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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 9  |  Issue : 1  |  Page : 45-50

Detection of antibodies against Prevotella Intermedia in patients with chronic periodontitis and periodontally healthy individuals


Department of Periodontology, Maratha Mandal's Nathajirao G Halgekar Institute of Dental Science's and Research Center, Rajiv Gandhi University of Health Sciences Belgaum, Karnataka, India

Date of Submission27-May-2020
Date of Acceptance11-Jan-2021
Date of Web Publication14-May-2021

Correspondence Address:
P T Dixitraj
Deepam, TC 6/897, VARA 696, Arappura Gardens, Vattiyoorkavu P.O, Thiruvananthapuram, - 695 013, Kerala
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/dmr.dmr_27_20

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  Abstract 


Background: Periodontitis, an inflammatory disease of multifactorial etiology, has bacteria playing an essential role in its pathogenesis. Prevotella intermedia plays an important role in disease initiation and progression. Objectives: The objective was to detect immunoglobulin G (IgG) antibodies against P. intermedia in blood of periodontally healthy individuals and patients with chronic periodontitis and compare their levels. Materials and Methodology: A total of 72 subjects were included, 36 subjects in the healthy group and 36 subjects in the chronic periodontitis group. Subgingival plaque sample and blood sample were obtained from each study subject. Samples were processed in the Department of Microbiology and Immunology. P. intermedia were confirmed using the culture method and serum IgG levels were assessed using ELISA (enzyme-linked immunosorbent assay) technique. Comparison between healthy and chronic periodontitis groups was done using independent t-test. Results: IgG levels against P. intermedia were more in the chronic periodontitis group compared to the healthy group, and the difference was statistically significant. Interpretations and Conclusions: Increased levels of IgG antibodies against P. intermedia are associated with periodontal disease. This elevated antibody activity might help to neutralize the effects of the bacterium. IgG antibody level against P. intermedia is a promising indicator in the serological diagnosis of periodontal disease. In chronic periodontitis, the antibody titer in the patient's serum against P. intermedia is raised and could be used as a diagnostic aid.

Keywords: ELISA, Prevotella intermedia, periodontal disease, serum immunoglobulin G


How to cite this article:
Dixitraj P T, Nayak A, Bansal S, Bhat K. Detection of antibodies against Prevotella Intermedia in patients with chronic periodontitis and periodontally healthy individuals. Dent Med Res 2021;9:45-50

How to cite this URL:
Dixitraj P T, Nayak A, Bansal S, Bhat K. Detection of antibodies against Prevotella Intermedia in patients with chronic periodontitis and periodontally healthy individuals. Dent Med Res [serial online] 2021 [cited 2021 Jun 17];9:45-50. Available from: https://www.dmrjournal.org/text.asp?2021/9/1/45/315963




  Introduction Top


Periodontitis, an inflammatory disease of multifactorial etiology, has bacteria playing an essential role in its pathogenesis. The bacteria studied, comprise predominantly of Gram-negative anaerobic rods.[1] Among them, Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, Bacteroides spp., and Selenomonas spp. have been reported as associated with chronic periodontitis.

In the oral cavity, along with other pathogens P. intermedia (formerly known as Bacteroides intermedius)[2] plays an important role in the onset and subsequent development of the polymicrobial periodontal diseases.[3] They are strict oral anaerobes and found in various niches and are seen in nonoral and oral infections. Oral infections include chronic periodontitis,[4] aggressive periodontitis,[5] puberty associated gingivitis,[6] and ulcerative gingivitis.[7] P. intermedia has been found in both periodontally healthy and diseased subjects[8] and studies[9] have shown that colonization of P. intermedia is more in periodontally diseased sites than in periodontally healthy sites.

