Efficacy of a chairside diagnostic test kit for estimation of C-reactive protein levels in periodontal disease.

BACKGROUND: C-reactive protein [CRP] levels increase to hundreds of mg/mL within hours following infection. Studies have shown that serum CRP levels were elevated in periodontal disease. However, in all the previous studies, CRP levels were measured by using high-sensitivity CRP assay kits with minimal detection limits of 0.1 to 3 mg/L, which was much below the normal value of 10 mg/L. These high-sensitivity CRP assays need a proper laboratory setup, and these methods cannot be used as a routine chair-side test in the dental office. AIM: The purpose of this study was to investigate the serum CRP levels in subjects with periodontal disease by using a rapid chair-side diagnostic test kit with a lower detection limit of 6 mg/L and to compare the CRP levels before and after periodontal therapy.
MATERIALS AND METHODS: A total of 45 systemically healthy subjects were selected for the study. Subjects were divided into three groups: group A: healthy controls, group B: gingivitis, group C: periodontitis. Serum levels of CRP were determined by using a latex slide agglutination method with commercially available kit with lower detection limit of 6 mg/L.
RESULTS: CRP was negative in all the 15 subjects in groups A and B at baseline, 7th and 30th day. CRP was positive only in 2 subjects in Group C at baseline and 7th day.
CONCLUSION: Estimation of serum CRP by using a rapid chair-side diagnostic test kit is not of any significance in subjects with periodontitis.

INTRODUCTION

The systemic acute phase refers to physiological and metabolic alterations that result immediately after the onset of infection or tissue injury, such as bacterial, viral or parasitic infection; mechanical trauma; tissue necrosis; or inflammation.[1] Acute-phase reactant proteins are defined as proteins whose serum concentration is altered by at least 25% in response to inflammation.[2] Most of the acute-phase proteins are synthesized primarily by the liver hepatocytes in response to pro-inflammatory cytokines. C-reactive protein [CRP] was the first protein to be discovered which behaves as an acute-phase reactant. CRP binds to phosphocholine, phospholipids, plasma lipoproteins and the plasma membranes of damaged or apoptotic but not intact cells.[3] Ligand-bound CRP activates the classical complement pathway 1 and can trigger inflammatory, opsonizing and complex solubilizing activities of the complement system. CRP levels reflect the extent and activity of disease.[45] CRP levels can be measured using immunoturbidimetric or immunoelectrophoretic assays[6-9] or latex slide agglutination method. The high-sensitivity CRP assay uses labeled monoclonal or polyclonal anti-CRP antibodies in an enzyme-linked immunosorbent assay (ELISA) or an immunofluorescent assay. CRP is normally present in ng/mL quantities but may increase dramatically to hundreds of mg/mL within 48 to 72 hours following tissue injury. CRP is a trace protein in healthy individuals, the median value of which was determined to be 0.8 mg/L in 483 healthy subjects, 90% of whom had CRP levels below 3 mg/L; and 99%, below 10 mg/L.[4] The latter finding has led to the suggestion that values of less than 10 mg/L should be regarded as clinically unimportant.[2] Periodontal disease is a chronic inflammatory process that occurs in response to a predominantly gram-negative bacterial infection originating from dental plaque. Periodontal pathogens affect local and systemic immune and inflammatory response.[5] Recent studies have shown that serum CRP levels were elevated in periodontal disease.[6-13] However, in all the previous studies,[6-13] CRP levels were measured by using high-sensitivity CRP assay kits with minimal detection limits of 0.1 to 3 mg/L, which was much below the normal value of 10 mg/L.[2] These high-sensitivity CRP assays need a proper laboratory setup, and these methods cannot be used as a routine chair-side test in the dental office. The purpose of this study was to investigate the serum CRP levels in subjects with gingival and periodontal disease by using a rapid chair-side diagnostic test kit with a lower detection limit of 6 mg/L and to compare the CRP levels before and after periodontal therapy.

