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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 7  |  Issue : 4  |  Page : 165-170

Comparison of salivary and serum C reactive protein levels in periodontitis and healthy patients using ELISA – A clinico pathological study


1 Assistant Professor, Department of Dentistry (Periodontics), Maharaja Suhel Dev Autonomous State Medical College and Mahrishi Balark Hospital, Bahraich, Uttar Pradesh, India
2 Assistant Professor, Department of Pathology, Autonomous State Medical College Firozabad, Bahraich, Uttar Pradesh, India

Date of Submission14-Jun-2020
Date of Decision19-Jun-2020
Date of Acceptance26-Jun-2020
Date of Web Publication30-Nov-2020

Correspondence Address:
Anshul Sawhney
Department of Dentistry (Periodontics), Maharaja Suhel Dev Autonomous State Medical College, Maharishi Balark Hospital, Bahraich - 271 801, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jdrr.jdrr_50_20

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  Abstract 


Background: Periodontitis is one of the most common chronic inflammatory diseases in the world. C-reactive protein (CRP) is important due to its crucial role in atherosclerosis as a marker. This study evaluates both salivary and serum CRP levels, Gingival Index, Plaque Index, Bleeding Index, and probing pocket depth. Methodology: A total of 150 participants were enrolled, out of which 50 were healthy, i.e., did not have periodontitis, 50 patients were suffering from chronic periodontitis previously called adult periodontitis, and 50 patients were suffering from aggressive periodontitis with features of rapid attachment loss and bone destruction. All these patients were divided into three groups, and salivary as well as serum samples were collected from each group which were analyzed using enzyme-linked immunosorbent assay. Results: The results showed that both salivary and serum CRP levels were highest in aggressive periodontitis patients followed by chronic periodontitis patients and least in healthy individuals. Conclusion: Both salivary and serum CRP are effective markers in evaluating the severity of periodontal disease.

Keywords: Atherosclerosis, attachment loss, indices, inflammatory, periodontitis


How to cite this article:
Sawhney A, Ralli M. Comparison of salivary and serum C reactive protein levels in periodontitis and healthy patients using ELISA – A clinico pathological study. J Dent Res Rev 2020;7:165-70

How to cite this URL:
Sawhney A, Ralli M. Comparison of salivary and serum C reactive protein levels in periodontitis and healthy patients using ELISA – A clinico pathological study. J Dent Res Rev [serial online] 2020 [cited 2021 Apr 21];7:165-70. Available from: https://www.jdrr.org/text.asp?2020/7/4/165/302052




  Introduction Top


Periodontitis is inflammation and loss of connective tissues supporting or surrounding the teeth. It is sub-classified into three major types based on clinical, radiographic, historical, and laboratory characteristics.[1],[2] It is caused as a reaction to plaque composed of bacteria in a matrix of salivary glycoproteins and extracellular polysaccharides, leading to chronic inflammation, gingival bleeding, increased pocket depth, and alveolar bone loss.[3] Chronic periodontitis is the most common form of periodontitis. It has slow-to-moderate rates of disease progression, but periods with rapid destruction may occur. Chronic periodontitis is associated with accumulation of plaque and calculus. Aggressive periodontitis, on the other hand, is characterized by a rapid rate of disease progression seen in an otherwise healthy individual, absence of large accumulation of plaque, and calculus. Family history is suggestive of a genetic trait. Various proteins have been identified which are secreted in response to inflammation by hepatocytes. They serve as mediators or inhibitors of the inflammatory processes. These include α1-antichymotrypsin, α-1 trypsin, α-macroglobulins, C-reactive protein (CRP), complement C-3, ceruloplasmin, complement C-3, fibrinogen, haptoglobins, lipocalin-2, and transferring. They have been used as a predictor of disease progression and serve as markers.[4],[5]

Its levels increase rapidly at the onset of inflammation and decrease as inflammation or infection subsides.[6] CRP was chosen as it is an important marker for cardiovascular diseases. Serum and salivary samples were collected from all the three groups for intergroup comparison and to evaluate and establish the predictive values of both the samples collected. The aim of this study was to evaluate the levels of serum and salivary CRP in periodontitis and nonperiodontitis groups at baseline, along with Gingival Index (GI), Plaque Index (PI), and Sulcus Bleeding Index among the selected three groups.