The host immune response to bacterial infection in periodontal disease has been exclusively studied earlier. In periodontal disease, the antibody levels to several bacteria are elevated and have been hypothesized that failure of the host to mount an effective immune response to periodontal pathogens may be an important mechanism in the pathogenesis of periodontitis.[10] Immunoglobulin G (IgG), the most abundant type of antibody, is found in all body fluids. The fundamental role of IgG is activating the humoral immune response, the complement system, and phagocytosis of microorganisms. IgG reacts with macrophages, neutrophils, and natural killer cells and can activate the complement system[11] and has a protective role in the pathogenesis of disease. In chronic periodontitis, antibodies are produced against various periodontopathogens and they activate cells of the immune system. Studies on various periodontopathogens and their major antigens have proved that in general, mostly IgG antibody titers are involved in investigations of humoral immune reaction.[12]

The humoral immune response, in which IgG and IgA antibodies are produced, is considered to have a protective role in the pathogenesis of periodontal disease,[13] but the mechanisms are not fully understood. Failure of the host to mount an effective immune response to periodontal pathogens may be an important mechanism in the pathogenesis of periodontitis.[14]

Even though P. intermedia has been regarded as a major pathogen contributing to periodontitis, less research has been done on this particular organism compared to other organisms. Very few studies have examined the immune responses in chronic periodontitis to the P. intermedia and the antibody levels in serum against these organisms in health. We were therefore prompted to take up a study which aimed at detecting and comparing the antibody levels against P. intermedia in patients with chronic periodontitis and periodontally healthy individuals.


  Materials and Methodology Top


This study included 36 periodontally healthy subjects and 36 patients with chronic periodontitis from both sexes. The criteria of the patients selected for the periodontally healthy group had the absence of gingival inflammation, absence of bleeding on probing, probing depth ≤3 mm, no clinical attachment loss and for chronic periodontitis group had the presence of gingival inflammation, presence of bleeding on probing, probing depth ≥5 mm, and clinical attachment loss >3 mm. Patients on any antibiotic therapy and periodontal treatment up to 3 months before this study were excluded. Patients with any systemic diseases or conditions, pregnant, lactating women, smokers, and patients below 30 years of age were excluded from the study. Written informed consent was obtained from each subject enrolled in the study. The ethical clearance for the study was obtained from the Institutional Ethics Committee. All microbiological and immunological procedures were carried out in the laboratory of the Department of Molecular Biology and Immunology at our institute.

A pooled subgingival plaque sample was collected from each subject. Randomly selected sites were isolated with sterile cotton rolls and areas were air-dried to prevent contamination. In periodontally healthy subjects, subgingival plaque samples were collected from the gingival sulcus. In chronic periodontitis subjects, after, supragingival plaque and calculus were removed carefully, a sterile curette was inserted into the selected site and the subgingival plaque sample was obtained by moving the curette along the root surface coronally. Plaque samples were immediately transferred in a RTF. About 10 ml of venous blood was collected from the patient. Blood samples were allowed to clot for 30 min at room temperature. The samples were centrifuged at 3000 RPM for 15 min. Then, the serum samples were removed and kept in append of tubes. These append of tubes were stored at –80ºc until laboratory analysis.[15]

Culturing technique using Kanamycin Blood Agar as anaerobic selective medium was used and gram staining and sugar fermentation tests were performed to confirm the organism.[16]

ELISA procedure

The protein estimation of the antigen was done using Lowry's method[17] and the antigen concentration obtained was 120 µg/ml using a biophotometer. This antigen was diluted in 1:6 ratio to obtain a concentration of 20 µg/ml and was then mixed with coating buffer to prepare coating solution. A polystyrene microtiter plate was used for coating antigen. Hundred microliters of coating solution was added to each well. Then, the plate was covered with adhesive and kept at 4°C overnight. The coating solution was removed after incubation and wash buffer was used to clean the wells. Then, 200 µl blocking buffer was added to block interference from unwanted antigens and was incubated for 2 h at room temperature. After incubation, the plates were washed with PBS. The serum sample collected was diluted in 1:100 ratio with PBS and 100 µl of this was added to the wells and kept for 1 h at room temperature. After 1 h, the wells were washed three times with PBS. Hundred microliters of conjugate (goat antihuman IgG HRP diluted with blocking buffer in a ratio 1:5000) was added and kept for 1 h at room temperature. After incubation, the wells were washed three times with PBS. Hundred microliters of the substrate (tetramethylbenzidine diluted with distilled water in ratio 1:20) was added and kept for half an hour. After half an hour, 100 µl of stop solution was added and the reaction was stopped. Readings were recorded at 450 nm with BIORAD I-mark microplate reader.[18]

Statistical analysis

Software Statistical Program for Social Science 9 (Spss Inc, Chicago, USA) was used for data analysis. Independent t-test was used to compare IgG levels against P. intermedia in the study groups (healthy and chronic periodontitis).


