Journal of Clinical and Diagnostic Research, ISSN - 0973 - 709X

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Dr Bhanu K Bhakhri

"The Journal of Clinical and Diagnostic Research (JCDR) has been in operation since almost a decade. It has contributed a huge number of peer reviewed articles, across a spectrum of medical disciplines, to the medical literature.
Its wide based indexing and open access publications attracts many authors as well as readers
For authors, the manuscripts can be uploaded online through an easily navigable portal, on other hand, reviewers appreciate the systematic handling of all manuscripts. The way JCDR has emerged as an effective medium for publishing wide array of observations in Indian context, I wish the editorial team success in their endeavour"



Dr Bhanu K Bhakhri
Faculty, Pediatric Medicine
Super Speciality Paediatric Hospital and Post Graduate Teaching Institute, Noida
On Sep 2018




Dr Mohan Z Mani

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I wish all success to your journal and look forward to sending you any suitable similar article in future"



Dr Mohan Z Mani,
Professor & Head,
Department of Dematolgy,
Believers Church Medical College,
Thiruvalla, Kerala
On Sep 2018




Prof. Somashekhar Nimbalkar

"Over the last few years, we have published our research regularly in Journal of Clinical and Diagnostic Research. Having published in more than 20 high impact journals over the last five years including several high impact ones and reviewing articles for even more journals across my fields of interest, we value our published work in JCDR for their high standards in publishing scientific articles. The ease of submission, the rapid reviews in under a month, the high quality of their reviewers and keen attention to the final process of proofs and publication, ensure that there are no mistakes in the final article. We have been asked clarifications on several occasions and have been happy to provide them and it exemplifies the commitment to quality of the team at JCDR."



Prof. Somashekhar Nimbalkar
Head, Department of Pediatrics, Pramukhswami Medical College, Karamsad
Chairman, Research Group, Charutar Arogya Mandal, Karamsad
National Joint Coordinator - Advanced IAP NNF NRP Program
Ex-Member, Governing Body, National Neonatology Forum, New Delhi
Ex-President - National Neonatology Forum Gujarat State Chapter
Department of Pediatrics, Pramukhswami Medical College, Karamsad, Anand, Gujarat.
On Sep 2018




Dr. Kalyani R

"Journal of Clinical and Diagnostic Research is at present a well-known Indian originated scientific journal which started with a humble beginning. I have been associated with this journal since many years. I appreciate the Editor, Dr. Hemant Jain, for his constant effort in bringing up this journal to the present status right from the scratch. The journal is multidisciplinary. It encourages in publishing the scientific articles from postgraduates and also the beginners who start their career. At the same time the journal also caters for the high quality articles from specialty and super-specialty researchers. Hence it provides a platform for the scientist and researchers to publish. The other aspect of it is, the readers get the information regarding the most recent developments in science which can be used for teaching, research, treating patients and to some extent take preventive measures against certain diseases. The journal is contributing immensely to the society at national and international level."



Dr Kalyani R
Professor and Head
Department of Pathology
Sri Devaraj Urs Medical College
Sri Devaraj Urs Academy of Higher Education and Research , Kolar, Karnataka
On Sep 2018




Dr. Saumya Navit

"As a peer-reviewed journal, the Journal of Clinical and Diagnostic Research provides an opportunity to researchers, scientists and budding professionals to explore the developments in the field of medicine and dentistry and their varied specialities, thus extending our view on biological diversities of living species in relation to medicine.
‘Knowledge is treasure of a wise man.’ The free access of this journal provides an immense scope of learning for the both the old and the young in field of medicine and dentistry as well. The multidisciplinary nature of the journal makes it a better platform to absorb all that is being researched and developed. The publication process is systematic and professional. Online submission, publication and peer reviewing makes it a user-friendly journal.
As an experienced dentist and an academician, I proudly recommend this journal to the dental fraternity as a good quality open access platform for rapid communication of their cutting-edge research progress and discovery.
I wish JCDR a great success and I hope that journal will soar higher with the passing time."



Dr Saumya Navit
Professor and Head
Department of Pediatric Dentistry
Saraswati Dental College
Lucknow
On Sep 2018




Dr. Arunava Biswas

"My sincere attachment with JCDR as an author as well as reviewer is a learning experience . Their systematic approach in publication of article in various categories is really praiseworthy.
Their prompt and timely response to review's query and the manner in which they have set the reviewing process helps in extracting the best possible scientific writings for publication.
It's a honour and pride to be a part of the JCDR team. My very best wishes to JCDR and hope it will sparkle up above the sky as a high indexed journal in near future."



