Oral Microbiota Transplant in Dogs with Naturally Occurring Periodontitis.

In periodontitis patients, dysbiosis of the oral microbiota is not only found at clinically diseased periodontal sites but also at clinically healthy periodontal sites, buccal mucosae, tongue, and saliva. The present study evaluated the safety and efficacy of an oral microbiota transplant (OMT) for the treatment of periodontitis in dogs. Eighteen systemically healthy beagle dogs with naturally occurring periodontitis were enrolled in the study and randomly assigned to a test or control group. A 4-y-old, periodontally healthy female beagle dog served as a universal OMT donor. To reduce periodontal inflammation, all dogs received full-mouth mechanical debridement of teeth and mucosae 2 wk before baseline. At baseline, full-mouth mechanical debridement was repeated and followed by adjunctive subgingival and oral irrigation with 0.1% NaOCl. Subsequently, test dogs were inoculated with an OMT from the healthy donor. No daily oral hygiene was performed after OMT transplantation. Adverse events were assessed throughout the observation period. Clinical examinations were performed and whole-mouth oral microbiota samples were collected at week 2, baseline, week 2, and week 12. The composition of oral microbiota samples was analyzed using high-throughput 16S ribosomal RNA gene amplicon sequencing followed by taxonomic assignment and downstream bioinformatic and statistical analyses. Results demonstrated that the intergroup difference in the primary outcome measure, probing pocket depth at week 12, was statistically insignificant. However, the single adjunctive OMT had an additional effect on the oral microbiota composition compared to the full-mouth mechanical and antimicrobial debridement alone. The OMT resulted in an "ecological shift" toward the composition of the donor microbiota, but this was transient in nature and was not observed at week 12. No local or systemic adverse events were observed throughout the study period. The results indicate that OMT may modulate the microbiota composition in dogs with naturally occurring periodontitis and can be applied safely.

Long-term impact of oral surgery with or without amoxicillin on the oral microbiome-A prospective cohort study.

Routine postoperative antibiotic prophylaxis is not recommended for third molar extractions. However, amoxicillin still continues to be used customarily in several clinical practices worldwide to prevent infections. A prospective cohort study was conducted in cohorts who underwent third molar extractions with (group EA, n = 20) or without (group E, n = 20) amoxicillin (250 mg three times daily for 5 days). Further, a control group without amoxicillin and extractions (group C, n = 17) was included. Salivary samples were collected at baseline, 1-, 2-, 3-, 4-weeks and 3 months to assess the bacterial shift and antibiotic resistance gene changes employing 16S rRNA gene sequencing (Illumina-Miseq) and quantitative polymerase chain reaction. A further 6-month follow-up was performed for groups E and EA. Seven operational taxonomic units reported a significant change from baseline to 3 months for group EA (adjusted p < 0.05). No significant change in relative abundance of bacteria and ß-lactamase resistance genes (TEM-1) was observed over 6 months for any group (adjusted p > 0.05). In conclusion, the salivary microbiome is resilient to an antibiotic challenge by a low-dose regimen of amoxicillin. Further studies evaluating the effect of routinely used higher dose regimens of amoxicillin on gram-negative bacteria and antibiotic resistance genes are warranted.

Evaluation of peer-generated MCQs to assess and support learning in a problem-based learning programme.

PURPOSE: Problem-based learning (PBL) students report uncertainty on the depth and breadth of learning required, and this is a significant stressor and challenge. Student-generated MCQ questions were trialled and evaluated as a way to support depth and breadth of learning. METHODS: Students set MCQs relating to specified learning issues, and an analysis and evaluation of setting and answering the MCQs were performed. The Revised Study Process Questionnaire (R-SPQ-2F) and final written examination scores were correlated to question setting and answering. Students were asked to rate the impact of the MCQs on their learning in PBL. RESULTS: A total of 147 questions were created and 2373 answered. Students reported challenges with setting questions, although these made them think more deeply and helped their learning and affirming their learning progress. MCQs authored indicated significant associations with Understanding, and examination scores were associated with MCQs authored. Students reported a moderate response to how the MCQs supported their depth and breadth of learning. CONCLUSIONS: While MCQ setting was perceived as a useful learning exercise, students engaged to different levels and experienced challenges. Students were uncertain whether the MCQs helped clarify the depth and breadth of learning in PBL, as they were not clear whether the questions set by their peers were relevant to the required learning outcomes.

Oral treponeme major surface protein: Sequence diversity and distributions within periodontal niches.

