| |
|
|
| Year : 2013 | Volume
: 24
| Issue : 3 | Page : 289-293 |
|
| Prevalence of archaea in chronic periodontitis patients in an Indian population |
|
|
Nipun Ashok1, Shivaraj Warad2, VP Prabath Singh3, Harshal Chaudhari2, Arun Narayanan4, Jean Rodrigues5
1 Department of Periodontics, Alfarabi College of Dentistry, Riyadh, Saudi Arabia
2 Department of Periodontics, P.M.N.M Dental College and Hospital, Bagalkot, India
3 Department of Endodontics, Amritha Institute of Dental Sciences, Cochin, India
4 Department of Periodontics, Kannur Dental College and Hospital, Kannur, India
5 Department of Endodontics, Alfarabi College of Dentistry, Riyadh, Saudi Arabia
Click here for correspondence address and email
| Date of Submission | 26-Apr-2012 |
| Date of Decision | 12-Jul-2012 |
| Date of Acceptance | 09-Sep-2012 |
| Date of Web Publication | 12-Sep-2013 |
|
|
 |
|
 Abstract | | |
Aim: The aim of this study was to investigate the prevalence of archaea in the subgingival crevices of patients with chronic periodontitis in an Indian population.
Materials and Methods: Thirty four chronic periodontitis patients and 16 healthy subjects were included in the study. Thirty four subgingival plaque samples were collected from chronic periodontitis patients, of which 17 samples were from deep pockets and 17 were from shallow pockets. Sixteen subgingival plaque samples were collected from healthy subjects. The presence of archaea in plaque samples was detected by polymerase chain reaction.
Results: Prevalence of archaea in chronic periodontitis patients was 29.4% and in healthy subjects was 11.8%, which was not a statistically significant difference. However, prevalence of archaea, in deep periodontal pockets was 47.1%, in shallow periodontal pockets was 11.8% and in healthy sulcus was 12.5%, respectively. Thus, showing a statistically significant difference between prevalence of archaea in deep periodontal pockets (47.1%) and healthy sulcus (12.5%) and also between deep periodontal pockets (47.1%) and shallow pockets (11.8%), respectively.
Conclusion: Archaea were detected commonly in severe periodontitis suggesting that these microorganisms might be involved in the pathogenesis of periodontal diseases. Keywords: Archaea, attachment loss, chronic periodontitis, gingival index, prevalence
How to cite this article:
Ashok N, Warad S, Singh VP, Chaudhari H, Narayanan A, Rodrigues J. Prevalence of archaea in chronic periodontitis patients in an Indian population. Indian J Dent Res 2013;24:289-93 |
How to cite this URL:
Ashok N, Warad S, Singh VP, Chaudhari H, Narayanan A, Rodrigues J. Prevalence of archaea in chronic periodontitis patients in an Indian population. Indian J Dent Res [serial online] 2013 [cited 2023 Jun 6];24:289-93. Available from: https://www.ijdr.in/text.asp?2013/24/3/289/117988 |
The primary cause of periodontitis is the cumulative effect of interaction between bacterial challenge and the immuno-inflammatory response of the host. [1] Even though specific infectious agents are of key importance in the development of periodontitis, it is highly unlikely that a single agent or even a small group of pathogens are the sole cause or modulators of this heterogeneous disease. [2] Moreover, a possibility exists that novel bacteria or viruses that induce severe periodontitis may yet be found. Since there is no direct evidence to conclude which microbes initiate the first step of subgingival pocket formation, the search for non-cultivable microorganisms in periodontitis must be continued to understand the pathogenicity of subgingival microflora. [3]
Archaea are a group of microorganisms that differ fundamentally from eukaryotes and bacteria in several genetic, biochemical and structural features and thrive in extreme environments such as hot springs, salt lakes or submarine volcanic habitats. [4],[5],[6] The distinction between the domains bacteria and archaea is based mainly on the different types of ribosomal RNA and the chemical nature of the membrane lipids i.e. diacyl D-glycerol diester in bacteria versus isoprenoid L-glycerol diethers or di-L-glycerol tetraethers in archaea. Archaea possess unique flagellins and ether-linked lipids and lack murine, a peptidoglycan that forms rigid cell wall sacculi in almost all bacteria. [7] Almost 15% of the proteins encoded by each archaeal genome are unique to archaea. A set of archaeal signature genes support the phylogenetic conclusion that archaea are an anciently diverged major lineage containing a substantial proportion of unique genes. [8]
