TRAF3IP2-IL-17 Axis Strengthens the Gingival Defense against Pathogens.

Recent genome-wide association studies have suggested novel risk loci associated with periodontitis, which is initiated by dysbiosis in subgingival plaque and leads to destruction of teeth-supporting structures. One such genetic locus was the tumor necrosis factor receptor-associated factor 3 interacting protein 2 (TRAF3IP2), a gene encoding the gate-keeping interleukin (IL)-17 receptor adaptor. In this study, we first determined that carriers of the lead exonic variant rs13190932 within the TRAF3IP2 locus combined with a high plaque microbial burden was associated with more severe periodontitis than noncarriers. We then demonstrated that TRAF3IP2 is essential in the IL-17-mediated CCL2 and IL-8 chemokine production in primary gingival epithelial cells. Further analysis suggested that rs13190932 may serve a surrogate variant for a genuine loss-of-function variant rs33980500 within the same gene. Traf3ip2 null mice (Traf3ip2-/-) were more susceptible than wild-type (WT) mice to the Porphyromonas gingivalis-induced periodontal alveolar bone loss. Such bone loss was associated with a delayed P. gingivalis clearance and an attenuated neutrophil recruitment in the gingiva of Traf3ip2-/- mice. Transcriptomic data showed decreased expression of antimicrobial genes, including Lcn2, S100a8, and Defb1, in the Traf3ip2-/- mouse gingiva in comparison to WT mice prior to or upon P. gingivalis oral challenge. Further 16S ribosomal RNA sequencing analysis identified a distinct microbial community in the Traf3ip2-/- mouse oral plaque, which was featured by a reduced microbial diversity and an overabundance of Streptococcus genus bacteria. More P. gingivalis was observed in the Traf3ip2-/- mouse gingiva than WT control animals in a ligature-promoted P. gingivalis invasion model. In agreement, neutrophil depletion resulted in more local gingival tissue invasion by P. gingivalis. Thus, we identified a homeostatic IL-17-TRAF3IP2-neutrophil axis underpinning host defense against a keystone periodontal pathogen.

Distinct Microbial Signatures between Periodontal Profile Classes.

Precise classification of periodontal disease has been the objective of concerted efforts and has led to the introduction of new consensus-based and data-driven classifications. The purpose of this study was to characterize the microbiological signatures of a latent class analysis (LCA)-derived periodontal stratification system, the Periodontal Profile Class (PPC) taxonomy. We used demographic, microbial (subgingival biofilm composition), and immunological data (serum IgG antibody levels, obtained with checkerboard immunoblotting technique) for 1,450 adult participants of the Dental Atherosclerosis Risk in Communities (ARIC) study, with already generated PPC classifications. Analyses relied on t tests and generalized linear models with Bonferroni correction. Men and African Americans had higher systemic antibody levels against most microorganisms compared to women and Caucasians (P < 0.05). Healthy individuals (PPC-I) had low levels of biofilm bacteria and serum IgG levels against most periodontal pathogens (P < 0.05). Subjects with mild to moderate disease (PPC-II to PPC-III) showed mild/moderate colonization of multiple biofilm pathogens. Individuals with severe disease (PPC-IV) had moderate/high levels of biofilm pathogens and antibody levels for orange/red complexes. High gingival index individuals (PPC-V) showed moderate/high levels of biofilm Campylobacter rectus and Aggregatibacter actinomycetemcomitans. Biofilm composition in individuals with reduced periodontium (PPC-VI) was similar to health but showed moderate to high antibody responses. Those with severe tooth loss (PPC-VII) had significantly high levels of multiple biofilm pathogens, while the systemic antibody response to these microorganisms was comparable to health. The results support a biologic basis for elevated risk for periodontal disease in men and African Americans. Periodontally healthy individuals showed a low biofilm pathogen and low systemic antibody burden. In the presence of PPC disease, a microbial-host imbalance characterized by higher microbial biofilm colonization and/or systemic IgG responses was identified. These results support the notion that subgroups identified by the PPC system present distinct microbial profiles and may be useful in designing future precise biological treatment interventions.

Association or Causation? Exploring the Oral Microbiome and Cancer Links.

