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Afro-Egyptian Journal of Infectious and Endemic Diseases
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Meabed, E., Henin, R. (2022). Prevalence of Entamoeba gingivalis and Trichomonas tenax among Patients ‎suffering from Chronic Systemic Diseases in Egypt ‎. Afro-Egyptian Journal of Infectious and Endemic Diseases, 12(4), 390-401. doi: 10.21608/aeji.2022.171351.1264
Eman MH Meabed; Ramy W Henin. "Prevalence of Entamoeba gingivalis and Trichomonas tenax among Patients ‎suffering from Chronic Systemic Diseases in Egypt ‎". Afro-Egyptian Journal of Infectious and Endemic Diseases, 12, 4, 2022, 390-401. doi: 10.21608/aeji.2022.171351.1264
Meabed, E., Henin, R. (2022). 'Prevalence of Entamoeba gingivalis and Trichomonas tenax among Patients ‎suffering from Chronic Systemic Diseases in Egypt ‎', Afro-Egyptian Journal of Infectious and Endemic Diseases, 12(4), pp. 390-401. doi: 10.21608/aeji.2022.171351.1264
Meabed, E., Henin, R. Prevalence of Entamoeba gingivalis and Trichomonas tenax among Patients ‎suffering from Chronic Systemic Diseases in Egypt ‎. Afro-Egyptian Journal of Infectious and Endemic Diseases, 2022; 12(4): 390-401. doi: 10.21608/aeji.2022.171351.1264

Prevalence of Entamoeba gingivalis and Trichomonas tenax among Patients ‎suffering from Chronic Systemic Diseases in Egypt ‎

Article 11, Volume 12, Issue 4, December 2022, Page 390-401  XML PDF (481.76 K)
Document Type: Original Article
DOI: 10.21608/aeji.2022.171351.1264
Authors
Eman MH Meabed; Ramy W Henin email
Department of Parasitology., Faculty of Medicine, Fayoum University.
Abstract
Background and study aim: The Oral cavity harbors many microorganisms and their balance is essential for its ‎health. Entamoeba gingivalis and Trichomonas tenax are the most commonly ‎found oral protozoa. This study aims to explore their prevalence in patients with ‎chronic systemic diseases and the associated risk factors.‎‎‎‎.
Materials and Methods: This case-control study included 150 chronic patients from Fayoum Governorate, ‎Egypt, designated into three groups (50 each). These are; a group of diabetic ‎patients, a group of chronic renal failure on regular hemodialysis, and a group of ‎chronic liver diseases with Child-Pugh Class B and C. In addition to 50 healthy ‎volunteer subjects were enrolled as a control group. A specialized dentist collected ‎dental plaques and saliva samples from all subjects. A designed questionnaire was ‎taken for personal, demographic data, oral risk factors as regular oral hygiene, ‎smoking, halitosis, and history of chronic or recurrent gum and teeth complaints‎. Oral samples were examined using direct microscopy, saline wet mount, and ‎Giemsa staining.  Each sample was cultured on Diamond's medium [TYM] for ‎detection of T. tenax, and on Locke’s-egg medium for detection of E. gingivalis. ‎HbA1c as indicator for diabetic control was measured in sera drawn from cases and controls.‎
Results: The prevalence of oral protozoa was significantly increased in chronic diseases, ‎as E. gingivalis was reported in 80%, 76, and 74% of diabetic, renal, and hepatic ‎groups of patients respectively compared to 20% in the control healthy group. ‎While T. tenax was reported in 70%, 62%, and 64% respectively compared to 16 ‎‎% in the control group < em>‎‎‎‎‎‎‎‎‎.
Conclusion: This high prevalence in chronic systemic disease needs more investigations ‎concerning its pathogenesis, immunological mediators that may affect systemic ‎diseases and the interplay between them‎ ‎‎‎‎‎‎‎.

Highlights
  • The study detected significant increase in prevalence of E. gingivalis, ‎T. tenax in patients with chronic diseases.‎
  • There is significant association of oral protozoa and HbA1c level, ‎gum inflammation, halitosis and dental Prosthesis.‎
  • The most important risk factors are residence, socioeconomic status, ‎literacy, and oral hygiene.‎
Keywords
Diabetic; Entamoeba; trichomonas
Main Subjects
Infectious diseases
Supplementary Files
download figure-1.jpg
Full Text

INTRODUCTION

The oral cavity is the second largest gathering of microorganisms after the colon, it shelters 700 species [1]. Many bacteria, fungi, and protozoa inhabit the mouth cavity in equilibrium. Disruption of this balance can lead to various diseases such as oral thrush, dental caries, gingivitis, and periodontitis.