Treponema denticola and other species (phylotypes) of oral spirochetes are widely considered to play important etiological roles in periodontitis and other oral infections. The major surface protein (Msp) of T. denticola is directly implicated in several pathological mechanisms. Here, we have analyzed msp sequence diversity across 68 strains of oral phylogroup 1 and 2 treponemes; including reference strains of T. denticola, Treponema putidum, Treponema medium, 'Treponema vincentii', and 'Treponema sinensis'. All encoded Msp proteins contained highly conserved, taxon-specific signal peptides, and shared a predicted 'three-domain' structure. A clone-based strategy employing 'msp-specific' polymerase chain reaction primers was used to analyze msp gene sequence diversity present in subgingival plaque samples collected from a group of individuals with chronic periodontitis (n=10), vs periodontitis-free controls (n=10). We obtained 626 clinical msp gene sequences, which were assigned to 21 distinct 'clinical msp genotypes' (95% sequence identity cut-off). The most frequently detected clinical msp genotype corresponded to T. denticola ATCC 35405T , but this was not correlated to disease status. UniFrac and libshuff analysis revealed that individuals with periodontitis and periodontitis-free controls harbored significantly different communities of treponeme clinical msp genotypes (P<.001). Patients with periodontitis had higher levels of clinical msp genotype diversity than periodontitis-free controls (Mann-Whitney U-test, P<.05). The relative proportions of 'T. vincentii' clinical msp genotypes were significantly higher in the control group than in the periodontitis group (P=.018). In conclusion, our data clearly show that both healthy and diseased individuals commonly harbor a wide diversity of Treponema clinical msp genotypes within their subgingival niches.

Salivary microbiome of an urban Indian cohort and patterns linked to subclinical inflammation.

OBJECTIVE: To profile salivary microbiomes of an urban-living, healthy Indian cohort and explore associations with proinflammatory status. METHODS: Fifty-one clinically healthy Indian subjects' salivary microbiomes were analyzed using 16S rRNA Illumina MiSeq sequencing. Community distribution was compared with salivary data from the Human Microbiome Project (HMP). Indian subjects were clustered using microbiome-based "partitioning along medoids" (PAM), and relationships of interleukin-1 beta levels with community composition were analyzed. RESULTS: Indian subjects presented higher phylogenetic diversity than HMP. Several taxa associated with traditional societies gut microbiomes (Bacteroidales, Paraprevotellaceae, and Spirochaetaceae) were raised. Bifidobacteriaceae and Lactobacillaceae were approximately fourfold greater. A PAM cluster enriched in several Proteobacteria, Actinobacteria, and Bacilli taxa and having almost twofold higher Prevotella to Bacteroides ratio showed significant overrepresentation of subjects within the highest quartile of salivary interleukin-1 beta levels. Abiotrophia, Anaerobacillus, Micrococcus, Aggregatibacter, Halomonas, Propionivivrio, Paracoccus, Mannhemia, unclassified Bradyrhizobiaceae, and Caulobacteraceae were each significant indicators of presence in the highest interleukin-1 beta quartile. 2 OTUs representing Lactobacillus fermentum and Cardiobacterium hominis significantly correlated with interleukin-1 beta levels. CONCLUSION: The salivary microbiome of this urban-dwelling Indian cohort differed significantly from that of a well-studied Western cohort. Specific community patterns were putatively associated with subclinical inflammation levels.

Prevalence and diversity of Synergistetes taxa in periodontal health and disease.

BACKGROUND AND OBJECTIVE: Members of the phylum Synergistetes have previously been identified within periodontitis subgingival plaque and are considered putative periodontopathogens. This study compared the diversity of subginigval Synergistetes in a cohort of subjects with periodontitis (n = 10) vs. periodontitis-free controls (n = 10). MATERIAL AND METHODS: Pooled subgingival plaque samples from all deep periodontal pockets or all sulci were collected from the periodontitis and periodontitis-free subjects, respectively. Bacterial 16S rRNA genes were PCR-amplified from purified subgingival plaque DNA using a Synergistetes 'selective' primer set. PCR products were cloned and sequenced to analyze the prevalence and diversity of Synergistetes operational taxonomic units (OTUs) present in plaque samples of both subject groups. RESULTS: A total of 1030 non-chimeric 16S rRNA clones were obtained, of which 162 corresponded to members of the phylum Synergistetes. A significantly larger number of Synergistetes clones were obtained from periodontitis subgingival plaque than from periodontitis-free controls (25.4% vs. 5.9%, p < 0.001). All Synergistetes clones corresponded to cluster A oral Synergistetes, and fell into 31 OTUs (99% sequence identity cut-off). Twenty-nine Synergistetes OTUs were detected in the periodontitis group while eight were detected in the periodontitis-free group (p < 0.001). Five Synergistetes OTUs; including one OTU corresponding to the recently-characterized species Fretibacterium fastidiosum, were more prevalent in the periodontitis subjects (p < 0.05). CONCLUSION: OTUs belonging to oral Synergistetes cluster A were more readily detectable and were more diverse in subgingival plaque from periodontitis subjects compared with periodontitis-free controls. Specific Synergistetes OTUs appear to be associated with periodontitis.

