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Dental cavities is the most infective and catching dental disease of all age groups. Which consequence overall wellness of persons. Childhood cavities is a multifactorial disease in teething and if left untreated it leads to trouble, uncomfortableness, and deficiency of involvement in everyday activities and finally destroys tooth construction and early loss of tooth. Steptococcus mutans ( S.mutans ) is a chief cariogenic micro-organism. This S.mutans breaks down sugar for energy and bring forth acidic environment, which causes demineralisation of superficial construction of tooth as enamel and dentin ensuing in dental cavities. It can be transmitted horizontally and vertically. Harmonizing to the recent surveies perpendicular manner of transmittal is more common in preschool kids than horizontal.

Introduction and Literature Review

Dental cavities is the most common infective disease, and remains a important unwritten wellness job worldwide for both kids and grownups. 1

Etiological Factors Involving in Dental Caries

Dental cavities is the consequence of interaction between micro-organisms chiefly Streptococcus mutans ( S.mutans ) , teeth and fermentable saccharides. 2

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Pathophysiology of Dental Caries

S.mutans is a facultative anaerobiotic Gram-positive coccus, stick to the tooth surface, interrupt down sugar for energy, lower the Ph and bring forth acidic environment, which causes demineralisation of superficial constructions of tooth as ( enamel and dentine ) , and if non treated earlier the procedure continues and consequence in future dental cavities. 3

Childhood Caries and Its Etiology

The common aetiologic factors involved in Childhood Caries ( CC ) are prolonged usage of a bottle with high sugar incorporating milk, juice or liquid, consumption of high cavities risk diet, low socio-economical groups ( e.g. low degree of maternal instruction ) and pregnant female parents holding carious dentitions with high salivary S.mutans count. 5 It besides has been shown that these micro-organisms strains besides found in female parents of Childhood Caries kids. This shows that female parents are the chief beginning of transmittal of dental cavities to their kids and the frequence of perpendicular transmittal is more common in pregnant female parents. 3

Manners of its Transmission

Dental cavities is a catching disease and S.mutans bacteria can be transmitted by both horizontally and vertically. 4, 5 Horizontal transmittal is more common in siblings, kids in same schoolroom, baby’s room or twenty-four hours attention centres. Children in same nursery school have reported to transport similar bacterial strains in their spit. 6

On the other manus, perpendicular transmittal is from parents to kids. The term is restricted by some to familial transmittal and extended by others to include besides transmittal of infection from one coevals to the following, by fluid as spit, milk or through the placenta. 7 In perpendicular transmittal S.mutans spread from female parents to their kids. 8 Exact method of perpendicular transmittal is still problematic, but it is believed harmonizing to the literature that there may be close contact between female parent and kids by sharing of nutrient and utensils. 9

Pregnant Mothers as a Main Source of Vertical Transmission

The inclination of dental cavities transmittal is to be reported, higher in those kids whose female parents are pregnant and have carious dentitions with high S.mutans count in their spit. 8

Pregnancy leads to many impermanent adaptative alterations in the organic structure, due to let go of of figure of endocrines as estrogen, Lipo-Lutin, relaxin and gonadotrophin. 10 And during this period the opportunities and hazard of cavities, gingival, periodontic and dental infections become higher than normal. The unwritten pit is affected by such endocrinal actions, and may show both transient and irreversible alterations every bit good as alterations that are considered pathological. 10

Window of Infectivity

High salivary S.mutans degree in pregnant female parent might do transmittal through a figure of day-to-day saliva contacts between the kid and the female parent. 11 The acquisition of S.mutans suggested to happen during a distinguishable age period: a “ window of infectivity ” between 19 and 31 months, in which the proportion of kids with S.mutans additions from 25 % to 75 % . 11 Studies utilizing genotypic and phenotypic methods strongly suggest that female parent is the primary beginning of infection for kids who carry S.mutans strains. 12, 10, 13-19 and spit is the chief tool for the transportation of strains. 20