Entamoeba gingivalis and Trichomonas tenax are the most important protozoa that inhabit the oral cavity of humans, living in the gingival tissues near the ‎base of the teeth without the cyst stage. Some researchers consider them as commensals. However, they are more predominant in persons with oral infections such as gingivitis, and periodontitis as they flourish with conditions of bad oral hygiene, and inflammatory anaerobic and suppurative conditions. Transmission occurs by saliva, droplet spray, close contact, and kissing or sharing eating utensils [2].

Known risk factors for these parasites are identified as smoking, alcohol consumption, diabetes, bad oral hygiene, aging, genetic predisposition, and systemic diseases [3].

Chronic non-communicable diseases such as diabetes, chronic liver, and kidney diseases are major public health problems causing 60% of global deaths and accounts for 82% of all mortality rate in Egypt, and 67% of premature deaths [4].

Chronic liver diseases especially viral hepatitis, and its consequences as cirrhosis are global health problems that affect hundreds of millions of people worldwide. In Egypt, over 6 million people are chronically infected with about 150,000 new cases every year. Other common chronic liver conditions as non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH) [5]. Recent studies have linked these conditions to intestinal dysbiosis. A significant increase in the phylum Proteobacteria was detected in these patients compared to the healthy controls, indicating that there is an association between intestinal microbiota, and hepatic pathology. Interestingly germ-free mice colonized with gut microbes from NAFLD mice developed severe fatty liver, and cirrhosis supporting that gut dysbiosis has a causal effect on liver diseases [6].

This interplay between mouth microbiota causing periodontal diseases, and systemic diseases was investigated regarding stroke, diabetes, atherosclerosis, coronary heart diseases, and chronic kidney diseases as it seems that there is a certain microbiota signature to be associated with the development of these diseases. However, whether these changes are causal factors or effects is still under debate. [7,8].

Diabetes is the most common metabolic disease worldwide and affects 10.7 % of the population in the Middle East and 15.2% in Egypt [9]. Many studies have investigated oral dysbiosis as a contributing factor to distal organ damage caused by diabetes through stimulation of innate immune response which relies on pathogen-associated molecular patterns (PAMPs) leading to the release of proinflammatory mediators that increase the insulin resistance and contribute to distal organ pathology [10].

Renal failure or end-stage renal disease is the fifth leading cause of death in Egypt with a prevalence of 10.6%. The associated uremia, dietary modification, metabolic acidosis, anemia, and electrolyte imbalance contribute to oral dysbiosis with a higher prevalence of gingival hyperplasia, chronic periodontitis, xerostomia, and root canal calcification [11].

Increasing data are accumulating regarding the bacterial component of oral microbiota but few studies discussed the oral protozoa. Its relation to systemic diseases and general health should be investigated and highlighted. The present study aimed to investigate the prevalence of two oral protozoa E. gingivalis and T. tenax in some ‎common systemic diseases found in Egypt and to study the associated risk factors.

SUBJECTS AND METHODS

Subjects:

The sample size was calculated using OpenEpi. The hypothetical power of the study was 80%, and the ratio of cases to control was 1:1. Patients were attendee of Fayoum general hospital and Fayoum health insurance hospital. Samples were collected in the period from July 2022 to October 2022. They were selected after their approval to participate in the study, by careful studying of their hospital case file for assessment of their systemic condition. This included previous recent clinical, imaging, and laboratory examination within the same week of oral sampling. On the other hand, the normal control subjects were selected from healthy University employees and their relatives.  They underwent general clinical examination and routine laboratory examination as glycated HB, liver enzymes, and renal function tests to exclude any general diseases.

Exclusion criteria: patients receiving anti-parasitic or ‎antibiotic treatment for 1 month before the study, Subjects with multiple chronic diseases were excluded.