Pseudomonas aeruginosa lipopolysaccharide inhibits Candida albicans hyphae formation and alters gene expression during biofilm development.

Elucidation of bacterial and fungal interactions in multispecies biofilms will have major impacts on understanding the pathophysiology of infections. The objectives of this study were to (i) evaluate the effect of Pseudomonas aeruginosa lipopolysaccharide (LPS) on Candida albicans hyphal development and transcriptional regulation, (ii) investigate protein expression during biofilm formation, and (iii) propose likely molecular mechanisms for these interactions. The effect of LPS on C. albicans biofilms was assessed by XTT-reduction and growth curve assays, light microscopy, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM). Changes in candidal hypha-specific genes (HSGs) and transcription factor EFG1 expression were assessed by real-time polymerase chain reaction and two-dimensional gel electrophoresis, respectively. Proteome changes were examined by mass spectrometry. Both metabolic activities and growth rates of LPS-treated C. albicans biofilms were significantly lower (P < 0.05). There were higher proportions of budding yeasts in test biofilms compared with the controls. SEM and CLSM further confirmed these data. Significantly upregulated HSGs (at 48 h) and EFG1 (up to 48 h) were noted in the test biofilms (P < 0.05) but cAMP levels remained unaffected. Proteomic analysis showed suppression of candidal septicolysin-like protein, potential reductase-flavodoxin fragment, serine hydroxymethyltransferase, hypothetical proteins Cao19.10301(ATP7), CaO19.4716(GDH1), CaO19.11135(PGK1), CaO19.9877(HNT1) by P. aeruginosa LPS. Our data imply that bacterial LPS inhibit C. albicans biofilm formation and hyphal development. The P. aeruginosa LPS likely target glycolysis-associated mechanisms during candidal filamentation.

Bacterial lipopolysaccharides variably modulate in vitro biofilm formation of Candida species.

The objective of this study was to evaluate the effect of the bacterial endotoxin LPS on Candida biofilm formation in vitro. The effect of the LPS of Pseudomonas aeruginosa, Klebsiella pneumoniae, Serratia marcescens and Salmonella typhimurium on six different species of Candida, comprising Candida albicans ATCC 90028, Candida glabrata ATCC 90030, Candida krusei ATCC 6258, Candida tropicalis ATCC 13803, Candida parapsilosis ATCC 22019 and Candida dubliniensis MYA 646, was studied using a standard biofilm assay. The metabolic activity of in vitro Candida biofilms treated with LPS at 90 min, 24 h and 48 h was quantified by XTT reduction assay. Viable biofilm-forming cells were qualitatively analysed using confocal laser scanning microscopy (CLSM), while scanning electron microscopy (SEM) was employed to visualize the biofilm structure. Initially, adhesion of C. albicans was significantly stimulated by Pseudomonas and Klebsiella LPS. A significant inhibition of Candida adhesion was noted for the following combinations: C. glabrata with Pseudomonas LPS, C. tropicalis with Serratia LPS, and C. glabrata, C. parapsilosis or C. dubliniensis with Salmonella LPS (P<0.05). After 24 h of incubation, a significant stimulation of initial colonization was noted for the following combinations: C. albicans/C. glabrata with Klebsiella LPS, C. glabrata/C. tropicalis/C. krusei with Salmonella LPS. In contrast, a significant inhibition of biofilm formation was observed in C. glabrata/C. dubliniensis/C. krusei with Pseudomonas LPS, C. krusei with Serratia LPS, C. dubliniensis with Klebsiella LPS and C. parapsilosis/C. dubliniensis /C. krusei with Salmonella LPS (P<0.05). On further incubation for 48 h, a significant enhancement of biofilm maturation was noted for the following combinations: C. glabrata/C. tropicalis with Serratia LPS, C. dubliniensis with Klebsiella LPS and C. glabrata with Salmonella LPS, and a significant retardation was noted for C. parapsilosis/C. dubliniensis/C. krusei with Pseudomonas LPS, C. tropicalis with Serratia LPS, C. glabrata/C. parapsilosis/C. dubliniensis with Klebsiella LPS and C. dubliniensis with Salmonella LPS (P<0.05). These findings were confirmed by SEM and CLSM analyses. In general, the inhibition of the biofilm development of LPS-treated Candida spp. was accompanied by a scanty architecture with a reduced numbers of cells compared with the profuse and densely colonized control biofilms. These data are indicative that bacterial LPSs modulate in vitro Candida biofilm formation in a species-specific and time-dependent manner. The clinical and the biological relevance of these findings have yet to be explored.

