographs of two weeks before treatment. The calculation also included the twenty-eighth day and finally the ninety-eighth day (or 14th weeks) after application. All patients were assessed with the frontalis muscle at rest and in motion. In the group of patients who received incobotulinumtoxin A solubilized in sodium chloride, within 14 weeks of application there was a 66% reduction of effects in patients with frontal wrinkles at rest. On the other hand, the patients who were asked to move the frontal muscle (movement) there was a reduction of 75% of the desired effect within 98 days of application. Moving wrinkles seem to tend to return to the initial moment earlier compared to the resting wrinkles. The results of this study are consistent with previous6 research that demonstrated that intramuscular treatment with botulinum toxin type improves the appearance of lines at rest and overall skin quality. One possible explanation for this effect is that muscle weakness eliminates the fold the repetitive fold of the skin and relieves chronic stress applied to overlapping skin, which causes elastin and collagen to be strengthened in these places over time [6]. In the group of patients who received incobotuliniumtoxin A solubilized in gluconate zinc, after 14 weeks of application there was a 100% reduction in the effects on patients with frontal wrinkles at rest. The patients who were asked to move the frontalis muscle there was also a 75% reduction in the effect within 98 days of application, corroborating with a study [7] that compared two presentations of Botulinum Toxin Type A (onabotulinium toxin A and incobotulinium toxin A). There are many myths that involve how to handle the botulinum toxin, many doctors use it only if they are reconstituted on the day of application, however, a study [8] confirmed that Botulinum Toxin Type A is an extremely stable molecule, and vigorous stirring does not impair its potency, even after 6 weeks of dilution. The results of the present study also showed that individuals who consume more zinc-rich foods, regardless of whether they were treated with incobotulinumtoxin A diluted in chloride sodium or zinc gluconate, had no more lasting effect. This variant was not evaluated by the author who described the oral use of zinc to increase the duration of effects of botulinum toxin [2]. This study evaluated whether some factors such as alcoholism, smoking, use of sunscreen and race could influence the results, considering the frontal wrinkles at rest and movement. It is known that wrinkles are also associated with aging, hormonal status, smoking and illness complications [9] and that there is a link between perioral wrinkles and smoking and that smoking is a risk factor for premature skin aging. Studies have proposed that cigarette exposure increases matrix-1 and 3 metalloproteinase activity and decreases the production of pro-collagen in the skin, possibly due to the production of free radicals induced by tobacco [10,11]. However, this study has not demonstrated therapeutic success or shorter duration in current smoking patients who received botulinum toxin applications with both dilutions.

Conclusions

The data identified that the duration of the effect of incobotulinumtoxin A diluted with Zinc gluconate was not superior to dilution with 0.9% sodium chloride. However, the study demonstrated that in patients who drink alcohol and who used botulinum toxin diluted in zinc gluconate 0.02% there seems to be a better efficiency. Another finding was that the habit of not using sunscreen does not influence the effect of Botulinum Toxin duration. Patients with low or high phototypes have the same duration of effect as botulinum toxin.

Acknowledgments

We thank all patients who participated in this study.

Financial Disclosure

None to declare.

Conflict of Interest

All cited authors declare that they have no conflict of interest to disclose.

Informed Consent

All participants agreed to the survey and signed a free and informed consent form.

Author Contributions

Leonardo Bianquini wrote the manuscript; Denise Cantarelli approved the final version; Paula Dellacqua collected data for review; Lúcia Pimassoni did a statistical analysis.

Data Availability

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

References

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4. RAPPL T, et al. (2013) Onset and duration of effect of incobotulinumtoxinA, onabotulinumtoxinA, and abobotulinumtoxinA in the treatment of glabellar frown lines: a randomized, double-blind study. Clinical, Cosmetic and Investigational Dermatology 24(6): 211-9. [crossref]
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8. SHOME D, et al. (2010) Botulinum toxin A: is it really that fragile a molecule? Dermatologic Surgery 36(4): 2106-2110. [crossref]
9. MANRÍQUEZ JJ, et al. (2014) Wrinkles. BMJ Clin Evid. [crossref]
10. CHIEN AL, et al. (2016) Perioral wrinkles are associated with female gender, aging, and smoking: development of a gender-specific photonumeric scale. Journal of the American Academy of Dermatology, 74(5): 924-930. [crossref]
11. Development of the World Health Organization WHOQOL-BREF quality of life assessment. The WHOQOL Group. Psychol Med 1998 28(3): 551-8. [crossref]

DOI: 10.31038/JCRM.2021413

Abstract

The COVID 19 pandemic started as a cluster of unexplained pneumonia in Wuhan, China and till now more than 111 million cases have been reported. Due to stringent public health measures, including lockdown strategies, the international travels were tremendously reduced. Hopes rise for end of pandemic as Corona virus vaccinations proved to have high efficacy and the true real-world effectiveness is estimated to be very good. International travels will probably start and the many safety issues should be remembered and emphasized for all travelers and any destination. The most predictable and avoidable travel related illness is infectious diarrhea that may be reduced by simple measures as are hand hygiene, food and water safety and less by antibiotic use or other pharmacologic options.

