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Rajiv Gandhi University of Health Sciences, Karnataka

Bangalore

ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR

DISSERTATION

1. Name of the candidate and address

(in block letters)

DR. SANYUKTA REGE

DEPARTMENT OF PERIODONTICS

A.E.C.S. MAARUTI COLLEGE OF

DENTAL SCIENCES AND RESEARCH

CENTRE, BANGALORE.

2. Name of the institution A.E.C.S. MAARUTI COLLEGE OF

DENTAL SCIENCES AND RESEARCH

CENTRE,

BANGALORE.

3. Course of study and subject MASTER OF DENTAL SURGERY (MDS)

PERIODONTICS.

4. Date of admission to course 31st May 2008

5. Title of the topic TRANSMISSION OF BANA POSITIVE

BACTERIAL SPECIES FROM PARENT

TO CHILD. A CLINICO

MICROBIOLOGICAL STUDY.

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BRIEF RESUME OF THE INTENDED WORK:

6.1 Need for the study

The gingival crevice of young children becomes colonized by a variety of bacteria,

including spirochetes. In recent years, considerable data has become available that

suggest that the periodontal disease may be related either to the presence or to the

overgrowth of one or more bacterial subtypes in the subgingival plaque. There is

evidence that Spirochetes, Porphyromonas gingivalis, Bacteroides forsythus,

Prevotella intermedia and Aggregatibacter actinomycetemcomitans are strongly

associated with gingivitis and / or periodontitis, which are also associated with adult

periodontal disease. 1 This finding is evidence that the bacterial species implicated in

adult periodontal diseases actually colonizes the teeth in childhood. These early forms

of periodontal disease may have serious medical consequences later in life. An infants

oral flora is acquired from his or her primary caregiver, almost always the mother. 2

Kononen and colleagues3 reported that various anaerobic species colonize the

edentulous mouth of infants, and maternal saliva may act as a source of some gram

negative anaerobes. T. denticola,P. gingivalis and T. forsythiapossess a trypsin like

enzyme that hydrolyzes the synthetic substrate N-benzoyl-DL-arginine-2-

naphthylamide (BANA). Clinicians can detect this enzyme within five to ten minutes

at chair side using the BANA test.

In previous studies, children whose parents or caregivers had a history of periodontal

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disease were significantly more likely to have dentitions that were colonized by the

BANA-positive species than were children whose parents or caregivers did not report

having a history of periodontal disease. Previous findings are compatible with the

hypothesis that children acquire the BANA-positive species from their parents or other

caregivers, especially if any of their caregivers has periodontitis.2,3,4

The purpose of this study is to use N- benzoyl-DL-arginine-2-naphthylamide (BANA)

test to obtain information regarding the prevalence of an enzyme unique to certain

periodontal pathogens in plaque samples of children as well as the transmission of

these pathogens from the parent to child.

6.2 Review of literature

6.21 A study5 was conducted to determine the anaerobic species Porphyromonas

gingivalis, Treponema denticola and Tannerella forsythia that are frequently associated

with periodontal disease by comparing results with the liquid BANA assay and a

commercially available kit. These organisms hydrolyze the synthetic peptide N-

benzoyl- DL- arginine-2- napthalamide (BANA), and such an enzyme can be detected

in plaque and is related to the clinical disease. The proportional agreement between

BANA positiveness and clinical disease was similar for both the liquid and paper

assay. The sensitivity, specificity and accuracy were 81, 78 and 80% respectively.

These findings indicate that a rapid paper assay for BANA hydrolysis gives data

comparable as with the liquid BANA assay.

6.22 A study2 conducted, to show the prevalence / transmission of Actinobacillus

actinomycetemcomitans within family members. The culture studies done in this study

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showed that 51% of all patients suffering from early periodontal disease carried oral

Actinobacillus actinomycetemcomitans. And 50% of their spouses and 30% of their

children harboured the bacterium. Comparison of the PCR generated amplitypes

showed that 26 out of 27 individuals had strains exhibiting a single amplitype of

Actinobacillus actinomycetemcomitans. They also showed that in 6 out of 7 families,

the husband and wife did not harbor the same Actinobacillus actinomycetemcomitans

amplitype. Furthermore, most often children carried an amplitype identical to one of

the parent. However, one of the 10 children harboured two amplitypes each which was

identical to the parents. Thus the authors propose that Actinobacillus

actinomycetemcomitans is acquired through contact with one of the parent at some

time during childhood.