Pseudomonas aeruginosa inhibits in-vitro Candida biofilm development.

BACKGROUND: Elucidation of the communal behavior of microbes in mixed species biofilms may have a major impact on understanding infectious diseases and for the therapeutics. Although, the structure and the properties of monospecies biofilms and their role in disease have been extensively studied during the last decade, the interactions within mixed biofilms consisting of bacteria and fungi such as Candida spp. have not been illustrated in depth. Hence, the aim of this study was to evaluate the interspecies interactions of Pseudomonas aeruginosa and six different species of Candida comprising C. albicans, C. glabrata, C. krusei, C. tropicalis, C. parapsilosis, and C. dubliniensis in dual species biofilm development. RESULTS: A significant reduction in colony forming units (CFU) of C. parapsilosis (90 min), C. albicans and C. tropicalis (90 min, 24 h and 48 h), C. dubliniensis and C. glabrata, (24 h and 48 h) was noted when co-cultured with P. aeruginosa in comparison to their monospecies counterparts (P < 0.05). A simultaneous significant reduction in P. aeruginosa numbers grown with C. albicans (90 min and 48 h), C. krusei (90 min, 24 h and 48 h),C. glabrata, (24 h and 48 h), and an elevation of P. aeruginosa numbers co-cultured with C. tropicalis (48 h) was noted (P < 0.05). When data from all Candida spp. and P. aeruginosa were pooled, highly significant mutual inhibition of biofilm formation was noted (Candida P < 0.001, P. aeruginosa P < 0.01). Scanning Electron Microscopy (SEM) and Confocal Laser Scanning Microscopy (CLSM) analyses confirmed scanty architecture in dual species biofilm in spite of dense colonization in monospecies counterparts. CONCLUSIONS: P. aeruginosa and Candida in a dual species environment mutually suppress biofilm development, both quantitatively and qualitatively. These findings provide a foundation to clarify the molecular basis of bacterial-fungal interactions, and to understand the pathobiology of mixed bacterial-fungal infections.

Escherichia coli and its lipopolysaccharide modulate in vitro Candida biofilm formation.

Demystification of microbial behaviour in mixed biofilms could have a major impact on our understanding of infectious diseases. The objectives of this study were to evaluate in vitro the interactions of six different Candida species and a Gram-negative coliform, Escherichia coli, in dual-species biofilms, and to assess the effect of E. coli LPS on Candida biofilm formation. A single isolate of E. coli ATCC 25922 and six different species of Candida, Candida albicans ATCC 90028, Candida glabrata ATCC 90030, Candida krusei ATCC 6258, Candida tropicalis ATCC 13803, Candida parapsilosis ATCC 22019 and Candida dubliniensis MYA-646, were studied using a standard biofilm assay. Each Candida species was co-cultured with E. coli on a polystyrene surface and biofilm formation was quantified by a c.f.u. assay. The biofilm was then analysed by Live/Dead staining and fluorescence microscopy (confocal laser-scanning microscopy, CLSM), whilst scanning electron microscopy (SEM) was employed to visualize the biofilm architecture. The effect of E. coli LPS on Candida biofilm cell activity at defined time intervals was assessed with an XTT reduction assay. A significant quantitative reduction in c.f.u. counts of C. tropicalis (after 90 min), C. parapsilosis (after 90 min and 24 h), C. krusei (after 24 h) and C. dubliniensis (after 24 and 48 h) was noted on incubation with E. coli in comparison with their monospecies biofilm counterparts (P <0.05). On the other hand, a simultaneous and significant reduction in E. coli cell numbers occurred on co-culture with C. albicans (after 90 min), and an elevation of E. coli cell numbers followed co-culture with C. tropicalis (after 24 h) and C. dubliniensis (after 24 h and 48 h) (P <0.05). All quantitative findings were confirmed by SEM and CLSM analyses. By SEM observation, dual-species biofilms demonstrated scanty architecture with reduced visible cell counts at all stages of biofilm development, despite profuse growth and dense colonization in their single-species counterparts. Significantly elevated metabolic activity, as assessed by XTT readings, was observed in E. coli LPS-treated C. tropicalis and C. parapsilosis biofilms (after 48 h), whilst this had the opposite effect for C. dubliniensis (after 24 h) (P <0.05). These data indicate that E. coli and Candida species in a mixed-species environment mutually modulate biofilm development, both quantitatively and qualitatively, and that E. coli LPS appears to be a key component in mediating these outcomes.

