Demonstration of Urease Activity in Subgingival Plaque Sample of Periodontitis Patients at a Tertiary Care Centre of Central Nepal

Introduction: The urease activity is produced by many oral and gastric microorganisms, which have demonstrated systemic implications as well. Urea can be detected both from saliva and gingival crevicular fluid, which could suggest that the oral cavity can act as an extragastric reservoir for many microbes leading to serious systemic diseases. Objective: The main objective of this current study was to find out the urease activity in human dental plaque. Methods: An analytical cross-sectional study was conducted from September to November 2023 in patients visiting the Department of Periodontology and Oral Implantology. The urease activity was detected using a rapid urease test (RUT) kit from a hundred cases diagnosed with periodontitis. All systemically healthy patients excluding patients on ongoing proton-pump inhibitor therapy were selected for the presence/absence of periodontitis as per the 2017 World Workshop classification. Data were collected and entered into Microsoft Excel, and further analysis was done using SPSS v.20. Results: Out of 100 patients, urease activity was found positive in 85 (85%) patients. Regarding gender and age, the urease activity was not much different and was not statistically significant (P value= 0.163 and 0.382 respectively). Conclusions: The results of this study suggest there is a high urease activity in dental plaque samples whose removal is essential to prevent our body from systemic threats like bacterial endocarditis, gastric carcinoma, etc. caused by urease-producing microorganisms.


INTRODUCTION
Urease is an enzyme that hydrolyses urea and is produced by several bacterial species including oral microorganisms like Streptococcus salivarius, Actinomyces naselundii, Haemophilus parainfluenzae, Staphylococcus epidermidis, etc. 1,2 Urea is delivered in the gingival crevicular fluid and salivary secretions even in normal healthy individuals but was found in greater concentration in the presence of gingival inflammation. 3,4Dental plaque is a complex structure with different microbial colonies protected by a resistant sheath.So, dental plaque can act as a suitable platform for the survival of many microorganisms.
Interestingly, oral microorganisms commonly found in dental plaque and generating the urease activity have been linked with systemic diseases like bacterial endocarditis (H.[7] Thus, it is paramount to understand the link between or buccal loss of attachment ≥3 mm with pocketing >3 mm detectable at ≥2 teeth as per the 2017 classification. 8The patients with systemic disease, patients consuming proton pump inhibitors, and patients who had taken antimicrobials within the previous two months are excluded from the current study. 9e sampling method used was purposive convenient sampling.The sample size was determined based on the prevalence method using data derived from a similar study done by Akshit et al., 83.3%. 10 The sample size obtained was 105 using a 94% confidence interval.The sample size was calculated using the standard formula as mentioned below:

RESULTS
The  2, 3).The comparison was done using Pearson   been linked with more than 50 systemic diseases. 12e dental plaque is a complex microbial structure which can harbour plenty of microorganisms that could lead to serious systemic complications. 13,14 ase is an enzyme that hydrolyses urea into ammonia and carbon dioxide. 7Urease activity is shown by numerous microorganisms including those present in the oral cavity. 1,26][17] In addition, urea is believed to increase the baseline pH of dental plaque. 18,19The pH of around 7.6 is required for the growth of dental plaque crystals causing periodontal disease thus suggesting the alkaline nature of plaque is important for disease causation. 20ease activity in dental plaque is measured by three common methods i.e., RUT, Culture, and polymerase chain reaction (PCR) among which rapid urease test is a simple, reliable, and cheap method for detection of urease activity.RUT has a sensitivity and specificity rate of around 80-100% and 97-99% respectively. 21,22 is commonly used for the detection of H. pylori in gastric mucosa as it could lead to gastric ulcers and gastric carcinomas.and a few other studies yielded a prevalence of RUT positivity of around 50% which is contradictory to the results of our current study. 29,30The high urease activity might be because of above-mentioned pathogens like H. pylori and H. parainfluenza.Thus, every attempt should be made for dental plaque the urease activity which could indicate the presence of potential bacteria leading to perio-systemic diseases.Rapid urease test is a simple, cost-effective way to evaluate the urease activity in a given sample.The current study is hence believed to reveal the presence of urease activity in oral cavity that might relate it to the causation of systemic disease as mouth is a known doorway for contamination.Correspondence Dr. Simant Lamichhane Email: drsimant@kusms.edu.npCitation Lamichhane S, Thapa S, Rijal AH, Marattha P, Rupakhety P, Humagain M et al.Demonstration of Urease Activity in Subgingival Plaque Sample of Periodontitis Patients at a Tertiary Care Centre of Central Nepal.J Nepal Soc Perio Oral Implantol.2023 Jul-Dec;7(14):60-5.METHODS The study was conducted in the Department of Periodontology and Oral Implantology, Kathmandu University School of Medical Sciences (KUSMS), Dhulikhel, Kavreplanchok, Nepal after obtaining the ethical clearance letter from the Institutional Review Committee of KUSMS (KUSMS-IRC Ref. 148/23).It was three-month (September-November 2023), analytical cross-sectional study conducted on a patient fulfilling the definitive set of criteria.The inclusion criteria were patients above 20 years of age, systemically healthy patients with periodontitis diagnosed as per World Workshop 2017 classification 8 of periodontal and peri-implant health and diseases.Periodontitis was defined as the presence of interdental loss of attachment present in ≥2 non-adjacent teeth and/ plaque samples were collected from an interproximal area of the posterior tooth of a diagnosed periodontitis case using area-specific curette (Figure1).Study participants were screened and diagnosed as periodontitis cases by the principal investigator and consultant periodontists of KUSMS.Informed consent was obtained from the participants agreeing for participating to the study.The area of sample collection was first air-dried using dry cotton gauze and three-way syringe to avoid contamination.Any samples contaminated with blood were discarded.The urease activity was measured using Rapid Urease Test (RUT) kit from Gastrohub, Kolkata, India with an ISO certification ISO 13485: 2016 (Figure2).First, the sample was collected, and then the label of RUT kit was peeled off to place the plaque sample in the urea broth.The urea broth was then moistened with the addition of 1-2 drops of distilled water provided in the test kit (Figure3).The test kit was covered with the label again as it was at the beginning.Finally, the evaluation of colour change was done at 10-180 minutes time frame as per the manufacturer's instruction.The colour changes from yellow to pink or red if the test is positive and remains yellow if Lamichhane et al: Demonstration of Urease Activity in Subgingival Plaque Sample of Periodontitis Patients at a Tertiary Care Centre of Central Nepal

Figure 1 :
Figure 1: Collection of plaque sample for rapid urease test.

Figure 3 :
Figure 3: Process of sample evaluation in urea broth.

Figure 4 :
Figure 4: Colour change-pink indicating positive and yellow indicating negative results.
removal as soon as possible to remove any infectious and inflammatory pathway that can hamper other distant body parts.There are certain limitations of this study like we could not isolate and identify the possible microorganisms leading to the urease activity in dental plaque.Further, culture-based methods and polymerase chain reaction techniques should be used in future studies.

Table 1 :
Distribution of frequency of demographic parameters.

Table 2 :
Association between rapid urease test results and gender.Lamichhane et al: Demonstration of Urease Activity in Subgingival Plaque Sample of Periodontitis Patients at a Tertiary Care Centre of Central Nepal

Table 3 :
Association between rapid urease test results and age groups.