IMPLANTOLOGY Presence of tenax and gingivalis in peri-implantitis lesions

Osman Fatih Arpağ, PhD/Özlem Makbule Kaya, PhD

Objective: The aim was to investigate the presence of Entam- Results: Although there was no presence of parasite around oeba gingivalis and Trichomonas tenaxx in peri-implantitis le- the healthy implants, two parasites were detected in peri- sions. Method and materials: A total of 141 individuals were implantitis lesions. Out of 101 lesions, 31 (30.7%) showed included in this study, of which 40 had clinically healthy im- E gingivalis, and 34 (33.6%) presented with T tenax. There was a plants (group H); the remaining were associated with peri-im- statistically significant difference between the presence of plantitis (group P). Gingival crevicular fluid was collected using E gingivalis and demographic data including gender, education absorbent paper, followed by a sample from the status, frequency of dental visits, and brushing frequency. Pres- peri-implant sulcus/pocket using a titanium curette. The sam- ence of T tenaxx in lesions was correlated with frequency of den- ples were transferred into an Eppendorf tube. Each specimen tal visits (P < .05). It was observed that E gingivalis and T tenax was divided into two parts. One part was examined under a were mostly detected in the mandible (P = .004 and .014, re- light microscope at a 10 × and 40 × magnification to detect spectively) in comparison with the maxilla. Conclusion: This parasites. The other part was spread on a microscope slide, study showed that peri-implantitis lesions were involved with stained with Giemsa stain, and examined under a microscope E gingivalis and T tenax, in contrast to the healthy areas. at 100 × magnification. Pearson chi-square test was used in the (Quintessence Int 2020;51:212–218; doi: 10.3290/j.qi.a43948) statistical analysis of data, with a significance level of P < .05.

Key words: dental implants, direct microscopy, Giemsa staining, microbiology, parasite, peri-implantitis

In recent years, dental implants have been widely used in den- some of the common types of bacteria found in peri-implantitis tistry to replace missing teeth. Accumulation of microbial bio- lesions.2-4 film around the causes a breakdown of the Periodontal lesions also contain viruses, and fungal and implant-supporting tissues such as alveolar bone and gingiva. protozoa species.5 However, the effect of parasites as a part of This condition, which is called peri-implantitis, clinically pres- the pathogenesis of is still debated. Previous ents as gingival and alveolar bone loss, studies have reported that two protozoans, eventually leading to implant loss.1 Several microorganisms are and , are among the types of parasites involved in the pathogenesis of peri-implantitis lesions. Some that are found in the dental plaque biofilm of patients with studies have reported that microbial plaque samples around .6,7 T tenax is an anaerobic motile-flagel- the healthy implant contain bacilli and cocci, and the samples lated protozoan that is classified in the same genus as Tricho- obtained from peri-implantitis lesions contain numerous fusi- monas vaginalis. T tenax plays a role in gland infections, pleuro- form bacteria, spirochetes, and motile rods. Porphyromonas pulmonary infections, sinusitis, and tonsillitis, which occur gingivalis, , Prevotella nigrescens, Tannerella outside the oral cavity. T tenax can be transmitted through forsythia, , and saliva, droplet spray, kissing, drinking water, and contaminated play a role in the progression of periodontal diseases, and are dishes. The prevalence of T tenax is correlated with demo-

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Table 1 Comparison of demographic data in the groups

Parameter Variable Group H Group P P value*

Gender, n (%) Male 14 (35.0%) 52 (51.5%) .077 Female 26 (65.0%) 49 (48.5%) Total 40 (100.0%) 101 (100.0%) Age, y (mean) Male 57.14 53.86 .594 Female 53.16 56.53 Overall 55.15 55.19 Education status, n (%) University 9 (22.5%) 16 (15.8%) .115 High school 13 (32.5%) 18 (17.8%) Primary school 10 (25.0%) 42 (41.6%) Illiterate 8 (20.0%) 25 (24.8%) Frequency of dental visit, n (%) Regular 11 (27.5%) 45 (44.6%) .062 Irregular 29 (72.5%) 56 (55.4%) Brushing frequency (daily), n (%) Twice or more 4 (10.0%) 9 (8.9%) .867 Once 24 (60.0%) 57 (56.4%) Sporadic 12 (30.0%) 35 (34.7%) n, count of individuals in same row; Group H, healthy patients; Group P, patients with peri-implantitis. *P value for significance level is < .05.

