New methods promise faster and more accurate detection of MDROs
By Elena V. Grigorenko, PhD, and Donald R. Stalons, PhD, D(ABMM), MPH
Antibiotics are marvels of modern medicine that have helped healthcare professionals fight infections caused by bacteria for the past 70 years. However, there is concern for the current use and future benefits of antibiotics. According to the World Health Organization (WHO), widespread inappropriate use of antibiotics has created global resistance that may soon drive every nation into a post-antibiotic era.1
Antibiotic resistance occurs when bacteria are no longer responsive to the antibiotics used to treat the infections they cause. The impact is startling when seen through statistics from the US Centers for Disease Control and Prevention (CDC). CDC reports that highly resistant bacterial infections cause an estimated 23,000 deaths and 2 million illnesses in the United States annually.2
The four main classes of broad-spectrum antibiotics—including carbapenems, extended spectrum beta-lactams, fluoroquinolones, and macrolides—have become less effective against such multidrug-resistant organisms (MDROs) as Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA), and salmonella, to name a few. The emergence and steady increase of MDROs presents a significant challenge because of the limited availability of therapeutic options; there are simply not enough new antibiotic therapies being developed. With standard treatments becoming increasingly ineffective, infections persist and more are likely to be passed on to others.
Moreover, according to WHO, antibiotic resistance is now present in all parts of the world. New resistance mechanisms emerge and spread globally, hampering the ability of healthcare professionals to treat common infectious diseases. WHO has named the rise of MDROs as one of the most serious threats facing global public health today.
A review of antibiotic resistance published earlier this year by the UK’s independent Review on Antimicrobial Resistance, estimated that by 2050 “superbugs” could kill 10 million people annually and cost the world $100 trillion in lost economic output every year.3
Antimicrobial stewardship is designed to promote the appropriate use of antimicrobials (including antibiotics), improve patient outcomes, reduce microbial resistance, and decrease the spread of infections caused by MDROs. In hospitals and other healthcare settings, antimicrobial stewardship is a pressing area of focus for hospital administrators. According to CDC, on any given day, about one in 25 hospital patients has at least one healthcare-associated infection.
If antibiotic resistance goes undetected, it can result in poor therapeutic outcomes, the spread of resistance, longer hospital stays, and increased costs. If these infections are detected more rapidly, the correct course of antibiotics will be delivered to the patient sooner, reducing the risk for the spread of infection and improving the patient’s healthcare experience.
Several initiatives worldwide are aiming to reduce the spread of antibiotic-resistant organisms. In 2014, President Obama issued an executive order to push forward a national plan for combating antibiotic-resistant bacteria, investing $4.6 billion in active programs to address the issue. Some of the significant outcomes of the plan’s first goals are the establishment of antimicrobial stewardship programs in acute care hospitals and improved antibiotic stewardship across all healthcare settings. Specifically, the plan has outlined steps to reduce inappropriate antibiotic use by 50% in outpatient settings, and by 20% in inpatient settings, by the year 2020.4 These measures to promote appropriate antibiotic use will also decrease healthcare costs by reducing unnecessary prescriptions and the incidence of resistant infections. To put the magnitude in perspective, in 2009, the United States spent $10.7 billion on antibiotics.5
This September, the United Nations held a high-level meeting in New York, during which global leaders committed to joining the fight against antimicrobial resistance. The primary objectives of the meeting were to summon and maintain strong national, regional, and international political commitments to addressing antimicrobial resistance, and to increase public awareness of this looming problem. The meeting emphasized the important roles and responsibilities of governments, as well as the roles of relevant intergovernmental organizations—particularly WHO—in responding to the challenges of antimicrobial resistance. The meeting also sought to encourage the engagement of all relevant sectors of society—including both consumers and organizations with interests in agriculture, environmental monitoring, and finance, as well as human and veterinary medicine.6
Diagnostic testing for antibiotic resistance has historically been culture-based, employing identification of bacterial isolates followed by antimicrobial susceptibility profiling. There are several problems inherent with using culture-based testing methods. The first is related to time. Because the culture must be grown, obtaining conclusive results can take 3 days or more. In the meantime, the patient is given an antibiotic based on an empirical diagnosis. This approach delays focused therapy and may result in the wrong course of action for the patient.