Like other bacteria, archaea are commonly mesophilic and some members are known to be closely associated with eukaryotic hosts, including humans. High number of methane producing archaea are found in gastrointestinal tract, vagina and oral cavity. [9],[10],[11] Methanobrevibacter is the major genus isolated from human oral cavity, of which majority were Methanobrevibacter oralis like species. [12]
There has been a controversy regarding the existence of pathogenic archaea. Archaea share some characteristics with known pathogens, which include ample access to host and capabilities of long-term colonization with an endogenous host, recognition by host defenses and evasion of host defenses. [12],[13],[14],[15]
Lepp et al. (2004) revealed a relationship between the severity of periodontitis and abundance of archaea in subgingival crevice. It was also seen that the relative abundance of archaea decreased at treated sites in association with clinical improvement. [16] Archaea has also been detected in infected root canals in teeth with apical periodontitis. [17]
Previous studies have shown the presence of these microorganisms in periodontal pockets, especially in deep pockets and their absence in the healthy sulcus. [12],[16] Moreover, serum IgG antibodies against M. oralis were detected in patients with considerable periodontitis indicating that M. oralis have highly antigenic molecules and may be recognized by immune system as potential pathogens. [13],[18] The inflammatory response, which is essentially protective in design, is responsible for much of the breakdown of the soft and hard periodontal tissues. [19] These suggest that archaea may have the potential to be involved in the pathogenesis of periodontitis. There seems to be a variation in the distribution of these methanogenic species in different communities. [20]
The aim of this study was to investigate the prevalence of archaeal DNA in subgingival plaque samples of chronic periodontitis patients in an Indian population, to correlate the severity of periodontitis with the presence of archaeal DNA and also to compare the clinical periodontal parameters in sites where archaeal DNA was present or absent.
| Materials and Methods | |  |
The present study included 34 chronic periodontitis patients (18 males and 16 females, 25-50 years in age: mean 36.1) and 16 healthy controls (7 males and 9 females, 25-42 years in age: mean 34.2). The study was conducted between March 2010 and December 2010 in the Department of Periodontology of P.M.N.M Dental College, Bagalkot, Karnataka, in accordance with the Helsinki Declaration of 1975, as revised in 2002. The ethical approval was obtained from the Institutional Review Board of P.M.N.M Dental College and Hospital, Bagalkot, Karnataka, India.
An informed consent was obtained from all subjects and all procedures were fully explained prior to the study. All participants were systemically healthy and had not received any antibiotic treatment 6 months prior to sampling and recording. Chronic periodontitis patients were selected based on the criteria of American Academy of Periodontology classification of periodontal diseases. Healthy controls selected did not have loss of attachment at any site. Patients with periapical periodontitis and oral soft tissue lesions were excluded from the study.
The clinical evaluations were based on the following; Gingival index (GI), [21] probing depth (PD) and clinical attachment loss (CAL) of the site selected. All parameters were measured using a Williams probe calibrated in millimeters.
Sample collection and preparation
Prior to sampling, supragingival plaque was gently removed with a sterile cotton pellet and the sample sites were isolated with cotton rolls. Subgingival plaque samples were collected from periodontal pockets in chronic periodontitis patients without site selection using a sterile curette. In healthy controls, subgingival plaque samples were collected from gingival crevices. Each site was classified as healthy sulcus (PD ≤ 3 mm), shallow pocket (4 mm ≤ PD ≤ 5 mm) and deep pocket (PD ≥ 6 mm).
Thirty four subgingival plaque samples were collected from 34 periodontitis patients of which 17 were from shallow periodontal pockets and 17 were from deep periodontal pockets. Sixteen subgingival plaque samples were collected from 16 healthy controls.
