Application of qualitative and quantitative real-time PCR, direct sequencing, and terminal restriction fragment length polymorphism analysis for detection and identification of polymicrobial 16S rRNA genes in ascites.

Qualitative and quantitative 16S rRNA gene-based real-time PCR and direct sequencing were applied for rapid detection and identification of bacterial DNA (bactDNA) in 356 ascites samples. bactDNA was detected in 35% of samples, with a mean of 3.24 log copies ml(-1). Direct sequencing of PCR products revealed 62% mixed chromatograms predominantly belonging to Gram-positive bacteria. Terminal restriction fragment length polymorphism (T-RFLP) results of a sample subset confirmed sequence data showing polymicrobial DNA contents in 67% of bactDNA-positive ascites samples.

Use of Bacterial DNA Concentration in Ascites as a Marker for Spontaneous Bacterial Peritonitis.

Background and aims: Spontaneous bacterial peritonitis (SBP) is a common and serious complication in patients with decompensated cirrhosis. Precise quantification of bacterial DNA (bactDNA) and the related inflammatory response might add further information on the course of disease. The aim of the study was to evaluate the association between bactDNA, cytokine levels and clinical outcome. Methods: Ascites and serum samples of 98 patients with decompensated liver cirrhosis (42 with SBP and 56 without SBP) as well as serum samples of 21 healthy controls were collected. BactDNA in ascites and serum was detected and quantified by 16S rRNA PCR. Concentrations of IL-1ß, TNF-α, IL-6, IL-8 and IL-10 were measured by a LEGENDplexTM multi-analyte flow assay. Clinical data were collected and analyzed retrospectively. Results: BactDNA was detected more frequently in ascites of patients with SBP (n = 24/42; 57.1%) than in ascites of patients without SBP (n = 5/56; 8.9%; P < 0.001). Additionally, IL-6 levels in both ascites and serum were significantly higher in patients with SBP (ascites P < 0.001, serum P = 0.036). The quantity of bactDNA in ascites was strongly correlated with polymorphonuclear neutrophil count in ascites (r = 0.755; P < 0.001) as well as ascites IL-6 levels (r = 0.399; P < 0.001). Receiver operating characteristic (ROC) curve analysis to diagnose SBP provided an AUC of 0.764 (95% CI: 0.661-0.867) for serum IL-6 levels, an AUC of 0.810 (95% CI: 0.714-0.905) for ascites IL-6 levels, and an AUC of 0.755 (95% CI: 0.651-0.858) for bactDNA levels in ascites. Conclusions: The correlation between the amount of bactDNA and IL-6 confirms the pathophysiological relevance of bactDNA and IL-6 as potential biomarkers for the diagnosis of SBP.

User-Centered Development of a Mobile App to Assess the Quality of Life of Patients With Cancer: Iterative Investigation and Usability Testing.

BACKGROUND: The treatment for cancer can have a negative impact not only on physical well-being but also on mental health and the quality of life (QoL). Health apps enable the monitoring of different parameters, but to date, there are only few that support patients with cancer and none that focuses on the assessment of QoL. Furthermore, patients as stakeholders are often only integrated at the late stage of the development process, if at all. OBJECTIVE: The aim of this research was to develop and evaluate a smartphone app (Lion-App) to enable patients with cancer to autonomously measure the QoL with an iterative, user-centered approach. METHODS: Patients with cancer were involved in a 3-stage process from conceptualization to the point when the app was available on the tester's private device. First, focus groups with members (N=21) of cancer support groups were conducted to understand their expectations and needs. Thereafter, individual tests were performed. After developing a prototype that incorporated findings from the focus groups, a second test cycle was conducted, followed by a beta test lasting 2 months. In our app, the QoL can be assessed via a patient diary and an integrated questionnaire. Through all stages, usability was evaluated using the modular extended version of the User Experience Questionnaire (UEQ+), including the calculation of a key performance indicator (KPI). If possible, the impact of sex on the results was evaluated. As part of the beta test, usage rates as well as age-dependent differences were also assessed. RESULTS: A total of 21 participants took part in the initial 3 focus groups. In the subsequent usability testing (N=18), 17 (94%) participants rated their impression through the UEQ+, with a mean KPI of 2.12 (SD 0.64, range: -3 to 3). In the second usability test (N=14), the mean KPI increased to 2.28 (SD=0.49). In the beta test, the usage rate of 19 participants was evaluated, of whom 14 (74%) also answered the UEQ+ (mean KPI 1.78, SD 0.84). An influence of age on the number of questionnaire responses in Lion-App was observed, with a decrease in responses with increasing age (P=.02). Sex-dependent analyses were only possible for the first usability test and the beta test. The main adjustments based on user feedback were a restructuring of the diary as well as integration of a shorter questionnaire to assess the QoL. CONCLUSIONS: The iterative, user-centered approach for development and usability testing resulted in positive evaluations of Lion-App. Our app was rated as suitable for everyday use to monitor the QoL of patients with cancer. Initial results indicated that the sex and age of participants seem to play only a minor role.

