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A Newfangled Jungle Tour Guide

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Microbiologists will surely appreciate the ironic quirk of history that spawned the era of modern molecular biology. In 1976, researchers at the University of Ghent unraveled the relatively humble 3,569-base genome of Escherichia phage MS2 (common name: Bacteriophage MS2). The first genome ever sequenced and arguably one of the greatest feats of technological and scientific achievement in human history was only possible with the help of a diminutive microorganism with a penchant for attacking and infecting other microbes. And with it, the genomics revolution was born1.

Compass Sketch

Over the next 25 years, steady improvements were made in sequencing techniques, largely fueled by use of more complex organisms with increasingly larger genomes. Microorganisms such as bacteriophages, bacteria and yeast formed the genomic backbone of much of this early work and enabled increasingly more ambitious feats of sequencing. Culminating in 2001, researchers published the first full draft sequence of the approximately 3.2 billion bases of the Homo sapiens genome (common name: Human)1. Since then, a dizzying array of organisms large and small have been sequenced, providing researchers with a genomic menagerie that has enabled some of the most important advances in modern medicine.

In particular, the advent of genomic techniques has allowed researchers to plumb the depths and scale the heights of microbial ecology and evolution in unprecedented ways. Using this newfangled tour guide, researchers have been able to peer into the vast biodiversity of microbes colonizing every ecological strata of the planet with unparalleled accuracy and sensitivity. Genomic techniques have been critical to our understanding of the microbial world because microbial species and interactions that really count in nature do not occur in pure cultures. Learn more about the microbial world »

DNA Sketch Genomic techniques have been critical to our understanding of the microbial world because microbial species and interactions that really count in nature do not occur in pure culture. Tire Marks

Genomic techniques have brought an astounding diversity and complexity of microbes both in natural and non-natural environs into sharp focus. It has also spawned entire fields of study dedicated to understanding how microbial diversity and interactions can precipitate certain disease states (known as pathogenomics). Last but not least, these techniques have provided the accelerant for development of a new generation of rapid and sensitive tests that detect (potentially) the full complement of microbes underlying various infectious diseases of concern – many of which are currently available to clinicians for routine use.

As is often the case with disruptive innovations, the pace of technological development may often outstrip the clinician end-users understanding of how to use this type of diagnostic information. For wound clinicians interested in learning how the recent genomic advances may benefit their patients, it is important to understand both the advantages and disadvantages these tests offer. When assessing the microbial jungle of the wound, your best bet is to know your tour guide.

Welcome aboard & please note the exits

Sketch of a Map

Genomic technologies can come in many different flavors, sizes and price points; however, they are all based on the ability to isolate and detect the nucleic acid building blocks of microbes (e.g. DNA or RNA). Positive microbial detection therefore occurs when specific pieces of DNA or RNA are detected in the clinical sample being analyzed.

The workhorse of many microbiology labs is nucleic acid probes, which are available in a variety of commercial kit types and applications. This type of direct microbial detection is rapid and simple. However, lack of sensitivity may be an issue because these assays often require large starting amounts of nucleic acids for reliable detection. Most direct probe detection assays require at least 104 copies of nucleic acid per microliter for reliable detection. Most clinical samples will rarely meet this requirement without some form of nucleic acid amplification beforehand2.

Tire Marks PCR can be applied to almost any source that contains DNA and can be used to identify microorganisms that were alive thousands, perhaps millions of years ago.

Polymerase chain reaction (PCR) was the first amplification technique developed and remains the most widely used in both clinical and research applications. PCR selectively amplifies low abundance nucleic acid targets to more detectable levels. PCR is a stupendously elegant solution because it exploits the natural and remarkable function of enzymes known as polymerases.

Polymerases are found in all living organisms. They function as “molecular photocopiers” that can faithfully reproduce copies of genetic material while also proofreading and correcting copies3. PCR can be applied to almost any source that contains DNA, including blood, hair, and tissue; and can be used to identify microorganisms, animals, or plants, even those that were alive thousands, perhaps millions of years ago3.