  Methodology Top


This study was carried out in the Department of Dentistry, Autonomous State Medical College, Bahraich (Uttar Pradesh). Patients selected were those who visited the dental outpatient department in the Medical College. A total of 150 patients were selected for the study, out of which 50 patients were healthy controls, that is, nonperiodontitis group (Group I), 50 patients with probing depth (PD) ≥5 mm and at least four sites with attachment loss >2 mm were classified into chronic periodontitis (Group II), 50 patients having eight teeth with PD >6 mm and radiographic evidence of alveolar bone loss, with at least three of these teeth were not first molars or incisors were classified into aggressive periodontitis (Group III). 1999 International Workshop for the Classification of Periodontal Disease and Conditions[3] was followed.

Group I patients were healthy controls and were not having any evidence of periodontitis. Patients who were smokers, diabetics, those who had systemic diseases, were on antibiotics from the last 6 months, and those who were on periodontal therapy from the last 1 year were excluded from the study[Figure 1], [Figure 2], [Figure 3], [Figure 4]. The total duration of the study was 1 year.
Figure 1: Chronic periodontitis (Group II) pretreatment

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Figure 2: Orthopantomograph of chronic periodontitis patients

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Figure 3: Aggressive periodontitis (Group III) pretreatment

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Figure 4: Orthopantomograph of aggressive periodontitis patients

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Use of acrylic stents, prepared on study models in Group II and III patients, was done for recording probing pocket depth with the help of PCP UNC 15 Probe (Hu Friedy's). Hu-Friedy Mfg. Co., LLC 3232 N. Rockwell St. Chicago, IL 60618-5935. At baseline, clinical parameters, that is, Plaque Index[7] (Silness P and Loe H, 1964), Gingival Index[8] (Loe H and Silness P, 1963) and Bleeding Index were recorded.

Saliva collection

The patients were refrained from any brushing and chewing habit till samples were collected. 2 mm of their salivary samples was put in capped tubes gently. All the samples were transported by the technician to the laboratory in an ice-chilled test tube in a thermocol box and stored at −80°C refrigeration. Enzyme-linked immunosorbent assay method was used using Cobas C 111 kit (Roche Diagnostics India Private limited Andheri, Mumbai Maharashtra) [Figure 5]. After all samples were collected, all patients were given oral hygiene instructions, proper brushing technique was demonstrated on dentoform models, and Phase I therapy was completed.
Figure 5: Biochemical analyzer (Cobas C 111) for the estimation of C-reactive protein

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Venipuncture

Patients were asked to make a fist so that veins are palpable and prominent, and their arm is grasped with the thumb to make the skin taught. The antecubital region is cleaned in a circular motion, beginning at the site and moving outward with alcohol wipes and allowed to air dry. Tourniquet is applied to the mid-upper arm to make pressure buildup in veins, which is loosened and removed just at the time of drawing blood. A 5 cc 23G syringe is inserted swiftly through the skin into the lumen of the vein at an angle of 15°–30° with the forearm to draw 3 ml of blood from the median cephalic vein. Gauze is applied on the puncture site with adequate pressure for 1–2 min once the syringe is removed. Blood is taken in ethylenediaminetetraacetic acid vacutainer and centrifuged for testing [Figure 6] and [Figure 7].
Figure 6: Armamentarium for collection of blood samples

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Figure 7: Centrifuge used for the preparation of serum

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The values were read out using a digital readout [Figure 8].
Figure 8: Digital readout

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Method of statistical analysis

Kruskal–Wallis test was applied to find out the significant difference between the study groups. The Wilcoxon signed-rank test was used to compare two related samples or repeated measurements on a single sample to assess whether their population mean ranks differ (i.e., it is a paired difference test). From the above tests, P value was calculated. P < 0.05 was taken to be statistically significant. The data were analyzed using SPSS software IBM SPSS Statistics, India.