There are also problems with accuracy. When the specimen contains more than one microbe, it becomes difficult to pinpoint what is causing the infection and what may have been living on the plate. Making such a determination relies on the skillset of the technician reading the plates and making decisions about what’s important and what’s not. Obtaining a pure culture is critical to accuracy in identifying and looking for antibiotic resistance susceptibility.
The technical restrictions on conventional bacteriologic screening approaches and their interpretation are challenging. As a result, manufacturers of in vitro diagnostics are tasked with finding technologies and test solutions that can achieve high sensitivity and specificity with a fast turnaround time.
MOLECULAR-BASED TESTING APPROACHES
Because accurate and timely detection of antibiotic-resistance patterns are crucial for all aspects of antimicrobial stewardship, the field is moving toward molecular-based approaches. These newer technologies enable diagnostic tests to help distinguish between viral and bacterial infections, and identify bacterial drug susceptibilities. Gene testing using polymerase chain reaction (PCR)-based tests is currently the best approach to screen for MDROs. The main advantage of molecular tests is that they identify specific resistance mechanisms within the same day. These tools allow clinicians to make better informed decisions more quickly—and deliver the most effective antibiotic to the patient.
Molecular-based testing has a few barriers to overcome before it is adopted as a mainstream tool. To date, there is not a reimbursement code for drug-resistance gene testing, which hampers its adoption due to financial implications. In addition, before molecular tests are integrated into the clinical workflow, education is required to help physicians understand the benefits of the test and trust its results over those of the traditional methods to which they are accustomed. There is also a need to train lab personnel to understand how to perform the test.
There is still a place for culture-based tests and, in fact, the two methods can often be used in conjunction to clearly establish genotypic and phenotypic linkage. Combining the new, molecular tests with traditional culture-based assays can make it possible to identify new resistance mechanisms, and will help to detect and control resistant bacteria.
A major motivator for hospitals to use molecular diagnostic tests for identifying antibiotic resistance is their potential for reducing healthcare costs. If a patient who is infected with resistant bacteria is not treated with the right antibiotic, for example, that patient will then consume more healthcare resources than patients infected with the same bacteria that are not resistant.
Another financial incentive is avoiding penalties that payors may levy based on the patient population that acquires infections while in the hospital. If a patient is given an ineffective antibiotic treatment, for example, it is not uncommon for them to contract Clostridium difficile, a bacterium that causes diarrhea. Patients who return to the hospital for treatment for an infection within 30 days of discharge can dramatically affect the hospital’s profit margin, since the hospital receives a set reimbursement for treating the patient, regardless of the number of visits the treatment may require.
The speed and sensitivity of a molecular-based approach provides beneficial options to help hospitals with their antimicrobial stewardship efforts.
PROFILING MULTIDRUG RESISTANCE GENES
In order to address the limitations of conventional tests, Thermo Fisher Scientific developed a screening panel of molecular assays for multidrug-resistant genes that can be performed on the company’s OpenArray platform. The high-throughput screen on this nanofluidic platform generates same-day results and can be run in conjunction with multiple identification technologies (see Figure 1). By detecting 17 gene groups associated with antibiotic resistance, the panel provides broad, clinically relevant coverage. Currently, the panel is for research use only, but the platform is poised to dramatically increase speed and accuracy for improved patient outcomes in the near future.
Results from clinical research conducted by Diatherix Laboratories LLC, Huntsville, Ala, in collaboration with Huntsville Hospital have shown how the molecular technique can be used to identify the presence of MDROs in the respiratory tracts and stool samples of hospitalized patients. The researchers employed an OpenArray TaqMan panel using a high-throughput real-time platform, the QuantStudio 12K Flex, to detect genes associated with resistance to three different classes of antibiotics, including beta-lactams, fluoroquinolones, and macrolides (see Table 1).
To obtain samples from which DNA could be extracted, the Huntsville researchers began with samples collected at an infection site, such as the respiratory tract or, for screening purposes, a rectal or stool swab. Nucleic acid extraction was performed using the Applied Biosystems MagMax Express 96 magnetic particle processor by Thermo Fisher Scientific (see Figure 2). The isolated DNA samples were then run through the QuantStudio system, and were ready to be analyzed in 4 to 5 hours (see Figure 3).
