Are There any Association Between Entamoeba gingivalis and Trichomonas tenax in Dental and Periodontal Diseases? A Systematic Review and Meta-analysis

There are frequent epidemiological and experimental proofs to propose that Entamoeba gingivalis and Trichomonas tenax infections are ignored factors for the development of dental and periodontal diseases. For a more comprehensive review, the present study was designed and conducted, including a systematic search in PubMed, Scopus, Web of Science, and Google Scholar databases for retrieving related reports up to December 01, 2019. Finally, 48 studies (a total of 70 datasets), including 30 datasets with case-control designs (1711 patients and 614 controls) and 40 datasets with cross-sectional designs (a total of 7257 participants) met the eligibility criteria. Based on the random-effects model, the pooled prevalence of protozoan infections in dental and periodontal patients was estimated to be 25.2% (95% CI, 21.2-29.8%; 2210/8968). In case-control designed studies, the prevalence of protozoan infections in dental and periodontal patients (35.8%, 614/1711) was found to be significantly higher than in controls (14.4%, 198/1371; OR: 4.15, 95% CI: 2.92-5.92). In addition, E . gingivalis (OR: 3.81, 95% CI: 2.36-6.14) and T . tenax (OR: 5.08, 95% CI: 2.81-9.20) were significantly higher in dental and periodontal patients compared to the controls. Our results demonstrated an apparently significant association between these protozoan infections in dental and periodontal patients. In conclusion, more standardized experimental designs are proposed using the animal model, and longitudinal (cohort) studies support the association between these protozoan infections in dental and periodontal patients to understand whether E. gingivalis and T. tenax infections are a co-factor in the development of dental and periodontal patients.

pathophysiology of oral tissue damage is obscure, and little information is available about their pathogenic role in DD and PD. 16 There are numerous reports of the prevalence of E. gingivalis and T. tenax infections among DD and PD patients throughout the world; nonetheless, up to now, there is no comprehensive study with statistical analysis in this regard. Henceforward, due to the important association of DD and PD with the presence of T. tenax and E gingivalis in the oral cavity, we conducted a systematic review and meta-analysis in order to shed light on whether E. gingivalis and T. tenax play a role in the occurrence of the DD and PD.

Search Strategy
Our comprehensive study was followed in agreement with the "Preferred Reporting Items for Systematic Reviews and Meta-Analyses" guidelines. 17 To assess the pooled prevalence and odds ratio (OR) of E. gingivalis and T. tenax infections in the DD and PD subjects, we systematically searched Google Scholar as a search motor engine and major databases (i.e., Scopus, PubMed, and Web of Science) for appropriate records until December 01, 2019. The searching process was performed using several medical subject heading (MeSH) terms; they included ("Entamoeba gingivalis" OR "Entamoeba buccalis" OR "Entamoeba maxillaris" OR "Amoeba pyogenes" OR "Trichomonas tenax" OR "Trichomonas buccalis") AND ("prevalence" OR "epidemiology") AND ("periodontal disease" OR "periodontitis" OR "dental disease" OR "dental caries" OR "dental decay"). Further, the references of the eligible records were manually searched to retrieve associated studies not recovered through the initial screening.

Eligibility Criteria
In the present research, four criteria were considered for including the studies. They were original research with case-control and cross-sectional designs and full texts about the prevalence of T. tenax and E. gingivalis infections in DD and PD patients and short reports and/ or abstracts having relevant data published in the English language. The other inclusion criteria included reports with information on the total sample size and positive samples and available published online data up to December 01, 2019. On the other hand, articles without the above-mentioned criteria, including non-human or animal studies, studies that only included healthy people or the general population, case reports, all types of reviews, editorials, and/or letters were excluded from the study.

Study Selection and Data Extraction
The initial screening was conducted via the title (topics)/ abstracts of the records, and the records were achieved accordingly; then, duplicated papers were removed by EndNote software. Two authors (A. F. and A. T.) screened and retrieved records through study criteria consideration. Next, two other researchers (Z. H. and S. B.) extracted the items of the studies, including the author's name, research publication year and execution time period, study design, geographical zone (continent/ country), diagnostic method, precipitants age (mean or range), total sample size, positive case number, and the detected protozoans types in every study. All data were imported into the Microsoft Excel Worksheet, and two researchers (K. S. and H. R.) double-checked them. All controversial issues were resolved by the corresponding author's consensus (S. B).

Statistical Analysis and Data Synthesis
All statistical analyses were directed via comprehensive meta-analysis statistical software (Version 2.2, BioStat). The prevalence of E. gingivalis and T. tenax in DD and PD patients was estimated by using the random effects model. Moreover, for the possible association assessment, the OR with a 95% confidence interval (CI) was calculated for case-control studies. The I 2 method was applied to the heterogeneity assessment of the studies. Likewise, the small sample size effects and publication bias of the studies were determined by Egger's regression test; the probable publication bias was presented as the funnel plot. In all statistical analyses, P < 0.05 was considered statistically significant. The pooled prevalence of E. gingivalis and T. tenax in people with DD and PD was displayed as the forest plot, and the difference in the prevalence rate of E. gingivalis and T. tenax in DD and PD individuals and controls was presented by an OR and 95% CI. Finally, gender prevalence rates in DD and PD patients were compared using the OR.

