Prevalence and microbiology of localized prepubertal periodontitis

Loss of crestal alveolar bone at primary teeth was ascertained radiographically in a dental school clinical population of 2264 children. 19 patients (0.84%) demonstrated distinct periodontal bone destruction around one or more primary teeth; in only 2 of these patients had periodontal disease been identified in previous clinical examinations. A microbiological study of 35 subgingival samples from 9 available patients revealed a high prevalence of black-pigmented Bacteroides spp., mainly Bacteroides intermedius. Actinobacillus actinomycetemcomitans and Capnocytophaga spp. were predominant organisms in some samples. The present data indicate that localized prepubertal periodontitis is more common than previously realized and is associated with bacteria generally regarded as major periodontal pathogens.     

Comparison of the microbiota of supra- and subgingival plaque in health and periodontitis

BACKGROUND, AIMS: The purpose of the present investigation was to compare the microbial composition of supra and subgingival plaque in 22 periodontally healthy (mean age 32+/-16 years) and 23 adult periodontitis subjects (mean age 51+/-14 years). METHODS: A total of 2358 supra and separately subgingival plaque samples were collected from the mesial aspect of all teeth excluding 3rd molars in each subject. Samples were examined for the presence and levels of 40 bacterial taxa using whole genomic DNA probes and checkerboard DNA-DNA hybridization. Clinical assessments including dichotomous measures of gingival redness, bleeding on probing, plaque accumulation and suppuration, as well as duplicate measures of pocket depth and attachment level, were made at 6 sites per tooth. Mean counts (x10(5), % DNA probe count and % sites colonized for each species were determined separately for supra and subgingival samples in each subject and then averaged across subjects in the 2 clinical groups. Significance of differences between healthy and periodontitis subjects was determined using the Mann-Whitney test and adjusted for multiple comparisons. RESULTS: Mean total DNA probe counts (x10(5), +/-SEM) for healthy and periodontitis subjects in supragingival plaque were 72.1+/-11 and 132+/-17.5, respectively (p<0.01), and in subgingival plaque 22.1+/-6.6 and 100.3+/-18.4, (p<0.001). Porphyromonas gingivalis, Bacteroides forsythus and Treponema denticola could be detected in supragingival plaque samples of both healthy and periodontitis subjects. Actinomyces species were the dominant taxa in both supra- and subgingival plaque from healthy and periodontitis subjects. 4 Actinomyces species accounted for 63.2%, of supragingival and 47.2% of subgingival plaque in healthy subjects and 48.% and 37.8% in periodontitis subjects respectively. Increased proportions of P. gingivalis, B. forsythus, and species of Prevotella, Fusobacterium, Campylobacter and Treponema were detected subgingivally in the periodontitis subjects. P. gingivalis, B. forsythus and T. denticola were significantly more prevalent in both supra- and subgingival plaque samples from periodontitis subjects. CONCLUSIONS: The main differences between supra and subgingival plaque as well as between health and disease were in the proportions and to some extent levels of Actinomyces, "orange" and "red" complex species.     

Microbial composition of supra- and subgingival plaque in subjects with adult periodontitis

Background, aims: The purpose of the present study was to compare and relate the microbial composition of supra and subgingival plaque in 23 adult periodontitis subjects (mean age 51±14 years). Methods: A total of 1,170 samples of supra and subgingival plaque were collected from the mesial aspect of every tooth (up to 28 supra and 28 subgingival samples) from each subject and evaluated for the presence and levels of 40 bacterial taxa using whole genomic DNA probes and checkerboard DNA‐DNA hybridization. Clinical assessments including dichotomous measures of gingival redness, bleeding on probing, plaque accumulation and suppuration, as well as duplicate measures of pocket depth and attachment level, were made at 6 sites per tooth. The counts (levels), % DNA probe count (proportion) and % of sites colonized (prevalence) of each species in supra and separately in subgingival plaque were computed for each subject. Significance of differences between supra and subgingival plaque for each species was sought using the Wilcoxon signed ranks test and adjusted for multiple comparisons. Results: All 40 taxa were detected in both supra and subgingival plaque. Actinomyces species were the most prevalent taxa in both habitats. 75 to 100% of supra and 62 to 100% of subgingival sites were colonized by at least one of the 5 Actinomyces species. Supragingival samples exhibited significantly higher counts of Actinomyces naeslundii genospecies 1, Actinomyces israelii, Actinomyces odontolyticus, Neisseria mucosa, Streptococcus gordonii, Capnocytophaga ochracea and Capnocytophaga sputigena when compared with mean counts in subgingival samples taken from the same tooth surfaces. Subgingival plaque samples presented significantly higher counts of Prevotella nigrescens, Prevotella intermedia, Bacteroides forsythus and Porphyromonas gingivalis. Subgingival samples exhibited a significantly higher proportion of “red” and “orange complex” species, while supragingival plaque exhibited higher proportions of “green” and “purple” complex species as well as Actinomyces species. Suspected periodontal pathogens could be detected in supragingival plaque from sites where subgingival samples were negative for the same species. Conclusions: The data indicate that supragingival plaque can harbor putative periodontal pathogens, suggesting a possible rôle of this environment as a reservoir of such species for the spread or reinfection of subgingival sites.     

