J Bagh College Dentistry Vol. 24(3), 2012 Evaluation of

Evaluation of Entamoeba gingivalis and in patients with periodontitis and and its correlation with some risk factors

Sumaiah Ibrahim, B.Sc, M.Sc. (1) Rasha Abbas, B.Sc., M.Sc. (2)

ABSTRACT Background: It was shown that two protozoans, Entamoeba gingivalis and Trichomonas tenax may be responsible for oral parasitic infection. This study was designed to detect these parasites in oral cavity of patients with periodontitis and gingivitis with some risk factors and compare it with healthy oral persons. Material and Method: A total of 60 patients with periodontitis and gingivitis and 25 healthy person (control group) enrolled in the present study. Two samples were collected from each patient, and saliva samples. These samples were stained with Giemsa stain and examined under light microscope, some factors were taken such as PH saliva, age, sex, smoking habits, diabetes mellitus, blood pressure, heart disease and any history of antibiotic consumption during the last six months. Results: This result shown Entamoeba gingivalis and Trichomonas tenax in dental plaque samples was higher than in saliva samples in patients with periodontitis (70%, 60%), (33%,20%) respectively. While in gingivitis it was shown that these two parasites higher in saliva than the dental plaque (60%,46.6%),(46.6%,30.9%)respectively. these two parasites are present in healthy individuals less than in patients with periodontitis and gingivitis . The presence of these protozoans was related to the type of (p=0.0257),sex(p=0.043),age(p=0.0058),PH of saliva(p=0.043) and risk factors (p=0.0168). Conclusion: This study showed Entamoeba gingivalis more common in patients with periodontal disease whereas Trichomonas tenax is that considered a protozoan in oral cavity. There was relationship between the presence of these parasites and the type of periodontal disease and the risk factors. Key words: Entamoeba gingivalis, Trichomonas tenax, periodontitis, gingivitis, risk factors. (J Bagh Coll Dentistry 2012;24(3):158-162).

INTRODUCTION The trophozoites of Entamoeba gingivalis and The oral cavity is suitable for invasion of many Trichomonas tenax are most probably transmitted microorganism, the protozoan Entamoeba from person to person by close contact,since they gingivalis and Trichomonas tenax are recognized exhibit only sligth resistance to the enviroment. eukaryotic representatives. Although these forms Kissing may be play a role in transmission, but are not generally associated with pathogenesis, direct passage by many ways such as mutual their presence in the oral cavity is taken as a sign usages of cups, spoon, fork, and subjects (1,2,3) contaminated by an infected person probably is a of poor dental hygiene . (7) Since mouth infection symptoms derive from the mode of transmission also . This study was interaction between pathogenic microbiota and the designed to detected of these parasites in oral host’s defense mechanisms, it is extremely cavity of patients with periodontitis and gingivitis important to study the microorganisms that cause with some oral risk factors and compare it with periodontitis and gingivitis in human(4). healthy oral persons. Entamoeba gingivalis and Trichomonas tenax were the first commensal foud in human oral MATERIALS AND METHODS cavity, they occure only as a trophozoit, and these Patients are found in gingival tissues, particularly in This study was conducted on 60 patients(39 suppurative, inflammatory processes, due to there (5) males and 21 females)with gingivitis and are preference for anaerobic environments . periodontitis and 25 samples presented a healthy Some authors believe that these commensal could attending the Teaching Hospital of be opportunistic, that these, capable of the College of Dentistry/Baghdad University proliferating in a gingival environment modified (6) during the period first of February to the end of by periodontal and gingivites disease . April 2012. Samples collection and parasitological examination The samples of saliva and dental plaque were (1) Lecturer, Department of Basic sciences, College of Dentistry, University of Baghdad collected from all patients at the morning, in the (2) Assistant Lecturer .Department of Basis sciences, College of first visit for each patient.Saliva samples were Dentistry, University of Baghdad. placed in sterile cup and diluted with normal