Changes of the bacterial composition in duodenal fluid from patients with liver cirrhosis and molecular bacterascites.

Small intestinal bacterial overgrowth and compositional changes of intestinal microbiota are pathomechanistic factors in liver cirrhosis leading to bacterial translocation and infectious complications. We analyzed the quantity and composition of duodenal bacterial DNA (bactDNA) in relation to bactDNA in blood and ascites of patients with liver cirrhosis. Duodenal fluid and corresponding blood and ascites samples from 103 patients with liver cirrhosis were collected. Non-liver disease patients (n = 22) served as controls. BactDNA was quantified by 16S-rRNA gene-based PCR. T-RFLP and 16S-rRNA amplicon sequencing were used to analyze bacterial composition. Duodenal bacterial diversity in cirrhosis was distinct to controls showing significantly higher abundances of Streptococcus, Enterococcus and Veillonella. Patients with bactDNA positive ascites revealed reduced spectrum of core microbiota with Streptococcus as key player of duodenal community and higher prevalence of Granulicatella proving presence of cirrhosis related intestinal dysbiosis. Regarding duodenal fluid bactDNA quantification, no significant differences were found between patients with cirrhosis and controls. Additionally, percentage of subjects with detectable bactDNA in blood did not differ between patients and controls. This study evaluated the diversity of bacterial DNA in different body specimens with potential implications on understanding how intestinal bacterial translocation may affect infectious complications in cirrhosis.

Antibacterial effect of a brominated self-etch adhesive on carious dentin - An in vivo study.

OBJECTIVES: This in vivo study compared the antibacterial effect of a self-etch adhesive with and without the brominated monomer 12-methacryloyloxydodecyl-pyridinium bromide (MDPB) on carious dentin after selective caries removal. METHODS: 10 patients showing deep primary carious lesions at two posterior teeth without pulpal symptoms were included. At visit I, carious tissue was selectively removed and carious dentin was sampled with a sterile roundbur (Komet No. 18). One cavity was restored with composite (SDR, Ceram X; DENTSPLY DeTrey) using an MDPB-containing self-etch adhesive (Clearfil Protect Bond, Kuraray Noritake; PB). The other restoration served as a control (Clearfil SE Bond II, Kuraray Noritake; SE). At visit II after 8 weeks, carious dentin was sampled again. Bacterial growth in carious dentin was differentiated using microbial cultivation. Bacterial DNA from intact cells and cell-free DNA were quantified using 16S rRNA gene-based real-time PCR and the microbial community composition was analyzed by amplicon deep-sequencing. Wilcoxon test was applied for statistical analysis. RESULTS: Both treatments showed a decrease of intact bacterial cells in carious dentin at visit II compared to visit I (PB: visit I: 1.1*106, visit II: 1.7*105 (p = 0.03); SE: visit I: 1.1*107, visit II = 2.4*105 (p = 0.002)). No statistically significant reduction of cell-free bacterial DNA was detected (PB: visit I: 6.1*105, visit II: 1.6*105 (p = 0.08); SE: visit I: 5.3*105, visit II: 2.9*105 (p = 0.10)). The decrease of intact cell-derived (p = 0.371) and cell-free DNA (p = 0.455) did not differ significantly between PB and SE. Lactobacillus was most abundant within the microbial community at both visits. Alpha-diversity was not affected by treatment and samples showed high intra- and interindividual diversity. CONCLUSION AND CLINICAL SIGNIFICANCE: Both self-etch adhesives have an antibacterial effect due to a decrease of bacterial DNA after selective caries removal. However, the results do not reveal any additional antibacterial effect by MDPB. The study is registered with the German Clinical Trials Register (DRKS00011532).

