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University of Khartoum

Graduate College
Medical and Health Studies Board

Saliva Composition and Candidal Counts among Type-2 Diabetic


Patients at Khartoum

By:
Yousif Osman Yousif
BDS , MSc (Uof K)

A Thesis Submitted for the Degree of PhD in Oral and Maxillofacial Surgery

Supervisor:
Prof. A. M. SULIMAN
BDS, MSc, FRCDS, Ph.D

2014

Dedication

This effort is dedicated to:

My ever father & teacher: El Shakhe Safe Eldin Abu El Azime,

To the soul of my father,

My mother, my ever-Supervisor Prof. A.M. Suliman

and

To all whom I love and they love me, too.

Yousif

ACKOWLEDGEMENTS
I raise my heart in gratitude to Allah Almighty for all the blessings Allah has
showered on me throughout this study.
I sincerely acknowledge with deep sense of gratitude and humble respect, the
valuable guidance and suggestions from my respected teacher and supervisor
Prof. A.M.Suliman. I am deeply indebted to him for his help, guidance, endless
support and constant encouragement throughout the period of this study.
My sincere thanks and appreciation are due to the Ministry of High Education &
Scientific Research for funding this research work. Without their help, this work
would not have come to existence.
I would like to extend my sincere thanks to the staff of Jaber Abu El Izz Diabetic
Centre & El Ribat University Hospital for their endless co- operation.
I express my sincere gratitude to Dr. Yasir Osman, Dr.Yasir Siddig and
Dr.Inaam for their help in the collection of the data.
Special thanks & gratitude to Dr.Mohamed Ali & miss Wafa for their continuous
support and encouragement.
My sincere gratitude to Miss Aisha Mohammed and Dr.Marium EL Hadi for
their great effort in the statistical analysis and for finalizing the computer work.
I heart- fully thank my senior colleagues at the department of Oral &
Maxillofacial Surgery for ever being helpful and supportive.
Last but not the least; I thank all the participants who formed an important part of
this study, without whose co-operation this study would not have been possible.
II

LIST OF ABBREVIATION

AGEs
BEC
BMI
C
CFU
D.M
EDTA
G
GLUT
HbA1c
IDDM
Ig
IL
NIDDM
P
r
RAGE
RT-PCR
SS
SD
SPSS
T
WHO

Advanced Glycation End Products


Buccal Epithelial Cells
Body Mass Index
Candida
Colony Forming Units
Diabetes Mellitus
Ethylene Diamine Triacetic Acid
Group
Glucose Transporter
Glycated Haemoglobin A1c
Insulin dependent diabetes mellitus
Immunoglobulin
Interleukin
Non Insulin dependent diabetes mellitus
Probability
Correlation Co efficient
Receptor for Advanced Glycation End Products
Real Time Polymerase Chain Reaction
Sjogren Syndrome
Standard Deviation
Statistical package for the Social Sciences
Table
World Health Organization

III

LIST OF CONTENTS
Item

Page no

CHAPTER ONE
1.1 Introduction & Literature Review

1 33

1.6. Rationale and Objectives

34 36

CHAPTER TWO
2.1 Methodology of the Biochemical Analysis of Saliva

37 42

2.2. Methodology of the Salivary Candidal Counts

43 46

CHAPTER THREE
3.1 Results of the Biochemical Analysis of Saliva

47 52

3.2. Results of the Salivary Candidal Counts

53 54

CHAPTER FOUR
4.1. Discussion-The Biochemical Analysis of Saliva

55 65

4.2 Discussion-The Salivary Candidal Counts

66 72

4.3. Conclusions:

73

4.4 Recommendations:

74

Annexures
References

75 98

Data Filling Form

99 101
IV

List of Tables and Figures


Table/Figure No

Title

Page No

1.1

Classification of D.M

1.2

Criteria for Diagnosis of D.M

1.3

Bacterial Genera of the Oral Cavity

31

3. 1
3.2
3.3
3. 4
3. 5
3. 6
3. 7

Groups and Gender


Mean age of the study groups
Duration of diabetes mellitus
Mean levels of salivary glucose
Mean levels of salivary amylase
Mean levels of salivary total protein
Mean levels of salivary urea

49
49
49
50
50
50
51

3. 8

Intergroup comparisons of mean levels of


salivary glucose

51

3.9

Intergroup comparisons of mean levels of


salivary amylase

51

3.10

Intergroup comparisons of mean levels of


salivary total protein

52

3. 11

Intergroup comparisons of mean levels of


salivary urea

52

3. 12
Fig. 3. 1

Mean colony-forming units (CFU) of the


study groups
Positive correlation between candidal forming
units and salivary glucose level

53
54

ABSTRACT
Background:
Diabetes mellitus is a group of metabolic disorders that share the common
underlying feature of hyperglycemia. Hyperglycemia in diabetes results from
defects in insulin secretion, insulin action, or most commonly from both of them.
In Sudan the prevalence of the disease in 1996 was 3.4% but it has increased
dramatically to 14.5 % in 2010.
Saliva is a complex fluid consisting mainly of water, essential electrolytes,
glycoproteins, antimicrobial enzymes and numerous other important constituents.
Diabetes has been consistently documented to be associated with altered salivary
composition and function. This disrupts the homeostasis of the oral cavity, making
it susceptible to various oral infections, with candidosis reported to be the
commonest one.
The present study consisted of two parts: the first one was designed to investigate
the saliva composition; mainly glucose, amylase, total protein and urea; among
type- 2 diabetic patients in comparison with non-diabetics. The second part aimed
to estimate the salivary candidal counts among both groups.
Methodology:
The study was conducted at Jabir Abu EL Iz Diabetic Centre-Khartoum during the
period May to August 2010. A total of 120 age and sex matched participants were
divided into 3 groups: uncontrolled diabetics, controlled diabetics and healthy nondiabetics. Salivary glucose was measured using glucose oxidase end-point method,
while amylase was measured by direct substrate kinetic enzymatic method.

VI

Pyrogallol red dye and diacetyl monoxime, were the methods used to measure total
protein and urea levels respectively.
Salivary Candida albicans counts were estimated using real- time PCR.
Results:
Salivary glucose and urea levels were found to be significantly elevated in both
uncontrolled and controlled diabetics, as compared to non-diabetics. (P < 0.05).
There was no significant difference when diabetics and non-diabetics were
compared for salivary amylase and total protein. (P > 0.05).
The study showed an increased Candida albicans counts among diabetics, in
comparison to non-diabetics, with a positive correlation between salivary candidal
counts and salivary glucose levels. (P < 0.0.1, r= 0.5).
Conclusion:
Diabetics saliva showed higher levels of glucose and urea in addition to increased
candidal counts. The latter, might be a factor behind the frequent candidosis seen
among those patients. Saliva may be used as a non-invasive diagnostic, as well as a
monitoring tool to assess the glycemic status of diabetic patients. Further studies
on larger populations and in different geographic areas, are required to establish
such a role.

VII


:

.
1996 3.4 14.5
.2010

.
.
: :
.
.
:

.2010 120
. :
.

.
.
.
:

.

.

.
:

.

.
.

VIII

CHAPTER ONE
Introduction and Literature Review
1. Diabetes Mellitus
1.1.1 Definition of Diabetes Mellitus
Diabetes mellitus is defined by the American Diabetes Associations Expert
Committee as a group of metabolic disorders characterized by hyperglycemia
resulting from defects in insulin secretion or insulin action or both. The chronic
hyperglycemia is associated with long-term damage, dysfunction and failure of
various organs, especially the eyes, kidney, nerves, heart and blood vessels. Thus,
Diabetes covers a wide range of heterogeneous diseases. (1, 2)

1.1.2 Epidemiology of Type-2 Diabetes


The world prevalence of diabetes in 2010 among adults aged 20-79 years was
estimated to be 6.4%, affecting 285 million adults (3). Between the year 2010 and
2030, there is an expected 70% increase in numbers of adults with diabetes in
developing countries and a 20% increase in developed countries(3). Each year
more than 231,000 people in the United states and more than 3.96 million people
worldwide die from diabetes and its complications(4). This number is expected to
increase by more than 50 percent over the next decade (5). Estimated global
1

healthcare expenditures to treat and prevent diabetes and its complications were at
least 376 billion US Dollars (USD) in 2010. By the year 2030, this number is
projected to exceed some 490 billion USD (6).
The reported prevalence of type 2 diabetes varies from zero in Togo (7), 9% in
Egypt (8) to 50% in pima Indians (USA) (9). Particular ethnic groups (e.g. South
Asian, Native American and Mexican- Americans) are highly susceptible to type 2 Diabetes, and this may be revealed when such groups migrate into relatively
affluent settings. Regional and ethnic differences in the prevalence of type-2
diabetes probably related to both the lifestyle and the underlying genetic
susceptibility (10).
Prevalence rates are relatively high (9-8%) in Tunisia as well as Egypt (8).
However, recently most Arabic countries have experienced an increase in the
disease prevalence, with the evolving trend particularly alarming in Jordan and
Egypt. (8, 11)
In the Kingdom of Saudi Arabia the prevalence of 4.95% and 4.30% was reported
during 1985 to 1987(12). In Yemen, the crude prevalence of known diabetes was
6.57% (13).