Dr. Arunava Biswas
MD, DM (Clinical Pharmacology)
Assistant Professor
Department of Pharmacology
Calcutta National Medical College & Hospital , Kolkata




Dr. C.S. Ramesh Babu
" Journal of Clinical and Diagnostic Research (JCDR) is a multi-specialty medical and dental journal publishing high quality research articles in almost all branches of medicine. The quality of printing of figures and tables is excellent and comparable to any International journal. An added advantage is nominal publication charges and monthly issue of the journal and more chances of an article being accepted for publication. Moreover being a multi-specialty journal an article concerning a particular specialty has a wider reach of readers of other related specialties also. As an author and reviewer for several years I find this Journal most suitable and highly recommend this Journal."
Best regards,
C.S. Ramesh Babu,
Associate Professor of Anatomy,
Muzaffarnagar Medical College,
Muzaffarnagar.
On Aug 2018




Dr. Arundhathi. S
"Journal of Clinical and Diagnostic Research (JCDR) is a reputed peer reviewed journal and is constantly involved in publishing high quality research articles related to medicine. Its been a great pleasure to be associated with this esteemed journal as a reviewer and as an author for a couple of years. The editorial board consists of many dedicated and reputed experts as its members and they are doing an appreciable work in guiding budding researchers. JCDR is doing a commendable job in scientific research by promoting excellent quality research & review articles and case reports & series. The reviewers provide appropriate suggestions that improve the quality of articles. I strongly recommend my fraternity to encourage JCDR by contributing their valuable research work in this widely accepted, user friendly journal. I hope my collaboration with JCDR will continue for a long time".



Dr. Arundhathi. S
MBBS, MD (Pathology),
Sanjay Gandhi institute of trauma and orthopedics,
Bengaluru.
On Aug 2018




Dr. Mamta Gupta,
"It gives me great pleasure to be associated with JCDR, since last 2-3 years. Since then I have authored, co-authored and reviewed about 25 articles in JCDR. I thank JCDR for giving me an opportunity to improve my own skills as an author and a reviewer.
It 's a multispecialty journal, publishing high quality articles. It gives a platform to the authors to publish their research work which can be available for everyone across the globe to read. The best thing about JCDR is that the full articles of all medical specialties are available as pdf/html for reading free of cost or without institutional subscription, which is not there for other journals. For those who have problem in writing manuscript or do statistical work, JCDR comes for their rescue.
The journal has a monthly publication and the articles are published quite fast. In time compared to other journals. The on-line first publication is also a great advantage and facility to review one's own articles before going to print. The response to any query and permission if required, is quite fast; this is quite commendable. I have a very good experience about seeking quick permission for quoting a photograph (Fig.) from a JCDR article for my chapter authored in an E book. I never thought it would be so easy. No hassles.
Reviewing articles is no less a pain staking process and requires in depth perception, knowledge about the topic for review. It requires time and concentration, yet I enjoy doing it. The JCDR website especially for the reviewers is quite user friendly. My suggestions for improving the journal is, more strict review process, so that only high quality articles are published. I find a a good number of articles in Obst. Gynae, hence, a new journal for this specialty titled JCDR-OG can be started. May be a bimonthly or quarterly publication to begin with. Only selected articles should find a place in it.
An yearly reward for the best article authored can also incentivize the authors. Though the process of finding the best article will be not be very easy. I do not know how reviewing process can be improved. If an article is being reviewed by two reviewers, then opinion of one can be communicated to the other or the final opinion of the editor can be communicated to the reviewer if requested for. This will help one’s reviewing skills.
My best wishes to Dr. Hemant Jain and all the editorial staff of JCDR for their untiring efforts to bring out this journal. I strongly recommend medical fraternity to publish their valuable research work in this esteemed journal, JCDR".



Dr. Mamta Gupta
Consultant
(Ex HOD Obs &Gynae, Hindu Rao Hospital and associated NDMC Medical College, Delhi)
Aug 2018




Dr. Rajendra Kumar Ghritlaharey

"I wish to thank Dr. Hemant Jain, Editor-in-Chief Journal of Clinical and Diagnostic Research (JCDR), for asking me to write up few words.
Writing is the representation of language in a textual medium i e; into the words and sentences on paper. Quality medical manuscript writing in particular, demands not only a high-quality research, but also requires accurate and concise communication of findings and conclusions, with adherence to particular journal guidelines. In medical field whether working in teaching, private, or in corporate institution, everyone wants to excel in his / her own field and get recognised by making manuscripts publication.