Study design

This is a case-control study. The study included 200 subjects designated into 3 groups suffering from chronic diseases (50 patients each). These were;  a group of diabetic cases, a group of chronic renal failure on regular hemodialysis, and a group of chronic liver diseases with Child-Pugh Class B and C. In addition, a fourth group of 50 systemically healthy volunteer subjects was enrolled as a control group.

Methods:

A questionnaire sheet was designed to include personal, demographic data as age, sex, residence, and literacy or educational level, where high education means a university degree, any degree between that and illiterate is considered moderate education. The socioeconomic status (SES) of cases was evaluated. in relation to the education and occupational levels  according to Revised Kuppuswamy’s SES Scale [12].

Oral risk factors a regular oral hygiene (subjects gave history about the rate and the regularity of using oral brush per day), smoking, bad oral smell (halitosis), and history of chronic or recurrent gum complaints such as gingivitis, loose teeth, caries, and the use of dental prosthesis .

A blood sample was drawn from each subject and underwent chemical analysis for the level of glycated hemoglobin (HbA1c), using kits (Human Inc.), according to the manufacturer's instructions. HbA1c indicates the average blood glucose level for the last two to three months and measure of glycaemic control in different subjects.

Oral sampling and diagnosis of oral protozoa by staining and culture techniques:

Dental plaques and calculus with saliva were collected by a specialized dentist using a curette in the morning without prior washing or after at least 3 hours after the last oral hygiene and placed in 2 sterile Eppendorf tubes according to [13]. One sample was used for direct microscopic examination and the other was used immediately for cultures.

The 1st sample was preserved in 0.5 ml saline 0.9% and used for wet mount microscopy and Giemsa staining [14]. Microscopic examination were made three times under dry magnification (400x) and then the specimens were fixed with methyl alcohol for 5 minutes and stained with Giemsa stain for 20 minutes at a dilution of 1:20. The preparations were examined at 1000x to detect violet, pear-shaped trophozoites with characteristic morphologic features. T. tenax was identified as a flagellated trophozoite, pear-shaped sized 5-16 μ,‎ and with characteristic movement. Entamoeba gingivalis was differentiated by its size (10-20μ), the presence of a single nucleus with a small central karyosome, prominent pseudopodia, and sluggish movement. Micrometry was done to confirm the size.

For the cultivation of T. tenax,a part from the oral sample was inoculated immediately into Diamond's medium (TYM) [14], TYM is a nutrient broth of trypticase, yeast, and iron to which inactivated horse serum and antibiotics are added] in standard culture tubes (16 by 125 mm) and incubated vertically at 37°C.

For the cultivation of E. gingivalis, another part of oral sample was immediately inoculated on diphasic Locke’s-egg ‎Medium (modified Boeck and Drbohlav’s medium) [15]. After 24 h incubation, ‎A drop of culture media was examined daily for 72 hours by light microscope using a 400X.

Statistical Analysis:

All Data are tabulated and analyzed using SPSS software version 22.0. The sensitivity, specificity, and diagnostic accuracy were calculated. The comparison between each diseased group and the control group was performed using Chi-square test. Comparison between more than 2 groups was done using Kruskal Wallis test. P value ≤ 0.05 is considered significant.

RESULTS

The demographic data of examined groups are shown in table1. The table shows the distribution of cases according their age, sex, residence, educational level, and the SES. The table also shows the related oral risk factors as smoking habit, halitosis, the mouth hygiene level, presence of dental prosthesis, the history of chronic gum inflammation. The level of HbA1c was measured indicating the average blood glucose level for the last two to three months and measure of glycemic control in different groups.

Table 2 illustrates that E. gingivalis was found in 80%, 76, and 74%  of diabetic, renal, and hepatic groups respectively compared to 20% in control group while T. tenax was found in 70%, 62%, and 64% respectively compared to 16 % in control group.

Table (3)compares wet mount and culture in detection of oral protozoa, wet mount microscopy detected 38.5% of E. gingivalis and 31% of T. tenax cases compared to 62.5% and 53% for culture. In addition, when taking culture as a confirmatory test, the wet mount had a sensitivity of only 58.5% - 61.6% and 100% specificity. Figure (1) shows some of the detected organisms during examinations.

