Rashidi Maybodi F., et al. J Dent Shiraz Univ Med Sci., 2016 September; 17(3): 171-176.

Original Article

The Effect of Nonsurgical Periodontal Therapy on and Gingivalis in Patients with

1 1 2 3 4 Fahimeh Rashidi Maybodi , Ahmad Haerian Ardakani , Ali Fattahi Bafghi , Alireza Haerian Ardakani , Akram Zafarbakhsh

1 Dept. of , Shahid Sadoughi University of Medical Sciences, Yazd, Iran. 2 Dept. of Parasitology & Mycology, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. 3 Dept. of Orthodontics, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. 4 Dept. of Periodontology, Shahrekord University of Medical Sciences, Shahrekord, Iran.

KEY WORDS ABSTRACT Trichomonas Tenax; Statement of the Problem: Trichomonas tenax and Entamoeba gingivalis are com- Entamoeba Gingivalis; mensal protozoa which inhabit the human oral cavity. These parasites are found in Periodontitis; patients with poor and might be a reason for progressive periodontal dis- Parasite eases.

Purpose: The aim of this study was to evaluate the effect of nonsurgical periodontal

treatment on the frequency of these protozoa in saliva and plaque samples. Materials and Method: In this clinical trial, samples of saliva and were

collected from 46 patients with moderate to severe chronic periodontitis before and after periodontal therapy. The samples were assessed for the frequency of parasites. Results: The frequency of Entamoeba gingivalis was reduced in saliva (p= 0.007) and

plaque (p= 0.027) three weeks after the treatment. Likewise, the frequency of Trichomonas tenax reduced in saliva (p= 0.030); however, the decrease was not signifi-

cant in plaque (p= 0.913). Trichomonas tenax frequency in dental plaque directly relat-

ed to the severity of periodontitis (r= 0.565, p≤ 0.000). In contrast, the number of Enta- moeba gingivalis in both saliva (r= -0.405, p≤ 0.005) and plaque (r= -0.304, p= 0.040)

was inversely related with the severity of the . Conclusion: Nonsurgical periodontal treatment could reduce the number of Trichomo- Received June 2015; nas Tenax and Entamoeba gingivalis in the oral environment of patients with chronic Received in revised form August 2015; Accepted December 2015; periodontitis.

Corresponding Author: Zafarbakhsh A., Dept. of Periodontology, Shahrekord University of Medical Sci- ences, Shahrekord, Iran. Email: [email protected] Tel: +98-3536255881

Cite this article as: Rashidi Maybodi F., Haerian Ardakani A., Fattahi Bafghi A., Haerian Ardakani A., Zafarbakhsh A. The Effect of Nonsurgical Periodontal Therapy on Trichomonas Tenax and Entamoeba Gingivalis in Patients with Chronic Periodontitis. J Dent Shiraz Univ Med Sci., 2016 September; 17(3): 171-176.

Introduction Trichomonas is also limited [4] and have been conduct- Presenting in several various clinical forms, periodonti- ed only in few countries. [5] tis is considered as one of the most widespread oral dis- Trichomonas are mostly parasitic or commensal eases. Approximately 5 to 20 percent of the world’s flagellates that live in low-oxygen environments. [6-8] population suffers from severe generalized periodontitis. T. tenax is currently considered as a member of the oral [1] Periodontal lesions contain numerous neutrophils, biofilm. [9, 10] Its prevalence in the oral cavity ranges motile bacteria, spirillae, spinning rods, and protozoa. from 4 to 53% worldwide; [11] however, in patients [2] Yet, the etiology of this multi-factorial disease is still with periodontitis; it is 3 to 4 times more than healthy unclear. The possible role of parasites in the develop- individuals. [12] Investigations have been performed on ment of periodontitis has been poorly studied. [3] Data the correlation between the prevalence of T. tenax and from previous studies about Entamoeba gingivalis and the status of periodontitis, [6, 13] which is found partic-