The samples from each subject were pooled in a sterile tube containing 0.5 ml of Tris-EDTA (10 mM tris-hydrochloride, 1 mM EDTA, pH 8) buffer, which maintains the integrity of DNA in the sample and was stored at -20°C until the molecular analysis. DNA was extracted from the samples using modified proteinase K method. Plaque in the sample was disrupted by vortexing, then centrifuged at 10,000 rpm for 5 min and the supernatant was discarded. Five hundred microliters of TE buffer (Tris-EDTA buffer) was added and then centrifuged at 10,000 rpm for 3 to 4 min and supernatant again discarded. This step was repeated four times. To this precipitate, 500 μl of Lysis buffer 1 (1 M tris HCL 5 μl, Triton 5 μl, 0.5 M EDTA 1 μl and distilled water 489 μl) was added, centrifuged at 10,000 rpm for 3 to 4 minutes and supernatant again discarded. Fifty microliter of Lysis buffer 2 (Tris HCl 50 mM, Nonidet p-40 0.45%, Tween-20 0.45%,) was added and to it 100 μg/ml of freshly prepared proteinase K was also added. It was kept in a water bath at 75°C for 2 h and then kept in a boiling water bath for 10 min and deep frozen at -80°C till amplification. [22]
Polymerase chain reaction
A conventional PCR was used to detect the DNA of archaea in subgingival plaque samples. 16 S ribosomal DNA fragments of oral archaea were amplified using an archaeal primer for which the specificity and sensitivity had been determined previously. [12],[16] The archaeal primer used was SDArch0333aS15 (5′-TCCAGGCCCTACGGG-3′) and SDArch0958aA19 (5′-YCCGGCGTTGAMTCCAATT- 3′).
The contents of the PCR mixture (50 μl) include 2.5U of Taq polymerase (Chromous Biotech, Bangalore, India), 1× included buffer (Chromous Biotech, Bangalore, India), 2.5 μM MgCl 2 , 0.4 μM of forward and reverse primers (Bioserve, Beltsville, USA), 0.2 μM deoxynucleotide triphosphate (dNTPs) and 2 μl of extracted DNA template. Amplification was performed with PCR system (Corbett research palm cycler version 2.2). Steps involved were 35 cycles of 94°C (30 s), 60°C (30 s) and 72°C (40 s) followed by a 7-min extension at 72°C. This was done on an automated cycler, which can heat and cool the tubes with the reaction mixture in a very short time. [13]
The PCR products were separated by electrophoresis in 1.5% agarose gel in 1-tris acetateethylenediaminetetraacetic acid buffer and visualized under ultraviolet light, following ethidium bromide staining. The positive samples were recorded, and the prevalence of archaea in plaque samples was calculated.
Statistical analysis
Chi-square test was used to test the independent samples. Z-proportionality test was used to compare the prevalence between the two samples. Mann-Whitney U test was used to compare the clinical parameters of archaea positive and negative groups. Differences were considered significant when P < 0.05.
| Results | | |
[Table 1] shows the distribution of archaea in subgingival plaque of periodontitis patients and healthy controls. A total of 50 subjects participated in the study in whom archaeal DNA was detected in 12 subjects (24%). Prevalence of archaea in chronic periodontitis patients was 29.41% (10/34) and in healthy controls was 12.50% (2/12). No statistical significant difference was found between the prevalence of archaea in chronic periodontitis patients and healthy subjects. | Table 1: Prevalence of archaea in chronic periodontitis patients and healthy subjects
Click here to view |
Prevalence of archaea in healthy sulcus, shallow periodontal pockets and deep periodontal pockets was 12.5% (2/16), 11.76% (2/17) and 47.06% (8/17), respectively [Table 2]. When distribution of archaea in healthy sulcus (H), shallow periodontal pockets P (S) and deep periodontal pockets P (D) were compared, there was a statistically significant difference between prevalence of archaea in healthy sulcus (H) and deep periodontal pockets P (D) and also between shallow periodontal pockets P (S) and deep periodontal pockets P (D). However, there was no statistically significant difference between prevalence of archaea in shallow periodontal pockets and healthy sulcus. | Table 2: Prevalence of archaea based on depth of the gingival sulcus and periodontal pocket
Click here to view |
When comparison was done between clinical parameters of archaea detected and undetected sites [Table 3], there was a statistically significant difference between PD in archaea detected and undetected sites. There was no statistically significant difference seen in CA loss and GI of archaea detected and undetected sites.
| Discussion | | |
The present study evaluated the prevalence of archaea in chronic periodontitis patients and periodontally healthy controls in an Indian population. Possible association between these microorganisms and the periodontal condition was also evaluated. Recently, archaea have been detected in periodontal pockets, but not in healthy gingival sulci using PCR suggesting that archaea may have the potential to be involved in pathogenesis of periodontitis. [12],[16],[18],[23]
To the best of our knowledge, this is the first time a study has been conducted to assess the prevalence of archaea in chronic periodontitis patients in an Indian population. Prevalence of archaea in subgingival sites of total subjects was 24% (12/50) and in chronic periodontitis patients was 29.4% (10/34). The findings of this study were similar to the study done by Yamabe et al. [18] in a Japanese population, in which the prevalence of archaea in total subjects and chronic periodontitis patients were 22.4% and 18.8%, respectively.
