Healthcare-associated infections (HAIs) are a leading cause of morbidity and mortality in the United States, with the latest figures estimating that one in 25 patients suffer from at least one HAI at any given moment.1 The sources of such infections are typically at sites of surgery or where indwelling medical devices have been used—for example, catheters, central lines, and ventilators. The young, the elderly, and patients with compromised immune systems are at greatest risk of developing these infections.
Numerous government agencies, such as the Agency for Healthcare Research and Quality (AHRQ), which is part of the U.S. Department of Health and Human Services (HHS), have developed programs to increase awareness among healthcare specialists in order to implement effective protocols and treatment regimens to decrease the occurrence of HAIs. In 2009, an initial Action Plan was created to tackle the problem of HAIs, and many healthcare facilities developed and implemented strategies to achieve their elimination. By 2014, reductions in central line-associated bloodstream infections and surgical site infections had been reported.2
Despite that progress, multidrug-resistant organisms (MDROs) such as methicillin-resistant staphylococcus aureus (MRSA) and Clostridium difficile have hampered efforts to prevent nosocomial infections. The proliferation of HAIs has been exacerbated by improper use or overuse of antimicrobials, including antibiotics. In response, the Obama administration published the National Action Plan for Combating Antibiotic-Resistant Bacteria in 2015, setting out five key goals in the U.S. strategy, and heads of state met at a high-level meeting of the United Nations General Assembly in September 2016 to commit to a coordinated aggressive approach on a global scale.
Antimicrobial resistance and stewardship programs
In response to this public health threat, the Centers for Disease Control and Prevention (CDC) published a list in 2013 of the top drug-resistant threats in the U.S., ranking the microorganisms with a hazard level of urgent, serious, or concerning. Urgent denotes high-consequence threats that require immediate attention even though they may not be currently widespread. Serious threats are significant ones that without monitoring and prevention efforts may become urgent. Concerning threats can currently be treated by antimicrobials, but cause severe illness, and therefore outbreaks require a rapid response to limit transmission.
The majority of MDROs on the list are responsible for HAIs, including two out of three in the urgent category—Clostridium difficile, which causes 15,000 deaths a year, and carbapenem-resistant Enterobacteriaceae (CRE), which are resistant to all or nearly all available antibiotics.3 At the serious threat level, organisms such as multidrug-resistant Acinetobacter spp., fluconazole-resistant Candida spp., extended-spectrum β-lactamase (ESBL) Enterobacteriaceae, vancomycin-resistant enterococci (VRE), and MRSA are responsible for more than 15,000 combined deaths each year.
Healthcare facilities have developed antimicrobial stewardship programs that have been shown to be effective at reducing patient morbidity and mortality caused by HAIs.4-5 As an example of the work conducted at an agency level, the CDC has produced a CRE prevention toolkit to assist healthcare facilities in tackling this spreading threat.6 In addition, as part of its Emerging Infections Program and with the National Healthcare Safety Network (NHSN), there is now a monitoring service to track the extent of CRE infections. As of February 2016, all but two states (Idaho and Maine) had reported their incidence rates of Klebsiella pneumoniae carbapenemase (KPC) to the CDC, and by April 2016, 21 states had additionally reported the prevalence of the β-lactamase OXA-48.
Rapid diagnostics and antibacterial stewardship
The accurate identification and antimicrobial susceptibility testing of pathogens is critical for an appropriate and effective clinical response to infection. Rapid diagnostic tests, along with the emergence of molecular testing, have much to offer the microbiology lab, with isolate identification possible in a matter of hours. This swiftness in turn reduces the time it takes to switch from empiric to targeted therapy, improving patient outcomes and minimizing the overuse of ineffective antibiotics and the chance for resistance to develop.
