Chairside quantitative immunochromatographic evaluation of salivary cotinine and its correlation with chronic periodontitis.

BACKGROUND: Cigarette smoking is an established and modifiable risk factor for periodontitis. Periodontitis appears to be dose-dependent on smoking. The purpose of this study was to assess a reliable marker of tobacco smoke exposure (salivary cotinine) chairside and to confirm the quantitative association between smoking and chronic periodontitis.
MATERIALS AND METHODS: Saliva samples from 80 males, aged 30-60 years, with chronic periodontitis, were evaluated chairside using NicAlert™ cotinine test strips (NCTS). Patients were divided into two groups: A (cotinine negative) and B (cotinine positive). Plaque index (PI), Gingival index (GI), gingival bleeding index (GBI), probing pocket depth (PPD), clinical attachment level (CAL), and gingival recession (GR) were compared between the two groups and among the subjects of group B.
RESULTS: Comparison showed that the severity of PPD (P<0.001), CAL (P<0.001), and GR (P<0.001) was more in group B than in group A. Severity of all periodontal parameters increased with increased salivary cotinine among the subjects in group B.
CONCLUSION: Quantitative direct association can be established between salivary cotinine and the severity of periodontitis. Immunochromatography-based cotinine test strips are a relatively easy method for quantification of salivary cotinine chairside. Immediate and personalized feedback from a chairside test can improve compliance, quit rates, and ease reinforcing smoking cessation.

INTRODUCTION

Evidence supports that periodontitis is more prevalent in smokers than in non-smokers. Tobacco metabolites suppress neutrophil function, influencing host defense mechanism, and inhibit the immune response.[1] Exposure to tobacco smoke and periodontal disease is an active area of research. Biochemical measure of tobacco use is desirable for determining disease activity and treatment outcome.[2] A widely used biochemical measure of tobacco smoke exposure is cotinine, a proximate metabolite of nicotine. It can be measured in blood, saliva, or urine, and is considered an accurate measure of smoking. Cotinine is formed by cytochrome P450 mediated C-oxidation of nicotine and is more stable.[3] Cotinine has a longer half-life of average 17 h[4] and remains relatively constant. An easy to obtain, economic, reliable, highly sensitive, and specific assay for cotinine status, that can be performed by untrained personnel at the point of care, will have a significant role in clinical and research settings.[5] Recently, simple screening devices that can provide semi-quantitative estimates of cotinine concentrations in a non-laboratory setting have been developed. An advantage of these kits is that large, expensive equipment is not required.[2] Saliva is a preferred source to assess the levels of cotinine as it correlates well with that of serum. Also, the samples are easy to obtain and more compliant for the patient in a field or clinical setting.[6] The purpose of this study was to assess salivary cotinine levels using a rapid chairside semi-quantitative immunochromatographic assay, and to establish a quantitative relation between salivary cotinine levels and chronic periodontitis, and further to highlight its importance in periodontal management.

MATERIALS AND METHODS

Eighty male patients, within the age group of 30-60 years, attending the Department of Periodontics, SIBAR Institute of Dental Sciences, Guntur, Andhra Pradesh, India, were selected for the study. Patients with chronic periodontitis and having two or more teeth with probing pocket depth (PPD) ≥4 mm and clinical attachment level (CAL) ≥4 mm were included.[7] Forty patients not known to be exposed to tobacco smoke were included in Group A and 40 patients known to be exposed to tobacco smoke either directly or passively were included in Group B. Patients on systemic antibiotic therapy or any other drug within the past 6 months, patients with any systemic disease, patients with history of oral prophylaxis within the past 6 months, subjects consuming tobacco in any form other than smoking, and former smokers were excluded.

A comparative clinical correlation study was planned with the subjects of the two groups. The study was approved by the ethical committee of the institution, and following the selection of subjects, a written informed consent was obtained.

Cotinine levels in saliva of both groups were measured following the guidelines of the users’ instruction manual of NicAlert™ (NYMOX Sales Corporation, New Jersey, USA).

Specimen collection

Unstimulated whole saliva was collected by asking the patient to drool the saliva. The participants’ saliva was directed through a small funnel into the collection tube (both included in the NicAlert kit) until the tube was half full. The funnel was then discarded and the tube was capped.