Several epidemiological investigations have found associations between poor oral health and different types of cancer, including colorectal, lung, pancreatic, and oral malignancies. The oral health parameters underlying these relationships include deficient oral hygiene, gingival bleeding, and bone and tooth loss. These parameters are related to periodontal diseases, which are directly and indirectly mediated by oral bacteria. Given the increased accessibility of microbial sequencing platforms, many recent studies have investigated the link between the oral microbiome and these cancers. Overall, it seems that oral dysbiotic states can contribute to tumorigenesis in the oral cavity as well as in distant body sites. Further, it appears that certain oral bacterial species can contribute to carcinogenesis, in particular, Fusobacterium nucleatum and Porphyromonas gingivalis, based on results from epidemiological as well as mechanistic studies. Yet, the strength of the findings from these investigations is hampered by the heterogeneity of the methods used to measure oral diseases, the treatment of confounding factors, the study design, the platforms employed for microbial analysis, and types of samples analyzed. Despite these limitations, there is an overall indication that the presence of oral dysbiosis that leads to oral diseases may directly and/or indirectly contribute to carcinogenesis. Proper methodological standardized approaches should be implemented in future epidemiological studies as well as in the mechanistic investigations carried out to explore these results.

The Salivary Microbiome and Oral Cancer Risk: a Pilot Study in Fanconi Anemia.

Fanconi anemia (FA) is a rare genetic disease characterized by chromosomal instability and impaired DNA damage repair. FA patients develop oral squamous cell carcinoma (OSCC) earlier and more frequently than the general population, especially after hematopoietic stem cell transplantation (HSCT). Although evidence of an etiological role of the local microbiome and carcinogenesis has been mounting, no information exists regarding the oral microbiome of FA patients. The aim of this study was to explore the salivary microbiome of 61 FA patients regarding their oral health status and OSCC risk factors. After answering a questionnaire and receiving clinical examination, saliva samples were collected and analyzed using 16S rRNA sequencing of the V3-V4 hypervariable region. The microbial profiles associated with medical and clinical parameters were analyzed using general linear models. Patients were young (mean age, 22 y) and most had received HSCT ( n = 53). The most abundant phyla were Firmicutes [mean relative abundance (SD), 42.1% (10.1%)] and Bacteroidetes [(25.4% (11.4%)]. A history of graft-versus-host disease (GVHD) ( n = 27) was associated with higher proportions of Firmicutes (43.8% × 38.5%, P = 0.05). High levels of gingival bleeding were associated with the genera Prevotella (22.25% × 20%), Streptococcus (19.83% × 17.61%), Porphyromonas (3.63% × 1.42%, P = 0.03), Treponema (1.02% × 0.28%, P = 0.009), Parvimonas (0.28% × 0.07%, P = 0.02) and Dialister (0.27% × 0.10%, P = 0.04). Finally, participants transplanted over 11 y ago showed the highest levels of Streptococcus (18.4%), Haemophilus (12.7%) and Neisseria (6.8%). In conclusion, FA patients that showed poor oral hygiene harbored higher proportions of the genera of bacteria compatible with gingival disease. Specific microbial differences were associated with a history of oral GVHD and a history of oral mucositis.

Levels of Candidate Periodontal Pathogens in Subgingival Biofilm.

