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Background: Oropharyngeal candidiasis is the most common opportunistic infection affecting patients with human immunodeficiency virus (HIV) infection. Because of convenience, cost, and reluctance to complicate antiretroviral treatment regimens, single-dose fluconazole may be a favorable regimen for treatment of moderate to severe oropharyngeal candidiasis. We conducted a prospective, randomized, double-blind, placebo-controlled trial to compare the clinical and mycological responses, relapse rates, and safety of a single 750-mg dose and a 14-day course of treatment with fluconazole., Methods: A total of 220 HIV-infected patients with clinical and mycological evidence of oropharyngeal candidiasis were randomly assigned in a 1:1 ratio to receive either a 750-mg single dose of orally administered fluconazole (110 patients) or 150 mg of orally administered fluconazole once per day for 2 weeks (110 patients). The primary efficacy analysis was based on clinical and mycological responses at the end of treatment. Secondary parameters were safety and relapse rate., Results: Single-dose fluconazole was equivalent to a 14-day course of fluconazole in achieving clinical and mycological cure, with clinical cure rates of 94.5% and 95.5%, respectively (odds ratio, 0.825; 95% confidence interval, 0.244-2.789; P= .99), and mycological cure rates of 84.5% and 75.5%, respectively (odds ratio, 1.780; 95% confidence interval, 0.906-3.496; P= .129). Drug-related adverse events were uncommon and were not different between the treatment groups., Conclusion: A single dose of 750 mg of fluconazole was safe, well tolerated, and as effective as the standard 14-day fluconazole therapy in patients with HIV infection and acquired immunodeficiency syndrome who had oropharyngeal candidiasis coinfection.

Background: In Tanzania, little is known on the species distribution and antifungal susceptibility profiles of yeast isolates from HIV-infected patients with primary and recurrent oropharyngeal candidiasis., Methods: A total of 296 clinical oral yeasts were isolated from 292 HIV-infected patients with oropharyngeal candidiasis at the Muhimbili National Hospital, Dar es Salaam, Tanzania. Identification of the yeasts was performed using standard phenotypic methods. Antifungal susceptibility to fluconazole, itraconazole, miconazole, clotrimazole, amphotericin B and nystatin was assessed using a broth microdilution format according to the guidelines of the Clinical and Laboratory Standard Institute (CLSI; M27-A2)., Results: Candida albicans was the most frequently isolated species from 250 (84.5%) patients followed by C. glabrata from 20 (6.8%) patients, and C. krusei from 10 (3.4%) patients. There was no observed significant difference in species distribution between patients with primary and recurrent oropharyngeal candidiasis, but isolates cultured from patients previously treated were significantly less susceptible to the azole compounds compared to those cultured from antifungal naïve patients., Conclusion: C. albicans was the most frequently isolated species from patients with oropharyngeal candidiasis. Oral yeast isolates from Tanzania had high level susceptibility to the antifungal agents tested. Recurrent oropharyngeal candidiasis and previous antifungal therapy significantly correlated with reduced susceptibility to azoles antifungal agents.

Using the ethnobotanical approach, some Tanzanian plants reported to be used by traditional healers for the treatment of oral candidiasis and fungal infections of the skin were collected and screened for their antifungal activity against Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida krusei and Cryptococcus neoformans. A total of 65 crude methanol extracts belonging to 56 plant species and 38 families were screened using the broth microdilution method, according to the guidelines of the Clinical and Laboratory Standard Institute (CLSI) (formerly, National Committee for Clinical and Laboratory Standards) [National Committee for Clinical Laboratory Standards, 2002. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. Approved Standard-2nd Edition M27-A2, National Committee for Clinical Laboratory Standards, Wayne, PA, USA]. Among the tested plant species, 45% (25 species) showed antifungal activity against one or more of the test fungi. The most susceptible yeasts were Cryptococcus neoformans, followed by Candida krusei, Candida tropicalis, and Candida parapsilosis. The least susceptible were Candida albicans and Candida glabrata. Strong antifungal activity was exhibited by extracts of Clausena anisata Oliv., Sclerocariya birrea Sond, Turraea holstii Gurk, Sterculia africana (Lour) Fiori, Acacia robusta subsp. Usambarensis (Taub) Brenan, Cyphosterma hildebrandti (Gilg), Desc, Elaeodendron buchannanii (Lows), Acacia nilotica (L.) Wild ex Del, Jatropha multifida L., and Pteridium aquilinum (L.) Kuhn.