Assessment of salivary cotinine

Cotinine levels were measured by NicAlert which is based on the principle of enzyme-linked immunosorbent assay (ELISA). In this assay, mouse monoclonal antibody coated gold particles were used with a series of avidity traps that allows quantification. The distance that gold migrates on the strip was known by a clear color change and provided an accurate measure of the amount of cotinine in the saliva sample. The test strip displayed seven zones with each zone representing a range of level of cotinine/smoking [e.g. zone “0” (0-10 ng/ml, a nonsmoker) to zone “6” (>1000 ng/ml, a heavy smoker)]. The results were recorded as values from 0 to 6 as shown in Table 1.

Table 1

Interpretation of the developed test strip

Results were read after 15-30 min, when the blue band disappeared or faded substantially. The test results were compared to the cotinine range chart provided. The strip was read as the lowest band appearing with red color as shown in Figure 1. The strip reading zero indicated as sample with cotinine level that confirmed a nonsmoker. If the color appeared as an indistinct smear, through the strip, results were not valid and the test was repeated with another test strip. The strip has more than one level in which there is red band formation or color. Different levels have different shades or colors in them. The presence of a reddish band in the lowest numbered level is the test result. It need not have to be the darkest.

Figure 1

Illustration of appearance of red bands and disappearance of blue band on the test strip

All subjects received a clinical periodontal examination by one single examiner using a manual calibrated probe (UNC-15, HuFriedy, Chicago, USA). Gingival index (GI),[8] Plaque index (PI),[9] gingival bleeding index (GBI),[10] PPD, CAL and gingival recession (GR) were recorded.

Results of continuous measurements were presented as Mean±SD (Min.-Max.) and results of categorical measurements were presented in number (%). Significance was assessed at 5%. Student's t-test (two-tailed, independent) was used to find the significance of study parameters on continuous scale. Chi-square/ 2 × 2, 2 × 3, 2 × 4 Fisher exact test was used to find the significance of study parameters on categorical scale. Analysis of variance (ANOVA) was used to find the significance of study parameters between groups. The statistical software programs, SPSS 15.0, Stata 8.0, MedCalc 9.0.1, and Systat 11.0 were used for the analysis of the data. Microsoft word and Excel have been used to generate graphs, tables etc.

RESULTS

The mean±SD for GI was 1.75±0.59 for Group B and 2.04±0.46 for Group A. Mean GI was significantly lower in Group B than in Group A, with P=0.016 [Table 2].

Table 2

Comparison of periodontal parameters

PI of both groups showed no statistically significant difference at the point of examination when compared using the Student's t-test (two-tailed, independent). The mean±SD for PI was 2.28±0.62 for Group B and 2.16±0.57 for Group A, with P=0.351 [Table 2].

GBI was significantly higher in Group A when compared to Group B. The mean±SD of bleeding index was 63.16±42.50 for Group B and 95.25±17.34 for Group A, with P<0.001 as shown in Table 2.

Table 2 shows that PPD was significantly more in Group B when compared to Group A, with P<0.001. The mean±SD for Group B was 5.09±0.55 mm and for Group A was 4.03±1.13 mm.

As shown in Table 2, the CAL was found to be significantly more in Group B when compared to Group A, with P<0.001. The mean±SD for Group B was 6.03±1.31 mm and for Group A was 4.49±0.97 mm.

GR was found to be significantly more in Group B when compared to Group A, with P<0.001 as shown in Table 2. The mean±SD for Group B was 3.76±1.49 mm and for Group A was 26.4±1.30 mm.

Table 3 shows the correlation of clinical parameters with cotinine levels among subjects of Group B and suggests that GI (P=0.019), PI (P=0.002), GBI (P<0.001), PPD (P<0.001), CAL (P=0.027), and GR (P=0.018) positively correlated with cotinine, inferring that all the above parameters increased with increase in salivary cotinine levels.

Table 3

Correlation of clinical parameters among Group B subjects with salivary cotinine

DISCUSSION

Exposure to tobacco smoke is generally accepted as a major environmental risk factor for periodontal diseases. As reviewed by Gelskey,[11] smoking meets most of the criteria for causation proposed by Hill. The specificity of the association between smoking and periodontitis is evident from the studies of Bergstrom et al.,[12] Jansson et al.,[13] and Tomar et al.,[14] who have shown that disease progression slows down in patients who quit smoking when compared to those who continue to smoke.