Authors are the souls of any journal, and deserve much respect. To publish a journal manuscripts are needed from authors. Authors have a great responsibility for producing facts of their work in terms of number and results truthfully and an individual honesty is expected from authors in this regards. Both ways its true "No authors-No manuscripts-No journals" and "No journals–No manuscripts–No authors". Reviewing a manuscript is also a very responsible and important task of any peer-reviewed journal and to be taken seriously. It needs knowledge on the subject, sincerity, honesty and determination. Although the process of reviewing a manuscript is a time consuming task butit is expected to give one's best remarks within the time frame of the journal.
Salient features of the JCDR: It is a biomedical, multidisciplinary (including all medical and dental specialities), e-journal, with wide scope and extensive author support. At the same time, a free text of manuscript is available in HTML and PDF format. There is fast growing authorship and readership with JCDR as this can be judged by the number of articles published in it i e; in Feb 2007 of its first issue, it contained 5 articles only, and now in its recent volume published in April 2011, it contained 67 manuscripts. This e-journal is fulfilling the commitments and objectives sincerely, (as stated by Editor-in-chief in his preface to first edition) i e; to encourage physicians through the internet, especially from the developing countries who witness a spectrum of disease and acquire a wealth of knowledge to publish their experiences to benefit the medical community in patients care. I also feel that many of us have work of substance, newer ideas, adequate clinical materials but poor in medical writing and hesitation to submit the work and need help. JCDR provides authors help in this regards.
Timely publication of journal: Publication of manuscripts and bringing out the issue in time is one of the positive aspects of JCDR and is possible with strong support team in terms of peer reviewers, proof reading, language check, computer operators, etc. This is one of the great reasons for authors to submit their work with JCDR. Another best part of JCDR is "Online first Publications" facilities available for the authors. This facility not only provides the prompt publications of the manuscripts but at the same time also early availability of the manuscripts for the readers.
Indexation and online availability: Indexation transforms the journal in some sense from its local ownership to the worldwide professional community and to the public.JCDR is indexed with Embase & EMbiology, Google Scholar, Index Copernicus, Chemical Abstracts Service, Journal seek Database, Indian Science Abstracts, to name few of them. Manuscriptspublished in JCDR are available on major search engines ie; google, yahoo, msn.
In the era of fast growing newer technologies, and in computer and internet friendly environment the manuscripts preparation, submission, review, revision, etc and all can be done and checked with a click from all corer of the world, at any time. Of course there is always a scope for improvement in every field and none is perfect. To progress, one needs to identify the areas of one's weakness and to strengthen them.
It is well said that "happy beginning is half done" and it fits perfectly with JCDR. It has grown considerably and I feel it has already grown up from its infancy to adolescence, achieving the status of standard online e-journal form Indian continent since its inception in Feb 2007. This had been made possible due to the efforts and the hard work put in it. The way the JCDR is improving with every new volume, with good quality original manuscripts, makes it a quality journal for readers. I must thank and congratulate Dr Hemant Jain, Editor-in-Chief JCDR and his team for their sincere efforts, dedication, and determination for making JCDR a fast growing journal.
Every one of us: authors, reviewers, editors, and publisher are responsible for enhancing the stature of the journal. I wish for a great success for JCDR."



Thanking you
With sincere regards
Dr. Rajendra Kumar Ghritlaharey, M.S., M. Ch., FAIS
Associate Professor,
Department of Paediatric Surgery, Gandhi Medical College & Associated
Kamla Nehru & Hamidia Hospitals Bhopal, Madhya Pradesh 462 001 (India)
E-mail: drrajendrak1@rediffmail.com
On May 11,2011




Dr. Shankar P.R.

"On looking back through my Gmail archives after being requested by the journal to write a short editorial about my experiences of publishing with the Journal of Clinical and Diagnostic Research (JCDR), I came across an e-mail from Dr. Hemant Jain, Editor, in March 2007, which introduced the new electronic journal. The main features of the journal which were outlined in the e-mail were extensive author support, cash rewards, the peer review process, and other salient features of the journal.
Over a span of over four years, we (I and my colleagues) have published around 25 articles in the journal. In this editorial, I plan to briefly discuss my experiences of publishing with JCDR and the strengths of the journal and to finally address the areas for improvement.
My experiences of publishing with JCDR: Overall, my experiences of publishing withJCDR have been positive. The best point about the journal is that it responds to queries from the author. This may seem to be simple and not too much to ask for, but unfortunately, many journals in the subcontinent and from many developing countries do not respond or they respond with a long delay to the queries from the authors 1. The reasons could be many, including lack of optimal secretarial and other support. Another problem with many journals is the slowness of the review process. Editorial processing and peer review can take anywhere between a year to two years with some journals. Also, some journals do not keep the contributors informed about the progress of the review process. Due to the long review process, the articles can lose their relevance and topicality. A major benefit with JCDR is the timeliness and promptness of its response. In Dr Jain's e-mail which was sent to me in 2007, before the introduction of the Pre-publishing system, he had stated that he had received my submission and that he would get back to me within seven days and he did!
Most of the manuscripts are published within 3 to 4 months of their submission if they are found to be suitable after the review process. JCDR is published bimonthly and the accepted articles were usually published in the next issue. Recently, due to the increased volume of the submissions, the review process has become slower and it ?? Section can take from 4 to 6 months for the articles to be reviewed. The journal has an extensive author support system and it has recently introduced a paid expedited review process. The journal also mentions the average time for processing the manuscript under different submission systems - regular submission and expedited review.
Strengths of the journal: The journal has an online first facility in which the accepted manuscripts may be published on the website before being included in a regular issue of the journal. This cuts down the time between their acceptance and the publication. The journal is indexed in many databases, though not in PubMed. The editorial board should now take steps to index the journal in PubMed. The journal has a system of notifying readers through e-mail when a new issue is released. Also, the articles are available in both the HTML and the PDF formats. I especially like the new and colorful page format of the journal. Also, the access statistics of the articles are available. The prepublication and the manuscript tracking system are also helpful for the authors.
Areas for improvement: In certain cases, I felt that the peer review process of the manuscripts was not up to international standards and that it should be strengthened. Also, the number of manuscripts in an issue is high and it may be difficult for readers to go through all of them. The journal can consider tightening of the peer review process and increasing the quality standards for the acceptance of the manuscripts. I faced occasional problems with the online manuscript submission (Pre-publishing) system, which have to be addressed.
Overall, the publishing process with JCDR has been smooth, quick and relatively hassle free and I can recommend other authors to consider the journal as an outlet for their work."



