Pulse-Controlled Amplification-A new powerful tool for on-site diagnostics under resource limited conditions.

BACKGROUND: Molecular diagnostics has become essential in the identification of many infectious and neglected diseases, and the detection of nucleic acids often serves as the gold standard technique for most infectious agents. However, established techniques like polymerase chain reaction (PCR) are time-consuming laboratory-bound techniques while rapid tests such as Lateral Flow Immunochromatographic tests often lack the required sensitivity and/or specificity. METHODS/PRINCIPLE FINDINGS: Here we present an affordable, highly mobile alternative method for the rapid identification of infectious agents using pulse-controlled amplification (PCA). PCA is a next generation nucleic acid amplification technology that uses rapid energy pulses to heat microcyclers (micro-scale metal heating elements embedded directly in the amplification reaction) for a few microseconds, thus only heating a small fraction of the reaction volume. The heated microcyclers cool off nearly instantaneously, resulting in ultra-fast heating and cooling cycles during which classic amplification of a target sequence takes place. This reduces the overall amplification time by a factor of up to 10, enabling a sample-to-result workflow in just 15 minutes, while running on a small and portable prototype device. In this proof of principle study, we designed a PCA-assay for the detection of Yersinia pestis to demonstrate the efficacy of this technology. The observed detection limits were 434 copies per reaction (purified DNA) and 35 cells per reaction (crude sample) respectively of Yersinia pestis. CONCLUSIONS/SIGNIFICANCE: PCA offers fast and decentralized molecular diagnostics and is applicable whenever rapid, on-site detection of infectious agents is needed, even under resource limited conditions. It combines the sensitivity and specificity of PCR with the rapidness and simplicity of hitherto existing rapid tests.

MinION as part of a biomedical rapidly deployable laboratory.

Fast turnaround times are of utmost importance for biomedical reconnaissance, particularly regarding dangerous pathogens. Recent advances in sequencing technology and its devices allow sequencing within a short time frame outside stationary laboratories close to the epicenter of the outbreak. In our study, we evaluated the portable sequencing device MinION as part of a rapidly deployable laboratory specialized in identification of highly pathogenic agents. We tested the device in the course of a NATO live agent exercise in a deployable field laboratory in hot climate conditions. The samples were obtained from bio-terroristic scenarios that formed part of the exercise and contained unknown bacterial agents. To simulate conditions of a resource-limited remote deployment site, we operated the sequencer without internet access. Using a metagenomic approach, we were able to identify the causative agent in the analyzed samples. Furthermore, depending on the obtained data, we were able to perform molecular typing down to strain level. In our study we challenged the device and discuss advances as well as remaining limitations for sequencing biological samples outside of stationary laboratories. Nevertheless, massive parallel sequencing as a non-selective methodology yields important information and is able to support outbreak investigation - even in the field.

Planning for exercises of chemical, biological, radiological, and nuclear (CBRN) forensic capabilities.

A forensic capability to help identify perpetrators and exclude innocent people should be an integral part of a strategy against terrorist attacks. Exercises have been conducted to increase our preparedness and response capabilities to chemical, biological, radiological, and nuclear (CBRN) terrorist attacks. However, incorporating forensic components into these exercises has been deficient. CBRN investigations rely on forensic results, so the need to integrate a forensic component and forensics experts into comprehensive exercises is paramount. This article provides guidance for planning and executing exercises at local, state, federal, and international levels that test the effectiveness of forensic capabilities for CBRN threats. The guidelines presented here apply both to situations where forensics is only a component of a more general exercise and where forensics is the primary focus of the exercise.

Nonparallel secretion of antibacterial activity and protein in porcine pancreatic juice.

The antimicrobial activity of exocrine pancreatic juice is an important component of gastrointestinal tract innate defenses, yet little is known about whether secretion is regulated in parallel with digestive enzymes. In this study, we used 8 pigs with pancreatic catheters to quantify antibacterial activity and measure protein content (indicator of enzyme secretion) of pancreatic juice collected hourly from 0700 to 1900, with the animals adapted to being fed at 0800 and 1600. Antibacterial activity in the samples of pancreatic juice was quantified by comparing the growth inhibition of Staphylococcus aureus subsp. aureus strain ATCC 6538P relative to a known concentration of gentamicin. Antibacterial activity (U/mL and /min) was highest in samples collected 1 hour prior to feeding (equivalent to 0.6 microgram gentamicin/mL), declined as the meal was consumed, and was lower (P < 0.05) in samples collected while the meals were being digested (0.41 microgram gentamicin/mL). Protein content was negatively correlated with antibacterial activity, with protein secretion lower (mg/mL and /min) before feeding, with an increase as the pigs ate and digested the meals (P < 0.05). The results indicate that the antibacterial activity in pancreatic juice is not secreted in parallel with protein secretion, suggesting that regulation involves alternative signaling pathways or contrasting responses to shared signals.

The enzyme levels in blood are not affected by oral administration of a pancreatic enzyme preparation (Creon 10,000) in pancreas-insufficient pigs.

After oral intake, small amounts of intact protein may be absorbed into the blood circulation. The current study investigated whether orally administered pancreatic enzymes were absorbed from the intestine. The study included 28 pigs; 3 control pigs with intact pancreatic function and 25 pigs that were made exocrine pancreas insufficient by duct ligation (20 pigs) or total pancreatectomy (5 pigs). The pigs received a pancreatic enzyme preparation (0, 2, 4, or 8 g of Creon 10,000) together with the feed. The blood plasma was analyzed for pancreatic lipase activity with a [3H]-triolein substrate assay, while (pro)colipase and cationic trypsin(ogen) levels were measured with enzyme-linked immunosorbent assay (ELISA). Administration of Creon (0-8 g) caused no significant changes in plasma (pro)colipase or cationic trypsin(ogen) levels. Lipase activity peaks in plasma samples were found, but they did not correspond to the administration of Creon. The potential source of these plasma lipase activity peaks is discussed. The results showed no absorption into blood of pancreatic enzymes after oral administration (0, 2, 4, or 8 g of Creon mixed with 100 g of feed) to pancreas-insufficient pigs.

A kinetic microplate method for quantifying the antibacterial properties of biological fluids.

The microplate assay for measuring antibacterial activity was adapted by incorporating a known concentration range of gentamicin as an internal standard. Staphylococcus aureus subsp. aureus strain ATCC 6538P, Escherichia coli ATCC 25922, and Lactobacillus spp. were used as target organisms, although other indicator organisms and antibiotics can be examined. Assay range and sensitivity were dependent on the species and density of indicator organism, and conditions (e.g., type, concentration, and pH of growth medium). Plotting the area under the growth curve (AUGC) versus gentamicin concentration (log transformed) yielded a linear curve that was used to quantify in units of gentamicin the antibacterial activity of a secretory fluid (SCF; pancreatic juice) and for comparisons of samples collected at different times, analysed on different days, and from different studies. This adaptation of the microtiter broth method will be useful for investigating man-made compounds, and the antibacterial activity of secretory fluids and the influences of age, diet, and health status.