graphic and systemic diseases such as diabetes and Down syn- dental implants (group H) were enrolled in the study. When a drome.8 E gingivalis is an opportunistic pathogen; it may cause patient had more than one implant, only one implant, consis- periodontal diseases in immunocompromised patients.9 More- tent with the inclusion criteria, was evaluated for that patient. over, E gingivalis has been identified in patients with advanced Patients who had used any antibiotic, antimicrobial, or anti- periodontitis, and it was detected in the dental plaque samples protozoal drugs in the previous 3 months and who had under- of disease-free individuals. Some studies have reported that gone any periodontal treatment within the last 6 months were the presence of E gingivalis in the oral cavity impairs gingival excluded. Systemically compromised patients, pregnant pa- health over time.10,11 tients, and smokers were also excluded. As previously noted, peri-implantitis is an infectious condi- The following criteria were used for inclusion: tion caused by several periodontopathic bacteria. The microbi- ■ individuals who were 18 years of age or older ota of peri-implantitis lesions was examined in previous stud- ■ for both groups, having implants loaded with a prosthetic ies.2,3 In the literature, no study investigating the presence of restoration at least 1 year before the study began parasites in peri-implantitis cases was found. Thus, the aim of ■ for group H, having implants without any peri-implant the present study was to investigate the presence of E gingivalis mucositis or peri-implantitis lesion and T tenax in peri-implantitis lesions. ■ for group P, having implants with a probing depth > 5 mm, gingival bleeding on probing, and radiographically detect- able bone loss distance > 2 mm between the implant collar Method and materials and the alveolar crest. Patient selection and study groups Sample collection Before the study began, all the ethical requirements were ful- filled (ethical approval number: 17.01.2019/08). Informed con- In the patients in both groups, the implant area was isolated sent was obtained from all the participants. In total, 101 patients with rolled cotton, and saliva was aspirated using suction. First, with peri-implantitis (group P) and 40 individuals with healthy gingival crevicular fluid was collected with an absorbent paper

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Table 2 Number of lesions with and without parasite in the groups Table 3 Distribution of species detected in combination or separately Group H (n = 40) Group P (n = 101) Species Count of lesions n (%) Species (+) (−) (+) (−) Entamoeba gingivalis (alone) 7 (17.1%) 0 (0.0%) 40 31 70 Entamoeba gingivalis Trichomonas tenaxx (alone) 10 (24.4%) (100.0%) (30.7%) (69.3%) Both species (together) 24 (58.5%) 0 (0.0%) 40 34 67 Trichomonas tenax (100.0%) (33.6%) (66.4%) Number of all cases detected 41 (100.0%) n, number of cases; +, presence of parasite; −, absence of parasite. n, count of lesions in which parasites were present together or alone.