Current rapid test methods for pathogen identification include polymerase chain reaction (PCR) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). PCR can routinely detect Gram-positive, Gram-negative, and yeast species as well as resistance markers, including carbapenem-resistance markers such as KPC, VIM, IMP, and OXA genes, mecA for MRSA, and van A/B for VRE detection. Results are provided in a couple of hours, but samples can be prone to contamination. MALDI-TOF MS is a well-established analytical chemistry technique, currently gaining widespread popularity in microbiology labs. The technique still requires overnight culturing of isolates, but results can be achieved within minutes of starting a run. In practice, samples tend to be batched for efficiency owing to staff limitations, and therefore the full advantages of rapid results may not be realized by clinicians.7
Next-generation rapid diagnostic tests
The five goals of the National Action Plan for Combating Antibiotic-Resistant Bacteria cover areas such as preventing and controlling the spread of infections, improving international collaboration, and accelerating new antibiotic development. Goal 3 is devoted to progressing the development, validation, and utilization of innovative rapid diagnostic tests by 2020. In conjunction with the HHS, funding and prizes to incentivize work on this goal have been created.
The next-generation of rapid diagnostics, featuring faster identification and antibacterial susceptibility testing (AST), will soon be commercially available in the U.S. These include the combination of PCR with electrospray ionization mass spectrometry (ESI-MS), miniaturized magnetic resonance for rapid identification and resistance marker detection, and automated microscopy utilizing fluorescent in situ hybridization (FISH) for identification and morphokinetic cellular analysis (MCA) for AST. One system can identify more than 1,000 bacteria, fungi, and viruses along with four resistance markers directly from patient specimens, with results produced within six hours. The full sample workup, including sample preparation, can be completed on a series of five instruments. This technology is currently CE marked but not commercially available in the U.S.
Another platform is a highly sensitive system that uses superparamagnetic labels to selectively bind to the target species, altering the magnetic properties of the sample. Any type of patient sample can be tested with this system. The change in the magnetic properties is detected, and a positive identification from whole blood samples can be made in three to five hours. A whole blood test panel for five Candida species has received FDA clearance, and clinical trials for a whole blood bacterial panel are planned to support an FDA submission by mid-2017.
Although rapid identification and genotypic resistance marker tests are useful, they provide only part of the information needed clinically—for example, information about which antimicrobials will not work for targeted patient therapy—rather than indicating which antimicrobials will. According to the Deputy Director of the CDC’s Office of Antimicrobial Resistance, Dr. Jean Patel, the way forward is phenotypic testing, which will provide the whole susceptibility picture for clinicians. Her hope is that the next generation of these tests will produce results on a time-scale similar to those seen for present molecular tests.8 One such rapid phenotypic susceptibility testing device is poised to hit the U.S. market. It uses MCA-based time-lapse imaging of bacterial cells in the presence of antibiotics to provide AST results within about seven hours, and its fully-automated integrated platform also provides species identification within about 90 minutes using multiplexed FISH assays. The current CE marked blood culture panel tests 17 bacterial and yeast targets, 23 antibiotics, and two resistance phenotype tests. Clinical trials for a blood culture panel have concluded in the U.S. and the product is now awaiting FDA clearance.
Future rapid AST techniques currently under development include the use of light-scattering to monitor the growth of bacteria in broth and engineered bioparticles to produce light when resistant bacteria are exposed to antibiotics. While these technologies demonstrate significant advancements and innovation in the fields of pathogen detection and antimicrobial susceptibility, they have yet to demonstrate their effectiveness in clinical trials for use in the U.S.
In order to be most effective against HAIs, new technologies will need to offer the ability to test not only blood but respiratory, urine, and wound samples as well. Also, a systematic development strategy will be necessary in order to anticipate and prepare for emerging resistance mechanisms. Although current antibiotic development has stagnated, the realization of this impending resistance crisis has inspired new interest, and rapid susceptibility devices will be needed to respond to it.