1.1.3 Diabetes Mellitus in Sudan:


Sudan has had contact with Middle East and Mediterranean civilizations since
ancient times. The Western part has many contacts with West Africa, and the
Eastern part have maintained close links with the countries of the Indian Ocean.
The population in the north-eastern parts of the country had undergone ethnic
absorption of immigrant Arabs during times of Islamization, and culturally became
Arabised. About 70% of the Sudanese population lives in the northern part of the
country. Twenty one percent of the populations are urban settlers. And an
estimated 10% of the rural inhabitants are nomads. Regular internal migration in
different parts of Sudan has taken place from rural areas and small towns to big
cities, particularly to the capital, Khartoum. This has been accompanied by
displacement of a large proportion of populations from drought and famine prone
areas in western and southern regions. In recent years permanent external
migration has also occurred (14). These cultural changes have lead to considerable
progress in educational and health establishment as well as improvement in the
standards of living. These social and economic advances were accompanied by a
change to a modern life style, characterized by higher caloric intake and less
physical activity, and the emergence of non communicable diseases such as
diabetes mellitus, which is a major health problem causing high morbidity and
mortality. It can be estimated from the hospital records that the number of diabetic
3

patients is increasing in all socioeconomic classes. Type 2 DM particularly


accounts for 75% of all diabetic patients attending the outpatient diabetic clinic in
Khartoum (15). The overall crude prevalence of diabetes mellitus and impaired
glucose tolerance in the total population of the northern part of Sudan in1996 was
3.4 % and 2.9% respectively. The highest overall prevalence was in the northern
parts of Sudan (5.5%) and the lowest was in the western region (0.9 %.) (16).
The prevalence of the undiagnosed diabetes was 2.2% of the total population. The
high prevalence of undiagnosed diabetes will have a profound impact on the
community in general and on the public health services in particular In Dongla
community in the northern state the prevalence was 8.3%. While in a sub group of
this community, with Egyptian descent the prevalence was 10.8%. There were no
urban /rural or male/ female differences in prevalence (16).
In the year 2006, the Federal Ministry of Health reported a dramatic increase in the
prevalence of the disease in Khartoum state reaching a 19.2% and in 2010 the
estimated prevalence of the disease was 14.5% all over the country (17).

1.1.4 Classification of D.M:


Diabetes

mellitus

is

classified

by

four

distinct

categories

based

on

aetiopathogenesis, although two main categories of diabetes make up the bulk of


the cases. Type-1 diabetes mellitus (T1DM) (previously known as insulin
dependent diabetes mellitus (IDDM)) and Type-2 diabetes mellitus (previously
known as non-insulin dependent diabetes mellitus (NIDDM)).
Other categories include gestational diabetes and other specific types of diabetes.
The latter are made up of those associated with gene defects of pancreatic cell
function and insulin resistance; syndromes associated with diabetes; diseases of the
exocrine pancreas; endocrinopathies and diabetes induced by drugs, chemicals or
infective agents (T.1.1) (18).
The above classification includes changes to reflect the aetiopathogenesis rather
than the therapeutic implications of the groups. It also reflects the fact that there is
a range of presentations, as well as therapeutic treatments, all of which can change
with time, meaning that patients should not be classified according to these
overlapping criteria. The terms insulin-dependent diabetes mellitus and noninsulin-dependent diabetes mellitus and their acronyms, IDDM and NIDDM, were
therefore removed from the classification as a result of the confusion that their use

generated. The terms type-1 and type-2 diabetes mellitus were retained, with
Arabic number being used. (18).
Type- 2 diabetes mellitus is the most prevalent form of diabetes, which results from
insulin resistance, with or without a secretory defect. It primarily occurs with
increasing age and is associated with genetic and environmental risk factors. Type-2
diabetes is commonly preceded by a long period of abnormal glycaemic control and
is part of the metabolic syndrome associated with hypertension, dyslipidaemia and
hyperglycaemia. The condition has a stronger genetic aetiology than Type-1 DM
although environmental factors such as diet, exercise, obesity and smoking have an
impact on the development of type 2 diabetes (19).

Table 1-1 Classification of Diabetes Mellitus (American Diabetes


Association, 2011)

* Maturity onset diabetes of the young


7

1.1.5 Diagnosis of DM:


The criteria for diagnosis of hyperglycemia and the classification of diabetes
mellitus are not uniformly accepted. Some physicians use the criteria of the United
States National Diabetes Data Group of 1979, which was endorsed by the World
Health Organization Study Group on Diabetes Mellitus in 1985 (20), or the WHO
report 1999 (21), while others prefer the criteria of Diabetes Mellitus of the
American Diabetes Association 1998 (22). T.1.2.
Table 1. 2 : Criteria for the diagnosis of diabetes according to the
World Health Organization (21) and the American
Diabetes Association (22)
World Health Organization

American Diabetes Association

Clinical:

Clinical:

Increased thirst and urine volume

1/ Polyurea, polydepsia and unexplained


weight loss plus casual plasma or capillary
Unexplained weight loss, established by
blood glucose 126 mg/dl (11.1mnol/1)
casual blood glucose
Or
Or
Biochemical :
Biochemical:
2/ Fasting plasma glucose 126 mg/dl. (7.0
The casual blood glucose, 5.5-11.1 mmol/
mmol/L) or capillary glucose 11.0 mg/dL
(100-200mg/dL) fasting blood glucose
level 7.0 mmol/L and/or a 2- h glucose Or 3/ 2 h plasma or capillary blood glucose
11.1 mmol/L (200mg/dL) after glucose 200mg/dL (11.1mmol/1) during an oral
load.
glucose tolerance test.

Casual is defined as any time of the day without regard to time since last
meal.
Fasting is defined as no caloric intake for at least 8hours.
Criteria 2 and 3 should be confirmed by repeat testing on a separate day.
The disease usually has a gradual and insidious onset. The diagnosis is made
incidentally (during insurance screening or during hospital visits for other medical
problems) in almost one-third of cases, and almost one-half do not complain of
obvious diabetic symptoms. Common presentations are with genital candidosis
(particularly in women), or urinary tract or skin infections (23). A significant
number of type-2 diabetic patients already have one or more chronic complications
at time of diagnosis notably diabetic retinopathy and macro vascular disease (24,
25). It has been observed that type -2 diabetes generally starts 4-7 years before the
diagnosis is made (26).

1.1.6 Risk Factors in Type- 2 DM:


Type -2 diabetes is a heterogeneous disorder due to a combination of genetic and
environmental factors that adversely affects -cells function and tissue insulin
sensitivity. (27, 28).

Major risk factors:


Race and ethnic background e.g. Africa-American, Hispanic- American, AsianAmerican and Native American; Australian aborigines, Polynesian islanders.
Age 45% years.
Family history: first-degree relatives with type-2 diabetes.
Obesity: Body Mass Index (BMI) 27 kg/m2, with central fat distribution.
History of impaired fasting glucose or impaired glucose tolerance.
History of gestational diabetes or delivery of a baby weighting > 4 kg.
Hypertension (blood pressure 140/90 mmHg).
Triglyceride level 2.8 mmol/L

10

Other risk factors


1- Malnutrition in the first year of life, and particularly in utero.
2- Lifestyle factors:
Physical inactivity.
High-fat, low-carbohydrate diet.
Alcohol.
Smoking.
Nutritional and lifestyle factors have long been recognized as important factors in
the pathogenesis of type-2 diabetes. There are powerful epidemiological
associations between type- 2 diabetes and obesity; together, overweight and obesity
account for about two- thirds of cases of type-2 diabetes (29). The risk of diabetes
begins to rise once the (BMI) is greater than 23 kg/m2, and the relative risk is 4090 fold higher in subject with a BMI > 40 kg/m2. Weight gain in adult life is also a
significant risk of developing the disease (30, 31). Over all, obesity is a major risk
factor for the development of type- 2 diabetes, but it is neither necessary nor
sufficient to cause the disease (29).

11

Recent evidence suggests that accumulation of triglyceride at sites other than in


adipose tissue notably skeletal muscle, liver and pancreatic -cells is also
associated with the development of diabetes ( 32, 33).
Weight loss has been shown in several studies to decrease the risk of progressing
from impaired glucose tolerance to diabetes; this is particularly the case if
combined with increased physical activity.

Weight loss is also an effective

component of treatment of patients with type- 2 diabetes. Prevention of weight gain


in childhood and adult life through dietary restraint and increased physical activity
are likely to protect against the development of type -2 diabetes (34, 35).

1.1 .7. Pathogenesis of Type-2 Diabetes:


Type- 2 diabetes is more complex in etiology and is characterized by a relative
insulin deficiency, reduced insulin action and insulin resistance of glucose
transport in skeletal muscle and adipose tissue. The manifestation of frank type- 2
diabetes is a continuum of insulin resistance, and the progression to full diabetes
ensues when pancreatic -call hyper secretion of insulin fails to compensate for
insulin resistance (36). Commonly found alterations of insulin secretion in type- 2
diabetic patients include release after ingestion of mixed meals, alterations in rapid
pulses, and ultrafine oscillations. In addition, it was found that type- 2 diabetic

12

islets release less insulin in response to glucose and this is accompanied by altered
expression of glucose transporters (in particular GLUT2) and that of glucokinase (37).

It was found that arginine and glipenclamide induced an early insulin secretion
phase from the diabetic islets. Both arginine (by increasing intracellular positive
charge) and sulfonylurea (by inhibiting K+ loss from -cell) cause depolarization of
-cell membrane. This event is followed by Ca2+ entry, increased cytosolic Ca+
concentration, translocation and exocytosis of insulin granules. In summary it is
believed that the defects of glucose-stimulated insulin release in type 2 diabetic cells lie between glucose transport and depolarization of cellular membrane (36).

1.2. SALIVARY GLAND AND THE COMPOSITION OF SALIVA:


The major salivary glands are the parotid glands, submandibular glands and
sublingual glands. Minor salivary glands are situated on the tongue, palate, and
buccal and labial mucosa. They are small mucosal glands with primarily a mucous
secretion (38). The secretory part of the salivary gland tissue consists of the
secretory end pieces (acini) and the branched ductal system. The fluid first passes
through intercalated ducts which have low cuboidal epithelium and narrow lumen.
Then the secretions enter the striated ducts which are lined by more columnal cells
with many mitochondria. Finally the saliva passes through the excretory ducts
where the cell type is cuboidal with stratified squamous epithelium (39). The
13

acinar cells first secrete isotonic primary saliva and then the striated duct cells
actively extract ions to render the saliva progressively more hypotonic as it passes
down the ducts towards the mouth (40).
Inorganic Composition of Saliva:
The most abundant component in saliva is water (approximately 99%), followed
by ions Na+,Cl Ca2+,K+,HCO3 ,H2PO4 , F, I,Mg2+ and thiocyanate.
The hypotonicity facilitates taste sensitivity and hydrates various organic
compounds that form a protective coating on the oral mucosa.
The bicarbonate serves as a buffering agent and calcium and phosphate neutralize
acids that would otherwise compromise tooth mineral integrity (41, 42).
The Organic Composition of Saliva:
Saliva includes a large number of organic compounds such as: urea, ammonia,
uric acid, glucose, cholesterol fatty acids, mono, di, and triglycerides, phosphor
and neutral lipids, glycolipids, amino acids, steroid hormones and proteins that aid
in the protection of the oral cavity tissues, including mucins, amylases, agglutinins,
glycoproteins, lysozymes, peroxidases, lactoferrin and secretory IgA. Non-immune
factors include lactoferrin, lysozyme, myeloperoxidase, histatins, cystatins, mucin
G1 and G2, and defensins (43-49). In addition, these macromolecules form a
viscoelastic mucosal coat and tooth enamel pellicle and aggregate and cleanse

14

bacteria and debris from the oral cavity (50). Saliva also contains a variety of
antimicrobial constituents and growth factors (51, 52).