Dr. P. Ravi Shankar
KIST Medical College, P.O. Box 14142, Kathmandu, Nepal.
E-mail: ravi.dr.shankar@gmail.com
On April 2011

Important Notice

Dentistry
Year : 2012 | Month : May | Volume : 6 | Issue : 4 | Page : 747 - 752

Prevalence of Periodontopathic Bacteria in the Subgingival Plaque of a South Indian Population with Periodontitis

Krishnan Mahalakshmi, Padma Krishnan, S.C. Chandrasekaran, K.H. Panishankar, Natarajan Subashini

1. Associate Professor, Department of Microbiology, Sree Balaji Dental College and Hospital, India. 2. Assistant Professor, Department of Microbiology, Dr. ALM PGIBMS, University of Madras, Chennai, India. 3. Department of Periodontics and Implantology, Sree Balaji Dental College and Hospital, Chennai, India. 4. Professor, Department of Periodontics and Implantology, Tamilnadu Government Dental College and Hospital, Chennai, India. 5. PG student, Department of Periodontics and Implantology, Tamilnadu Government Dental College and Hospital, Chennai, India.

Correspondence Address :
Padma Krishnan
Assistant Professor, Department of Microbiology,
Dr. ALM PGIBMS, University of Madras,
Sekkizhar campus, Taramani.
Chennai, India - 600 113.
Phone: + 919840742105; Fax No: 91-44-24540709
E-mial: padma.abpkn@gmail.com

Abstract

Background: Periodontitis is an important global public health problem which involves mostly the adult population over 35-40 years of age. Strongly considering that periodontitis is a polymicrobial infection, the screening of the selective microbial population, rather than the isolation of a single member of the subgingival flora, should give a more wide-ranging perception in the aetiology of periodontitis. Different compositions of the bacterial species in the subgingival microflora of the periodontitis patients have been reported in diverse ethnicity. Similar studies on the bacterial aetiology of periodontitis is completely lacking in the Indian population.

Objectives: To detect and compare the prevalence of the eight putative, periodontal bacterial pathogens (Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Aggregatibacter actinomycetemcomitans, Campylobacter rectus, Eikenella corrodens, Prevotella intermedia and Prevotella nigrescens) among periodontitis patients and healthy subjects.

Materials and Methods: A total of four hundred subgingival plaque samples which were collected from two hundred periodontitis patients (chronic periodontitis patients-ChP, n=128, aggressive periodontitis patients-AgP, n=72) and two hundred healthy subjects was subjected to Polymerase Chain Reaction (PCR) with the help of species specific primers which targeted the 16S rRNA gene of the bacterial species. The statistical analysis was performed by using the Chi-square test.

Results: The prevalence of various microorganisms in chronic periodontitis (n=128), aggressive periodontitis (n=72) and in healthy subjects (n=200) was 80.5%, 73.6% and 11% for P. gingivalis, 73.4%, 59.7% and 10.5% for T.forsythia, 71.1%, 33.3% and 5.5% for T. denticola, 32.0%, 61.1% and 2.5% for A. actinomycetemcomitans, 17.2%, 11.1% and 8.5% for C. rectus, 15.6%, 11.1% and 6% for E.corrodens, 16.4%, 25.0 % and 7.5% for P. intermedia and 13.3%, 13.9 and 14% for P. nigrescens respectively.