Keywords

Traveler’s diarrhea, Guidelines, Antibiotics

The COVID-19 pandemic forced tourism to shift from the global to an idyllic local pattern within country’s borders but the Coronavirus vaccination strategies applied all over the world bring the expectation for less travel restrictions.

Travelers’ diarrhea (TD) was the most predictable travel-related illness with rates (30-70%) depending on destination, season, adventurous eating or sexual practices and resulting in unpleasant holiday, hospitalization and eventually prolonged recovery mainly in immunosuppresed patients [1-3].

The etiology is dominated by bacteria (80%-90%), less by viruses and protozoa. Guidelines are published in the Yellow Book by CDC, the International Society of Travel Medicine, providing relevant data, clinical evidence and consensus statements [2,3]. Diarrhea often occurs abruptly and is accompanied by abdominal cramping, fever, nausea, or vomiting. The previous severity definitions based on the number of unformed stools per day were revised by using the relevant criteria of functional impact. This therapeutic approach depending on severity, safety and the effectiveness of treatment classifies TD in: mild (acute diarrhea that is tolerable, is not distressing, and does not interfere with planned activities), moderate (acute diarrhea that is distressing or interferes with planned activities) and severe (acute diarrhea that is incapacitating or completely prevents planned activities). Acute severe diarrhea also includes dysentery (grossly bloody stools) and persistent diarrhea (lasting>2 weeks) [3].

The main exposures, epidemiological entities and etiologies are expressed as:

• Foodborne outbreaks associated with many food items [noroviruses, nontyphoidal Salmonella, Clostridium perfringens, Bacillus cereus, Staphylococcus aureus, Campylobacter spp, coli pathotypes (enteroaggregative, enterotoxigenic, enteroinvasive), Listeria, Shigella, Yersinia, Cyclospora cayetanensis, Cryptosporidium spp, hepatitis A virus],
• Waterborne (drinking or swimming) – Campylobacter, Cryptosporidium, Giardia, Shigella, Salmonella, STEC, Plesiomonas shigelloides,
• Travel to resource-challenged countries – coli (enteroaggregative, enterotoxigenic, enteroinvasive), Shigella, Typhi and nontyphoidal Salmonella, Campylobacter, Vibrio cholerae, Entamoeba histolytica, Giardia, Blastocystis, Cyclospora, Cystoisospora, Cryptosporidium) [3,4].

Any traveler should be advised about the probable exposures, food, water safety and hygiene, and informed about individual and other population consequences related to the travel (dissemination of antimicrobial resistance), the self assessment of disease severity and treatment [3,5].

High-risk destinations (TD incidence >20%) include Africa (exception of South Africa), South and Central America, South and Southeast Asia, Mexico, Haiti, and the Dominican Republic, intermediate-risk destinations (TD incidence 8 to < 20%) include Southern and Eastern Europe, Central and East Asia (including China and Russia), the Middle East (including Israel), South Africa, and Caribbean Islands and low-risk destinations (TD incidence < 8%) include North America, Northern and Western Europe, Australia, New Zealand, Singapore, and Japan [1,6]. Foodborne outbreaks may occur in well developed countries affecting local population and travelers as it happened in Spain in 2019, Listeria monocytogenes was linked to the consumption of domestically produced chilled pork roast from a single manufacturer in the municipality of Sevilla [7].

The incubation period is short for viruses and bacteria (6-72 hours) and much longer for intestinal parasites (1-3 weeks). Untreated bacterial diarrhea usually lasts 3-7 days, viral diarrhea 2-3 days and parasitic enteritis lasts a couple of weeks. Usually, the microbiologic testing is not necessary except for persistent or severe diarrhea in returning travelers (strong recommendation, low/very low level of evidence). Molecular testing may confirm frequent or rare etiologies when needed [2-4]. Klem et al. found that the most frequent long term complication is postinfectious irritable bowel syndrome, 41.9% after enteritis caused by parasites and protozoa, and 13.8% after bacterial infection [8].

There were many studies and meta-analyses upon prophylaxis and treatment options in TD with the conclusion that antibiotics are not to be considered routinely in mild and moderate acute diarrhea [2,3]. No vaccines are available for common enteric pathogens causing TD, except for cholera and typhoid fever. Live attenuated cholera vaccines are recommended to adults, as a single dose given orally with a good efficacy (90% at 10 days and 80% at 3 months). The vaccines are not recommended to immunosuppressed patients, except for asplenic patients or those having chronic kidney disease. The commonly used typhoid fever vaccine is an inactivated Vi capsular polysaccharide vaccine given intramuscularly, in children ≥ 2 years and adults with an efficacy of 50-80%, the booster dose can be given after 2 years after primary vaccination. Both vaccines are recommended only for travelers to high risk regions, unconventional itineraries and housing (Table 1) [2].