Clustering of subgingival microbial species in adolescents with periodontitis

It has become increasingly recognized that groups of microorganisms interact within the subgingival plaque of adult subjects with periodontitis. It is much less clear, however, whether the consortia of microorganisms associated with periodontitis are different in early and more advanced cases of periodontitis. To investigate this point further, subgingival plaque was collected from six sites in 87 adolescents with periodontitis and 73 controls and the samples were analyzed for the detection of 18 microbial species using the DNA-DNA hybridization technique. Actinomyces oris accounted for the highest proportion of the flora and was more predominant among controls. Prevotella nigrescens, Prevotella intermedia, Porphyromonas gingivalis, and Tannerella forsythia were present at higher levels among the subjects with periodontitis. Factor analyses identified one factor characterized by highly positive loadings for T. forsythia, Campylobacter rectus, P. gingivalis, P. intermedia, P. nigrescens, Parvimonas micra, and Treponema denticola, and another factor characterized by highly positive loadings of A. oris, Capnocytophaga ochracea, Eikenella corrodens, Streptococcus intermedius, Selenomonas noxia, Streptococcus oralis, Streptococcus sanguinis, and Veillonella parvula. Aggregatibacter actinomycetemcomitans and Streptococcus mutans did not load on any of the two factors, while Fusobacterium nucleatum loaded on both. These findings confirm the occurrence of clustering of subgingival bacteria according to case status also among young individuals.     

Periodontal pathogens in subgingival plaque of HIV-positive subjects with chronic periodontitis

Many putative periodontal pathogens associated with periodontal disease in human immunodeficiency virus (HIV)-infected patients also occur in non-HIV-infected individuals. This study examined the prevalence of eight periodontal pathogens in HIV-positive and HIV-negative patients with chronic periodontitis using the 16s RNA polymerase chain reaction technique. The results showed a significant prevalence of Porphyromonas gingivalis and Treponema denticola among HIV-negative patients compared to HIV-positive patients. Sixty percent of the patients in both groups were colonized by five to six species. Odds ratio analysis revealed a statistically significant positive association between three of the 28 possible combinations in the HIV-positive group. They included Prevotella nigrescens/Campylobacter rectus, P. nigrescens/P. gingivalis and P. nigrescens/T. denticola. Although the prevalence of periodontal pathogens is similar in both the groups, the combination of certain periodontal pathogens may be responsible for chronic periodontitis seen in HIV-infected adults.     

Lethal photosensitization of bacteria in subgingival plaque from patients with chronic periodontitis

Subgingival plaque samples from patients with chronic periodontitis were exposed to light from a 7.3 mW Helium/Neon laser for 30 s in the presence and absence of 50 μg /ml toluidine blue O as a photosensitizer. Viable counts of various groups and species of bacteria were carried out before and after irradiation. The median numbers of viable bacteria initially present in the 30-μl aliquots irradiated were 1.13 × 10⁵ cfu (aerobes), 4.08 × 10⁵ cfu (anaerobes), 4.92 × 10³ cfu (black-pigmented anaerobes), 4.75 × 10² cfu ( Porphyromonas gingivalis ), 6.15 × 10³ cfu ( Fusobacterium nucleatum ) and 1.7 × 10⁴ cfu (streptococci). The dye/laser combination achieved significant reductions in the viability of these organisms, the median reductions in the viable counts being 91.1% for aerobes, 96.6% for anaerobes, 100% for black-pigmented anaerobes, P. gingivalis and F. nucleatum and 94.2% for streptococci. Overall, the viability of bacteria in the 20 plaque samples was not significantly decreased by the dye alone. However, in a small minority of samples there were indications of light-independent, dye-induced toxicity. Low-power lasers, in conjunction with appropriate photosensitizers, may be a useful adjunct to mechanical debridement in the treatment of inflammatory periodontal diseases if a similar effectiveness against subgingival plaque bacteria can be achieved in vivo .     