1.2.1. The Diagnostic uses of Saliva:


Salivary diagnosis is an increasingly important field in dentistry, physiology,
internal medicine, endocrinology, pediatrics, immunology, clinical pathology,
forensic medicine, psychology and sports medicine (53). A growing number of
drugs, hormones and antibodies can be reliably monitored in saliva, which is an
easily obtainable, non-invasive diagnostic medium (54, 55, 56, 57). Thus, salivary
diagnosis is anticipated to be particularly useful in cases where repeated samples of
body fluid are needed but where drawing blood is impractical, unethical, or both.
Salivary concentrations of drugs and hormones also represent the free fractions of
serum in many instances, with good correlations with the respective total
concentrations in serum (58, 59).
Assays of steroid hormones from saliva are widely used and well validated ((60),
providing an unstressful sampling instead of venipuncture. Multiple specimens of
saliva for steroid hormone analysis can be easily collected by the patient, at home,
to monitor fertility cycles, menopausal fluctuations, stress and other diurnal
variations (59). Also, some hormones other than steroids have been found to be
reflective of their plasma levels and could be considered for saliva monitoring.
15

Salivary antibody levels can be determined to screen for infectious diseases. AntiHIV antibody immunocapture assays have also been developed and tested for
saliva, which could be useful in high-risk groups under field conditions in
developing countries (61). Salivary assays have been used for monitoring of
hepatitis A, B and C, measles, Epstein-Barr virus, rubella, parvovirus B 19, human
herpes virus 6, Helicobacter pylori and rotavirus infection (55, 62). In addition to
measuring antibody, it is possible to identify a number of viral antigens in saliva,
for example mumps and cytomegalo virus. Saliva has also proven to be a
convenient source of host and microbial DNAs (57).
There has been growing interest in the use of saliva in pharmacokinetic studies of
drugs and in therapeutic drug monitoring in a variety of clinical situations. It has
been suggested that drug levels in saliva reflect the free, non-protein-bound portion
in plasma and hence may have a greater therapeutic implication than the total
blood levels (55).
Lipid solubility is a determining factor in saliva excretion of drugs, and the degree
of acidity and basicity of a drug will determine its salivary/plasma ratio. The
salivary flow rate, pH, sampling conditions, contamination and many other
pathophysiological factors may influence the concentrations of drugs in saliva (63).
Drugs currently monitored in saliva include anticonvulsants, theophylline,

16

salicylate, digoxin, anti-arrhythmic drugs, lithium, benzodiazepines, amitriptyline,


chlorpromazine, methadone, ethanol, marijuana, cocaine and caffeine (64, 63).
It has become apparent that many systemic diseases affect salivary gland function
and salivary composition. Studies of the effects of systemic diseases on salivary
variables have been valuable in understanding the pathogenesis of the diseases, but
their use as diagnostic markers has been limited. Primary Sjgrens syndrome (SS)
is a common autoimmune disorder characterized by generalized exocrine
hypofunction and serologic abnormalities. More than 90% of patients are women,
and one of the main diagnostic procedures is biopsy of the minor salivary glands of
the lip. It has been suggested that whole saliva flow rate and gland-specific
sialometry and sialochemistry could be used to provisionally diagnose SS (65, 66).
In SS, salivary glands are indeed affected, resulting in a diminished salivary flow.
It has been suggested that diminished output of salivary defense factors, rather than
their absolute concentrations, may be related to the oral health problems seen in SS
patients (67).
Cystic fibrosis affects all of the exocrine glands to varying degrees (68). The most
dramatic changes in the composition of saliva reported have been an elevation in
calcium (Ca) and proteins, and this reduces the flow rate of minor salivary glands
to virtually zero. Normally the flow rate of single labial gland is 0.1 l/min (68).
17

This phenomenon can be used as a diagnostic test by measuring the flow from
labial glands of the lower lip (55). The sodium (Na) and potassium (K)
concentrations of saliva are markedly affected by corticosteroids, especially
aldosterone. The Na/K ratio of stimulated whole saliva can be used in diagnosing
and monitoring Cushings syndrome and Addisons disease. Investigators have also
demonstrated the diagnostic value of Na/K ratio in primary aldosteronism (69).

In several clinical situations salivary analysis has provided valuable information


for both the clinician and the investigator. These situations include digitalis
toxicity, affective disorders, stomatitis in chemotherapy, specific secretory IgA
deficiency, smoking, ovulation time, relation of dietary factors to cancer and
chronic pain syndromes (55, 70).
Human saliva contains a large number of enzymes derived from the salivary
glands, oral microorganisms, crevicular fluid, epithelial cells, and other sources.
However, the use of whole saliva enzymes for diagnostic purposes has been more
difficult than the use of serum enzymes. It has been difficult to standardize saliva
collection methods and enzyme analytical procedures so that direct comparisons
between different laboratories would be possible (71).
Interpretation of results has also proved to be difficult. However, various studies
have been made to find correlations between diseases or clinical situations and

18

salivary enzyme levels. Saliva is essential for remineralization of teeth, protection


and lubrication of oral mucosal tissues (72). Measurement of the patients saliva
flow is of primary importance in oral medicine and dentistry (73). For many years
dental investigators have been exploring changes in salivary flow rate and
composition as means of diagnosing and monitoring a number of oral diseases. It
has even been suggested that analysis of saliva may also offer a cost-effective
approach to the assessment of periodontal diseases in populations (74), even
though no specific salivary marker of periodontal disease activity has been found
so far.
A great number of studies with conflicting results have been published regarding
various individual salivary agents and their possible association with oral health,
particularly dental caries (75). However, it appears that no single chemical agent is
much more important than others. Many of the various defense factors show
additive or even synergistic interactions against oral pathogens (76).

19

1.2.2 SALIVARY ANTIMICROBIAL PROTEINS


1.2.2.1. Salivary immunoglobulins
Salivary secretory immunoglobulins (sIgA and sIgM) originate from immune cells
which are produced as a host response to an antigenic stimulus (77). The
immunoglobulins may be directed at specific bacterial molecules, including cell
surface molecules such as adhesins, or against enzymes. By binding to such
molecules, adhesion of specific bacteria to oral surfaces may be blocked, so
preventing colonization by the affected species (78, 79).
Several studies have confirmed that sIgA is mainly dimeric rather than
monomeric, and it is associated with an epithelial glycoprotein called SC
(secretory component) (80). At least 95% of the IgA normally appearing in saliva
is produced by the local gland-associated immunocytes rather than being derived
from the serum (77).

1.2.2.2 Nonimmunoglobulin proteins


1.2.2.2.a. Lysozyme
Lysozyme represents the main enzyme of the nonspecific salivary immune defense
(81), and it is secreted mainly by the submandibular and sublingual glands (82).
Salivary lysozyme hydrolyses specific bonds in exposed bacterial cell walls,
20

causing cell lysis and death. Many organisms, however, have cell capsules or other
cell wall protective material, which confer resistance against lysozyme attack (83).
Lysozyme has been proposed as a lytic factor for bacteria which immunoglobulins
have bound, mimicking in some respects the complement system in serum.
Lysozyme aggregates cell suspensions of some bacterial species (78). It is also
known that lysozyme contributes to mucosal protection and modulates Candida
populations in the oral cavity (84).

1.2.2.2 .b. Peroxidase systems


Salivary peroxidase and myeloperoxidase, catalyze a reaction involved in the
inhibition of bacterial growth and metabolism, and the prevention of hydrogen
peroxide accumulation, thus protecting proteins from the action of oxygen and
reactive oxygen species (85, 86). More precisely, salivary peroxidase catalyses the
oxidation of thiocyanate ion (SCN) to generate oxidation products that inhibit the
growth and metabolism of many microorganisms (86). The primary oxidizer,
hydrogen peroxide, is produced by the bacteria, and the production of the toxic
agents is highly localized and occurs close to the bacterial target (78).

21

1.2.2.2.c Lactoferrin
Lactoferrin is present in plasma and in mucosal secretions (87). Salivary lactoferrin
has antibacterial activity. Lactoferrin binds iron, making it unavailable for
microbial use (83). Lactoferrin, in its unbound state, also has a direct bactericidal
effect on some microorganisms including Streptococcus mutans strains.

1.2.2.2 .d.Histatins
Histatins comprise a group of small histidine-rich proteins present in the saliva.
The most significant function of histatins may be their anti-fungal activity against
Candida albicans (88, 89). Oral candidiasis may also modulate the levels of
salivary histatin (90, 91). It has been suggested that histatins could be used as
components of artificial saliva for patients with salivary dysfunction (88).
Histatins have been shown to be tannin-binding proteins in human saliva (92, 93).
Histatins also bind to enamel surfaces and hydroxyapatite in a complex manner (94).

1.2.2.2.e. Agglutinins
Salivary agglutinins are glycoproteins which have the capacity to interact with
unattached bacteria, resulting in clumping of bacteria into large aggregates which
are more easily flushed away by saliva and swallowed (95). Bacterial binding to
salivary proteins may in part account for individual differences in the colonization
22

of tooth surfaces. Agglutinins induce the aggregation and clearance of streptococci


from the oral cavity and are also important modulators of initial plaque formation
(96). On the other hand, it seems that salivary agglutinins may mediate the
adherence of various bacterial species to the tooth surfaces (97, 98).
A number of salivary proteins with an agglutinating capacity have been identified:
parotid saliva glycoproteins, mucins, sIgA, 2-microglobulin, fibronectin and
lysozyme (83).