Colonization of S.mutans

Once transmitted, S.muatns can colonise in the channels of babies lingua. 6 On the ages of one and two, bacteriums start developing settlements on dentitions that eventually cause Childhood Caries ( CC ) . Berkowitz et Al ( 2003 ) reported in a survey that female parents with high salivaryA S.mutansA count, that is exceeded 105 colony-forming units ( CFU ) were approximately nine times more likely to go through the causative bacteriums on to their kids than female parents with low salivary S.mutans count. 6 High cavities prevalence has been shown in Pre-school kids with high colonisation of S.mutans than those kids with low degree of S.mutans. 6 So at early age colonisation with S.mutans is an of import factor for the beginning of early cavities. ( 8,9 ) ( 7,8 ) . This clip of colonisation of micro-organism is of import to understand the cavities risk factors every bit good as the right clip of preventative steps should be implemented.

Klein et Al. ( 2004 ) studied the form of perpendicular transmittal of S.mutans from female parent to child, genotypic diverseness and stableness, in this study16 mother-child brace monitored over a 20-months period. The kids harbored one to four distinguishable genotypes of S.mutans, presence of fiting genotypes of S.mutans was similar in 81.25 % of mother-child braces, and 16 genotypes transmitted out of 52 genotypes. 19

Genotypic diverseness of S.mutans

It has implicated as a virulent factor, more of import than bacterial count in doing dental cavities. 21 14 distinguishable genotypes have identified from 88 isolates of S.mutans in spit samples from immature grownups. 21

Different genotypes of S.mutans have been detected at different age groups. 22, 23, 12 Emanuelsson et Al. ( 2003 ) found a upper limit of seven genotypes in the survey with immature grownups who had dental cavities experienced in the yesteryear. 24 Napimoga et Al. ( 2004 ) besides detected eight genotypes in caries-active topics by utilizing Arbitrary Primer Polymerase Chain Reaction. 25 Though, harmonizing to surveies it has observed that kids harbor merely one to five different genotypes of S.mutans. 8, 13, 26-28

Napimoga et Al. ( 2004 ) survey found describing correlativity between genotypic diverseness in cavities active and cavities free kids and have shown at odds consequences. 25The findings of Pieralisi, et Al. ( 2010 ) survey showed a favourable relationship between cavities activity and the genotypic diverseness of S.mutans. 29 On the other manus, Kreulen et Al. ( 1997 ) detected a negative correlativity between cavities activity and genotypic diverseness. 8 While survey by Lembo et Al. ( 2000 ) have found notable differences in the figure of genotypes identified in caries-free and caries-active kids. 30

Polymerase Chain Reaction Method versus Culture Method

Bacterial civilization is the gilded criterion for the sensing of S.mutans in spit samples. Selective media as mitis-salivaris ( MS ) or MS-Bacitracin ( MSB ) agar have used to analyze settlement morphology for the sensing of S.mutans. 31-32 Disadvantages of civilization are, clip consuming, hard and non easy to distinguish the micro-organism among other species. 33 For the sensing of S.mutans in spit, direct microscopy, enzyme trial, species-specific DNA investigations, PCR and Enzyme Linked Immunosorbent check ( ELISA ‘s ) cultivation. Polymerase Chain Reaction ( PCR ) methods have reported to be more sensitive and specific for the sensing of S.mutans than conventional cultural technique. 34-35 By this method, low Numberss of bacterial species with a sensing bound of every bit few as 25-100 cells can be detected. 35

Study Aims:

To detect the chief cariogenic genotypes of S.mutans.

To detect the perpendicular transmittal of S.mutans genotypes in mother-child brace.

Material and Methods:

Study Design:

Cross sectional Analytical survey.

Topographic point of survey:

Study will be conducted in Basic Health Unit ( BHU ) Ali Raza Bad in Peri-Urban country of Lahore.

Duration of survey:

8 months.