Background: The aim of the study was to compare the prevalence and types of HIV-related oral lesions between children and adult Tanzanian patients on HAART with those not on HAART and to relate the occurrence of the lesions with anti-HIV drug regimen, clinical stage of HIV disease and CD4+ cell count., Methods: Participants were 532 HIV infected patients, 51 children and 481 adults, 165 males and 367 females. Children were aged 2-17 years and adults 18 and 67 years. Participants were recruited consecutively at the Muhimbili National Hospital (MNH) HIV clinic from October 2004 to September 2005. Investigations included; interviews, physical examinations, HIV testing and enumeration of CD4+ T cells., Results: A total of 237 HIV-associated oral lesions were observed in 210 (39.5%) patients. Oral candidiasis was the commonest (23.5%), followed by mucosal hyperpigmentation (4.7%). There was a significant difference in the occurrence of oral candidiasis (chi2 = 4.31; df = 1; p = 0.03) and parotid enlargement (chi2 = 36.5; df = 1; p = 0.04) between children and adults. Adult patients who were on HAART had a significantly lower risk of; oral lesions (OR = 0.32; 95% CI = 0.22-0.47; p = 0.005), oral candidiasis (OR = 0.28; 95% CI = 0.18-0.44; p = 0.003) and oral hairy leukoplakia (OR = 0.18; 95% CI = 0.04-0.85; p = 0.03). There was no significant reduction in occurrence of oral lesions in children on HAART (OR = 0.35; 95% CI = 0.11-1.14; p = 0.15). There was also a significant association between the presence of oral lesions and CD4+ cell count < 200 cell/mm3 (chi2 = 52.4; df = 2; p = 0.006) and with WHO clinical stage (chi2 = 121; df = 3; p = 0.008). Oral lesions were also associated with tobacco smoking (chi2 = 8.17; df = 2; p = 0.04)., Conclusion: Adult patients receiving HAART had a significantly lower prevalence of oral lesions, particularly oral candidiasis and oral hairy leukoplakia. There was no significant change in occurrence of oral lesions in children receiving HAART. The occurrence of oral lesions, in both HAART and non-HAART patients, correlated with WHO clinical staging and CD4+ less than 200 cells/mm3.

As a result of the caries prevalence of the Dutch population the character of the routine dental examination is changing. Caries screening remains relevant for high caries risk individuals but the interpretation of early caries lesions deserves more attention, especially the assessment of progression of caries lesions in time. Diagnosis of caries by dental practitioners in the Netherlands mainly consists of visual inspection in combination with bitewing radiography and both methods play an important role in the timing of bitewing radiographs and forthcoming recall visits for oral health examination. The content and frequency of the routine dental examination are discussed in relation to the individual risk prediction as relevant aspects of quality of dental care in the Netherlands.

This study aimed to determine whether salivary mutans streptococci and lactobacilli counts could add any value to a combination of caries experience variables that was recently presented for the prediction of caries. Sixty-nine children at the age of 7.5 yr participated in this longitudinal study. Microbiological data were obtained at the ages of 7.5, 9.5 and 11.5 yr and caries data at the ages of 7.5. 9.5. 11.5, 13.5 and 15.5 yr. Spearman's correlation coefficients were computed, and forward multiple regression analyses were carried out using bacterial counts and caries experience parameters as explanatory variables and caries increment as a dependent variable. The explained variance (adjusted R2 value) was the measure used to assess the additional value of bacterial counts to the caries predictive potential of the combined parameters of the past caries experience. Correlation coefficients between bacterial counts and 4-yr caries increment were from 0.22 tip to 0.54. In all cases, the simultaneous streptococcus mutans-lactobacilli counts showed a statistically non-significant additional adjusted R2 value of <0.06. The results do not lend support to the concept that these salivary bacterial counts are useful additional caries predictors for the mixed dentition, when a combination of caries experience parameters is used.

Subgingival dental plaque consists mainly of microorganisms that derive their energy from amino acid fermentation. Their nutrient requirements are met by the subgingival proteolytic system, which includes proteases from microorganism and inflammatory cells, and substrate proteins from sulcus exudate, including albumin. To determine the selective effect of individual proteins on microbiota, we used albumin as the main substrate for growth. Eight subgingval plaque samples from untreated periodontal pockets of patients with adult periodontitis were inoculated in peptone yeast medium with bovine albumin (9 g/l). After three subculture steps, cell yields of the enrichment cultures at the medium with 0, 1.25, 2.5, 5, 10, and 20 g/l albumin were determined. Proteolytic activity (U/absorbance at 550 nm) of the enrichment cultures and different isolates derived from the cultures was estimated by the degradation of resorufin-labeled casein. It was observed that the yield of the mixed culture was albumin limited, and the proteolytic activities of the cultures in albumin broth were higher than in control (peptone broth). Among the isolates from the enrichment cultures, Peptostreptococcus micros, Prevotella melaninogenica, Prevotella buccae and Prevotella bivia demonstrated proteolysis. The frequent occurrence of Streptococcus gordonii and Streptococcus anginosus in the albumin cultures is explained by their ability to utilize arginine as an energy source for growth. Albumin in the medium was partly degraded by pure cultures but completely consumed in enrichment cultures, indicating synergy of bacterial proteinases. It is concluded that the subgingival microbiota possesses proteolytic activity and may use albumin as a substrate for their growth. Enrichment cultures on albumin may serve as a relatively simple in vitro model to evaluate the effects of proteinase inhibitors.