PCR-based commercial kits are available for many clinically relevant bacterial, fungal and viral species, and enabled microbiology laboratories to routinely harness the precision of DNA-based detection4. Nonetheless, they are limited in scope and microbial coverage to those targets offered by the manufacturer. Some investigators have circumvented this bottleneck by developing in-house or home-brew PCR assays to expand their laboratory’s microbial coverage, but this may not be an option for every group as there can be significant cost and resource considerations.

DxWound harnesses the precision and sensitivity of genomic technology to provide a consolidated solution geared specifically to wound care providers. Learn How »

Getting a handle on bugs... and drugs?

Sketch of a Bug

Conventional antimicrobial susceptibility testing relies on growth in culture and how well a given microbe will respond to an array of agents. While these methods can prove useful for selecting potentially efficacious therapeutic agents, they are slow and have limitations5. One common and clinically relevant example is detection of methicillin resistance in staphylococci. Because methicillin resistance can be expressed in a heterogeneous fashion, phenotypic characterization of resistance can be complicated6.

Molecular techniques offer the ability to detect specific antimicrobial-drug resistance genes for many clinically relevant organisms2. These approaches have significantly improved understanding of the spread and genetics of resistance. In some cases, such as with mecA, detection of the resistance gene is used for confirming resistance identified using phenotypic methods5-7.

Molecular detection of antimicrobial resistance offers many advantages. Cultivation or growth of microbes is not required for detection. In fact, it’s possible to simultaneously detect the microbe and identify resistance genes from a single specimen without prior culture or growth steps2, 5. This is a significant advantage for a variety of organisms that are challenging to culture or non-viable; and those displaying resistance mechanisms that are not reliably detected by phenotypic methods5.

Nonetheless, it is unlikely that molecular methods will entirely replace traditional susceptibility testing methods for assessing antimicrobial resistance anytime soon2. Molecular tests are based on identifying genes with well-characterized sequences and validated associations with observed drug resistance patterns. They may not be able to detect newly emerging or previously uncharacterized resistance mechanisms and/or genes2.

These tests are also not crystal balls that have perfect predictive power. Instead, like all molecular tests, they provide probabilistic information that can help a clinician understand the likelihood of an outcome. This is because the presence of a resistance gene does not guarantee expression of the gene; conversely, the absence of a resistance gene does not exclude the possibility of other resistance mechanisms2.

Gene Sketch It's possible to simultaneously detect the microbe and identify resistance genes from a single specimen without prior culture. This is a significant advantage for organisms that are challenging to culture. Tire Marks

Instead, positive detection of a resistance gene indicates the likelihood of antimicrobial resistance. In fact, traditional susceptibility tests also have a probabilistic nature. Susceptibility testing does not guarantee that a specific antimicrobial will effectively treat the causative organism in 100 percent of infection cases. Instead, susceptibility results indicate a high likelihood that a certain antimicrobial will be effective – based on many important assumptions.

For example, susceptibility presumes that the causative organism has been correctly identified, no contamination was present during culture, and resistance mechanisms expressed in the wound biofilm are replicated in the petri dish by the isolated microbes. These are important caveats that must be understood by the clinician using results to guide therapy.

For now, the greatest value may be realized when susceptibility testing and molecular methods are used synergistically – and coupled to clinical presentation and other host factors – to help clinicians and microbiologists gain a more holistic understanding of antimicrobial resistance.

The price of admission

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Molecular diagnostics offer not only the potential for improved patient care and outcomes, but potential opportunities for cost reductions and/or cost avoidance. It is assumed that these tests can help clinicians avoid unnecessary diagnostic procedures, particularly those that are less sensitive or specific, reduce use of ineffective therapies and lower rates of hospital-acquired infections2, 8.

This may be true, but an often overlooked component to this formula is the inherent cost of implementing and conducting molecular testing. Not all molecular tests are extremely expensive; however, more complex and sophisticated tests often carry higher price tags in terms of direct costs. Tests based on kits and that require minimal instrumentation are typically quite inexpensive. Examples include DNA probe methods that detect C. trachomatis or N. gonorrhoeae3.

