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.

Compound K Production: Achievements and Perspectives.

Compound K (CK) is one of the major metabolites found in mammalian blood and organs following oral administration of Panax plants. CK, also known as minor ginsenoside, can be absorbed in the systemic circulation. It has garnered significant attention in healthcare and medical products due to its pharmacological activities, such as antioxidation, anticancer, antiproliferation, antidiabetics, neuroprotection, and anti-atherogenic activities. However, CK is not found in natural ginseng plants but in traditional chemical synthesis, which uses toxic solvents and leads to environmental pollution during the harvest process. Moreover, enzymatic reactions are impractical for industrial CK production due to low yield and high costs. Although CK could be generated from major ginsenosides, most ginsenosides, including protopanaxatriol-oleanane and ocotillol-type, are not converted into CK by catalyzing ß-glucosidase. Therefore, microbial cell systems have been used as a promising solution, providing a safe and efficient approach to CK production. This review provides a summary of various approaches for the production of CK, including chemical and enzymatic reactions, biotransformation by the human intestinal bacteria and endophytes as well as engineered microbes. Moreover, the approaches for CK production have been discussed to improve the productivity of target compounds.

Selfish personalities influencing start-up intention and motivation: a study of Vietnam.

This research explores the influences of selfish personalities of the Dark Triad on start-up intention and motives based on a sample of 400 university students in Vietnam, discovering mixed effects of narcissism, psychopathy, and Machiavellianism. A high level of narcissism and Machiavellianism leads to high start-up intention. There is a negative relationship of Machiavellianism with pro-social motive and a positive association with selfish entrepreneurship. In addition, narcissism is positively associated with pro-social start-up motives. This study has found no effect of psychopathy but a positive link to selfish entrepreneurial motivation. Implications have been suggested for educators and investors.

Disability from Japanese encephalitis in Cambodia and Viet Nam.

A cohort of Japanese encephalitis (JE) survivors in Cambodia and Viet Nam were assessed at least 4 months after hospital discharge in order to understand the extent of disability after JE. We used a simple assessment tool which focuses on the impact on daily life. In total, 64 disability assessments were conducted: 38 in Cambodia and 26 in Viet Nam. In Cambodia, 4 (11%) children had severe sequelae, suggesting the children would likely be dependent, 15 (39%) had moderate sequelae and 17 (45%) had mild sequelae. In Viet Nam, two (8%) persons had severe sequelae, five (19%) had moderate sequelae and eight (31%) had mild sequelae. In many JE-endemic areas there are no multi-disciplinary teams with sophisticated equipment to assess patients after JE disease. This assessment tool can assist with patient management and generate data to support the need for programmes to prevent disease and improve outcomes for survivors.

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.

Clinical utility of an optimised multiplex real-time PCR assay for the identification of pathogens causing sepsis in Vietnamese patients.

INTRODUCTION: For the identification of bacterial pathogens, blood culture is still the gold standard diagnostic method. However, several disadvantages apply to blood cultures, such as time and rather large volumes of blood sample required. We have previously established an optimised multiplex real-time PCR method in order to diagnose bloodstream infections. MATERIAL AND METHODS: In the present study, we evaluated the diagnostic performance of this optimised multiplex RT-PCR in blood samples collected from 110 septicaemia patients enrolled at the 108 Military Central Hospital, Hanoi, Vietnam. RESULTS: Positive results were obtained by blood culture, the Light Cylcler-based SeptiFast® assay and our multiplex RT-PCR in 35 (32%), 31 (28%), and 31 (28%) samples, respectively. Combined use of the three methods confirmed 50 (45.5%) positive cases of bloodstream infection, a rate significantly higher compared to the exclusive use of one of the three methods (P=0.052, 0.012 and 0.012, respectively). The sensitivity, specificity and area under the curve (AUC) of our assay were higher compared to that of the SeptiFast® assay (77.4%, 86.1% and 0.8 vs. 67.7%, 82.3% and 0.73, respectively). Combined use of blood culture and multiplex RT-PCR assay showed a superior diagnostic performance, as the sensitivity, specificity, and AUC reached 83.3%, 100%, and 0.95, respectively. The concordance between blood culture and the multiplex RT-PCR assay was highest for Klebsiella pneumonia (100%), followed by Streptococcus spp. (77.8%), Escherichia coli (66.7%), Staphylococcus spp. (50%) and Salmonella spp. (50%). In addition, the use of the newly established multiplex RT-PCR assay increased the spectrum of identifiable agents (Acintobacter baumannii, 1/32; Proteus mirabilis, 1/32). CONCLUSION: The combination of culture and the multiplex RT-PCR assay provided an excellent diagnostic accomplishment and significantly supported the identification of causative pathogens in clinical samples obtained from septic patients.