1.2.3. AMYLASES
Amylase ( amylase) is one of the most important salivary digestive enzymes. It
consists of two families of isoenzymes, of which one set is glycosylated and the
other contains no carbohydrate (71). Salivary amylase is a calcium metalloenzyme
which hydrolyses the alpha bonds of starches, such as amylose and amylopectin
(78). Maltose is the major end-product.
It has been suggested that amylase accounts for 40 to 50% of the total salivary
gland-produced protein, most of the enzyme being synthesized in the parotid gland
(82). Human parotid saliva and submandibular saliva contain about 45 mg and 30
mg of amylase, respectively, per 100 mg of protein (71). However, it has also been
claimed that amylase makes up about 1/3 of the total protein content in parotid

23

saliva, and the content in whole saliva would be lower (99). The concentration of
amylase increases with the salivary flow rate, and it is generally considered to be a
reliable marker of serous cell function (100).
In addition to its well-known function as a digestive enzyme, amylase has been
reported to act as an antimicrobial enzyme. Amylase activity exists also in tears,
nasal and bronchial secretions, milk, serum, urine and in the secretions of the
urogenital tract (83). Amylase also interacts specifically with certain oral bacteria
and may play a role in modulating the adhesion of those species to teeth (101). It
has been found that salivary amylase inhibits the growth of Legionella
pneumophila and Neisseria gonorrhoeae (83). Amylase is also present in human
acquired pellicle in vivo (102). Fasting has been found to decrease whole saliva
amylase levels and activity (71). The amylase concentrations in radiation-induced
hyposalivation have been found to be reduced (100).

1.2. 4. SALIVARY ALBUMIN


Albumin is the most abundant serum protein, accounting for more than 50% of all
plasma proteins. Its molecular mass is 69 kDa and the normal serum reference
limits are 40 - 52 mg/l. Albumin is synthesized exclusively in the liver at a rate of
100 - 200 mg/kg/day. Factors that regulate albumin synthesis are nutrition,

24

hormonal balance, and osmotic pressure. The half life of albumin is approximately
15 - 20 days. About 4% of albumin is degraded per day, but synthesis can be
increased by as much as 100% by conditions that decrease serum albumin or lower
intravascular osmotic pressure (103). Nephrotic syndrome is the best known
example of systemic disorders with characteristic proteinuria and subsequent
hypoalbuminaemia which lead to oedema (104). In the oral cavity, albumin is
regarded as a serum ultrafiltrate to the mouth (105), 106) and it may also diffuse
into the mucosal secretions (107).
Salivary albumin is selectively adsorbed by different materials in the oral cavity,
which may enable the attachment of specific bacteria and thus alter the
composition of dental plaque (108). Salivary albumin has been shown to increase
in medically compromised patients whose general condition gets worse (109).
Immunosuppression, radiotherapy, and diabetes are examples of states where high
concentrations of salivary albumin have been detected (110, 111).
Salivary albumin levels have been used as a marker for the degree of mucositis and
inflammations in salivary glands (112). Butler et al. (113) found that albumin
levels in whole saliva fluctuated in most of the elderly patients in their study.
Cuida et al. (114) found that albumin concentrations were higher in both parotid
and whole saliva in patients with primary Sjgrens syndrome (SS) than in the
control group. However, the output/min of albumin was lower in SS patients.
25

Lenander- Lumikari et al. (115) found that albumin concentrations were higher in
patients with celiac disease than in healthy controls. It may be hypothesized that
salivary albumin can be used to assess the integrity of mucosal function in the
mouth (116). In periodontitis patients, significantly increased levels of salivary
albumin have been reported, and a significant correlation between salivary albumin
and gingival index in diabetic patients has been found (110).
On the other hand, Sweeney and co-workers (117) did not find any difference in
serum albumin concentrations in elderly patients with mucosal pathology in the
mouth when compared with those with healthy mouths.
Yoshihara et al. (118) found that there is a relationship between root caries and
serum albumin concentrations in elderly subjects. Terrapon et al. (119) found that
the low salivary albumin of old edentulous people was similar to that in a group of
younger individuals with a healthy periodontium.

1.2.5. SALIVARY UREA


Urea is metabolized by the oral bacteria to ammonia and CO, resulting in an
increase in the pH of the oral environment (120, 121). It has been suggested that
plaque cariogenicity may be inversely related to salivary urea concentrations (121).

26

In certain metabolic conditions, mainly patients with renal dysfunction, urea is


increased and it could be easily monitored in saliva. (122, 123).

1.3 SALIVARY COMPOSITION & D.M:


Lower stimulated salivary flow rates in poorly controlled diabetic patients
compared to well controlled diabetic patients and controls were reported by
Harrison & Bowen (124). In another study by Sreebny et al on adult diabetic
patients, inverse relationships were observed between salivary flow rates and
HbA1c levels (125). Based on the equal flow rates between subjects with diabetes,
those with impaired glucose tolerance and controls, Cherry-Peppers et al (126)
concluded that salivary gland function was not significantly impaired in well
controlled subjects with altered glucose metabolism.
Reuterving et al (127) examined the salivary factors of 11 diabetic patients at
baseline and after improvement of their level of metabolic control one to five
months later. They reported that individual variations in salivary flow rates were
conspicuous and were not affected by improved metabolic control. Salivary
glucose levels, instead, were lower during better metabolic control. During poor
metabolic control with high blood glucose values, high blood glucose levels could
possibly be reflected as high salivary glucose levels. There is, however, little
evidence of this since: some weak correlations between saliva glucose and blood
27

glucose levels have been reported (127, 128, 129, 130), but no such correlations
have been found by others (131, 132, 133, 134, 135). Tenovuo et al. (1986) (136)
analyzed glucose levels in more than one hundred simultaneously taken stimulated
whole saliva samples and blood samples of seven patients. The variations in
salivary glucose were found to be extensive, and the correlation between salivary
and blood glucose was highly individual: some subjects showed high correlations,
while some others showed no change in salivary glucose, even when their blood
glucose levels were very high.
Glucose in parotid saliva or in gingival crevicular fluid is more strongly related to
blood glucose levels than glucose in mixed saliva (137, 138). Borg Andersson et
al. (130) reported that, after a standardized carbohydrate load, glucose levels in
parotid saliva increased in diabetic patients.
In an experimental animal study by Reuterving (139), the salivary secretion rate
was found to be decreased in diabetic rats, and the flow rates correlated inversely
with blood glucose levels.
Salivary microbial counts in relation to glucose control have hardly been
investigated. Reuterving et al. (127) observed that the counts of mutans
streptococci in mixed saliva decreased significantly as the metabolic control of 11
diabetic patients improved, but the counts of lactobacilli remained stable. Twetman
28

et al. (140) reported an increase of salivary lactobacilli counts along with a rise of
salivary glucose levels. During a two-year follow-up of newly diagnosed patients
with type 1 diabetes, salivary glucose levels tended to be lower during the second
than the first year, and the counts of lactobacilli dropped significantly during the
first six months, while the counts of mutans streptococci remained stable (141).
High salivary glucose levels might, however be connected with an increase of
microbial colonization, as also indicated by Darwazeh et al. (129).

1.4 THE MOUTH AS A MICROBIAL HABITAT:


The oral cavity, like other parts of the gastrointestinal tract, possesses natural
microflora. It has a number of features that makes it a unique microbial habitat.
Teeth characteristically provide hard non-shedding surfaces that allow
accumulation of large masses of microorganisms (dental plaque), especially in
stagnant areas. Such accumulation is restricted on mucosal surfaces due to
continuous epithelial desquamation; the only exception is the dorsum of the tongue
that is highly papillated and thus supports higher densities of microbes (142).
Another important feature is that the oral cavity is continuously bathed with saliva,
which has a profound effect on the ecology of the mouth. Saliva has a pH range
(6.75-7.25) that favors growth of many microorganisms. Salivary components
influence oral microbes by one of four mechanisms: aggregating microbes to
29

facilitate their clearance from the mouth, adsorbing to teeth surface to form an
acquired pellicle to which microorganisms can attach, serving as a primary source
of nutrients, and mediating microbial inhibition or killing (143). In addition to
saliva, the gingival crevicular fluid, a plasma derived fluid that flows through the
junctional epithelium, provides microbes in the gingival crevice with nutrients and
carries host immune components that play an important role in regulating the
microflora (142).
Oral microbes are predominantly bacteria but fungi, viruses, mycoplasmas and
even protozoa (142) and archaea (144) can also be found.
The bacteria found in the oral cavity are presented in T.1.3. Below:

Table1.3. Bacterial genera found in the oral cavity (145).

30

1.5 CANDIDA ALBICANS & D.M:


Yeasts are conventional constituents of the oral microflora in humans; conversely,
they are also recognized as opportunistic pathogens. Candida albicans, the most
common yeast species in the human oral cavity, and several related Candida
species are strategic pathogenic fungi, which reside in the oral cavities of healthy
humans without causing detrimental effect (146). The innate and acquired host
defense mechanisms act in concert with the resident bacterial flora, such that
Candida grows and survives as commensals. Although, secretory IgA is a major
defender of the oral mucosa, the inhabitant microbiota still persists in the oral
cavity. Survival of native oral bacteria may be due to their ability to neglect the
immune system or perhaps, over a period of evolutionary adaptation, they have
achieved a symbiotic state with the host (147). Aspartic proteinase, produced by C.
albicans can hydrolyze almost all immunoglobulins, including IgA, and several
host defence anti-fungal proteins, such as salivary Iactoferrin (148). Other
Candidal species, including C. dublinensis, C. tropicalis and C. parapsilosis have
also shown similar proteolytic activity (149).
The virulence of Candida species, especially C. albicans, increases in
immunocompromized subject. Immunosppression provides an excellent platform
for fungal proliferation. Diseases where the immune system is suppressed include

31

DM, liver cirrhosis, acquired immune deficiency syndrome and nutritional


deficiency syndrome and nutritional deficiencies (150). Xersotomia or dry mouth,
a common complaint in diabetic subjects predisposes the oral mucosa to
opportunistic infections by microorganisms especially C. albicans (151). It has
been documented that patients with DM harbor high levels of C. albicans in their
oral mucosa while intensive Candidal colonization can be seen in diabetic patients
with elevated glycemic levels (152).
A study on the prevalence of pathogenic yeasts and humoral antibodies to Candida
in diabetic patients done by Odds et al, (153) showed that the extent of yeast
colonization was related to the blood glucose and urine sugar levels at the time of
sampling.
Lamey et al, (154) conducted a study on secretor status, candidal carriage and
candidal infection in 109 patients with diabetes mellitus and 100 healthy
individuals. The results showed a significantly higher prevalence of oral candidal
carriage and infection in diabetics than non-diabetic individuals.
Fisher et al, (155) studied the relationship between the quality of glycemic control
in diabetes mellitus and the carriage of Candida species, no association was
identified between carriage rates and the type of treatment, or with the quality of
glycemic control. Torres et al (156) investigated the relationship between salivary
32

flow rates and Candida counts in 112 subjects who reported xerostomia.
Stimulated whole saliva was collected and streaked in Candida agar plates and
counted over 72 hours. The results showed a significant inverse relationship
between salivary flow and Candida colony forming units (CFU). Salivary
alterations in type-2 diabetes mellitus were investigated by Dodds et al. (157). The
authors assessed the salivary flow rates and yeast carriage in 233 subjects with type
2 diabetes mellitus and 240 healthy control subjects. The results showed that
diabetic patients had reduced output of both stimulated and unstimulated saliva;
and had high oral yeast counts compared with healthy subjects. The authors
concluded that diabetics may be more prone to oral dryness and infections than
non-diabetics.

