Tests that can distinguish whether HIV-positive people are infected with a drug-resistant strain or a non-resistant strain allow patients to get the most effective treatment as quickly as possible. A team of Brown University researchers describes a new method that works faster and more sensitively in lab testing than the current standard technologies.

The main advance enabling that improved performance is that the system operates directly on the virus’ more readily available RNA rather than requiring extra, potentially error-prone steps to examine DNA derived from RNA. In a single tube, the system can first combine two engineered probes (ligation) if a mutation is present and then make many copies of those combined probes (amplification) for detection.

“LRA (ligation on RNA amplification) uniquely optimizes two enzymatic reactions — RNA-based ligation, and quantitative PCR (polymerase chain reaction) amplification — into a single system,” said Anubhav Tripathi, professor of engineering at Brown and corresponding author on the paper. “Each HIV contains about 10,000 nucleotides, or building blocks, in its genetic material, and a drop of blood from a patient with resistant HIV can contain thousands to millions of copies of HIV. To find that one virus, out of thousands to millions, which is mutated at just a single nucleotide is like finding a needle in a haystack.”

The experiments reported in the paper show that the LRA test was sensitive enough to find a commonly sought K103N mutation in concentrations as low as one mutant per 10,000 strands of “normal” viral RNA. The LRA detection worked within two hours, while alternative technologies such as ASPCR or pyrosequencing, can take as long as eight.

LRA works by sending in many copies of a pair of short engineered probes of genetic material to complement the RNA in the HIV sample. Under optimized conditions, those pairs that perfectly match the target HIV RNA containing a mutation that causes drug resistance can rapidly become fused together, or ligated, by an enzyme. If there is a single nucleotide difference, the pair won’t fuse.

The fusing of the engineered genetic probes is designed to happen at room temperature. After a short period, the LRA system then heats the slightly alkaline solution, which shuts off the fusing reaction but turns on the amplification (copying) of fused pairs. That allows the LRA system to produce a strong signal of fused pairs, if there are any. All this happens in a single step, without any need to change solution. Brown University

Combining the investigative tools of genetics, transcriptomics, epigenetics and metabolomics, a Duke Medicine research team has identified a new molecular pathway involved in heart attacks and death from heart disease.

The researchers found that stress on a component of cells called the endoplasmic reticulum (ER) is associated with risk of future heart events, and it can be detected in bits of molecular detritus circulating in the blood.

“ER stress has long been linked to Type 1 diabetes and Parkinson’s disease, among others, but this is the first indication that it is also playing a role in common heart attacks and death from heart disease,” said senior author Svati H. Shah, M.D., associate professor of medicine and faculty at the Molecular Physiology Institute at Duke. “It’s also exciting that we are able to measure this ER stress in a small drop of blood, providing a potential way to intercede and lower the risk of a major cardiovascular event.”

Even after mapping the human genome and finding genetic traits associated with cardiovascular disease, the mechanisms underlying the inherited susceptibility to this disease have not been fully understood. Shah said the Duke team’s research approach — using a variety of analytical methods measuring over a million data points in 3,700 patients — enabled them to fill in some of the missing steps leading to cardiovascular disease, which is often inherited.

“With genetics, everyone is lumped together if they share a trait,” Shah said. “But everyone knows if you have two people with the same trait, but one is overweight, smokes and has a bad lifestyle, that person has a different pathway that led to heart disease than someone who is normal weight, doesn’t smoke, eats right and exercises.”

The Duke team focused on the intermediates between the genes and the disease pathway. This involved metabolomics — an analysis of the metabolites, or trace chemicals, left behind as the by-products from cellular processes.

Among a group of 3,700 patients referred for cardiac catheterization in the CATHGEN study, Shah and colleagues performed a genome-wide analysis of specific metabolite levels that had previously been identified as predictors of cardiovascular disease.

In their earlier work, the researchers had flagged these metabolites as markers for cardiovascular disease, but had not known how they were generated or what the underlying biological pathways were. The current study resolved that question, finding that these genes were directly linked to ER stress, which occurs when the endoplasmic reticulum organelle becomes overworked in its job managing excess and damaged cellular proteins.

Shah and colleagues then took an epigenetics and transcriptomics approach to determine what the differences were between patients with high or low levels of metabolites. Once again, the ER stress pathway came up as a key component.

