Abstract
Investigators using light microscopy have identified the protozoan parasite Entamoeba gingivalis from diseased gingival pockets for nearly 100 years. The objective of the present investigation was to develop a molecular biology approach for determining the presence of E. gingivalis in both diseased gingival pockets and healthy gingival sites. For this, a previously developed conventional polymerase chain reaction (PCR) was evaluated and a real-time polymerase chain reaction assay was developed. Paper points were inserted into the base of the sulcus of both diseased gingival pockets and healthy gingival sites. DNA was extracted using the QIAamp DNA mini kit, and subsequently analyzed using conventional and real-time PCR analysis. A previously described primer set specific for the small subunit ribosomal RNA gene (SSU rDNA) of E. gingivalis was used for the conventional PCR. For the real-time PCR, a primer set was designed to amplify a 135-bp fragment inside the SSU rDNA of E. gingivalis. A conventional PCR assay detected E. gingivalis in 27% of diseased gingival pockets. The real-time PCR using a different primer set detected protozoa in 69% of diseased pocket sites. Thus, the latter technique proved more sensitive for detection of E. gingivalis. No E. gingivalis were detected in any of the healthy gingival pocket sites using either type of PCR assay. Results support a concept that the presence of E. gingivalis is associated only with diseased gingival pocket sites. The newly described methodology may also serve to provide a novel eukaryotic cell marker of disease status in gingival pockets.
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References
Armitage GC (1999) Development of a classification system for periodontal diseases and conditions. Northwest Dent 79:31–35
Ashimoto A, Chen C, Bakker I, Stots J (1996) Polymerase chain reaction detection of 8 putative periodontal pathogens in subgingival plaque of gingivitis and advanced periodontitis lesions. Oral Microbiol Immunol 4:266–273
Barrett MT (1914) The protozoa of the mouth in relation to pyorrhea alveolaris. Dent Cosm 56:948–953
Bass CC, Johns FM (1915) Alveolodental pyorrhea. WB Saunders, Philadelphia
Bruchhaus I, Loftus BJ, Hall N, Tannich E (2003) The intestinal protozoan Entamoeba histolytica contains 20 cysteine protease genes, of which only a small subset is expressed during in vitro cultivation. Eukaryot Cell 2:501–509
Burt B (2005) Position paper: epidemiology of periodontal diseases. J Periodontol 76:1406–1419
Cirillo JD, Falkow S, Tompkins LS (1994) Growth of Legionella pneumophilia in Acanthamoeba castellanii enhances invasion. Infect Immun 62:3254–3261
Cirillo JD, Falkow S, Tompkins LS, Bermudez LE (1997) Interaction of Mycobacterium avium with environmental amoebae enhances virulence. Infect Immun 65:3759–3767
Clark CG, Diamond LS (1991) The Laredo strain another Entamoeba histolytica-like amoebae are Entamoeba moshkovskii. Mol Biochem Parasitol 46:11–18
Clark CG, Diamond LS (1997) Intraspecific variation and phylogenetic relationships in the genus Entamoeba as revealed by riboprinting. J Eukaryot Microbiol 44:142–154
Dao AH (1985) Entamoeba gingivalis in sputum smears. Acta Cytol 29:623–633
Dao AH, Robinson DP, Wong SW (1983) Frequency of Entamoeba gingivalis in human gingival scrapings. Am J Clin Pathol 80:380–383
Derderian GM (1991) An in vitro study of the co-cultivation of Actinobacillus actinomycetemcomitans (MS thesis). Indiana University School of Dentistry, Indianapolis
Fields MW, Yan T, Rhee SK, Carroll SL, Jardine PM, Watson DB, Criddle CS, Zhou J (2005) Impacts on microbial communities and cultivable isolates from groundwater contaminated with high levels of nitric acid-uranium waste. FEMS Microbiol Ecol 53:417–428
Gannon JT, Linke HA (1992) Synergistic growth studies of Entamoeba gingivalis using an ecologen. Int J Parasitol 22:927–931