Lepp et al. [16] detected archaeal DNA in 36% of periodontitis patients in the United States of America. In contrast to the previous studies by Lepp et al., [16] Li et al., [12] Yamabe et al., [18] the present study detected the presence of archaea in healthy sulcus. Prevalence of archaea in healthy sulcus was 12.5% (2/16). This variation in the result may be due to ethnic difference within the study population or host genetics, which may predispose some individual to colonization by these methanogens. There was no statistically significant difference between prevalence of archaea in healthy controls and chronic periodontitis patients.
Prevalence of archaea in healthy sulcus, shallow periodontal pockets and deep periodontal pockets were 12.5%, 11.8% and 47.1%, respectively. These findings are in accordance with the study done by Yamabe et al., [18] in which archaea were more commonly detected in sampling sites where pocket depth was more than 6 mm. Though, archaea were detected in shallow pockets and healthy sulcus, they were more frequent in deep periodontal pockets. This shows that pocket depth is a more important criteria for colonization of archaea than disease status of the individual.
Methanobrevibacter, the major genus of archaea isolated from the oral cavity are anaerobes, and pocket depth provides an anaerobic environment that is required for their colonization. [12] It has been postulated that syntropy occurs in the anaerobic microbial community of deep periodontal pockets, where methanogens consume H 2 produced by secondary fermenters, and contributes to periodontal disease. [15]
The mean PD in archaea detected sites was 6.25 ± 2.38 mm and in archaea undetected sites was 4.45 ± 2.38 mm and mean CAL in archaea positive sites was 5.67 ± 3.26 mm and in archaea negative sites was 3.71 ± 3.24 mm. There was a statistically significant difference between PD of archaea detected and undetected sites, but there was no statistically significant difference between loss of attachment of archaea detected and undetected sites. This implies that presence of archaea is more dependent on PD rather than the CAL.
Mean GI in archaea detected sites was 2.08 ± 0.29 and in archaea undetected sites was 1.66 ± 0.63. There was no statistically significant difference in GI of archaea detected and undetected sites. These data suggests that bleeding may not be a prerequisite for the presence of archaea in the sites.
Archaea are closely associated with human beings, but none of them have been described as a human pathogen. Archaea share many characteristics with known pathogens. The paracrystalline cell surface S-layer of many archaea may play an important role in evading an immune response. It is plausible that archaea, in the context of a complex population of human bacterial flora, have the potential for virulence gene acquisition. [15] Kachlany et al. reported that tad gene, which is required for nonspecific adherence by Aggregatibacter actinomycetemcomitans is widespread in bacteria and archaea suggesting these genes are conserved by descent or acquired by horizontal gene transfer. [24] Though they were frequently isolated from sites of severe periodontal disease, it is still not clear whether archaea are a causative agent of periodontitis or they exist as syntropic partners with other members of the subgingival biofilm community.
Methanogens are strict anaerobes characterized by the ability to produce methane from CO 2 and H 2 and in some cases from formate, acetate or methanol. Hydrogen is the waste end product of the metabolism of microorganisms in anoxic environment. Maintaining a low hydrogen concentration is necessary for anaerobic fermentative process. Methanogens depend on hydrogen and carbon dioxide produced by other species; in return some species grow better in the presence of the methanogens because of the altered patterns of the redox balance associated with reduced partial pressure of hydrogen due to interspecies hydrogen transfer. [25]
Pathology due to microbial infection usually results either from direct damage to the host by the microorganism or from a host inflammatory response. It has been recognized that although pathogens initiate periodontal inflammation, the host response to pathogens is equally if not more important in mediating connective tissue breakdown including bone loss in periodontitis. [19] According to Krishnan et al., archaeosomes (archaeal liposomes) can induce strong humoral T-helper response and long-term cytotoxic T cell response. Archaeosomes are also shown to enhance antigen presenting cell recruitment and activation in vivo. [26],[27] Yamabe et al. [18] demonstrated that patients with severe periodontitis had IgG antibodies against M. oralis, suggesting that patient's sera recognized M. oralis specific antigens and therefore, the patients with severe periodontitis were exposed to M. oralis. Since archaea can induce a host response in patients with periodontitis, they are likely inflammatory modifiers and might play a role in pathogenesis of periodontitis.