The effect of pH, redox potential, O2 and H2 on the growth and proteolytic activity of Treponema denticola ATCC 33520 was studied in a chemostat at different growth rates. The peptidase and protease activities were estimated using different amido-methyl coumarin derivatives and azocasein. The maximum growth rate of T. denticola ATCC 33520 was 0.14 h-1. Reduction of the growth rate of T. denticola by 50-60% gave: an increase in cell mass of 150-200%, a higher acetogenesis and a shift of the pH optimum. The protease and phenylalanine peptidase activities seemed to be of greater importance for the growth of T. denticola ATCC 33520 than the rather low arginine and proline peptidase activities. The redox potential (Eh) played a secondary role. At microaerophilic conditions with 1-5% O2, the cultures maintained a redox potential below -311 mV and an optimal acetogenesis. The presence of H2 induced a marked growth stimulation of T. denticola ATCC 33520. It is concluded that the cell mass and proteolytic activity of T. denticola ATCC 33520 are modulated by the growth rate and the pH and to a lesser extend by the redox potential and presence of O2. Stagnation of the exudate-flow influences these factors and will lead to an increase of the spirochetal population and proteolysis in the periodontal pocket.

The purpose of this study was to compare a commercially available DNA probe technique with conventional cultural techniques for the detection of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia in subgingival plaque samples. Samples from 20 patients with moderate to severe periodontitis were evaluated at baseline and during a 15 months period of periodontal treatment. Paperpoints from 4 periodontal pockets per patient were forwarded to Omnigene for DNA probe analysis, and simultaneously inserted paperpoints from the same pockets were analyzed by standard culture techniques. In addition, mixed bacterial samples were constructed harbouring known proportions of 25 strains of A. actinomycetemcomitans, P. gingivalis and P. intermedia each. A relatively low concordance was found between both methods. At baseline a higher detection frequency was found for A. actinomycetemcomitans and P. gingivalis for the DNA probe technique; for P. intermedia the detection frequency by culture was higher. For A. actinomycetemcomitans, 21% of the culture positive samples was positive with the DNA probe. Testing the constructed bacterial samples with the DNA probe method resulted in about 16% false positive results for the 3 species tested. Furthermore, 40% of P. gingivalis strains were not detected by the DNA probe. The present data suggest that at least part of the discrepancies found between the DNA probe technique used and cultural methods are caused by false positive and false negative DNA probe results. Therefore, the value of this DNA probe method for the detection of periodontal pathogens is questionable.

The sensitivity of the subgingival plaque for amoxicillin, doxycycline, ofloxacin and clindamycin was estimated by agar diffusion disks and Etest in 22 periodontal patients. The sensitivity of the subgingival plaque varied per patient. Resistance of the subgingival plaque for amoxicillin was observed in 50% of the patients and was significantly related to the use of antibiotics in the previous 6 months. The resistance to clindamycin was 68% and to doxycycline 82%. The resistance of the subgingival plaque to these antibiotics could not be related to the previous use. A decision-tree was constructed including a step wise use of a microscopical spirochete test and an antibiotic sensitivity test of the subgingival microflora for the above mentioned antibiotics. The clinical implications of the observed long lasting antibiotic resistance and the practical use of a decision-tree including a spirochete test and antibiotic sensitivity tests need further investigation.

This review aims to compare the occurrence and distribution of mutans streptococci in Africa, Europe, and North America and in addition will try to offer explanations for existing relationships among salivary mutans streptococci counts, dietary patterns, and dental caries. The literature reveals that salivary mutans streptococci counts in child populations of the three continents are comparable. The distribution of mutans streptococci species, with a predominance of S. mutans followed by S. sobrinus, and the virtual absence of other mutans streptococci species are also comparable. Although it is widely believed that diet has an important effect on mutans streptococci counts, this review provides evidence that this does not hold true when variations in dietary patterns are moderate, as they normally are in real-life situations. Since the diets of the child populations in the three continents vary moderately, a strong dietary-induced effect on salivary mutans streptococci counts cannot be expected. The observed analogous salivary mutans streptococci counts in these child populations are thus 'not surprising' but are in accordance with the conceptual expectation. The differences in caries experience in children of the three continents cannot be explained by the prevailing mutans streptococci species but instead should be attributed to differences in the cariogenicity of the various diets. The fact that the cariogenicity of the diet determines the development of dental caries while hardly affecting the mutans streptococci counts explains the limited value of the latter as an indicator of dental caries. The reviewed literature shows that mutans streptococci are ubiquitous in children aged 7 years and older in Africa, Europe, and North America. Mutans streptococci should therefore be considered as belonging to the indigenous microflora of the human mouth.

In 1993 a representative sample of dental practitioners in the Netherlands was questioned on the use of antibiotics for the prevention of endocarditis in the previous year. Most of the responding dentists (67%) reported prescription of antibiotics for patients at risk. Some of them prescribed an antibiotic regimen that differed from the guidelines drawn up by the Dutch Heart Foundation. In 1992 only 47% of the responding dentists prescribed antibiotics for the prevention of endocarditis entirely according to these guidelines.