In recent years, several new periodontal taxa have been associated with the etiology of periodontitis. A recent systematic review provides further support for the pathogenic role of 17 species/phylotypes. Thus, the aim of this study was to assess the prevalence and levels of these species in subjects with generalized chronic periodontitis (GChP; n = 30), generalized aggressive periodontitis (GAgP; n = 30), and periodontal health (PH; n = 30). All subjects underwent clinical and microbiological assessment. Nine subgingival plaque samples were collected from each subject and analyzed for their content of 20 bacterial species/phylotypes through the RNA-oligonucleotide quantification technique. Subjects from the GChP and GAgP groups presented the highest mean values for all clinical parameters in comparison with the PH group (P < 0.05). Subjects with GChP and GAgP showed significantly higher mean levels of Bacteroidetes sp. human oral taxon (HOT) 274, Fretibacterium sp. HOT 360, and TM7 sp. HOT 356 phylotypes, as well as higher mean levels of Filifactor alocis, Fretibacterium fastidiosum, Porphyromonas gingivalis, Tannerella forsythia, and Selenomonas sputigena species than PH subjects (P < 0.05). GAgP subjects presented higher mean levels of TM7 sp. HOT 356 and F. alocis than GChP subjects (P < 0.05). A significantly higher mean prevalence of Bacteroidales sp. HOT 274, Desulfobulbus sp. HOT 041, Fretibacterium sp. HOT 360, and Fretibacterium sp. HOT 362 was found in subjects with GChP and GAgP than in PH subjects. Mean levels of P. gingivalis (r = 0.68), T. forsythia (r = 0.62), F. alocis (r = 0.51, P = 0.001), and Fretibacterium sp. HOT 360 (r = 0.41) were correlated with pocket depth (P < 0.001). In conclusion, Bacteroidales sp. HOT 274, Desulfobulbus sp. HOT 041, Fretibacterium sp. HOT 360, Fretibacterium sp. HOT 362, and TM7 sp. HOT 356 phylotypes, in addition to F. alocis, F. fastidiosum, and S. sputigena, seem to be associated with periodontitis, and their role in periodontal pathogenesis should be further investigated.

HIV/AIDS-Associated Cryptococcosis in Portugal Spanning the Pre- to Post-HAART Era: A Retrospective Assessment at the Genotypic Level Based on URA5-RFLP.

Cryptococcosis caused by the fungus Cryptococcus neoformans is an opportunistic mycosis, infecting mainly immunodepressed individuals. Molecular epidemiology studies of cryptococcosis in Europe are limited. This paper presents a retrospective study of cryptococcosis in 105 cryptococcal isolates from two hospitals in Lisbon, Portugal, among HIV/AIDS patients, from 1991 to 2007. Among these patients, the number of cases of cryptococcosis increased from 5.1 to 6.9 cases per year from the pre- to post-highly active antiretroviral therapy (HAART) era. As expected, the median age of the patients increased, from 32 (mean: 33 ± 8) to 39 (mean: 41 ± 10) years, and the ratio of male to female patients remained high (7.7 and 7.6, respectively). Strain genotyping based on restriction fragment length polymorphism of the orotidine monophosphate pyrophosphorylase (URA5-RFLP) gene showed that, in general, the relative frequencies of the genotypes VNI-IV are similar to those from other European countries. These frequencies were, respectively, for the pre- and post-HAART periods: 41.7 and 43.5 % for VNI; 2.8 and 17.4 % for VNII; 38.9 and 30.4 % for VNIII; 16.7 and 7.2 % for VNIV and 0 and 1.4 % for VGII. Some apparent although statistically insignificant differences among these values were observed between both periods. The genotypic frequencies were not also statistically different according to the patients' gender or age range. Of note are the high proportion of VNIII isolates (common in Europe) and the high increase in the frequency of the VNII genotype in the post-HAART. Ultimately, these results may have implications in disease therapy, management and control.

Immunological profile of periapical endodontic infections from HIV- and HIV+ patients.

AIM: To evaluate CD4(+) CD28(+) and CD8(+) T-cell genes and the gene expression of IFN-γ, TNF-α, IL-1-ß, IL-17A, IL-10, CCL-2/MCP-1, CCL-4, CCL-5 (RANTES), CXCR4, CCR5 and RANKL from cells in the periapical interstitial fluid from root canal infections in healthy patients (HIV-) and HIV-positive individuals (HIV+). METHODOLOGY: Subjects included 20 HIV- and 23 HIV+ patients referred to the School of Dentistry at the Universidade Federal de Minas Gerais (Belo Horizonte, MG, Brazil). Almost all HIV+ patients were undergoing highly active antiretroviral therapy (HAART). Clinical samples were taken from teeth with pulp necrosis, and no patients had acute periapical symptoms at the time of the appointments. After cleaning and drying, 3 paper points were introduced into the root canal, passing passively through the root apex (2 mm) into the periapical tissues for 1 min. The samples were collected immediately after root canal cleaning and 7 days later (restrained root canal bacterial load) to characterize those gene expressions using real-time PCR. RESULTS: Significantly higher levels of CD4(+) CD28(+) and CD8(+) T cells in teeth with restrained bacterial loads (second collection) compared with the first collection were observed in both HIV- and HIV+ samples. In HIV- patients, an increase in IL-10 and CXCR4 expression was demonstrated as well as a decrease in pro-inflammatory cytokines such as RANKL, IFN-γ, IL1-ß and CCL5. However, in HIV+ patients an increase in cytokines IFN-γ, IL-1-ß, TNF-α and IL-17A, and chemokines CCL-2, CXCR4 and CCR5 were observed. The chemokine CCL-5 was not detected in HIV+ individuals. CONCLUSIONS: These findings suggest that after reducing the root canal bacterial load in HIV- individuals an anti-inflammatory response is generated whilst in HIV+ patients a pro-inflammatory response is sustained in the periapical area.

