Catherine Bisson
email: catherine.bisson@univ-lorraine.fr

Sophie-Myriam M Dridi

Marie-Claire Machouart

Assessment of the role of Trichomonas tenax in the etiopathogenesis of human periodontitis: A systematic review

published or not. The documents may come

Assessment of the role of Trichomonas tenax in the etiopathogenesis of humain periodontitis: A systematic review

Introduction

The global burden of oral disorders and their associated sequelae have been assessed at various dates (1990, 2005 and 2010) by Marcenes et al. [START_REF] Marcenes | Global burden of oral conditions in 1990-2010: a systematic analysis[END_REF]. These authors concluded that DALYS (Disability-Adjusted Life Years) induced by oral conditions had increased by 20.8% between 1990 and 2010 due to population growth, the ageing process and severe periodontitis. Severe periodontitis is the sixth most prevalent disease affecting millions of people worldwide and because of its dental and systemic impacts should therefore be a public health priority [START_REF] Kassebaum | Global burden of severe periodontitis in 1990-2010: a systematic review and meta-regression[END_REF].

Periodontitis is recognized as an inflammatory disease, mainly induced by pathobionts such as Porphyromonas gingivalis in synergy with other putative pathogens present in a complex and diversified biofilm. These periodontopathogens cause an impaired immune response leading to the loss of periodontal tissue [START_REF] Hajishengallis | Immune and regulatory functions of neutrophils in inflammatory bone loss[END_REF]. But neither immune deficiencies nor the virulence of periodontal pathogens can explain why, despite the identification of periodontal pathogenic bacteria in the saliva, only a few teeth are severely affected by periodontitis in patients, or why one tooth presented alveolar bone and soft tissue destruction while the periodontium of its neighbour was barely affected.

In order to explain these clinical observations, but also the failure in stabilizing periodontitis in certain patients, the authors attempted to explore other potential etiologies of periodontal diseases. The oral cavity harbours a rich and complex biofilm that includes bacteria, but also viruses, fungi and protozoans [START_REF] Avila | The oral microbiota: living with a permanent guest[END_REF]. All these microorganisms could be implicated directly or indirectly in the development of periodontitis. A systematic review with meta-analysis evaluating the microbiota from patients with periodontitis has shown a significant presence of some herpesviruses and other authors have identified fungi (Candida genus), both of which could have a potential pathological role in this disease [START_REF] Li | Herpesviruses in etiopathogenesis of aggressive periodontitis: A meta-analysis based on case-control studies[END_REF][START_REF] Canabarro | Association of subgingival colonization of Candida albicans and other yeasts with severity of chronic periodontitis[END_REF]. Moreover, two main protozoans have been identified in the oral cavity: Entamoeba gingivalis belonging to the rhizopod class and Trichomonas tenax to the zoomastigophora one. As regards Trichomonas tenax, some authors suggested its implication in periodontal diseases [START_REF] Feki | Protozoa of the human oral cavity (epidemiological correlations and pathogenic possibilities[END_REF][START_REF] Benabdelkader | Specific clones of Trichomonas tenax are associated with periodontitis[END_REF]. A recent review concerning T. tenax and periodontal diseases has been published in which the authors concluded that there was a correlation between the presence of the flagellate and the periodontal diseases and that this protozoan was involved in inflammatory process of gingivitis [START_REF] Marty | Trichomonas tenax and periodontal diseases: a concise review[END_REF]. Nevertheless, this review did not follow the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines and the PICO (Patient Intervention Comparison Outcome) framework as recommended by the Centre for Evidence-Based Medicine [START_REF] Moher | Preferred reporting items for systematic reviews and meta-analyses: the PRISMA Statement[END_REF][START_REF] Hardi | Evidence-Based Medicine and Systematic Review Services at Becker Medical Library[END_REF]. In this review, 47 clinical studies about prevalence of the protozoan in periodontal diseases were not classified according to population (adults, children), presence of systemic disease, previous antibiotic consumption, and the keywords used for the literature research on periodontal diseases assessment were not detailed.

Therefore, we propose here the first systematic review of literature to investigate the presence of T. tenax in periodontitis in adult patients and to assess its potential role in the etiopathogenesis of periodontitis, according to Koch's postulates revisited by Socransky [START_REF] Socransky | The bacterial etiology of destructive periodontal disease: current concepts[END_REF]: (i) postulate "Association" (elevated level of microorganism in lesions of periodontitis), (ii) postulate "Elimination" (microorganism elimination resulted in successful therapy), (iii) postulate "Host response" (microorganism induces immune response: elevated serum and local antibodies, cytokines production), (iv) postulate "Virulence factors" (microorganism produces enzymes inducing a periodontal degradation) and (v) postulate "Animal studies" (microorganism induces disease in gnotobiotic animal).

Firstly, we investigate through literature data the presence of T. tenax in diseased sites and its absence in healthy sites in adult patients with periodontitis, secondly the impact of non-surgical periodontal treatment on the decrease or elimination of this flagellate in healed sites and thirdly the potential virulence factors of this protozoan which could cause the destruction of periodontal tissue.

Methods

Protocol and study design

This systematic review was elaborated by following the PRISMA guidelines [START_REF] Moher | Preferred reporting items for systematic reviews and meta-analyses: the PRISMA Statement[END_REF] (S1 Table ).

Eligibility criteria

In this review, the focused questions addressed using the PICO framework, aimed to evaluate the potential pathogenicity of Trichomonas tenax in the development of periodontitis according to Koch's postulates revisited by Socransky [START_REF] Socransky | The bacterial etiology of destructive periodontal disease: current concepts[END_REF]:

1. Is T. tenax (I) identified in high numbers in sites with periodontitis (P) and in lesser numbers or not at all in healthy sites? (postulate "Association")

2. Does the non-surgical treatment of periodontitis induce the decrease or the absence of T. tenax in the healed periodontal pocket and/or periodontal pocket with depth reduction? (C) (postulate "Elimination")

3. Does T. tenax produce virulence factors (adhesion capacity and tissue invasion, immunomodulating molecules, toxicity) and can it induce similar periodontal diseases in an appropriate animal model? (O) (postulates "Virulence factors", "Host response" and "Animal studies"). No publication was selected prior to the 1960s, because terms used to evaluate periodontal status were confusing (pyorrhoea, dying periosteum. . .). The electronic research was conducted independently by two authors (CB, SMD).

Information sources and research strategy for relevant studies

Study selection

Only full text articles published in the English, French, Italian and Spanish/Portuguese languages were considered.

All studies relating to Trichomonas tenax were considered eligible for inclusion if they were original articles in peer-reviewed journals conducted in humans and were:

• Cross sectional, case-control studies, controlled trials • Case reports or case series (accepted for research pathogenicity factors).

• In vitro studies with evaluation of potential virulence factors and animal models of periodontitis.

All reviews of literature were excluded from the selected articles because they give redundant informations and are not detailed enough.

Inclusion criteria. Studies that were selected for this analysis include the following criteria:

• Adult population (� 18 years)

• Patients diagnosed with periodontitis using one or more of the following terms: pocket > 3 mm and/or CAL > 2 mm (Clinical Attachment Level), bone loss, grade of the periodontal tissue degradation (moderate or severe periodontitis, Armitage classification, 1999) [START_REF] Armitage | Development of a classification system for periodontal diseases and conditions[END_REF].