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saline at room temperature (25-28C),immediately positive for Entamoeba gigivalis,where18 cases after dilution, these samples were stained with (60%) were from patients with periodontitis, 18 Giemsa stain and examined under a light (60%) were from patients with gingivitis while it microscope (40x,100x,200x,400x)(8) found in 6 cases (24%) in control individuals. The dental plaque samples were collected by Trichomonas tenax was identified in 20 saliva scraping the area with sterile swab rubbed around samples (33.3%), where 6 cases (20%) were from the surface of teeth from caries and around the patients with periodontitis, 14 cases (46.6%) were gingival crevices, the swabs were dipped in sterile from patients with gingivitis and only in 3 cases vials containing normal saline, after that the swab (12%) in control group.There was a significant rolled on the slide, also stained with Giemsa stain correlation between presence of these two and examined under light microscope parasites and type of periodontal disease (χ2 (40x,100x,200x,400x). =4.73,p=0.0257). The parasites of Entamoeba gingivalis were Table (2)showed higher Entamoeba gingivalis identified by their shape depending on the and Trichomonas tenax in males than in expansion of the formation and females,(38.4%,28.5%)(5.1%,0) respectively, so presence of vacuoles while Trichomonas tenax there was a significant correlation between the sex was identified by its flagella and characteristic of the patients and the presence of these protozoan locomotion parasites (χ2 =3.860,p=0.043) Questionnaire for these patients who entered in Regarding the relationship between patients age the present study, this includes: general health, and presence of these protozoan, Entamoeba age, sex, smoking habits, diabetes mellitus, Blood gingivalis was more common in patients age 61 to pressure, heart disease and any history of any type 70 years(60%), whereas Trichomonas tenax was of antibiotic consumption during the last six more common in patients age 31 to months for each patients(most antibiotics used in 40years(28.5%), Both commensals were found in this country). higher rate in age 21 to 30 years(80%) (table Determination of saliva PH 3),There was statistically significant correlation Universal indicator strips (PH 0-14) were used to between the age of patients and presence of these determine the saliva PH and a possible protozoan (χ2 = 10.307, P=0.0058). relationship between PH and presence of the The saliva PH of the participants ranged from 5 study microorganisms. to 7.5,Entamoeba gingivalis was found in higher Statistical analyses rate in PH 6-6.5 (44.4%)while Trichomonas tenax The data were analyzed by the non parametric chi was found in higher rate in PH 7-7.5(5%),Both square test(χ2 test).The significance level was set protozoan were found in higher rate in PH 5- at (p< 0.05). 5.5(100%) (table 4), There was statistically significant correlation between the saliva PH and RESULTS presence of these parasites(χ2 =3.563,P=0.043). Table (1) showed that the microscopical 52 of 60 patients examined in this study examination of the fresh samples revealed a total presented risk factors,9 individuals were negative 35 dental plaque samples(58.3%) were positive for these parasites and 43 were positive, for Entamoeba gingivalis, where 21cases (70%) Entamoeba gingivalis was found in higher rate in were taken from patients with periodontitis,14 patients with hypertension in periodontitis and cases (46.6%) were taken from patients with gingivitis (60%), while the parasite Trichomonas gingivitis and it found in 4cases (16%) in control tenax was founed in higher rate in patients who individuals,19 cases (31.6%)of dental plaque taken antibiotics at last six months (7.6%),Both samples were positive for Trichomonas tenax, parasites were present in higher rate in patients where 10 cases(33.3%)were taken from patients with diabetic (56.2%) (table5),There was with periodontitis and 9 cases (30%)from patients statistically significant correlation between the risk factors of patients and presence of these with gingivitis and 5cases (20%) in control 2 group.As for the saliva samples,36 (60%) were parasites(χ =8.175,P=0.0168).

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Table 1: Detection rate of Entamoeba gingivalis and Trichomonas tenax in dental plaque and saliva samples according to the periodontal status of the patients. Entamoeba gingivalis Trichomoas tenax Clinical status No.of samples

Dental plaque saliva Dental plaque saliva Periodontitis No % No % No % No % 30 21 70 18 60 10 33.3 6 20 Gingivitis 30 14 46.6 18 60 9 30 14 46.6 Total 60 35 58.3 36 60 19 31.6 20 33.3 Control 25 4 16 6 24 5 20 3 12 χ2 =4.73 P=0.0257 P < 0.05 significant

Table 2: Detection rate of Entamoeba gingivalis and Trichomonas tenax in dental plaque and /or saliva samples according to the patients sex. Entamoeba Trichomonas No. of both Sex gingivalis tenax samples No % No % No % Male 39 15 38.4 2 5.1 16 41 Female 21 6 28.5 0 0 10 47.6 Total 60 21 35 2 3.3 26 43.3 χ2 =3.860 P=0.043 P < 0.05 significant

Table 3: Detection rate of Entamoeba gingivalis and Trichomnas tenax in dental plaque and /or saliva samples according to the patients age. Entamoeba Trichomonas Age Both Total gingivalis tenax (years) No. % No. % No. % >10 11 3 27.2 0 0 3 27.2 11-20 9 5 55.5 0 0 3 33.3 21-30 10 1 10 0 0 8 80 31-40 7 1 14.2 2 28.5 3 42.8 41-50 7 3 42.8 0 0 3 42.8 51-60 11 5 45.4 0 0 5 45.4 61-70 5 3 60 0 0 1 20 Total 60 21 35 2 3.3 26 43.3 χ2=10.307 P=0.0058 P < 0.05 significant