Levels of Selenomonas species in generalized aggressive periodontitis.

BACKGROUND AND OBJECTIVE: To compare the levels of Selenomonas sputigena and uncultivated/unrecognized Selenomonas species in subgingival biofilms from periodontally healthy subjects and from subjects with generalized aggressive periodontitis. MATERIAL AND METHODS: Fifteen periodontally healthy subjects and 15 subjects with generalized aggressive periodontitis were recruited and their clinical periodontal parameters were evaluated. Nine subgingival plaque samples were collected from each subject and all were individually analyzed for the levels of 10 bacterial taxa, including cultured and uncultivated/unrecognized microorganisms, using the RNA-oligonucleotide quantification technique. Between-group differences in the levels of the test taxa were determined using the Mann-Whitney U-test. RESULTS: Subjects with generalized aggressive periodontitis showed significantly higher mean counts of Porphyromonas gingivalis, S. sputigena and the Mitsuokella sp. Human Oral Taxon (HOT) 131 (previously described as Selenomonas sp. oral clone CS002), while higher mean counts of Actinomyces gerencseriae and Streptococcus sanguinis were found in periodontally healthy subjects (p < 0.01). Selenomonas sp. HOT 146 was only detected in the generalized aggressive periodontitis group. In the generalized aggressive periodontitis group, the levels of P. gingivalis and S. sputigena were higher in deep sites (probing depth ≥ 5 mm) than in shallow sites (probing depth ≤ 3 mm) (p < 0.01). Furthermore, in subjects with generalized aggressive periodontitis, sites with probing depth of ≤ 3 mm harbored higher levels of these two species than sites with the same probing depth in periodontally healthy subjects. There were positive correlations between probing depth and the levels of P. gingivalis (r = 0.77; p < 0.01), S. sputigena (r = 0.60; p < 0.01) and Selenomonas dianae (previously described as Selenomonas sp. oral clone EW076) (r = 0.42, p < 0.05). CONCLUSION: S. sputigena and Mitsuokella sp. HOT 131 may be associated with the pathogenesis of generalized aggressive periodontitis, and their role in the onset and progression of this infection should be investigated further.

Rediscovering Sig Socransky, the genius and his legacy.

Some individuals make contributions so vital to their field of knowledge that their names become almost synonymous with that field. This is the case of Sig Socransky and the field of periodontal microbiology. Sig Socransky, or simply Sig, was born in Toronto, Canada and received his DDS degree from the University of Toronto in 1957. He studied microbiology and periodontology at Harvard, receiving a certificate in 1961. That same year he was recruited to work as a Research Associate at the Forsyth Dental Center. In 1968, he was nominated Senior Member of the Staff and Head of the Department of Periodontology. During his 50-year career at Forsyth, Sig published over 300 manuscripts, keeping an average of 7 publications per year. His work had an indelible impact in the fields of periodontology and oral microbiology. All these accomplishments pale in comparison with the impact that Sig had on a personal level. We have collected testimonials from some of his former students, closest collaborators, and friends in an attempt to give readers an insight into Sig's personality. We hope we can offer those who knew him through his work a glimpse of how it felt to interact with this remarkable individual.

Comparison of microbial changes in early redeveloping biofilms on natural teeth and dentures.

