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Dental caries is the most infectious and communicable dental disease of all age groups. Which effect overall health of individuals. Childhood caries is a multifactorial disease in dentition and if left untreated it leads to pain, discomfort, and lack of interest in routine activities and ultimately destroys tooth structure and early loss of tooth. Steptococcus mutans (S.mutans) is a main cariogenic microorganism. This S.mutans breaks down sugar for energy and produce acidic environment, which causes demineralization of superficial structure of tooth as enamel and dentin resulting in dental caries. It can be transmitted horizontally and vertically. According to the recent studies vertical mode of transmission is more common in preschool children than horizontal.
Introduction and Literature Review
Dental caries is the most common infectious disease, and remains a significant oral health problem worldwide for both children and adults. 1
Etiological Factors Involving in Dental Caries
Dental caries is the result of interaction between microorganisms mainly Streptococcus mutans (S.mutans), teeth and fermentable carbohydrates. 2
Pathophysiology of Dental Caries
S.mutans is a facultative anaerobic Gram-positive cocci, stick to the tooth surface, break down sugar for energy, lower the Ph and produce acidic environment, which causes demineralization of superficial structures of tooth as (enamel and dentine) ,and if not treated earlier the process continues and result in future dental caries. 3
Childhood Caries and Its Etiology
The common etiological factors involved in Childhood Caries (CC) are prolonged use of a bottle with high sugar containing milk, juice or liquid, intake of high caries risk diet, low socio-economical groups (e.g. low level of maternal education) and pregnant mothers having carious teeth with high salivary S.mutans count. 5 It also has been shown that these micro-organisms strains also found in mothers of Childhood Caries children. This shows that mothers are the main source of transmission of dental caries to their children and the frequency of vertical transmission is more common in pregnant mothers. 3
Modes of its Transmission
Dental caries is a transmissible disease and S.mutans bacterium can be transmitted by both horizontally and vertically. 4, 5 Horizontal transmission is more common in siblings, children in same classroom, nursery or day care centers. Children in same nursery school have reported to carry similar bacterial strains in their saliva. 6
On the other hand, vertical transmission is from parents to children. The term is restricted by some to genetic transmission and extended by others to include also transmission of infection from one generation to the next, by fluid as saliva, milk or through the placenta. 7 In vertical transmission S.mutans spread from mothers to their children. 8 Exact method of vertical transmission is still debatable, but it is believed according to the literature that there may be close contact between mother and children by sharing of food and utensils. 9
Pregnant Mothers as a Main Source of Vertical Transmission
The tendency of dental caries transmission is to be reported, higher in those children whose mothers are pregnant and have carious teeth with high S.mutans count in their saliva. 8
Pregnancy leads to many temporary adaptive changes in the body, due to release of number of hormones as estrogen, progesterone, relaxin and gonadotropin. 10 And during this period the chances and risk of caries, gingival, periodontal and dental infections become higher than normal. The oral cavity is affected by such endocrine actions, and may present both transient and irreversible changes as well as modifications that are considered pathological. 10
Window of Infectivity
High salivary S.mutans level in pregnant mother might cause transmission through a number of daily saliva contacts between the child and the mother. 11 The acquisition of S.mutans suggested to occur during a distinct age period: a "window of infectivity" between 19 and 31 months, in which the proportion of children with S.mutans increases from 25% to 75 %. 11 Studies using genotypic and phenotypic methods strongly suggest that mother is the primary source of infection for children who carry S.mutans strains. 12, 10, 13-19 and saliva is the main tool for the transfer of strains. 20
Colonization of S.mutans
Once transmitted, S.muatns can colonize in the grooves of infants tongue. 6 On the ages of one and two, bacteria start developing colonies on teeth that finally cause Childhood Caries (CC). Berkowitz et al (2003) reported in a study that mothers with high salivary S.mutans count, that is exceeded 105 colony-forming units (CFU) were about nine times more likely to pass the causative bacteria on to their children than mothers with low salivary S.mutans count. 6 High caries prevalence has been shown in Pre-school children with high colonization of S.mutans than those children with low level of S.mutans. 6 So at early age colonization with S.mutans is an important factor for the beginning of early caries. (8,9)(7,8). This time of colonization of microorganism is important to understand the caries risk factors as well as the correct time of preventive measures should be implemented.