Table 1: Treatment options in TD diarrhea [2,3].

*Loperamide, 4 mg (2 mg/tb), as soon as possible then 2 mg after each lost stool, maximum 12 mg/day. Not recommended for children <12 years, in febrile or bloody diarrhea [9].
**BSS 524 mg (262 mg/tb) every 30 minutes to 1 hour as needed (maximum of 8 doses/24 hours). Not recommended for children <12 years, pregnant women, travelers taking aspirine or methotrexate [10,11].

Antibiotics in TD

• Azithromycin may be used to treat moderate TD and should be used in severe TD as a single dose regimen (1,000 mg) or two doses of 500 mg 12 hours apart (better tolerated) or 500 mg/day for 3 days (if no resolution in 24 hours). It is the preferred regimen for invasive and febrile diarrhea and in regions where there are suspected or demonstrated fluoroquinolone resistant coli pathotypes and Campylobacter. Can be given to pregnant women and children.
• Fluoroquinolones (orally) may be used in moderate noninvasive TD and less in severe TD (weak recommendation, moderate level of evidence). There is some evidence about the emergence of antimicrobial resistance and the risk of dysbiosis beyond the well-known musculoskeletal adverse events that makes the benefit/risk ratio doubtful. Levofloxacin may be used as a single dose of 500 mg or in a 3 day course, ciprofloxacin 750 mg as a single dose or 500 mg in a 3 day course and ofloxacin 400 mg as a single dose or in a 3 day course. The 3 day course is considered when symptoms persist > 24 hours [3,4].
• Rifaximin may be used in non-invasive moderate and severe TD (weak recommendation, moderate level of evidence). Rifaximin is not recommended in invasive TD (Campylobacter, Salmonella, Shigella, invasive coli). Since it is a non-absorbable oral antibiotic, the safety profile is excellent. In TD rifaximin is given as 200 mg three times daily for three days [9-12].

Antibiotic regimens may be combined with loperamide because the anti-motility action is the fastest then completed by the curative antibiotic treatment and there are no more adverse effects with the combined strategy [3,9]. Doxycycline might be recommended for malaria prophylaxis and was associated with lower TD risk, suggesting bacterial enteropathogen susceptibility similar to previous observations and additional benefit in infection prevention [13].

Some studies showed higher rates of extended-spectrum β-lactamase producing Enterobacteriaceae (ESBL-E) if combined therapy (loperamide and antibiotic) was used in TD [3,14]. Arcilla et al. found that the most important predictors for the acquisition of ESBL-E during international travel were: antibiotic use during travel (adjusted odds ratio 2.69, 95% CI 1.79-4.05), persistent TD after return (2.31, 1.42-3.76), and pre-existing chronic bowel disease (2.10, 1.13-3.90) [5]. Ghandi et al. evaluated the patterns of empiric antibiotic self-treatment in international travelers from US using 31 Global TravEpiNet (GTEN) sites (Centers for Disease Control and Prevention sponsored consortium of clinics that provide pretravel health consultations). Between 2009 and 2018 the rate of antibiotic prescription declined steadily from >75%, mainly for fluoroquinolones showing that doctors and travelers are less prone to antibiotic treatment or prevention [15].

TD Prevention

Beyond hand hygiene, sanitation and food safety recommendations, antimicrobial prophylaxis is not routinely considered in international travelers (strong recommendation, low/very low level of evidence) being prescribed only for travelers at high risk of health-related complications of TD (strong recommendation, low/very low level of evidence). The most commonly recommended antibiotic is rifaximin which has an excellent safety profile [3,4,12].

BSS (two tabs 4 times a day) may be used for prophylaxis and can reduce the incidence of travelers’ diarrhea by almost half, though it should be avoided in children and pregnant women due salicylate side effects (strong recommendation, high level of evidence) [3,10,11]. Regarding probiotics and prebiotics there is insufficient evidence to recommend their use as preventive or treatment measure in TD. A recent systematic Cochrane review found that probiotics may not affect the duration of diarrhea [16].

Foodborne and waterborne infections, may be severe in immunocompromised people. Travelers with liver disease should avoid direct exposure to salt water that may expose them to Vibrio spp., and all immunocompromised hosts should avoid raw seafood. Drug interactions should be evaluated before considering antibiotic prophylaxis or self treatment. Fluoroquinolones and rifaximin have significant interactions with antiviral HIV treatment. Macrolides may have significant interactions with antiviral HIV medication and transplant-related immunosuppressive drugs. Fluoroquinolones and azithromycin in combination with calcineurin inhibitors and mTor inhibitors (tacrolimus, cyclosporine, sirolimus, everolimus) may cause prolonged QT interval [2].

