Use of Molecular Diagnostics in the Wound Clinic

  Wound clinics are being assailed from many different directions in today’s healthcare environment to heal wounds more quickly. Third-party payers such as Medicare are now limiting the number of debridements and other services that can be provided to the patient, making it imperative to heal wounds efficiently. Also, for a wound clinic to differentiate itself from the competitive pressure of other providers, wound-healing outcomes must improve: To best serve each individual patient, wound healing should be as rapid as possible. Therefore, it is necessary for each wound clinic to utilize the emerging technologies that will accomplish this goal.   Generally, wound care providers believe bacteria are an important reason why wounds don’t heal. More often than not (more than 68% of the time), we prescribe at least one course of antibiotics to a patient with a chronic wound.¹ Such approaches work just enough of the time to keep us treating wounds with antibiotics and/or antimicrobial dressings. Plus, three of the top four wound dressings worldwide are antimicrobial.   However, the literature does not support our actions. A recent Cochran analysis² concludes there is no evidence to support the use of antibiotics for the treatment of venous leg ulcers, and the Infectious Disease Society of America guidelines for diabetic foot ulcers strongly discourage the general use of antibiotics, recommending only the most limited use of antibiotics when absolutely necessary. A literature review by Landis³ found no evidence for the prophylactic or routine use of antibiotics when there are no signs of clinical infection or increased bacterial bioburden in chronic wounds. Several studies^1,4,5 show that, based on culture results, antibiotic use does not improve wound-healing outcomes. Thus, objective data do not support clinician belief that microorganism management is crucial to wound healing.   The issue may not be so much the limitation of the antibiotics or the dressings as the diagnostic tool we use to direct the use of these treatments.   Wound care providers have long known that many wounds, especially diabetic foot ulcers, are polymicrobial.⁶ Only recently have we come to understand that wound microbial burden is usually organized as a biofilm on the surface of the wound.⁷ Biofilm in nature and in human chronic infections is generally polymicrobial and very resistant to antibiotics and its host immunity.⁸ There is some concern that the minor species in an individual wound biofilm may just be contaminants and as such do not contribute to the virulence of the wound bioburden. However, until the science regarding the subject is better understood, it may be prudent not to ignore any of the constituents of the polymicrobial infection.⁹ Therefore, identifying all the different bacterial and fungal species of microorganisms that make up the wound biofilm, and securing accurate relative quantification of each species is important for today’s wound clinics.   Because “time is tissue,” a test for microbial identification and quantification should be rapid. Any new diagnostic test also should be more accurate in identifying microbes than current clinical cultures. The test should be sensitive enough to identify all different microorganisms regardless of their percentage of the whole community. The diagnostic test should be as free from bias (able to evaluate one group of bugs the same as another group of bugs) as possible. Plus, the microbial identification test must not break the bank.

Limits of Clinical Cultures

  Clinical cultures do not meet the need to evaluate chronic wounds. Clearly, using cultivation methods to identify bacteria does not improve the wound management outcomes.¹⁰ This is not to say that bacteria play an unimportant role in nonhealing wounds; rather, the culture method itself is in question.   Cultures fail to grow more than 85% of organisms identified in chronic wounds.¹¹ When cultures do grow microorganisms, culture results are wrong 25% of the time¹⁰ and take a long time (when diagnostic speed is important) to yield results. Most importantly, clinical cultures are geared to report one organism and fail to demonstrate the amazing diversity of the bioburden of most chronic wounds.

