Microbiology’s shifting role in war on sepsis

With the launch of its MCA instrument, Geisinger performs rapid susceptibility testing for Gram-positive and Gram-negative infections found in pediatric patients and neonates who are at risk for antimicrobial toxicity. “We strive to provide results upon which antibiotics can be rapidly adjusted to limit toxicity, by selecting the best antibiotic based on rapid susceptibility test results,” she says.

She hopes an integrated team approach, supported with data and metrics, “will provide a positive impact in pediatric patients, as it did in adults. If improvements are not documented, we will re-examine our approach.”

Dr. Wolk’s laboratory is performing some NGS testing, more from the adult population than pediatric, and more on tissues and normally sterile sites than from others. “It’s not yet routine for direct specimens, and we have not yet deployed it for blood culture positive samples. It’s used in specialized cases in which the source of the infection cannot be found by routine culture or PCR methods.” The Geisinger algorithm for NGS includes a doctoral microbiology consult along with support by infectious disease physicians. To conserve resources, “We always consider the Gram stain and the stains from pathology tissue blocks to make sure we’re not paying for a sequence analysis on a sample that would yield results in our normal course of events.” In some cases, acridine orange stain is deployed in an attempt to identify microbes that are not evident on the Gram stain. “Using this approach, we’ve identified Legionella spp. and Mycoplasma spp. in cases of endocarditis.”

Her laboratory has automated molecular testing and specimen inoculation and is planning to deploy total automation when funding is confirmed. For the handling of sepsis specimens, automation doesn’t necessarily have an impact on specimen inoculation of the blood culture, “but it does allow the laboratory to aliquot and store positive blood culture broths efficiently and greatly decreases our footprint for storage space for positive broths.”

Dr. Wolk makes a case for including pediatric samples in upcoming clinical trials. “From my perspective, it’s troublesome that, in some cases, the pediatric blood culture bottles were not included in the original FDA clinical trials for rapid molecular methods, thus requiring individual laboratories to fund the costs of verification. I urge commercial suppliers to consider including pediatric blood culture bottles for rapid diagnostic testing in the pediatric population, so that benefit can be documented.” Dr. Wolk is hopeful that the direct-from-whole-blood testing will prove to be beneficial in pediatric patients.

Sepsis in the totally automated lab

Total laboratory automation is a central feature of NorthShore University HealthSystem’s approach to sepsis, says Erin McElvania, PhD, D(ABMM), director of microbiology. NorthShore, located in Chicago’s northern suburbs, performs about 300,000 microbiology-specific tests per year in its main laboratory.

“Our general protocol is to have our physicians submit blood cultures, then we incubate them in our BD Bactec automated blood culture system until they signal positive, and then we use our Kiestra total laboratory automation to process specimens for culture.” In addition, the Verigene assay is used for Gram-negative and Gram-positive organisms. “That gives us rapid identification of the most common agents of sepsis and several resistance determinants as well.”

“Because of the smart incubators in our TLA, which are more of a closed system, the agar plates stay at a constant temperature and we see bacterial growth much sooner. We check the plates by imaging them often, and if there is any bacterial growth, we are able to run MALDI-TOF mass spectrometry on those isolates and get a rapid identification when compared to growth of isolates in traditional incubators and biochemical testing.”

Optimizing the Kiestra TLA to work better for NorthShore patients is an ongoing project, she says. “We initially did studies to figure out the earliest time point at which we should look at the positive blood cultures we plated, and we settled on four hours. So now that is the first time they are imaged and viewed. Oftentimes that’s a little early, but then we follow up with additional imaging every hour or two to see if we can detect growth.

“Since it’s automated,” she continues, “we can automatically image the plate at many time points so no one has to take anything out of the incubator. The plates are out of the incubator only for about 20 seconds. So with Kiestra TLA we are able to check them frequently without slowing their growth.” In fact, Dr. McElvania says she is pleasantly surprised at how fast organisms grow in the Kiestra, contributing to an efficient workflow.

A rapid resistance screen the laboratory has developed and is testing now will be used in addition to traditional susceptibility testing, Dr. McElvania says. “We are able to use our Kiestra TLA with its smart incubators and a heavier inoculum to achieve rapid bacterial growth. By imaging the plates every hour, we are able to determine the earliest time points in which you can reliably read the zone diameters for our modified disk diffusion assay.” The cost is reasonable: Even including the agar plates and disks, it amounts to about $1 or $2 per culture.

Verigene is fast, she says, but there are a lot of drugs for which the resistance cannot be identified by a molecular target. “The Verigene gives the microbiology lab a few resistance determinants where antibiotic decisions may be made. But it’s not a full antimicrobial susceptibility panel, so that’s an area where we’re trying to help physicians and get them information to optimize their antibiotics earlier. For additional information, we plan to use our rapid phenotypic resistance screening assay, and we are in the process of validating it for a number of antibiotics.”

NorthShore’s sepsis population tends to include many older patients from nursing homes or long-term care facilities, often with many comorbidities, along with a smaller pediatric and NICU population. “We generally encounter the routine sepsis-causing organisms, and we have a low rate of antimicrobial resistance compared to other academic centers that have more transplant patients.”

Dr. McElvania has considered the Accelerate Pheno as a way to improve ID and susceptibility testing. “I think it has a lot of promise, but it’s also incredibly expensive and takes up a lot of space. Each instrument can only run one sample at a time, and we would need many of them based on our blood culture volume.”