6.23 The authors6, conducted a study was to determine if a relationship could be found

between the presence of either organisms or periodontitis in the parent and the

presence of BANA positive species in the child. Children whose parents were

colonized by BANA positive bacteria were 9.8 times more likely to be colonized by

BANA positive species. Children whose parents had clinical evidence of periodontitis

were 12 times more likely to be colonized by these BANA positive species. This data

is compatible with the hypothesis that children may acquire these BANA positive

species from their parents, especially if the parent has periodontitis.

6.24 The study7 the use of a chair side BANA kit to diagnose anaerobic periodontal

infection in patients with advanced form of periodontal disease. Porphyromonas

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gingivalis, Treponema denticola, Tannerella forsythia have been indicated in

periodontal disease and each possess an enzyme capable of hydrolyzing the synthetic

trypsin substrate BANA. The BANA test is dependent on the length and temperature

of incubation. Therefore the authors have evaluated a 5 min/35oC, a 5 min / 55oC and a

15 min / 55oC incubation protocol to determine whether the performance of BANA test

could be optimized using samples obtained from subjects seeking dental treatment.

These findings suggested that the best fitting model was the 5 min / 35 o C incubation

protocol.

6.25 The authors4, conducted a study was to use N-benzoyl-DL-arginine- 2-

naphthalamide (BANA) test to obtain information regarding the prevalence of an

enzyme unique to certain periodontal pathogens in plaque samples of children as well

as the potential transmission of these pathogens from care givers to children. The

authors tested 218 subjects between 3 to 10 years and 44% of the children had at least

one plaque sample that tested positive or weakly positive. Positive results were more

frequent in the mixed dentition as well as in children with gingivitis. These findings

suggest that the BANA test results were positive for almost one half of the children. A

positive reaction was associated with gingivitis, a mixed dentition, a BANA positive

care giver or a care giver with a history of periodontal disease in the family.

6.3 Objectives of the study

The purpose of the study was to determine the potential transmission of BANA

positive species from parent to child using the BANA test, in young Indian children.

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7. MATERIAL AND METHODS

7.1 Source of data

120 children and their parent/s visiting the Department of Pedodontics / Periodontia,

AECS Maaruti College of Dental Sciences and Research Centre, Bangalore will be

taken as subjects for the said study.

7.2 Method of collection of data (including sampling procedures if any)

Children between the age group 5 years to 13 years will be examined. The inclusion

criteria will be 1) All of whom have posterior teeth , 2) Are in good general health ,

3) Co-operative with dental treatment and 4) Who agree to undergo the BANA test.

The exclusion criteria will be 1) parents or child who are undergoing antibiotic therapy

or have been in the last 6 months.

The Ethics Committee of AECS Maaruti College of Dental Sciences reviewed and

approved all procedures included in this research.

Experimental protocol: All children and their parent/s that come to the Department

of Pedodontics / Periodontia will participate in the study. The purpose and design of

the study will be explained to the parent. In addition to the dental examination, BANA

test procedure and possible discomfort, as well as the benefits of the test will be

described to the parent. If he or she agrees to allow his or her child to participate in the

study, as well as himself or herself, a signed consent will be obtained before the

examination. An oral consent from the 6- to 10-year-old children will be obtained

before the test. The parent will be asked for relevant data about the child and himself

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or herself (such as birth date, sex and age. The child will be examined and the type of

dentition (primary, mixed or permanent), decayed, missing and filled teeth (dmft or

DMFT ) index, caries status (caries free, small pit and- fissure caries, moderate

occlusal caries, proximal caries or rampant caries) and the patients level of oral

hygiene will be recorded. The plaque index (PI) at the interproximal site to be

sampled for the BANA test will be determined. Interproximal/subgingival plaque

samples will be collected for the BANA test. A plaque remover (STIM tooth pecker)

will be used to obtain plaque samples from each quadrant. Any supragingival plaque if

present will be removed from the site and the plaque remover will be inserted between

the first and second molars in children with a primary dentition; between the second

primary molar and first permanent molar in children with a mixed dentition; and

between the second premolar and first permanent molar in children with a late mixed

dentition or a permanent dentition. If the child has any missing teeth, the plaque

sample from the mesial or distal side of the remaining tooth will be taken. This

procedure will be repeated for the parent and plaque sample will be taken from the site

between the first molar and second premolar.