• Patients diagnosed with periodontitis with no surgical treatment in the last 3 months (to evaluate the prevalence of T. tenax in periodontal pockets)

• Patients diagnosed with periodontitis treated by non-surgical treatment (to evaluate the impact of the therapy on protozoan in the healed sites).

• Studies with collection of dental biofilm (supra and/or subgingival)

• In vitro studies with detailed protocol Exclusion criteria. Articles were excluded from the selection because of the presence of:

• Adolescent and children population (< 18 years)

• Unclear periodontal diagnosis of patients included, no precise description of periodontal parameters

• Periodontitis as manifestations of systemic disease

• Patients who received surgical periodontal therapy

• Patients taking medication that could modify the oral microbiota

• Studies relating only to the collection of saliva, tooth decay or oral mucosa.

• In vitro studies with other trichomonads species than T. tenax.

Data selection process and elements

Data were collected independently by two authors (CB, SMD) and definitively included by these two authors, and all disagreements were resolved after discussion and/or with the intervention of a third author (MM). Reasons of exclusion of the articles were fully detailed in S1

Risk of bias in individual studies and across studies

Risk of bias was assessed through the Newcastle-Ottawa Scale (NOS) for case-control studies and modified NOS for cross-sectional studies [START_REF] Wells | Osteoporosis Methodology Group and The Osteoporosis Research Advisory Group. Meta-analyses of therapies for postmenopausal osteoporosis. V. Meta-analysis of the efficacy of hormone replacement therapy in treating and preventing osteoporosis in postmenopausal women[END_REF]. The following criteria were assessed:

• Representativeness of the exposed cohort • Ascertainment of exposure • Quality of measurements/protocol and outcomes: periodontal parameters and precise protocol of collection of biofilm are evaluated.

For in vitro research articles, protocols [time of incubation, strain of T. tenax, different MOI (Multiplication of Infection), different cells line] used by each author are detailed.

Results

Study selection

As shown in Fig 1, a total of 376 articles were identified from the previously described electronic database search. After duplicate removal and language exclusion, a total of 156 articles were obtained. Finally, after the removal of non-full text and non-relevant papers, 25 articles were deemed appropriate to be included. The main reasons for exclusion were: the absence of periodontal status, lack of precise periodontal parameters, the identification of trichomonads in animal studies but not in human studies, the studies included patients with medication modifying microbiota, the diagnosis of periodontitis as a manifestation of systemic disease, other trichomonads species than T. tenax, the absence of a T. tenax factor of virulence. Some clinical studies were discarded because essential details were missing:

1. One study included patients with periodontitis as well as patients with periodontitis and diabetes and did not specify the proportion of patients T. tenax-positive among those with diabetes.

2. one study includes both children and adults but did not specify the T. tenax positivity according to the population age.

3. In other studies, some included patients had already been treated with antibiotics, without anyone knowing the proportion of patients T. tenax positive among those receiving or not antibiotics.

4. Finally, in some papers the origin of the sample was unknown such as "rinsing sample" and/or samples from saliva, mucosa, decay.

Characteristics of included studies

• Presence of T. tenax in periodontitis or gingivitis sites and in healthy sites from controls (Table 1). Three case-control studies were selected to evaluate the presence of T. tenax in periodontitis (Table 1). Two of them have used the microscope to detect the flagellates, and only one the molecular biology tools. Whatever the technique used, the authors showed a higher frequency of this flagellate in patients with periodontitis (from 42.5 to 32.3%) than those with gingivitis (from 32.3% to 0%). The controls with healthy periodontium had positivity to T. tenax from 28.2% to 7.1%. The frequency of the flagellate in the control group doubled depending on authors, but its frequency in periodontitis sites was similar [START_REF] Feki | Protozoa of the human oral cavity (epidemiological correlations and pathogenic possibilities[END_REF][START_REF] Benabdelkader | Specific clones of Trichomonas tenax are associated with periodontitis[END_REF][START_REF] Ferrara | Possible pathogenic role of Trichomonas tenax in chronic periodontitis[END_REF].

• Impact of non-surgical treatment of flagellate in healed sites (Table 1). Only one randomized controlled trial evaluated the impact of scaling and root planing (SRP) on the frequency of T. tenax in periodontitis sites [START_REF] Rashidi Maybodi | The Effect of Nonsurgical Periodontal Therapy on Trichomonas Tenax and Entamoeba Gingivalis in Patients with Chronic Periodontitis[END_REF]. The authors selected patients with moderate to severe periodontitis (CAL > 5 mm) and randomly assigned them to the experimental and control group. Before receiving an oral hygiene instruction, a sample of dental biofilm was collected from the patients in each group. Three weeks after SRP, a second biofilm sample was collected from patients in the experimental group. The authors concluded that the SRP could reduce the number of T. tenax in saliva but not in the dental plaque of patients with moderate to severe periodontitis. No detailed description of PPD (Periodontal Pocket Depth) and CAL before and after SRP was reported.

• Potential virulence factors from protozoans (Table 2). Authors have detected the capacity of T. tenax ATCC 30207 to adhere to diverse cells and induce cytotoxicity [START_REF] Ribeiro | Is Trichomonas tenax a Parasite or a Commensal?[END_REF], while others studies showed no adherence or cytotoxic property [START_REF] Alderete | Specific nature of Trichomonas vaginalis parasitism of host cell surfaces[END_REF][START_REF] Alderete | Pathogenic Trichomonas vaginalis cytoxicity to cell culture monolayers[END_REF]. Moreover, flagellates produce numerous enzymes, such as cysteine protease, cathepsin B like protein, haemolysins, metalloproteinase and other proteinases that could participate in the breakdown of collagen, gelatin observed in periodontal diseases [START_REF] Sibaei | Growth kinetics, antigen profiling, and proteinase activity of Egyptian Trichomonas tenax isolates derived from patients having oral infections[END_REF][START_REF] Yamamoto | Characterization of the Cathepsin B-like Proteinases of Trichomonas Tenax ATCC 30207[END_REF][START_REF] Nagao | Two Distinct Hemolysins in Trichomonas Tenax ATCC 30207[END_REF][START_REF] Segović | Trichomonas Tenax Proteolytic Activity[END_REF][START_REF] Bo ´zner | Degradation of Collagen Types I, III, IV and V by Extracellular Proteinases of an Oral Flagellate Trichomonas Tenax[END_REF][START_REF] Bo ´zner | Cell-associated and extracellular proteolytic activity of an oral flagellate, Trichomonas tenax[END_REF]. Live trophozoites of the Hs-4 strain of T. tenax failed to induce secretion of IL-1β, IL-8, IL-10 or TNFα from macrophages. Lysed trophozoites stimulated synthesis of IL-8 from phagocytic cells [START_REF] Govro | Cytokine response of human THP-1 macrophages to Trichomonas tenax[END_REF]. The intravenous injection of T. tenax antigen and/or live flagellates in the rabbit elicits the production of antibodies [START_REF] Ioli | Trichomonas tenax in haemodyalized patients-serologic research[END_REF][START_REF] Kott | A Serological Study of Trichomonas Sp. Parasitic in Man[END_REF]. The observation by transmission electron microscopy revealed the presence of bacteria inside the T. tenax cytoplasm: some were dividing while others were dead.