point that was inserted into the for 10 seconds. study groups. Differences were considered to be statistically A dental plaque sample was then taken from the same site significant when P < .05. using a titanium curette.12 In group P, the deepest site of the pocket around the implant was selected for the samples. The Results samples were transferred into an Eppendorf tube filled with 0.9% NaCl solution (0.5 mL). Each sample was delivered to the No problems occurred when collecting and transferring the parasitology laboratory for examination, as quickly as possible. samples. Thus, a total of 144 individuals were evaluated for the presence of parasites. Table 1 presents the patients’ demographic data, including Microscopic determination their gender, age, education status, frequency of dental visits, The dilution sample drawn with a pipette was incubated at and brushing frequency. The distribution of the variables in the 37°C for 20 minutes. Two different methods were used to detect two groups was not statistically significant (P > .05). the parasites. In the first method, one droplet of the sample The prevalence of E gingivalis and T tenax in the two groups taken from the incubated dilution was placed on a microscope is presented in Table 2. From the incidence rate, it is clear that slide and closed with a glass cover. The specimen was exam- both parasites were associated with the peri-implantitis lesions. ined under a light microscope at 10 × magnification and 40 × E gingivalis and/or T tenax were found in almost one-third of magnification. In the second method, the specimen on a micro- the peri-implantitis cases. Forty-one out of 101 peri-implantitis scope slide was stained with a Giemsa stain for 45 minutes. lesions involved at least one of the parasites. The presence of Then, the slide was gently washed under clean water and air- both parasites in the same lesion was observed in 24 patients dried. After immersion oil was dropped onto the slide, the (58.5%) with peri-implantitis (Table 3). specimen was examined under a microscope at 100 × magnifi- Although the incidence of E gingivalis was greater in women cation.13 After the microscopic examinations, a dichotomous than in men (P < .05), the incidence of T tenax was not (P > .05). scoring model was used to indicate the presence or absence of The incidence rate of the parasites with respect to gender was parasites. All observations were performed by a blinded para- independent of the mean age (P > .05). The incidence of E gin- sitologist with at least 10 years of experience. givalis decreased as the level of education increased (P < .05), but the incidence of T tenax was not associated with different educational levels. E gingivalis was found in eight out of 45 Statistical analysis patients (17.8%) with peri-implantitis (group P) who regularly Statistical analysis was performed using SPSS software (version visited the dental practitioner. In the patients in group P who 20.0, IBM). Pearson chi-square test was used to determine the did not regularly visit the dental practitioner, E gingivalis was correlation between the demographic data and the presence found in 23 out of 56 lesions (41.1%). Although the proportion of parasites; it was also used to determine the distribution of of E gingivalis present in the lesions of the group P patients who the parameters, including age, gender, education status, fre- brushed once per day was 22.8%, the proportion of E gingivalis quency of dental visits, and brushing frequency in the two present in the lesions of group P patients who brushed two or

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Table 4 Distribution of lesions with or without parasite and demographic data in group P

Entamoeba gingivalis Trichomonas tenax

Parameter Variable (+) (−) P value (+) (−) P value

Female 20 (59.2%) 29 (40.8%) 15 (30.6%) 34 (69.4%) Gender, n (%) Male 11 (21.2%) 41 (78.8%) .032* 19 (36.5%) 33 (63.5%) .529 Total 31 (30.7%) 70 (69.3%) 34 (33.7%) 67 (66.3%) Female 57.35 58.34 61.93 56.17 Age, y (mean) Male 65.18 52.70 .735 53.21 54.27 .467 Overall 61.26 55.52 57.57 55.22 University 3 (9.7%) 22 (31.4%) 8 (32.0%) 17 (68.0%) High school 10 (32.3%) 32 (45.7%) 11 (26.2%) 31 (73.8%) Education status, n (%) .003* .432 Primary school 11 (35.5%) 7 (10.0%) 8 (44.4%) 10 (55.6%) Illiterate 7 (22.5%) 9 (12.9%) 7 (43.8%) 9 (56.2%) Regular 8 (17.8%) 37 (82.2%) 9 (20.0%) 36 (80.0%) Frequency of dental visit, n (%) .012* .009* Irregular 23 (41.1%) 33 (58.9%) 25 (44.6%) 31 (55.4%) Sporadic 7 (77.8%) 2 (22.2%) 5 (55.6%) 4 (44.4%) Brushing frequency (daily), n (%) Once 13 (22.8%) 44 (77.2%) .004* 19 (33.3%) 38 (66.7%) .310 Twice or more 11 (31.4%) 24 (68.6%) 10 (28.6%) 25 (71.4%) n, number of individuals; %, percentage of patients in same row; +, presence of parasite; −, absence of parasite. *Statistically significant (P < .05)