Mechanism of 8-amino-7-oxononanoate synthase: spectroscopic, kinetic, and crystallographic studies.

8-Amino-7-oxononanoate synthase (also known as 7-keto-8-aminopelargonate synthase, EC 2.3.1.47) is a pyridoxal 5'-phosphate-dependent enzyme which catalyzes the decarboxylative condensation of L-alanine with pimeloyl-CoA in a stereospecific manner to form 8(S)-amino-7-oxononanoate. This is the first committed step in biotin biosynthesis. The mechanism of Escherichia coli AONS has been investigated by spectroscopic, kinetic, and crystallographic techniques. The X-ray structure of the holoenzyme has been refined at a resolution of 1.7 A (R = 18.6%, R(free) = 21. 2%) and shows that the plane of the imine bond of the internal aldimine deviates from the pyridine plane. The structure of the enzyme-product external aldimine complex has been refined at a resolution of 2.0 A (R = 21.2%, R(free) = 27.8%) and shows a rotation of the pyridine ring with respect to that in the internal aldimine, together with a significant conformational change of the C-terminal domain and subtle rearrangement of the active site hydrogen bonding. The first step in the reaction, L-alanine external aldimine formation, is rapid (k(1) = 2 x 10(4) M(-)(1) s(-)(1)). Formation of an external aldimine with D-alanine, which is not a substrate, is significantly slower (k(1) = 125 M(-)(1) s(-)(1)). Binding of D-alanine to AONS is enhanced approximately 2-fold in the presence of pimeloyl-CoA. Significant substrate quinonoid formation only occurs upon addition of pimeloyl-CoA to the preformed L-alanine external aldimine complex and is preceded by a distinct lag phase ( approximately 30 ms) which suggests that binding of the pimeloyl-CoA causes a conformational transition of the enzyme external aldimine complex. This transition, which is inferred by modeling to require a rotation around the Calpha-N bond of the external aldimine complex, promotes abstraction of the Calpha proton by Lys236. These results have been combined to form a detailed mechanistic pathway for AONS catalysis which may be applied to the other members of the alpha-oxoamine synthase subfamily.

Treatment of congenital and acquired hemophilia patients by extracorporeal removal of antibodies to coagulation factors: a review of US clinical studies 1987-1990. Hemophilia Study Group.

This paper reviews the use of extracorporeal immunoadsorption with immobilized Staphylococcal Protein A in attempts to lower the inhibitor titer in 22 patients with either congenital hemophilia or with acquired inhibitors. Eighty-five immunoadsorption procedures were performed at 13 locations in the United States between June, 1987 and February, 1990. In general, immunoadsorption was shown to efficiently remove IgG and, in eight congenital hemophilia patients, it also produced a clinically significant lowering of inhibitors allowing effective conventional factor replacement therapy. Three of thirteen congenital hemophilia patients treated received factor concentrate prior to immunoadsorption and were anamnestic at the time of treatment. Although they experienced substantial lowering of their inhibitor titers, it was not sufficient to allow effective factor replacement. The effectiveness of immunoadsorption therapy in the 9 patients with acquired inhibitors was more difficult to evaluate due to the wide variety of concomitant medications which were employed, although in several patients serious bleeding episodes were substantially improved (or halted) following immunoadsorption. Side effects associated with immunoadsorption were slight. These findings suggest that immunoadsorption can be a significant benefit to patients with inhibitors, particularly if it is instituted prior to factor administration.

Extracorporeal removal of anti-HLA antibodies in transplant candidates.