With or without COVID-19 vaccine passports, probably more and more people will travel all over the world in the next years needing protection for the most frequent unpleasant event during the travel, TD. Somehow, a blessing in disguise, the COVID-19 pandemic imposed the highest hygiene rules and probably lower rates of infectious diarrhea in international travelers will be observed.

Author Contributions

Concept and writing of the manuscript (A.R.). The author approved the final version of the manuscript.

Funding

No external financial support was received.

Conflict of Interest

Nothing to declare for the author.

References

1. Steffen R, Hill DR, DuPont HL (2015) Traveler’s diarrhea: a clinical review. JAMA 313(1):71-80. [crossref]
2. Centers for Disease Control and Prevention. CDC Yellow Book 2020: Health Information for International Travel. New York: Oxford University Press.
3. Riddle MS, Connor BA, Beeching NJ, DuPont HL, Hamer DH, et al. (2017) Guidelines for the prevention and treatment of travelers’ diarrhea: a graded expert panel report. J Travel Med 24(suppl_1):S57-S74. [crossref]
4. Shane AL, Mody RK, Crump JA, et al. (2017) Infectious Diseases Society of America Clinical Practice Guidelines for the Diagnosis and Management of Infectious Diarrhea. Clin Infect Dis 65(12):1963-1973. [crossref]
5. Arcilla MS, van Hattem JM, Haverkate MR, Bootsma MCJ, van Genderen PJJ, et al. (2017) Import and spread of extended-spectrum β-lactamase-producing Enterobacteriaceae by international travellers (COMBAT study): a prospective, multicentre cohort study. Lancet Infect Dis 17(1):78-85. [crossref]
6. LaRocque R, Harris J.B. Travelers’ diarrhea: Microbiology, epidemiology, and prevention.
7. European Centre for Disease Prevention and Control, European Food Safety Authority, 2019. Multi-country outbreak of Listeria monocytogenes sequence type 6 infections linked to ready-to-eat meat products – 25 November 2019.
8. Klem F, Wadhwa A, Prokop LJ, Sundt WJ, Farrugia G, et al. (2017) Prevalence, Risk Factors, and Outcomes of Irritable Bowel Syndrome After Infectious Enteritis: A Systematic Review and Meta-analysis. Gastroenterology 152:1042–1054. [crossref]
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10. Brum JM, Gibb RD, Ramsey DL, Balan G, Yacyshyn BR (2020) Systematic Review and Meta-Analyses Assessment of the Clinical Efficacy of Bismuth Subsalicylate for Prevention and Treatment of Infectious Diarrhea. Dig Dis Sci, [crossref]
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13. Lago K, Telu K, Tribble D, Ganesan A, Kunz A, et al. (2020) For The Infectious Disease Clinical Research Program TravMil Study Group. Doxycycline Malaria Prophylaxis Impact on Risk of Travelers’ Diarrhea among International Travelers. Am J Trop Med Hyg 103(5):1864-1870. [crossref]
14. Kantele A, Mero S, Kirveskari J, Lääveri T (2016) Increased Risk for ESBL-Producing Bacteria from Co-administration of Loperamide and Antimicrobial Drugs for Travelers’ Diarrhea. Emerg Infect Dis 22(1):117-120. [crossref]
15. Gandhi AR, Rao SR, Chen LH, Nelson MD, Ryan ET, et al. (2020) Prescribing Patterns of Antibiotics for the Self-Treatment of Travelers’ Diarrhea in Global TravEpiNet, 2009–2018, Open Forum Infectious Diseases, [crossref]
16. Collinson S, Deans A, Padua-Zamora A, Gregorio GV, Li C, et al. (2020) Probiotics for treating acute infectious diarrhoea. Cochrane Database of Systematic Reviews, Art. No.: CD003048. [crossref]

DOI: 10.31038/JCRM.2021412

Abstract

Introduction: Er:YAG laser has the potential to reach areas of the periodontal pocket that are not accessible with traditional scaling and root planing (SRP). However the clinical and microbiological additional benefits of Er:YAG lasers as an adjunct to conventional SRP are not well documented.

Objective: Evaluate the efficacy of Er:YAG laser as an adjunct in non-surgical periodontal therapy (NSPT).

Materials and methods: A prospective randomized, controlled, split mouth single blinded clinical trial was performed on eleven participants diagnosed with moderate to severe periodontitis with probing depths (PD) ≥ 5 mm. One quadrant received SRP with ultrasonic and hand instrumentation only, while in the other quadrant Er:YAG was used in addition to SRP. Clinical data were collected at baseline, 6 weeks and 3 months following SRP+/-Er:YAG. Microbial samples were collected, analyzed numerically, and cultured for a panel of periodontal pathogens at baseline and 3 months.