• Research on the potential pathogenicity and invasive power of T. tenax in extra-oral location (Table 3). Flagellates have been isolated from sputum, pleural sample, bronchoalveolar lavage, cervical lymph nodes, vaginal swabs, pharyngeal samples, urines and subhepatic abscess pus [START_REF] Brosh-Nissimov | A false-positive Trichomonas vaginalis result due to Trichomonas tenax presence in clinical specimens may reveal a possible T. tenax urogenital infection[END_REF][START_REF] Dimasuay | First report of Trichomonas tenax infections in the Philippines[END_REF][START_REF] Leterrier | Trichomonads in Pleural Effusion: Case Report, Literature Review and Utility of PCR for Species Identification[END_REF][START_REF] Crucitti | Trichomonas vaginalis is highly prevalent in adolescent girls, pregnant women, and commercial sex workers in Ndola, Zambia[END_REF][START_REF] Chiche | Trichomonas tenax in pulmonary and pleural diseases[END_REF][START_REF] Duboucher | Lymph Node Infection by Trichomonas Tenax: Report of a Case with Co-Infection by Mycobacterium Tuberculosis[END_REF][START_REF] Jakobsen | Trichomonas Species in a Subhepatic Abscess[END_REF]. Generally, the identification was a microscope observation with staining or PCR. The authors concluded an aspiration of the parasite from the oral cavity to the lung location, and autoinoculation via hands or through oral sex for vaginal and urine identification. However, in two case reports, T. tenax could have migrated via the lymphatic vessels to the cervical lymph node and via bloodstream to subhepatic abscess. Some authors suggested a potential pathogenic role and/or flagellate co-infection agent in various infections. But most of these publications are case reports presenting infectious disease in immunocompromised patients.

Risk of bias and quality assessment of selected studies and across studies

• Regarding the case-control studies (Table 1). For some studies, the absence of detailed protocol of collection of the microbiota makes it difficult to draw any conclusions on the localization of the periodontal colonisation of this protozoan: in sub-gingival and/or supragingival biofilm? Even if no study has selected an appropriate sample size of patients to provide sufficient statistical power to detect the flagellate prevalence in periodontitis, studies showing its presence in periodontal pocket could not be discarded. Two of three case-control studies used the microscope to detect T. tenax which may be biased because its phenotypic characteristics are quite similar to those of T. vaginalis and T. hominis, but these species are very rarely described in the mouth [START_REF] Crucitti | Trichomonas vaginalis is highly prevalent in adolescent girls, pregnant women, and commercial sex workers in Ndola, Zambia[END_REF]. Quality assessment of clinical studies is detailed in S2 Table.

• Regarding the randomized controlled trials (Table 1). No periodontal parameters have been established and measurable before and after SRP, periodontal healing cannot be evaluated according to the protozoan decrease. The authors only specified the degree of periodontal degradation with CAL parameter but not the infected periodontal status of the patients (no PPD data). The researcher did not discuss the credibility of their findings with the healing or not of sites with periodontitis, and only reported differences in the frequency of flagellate detection before and after the treatment in both saliva and biofilm sample. S2 Table shows the details of the quality assessment.

• As regards the laboratory studies (Table 2). Numerous publications have explored the potential pathogenicity of T. tenax. However, the authors generally used an ATCC strain that could not fully reflect the virulence of clinical isolates and used cell lines for cytotoxicity testing and cytokine production. Certainly, cell lines can react differently from primary cell lines that constitute the cellular model with the closest similarity to the patient cells. Moreover, the methodological heterogeneity of protocols, the few T. tenax strains available and the xenic culture medium used in some studies make it difficult to evaluate results. No animal model of periodontitis has been found with T. tenax.

Medium used in some studies make it difficult to evaluate results. No animal model of periodontitis has been found with T. tenax.

• As regards the extra-oral location of T. tenax to research the pathogenic potential and invasive power (Table 3). Some studies identified flagellates under the microscope, which is not the most appropriate technique to confirm the trichomonads species. The authors postulated the inhalation of flagellates from the oral cavity to the respiratory tract without any dental sample analysis [START_REF] Dimasuay | First report of Trichomonas tenax infections in the Philippines[END_REF][START_REF] Leterrier | Trichomonads in Pleural Effusion: Case Report, Literature Review and Utility of PCR for Species Identification[END_REF][START_REF] Chiche | Trichomonas tenax in pulmonary and pleural diseases[END_REF]. The majority of publications were case reports or case report series; further investigations are required to conclude of any T. tenax pathogenicity in an extra-oral location.

Discussion

This systematic review has evaluated the role of T. tenax in the etiopathogenesis of periodontitis using Koch's postulates revisited by Socransky as PICO framework. The principle findings were discussed for each postulate and concerned in particular two of them: "Association" and "Virulence factors". Among the various postulates and causal models that could provide evidence of pathogenicity for a putative periodontal pathogen, Koch's postulates revisited by Socransky is the most well-known and used. Unfortunately, the implementation of these postulates is limited to cultivable microbiota and has never be adapted to uncultivable microorganisms identified by molecular biology that could also be suspected of initiating or participating in the pathogenesis of the disease. These Koch's postulates must be adapted to new technologies and could be based on the detection of nucleic acid sequence as suggested by Fredricks and Relman [START_REF] Fredericks | Sequence-based identification of microbial pathogens: a reconsideration of Koch's postulates[END_REF]. But these last postulates cannot be applied to this literature review, because in most of the clinical studies the authors identified T. tenax using microscopy.

It is increasingly clear that periodontitis is the result of the synergy of microorganisms embedded in a biofilm [START_REF] Perez-Chaparro | Newly identified pathogens associated with periodontitis: a systematic review[END_REF]. But among all the detected microorganisms the Socransky modified postulates remains a way to discriminate pathogenic bacteria in periodontitis. In this review, these postulates are applied to protozoan to compare their virulence factors to those of well-known periodontopathogens.

The prevalence of Trichomonas tenax in periodontitis

Periodontitis is an inflammatory disease resulting from dysbiotic microbial communities of pathobiont and keystone pathogens, which develop virulence factors inducing periodontal tissue degradation. The dysbiosis of periodontal microbiota results in the breakdown of the bacterial balance in favour of periodontopathogens and to the detriment of commensal bacteria [START_REF] Hajishengallis | Immunomicrobial pathogenesis of periodontitis: keystones, pathobionts, and host response[END_REF]. The pathogens are present in relative low abundance in the healthy sites and their quantity is proportional to the PPD in diseased sites.