Table (4) shows the distribution of cases of diagnosed E. gingivalis in the different studied groups and the relation to different risk factors. The table shows that 80% of the diabetic groups, 76% in the renal group, and 74% in the hepatic group were positive for E. gingivalis compared to 20% in the control group.   E. gingivalis was significantly detected in cases with age range from 41-60 and >60 group in diseased and control group [p value <0.05]. It was significantly detected in renal cases who had rural residence and illiteracy. The parasite was significantly detected in hepatic and renal cases with bad oral hygiene. E. gingivalis was significantly detected in diabetic and renal cases with HbA1c ‎>7.0, ‎who gave history of halitosis and chronic gum inflammation. Factors as literacy, SES, mouth hygiene, having dental prosthesis , halitosis, and history of gum inflammation are significantly associated with infection in all groups.

Table (5) shows the prevalence of T. tenax in different groups and in relation to different risk factors. T. tenax  was  detected  in 70% of diabetic group, 62% of renal group, 64% of hepatic group and16  of control group. In diabetic group, the parasite was significantly associated with HbA1c ‎>7.0, in relation to age more than 40 years old, rural residence, being smoker, halitosis and chronic gum inflammation, and having dental prosthesis .  Diagnosed cases in renal group showed significant risk factors of rural residence, and halitosis. Diagnosed cases in hepatic group showed significant risk factors of being smoker, halitosis, and having dental prosthesis.

Table (1): Demographic data and some studied risk factors among the study groups.

Studied factor

Study groups

Control

Diabetic ‎

Renal ‎

Hepatic ‎

p-value*

Total

N

‎%‎

N

%

N

‎%‎

N

‎%‎

N

‎%‎

Age

20 – 40

15

30

15

30

10

20

5

10

0.340

‎45‎

‎22.5‎

41- 60

17

34

22

44

25

50

20

40

‎84‎

‎42‎

>61

18

36

13

26

15

30

25

50

‎71‎

‎35.5‎

Sex

Male

24

48

23

46

34

68

30

60

0.288

‎71‎

‎55.5‎

female

26

52

27

54

16

32

20

40

‎89‎

‎44.5‎

Residence

Rural

20

40

22

44

10

20

37

74

0.231

‎89‎

‎44.5‎

Urban

30

60

28

56

40

80

13

26

‎111‎

‎55.5‎

Literacy (education)

Illiterate

10

20

20

40

11

22

31

62

0.121

‎72‎

‎36‎

Moderate

28

56

19

38

23

46

12

24

‎82‎

‎41‎

Higher

12

24

11

22

16

32

7

14

‎46‎

‎23‎

Socioeconomic status

Low

14

28

23

46

11

22

30

60

0.112

‎78‎

‎39‎

Moderate

25

50

16

32

27

54

12

24

‎80‎

‎40‎

Higher

11

22

11

22

12

24

8

16

‎42‎

‎21‎

Regular tooth brushing (at least once daily)

Good hygiene

20

40

15

30

18

36

16

32

0.727

‎69‎

‎34.5‎

Bad hygiene

30

60

35

70

32

64

34

88

‎131‎

‎60.5‎

Smoking (current or in last one year)

Smoker

23

46

34

68

6

12

4

8

0.001

‎67‎

‎33.5‎

Non- smoker

27

54

16

32

44

88

46

92

‎133‎

‎66.5‎

Dental Prosthesis

Yes

29

58

34

68

32

64

30

60

0.738

‎125‎

‎62.5‎

No

21

42

16

32

18

36

20

40

‎75‎

‎37.5‎

HbA1c

<7.0

50

100

19

38

50

100

50

100

0.001

‎169‎

‎84.5‎

>7.0

0

0

31

62

0

0

0

0

‎31‎

‎15.5‎

Halitosis

Yes

34

68

46

92

44

88

42

84

0.001

‎166‎

‎83‎

No

16

32

4

8

6

12

8

16

‎34‎

‎17‎

Chronic Gum ‎inflammation

Yes

33

66

37

74

34

68

33

66

0.001

‎121‎

‎60.5‎

No

17

34

13

26

16

32

17

34

‎79‎

‎39.5‎

*Significant differences at p < 0.05, using Kruskal Wallis test.

Table (2): Prevalence of oral protozoa in the study groups.