Table 4: Detection rate of Entamaeba gingivalis and Trichomonas tenax in dental plaque and/or saliva samples according to the salivary PH. Entamoeba Trichomonas. Both No. of PH gingivalis tenax samples No. % No. % No. % 5-5.5 2 0 0 0 0 2 100 6-6.5 18 8 44.4 0 0 6 33.3 7-7.5 40 13 32.5 2 5 18 45 Total 60 21 35 2 3.3 26 43.3 χ2=3.563 P=0.043 P < 0.05 significant

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Table 5: Detection rate of Entamoeba gingivalis and Trichomonas.tenax in dental plaque and/or in saliva samples according to the risk factors of patients. Entamoeba Trichomonas Risk No. Both Negative gingivalis tenax factor sample sample No. % No. % No. % Smoking 14 5 35.7 1 7.1 5 35.7 3 Diabetic 16 6 37.5 0 0 9 56.2 1 B.pressure 5 3 60 0 0 2 40 1 Herat.Dis 4 2 50 0 0 1 25 1 Antibiotic 13 7 53.8 1 7.6 2 15.3 3 Total 52 21 40.3 2 3.8 19 36.5 9 χ2 =8.175 P=0.0168 P < 0.05 significant

53% (15),but some reports found this parasite much DISCUSSION (3) There are only few reports on the role of oral less (2.17%) . the role of Trichomonas tenax in commensals in the pathogenesis of periodontitis periodontitis and gingivitis is still controversial. and gingivitis despite the high incidence of certain Although a relationship between the increased occurrence of this protozoan and progression of protozoa, such as Entamoeba gingivalis and (15) Trichomonas tenax. In the present study,the result this disease has been demonstrated recently , revealed that the incidence of Entamoeba the precise mechanism of tissue damage is still unknown. However, further studies are necessary gingivalis was higher than the parasite Trichomonas tenax, very similar results were for the complete elucidation of this mechanism. found by other studis (3,4,9,10.11),and the rate of oral Saliva PH ranged from 5.5-7.5, other research suggested that the peak incidence of commensals parasites higher than in control individuals, this (4) also in agreement with many studies(4,7,11).this in salivary samples occurred between 6-6.5 , study indicating that the incidence rate of while other study have not found a relationship between saliva PH and the presence of these Entamoeba gingivalis was (35%), this rate (10) approximately for many studis (4,7,9,12). It has been protozoa . suggested that these protozoans could affect the Regarding gender both parasites were more formation of contribute to the development and common in male, these data are in agreement with those of other studies(7,16)and the difference was progression of periodontal disease. The other researchers state that this protozoans may be statistically. opportunistic, since they are capable of Regarding age, several researchers reported that the presence of oral protozoan increases with proliferating in the microenvironment of the (16,17) mucobuccal fold affected by periodontal disease age as such in this study, where Entamoeba (6). gingivalis was found in higher rate in age group Therefore, if Entamoeba gingivalis helps the (61-70), this finding support the idea that development and progression of periodontitis and Entamoeba gingivalis may play an active role in gingivitis, these diseases increasingly facilitate the the mouth disease, the both parasites were found proliferation of these protozoa, this vicious circle in higher rate in age group(21-30),the parasite could explain the increased incidence of these Trichomonas tenax was found in high rate in age group(31-40),this results are in general agreement microorganisms in the dental plaque and saliva (12,19). samples of patients with periodontitis and with other studies However, statistically gingivitis. A suspension of Entamoeba gingivalis significant correlation between age and presence was spread on the gingival margins of rats of oral commensals was observed in this study. immunosuppressed with (prednisolone acetate), the complexity of the oral environment and the leading to the development of the clinical signs multifactorial nature of the caries lesion, with the and inflammatory process of periodontal disease periodontal disease , requires the cooperation of other disciplines such as microbiology, chemistry much faster than that observed in immunocompetent rats(13) ,these data indicate that and dietetics. immunosuppression may play role in the Other factors that increase the risk of pathogenesis of periodontitis and gingivitis periodontitis and gingivitis disease are diabetes, induced by this commensal. hypertension, smoking, heart disease and The rate of incidence of Trichomonas tenax in antibiotic consumption, in this study, there is this study was a little(3.3%),this approximately to statistically significance between these factors and other researche(14). Prevalence of patients with the presence of these parasite, the reasons may be Trichomonas tenax world wide ranges from 4.0 to that the immunity which reduced in these patients,