The real prevalence of T. tenax in periodontitis could not be established for two reasons: (i) the patients sample size seems to be insufficient to achieve a statistical significance, (ii) the periodontal parameters were not detailed enough. Nevertheless, this protozoan has been found in healthy sites as well as sites with gingivitis and periodontitis, from 7.1 to 28.2%, 0 to 32.3% and 32.3 to 42.5% respectively. No indication on the quantity of flagellates in the various sites is known. The implementation of the dysbiosis concept for T. tenax cannot be totally excluded as the flagellate is detected in both pathological and healthy sites as indicated for periodontal pathogens [START_REF] Feki | Protozoa of the human oral cavity (epidemiological correlations and pathogenic possibilities[END_REF][START_REF] Benabdelkader | Specific clones of Trichomonas tenax are associated with periodontitis[END_REF][START_REF] Ferrara | Possible pathogenic role of Trichomonas tenax in chronic periodontitis[END_REF][START_REF] Chen | Oral microbiota of periodontal health and disease and their changes after nonsurgical periodontal therapy[END_REF]. The identification method (direct observation or observation of the culture after the incubation time) and the heterogeneity of the sampling protocol make it difficult to conclude on the specific identification of the protozoan in periodontitis due to the morphological similarities with T. hominis and T. vaginalis. Nevertheless, one case-control study used the PCR assay method and confirmed the presence of trichomonad species in samples from both patients with or without periodontitis. Some clinical studies have identified the flagellate with PCR, but they could not be included in this review because authors have selected patients with periodontal diseases as manifestation of systemic disease and/or patients without any description of periodontium [START_REF] Mehr | Prevalence of Oral Trichomonas tenax in Periodontal Lesions of Down Syndrome in Tabriz, Iran[END_REF][START_REF] Dybicz | Studies on prevalence of infection with Trichomonas tenax identified by molecular techniques-in respect to oral health of patients with various systemic disease requiring immunosuppressive therapy[END_REF][START_REF] Crucitti | Trichomonas vaginalis is highly prevalent in adolescent girls, pregnant women, and commercial sex workers in Ndola, Zambia[END_REF].

The postulate "Elimination"

The postulate "Elimination" evaluates the impact of non-surgical periodontal therapy on the decrease or elimination of a putative pathogen in a healed site. Only one randomly controlled trial evaluates the impact of scaling and root planing (SRP) on the frequency of protozoan identification in patients with periodontitis (CAL > 5 mm) [START_REF] Rashidi Maybodi | The Effect of Nonsurgical Periodontal Therapy on Trichomonas Tenax and Entamoeba Gingivalis in Patients with Chronic Periodontitis[END_REF]. Surprisingly, the SRP treatment induced a decrease of T. tenax in saliva but not in dental plaque. Some authors found a high frequency of the flagellate in patients with poor hygiene [START_REF] Wantland | Correlation of some oral hygiene variables with age, sex, and incidence of oral protozoa[END_REF].

One might expect that improved hygiene and a debridement of the periodontal pocket would reduce the number of flagellates, as demonstrated for bacteria [START_REF] Rosalem | Effect of non-surgical treatment on chronic and aggressive periodontitis: clinical, immunologic, and microbiologic findings[END_REF]. Only one periodontopathogen was not eliminated after SRP: Aggregatibacter actinomycetemcomitans [START_REF] Mu ¨ller | Eradication of Actinobacillus actinomycetemcomitans from the oral cavity in adult periodontitis[END_REF]. This pathobiont presents numerous virulence factors and a tissue and cell invasiveness capacity that could explain "its resistance" to non-surgical treatment, but these properties have not been described for flagellates. In the absence of periodontal parameters description before and after treatment and in the absence of dental sample location (subgingival and/or supragingival), it is difficult to draw solid conclusions from this study.

The postulate "Virulence factors"

Numerous authors have evaluated the factors that may respond to the postulate "Virulence factors" in relation to T. tenax. Cellular adhesion is the first step in tissue colonisation for any microorganism. The adhesion and colonisation capacity has clearly been described for T. vaginalis but not for T. tenax [START_REF] Alderete | Specific nature of Trichomonas vaginalis parasitism of host cell surfaces[END_REF][START_REF] Alderete | Pathogenic Trichomonas vaginalis cytoxicity to cell culture monolayers[END_REF]. In 1983, Ribaux et al. described fibronectin-like protein located on plasma membrane and protozoan axostyle, which could potentially have a role in T. tenax adhesion to eukaryotic cells and in the phagocytosis of bacteria. These authors demonstrated a particularly intense labelling localised in the contact zone between bacteria and two strains of T. tenax by indirect immunostaining, one isolated from a patient and the other from a laboratory strain, cultured in xenic medium [START_REF] Ribaux | Immunohistochemical localization of fibronectin-like protein on the cell surface of the oral flagelatte Trichomonas tenax[END_REF]. This aspect was confirmed by Ribeiro et al. who demonstrated the ability of T. tenax ATCC 30207 to adhere to both cell lines and primary cells (patient epithelial cells) [START_REF] Ribeiro | Is Trichomonas tenax a Parasite or a Commensal?[END_REF].

The explanation of the inconsistencies between the Alderete and Ribeiro adhesion results could be attributed to the different times of protozoan cell interaction: thirty minutes (no contact between the flagellates and cells) and 2 hours (visible interaction between the flagellates and the epithelial cells) respectively [START_REF] Ribeiro | Is Trichomonas tenax a Parasite or a Commensal?[END_REF][START_REF] Alderete | Specific nature of Trichomonas vaginalis parasitism of host cell surfaces[END_REF][START_REF] Alderete | Pathogenic Trichomonas vaginalis cytoxicity to cell culture monolayers[END_REF].

Moreover, Ribeiro has also shown the ability of T. tenax to induce cytotoxic effects such as damage and disruption of cell monolayers. The oral protozoan appears to phagocytose membrane portions of MDCK (Mardin-Darby Canine Kidney) epithelial cells and induces membrane blebbing and apoptotic bodies in HeLa cells. The epithelial tissue is the first barrier protecting the periodontium. The potential capacity of T. tenax to induce damage in cell monolayers could promote and facilitate its invasion into the deepest connective tissue. This hypothesis should be confirmed in animal models and with the analysis of gingival tissue of patients with periodontitis. This cytotoxicity would prove the pathogenic role of T. tenax and its potential participation in the cell breakdown described in periodontal diseases [START_REF] Bugueno | Porphyromonas gingivalis Differentially Modulates Cell Death Profile in Ox-LDL and TNF-α Pre-Treated Endothelial Cells[END_REF]. Likewise, Alderete observed no measurable cytotoxicity due to the too short contact of T. tenax with epithelial cell lines [START_REF] Alderete | Specific nature of Trichomonas vaginalis parasitism of host cell surfaces[END_REF].

