BR Biomedical Reports 2049-9434 2049-9442 D.A. Spandidos 10.3892/br.2019.1245 BR-0-0-1245 Articles Red complex periodontal pathogens are risk factors for liver cirrhosis NagaoYumiko 1 2 TanigawaTakeshi 1 Department of Public Health, Graduate School of Medicine, Juntendo University, Tokyo 113-8421, Japan Department of Pathology and Microbiology, Faculty of Medicine, Saga University, Saga 849-8501, Japan Correspondence to: Professor Yumiko Nagao, Department of Public Health, Graduate School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan y.nagao.qd@juntendo.ac.jp

Abbreviations: HCV, hepatitis C virus; HBV, hepatitis B virus; CH-C, chronic hepatitis C; HCC, hepatocellular carcinoma; LC, liver cirrhosis; PBC, primary biliary cholangitis; OLP, oral lichen planus; IFN, interferon; DM, diabetes mellitus; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; BMI, body mass index; RBC, red blood cell; WBC, white blood cell; Plt, platelet; Hb, hemoglobin; FBS, fasting blood glucose; ALT, serum alanine aminotransferase; AST, aspartate aminotransferase; LDH, lactate dehydrogenase; ALP, alkaline phosphatase; PT, prothrombin time; T. Bil, total bilirubin; T. cho, total cholesterol; T. pro, total protein; Alb, albumin

 11 2019 03 10 2019 11 5 199 206 24 06 2019 23 09 2019 Copyright: © Nagao et al. 2019 This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Periodontal disease has been associated with liver disease; however, the identities of the periodontal disease-causing bacteria in patients with viral liver disease remain unknown. The aim of the present study was to determine the counts of the 3 periodontal pathogens that form the red complex in chronic periodontitis (Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola) and 3 other bacteria (Aggregatibacter actinomycetemcomitans, Prevotella intermedia and Fusobacterium necrophorum) in patients with liver disease. A total of 47 patients with liver disease were divided into two groups based on the counts of the red complex bacteria: group A (high counts of red complex bacteria) and group B (low counts of red complex bacteria). The counts of the 6 types of bacteria in saliva and the prevalence of P. gingivalis-specific fimA genotype were determined. In addition, salivary occult blood tests and serological assays were performed. Univariate and multivariate logistic regression analyses were used to analyze the risk factors between the two groups of patients. Hepatitis C virus-related liver disease was the most frequent (41/47; 87.2%) occurrence followed by liver cirrhosis (LC; 12/47; 25.5%) and oral lichen planus (32/47; 68.1%). The significant risk factors between the two groups were LC, albumin (Alb) level, ratios of each bacteria and prevalence of the fimA II genotype. The 3 factors identified in the multivariate analysis to be associated with the red complex bacteria count were low Alb level (<3.7 g/dl), LC and fimA II genotype, with adjusted odds ratios of 6.93, 4.72 and 4.08, respectively (P<0.05). These data indicated that patients with LC were at increased risk of presenting with the red complex bacteria leading to periodontal disease progression. Therefore, these patients may need to take additional care of their oral health compared with patients without LC, which may prove beneficial for the maintenance of their general health.

 hepatitis C virus liver cirrhosis periodontitis red complex oral lichen planus Introduction

According to the World Health Organization, in 2015 the estimated number of people infected with hepatitis B virus (HBV) and the number of mortalities reported due to hepatitis B-related complications such as cirrhosis and hepatocellular carcinoma (HCC) were 257 and 88.7 million, respectively (1). In addition, it is estimated that 71 million people are persistently infected with hepatitis C virus (HCV), and the number of deaths due to hepatitis C, mostly from liver cirrhosis (LC) and HCC, is 399,000 annually (2).

In Japan, the annual number of deaths from HCC is ~30,000, 80% of which are caused by HBV and HCV (3). The HCV-infected patients in Japan are generally older compared with those in other countries, and a high percentage of them present with complications such as LC and/or HCC (4,5).

However, in recent years, the incidence of HCC caused by viral hepatitis has decreased, whereas that of hepatitis virus-negative HCC, or so-called non-B, non-C HCC, has increased (6,7). In a nationwide multicenter study by Tateishi et al (7), the proportion of non-B, non-C HCC had increased from 10.0% in 1991 to 32.5% in 2010. This has been attributed to the increase in alcohol consumption and the incidence of lifestyle-associated diseases, especially diabetes mellitus (DM) and non-alcoholic steatohepatitis (NASH). The number of patients with non-B, non-C HCC is expected to increase in the future.

Patients with HCV-associated liver diseases are known to have poor periodontal health with a higher incidence of oral lichen planus (OLP) and oral cancer compared with healthy people (8-10). Periodontitis is a pathological inflammatory disease of the gums and supporting bone tissues, which leads to tissue destruction and alveolar bone loss in response to bacterial dental plaque (11). In particular, the so-called ‘red complex’ bacteria comprising Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola, is directly associated with the severity of periodontal diseases (12). P. gingivalis is a gram-negative oral anaerobe known to have a number of virulence factors, such as cysteine proteinases (gingipain), lipopolysaccharide, capsule, and fimbriae (13). T. forsythia, formerly Bacteroides forsythus, is thought to exacerbate periodontal disease via symbiosis with the highly pathogenic P. gingivalis and T. denticola.