Study population:

188 mother-child brace topics ( pregnant female parents and their kids up to 5 old ages of age ) would be selected by convenience trying from Ali Raza Bad in Peri-urban country of Lahore ( catchment country of BHU ) . Consent would be obtained from female parents for themselves and besides for their enrolled kids prior to the survey harmonizing to ethical consideration, the topics will have a dental scrutiny performed by utilizing WHO 1987 cavities diagnostic standards to find the decayed, losing, filled dentitions ( dmft ) index. 36 Subjects holding history of systemic diseases and disposal of antibiotics within 3 months will be excluded from survey. Institutional Review Board ( IRB ) at Shaikh Zayed Medical Complex approved the survey design, protocol, and informed consent.

Inclusion standards:

Informed consent from enrolled pregnant female parents.

Consent of up to 5 old ages old kids of pregnant female parents will be obtained from their female parents prior to the survey.

Pregnant female parents holding at least 3 carious dentitions.

Children of pregnant female parents up to 5 old ages old with at least 3 carious dentitions.

Exclusion Standards:

Missing of any anterior dentitions.

Absence of any implicit in systemic disease.

History of antibiotic usage within 3 months.

Sample aggregation:

1-Study population would be pregnant female parents and their pre-school up to 5 old ages old kids recruited from Ali Raza Bad in Per-urban country of Lahore ( catchment country of BHU ) .

2- Saliva would be stimulated by masticating gum at any clip.

3- Subjects stimulated saliva sample would be collected in a 1.5 milliliter unfertile tubing, stored in cold concatenation ( isotherm box ) within 6 hours and will be transported to Division of Molecular Virology and Molecular Diagnostics, National Center of Excellence in Molecular Biology ( CEMB ) University of The Punjab Lahore, for laboratory analysis.

Sample Size computation:

Sample size was estimated 188 by utilizing 5 % degree of significance with expected frequence of vertically transmitted S.mutans 60 % in mother-child brace with 7 % border of mistake.

95 % Confidence Interval ZI±/2=1.96

Expected prevalence of S.mutans P=60 % =0.6

Margin of mistake I±=0.07

Sample size n= ?

n = Z 2 P ( 1-P )


n = ( 1.96 ) 2 A-0.6A-0.4

( 0.07 ) 2

n = 0.921


n = 188

Data aggregation:

The information of patients sing their name, age and socioeconomic position ( SES ) will be collected on a Performa ( Annexure-II ) .

Subjects and Samples

Informed Consent:

Isolate S.mutans strains from 188 pregnant female parents and their pre-school kids up to 5 old ages of age with primary teething, will choose for this survey by convenience sampling. Consent would be taken from the female parents after stating them about the intent of the survey and besides take consent for their enrolled kids prior to the survey. The consent will be saved in composing with the signature or thumb-mark of the accepting individual harmonizing to the ethical guidelines of the Institutional review board commission ( IRB ) at Shaikh Zayed Medical Complex.

Polymerase Chain Reaction ( PCR ) Method:

Genomic DNA readying:

Entire nucleic acid will be extracted from stimulated spit utilizing NecleoSpin Nucleic Acid Extraction Kits ( MHCHERAV NAGEL ; Germany ) as per process process given in kit protocol.

Primer Design:

Primers will be design by utilizing primer 3 package ( hypertext transfer protocol: // ) aiming 16S rDNA. In add-on PCR sensing of the tried species will besides be performed utilizing published primers as described by Igarashi et Al. ( 1996, 2000 ) will be done by the method of Goncharoff et Al. ( 1993 ) . 37, 38

PCR elaboration and sensing:

The mark cistron ( 16S rDNA ) will be amplified with cistron specific primers. We will utilize nested PCR for elaboration due to its higher sensitiveness and specificity. PCR will be run at different temperatures to optimise the needed cistrons. PCR elaboration will be performed in a reaction mixture ( 20 I?l ) with 2 units of polymerase enzyme, along with the needed reagents, 20 pmol of each forward and contrary primers and 20 50 nanogram of templet DNA solution in a thermic cycler ( ABI 2700 ; Applied Biosystem ) . Each set of PCR analyses will include proper negative control ( H2O space ) and positive controls.

