Enrichment of bacterial DNA for the diagnosis of blood stream infections.

BACKGROUND: Blood cultures are commonly employed to identify bacterial pathogens causing sepsis. PCR assays to diagnose septicemia require extraction of bacterial DNA from blood samples and thus, delay the initiation of appropriate antimicrobial treatment. The presence of abundant human DNA may hamper the sensitivity of PCR in the detection of bacteria. METHODS: We used serial dilutions of E. Coli spiked pseudo-blood-sepsis samples to develop a simple method that combines the use of a polar detergent solvent and adjustment of the basic pH to remove human DNA. A 16S rRNA gene-based screening algorithm was established to differentiate Gram-positive and Gram-negative groups of bacteria and the family of Enterobacteriaceae. A stringent validation with appropriate controls was implemented. The method of human DNA removal was then applied on 194 sepsis blood samples and 44 cerebrospinal fluid (CSF) samples by real-time PCR. RESULTS: This uncomplicated and straightforward approach allows to remove up to 98 % of human DNA from peripheral blood of septic patients. The inhibitory effect of human DNA is efficiently prevented and the detection limit of real-time PCR is increased to 10 E. Coli CFUs/ml. This sensitivity is 10 times higher compared to conventional real-time PCR assays. The classical blood culture detected 58/194 (30 %) of sepsis and 9/44 (21 %) of CSF samples. Out of the 194 blood samples tested, the conventional real-time PCR targeting 13 common sepsis causing pathogens correctly detected the bacterial DNA in 16/194 (8 %) only and 14/44 (32 %) in cerebrospinal fluid samples. Our newly established approach was able to provide correct diagnoses in 78 (40 %) of the 194 blood samples and in 14 (32 %) of the CSF samples. The combination of both blood cultures and our technique raised the rate of sepsis diagnoses to 112/194 (58 %). Of the total group tested positive, 46 (24 %) cases showed overlap with the classical methodology. CONCLUSION: We report a simple optimized in-house protocol for removal of human DNA from blood sepsis samples as a pre-analytical tool to prepare DNA for subsequent PCR assays. With the detection increase of our in-house DNA removal approach, subsequent PCR assays can reach detection limits of 10 E. coli CFUs/ml and significantly improve the diagnostic rate in blood sepsis cases.

Simple multiplex PCR assays to detect common pathogens and associated genes encoding for acquired extended spectrum betalactamases (ESBL) or carbapenemases from surgical site specimens in Vietnam.

UNLABELLED: Surgical site infection (SSI) is common in Vietnamese post-operative patients. It contributes to increased morbidity, mortality, hospitalization time and health care expenditure. Bacterial culture is considered the gold standard procedure to identify SSI pathogens and antibiotic resistant properties; however, it can detect microbes that can readily grow and is time-consuming. We propose optimized multiplex PCR assays to diagnose the most relevant microbes and associated genes encoding for acquired extended spectrum betalactamases (ESBL) or carbapenemases from Vietnamese patients with SSI in a hospital setting in Hanoi. METHODS: Ninety-one patients (n = 91) were collected in order to identify microbial pathogens and associated genes encoding for acquired extended spectrum betalactamases (ESBL) or carbapenemases by both conventional bacterial culture and in-house multiplex PCR assays. RESULT AND CONCLUSION: The novel in-house multiplex PCR assays are comparable to the bacterial culture approach in screening for common pathogens causing SSI and for relevant genotypes conferring betalactam/carbapenem resistance for bacteria. This is the first report of Turkey-specific ESBL gene (PER-1) and two Oxacilinase families (Oxa23 and Oxa 58) in Vietnam.

Large-Scale Synthesis of Nanosilica from Silica Sand for Plant Stimulant Applications.

Nanosilica is a versatile nanomaterial suitable as, e.g., drug carriers in medicine, fillers in polymers, and fertilizer/pesticide carriers and potentially a bioavailable source of silicon in agriculture. The enhanced biological activity of nanosilica over quartz sand has been noted before; it is directly related to the altered physicochemical properties of the nanoparticles compared to those of the bulk material. Therefore, it is feasible to use nanosilica as a form of plant stimulant. Nanosilica synthesis is a relatively cheap routine process on the laboratory scale; however, it is not easily scalable. Largely for this reason, studies of nanosilica fertilizers are scarce. This study will focus on industrial-scale silica nanoparticle production and the application of nanosilica as a plant stimulant in maize. A variant of the sol-gel method is used to successfully synthesize nanosilica particles starting from silica sand. The resulting particles are in the size range of 16-37 nm with great purity. The potential of nanosilica as a plant stimulant is demonstrated with the increased quantity and quality of maize crops.

Circulating miR-147b as a diagnostic marker for patients with bacterial sepsis and septic shock.

