The world health organization (WHO) considered periodontitis as a handicapping disease due to its damaging consequences affecting a population’s health (WHO, 2016). Entamoeba gingivalis parasitic trophozoites were frequently detected in oral cavities, yet the association of periodontitis with this protozoan is still debatable, fluctuating from complete accusation to intense rejection of such an association (Bonner et al., 2018). Very few published scientific studies relied on the specific parasitological parameters to identify the oral protozoan microscopically, yet nearly all of them confirmed its 100% occurrence within progressive periodontal pockets and from 0 to 26% within healthy periodontal sites (Fisher, 1927; Hinshaw and Simonton, 1928; Wantland and Wantland, 1960; Wantland and Lauer, 1970; Gottlieb and Miller, 1971; Keyes and Rams, 1983; Lange, 1984; Linke et al., 1989 and Hassan et al., 2019). Unfortunately, for unknown reasons, all these articles neglected the difference in morphological characteristics between the detected trophozoites among the diseased and healthy periodontal sites, except Hassan et al. (2019) in which detailed information was reported, yet not specifically with the different conditions. That is why this study aimed to investigate this structural issue in addition to the molecular investigation proposed to detect genetic variability within the isolates.
The current study revealed significant morphological differences between E. gingivalis trophozoites within diseased and healthy sites including the size of parasitic stages, specific morphological variations in addition to the mean number of trophozoites between the 2 categories. Detailed morphological features reported in this study were sufficient to differentiate the parasitic stages within the diseased and healthy periodontal sites. This was in contrast to the findings reported by García et al. (2018) in which morphological characteristics were ignored and excluded by the authors. They attributed this to the ineffectuality of microscopic examination to differentiate between amoebas. Similarly, other authors reported similar findings concerning morphology, in which no characteristic findings were reported (Clark et al. 2006; Jacob et al. 2016; Ponce-Gordo and Martinez-Diaz 2010; Stensvold et al. 2011; Weedall and Hall 2011).
García et al. (2018) highlighted an example for such a problem, however, it was related to E. histolytica and E. dispa. They stated that both amoebas are morphologically similar, but their genetic and pathogenic differences allow them to be classified as independent, although closely related species (Clark 2000). However, many authors recorded a clear microscopic difference related to erythrophagocytic activity which was observed in the former species but never observed in the latter one.
These unique microscopic findings are certainly in need of professional specialists, not to be misdiagnosed or misinterpreted. Thereby supporting the urgent need for real engagement of multidisciplinary diagnostic strategies, involving specialists from a variety of medical fields to reach truthful scientific outcomes concerning the association of certain microorganisms with particular diseases or disorders.
Morphologically in this study, in addition to the observed characteristic nuclei, leuckophagocytic vacuoles were found within all the trophozoites isolated from affected periodontal sites. This may be related to certain virulence factors, facilitating the invasive power of E. gingivalis trophozoites, which is more or less similar to what is reported by Bansal et al. (2009), but in connection with erythrophagocytic ability of Entamoeba histolytica. This ability was not documented within the non-pathogenic E. dispar which has morphological features similar to E. histolytica. Phagocytosis of blood elements has been proposed as a pathogenicity indicator as well as a prominent virulence marker for Entamoeba species (Trissl et al., 1978 and Bhattacharya et al., 2002).
The significant difference regarding the larger size of E. gingivalis trophozoites identified within the diseased sites, possibly resulting from engulfing several WBCs which may have cause cytoplasmic expansion. E. gingivalis can engulf one or more human cells at a time, mainly polymorphonuclear cells and neutrophils. These are the chief cells within the periodontal pockets, so engulfing these cells may expose the first line of the innate immune mechanism to intense danger by consuming its powerful weapon (Bonner et al., 2014). Thus, E. gingivalis finds a perfect environment involving damaged tissue, bacteria, and fungi, permitting its safe embellishment and colonization. This may explain the significantly higher number within samples isolated from the diseased cases, taking into consideration that all the enrolled subjects were immunocompetent. This intense colonization within immunocompetent subjects was previously reported in other work published by Hassan et al. (2019).
Garcia et al. (2018) suggested the existence of different parasitic subtypes with the higher proliferative ability and/or proteolytic activity. This explains why this study intended to molecularly scan the different isolates, performing post-real-time PCR HRM to detect possible genetic variability between them. In this study, a significantly higher number of wild-type trophozoites was reported within healthy subjects. While a higher number of mutants was reported within diseased cases which were related to the known H57 strain of Cembranelli et al. (2013), plus unknown mutants revealed by HRM analysis (14 out of 26). Mutations observed in this work may have been triggered as a result of changes within the oral environment, which possibly turn the commensal amoebae into a pathogenic one, creating a vicious circle that facilitates more periodontal damage without cellular immune controlling mechanism, resulting in acceleration of amoebic colonization and so on. This is in concordance with the report of Tonetti et al. (2018) who documented the presence of E. gingivalis in some sulci that were not causing any symptoms as well as in diseased sites. However, certain conditions that change the surrounding environment can cause pathogenic conditions in the oral cavity.