33

1.6. RATIONALE AND OBJECTIVES

Diabetes mellitus is a widespread metabolic disease causing well-documented


deleterious effects on the general health of an individual. Multiple epidemiologic
studies have suggested that diabetes is a risk factor for the development of oral
diseases with oral candidosis represents the commonest one. The condition is
probably the most frequent metabolic disease with salivary implication.
The understanding of the role of each salivary component in the oral cavity
homeostasis is crucial to perceive how its changes or absence may be linked with
pathological conditions. The most commonly used laboratory diagnostic
procedures involve the analyses of the cellular and chemical constituents of blood.
Other biologic fluids are utilized for the diagnosis of disease, while saliva offers
many distinctive advantages.
Biochemical and microbial analysis of saliva would be of great biomedical
importance, since saliva is very easy to collect offering a cost-effective approach
for screening of large populations, and could represent an alternative for the patient
whose blood is difficult to obtain when compliance is a problem.
The present study is first study in Sudan relating saliva composition & candidal
counts to diabetes.

34

Study (1): General Objective:


To investigate the biochemical composition of saliva among Type-2 diabetic
patients.
Study (1): Specific Objectives:
1- To investigate salivary:
Glucose level.
Amylase level.
Total protein level.
Urea level.
Among Type-2 diabetic patients and to compare them with healthy nondiabetic subjects.
2- To compare the study findings with those available in literature.

35

Study (2) General Objective:


To estimate salivary candidal counts among type-2 diabetic patients.
Study (2) Specific Objectives:
To estimate salivary Candida albican counts among type-2 diabetic patients
using Real-Time PCR and compare them with a healthy non-diabetic
subjects.
To compare the study findings with previous reports.

36

CHAPTER TWO
2.1 STUDY (1): METHODOLOGY
The Biochemical Analysis of Saliva

2.1.1 Study Design:


- Prospective analytical hospital based case-control study.
2.1.2 Study Area:
-The study was carried out at Jaber Abu El Izz Diabetic centre & EL Ribat
University Hospital in the period May to August 2010.
2.1.3 Study population:
-One hundred and twenty age- and sex-matched study subjects in an age range from
40 to 69 years.
The study population was divided into 3 groups:
Group (1):
-An uncontrolled diabetic group (40 patients).
-21 males, 19 females with a mean age: 48.50 7.86 years.
(T. 2.1 & 2.2).

37

Group (2):
-A controlled diabetic group (40 patients).
-23 males, 17 females with a mean age: 49.50 10.88 years.
(T. 2.1& 2.2).
Group (3):
-A healthy non-diabetic group (40 subjects).
-20 males, 20 females with a mean age: 46.12 10. 25 years.
(T.2.1 & 2.2).
The diabetic patients were divided according to the disease duration into:
-Short duration diabetics:
-included those patients who have been diabetic up to 6 years.
-long duration diabetics:
-included those patients who have been diabetic for more
Than 6 years. (T.2.3).
2.1.4 Inclusion and exclusion criteria:
- Cases of type-2 diabetes mellitus who were diagnosed clinically with features of
polyuria, polydypsia and polyphagia and elevated blood glucose levels, as per the
criteria established by the Expert Committee on Diagnosis and Classification of
Diabetes Mellitus in 1998 (1) were included in the study.
- Volunteers workers at EL Ribat University Hospital, with no features of diabetes
38

mellitus and blood glucose levels within normal limits, confirmed by checking their
fasting blood glucose level twice within a two weeks interval were included as a
control group (3).
-Patients with severe diabetic complications, with any other systemic illnesses or on
medications other than those for diabetes were excluded.
- Individuals who were:
-Alcoholics.
- Tobacco users.
-Denture wearers.
Were excluded.
2.1.5 Ethical Considerations:
- Ethical clearance & approval from the Postgraduates Board of the Faculty of
Dentistry-University of Khartoum, and the administrators of Jaber Abu EL
Izz Diabetic Centre and EL Ribat University Hospital were received.
- The study protocol was explained for the subjects involved and an informed
consent was obtained.

2.1.6 Salivary sample collection for biochemical analysis:


1- Participants were instructed not to brush their teeth, eat or drink for 2 hours
before the time of saliva collection.
39

2- The study subjects were instructed to sit in the upright

position in a quiet

and cool isolated room.


3- Unstimulated whole saliva samples were used for salivary estimations.
4- Subjects were asked to spit out or swallow saliva that was already present in
the mouth.
5- Salivary sample collection was performed in the morning between 8:0011:00 a.m.
6- Saliva was collected using the spitting method for at least 5 minutes.
7- Saliva collected in the initial 30 seconds was discarded.
8- Samples were collected in an ice-chilled graduated saliva container.
9- One milliliter of each unstimulated saliva sample was centrifuged at 3,000
rpm for 20 min.
10- The clear supernatant was processed immediately for estimation of glucose,
amylase, total protein and salivary urea using a semiautomatic analyzer (Biotron
BTR 830).
2.1.7 Materials used:
1- Kit for glucose estimation (Glucose oxidase, End point assay).
2- Alpha-Amylase Kit (Direct Substrate Method, Kinetic Enzymatic).
3- Microprotein Estimation (Pyrogallol Red Dye, End point method).

4-PCR Real time Kit for Candida albicans of Primer Design Lt. Company (U.K).
40

2.1.8 Salivary glucose estimation:


-Salivary glucose estimation was performed using the glucose oxidase end-point
method.
-1,000 l of reagent solution was pipetted into each of 3 test tubes labeled Blank,
Standard and Test.
- 10 l of standard was added to the test tube marked as Standard, followed by 10
l of test sample to the Test test tube.
-The solutions were mixed well and all the test tubes were kept in an incubator at
37C for 10 min before aspiration.
- Reagent blank was first aspirated in the analyzer, followed by standard solution,
for which the reading was noted.
-Finally, the test sample was aspirated and the reading was noted.
-Results were calculated and values were expressed as milligrams per deciliter
(mg/dl). (T. 2.4).
2.1.9 Salivary amylase estimation:
-Salivary-amylase estimation was performed using the direct substrate kinetic
enzymatic method.
-The mean absorbance change per minute (A/min) was calculated in terms of units
per liter (u/l). (T. 2.5).

41

2.1.10 Salivary total protein estimation:


-Salivary total protein estimation was performed using the pyrogallol red dye.
-Results were calculated and values were expressed in terms of milligram per
deciliter (mg/dl). (T. 2.6).
2.1.11 Salivary urea:
-Salivary urea was measured by diacetyl monoxime method.
-Results were calculated and values were expressed in terms of milligram per
deciliter (mg/dl). (T. 2.7).

2.1.12 Statistical analysis:


-Study variables were statistically analyzed using the SPSS 11:00 software package.
- Values are expressed as means standard deviation (SD) and P (probability)
values of 0.05 were considered to be significant with a 95% confidence interval.
- Diabetic groups were compared with the healthy non- diabetic group using
Dunnetts d-test.
- Differences among uncontrolled and controlled diabetic groups were analyzed by
Z-test.

42

2.2 Study (2): Salivary Candidal Counts -Methodology


2.2.1 Saliva sampling and assessment of salivary yeast count:
-Saliva sampling for estimation of colony-forming units (CFUs) of Candida was
performed using the oral rinse technique.
-All subjects rinsed their mouth for 60 seconds with 10 mL sterile phosphatebuffered saline (PBS, pH 7.2, 0.1 mol/L).
-Each subject returned the rinse into a sterile container.
- The rinse was immediately centrifuged at 1,700 rpm for 10 minutes.
- The supernatant was removed and the deposit was re-suspended in 1 ml of PBS.
- Samples were stored at -20C prior to DNA extraction.
- Confirmation of yeast identity was achieved with the API 32C system
(BioMerieux).
2.2.2 Extraction of DNA from concentrated rinse samples.
-DNA was extracted from 950 ul of CRC concentrate and spiked control samples
by the use of the QIAamp DNA Mini kit (Qiagen, Crawley, United Kingdom).
-The extracted DNA was resuspended in 50 micro l of Tris-EDTA buffer.
-This template DNA was then used for estimation of salivary Candidal counts by
PCR Real time.
2.2.3 Detection and estimation Candida albicans counts by PCR Real Time:
-We used the primer designed by the Primer Design Ltd. Company. (U.K).
43

-A reaction mix was prepared according to manufacturer instructions for the


standard cure well as the followings:
Component

Volume

2 x Precision TM Master Mix

10 l

Pathogen Primer/Probe Mix

1 l

Internal extraction control Primer/Probe Mix

1 l

RNAse/DNAse free water

4 l

Final Volume

15 l

Then a 15 l was pipetted into each well according to real-time PCR experimental
plate set up.
- RNAse/DNAse free water has been prepared in the concentration of (10ul of
sample DNA and 190ul of water),
- An moment of 5l of this preparation was pipetted in to the wells.
-The final volume in each well is 20 l.
-A series of standard curve dilution were done in the following manner:
1) 900ul of RNAse/DNAse free water pipetted into 5 tubes and labeled 2-6.
2) 100ul positive control Template pipetted into tube 2.
3) The volume was vortexed thoroughly.
4) Pipette tip was changed and 100ul pipette from tube 2 into tube 3
44

5) The volume was vortexed thoroughly.


