Conventional methods and future trends in antimicrobial susceptibility testing

Antimicrobial susceptibility testing is an essential task for selecting appropriate antimicrobial agents to treat infectious diseases. Constant evolution has been observed in methods used in the diagnostic microbiology laboratories. Disc diffusion or broth microdilution are classical and conventional phenotypic methods with long turnaround time and labour-intensive but still widely practiced as gold-standard. Scientists are striving to develop innovative, novel and faster methods of antimicrobial susceptibility testing to be applicable for routine microbiological laboratory practice and research. To meet the requirements, there is an increasing trend towards automation, genotypic and micro/nano technology-based innovations. Automation in detection systems and integration of computers for online data analysis and data sharing are giant leaps towards versatile nature of automated methods currently in use. Genotypic methods detect a specific genetic marker associated with resistant phenotypes using molecular amplification techniques and genome sequencing. Microfluidics and microdroplets are recent addition in the continuous advancement of methods that show great promises with regards to safety and speed and have the prospect to identify and monitor resistance mechanisms. Although genotypic and microfluidics methods have many exciting features, however, their applications into routine clinical laboratory practice warrant extensive validation. The main impetus behind the evolution of methods in antimicrobial susceptibility testing is to shorten the overall turnaround time in obtaining the results and to enhance the ease of sample processing. This comprehensive narrative review summarises major conventional phenotypic methods and automated systems currently in use, and highlights principles of some of the emerging genotypic and micro/nanotechnology-based methods in antimicrobial susceptibility testing.     

Peripherally administrated prions reach the brain at sub-infectious quantities in experimental hamsters

The mechanisms implicated in prion infection and tissue distribution are not completely understood. In this study we investigated the levels of 263K prions in brain and spleen of Syrian hamsters few days after intra-peritoneal challenge. For this purpose we utilized the PMCA technology which permits to detect as little as few PrP^Sc molecules. Our results show that peripherally administered prions directly reach the brain, although at levels below the minimum necessary to produce disease. PrP^Sc remains in the brain several days after administration suggesting inefficient clearance or early replication. Understanding the fate of the infectious agent after administration and its uptake in different organs and fluids may provide useful information to develop strategies to minimize further spreading of prion diseases.     

Simple procedure for estimating the efficiency of PCR

The extension efficiency of PCR-driven DNA amplification was examined by step-controlled limiting dilution method using as internal controls 8E5 lymphocytes carrying a single copy of HIV genome. The results reveal that under standard conditions recommended by the PCR kit manufacturer the exponential growth of DNA template is approximately one third of the theoretical doubling rate.     

IL-32θ negatively regulates IL-1β production through its interaction with PKCδ and the inhibition of PU.1 phosphorylation

It has been well known that IL-32 exerts pro-inflammatory effects on the various inflammatory diseases in clinical studies. Here, we confirmed that IL-32θ, a new isoform of IL-32, decreased the phorbol 12-myristate 13-acetate (PMA)-induced IL-1β expression in THP-1 human myelomonocyte. We previously reported that the IL-32 isoforms control expressions of other cytokines via novel PKCs. Likewise, IL-32θ interacted with PKCδ, and consequently inhibited PKCδ-mediated phosphorylation of PU.1. Moreover, IL-32θ attenuated the localization of PU.1 into the IL-1β promoter region. These findings reveal that IL-32θ reduces PKCδ-mediated phosphorylation of PU.1, resulting in attenuation of IL-1β production.     

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.     

Specialist versus generalist parasites: the interactions between host diversity,environment and geographic barriers in avian malaria

The specialist versus generalist strategies of hemoparasites in relation to their avian host, as well as environmental factors, can influence their prevalence, diversity and distribution. In this paper we investigated the influence of avian host species, as well as the environmental and geographical factors, on the strategies of Haemoproteus and Plasmodium hemoparasites. We determined prevalence and diversity by targeting their cytochrome b (Cytb) in a total of 2,590 passerine samples from 138 localities of Central and South America, and analysed biogeographic patterns and host-parasite relationships. We found a total prevalence of 23.2%. Haemoproteus presented a higher prevalence (15.3%) than Plasmodium (4.3%), as well as a higher diversity and host specificity. We determined that Plasmodium and Haemoproteus prevalences correlated positively with host diversity (Shannon index) and were significantly influenced by bird diversity, demonstrating a possible “amplification effect”. We found an effect of locality and the avian family for prevalences of Haemoproteus and Plasmodium. These results suggest that Haemoproteus is more specialist than Plasmodium and could be mostly influenced by its avian host and the Andes Mountains.     

The typology of syncretisms and the status of feature structure. Verbal paradigms across 355 Dutch dialects

In this article syncretic patterning in the present indicative paradigm of the verb kloppen (‘to knock’) is described for 355 Dutch dialects taken from the morphological atlas of Dutch dialects (Van den Berg 2003). Following Baerman et al. (2005, The syntax-morphology interface. A study of syncretism. Cambridge: Cambridge University Press), I distinguish syncretisms driven by (universal) feature structure and language specific sources of syncretism. I present independent evidence for the role of phonology, pragmatics and amplification in the formation of syncretic patterns of Dutch. The benefit of the study of the interaction between language specific routes to syncretism and feature structure is threefold. We know language specific routes to syncretism can obscure feature structure. By distinguishing the different routes to syncretism we canalsorevealthe strength of feature structure. Secondly, distinguishing sources of syncretisms enables us to understand similarities and differences in the cross-linguistic patterning of syncretisms. Thirdly, we can link typological data to language acquisition patterns.     

Identification and genomic analysis of a novel member of Microviridae,IME-16, through high-throughput sequencing

<正>Dear Editor,Bacteriophages predominate in the biosphere and outnumber their hosts by at least one order of magnitude(Srinivasiah et al.,2008).They have been used for over90 years as an alternative to antibiotics in Eastern Europe(Deresinski,2009).With the increasing emergence of antibiotic resistance,the therapeutic potential of bacteriophages is being reevaluated(Kutter et al.,2010).Until recently,phages possessing ds DNA genomes have been