In most investigations, tobacco smoke exposure was examined exclusively via a self-administered questionnaire, the validity of which is often questioned because of underestimation.[7] On the other hand, self-reported measures are likely to be imprecise indicators of intake of tobacco smoke.[7] A quantitative assessment of tobacco smoke exposure through evaluation of its metabolites would help overcome such drawbacks. Hence, in our study, semi-quantitative assessment of salivary cotinine levels was done.

Markers of exposure to cigarette smoke include carbon monoxide (carboxyhemoglobin), thiocyanate ion, nicotine, and cotinine.[15] A general consensus is that cotinine has the prerequisites of specificity, retention time in the body, and detectable concentration levels that make it the analyte of choice for quantifying tobacco smoke exposure.[16] At present, cotinine is generally regarded as the best marker for monitoring tobacco exposure in either actively or passively exposed individuals. Moreover, cotinine has a much longer half-life of 18-20 h, making it more appropriate for use as an exposure marker.[15] In studying the long-term effects of tobacco smoke exposure, cotinine levels may represent an alternative measure to complement, confirm, or better quantify self-reported smoking status.[17] However, few studies have demonstrated an association between cotinine level in body fluids and periodontitis.[7] Hence, in our study, cotinine levels were assessed.

Noninvasive samples, like saliva, are of particular importance in field settings. Saliva and blood cotinine levels are highly correlated, with a ratio of 1.1-1.4 for saliva to blood.[18] An investigation by Bernert et al.[6] reported that the serum levels of cotinine were more closely correlated with unstimulated saliva (> by 4%) than with stimulated saliva (> by 41%). Hence, unstimulated salivary cotinine levels were assessed in the current study.

Cotinine has been measured in the biologic matrices using various methods like chromatography, colorimetry, raidoimmunoassay, ELISA, etc.[19] ELISA with monoclonal antibodies has been proven useful in detecting cotinine in saliva.[20] Studies of ELISA tests for urine cotinine showed almost total agreement with the confirmatory tests like gas chromatography.[21] The NicAlert cotinine test strips (NCTS) used in this study contain cotinine-specific monoclonal antibodies and is an immunochromatographic strip that works on the principle of ELISA. It is designed to provide a semi-quantitative estimate and can be assessed conveniently by non-specialists, based solely on visual inspection. Studies by Fiona Cooke et al.[22] have shown that NCTS are a valid alternative to the more expensive and time-consuming gas chromatographic tests when testing saliva to verify tobacco smoke exposure. NCTS detect exposure to nicotine from all sources. NCTS have a specificity of 95%, sensitivity of 93%, positive predictive value of 95%, and a negative predictive value of 93%.

On evaluation of gingivitis and bleeding on probing, in smokers and non-smokers, most investigations have found that smokers had less bleeding on provocation than non-smokers.[23] GI was higher in Group A than in Group B, and the difference was found to be statistically significant (P=0.016). These findings are in consistence with the data of Newbrun et al.[23] The mean GBI was higher in Group A than in Group B and the difference was statistically significant (P<0.001). These findings are also in consistence with the report of Newbrun et al.[23] Decreased gingival bleeding in smokers has been explained as being due to nicotine, which causes vasoconstriction of peripheral blood vessels such as in forearm, skin, and hands.[24] Lang et al.[25] demonstrated that the absence of bleeding on probing was an indicator of periodontal stability. However, the lack of bleeding on probing in smokers with advanced periodontal disease represents a false-negative predictor, thus making the presently accepted clinical significance of bleeding on probing of uncertain value in smokers.[24]

One of the variables commonly associated with prevalence and severity of periodontitis is plaque. In our study, there was no significant difference in PI between the two groups. These results are in consistence with the findings of Bastian and Waite.[26] This implies that the harmful effects of smoking on periodontal health may not be associated with plaque accumulation and poor oral hygiene.[27]

PPD and CAL estimate the degree of periodontal destruction, and therefore are measures of periodontal morbidity. Further, as these measures are normally found from a large number of single measurements in the individual, they must be considered to provide robust and reliable data.[28] In the present study, mean±SD for PPD was 5.09±0.55 mm for Group B and 4.03±1.13 mm for Group A, and mean±SD for CAL was 6.03±1.31 mm for Group B and 4.49±0.97 mm for Group A. They were found to be significantly more in Group B than in Group A, in spite of the plaque scores showing no significant statistical difference. Similar findings have been reported in studies by Bergstrom et al.[29] These findings which were independent of plaque levels suggest that the effect of tobacco smoke exposure on periodontal conditions may be direct rather than being related to plaque infection.[27]