Studied factor

Study groups

Control

Diabetic ‎

Renal ‎

Hepatic ‎

Total Diseased

total

N

‎%‎

N

%

N

‎%‎

N

‎%‎

N

‎%‎

N

‎%‎

E. gingivalis

Positive

10

20

40

80

38

76

37

74

115

76.7

125

62.5

P value*

 

 

0.001

0.001

0.001

0.001

 

 

T. tenax

Positive

8

16

35

70

31

62

32

64

98

65.3

106

53

P value*

 

 

0.001

0.001

0.001

0.002

 

 

Mixed infections

 

2

4

15

30

7

14

8

16

30

20

32

16

 

P value**

 0.046*

 

 

 

* comparing diseased group with control group using Chi-Square test, Significant difference at p < 0.05.

** Comparing the four groups using Kruskal Wallis Test, Significant difference at p < 0.05

Table (3): Comparison between wet mount and culture in detection of oral protozoa among cases and controls.

 

Wet mount

Culture

Positive

N (%)

Negative

N (%)

Positive

N (%)

Negative

N (%)

E. gingivalis

77(38.5)

123(61.5)

125(62.5)

75(37.5)

Sensitivity 61.60%   

Specificity100.00%   

Positive Predictive Value 100.00%

Negative Predictive Value 87.29%

Accuracy  89.44%

T. tenax

62 (31)

138(69)

106 (53)

94(47)

Sensitivity                             58.49%

Specificity                             100.00%

Positive Predictive Value    100.00%               

Negative Predictive Value 86.4%    

 Accuracy                              88.58%

 Table (4): Prevalence of E. gingivalis in relation to risk factor.

Characteristic

Study group

Control group

Diabetic group

Renal group

Hepatic group

Total diseased

E.gingivalis

Positive

N        %

P ‎value

Positive

N     %

P ‎value

Positive

N       %

P ‎value

Positive

N     %

P ‎value

‎Positive

N     %

P ‎value

Age

20 - 40

1

6.3

0.048

6

40.0

0.001

3

30.0

0.023

1

20.0

0.045

10

22.2

0.038

41- 60 

5

18.5

21

95.5

20

80.0

16

80.0

62

73.8

>60

4

57.1

13

100.0

15

100.0

20

80.0

52

73.2

Sex

Male

6

25

0.489

19

82.6

0.372

24

70.6

0.162

19

63.3

0.548

62

87.3

0.006

Female

4

25.4

21

77.8

14

87.5

18

90.0

53

59.6

Residence

Rural

7

35

0.067

22

100.0

0.125

10

100.0

0.001

30

81.1

0.499

62

69.7

0.001

Urban

3

10

18

64.3

28

70.0

7

53.8

53

47.7

Literacy

Illiterate

6

60

0.041

20

100.0

0.047

10

90.9

0.025

27

87.1

0.046

57

79.2

0.001

Moderate

3

10.7

14

73.7

19

60.9

8

66.7

36

43.9

Higher

1

8.3

6

54.5

9

25.0

2

28.6

12

26.1

Socioeconomic status

Low

4

28.6

0.049

20

87.0

0.035

10

90.9

0.037

30

100.0

0.039

60

76.9

0.004

Moderate

5

20

15

93.8

23

85.2

5

41.7

43

53.8

Higher

1

9.1

5

45.5

5

41.7

2

25.0

12

28.6

Regular tooth brushing

Good ‎hygiene

0

0

0.003

12

80.0

0.037

6

33.3

‎0.001‎

3

18.8

0.004

21

30.4

0.001

Bad ‎hygiene

10

33.3

28

80.0

32

100.0

34

100.0

104

79.4

Smoking

Smoker

7

30.4

0.154

27

79.4

0.028

6

100.0

0.620

4

100.0

0.107

37

55.2

0.124

Non- ‎smoker

3

11.1

13

81.3

32

72.7

33

71.7

78

58.6

Dental Prosthesis

Yes

7

24.1

0.023

30

88.2

0.04

30

93.8

0.032

29

96.7

0.027

89

69.0

0.030

No

3

14.3

10

62.5

8

44.4

8

40.0

26

40.0

HbA1c

‎<7.0‎

10

20

 

10

52.6

0.018

38

76.0

 

37

74.0

 

85

50.3

0.001

‎>7.0‎

0

0

30

96.8

0

 

 

 

30

96.8

Halitosis

Yes

9

26.5

0.038

38

82.6

0.001

38

86.4

0.004

37

88.1

0.001

113

68.1

0.001

No

1

6.3

2

50.0

0

0.0

0

0.0

2

5.9

Gum inflammation

Yes

8

24.2

0.076

31

83.8

0.027

34

100.0

0.001

33

100.0

0.007

98

81.0

0.001

No

2

11.7

9

69.2

4

25.0

4

23.5

17

21.5

Total

 

10

20

 

40

80

 

38

76

 

37

74

 

115

57.5

 

Comparing each diseased group with control group using Chi-square test Significant differences at p < 0.05.