Effect of a bioactive cement on the microbial community in carious dentin after selective caries removal - An in-vivo study.

OBJECTIVES: Selective caries removal in deep lesions means that soft carious affected dentin is left in the center of the cavity. Thus, using a tricalcium silicate cement Biodentine™ (Septodont, Paris) to seal the remaining soft dentin could have an antibacterial effect. This in-vivo study aimed to do quantitative and qualitative analyses on the bacterial composition within carious dentin before and after selective caries removal when applying Biodentine. METHODS: Eleven patients with deep primary carious lesions at two posterior teeth without pulpal symptoms were included. Carious dentin was selectively removed and sampled with a sterile round bur (Komet No. 18) at baseline visit and eight weeks later. On the first visit, one lesion per patient, the remaining carious dentin was covered with Biodentine before adhesive restoration. Caries samples were investigated by microbial cultivation, molecular analysis and amplicon deep-sequencing of 16S rRNA genes. Bacterial DNA from intact cells was differentiated from cell-free DNA by DNase degradation prior to DNA isolation. RESULTS: Reduction of cell-derived as well as cell-free bacterial DNA eight weeks after selective caries removal was significantly higher when Biodentine was applied. Lactobacillus was most abundant within the microbial community of deep carious dentin lesions at the first visit. After intervention with Biodentine application, Lactobacillus was diminished to a high degree. In general, the diversity in samples, as well as bacterial composition differed interindividually as well as intraindividually. CONCLUSION AND CLINICAL SIGNIFICANCE: Despite the heterogenous and diversity of microbial composition in patients, Biodentine can have beneficial antibacterial effects when applied to residual carious dentin, offering an alternative and safe treatment option. The study is officially registered with German Clinical Trials Register (DRKS00011067).

Molecular characterization of intact cell-derived and cell-free bacterial DNA from carious dentine samples.

Microbial analyzes of carious dentine samples, especially in terms of interventions, represent a challenge due to difficulties in carious dentine sampling particularly with small bacterial DNA contents. Therefore, information about the quantitative reduction of bacterial DNA as well as microbial shifts and differences in diversity correlating with treatment interventions are scarce. In this study, carious dentine samples were collected in a first step in the course of a selective caries excavation at two different deep dentine caries lesions in three patients. Second, after selective caries excavation and sampling of carious dentine, an intervention was performed by applying dental materials onto the remaining carious dentine followed by a restoration of the study teeth with composite fillings. After 8 weeks, remaining carious dentine was sampled and analyzed as described above. The microbial community before and after therapy was analyzed by conventional culture compared to bacterial DNA analyses using 16S rRNA gene based real-time PCR and terminal restriction fragment length polymorphism (T-RFLP) for fingerprinting community changes within carious dentine samples. An ultra-pure workflow allowed the valid comparison of even small carious dentine samples with low DNA contents and the differentiation between intact cell-derived and cell-free bacterial DNA. Intra- and inter-subject related differences in the bacterial DNA content and its composition in deep dentine caries were determined considering the first visits. The ratio of cell-free bacterial DNA and DNA from intact cells decreased in two of three subjects included in the current study from visit 1 to visit 2 with the test substance (1:200 to 1:17) and the control substance (1:82 to 1:7). T-RFLP revealed changes in the bacterial diversity and composition shifts after treatment as well as between cell-free bacterial DNA and DNA derived from intact cells. The approach of differentiation and quantification of cell-free and intact cell-derived bacterial DNA is reasonable within the investigation of carious dentine samples, especially when considering the effect of an intervention. T-RFLP is principally suitable for the analysis of microbial shifts within carious dentine samples.