In addition, the advantages of using rapid diagnostic tests in conjunction with an effective antimicrobial stewardship plan should not be underestimated. Any time benefits afforded by new technologies will be wasted if slow or ineffective results reporting leads to delays in changing from empiric to targeted therapy. When assessing techniques, the need for batching of samples for the convenience of staffing shifts or reduction of cost-per-test must be weighed, as these, too, mitigate any benefits derived from the rapidity of new tests.9 The deficiency of information pertaining to instrument performance as new resistance threats emerge is also a concern.10 Clinical laboratories should not only seek analytical reproducibility when it comes to new instrumentation, but also rigorous, data-driven objective studies to provide evidence for the impact of these new systems on patient care.11
Despite these challenges, the development of rapid diagnostic tests is a major leap forward in the battle against MDROs and HAIs, alongside antimicrobial stewardship programs, improved hygiene practices, improved antibiotic prescribing, and better reporting and tracking of drug-resistant outbreaks. As researchers and industry rise to meet the challenge of developing and rigorously assessing the next generation of rapid diagnostic tests, patient care could become even more effective, with shorter recovery times, lower healthcare costs for both patients and healthcare facilities, and ultimately reduced numbers of deaths.
- Centers for Disease Control and Prevention. National and state healthcare-associated infections progress report. http://www.cdc.gov/hai/surveillance/progress-report/index.html. Published March 2016.
- Zimlichman E, Henderson D, Tamir O, et al. Health care-associated infections: a meta-analysis of costs and financial impact on the US health care system. JAMA Intern Med. 2013;173(22):2039-2046.
- Centers for Disease Control and Prevention. Biggest threats. http://www.cdc.gov/drugresistance/biggest_threats.html. Updated September 8, 2016.
Carling P, Fung T, Killion A, Terrin N, Barza M. Favorable impact of a multidisciplinary antibiotic management program conducted during 7 years. Infect Control Hosp Epidemiol. 2003;24(9):699-706.
- Bradley S. Antibiotic stewardship in hospitals and long-term care facilities: building an effective program. Pa Patient Saf Advis. 2015;12(2):71-78.
- Centers for Disease Control and Prevention. Facility guidance for control of carbapenem-resistant enterobacteriaceae (CRE) – November 2015 update CRE Toolkit. http://www.cdc.gov/hai/organisms/cre/cre-toolkit/index.html. Updated November 2015.
- Titus K. Labs enter a MALDI-TOF state of mind. http://www.captodayonline.com/labs-enter-maldi-tof-state-mind/. Published October 2016.
- Griswold A. Carbapenem resistance: advice from the frontline. http://www.captodayonline.com/carbapenem-resistance-advice-frontline/. August 2015.
- Buehler SS, Madison B, Snyder SR, et al. Effectiveness of practices to increase timeliness of providing targeted therapy for inpatients with bloodstream infections: a laboratory medicine best practices systematic review and meta-analysis. Clin Microbiol Rev. 2015; 29(1):59-103.
- Humphries R, Hindler JA. Emerging resistance, new antimicrobial agents…but no tests! The challenge of antimicrobial susceptibility testing in the current U.S. regulatory landscape. Clin Infect Dis. 2016;63(1):83-88.
- Doern GV. The value of outcomes data in the practice of clinical microbiology. J Clin Microbiol.2014;52(5):1314-1316.
Angharad Laetsch, PhD, serves as Medical Writer for Tucson, Arizona-based Accelerate Diagnostics, Inc. She holds a doctorate in chemistry from Imperial College London for her research into luminescent labels for use in miniaturized diagnostic devices.
Lessons from research offer new perspectives on sepsis
By MLO Staff
There probably was a time when it could fairly have been said that researchers and clinicians were not sufficiently engaged with the problem of sepsis—when sepsis was a somewhat under-recognized killer of patients in hospitals. But that time is long past. The severity of the problem is now fully appreciated, and efforts to combat it, both in the research lab and the clinical setting, are ongoing. Not long ago, two recent studies came across our desk, regarding the cellular events that precipitate sepsis and a possible new approach to treatment.
Confronted with sepsis, a key immune mechanism breaks
When the body encounters an infection, a molecular signaling system ramps up the body’s infection-fighting system to produce more white blood cells to attack invading bacteria. Now researchers have discovered that when the body is facing a massive bacterial infection resulting in sepsis, that same signaling system malfunctions, damaging the body’s ability to fight the invaders.
In addition to suppressing the mature blood cells battling against the infection, malfunctioning of this signaling system results in permanent damage to the body’s blood-producing cells—called hematopoietic stem cells—that are located in the bone marrow. The research, by scientists at the Indiana University School of Medicine, was published recently in the journal Stem Cell Reports.