Numerous articles dealt with the capacity of T. tenax to produce enzymes. The authors have identified some of them and assessed their properties. In 1991, Bo ´zner and Demes, and more recently El Sibaei, identified a temperature-dependant proteinase capable of degrading types I, III, IV and V collagen, that would be cysteine proteinases or metallo-proteinases [START_REF] Sibaei | Growth kinetics, antigen profiling, and proteinase activity of Egyptian Trichomonas tenax isolates derived from patients having oral infections[END_REF][START_REF] Bo ´zner | Degradation of Collagen Types I, III, IV and V by Extracellular Proteinases of an Oral Flagellate Trichomonas Tenax[END_REF][START_REF] Bo ´zner | Cell-associated and extracellular proteolytic activity of an oral flagellate, Trichomonas tenax[END_REF]. These enzymes are cell-associated and extracellular proteinases released from alive T. tenax [START_REF] Bo ´zner | Cell-associated and extracellular proteolytic activity of an oral flagellate, Trichomonas tenax[END_REF]. Moreover, Yamamoto et al. have shown that an ATCC T. tenax strain can produce a specific enzyme that hydrolyses acid soluble type I collagen as well as gelatin, described as cathepsin B [START_REF] Yamamoto | Characterization of the Cathepsin B-like Proteinases of Trichomonas Tenax ATCC 30207[END_REF]. Numerous publications have described one of the main virulence factors in periodontitis: cysteine proteinases, mainly produced by P. gingivalis but also by Tannerella forsythia, are capable of directly degrading host tissues, activating the host proenzymes or neutralizing the host immune system [START_REF] De Diego | Structure and mechanism of cysteine peptidase gingipain K (Kgp), a major virulence factor of Porphyromonas gingivalis in periodontitis[END_REF][START_REF] Sahingur | DNA from Porphyromonas gingivalis and Tannerella forsythia induce cytokine production in human monocytic cell lines[END_REF]. The collagenolytic activities of T. tenax proteases could also, as indicated for bacterial cysteine proteases, participate in host matrix protein damage and play a role in the etiopathogenesis of periodontal diseases [START_REF] Tokuda | Role of Porphyromonas gingivalis protease activity in colonization of oral surfaces[END_REF]. But Bo ´zner et al. concluded that it would be of interest to purify T. tenax enzymes and test their effect on extracellular matrix proteins in the oral cavity [START_REF] Bo ´zner | Cell-associated and extracellular proteolytic activity of an oral flagellate, Trichomonas tenax[END_REF]. In addition to these enzymes, T. tenax seems to be able to produce haemolysins, which are also synthesised by many pathobionts such as P. gingivalis, Prevotella intermedia, A. actinomycetemcomitans and Treponema denticola [START_REF] Hosogi | Monoclonal antibody against Porphyromonas gingivalis hemagglutinin inhibits hemolytic activity[END_REF][START_REF] Balashova | Leukotoxin confers beta-hemolytic activity to Actinobacillus actinomycetemcomitans[END_REF][START_REF] Kachlany | Aggregatibacter actinomycetemcomitans leukotoxin: from threat to therapy[END_REF][START_REF] Chu | Cystalysin, a 46-kilodalton cysteine desulfhydrase from Treponema denticola, with hemolytic and hemoxidative activities[END_REF][START_REF] Nagao | Two Distinct Hemolysins in Trichomonas Tenax ATCC 30207[END_REF].

The postulate "Host responses"

All bacteria regarded as periodontal pathogens can trigger a "Host response" to both innate and adaptative immunity. The only study assessing the response of macrophages to contamination with live ATCC T. tenax strain failed to show pro-inflammatory cytokine production [START_REF] Govro | Cytokine response of human THP-1 macrophages to Trichomonas tenax[END_REF]. Nevertheless, interleukine-8 is the only macrophage-produced cytokine after 6 hours of incubation with flagellate lysates. The absence of cytokine synthesis with live flagellate could be the result of the low ratio of trichomonads: macrophage, 1:5. Certainly, Ribeiro's cytotoxicity assay used an inverted ratio (5:1). T. tenax seems capable of inducing antibody production as shown in the blood analysis of haemodialysis patients [START_REF] Ioli | Trichomonas tenax in haemodyalized patients-serologic research[END_REF]. Moreover, Kott and Adler detected two types of antibodies: one targeting the T. tenax flagella and another an agglutinin, but this result is not relevant to show the potential pathogenicity of T. tenax [START_REF] Kott | A Serological Study of Trichomonas Sp. Parasitic in Man[END_REF]. Indeed, intravenous injection of a commensal bacteria could induce the production of systemic antibodies [START_REF] Macpherson | Interactions between commensal intestinal bacteria and the immune system[END_REF]. Ioli et al. have detected antibodies against T. tenax in the blood of haemodialysis patients without controlling the presence of bone loss around teeth of these patients. So, the conclusion of the flagellate involvement in periodontitis should be cautiously considered. All of these in vitro studies provided some possible responses to the question of the pathogenicity of T. tenax. But these in vitro cellular models generally used cell lines and ATCC T. tenax strain that cannot fully replicate the in vivo interaction between the T. tenax wild-type strain and primary human gingival cells.

Potential pathogenicity in extra-oral location of Trichomonas tenax

T. tenax is described in the literature as an inhabitant of the oral cavity, but some studies have shown this flagellate in extra-oral locations such as the vagina, the respiratory system, the liver and the lymph node of an immunocompromised patient with severe infectious disease. Most of these articles are case reports which have the lowest level of evidence of all clinical studies and conclude that T. tenax has no invasive power (via bloodstream) or pathogenicity. The authors hypothesized that the flagellate moved from the oral cavity to the lung or was self-inoculated via the hands or through oral sex to the vagina and was only a co-factor aggravating the underlying infection. Duboucher et al. could not explain the lymphatic colonization of the flagellate, although an invasion via the lymphatic vessels, and postulated a putative role in the cervical adenopathy of the patient as this protozoan was the only microorganism identified in the lymph node [START_REF] Duboucher | Lymph Node Infection by Trichomonas Tenax: Report of a Case with Co-Infection by Mycobacterium Tuberculosis[END_REF]. Even if three weeks later a culture of the excised lymph node showed Mycobacterium tuberculosis, the authors suggest that the observed caseous necrosis could be attributed not only to M. tuberculosis but also to T. tenax [START_REF] Duboucher | Lymph Node Infection by Trichomonas Tenax: Report of a Case with Co-Infection by Mycobacterium Tuberculosis[END_REF]. Jakobsen speculated about a possible migration through the blood to explain the presence of T. tenax in subhepatic abscess [START_REF] Jakobsen | Trichomonas Species in a Subhepatic Abscess[END_REF]. Only Brosh-Nissimov et al. have reported the presence of the flagellate in extra-oral locations in immunocompetent patients with urogenital infection. These authors concluded that T. tenax could be one of the potential etiologies of this dysuria [START_REF] Brosh-Nissimov | A false-positive Trichomonas vaginalis result due to Trichomonas tenax presence in clinical specimens may reveal a possible T. tenax urogenital infection[END_REF].

Strengths and limitations

As far as is known, this review is the first one to investigate the role of T. tenax in periodontitis using PRISMA guidelines and Koch's postulates revisited by Socransky. These results shown an interesting evaluation of the role of this flagellate in the periodontitis of human adult without any systemic nor genetic disease. Nevertheless, this review presents some limitations: (i) language restriction, (ii) limited number of papers with precise periodontal parameters detecting T. tenax, (iii) majority of the included articles identified this trichomonads by microscope which is not the most accurate technique, (iv) regarding research articles, authors used either ATCC or patient strain which may not be representative of all wild strains in patients with periodontitis (v) and the use of different protocols (MOI, time of incubation, cell lines) makes comparison difficult and does not allow a firm conclusion.