  Results Top


[Table 1] illustrates the age and sex of subjects of both healthy and chronic periodontitis group.
Table 1: Age and sex of the study population

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[Table 2] illustrates the IgG values (relative units/ml) of samples analyzed by the ELISA method of both healthy and chronic periodontitis groups.
Table 2: Enzyme-linked immunosorbent assay results

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[Table 3] and [Graph 1] show the distribution of samples in healthy and chronic periodontitis groups with respect to age. The mean age of the healthy group (±standard deviation) was 36.36 ± 7.86. The mean age of the chronic periodontitis group was 44.97 ± 11.77. 77.78% of subjects in the healthy group were in the age group of 30–39 years; 13.89% of subjects in the healthy group were in the age group of 40–49 years; 8.33% of subjects in the healthy group were in the age group >50 years; 33.33% of subjects in the chronic periodontitis group were in the age group of 30–39 years; 33.33% of subjects in the chronic periodontitis group were in the age group of 30–39 years; and 33.33% of subjects in the chronic periodontitis group were in the age group >50 years.
Table 3: Distribution of samples in healthy and chronic periodontitis groups with respect to age

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[Table 4] and [Graph 2] show the distribution of males and females in healthy and chronic periodontitis groups. Of the total 36 subjects in the healthy group, 23 were male and 13 were female, i.e., 63.89% of the healthy patients were male and 36.11% were female. Of the total 36 subjects in the chronic periodontitis, 12 were male and 24 were female, i.e., 33.33% of the chronic periodontitis patients were male and 66.67% were female.
Table 4: Distribution of males and females in healthy and chronic periodontitis groups

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[Table 5] and [Graph 3] show the comparison of healthy and chronic periodontitis groups with IgG levels (relative units/ml) against P. intermedia by independent t-test. The mean IgG value (±SD) against P. intermedia in the healthy group was 8.93 ± 2.88 and in the chronic periodontitis group was 28.67 ± 13.85. P value of 0.0001 was obtained, which implies that the results are statistically significant (P < 0.05 - results are statistically significant).
Table 5: Comparison of healthy and chronic periodontitis groups with immunoglobulin G levels (relative units/ml) against Prevotella intermedia by independent t-test

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  Discussion Top


P. intermedia has been isolated frequently from odontogenic abscesses, saliva, the tongue, and both healthy and diseased subgingival sites. Although its precise role in the infectious process remains unknown, it is becoming increasingly clear that P. intermedia is one of a group of microorganisms that cause periodontal disease.[19]

Maeda et al.[20] studied the incidence of black-pigmented rods (BPRs), especially P. intermedia and P. nigrescens, in periodontal health and disease. Microbiological specimens were collected from subgingival crevice or periodontal pocket by paper point. BPR's appeared in 71.1% of healthy subjects and 87.8% in active sites of periodontitis patients, although healthy sites of the periodontitis group showed lesser BPR's than healthy subjects. The results of our study indicate that P. intermedia is present in both healthy and diseased sites. Our study results are in agreement with Maeda et al.

In a study by Schenck[21] in 1985, ELISA was used to assess IgG, IgA, and IgM serum antibodies against lipopolysaccharide from Bacteroides gingivalis in periodontal health and disease. They found that patients with periodontitis had significantly higher levels of specific antibodies than patients with healthy gingiva. In the current study, the mean IgG value (±SD) against P. intermedia in the healthy group was 8.93 ± 2.88 (relative units/ml) and in the chronic periodontitis group was 28.67 ± 13.85 (relative units/ml). Although similar findings were observed in our study using ELISA, the organisms were different. Our study results suggest that IgG levels are increased during disease, which can be attributed to changes in the immunoregulatory mechanism.