Conclusion: The present study demonstrated a high prevalence of the red complex group (P. gingivalis, T. forsythia and T. denticola) in the ChP patients. The high odds ratio for P. gingivalis, T.forsythia, T.denticola and A.actinomycetemcomitans suggested a strong association between them and periodontitis. The incidence of A.actinomycetemcomitans along with P.gingivalis and T.forsythia was high in AgP. An appropriate therapeutic strategy can be considered in view of these bacterial consortiums. In addition, two formerly unreported symbiotic relationships were found between the 8 bacterial species which were analyzed.

Keywords

Aggressive periodontitis, Anaerobic bacteria, Chronic periodontitis, PCR, Red complex group, Sub-gingival plaque

Introduction
Periodontitis is a progressive disease which is widely regarded as the second most common disease worldwide after dental decay. It is caused by microorganisms that adhere to and grow on the tooth surfaces, along with an excessively aggressive immune response against these microorganisms. It involves the progressive loss of the alveolar bone around the teeth and if left untreated, it can lead to the loosening and the subsequent loss of teeth. It had been previously estimated that about 500 bacterial species colonized the human oral cavity (1), (2). A majority of these organisms were found to be commensals and they existed in complex communities, forming oral biofilms on the tooth surfaces, although only a selective number of the anaerobes had been implicated as periodontal pathogens. The predominating microorganisms which are isolated from the teeth and the gingival sulcus of the individuals with a healthy periodontium include mainly gram-positive, facultative, anaerobic bacteria and rarely, gram-negative, anaerobic bacilli (3). The gramnegative anaerobic bacteria, on the other hand, have been found to predominate in the subgingival niche with increasing severity of the Original Article periodontal disease (4),(5). Among these gram-negative bacteria, Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola have been designated as the red complex (6). The anaerobic culture methods are arduous and they may fail in identifying all the organisms due to the diverse growth requirements of the bacteria which inhabit the subgingival microflora. The 16S rRNA gene detection by PCR for the organisms that are uncultivable and are difficult to identify can eliminate the ambiguity in the diagnostic microbiology. As the literature on the bacterial aetiology of periodontitis is completely lacking in the Indian scenario, this study was planned, to detect the eight putative periodontal pathogens.

Material and Methods

The Study Population Two hundred periodontitis patients (chronic periodontitis patients- ChP, n=128, aggressive periodontitis patients-AgP, n=72) were recruited from the Department of Periodontics and ImplantologyTamil Nadu, Government Dental College and Hospital and the Sree Balaji Dental College and Hospital for the study. The age of the study population ranged between 20-60 years. More than 3 teeth with a pocket probing depth (PPD) of ≥ 5mm and bleeding on probing were the inclusion criteria. Patients who were on periodontal therapy and dental hygiene procedures in the past year, those who were on antibiotic therapy during the past six months, pregnant women and those with a history of diabetes, other systemic conditions and smoking were the exclusion criteria. 200 healthy subjects with a PPD of ≤ 3mm and ≤ 3.6% of sites which exhibited gingival bleeding were included as the control group. A written informed consent was obtained from the patients and the healthy subjects. This study was reviewed and approved by the human ethical committee of the Dr A.L.M Post Graduate Institute of Basic Medical Sciences, University of Madras, Chennai.

Sample collection and DNA isolation
The PPD and the clinical attachment level CAL (mm) was recorded. The PPD was measured with a graduated Williams periodontal probe. The subgingival plaque was collected from the periodontitis patients and the healthy subjects, from three different sites, by using a sterile Gracy curette after the careful removal of the supragingival plaque with a sterile cotton roll. The samples from each patient were pooled into 500μl of phosphate buffered saline (PBS, pH 8.0), transported in ice and stored at -20oC till they were assayed. DNA was extracted from them as per the method of Wu et al. (7)

PCR Assays
All the PCR analyses were performed in duplicate. Positive and negative controls were incorporated in all the batches of the samples which were examined. The positive controls consisted of DNA which was extracted from clinical samples (subgingival plaque), which was previously tested and was found to be positive for each of the bacterial species, as was confirmed by PCR and sequencing. Sterile millipore water was included as a negative control. One negative control was included for a batch of ten samples which were analyzed. The PCR reactions were run in a thermal cycler (Eppendorf Mastercycler Gradient; Eppendorf AG, Hamburg, Germany). The PCR reaction mixture (50μl) for the detection of the 16S rRNA gene of P. gingivalis, T. forsythia, T.denticola, E. corrodens, C. rectus, A.actinomycetemcomitans, P. intermedia and P. nigrescens contained 5μl of 10X PCR buffer (20 mM/L Tris-HCl, 50 mM/L KCl, pH 8.4), 1.25 U Taq DNA polymerase (Bangalore Genei, India.), 0.25 mMol/L of each dNTP (Medox Biotech India Pvt Ltd), 1.5mM MgCl2(Sigma-Aldrich Pvt Ltd, India), 0.5 μM of each primer (Sigma-Aldrich Pvt Ltd, India) and 5 μl of the template. The PCR thermocycling conditions for the detection of the 16S rRNA of P. gingivalis, T. forsythia, T. denticola, E. corrodens and C. rectus included an initial denaturation at 94oC for 2 min, followed by 36 cycles of denaturation at 95oC for 30 sec, primer annealing at 60oC for 1 min and extension at 72oC for 1min, and subsequently, a final extension step at 72oC for 2 min. The temperature profile for the detection of the 16S rRNA of A.actinomycetemcomitans, P.intermedia and P.nigrescens included an initial denaturation at 95oC for 2 min, followed by 36 cycles at 94oC for 30 sec, at 55oC for 1 min and at 72oC for 2 min, and a final extension step at 72oC for 10min. The PCR products were fractionated in a 1.5% agarose gel electrophoresis in Tris-Borate EDTA buffer. The gel was stained with 0.5μg/ml ethidium bromide and it was photographed by using a BioRad UV gel documentation system. A 100 bp ladder (Medox Biotech India Pvt Ltd) served as the molecular weight marker.