“Using this multi-platform ‘omics’ approach, we identified these novel genetic variants associated with metabolite levels and with cardiovascular disease itself,” Shah said. “We don’t believe that the metabolites themselves are causing heart attacks — they might just be by-products of a dysregulated process that people are genetically susceptible to — but that’s something we need to study further.” Duke Medicine

Randox Quality Control announces the launch of 8 new RIQAS EQA Programmes, with cycles scheduled to begin in March 2016. The new programmes are: CSF, Sweat Testing, Immunosuppressants, Trace Elements in Serum, Trace Elements in Urine, Trace Elements in Blood, Anti-TSH Receptor, Cyfra 21-1. These new RIQAS programmes will provide clinical laboratories with the ability to review calibration issues, systematic errors and monitor accuracy and bias. Furthermore these laboratories will be able to assess their analytical performance in comparison with other laboratories which are employing the same instrument or methods. The new programmes are available in liquid and lyophilized formats, covering the full clinical decision range. Monthly reporting supports the rapid identification of errors and allows implementation of the necessary corrective actions therefore saving the need for expensive and time consuming patient sample retests.  Finally the rapid report turnaround will ensure results are received within 24-72 hours and, if required, corrective actions can then be implemented before the next cycle. RIQAS is the largest international EQA scheme used by more than 32,000 laboratory participants in 123 countries. Such large, international peer groups guarantee the statistical validity of the company’s extensive database of instrument and method results.

www.randoxqc.com

The Labquality Days Congress will be held at the Messukeskus Expo and Convention Centre in Helsinki on 11th-12th of February 2016. Labquality Days is one of the largest annual congresses in Scandinavia focused on quality and laboratory medicine. The congress inspires clinical chemistry, laboratory medicine professionals, researchers, healthcare experts, users of point-of-care devices, medical staff working with quality issues, managers and higher level personnel administration of social- and or healthcare sectors. The 2016 congress themes are now announced: Point-of-Care Testing (POCT) and preanalytics. POCT has already a major role in healthcare workflow. Test sensitivity or specificity, price, speed and patient convenience are some heavily discussed topics in scientific meetings. In preanalytics, various disciplines such as microbiology, clinical chemistry and hematology have their own characteristic variables. Individual analyses have some unique factors that should also be taken into account in order to obtain reliable results. Labquality Days will bring together leading international speakers and opinion leaders. The programme consists of scientific lectures and panel discussions. During the congress participants have the opportunity to meet colleagues, share ideas and experience the vast clinical laboratory exhibition.

www.labqualitydays.com

Alex Travis, associate professor at the Baker Institute for Animal Health, co-authored the study that led to the development of a new stroke diagnosis tool.
Minutes count when treating stroke, but current diagnostics take as long as three hours, careful lab work and skilled technicians to arrive at a conclusive diagnosis. Scientists at Cornell’s Baker Institute for Animal Health have developed a device that helps diagnose stroke in less than 10 minutes using a drop of blood barely big enough to moisten your fingertip.

Having demonstrated proof of principle, the technology eventually could be expanded and used in point-of-care testing devices to diagnose other conditions in humans and animals, including traumatic brain injury (concussion), some forms of dementia, and even some types of cancer and heart disease.

The study’s lead author, Roy Cohen, a research scientist at the Baker Institute, says the technology represents the successful pairing of two big goals in medical diagnostics – small size and simplicity, a combination that means testing could be carried out at a patient’s bedside.

“Three-quarters of stroke patients suffer from ischemic stroke – a blockage of a blood vessel in the brain. In those cases, time is of the essence, because there is a good drug available, but for a successful outcome it has to be given within three or four hours after the onset of symptoms,” says Cohen. “By the time someone identifies the symptoms, gets to the hospital and sits in the emergency room, you don’t have much time to obtain the full benefit of this drug.” Enhancing the speed of diagnosis could save many people from suffering lasting effects of ischemic stroke, he says.

To diagnose stroke, a condition in which blood flow to an area of the brain is limited or cut off, the technology will one day detect several bloodborne biomarkers, molecules that appear in the blood when the stroke occurs. The technology uses enzymes attached to nanoparticles to detect the biomarker molecules and convert that detection into light.

To demonstrate the effectiveness of this new approach, the researchers focused on the biomarker neuron-specific enolase (NSE), a substance found in higher concentrations in the blood of victims of stroke and other conditions. By measuring the amount of light produced from various samples, Cohen and his colleagues can determine the concentration of NSE in the sample. At each step of the way, the signal from the NSE is amplified, so even minute quantities give off enough light for detection.

The idea to tether the enzymes, says co-author Alex Travis, associate professor of reproductive biology at the Baker Institute for Animal Health, came from the hardworking enzymes tethered to the shafts of sperm tails. These sperm enzymes efficiently turn sugars into energy that powers the flagellum and moves the sperm along. The fact that they’re attached to the sperm tail instead of floating around in solution enables the enzymes to efficiently pass the substrate along from point to point and get the most “bang for the buck” from a sugar molecule, according to Travis.

Going forward, Travis and his team will collaborate with a private company to develop the stroke-detecting technique for clinical testing and eventually make it available for use in hospitals. But he’s also excited to expand the system to diagnose other conditions.

“This system could be tailored to detect multiple biomarkers,” says Travis. “That’s the strength of the technique. You could assemble a microfluidic card based on this technology that could detect 10 biomarkers in different wells, and the readout would be the same for each one: light.” Using the same detection system for multiple different biomarkers would make for a simple system in a relatively small package, he says. Cornell University