We report on the results of a clinical trial in which 14 transplant candidates were treated with an extracorporeal immunoadsorption system using Protein A that selectively removes immunoglobulin from plasma; we also assessed the dynamics of anti-HLA antibody as a model of IgG removal and re-equilibration, as well as the clinical safety of the procedure. At the end of a treatment course, plasma IgG levels were reduced by 90% +/- 8% of control values (P less than 0.01). In contrast, albumin levels were reduced by only 15% (P less than 0.05). Specific cytotoxic anti-HLA antibody titers were reduced by approximately 18-fold. Panel reactivity was measured as the proportion of a 40-member cell donor panel killed by patients' serum in the presence of complement; in nine of the 14 patients, there was a significant reduction in this parameter (range, 23% to 87%). During the 4-week follow-up period, anti-HLA antibody titers returned to baseline levels. There were no remarkable changes observed in blood chemistries, nor were there any unanticipated adverse reactions seen in the patients treated. We conclude that selective extracorporeal immunoadsorption is a safe and effective way of removing IgG-type antibodies, with potential application to reduction of HLA antibodies in transplant candidates.

The effect of pH on the aggregation of biotinylated antibodies and on the signal-to-noise observed in immunoassays utilizing biotinylated antibodies.

During the development of immunoassays to detect gram-negative bacteria, an effect of pH on the aggregation of some murine monoclonal antibodies directed to Neisseria gonorrhoeae was observed. By reacting positively charged primary amines on these antibodies with the neutral NHS-biotin (N-hydroxy-succinimidobiotin), the surface charge on the antibodies was altered and a concomitant change in the solubility of these antibodies noted. This derivatization produced not only a pH-dependent change in the solubility properties of the antibodies, but also affected the response of immunoassays in which these antibodies were used. Data presented suggests that the signal-to-noise (S/N) observed in these assays is maximized under conditions where the biotinylated antibody is introduced into the assay at a pH at least 2 U above its pI. Our hypothesis is that as the pH of the solution approaches the biotinylated antibodies' isoelectric point, they become 'stickier', perhaps by aggregation (which we have directly measured), leading to high non-specific binding and hence a lower S/N.

Immunoglobulin isotypes of anti-Trichomonas vaginalis antibodies in patients with vaginal trichomoniasis.

Studies of anti-Trichomonas vaginalis antibodies in patients with vaginal trichomoniasis were undertaken in attempts to identify the predominant antibody isotype produced and to delineate clinically significant antigens. The total antibody content of serum samples from 23 patients was determined by an enzyme-linked immunosorbent assay (ELISA) that employed anti-human immunoglobulin and isotype-specific antisera. The immunochemical reactivity of these antibodies was examined by Western blot analysis. The anti-T. vaginalis titer of all but two of these serum samples was greater than 200 (range, greater than 200 to 12,800). By using an ELISA titer of at least 200 as a criterion, 21 of the serum samples contained antibodies of the immunoglobulin G (IgG) isotype, 17 contained IgM antibodies, and 6 contained IgA antibodies directed to the protozoan. Western blot analyses of these serum samples revealed approximately 29 antigenic trichomonad polypeptides, with apparent molecular sizes ranging from 14 to greater than 100 kilodaltons and with individual serum samples possessing different patterns of reactivity. These results add to the current understanding of the serological and secretory immune responses to T. vaginalis, as well as define potential antigens for use in immunodiagnostics.

Rapid assay for immunological detection of Trichomonas vaginalis.

Trichomoniasis is a common sexually transmitted disease with an estimated incidence of 4 million to 8 million cases a year in the United States. The most commonly used method of diagnosis is a direct microscopic observation (wet mount) of vaginal secretions and, although both rapid and inexpensive, the sensitivity of this technique is generally 50 to 70%. We developed an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of Trichomonas vaginalis which is both rapid and sensitive (detection limit of approximately 100 trichomonads per ml). This assay, which employs affinity-purified rabbit anti-T. vaginalis antibodies in a "sandwich" configuration, is simple to perform and is neither interfered with nor appears to cross-react with other microorganisms which are common inhabitants of the urogenital tract. One hundred and seventy-seven consecutive unselected patients attending a clinic for sexually transmitted diseases were evaluated for trichomoniasis by a broth culture technique monitored for up to 7 days (and considered here to be the standard for positivity), the conventional wet mount method, a solid culture procedure, and the ELISA. Of these, 84 were positive by culture; 33 were positive by the wet mount; and despite the fact that the vaginal specimens were diluted 20-fold during the culture procedures prior to testing in the ELISA, 65 were positive by ELISA. In addition to exhibiting a sensitivity of 77%, the specificity of the ELISA was 100%. These results demonstrate that the ELISA is a significant improvement over the wet mount method for the diagnosis of trichomoniasis.