New method to diagnose sepsis is faster, cheaper

A new method could cut hours off the time it takes to diagnose blood infections while also eliminating the need for complicated manual processing and expensive equipment, according to a report to be published in mBio®, the online open-access journal of the American Society for Microbiology, on November 19. The method combines a selective lysis step in which blood cells in the sample are destroyed, a centrifugation step to collect any bacteria or fungi in the sample, and a fluorescence step that analyzes the particular fingerprint of any pathogens present in the sample. Tests show the method correctly identifies the species of bacteria or fungi in 96.5 % of positive blood culture samples, crucial information for doctors to provide the appropriate drugs for their patients.

"The primary benefit of getting a rapid identification is making sure the patient is on the right [antibiotic] therapy and to quickly make any needed adjustments to the initial therapy," says co-author John Walsh of bioMérieux, Inc. in Durham, North Carolina. Patients with bloodstream infections are usually in very serious condition, says Walsh, and faster identification of the organism causing the infection can help get patients on the most effective antibiotics faster and save lives. Proper diagnosis is also important from the perspective of antibiotic stewardship: using more appropriate, targeted antibiotics reduces the risk of contributing to the spread of resistance to broad-spectrum antibiotics.

Walsh says the current standard approach to diagnosing bloodstream infections, Gram staining and overnight sub-culture followed by phenotypic ID tests, have limitations that can prevent timely treatment. Gram staining provides early, low level information about the type of microorganism present, but it sometimes takes hours to deliver a result, and technicians can make mistakes in the process that provide misleading results. Other more specific identification methods are also available for diagnosis, but they can take at least a day or two to produce results and many require expensive equipment.

In the technique developed by Walsh et al., a sample of positive blood culture is treated with lysis buffer to "pop" the blood cells, then transferred to a specialized optical tube. The tube is centrifuged, which drives bacteria or fungi, which are denser than the solution, down through a liquid density cushion to form a pellet at the bottom of the tube.

Then comes the intrinsic fluorescence spectroscopy (IFS): the microbial pellet is irradiated with light ranging from the deep ultraviolet to infrared, which excites certain organic molecules in the microorganisms, including tryptophan, NADH, FAD, porphyrins, and others, and causes them to fluoresce in a characteristic way depending on the identity of the microbe. The exact pattern of fluorescence is compared with a database of 37 of the most common known pathogens to identify the organism present.

Sepsis (sometimes referred to as 'blood poisoning') is a life-threatening condition that arises when the body's inflammatory response to a bacterial infection injures its own tissues and organs. Costing the NHS over £2billion annually, the condition kills more people than breast and bowel cancer combined (approximately 37,000 a year). Rapid diagnosis and treatment with antibiotics saves lives, but as there are currently no biomarkers in clinical use to enable fast diagnosis, it can take up to two days to analyse samples in the laboratory.