-Steps 4 and 5 were repeated to complete the dilution series.
Standard Curve

Copy Number

Tube 1 Positive control

2 105 Per l

Tube 2

2 104 Per l

Tube 3

2 103 Per l

Tube 4

2 102 Per l

Tube 5

20 Per l

Tube 6

2 Per l

-Finally a 5 l standard template was pipetted into each well, according to the
experimental plate set up. Making the final volume in each well 20l
Amplification Protocol:
The amplification protocol proceeded according to the table below:

50 Cycles

Step

Time

Temp

Denaturation

10s

95 C

DATA COLLECTION

60s

60 C

45

2.2.4 Statistical analysis:


- Statistical analysis was performed using the Statistical Package for the Social
Sciences (SPSS version 11) software.
-Differences in means between groups were assessed using Mann-Whitney U test
-Relationships between variables were evaluated by Pearson correlation coefficient.
A P value of .05 was considered to be statistically significant.

46

CHAPTER THREE
3.1 : Results of The Biochemical Analysis of Saliva
3.1.1 Salivary glucose:
-Means salivary glucose level were:
-Group (1): The uncontrolled diabetics: 8.096.45 mg/dl.
-Group (2): The controlled diabetics:

7.646.44 mg/dl.

-Group (3): The healthy non-diabetic subjects: 1.89 1.44 mg/dl.


- Mean salivary glucose levels were higher in the uncontrolled and controlled
diabetic groups than in the healthy non-diabetic group. T. 2.4.
- The difference between group (1) & (3) was highly significant. P < 0.01. T. 2.8.
- The difference between group (2) & (3) was highly significant. P < 0.01. T. 2.8.
- No significant difference was found between group (1) & (2). P > 0.05. T. 2.8.
3.2.1 Salivary amylase:
-Means salivary amylase level were:
-Group (1): 108.486.37 .u/ml.

T. 2.5.

-Group (2): 100.8360.77 u/ml. T. 2.5.


-Group (3): 146.72 10.70.u/ml. T. 2.5
- No significant difference was found between group (1) & (3). P > 0.05. T. 2.9.
- No significant difference was found between group (1) & (2). P > 0.05.T. 2.9.
-The difference between group (2) & (3) was significant. P < 0.05. T. 2.9.

47

3.3.1 Salivary total protein:


-Means salivary total protein level were:
-Group (1): 90.0144.22 mg/dl.

T. 6.

-Group (2): 88.9149.71 mg/dl.

T. 6.

-Group (3): 98.25 49.59 mg/dl. T. 6.


- No significant difference was found between group (1) & (3). P > 0.05. T. 2.10.
- No significant difference was found between group (2) & (3). P > 0.05. T. 2.10.
- No significant difference was found between group (1) & (2). P > 0.05. T.2.10.
3.4.1 Salivary urea:
-Means salivary urea level were:
-Group (1): 193.8 mg/dl. T. 2.7.
-Group (2): 189.0 mg/dl. T. 2.7.
-Group (3): 9.7 1.4 mg/dl. T. 2.7.
-The difference between group (1) & (3) was significant. P < 0.01. T. 2.11.
-The difference between group (2) & (3) was significant. P < 0.01. T. 2.11.
- No significant difference was found between group (1) & (2). P > 0.05. T.2.11.
3.5.1 Duration of diabetes mellitus:
-No statistically significant difference was noted between the uncontrolled and
controlled diabetic groups in relation to the duration of diabetes mellitus.
P>0.05. (T.2.3).
48

(Table 3.1) Groups and gender:


Variables

According to
gender (Chisquare-test, P
value=0.12-NS)

Male (n)
Female (n)

Group 1 (40)
Uncontrolled
Diabetic
group
21
19

Group 2 (40)
Controlled
Diabetic
group
23
17

Group 3 (40)
Healthy nondiabetic
group
20
20

P value, probability value; SD, standard deviation; NS, not statistically significant

(Table 3.2) Mean age of the study groups:


Variables

According to Age
(ANOVA test P
value=0.32-NS)

Group 1 (40)
Uncontrolled
Diabetic
group
Mean SD
48.507.86
(Age range in
years)

Group 2 (40)
Controlled
Diabetic
group

Group 3 (40)
Healthy nondiabetic
group

49.5010.88

46.1210.25

P value, probability value; SD, standard deviation; NS, not statistically significant

(Table 3.3) Duration of diabetes mellitus:


Variables

Duration of
diabetes mellitus

Group 1 (40)
Uncontrolled
Diabetic
group
08

Long duration
diabetics
Short duration 32
diabetics

Group 2 (40)
Controlled
Diabetic
group
07

Group 3 (40)
Healthy nondiabetic
group
-

33

P value, probability value; SD, standard deviation; NS, not statistically significant
49

(Table 3.4) Mean levels of salivary glucose:


Parameter
Salivary glucose (mg/ml)

Group

Mean Sd

Uncontrolled
group
Controlled group
Healthy group

8.096.45
7.646.44
1.891.44

Mg/dl, milligram per deciliter; u/mL, units per milliliter;

(Table3.5) Mean levels of salivary amylase:


Parameter
Salivary amylase (u/ml)

Group

Mean Sd

Uncontrolled
group
Controlled group
Healthy group

108.486.37
100.8360.77
146.7210.70

Mg/dl, milligram per deciliter; u/mL, units per milliliter;

(Table 3.6) Mean levels of salivary total protein:


Parameter
Salivary total protein (mg/dl)

Group

Mean Sd

Uncontrolled
group
Controlled group
Healthy group

90.0144.22

Mg/dl, milligram per deciliter; u/mL, units per milliliter;

50

88.9149.71
98.2549.59

(Table3.7) Mean levels of salivary urea:


Parameter
Salivary urea (mg/dl)

Group

Mean Sd

Uncontrolled
group
Controlled group
Healthy group

193.8
189.0
9.71.4

Mg/dl, milligram per deciliter; u/mL, units per milliliter;

(Table 3.8) Intergroup comparisons of mean levels of salivary glucose:


Parameter

Intergroup
comparisons

Salivary glucose
(mg/dl)

Uncontrolled
group
With healthy
group
Controlled group
With healthy
group

Dunnett
D test P
value
0.00
S

Intergroup
comparisons

Z-test
P value

Uncontrolled group

0.75

With controlled
group

NS

0.00
S

S, statistically significant; NS: not statistically significant

(Table 3.9) Intergroup comparisons of mean levels of salivary amylase:


Parameter

Intergroup
comparisons

Salivary amylase
(mg/dI)

Uncontrolled
group
With healthy
group
Controlled group
With healthy
group

Dunnett
D test P
value
0.10
NS
0.04
S

S, statistically significant; NS: not statistically significant


51

Intergroup
comparisons

Z-test
P value

Uncontrolled group

0.65

With controlled
group

NS

(Table3.10) Intergroup comparisons of mean levels of salivary total protein:


Parameter

Intergroup
comparisons

Salivary total
protein (mg/dl)

Uncontrolled
group
With healthy
group
Controlled group
With healthy
group

Dunnett
D test P
value
0.66
NS

Intergroup
comparisons

Z-test P
value

Uncontrolled
group
With controlled
group

0.91
NS

0.59
NS

S, statistically significant; NS: not statistically significant

(Table 3.11) Intergroup comparisons of mean levels of salivary urea:


Parameter

Intergroup
comparisons

Salivary urea
(mg/dl)

Uncontrolled
group
With healthy
group
Controlled group
With healthy
group

Dunnett
D test P
value
0.03
S
0.035
S

S, statistically significant; NS:not statistically significant

52

Intergroup
comparisons

Z-test
P value

Uncontrolled group

0.70

With controlled
group

NS

3.2 Study (2) :Results of the Salivary Candidal Counts


-The mean Candidal forming units (CFU) in group (1) was: 8,257935.0 CFU/mL.
- The mean CFU in group in group (2) was: 1,610250.0 CFU/mL.
- The mean CFU in group in group (3) was: 41075.0 CFU/mL.
- CFU was significantly higher in diabetic subjects than in non-diabetic subjects.
P <0.05. (T. 3.1).
-CFU of Candida showed a positive correlation with salivary glucose level.
(r= 0.519; P.<0.0.1 Fig. 3.1)
(Table 3.12) Mean colony-forming units (CFU) of the study groups (MannWhitney
U test):

Group
G1 (n 40)
G2 (n 40)
G3 (n 40)

MeanSD
CFU
8,257 935.0
1,610250.0
41075.0

P value
.000
.000
.000

53

Fig (3.1): Positive correlation between candidal forming units and salivary
Glucose level.