Periodontal disease affects up to 50% of the adult population worldwide and severe periodontitis has been reported as the 6th most prevalent disease worldwide (14). Furthermore, it has been demonstrated to be associated with several systemic diseases (15,16). Recently, studies indicating the frequent presence of P. gingivalis in patients with non-alcoholic fatty liver disease (NAFLD)/NASH have suggested a close association between periodontitis and NAFLD/NASH (17,18).

We previously reported that the periodontal inflammatory response may be associated with liver fibrosis and obesity in patients with HCV and HBV with liver disease (19). However, studies examining the association between red complex and liver fibrosis are lacking. Therefore, in the present study, the proportion of patients with red complex in addition to liver disease was examined.

 Materials and methods <sec> <title>Patients

A total of 47 patients with oral mucous membrane and liver disease who visited or were screened at the Saga University and Kurume University between January 2013 and February 2018 were included in this study. Informed consent was obtained from the patients, including all those who had been treated for liver disease by hepatologists, and each patient was examined by an oral surgeon. Patients on antiviral therapy [such as interferon (IFN), direct-acting antiviral therapy or nucleic acid analog for viral hepatitis infection] and antibiotic treatment were excluded from the study.

 Examination of oral mucosal disease

Information regarding the daily frequency of tooth brushing, smoking habits and alcohol consumption were collected from the patients. Mucosal examinations were performed using a headlight (Welch Allyn, Ltd.) and biopsies were conducted on patients with OLP, leukoplakia and oral cancer.

 Screening by salivary occult blood test

An occult blood test involves the assessment of the presence of blood, derived primarily from gingival tissues and released into the saliva. The presence of periodontal disease was considered as an indicator of periodontitis and ascertained using the Salivaster® reagent-based test (Showa Yakuhin Kako Co., Ltd.) (20). The Salivaster® is a colorimetric test based on a catalytic reaction of hemoglobin in saliva, which induces the formation of different colors ranging from yellow to dark blue. The occult blood reaction is classified as follows: Double-positive (++); positive (+) and negative (-). This method was demonstrated to have a sensitivity of 75.9% and a specificity of 90.5% for the detection of gingival inflammation (21).

 Saliva sample collection

Whole saliva (1 ml) was collected from the patients following chewing a non-flavored gum for 5 min. All samples were immediately sent to the Health Examination Laboratory (BML, Inc.) for bacterial testing (22).

 Identification of periodontitis bacteria and detection of P

gingivalis fimA genotypes. The total number of bacteria in saliva and the counts of 6 types of periodontal pathogens (Aggregatibacter actinomycetemcomitans; Prevotella intermedia; P. gingivalis; T. forsythensis; T. denticol; Fusobacterium necrophorum) were quantified using the modified polymerase chain reaction (PCR)-Invader assay as previously described (23). Based on the ratio of each periodontal pathogen to the total bacterial count, the following criteria were used to assess the risk: i) A. actinomycetemcomitans; no risk (<0.005%), moderate risk (0.006-0.009%) and high risk (>0.01%); ii) P. intermedia; no risk (<0.09%), moderate risk (0.1-0.49%) and high risk (>0.5%); iii) P. gingivalis; no risk (<0.09%), moderate risk (0.1-0.49%) and high risk (>0.5%); iv) T. forsythensis; no risk (<0.09%), moderate risk (0.1-0.49%) and high risk (>0.5%); v) T. denticol; no risk (<0.09%), moderate risk (0.1-0.49%) and high risk (>0.5%); and vi) F. necrophorum; no risk (<5%) and high risk (>5%).

The patients were divided into two groups based on the proportion of the red complex bacteria as follows: Group A (high bacterial counts) and group B (low bacterial counts). Group A comprised of patients who presented with increased proportions of at least one of the 3 types of red complex bacteria, according to the aforementioned risk criteria, whereas those who demonstrated no risks for all 3 bacterial types were categorized into group B. P. gingivalis-specific fimA genotypes ranging from types I-V was detected in all the patients.

 Measurement of obesity

Obesity was defined as a body mass index (BMI) ≥25 kg/m2.

 Serological assays

All subjects were assessed for red blood cell, white blood cell (WBC), and platelet (Plt) counts, hemoglobin (Hb), fasting blood glucose (FBS), and HbA1c levels; additionally, the following liver function tests were realized: Serum alanine aminotransferase (ALT); aspartate aminotransferase (AST); lactate dehydrogenase (LDH); alkaline phosphatase (ALP); prothrombin time (PT); total bilirubin (T. Bil); total cholesterol (T. cho); total protein (T. pro); and albumin (Alb). All the biochemical parameters were measured by standard clinical methods using venous blood samples taken the morning after 12 h overnight fast.

 Evaluation of liver diseases

Anti-HCV was measured using a chemiluminescent enzyme immunoassay kit (Lumipulse II HCV; Fujirebio). HCV RNA in the serum was analyzed by using the standardized automated quantitative PCR (COBAS AMPLICOR HCV MONITOR v2.0 Test; COBAS® AmpliPrep/COBAS® Taq-Man® HCV Test; Roche Molecular Diagnostics) as reported previously (24,25). Similarly, HCV genotype was determined by PCR as reported previously (26).

Ultrasonographic examinations were performed on all patients in order to investigate the shape of the liver and to identify the lesions in the organ. Computed tomography were performed on all patients. Liver biopsies were performed on patients without bleeding tendencies (i.e., those who did not present with low platelet counts). Other potential predictors of the progression of LC, including serum Alb, T. Bil, PT and Plt were also used. The fibrosis-4 (FIB-4) index, a biomarker that has been demonstrated to be effective in assessing liver fibrosis and LC, was calculated using the following formula: Age (years) x AST (U/l)/[Plt (109/l) x ALT (U/l)1/2] (27). An FIB-4 index ≥2.67 indicated the presence of liver fibrosis (28).