In 1993 a representative sample of dentists in the Netherlands was questioned on the use of antibiotics in the treatment of periodontitis. It was found dat 50% of the general practitioners had a positive attitude, 15% a negative one and 35% had an indifferent attitude towards the use of antibiotics in periodontal treatment. Almost 40% of them indicated the prescription of antibiotics for periodontal reasons in the previous year. Most of the dentists (71%) prescribed a combination of metronidazole and amoxicillin. The average prescription frequency in 1992 was 18 times. The prescription frequency and the choice of a combination of metronidazole and a broad spectrum penicillin in the treatment of periodontitis is discussed, emphasizing the importance of the traditional mechanical therapy, the disturbance of the colonization resistance by means of antibiotics and the worldwide concern about the usage of antibiotics.

The purpose of the study was to determine microscopically the %s of spirochetes at sites without periodontal destruction in subjects with destructive periodontal diseases (cases) and in subjects free of it (controls), who had not received professional prophylaxis. From a sample of 164 individuals aged between 30-44 years living in rural and urban areas of Tanzania, cases and controls were selected. Cases (n = 25) were selected who exhibited at least 3 teeth with pocket depth of > 5 mm. The controls (n = 28) had no pockets deeper than 3 mm. From each subject, 1 subgingival plaque sample was taken at the mid point of the lingual surface of 1 of the upper premolars which showed bleeding on probing but no calculus and no pockets. In addition, one subgingival sample was obtained from a pocket. Pockets contained the highest %s of spirochetes, which confirms earlier findings. A significant difference in % of spirochetes between cases and controls was found at non-destructive sites, indicating a host effect on the subgingival microflora. However, the spirochete counts at non-destructive sites did not provide a reliable measure to identify subjects with destructive periodontal disease.

Periodontitis is a mixed infection with subgingival bacteria that belong to the microflora of the host. The bacteriological tests (Microscopy, Culture, Omnigene, Affirm DP and Evalusite) are mainly aimed at spirochetes or A. actinomycetemcomitans, P. gingivalis and P. intermedia. The detection limits of the different tests vary and, therefore, the detection levels are between 0.01-10% of the subgingival microflora. However, threshold levels to distinguish between diseased and carrier state are not established and the low sensitivity and specificity of the tests limit their diagnostic usefulness. The correlation between the tested bacteria and periodontitis does not mean a causal relationship. As a result, the justification of the elimination of the tested bacteria is questionable. The tests of the subgingival plaque do not only add to the microbiological knowledge, but might also promote the overtreatment with antibiotics, such as the in the Netherlands popular combination of amoxicilline and metronidazole. The balance between host and microflora can be restored in most patients by mechanical elimination of the mainly Gram negative anaerobic subgingival plaque. In special occasions metronidazole can be empirically prescribed in order to support the subgingival debridement. In the relatively rare occasions of superinfection, a sensitivity test of the subgingival microbiota is recommended.

Dentistery recently has developed a new preventive treatment, that is the suppression of the acid-producing bacteria in dental plaque. The background and application of two chlorine hexidine lacquers, EC40 and Clorzoin are discussed.

The occurrence of mutans streptococci species was investigated in Tanzanian infants aged between 1.0 and 3.5 years with rampant caries and without caries using biochemical and immunological techniques. These infants came from Turiani and Singida, where the caries prevalence for this age group was 12.8% and 1.6% respectively. Streptococcus mutans was found to be the only mutans streptococcal species. The other species, including the previously reported Streptococcus rattus, were not found. Consequently, the differences in caries experience observed between and within the two populations could not be explained by differences in mutans streptococcal species.

We studied the nature of attachment of Treponema denticola ATCC 33520 to a microscopically distinct population of rounded rat palatal epithelial cells. The motility of the freshly harvested spirochetes appeared not be a prerequisite for attachment. Treatment of T. denticola ATCC 3350 with proteinase-K, heat, glutaraldehyde, formaldehyde and periodate oxidation decreased the attachment to the rounded rat palatal epithelial cells, indicating the involvement of protein and carbohydrate moieties. Trypsin treatment had no effect on the attachment. The attachment of T. denticola ATCC 33520 was decreased after treatment with native non-immune rabbit serum, native polyclonal rabbit serum, D-mannose, N-acetyl-D-galactosamine and sialic acid. The results indicate that the attachment of T. denticola ATCC 33520 to rounded rat palatal epithelial cells is mediated by trypsin-resistant adhesin(s) of protein and carbohydrate nature, with affinity for D-mannose, N-acetyl-D-galactosamine and sialic acid.

The ability of Streptococcus mutans to ferment carbohydrates and to produce acid was investigated in different flours in vitro. The amounts of acid produced suggest a possible ecological role of the tested flours in the occurrence of Strep. mutans in dental plaque.