Results: In sites with 5 mm probing depths or more, the SRP group had an initial mean PD of 5.9 ± 1.1 mm which decreased to 4.5 ± 1.7 mm at 6 weeks and remained the same at 3 months. The SRP+Er:YAG group had an initial mean PD of 6.0 ± 1.4 mm that decreased to 4.6 ± 2.0 mm and to 4.6 ± 1.8 mm at 6 weeks and 3 months respectively. The SRP group had an initial mean BOP of 92.7% which decreased to mean of 64.6% and 61.5% at the 6 weeks and 3 months respectively. The SRP+Er:YAG group had an initial mean BOP of 80.9%. BOP decreased to 58.4% and 43.8% at 6 weeks and 3 months respectively. There were no statistically significant differences between the SRP and SRP+Er:YAG group in clinical parameters and bacterial counts. There was an overall decrease in the percentage of the subgingival cultivable microflora at 3 months in both groups. There were no statistically significant differences between the SRP and SRP+Er:YAG group with respect to clinical changes, bacterial counts, and cultivability profiles.

Conclusion: Within the study limitations, the use of Er:YAG laser when used as an adjunct to SRP conferred no additional clinical and microbial benefits.

Introduction

Er:YAG (erbium-doped yttrium aluminum garnet) laser is one of the most frequently used lasers in non-surgical and surgical periodontal therapy. Er:YAG lasers emit infrared light at 2940 nm which is highly absorbed by water molecules, resulting in instant vaporization. Er:YAG laser light is well absorbed by all biological tissues with high water content and it can be used for soft tissue ablation and bacterial killing [1]. Er:YAG laser light is also absorbed by hydroxyapatite, thus making it suitable for ablation and calculus removal [2]. These properties are advantageous in periodontal therapy where removal of plaque and calculus is traditionally done by scaling and root planing (SRP).

Er:YAG is a suitable choice for effective calculus removal. Its wavelength and the use of water spray surface coolant effectively remove smear layers, calculus, and necrotic cementum from root surfaces without inducing tissue damage. The use of Er:YAG laser has demonstrated better wound healing, decreased swelling and scarring, less pain and better patient tolerance compared to conventional non- surgical and surgical methods [3].

Several other studies have shown improved periodontal clinical parameters after laser therapy. Schwarz et al. showed that laser therapy resulted in significant reduction in bleeding on probing and gains in clinical attachment levels compared to scaling and root planning alone. The clinical attachment gain in the laser group was maintained for more than 2 years [4]. Ando et al. concluded from an in vitro study that the Er:YAG laser has bactericidal effect at low energy levels [5] and Ishikawa et al. reported that Er:YAG laser may provide an antimicrobial advantage compared to conventional mechanical periodontal therapy [1]. The laser degrades and removes bacterial endotoxins without producing a smear layer [1]. However, a recent review of the literature was inconclusive regarding the clinical effects and microbiological outcomes of Er:YAG lasers when used as an adjunct to conventional scaling and root planing [3].

While scaling and root planing results in significant reduction of tissue inflammation [6], several studies have demonstrated that there is a limitation in the efficacy of curettes during scaling and root planing in periodontal pockets that are 5 mm or more [7,8]. Thus, there is clearly a need for developing effective methods of plaque and calculus removal in deep pockets and hard-to access locations. In addition, the evaluation of Er-YAG laser as an adjunct to SRP is needed, as the two treatments are not mutually exclusive.

The purpose of this study was to evaluate the efficacy of Er:YAG laser as an adjunct to scaling and root planing in non-surgical periodontal therapy on probing depth, clinical attachment levels, gingival bleeding and microbial colonization patterns.

Materials and Methods

Enrollment of Participants

This prospective randomized, controlled, split mouth single blinded clinical trial was conducted at Tufts University School of Dental Medicine (TUSDM). The protocol was approved by the Tufts University Medical Center Institutional Review Board (IRB #12321). Participants were patients enrolled for treatment in the Post-graduate Periodontology Clinic at TUSDM.

Potential participants were informed about the study and provided with ample time to ask any questions on study participation. Patients who decided to participate then provided informed. A copy of the informed consent form (ICF) was given to the participant. Demographic information was collected and a medical history was obtained.

Inclusion Criteria

To qualify for the study, each subject had to have all single rooted permanent maxillary and mandibular teeth from the central incisors to the second premolars, a recent diagnosis of moderate to severe chronic periodontitis [8] and a full mouth and vertical bite-wing series of diagnostic radiographs exposed at TUSDM within 6 months preceding entry of the study.