Antibiotic susceptibility testing of subgingival plaque samples

The in vitro inhibitory effect of several antimicrobial agents was determined against dispensed dental plaque samples taken from periodontally diseased sites as an aid in the selection of antibiotics for adjunctive use in periodontal therapy. 2 groups of patients were sampled. 1 group of 10 patients with severely advanced disease had received periodontal treatment which included the frequent adjunctive use of an antibiotic. The second group consisted of 15 individuals with less severe periodontal disease; only 4 individuals had been previously treated with antibiotics for their periodontal disease. Bacterial samples of subgingival plaque were taken from each patient and tested against a battery of antibiotics to determine which agent was the most effective in suppressing bacterial growth. Each antibiotic was incorporated into Trypticase-soy blood agar at a concentration equivalent to that achieved in either gingival fluid or blood following recommended oral dosages. The inhibitory effect was determined by comparing the number of bacterial recovered on the antibiotic-containing medium to the total number of bacteria recovered on the basal medium. Penicillins, with the exception of cloxacillin, were the most effective in inhibiting bacterial growth. Benzylpenicillin consistently inhibited the growth of 90% of the isolates recovered on media free of antibiotics while ampicillin and amoxicillin frequently inhibited 99% or more of the bacteria recovered. Tetracycline was generally inhibitory for at least 90% of the isolates if the patients had not been previously treated with this agent. However, resistance to this drug was common in samples taken from patients previously treated with tetracycline. Doxycycline, a tetracycline derivative, did not inhibit significantly more isolates than tetracycline. Clindamycin was inhibitory for 90% or more of the organisms in most of the samples; and, was usually effective in inhibiting isolates in samples which exhibited large numbers of isolates resistant to tetracycline. Erythromycin was relatively ineffective against the isolates recovered from samples from the severely diseased group but was inhibitory to isolates in some samples taken from the more moderately diseased group. Metronidazole, at the concentration tested, was largely ineffective against the isolates in bacterial samples from both groups. No single antimicrobial agent was found to be inhibitory for greater than 90% of the bacteria recovered from all of the subgingival plaque samples with the possible exception of some penicillins.     

Characterization of hemolytic bacteria in subgingival plaque

Three-quarters of the patients with periodontal diseases surveyed in this study had one or more distinct types of hemolytic bacteria in their subgingival plaque. Twelve different species of bacteria were identified, belonging to five genera (Actinomyces, Streptococcus, Staphylococcus, Prevotella, and Actinobacillus). Nine hemolytic isolates, consisting of four Prevotella denticola strains, two Actinomyces naeslundii genospecies 2 strains, and one each of P. melaninogenica, Streptococcus constellatus, and A. naeslundii genospecies 1 strains were characterized. Incorporation of pronase into blood agar medium inhibited hemolysis by all of the isolates, suggesting a proteinaceous component for each of their hemolysins. With one exception, hemolysin production appeared to be regulated by the concentration of environmental iron: exogenous hemin was found to inhibit hemolysin production, and the iron scavenging compound, 2,2′-dipyridyl, was found to promote hemolysin production by all of the strains except for the S. constellatus isolate. Genomic libraries of each of the hemolytic plaque isolates were prepared in Escherichia coli using pBR322. Hemolytic clones were isolated on blood agar medium containing ampicillin at frequencies ranging from 1–6.7 × 10^?4. Extensive restriction mapping revealed regions of homology in the case of clones derived from three P. denticola strains isolated from the same subject. Two of the P. denticola-derived clones were virtually identical throughout the entirety of their > 5 Kb inserts. The clone derived from the third strain showed good homology to the other two within a 1.3 Kb region, but the flanking DNA showed no homology even though all three P. denticola isolates were shown to be clonally related by ribotyping. The results indicate that hemolytic bacteria are frequently recovered from active disease sites of subjects with periodontal diseases. The hemolytic phenotype appears to be restricted to a small proportion of the total number of species normally resident in subgingival plaque. Restriction mapping suggested that a variety of hemolysin genes may be involved and that, at least in certain cases, they may be on mobile genetic elements.     

