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

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].

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 1^st 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.

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

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

* 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.

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

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.

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

DISCUSSION