In general, the reason attributed to the increased PPD and CAL in patients exposed to tobacco smoke is twofold. Both bacterial flora and host take part in the pathogenesis of periodontal diseases, and since no difference has been found in the periodontal pathogens between smokers and non-smokers,[27] it would appear that the deleterious effects of tobacco on the host occur through two mechanisms. On one hand, it systemically causes alteration of the immune response, and on the other hand, it acts locally through release of cytotoxic metabolites and vasoactive substances. These are produced by the combustion of tobacco and in turn affect the fibroblasts and vascular response.[30]

In the present study, GR was significantly more in Group B than in Group A, with P<0.0001. These results are in agreement with the results of Martinez Canut et al.[31] and Gunsolley et al.[32] who have reported that GR was twice as severe in smokers as in non-smokers. Recession in smokers could be due to vasoconstriction and less inflammatory response caused by nicotine.[24]

Our study also shows that Group B has significantly higher levels of salivary cotinine when compared to those of Group A. On correlation of periodontal parameters with those of salivary cotinine among Group B subjects, it was found that GI (P=0.019), PI (P=0.002), GBI (P<0.001), PPD (P<0.001), CAL (P=0.027) and GR (P=0.018) positively correlated with cotinine. This shows that as the salivary cotinine levels increased, the severity of periodontal parameters also increased. Tobacco smoke interacts with and compounds the effects of various systemic conditions, resulting in greater disease severity. These results are in consistence with the results of Gonzalez et al.[17] This finding also establishes a quantitative direct association between the level of tobacco smoke exposure and severity of periodontitis.

Our results show that categorization of subjects as smokers and non-smokers may not be sufficient to evaluate the role of smoking in the severity of periodontal disease. This is because smokers represent a highly heterogeneous (as they show variations in the types and methods of tobacco exposure, rate of metabolism, etc.) group of subjects. Therefore, a quantitative and more objective method to describe such a group would provide a better analytical tool to evaluate the association between tobacco smoke exposure and periodontal disease. On the other hand, biochemical markers are difficult to estimate and are not dependent on social pressures, recollection, or brand of tobacco used.[17]

Our data provide evidence to show that the use of cotinine levels is an objective, reliable, and quantitative method for measuring tobacco use. Our findings also confirm the relation between tobacco smoke exposure and periodontal disease, and establish a quantitative direct association between the level of tobacco smoke exposure (as determined by salivary cotinine) and severity of periodontal disease.

The findings indicate that NCTS are a valid alternative to the more expensive, laborious, and time-consuming gas chromatographic studies. NCTS also have the potential for use in large population-based trials, for evaluating the effectiveness of cessation programs, and in population prevalence studies. Immediate and personalized feedback from a point-of-care test such as NCTS improves compliance and also improves quit rates. It also helps in reinforcing cessation of smoking.[22]

The most important limitation of the present study is its cross-sectional design, as all information pertaining to periodontal disease and tobacco smoke exposure were collected simultaneously. A single spot evaluation of cotinine level may not reflect its long-term average, which may attenuate associations with self-reported measure of exposure to smoke.[33] Instances where non-smokers can test positive for cotinine and come under Group B include environmental tobacco smoke (ETS) exposure, passive smoking, dietary factors, etc.[17] In addition, individual metabolism, rate of absorption, time of smoking, and smoking habits, as well as ethnic differences,[34] all play a role in the estimation of tobacco exposure. Also, the sample included only males, and hence the results cannot be generalized. Longitudinal studies involving larger population and evaluation of more specific markers such as anabasine and anatabine are required.

CONCLUSION

Quantitative direct association can be established between salivary cotinine and severity of periodontitis. Immunochromatography-based cotinine test strips are a relatively easy method for quantification of salivary cotinine chairside. The results of this study suggest that cotinine test strips are a valid alternative to the more expensive, laborious, and time-consuming techniques like gas chromatography for the assessment of salivary cotinine. As the habit of quitting smoking is gradual, cotinine test strips can serve as simple, regular, and chairside reminders in a dental office.

Periodontists and other dental practitioners should try to discourage their patients from smoking for reasons of periodontal health as well as for better general health and quality of life. These findings should motivate periodontists promote tobacco cessation in their practice, as patients tend to visit them more regularly. The fact that periodontists are trained in preventive measures, patient education, and motivation will be an added advantage in tobacco cessation.