In the present study an assay for the attachment of T. denticola to epithelial cells is described. An indirect immunohistochemical staining method, using two native polyclonal antisera, revealed dark-brown coloured spirochetes attached to rat palatal epithelial cell (RPE) monolayers. In addition, two morphologically distinct populations of RPE cells could be distinguished in the monolayers when using phase contrast microscopy. One minor population consisted of isolated rounded RPE cells that were lying on top of a confluent monolayer of flattened RPE cells. The rounded RPE cells were more receptive for the attachment of T. denticola than the flattened cells. The rounded RPE cells were evenly distributed over the monolayer, but the attachment of spirochetes to the rounded cells was greater at the edge than in the centre of the monolayers. The percentage of rounded RPE cells with attached spirochetes depended on the incubation time (optimum 6 h), temperature (optimum 37 degrees C) and pH (optimum 7.0). It is speculated that the attachment of T. denticola is a physical/chemical process of yet unknown nature and that differences in the number of microvilli and/or the amount of available receptors, between the two morphologically distinct cell types, accounts for the differences in the numbers of attached spirochetes.

The introduction of Kodak's chairside test kit, Evalusite, for Actinobacillus actinomycetemcomitans (A.a.) and two other oral bacteria has stimulated this study of the relation between A.a. and periodontal destruction. A.a. has been associated with oral abscesses, brain abscesses, endocarditis and localized juvenile periodontitis. However, in all periodontal studies A.a. also has been observed in 'ordinary' periodontal pockets and subjects without active periodontal destruction. Based on longitudinal studies it is concluded, that there is no proof for a causal relationship between A.a. and periodontal destruction, that the detection of A.a. is not indicative for future alveolar bone loss and that the sensitivity of a test of A.a. is too low for the diagnosis of progressive periodontitis. The sheer presence of A.a. in spite of its virulence factors is not sufficient to cause periodontal destruction. It is stated that, in order to prevent overtreatment and medicalization, the use of bacteriological tests and broad spectrum antibiotics should be restricted to patients with progressive periodontal destruction not responding to standard treatment.

The attachment of 10 different Treponema denticola strains to monolayers of 4 types of epithelial cells derived from rat palatal epithelium, guinea pig ear, human buccal epithelium and human corneal epithelium was screened microscopically. Most T. denticola strains were able to attach to all four types of epithelial cells. The T. denticola strains seemed to attach better to epithelial cells derived from primary cultured material. The T. denticola strains showed different degrees of attachment. Scanning electron microscopy studies revealed that the attachment of T. denticola was not only tip-associated but occurred also at random points in close contact with microvilli of the epithelial cells. Attached spirochetes were non-uniformly distributed over the monolayers, indicating the presence of receptive subpopulations of epithelial cells in the monolayers.

The establishment of S. mutans together with A. viscosus was investigated in dental plaque of specific pathogen-free (SPF) rats fed different carbohydrate diets. Two Tanzanian S.mutans strains MM3 and MM24 and one Tanzanian A. viscosus strain MM13 were used for this purpose. The basic diet consisting of 32% skim-milk, 7% yeast extract and 1% soy bean oil was supplemented with either 10% lactose and 50% corn flour, or 10% glucose and 50% corn flour, or 60% amylum or 60% wheat flour. S. mutans and A. viscosus were enumerated twenty days after inoculation. S. mutans counts were high irrespective of the dietary regime. A. viscosus counts in the glucose, lactose and amylum groups were of the same magnitude and significantly higher than those of the wheat flour group. The hypothesis that the establishment of S. mutans in sucrose free diets could be facilitated by the extracellular polysaccharides produced by A. viscosus was not supported by the present data. The finding that S. mutans can establish in high number in dental plaque of SPF rats in the absence of sucrose corroborates previous reports indicating high S. mutans counts in African populations with a low sucrose intake.

The oral spirochaete Treponema denticola ATCC 33520 was grown at a mean generation time of 10 h in anaerobic continuous culture in a serum- and carbohydrate-free medium at pH 7.0. The extracellular proteolytic activities of this spirochaete were then investigated by incubating washed cells with 68 2-naphthylamide derivatives of the Extended API System. Chymotrypsin-like, trypsin-like, elastase-like and iminopeptidase activities were demonstrated. The phenylalanine peptidase or chymotrypsin-like activity of T. denticola ATCC 33520, estimated with N-succinyl-L-phenylalanyl-L-leucyl-L-phenylalanine-thiobenzyl ester (SPLP) had a pH optimum at pH 8.5, a specific activity of 36.6 nmol min-1 (mg dry wt)-1 and was inhibited only slightly by HgCl2. The trypsin-like activity, estimated with benzoyl-DL-arginine-7-amido-4-methylcoumarin (BAMC), had a pH optimum at pH9, and a specific activity of 0.3 nmol min-1 (mg dry wt)-1; inhibition by HgCl2 indicated the involvement of active thiol groups. The activity should preferably be termed arginine peptidase activity, according to the carboxy-terminal amino acid of the test substrate. The extracellular proline peptidase activity, estimated with L-proline-7-amido-4-methylcoumarin. HBr (PRAMC), had an activity of 1.5 nmol min-1 (mg dry wt)-1, an optimum at pH 8.5 and the properties of a thiol protease. The main cell-bound and extracellular active peptidase activities of fast-growing cells of T. denticola ATCC 33520 are phenylalanine peptidase, proline peptidase, arginine peptidase and an oligopeptide-dependent alanine peptidase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

The estimation of individual caries risk factors and the related preventive treatment in the dental practice are discussed. Salivary counts of mutans streptococci and lactobacilli reflect the bacteriological attack (mutans streptococci) and sugar intake (lactobacilli). Caries resistance can be estimated by measurement of the salivary secretion rate and buffer capacity. Treatment of high caries risk patients should be directed against etiological factors. Salivary flow rates and buffer capacity can be stimulated by daily (sugar-free) gum-chewing. For the use of fluoridated toothpaste and sugar intake the dentist is dependent upon the cooperation of the patient. The bacteriological factor in the caries process can be suppressed by application of chlorhexidine varnish.

