Evaluation of nonsurgical periodontal therapy in chronic periodontitis patients with anemia by estimating hematological parameters and high-sensitivity C-reactive protein levels.

BACKGROUND: Periodontal tissues mount an immune inflammatory response to bacteria and their products. Certain inflammatory cytokines produced during periodontal inflammation increase the production of acute phase proteins like high‑sensitivity C‑reactive protein (hs‑CRP) and can depress erythropoietin production leading to the development of anemia. AIM: The aim of this study is to investigate the efficacy of nonsurgical periodontal therapy on red blood cell (RBC) parameters and hs-CRP in chronic periodontitis patients with anemia.
MATERIALS AND METHODS: This is a longitudinal, interventional study with 6-month follow-up. A total of 30 subjects with anemia and chronic periodontitis with age group of 33-55 years were selected by screening hemoglobin (Hb) levels and examining periodontal findings. The clinical parameters plaque index (PI), gingival index (GI), probing pocket depth (PPD) and clinical attachment level (CAL) were recorded at baseline. Laboratory blood investigations were performed to evaluate RBC count, Hb, packed cell volume (PCV), erythrocyte sedimentation rate (ESR) and red cell indices, hs-CRP at baseline. Nonsurgical periodontal therapy was performed for all patients. Patients were recalled after 6 months. The clinical and hematological parameters were re-evaluated to analyze the changes after nonsurgical periodontal therapy.
RESULTS: The results showed that there was a significant increase in Hb levels, RBC count and PCV from baseline to 6 months after nonsurgical periodontal therapy. There is significant decrease in levels of ESR and hs-CRP levels after nonsurgical periodontal therapy indicating resolution of periodontal inflammation. There is a significant decrease in PPD, scores of PI and GI and significant increase in CAL gain. Minimal changes in mean corpuscular volume, mean corpuscular hemoglobin (MCH) and MCH concentration indicated that the lower values of red cell parameters are not due to any vitamin and mineral deficiencies, but secondary to the chronic inflammatory changes associated with chronic periodontal disease.
CONCLUSION: The present study strengthens the hypothesis that chronic periodontitis may lead to anemia and provides evidence that nonsurgical periodontal therapy can improve the anemic status and reduce levels of hs-CRP in patients with chronic periodontitis.

Periodontal disease is one of the most common oral diseases of humans. It occurs as a result of exposure of the periodontium to dental plaque, biofilms that accumulate on teeth to form bacterial masses.[1] Dental plaques are complex with more than 400 bacterial species having been collectively isolated from dental plaques of patients with periodontal disease.[2] Periodontal destruction results from the action of various toxic products released by the specific pathogenic sub gingival plaque bacteria, as well as host responses elicited against the plaque bacteria and their products. The inflammatory response results in gingival ulceration around the tooth, which can allow intact bacterial cells or their products, including lipopolysaccharides, peptidoglycan fragments, and hydrolytic enzymes into the systemic circulation. Host responses to periodontal infections result in local production of cytokines and biological mediators including interleukins (ILs) and prostaglandins.[3] It has therefore been speculated that periodontitis results in low grade systemic inflammation causing anemia and elevated C-reactive protein (CRP).[45]

The association between anemia and periodontal disease has been explored since 20th century. Siegel in their study have reported depression in the number of erythrocytes apparently secondary to the presence of periodontal disease.[6] Lainson et al. were one of the first authors to implicate anemia as a systemic cause of periodontitis.[7] Anemia of chronic disease (ACD) is defined as the anemia occurring in chronic infections, inflammatory conditions or neoplastic disorders that are not due to marrow deficiencies or other diseases, and occurring despite the presence of adequate iron stores and vitamins. As periodontal disease is a chronic infectious condition leading to immune responses which promotes iron sequestration and inhibition of production and survival of erythrocytes.[8]

C-reactive protein was originally discovered by Tillett and Francis in 1930 as a substance in the serum of patients acutely infected with pneumococcal pneumonia that formed a precipitate when combined with C-polysaccharide of the cell wall of streptococcus pneumoniae.[9]

C-reactive protein is a serum protein synthesized in liver only during an inflammatory disease and is normally present as a trace constituent of plasma.[10] The rate of synthesis and secretion of CRP increases within 12 h of an acute injury or onset of inflammation. The CRP level reaches peak levels within 24–48 h. The CRP level returns to normal when the inflammatory process terminates. Hence, CRP levels provide useful information for the diagnosis, monitoring and therapy of the inflammatory process and associated disease. CRP during its role in an inflammatory process binds to the surface of pathogens and opsonizes them for uptake of phagocytes. CRP can also activate the classic complement cascade by binding to “q” factor of complement factor 1. It acts as anti-inflammatory by decreasing neutrophils migration to the site of inflammation, preventing adhesion of neutrophils to endothelial cells, and affecting clearance of nuclear antigens released from apoptotic or necrotic cells.[11]