 Evaluation of extrahepatic diseases

The diagnosis of type II DM was based on the criteria adopted by the American Diabetes Association in 2011(29). Hypertension was defined as a systolic blood pressure of ≥140 mmHg or diastolic blood pressure of ≥90 mmHg.

 Statistical analysis

All data are expressed as n or mean ± standard deviation. Differences between the two groups were analyzed using the Mann-Whitney U, Wilcoxon's and Fisher's exact tests, as appropriate. Adjusted odds ratios were calculated using logistic regression analysis. All statistical analyses were conducted using JMP v.13 (SAS Institute, Inc.). P<0.05 was considered to indicate a statistically significant difference.

 Results <sec> <title>Characteristic of the patients

Patient characteristics are summarized in Tables I and II. The 47 patients (17 males and 30 females) ranged in age from 49-89 years, with an average age of 70.2±8.8 years. The most common oral mucosal disease was OLP (32/47; 68.1%), while the other oral diseases observed were head and neck squamous cell carcinoma (4/47; 8.5%) and Sjögren's syndrome (2/47; 4.3%).

The rates of anti-HCV antibody and HBsAg positivity were 87.2% (41/47) and 2.1% (1/47), respectively. The most common liver disease observed was chronic hepatitis C (CH-C; 16/47; 34.0%; Table II). In addition, 12 patients [LC and post-HCC (n=8); LC (n=3); and primary biliary cholangitis (PBC) (n=1)] were diagnosed with cirrhosis. Biopsies were not obtained from 3 out of 12 patients with LC, due to low platelet counts.

 Univariate analysis of factors associated with the two red complex groups (high vs low)

A total of 15 patients in group A exhibited high numbers of red complex bacteria, while 32 patients in group B had low numbers of the bacteria. Univariate analyses demonstrated significant associations between groups A and B (Table II). The significant factors associated with red complex bacteria count were the presence of LC (P=0.0229) and Alb levels (P=0.0399). No significant differences in the levels of AST, ALT, T. cho, FBS, HbA1c, WBC, Hb, Plt, AFP and FIB-4 index were observed between the two groups. Similarly, no differences in the presence of extrahepatic manifestations, such as OLP, DM, hypertension, heart disease, thyroid disease and past history of extrahepatic cancer, BMI, smoking habits, alcohol intake, daily frequency of tooth brushing, and salivary occult blood test results were identified.

 Identification and characteristics of periodontitis bacteria

Table III demonstrates the total number of bacteria in the saliva, the counts of the 6 types of periodontal bacteria, the ratios of each bacteria and the prevalence of the fimA II genotype of P. gingivalis. The presence of the fimA genotype II was a significant factor associated with red complex bacteria count (P=0.0310).

 Multivariate analysis of factors associated with high counts of red complex bacteria

All variables in the univariate analyses were included in the multivariate analysis (Table IV). Three factors were associated with a high number of red complex bacteria: Alb level <3.7 g/dl; presence of LC; and P. gingivalis fimA genotype II. The adjusted odds ratios for these factors were 6.93, 4.72 and 4.08, respectively, and each was statistically significant.

 Discussion

Periodontal disease is closely associated with general health: The chronic inflammatory burden of periodontal infection and the host response is associated with cardiovascular disease (30,31), type II DM and insulin resistance (32), gastrointestinal and colorectal cancer (33), low term birth weight in newborn babies (34), chronic obstructive pulmonary disease (35) and Alzheimer's disease (36).

Patients with viral liver disease have poorer periodontal health compared with healthy people (8). In particular, it has been demonstrated that patients with LC tend to exhibit significant amounts of clinical attachment loss (37) and poor periodontal status (38,39) when compared with healthy people. The results of a systematic review by Grønkjær (40) indicated that an increased number of patients with LC exhibited poor oral health; in addition, periodontal disease was reported in 25-69% and apical periodontitis was diagnosed in 49-79% of the patients with LC (40).

Red complex bacteria, composed of P. gingivalis, T. forsythia and T. denticola, serve a major role in the pathogenesis of periodontal disease (12). These bacteria are found in high numbers in periodontal lesions and deep pockets in adults (41). Recently, the association between P. gingivalis and digestive system cancer, including oral squamous cell carcinoma, esophageal squamous carcinoma and pancreatic cancer, has been demonstrated in several clinical and experimental studies (42-44).

A cross-sectional study analyzing the association between oral bacteria and BMI in a healthy Japanese population demonstrated that BMI and waist circumference were independently associated with the number of red complexes (45). However, in the present study, the BMI was not significantly associated with red complex counts in both univariate and multivariate analyses. This discrepancy in results may be due to differences in the backgrounds of the subjects between the two studies. The subjects in the present study comprised elderly people with liver disease (average age, 70.2 years), and the association was analyzed by dividing the number of subjects in each group by the quantity of the red complex bacteria. Conversely, Matsushita et al (45) used the multivariate analysis of covariance to calculate the effect size of BMI for the number of components of red complex (0-3) in the healthy population (average age, 52.0 years).