Table 5 Incidence of both parasites in quadrants and arches

Entamoeba gingivalis, n (%) Trichomonas tenax, n (%) Quadrant/arch (+) (−)P value (+) (−) P value

Maxillary right 1 (3.2%) 19 (20.5%) 0 (0.0%) 20 (21.5%) Maxillary left 3 (9.7%) 30 (25.2%) 6 (17.6%) 27 (23.7%) .000* .001* Mandibular left 16 (51.6%) 23 (21.3%) 15 (44.1%) 24 (22.5%) Mandibular right 11 (35.5%) 38 (33.0%) 13 (38.3%) 36 (32.3%) Maxilla 4 (12.9%) 49 (45.7%) 6 (17.6%) 47 (45.2%) .004* Mandible 27 (87.1%) 61 (54.3%) 28 (82.4%) 60 (54.8%) .014* n (%), number and percentage of quadrants (note that number of quadrants is equivalent to number of patients enrolled in the study). *Statistically significant (P < .05) +, presence of parasite; −, absence of parasite.

more times per day was 31.4%. The proportion was 77.8% for also significantly affected the incidence of T tenax, the effect of group P patients who did not brush daily. T tenax was detected brushing frequency was not statistically significant (Table 4). in the lesions of 9 of the 45 patients in group P (20.0%) who The parasites were frequently detected in the quadrants of maintained regular dental visits; it was detected in the lesions the mandible. E gingivalis and T tenax were present in the man- of 25 of 56 patients (44.6%) who infrequently visited the dental dible in 27 cases (87.1%) and 28 cases (82.4%), respectively. The practitioner. The frequency of brushing and dental visits had a parasites were significantly more likely to be present in the statistically significant effect on the existence of E gingivalis in peri-implantitis lesions found in the mandible than in lesions peri-implantitis lesions. Although frequency of dental visits found in the maxilla (Table 5).