Molecular quantification and differentiation of Candida species in biological specimens of patients with liver cirrhosis.

Patients with liver cirrhosis are susceptible to fungal infections. Due to low sensitivity of culture-based methods, we applied a real-time PCR assay targeting the 18S rRNA gene in combination with direct sequencing and terminal-restriction fragment length polymorphism (T-RFLP) in order to establish a novel tool to detect fungal DNA and to quantify and differentiate Candida DNA, also in polyfungal specimens. In total, 281 samples (blood n = 135, ascites n = 92, duodenal fluid n = 54) from 135 patients with liver cirrhosis and 52 samples (blood n = 26, duodenal fluid n = 26) from 26 control patients were collected prospectively. Candida DNA was quantified in all samples. Standard microbiological culture was performed for comparison. Blood and ascites samples, irrespective of the patient cohort, showed a method-independent low fungal detection rate of approximately 1%, and the Candida DNA content level did not exceed 3.0x10(1) copies ml-1 in any sample. In contrast, in duodenal fluid of patients with liver cirrhosis high fungal detection rates were discovered by using both PCR- and culture-based techniques (81.5% vs. 66.7%; p = 0.123) and the median level of Candida DNA was 3.8x10(5) copies ml-1 (2.3x10(2)-6.3x10(9)). In cirrhosis and controls, fungal positive culture results were confirmed by PCR in 96% and an additional amount of 44% of culture negative duodenal samples were PCR positive. Using T-RFLP analysis in duodenal samples, overall 85% of results from microbial culture were confirmed and in 75% of culture-negative but PCR-positive samples additional Candida species could be identified. In conclusion, PCR-based methods and subsequent differentiation of Candida DNA might offer a quick approach to identifying Candida species without prior cultivation.

Detection of molecular bacterascites in decompensated cirrhosis defines a risk with decreased survival.

BACKGROUND: The prognostic relevance of bacterial DNA (bactDNA) detection in ascitic fluid of patients with cirrhosis is still under debate. Using quantitative real-time PCR with broad-range primers targeting the V3 and V4 variable region of the 16S rRNA gene, we measured bactDNA concentrations in patients with and without leukocytic ascites and evaluated the impact on short-term survival. PATIENTS AND METHODS: Ascites samples from 173 patients with decompensated cirrhosis were consecutively collected between February 2011 and December 2012. BactDNA-positive ascites samples were sequenced and chromatograms were identified using RipSeq. Clinical data collection and survival analyses were carried out retrospectively and correlated with ascites bactDNA levels. RESULTS: BactDNA was detected qualitatively with a similar frequency in both nonleukocytic and leukocytic ascites [40% (57/144) and 43.5% (10/23), respectively; P=0.724]. However, the median bactDNA level was significantly higher in leukocytic ascites than in nonleukocytic ascites (1.2×10 vs. 5.7×10 copies/ml; P=0.008). Patients' survival was associated significantly with bactDNA level. The 30-day and 180-day survival was reduced if bactDNA was above the quantification limit of 520 copies/ml (84 and 63% vs. 72 and 43%, respectively; P<0.05) and worst if bactDNA was above 5000 copies/ml. The bacterial spectrum was dominated by Gram-positive strains as shown by direct sequencing. CONCLUSION: BactDNA quantification in ascitic fluid samples using culture-independent 16S rRNA gene-based methods seems to be an interesting approach to identify patients at risk of reduced survival. Our study warrants further evaluation of antibiotic treatment in patients with molecular bacterascites.