In another large-scale cross-sectional study involving 997 Japanese adults, the detection rates of P. gingivalis, T. denticola, and T. forsythia were demonstrated to be 46.3, 76.4 and 61.1%, respectively (46). P. gingivalis fimA fimbriae are classified into 6 genotypes (types I-V and Ib), based on the diversity of the fimA genes encoding the fimbrial subunits (47); they promote the adhesion of the bacteria to the gingival sites and their invasion into the tissues. It has been suggested that bacterial clones with types II/IV/Ib fimA are invasive, whereas those with types I/III/V fimA are non-invasive (48).

To the best of our knowledge, the detection rate of the red complex in patients with liver disease has not been evaluated so far. In the present study, the presence of LC, hypoalbuminemia and P. gingivalis fimA II type were associated with high detection rates of the red complex bacteria. Hypoalbuminemia is a feature of chronic and advanced LC; therefore, it is highly likely that liver fibrosis is associated with the severity of periodontal disease. No association between the number of red complexes and HCC was observed in the present study. Although the prevalence of diabetes and extrahepatic cancer was increased in group A when compared with group B, no statistically significant difference was noted. In addition, no statistically significant differences in the levels of AST, ALT, T. cho, FBS, HbA1c, WBC, Hb, Plt, AFP and FIB-4 index, BMI, smoking habits, alcohol intake, daily frequency of tooth brushing, and salivary occult blood test results were observed between the two groups.

Certain limitations of the present study include the small sample size and the fact that the presence of periodontitis was not evaluated by determining the probing depth and bleeding on probing. Additional studies using increased numbers of patients are required.

We have previously reported the association between periodontitis and the progression of liver disease in patients infected with HCV and/or HBV (19); the 5 factors associated with periodontal disease in the study were: Decreased platelet count; tooth brushing only once a day; current IFN treatment; ≥65 years of age; and obesity. Furthermore, the prevalence of P. gingivalis fimA genotype II was increased in patients with cirrhosis (50%) when compared with those with non-cirrhotic CH-C (21.4%).

Patients with LC have increased risk of developing bacterial infection and sepsis leading to sepsis-induced organ failure and mortality (49). Spontaneous bacterial peritonitis (SBP) is an infection characterized by the presence of LC. SBP pathogenesis in patients with cirrhosis is considered to be the primary consequence of bacterial translocation due to decreased intestinal peristalsis, overgrowth of enterobacteria, impaired intestinal barrier, decreased bile acid and diminished IgA levels (50). Ashare et al (51) demonstrated that 94% of the cirrhotic subjects in the study population developed bacteremia following tooth brushing; whereas the control subjects were able to overcome this condition, the subjects with cirrhosis had prolonged periods of bacteremia, indicating that patients with cirrhosis may be more susceptible to sepsis.

Oral bacteria can survive in the bloodstream and may adhere to non-oral organs and tissues, including the heart valves, carotid atheromatous plaque, brain, spleen, pancreas, liver and bone (52,53). There have been a series of studies suggesting an association between oral and intestinal flora (54,55). In 2014, it was demonstrated that a large proportion (54%) of bacteria derived from the oral cavity were frequently detected amongst the intestinal flora in patients with LC, and the ratio was correlated with the severity of the disease, suggesting an invasion of the gut from the mouth in LC (54).

The problems with oral hygiene that patients with viral liver disease suffer include decreased salivary flow, elicitation of periodontal disease by insulin resistance, prolonged bleeding, poor hygiene due to pain from OLP, and medical discrimination and prejudice (9,19,56,57), which may hinder radical dental treatment in patients with liver disease. In Japan, the most common place where 1,705 individuals with hepatitis felt discrimination or prejudice was the dental clinic (39.3%) followed by the hospital and other clinics (26.3%), nursing homes (8.7%), community (7.5%) and beauty salons (7.3%) (58). However, information disclosure and education about hepatitis to the dental personnel are not sufficient. It is necessary for dentists to raise awareness about oral diseases in patients with HCV/HBV infection.

In conclusion, the red complex bacteria involved in the pathogenesis of periodontal disease was associated with LC in the present study. These data indicated that maintenance of oral hygiene is important in patients with LC in order to prevent the occurrence of periodontal disease, thereby leading to improved general health.

 Acknowledgements

Not applicable.

 Funding

The present study was supported in part by a Grant-in-Aid for Scientific Research (C) (grant nos. 25463274 and 17K12012) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

 Availability of data and materials

All the datasets generated and analyzed in the present study are included in this published article.

 Authors' contributions

YN collected data from participants, performed the statistical analysis and wrote the manuscript. TT interpreted data and added intellectual revision. Both the authors have read and approved the final version of this manuscript for publication.

 Ethics approval and consent to participate

The study protocol was approved by the Ethics Committee of Kurume University (reference no. 12240) and the Ethics Committee of Saga University (reference no. 2015-02-16) in accordance with the Declaration of Helsinki. Written informed consent for participation in the study was obtained from each patient.

 Patient consent for publication

Not applicable.

 Competing interests

The authors declare that they have no competing interests.