Nonsurgical periodontal therapy includes scaling and root planning. Scaling is defined as the removal of plaque, calculus, and stain from the crown and root surfaces, whereas root planning specifies the removal of cementum or surface dentin that is rough or impregnated with calculus, toxins, or microorganisms. Nonsurgical periodontal therapy restores gingival health by removing elements that provoke gingival inflammation.[12]

The present interventional trial was carried out to find the improvement in periodontal status after nonsurgical periodontal therapy could result in any alteration of the hematological parameters which includes red blood cell (RBC), hemoglobin (Hb), packed cell volume (PCV), erythrocyte sedimentation rate (ESR), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), MCH concentration (MCHC), and high-sensitivity (hs)-CRP levels.

Materials and Methods

In this 6-month follow-up, longitudinal, interventional study, a total of 30 (age range: 30–50 years) with chronic periodontitis and anemia were selected from the outpatient section of the Department of Periodontics, St. Joseph Dental College. Nonsurgical periodontal therapy was performed to the patients. Patients who were pregnant; who suffered from any acute or chronic medical condition, except chronic periodontitis; who had a history of blood loss; who were smokers; or had undergone periodontal therapy or used antibiotics or oral supplementation in the previous 6 months prior to enrollment were excluded. Subjects were examined for chronic periodontitis on the basis of the following criteria: Probing depths (PDs) >5 mm at 30% of sites and clinical attachment levels (CALs) >2 mm at 30% of sites. Other signs of inflammation were recorded using the gingival index (GI) and plaque index (PI).[13]

After periodontal findings recorded as shown in the Figure 1, blood samples were obtained from all patients for RBC analyses and hs-CRP. Patients were instructed to follow the irroutine diet habits without any modification, including any iron or vitamin supplementation. Periodontal recordings [Figure 2] and RBC analyses were repeated after completion of 6 months.

Figure 1

Clinical photograph showing measurement of CAL at baseline

Figure 2

Clinical photograph showing measurement of CAL post-operatively

Hematological analyses

Under aseptic measures, 3 ml venous blood samples were drawn by venipuncture in antecubital fossa using a 5-ml syringe, collected into EDTA-containing vacuum tubes, and transported to a clinical laboratory for RBC analyses. Hb%, number of erythrocytes (RBC), PCV, MCV, MCH and MCHC were measured on a fully automated hematologicanalyzer [Figure 3]. The ESR was measured according to Westergren procedures as shown in Figure 4.

Figure 3

Fully automated Hematological Analyser which gives complete blood picture

Figure 4

Westergren method to measure ESR

Analysis of high-sensitivity-C-reactive protein

Under aseptic measures, 2 ml venous blood samples were drawn by venipuncture in antecubital fossa using a 5-ml syringe, collected into vacutainer tubes, and transported to a clinical laboratory for evaluation of hs-CRP by fully automated analyzer using immuno tubidimetry method as shown in Figure 5.

Figure 5

Fully automated Analyser to measure hs-CRP by immunoturbidometry

Statistical analyses

The collected data were subjected to statistical analysis through given by IBM SPSS Statistics. (www.ibm.com/software/analytics/sppp/). The statistical methods applied were paired sample Z-test, Wilcoxon Signed-rank test (paired test). The results were presented in text, tables, and graphs.

Results

A total of 30 patients (16 males and 14 females; mean age: Mean age 40.5 ± 4 years) participated. Mean and standard deviation (SD) values of hematological and clinical parameters at baseline and 6 months are shown in Table 1.

Table 1

Demonstrates the haematological and clinical parameters at baseline and after 6 months postoperatively

Table 1 shows the mean and SD of Hb, RBC count, hs-CRP, PCV, ESR. MCH. MCHC, MCV, PD, CAL, PI, and GI at baseline and 6 months. A statistically significant increase in values was observed from baseline to 6 months except for MCH. MCHC, and MCV.

Graph 1 shows values of PD, CAL, ESR, hs-CRP and RBC values at baseline and 6 months. Graph 2 shows the PI and GI preoperative and postoperative values. Graph 3 shows MCH. MCHC, MCV, PCV and Hb values at baseline and 6 months, respectively. There was statistically significant increase in values at baseline to 6 months except for MCH. MCHC, MCV.