171 The Effect of Nonsurgical Periodontal Therapy on Trichomonas Tenax and Entamoeba Gingivalis in … Rashidi Maybodi F., et al. ularly in patients with poor oral hygiene. [13-15] It can tics, Shiraz School of Dentistry, Iran, from March to be simply transmitted through saliva, droplet spray, October 2014. Informed consent was collected from the kissing, using contaminated dishes, and drinking water. patients. The participants were screened and examined [4, 7, 11] Oral cavity is the common residence of to make sure if they met the criteria for periodontal dis- Trichomonas species; although, it is occasionally found ease. The exclusion criteria were having a history of in the respiratory tract. [16-18] The significance of its consumption of systemic antibiotics within the three presence in the respiratory tract of humans is still un- preceding months, periodontal therapy during the pre- clear. Pleuropulmonary trichomoniasis has been ob- vious six months, medications that affect the periodon- served in bare cases. [19-21] A study in Egypt (2004) tium ( such as immunosuppressives, antihypertensives, showed that the prevalence of pulmonary trichomonia- anticonvulsants, contraceptives), systemic diseases such sis was 8% (20 cases) in a total of 250 individuals. [22] as diabetes, heart disease or respiratory diseases, preg- Entamoeba gingivalis (E. gingivalis) is found in nancy, smoking or other drug abuse. A demographic the oropharynx, but rarely in the head and neck lesions. form was filled out for each individual concerning age, [23-25] This microorganism is found more common in sex, smoking habits, and medical status. patients with poor dentition, [24] periodontal disease, As previously mentioned, the aim of this study and immune suppression. [26-28] It was the first com- was to evaluate the effect of nonsurgical periodontal mensal found in the human oral cavity. [12, 29] E. gin- treatment on the frequency of T. tenax and E. gingivalis givalis lives on the surface of the teeth and gum tissues. in saliva and plaque samples [5, 7, 35] before and 3 [30] Inflammatory process produces a propitious anaer- weeks after intervention. The first follow-up should be obic environment for their growth. [31] The trophozoite considered at least 3 weeks after treatment. This inter- is measured about 10-30 µm, actively motile with mul- val is needed for tissue healing. [36] To this end, the tiple , the cytoplasm contains food vacuoles patients were randomly divided into the case (23 pa- with ingested bacteria, leukocytes and epithelial cells. tients) and control (23 patients) groups by a table of [29] According to some studies, this is consid- random numbers. Considering the severity of periodon- ered as an important cause of periodontal disease. [32] tal disease by evaluating the mean clinical attachment E. gingivalis is an opportunistic pathogen whose associ- loss (CAL) in the patients’ , they were ation with synergistic symbiotic bacteria may cause per- also divided into one of the two subgroups of moderate iodontal diseases in hosts with low immunity. [33-34] (CAL=3-4mm) and severe periodontitis (CAL≥5mm). Conversely, some studies have reported the gen- [36] Sampling was done in two sessions (baseline and eral presence of E. gingivalis in disease-free individuals three week after the treatment). After collecting the first which was related to the amount of present on samples from all patients, the control group received the teeth. [35] As previously mentioned in several stud- only oral hygiene instructions. Scaling and root plan- ies, these parasites exist in the oral cavity and probably ning was done in case group via ultrasonic set (Wood- play a role in development of periodontal disease. Yet, pecker; China) for 20 to 60 minutes until eliminating no study has investigated the patients with moderate to deposits completely. Then face-to-face oral hygiene severe chronic periodontitis. Hence, the current study instructions were given by using a dental model. Three was designed to evaluate the effect of nonsurgical peri- weeks later, the second set of samples were prepared odontal treatment on the level of Trichomonas tenax and delivered to the lab. The examiner who studied the and Entamoeba gingivalis in dental plaques and saliva. saliva and plaque samples was blind to the groupings throughout the study. Materials and Method Dental plaque was collected from the teeth sur- In this randomized clinical trial (#IRCT2014071013167 faces by using sterile curettes (Gracey curette #13-14). N4), 46 patients with moderate to severe chronic perio- Non-stimulated saliva (0.1 ml) was collected by using dontitis (according to the Periodontology Association in sterile pipettes (Hirschmann graduated pipette; Class 1998) and the age range of 30-50 years old were select- AS, 10 mL volume, accuracy: 0.05 mL, Germany). The ed out of those referring to the Department of Periodon- labeled smears of saliva and dental plaques were fixed