Hemagglutination by different Treponema denticola strains was observed for erythrocytes of human, horse, bovine, and rabbit origin. The growth of T. denticola ATCC 33520 in serum-free medium in continuous culture enabled us to study the hemagglutinating activity of freshly harvested spirochetes of a defined physiological status. The hemagglutinating activity was cell bound and not related to motility or appendages, such as fimbriae. The activity was destroyed by proteolytic enzymes, heat, and alkylation, indicating that the agglutinin is of a proteinaceous nature. In addition, periodate oxidation of the spirochetes indicated the involvement of carbohydrate groups. Microscopic inspection of the hemagglutination mixtures at the titration endpoints revealed that only a part of the spirochete population was involved in the hemagglutination process. The hemagglutinating activity was found to be growth phase related. The activity was blocked by serum, while of all tested amino acids and carbohydrates, only sialic acid blocked the activity at low concentrations. In conclusion, we found a hemagglutinating activity in T. denticola which was cell bound and growth phase related. The agglutinin may be a glycoprotein, like lectin, that recognizes sialic acid as a receptor.

This study aimed to investigate the prevalence of selected components of the oral microflora in breast-fed children who developed rampant caries (resembling nursing caries) under hitherto unexplained circumstances. Dental plaque and saliva samples were collected from breast-fed children, aged between 1 and 2.5 years, with and without rampant caries. Mutans streptococci and lactobacilli were isolated from dental plaque of all children with rampant caries and from most caries-free children. None of the colonies of mutans streptococci resembled those of Streptococcus sobrinus. The mean counts of the mutans streptococci and lactobacilli were 100-fold higher in plaque samples from children with rampant caries as compared with caries-free children. No difference could be found between the numbers of mutans streptococci in plaque overlaying cavities and that from adjacent sound enamel. In contrast, the counts of lactobacilli in plaque were approximately 100-fold higher from cavities than from sound surfaces. The levels of mutans streptococci in saliva were directly related to the presence of rampant caries. The results show that caries-free and caries-active breast-fed children, aged 1 to 2.5 years, harbour mutans streptococci and lactobacilli on their teeth. Rampant caries in these children can occur in the absence of nursing bottles or any other feeding abuse during weaning and in the presence of an aciduric plaque microflora, as has been reported for children with nursing bottle caries.

The clinical and microbiological effect of locally-applied chlorhexidine gel 2%, amine fluoride gel 1.25%, stannous fluoride gel 4% or placebo gel in 40 periodontal pockets of 10 patients were studied. The gels were applied 3 x within 10 min. after mechanical debridement of the pockets. The treatment effect on the subgingival microflora was evaluated by microscopic and culture studies of the subgingival plaque samples. In addition, supragingival plaque, bleeding after probing and probing pocket depth were scored. Examination were carried out before and during a period of 36 weeks after treatment. At the start, the cultured microflora consisted mainly of anaerobic Gram-positive bacteria. Following treatment, the clinical parameters were significantly reduced. Concomitantly, the %s of spirochetes, motile rods and non-motile rods decreased significantly. A significant decrease was also found in the total anaerobic count, whereas the facultative counts remained at the same level found before treatment. This suggested that the treatment resulted in a mainly facultative subgingival microflora. The % Gram-negative rods showed a significant reduction after treatment, but returned to base line at week 12. Statistical analysis of the bacteriological and clinical examinations failed to demonstrate any significant differences between the 4 treatment groups. Thus, in comparison to the placebo gel, subgingival application of chlorhexidine gel 2%, amine fluoride gel 1.25% or stannous fluoride gel 4% did not augment the effect of mechanical debridement on bacteriological and clinical parameters during the experimental period of 36 weeks. However the indicated treatments resulted in a facultative subgingival microflora which is compatible with the host.

The short-term bactericidal effect of 2% chlorhexidine gel, 4% stannous fluoride gel or amine fluoride gel containing 1.25% fluoride on the subgingival microflora was determined in 40 periodontal pockets of 10 patients. The antimicrobial gels or placebo gel were applied in 5-9 mm deep periodontal pockets 3 times within 10 min. Before and 30 min after the applications, samples were taken of the subgingival microflora for determination of the total number of bacteria as well as the number of black pigmented Bacteroides. Reductions of the total number of bacteria were found in all test groups. The reductions found in the pockets treated with chlorhexidine gel or stannous fluoride gel were significantly greater than the reduction found in the pockets treated with a placebo gel. A significant reduction of black-pigmented Bacteroides was found after treatment with chlorhexidine gel or amine fluoride gel. It is concluded that 2% chlorhexidine gel or 4% stannous fluoride gel has a more than 99% reduction effect on the microflora of periodontal pockets within 30 min after application.