Detection of PCR Merchandises:

Following elaboration, 10 I?l of the PCR merchandises along with 2 ul of lading dye will be

analysed by cataphoresis on a 1-2 % agarose gel. Agarose or polyacrylamide gel will be used

for gel cataphoresis. Gel will be prepared by utilizing Tris- Ethylenediaminetetraacetic acid

( EDTA ) buffer ( TAE Buffer ) or Tris-Borate-EDTA Buffer ( TBE Buffer ) and Ethidium

Bromide will be used as tracking dye. The nested PCR elaboration merchandise will be run on gel.

After run gel will be visualized on UV Trans-illuminator after staining with Ethedium Bromider.

The size of the PCR merchandises will be estimated from the cataphoretic migration of merchandises

relation to a 100-bp DNA size ladder marker.

Constitution of Assay Conditions and Sensitivity:

Before analysing topics samples on big graduated table, optimisation of PCR protocol will be done to maximise sensitiveness, specificity and duplicability to avoid false negative consequences. Specificity of the reaction will be increased by I ) optimisation of dNTPs and primer concentration II ) optimising tempering temperature III ) optimising PCR rhythm figure. Sensitivity of the assay status will be established utilizing standard positive control DNA, to analyse the figure of transcripts of mark Deoxyribonucleic acid that are necessary to reproducibility give a noticeable and specific elaboration merchandise.


S.mutans genotyping will be carried out by utilizing the method described by Saarela, et Al ( 1996 ) . 39 Briefly, the Deoxyribonucleic acid from entire S.mutans isolates will be used for genotyping. The AP-PCR fingerprinting will be performed with two primers, OPA-02 ( 5aˆ?-TGCCGAGCTG-3aˆ? ) and OPA-13 ( 5aˆ?-CAGCACCCAC-3aˆ? ) . 40. Amplification merchandises will be analyzed electrophoretically with a 1.5 % agarose gel with Tris-borate-EDTA running buffer ( pH 8.0 ) . A 100-bp DNA ladder include in each gel. Ethidium bromide-stained gel images will be captured with the LISCAP digital imagination system. Molecular sizes for each set will be computed and analyzed by Bioinformatics package which will be used to bring forth similarity dendrograms ( Dice coefficient, & gt ; 95 % ; unweight brace group method with arithmetic mean ) will be used to demo the S.mutans genotypes.

Facilities Available:

Labs at Shaikh Zayed Hospital Lahore and Division of Molecular Virology and Molecular Diagnostics, National Center of Excellence in Molecular Biology ( CEMB ) University of The Punjab Lahore are good equipped to transport out work on about all the above-named aims. There is first-class Molecular Diagnostic Laboratory in the CEMB for the molecular sensing work. Equipment like thermic cycler and western blotting ( PAGE apparatus / Blot Transfer setup ) bench top extractors, ultracentrifuge, Real clip PCR, Arbitrary primer sequence-based polymerase concatenation reaction ( AP-PCR ) , Agarose gel cataphoresis setup, laminar flow goons etc. are available for the smooth running of the undertaking. Deoxyribonucleic acid synthesis installation is besides available, required for the synthesis of DNA- oligos, which will be used for the elaboration and quantification of viral cistrons. There is a well-established Serology and Heamatology Lab in Shaikh Zayed Hospital.


The purpose of this survey is to detect the chief cariogenic genotypes of S.mutans in ( Peri-Urban country of Pakistani Population ) up to 5 old ages old kids spit samples by PCR and genotype methods. Genotypes vary from population to population, and no related survey has been done in Pakistan. Besides to detect whether genotypes transfer vertically or non. Due to hapless unwritten hygiene, low socio-economic position, low maternal instruction and hormonal alterations, the opportunities of cavities infection additions from pregnant female parents to their kids instead than horizontal transmittal