In the Huntsville study, screening of patient samples revealed a complex pattern of up to 14 genes that encode for resistance to one of four major classes of antibiotics, with ermC and TEM being the most prevalent (91% and 48%, respectively, in respiratory and gastrointestinal samples). CTX Groups 1, 2, and 9; and qnrA and qnrS were also detected.7 Ongoing studies are in progress to assess the utility of molecular testing for antibiotic resistance genes in the screening and management of patients with increased risks of infection with highly resistant organisms.
To screen for and understand the antibiotic resistance gene pattern present in a sample, the Thermo Fisher Scientific technology permits users to combine 17 assays and one process-control assay, in triplicate, onto a single panel. Large numbers of samples can then be tested across a panel of antibiotic-resistance genes, reducing the cost per sample compared with traditional culture-based methods.
Thermo Fisher Scientific has programs underway to develop bacterial and fungal identification. The company’s nanofluidic plate permits laboratorians to merge the solution that can identify the presence of “superbugs” with genes that indicate susceptibility to MDROs, in order to provide even more information for therapeutic decisionmaking in the future.
IMPACT ON ANTIBIOTIC RESISTANCE
With the emergence of antibiotic-resistant “superbugs,” new technologies and research tools that can rapidly detect and analyze such bugs are taking on a critical role in improving our understanding of how such microorganisms affect human health.
The field of laboratory diagnostics is moving toward applications that employ molecular approaches for microbial detection, essentially providing clear insights into the appropriate therapeutic intervention in the case of antibiotic resistance. Moving forward, clinicians will be able to implement the tenets of precision medicine to use information derived from the genome to deliver the right medicine at the right time.
The timely and accurate identification of organisms causing infections, together with multidrug resistance gene profiles, offers the ability to improve patient outcomes, reduce costs for hospitals and healthcare systems, and address the growing threat of antibiotic resistance.
Elena V. Grigorenko, PhD, is vice president of research and development, and Donald R. Stalons, PhD, D(ABMM), MPH, is vice president of operations and clinical laboratory director, at Diatherix Laboratories LLC. For further information, contact CLP chief editor Steve Halasey via [email protected].
- Antimicrobial resistance: global report on surveillance, 2014. Geneva, Switzerland: World Health Organization. 2014. Available at: www.who.int/drugresistance/documents/surveillancereport/en. Accessed September 7, 2016.
- Antibiotic resistance threats in the United States, 2013. Atlanta: Centers for Disease Control and Prevention. 2013. Available at: www.cdc.gov/drugresistance/threat-report-2013/pdf/ar-threats-2013-508.pdf. Accessed September 7, 2016.
- Tackling drug-resistant infections globally: final report and recommendations. London: The Review on Antimicrobial Resistance, 2016. Available at: http://amr-review.org/sites/default/files/160525_Final%20paper_with%20cover.pdf. Accessed September 7, 2016.
- National action plan for combating antibiotic resistant bacteria. Washington, DC: The White House. 2015. Available at: www.whitehouse.gov/sites/default/files/docs/national_action_plan_for_combating_antibotic-resistant_bacteria.pdf. Accessed September 7, 2016.
- Suda KJ, Hicks LA, Roberts RM, Hunkler RJ, Danziger LH. A national evaluation of antibiotic expenditures by healthcare setting in the United States, 2009. J Antimicrob Chemother. 2013;68(3):715–718; doi: 10.1093/jac/dks445.
- High-level meeting on antimicrobial resistance [press release online]. New York: General Assembly of the United Nations, 2016. Available at: www.un.org/pga/70/events/high-level-meeting-on-antimicrobial-resistance. Accessed September 12, 2016.
- Malone L, Stonebraker M, Brzezinski S, et al. Molecular detection of antimicrobial susceptibility: changing paradigm of laboratory testing for multidrug-resistant organisms [ID Week 2015, poster abstracts, no. 1596]. Open Forum Infect Dis. 2015;2(suppl 1):S379; doi: 10.1093/ofid/ofv133.1149.