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Periodontitis: Scientific Method Report

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In the recent period there is a growing body of research interest on the incidence of Coronary heart disease and its association with chronic periodntitis. Research dissecting the connection between coronary heart disease and the associated problems like chronic periodntitis is still limited in the literature. There is need to address this problem through a study in order to strengthen earlier studies and validate the utility of appropriate markers, at an earlier stage. To this end, two related articles were described briefly here. Periodontitis is a dental disorder that accompanies systemic inflammatory response (D’Aiuto, Ready, & Tonetti, 2004). As such it could contribute to high levels of serum inflammatory markers (D’Aiuto, Ready, & Tonetti, 2004). Hence, researchers have explored the role of C- reactive protein (CRP) to determine the outcome of periodontal therapy. They have studied ninety four subjects who have received non-surgical periodontal therapy by employing interleukin-6 (IL-6) and CRP after therapy (D’Aiuto, Ready, & Tonetti, 2004).They found a reduction in the risk associated CRP levels in 40 subjects (D’Aiuto, Ready, & Tonetti, 2004). This study has indicated that cardiovascular risk is better determined by serum CRP concentrations in individuals with periodontitis (D’Aiuto, Ready, & Tonetti, 2004). In another study, the problem of cardiovascular disease was thoroughly investigated by gene polymorphism studies (Pai et al., 2008). Here, a method of single nucleotide polymorphism (SNPs) in CRP gene was devised in a large population consisting of 32,826 women and 18,225 men (Pai et al., 2008).They have further correlated the DNA analysis with plasma CRP levels. They have found that alleles were associated with high CRP levels (Pai et al., 2008). It was revealed that a significant association exists between variations in CRP gene and plasma CRP levels (Pai et al., 2008).Experimental Design Plan: The plan begins with screening dental and general hospitals and enquiring at the outpatient departments regarding the frequency of patient visits.

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The registry maintained in the hospitals will be thoroughly studied to consider the patients profile enough to suit for the proposed study. The patients profile involves their age, sex previous illness history, drug history, complaints at the time of attending the clinic etc. The information gathered from screening procedure would help us to select the best out- patient department that receives maximum number of patients. Patients attending the clinic will be approached and explained about the study. Those willing to participate would be asked to sign on a written consent. A brief questionnaire will be conducted and the details would be noted and entered into a computer database. Patients with complaints other than periodontitis will be excluded. These may include fever, neuromuscular complaints, diabetes, rheumatoid arthritis etc. This would enable us to decrease the impact of other possible factors. This could facilitate a strong relationship between periodontitis and the proposed study marker CRP.

All the patients will be selected before the initiation of the therapy. So the outline of the data collection is screening outpatient departments, making a retrospective analysis of old case sheets, providing informed consent and conducting a questionnaire. Here, a computer generated database like MS-excel or software that is compatible with the standards of Electronic Medical Records (EMR) will be used. Concomitantly, a program (C ++ or Oracle based) will also be developed to enter the data obtained from the various clinical settings.

The template will contain the contents as follows: Name and address of Hospital/Clinic, Patient Name, Age, Sex, Registration Number, Residential Address, Family history, previous illness history, Drug history, Present findings, Suggested Medication in a sequence one below the other. This information will be used as a data log sheet for documenting the data collection procedure

Investigation following the experimental design

The purpose of the experiment to test the experimental question whether periodontitis leads to altered CRP levels and gene polymorphisms in our population.

Here, the independent variable is inflammation where as the dependant variable is treatment. The control variables are age and sex. This experiment is useful as it helps to identify the altered serum CRP status and the associated gene polymorphisms that would otherwise predispose individuals with periodontitis to the risk of cardiovascular inflammation. This might help the patients to seek appropriate anti-inflammatory drugs and lessen the serum CRP levels thereafter. Ultimately, the societal burden of periodontits induced heart problems could be minimized. So, the hypothesis is treatment could minimize inflammation with low CRP levels and the risk of cardiovascular disease is minimized in patients with periodontitis. Here, we predict a reduction in inflammation and a low CRP index in patients with periodontitis and it may be highly unlikely for these patients to develop inflammation typical of cardiovascular disease.The hypothesis was devised keeping in view of the fact that the fatal cardiovascular disease accompanies inflammation. As periodontitis was also believed to contribute to inflammation, it was anticipated that there could be a rise in the serum CRP levels and a likely association with CRP gene polymorphism.

Results: The data was collected form 160 patients reported to be suffering from periodontitis. All the patients wee initially approached and explained about the significance of the study. After providing a written consent, a questionnaire was conducted to collect the information. The study was conducted after seeking a permission grant from the concerned hospital authorities. We made use of databases, softwares to store the data.

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To ensure the quality of the data, two dedicated nursing professionals were assigned. The data was rechecked by contacting the patients through telephone. The information given by the patients was correlated with the generated database. The sequential measurement was Buccal / oral swabbing will be done to isolate the infectious agent. Blood samples will be collected, and used for serum CRP and CRP gene analysis. For CRP gene analysis, genomic DNA will be isolated and subjected to PCR (polymerase chain reaction), RFLP (Restriction Fragment Length Polymorphism).

Agarose gel electrophoresis was carried out to look for the bands by transillumnator. We found elevated CRP levels in 140 patients. We also found alleles associated with higher CRP levels for SNPs at positions 1919A>T and 4741G>C. In contrast some of the alleles were associated with lower CRP levels for SNPs at positions 2667G>C and 3872C>T. This has strengthened a recent finding on CRP gene polymorphism (Pai et al., 2008). Here the variable serum CRP was initially higher before starting the therapy. We made a follow up of patients to recheck the serum CRP levels. As expected we found decreased CRP levels.

This has strengthened a previous finding on the role of CRP in periodontitis (D’Aiuto, Ready, & Tonetti, 2004). Hence the variable has shown variation corresponding to the treatment. However, the variation observed in serum RP levels was not correlated with that of CRP gene polymorphism which is a major limitation of our study. We report that as our findings are close to that described in earlier literature, our study indicates the accuracy of measurement used throughout the experiment. This was due to the fact that the variation in the CRP gene could not be considered as the predictor of coronary problems that accompany inflammation. This may indicate that further studies are required to rule out the efficacy of gene variables in dissecting the relationship between periodontitis and cardiovascular problems.

Conclusion: Periodontitis has emerged as a problem of systemic inflammation with respect to cardiovascular disease. Our results were in agreement with the experimental procedures/ methodologies that were intended to measure. This was evident with the high serum CRP levels and CRP gene polymorphisms. We have strictly excluded patients complaining of problems other periodontitis. Therefore, it an be inferred that our results are valid. Further, our results also confirm with the hypothesis. We are hopeful that our experiment could produce similar results if replicated provided the researchers are strict in following the inclusion / exclusion criteria during the selection of study participants. We hope that our experiment could aid other researchers who intend to study the burden of cardiovascular inflammation in patients with complaints typical of periodontitis or dental complaints that accompany cardiac inflammation. Hence, we anticipate that our methodology produces similar results.

References

D’Aiuto, F., Ready, D., Tonetti, M.S. (2004). Periodontal disease and C-reactive protein-associated cardiovascular risk. J Periodontal Res. 2004; 39(4):236-41.

Pai, J.K., Mukamal, K.J., Rexrode, K.M., Rimm, E.B. (2008). C-reactive protein (CRP) gene polymorphisms, CRP levels, and risk of incident coronary heart disease in two nested case-control studies. PLoS One, 3, e1395.

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