Microbial complexes in subgingival plaque

Abstract. It has been recognized for some time that bacterial species exist in complexes in subgingival plaque. The purpose of the present investigation was to attempt to define such communities using data from large numbers of plaque samples and different clustering and ordination techniques. Subgingival plaque samples were taken from the mesial aspect of each tooth in 185 subjects (mean age 51 ± 16 years) with (n= 160) or without (n= 25) periodontitis. The presence and levels of 40 subgingival taxa were determined in 13.261 plaque samples using whole genomic DNA probes and checkerboard DNA-DNA hybridization. Clinical assessments were made at 6 sites per tooth at each visit. Similarities between pairs of species were computed using phi coefficients and species clustered using an averaged unweighted linkage sort. Community ordination was performed using principal components analysis and correspondence analysis. 5 major complexes were consistently observed using any of the analytical methods. One complex consisted of the tightly related group: Bacteroides forsythus. Porphvromonas gingivalis and Treponema denticola. The 2nd complex consisted of a tightly related core group including members of the Fusobcterium nucleaturm/periodonticum subspecies, Prevotella intermedia. Prevotella nigrescens and Peptostreptucoccus micros. Species associated with this group included: Eubacterium nodatum, Campylobacter rectus, Campylobacter showae, Streptococcus constellatus and Campylobacter gracilis. The 3rd complex consisted of Streptococcus sanguis. S. oralis, S. mitis, S. gordonii and S. intermedius. The 4th complex was comprised of 3 Capnocytophaga species, Campylobacter concisus, Eikenella corrodens and Actinobacillus actinomycetemcomitans serotype a. The 5th complex consisted of Veillonella parvula and Actitwmyces odontotylicus. A. actinotnycetemcomitans serotype b, Selenomonas noxia and Actinomyces naeslundii genospecies 2 (A. viscosus) were outliers with little relation to each other and the 5 major complexes. The 1st complex related strikingly to clinical measures of periodontal disease particularly pocket depth and bleeding on probing.     

Periodontal pathogens in subgingival plaque of HIV‐positive subjects with chronic periodontitis

Many putative periodontal pathogens associated with periodontal disease in human immunodeficiency virus (HIV)‐infected patients also occur in non‐HIV‐infected individuals. This study examined the prevalence of eight periodontal pathogens in HIV‐positive and HIV‐negative patients with chronic periodontitis using the 16s RNA polymerase chain reaction technique. The results showed a significant prevalence of Porphyromonas gingivalis and Treponema denticola among HIV‐negative patients compared to HIV‐positive patients. Sixty percent of the patients in both groups were colonized by five to six species. Odds ratio analysis revealed a statistically significant positive association between three of the 28 possible combinations in the HIV‐positive group. They included Prevotella nigrescens/Campylobacter rectus, P. nigrescens/P. gingivalis and P. nigrescens/T. denticola. Although the prevalence of periodontal pathogens is similar in both the groups, the combination of certain periodontal pathogens may be responsible for chronic periodontitis seen in HIV‐infected adults.     

慢性牙周炎龈下菌斑中五种牙周可疑致病微生物的分布

目的研究五种牙周可疑致病微生物在慢性牙周炎患者龈下菌斑的分布。方法选择27例慢性牙周炎患者,每位患者选取牙周袋最深的两个位点作为观察位点,采集龈下微生物样本,采用多重聚合酶链反应和反杂交的方法对伴放线菌嗜血菌、牙龈卟啉单胞菌、福赛斯坦纳菌、中间普雷沃菌和齿垢密螺旋体五种微生物进行半定量检测。结果在所检测的54个位点中,牙龈卟啉单胞菌、中间普雷沃菌、福赛斯坦纳菌和齿垢密螺旋体均有较高的检出率,分别为98.15%、92.59%、100%和98.15%;伴放线菌嗜血菌检出率较低,为20.37%。牙龈卟啉单胞菌和福赛斯坦纳菌的检出量明显高于其他三种微生物,其差异有统计学意义（P<0.05）。结论慢性牙周炎患者多存在牙龈卟啉单胞菌、福赛斯坦纳菌、中间普雷沃菌和齿垢密螺旋体的同时感染,且牙龈卟啉单胞菌和福赛斯坦纳菌的感染量较高。     