  Results Top


The two methods, that is, serum and salivary CRP, were used to evaluate CRP as a sensitivity marker to be used as a valuable tool in identifying patients with enhanced disease susceptibility. For this, a total of 150 patients were selected and divided into three groups according to the inclusion and exclusion criteria. The mean age of the patients in Group I, II, and III was 20–24, 26–46, and 21–33 years [Table 1] and [Graph 1], respectively. The percentage of males and females in all the three groups was calculated [Table 2] and [Graph 2]. When the mean baseline concentration of serum CRP was compared, there was a statistically significant difference (P < 0.05) in the CRP levels between Groups I and II and between Group I and Group III, but there was no significant difference found between Groups II and III [Table 3] and [Graph 3]. Similarly, when the mean baseline concentration of salivary CRP was compared, a statistically significant difference (P < 0.05) was found in the CRP levels between Groups I and II and between Groups I and Group III [Table 4] and [Graph 4]. Among the periodontal parameters, at baseline, the mean GI scores in the Groups I, II, and III were 0.61 ± 0.21, 1.46 ± 0.34, and 1.42 ± 0.70, respectively. A statistically significant difference (P < 0.001) was found in the GI between Groups I and II and between Groups I and III but not between Groups II and III [Table 5] and [Graph 5]. The plaque control in all the patients was satisfactory. At baseline, the mean PI in the Groups I, II, and III was 0.75 ± 0.10, 1.43 ± 0.28, and 1.61 ± 0.63, respectively. A statistically significant difference (P < 0.001) was found in the PI between Groups I and II and between Groups I and III, but no significant difference was found between Group II and Group III [Table 6] and [Graph 6]. The mean Bleeding Index scores in the Groups I, II, and III at baseline were 0.69 ± 0.14, 2.12 ± 0.33, and 2.10 ± 0.54, respectively. A statistically significant difference (P < 0.001) was found in the Bleeding Index between Groups I and II and between Groups I and III, but no significant difference was found between Groups II and III [Table 7] and [Graph 7]. The mean PDs for the entire mouth at the beginning of the study for Groups II and III were 5.22 ± 0.78 and 6.09 ± 0.61 mm, respectively. A statistically significant difference (P < 0.001) was found in the mean PDs between Groups II and III [Table 8] and [Graph 8].
Table 1: Age distribution of the study

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Table 2: Sex/Gender distribution of the study

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Table 3: Mean Serum C-reactive protein (CRP) levels in group I, II, III before Treatment( At Baseline )

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Table 4: Mean Salivary C-reactive protein (CRP) levels in group I, II, III before Treatment( At Baseline )

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Table 5: Gingival Index among the groups I, II, III

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Table 6: Plaque index among the groups I, II, III

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Table 7: Sulcus Bleeding Index among the groups I, II, III

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Table 8: Comparison of mean probing depth among the groups II, III

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  Discussion Top


CRP is produced by the liver and is stimulated by circulating cytokines.[9] C-Reactive protein has recently been shown to be measurable in saliva from periodontal patients using 'lab-on-a chip' method 17 During acute phase of an inflammatory response. It may reach saliva via gingival crevicular fluid or through salivary glands.

Circulating C-reactive protein may reach saliva via salivary glands or gingival crevicular fluid.[7],[8] C- Reactive Protein (CRP) is produced by the liver and is stimulated by circulating cytokines.[9] C-reactive protein (CRP) is one of the major acute-phase proteins synthesized in response to pro-inflammatory cytokines. The concentration of C-reactive protein (CRP) increases with inflammation. Periodontitis has been related to the elevation of systemic levels of C-reactive protein (CRP) and Interleukin-6. In present study also C-reactive protein (CRP) levels were increased in patients with chronic and aggressive periodontitis patients with increased pocket depth and attachment loss. C-reactive protein (CRP) belongs to the pentraxin family of calcium-dependent ligand-binding plasma proteins, the other member of which in humans is serum amyloid P component (SAP).[10] CRP has been shown to predict cardiovascular mortality. The plasma levels in healthy controls are 1 mg/L with a normal value defined as <10 mg/L. The plasma levels increase within 4–6 h of initial tissue injury and continue to increase several 100 folds within 24–48 h. The doubling rate is every 8–9 h and the half-life is <24 h. CRP plays a role in the inhibition of chemotaxis and neutralizes the deleterious effects of histones. CRP has been found to activate complement in damaged vessel walls, promote the formation of foam cells during the initiation of atheroma formation, and modulate neutrophil function. Recent studies have demonstrated that periodontal patients display elevated concentration of serum CRP when compared with healthy individuals.[11],[12],[13] Our study showed elevated levels of CRP in both groups (II and III) of periodontitis, which reinforces the observations of the previous studies.[14],[15],[16]

CRP has recently been shown to be measurable in saliva from periodontal patients using “laboratory-on-a chip” method.[17] In accordance with studies by Giannobile et al.,[18] CRP levels increased with the severity of periodontal disease and were more in aggressive periodontitis as compared to chronic periodontitis patients as compared to periodontally healthy individuals in our study. Our study evaluated and compared both salivary and serum CRP Levels at baseline among both healthy and periodontitis patients. Salivary samples were easy to collect, were noninvasive, and were sensitive for screening periodontitis patients. Previous studies have shown an association of elevated CRP levels as a risk predictor for various cardiovascular diseases.[19] Early intervention is required, whether nonsurgical or surgical periodontal therapy, to decrease a load of harmful pathogens in the oral cavity and for further improvement in systemic well-being also. Further long-term studies are needed to establish the role of both serum and salivary CRP levels as a reliable marker and establish the association of periodontitis with other systemic diseases.