172 Rashidi Maybodi F., et al. J Dent Shiraz Univ Med Sci., 2016 September; 17(3): 171-176. with methanol and sent to the parasitology lab. Then, the abundance of parasites (signification level≤0.005). these Giemsa-stained smears were observed by using The changes in parasites abundance before and after the microscope (Nikon; Eclipse E200, Japan) at both 40X treatment were determined by using Mann-Whitney and 100X magnifications. Detection of T. tenax was test. established as a pear-shaped flagellated trophozoite of about 5-13µ (Figure 1). The other oral protozoan, E. Results gingivalis was differentiated by its size (10-20μ), pres- Out of all the 46 patients, 33 were females and 13 were ence of prominent pseudopodia and food vacuole (Fig- males. There were 29 patients with severe periodontitis ure 2). (15 cases and 14 controls), and 17 with moderate perio- dontitis (8 cases and 9 controls). The mean baseline CAL in the two groups was almost equal (case group= 5.74±1.711 mm, and control group= 5.64±1.677 mm). Generally, the frequency of T. tenax was less than E. gingivalis in both saliva and dental plaque (p≤ 0.005). The severity of periodontitis was directly related

with the baseline frequency of T. tenax in dental plaque Figure 1: Microscopic view of Trichomonas gingivalis (r= 0.565, p≤ 0.000); however, it had no relationship

with the frequency of this parasite in saliva. The fre- quency of E. gingivalis in saliva (r=- 0.405, p≤ 0.05) and dental plaque (r= -0.304, p= 0.040) had a signifi- cantly reverse relationship with the severity of periodon- tal disease. Performing the test of homogeneity of variances

of parasites showed that the frequency of T. tenax in Figure 2: Microscopic view of Entamoeba gingivalis saliva (p=0.039) and dental plaque (p= 0.017) was sta-

tistically different in patients with different education Microscopic observation was repeated twice to levels. Parasites were mostly found in no-education confirm the diagnosis and distinguishing the specific (illiterate) group. pattern of each parasite family. In the microscopic study Post-hoc test revealed no correlation between the of parasites, the presence of very few number of para- patient’s age and the frequency of T. tenax in dental sites was considered as level 1 (0-1 parasites), few plaque (p= 0.066) or saliva (p= 0.34). T. tenax in dental number as level 2 (2-4 parasites), moderate as level 3 plaque was detected only in females (p≤ 0.005). (5-10 parasites), high as level 4 (11-15 parasites), and The frequency of E. gingivalis in dental plaque very high (16≤parasites) as level 5 (the results were (p= 0.212) and saliva (p= 0.441) had no significant dif- reported qualitatively rather than quantitatively). ference between various groups with different levels of In this study, the probable correlation between education. It was found more frequently in males’ saliva demographic data such as age, sex, and education level rather than females’ (p≤ 0.005). No relationship was and the initial abundance of parasites were analyzed in noticed between the age and colonization of E. gingi- oral samples. Furthermore, the abundance of parasites valis in saliva (p= 0.13) and dental plaque (p= 0.314). after periodontal treatment was compared with the base- Three weeks after the treatment, E. gingivalis de- line values. creased in the in case group's saliva (p= 0.007) and The statistical analysis was done by using SPSS plaque samples (p= 0.027). T. tenax also reduced in software, version 16. The independent-samples t-test saliva (p= 0.030); nevertheless, it did not have a signifi- was used for variables with abnormal distribution (sig- cant reduction in dental plaque (p= 0.913). nificance level ≤0.05). Pearson Correlation test was employed to analyze the relationship of severity of the Discussion disease (on the basis of CAL levels), age and sex with The initial treatment to eliminate pathogenic bacteria in