Stannous fluoride and subgingival chlorhexidine irrigation in the control of plaque and chronic periodontitis

The purpose of this study was to investigate the possible benefits of stannous fluoride dentifrice (SnF2) and once daily chlorhexidine (CH) in the control of chronic periodontitis, following a single course of scaling and root planing. 14 adult patients with pockets 4 mm or deeper received a thorough scaling and root planing, followed by instruction in the irrigation of those pockets with CH. They were given either SnF2-containing or a placebo dentifrice, and a new multituft toothbrush, but no instruction in routine oral hygiene. Plaque Index (PII), Sulcus Bleeding Index (SBI) and pocket depth were recorded prior to scaling on day 0. The same parameters and gingival shrinkage were recorded at weeks 1, 2, 4, 8 and 12. CH irrigation was discontinued at day 28. Both groups showed highly significant improvements in all parameters except gingival shrinkage after 28 days. PII was significantly less at all stages in the SnF2 group. It reached baseline values by day 56 in the placebo and day 84 in the SnF2 group. Periodontitis as assessed by SBI remained reduced significantly below baseline values in both groups at the end of the study. At this time there was no significant difference between the groups as regards SBI. It was concluded that a single course of thorough scaling and root planing followed by once daily subgingival irrigation with CH for 1 month, produced a reduction in periodontitis still apparent 2 months after cessation of irrigation. This regime allows a 3-month interval between oral hygiene visits. SnF2-containing dentifrice had a plaque control effect additional to that produced by the regime but had no effect on periodontitis as assessed by SBI.     

光动力疗法对慢性牙周炎龈下牙周致病菌的影响

目的：应用PeriowaveTM光动力杀菌系统对慢性牙周炎患者进行治疗,通过龈下菌斑中牙周致病菌比例的变化,评价光动力疗法（photodynamic therapy,PDT）治疗慢性牙周炎的临床效果。方法：选取60名慢性牙周炎患者,分别给予SRP＋1次PDT（A组）、SRP＋2次PDT（B组）或单纯SRP（C组）治疗。利用real-time PCR技术检测A、B、C三组在治疗前、治疗后6周、治疗后12周龈下菌斑中牙周致病菌P.g、A.a、T.f所占比例的变化。结果：治疗后6周,A、B两组牙周致病菌P.g在总菌中的比例都有非常显著降低（p〈0.01）,C组有显著降低（p〈0.05）;治疗后12周,A、B组仍有非常显著的降低（p〈0.01）,与C组相比有非常显著差异（p〈0.01）,但A、B两组之间没有差异（p〉0.05）;而仅在治疗后12周,B组的A.a相对于总菌的比例与基线相比有显著降低（p〈0.05）,且这一变化显著大于A、C两组（p〈0.05）;在治疗后6周,A、B组T.f相对于总菌含量的下降与基线相比,明显大于C组（p〈0.05）,治疗后12周,A、B两组T.f相对于总菌的含量仍有非常显著的下降（p〈0.01）,但A组和B组之间都没有差异。结论：PDT对P.g、A.a和T.f3种牙周致病菌都有杀灭作用,但P.g、T.f对PDT更为敏感,PDT可以作为治疗慢性牙周炎的辅助方法。     

Application of denaturing gradient gel electrophoresis (DGGE) to the analysis of microbial communities of subgingival plaque