Primer Specificity
The species-specific primers for P. gingivalis, T. forsythia, T. denticola, E. corrodens, C. rectus, P.intermedia and P.nigrescens were as per Ashimoto et al’s protocol (8). The primer for A. actinomycetemcomitans was as per Slots et al’s protocol (9). The species specificity was confirmed by sequencing one PCR product from a clinical sample for each primer in an Applied Biosystem (ABI) 3130 Genetic Analyser, ABI PRISM Big Dye Terminators Version 3.1. The sequences which were generated were compared with those which were available in the GenBank database.

Nucleotide sequence analysis
All the sequenced products unveiled nucleotide identities of ≥99% with the corresponding taxa when they were blasted in the Genbank database, thus ensuring reliability in the detection protocol. The sequences for the 16S rRNA gene of P. gingivalis, T. forsythia, T. denticola, A. actinomycetemcomitans, C. rectus, E. corrodens, P. intermedia and P.nigrescens were submitted to GenBank under the accession nos, HQ112349, HQ112350, HQ202265, HQ188689, HQ269826, HQ269827, HQ202263 and HQ202264 respectively.

Statistical analysis
The mean clinical measurements such as PPD and CAL were computed for each subject and they were then averaged across the subjects within the groups. Any difference of P <0.05 was considered as statistically significant. The prevalence of the eight bacterial species among the periodontitis patients and the healthy subjects was compared with the Pearson’s Chi-square test. A P value of < 0.05 was considered as statistically significant. The odds ratio was calculated by using the Chi-square test with a 95% confidence interval, to analyze the association between two different bacterial species among the periodontitis patients and the eight bacterial species with disease and health. A P value of <0.0001 was considered as statistically significant.

Results

The periodontal clinical parameters of the two subject groups are shown in (Table/Fig 1). A significant difference was observed between the groups with respect to age (p <0.0001); No significant differences were observed between the groups with respect to the gender (male/female ratio) (p = 0.5). The subjects with periodontitis presented significantly higher values of the mean PPD (p < 0.0001) and CAL (p < 0.0001), than the periodontally healthy individuals. (Table/Fig 1) The species specific primers for all the eight bacterial species successfully detected a single band of the expected size. (Table/Fig 2) shows the prevalence of the eight bacterial species in ChP, AgP and in healthy subjects. A co-occurrence of P.gingivalis / T. forsythia was observed in (71) 55.5% of the ChP patients. Correspondingly, a co-occurrence of P.gingivalis / T. denticola was observed in (72) 56.3% of the ChP patients. Among the AgP patients, the co-occurrence of P.gingivalis / T. forsythia was found to be (32) 44.4%. Conversely, a co-occurrence of P. gingivalis / T. denticola was observed only in (13) 18% of the AgP patients. The co-occurrence of T. forsythia / T. denticola was (66)51.5% and (13)18% in the ChP and the AgP patients respectively. A cooccurrence of the red complex group (P.gingivalis, T. forsythia and T. denticola) was observed in (47) 36.7% of the ChP and in (5) 6.9%of the AgP patients. A striking colonization of the red complex group was completely absent among the healthy subjects. A majority of the ChP patients who harboured the red complex group showed a PPD of ≥6mm and a CAL of ≥8mm. Conversely, AgP patients who were positive for the red complex group revealed a PPD and a CAL of ≥ 8mm and ≥ 10mm respectively.