Exclusion Criteria

Participants who did not speak or write English were excluded from the study. Participants who had received mechanical debridement or any other professional periodontal therapy within 6 months prior to entering the study, who presented with significant chronic oral soft tissue pathologies, who presented with fixed or removable appliances partial dentures, who were current smokers, who ordinarily required prophylactic antibiotics prior to dental procedures, or who had taken systemic antibiotic medications within the previous 6 were excluded from the study.

Participants with uncontrolled chronic systemic conditions or diseases such as diabetes mellitus (HbA1C>7%), with immunological disorders, with known drug allergies or known adverse effects following the use of oral hygiene products, and who might be pregnant or lactating were excluded from the study.

Also, teeth with Miller grade III mobility or teeth with hopeless prognosis [9] indicated for extraction were excluded from the study.

Clinical Measurements

Using radiographs and the results of a complete periodontal examination, the diagnosis of generalized moderate to severe chronic periodontitis was confirmed, The examiner (I.K.) was previously calibrated to >90% accuracy in repeat probing depth measurements using the UNC probe [10]. Clinical measurements were taken at baseline and at 6 weeks and 3 months post treatment. The examiner was blind to all treatment assignments.

Two quadrants in each subject with single rooted teeth that demonstrated periodontal probings ≥ 5 mm were selected for the trial. A computer-generated randomization list was used to determine which quadrant would receive scaling and root planing only and which quadrant would receive SRP supplemented with the Er:YAG laser therapy.

Microbial Sampling and Transport

Before treatment, baseline microbial samples were obtained on single rooted, non-furcated teeth with 5-9 mm probing depths and bleeding on probing. The deepest two sites in each quadrant were chosen for microbial sampling giving a total of 4 microbial samples per participant. These same sites would be sampled again for microbial analysis post- treatment at the scheduled 3 month follow-up visit.

After air drying and isolation with cotton rolls and careful removal of supra-gingival plaque, one sterile, absorbent paper point, (Johnson & Johnson, East Windsor, NJ), was advanced into each of the selected periodontal sites for 10 seconds [11]. After removal, the two paper points per quadrant were pooled together in a glass vial of 2.0 mL anaerobically prepared viability medium Gothenburg anaerobic (VMGA) III transport medium [10]. Subgingival samples were then transported within 24 hours for microbial analysis to the Oral Microbiology Testing Service (OMTS) Laboratory located at the Temple University Kornberg School of dentistry. All laboratory microbiologic procedures were performed by OMTS personnel who were unaware of the participants’ overall health status, clinical diagnosis, and site specific conditions or that participants were part of a clinical trial [10].

Putative periodontal pathogens examined for in this study include Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia/nigrescens, Parvimonas micra, Fusobacterium nucleatum, Campylobacter rectus, Streptococcus constellatus, Streptococcus intermedius, Staphylococcus aureus, Enterococcus faecalis, Gram-negative enteric rods/pseudomonads, and Candida species [10]. The specimen vials were heated to 35°C [10,12]. The plaque samples were dispersed from the paper points using a vortex mixer for 45 seconds in Möller’s viability medium Gothenburg I anaerobic dispersion solution comprised of pre-reduced, anaerobically sterilized 0.25% tryptose, 0.25% thiotone E peptone, and 0.5% sodium chloride. The same medium was used to create a 10-fold dilutions of the bacterial suspensions [10]. Then, 0.1 mL dilution aliquots were spread with a sterile bent-glass rod onto non- selective enriched Brucella blood agar (EBBA) primary isolation plates [10]. EBBA was comprised of 4.3% Brucella agar supplemented with 0.3% bacto-agar, 5% defibrinated sheep blood, 0.2% hemolyzed sheep red blood cells, 0.0005% hemin, and 0.00005% menadione. EBBA plates were incubated at 35°C for 7 days in an anaerobic chamber containing 85% N2, 10% H2, and 5% CO₂, EBBA plates were used to determine the composition of predominant cultivable microbial species using established OMTS methods [10,11].

Total anaerobic viable counts were made on non-selective EBBA primary isolation plates. Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia/nigrescens, Parvimonas micra, Fusobacterium nucleatum, Campylobacter rectus, Streptococcus constellatus, Streptococcus intermedius, Staphylococcus aureus, Enterococcus faecalis were distinguished on EBBA using established microbial identification OMTS protocols [10]. Aggregatibacter actinomycetemcomitans, Gram-negative enteric rods, pseudomonads, and Candida species were quantitated on selective trypticase soy-bacitracin-vancomycin (TSBV) agar plates that were incubated at 35°C for 3 days in air plus 5% CO2 [10]. The proportional recovery of each test species was ascertained for each individual by calculating the percentage of positive test species colony forming units relative to total subgingival anaerobic viable counts as determined on non- selective EBBA primary isolation plates [10].