Conclusions

Even if T. tenax appears to be identified in patients with periodontitis, there are still many unanswered questions. The cellular model with flagellate has demonstrated its acknowledged adhesion, cytotoxicity and enzyme production that could contribute to periodontal tissue breakdown brought about by periodontopathogens. Its direct pathogenic role in periodontitis but also in extra-oral infections remains unclear and requires further investigations. Periodontal microbiota dysbiosis is not only an imbalance in the relative abundance of certain microbial species found in diseased sites, but also involves polymicrobial synergy leading to immune dysfunctions that result in alveolar bone and soft tissue destruction. In the absence of direct pathogenicity, T. tenax could be indirectly involved in the etiopathogenesis of periodontitis. Certainly, the bacteria phagocytized by the flagellate may be either lysed or alive inside its cytoplasm and be protected from immune cells and antibiotics action [START_REF] Ribaux | Study of an oral protozoan Trichomonas tenax using scanning and transmission electron microscopy[END_REF]. In this new context, the roles of individual bacteria and their interaction with host and also with other microorganisms such as protozoan must be evaluated.

Fig 1 .

 1 Fig 1. Study flow diagram. https://doi.org/10.1371/journal.pone.0226266.g001

  The five databases MEDLINE, Science Direct, Web of Science, Dentistry and Oral Sciences Source and Cochrane Central Register of Controlled Trials were consulted and our research was conducted on all relevant literature up to March 2019 using MeSH, and the following key-

words: Trichomonas tenax AND (periodontal diseases), Trichomonas tenax AND (periodontitis), Trichomonas tenax AND (antibody) OR (pathogenicity) OR (virulence) OR (enzyme) OR (cytokines) OR (toxicity) OR (adhesion) OR (adherence) OR (invasion) OR (invasive).

  All publications meeting the eligibility criteria were included, processed for data extraction and categorized according to the PICO framework:

	Population: adult patients with periodontitis
	Intervention: presence of trichomonads in sites with periodontitis
	Comparison: absence or reduction of the flagellates in diseased sites after non-surgical
	treatment
	Outcome: potential virulence factors of the protozoan (adhesion, tissue invasion, cytokines,
	antibody, toxicity).

Table, and quality appraisal of included clinical studies was described in S2 Table.

If the study was published twice, the article presenting the most complete data set was selected.

Table 1 . Identification of T. tenax in patient with or without periodontal disease. Postulates Authors Country Study design Population description Sample size (N) Diagnosis periodontal disease Criteria to define periodontal status Selection and identification methods Samples Results: positivity to T. tenax Outcomes-Limitations

 1

	Outcomes: more frequent	detection of T. tenax in patients	with periodontitis than healthy	patients.	Limitations:	� Periodontitis patients: no	trichomonads positivity according	to PPD a and CAL b parameters				Outcomes: patients T. tenax	positive: only periodontitis and	healthy patients.	Limitations:	� Periodontitis patients: no	description of PPD a and CAL b	parameters	� No control of flagellate species	by PCR c	Outcomes: highest percentage of	T. tenax for periodontitis patients,	followed by gingivitis and healthy	patients.	Limitations:	� Periodontitis patients: no	description of PPD a and CAL b	parameters	� Only inclusion of patients	consulting Hospital Dental	Department	� Matching of sample size of the	periodontitis, gingivitis and	healthy groups, but no	information for age and sex	group.	� No control of flagellate species	by PCR c	(Continued )
	� Culture qPCR result:	-34.9% periodontitis	patients	-18.8%% control patients	� Direct qPCR result	-34% periodontitis patients	-28% control patients	� Combination of both	results	-42.5% periodontitis	patients	-28.2% control patients			� 32,3% periodontitis	patients							� 13% patients with healthy	gum	� 32,3% patients with	gingivitis	� 38,5% patients with	periodontitis
	� Pool of multiple sub	gingival plaques	collection per patient	� qPCR c from direct	detection of collected	sample or after culture	of the collected sample.
	� No information on periodontal	indexes.	� Diagnosis of periodontitis according	to probing depth and attachment loss	(Mild, N = 19; Moderate, N = 27,	severe, N = 60)
	� 191 patients	(19 � years�87, 95 males	and 96 females):	-control (Heathy gum,	N = 85)	-Periodontitis (N = 106)						� 59 patients	(25 � years�60, 29 males	and 30 females):	-Heathy gum (N = 14)	-Gingivitis (N = 14)	-Periodontitis (N = 31)				� 300 patients (15 � years	�65)	� Periodontal status:	-healthy periodontium	(N = 100)	-gingivitis (N = 96)	-periodontitis (N = 104)
	Association Benabdelkader	et al. 2019 [8]	France	Case-control	study							Ferrara et al.	1986 [15]	Italy	Case-control	study					Feki et al. 1981	[7]	France	Case-control	study

�

No information on periodontal indexes. � Diagnosis of periodontal disease according to the tooth supporting tissue: Health, superficial (gingivitis) or profound inflammation (periodontitis) � 1 Supra and 1 sub gingival plaque per patient � Microscope observation of culture after incubation of 6 days. � 18,6% all patients � 7,1% healthy patients � 0% gingivitis patients � No information on periodontal indexes � Diagnosis of: -Periodontitis = bone resorption, pocket -Gingivitis = bleeding, modifications of colour and aspect of gingiva. -Health = no modification � One sample of dental plaque, tartric sample per patient � Microscope observation of culture after incubation of 48 hours. � 28,7% all patients (19 < years�65)

Table 1 .

 1 (Continued)

	Postulates Authors	Country	Study design

Population description Sample size (N) Diagnosis periodontal disease Criteria to define periodontal status Selection and identification methods Samples Results: positivity to T. tenax Outcomes-Limitations

	Outcomes: CAL b is representative	of the degree of degradation of	periodontal tissues	Limitations:	� No assessment of outcome, no	description of periodontal indexes	(except for CAL b ), no report of	correlation between periodontal	parameters and T. tenax presence.						� No control of flagellate species	by PCR c
				� Correlation between	severity of periodontitis and	frequency of T.tenax in	dental plaque: r = 0.565,	p�0.005)	� Identification of T.tenax	only in dental plaque of	female	� 3 weeks after SRP,	significant reduction of T.	tenax frequency in saliva but	not in dental plaque.
	For each patient,	collection of:	1 dental plaque and	1 saliva samples
					± 1.7mm
	Elimination Rashidi Maybodi 46 patients	et al.2016 [16] (30<years<50)	Iran � case group (15 with	Randomized severe and 8 with	Clinical trial moderate periodontitis)	� control group (14 with	severe and 9 with	moderate periodontitis)	� exclusion: previous	consumption of	antibiotics,	immunomodulators				PPD a : Periodontal Pockets Depth,	CAL b : Clinical Attachment Level,	PCR c : Polymerase Chain Reaction/	qPCR c : quantitative PCR.

�

Moderate (CAL = 3-4 mm) to severe (CAL� 5 mm) periodontitis (classification of

Armitage, 1998)

. � CAL of case group: 5.74 ± 1.7mm � CAL of control group: 5.64 � For control group, samples collected before OHI � For case group, samples collected before OHI and 3 weeks after SRP. � Giemsa staining and microscope observation of sample � Baseline frequency (%) of T.tenax for both group: not reported � No report of blindness of data collection � Only inclusion of patients consulting School of Dentistry. � No information on level of infection (PPD a not reported). https://doi.org/10.1371/journal.pone.0226266.t001

Table 2 . Potential virulence factors of T. tenax.