173 The Effect of Nonsurgical Periodontal Therapy on Trichomonas Tenax and Entamoeba Gingivalis in … Rashidi Maybodi F., et al. periodontal disease is the mechanical removal of dental Ibrahim and Abbas observed that E. gingivalis plaque and calculus. [37-39] consequently, it was sup- was more frequent in 61-70 year-old and T. tenax in 21- posed that these methods could help reduce parasites as 30 year-old patients, [26] while; the age parameter had much as it does for perio-pathogen bacteria. The ob- no significance in our study. Ghabanchi et al. detected tained results confirmed the probable efficacy of perio- that E. gingivalis could be found in dental plaque and dontal treatment in reducing the colonization of oral saliva of healthy subjects. [7] Our study showed a re- parasites in dental plaque except for T.tenax. No similar verse significant correlation between the severity of study was found about the effect of periodontal treat- periodontitis and E. gingivalis frequency, which may ment on the management of oral parasites; however, confirm the abundance of this parasite even in healthy some studies have evaluated the prevalence of these conditions. In contrast, Trim et al. [35] reported that E. protozoans. gingivalis was only present in pockets deeper than 4-7 Ghabanchi et al., [7] observed that E. gingivalis mm and this result was confirmed by Albuquerque et al. was more abundant than T. tenax in patients with either [12] and contrasted by the current study. This may be healthy periodontium or periodontitis, which was con- due to the differences in laboratory procedures. Hamad sistent with the results of the present study. In a study et al. [40] showed a positive relationship between the conducted by Bonner et al., 72 periodontal pockets and presence of parasite in the mouth and illiteracy or low 33 healthy gingival sulci were sampled to evaluate the education level which was partially confirmed by the correlation between E. gingivalis colonization and peri- present study. In our study, T. tenax was mostly found odontitis by using PCR (polymerase chain reaction). [3] in illiterate patients’ saliva or dental plaque, but there Although Bonner used a much more sensitive technique was no relationship between the amount of E. gingivalis (PCR) compared with this study (light microscope), and various education levels. In a study, Ullah et al. [41] both studies noted higher frequency of E. gingivalis reported a positive association between the prevalence compared with T.tenax. The only difference was that in of mouth parasites and poverty. The higher incidence our study, there was a significant reverse correlation was found among the lower class which was to some between the periodontal disease and E. gingivalis fre- extent in line with our study. The above-mentioned quency in saliva. Meanwhile, Bonner et al. stated that study also reported that the overall prevalence of these the presence of parasite was directly related to the peri- two parasites was higher in men than women. Our study

odontal disease in an overall view. Ibrahim and Abbas found T. tenax only in females’ dental plaque. This might reported a direct relationship between the severity of be attributed to the higher prevalence of T. vaginalis in periodontitis and abundance of E. gingivalis and T. females and the microscopic similarities of this species tenax in both saliva and plaque. [26] The current study and T. tenax. [6] E. gingivalis was more prevalent in detected a direct correlation between the frequency of T. males’ saliva than females’. Ullah et al. [41] included tenax in plaque and severity of periodontal disease; smokers, snuff dippers and participants with improper whereas, this direct relationship was not observed in brushing habits or poor oral hygiene in their study and salivary T. tenax or generally in E. gingivalis. It should as expected, the presence of these confounding factors be noted that the level of CAL was not considered as a was more frequent in males rather than females. determining factor in the severity of periodontal disease Gharavi et al. [5] found no relationship between T. in Ibrahim’s study, [26] only the pocket depth was tenax infection and sex or age, but E. gingivalis was measured. Hence, the relationship between the parasites related to age higher than 20 and male sex. Contrarily, and the disease severity cannot be confirmed with cer- Albuquerque et al. reported no correlation between age tainty. They also reported a higher abundance of E. gin- and the presence of both parasites, which was in agree- givalis in hypertensive patients and T. tenax in patients ment with the results of the present study. with increased use of antibiotics over the preceding 6 months. Unlike the present study, they did not exclude Conclusion patients with systemic complications and found that Regardless of patient’s demographic characteristics, it both parasites increased in diabetic patients. seems that oral hygiene instructions in combination with