BACKGROUND: Surfaces and fluids can affect oral bacterial colonization. The aim of this study is to compare redeveloping biofilms on natural teeth and dentures. METHODS: Supragingival plaque samples were taken from 55 dentate individuals and the denture teeth of 62 edentulous individuals before and after professional cleaning. Also, samples from seven "teeth" (samples included dentures) in randomly selected quadrants were collected after 1, 2, 4, and 7 days of no oral hygiene. Samples were analyzed using checkerboard DNA-DNA hybridization. Counts and proportions of 41 bacterial taxa were determined at each time point, and significant differences were determined using the Mann-Whitney U test. Ecological succession was determined using a modified moving window analysis. RESULTS: Mean total DNA probe counts were similar precleaning but were higher in dentate individuals at all post-cleaning visits (P <0.01). Precleaning edentate biofilms had higher counts and proportions of Streptococcus mitis, Streptococcus oralis, and Streptococcus mutans, whereas dentate individuals had higher proportions of Tannerella forsythia, Selenomonas noxia, and Neisseria mucosa. By day 2, mean counts of all taxa were higher in natural teeth, and most remained higher at day 7 (P <0.01). Succession was more rapid and complex in dentate individuals. Both groups demonstrated increased proportions of S. mitis and S. oralis by day 1. N. mucosa, Veillonella parvula, and Eikenella corrodens increased in both groups, but later in samples from edentate individuals. CONCLUSIONS: "Mature" natural and denture teeth biofilms have similar total numbers of bacteria but different species proportions. Post-cleaning biofilm redevelopment is more rapid and more complex on natural teeth than on denture teeth.

Microbiologic profile of endodontic infections from HIV- and HIV+ patients using multiple-displacement amplification and checkerboard DNA-DNA hybridization.

OBJECTIVE: To compare the microbiota of endodontic infections in necrotic pulp from HIV-negative and HIV-positive subjects. MATERIALS AND METHODS: Root canal samples from necrotic pulp were collected from 40 HIV- and 20 HIV+ subjects. Pulps were amplified using multiple displacement amplification (MDA). Then, checkerboard DNA-DNA hybridization was employed to assess the levels of 107 microbial taxa. The percentage of DNA probe count and the percentage of teeth colonized by each test species were investigated. Significant differences between groups regarding proportions of taxa and prevalence of the test species were sought using the Mann-Whitney test and the Chi-square analysis, respectively. RESULTS: The most prevalent taxa detected were Dialister pneumosintes, Stenotrophomonas maltophilia, Streptococcus sobrinus, Corynebacterium diphteriae, and Helicobacter pylori among HIV- subjects and D. pneumosintes, Prevotella tannerae, Porphyromonas gingivalis, Parvimonas micra, Prevotella nigrescens, and Corynebacterium diphtheriae among HIV+ individuals. D. pneumosintes, C. diphtheria, and C. albicans were the most abundant species in the HIV- group, whereas the predominant taxa in HIV+ samples were P. tannerae, D. pneumosintes and Olsenella uli. P. tannerae, O. uli, Veilonella dispar, Bacteroides fragilis, and Actinomyces meyeri were significantly more abundant in HIV+ samples. CONCLUSIONS: There were significant differences in the prevalence and proportions of specific microbial taxa between HIV- and HIV+ individuals. The root canal microbiota may represent a reservoir of important oral and medical pathogens, mainly in HIV+ individuals.

Early microbial succession in redeveloping dental biofilms in periodontal health and disease.

BACKGROUND AND OBJECTIVE: The development of dental biofilms after professional plaque removal is very rapid. However, it is not clear whether most bacterial species return at similar rates in periodontally healthy and periodontitis subjects or if there are differences in bacterial recolonization between supragingival and subgingival biofilms in periodontal health and disease. MATERIAL AND METHODS: Supragingival and subgingival plaque samples were taken separately from 28 teeth in 38 healthy and 17 periodontitis subjects immediately after professional cleaning. Samples were taken again from seven teeth in randomly selected quadrants after 1, 2, 4 and 7 d of no oral hygiene and analyzed using checkerboard DNA-DNA hybridization. The percentage of DNA probe counts were averaged within subjects at each time-point. Ecological succession was determined using a modified moving-window analysis. RESULTS: Succession in supragingival biofilms from subjects with periodontitis and from healthy individuals was similar. At 1 d, Streptococcus mitis and Neisseria mucosa showed increased proportions, followed by Capnocytophaga gingivalis, Eikenella corrodens, Veillonella parvula and Streptococcus oralis at 1-4 d. At 4-7 d, Campylobacter rectus, Campylobacter showae, Prevotella melaninogenica and Prevotella nigrescens became elevated. Subgingival plaque redevelopment was slower and very different from supragingival plaque redevelopment. Increased proportions were first observed for S. mitis, followed by V. parvula and C. gingivalis and, at 7 d, by Capnocytophaga sputigena and P. nigrescens. No significant increase in the proportions of periodontal pathogens was observed in any of the clinical groups or locations. CONCLUSION: There is a defined order in bacterial species succession in early supragingival and subgingival biofilm redevelopment after professional cleaning.