Treatment

Scaling and root planing was performed with an ultrasonic scaler (Varios 360 LUX Model: NE149) and hand scalers and Gracey curettes under local anesthesia using 2% lidocaine + 1:100 000 epinephrine.

Er:YAG laser treatment was delivered with AdvErL Evo Er:YAG Laser for Dentistry (Morita Model: MEY-1-A), with PS600TS tip using the manufacturer’s recommended settings (70 mJ, 25 pulses per second, PPS). The laser tip was inserted into the periodontal pockets measured prior treatment with PD ≥ 5 mm and was moved repeatedly in an apico-coronal direction without touching the root for 10 seconds.

Participants were given a prescription of 0.12% chlorhexidine (Peridex) mouthwash to use for 6 weeks after treatment. One operator (A.A) performed all scaling and root planing and Er:YAG laser treatments.

Statistical Analysis

Descriptive statistics (means and standard deviations for continuous items, counts and percentages of categorical items) were calculated.

Differences in probing depth reduction or attachment gain between laser treatment and control groups were analyzed with a nested mixed effects linear regression model. Normality of the data was assessed graphically. Statistical significance between the two groups’ gingival bleeding index was determined with a generalized estimating equation model. Differences in microbial load was investigated with the Wilcoxon signed-rank test. P-values less than 0.05 were considered statistically significant. The software Stata 13.1 (StataCorp LLC, College Station, TX) was used for the analysis.

Results

Seventeen study volunteers were screened and 11 participants (6 males and 5 females) were enrolled in this prospective, randomized single-blinded clinical trial. All 11 study participants complied fully with all study procedures and follow-up visits. The mean age of the study population was 48 ± 16 years. Table 1 shows the demographic background of the study population.

Table 1: Demographics of the study population.

Clinical parameters (PD, CAL and BOP) were improved after treatment in both the SRP and SRP+Er:YAG laser group (Table 2). However, there were no significant differences between the SRP and the SRP + Er:YAG groups at 3 months (P = 0.08).

Table 2: Mean probing depths (PD, mm) , clinical attachment levels (CAL, mm), and bleeding on probing (BOP, %) of sites with baseline probing depth of ≥ 5 mm in SRP only (Control) and SRP + Er:YAG (Test) groups at baseline, 6 weeks and 3 months for all study participants.

SD=Standard deviation.

Microbiological data was expressed as percentage of the cultivable microflora before and 3 months after SRP or SRP+Er:YAG treatment. A. actinomycetemcomitans, Enteric gram negative rods, E. faecalis, S. aureus and Candida species were not detected in any participant at baseline or at the 3 months follow up visit in either the test and control groups in all participants. Porphyromonas gingivalis was detected in one participant only in both treatment groups (Table 3). When comparing baseline and 3 months data, there was an overall reduction in the percentage of cultivable microflora for Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, Fusobacterium nucleatum and Parvimonas micra in both SRP and SRP+Er:YAG laser groups. There was a slight increase in Campylobacter rectus and Streptococcus constellatus in the SRP group and in Streptococcus intermedius in the SRP+Er:YAG laser group from baseline to 3 months follow up in one participant only. However no statistically significant differences in percentage of the cultivable microflora were found when comparing SRP and SRP-Er:YAG lase treated groups. Since the data is not normally distributed, the Wilcoxon signed-rank test was used. There was no statistically significant difference in the change in any bacteria between baseline and 3 months follow up (P > 0.05) (Table 4).

Table 3: Comparison between the mean percentage in the cultivable microflora between baseline and 3 months for the SRP and SRP+Er:YAG groups.

Table 4: Comparison between the difference in the mean of the cultivable microflora between baseline and 3 months for the SRP and SRP+Er:YAG groups.

SD=Standard deviation IQR: Inter-quartile range.
*Difference between baseline and 3 months data from 11 participants.
**Negative value indicates an increase from baseline to 3 months.

Discussion

The current literature is equivocal regarding the efficacy of Er:YAG laser as part of non-surgical periodontal therapy. The present prospective randomized single-blinded clinical trial was designed to better understand the efficacy of Er:YAG laser therapy on clinical periodontal parameters as well as microbial recolonization patterns in patients diagnosed with chronic moderate to severe chronic periodontitis.

Er:YAG laser in combination with SRP and ultrasonic instrumentation effectively reduced PD by 1.4 mm from baseline to 3 months in sites with PD ≥ 5 mm. This is comparable to results in a literature review by Cobb, who reported an average PD reduction of 1.29 mm in 4-6 mm sites [13]. However, we did not find statistically significant differences between SRP and SRP+Er:YAG treated pockets after 6 weeks and 3 months. Periodontal inflammation as measured by BOP also decreased in both of our treatment groups at 6 weeks and 3 months; however there was no significant difference between the SRP and SRP+Er:YAG groups. These observations are in line with previous studies [14-19].