Klein et al. (2004) studied the pattern of vertical transmission of S.mutans from mother to child, genotypic diversity and stability, in this study16 mother-child pair monitored over a 20-months period. The children harbored one to four distinct genotypes of S.mutans, presence of matching genotypes of S.mutans was similar in 81.25 % of mother-child pairs, and 16 genotypes transmitted out of 52 genotypes. 19
Genotypic diversity of S.mutans
It has implicated as a virulent factor, more important than bacterial count in causing dental caries. 21 14 distinct genotypes have identified from 88 isolates of S.mutans in saliva samples from young adults. 21
Different genotypes of S.mutans have been detected at different age groups. 22, 23, 12 Emanuelsson et al. (2003) found a maximum of seven genotypes in the study with young adults who had dental caries experienced in the past. 24 Napimoga et al. (2004) also detected eight genotypes in caries-active subjects by using Arbitrary Primer Polymerase Chain Reaction. 25 Though, according to studies it has observed that children harbor only one to five different genotypes of S.mutans. 8, 13, 26-28
Napimoga et al. (2004) study found reporting correlation between genotypic diversity in caries active and caries free children and have shown conflicting results. 25The findings of Pieralisi, et al. (2010) study showed a favorable relationship between caries activity and the genotypic diversity of S.mutans. 29 On the other hand, Kreulen et al. (1997) detected a negative correlation between caries activity and genotypic diversity. 8 While study by Lembo et al. (2000) have found noteworthy differences in the number of genotypes identified in caries-free and caries-active children. 30
Polymerase Chain Reaction Method versus Culture Method
Bacterial culture is the gold standard for the detection of S.mutans in saliva samples. Selective media as mitis-salivaris (MS) or MS-Bacitracin (MSB) agar have used to study colony morphology for the detection of S.mutans. 31-32 Disadvantages of culture are, time consuming, difficult and not easy to differentiate the microorganisms among other species. 33 For the detection of S.mutans in saliva, direct microscopy, enzyme test, species-specific DNA probes, PCR and Enzyme Linked Immunosorbent assay (ELISA's) cultivation. Polymerase Chain Reaction (PCR) methods have reported to be more sensitive and specific for the detection of S.mutans than conventional cultural technique. 34-35 By this method, low numbers of bacterial species with a detection limit of as few as 25-100 cells can be detected. 35
To observe the main cariogenic genotypes of S.mutans.
To observe the vertical transmission of S.mutans genotypes in mother-child pair.
Material and Methods:
Cross sectional Analytical study.
Place of study:
Study will be conducted in Basic Health Unit (BHU) Ali Raza Bad in Peri-Urban area of Lahore.
Duration of study:
188 mother-child pair subjects (pregnant mothers and their children up to 5 years of age) would be selected by convenience sampling from Ali Raza Bad in Peri-urban area of Lahore (catchment area of BHU). Consent would be obtained from mothers for themselves and also for their enrolled children prior to the study according to ethical consideration, the subjects will receive a dental examination performed by using WHO 1987 caries diagnostic criteria to determine the decayed, missing, filled teeth (dmft) index. 36 Subjects having history of systemic diseases and administration of antibiotics within 3 months will be excluded from study. Institutional Review Board (IRB) at Shaikh Zayed Medical Complex approved the study design, protocol, and informed consent.
Informed consent from enrolled pregnant mothers.
Consent of up to 5 years old children of pregnant mothers will be obtained from their mothers prior to the study.
Pregnant mothers having at least 3 carious teeth.
Children of pregnant mothers up to 5 years old with at least 3 carious teeth.
Missing of any anterior teeth.
Absence of any underlying systemic disease.
History of antibiotic use within 3 months.
1-Study population would be pregnant mothers and their pre-school up to 5 years old children recruited from Ali Raza Bad in Per-urban area of Lahore (catchment area of BHU).
2- Saliva would be stimulated by chewing gum at any time.
3- Subjects stimulated saliva sample would be collected in a 1.5 ml sterile tube, stored in cold chain (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 calculation:
Sample size was estimated 188 by using 5% level of significance with expected frequency of vertically transmitted S.mutans 60% in mother-child pair with 7% margin of error.
95% Confidence Interval ZÎ±/2=1.96
Expected prevalence of S.mutans P=60%=0.6
Margin of error Î±=0.07
Sample size n=?
n = Z 2 P(1-P)
n = (1.96) 2 Ã-0.6Ã-0.4
n = 0.921
n = 188
The data of patients regarding their name, age and socioeconomic status (SES) will be collected on a Performa (Annexure-II).