Graph 1

Erythrocyte sedimentation rate, high-sensitivity C-reactive protein, probing depth, clinical attachment level, red blood cell at preoperative and postoperative for male and female patients

Graph 2

Plaque index and gingival index at preoperative and postoperative for male and female patient

Graph 3

Various parameters of red blood cell analysis at preoperative and postoperative for male and female patients

Discussion

The main aim of this study is to find out the efficiency of nonsurgical periodontal therapy on RBC parameters and hs-CRP in chronic periodontitis patients with anemia. In this study, sample size of 30 with 16 males and 14 females of mean age 40.5 ± 4 were enrolled.

The Hb value at baseline (11.93 ± 1.42) was low in chronic periodontitis patients. This can be due to the presence of pathogenic bacteria or their products, which stimulate the release of inflammatory cytokines resulting in depression of erythropoiesis and a blunted erythropoietin response. Similar results were reported by Pradeep et al.,[14] Ali,[8] Gokhale et al.[4] The rise in Hb (12.86 ± 1.16) postoperatively could be due to removal of bacterial etiology by periodontal therapy which results in decrease of cytokines.

The RBC count at baseline (4.68 × 106 ± 0.40 × 106) was low. The down regulation of the erythropoiesis in bone marrow by pro-inflammatory cytokines such as IL-1, IL-6 and tumour necrosis factor-α could be responsible for a decrease in number of erythrocytes. This is in accordance with studies done by Malhotra et al.,[15] Yamamoto et al.[16] In this study, there was significant increase in RBC count (4.88 × 106 ± 0.42 × 106) postoperatively. This could be attributed to the effect of nonsurgical therapy, which caused resolution of inflammation.

In this study, there was significant increase in PCV levels (1.15 ± 0.22) from baseline to 6 months after nonsurgical periodontal therapy. Similar results were reported by Pradeep and Anuj.[14] The increase PCV is due to rise in levels of RBC count after nonsurgical periodontal therapy.

The mean value of hs-CRP at baseline (3.44 ± 2.10) was high. This can be due to the presence of pro-inflammatory cytokines at the site of local pathology, which activate hepatocytes to produce acute phase proteins including CRP. In this study, there was significant decrease in hs-CRP levels (0.71 ± 0.13) from baseline to 6 months after nonsurgical periodontal therapy. This could be due to resolution of inflammation by nonsurgical periodontal therapy. These results were in accordance with the studies done by Correa et al.,[17] Oliveria et al., (2011)[13] Radafshar et al., (2010)[15] Noack et al.,[5] Thakare et al.,[18] Gomes-Filho et al.,[19] Pitiphat et al.,[20] Herrera et al.[21] To assure greater reliability of the findings high sensitivity of the immunoturbidometry method used to assess hs-CRP, being capable of detecting variations of even 0.2 mg/L.

In this study, there was significant decrease in ESR levels (1.15 ± 0.22) from baseline to 6 months after nonsurgical periodontal therapy. These results were in accordance with study done by Agarwal et al.[22] It is explained by the fact that ESR is considered a valuable parameter for any inflammatory process. Elevated values of ESR at baseline suggested that chronic periodontitis has an inflammatory component in it and the decrease in ESR over time was due to a reduction of the periodontal inflammation after nonsurgical periodontal therapy.

There was a significant reduction of scores of GI and PI and decrease in probing pocket depth, increase CAL gain 6 months after nonsurgical periodontal therapy. The result was in accordance with study done by Correa et al.[17] This could be attributed that effective mechanical debridement, which reduced the bacteria load decreasing the local inflammation and oral hygiene maintenance of the patient postoperatively.

In this study, mean corpuscular Hb and MCHC followed a nonsignificant rise (P = 0.201 and P = 0.093 respectively) from baseline to 6 months after the periodontal therapy. The result was in accordance with study done Agarwal et al.[22] The small increment of change in mean corpuscular Hb and MCHC values compared with increase in Hb levels implied that anemia associated with periodontitis is of normochromic type.

In this study, MCV followed a statistically nonsignificant rise (P = 0.152) from baseline to 6 months after the periodontal therapy. This is in accordance with study done by Pradeep and Anuj.[14] MCV levels are the main determinants of certain types of anemia. A depressed level of MCV (microcytosis) relates anemia to iron deficiency and elevated level of MCV (macrocytosis) relates anemia to vitamin deficiency. In our study, MCV levels were between the reference values as seen in ACD and called as normocytosis. This indicated that anemia due to chronic periodontitis is not due to vitamin or mineral deficiencies, but secondary to the inflammatory changes present in periodontitis.

The present interventional study has paved the path for future studies, with a larger study population for a longer period of time to further validate the effect of nonsurgical periodontal therapy on RBC parameters and hs-CRP in anemic patients.