Laboratorial diagnosis of paracoccidioidomycosis and new insights for the future of fungal diagnosis.

Paracoccidioidomycosis (PCM) is the most prevalent mycosis in Latin-America. As for other mycosis, its importance of has been largely underestimated, partially due to the limited geographical distribution of the etiologic fungal agent (Paracoccidioides brasiliensis). However, the advent of AIDS and other immune suppressing conditions is creating an emergent need for improved diagnostic tests envisaging simpler, cheaper, faster and more sensitive and accurate detection of pathogenic fungi, especially those causing systemic and opportunistic diseases. Routine laboratorial diagnosis of PCM disease relies mainly on direct observation of the fungus. However, culture growing is slow and, too often, definite diagnosis can only be obtained at later growing stages. Immunodiagnosis is also widely employed, although usually cumbersome and complex. Enzyme-based immunoassays are more amenable to automation for high-throughput testing, but may lead to cross-reactivity with other fungi. Plus, molecular diagnosis relying on polymerase-chain reaction (PCR) and nucleic-acid hybridization, although still at early stages of application to routine diagnosis of P. brasiliensis, has triggered the development of techniques for its improved specific detection, thus contributing for epidemiological studies as well. In the future, microarrays and newer biosensing technologies, coupled to new bionanotechnological tools, will certainly improve diagnosis of PCM and other mycosis through very specific and sensitive pathogen biomolecular detection.

RNA-oligonucleotide quantification technique (ROQT) for the enumeration of uncultivated bacterial species in subgingival biofilms.

Approximately 35% of the species present in subgingival biofilms are as yet uncultivated, so their role in periodontal pathogenesis is unknown. The aim of the present study was to develop a high throughput method to quantify a wide range of cultivated and uncultivated taxa in subgingival biofilm samples associated with periodontal disease or health. Oligonucleotides targeting the 16S ribosomal DNA gene were designed, synthesized and labeled with digoxigenin. These probes were hybridized with the total nucleic acids of pure cultures or subgingival biofilm samples. Target species included cultivated taxa associated with periodontal health and disease, as well as uncultivated species, such as TM7 sp. OT 346, Mitsuokella sp. OT 131 and Desulfobulbus sp. OT 041. Sensitivity and specificity of the probes were determined. A Universal probe was used to assess total bacterial load. Sequences complementary to the probes were used as standards for quantification. Chemiluminescent signals were visualized after film exposure or using a CCD camera. In a pilot clinical study, 266 subgingival plaque samples from eight periodontally healthy people and 11 patients with periodontitis were examined. Probes were specific and sensitivity reached 10(4) cells. Fusobacterium nucleatum ss. polymorphum and Actinomyces gerencseriae were the most abundant cultivated taxa in clinical samples. Among uncultivated/unrecognized species, Mitsuokella sp. OT 131 and Prevotella sp. OT 306 were the most numerous. Porphyromonas gingivalis and Desulfobulbus sp. OT 041 were only detected in patients with periodontitis. Direct hybridization of total nucleic acids using oligonucleotide probes permitted the quantification of multiple cultivated and uncultivated taxa in mixed species biofilm samples.

Microbiota of deciduous endodontic infections analysed by MDA and Checkerboard DNA-DNA hybridization.