54

CHAPTER FOUR
Discussion
4.1 THE BIOCHEMICAL ANALYSIS OF SALIVA
Diabetes mellitus is a group of metabolic disorders that share the common
underlying feature of hyperglycemia. Hyperglycemia in diabetes results from
defects in insulin secretion, insulin action, or most commonly from both f them.
Chronic hyperglycemia and the attendant metabolic dysregulation may be
associated with secondary damage in multiple organ systems, especially the
kidneys, eyes, nerves, and blood vessels (158).
The disease is the fifth most common cause of death in the world and it is
estimated that one in eight deaths (12.2%) among adult individuals were
attributable to the condition (4).
Reports in 2011, showed that 366 million people worldwide were affected by
diabetes and the number is continuing to rise steeply and by the year 2030,
predictions suggest that the number of people with diabetes will reach 552 million
(159) . In Sudan the prevalence of the disease has risen dramatically from 3.4% at

55

the Northern part of the country in1996 (16) to 14.5% with a prevalence rate of
19.2% at Khartoum state in 2006 (17).
It has been shown that a wide spectrum of oral manifestations of DM has been
reported. These manifestations included: Xerostomia , Periodontal diseases ,
Dental caries , Taste impairment , Sialosis , Fungal infections, Lichen planus
,Geographic & Fissured tongue (160).
According to our knowledge there is only one study carried on diabetes mellitus in
relation to oral health in Sudan published by Kamil and Ghandour in 2013 (161).
This study investigated the periodontal health of diabetics compared to nondiabetics at Khartoum University Clinic. The authors found diabetics were at a
higher risk to develop periodontitis compared to non-diabetics.
The present study was the first one in the country investigating saliva composition
and candidal counts among type- 2 diabetic patients. The study was conducted at
Jabir Abu EL Izz Diabetic Centre Khartoum in the period May to August 2010.
4.1.1 SALIVARY GLUCOSE:
In the present study, it was found that the salivary glucose values were higher
among diabetics than in the controls. (Mean level among controlled diabetics:

56

7.64mg/dl, uncontrolled diabetics: 8.09mg/dl., non-diabetics: 1.89mg/dl), and the


difference was statistically highly significant . (P < 0.01).
Carlson et al. (162) reported the presence of sugar in saliva of diabetic patients
and other authors have reported increases in salivary glucose levels in patients with
diabetes mellitus in comparison to non-diabetics (163, 164). However, Forbat et al
(133) concluded that salivary glucose levels did not reflect blood glucose levels.
Similarly, Carda et al (165) concluded that the salivary glucose levels of the
majority of diabetic patients were in the normal range.
On the other hand many studies (166,167,168) showed a positive correlation
between salivary glucose and serum glucose. However, other trials (169, 133)
failed to establish a correlation between salivary and serum glucose. Even
Englander et al (163) expressed doubt regarding replacement of plasma with
parotid secretion in the diagnosis of diabetes mellitus, because of its lower levels
of glucose concentration. However, Mitsumori et al (170) manufactured a saliva
analyzing system using a glucose sensor and performed an in vivo evaluations,
concluding that this device of salivary glucose measurement could be used as an
indicator of blood glucose level.
It is known that glucose is present in the saliva of normal individuals; however, the
mechanism of its secretion is still obscure. Both paracellular and intercellular
57

pathways have been proposed (171), but this is still a hypothesis rather than an
established theory. Many authors have tried to explain the increased glucose
content in the salivary secretion of diabetic patients. Lopez et al (172) tried to show
that the salivary glands act as filters of blood glucose that are altered by hormonal
or neural regulation. And according to Qureshi et al (173) persistent hyperglycemia
leads to microvascular changes in the blood vessels, as well as basement
membrane alteration in the salivary glands. This leads to increased leakage of
glucose from the ductal cells of the salivary glands, thereby increasing the glucose
content in saliva. Sreedevi et al (168) showed that glucose is a small molecule that
easily diffuses through semi permeable membranes. Thus, large amounts of
glucose become available to saliva when blood glucose levels are elevated, as in
diabetes. Alterations in the permeability, occurring as a result of basement
membrane changes in diabetes, may be an a additional explanation for the
increased concentration of glucose in saliva.
On the other hand, Belazi et al (166) proposed that the increased permeability of
basement membrane in patients with diabetes mellitus may lead to enhanced
leakage of serum-derived components into whole saliva via gingival crevices. The
small glucose molecule can easily diffuse via the semi permeable basement
membrane. Therefore the authors blamed the gingival crevicular fluid as the culprit
for increased glucose levels in salivary secretion.
58

Depending on those different suggestions, it is likely that the presence of glucose


in saliva is multi-factorial and no single mechanism is to be blamed.
A recent study by Abikshyeet et al in 2012 (174) formulated equations which can
predict the fasting serum glucose level and HbA1c percentage if the fasting
salivary glucose level is known & hence stated this equation:
Serum glucose = 99.664 + 13.027 salivary glucose (patient)
HbA1c = 6.762 + 0.249 salivary glucose (patient)
The authors concluded that fasting salivary glucose level can be used as a
noninvasive diagnostic, as well as a monitoring tool to assess the glycemic status
of diabetes mellitus patients. However further studies on larger populations and in
different geographic areas are needed to establish salivary glucose estimation as a
diagnostic as well as a monitoring tool for diabetes mellitus.

59

4.1.2 SALIVARY AMYLASE AND SALIVARY TOTAL PROTEIN:


The study showed insignificant result when the salivary amylase levels of the
uncontrolled diabetics were compared with that of

the non-diabetics. (P >0.05).

However a significantly lower mean salivary amylase levels in the controlled


diabetics when compared to the healthy non-diabetics was detected.( P <0.05) .
(Mean

level

among

uncontrolled

diabetics:108.48u/ml.controlled

diabetics:100.83u/ml, non-diabetics:146.72u/ml). This is in agreement with what


was reported by Yavuzilmaz et al. (175) , who attributed them to the hormonal and
metabolic changes occurring in diabetic patient.
In contrast, significant increases in salivary amylase levels have been demonstrated
in diabetics according to earlier reports (176, 177).
The importance of amylase in the adhesion of microorganisms remains uncertain,
although several studies have supported amylase as a potential factor in
streptococcal adhesion to teeth and in plaque formation (178).
Pal et al. concluded that higher glucose concentrations leads to the proliferation of
microbes and high levels of amylase enhance accumulation of plaque with
consequent rises in total bacterial count in diabetics (176).

60

With regard to salivary total protein, the present study results revealed similarity to
many previous studies (136, 179, 177), which showed no significant differences
were detected between diabetics & non diabetics. [Mean level among controlled
diabetics:88.91mg/dl., uncontrolled diabetics: 90.01mg/dl., non- diabetics:
98.25mg/dl. P.>0.05]. But recent studies have reported higher salivary total protein
levels in diabetics (176, 180) ,while Streckfus et al. (181) estimated significant
lower protein concentrations in diabetics and emphasized protein utilization by other
biochemical metabolic pathways as an overall systemic response to glucose intolerance.
Also Insulin is known to have the potential to alter protein metabolism (177).

In the extensive study made by Dodds et al on the salivary alteration in type 2


diabetic patients, an elevated concentration of certain salivary protein components
in the diabetic subjects was found. Namely the concentrations of lactoferrin,
secretory IgA, Myloperoxidase and Salivary peroxidase were all higher in diabetics
than healthy controls.(157).
Increased leakage of proteins through basement membranes affected by diabetes
may be responsible for the elevated concentrations of certain proteins, and these
mechanisms have been suggested by some authors (182). Lactoferrin levels in
parotid saliva have been linked to acute and chronic episodes of recurrent parotitis
(183). According to Ortiz et al, (184), the salivary Myeloperoxidase produced by
infiltrating polymorphonuclear leukocytes raising the possibility that many of the
61

diabetics had a low grade infection of their parotid glands. Although it was not
evident that diabetics had elevated concentrations of albumin in their saliva,
albumin is substantially increased, like lactoferrin, with acute inflammation of the
salivary glands as reported by Mandle et al (185) & Tabak et al (183), suggesting
that low grade infection of the salivary glands causing increased leakage of serum
proteins into saliva might be a common finding in diabetics.
4.1.3 SALIVARY UREA:
The average value of urea in the saliva of the non-diabetic group was 9.7 mg/dl.
The mean value of urea in the saliva of patients with controlled diabetes was 19.0
mg/dl, and the mean value of urea in the saliva of the uncontrolled diabetic patients
was 19.0 mg/dl ,showing a significant difference.( P < 0.05) between diabetics &
non-diabetics.
This result is in consistent with Carda et al (165) & Ivanoviski et al (186) studies.
This elevation in salivary urea among diabetic patients could be due to the nature
of diabetes as a metabolic disease causing disruption of metabolic processes in the
human body, so it is likely that serum levels of urea in patients with diabetes are
elevated. These increased levels of serum urea in patients with diabetes may also
be due to the dietary regimen and the increased intake of protein among those
patients. Another factor to be considered is that the increased permeability of
62

acinar cells in the salivary glands of patients with diabetes may allow enhanced
ultra filtration of blood.
4.1.4 DURATION OF DIABETES MELLITUS:

In this study, there was no significant finding with regard to the duration of
diabetes mellitus and salivary glucose level. (P >0.05). No correlations were
previously found between salivary glucose levels and disease duration, according
to many studies (187, 129), Although few studies reported that with increased
duration of diabetes, glucose values in saliva decreased as a result of fatty
infiltration in the acini and diabetic microangiopathy of salivary glands (176, 188).
It is of interest to notice that the alterations in saliva composition in diabetic
subjects seen in the present study might be due to the presence of diabetes induced impairment of salivary gland function. Similar findings have also been
previously described in the literature and were associated with diabetes inducedneuropathic changes in the salivary parenchyma with Iymphocythic gland infiltrate
similar to the one occurring in the pancreas of these diabetic patients (189).
The above mentioned alterations in saliva composition may well explain the
pathogenesis of oral lesions seen among

diabetics such as xerostomia,

periodontitis & candidosis. These alterations, according to Tayler et al (190) were


secondary to the persistent hyperglycemia occurring in D.M .The authors pointed
63

that the so called

non-enzymatic glycosylation, which has recently attracted

increasing interest, appeared to play a crucial pathophysiologic role behind

the

development
Of diabetic complications. Proteins and lipids exposed to aldose sugars go through
reactions which are not enzyme-dependent reactions . Later, irreversible advanced
glycosylation end products (AGEs) are formed. This process also takes place
during normal ageing, but in diabetes their formation is accelerated to an extent
related to the level and duration of

hyperglycaemia (191, 192). The potential

pathophysiological significance of AGEs is associated with their accumulation in


plasma, cells and tissues and their contribution to the formation of cross-links,
generation of reactive oxygen intermediates and

interactions with particular

receptors on cellular surface (193).