Hamlet et al. in 2001[22] studied the natural distribution of the three putative periodontopathogens P. gingivalis, P. intermedia, and A. actinomycetemcomitans in an Australian population and the relationship between these organisms, pocket depths, and supragingival plaque scores. Subjects entered into the study were in good health and had at least 16 uncrowned teeth. The subgingival plaque was collected from the shallowest and deepest probing site in each sextant of the dentition. In total, 6030 subgingival plaque samples were collected from 504 subjects. The subject's ages ranged from 17 to 64 years with a mean age of 38.3 ± 10.8 years. An ELISA utilizing pathogen-specific monoclonal antibodies was used to quantitate bacterial numbers. A. actinomycetemcomitans was the most frequently detected organism followed by P. gingivalis and P. intermedia. A. actinomycetemcomitans presence was overrepresented in the youngest age group but under-represented in the older age groups. Conversely, P. gingivalis and P. intermedia presence was under-represented in the youngest age group but over-represented in the older age groups. The bacterial presence was strongly associated with pocket depth for both A. actinomycetemcomitans and P. gingivalis. In our study, samples were taken from patients with 30 years and above. The mean age (±SD) of subjects were 36.36 ± 7.86 and 44.97 ± 11.77 in healthy and chronic periodontitis groups, respectively, for our study. All samples showed the presence of P. intermedia irrespective of the age in both the study groups. Although the results are similar, a direct comparative assessment is difficult since study groups were of different age groups. Also, in our research, we used in-house indirect ELISA to detect the antibodies.

Haffajee et al. in 1995[23] conducted a study to group periodontitis subjects according to their elevated serum antibody levels to specific subgingival species. A total of 119 subjects with evidence of prior periodontal destruction were included. Serum samples were obtained from each subject at each visit and the level of antibody was determined by ELISA to 12 subgingival species. Subgingival plaque samples were taken and 14 different subgingival species were determined. Subjects were grouped by cluster analysis of their elevated antibody levels. Subjects in different cluster groups differed with respect to age, number of active sites, levels of gingivitis, prior periodontal destruction, and levels of 11 of 14 microbial species tested. Subjects with multiple elevated antibody levels had significantly more active sites. The study concluded that in chronic periodontitis, the serum antibody levels of different microorganisms will be elevated. Our study showed similar results where the serum antibody levels were increased in chronic periodontitis. Even though our study had a remarkable similarity with this study, it was confined to the fact that the samples were collected at the initial visit only, and IgG levels in the serum were assessed only at the initial visit.

Danielsen et al. in 1993[24] conducted an experimental gingivitis study in twenty-eight young, healthy adults in which blood and clinical recordings were obtained at baseline; after a 4-week period of thorough oral hygiene; after a subsequent 3-week period of plaque accumulation; and after another 2 weeks of thorough oral hygiene. Serum IgG antibodies against whole cells of P. gingivalis, P. intermedia, F. nucleatum, and S. sanguis were determined using enzyme-linked immunosorbent assay. Mean serum IgG antibody levels to P. intermedia, F. nucleatum and S. sanguis remained essentially constant during the experiment, whereas the IgG antibodies to P. gingivalis declined during the initial period of oral hygiene and the subsequent period of plaque accumulation to an average of 84.5% of the baseline value. This reduction could be attributed to the people who developed marked gingival inflammation during the period of plaque accumulation, indicating that the systemic host response may be associated with local tissue responses to variations in oral hygiene. These people were, however, also characterized by higher initial serum IgG responses to P. gingivalis than people who developed less pronounced gingival inflammation during the experiment. The variability and individuality noted in the host response to potential pathogens have important implications for attempts to use such measures for establishing a diagnosis or prognosis for the individual patient. The objective of our current study was only to assess pretreatment antibody levels against P. intermedia in both healthy subjects and subjects with chronic periodontitis.