Table (5): Prevalence of T. tenax in relation to risk factors.

Characteristic

Study group

Control group

Diabetic group

Renal group

Hepatic group

Total diseased

T. tenax

Positive

N      %‎

P ‎value

Positive

N    %   ‎

P ‎value

Positive

N     %‎

P ‎value

Positive

N     %‎

P ‎value

Positive

N     %‎‎

P ‎value

Age

20 - 40

0

0.0

0.244

‎4

26.7

0.001

1

10.0

0.002

1

20.0

0.007

6

13.3

0.016

41- 60

4

11.8

‎18‎

81.8

15

60.0

11

55.0

44

52.4

>60

4

11.1

‎13‎

100.0

15

100.0

20

80.0

48

67.6

Sex

Male

4

8.3

0.590

‎17‎

73.9

0.324

21

61.8

0.544

16

53.3

0.061

54

76.1

0.840

Female

4

7.7

‎18‎

66.7

10

62.5

16

80.0

44

49.4

Residence

Rural

5

12.5

0.098

‎1‎8

81.8

0.014

10

100.0

0.001

28

75.7

0.019

56

62.9

0.033

Urban

3

5.0

‎17‎

60.7

21

52.5

4

30.8

42

37.8

Literacy

Illiterate

6

30.0

0.027

‎20‎

100.0

0.023

10

90.9

0.286

24

77.4

0.013

54

75.0

0.030

Moderate

1

1.8

‎12‎

63.2

15

65.2

7

58.3

34

41.5

Higher

1

4.2

‎3‎

27.3

6

37.5

1

14.3

10

21.7

Socioeconomic status

Low

7

25.0

0.047

‎20‎

87.0

0.002

10

90.9

0.051

28

93.3

0.019

58

74.4

0.011

Moderate

1

2.0

‎12‎

75.0

17

63.0

4

33.3

33

41.3

Higher

0

0.0

‎3‎

27.3

4

33.3

0

0.0

7

16.7

Regular tooth brushing

Good ‎hygiene

0

0.0

‎0.001‎

 

‎8

53.3

0.036

3

16.7

0.658

0

0.0

0.009

11

15.9

0.003

Bad ‎hygiene

8

13.3

‎27‎

77.1

28

87.5

32

94.1

87

66.4

Smoking

Smoker

7

15.2

‎0.046

 

‎27‎

79.4

0.020

6

100.0

0.106

2

50.0

0.452

35

52.2

0.057

Non ‎smoker

1

1.9

‎8‎

50.0

25

56.8

30

65.2

63

47.4

Dental prosthesis

Yes

‎5‎

17.2

‎0.043‎

 

‎29‎

73.5

0.036

30

93.8

0.656

24

80.0

0.410

83

66.4

0.001

No

3

7.1

‎7‎

68.8

1

5.6

8

40.0

16

21.3

HbA1c

‎<7.0‎

10

10.0

0.244

‎5‎

26.3

0.001

31

62.0

 

32

64.0

 

68

40.2

0.001

‎>7.0‎

0

0

‎30‎

96.8

0

 

0

 

30

96.8

Halitosis

Yes

8

11.8

0.034

‎35‎

76.1

0.001

31

70.5

0.001

32

76.2

0.004

96

59.0

0.000

No

1

4.0

0

0.0

0

0.0

1

3.7

2

0.0

Gum inflammation

Yes

7

10.6

‎0.028‎

‎33‎

89.2

0.038

27

79.4

0.117

27

81.8

0.016

87

71.9

0.003

No

1

2.9

‎2‎

15.4

4

25.0

5

29.4

11

13.9

Total

 

8

16

 

35

70

 

31

62

 

32

64

 

98

94

 

Comparing each diseased group with control group using Chi-square test Significant differences at p < 0.05.

