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Spring 2022 - Safety

Advances in Diagnostic Testing for Antimicrobial Resistance

The threat of antimicrobial resistance has increased substantially since the start of the COVID-19 pandemic, and while a host of diagnostic tools are available, these life-threatening infections still pose a threat due to limitations of tests, especially those that are rapid.

ANTIMICROBIALS HAVE been revolutionizing medicine since their discovery in the 20th century, decreasing mortality and morbidity across the globe. However, their recent use and overuse have created an endemic of antimicrobial resistance (AMR) that now threatens the health that these drugs were designed to protect — intensified by nearly every aspect of the world in which we live: international travel, healthcare settings, wastewater and ground soil, food-producing animals and even the COVID-19 pandemic. Although COVID-19 has dominated headlines in the past couple of years, its likely contribution to worsening the AMR endemic has not widely been discussed. Prior to the pandemic, AMR was estimated to kill more than 68,000 people annually in the European Union and United States alone. Looking ahead, the global threat of AMR is projected to cause more deaths than all cancers combined by 2050,1 and complications from antibiotic resistant infections will cost as much as $100 trillion.2 The financial ramifications of AMR could be comparable to those of climate change by the year 2030.3

Efforts to treat the real-time threat of COVID-19, compounded by threats of AMR, have been herculean. Particularly in the early days of the pandemic when there were far more unknowns about this virus, empiric administration of antibiotics to prevent secondary bacterial infections were commonplace. Still today, anywhere from 56 percent to 92 percent of hospitalized COVID-19 patients receive antibiotics throughout their course of treatment even though only 6 percent to 15 percent are suffering from a bacterial co-infection. Further, a retrospective study shows some patients with secondary bacterial infections are now acquiring AMR infection strains.1

In the U.S. alone, the Centers for Disease Control and Prevention (CDC) acknowledges the burden of deaths and infections from antibiotic resistance is actually greater than initially thought, with estimates of more than 2.8 million antibiotic-resistant infections annually and more than 35,000 deaths.4

The challenge of AMR is nothing new. Four years after the introduction of commercially available penicillin in 1943, resistance was observed for Staphylococcus aureus. However, the urgency and proliferation of AMR, with genes developing singular and multiple resistance, has resulted in stepped-up efforts at mitigation, as well as development of more targeted therapies. Regrettably, innovation and production of new antibiotics are hampered by huge costs, including the expense of clinical trials and the risk that new drugs will soon be met with AMR, rendering them all but ineffective.

Healthcare associated infections, a major source of AMR infections, are stratified by the World Health Organization into three groups based on urgency of pathogens. High-priority bacteria include penicillin-resistant Streptococcus pneumoniae and so-called ESKAPE pathogens that are resistant to many antibiotics, including those considered last resort such as methicillin-resistant Staphylococcus aureus.

Through testing, antibiotic sensitivity can largely be determined, but results can take as long as a week. This delay perpetuates the problem of AMR since doctors may begin precautionary broad-spectrum antibiotics while awaiting results. It is estimated 50 percent of antibiotics are prescribed for the wrong strains due to incomplete or nonexistent testing.5

It is believed improved diagnostic testing, particularly rapid tests, will help to better identify infection strains and concurrent AMR.

AMR Diagnostics: The Current Marketplace

AMR can occur in a number of ways, including intrinsically or through development of mutations by the very bacterial strains these antibiotics target. The chance of AMR occurrence and the severity of a subsequent infection are directly related to the affected individual’s immune status.

Effectiveness of antimicrobial drugs are best tested using pure culture isolates with several cultivation rounds. According to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical Laboratory Standards Institute (CLSI) guidelines, phenotype testing provides the most reliable diagnostic because it answers which antibiotic should be used and at what dose regardless of the resistance mechanism.5 Minimum inhibitory concentrations (MIC) of antibiotic susceptibility tests (ASTs) under the EUCAST guidelines are generally higher than those of CLSI, even though CLSI’s are more widely used.6

Disk diffusion is considered the gold standard of phenotype AST. It is simple and cost-effective, but it requires an overnight incubation and bacteria susceptibility confirmation between 16 hours and 24 hours, so it is less than ideal for analyzing slow-growing and fastidious bacteria. Dilution, both micro and macro, are other options for AST, although for both, it is difficult to maintain the recommended testing parameters such as pH and temperature. Likewise, for both diffusion and dilution, laborious testing requirements, length of incubation time and high risk of cross contamination provide significant challenges. On the other hand, epsilometer testing is preferred over disk diffusion and dilution for assessing AST because it is reliable across a range of antibiotics, particularly slow-growing and fastidious bacteria.6

Rapid ASTs can benefit patient outcomes through more timely identification and administration of targeted treatments, resulting in shortened hospital stays and reduced overall healthcare expense, although the implementation costs are vast, which include test kits, instruments and reagents, and required laboratory staff.

A number of commercially available rapid AST systems are on the market today, with reported result times in about four hours and susceptibility testing in six hours to eight hours, including broth dilution-based systems that use ready-made AST cassettes or cards. However, these are expensive and oftentimes don’t take into account the total time needed for culture enrichment and isolation. There are also some doubts about whether accelerated cultures can fully substitute for traditional growth-based cultures. Therefore, in clinical settings, a more likely approach is the collection of pure isolates via culture samples followed by AST.

Genotype tests, with their sensitivity and specificity, make them generally better suited for rapid detection methods, with the polymerase chain reaction tests the most efficient. Genotype tests require shorter incubation times and have a reduced risk of contamination. However, they do have drawbacks, including specific assays for individual antimicrobial agents, and less sensitivity for latent infections. They also require skilled personnel to perform the tests.6

Most rapid AST diagnostics today offer end-point analysis only and, therefore, may not be ideal in outpatient settings.5 That said, diagnostic innovation continues, particularly for outpatient care settings where antibiotics are often prescribed.