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and the presence of these parasite are making disease and healthy population in Sthiraz, Southern facilitates. Iran. Indian J Dent Res 2010; 21(1):89-91. The present study recorded a relationship 12. Onyido AE, Amadi ES, Olofin I, Onwusmma AA, Okoh I, Chikwendu CI. Prevalence of Entamoeba between the presence of Entamoeba gingivalis gingivali and Trichomonas tenax among dental and Trichomonas tenax and these factors. attending Federal School of Dental Technology and In fact, risk factors for periodontitis and gingivitis Therapy Clinic, Enugu, Nigeria. Nature and Science were present in only a few participants, so any 2011; 9 (9):59-62. inference or deliberation on this subject should be 13. Al-Saeed WM. Pathogenic effect of Entamoeba cautious. Other studies did not find relationship gingivalis on gingival tissue of rats. Al-Rafidain Dent J 2003; 3(1):70-73. between the presence oral parasites and these (4,10) 14. Rodriguez DR, Garza-Ramot DM, Gonzalez DF, Mata factors . DB. Entamoeba gingivalis in children attending the The results of this study suggest that Entamoeba Dentistry School in Unal. Intern Assoc Dent Res 2010; gingivalis occur more frequently in the patients 996. with periodontal and gingivitis disease, whereas 15. Athari A, Soghandi L, Haghighi A, Kazemi B. Trichomonas tenax sees the oral cavity as the Prevalence of oral trichomoniasis` in patients with periodontitis and gingivitis using pcr and direct smear. natural habitat given its commonness in individual Iranian J Publ Health 2007; 36 (3) :33-7. with healthy . However, further studies are 16. Vrablic J, Tomova S, Cattar G, Randova L, Suttova S. necessary for determining the real nature of the Morphology and diagnosis of Entamoeba gingivalis relationship between these species and the and Trichomonas tenax and their occurrence in periodontal disease. children and adolescents. Bratisl Lek Listy 1991; 92(5):241-6. 17. Jawad SQ. Frequency of Entamoeba gingivalis and REFERENCES Trichomonas tenax among patients with dental prosthesis-fixed or removable. J of College of Basic 1. Brooks B, Schuster FL. Oral protozoa: survey, Education 2011; 68:97-100. isolation and ultrastructure of Trichomonas tanax from 18. Osorio EM, Ibanez AB, Ibenoz CM, Moro AM. clinical practice. American microscopictical society Entamoeba gingivalis and Trichomonas tenax en Inc 1984;103 (4): 376-82 cavided bucal de patients de la clinica intergral del 2. Jian BO, Kolansky AS, Baloach ZW. Entamoeba adulto de la faculated de odontologia,Maracaibo, gingivalis pulmonary abscess-dignosed by needle Venzula. Rev Soc Ven Microbiol 2009;29 (2):122-7. aspiration. Cyto J 2008; 5(12). 3. Abulqumsaan M,Toz SO, Yolasigmaz A. The investigation of Entamoeba gingivalis and Trichomonas tenax in a group of patients with periodontal disease.Turky Society for Parasitology 2010; 34(2):91-4. 4. Junior RL,Melo C,Santana W.Ribeiro J.Incidence of Entamoeba gingivalis and Trichomonas tenax in samples of dental biofilm and saliva with periodontal diseas. RGO-Rev Gaucha Odontal proto Alegre 2011; 59 (1):35-40. 5. Chen JF,WenWR, Liu GY, Chen WL, Lin LG Hong HY. Studies on periodontal disease caused by Entamoeba gingivalis and its pathogenic mechanism. Rev China Med J 2001; 114(12):12-15. 6. El-Azzoumi M, Al-Brady AM. Frequency of Entamoeba gingivalis among periodontal and patients under chemotherapy. J Egypt Soc Parasitlogy 1994; 24(3): 649-55. 7. Al- Najar S, Adnan E. The first record of Entamoeba gingisvalis in Iraqi patints. J Fac Med Baghdad 1986; 28 (2):73-80. 8. Navazesh M. Methods for collecting saliva. Ann N Y Acad Sci 1993;694:72-7. 9. Gharavi MJ, Hekmat S, Ebrahimi A, Jahani MR. Buccal cavity protozoa in patients referred to the faculty of Dentisry inTehran, Iran. Iranian J Parasitology 2006; 1(1):43-6. 10. Nocito MI, Vasconi CM. Entamoeba gingivalis and Trichomonas tenax in diabetic patients RCOE 2003; 8(1):13-23. 11. Ghabanchi J, Zibaei M, Afkar MD, Sarbazie AH. Prevalence of oral Entamoeba gingivalis and Trichomonas tenax in patients with periodontal

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