AIMS: To evaluate the microbiota of endodontic infections in deciduous teeth by Checkerboard DNA-DNA hybridization after uniform amplification of DNA in samples by multiple displacement amplification (MDA). METHODOLOGY: Forty samples from the root canal system of deciduous teeth exhibiting pulp necrosis with or without radiographically detectable periradicular/interradicular bone resorption were collected and 32 were analysed, with three individuals contributing two samples; these were MDA-amplified and analysed by Checkerboard DNA-DNA hybridization for levels of 83 bacterial taxa. Two outcome measures were used: the percentage of teeth colonized by each species and the mean proportion of each bacterial taxon present across all samples. RESULTS: The mean amount of DNA in the samples prior to amplification was 5.2 (±4.7) ng and 6.1 (±2.3) µg after MDA. The mean number of species detected per sample was 19 (±4) (range: 3-66) to the nearest whole number. The most prevalent taxa were Prevotella intermedia (96.9%), Neisseria mucosa (65.6%), Prevotella nigrescens (56.2%) and Tannerella forsythia (56.2%). Aggregatibacter (Haemophilus) aphrophilus and Helicobacter pylori were not detected. P. intermedia (10%), Prevotella tannerae (7%) and Prevotella nigrescens (4.3%) presented the highest mean proportions of the target species averaged across the positive samples. CONCLUSION: Root canals of infected deciduous teeth had a diverse bacterial population. Prevotella sp. were commonly found with P. intermedia, Prevotella tannerae and Prevotella nigrescens amongst the most prominent species detected.

Use of multiple-displacement amplification and checkerboard DNA-DNA hybridization to examine the microbiota of endodontic infections.

Multiple-displacement amplification (MDA) has been used to uniformly amplify bacterial genomes present in small samples, providing abundant targets for molecular analysis. The purpose of this investigation was to combine MDA and checkerboard DNA-DNA hybridization to examine the microbiota of endodontic infections. Sixty-six samples were collected from teeth with endodontic infections. Nonamplified and amplified samples were analyzed by checkerboard DNA-DNA hybridization for levels and proportions of 77 bacterial taxa. Counts, percentages of DNA probe counts, and percentages of teeth colonized for each species in amplified and nonamplified samples were computed. Significance of differences for each species between amplified and nonamplified samples was sought with Wilcoxon signed-rank test and adjusted for multiple comparisons. The amount of DNA in the samples ranged from 6.80 (+/- 5.2) ng before to 6.26 (+/- 1.73) mug after MDA. Seventy of the 77 DNA probes hybridized with one or more of the nonamplified samples. All probes hybridized with at least one sample after amplification. Most commonly detected species at levels of >10(4) in both amplified and nonamplified samples were Prevotella tannerae and Acinetobacter baumannii at frequencies between 89 and 100% of samples. The mean number of species at counts of >10(4) in amplified samples was 51.2 +/- 2.2 and in nonamplified samples was 14.5 +/- 1.7. The endodontic microbiota was far more complex than previously shown, although microbial profiles at teeth with or without periradicular lesions did not differ significantly. Species commonly detected in endodontic samples included P. tannerae, Prevotella oris, and A. baumannii.

Trends in dengue diagnosis.

The conventional diagnosis of dengue virus infections includes the detection of the virus in serum or tissue samples, both by isolation in culture or through detection of specific viral molecules (genome RNA or dengue antigens) and detection of specific anti-dengue antibodies (serology). Isolation of dengue virus provides the most direct and conclusive approach to diagnosis, despite the demand for high-level equipment, technical skills and manpower. However, it is useless in early diagnosis because several days are required to isolate and classify the virus. Serology, despite being simpler, is not able to afford an accurate early diagnosis in primary infections because 4-5 days are required for the immune system to produce a sufficient amount of antibodies. Moreover, it leads to misleading results in secondary infections owing to cross-reactivity among serotype-specific antibodies and with other flavivirus antibodies. The RT-PCR and other PCR-based techniques are fast, serotype-discriminating, more sensitive and easier to carry out than conventional nucleic-acid hybridisation, but are handicapped by easy sample contamination and high technological demands. Recently, advances in bioelectronics have generated commercial kits and new techniques for detection of dengue antibodies and RNA, based on biosensor technology. Most of them are rapid, easy to operate, reusable, cheap, sensitive and serotype-specific. Nevertheless, their accuracy is still questionable because most still lack validation and standardisation. This review summarises and describes the techniques currently employed and anticipated in the near future for diagnosis of dengue disease.