In general, molecular tools such as specific target gene amplification and sequencing certainly expand genetic information concerning E. gingivalis. The work of Yamamoto et al. (1995) succeeded to molecularly identify this oral protozoan, using a long amplicon of 1400 bp. Their work has invoked more debate concerning the molecular identity of the parasite documented by various investigators. Kikuta et al. (1996) and Trim et al. (2011) molecularly detected the parasite only in diseased periodontal sites. While Bonner et al. (2014) and Deng et al., (2017) molecularly identified the parasite within 33.3% and 60% of healthy subjects respectively. The authors explained their higher molecular results within the healthy subjects to be due to their accurate control of PCR inhibitors and matrix degradation.
Genetic variability of E. gingivalis has been recognized by Cembranelli et al. (2013) and Garcia et al. (2018b) by studying the genetic variability of 18S-SSU rDNA. Furthermore, genetically identical E. gingivalis species revealed different virulence factors as reflected by the transcriptomic study (Santi-Rocca et al., 2008). This may explain the discrepancies in their molecular detection when compared with microscopy or clinical diagnosis as reported in this study. García et al. (2018) documented the presence of a kamaktli E. gingivalis variant which is located within a closely related clade to ST1, but the authors confirmed its mutability and named it as ST2. However, they did not mention any link to pathogenesis or anything related to the periodontal manifestation. Formerly, Clark and Diamond (1997) characterized three samples of E. gingivalis by riboprinting the 18S rRNA region. Differential banding patterns observed after sample treatment with the restriction enzyme Rsa1 allowed them to categorize these 3 samples into two ribodemes: ribodeme-1 (two oral isolates) and ribodeme- 2 (one uterine isolate).
Dissimilar band patterns created by kamaktli variants (Garcia and his co-workers, 2018) suggested its disparity from both ribodemal strains 1 and 2. The authors suggested that the unidentified E. gingivalis genetic type described by Cembranelli et al. (2013) in immunocompromised patients is possibly related to the kamaktli variant.
On the other hand, no cyst stage was found within our samples and all the observed parasitic stages were related to the E. gingivalis trophozoite stage. This was in concordance with most of the aforementioned documented reports. The report of García et al. (2018) triggered a new debate about E. gingivalis when they suggested a possible ability of their kamaktli variant to produce a cyst stage which had never been suggested before, still leaving the morphology of such a variant in the pending situation. Before this, Stensvold et al. (2011) advised the investigators in this field to postpone any formal taxonomic categorization until clear morphological data are obtainable. However, the kamaktli variant was categorized by the authors without any identifiable morphological characteristics, even the parasitic stage’s level. Trim et al. (2011) discussed this issue and concluded that; E. histolytica is the only well-documented human pathogen among all known amoebae and recommended the urgent need of an accurate and efficient detection methodology to correctly document other types of amoebae. Unfortunately, the debate concerning the role or the ability of some amoebae to initiate pathogenic effects within certain tissues (as in E. gingivalis) is still considered as a big obstacle facing the deeper understanding of its role in the pathogenicity of periodontal infection.
In this study, samples were collected from at least three different sites in each subject and confirmed positive after repeated microscopic examination by specialists. Only samples with positive microscopic confirmation were included, otherwise, all samples proven to be negative were excluded. This may be due to the nature of our study which was not intentionally designed to compare variable diagnostic tools. The design was mainly targeting the detection of morphological and genetic diversity within different isolates. Therefore, parasitological confirmation, involving detailed morphological features was a must. This possibly facilities specific figuring of isolates from different subject categories and possibly supported distinguishing each of them. Real-time PCR is an extremely sensitive molecular technique that operates within a closed system, thus avoiding the possible contamination during the post-polymerization process in conventional PCR. EvaGreen dye was used in our technique which is a third-generation dye developed to avoid PCR inhibition which may occur during the use of other dyes (Martino et al., 2010). In the current study, another benefit was achieved by this molecular technique which was the facilitation of simultaneous discovery of mutations using HRM software analysis and synthetic reference strains. Together, parasitological and molecular results supported the concept that E. gingivalis is one of the contributors to periodontal diseases, hence necessitating proper management to avoid the unnecessary destructive impact of this oral protozoal infection.
In conclusion, this study confirmed the morphological and genetic variability between different isolates and recommends further in-depth molecular studies to investigate the role of this oral protozoan in the pathogenicity of periodontal affection. This study also highlighted the importance of real engagement of multidisciplinary diagnostic strategies, involving experts from a variety of medical fields to reach truthful scientific outcomes concerning the association of certain microorganisms with particular diseases or disorders.