Study Characteristics
The selection steps of the present systematic review and meta-analysis studies are presented in the flow diagram ( Figure 1). Briefly, 1242 papers were recovered in the initial search step; eventually, 48 records containing 70 datasets meeting the inclusion criteria remained for analysis after removing irrelevant and duplicated studies.  The included records were published between March 1970 and September 2019. The 48 studies were performed in 20 diverse geographical zones in four continents (12,6,6,6,5,5,4,4,4 Tables 1 and 2, alongside the microscopic examination and culture, PCR had been used for the detection of E. gingivalis and T. tenax infections in 15.7% (11/70) of the studies. Regarding the type of study design, 30 and 40 studies were of case-control and cross-sectional type, respectively. The main characteristics of the casecontrol and cross-sectional studies are summarized in Tables 1 and 2, respectively.

The Pooled OR of Entamoeba gingivalis and Trichomonas tenax Based on Case-control Studies
The meta-analysis outcomes demonstrated a significant positive association between exposure to both protozoan infections (E. gingivalis and T. tenax) and DD and PD (OR: 4.15; 95% CI: 2.92-5.92%, Figure 2). Subgroup analysis considering the causative protozoan revealed a significantly higher prevalence of E. gingivalis (OR: 3.81; 95% CI: 2.36-6.14%) and T. tenax (OR: 5.08; 95% CI: 2.81-9.20%) in DD and PD patients compared to healthy controls ( Figures S4 and S5).
In a subgroup analysis by gender in DD and PD patients, the prevalence in males was higher than in females (30.1% vs. 28.6%), but no significant association was found between males and females (OR: 1.15; 95% CI, 0.85-1.57%, Figure S6).

Publication Bias Results
According to Egger's regression test, a significant publication bias (t = 4.78, P = 0.00) was observed in the case-control studies, whereas publication bias was not significant in the cross-sectional studies (t = 1.73, P = 0.09). Figure 3 (funnel plot) shows the potential publication bias identification.

Discussion
Over the last century, it was postulated that only bacteria were microorganisms that contributed to formation of plaque and dental calculus. On the other hand, although  the main structure of these plaques is composed of a wide range of bacteria, some fungal, Mycoplasma, and protozoan species also have a neglected role in plaque formation and dental calculus. Recently, several epidemiological investigations have been performed on the prevalence of E. gingivalis and T. tenax infections in people with DD and PD in some countries; their presence in dental disorders led to the theory that these protozoans have a possible role in causing DD and PD. To fill this gap, the current systematic review and meta-analysis focused on the observational studies that evaluated the prevalence and association between E. gingivalis and T. tenax infections in subjects suffering from DD and PD. In this meta-analysis, it was found that the prevalence of E. gingivalis and T. tenax infections in DD and PD patients was significantly increased compared to the control group.
To date, the exact mechanism of the pathogenesis of these two protozoans in DD and PD has not been fully understood and is still under debate. Some virulence factors related to T. tenax have been partially discovered in recent papers. As it is evident, this flagellate has been documented that has the ability to secrete some proteolytic enzymes (e.g., metalloproteinase and cysteine proteinase), thereby leading to degrading collagen. 21 Moreover, no study has so far described the potential ability of this parasite in adhering to different eukaryotic cells through a wide range of iron-dependent surface adhesions, resulting in the cytotoxic effect by producing apoptotic bodies through the release of cysteine proteases. 66 These virulence factors can be indirectly involved in DD and PD so that conditions for the growth of periodontopathogenic such as anaerobic bacteria Tannerella forsythia, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Fusobacterium nucleatum have been prepared by inducing damage of the tooth-supporting tissue. 15,67 All the above-mentioned factors may support the possible impress of the mentioned protozoan in the initiation and progression of DD and PD. The information on the pathogenicity of E. gingivalis is vaguer than T. tenax. In this regard, the genetic variation in E. gingivalis species appears to be important, and the genetic distance between the ST1 and ST2-kamaktli variants may indicate many differences in their biology and the possible association of these subtypes with various pathologies. 40,68 Thus, studies have demonstrated that the ST2 "kamaktli" variant is more genetically diverse than E. gingivalis ST1, which may indicate the hypothesis that strains cannot have the same pathogenicity and tropism. 68,69 Given that most epidemiological studies on this subject were performed by microscopy and culture methods, there is little information on the molecular epidemiology of this protozoan, thus it is recommended that molecular studies be conducted to gain a deeper understanding of this issue.
In this comprehensive study, we faced limitations, including differences in the sensitivity and specificity of techniques used in analyzed studies for parasite identification that the estimated prevalence can be affected by these differences, as well as limited reports from some geographical regions of the world about the prevalence of E. gingivalis and T. tenax among DD and PD patients. The other limitation were small sample sizes in some included studies and no further information about precipitants' complications condition, as well as differences in the number and type of variables and the quality of the analyzed studies.
In conclusion, the present study presented a relatively more comprehensive picture of the prevalence of E. gingivalis and T. tenax in subjects with DD and PD. The findings of this meta-analysis indicated that patients suffering from PD and DD were significantly more likely to be positive for E. gingivalis and T. tenax than healthy controls, suggesting that there is a significant association between these protozoans in the oral cavity and the occurrence of DD and PD.