The enzyme profiles of 20 oral and non-oral Treponema strains were investigated using an API ZYM Complete Research kit. The test included 10 2-naphthyl derivatives of fatty acids, 20 p-nitrophenol derivatives of carbohydrates and 60 2-naphthylamide derivatives of amino acids and peptides. The oral Treponema species investigated were T. denticola, T. vincentii and T. Pectinovorum. The non-oral species examined were T. phagedenis, T. hyodysenteriae and intestinal spirochaetes of human and chicken origin. Esterase activities on C5 to C10 fatty acids were common among different Treponema species. Glycosidase activities were infrequently observed in T. vincentii, T. pectinovorum and T. phagedenis Reiter strain. Arabinosidase, lactosidase and xylosidase activity was observed in the T. hyodysenteriae strains but alpha-L-fucosidase activity was found only in T. denticola and T. phagedenis. More exo- and endo-peptidase activities were found in T. denticola than in other species. The enteropathogenic T. hyodysenteriae isolates had a very low proteolytic profile. Dipeptidyl prolyl amidase activity was observed in all species except in the T. phagedenis Reiter strain and the avian intestinal spirochaetes. The enzyme profiles did not discriminate between oral and non-oral Treponema species.

The Streptococcus- and Lactobacillus-tests available in The Netherlands for the selection of patients at risk are discussed. Attention is paid tot the calculation of sensitivity, specificity and predictive value of microbiological tests. Although the predictive values or diagnostic power of the saliva tests are rather low, the use of such tests in dental practice is advocated.

The specificity, sensitivity and predictive value of bacteriological tests in periodontology are reviewed. It is stated that the bacteriological aim in periodontal therapy is the change of the subgingival flora in a facultative flora which is compatible with the host. Bacteriological tests can be used to identify indicator bacteria. It is discussed how a microscopical or a bacteriological identification test can support periodontal diagnoses and indication of periodontal therapy.

The clearance of a fluorescein gel applied with an irrigation technique in periodontal pockets was studied. In order to evaluate the time a drug remains in a periodontal pocket, the alteration in concentration of this drug in a given time period was investigated. After application of the gel in 4 pockets of 10 patients, samples were taken from 1 of the 4 pockets at 5, 10, 20 and 40 min. The results show that in the 1st phase, directly after application, most of the gel disappeared from the pocket, while in the 2nd phase, the fluorescein gel is washed out with a calculated 50% reduction time of 12.5 min. The rapid clearance of the gel observed after irrigation might provide a partial explanation of the minor effect of pocket irrigation on the subgingival microflora.

The presence of spirochetes and other bacteria during the induction of necrotizing ulcerative gingivitis (NUG) in beagle dogs was studied by transmission electron microscopy. NUG was induced by application of dental debris from diseased dogs to 4 beagles treated with corticosteroid. Biopsies of interdental papillae were collected during a period of 7 weeks. Spirochetes and other bacteria were observed in enlarged intercellular spaces between non-keratinized epithelial cells. Intracellular bacteria were found with and without surrounding vesicular spaces. The invasive spirochetes were mainly intermediately sized and of flagellar "4-8-4" configuration or higher. Bacterial invasion of the sulcular epithelium occurred before clinical evidence of NUG could be observed. It is concluded that invasion by intermediate spirochetes and other bacteria plays a role in initiating ulcerative gingivitis in beagles.

In a 22-year-old female patient, advanced localized periodontal destruction was observed. The planned treatment consisted of oral hygiene instructions, professional plaque control, deep scaling and root planning and finally modified Widman flap surgery. One molar had to be extracted but was left untreated initially as a control. During the treatment period of 9 months and during 1 year thereafter, samples were taken of the subgingival plaque for dark-field microscopy. The unplanned use of amoxicillin by the patient for a middle ear infection resulted in a suppression below detection level of spirochetes at the investigated sites. At the nontreated control site, the absence of spirochetes was accompanied by a 3-mm reduction of pocket depth and a 2-mm gain in clinical probing attachment, while some formation of new alveolar bone was observed. At the treated sites, clinical improvement was observed. However, a distinction between the effect of the periodontal therapy and the nonscheduled use of amoxicillin could not be made at the treated sites. It is concluded that a single course of systematically administered amoxicillin changed the composition of the subgingival microflora over a long period of time (17 months) and had a beneficial effect upon the status of the periodontium.