 References WHO: Hepatitis B Fact sheet July 2018. http://www.who.int/mediacentre/factsheets/fs204/en/. WHO: Hepatitis C Fact sheet July 2018. http://www.who.int/mediacentre/factsheets/fs164/en/. UmemuraTIchijoTYoshizawaKTanakaEKiyosawaKEpidemiology of hepatocellular carcinoma in JapanJ Gastroenterol44 (Suppl 19)10210720091914880210.1007/s00535-008-2251-0 AsahinaYTsuchiyaKTamakiNHirayamaITanakaTSatoMYasuiYHosokawaTUedaKKuzuyaTEffect of aging on risk for hepatocellular carcinoma in chronic hepatitis C virus infectionHepatology5251852720102068395110.1002/hep.23691 TanakaJKoyamaTMizuiMUchidaSKatayamaKMatsuoJAkitaTNakashimaAMiyakawaYYoshizawaHTotal numbers of undiagnosed carriers of hepatitis C and B viruses in Japan estimated by age- and area-specific prevalence on the national scaleIntervirology5418519520112145495610.1159/000324525 TateishiROkanoueTFujiwaraNOkitaKKiyosawaKOmataMKumadaHHayashiNKoikeKClinical characteristics, treatment, and prognosis of non-B, non-C hepatocellular carcinoma: A large retrospective multicenter cohort studyJ Gastroenterol5035036020152492963810.1007/s00535-014-0973-8 TateishiRUchinoKFujiwaraNTakeharaTOkanoueTSeikeMYoshijiHYatsuhashiHShimizuMTorimuraTA nationwide survey on non-B, non-C hepatocellular carcinoma in Japan: 2011-2015 updateJ Gastroenterol5436737620193049890410.1007/s00535-018-1532-5 Al-SharqiAJPeriodontal status of patients with hepatitis B viruses and B and C virus (comparative study)J Pure Appl Microbiol12293300201810.22207/JPAM.12.1.35 NagaoYSataMTanikawaKItohKKameyamaTLichen planus and hepatitis C virus in the northern Kyushu region of JapanEur J Clin Invest259109141995y10.1111/j.1365-2362.1995.tb01966.x NagaoYSataMTanikawaKItohKKameyamaTHigh prevalence of hepatitis C virus antibody and RNA in patients with oral cancerJ Oral Pathol Med243543601995750029110.1111/j.1600-0714.1995.tb01199.x WilliamsRCPeriodontal diseaseN Engl J Med3223733821990240526810.1056/NEJM199002083220606 SocranskySSHaffajeeADCuginiMASmithCKentRL JrMicrobial complexes in subgingival plaqueJ Clin Periodontol251341441998949561210.1111/j.1600-051x.1998.tb02419.x LamontRJJenkinsonHFLife below the gum line: Pathogenic mechanisms of Porphyromonas gingivalisMicrobiol Mol Biol Rev621244126319989841671 TonettiMSChappleILJepsenSSanzMPrimary and secondary prevention of periodontal and peri-implant diseases: Introduction to, and objectives of the 11th European Workshop on Periodontology consensus conferenceJ Clin Periodontol42 (Suppl 16)S1S420152568324210.1111/jcpe.12382 FalcaoABullónPA review of the influence of periodontal treatment in systemic diseasesPeriodontol 20007911712820193089276410.1111/prd.12249 BuiFQAlmeida-da-SilvaCLCHuynhBTrinhALiuJWoodwardJAsadiHOjciusDMAssociation between periodontal pathogens and systemic diseaseBiomed J42273520193098770210.1016/j.bj.2018.12.001 YonedaMNakaSNakanoKWadaKEndoHMawatariHImajoKNomuraRHokamuraKOnoMInvolvement of a periodontal pathogen, Porphyromonas gingivalis on the pathogenesis of non-alcoholic fatty liver diseaseBMC Gastroenterol121620122234081710.1186/1471-230X-12-16 AlazawiWBernabeETaiDJanickiTKemosPSamsuddinSSynWKGillamDTurnerWPeriodontitis is associated with significant hepatic fibrosis in patients with non-alcoholic fatty liver diseasePLoS One12e018590220172922036710.1371/journal.pone.0185902 NagaoYKawahigashiYSataMAssociation of periodontal diseases and liver fibrosis in patients with HCV and/or HBV infectionHepat Mon14e2326420142573772910.5812/hepatmon.23264 YasuiTTanakaSOnozawaHMatsumotoMTanakaSNakaoSMass screening of periodontal diseases using new test paper for detecting salivary occult boldNippon Shika Hyoron5871891971991 KaufmanELamsterIBAnalysis of saliva for periodontal diagnosis - a reviewJ Clin Periodontol2745346520001091488510.1034/j.1600-051x.2000.027007453.x TadaATakeuchiHShimizuHTadokoroKTanakaKKawamuraKYamaguchiTEgashiraTNomuraYHanadaNQuantification of periodontopathic bacteria in saliva using the invader assayJpn J Infect Dis6541542320122299621510.7883/yoken.65.415 TadokoroKYamaguchiTKawamuraKShimizuHEgashiraTMinabeMYoshinoTOguchiHRapid quantification of periodontitis-related bacteria using a novel modification of Invader PLUS technologiesMicrobiol Res165434920101871874810.1016/j.micres.2008.06.001 LeeSCAntonyALeeNLeibowJYangJQSovieroSGutekunstKRosenstrausMImproved version 2.0 qualitative and quantitative AMPLICOR reverse transcription-PCR tests for hepatitis C virus RNA: Calibration to international units, enhanced genotype reactivity, and performance characteristicsJ Clin Microbiol3841714179200011060086 SizmannDBoeckCBoelterJFischerDMiethkeMNicolausSZadakMBabielRFully automated quantification of hepatitis C virus (HCV) RNA in human plasma and human serum by the COBAS AmpliPrep/COBAS TaqMan systemJ Clin Virol3832633320071734409310.1016/j.jcv.2006.12.021 DusheikoGSchmilovitz-WeissHBrownDMcOmishFYapPLSherlockSMcIntyreNSimmondsPHepatitis C virus genotypes: An investigation of type-specific differences in geographic origin and diseaseHepatology1913181994827634910.1002/hep.1840190104 Vallet-PichardAMalletVNalpasBVerkarreVNalpasADhalluin-VenierVFontaineHPolSFIB-4: An inexpensive and accurate marker of fibrosis in HCV infection. comparison with liver biopsy and fibrotestHepatology46323620071756782910.1002/hep.21669 ShahAGLydeckerAMurrayKTetriBNContosMJSanyalAJNash Clinical Research Network: Comparison of noninvasive markers of fibrosis in patients with nonalcoholic fatty liver diseaseClin Gastroenterol Hepatol71104111220091952353510.1016/j.cgh.2009.05.033 American Diabetes Association: Diagnosis and classification of diabetes mellitus. Diabetes Care 34 (Suppl 1): S62-S69, 2011 