PCR-based genotyping tests or even more advanced DNA sequencing methods are considered high-complexity tests and are often much more expensive2. Hospitals or institutions interested in establishing these types of molecular tests in their microbiology laboratory must consider a number of cost factors. These include high labor costs due to requirements for experienced, licensed clinical laboratory scientists, expensive equipment and reagents, licensing fees and R&D costs2, 9.

Advances in automation and production of less expensive reagents are helping drive down these costs and reduce technician time and overhead2. Furthermore, an emerging segment of independent laboratory service providers are increasingly catering their test menu to infectious disease management. These reference laboratories offer health systems and hospitals the opportunity to leverage molecular diagnostics to improve patient care without incurring the expense of in-house development.

Laboratory developed tests (LDTs) are proprietary tests that are offered exclusively by a lab entity to clinicians and hospitals10. They often undergo a rigorous process of discovery, R&D, production scale-up and finally, quality assurance and validation; all of which involve the dedicated efforts of research scientists, clinical laboratory technicians, and often, data analysts.

Tire Marks Reference laboratories offer health systems and hospitals the opportunity to leverage molecular diagnostics to improve patient care but without incurring the expense of in-house development. Flask Sketch

The Centers for Medicare and Medicaid Services (CMS) regulate these tests and require laboratories to meet mandated testing standards established by the Clinical Laboratory Improvement Amendments (CLIA). Labs offering clinical testing for infectious disease must have CLIA certification but currently do not require FDA approval. Importantly, many elements required to demonstrate adequate test performance, validity and quality control overlap between FDA and CLIA standards10.

For the infectious disease clinician, wound care provider or microbiologists, molecular diagnostics offer an innovative and extremely useful tool to help guide diagnosis, therapy and management of patients. Options are available to help institute testing within the microbiology laboratory as well as to outsource the cost and resource considerations to third party laboratory vendors. In either case, the best bet for the end user is to know your tour guide and understand where the journey will take you.

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Stay tuned. There’s more.

Don't miss our next article: Applying Molecular Testing in Wound Care: The Good, Bad, and Ugly

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  1. yourgenome website. Accessed September 20, 2017. https://www.yourgenome.org/facts/timeline-organisms-that-have-had-their-genomes-sequenced
  2. Pfaller, MA. Molecular Approaches to Diagnosing and Managing Infectious Diseases: Practicality and Costs. Emerging Infec Dis 2001;7(2):312-318.
  3. Powledge, Tabitha M. The polymerase chain reaction. Adv Physiol Educ 2004;28: 44–50,
  4. Medical Laboratory Observer. Accessed September 20, 2017. https://www.mlo-online.com/direct-nucleic-acid-diagnostic-tests-for-bacterial-infectious-diseases.php
  5. Cockerill FR III. Genetic methods for assessing antimicrobial resistance. Antimicrob Agents Chemother 1999; 43:199-212.
  1. Kim C, Mwangi M, Chung M, et al. The mechanism of heterogeneous beta-lactam resistance in MRSA: key role of the stringent stress response. PloS one [09 Dec 2013, 8(12):e82814]
  2. Hussain Z, Stoakes L, Massey V, Diagre D, Fitzgerald V, El Sayed S, et al. Correlation of oxacillin MIC with mec A gene carriage in coagulase-negative staphylococci. J Clin Microbiol 2000;38:752-4
  3. Ross JS. Financial determinants of outcomes in molecular testing. Arch Pathol Lab Med 1999;123:1071-5.
  4. Ferreira-Gonzalez A, Garrett CG. Pitfalls in establishing a molecular diagnostic laboratory. Hum Pathol 1996;27:437-40.
  5. Katsanis SH, Katsanis N. Molecular genetic testing and the future of clinical genomics. Nat Rev Genetics. 2013;14:415-426.