The laboratory is in contact throughout the day with the clinicians in the infectious disease group and with the infectious disease pharmacists, she says. “Usually, if something isn’t identified by our Verigene, they will call and have us review the Gram stain and see if we have any clues, or they frequently come to the lab to look at Gram stains themselves and discuss patient cultures, so we have a very good relationship with them.”

There is a lot of concern about culture-negative sepsis, of course, and Dr. McElvania says many new culture-independent methods are coming up that could increase sensitivity of pathogen detection. “But they’re not without their own limitations.”

One of her biggest concerns about culture-independent methods is the lack of susceptibility testing on organisms if only molecular methods are used for identification. “They don’t have the ability to do susceptibility testing,” she says.

NGS instrumentation is available in the molecular laboratory, which is adjacent to microbiology. “They’re mainly using it for cancer diagnostics so far, and they are just starting to use it to look for pathogens directly from patient specimens that did not grow in routine bacterial culture.”

Optimizing pathogen recovery through traditional methods and then using molecular testing for culture-independent pathogen detection is a promising strategy, in Dr. McElvania’s view. “I’m most excited and interested in those two things. New technologies, new assays are coming on the market that will help us with these quandaries. But for now, collecting optimal blood volume for culture before antibiotics are initiated is still the gold standard.”

Sepsis diagnosis from whole blood

A notable recent advance was the FDA’s approval, in May, of T2 Biosystems’ bacteria panel. Lee Health in Florida, an early installer of the T2, tests for Candida only (the panel was approved in 2014); it has been conducting those tests successfully for more than a year, says Fran Cioffi, MT(ASCP), microbiology supervisor. “There is a T2 instrument that can do both Candida and bacteria, and I’m hopeful we will be moving to that.”

Use of the T2 is restricted at Lee Health so that only ID physicians or intensivists can order it for patients in the ICU, those on multiple antibiotics, or cancer and other immunosuppressed patients. “Those are the ones who are going to be subject to having Candida in their blood,” Cioffi notes. “Lee Health system placed the T2 in its trauma hospital because its lab had space and the other hospitals didn’t. But if we continue taking the technology, we will have all of our instruments on all campuses. That would be our goal.”

The T2 boasts distinct advantages over traditional blood culture, Cioffi points out. “With T2, you need one to three colony-forming units, which means you can draw right from a blood specimen tube, and in two-and-a-half to three hours you can see whether it’s positive for Candida. Yeast grows slowly and on a blood culture you’d need 60 colony-forming units. So we’re able, with the T2 test, to take patients off their antifungal drugs, which are toxic and expensive, or put them on. And with candidiasis in patients, the mortality rate is high. So this has been very successful for us.”

Expense—about $130 per test—can be a stumbling block, she says. “You cannot run the test on every patient. Any test we bring in that’s molecular, you have to have control over the ordering. You can’t put the tests out there for hospitalists or anybody to order it. Right now, providers will order blood cultures if the patients have an elevated fever. You can’t do T2 on those. We have test utilization curbs in place with Candida and will do the same with bacteria.”

On the flip side, some bacteria would be missed if only a blood culture were done. Organisms like Acinetobacter would cause a change in empiric therapy, Cioffi says. “That’s an organism you don’t get very often.” One patient in a study conducted at her hospital was positive for Acinetobacter with the T2 but would have been treated only for S. aureus with a regular blood culture. “Normal protocol in the lab is that once a blood culture dings positive, you take it out, you subculture it, you know you have the organism, and you treat for it.”

“But with sepsis, it used to be that single blood cultures were always single organisms. And that’s long gone. Now, with IV drug users, you might get two or three organisms that come up in their blood culture. We’ve seen that increase over the last few years.” In another case, an E. coli was never recovered. “Sometimes when a patient is on antibiotics, you have a slower amount of growth with a lower colony count. With blood cultures, because you need a higher colony count, you might not pick that up, but you’re going to pick it up with the T2.” Still, that the T2 detects only 10 organisms means it is a long way from replacing culture, she says.

Control of contamination is an area in which her lab is making strides. “We run about 6,500 blood cultures a month and our rate of positives is about five to 10 percent. But the CAP standards require that your contamination rate be below three percent, and our rate in the ED, where the nurses were drawing, was getting high.”

Installing Magnolia Medical Technologies’ Steripath helped reduce contamination, she says. “The technology was discovered by a pathologist who noticed, when he was analyzing bone marrow, there was an epidural plug. He concluded that the same thing probably happens when we do blood cultures. With Steripath, the first 1 or 2 mL of blood when cultures are being drawn goes into a trap and then the rest of the blood goes into the bottle.” Use of Steripath has brought the contamination rates for the ED down to the CAP-recommended level, Cioffi reports.

The challenge of increasing resistance

Dr. McElvania, Dr. Wolk, and Dr. Graf agree that for purposes of treating sepsis, the worldwide concern about the emergence of resistant pathogens is warranted.

“The clinicians worry about the increasing pool of bugs that are resistant to frontline agents, and now those are just going to become more and more prevalent,” Dr. Graf says. “We are kind of lucky in the pediatric hospital that we don’t see carbapenem-resistant Enterobacteriaceae that often. But that’s likely going to change. The adult hospitals see them a lot more, and it’s just a matter of time before they become more common in our community. With a lack of new antibiotics coming down the pipeline, the array of options we will have for treatment of multidrug-resistant bugs like Candida auris and carbapenem-resistant Enterobacteriaceae is very narrow.”

Pair those trends with an increasing population of susceptible hosts and the problem is compounded. “We’re all treating kids more and more these days,” she says, “and so our population of immunocompromised hosts is just continuing to grow, and they are much more at risk for these kinds of infections.”

Anne Paxton is a writer and attorney in Seattle.