174 Rashidi Maybodi F., et al. J Dent Shiraz Univ Med Sci., 2016 September; 17(3): 171-176. scaling and root planning can help controlling excessive Whose Attended to Clinics Periodontics, Dentistry Col- colonization of parasites, particularly E. gingivalis and lege\ Babylon Univ. IASJ. 2011; 3: 179-184. T. tenax and their probable opportunistic infestation. [9] Kurnatowska AJ, Dudko A, Kurnatowski P. Invasion of Trichomonas tenax in patients with periodontal diseases. Acknowledgments Wiad Parazytol. 2004; 50: 397-403. This study was supported by Shahid Sadoughi Universi- [10] Hersh SM. Pulmonary trichomoniasis and Trichomonas ty of Medical Sciences and the article was derived from tenax. J Med Microbiol. 1985; 20: 1-10. a doctorate thesis. [11] Mallat H, Podglajen I, Lavarde V, Mainardi JL, Frappier J, Cornet M. Molecular characterization of Trichomonas Conflicts of Interest tenax causing pulmonary infection. J Clin Microbiol. The authors declare that there are no conflicts of inter- 2004; 42: 3886-3887. est. [12] Albuquerque RLC, Moura C, Alcântara W, Lopes A, Albuquerque F. Incidence of Entamoeba gingivalis and References Trichomonas tenax in samples of dental biofilm and sali- [1] Burt B; Research, Science and Therapy Committee of the va from patients with periodontal disease. RGO. 2011; American Academy of Periodontology. Position paper: 59: 35-40. epidemiology of periodontal diseases. J Periodontol. [13] Lewis KL, Doherty DE, Ribes J, Seabolt JP, Bensadoun 2005; 76: 1406-1419. ES. Empyema caused by trichomonas. Chest. 2003; 123: [2] Bonner M. Medical Implication of Oral . 291-292. Canada. 5th International Congress on Tropical Medicine [14] Abualqomsaan M, Töz SO, Yolasiğmaz A, Turgay N. and International Health. Amsterdam. The Netherlands. The investigation of Entamoeba gingivalis and 2007; p. 24-28. Available at: www.calameo.com/books/ Trichomonas tenax in a group of patients with periodontal 0000884407729fb05e363 disease. Turkiye Parazitol Derg. 2010; 34: 91-94. [3] Bonner M, Amard V, Bar-Pinatel C, Charpentier F, [15] Athari A, Soghandi L, Haghighi A, Kazemi B. Prevalence Chatard JM, Desmuyck Y, et al. Detection of the amoeba of oral trichomoniasis in patients with periodontitis and Entamoeba gingivalis in periodontal pockets. Parasite. using PCR and direct smear. Iranian J Public 2014; 21: 30. Health. 2007; 36: 33-37. [4] Haghighi A, Soghandi L, Athari A, Kazemi B. Prevalence [16] Porcheret H, Maisonneuve L, Estève V, Jagot JL, Le of Oral Trichomoniasis in Patients with Periodontitis and Pennec MP. Pleural trichomoniasis due to trichomonas Gingivitis Using PCR and Direct Smear. Iran J Public tenax. Rev Mal Respir. 2002; 19: 97-99. Health. 2007; 36: 33–37. [17] Walzer PD, Rutherford I, East R. Empyema with [5] Gharavi MJ, Hekmat S, Ebrahimi A, Jahani MR. Buccal Trichomonas species. Am Rev Respir Dis. 1978; 118: Cavity Protozoa in Patients Referred to the Faculty of 415-418. Dentistry in Tehran, Iran. Iranian Journal of Parasitology [18] Dimasuay KG, Rivera WL. First report of Trichomonas 2006. 1: 43-46. tenax infections in the Philippines. Parasitol Int. 2014; 63: [6] Kucknoor AS, Mundodi V, Alderete J. Genetic identity 400-402. and differential gene expression between Trichomonas [19] Chiche L, Donati S, Corno G, Benoit S, Granier I, vaginalisand Trichomonas tenax. BMC Microbiol. 2009; Chouraki M, et al. Trichomonas tenax in pulmonary and 9: 58. pleural diseases. Presse Med. 2005; 34(19 Pt 1): 1371- [7] Ghabanchi J, Zibaei M, Afkar MD, Sarbazie AH. 1372. Prevalence of oral Entamoeba gingivalis and [20] Wang HK, Jerng JS, Su KE, Chang SC, Yang PC. Trichomonas tenax in patients with periodontal disease Trichomonas empyema with respiratory failure. Am J and healthy population in Shiraz, southern Iran. Indian J Trop Med Hyg. 2006; 75: 1234-1236. Dent Res. 2010; 21: 89-91. [21] Lopez-Escamilla E, Sanchez-Aguillon F, Alatorre- [8] Al-hamiary AK, Kezar MY, AlKhafaji YA. Prevalence of Fernandez CP, Aguilar-Zapata D, Arroyo-Escalante S, Oral Protozoa in Periodontitis and Gingivitis Patients Arellano T, et al. New tetratrichomonas species in two

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