MATERIALS AND METHODS

A total of 45 systemically healthy, nonsmoking subjects, aged 25 to 48 years [31 males and 14 females], were selected from the outpatient department of SDM College of Dental Sciences and Hospital, Dharwad, India. Inclusion criteria required that subjects have a minimum of 20 natural teeth; have not received antibiotic or periodontal therapy within the past 6 months; and do not require any dental treatment which needs antibiotic prophylaxis. Patients were not included in the study if they were pregnant; or if they were suffering from any chronic inflammatory or immunological conditions such as arthritis or gastrointestinal disorders. Fifteen subjects were selected and assigned each to group A, group B and group C. Group A comprised healthy controls (no sign of gingival or periodontal disease); group B, subjects with gingivitis (Loe and Silness Gingival Index score of ≥2); group C, subjects with periodontitis (clinical probing pocket depth of ≥6 mm in minimum of 6 teeth). The study protocol was approved by the institutional ethical and review board. All the subjects were informed about the purpose of the study, and they signed an informed consent document prior to participation.

Comprehensive medical and dental histories were obtained from all the subjects at baseline. Clinical measurements such as those obtained using Loe and Silness Gingival Index and Turesky Gilmore Glickman modification of the Quigley Hein Plaque Index were recorded at 4 sites per tooth [mesio-buccal, buccal, disto-buccal and lingual / palatal] on all teeth except third molars at baseline in all the 3 groups, and on the 7th and 30th day post-baseline in groups B and C.Probing pocket depth was measured with Williams graduated periodontal probe on 4 sites per tooth of all teeth except third molars at baseline in group C. All the clinical measurements were carried out by the same examiner to minimize inter-examiner variation.

Venous blood samples were obtained from the antecubital vein by venipuncture using a standard 2-mL syringe from each subject in all the 3 groups at baseline; and on the 7th day and 30th day post-baseline, blood samples were obtained only from subjects in groups B and C. Samples were allowed to coagulate for 1 to 2 hours at room temperature in a serum-collecting tube [Figure 1]. Serum was then separated by using micropipette. Serum levels of CRP were determined by using a latex slide agglutination method with commercially available kit obtained from Span Diagnostic Ltd. [Figures 2 and 3], as it has the advantage of rapid performance (within 2 minutes) in comparison to other methods for detection of CRP.[6-9] Since the lower detection limit of CRP was 6 μg/mL, subjects with CRP values <6 μg/mL were regarded sero-negative. After recording baseline clinical and biochemical measurements, only oral hygiene instructions were given to subjects belonging to group A. Full-mouth scaling and root planing was carried out in subjects belonging to groups B and C. Recall visits were scheduled for all the subjects belonging to groups B and C on 7th and 30th day to record both clinical and biochemical measurements. Descriptive statistics included mean and standard deviation for each of the measured clinical parameters. Clinical parameters at baseline and reassessment were compared using t tests. Since CRP levels were negative in most of the cases and as there were no variables present, CRP values were not subjected to statistical analysis.

Figure 1

Serum samples

Figure 2

Commercially available test kit for estimation of CRP

Figure 3

Negative and positive slide agglutination test

RESULTS

A total of 45 subjects completed the study, which was of 1-month duration. Descriptive statistics, including mean, standard deviation and standard error for Gingival Index, Plaque Index and CRP, are listed in Table 1. There was a significant improvement in Gingival Index and Plaque Index in groups B and C when compared to baseline values, as shown in Table 1. In this study, CRP was negative in all the 15 subjects in group A; it was also negative in all the 15 subjects in group B at baseline, 7th and 30th day, as shown in Figure 4. In only 2 subjects, CRP was positive in group C at baseline and 7th day; in 1 of these subjects, CRP turned negative on 30th day.