About (9) 4.5% ChP patients colonized A.actinomycetemcomitans along with the presence of the red complex group. Such an association was totally absent among the AgP patients. Two ChP patients harboured all the seven organisms except E.corrodens. Four ChP patients colonized 5 species (P. gingivalis, T. forsythia, T. denticola A. actinomycetemcomitans and P. intermedia). Two healthy subjects harboured four organisms, a combination of T. forsythia, A.actinomycetemcomitans, P. intermedia and P. nigrescens/ P. gingivalis T.forsythia, C.rectus and P. nigrescens. P. gingivalis and T. forsythia co-occurred in (5) 2.5% of the healthy subjects. P. gingivalis, T.forsythia and C.rectus was colonized in 2 healthy subjects. Overall, the prevalence of P. gingivalis was high in both the ChP and the AgP patients, followed by T. forsythia and T. denticola in the ChP patients and A. actinomycetemcomitans and T. forsythia in the AgP patients respectively.

The odds ratio was calculated to assess the association between the bacterial species. Twenty eight bacterial combinations were tested for the periodontitis group (both ChP and AgP) by using the Chi-square test (Table/Fig 3). A statistically significant odds ratio (p<0.01) was obtained for 19 of the 28 bacterial combinations. A low odds ratio occurred for C.rectus/ P.intermedia and E.corrodens / P.intermedia at 0.72 and 0.67 respectively. The odds ratio which was calculated by using the Chi-square test for the bacterial species for periodontitis (ChP and AgP) and health revealed a statistically significant positive association for P.gingivalis, T. forsythia, T.denticola and A.actinomycetemcomitans towards both the groups of periodontitis. A high odds ratio occurred for P. gingivalis and A. actinomycetemcomitans The lowest odds ratio occurred for P.nigrescens (Table/Fig 4). No negative association (odds ratio <0.5) was observed. The odds ratio which was calculated to assess the association of the eight bacterial species only for ChP or AgP (Table/Fig 5) revealed a positive association for T.denticola towards ChP and for A. actinomycetemcomitans towards AgP (P <0.0001). None of the other bacterial species showed a significant odds ratio solely for ChP or AgP.

Discussion

Owing to the low sensitivity and the ambiguity in the identification of anaerobes to the species level by cultivation, the present study used the 16S rRNA- based PCR method of detection. Among the eight putative periodontal pathogens which were screened, the red complex group (P. gingivalis, T. forsythia and T. denticola) showed a higher prevalence in the periodontitis group as compared to other gram-negative anaerobes, which is well in agreement with several other existing data, which suggested that this group was associated with the severity of periodontal tissue destruction (6),(10),(11). On the other hand, the percentage prevalence of the red complex in the healthy periodontium was low. Our study showed an association of only the red complex group and A. actinomycetemcomitans to adult periodontitis as against Ashimoto et al’s findings (8), who reported this association for all the eight bacterial species. The present study supported the fact that the red complex group of bacteria were putative periodontopathic bacteria, which was in concurrence with the findings of existing studies in a diverse population. In the present study, the members of the red complex were frequently detected together, as was also reported by previous studies (12), (13), (2), (14) and this exhibited a very strong correlation with the pocket depth, which is in accordance with the earlier findings of Socransky et al (6). The presence of the red complex consortium was observed in a majority of the ChPpatients. They were totally absent among the healthy subjects. Our findings showed such a consortium only in five AgP patients. A pathogen may more readily colonize the subgingival sites which are already occupied by other organisms, due to gingival inflammation or the growth factors which are produced by other organisms. However, some organisms may occur together in the periodontitis lesions, merely because they both induce destructive disease without interacting with each other. P. gingivalis can also be isolated from healthy individuals, but in a very low frequency and it is therefore recognized as a part of the commensal oral microbiome. A good statistical significance was observed between the healthy and the periodontitis group with respect to the P.gingivalis prevalence (p<0.0001). The prevalence of P.gingivalis (11%) in the present study among the healthy subjects was very well in accordance with Takeuchi et al. (15) and Botero et al’s findings (16). As compared to the present study, few previous studies reported a higher prevalence of P.gingivalis among the healthy subjects in diverse ethnic groups (17), (18), (19).

in the present study was consistent with quite a number of the earlier reports (8), (16), (20), (17), (21). It was found to be moderately low as compared to a number of previous reports (22), (18), (23), (15), (7). The prevalence of P. gingivalis among the AgP patients in the present study was high as compared to the finding of Mullally et al. (24) who reported a 16.7% prevalence. Conversely, Botero et al. (16) reported a very high prevalence of 91.6% amongst the AgP group. In agreement with the findings of previous studies (25), (26), (11), (23), (27), (28), the data of the present study supported the notion thatA. actinomycetemcomitans was a predominant pathogen in the aetiology of AgP. The occurrence of A. actinomycetemcomitans in ChP and AgP was associated with the severity of the disease with respect to PPD and CAL. A. actinomycetemcomitans was most frequently detected in sites with a pocket depth of ≥5 mm and a clinical attachment loss of ≥7 mm. A statistical significance (p<0.0001) was observed for the prevalence of A. actinomycetemcomitans among the AgP patients. The association of A. actinomycetemcomitans with the red complex group was completely absent among the AgP patients, thus suggesting its pronounced role in AgP. The prevalence of A. actinomycetemcomitans among the ChP group in the present study was consistent with few other reports (8), (29), (30), although its prevalence among ChP and the healthy subjects was inconsistent with the findings of Kumar et al. (18) and Junior et al (31). The prevalence of A. actinomycetemcomitans in the present study was high among the periodontitis group (both ChP and AgP) as compared to previous reports from different ethnicity (32), (33), (34), (24), (15), (35). Conversely, few studies have reported a higher prevalence among the ChP and the AgP groups for A. actinomycetemcomitans (20), (7), (23), (31). The prevalence A. actinomycetemcomitans in AgP was very high as compared to that in healthy subjects, which was similar to the findings of Faveri et al (36).