BeckJDSladeGOffenbacherSOral disease, cardiovascular disease and systemic inflammationPeriodontol 20002311012020001127675710.1034/j.1600-0757.2000.2230111.x BeukersNGvan derHeijden GJvan WijkAJLoosBGPeriodontitis is an independent risk indicator for atherosclerotic cardiovascular diseases among 60 174 participants in a large dental school in the NetherlandsJ Epidemiol Community Health71374220172750278210.1136/jech-2015-206745 MealeyBLOatesTWAmerican Academy of Periodontology: Diabetes mellitus and periodontal diseasesJ Periodontol771289130320061688179810.1902/jop.2006.050459 WhitmoreSELamontRJOral bacteria and cancerPLoS Pathog10e100393320142467639010.1371/journal.ppat.1003933 UppalAUppalSPintoADuttaMShrivatsaSDandoluVMupparapuMThe effectiveness of periodontal disease treatment during pregnancy in reducing the risk of experiencing preterm birth and low birth weight: A meta-analysisJ Am Dent Assoc1411423143420102111912610.14219/jada.archive.2010.0104 ScannapiecoFAHoAWPotential associations between chronic respiratory disease and periodontal disease: Analysis of National Health and Nutrition Examination Survey IIIJ Periodontol72505620011121007310.1902/jop.2001.72.1.50 SparksStein PSteffenMJSmithCJichaGEbersoleJLAbnerEDawsonD IIISerum antibodies to periodontal pathogens are a risk factor for Alzheimer's diseaseAlzheimers Dement819620320122254635210.1016/j.jalz.2011.04.006 NovacekGPlachetzkyUPötziRLentnerSSlavicekRGanglAFerenciPDental and periodontal disease in patients with cirrhosis - role of etiology of liver diseaseJ Hepatol225765821995765033810.1016/0168-8278(95)80453-6 MovinSRelationship between periodontal disease and cirrhosis of the liver in humansJ Clin Periodontol84504581981694991810.1111/j.1600-051x.1981.tb00894.x FunatsuKYamadaMKawishimaYNishidaJUenoMEbiharaYMizunoYOdaMTsuchiyaMMicrocirculatory disturbances of oral mucosa and periodontal disease in patients with liver cirrhosisJ Gastroenterol Hepatol4 (Suppl 1)9910219892519101 GrønkjærLLPeriodontal disease and liver cirrhosis: A systematic reviewSAGE Open Med3205031211560112220152677079910.1177/2050312115601122 FariasBCSouzaPRFerreiraBMeloRSMachadoFBGusmãoESCimõesROccurrence of periodontal pathogens among patients with chronic periodontitisBraz J Microbiol4390991620122403190610.1590/S1517-83822012000300009 KatzJOnateMDPauleyKMBhattacharyyaIChaSPresence of Porphyromonas gingivalis in gingival squamous cell carcinomaInt J Oral Sci320921520112201057910.4248/IJOS11075 AhnJSegersSHayesRBPeriodontal disease, Porphyromonas gingivalis serum antibody levels and orodigestive cancer mortalityCarcinogenesis331055105820122236740210.1093/carcin/bgs112 MichaudDSIzardJWilhelm-BenartziCSYouDHGroteVATjønnelandADahmCCOvervadKJenabMFedirkoVPlasma antibodies to oral bacteria and risk of pancreatic cancer in a large European prospective cohort studyGut621764177020132299030610.1136/gutjnl-2012-303006 MatsushitaKHamaguchiMHashimotoMYamazakiMYamazakiTAsaiKYamoriMBesshoKTodaHHasegawaGThe novel association between red complex of oral microbe and body mass index in healthy Japanese: A population based cross-sectional studyJ Clin Biochem Nutr5713513920152638867110.3164/jcbn.15-19 ChigasakiOTakeuchiYAokiASasakiYMizutaniKAoyamaNIkedaYGokyuMUmedaMIshikawaIA cross-sectional study on the periodontal status and prevalence of red complex periodontal pathogens in a Japanese populationJ Oral Sci6029330320182992571410.2334/josnusd.17-0223 HamadaSFujiwaraTMorishimaSTakahashiINakagawaIKimuraSOgawaTMolecular and immunological characterization of the fimbriae of Porphyromonas gingivalisMicrobiol Immunol389219301994772368410.1111/j.1348-0421.1994.tb02148.x AmanoANakagawaIKataokaKMorisakiIHamadaSDistribution of Porphyromonas gingivalis strains with fimA genotypes in periodontitis patientsJ Clin Microbiol3714261430199910203499 GustotTDurandFLebrecDVincentJLMoreauRSevere sepsis in cirrhosisHepatology502022203320091988587610.1002/hep.23264 ThalheimerUTriantosCKSamonakisDNPatchDBurroughsAKInfection, coagulation, and variceal bleeding in cirrhosisGut5455656320051575354410.1136/gut.2004.048181 AshareAStanfordCHancockPStarkDLilliKBirrerENymonADoerschugKCHunninghakeGWChronic liver disease impairs bacterial clearance in a human model of induced bacteremiaClin Transl Sci219920520092044389310.1111/j.1752-8062.2009.00122.x Van DykeTEvan WinkelhoffAJInfection and inflammatory mechanismsJ Periodontol84 (Suppl 4)S1S720132362732110.1111/jcpe.12088 ScannapiecoFAThe oral microbiome: Its role in health and in oral and systemic infectionsClin Mic News35163169201310.1016/j.clinmicnews.2013.09.003 QinNYangFLiAPriftiEChenYShaoLGuoJLe ChatelierEYaoJWuLAlterations of the human gut microbiome in liver cirrhosisNature513596420142507932810.1038/nature13568 NakajimaMArimatsuKKatoTMatsudaYMinagawaTTakahashiNOhnoHYamazakiKOral administration of P. gingivalis induces cysbiosis of gut microbiota and impaired barrier function leading to dissemination of enterobacteria to the liverPLoS One10e013423420152621806710.1371/journal.pone.0134234 NishimuraFMurayamaYPeriodontal inflammation and insulin resistance--lessons from obesityJ Dent Res801690169420011166947610.1177/00220345010800080201 NagaoYHanadaSShishidoSIdeTKumashiroRUenoTSataMIncidence of Sjögren's syndrome in Japanese patients with hepatitis C virus infectionJ Gastroenterol Hepatol1825826620031260352510.1046/j.1440-1746.2003.02982.x TatsuokaSIssue record of ‘Research to understand the actual situation of prejudice and discrimination against hepatitis virus infected people, and to prepare guidelines for prevention of the damage’ by Health, Labour and Welfare Sciences Research Grants2011-2013(In Japanese)