Table 1

Descriptive statistics, including mean, standard deviation and standard error for Gingival Index, Plaque Index and CRP

Figure 4

C-reactive protein levels in groups A, B and C

DISCUSSION

C-reactive protein is a well-known acute-phase reactant produced by the liver in response to inflammation due to various stimuli.[1] In acute inflammation, serum CRP levels exceed 100 mg/L, and the level decreases in chronic inflammation. Consequently, CRP has been used as a marker of the course of infection. There have been several cross-sectional studies demonstrating elevated levels of CRP in periodontitis patients.[6-13] These studies fall short in indicating that periodontitis was the cause for the observed serum CRP levels,[6-13] as CRP levels fluctuate with aging, high blood pressure, alcohol use, smoking, low levels of physical activity, chronic fatigue, coffee consumption, having elevated triglycerides, insulin-resistance diabetes, taking estrogen, eating a high-protein diet, suffering from sleep disturbances, and depression.

However, to fully confirm that elevation in CRP is due to periodontal infection, it is essential to see whether periodontal treatment is effective in reducing CRP levels following successful periodontal therapy. Ebersole et al.,[6] Slade et al.,[7] Ide et al. [14] and Armitage et al.[15] found a positive correlation between CRP and periodontitis, but they failed to illustrate changes in CRP following treatment.

In the present study, CRP was negative in all the 15 subjects in groups A and B. CRP was also negative among the 13 subjects in group C, in spite of the fact that subjects in group C had probing pocket depth of ≥6 mm in 6 or more sites. This was in contrast to the studies conducted by Ebersole et al.,[6] Slade et al.,[7] Ide et al.[14] and Beck et al.,[16] where they found a positive correlation between CRP and probing pocket depth of ≥4 mm in periodontitis patients. In our study, CRP was positive only in 2 subjects in group C at baseline. In 1 subject, CRP remained positive on 7th and 30th days even after full-mouth scaling and root planing (SRP). In other subjects, CRP was positive on 7th day, but it turned negative on 30th day, as the patients had undergone extraction of few teeth which were periodontally compromised. This was in contrast to the study conducted by Slade et al.,[7] in which they found similar CRP values in edentulous patients and patients with severe periodontitis. In this study, we observed a significant improvement in gingival and plaque scores following SRP but failed to observe any change in CRP. Some other authors also observed similar findings.[561415] The results of our study suggest a negative correlation between gingivitis, periodontitis and CRP.

Possible explanations for the negative CRP-periodontal disease relationship are as follows:

Periodontal disease in the study subjects may be in an inactive state; since periodontal disease is a chronic one, it may remain asymptomatic for decades, during which it can be detected by clinical and radiological examination. Therefore, possibilities exist that CRP concentration may be more closely associated with severe or progressive periodontal disease than simply disease status alone.[671415]

There is no standardization of CRP values in healthy subjects and those with periodontal disease, as observed from previous studies.[6-13] Roberts et al.[17] recorded median CRP levels of 0.9 mg/L in 388 healthy subjects, 75% of whom had CRP levels below 2 mg/L; and 95%, below 8.4 mg/L. Wu et al.[18] recorded CRP values of below 3 mg/L in 73% of 5,342 healthy subjects; and 94%, below 9 mg/L. Based on these studies and the studies done by Shine et al.[4] and Morey et al.,[2] CRP values <10 mg/L were considered normal.

There is no standardization of the lower detection limit of commercially available CRP test kits. In the present study, we have used a chair-side diagnostic test kit for the estimation of CRP, which had a lower detection limit of 6 μg/mL, which is equivalent to 6 mg/L. In this study, subjects who had negative CRP readings could actually have CRP value between 0 and 6 mg/L, which may also be one of the reasons for getting negative results. During the present study, authors have observed the following advantages of using chair-side diagnostic test kit for the estimation of CRP: less time consuming, easy to use in dental clinics and economical.

CONCLUSIONS

Based on the results of this study, we infer that estimation of serum CRP using a chair-side diagnostic test kit with a lower detection limit of 6 mg/L is not of any significance in subjects with periodontitis. Even though the detection limit of CRP test kit was much below the normal value, i.e., 10 mg/L, the negative CRP values in the present study suggest that periodontal infection may not be severe enough to raise systemic CRP values to a significant level.