Microbial and clinical parameters were studied in 11 subjects with chronic inflammatory periodontitis. 2 periodontal pockets per subject were studied longitudinally. The microbial parameters included counts of different subgingival micro-organisms by dark field microscopy, counts of the total colony forming units (c.f.u.) on anaerobic blood agar, the facultative anaerobic counts and counts of black-pigmented Bacteroides, Fusobacterium and E. corrodens. The clinical parameters were probing pocket depth, bleeding after probing and crevicular fluid production. Clinical and microbial observations were compared during 3 consecutive periods of non-treatment, debridement and metronidazole therapy. The experimental sites were debrided by deep scaling while no debridement was carried out at the control sites. The effect of this treatment was studied over a period of 3 months. Then, at the experimental sites, a 2nd session of debridement was followed by administration of metronidazole. The effect of metronidazole alone and combined with mechanical debridement was studied during a subsequent 3-month period. The disease activity did not correlate with the microbial parameters and was evident in the presence as well as in the absence of black-pigmented Bacteroides. A single session of subgingival debridement resulted in significant reductions in probing depth, spirochetes, motile organisms, black-pigmented Bacteroides and E. corrodens. Repopulation of the subgingival sites was observed. However, the composition of the subgingival microbiota remained significantly changed during the 3 months after debridement. The re-isolation of the same Bacteroides-species and the same B. gingivalis type after treatment indicated an outgrowth of micro-organisms remaining at these sites.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of bacteriocins produced by strains of Streptococcus mutans on the microbial composition of dental plaque was studied in gnotobiotic rats. In one set of tests, using S. mutans strains T2 and SW31, rats were simultaneously infected with the bacteriocinogenic parent (bac(+)) and corresponding bac(-) mutants, and the relative proportions of each strain present in three different tooth sites were determined at various time intervals after inoculation. Some animals were monoinfected with bac(+) and bac(-) strains, and in vivo properties, such as plaque fermentation end products, amount of plaque formed, and cariogenicity, were measured. In another series of tests with strains T2 bac(+) and T2 bac(-), animals were sequentially inoculated with a time interval of 1 week. On simultaneous inoculation, the bac(-) mutant, which was sensitive in vitro to the bacteriocin produced by the parent, was totally unable to establish in a plaque consisting of the bac(+) strain even when the challenging dose was increased 100-fold. In contrast, the bac(+) strain was successful not only in invading an established bac(-) plaque but also in eventually almost eliminating the mutant. In monoinfected animals, there were no significant differences in plaque-forming ability, fermentation end products, or cariogenicity between the two strains. Also, bacteriocin activity was detected in plaque harboring the bac(+) but not the bac(-) strain. The results not only indicate that bacteriocin activity can occur in vivo but also suggest that bacteriocinogeny might confer an ecological advantage upon a strain attempting to establish itself in an ecosystem.

The gingival microbiota in beagle dogs was investigated during a period of 42 days after the placement of continuous wire and arch bar splints. The splints were found to induce an increase in helicoidal-shaped micro-organisms and black-pigmented Bacteroides. Two different asaccharolytic Bacteroides types were isolated and tentatively identified as B. gingivalis and B. asaccharolyticus. In addition, B. intermedius-like organisms were isolated. The last two species were observed only after splint placement. The local accumulation of helicoidal-shaped micro-organisms in the epithelial tissue rather than in the micro-ulcerations was regarded as an indication of an active role for these spirochetes in the disease process. The observed shift toward tissue-invading and potentially pathogenic micro-organisms after splinting stresses the need for antimicrobial measures during splint placement in order to minimize periodontal destruction.

Antibiograms with 20 different antimicrobial disks were studied for antibiotyping of Bacteroides gingivalis isolates. The stability of the antibiotypes was tested by passage in mice. Several B. gingivalis isolates of the same subject were used to investigate the presence of different antibiotypes in one individual, while isolates from different subjects were used to investigate individual differences. The antibiotypes were found to remain stable after animal passage. All tested strains of different origin represented different antibiotypes. The isolates from one subject all belonged to the same antibiotype. Principal component analysis of the data showed that two factors were important in the discrimination of the strains of B. gingivalis. One included beta-lactam antimicrobial agents that affect the cell wall. The other included antimicrobial agents that inhibit synthesis of protein and nucleic acid. Both principal component analysis and discriminant analysis proved to be of great use in the reduction of the amount of data and the visualization of the relations between different antibiotypes of B. gingivalis in a linear map. Among the investigated subjects, different antibiotypes of B. gingivalis were found, indicating that in the mouth of an individual, one antibiotype of B. gingivalis predominates and that different persons harbor different antibiotypes of B. gingivalis.

Metronidazole concentrations were estimated in four human volunteers after a single dose of 750 mg taken orally. Samples of blood, saliva, and gingival crevice fluid were collected before intake and during the following 24 hours. The concentrations of metronidazole in plasma and saliva were measured by high-performance liquid chromatography (HPLC). The concentrations in gingival fluid were estimated by a capillary agar-diffusion assay. The results of the metronidazole measurements as obtained by both methods were significantly correlated. The peak concentrations of metronidazole in plasma and saliva were in the same range, 8.7-13.8 micrograms/mL, and similar concentrations were found in the gingival fluid samples. It is concluded that metronidazole taken orally has similar pharmacokinetics in both saliva and plasma, and that a single oral dose of 750 mg metronidazole leads to a concentration of the drug in the gingival crevice fluid that exceeds the minimal inhibitory concentration for most anaerobic oral micro-organisms.