 2

	Postulates	Authors	Experiment	Results	Outcomes-Limitations
	Virulence	Ribeiro et al.	� Adhesion T. tenax ATCC a 30207 (axenic	tenax	Outcomes: capacity to adhere on different cells,
	factors	2015 [17]	medium)-mammalian cell monolayer	cytotoxicity on MDCK c and HeLa e	to penetrate inside 3D spheroids and to induce
	Adhesion,		(epithelial cells, HeLa e , MDCK c ) (SEM study).	viability.	cytotoxic effects and damages of cells monolayer
	Tissue		� Cytotoxicity assays (MOI 5:1, flagellates: cells	� Significant damage of 3D spheroid by	(disrupting of cells monolayer, phagocytose of
	invasion,		HeLa, MDCK) MTT b evaluation.	T. tenax	membrane portions of MDCK c cells, induction
	Cytotoxicity		� Interactions (MOI h 1:1) T. tenax-3D	� 3D spheroid penetration of T. tenax	of apoptotic bodies and membrane blebbing of
			spheroid (epithelial cells and fibroblasts) SEM f	� Damage and disrupting of MDCK c	HeLa e cells) = evidence of virulence supporting
			study, during 2,6,12 & 24h.	and HeLa e monolayers by T. tenax.	a pathogenic role for T. tenax.
				� Adhesion of T. tenax on different cells	Limitations: in vitro cellular model not fully
				(MDCK c , HeLa, gum cells, 3D	reflect clinical reality. Differences between
				spheroid)	virulence factors of ATCC a and wild clinical
					strains.
		Alderete et al.	� Adhesion T. tenax ATCC a (axenic medium.)	� T. tenax: no adhesion, no damage to	Outcomes: no adhesion and toxicity capacity of
		1985 [18]	or T. vaginalis-Cells monolayer (HEp-2 d ,	HeLa	T. tenax ATCC (lack of cell adhesion and HeLa e
			HeLa e and human fibroblast cell lines).	� T. vaginalis: adhesion to cell	cytotoxicity capacities).
			� Interactions (MOI h 5:1) T. tenax-cells,	monolayers, cytotoxicity on HeLa e	Limitations: time of contact for test adherence
			during 30 minutes.		too fast. Differences between virulence factors
					of ATCC a and wild clinical strains.
		Alderete et al.
		1984 [19]
		Ribaux et al.
		1983 [45]
	Virulence	El Sibaei et al.			Outcomes: capacity of clinical isolate of T.
	factors	2012 [20]			tenax to have proteolytic activity, putative
	Enzymes				virulence factor involving in degradation of
					periodontal tissues.
					Limitations: in vitro study not always reflect of
					clinical reality, microscopic identification of
					trichomonads without control by PCR j of
					species.
		Yamamoto			Outcomes: T. tenax capacity to produce
		et al. 2000			proteinase potentially hydrolysing acid soluble
		[21]			type I collagen, gelatin and participating to
					degradation of periodontal tissues.
					Limitations: T. tenax ATCC strain no
					representative of all virulence factors of clinical
					wild strains.
		Nagao et al.	� Test of hemolytic activities of T. tenax ATCC a		Outcomes: T. tenax ATCC capacity to produce
		2000 [22]	30207 (axenic medium) (intact cell, culture		hemolysins: potential virulence factor
			supernatant, culture filtrate, cells debris and	inhibited by various cysteine-proteinase	contributing to the periodontal disease process.
			lipid enriched fractions) on animal and human	inhibitors	Limitations: in vitro study and ATCC strain: no
			erythrocytes.	� Other hemolysin lipid-like (in intact	representative of all virulence factors of clinical
			� Identification of enzyme with chemical and	cells, cell-debris, lipid enriched	wild strains.
			proteinase inhibitors.	fractions)
		Segovic et al.	� T. tenax (dental plaque, axenic broth	� Major proteolytic activity at acid pH	Outcomes: capacity of clinical isolates to
		1998 [23]	medium.)	and weak one at basic pH	synthesize endopeptidases: potential virulence
			� Analyse of protozoan lysates	� Identification of different	factor in the periodontal disease process
				endopeptidases in cell lysates	Limitations: in vitro study = not fully
					representative of mechanisms of patient disease.
					(Continued )

� Significant time dependent T.

� Cytotoxicity: T. tenax ATCC a (axenic medium) or T. vaginalis NHY 286 -epithelial cells (HEp-2 d and HeLa e ) � Interactions (MOI h 1:1, 5:1), T. tenax-cells, during 20h. � Evaluation of viability of host cells (trypan blue uptake and release of 3 H-thymidine of labelled cells)

� No or little cytotoxicity of HeLa e cell monolayer due to T. tenax. � Extensive disruption of monolayers and irreversible cellular damage due to alive T. vaginalis.

Outcomes: no measurable cytotoxicity on HeLa e cells by T. tenax = commensal protozoan of normal flora of oral cavity. Limitations: in vitro study not always reflect clinical reality, cytotoxicity from only one strain of T. tenax, cell lines no representative of cytotoxicity on primary cell line.

� Two strains of T. tenax (one from patient ulcerative gingivitis, other from laboratory, xenic medium) � Research of fibronectin (immunofluorescence technique)

� Fibronectin like protein: localisation on plasma membrane and axostyle. � Areas of intense labelling at contact zones between protozoa and bacteria.

Outcomes: capacity to produce fibronectin like protein with potential role in adhesion of T. tenax to gingival cells, connective tissues and in phagocytosis of bacteria = potential evidence of involvement in microbiota dysbiosis of periodontal diseases. Limitations: in vitro study of protozoan interactions not always reflect of clinical reality.

� T. tenax from patients with periodontitis, gingivitis. � Analysis of protein profile of lysates by SDS-PAGE i and proteinases by nondenaturing gelatin-SDS-PAGE.

� Identification of 19 proteinases bands, among them detection of cysteineproteinase.

� T. tenax ATCC a 30207 (axenic medium). � Identification of enzyme by electrophoresis and effect of enzyme inhibitors � Identification of cathepsin B-like protein � One hemolysin protein-like (supernatant, filtrate of T. tenax), � Culture filtrate of T. tenax (private strain, axenic medium). � � Lysates and culture filtrate of T. tenax (private strain, axenic medium). � Test of specific inhibitors on T. tenax proteolysis activity. � Electrophoretic analysis of T. tenax proteinases.

� Macrophage (THP-1 cells stimulated) � Hs-4 strain T. tenax (axenic medium) � MOI h T. tenax: THP (1:5, 1:10, 1:20 with alive and lysed T. tenax) during 4,8 & 16h. � Analysis of cytokines (TNFα, IL-1β, IL-8 & IL-10) � No cytokine production with alive T. tenax � Higher IL-8 after 16h with dead T. tenax (higher MOI h , 1:5) Outcomes: no production of cytokines such as TNFα, IL-1β & IL-10 after interactions of macrophage with live or lysed T. tenax.