Molecular Approaches

  Molecular identification and quantification of microorganisms in host infections is an emerging science. Numerous technologies are available for evaluating microbial DNA. Each method has its own strengths and weaknesses, but, used in combination, molecular methods can easily meet the requirements necessary for evaluating wound biofilm.   Molecular methods address the subcellular level — ie, look at genes and the molecular products and pathways related to those genes. For microbial identification, the technologies examined here deal solely with the microbial DNA. These technologies include established, well-understood methods such as polymerase chain reaction (PCR) testing, as well as cutting-edge technology including next-generation sequencing and next-next-generation platforms soon available for clinical use.   Polymerase Chain Reaction. PCR testing is “ancient” (more than 20 years old) in the world of molecular technologies. It encompasses different methods that each rely on the copying segments of DNA using polymerase. Real-time PCR testing is employed for microorganism identification; this method comprises utilizing a small primer that is an exact match to a target area of DNA within the microbe of interest. This means a specific primer has to be carefully developed for the specific species of microorganism to be identified that does not cross-react with any other microbial species (and at the same time binds to the DNA target and amplifies that target efficiently with each cycle of the test). With each cycle, the target doubles, quickly providing enough for the instrument to sense the presence of the target. The more of the target DNA present at the start, the fewer the doublings necessary to identify the presence of the microorganism.   PCR provides three important pieces of information for the clinician: 1) whether the sample contains the specific microorganism the primer tested for 2) if present, in what quantity, and 3) if not present (no reaction), how this influences choice of initial therapies.   PCR Strengths and Weaknesses. The strength of PCR testing is that it is very quick, only taking from 2 to 3 hours to yield results.   The technology of specific PCR is very evolved; a number of tools exist to optimize its processes and results. A number of new platforms (machines) are available, one of which allows evaluation of up to 96 different targets in a single sample. Importantly, PCR costs have decreased almost to the same price as a clinical culture and will continue to fall.   The most glaring weakness is that PCR can identify a microorganism only if a specific primer is used. In wounds, the number of species of bacteria and fungus reaches well into the thousands. It is impractical for PCR to attempt to identify this type of breadth of bacterial and fungal diversity.   Also, PCR limits how well it amplifies (copies) one species compared to another; this bias makes PCR less suited to determining the relative contributions of one species of microorganisms to another in the wound biofilm. Significant advances in other molecular technologies can overcome this limitation in breadth of microbe identification, as well as relative quantification of the microbes within the polymicrobial infection.   Ribosomal DNA. Several years ago, scientists turned to the 16S ribosomal DNA gene present in all bacteria and the 18S ribosomal gene present in fungus to solve the problem of diagnosing multiple different microorganisms. The 16S rDNA gene in bacteria was found to be a microbial fingerprint — ie, the 16S rDNA gene contains nine hypervariable regions which, when sequenced, reveal the bacteria’s identity.   An innovative technology developed to exploit this property of the 16S ribosomal-DNA gene is a hybrid technology utilizing PCR and mass spectroscopy. This platform utilizes PCR to amplify the 16S region of all the different microbes in the sample until a sufficient quantity for analysis is accumulated. To analyze the 16S rDNA, the target material is electrosprayed into the mass spectrometer and the fragments analyzed by advanced mathematical algorithms.   The results are reproducible identification of microorganisms known and in the database, and provides some quantification. This rapid molecular method to identify unknown bacteria without specific primers makes it much broader than PCR and can be accomplished in 8 hours, comparable to the speed of PCR.   However, this hybrid methodology has some weaknesses. Because the exact sequence of the 16S region is not determined, the microorganisms can only be identified if their information is registered in the database. Some primer bias in the universal primers utilized within the hybrid method allows identification of some microorganisms better than others — ie, the universal primer fails to copy the 16S ribosomal DNA of some organisms efficiently and they are not detected by the mass spectroscopy portion of the test. Also, the cost of PCR/mass spectroscopy is significantly greater than real-time PCR.   To truly know the exact sequence of the 16S rDNA or the 18S rDNA and subsequently to identify microorganisms within the wound bioburden, technologies that determine the order of the DNA bases (ATCG) of this region are necessary. Early sequencing technologies were time-intensive and extremely costly, but over the last 5 years, sequencing technology has exploded into a number of different methods for determining DNA sequences. Only a few are currently clinically relevant.   Pyrosequencing. Pyrosequencing is based on recording a light signal (pyro) each time a DNA base is added to the copy strand of the template of the 16S rDNA strand.   The light signals are specific for each base added; therefore, an accurate sequence can easily be determined. Pyrosequencing methods have the advantage of being able to sequence long segments of the 16S and 18S regions.   Utilizing advanced methods of error suppression such as chimera checking, noise reduction, and other bioinformatics, up to 700 bases of the 16S and 18S region can be determined with 99% accuracy.¹² These segments then can be compared to a national database that records the entire rDNA sequence for known microorganisms. These public databases contain the 16S sequences for thousands of bacteria. By comparing to a database, the segment of the 16S region sequenced by the pyrosequencing method can be assigned to a specific microorganism at a species level with a high level of confidence.   Pyrosequencing is massively parallel — it can look at millions of different bacteria at the same time. This means the rDNA region from individual bacteria and fungus throughout the sample can be captured, sequenced, and identified.   The number of copies of each segment identified can be utilized to determine the relative contribution of each species to the sample. This relative quantification is much better than PCR for determining the relative proportion of the species present, but is still not perfect.   The strength of pyrosequencing is that the evaluation yields a scientifically sound identification of the microorganism with DNA certainty.   Also, a large database of thousands of bacteria and fungus means that a micro-organism rarely goes unidentified. This allows pyrosequencing to be truly universal in its ability to identify all bacteria (>98%)¹³ and all fungus (>95%).¹⁴   Additionally, pyrosequencing is able to give us an understanding of the relative amounts of each of these organisms in the wound microbiota. In addition, pyrosequencing is not subject to dynamic range issues (major populations covering up the presence of minor populations).   However, pyrosequencing is a massive undertaking, yielding a terabyte (ie, huge amount) of information in a single run. This information must be reduced and compared to databases, which is very time-intensive.   The turnaround time for pyrosequencing is longer than PCR testing but still less than current clinical culture methods. For such a powerful universal diagnostic, the wait is worthwhile.

Looking Ahead

  The future holds amazing possibilities for microorganism identification. New platforms, such as Ion Torrent (Ion Torrent Systems, Inc., Guilford, CT; San Francisco, CA; and Beverly, MA) and the PacBio (Pacific BioSciences, Inc., Menlo Park, CA) are emerging. These sequencing methods utilize the same advantages of pyrosequencing in that they will yield the same lengths of segments, which is necessary for microbial identification, but at much faster speeds and at much lower costs.   Molecular methods are in the pioneer stage. The identification of microbe presence and relative quantity by molecular methods makes a difference in wound outcomes, whereas culture methods have not improved outcomes.¹⁵ With new technologies emerging, the cost of molecular methods will be less than the current price of cultures. The results will be far more accurate, more sensitive and, just as importantly, far more complete by identifying and quantitating all microbes present. The pushback will be that microorganisms that are not contributing to the nonhealing of the wound will be identified and may be inappropriately treated. However, this is a common concern. Just as when new imaging methods emerged that revealed smaller lesions with higher definition, the pushback was concern that we may be identifying lesions of scant clinical concern. The point to remember is that medical care does better with a fuller understanding, more complete, and more accurate diagnosis of the problem. Molecular methods provide a robust, cost-effective diagnosis. The clinician must decide how that diagnostic tool is best used.   Dr. Wolcott is Medical Director, Research and Testing Laboratory of the South Plains Medical Director, Southwest Regional PCR Laboratory; Adjunct Professor, Department of Microbiology and Immunology, and Department of Surgery, Texas Tech University Health Sciences Center, School of Medicine, Lubbock, TX; Medical Director, Southwest Regional Wound Care Center. He may be reached at randy@randallwolcott.com.

References

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