AGEs have direct effects on the host response by affecting tissue structures, e.g. by
increasing collagen cross-links, which is followed by changes in collagen solubility
and turnover

(194). Thickening of basement membranes is partly due to

glycosylation of membrane proteins or entrapment of glycosylated serum proteins


into basement membranes (195).
AGEs are bound to the specific cell surface receptors for AGEs, within which
family of receptors RAGE is the most well defined and resembles macrophage
scavenger receptors.
64

This interaction results in oxidant stress of the target cells, inducing production of
different patterns of cytokines and growth factors depending on the type of cells
involved. Excess production of growth factors and cytokines plays an essential
role in both micro- and macrovascular alterations. AGEs, by themselves, appear to
generate reactive oxygen intermediates and the interaction between AGE and
RAGE further induces production of intra- and extracellular oxidants.
Oxidative modifications of lipoproteins, in turn, accelerate atherogenesis (196).
Free oxygen radicals cause tissue destruction directly and exaggerate the
inflammation related tissue destruction because activated monocytes produce
proinflammatory cytokines, such as IL-1a, IL-6 and TNF-a.
In conclusion, hyperglycaemia, either directly or through AGE formation, causes
various structural and functional modifications of cells as well as quantitative and
qualitative alterations of the extracellular matrix, which may modify the host
response and alter all tissues homeostasis including the oral tissues.

65

CHAPTER FOUR

Discussion
4.2 SALIVARY CANDIDAL COUNTS
Candidosis is a superficial opportunistic infection, essentially facilitated by local
and systemic predisposing factors. It is the most common mycosis of the oral
cavity in both healthy & Immunosuppresed persons (197).
One reason for commonality of this disease is probably because 4060% of
healthy adults harbor commensal Candida in the oral cavity, without any signs or
symptoms of candidosis (198). Candida species have been frequently isolated from
the oral cavities of patients with diabetes mellitus (DM) (199) and it has been
reported that up to 77% of diabetic patients harbor oral Candida (200). A number
of candidal species could be recovered from the oral cavities of DM patients, with
C. albicans representing the most commonly recovered species, in more than 80%
of diabetic patients (155).
Most of the previous studies on salivary microbial counts focused on the bacterial
counts and controversial results were reported. Sandholm et al (201) reported more
gram-negative rods and a higher proportion of total gram-negative bacteria in
samples of patients with diabetes than in control samples. Later studies have not
conclusively supported this, as they have revealed no significant differences in
66

microbial species (202). However, a depressed humoral immune response among


diabetics was suggested by Smith et al. (1996) (203), who detected lower IgG
antibody titers against Porphyromonas gingivalis and Bacteroides forsythus in
sera of diabetic patients compared to controls.
It is well realized that acidogenic bacteria (mutans streptococci & lactobacilli)
were the commonest bacteria that have been estimated among diabetics, but most
studies have not detected

differences in these bacterial counts (136,204).

However, Swanljung et al. (1992) (205) reported high counts of

mutans

streptococci and lactobacilli in diabetic patients than in controls.


The present study focused on the estimation of Candida albicans among type 2
D.M patients compared to a healthy control group.
In this study the oral rinse sampling method was used. According to Samaranayke
(206) it is the most appropriate and sensitive technique for evaluating the overall
oral yeast carriage.
In the present study the real-time polymerase chain reaction (RT.PCR) was used.
To the best of our knowledge this is the first study that utilized this technology in
the estimation of candidal counts in the saliva of diabetic patients.
RT.PCR uses fluorescent reporter molecules to monitor the production of
amplification products during each cycle of the PCR reaction. This combines the
DNA amplification and detection steps into one homogeneous assay and obviates
67

the need for gel electrophoresis to detect amplification products. Appropriate data
analysis and/or use of opposite chemistries also eliminate the need for Southern
blotting or DNA sequencing for amplicon identification.
According to Stephen (207) the technique is simple, specific & sensitive. Its
reliable instrumentation, and improved protocols, has made it the benchmark
technology for the detection of DNA.
In the present study, Candidal CFUs were significantly higher in diabetic subjects
(group I & group I I) compared with non- diabetic subjects (group III). [Mean CFU
among uncontrolled diabetics: 8.200CFU/ml, controlled diabetics: 1.600CFU/ml,
non-diabetics: 400CFU/ml]. This is similar to the findings of Dodds et al & Jones
et al studies (157,208). There was a significant positive correlation(r=0.519,
P<0.05) between salivary glucose and CFU of Candida in the overall study
population, in line with the observation of other investigators that increased
Candida reflects increased salivary glucose levels (199,209).
Varying rates of oral carriage of Candida in diabetic patients ranging from 18 to
80% , were reported in literature since 1958.Some of these studies have used crude
estimates for the degree of glycosuria and the measurement of blood glucose, to

68

delineate the diabetic patients and the control subjects. Furthermore, variations in
the therapeutic regimens from one study to another were present, as some patients
were controlled by insulin whereas others were controlled by oral hypoglycaemic
agents or diet alone.
Aly et al (210) found an increased oral carriage of yeasts associated with plasma
glucose level in patients with type 2 D.M. However, Fisher et al (155), Manfredi
et al (160), failed to show such an association. Interestingly Al-Karaawi et al (211)
observed lower rates of candidal counts among diabetics.
Many reasons seem to exist behind the differences in the results of those studies,
ranging from patient selection, inappropriate control subjects, varying and
unsuitable sampling or counting methods. A number of studies have now clearly
shown that the oral rinse sampling method of Samaranayake et al (206), which is
the method adopted in this study is the most appropriate and sensitive technique for
evaluating overall oral yeast carriage compared with imprint culture, swab or
saliva sampling.
Future workers in this field should pay great attention to the sampling as well as
the counting methods and above all , appropriate selection of patient and control in
order to obtain globally comparable data.

69

Denture wearers were excluded in the present study due to the high prevalence of
oral candidal carriage associated with denture wearing, particularly in diabetic
patients. Dentures may act as an additional reservoir for these organisms.
Furthermore, denture-induced trauma may reduce tissue resistance against
infection, thus increasing the permeability of the epithelium to soluble candidal
antigens and toxins, exposing molecules such as fibronectin which act as candidal
receptors (212).
Tobacco users were also excluded because the rate of oral candidal carriage in
tobacco users was higher than in non-tobacco users. (213). In addition to that,
blood glucose levels in diabetic patients who were tobacco users were significantly
higher than in those without this habit. (209), which may be as a result of the effect
of tobacco on adrenaline levels.
Broad spectrum antimicrobial therapy may suppress oral bacteria and cause C.
albicans colonization even in non-diabetic individuals. The present study excluded
patients under medications other than those for diabetes especially those under
antimicrobial therapy.
The mechanism by which diabetes predisposes to high oral carriage of Candida is
not yet established. However, it is widely recognized that high salivary glucose
levels in diabetic patients favor yeast growth. (129).
70

Yeast adhesion to epithelial cell surfaces is recognized as an essential first step in


the process of candidal colonization and subsequent infection. The nature of the
host cell receptors is also important for candidal species to adhere to the epithelial
surface. Salivary glucose may form chemically reversible glycosylation products
with proteins in tissues during hyperglycaemic episodes (214). It is possible that
accumulation of such glycosylation products on buccal epithelial cells (BEC) may
increase the number of available receptors for Candida . The fact that Candida
adherence to BEC from diabetic patients is greater than in the control individuals
implies that the high levels of glycogen in the former may play a role in this
process.
A second factor to be considered behind the high candidal counts encountered in
the present study is the fact that salivary flow rates and pH values of diabetic
patients were significantly lower than non-diabetic control subjects (210).
Therefore, it is likely that a decrease in salivary flow rate consequent to diabetes
may further enhance candidal colonization due to the reduction in secretory
immunoglobulin A (sIgA) and free secretory component (SC) which inhibits
Candida cell adhesion to the epithelial cells.
A third host factor which may promote the oral carriage of Candida in diabetes is
the possible defects in candidacidal activity of neutrophils, particularly in the
presence of glucose (215). For instance, when the Candida killing ability is
71

correlated with the production of superoxide, the polymorphonuclear leucocytes


(PMN) from diabetic patients with candidosis produced less free oxygen radicals
and reduced phagocytosis and intracellular killing of Candida cells (216). Hence,
decreased phagocytosis, intracellular killing, bactericidal activity and chemotaxis
associated particularly with poorly controlled diabetes may render the diabetic
person more prone to candidal infection.

72

CHAPTER FOUR
4.3 CONCLUSIONS

1. Salivary glucose level is higher among diabetic patients in comparison to


healthy individuals.
2. Salivary urea level is also higher among diabetic groups.
3. The study findings are not significantly different when diabetics & healthy
subjects were compared for salivary amylase and total protein.
4. The study showed an increased Candida albicans counts among diabetics in
comparison to non-diabetics.
5. A positive correlation between salivary candidal counts & salivary glucose
level was detected.
6. The simplicity, sensitivity and specificity of Real- Time PCR , made it the
counting method of choice when salivary candidal count is considered.

73

CHAPTER FOUR
4.4 RECOMMENDATIONS

1. Further studies with larger sample size and in different geographic areas are
needed to establish saliva as a diagnostic as well as a monitoring tool for
diabetes mellitus.
2. Other saliva compositions e.g.: electrolytes and hormones, and other
microbial counts e.g. bacteria and other fungal species need to be
investigated.
3. Research centers in Sudan have to focus on saliva-related researches to
highlight its significance as an important diagnostic fluid.
4. Raising public awareness including the medical staff and health workers
about the orofacial manifestations and complications of D.M is highly
recommended.
5. For all wholistic programs in the management of D.M the role of oral health
professionals must be emphasized.
6. The interaction between D.M and oral health is an area of importance that
needs to be investigated comprehensively in the future.

74

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Saliva Composition and Candidal Counts among Type-2


Diabetic Patients
Data Filling Form

Form no. ..
Name:

Gender: .

Age: ..

Address:

Occupation:Tribe

Diabetic status:

FSBHbA1c.

Diabetic

non-Diabetic

If Diabetic:

Controlled

uncontrolled

99

Diabetes mellitus type:

Type 2

Type 1

Duration of Diabetes mellitus:

< 6 years

> 6 years

Social habits: Tobacco user.


Alcoholic.
Denture wearer.
Others.

100

Other

Current medication (including those taken in the past 3 months)


* Antidiabetic medication:
1. Oral hypoglycemic
2. Insulin
3. Others

* Antimicrobial
* Other medication (specify) .

Salivary Parameters investigated:

1. Glucose level

..

2. Amylase level

..

3. Total protein level ..


4. Urea level

..

5. Candida count

..

101