Oral mucosal disease among the patient cohort in the present study (n=47).

Characteristics N
Sex (male/female) 17/30
Age, years 70.2±8.8
Diagnosis of oral diseases  
     OLP 30
     OLP, oral cancer (SCC) 1
     OLP, post op of laryngeal cancer (SCC)a 1
     Leukoplakia, post op of tongue cancer (SCC) 2
     Sjögren's syndrome 1
     Sjögren's syndrome, traumatic ulcer of tongue 1
     Burning mouth syndrome 1
     Cheilitis 1
     No abnormality in screening for oral cancer 9

Data are presented as n or the mean ± SD. aHistory of laryngeal cancer treated operatively 8 years previously. OLP, oral lichen planus; SCC, squamous cell carcinoma; SD, standard deviation.

Univariate analysis of factors associated with red complex bacteria.

Factors Total High counts of red complex bacteria (group A) Low counts of red complex bacteria (group B) P-value
No. subjects 47 15 32  
Sex (male/female) 17/30 6/9 11/21 NS
Age, years 70.2±8.8 72.9±9.7 69.0±8.2 NS
Smoking history (yes) 16 (34.0) 6 (40.0) 10 (31.3) NS
Alcohol intake (yes) 19 (40.4) 7 (46.7) 12 (37.5) NS
Diagnosis of liver diseases        
 CH-C 16 4 12 NS
 CH-C SVR 8 1 7 NS
 CH-C, post HCC 4 2 2 NS
 CH-C SVR, post HCC 1 0 1 NS
 CH-C, fatty liver 1 0 1 NS
 LC, post HCC 8 4 4 NS
 LC 3 2 1 NS
 Fatty liver 3 1 2 NS
 PBC following liver transplantation 1 1 0 NS
 Asymptomatic HBV carrier 1 0 1 NS
 Polycystic liver disease 1 0 1 NS
Liver cirrhosis 12 (25.5) 7 (46.7) 5 (15.6) 0.0229
OLP 32 (68.1) 10 (66.7) 22 (68.8) NS
Diabetes mellitus 13 (27.7) 6 (40.0) 7 (21.9) NS
Hypertension 19 (40.4) 6 (40.0) 13 (40.6) NS
Heart disease 5 (10.6) 1 (6.7) 4 (12.5) NS
Thyroid disease 6 (12.8) 1 (6.7) 5 (15.6) NS
Past history of extrahepatic cancer (%) 11 (23.4) 4 (26.7) 7 (21.9) NS
BMI, kg/m2 23.2±3.2 24.0±3.1 22.8±3.2 NS
Obesity (BMI ≥25) 14 (29.8) 5 (33.3) 9 (28.1) NS
AST, U/l 39.7±23.1 49.0±27.9 35.3±19.6 NS
ALT, U/l 35.6±27.9 45.3±36.6 31.1±22.1 NS
LDH, U/l 205.6±51.1 221.1±57.4 198.6±47.3 NS
ALP, U/l 306.1±193.9 330.3±149.3 294.4±213.4 NS
T.pro, g/dl 7.28±0.51 7.31±0.65 7.26±0.45 NS
Alb, g/dl 3.95±0.48 3.74±0.46 4.05±0.46 0.0399
T. Bil, mg/dl 0.82±0.31 0.89±0.35 0.79±0.28 NS
Fasting glucose, mg/dl 114.4±47.2 110.4±31.1 116.5±53.9 NS
HbA1c, % 5.9±1.0 5.9±1.0 6.0±1.0 NS
T. cho, mg/dl 168.6±33.4 178.5±37.6 163.6±30.6 NS
RBC, 104/µl 422.3±46.2 424.9±37.0 421.1±50.7 NS
Hb, g/dl 13.1±1.6 13.5±1.3 13.0±1.8 NS
WBC, 102/µl 44.0±14.0 45.3±16.0 43.4±13.2 NS
Plt, 104/µl 13.9±5.6 12.6±5.2 14.5±5.8 NS
AFP, ng/ml 23.9±68.9 28.2±66.7 22.0±71.0 NS
FIB-4 index 4.397±3.03 5.23±2.89 4.00±3.07 NS
Daily frequency of tooth brushing (≤1 per day) 7 (14.9) 2 (13.3) 5 (15.6) NS
Positive salivary occult blood test 38 (80.9) 13 (86.7) 25 (78.1) NS