  Conclusion Top


Our study concludes that CRP is an essential marker in assessing the periodontal disease status apart from its role in various other systemic diseases. Further studies are warranted to confirm the reliability of both salivary and serum CRP levels for screening periodontitis patients. Our study confirms the reliability of both serum and salivary CRP levels and among the two, the method of collection of saliva is easy and noninvasive.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Pitiphat W, Savetsilp W, Wara-Aswapati N. C-reactive protein associated with periodontitis in a Thai population. J Clin Periodontol 2008;35:120-5.  Back to cited text no. 5
    
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Ersin Kalkan R, Öngöz Dede F, Gökmenoǧlu C, Kara C. Salivary fetuin-A, S100A12, and high-sensitivity C-reactive protein levels in periodontal diseases. Oral Dis 2018;24:1554-61.  Back to cited text no. 6
    
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Dekker RL, Lennie TA, Moser DK, Miller CS, Ebersole JL, Chung ML, et al. Salivary Biomarkers, Oral Inflammation, and Functional Status in Patients With Heart Failure. Biol Res Nurs 2017;19:153-61.  Back to cited text no. 7
    
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Korte DL, Kinney J. Personalized medicine: An update of salivary biomarkers for periodontal diseases. Periodontol 2000 2016;70:26-37.  Back to cited text no. 8
    
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Pradeep AR, Priyanka N, Prasad MV, Kalra N, Kumari M. Association of progranulin and high sensitivity CRP concentrations in gingival crevicular fluid and serum in chronic periodontitis subjects with and without obesity. Dis Markers 2012;33:207-13.  Back to cited text no. 9
    
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Grover HS, Saini R, Bhardwaj P, Bhardwaj A. Acute-phase reactants. J Oral Res Rev 2016;8:32-5.  Back to cited text no. 10
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Rohde LE, Hennekens CH, Ridker PM. Survey of C-reactive protein and cardiovascular risk factors in apparently healthy men. Am J Cardiol 1999;84:1018-22.  Back to cited text no. 11
    
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Ridker PM, Hennekens CH, Buring JE, Rifai N. C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. N Engl J Med 2000;342:836-43.  Back to cited text no. 12
    
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Shojaee M, Fereydooni Golpasha M, Maliji G, Bijani A, Aghajanpour Mir SM, Mousavi Kani SN. C-reactive protein levels in patients with periodontal disease and normal subjects. Int J Mol Cell Med 2013;2:151-5.  Back to cited text no. 13
    
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Iyengar A, Paulus JK, Gerlanc DJ, Maron JL. Detection and potential utility of C-reactive protein in saliva of neonates. Front Pediatr 2014;2:131.  Back to cited text no. 14
    
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Mathew S, Sharma P, Dahiya S. Validation of salivary C reactive protein as a marker of inflammation in humans. Rheumatology 2018;57:255.  Back to cited text no. 15
    
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Dholey MK, Kole D, Rambabu D, Sen S. Comparative estimation of salivary and serum C- reactive protein levels in chronic periodontitis with or without Type II diabetes mellitus: A clinico-biochemical study. SRM J Res Dent Sci 2017;8:99-104.  Back to cited text no. 16
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Christodoulides N, Mohanty S, Miller CS, Langub MC, Floriano PN, Dharshan P, et al. Application of microchip assay system for the measurement of C-reactive protein in human saliva. Lab Chip 2005;5:261-9.  Back to cited text no. 17
    
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Giannobile WV, Beikler T, Kinney JS, Ramseier CA, Morelli T, Wong DT. Saliva as a diagnostic tool for periodontal disease: Current state and future directions. Periodontol 2000 2009;50:52-64.  Back to cited text no. 18
    
19.
Haverkate F, Thompson SG, Pyke SD, Gallimore JR, Pepys MB. Production of C-reactive protein and risk of coronary events in stable and unstable angina. European Concerted Action on Thrombosis and Disabilities Angina Pectoris Study Group. Lancet 1997;349:462-6.  Back to cited text no. 19
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8]



 

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