Albandar et al., 2001[10] studied the associations between serum antibody levels to periodontal pathogens and early-onset periodontitis (EOP). The association between serum IgG, IgA, and IgM antibodies to 6 periodontal microorganisms and clinical subtypes of varying severity of EOP was studied by using ELISA. The study was done on 159 samples aged 19–25 years and included 97 cases with EOP and 62 controls with no clinical signs of EOP. Serum levels of A. actinomycetemcomitans, P. gingivalis, P. intermedia, C. rectus, E. corrodens, and F. nucleatum were assessed. Serum levels of IgG and IgA antibody reactive to P. gingivalis, A. actinomycetemcomitans, and P. intermedia were significantly higher in generalized EOP cases compared to healthy controls. IgM antibody levels did not show any significant associations with EOP for any of the 6 bacterial species tested. There were no significant differences in antibody levels between controls and the 13 subjects who were classified with localized EOP. The study concluded that the antibodies to P. gingivalis, P. intermedia, and A. actinomycetemcomitans may play a significant role in the pathogenesis of EOP. In the current study, only IgG levels were assessed against P. intermedia in chronic periodontitis patients and found that the IgG levels increase during disease than in health.

In the current study, when the quantification of IgG antibodies against P. intermedia was done, it ranged from 2 to 50.5 relative units/ml in the periodontitis group. Even though many patients had elevated antibody levels, out of the 36 samples, 7 samples showed values corresponding to those of healthy subjects. The low values suggest that though some of the patients were infected with P. intermedia, they were not producing high amounts of antibodies against the organism. Perhaps antibody production was kept down by immunoregulation in some of the patients. Hence, difference in the individual host immune response, extent, and duration of the infection could be the factors responsible for observed lack of correlation.


  Conclusions Top


In our present study, IgG antibodies against P. intermedia were exhibited more in the chronic periodontitis group compared to the healthy group, and the difference was statistically significant. The various studies discussed here point toward varying data. This difference can be attributed to multiple factors such as immunoregulation, demographic, ethnicity, behavioral, oral, and general health-related characteristics and distribution of the organism. Future studies on IgG levels to periodontopathogens during disease initiation, progression, and also the IgG levels before and after treatment and their comparison with healthy individuals can enlighten immunoregulation mechanisms in patients with chronic periodontitis. Long-term follow-up randomized clinical trial is essential to be able to convincingly predict the effect of periodontal treatment on IgG levels and hence the remission of the disease process. The variation in the IgG levels after treatment if any, could point toward the possibility of using IgG levels against periodontopathogens in order to assess the disease activity. Having said this, it still could help furthering research on this subject.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
van Winkelhoff AJ, Loos BG, van der Reijden WA, van der Velden U. Porphyromonas gingivalis, Bacteroides forsythus and other putative periodontal pathogens in subjects with and without periodontal destruction. J Clin Periodontol 2002;29:1023-8.  Back to cited text no. 1
    
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21.
Schenck K. IgG, IgA and IgM serum antibodies against lipopolysaccharide from Bacteroides gingivalis in periodontal health and disease. J Periodontal Res 1985;20:368-77.  Back to cited text no. 21
    
22.
Hamlet SM, Cullinan MP, Westerman B, Lindeman M, Bird PS, Palmer J, et al. Distribution of Actinobacillus actinomycetemcomitans, Porphyromonasgingivalis and Prevotella intermedia in an Australian population. J Clin Periodontol 2001;28:1163-71.  Back to cited text no. 22
    
23.
Haffajee AD, Socransky SS, Taubman MA, Sioson J, Smith DJ. Patterns of antibody response in subjects with periodontitis. Oral Microbiol Immunol 1995;10:129-37.  Back to cited text no. 23
    
24.
Danielsen B, Wilton JM, Baelum V, Johnson NW, Fejerskov O. Serum immunoglobulin G antibodies to Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum and Streptococcus sanguis during experimental gingivitis in young adults. Oral Microbiol Immunol 1993;8:154-60.  Back to cited text no. 24
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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