“Our goal is to find out what causes this bone marrow failure during serious infections, and find ways to prevent it,” says Nadia Carlesso, MD, PhD, professor of pediatrics and of medical and molecular genetics at the IU School of Medicine.
Most research has focused on understanding and managing the late consequences of sepsis, while little is known about the changes occurring in the bone marrow at early stages of the response to bacterial infection, when the opportunity for effective treatment might still be available. Carlesso’s group has pioneered the study of bone marrow responses during acute infection. Using laboratory models of severe sepsis, they have discovered that the blood-producing stem cells fail to continuously generate mature neutrophils, which are the most critical bacteria-fighting cells.
“In this research we determined that in cases of severe infection and sepsis, a key mechanism in the body’s response to infection is broken. These findings point to potential new targets for protecting the immune system during major infections,” Carlesso says.
The researchers focused on a set of proteins called toll-like receptors, which function as sentinels on the surfaces of cells. When the receptors detect the presence of invading bacteria, they send signals to the body’s immune response system. The researchers looked at toll receptor 4 (TLR4), which activates two signaling pathways that stimulate the production of more neutrophils during common infections, but suppress it during severe infections.
In a laboratory model of sepsis using mice, the researchers found that two abnormal effects activated by toll receptor 4 during severe infection—the suppression of neutrophil production and the damage to the bone marrow’s blood-producing stem cells—are mediated by two different molecules downstream of TLR4.
“This study is a good start, as it provides a more precise map to follow, but more research is needed to better understand this process and develop better, and much needed, therapeutic strategies for sepsis,” says Carlesso.
Countering severe bacterial infections and sepsis
Bacterial infections that don’t respond to antibiotics are of rising concern, as is sepsis—the immune system’s last-ditch, failed attack on infection, which ends up being lethal itself. Reporting online in Nature, researchers at Boston Children’s Hospital describe new potential avenues for controlling both sepsis and the runaway bacterial infections that provoke it. Through meticulous experiments, scientists in Boston Children’s Program in Cellular and Molecular Medicine (PCMM) revealed the final cellular events necessary for both sepsis and stemming the bacterial attack.
Recent research has shown that at any sign of bacterial invasion, protein complexes called inflammasomes are activated. This activation triggers a process called pyroptosis: the infected cells explode open, releasing bacteria as well as chemical signals that sound an immune alarm. But there’s a balance: too strong an alarm can trigger sepsis, causing fatal blood-vessel and organ damage.
“The immune system is trying to control the infection, but if the bacteria win out, the immune response can kill the patient,” explains Judy Lieberman, MD, PhD, senior investigator on the study together with Hao Wu, PhD. “Most attempts to quiet the immune response haven’t worked in treating sepsis in the clinic, because the parts that trigger it haven’t been well understood.”
Once activated, inflammasomes activate enzymes called caspases that cut a molecule called gasdermin D in two. This cleavage unleashes gasdermin D’s active fragment, known as gasdermin-D-NT. But how this causes pyroptosis hasn’t been known.
Lieberman, Wu and their colleagues showed that gasdermin-D-NT packs a one-two punch. On the one hand, it perforates the membranes of the bacteria that are infecting cells and kills them. It also punches holes in the membrane of the host cell, causing pyroptosis—killing the cell and releasing bacteria and immune alarm signals. Nearby uninfected cells are left unscathed, the team found.
Second, the team discovered that gasdermin-D-NT directly kills bacteria outside of cells, including E. coli, S. aureus, and Listeria. In a dish, this happened quickly—within five minutes.
The results now need to be replicated in animal models of infection and sepsis, but Lieberman believes that understanding how gasdermin-D-NT works could be harnessed to help treat highly dangerous bacterial infections.
“Because of widespread antibiotic resistance, we have to think about other strategies,” Lieberman says. “Since the fragment kills bacteria but not uninfected host cells, one can imagine injecting the fragment directly, especially to treat a localized infection involving antibiotic-resistant bacteria.”
For sepsis, Lieberman speculates about ways of inhibiting or blocking gasdermin-D-NT, such as with antibodies or strategies targeting caspase enzymes.