� Antigen (centrifugation of cyclic cryolysis laboratory strain of T. tenax). � Antibody production (administration of T. tenax antigen in rabbit) � Analysis of T.tenax antibody in haemodialyzed blood of patients. � Detection of antibodies against T. � T. vaginalis and T. hominis (patient strains, axenic medium). � T. tenax (xenic medium). � Intravenous injections of alive flagellate suspension in rabbits. � Agglutination tests � Detection of 2 types of T. tenax i : Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis, PCR j : Polymerase Chain Reaction. https://doi.org/10.1371/journal.pone.0226266.t002

Table 3 . Extra-oral location of T. tenax: Research of invasion, colonization and pathogenicity capacity.

 3

	Authors	Population-Case Report	Sample	Results	Outcomes-Limitations
		Medical history	Protozoan identification
	Brosh-Nisimov	22 soldiers T. vaginalis + (8	� Pharyngeal and urine	� 3/14 (21%) urine samples T.tenax	Outcomes: presence of T. tenax in urine = possible
	et al. 2018 [29]	pharyngeal samples, 14	samples	+ with 2 patients with symptoms	role in urogenital infection (patients suffering from
	Case report	urines)	� Anyplex™ kit and Xpert-	� 100% pharyngeal samples T.	dysuria, hematuria, urolithiasis), and presence in
	series	using Multiplex PCR a tests	TV™ tests	tenax +	pharyngeal sample with only one patient suffering
			To detect false sample T.	Only 1 patient with symptoms	from tonsillitis. Hypothesis of urine contamination
			vaginalis +		from saliva.
					Limitations: no T. tenax research in saliva to
					confirm contamination.
	Dimasuay and	� 44 patients suffering from	� Sputum		Outcomes: detection of T. vaginalis in vaginal and
	Rivera 2014	respiratory ailment	� PCR a (amplification of		T. tenax in sputum samples. Assumption authors:
	[30]		18S rRNA b )		migration of T. tenax from oral cavity to the
	Philippines Cohort study	� 186 sex workers	� Microscope observation /patient � 2 vaginal swab samples	� 19 vaginal swabs T. vaginalis +	respiratory tract, involvement in aggravation of Limitations: no research of T. tenax in oral cavity. respiratory infection, no capacity to cause infection.
			� PCR (amplification of
			18S rRNA b )
	Leterrier et al.	Case report: woman	� Pleural fluid sample	� Identification of T. tenax and	Outcomes: T. tenax identification in
	2012 [31]	(67years)	� Microscope observation	aero-anaerobic flora in sample	immunocompromised patient (high corticotherapy
	France	� medical history:	� Culture	� Improvement with amoxicillin	dose). Assumption authors: possible migration of
	Case report	glioblastoma	� PCR a	after metronidazole treatment	flagellates to the respiratory tract by aspiration of
		� After excision of tumoural		� Finally, patient died	oropharyngeal secretions. Possible role of co-
		cyst and corticotherapy,			infecting agent of T. tenax.
		detection of pleural effusion			Limitations: no identification of pathogen bacterial
					flora in sample.
	Crucitti et al.		� Self-collection from	� Identification in vaginal
	2010 [32]		vagina, rectum and oral	infection of:
	Zambia		cavity	-T. vaginalis: 24,6% ado, 32,2%
	Cohort study		� PCR a	pregnant, 33,2 sex
				-T. tenax: 0.7% ado, 0.3% pregnant
	Chiche et al.	Case report: men (84 years)		� Direct examination of	Outcomes: putative pathogenicity of trichomonads
	2005 [33]	� medical history: chronic		bronchoalveolar lavage was T.	in older immunocompromised patient. Assumption
	France	lymphoid leukemia with		tenax +	authors: flagellates colonization of tracheo-
	Case report	respiratory and fever		� Decrease of fever with	bronchial tree after inhalation from oral cavity.
		� ventilator dependent		metronidazole treatment	Limitations: no control of trichomonads' species by
				� Finally, patient died	PCR a , no research of its presence in oral cavity.
	Duboucher	Case report: woman (82	� Needle aspiration of	lymph
	et al. 2000 [34]	years)	cervical lymph node and	liquid, cytologic preparation,
	France	� medical history: fever,	dental scraping	dental scraping	tenax
	Case report	asthenia and cervical	� May-Gru ¨nwald-Giemsa	� Identification Mycobacterium	potential invasion of lymphatic vessels after passing
		adenopathy	staining Microscope	tuberculosis in culture of excised	through oral mucosa. Possible facilitation of
		� edentate patient without	observation	lymph node.	flagellate colonization by tuberculosis.
		any mucosal wounds nor	� Culture of cervical	� With metronidazole therapy,	Limitations: low-level evidence of invasion and
		fistula	effusion after lymph node	healing of cervical lesion,	pathogenicity capacity of T. tenax in
			excision	persistence of fever	immunocompromised patient (case-report). No
			� Transmission electron		explanation about origin of T. tenax. No control of
			microscopy observation.		trichomonads' species by PCR a .
	Jakobsen et al.	Case report: man (52year-	� Aspirated pus from	� Identification of numerous	Outcomes: detection of flagellate microorganism
	1987 [35]	old)	subhepatic abscess.	bacteria and flagellates (T. tenax or	morphological consistent with T. tenax or T.
	Case report	� medical history: abdominal	� Microscope observation	T. hominis).	hominis in pus and numerous bacteria
		pain, alcoholic,		� No improvement with	(Streptococcus salivarius, S. milleri, Prevotella
		� subhepatic abscess		intravenous antibiotic	melanogenica (formerly Bacteroides melanogenicus)
		� partially edentulous		(metronidazole + penicillin).	and B. capillosus.
				� Finally, patient died	Limitations: no PCR a to control trichomonads'

� 14 sputums T. tenax + � 1 sputums T. gallinae + � 460 adolescent girls � 307 pregnant women � 197 sex workers (= sex) � Identification in oral cavity of: -T. vaginalis: 0.7% pregnant, 1.1% sex -T. tenax: 5.3% pregnant, 4.3% sex Outcomes: colonization of pregnant woman and sex workers by T. tenax and T. vaginalis both in vaginal and oral samples. Assumption authors: identification of T. tenax in vaginal swab after autoinoculation via hands or through oral sex. Limitations: no report of data about periodontal status of patients and about simultaneous presence of T. tenax in mouth and vagina. � Bronchoalveolar lavage � Microscope observation � Detection of T. tenax in � Finally, patient died Outcomes: detection of T. tenax in lymph node. Role of M. tuberculosis (possible role for T. tenax) in caseous necrosis. Assumption authors: T. species. Low-level evidence of migration from oral cavity to liver and of pathogenicity of Trichomonads (no search of T. tenax in dental sample). PCR a : Polymerase Chain reaction; rRNA b : ribosomal ribonucleic acid. https://doi.org/10.1371/journal.pone.0226266.t003

PLOS ONE | https://doi.org/10.1371/journal.pone.0226266December 17, 2019

This systematic review was supported by a grant from the Conseil Regional du Grand Nancy.

All relevant data are within the paper and its Supporting Information files.

Supporting information