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Discussion present study ranged from 27 to 75. No correlation was found between the presence of the protozoans and age. Many studies To the best of the present authors’ knowledge, this is the first have reported a negative association between the existence of study to examine the parasitic contamination of dental these two parasites and the gender of the subject.20,22,23 How- implants. Therefore, the findings will be discussed in light of ever, it has been reported that T tenax was more prevalent in studies that have examined the associations between parasites women than in men and that E gingivalis was more prevalent in and periodontal diseases. It has been reported that protozoans men than in women.9 The present research showed that the can be involved in the pathogenesis of periodontitis. For exam- incidence rate for E gingivalis in peri-implantitis lesions was ple, T tenax contributes to collagenolytic activity through vari- higher in women than in men, but no correlation was found ous proteinases.14 E gingivalis is an opportunistic pathogen that between the incidence rate for T tenax and gender. creates synergy with some bacteria in a way that may result in Peri-implantitis is an inflammatory condition caused by periodontal diseases.15 Although the presence of both T tenax anaerobic bacteria, such as P gingivalis, T forsythia, and P interr- and E gingivalis in patients with periodontitis has been associ- media. Lack of education, poor , low socioeco- ated with the pathogenesis of the periodontal diseases, some nomic status, and irregular dental visits are correlated with the researchers have reported that both E gingivalis and T tenax development of implant diseases. The data show that demo- might colonize dental plaque biofilm, dental , and oral graphic factors make it more likely that peri-implant microbiota fluids, such as saliva and gingival crevicular fluid, in healthy will change from an aerobic environment to an anaerobic envi- individuals without any periodontal disease.11,16 These data are ronment.1,24,25 In the present study, the presence of E gingivalis, not consistent with the present results, in which neither E gin- an opportunistic pathogen protozoon, was strongly associated givalis nor T tenax could be detected in the samples from the with brushing frequency, frequency of dental visits, and the patients in group H. A linear relationship between the parasites education status of the patients with peri-implantitis. T tenax and peri-implantitis lesions may be a result of the deepening of was more prevalent in patients who visited the dental practi- peri-implant pockets.8 As is true of deep periodontal pockets, tioner infrequently than in those who visited regularly. Other- the microbiota around diseased dental implants is more anaer- wise, there was no an association between T tenax and the obic than it is in narrow pockets. Thus, decreasing the oxygen other parameters. ratio in deeper pockets can result in an increase in the coloni- Of the 101 peri-implantitis lesions, 68 (67.3%) were ob- zation and growth of anaerobic parasites.17 Protozoa, such as E served in the mandible and 33 (32.7%) were observed in the gingivalis, are associated with diabetes, drinking water, and maxilla. Similarly, both E gingivalis and T tenax were more fre- they can be found in saliva, droplet spray, kissing, and contam- quently detected in the lesions of the mandible than in the le- inated dishes. One study reported that the prevalence of E gin- sions of the maxilla. A study by Fransson et al26 showed that the givalis and T tenax were increased in the mouths of diabetic prevalence of peri-implantitis was higher in the mandibular patients.8 Therefore, individuals with any systemic disease were anterior region, due to the anatomical challenges of this re- excluded from the present study. gion; the lack of keratinized tissue and inadequate bone vol- Parasites can be detected in a laboratory by using several ume may increase the risk for peri-implant diseases. However, techniques, including direct microscopy, staining, serology- other studies have reported that peri-implant diseases more based assays, molecular-based assays, and proteomic examina- commonly occur in the maxilla because its bone density is tions. The present study used direct microscopy and Giemsa lower than that of the mandible.27,28 Because the present study staining. Although other diagnostic tests are more sensitive, is not a prevalence study, it cannot be inferred from the find- direct microscopy can be easily used to detect; moreover, this ings that peri-implantitis lesions occur more frequently in the cost-effective technique can be used to obtain an immediate mandible. The high prevalence of the two studied parasites in diagnosis.6,18,19 the lesions of the mandible was due to the fact that the dental The effect of age on the prevalence of parasitic contamina- implants that met the specific criteria required for inclusion in tion is controversial. On the one hand, the presence of E gingi- the present study were mostly found in the mandible when valis and T tenax increases with age; on the other hand, this compared to the maxilla. presence is more often observed among children between the The present study has a few limitations. As this was not a ages of 5 and 10 than among young adults and older members multi-center study and the participants enrolling in the study of the population.20,21 The age of the individuals included in the were mostly local inhabitants, the effect of the geography on

216 QUINTESSENCE INTERNATIONAL | volume 51 • number 3 • March 2020 Arpağ/Kaya the prevalence of the parasitic contamination was not ana- Declaration lyzed. Perhaps the healthy group (group H) should have included more individuals. Moreover, the study did not include The authors declare that they have no conflict of interest. periodontal/peri-implant scores and measurements, such as Plaque Index, Gingival Index, probing depth, and level of Acknowledgments attachment loss, for the sampled sites and/or for the full mouth. The authors thank the staff members, especially Muhammet Atılgan, at the clinic of , and at the Parasitology Laboratory of Health Practices and Research Hospital in Hatay Conclusion Mustafa Kemal University for their kind help in collection and In this study, the presence of E gingivalis and T tenaxx was asso- transfer of samples. Thanks also to Dr Caner Öztürk for his ciated with the lesions of patients suffering from peri-implanti- efforts in statistical analysis of the study data. tis in comparison to individuals whose peri-implant areas were clinically healthy. However, further studies are needed in order to identify the nature of the causal relationship between the two parasites and the development of peri-implantitis.

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Osman Fatih Arpağ Osman Fatih Arpağ Assistant Professor, Department of Peri- odontology, Faculty of Dentistry, Hatay Mustafa Kemal University, Hatay, Turkey. ORCID ID: 0000-0002-1510-8917

Özlem Makbule Kaya Professor, Department of Medical Parasi- tology, School of Medicine, Hatay Mustafa Kemal University, Hatay, Turkey. ORCID ID: 0000-0002-8382-3357

Correspondence: Osman Fatih Arpağ, Faculty of Dentistry, Hatay Mustafa Kemal University, 31040, Hatay, Turkey. Email: [email protected]

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