Subjects and Samples
Isolate S.mutans strains from 188 pregnant mothers and their pre-school children up to 5 years of age with primary dentition, will select for this study by convenience sampling. Consent would be taken from the mothers after telling them about the purpose of the study and also take consent for their enrolled children prior to the study. The consent will be saved in writing with the signature or thumb-mark of the consenting person according to the ethical guidelines of the Institutional review board committee (IRB) at Shaikh Zayed Medical Complex.
Polymerase Chain Reaction (PCR) Method:
Genomic DNA preparation:
Total nucleic acid will be extracted from stimulated saliva using NecleoSpin Nucleic Acid Extraction Kits (MHCHERAV NAGEL; Germany) as per procedure procedure given in kit protocol.
Primers will be design by using primer 3 software (http://frodo.wi.mit.edu/primer3/) targeting 16S rDNA. In addition PCR detection of the tested species will also be performed using published primers as described by Igarashi et al. (1996, 2000) will be done by the method of Goncharoff et al. (1993). 37, 38
PCR amplification and detection:
The target gene (16S rDNA) will be amplified with gene specific primers. We will use nested PCR for amplification due to its higher sensitivity and specificity. PCR will be run at different temperatures to optimize the required genes. PCR amplification will be performed in a reaction mixture (20 Î¼l) with 2 units of polymerase enzyme, along with the required reagents, 20 pmol of each forward and reverse primers and 20 50 ng of template DNA solution in a thermal cycler (ABI 2700; Applied Biosystem). Each set of PCR analyses will include proper negative control (water blank) and positive controls.
Detection of PCR Products:
Following amplification, 10 Î¼l of the PCR products along with 2 ul of loading dye will be
analysed by electrophoresis on a 1-2 % agarose gel. Agarose or polyacrylamide gel will be used
for gel electrophoresis. Gel will be prepared by using 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 amplification product 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 products will be estimated from the electrophoretic migration of products
relative to a 100-bp DNA size ladder marker.
Establishment of Assay Conditions and Sensitivity:
Before analyzing subjects samples on large scale, optimization of PCR protocol will be done to maximize sensitivity, specificity and reproducibility to avoid false negative results. Specificity of the reaction will be increased by I) optimization of dNTPs and primer concentration II) optimizing annealing temperature III) optimizing PCR cycle number. Sensitivity of the assay condition will be established using standard positive control DNA, to analyze the number of copies of target DNA that are necessary to reproducibility yield a detectable and specific amplification product.
S.mutans genotyping will be carried out by using the method described by Saarela, et al (1996). 39 Briefly, the DNA from total S.mutans isolates will be used for genotyping. The AP-PCR fingerprinting will be performed with two primers, OPA-02 (5â€²-TGCCGAGCTG-3â€²) and OPA-13 (5â€²-CAGCACCCAC-3â€²). 40. Amplification products 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 imaging system. Molecular sizes for each band will be computed and analyzed by Bioinformatics software which will be used to generate similarity dendrograms (Dice coefficient, >95%; unweight pair group method with arithmetic mean) will be used to show the S.mutans genotypes.
Laboratories 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 well equipped to carry out work on almost all the above-mentioned objectives. There is excellent Molecular Diagnostic Laboratory in the CEMB for the molecular detection work. Equipment like thermal cycler and western blotting (PAGE apparatus / Blot Transfer apparatus) bench top centrifuges, ultracentrifuge, Real time PCR, Arbitrary primer sequence-based polymerase chain reaction (AP-PCR) , Agarose gel electrophoresis apparatus, laminar flow hoods etc. are available for the smooth running of the project. DNA synthesis facility is also available, required for the synthesis of DNA- oligos, which will be used for the amplification and quantification of viral genes. There is a well-established Serology and Heamatology Lab in Shaikh Zayed Hospital.
The aim of this study is to observe the main cariogenic genotypes of S.mutans in (Peri-Urban area of Pakistani Population) up to 5 years old children saliva samples by PCR and genotype methods. Genotypes vary from population to population, and no related study has been done in Pakistan. Also to observe whether genotypes transfer vertically or not. Due to poor oral hygiene, low socio-economic status, low maternal education and hormonal changes, the chances of caries infection increases from pregnant mothers to their children rather than horizontal transmission