Differences in the outcomes of published clinical trials on the effect of Er:YAG laser may be attributable to the various treatments the control groups have received: no treatment, SRP alone, ultrasonic scaling alone or a combination of SRP and ultrasonic; and whether the laser was used alone or in combination with other treatment modalities. A number of studies tested Er:YAG laser against SRP alone, and found no significant Er:YAG laser effect in terms of PD reduction at 3, 6 or 12 months [14-18]. On the other hand, Schwarz et al. [4] reported superior outcomes with Er:YAG laser alone compared to SRP in a randomized controlled split mouth clinical trial. Recently, Zhou et al. found statistically significant effect of Er:YAG laser when compared to SRP alone, albeit the effect was judged clinically minimally important by the authors [20]. Compared to ultrasonic debridement alone, adjunct application of Er:YAG laser induced significant PD reduction 12 months after treatment [19].

In comparison, our approach was to use the common standard of care (SRP combined with ultrasonic scaling) as control, and add Er:YAG laser as an adjunct. Findings presented here indicate that Er:YAG laser does not confer additional benefits in terms of pocket reduction or elimination of inflammation when used in combination with SRP and ultrasonic scaling. An important factor to be considered when comparing studies is various laser energy and pulse settings. In our study we applied 70 mJ at 25 Hz, following manufacturer’s recommendations. Other investigations employed different laser sources and different pulse settings: Schwarz et al. used 160 mJ at 10 Hz [4] and Zhou et al. used 50-100 mJ at 15-30 Hz [20].

We found an overall decrease of the subgingival microbial load at 3 months compared to baseline but that decrease was not significant. This is consistent with other in literature where the total number of the subgingival microbiota was reduced after therapy but the gram-negative species returned to baseline 3 – 6 months post therapy [21,22]. The literature is inconsistent when microbial outcomes were measured. Some studies have shown that there was no significant difference in terms of microbial outcomes between the SRP only group and SRP+Er:YAG laser groups [14,18,19]. One study found significantly greater reduction in A. actinomycetemcomitans, Porphyromonas gingivalis, Prevotella nigrescens and Tannerella forsythia at 6 and 12 months in the SRP + Er:YAG laser group [16]. In that study, patients were enrolled in a rigorous plaque-control program prior to laser treatment.

One of the limitations of our present study is that bacterial samples were collected only after 3 months. By that time the microbial load may have returned to its pre-treatment baseline [21]. Also, using bacterial cultures only enables us to detect live bacteria. Utilizing more sensitive approaches in the microbial analysis might have enabled us to detect more cultivable microflora. It cannot be excluded that the split mouth design might have resulted in translocation of bacteria between the test and control quadrants that influenced the clinical and microbial results. Increasing the sample size might have permitted us to detect treatment effects that did not reach statistical significance. Also, longer follow up periods could have added to our knowledge on the long-term efficacy of laser therapy.

Contradictory data in the literature may be due to heterogeneity in study design such as the different methods used for bacterial sampling and detection, the time it took for samples to be analyzed after they were obtained, different equipment and settings, the method and efficacy of SRP, the uneven root surface topography, the thickness of the subgingival biofilm and the timing and duration of laser therapy. It appears that both the laser type and settings impact the outcomes achieved in different studies. Further studies are needed to establish appropriate settings and the use of water coolant for periodontal nonsurgical therapy with the laser as monotherapy or as an adjunct to SRP [24-26].

Although patient-centered outcomes were not measured in this study, several participants reported less post-treatment sensitivity on the Er:YAG laser treated side. This coincides with studies that have shown significant reduction in dentin hypersensitivity following Er:YAG application [27,28]. Likewise, bleeding during instrumentation appeared to be less on the Er:YAG laser treated sites, suggestive of Er:YAG laser’s hemostatic properties, which may lead to improved post-operative patient experience, particularly in those taking anticoagulants [2]. It is worth noting that none of the participants reported adverse outcomes. Thus, for future considerations, an evaluation of the post-treatment experience by the participants may add value to the results.

Conclusion

Scaling and root planing with or without Er:YAG laser treatment reduced periodontal inflammation as measured by probing depth reduction, gain in clinical attachment and decreased bleeding on probing. Within the limitations of this study, the use of Er:YAG laser as an adjunctive therapy to scaling and root planing in patients diagnosed with moderate to severe chronic periodontitis did not provide clinical and microbial benefit over a combination of ultrasonic and hand instrumentation.

Acknowledgements

Thomas Rams, DDS, MHS, PhD, Professor – Director of Oral Microbiology Testing Service Laboratory, Department of Periodontology and Oral Implantology, Kornberg School of Dentistry.

Conflict of Interest

The authors declare that they have no conflict of interests in this study.

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