In the present study, there was a strong association of T. forsythia with periodontitis and a good statistical significance was observed between the periodontitis patients and the healthy subjects (p< 0.0001). These findings were consistent with several previous reports (8), (16), (22), (18), (37), (38). They were not in agreement with Conrads et al’s (39) and Lee et al’s reports (23). Among the different species of spirochetes, T.denticola has been extensively characterized in terms of its pathogenicity and involvement in the development of periodontitis. T. denticola has been shown to be associated with severe periodontal disease in adult humans and it can serve as a prognostic marker for the disease recurrence (40). A good statistical significance was observed between the patients and the healthy group (p< 0.0001) for T. denticola. A high prevalence among the periodontitis patients suggested that this spirochete was associated with alveolar bone loss, particularly in the ChP group. Our study reported a moderately high prevalence of T. denticola among the ChP patients as compared to the findings of Ashimoto et al (8) and Moter et al (41). The prevalence of T. denticola was in concurrence to the findings of a previous study that had used the real -time PCR assay (42). The prevalence of this species was relatively low in comparison to that in earlier studies (18), (23), (15). The data of the healthy population corroborated the findings of Takeuchi et al (15).

A statistical significance was observed between the patients and the healthy group for C.rectus (p=0.044) and E.corrodens (p=0.008). The present study reported a low prevalence of C.rectus and E.corrodens in the adult periodontitis group as compared to Ashimoto et al’s (8) and Botero et al’s findings (16). A statistical significance was observed between the patients and the healthy group for P.intermedia (p=0.044), but not for P. nigrescens (p=0.885). In few previous studies, a high prevalence of P. intermedia and P.nigrescens, which ranged between 52- 85% was reported (8), (16). Colombo et al (43) reported a 37 % prevalence of P. intermedia. A very low prevalence of 2.6% was reported by Conrads et al (39).

The odds ratio which was calculated to assess the association between the bacterial species among the periodontitis patients in this study revealed a positive association for 19 of the 28 bacterial combinations. Previous positive associations between periodontopathic bacteria have been reported for T. forsythia/ Crectus, P. gingivalis/P. intermedia, P. intermedia/C. rectus, E. corrodens/C. rectus (6) and T.denticola/P. gingivalis (44), (45) and amongst P. gingivalis, P. intermedia and T. forsythia (46), (47). Earlier, in a similar study, a positive association was reported for 17 of the 28 bacterial combinations (8). The present study reported an additional positive association for P. gingivalis/E. corrodens and T. denticola/E. corrodens. The high odds ratio between the bacterial species perhaps showed a symbiotic association in the periodontal pockets. The low odds ratios for C.rectus/ P.intermedia and E.corrodens / P.intermedia suggested their negative association.

Conclusion

The odds of detecting P. gingivalis, T.forsythia, T.denticola and A.actinomycetemcomitans were 25.68, 15.53, 20.24 and 25.82 times high in individuals with periodontitis as compared to those in the healthy subjects. The high odds ratio for P. gingivalis, T.forsythia, T.denticola and A.actinomycetemcomitans suggested a strong association between them and periodontitis (ChP and AgP). In aggressive periodontitis, in addition to A.actinomycetemcomitans, the prevalence of P.gingivalis and T.forsythia was also high. The significant odds ratio for T.denticola and A.actinomycetemcomitans showed a positive association exclusively with ChP and AgP respectively. A significant difference in the prevalence of P.nigrescens was not observed among periodontitis and the healthy group, thus suggesting their symbiotic role in the periodontal pockets. A large cohort study may help in further substantiating the aetiology of this polymicrobial infection.

Acknowledgement

The authors thank Dr. Eiko Nakagawa Itano, Department of Pathology Science, Biological Science Center, State University of Londrina, Londrina, PR, Brazil for generously providing the genomic DNA of A. actinomycetemcomitans strain ATCC 43718, which helped in the initial standardization for detecting A. actinomycetemcomitans by PCR. This work was supported by the funds which were generated through the university- industry collaborative research programme of the University of Madras.

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