Data are presented as n (%) or the mean ± SD. CH-C, chronic hepatitis C; SVR, sustained virological response; LC, liver cirrhosis; HCC, hepatocellular carcinoma; HBV, hepatitis B virus; PBC, Primary biliary cholangitis; OLP, oral lichen planus; BMI, body mass index; AST, aspartate aminotransferase; ALT, serum alanine aminotransferase; LDH, lactate dehydrogenase; ALP, alkaline phosphatase; T. pro, total protein; Alb, albumin; T. Bil, total bilirubin; T. cho, total cholesterol; RBC, red blood cell; Hb, hemoglobin; WBC, white blood cell; Plt, platelet; AFP, alpha-fetoprotein; NS, not significant; SD, standard deviation.

Comparison of red complex bacteria between the two groups.

Factors Total High counts of red complex bacteria (group A) Low counts of red complex bacteria (group B) P-value
Total number of bacteria in saliva, copy/10 µl 13,210,426±10,941,106 13,259,333±16,269,145 13,187,500±7,621,944 NS
Number of A. actinomycetemcomitans in saliva, copy/10 µl 10±0 10±0 10±0 NS
Number of P. intermedia in saliva, copy/10 µl 6,451±15,798 13,058±20,338 3,486±12,384 NS
Number of P. gingivalis in saliva, copy/10 µl 4,249±7,420 9,537±10,400 1,770±3,601 0.0007
Number of T. forsythensis in saliva, copy/10 µl 8,020±13,913 17,155±21,323 3,738±4,755 0.0002
Number of T. denticol in saliva, copy/10 µl 1,586±3,957 2,370±4,082 1,218±3,908 NS
Number of F. necrophorum in saliva, copy/10 µl 498,704±719,688 811,007±1,151,579 352,313±316,793 NS
Risk of A. actinomycetemcomitans (ratio of the total bacterial count >0.006%) 0 (0) 0 (0) 0 (0) NS
Risk of P. intermedia (ratio of the total bacterial count >0.1%) 7 (14.9) 5 (33.3) 2 (6.3) 0.0151
Risk of P. gingivalis (ratio of the total bacterial count >0.1%) 7 (14.9) 7 (46.7) 0 (0) <0.0001
Risk of T. forsythensis (ratio of the total bacterial count >0.1%) 11 (23.4) 11 (73.3) 0 (0) <0.0001
Risk of T. denticol (ratio of the total bacterial count >0.1%) 3 (6.4) 3 (20.0) 0 (0) 0.0089
Risk of F. necrophorum (ratio of the total bacterial count >5%) 11 (23.4) 7 (46.7) 4 (12.5) 0.0099
P. gingivalis fimA genotype II (positive) 15 (31.9) 8 (53.3) 7 (21.9) 0.0310
P. gingivalis fimA        
Genotype I 10 (21.3) 3(20) 7 (21.9) NS
Genotype II 13 (27.7) 6(40) 7 (21.9) NS
Genotype I and II 2 (4.3) 2 (13.3) 0 (0) 0.0348
Genotype III 1 (2.1) 0 (0) 1 (3.1) NS
Genotype IV 4 (8.5) 1 (6.7) 3 (9.4) NS
Genotype V 1 (2.1) 1 (6.7) 0 (0) NS
Below detection sensitivity limits 16 (34.0) 2 (13.3) 14 (43.8) 0.0402

Data are presented as n (%) or the mean ± SD. A. actinomycetemcomitans, Aggregatibacter actinomycetemcomitans; P. intermedia, Prevotella intermedia; P. gingivalis, Porphyromonas gingivalis; T. forsythensis, Tannerella forsythia; T. denticol, Treponema denticola; F. necrophorum, Fusobacterium necrophorum; NS, not significant; SD, standard deviation.

Multivariate analysis of factors associated with high counts of red complex bacteria.

Factor Odds ratio 95% confidence interval P-value
Albumin <3.7 g/dl 6.93 1.74-31.30 0.0059
Presence of liver cirrhosis 4.72 1.20-20.27 0.0263
Porphyromonas gingivalis fimA genotype II 4.08 1.12-15.96 0.0336