Gilchrist CA, Houpt E, Trapaidze N, Fei Z, Crasta O, Asgharpour A, Evans C, Martino-Catt S, Baba DJ, Stroup S, Hamano S, Ehrenkaufer G, Okada M, Singh U, Nozaki T, Mann BJ, Petri WA Jr (2006) Impact of intestinal colonization and invasion on the Entamoeba histolytica transcriptome. Mol Biochem Parasitol 147:163–176
Gottlier DS, Miller JH (1971) Entamoeba gingivalis in periodontal disease. J Periodontol 42:412–415
Greub G, Raoult D (2004) Microorganisms resistant to free-living amoebae. Clin Microbiol Rev 17:413–433
Haake SK, Meyer DH, Fives-Taylor PM (2006) Periodontal diseases. ASM Press, Washington
Jervoe-Storm PM, Koltzscher M, Falk W, Dorfler A, Jepsen S (2005) Comparison of culture and real-time PCR for detection and quantification of five putative periodontopathogenic bacteria in subgingival plaque samples. J Clin Periodontol 32:778–783
Keyes PH, Rams TE (1983) A rational for management of periodontal diseases: rapid identification of microbial ‘therapeutic targets’ with phase microscopy. J Am Dent Assoc 106:803–812
Kikuta N, Yamamoto A, Goto N (1996) Detection and identification of Entamoeba gingivalis by specific amplification of rRNA gene. Can J Microbiol 42:1248–1251
Krogstad DJ, Spencer HC Jr, Healy GR, Gleason NN, Sexton DL, Herron CA (1978) Amebiasis: epidemiologic studies in the United States, 1971-1974. Ann Intern Med 88:89–97
Li E, Yang WG, Zhang T, Stanley SL Jr (1995) Interaction of laminin with Entamoeba histolytica cysteine proteinases and its effect on amebic pathogenesis. Infect Immun 63:4150–4153
Linke HA, Gannon JT, Obin JN (1989) Clinical survey of Entamoeba gingivalis by multiple sampling in patients with advanced periodontal disease. Int J Parasitol 19:803–808
Lyons T, Scholten T, Palmer JC, Stanfield E (1983) Oral amoebiasis: the role of Entamoeba gingivalis in periodontal disease. Quintessence Int 12:1243–1248
Petri WA Jr, Haque R, Mann BJ (2002) The bittersweet interface of parasite and host: lectin-carbohydrate interactions during human invasion by the parasite Entamoeba histolytica. Ann Rev Microbiol 56:39–64
Pimenta PFP, Diamond LS, Mirelman D (2002) Entamoeba histolytica Schaudinn, 1903 and Entamoeba dispar Brumpt, 1925: differences in their cell surfaces and in the bacteria-containing vacuoles. J Eukaryot Microbiol 49:209–219
Ravidn JI (1986) Pathogenesis of disease caused by Entamoeba histolytica: studies of adherence, secreted toxins, and contact-dependent cytolysis. Rev Infect Dis 8:247–260
Roy S, Kabir M, Mondal D, Ali IK, Petri WA Jr, Haque R (2005) Real-time PCR assay for diagnosis of Entamoeba histolytica infection. J Clin Microbiol 43:2168–2172
Sajid M, McKerrow JH (2002) Cysteine proteases of parasitic organisms. Mol Biochem Parasitol 120:1–21
Socransky SS (1977) Microbiology of periodontal disease—present status and future considerations. J Periodontol 48:497–504
Stanley SL Jr, Zhang T, Rubin D, Li E (1995) Role of the Entamoeba histolytica cysteine proteinase in amebic liver abscess formation in severe combined immunodeficient mice. Infect Immun 63:1587–1590
Tillack M, Biller L, Irmer H, Freitas M, Gomes MA, Tannich E, Bruchhaus I (2007) The Entamoeba histolytica genome: primary structure and expression of proteolytic enzymes. BMC Genomics 8:170
Wantland WW, Laurer D (1970) Correlation of some oral hygiene variables with age, sex, and incidence of oral protozoa. J Dent Res 49:292–297
Zhang H, Bhattacharya D, Lin S (2004) Phylogeny of dinoflagellates based on mitochondrial cytochrome B and nuclear subunit rDNA sequence comparisons. J Phycol 41:411–420
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Trim, R.D., Skinner, M.A., Farone, M.B. et al. Use of PCR to detect Entamoeba gingivalis in diseased gingival pockets and demonstrate its absence in healthy gingival sites. Parasitol Res 109, 857–864 (2011). https://doi.org/10.1007/s00436-011-2312-9
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DOI: https://doi.org/10.1007/s00436-011-2312-9