Papillary bleeding score (PBS): The PBS indicates gingival inflammation and can be

calculated at the same time that the plaque sample for the BANA test is obtained. After

removing the plaque with help of a plaque remover from the subjects mouth, any

bleeding in the interproximal area will be recorded on a scale from 0 to 5. 4

BANA test: After removing the plaque remover from the interproximal site, it will be

wiped onto the lower portion of the strip of the BANA test card to transfer any

adherent plaque. A separate plaque remover will be used for each plaque sample, so

four distinct samples will be obtained for each subject. After all tooth sites are

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sampled, a cotton swab will be used to slightly moisten the upper portion of the strip

with distilled water. The BANA test card will be folded in half and placed in an

incubator at 55o C for five minutes5. Then on removing the card the lower portion of

the strip will be discarded in a manner appropriate for contaminated material. The

color on the upper end of the strip will be recorded as follows: no blue color

(negative), a faint blue color (weakly positive) or a distinct blue color (positive).

Statistical analysis: A statistical software (SPSS) will be used for statistical analysis.

Frequency tables will be generated for each categorical variables and descriptive

statistics for continuous variable. Chi square test will be used to assess the significance

of the relationship of BANA test results to each categorical variables. Independent

sample t- test will be performed to determine if there is a difference in the means of

continuous variables for BANA positive and BANA negative patients. A step wise

logistic regression analysis will be used to determine the association between the

various independent variables and BANA positive status of the child.

7.3 Does the study require any investigation or intervention to be conducted on

patients or other humans or animals? (If so please describe briefly)

Yes.

1. Subgingival plaque samples will be collected from the patients after removing

supragingival plaque, with a separate disposable plaque remover for each

quadrant.

2. Papillary bleeding scores will be calculated from 0 to 5.

3. The DMFT and dmft scores will be calculated.

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4. Plaque Index.

7.4 Has ethical clearance been obtained from your institution in case of 7.3

Yes.

7.5 Any trial test have been carried out?

Yes

8. LIST OF REFERENCES

1. Page R. C. Gingivitis Journal of Clinical Periodontology 1986 ; 13 (5) :

345- 359.

2. Preus Hans R, Zambon Joseph J, Dunford Robert G and Genco Robert J.

The distribution and transmission of Actinobacillus actinomycetemcomitans

in families with established periodontitis Journal of Periodontology1994;

65(1): 2-7.

3. Kononen E, Saarela M, Karjalainen J et al Transmission of oral Prevotella

melaninogenica between mother and her young child. Oral Microbiology and

Immunology 1994; 9 (5) : 310 314.

4. Lee Y, Tchaou Wen Shiun S, Welch K B and Loesche W J The

transmission of BANA positive periodontal bacterial species from caregivers to

children JADA, Vol 37, Nov 2006: 1539-1546.

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5. Loesche W J, Kazor C E and Taylor G W. The optimization of BANA test

as a screening instrument for gingivitis among subjects seeking dental

treatmentJournal of Clinical Periodontology; 1997; 24(10):718-726.

6. Loesche W J, Bretz W A, Kerschensteiner D et. al. Development of a

diagnostic test for anaerobic periodontal infections based on plaque hydrolysis

of benzoyl DL arginine naphalamide Journal of Clinical Microbiology;

July 1990, 1551 1559.

7. Watson M R, Bretz W A and Loesche W J Presence of Treponema

denticola and Porphyromonas gingivalis in children correlated with periodontal

disease of their parent Journal of Dental Research;October 73(10): 1636-

1640.

9. Signature of candidate

10. Remarks of guide This is a genuine study which will be

carried out by the post graduate student

under my supervision and guidance.

11. Name and designation (in block

letters)

11.1 Guide DR. V. RANGANATH

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11.2 Signature

11.3 Co Guide (if any)

11.4 Head of department

11.5 Signature

PROFESSOR & HEAD

DEPARTMENT OF PERIODONTICS

A.E.C.S. MAARUTI COLLEGE OF

DENTAL SCIENCES AND RESEARCH

CENTRE,

BANGALORE

DR. ASHISH NICHANI

PROFESSOR

DEPARTMENT OF PERIODONTICS

A.E.C.S. MAARUTI COLLEGE OF

DENTAL SCIENCES AND RESEARCH

CENTRE,

BANGALORE

DR. V. RANGANATH

12. 12.1 Remarks of chairman and

principal

12.2 Signature

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