DISCUSSION

This study aimed to investigate the prevalence of oral protozoa in the ‏most common systemic disease found in Egypt compared to a healthy group and to assess the relationship between ‏these organisms and different risk factors.‏‎ The bacterial component of‎ ‎‏‎ the oral microbiome has been extensively reviewed but the ‎‏protozoal ‎members and their relation to systemic diseases are rarely investigated.‎

Table (1) shows there is no significant association regarding age, sex, residency, literacy, SES, and oral hygiene but the other factors as HbA1c, halitosis and gum inflammation showed significant distribution, partially because of the specific groups selected in this study.

The present study showed that 80% of the diabetic groups, 76% in the renal group, and 74% in the hepatic group were positive for E. gingivalis compared to 20% in the control group (Table 2).  For T. tenax 70%, 62%, 64% and 16% were detected in the studied groups respectively.

Most previous studies were concentrating on the prevalence among oral diseases such as gingivitis and periodontitis [16,17]. A Previous studyreported the prevalence of E. gingivalis as 47.9% among healthy subjects compared to 88.9% among patients with periodontitis, the prevalence was lower for T. tenax  25.6%  in the periodontitis group and 3.2% in the healthy group [18]. Another study reported that the prevalence of E. gingivalis in the periodontitis group ranges from 30% and 80% [19]. Recently, a study that compared the prevalence in patients with chronic diseases attending the university dental clinic in Iraq, found that the prevalence of E. gingivalis and T.tenax was 20%, 18.9%, respectively among diabetics, and 3.3% and 5.4% among patients with renal disease [7]. This wide range may be due to the location of each study, the sample selection, and prior use of antibiotics.

In the present study, wet mount microscopy detected 38.5% of E. gingivalis and 31% of T. tenax cases compared to 62.5% and 53% for culture. Wet mount has 100% specificity and a sensitivity rate of only 58.4% to 61.6 %, which is slightly lower than previous study that reported its sensitivity 64.28% and specificity 97.2%, compared to PCR as a gold standard [14].  For culture, the same authors reported a sensitivity rate of (85.7%), and specificity (97.2%). They reported that wet mount microscopy is the cheapest, quickest, and the most specific when done on a fresh sample for detection of the motile trophozoite [14]. However, wet mount depends on the examiner’s expertise and the immediate transfer to the laboratory and processing the sample. Lugol’s iodine was not used in wet preparation as it kills the trophozoite and causes shape distortion so motile organisms cannot be seen [20].

Culturing is a reliable method; however, it can be affected by many factors as the disintegration of the trophozoite before culturing, the low number of organisms in the sample, absence or presence of chemicals that is essential or harmful to the organisms. A previous study reported that less than 6 trophozoites led to negative culture despite positive PCR [21].

Results of this study showed a significant relationship regarding age for both protozoa with more prevalence in the older group with 67.5% ( > 61 Y.) and 52.4% in (41-60 Y.).  This is in agreement with previous studies that the prevalence of these protozoa increases with age as 35% (60Y) [22]. A study performed on diabetic patients concluded that prevalence increases with age with maximum prevalence in (61 – 70 age group) [7]. There was no significant association regarding sex as described by [23,24].

This study found a significant association of parasitic prevalence in the diabetic group with 80% of diabetics having E. gingivalis and 70%  having T. tenax, Also infection is significantly associated with HbA1c, Mixed infection increased significantly in the diabetic group (30%) compared to other diseased groups.

Results of this study showed that the prevalence of oral protozoa was significantly associated with HbA1c >7 as shown in previous studies [25,26]. ‎ There is a mutual relationship between these parasites and diabetes. Uncontrolled diabetes creates a good environment for microbes in part due to decreased mucosal membrane immunity, poor repair, impaired secretion of saliva or xerostomia, decreased capillary perfusion, and peripheral neuropathy. In addition, increased sugar concentration in saliva makes the dental plaque thicker and sticky enough to allow more organisms to flourish. So oral infections as gingivitis and periodontitis are more prevalent in uncontrolled diabetics as the present study confirmed. On the other hand, the presence of these protozoa can aggravate glycemic control as the associated inflammation can contribute to insulin resistance and distal organ damage. It wasfound that healthy subjects with oral inflammatory conditions have higher HbA1c so have an increased risk to develop diabetes, also diabetic patients with periodontitis have less efficient diabetic control and more incidence of complications than healthy diabetics [27].