OBJECTIVES: Denaturing gradient gel electrophoresis (DGGE) was applied to the microbiologic examination of subgingival plaque. MATERIALS AND METHODS: The PCR primers were designed from conserved nucleotide sequences on 16S ribosomal RNA gene (16SrDNA) with GC rich clamp at the 5'-end. Polymerase chain reaction (PCR) was performed using the primers and genomic DNAs of typical periodontal bacteria. The generated 16SrDNA fragments were separated by denaturing gel. RESULTS: Although the sizes of the amplified DNA fragments were almost the same among the species, 16SrDNAs of the periodontal bacteria were distinguished according to their specific sequences. The microflora of clinical plaque samples were profiled by the PCR-DGGE method, and the dominant 16SrDNA bands were cloned and sequenced. Simultaneously, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia were detected by an ordinary PCR method. In the deep periodontal pockets, the bacterial community structures were complicated and P. gingivalis was the most dominant species, whereas the DGGE profiles were simple and Streptococcus or Neisseria species were dominant in the shallow pockets. The species-specific PCR method revealed the presence of A. actinomycetemcomitans, P. gingivalis and P. intermedia in the clinical samples. However, corresponding bands were not always observed in the DGGE profiles, indicating a lower sensitivity of the DGGE method. CONCLUSION: Although the DGGE method may have a lower sensitivity than the ordinary PCR methods, it could visualize the bacterial qualitative compositions and reveal the major species of the plaque. The DGGE analysis and following sequencing may have the potential to be a promising bacterial examination procedure in periodontal diseases.     

Treatment of prepubertal periodontitis

This paper reports the treatment of prepubertal periodontitis in a 3-year-old girl with Papillon-Lefèvre Syndrome. Initially, the patient was found to have a myeloperoxidase deficiency and microbiological tests have identified Bacteroides and Fusobacterium, in 60% and 25%, respectively of the total number of microbial flora cultivated. The initial treatment was extraction of all the primary teeth with grade 3 mobility, scaling, root planing and daily subgingival irrigation with a 0.2% solution of chlorhexidine. Several months before the eruption of the first permanent molars, the rest of the primary teeth were extracted. The patient was treated with daily subgingival irrigation of chlorhexidine and weekly professional oral hygiene. At the age 6 1/2 years, the permanent teeth have normal gingiva and crevice depths; microbiological investigation reveals a prevalence of the coccoid forms, and radiographs show no evidence of periodontal pathology.     

Proportion of antibiotic resistance in subgingival plaque samples from Mexican subjects

AIM: To determine the proportion of bacteria resistant to amoxicillin and doxycycline in subgingival plaque samples from Mexican subjects. MATERIALS AND METHODS: Two subgingival plaque samples were taken from 20 Mexican subjects. Samples were dispersed, diluted and plated on non-antibiotic agar plates and on plates containing 0.5, 1, 2, 4, 8 and 16 microg/ml of either amoxicillin or doxycycline. The proportion of resistant bacteria was calculated based on the total number of colony-forming units present in the non-antibiotic containing plates. RESULTS: On average, 0.4-13.4% and 0.9-20.4% of the total cultivable subgingival microbiota was resistant to the concentrations tested of amoxicillin and doxycycline, respectively. The differences between antibiotics were statistically significant for the 0.5, 2 and 4 mug/ml concentrations (p < 0.05, Wilcoxon's test). CONCLUSIONS: Our findings revealed that a relatively small proportion of the total cultivable subgingival microbiota from Mexican subjects was resistant to amoxicillin and doxycycline.     

Isolation and characterization of subgingival staphylococci from periodontitis patients and controls

OBJECTIVES: To isolate and characterize subgingival staphylococci from patients with periodontal disease and from periodontally healthy controls, to evaluate the periodontal environment as a potential source for systemic staphylococcal infections. METHODS: Periopaper strips were used to isolate subgingival staphylococci from 28 patients with chronic periodontitis and 28 periodontally healthy age and sex-matched controls. Staphylococci were identified by microbiological methods and antibiotic resistance profiles determined. RESULTS: Staphylococci were isolated from 54% diseased subgingival and 43% healthy subgingival sites in over 50% periodontitis patients and from 29% healthy subgingival sites in 54% controls. No significant differences in the frequency of isolation or numbers of staphylococci isolated from diseased and healthy sites were noted. Staphylococcus epidermidis was the predominant oral species. Seventy per cent (115 of 165) of all isolates were penicillin-resistant. CONCLUSIONS: Subgingival staphylococci are present in both periodontitis patients and controls. In periodontitis there is an increased risk of bacteraemia because of the increased dentogingival surface area. The dental and periodontal health of patients at risk from haematogenous infections should therefore be maintained at a high level. Antibiotic resistance profiles of the oral staphylococcal isolates suggest that amoxicillin may no longer be a suitable antibiotic for prophylaxis against systemic infections such as prosthetic valve endocarditis.