Microfluidics-based diagnostics are some of the most promising devices on the horizon because they are portable, cost-effective and reproducible, and they use a minimal number of samples. When coupled with optical sensors, AST tests can detect MIC in just a few hours and, in one reported case using a single bacterial cell analysis, within 30 minutes.

ATP bioluminescence assay is an enzyme-based approach to AST in which resistant bacteria result in bioluminescence, whereas susceptible bacteria stay neutral and can produce identification and susceptibility results of urinary infections within three hours to six hours.

Simplified blood culture system (SBCS) can be used for testing blood infections. These samples require no processing and can provide susceptibility within eight hours to 12 hours compared to a standard blood culture turnaround of up to 48 hours.6

The Microbiome

The human microbiome is an important area of research as a harbinger of AMR bacteria. Even in the absence of antibiotic exposure, some studies show multidrug resistance in as much as 20 percent to 30 percent of the human gut microbiota, and drug-resistant genes have been detected in newborn meconium (the first feces or stool of the newborn), in some cases in rates higher than their mother’s. Determining a predictive likelihood of carrying AMR bacteria, as well as becoming infected with one, is being looked at via high throughput DNA sequencing and bioinformatics. For example, homology-based methods can be used to predict antimicrobial-resistant genes using computers. The taxonomy of antimicrobial-resistant genes can also be studied to determine the source (i.e., whether it was passed vertically as part of microbial cell divide or horizontally by unrelated groups).7

Of course, the optimal antimicrobial testing mechanism will differ by healthcare setting based on availability, cost (including staffing) and accuracy. This may be particularly true in outpatient settings. However AST is assessed, communication of results and countering the effects of AMR infections are key.

Federal Efforts and Innovations

The Antibiotic Resistance Solutions Initiative, part of the CDC’s One Health approach under the National Action Plan for Combating Antibiotic-Resistant Bacteria, is one of many domestic and international efforts aimed at studying AMR and supporting innovation for diagnostics and mitigation.

A network of Antibiotic Resistance Laboratories, established in 2016, routinely tests and tracks emerging antibiotic resistance. When a germ of significance is identified, state and local health departments work with healthcare facilities to isolate patients and begin infection control procedures to reduce and stop further transmission.4 In addition, a Global Antimicrobial Resistance Laboratory & Response Network was launched in 2021, which will span 50 countries and improve the detection of emerging AMR threats, as well as identify risk factors that drive the emergence and spread of AMR across healthcare, the community and the environment.

CDC and the U.S. Food and Drug Administration house an extensive isolate bank, providing samples at no cost (excluding shipping) to approved institutions for diagnostics and drug development, including validation of laboratory results and assays. As of February 2021, the isolate bank housed 29 panels and 952 isolates gathered from national reference labs and tracking activities, as well as from specimens in healthcare, food and the community.

Further spurring innovation is the Antimicrobial Resistance Diagnostic Challenge, a joint effort between the National Institutes of Health and the Health and Human Services Office of the Assistant Secretary for Preparedness and Response in support of the National Action Plan for Combating Antibiotic Resistant Bacteria. In 2021, the challenge awarded a $19 million federal innovation prize for rapid point-of-care laboratory diagnostic tests to combat the development and spread of drug-resistant bacteria. Funding for the prize was split between the National Institute of Allergy and Infectious Diseases and Biomedical Advanced Research and Development Authority. The winner was Visby Medical’s single-use disposable rapid test for gonorrhea.8

The challenges of AMR are unrelenting, but so too are efforts to detect and combat it. Through prioritized global awareness and innovation in diagnostics, perhaps one day soon the etiology of its spread and identification of appropriate targeted interventions will provide for a better understanding and further development of a strong and meaningful intervention.


1. Avershina E, Shapovalova V, and Shipulin G. Fighting Antibiotic Resistance in Hospital-Acquired Infections: Current State and Emerging Technologies in Disease Prevention, Diagnostics and Therapy. Frontiers in Microbiology, July 21, 2021. Accessed at

2. Jones E. The Missing Link in Fighting Antibiotic Resistance Rapid Diagnostics Can Flag Outbreaks of “Superbugs” Before They Spread. Scientific American, Dec. 4, 2019. Accessed at

3. Rosini R, Nicchi S, Pizza M, and Rappuoli R. Vaccines Against Antimicrobial Resistance. Frontiers in Immunology, June 3, 2020. Accessed at

4. United States Taskforce for Combating Antibiotic Resistant Bacteria. National Action Plan for Combatting Antibiotic-Resistant Bacteria, Progress Report: Year 4, September 2019. Accessed at

5. Vasala A, Hytönen VP, and Laitinen OH. Modern Tools for Rapid Diagnostics of Antimicrobial Resistance. Frontiers in Cellular Infection and Microbiology, July 15, 2020. Accessed at

6. Khan ZA, Siddiqui MF, Park S. Current and Emerging Methods of Antibiotic Susceptibility Testing. Diagnostics, May 3, 2019. Accessed at

7. Brinkac L, Voorhies A, Gomez A, and Nelson KE. The Threat of Antimicrobial Resistance on the Human Microbiome. Microbial Ecology, November 2017. Accessed at

8. National Institutes of Health. Antimicrobial Resistance Diagnostic Challenge. Accessed at

Amy Scanlin, MS
Amy Scanlin, MS, is a freelance writer and editor specializing in medical and fitness topics.