Detection of pneumonia associated pathogens using a prototype multiplexed pneumonia test in hospitalized patients with severe pneumonia

Severe pneumonia remains an important cause of morbidity and mortality. Polymerase chain reaction (PCR) has been shown to be more sensitive than current standard microbiological methods--particularly in patients with prior antibiotic treatment--and therefore, may improve the accuracy of microbiological diagnosis for hospitalized patients with pneumonia. Conventional detection techniques and multiplex PCR for 14 typical bacterial pneumonia-associated pathogens were performed on respiratory samples collected from adult hospitalized patients enrolled in a prospective multi-center study. Patients were enrolled from March until September 2012. A total of 739 fresh, native samples were eligible for analysis, of which 75 were sputa, 421 aspirates, and 234 bronchial lavages. 276 pathogens were detected by microbiology for which a valid PCR result was generated (positive or negative detection result by Curetis prototype system). Among these, 120 were identified by the prototype assay, 50 pathogens were not detected. Overall performance of the prototype for pathogen identification was 70.6% sensitivity (95% confidence interval (CI) lower bound: 63.3%, upper bound: 76.9%) and 95.2% specificity (95% CI lower bound: 94.6%, upper bound: 95.7%). Based on the study results, device cut-off settings were adjusted for future series production. The overall performance with the settings of the CE series production devices was 78.7% sensitivity (95% CI lower bound: 72.1%) and 96.6% specificity (95% CI lower bound: 96.1%). Time to result was 5.2 hours (median) for the prototype test and 43.5 h for standard-of-care. The Pneumonia Application provides a rapid and moderately sensitive assay for the detection of pneumonia-causing pathogens with minimal hands-on time

Detection and elimination of cellular bottlenecks in protein-producing yeasts

Yeasts are efficient cell factories and are commonly used for the production of recombinant proteins for biopharmaceutical and industrial purposes. For such products high levels of correctly folded proteins are needed, which sometimes requires improvement and engineering of the expression system. The article summarizes major breakthroughs that led to the efficient use of yeasts as production platforms and reviews bottlenecks occurring during protein production. Special focus is given to the metabolic impact of protein production. Furthermore, strategies that were shown to enhance secretion of recombinant proteins in different yeast species are presented

Detection of pneumonia associated pathogens using a prototype multiplexed pneumonia test in hospitalized patients with severe pneumonia

Severe pneumonia remains an important cause of morbidity and mortality. Polymerase chain reaction (PCR) has been shown to be more sensitive than current standard microbiological methods--particularly in patients with prior antibiotic treatment--and therefore, may improve the accuracy of microbiological diagnosis for hospitalized patients with pneumonia. Conventional detection techniques and multiplex PCR for 14 typical bacterial pneumonia-associated pathogens were performed on respiratory samples collected from adult hospitalized patients enrolled in a prospective multi-center study. Patients were enrolled from March until September 2012. A total of 739 fresh, native samples were eligible for analysis, of which 75 were sputa, 421 aspirates, and 234 bronchial lavages. 276 pathogens were detected by microbiology for which a valid PCR result was generated (positive or negative detection result by Curetis prototype system). Among these, 120 were identified by the prototype assay, 50 pathogens were not detected. Overall performance of the prototype for pathogen identification was 70.6% sensitivity (95% confidence interval (CI) lower bound: 63.3%, upper bound: 76.9%) and 95.2% specificity (95% CI lower bound: 94.6%, upper bound: 95.7%). Based on the study results, device cut-off settings were adjusted for future series production. The overall performance with the settings of the CE series production devices was 78.7% sensitivity (95% CI lower bound: 72.1%) and 96.6% specificity (95% CI lower bound: 96.1%). Time to result was 5.2 hours (median) for the prototype test and 43.5 h for standard-of-care. The Pneumonia Application provides a rapid and moderately sensitive assay for the detection of pneumonia-causing pathogens with minimal hands-on time

Insertional Mutagenesis in the n-Alkane-Assimilating Yeast Yarrowia lipolytica: Generation of Tagged Mutations in Genes Involved in Hydrophobic Substrate Utilization

Tagged mutants affected in the degradation of hydrophobic compounds (HC) were generated by insertion of a zeta-URA3 mutagenesis cassette (MTC) into the genome of a zeta-free and ura3 deletion-containing strain of Yarrowia lipolytica. MTC integration occurred predominantly at random by nonhomologous recombination. A total of 8,600 Ura(+) transformants were tested by replica plating for (i) growth on minimal media with alkanes of different chain lengths (decane, dodecane, and hexadecane), oleic acid, tributyrin, or ethanol as the C source and (ii) colonial defects on different glucose-containing media (YPD, YNBD, and YNBcas). A total of 257 mutants were obtained, of which about 70 were affected in HC degradation, representing different types of non-alkane-utilizing (Alk(−)) mutants (phenotypic classes alkA to alkE) and tributyrin degradation mutants. Among Alk(−) mutants, growth defects depending on the alkane chain length were observed (alkAa to alkAc). Furthermore, mutants defective in yeast-hypha transition and ethanol utilization and selected auxotrophic mutants were isolated. Flanking borders of the integrated MTC were sequenced to identify the disrupted genes. Sequence analysis indicated that the MTC was integrated in the LEU1 locus in N083, a leucine-auxotrophic mutant, in the isocitrate dehydrogenase gene of N156 (alkE leaky), in the thioredoxin reductase gene in N040 (alkAc), and in a peroxine gene (PEX14) in N078 (alkD). This indicates that MTC integration is a powerful tool for generating and analyzing tagged mutants in Y. lipolytica

Pathogen performance of the multiplexed prototype assay [including discrepant results resolution in brackets].

a<p>true positive: positive in microbiological standard-of-care testing and positive in the multiplexed assay.</p>b<p>false negative: positive in microbiological standard-of-care testing and negative in the multiplexed assay.</p>c<p>false positive: negative in microbiological standard-of-care testing and positive in the multiplexed assay.</p>d<p>true negative: negative in microbiological standard-of-care testing and negative in the multiplexed assay.</p>e<p>positive predictive value.</p>f<p>negative predictive value [confirmed FN and FP in brackets].</p><p>* confirmation of <i>Streptococcus</i>: only as "<i>Streptococcus</i> spp."</p><p>Additional pathogens not covered by the multiplexed assay: 114 yeasts (including 85 <i>Candida</i> spp.), 7 other fungi, 9 <i>Citrobacter</i> spp., 49 coagulase neg. staphylococci, 34 enterococci, 35 streptococci (mostly <i>viridans</i> group), 8 other Gram-positive bacteria (<i>Leuconostoc</i> spp., <i>Rothia</i> spp., <i>Corynebacterium</i> spp.), 3 <i>Pseudomonas</i> spp., 10 <i>Neisseria</i> spp., 3 <i>Haemophilus</i> spp., 10 <i>Citrobacter</i> spp., 7 other Gram-negative bacteria (<i>Ralstonia</i> spp., <i>Achromobacter</i> spp., <i>Burkholderia</i> spp., <i>Raoultella</i> spp., <i>Serratia</i> spp.).</p><p>Pathogen performance of the multiplexed prototype assay [including discrepant results resolution in brackets].</p