Dried blood spots perform well to identify patients with active HCV infection in Vietnam.

Recently, treatment advances in direct-acting antivirals have radically changed the management of HCV patients. However, in resource-limited countries, identification of patients with active HCV infection is still challenging in remote settings due to the limited access to laboratories able to measure HCV viral load. This study evaluated whether dried blood spots (DBS) transferred to a central laboratory could overcome this challenge. A total of 315 HCV-infected patients, naïve to anti-HCV treatment, provided each three type of samples: plasma, DBS with calibrated quantities of venous blood and DBS with uncalibrated quantities of capillary blood. Qualitative comparison was conducted in terms of detection of HCV viral load on DBS as opposed to plasma to estimate sensitivity and specificity. Quantitative comparisons were conducted by means of correlation estimation. Of the 250 patients with detected plasma HCV viral load, 245 also had detectable DBS HCV viral load (capillary or venous) leading to a sensitivity of 98.0% (95% confidence interval (CI): 95.4%-99.3%); importantly, all measurements with a plasma HCV viral load >118 IU/mL were also detected in DBS. When HCV was not detected in plasma, it was also not detected in DBS resulting in 100% specificity (95% CI: 94.5%-100%). Quantitative HCV viral load results were very similar when utilizing plasma or DBS sample types as illustrated by correlations >0.99. In conclusion, DBS sample types, with either uncalibrated capillary blood or calibrated venous blood, performed well to distinguish patients with active HCV infection, and who therefore need treatment, from other patients.

Segregostat: a novel concept to control phenotypic diversification dynamics on the example of Gram-negative bacteria.

Controlling and managing the degree of phenotypic diversification of microbial populations is a challenging task. This task not only requires detailed knowledge regarding diversification mechanisms but also advanced technical set-ups for the real-time analyses and control of population behaviour on single-cell level. In this work, set-up, design and operation of the so called segregostat are described which, in contrast to a traditional chemostat, allows the control of phenotypic diversification of microbial populations over time. Two exemplary case studies will be discussed, i.e. phenotypic diversification dynamics of Eschericia coli and Pseudomonas putida based on outer membrane permeabilization, emphasizing the applicability and versatility of the proposed approach. Upon nutrient limitation, cell population tends to diversify into several subpopulations exhibiting distinct phenotypic features (non-permeabilized and permeabilized cells). Online analysis leads to the determination of the ratio between cells in these two states, which in turn triggers the addition of glucose pulses in order to maintain a predefined diversification ratio. These results prove that phenotypic diversification can be controlled by means of defined pulse-frequency modulation within continuously running bioreactor set-ups. This lays the foundation for systematic studies, not only of phenotypic diversification but also for all processes where dynamics single-cell approaches are required, such as synthetic co-culture processes.

Growth-dependent recombinant product formation kinetics can be reproduced through engineering of glucose transport and is prone to phenotypic heterogeneity.

BACKGROUND: Escherichia coli W3110 and a group of six isogenic derivatives, each displaying distinct specific rates of glucose consumption were characterized to determine levels of GFP production and population heterogeneity. These strains have single or combinatory deletions in genes encoding phosphoenolpyruvate:sugar phosphotransferase system (PTS) permeases as PtsG and ManX, as well as common components EI, Hpr protein and EIIA, also the non-PTS Mgl galactose/glucose ABC transporter. They have been transformed for expressing GFP based on a lac-based expression vector, which is subject to bistability. RESULTS: These strains displayed specific glucose consumption and growth rates ranging from 1.75 to 0.45 g/g h and 0.54 to 0.16 h-1, respectively. The rate of acetate production was strongly reduced in all mutant strains when compared with W3110/pV21. In bioreactor cultures, wild type W3110/pV21 produced 50.51 mg/L GFP, whereas strains WG/pV21 with inactive PTS IICBGlc and WGM/pV21 with the additional inactivation of PTS IIABMan showed the highest titers of GFP, corresponding to 342 and 438 mg/L, respectively. Moreover, we showed experimentally that bistable expression systems, as lac-based ones, induce strong phenotypic segregation among microbial populations. CONCLUSIONS: We have demonstrated that reduction on glucose consumption rate in E. coli leads to an improvement of GFP production. Furthermore, from the perspective of phenotypic heterogeneity, we observed in this case that heterogeneous systems are also the ones leading to the highest performance. This observation suggests reconsidering the generally accepted proposition stating that phenotypic heterogeneity is generally unwanted in bioprocess applications.