BACKGROUND: Early diagnosis, precise antimicrobial treatment and subsequent patient stratification can improve sepsis outcomes. Circulating biomarkers such as plasma microRNAs (miRNAs) have proven to be surrogates for diagnosis, severity and case management of infections. The expression of four selected miRNAs (miR-146-3p, miR-147b, miR-155 and miR-223) was validated for their prognostic and diagnostic potential in a clinically defined cohort of patients with sepsis and septic shock. METHODS: The expression of plasma miRNAs was quantified by quantitative PCR (qPCR) in patients with bacterial sepsis (n = 78), in patients with septic shock (n = 52) and in patients with dengue haemorrhagic fever (DHF; n = 69) and in healthy controls (n = 82). RESULTS: The expression of studied miRNA was significantly increased in patients with bacterial sepsis and septic shock. The plasma miR-147b was able to differentiate bacterial sepsis from non-sepsis and septic shock (AUC = 0.77 and 0.8, respectively, p≤ 0.05), while the combination of plasma miR-147b and procalcitonin (PCT) predicted septic shock (AUC = 0.86, p≤ 0.05). CONCLUSIONS: The plasma miR-147b may be an useful biomarker independently or in combination with PCT to support clinical diagnosis of sepsis and equally prognosis of patients with septic shock.

Design, synthesis and bioevaluation of novel 6-substituted aminoindazole derivatives as anticancer agents.

In the present study, a series of 6-substituted aminoindazole derivatives were designed, synthesized, and evaluated for bio-activities. The compounds were initially designed as indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors based on the structural feature of five IDO1 inhibitors, which are currently on clinical trials, and the important anticancer activity of the indazole scaffold. One of them, compound N-(4-fluorobenzyl)-1,3-dimethyl-1H-indazol-6-amine (36), exhibited a potent anti-proliferative activity with an IC50 value of 0.4 ± 0.3 µM in human colorectal cancer cells (HCT116). This compound also remarkably suppressed the IDO1 protein expression. In the cell-cycle studies, the suppressive activity of compound 36 in HCT116 cells was related to the G2/M cell cycle arrest. Altogether, the current findings demonstrate that compound 36 would be promising for further development as a potential anticancer agent.

In vitro and in silico determination of glutaminyl cyclase inhibitors.

Alzheimer's disease (AD) is the most common form of neurodegenerative disease currently. It is widely accepted that AD is characterized by the self-assembly of amyloid beta (Aß) peptides. The human glutaminyl cyclase (hQC) enzyme is characterized by association with Aß peptide generation. The development of hQC inhibitors could prevent the self-aggregation of Aß peptides, resulting in impeding AD. Utilizing structural knowledge of the hQC substrates and known hQC inhibitors, new heterocyclic and peptidomimetic derivatives were synthesized and were able to inhibit the hQC enzyme. The inhibiting abilities of these compounds were evaluated using a fluorometric assay. The binding mechanism at the atomic level was estimated using molecular docking, free energy perturbation, and quantum chemical calculation methods. The predicted log(BBB) and human intestinal absorption values indicated that these compounds are able to permeate the blood-brain barrier and be well-absorbed through the gastrointestinal tract. Overall, 5,6-dimethoxy-N-(3-(5-methyl-1H-imidazol-1-yl)propyl)-1H-benzo[d]imidazol-2-amine (1_2) was indicated as a potential drug for AD treatment.

Optimisation of quantitative miRNA panels to consolidate the diagnostic surveillance of HBV-related hepatocellular carcinoma.

BACKGROUND: Circulating microRNAs (miRNA) are biomarkers for several neoplastic diseases, including hepatocellular carcinoma (HCC). We performed a literature search, followed by experimental screening and validation in order to establish a miRNA panel in combination with the assessment of alpha-fetoprotein (AFP) levels and to evaluate its performance in HCC diagnostics. METHODS: Expression of miRNAs was quantified by quantitative PCR (qPCR) in 406 serum samples from 118 Vietnamese patients with hepatitis B (HBV)-related HCC, 69 patients with HBV-related liver cirrhosis (LC), 100 chronic hepatitis B (CHB) patients and 119 healthy controls (HC). RESULTS: Three miRNAs (mir-21, mir-122, mir-192) were expressed differentially among the studied subgroups and positively correlated with AFP levels. The individual miRNAs mir-21, mir-122, mir192 or the triplex miRNA panel showed high diagnostic accuracy for HCC (HCC vs. CHB, AUC = 0.906; HCC vs. CHB+LC, AUC = 0.81; HCC vs. CHB+LC+HC, AUC = 0.854). When AFP levels were ≤20ng/ml, the triplex miRNA panel still was accurate in distinguishing HCC from the other conditions (CHB, AUC = 0.922; CHB+LC, AUC = 0.836; CHB+LC+HC, AUC = 0.862). When AFP levels were used in combination with the triplex miRNA panel, the diagnostic performance was significantly improved in discriminating HCC from the other groups (LC, AUC = 0.887; CHB, AUC = 0.948; CHB+LC, AUC = 0.887). CONCLUSIONS: The three miRNAs mir-21, mir-122, mir-192, together with AFP, are biomarkers that may be applied to improve diagnostics of HCC in HBV patients, especially in HBV-related LC patients with normal AFP levels or HCC patients with small tumor sizes.