The present study showed a significant association between infection and having dental prosthesis for 69% of E. gingivalis and 66.4% for T. tenax. This finding comes in agreement with other studiesthat reported a prevalence of 70.37% and 64.81%, respectively. The two recently emergent diseases Peri-implant mucositis and peri-implantitis which represent gingivitis and periodontitis for natural teeth, these conditions affect 80% and 56% respectively of patients baring dental implants.

The present study showed a significant association between infection and smoking in the diabetic group only.  Smoking is a well-known risk factor for oral and systemic diseases, as it leads to disruption of oral symbiosis through mucosal immunosuppression, decreases salivation, and decreased oxygen and biofilm formation. Most previous studies reported an increased prevalence of oral protozoa as well as oral inflammatory conditions among smokers [29]. Diabetic smokers are at high risk for poor periodontal prognosis than non-smokers [30]. On the contrary, few studies showed no significant association related to smoking. [31]

Halitosis is a common problem affecting 20 – 60 of the population. This study showed a significant association with both E. gingivalis and T. tenax in all tested groups. Oral causes account for 80 to 85% of all cases, etiology includes overgrowth of microorganisms, and oral diseases such as periodontitis, alveolitis, deep carious lesions, tongue biofilm, and candidiasis. Extra-oral causes such as gastroesophageal reflux disease, gastritis, sinusitis, or systemic diseases as diabetes, renal and hepatic patients [32]. Aerobic and anaerobic bacteria were studied as etiological factors,these microorganisms tend to produce volatile sulfur compounds (VSCs) that are responsible for bad odor [33]. Conversion of methionine to VSCs was discovered in E. histolytica and T. vaginalis as they own the gene encoding methionine-γ-lyase (MGL) [34]. Whether oral protozoa can Produce VSCs or not is not yet investigated.

In the present study, E. gingivalis was detected in 76%, and T. tenax was detected in 62% in the renal group.  Chronic kidney disease (CKD) is a global public health problem, affecting 10% of the population worldwide [35]. Patients with chronic renal insufficiency and those on dialysis are more prone to periodontitis and other oral pathology,Studies have shown that chronic gingivitis can adversely affect renal diseases through oxidative stresses and inflammatory cytokines [36].

Among renal group a significant association was found regarding residence, literacy, and oral hygiene. Common findings in this group include dry mouth, taste disturbance, uremic odor, whitish discoloration of the tongue, parotid gland dysfunction, and mucosal inflammation.

Recently, several studies have reported the link between non-alcoholic fatty liver diseases (NAFLD), non-alcoholic steatohepatitis (NASH), and chronic periodontal diseases. It was suggested that 3 months of periodontal treatments in patients with NAFLD led to improvement in liver enzymes [37]. It is thought that chronic periodontitis and the related pathogens release endotoxins and lipopolysaccharides, bacterial DNA, interleukin 1alpha, and various cytokines that lead to systemic inflammatory response aggravating liver diseases (steatosis, fibrosis, and hepatocellular carcinoma) [38,39]. Most fingers point at Porphyromonas gingivalis, a major pathogenic bacteria in periodontitis [40].

CONCLUSION

E. gingivalis was found in 76.7% in chronic disease groups compared to 20% in the control group. T. tenax was found in 65.3% in the study groups compared to 16% in the control group. There is a significant association between oral protozoa and HbA1c level, gum inflammation, halitosis, and dental prosthesis. The most important risk factors are residence, socioeconomic status, literacy, and oral hygiene. This high prevalence in systemic diseases needs to be investigated concerning its pathogenesis, immunological mediators that affect systemic diseases, and the interplay between them.

Acknowledgment: Special thanks go to all hospital staff members and patients who agreed to ‎participate in this study. Great thanks to Dr. Maged Fahmy, a consultant of ‎Dentistry who helped in collecting the samples‎.

Funding: None. Author funded

Conflicts of interest: None.

Ethical consideration:

This work has been done according to the declaration of Helsinki and the sound practice. The present study was approved by the Ethics Committee of the Faculty of Medicine, Fayoum University. Informed consent was taken from each patient prior included in this study and after an explanation of the purpose of the study.

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