| Conclusion | | |
Though, it is difficult to conclude that archaea is a causative agent of periodontitis, these organisms have the ability to induce an immune response in the host. And since the pathogenic process of periodontal disease is largely the result of the host response to the microbial challenges, archaea, which commonly reside in deep periodontal pockets, might play a role in the pathogenesis of periodontitis. So, further studies are needed to be carried out to understand the role of these microorganisms in periodontal diseases.
| References | | |
| 1. | Palmer RM, Scott DA, Meekin TN, Poston RN, Odell EW, Wilson RF. Potential mechanisms of susceptibility to periodontitis in tobacco smokers. J Periodontal Res 1999;34:363-9. 
[PUBMED] |
| 2. | Slots J. Herpes viruses in periodontal diseases. Periodontol 2000:2005;38:33-62.
|
| 3. | Nishihara T, Koseki T. Microbial etiology of periodontitis. Periodontol 2000:2004;36:14-26.
|
| 4. | Woese CR, Kandler O, Wheelis ML. Towards a natural system of organism: Proposal for the domains archaea, bacteria and eucarya. Proc Natl Acad Sci USA 1990;87:4576-9.
[PUBMED] |
| 5. | Barns SM, Fundyga RE, Jeffries MW, Pace NR. Remarkable archaeal diversity detected in a Yellowstone: National Park hot-spring environment. Proc Natl Acad Sci USA 1994;91:1609-13.
[PUBMED] |
| 6. | DeLong E. Archaeal means and extremes. Science 1998;280:542-3.
[PUBMED] |
| 7. | Kandler O, Konig H. Cell wall polymers in archaea. Cell Mol Life Sci 1998;54:305-8.
|
| 8. | Graham DE, Overbeek R, Olsen GJ, Woese CR. An archaeal genomic signature. Proc Natl Acad Sci USA 2000;97:3304-8.
|
| 9. | Karlin DA, Jones RD, Stroehlein JR, Mastromarino AJ, Potter GD. Breath methane excretion in patients with unresected colorectal cancer. J Natl Cancer Inst 1982;69:573-6.
|
| 10. | Belay N, Mukhopadhyay B, Conway de Macario E, Galask R, Daniels L. Methanogenic bacteria in human vaginal samples. J Clin Microbiol 1990;28:1666-8.
|
| 11. | Belay N, Johnson R, Rajagopal BS, Conway de Macario E, Daniels L. Methanogenic bacteria from human dental plaque. Appl Environ Microbiol 1988;54:600-3.
[PUBMED] |
| 12. | Li CL, Liu DL, Jiang YT, Zhou YB, Zhang MZ, Jiang W, et al. Prevalence and molecular diversity of archaea in subgingival pockets of periodontitis patients. Oral Microbiol Immunol 2009;24:343-6.
[PUBMED] |
| 13. | Reeve JN. Archaebacteria then Archaeas now. Are there really no archaeal pathogens? J Bacteriol 1999;181:3613-7.
[PUBMED] |
| 14. | Caviccholi R, Curmi PM, Saunders N, Thomas T. Pathogenic archaea: Do they exist? Bioessays 2003;25:1119-28.
|
| 15. | Eckburg PB, Lepp PW, Relman DA. Archaea and Their Potential Role in Human Disease. Infect Immun 2003;71:591-6.
[PUBMED] |
| 16. | Lepp PW, Brinig MM, Ouverney CC, Palm K, Armitage GC, Relman DA. Methanogenic archaea and human periodontal disease. Proc Natl Acad Sci USA 2004;101:6176-81.
[PUBMED] |
| 17. | Vianna ME, Conrads G, Gomes BP, Horz HP. Identification and quantification of archaea involved in primary endodontic infections. J Clin Microbiol 2006;44:1274-82.
[PUBMED] |
| 18. | Yamabe K, Maeda H, Kokeguchi S, Tanimoto I, Sonoi N, Asakawa S, et al. Distribution of Archaea in Japanese patients with periodontitis and humoral immuneresponse to the components. FEMS Microbiol Lett 2008;287:69-75.
[PUBMED] |
| 19. | Page RC. Milestones in periodontal research and the remaining critical issues. J Periodontal Res 1999;34:331-9.
[PUBMED] |
| 20. | Morii H, Oda K, Suenaga Y, Nakamura T. Low methane concentration in the breath of Japanese. J UOEH 2003;25:397-407.
[PUBMED] |
| 21. | Loe H. The gingival index, plaque index and the retention index systems. J Periodontol 1967;38:610-6.
|
| 22. | Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM, Van Der Noordaa J. Rapid and simple method of purification of nucleic acids. J Clin Microbiol 1990;28:495-503.
[PUBMED] |
| 23. | Kulik EM, Sandmeier H, Hinni K, Meyer J. Identification of archaeal rDNA from subgingival dental plaque by PCR amplification and sequence analysis. FEMS Microbiol Lett 2001;196:129-33.
[PUBMED] |
| 24. | Kachlany SC, Planet PJ, Bhattacharjee MK, Kollia E, DeSalle R, Fine DH, et al. Nonspecific adherence by actinobacillus actinomycetemcomitans requires genes widespread in bacteria and archaea. J Bacteriol 2000;182:6169-76.
[PUBMED] |
| 25. | Jiang YT, Xia WW, Li CL, Jiang W, Liang JP. Preliminary studies of the presence and association of bacteria and archaea in teeth with apical periodontitis. Int Endod J 2009;42:1096-103.
[PUBMED] |
| 26. | Krishnan L, Sad S, Patel GB, Sprott GD. The potent adjuvant activity of archaeosomes correlates to the recruitment and activation of macrophages and dendritic cells in vivo. J Immunol 2001;166:1885-93.
[PUBMED] |
| 27. | Krishnan L, Sad S, Patel GB, Sprott GD. Archaeosomes induce long-term CD81 cytotoxic T cell response to entrapped soluble protein by the exogenous cytosolic pathway, in the absence of CD41 T cell help. J Immunol 2000;165:5177-85.
[PUBMED] |
Correspondence Address:
Nipun Ashok
Department of Periodontics, Alfarabi College of Dentistry, Riyadh
Saudi Arabia
 Source of Support: This study was self funded by authors, Conflict of Interest: None  | Check |
DOI: 10.4103/0970-9290.117988
[Table 1], [Table 2], [Table 3] |
|
| This article has been cited by | | 1 |
Functional biomes beyond the bacteriome in the oral ecosystem |
|
| A.S. Smiline Girija, Pitchaipillai Sankar Ganesh | | Japanese Dental Science Review. 2022; 58: 217 | | [Pubmed] | [DOI] | | | 2 |
The Oral Archaeome: A Scoping Review |
|
| A. Belmok, J.A. de Cena, C.M. Kyaw, N. Damé-Teixeira | | Journal of Dental Research. 2020; 99(6): 630 | | [Pubmed] | [DOI] | | | 3 |
Archaea associated with human surfaces: not to be underestimated |
|
| Corinna Bang,Ruth A. Schmitz,Franz Narberhaus | | FEMS Microbiology Reviews. 2015; 39(5): 631 | | [Pubmed] | [DOI] | | | 4 |
Biofilm formation of mucosa-associated methanoarchaeal strains |
|
| Corinna Bang,Claudia Ehlers,Alvaro Orell,Daniela Prasse,Marlene Spinner,Stanislav N. Gorb,Sonja-Verena Albers,Ruth A. Schmitz | | Frontiers in Microbiology. 2014; 5 | | [Pubmed] | [DOI] | | | 5 |
Biofilm formation of mucosa-associated methanoarchaeal strains |
|
| Corinna Bang,Claudia Ehlers,Alvaro Orell,Daniela Prasse,Marlene Spinner,Stanislav N. Gorb,Sonja-Verena Albers,Ruth A. Schmitz | | Frontiers in Microbiology. 2014; 5 | | [Pubmed] | [DOI] | |
|
|
|
|
|
|
|
|
|
|
|
|
| Article Access Statistics | | | Viewed | 7026 | | | Printed | 398 | | | Emailed | 0 | | | PDF Downloaded | 151 | | | Comments | [Add] | | | Cited by others | 5 | | |
|
|
Users online:
