Molecular Diagnosis: A New Era in Wound Care

Many host impairments interfere with the healing trajectory of chronic wounds, but even when these are controlled, healing may fail to progress. There is a growing realization that bacterial bioburden, present in all chronic wounds, is a contributor to healing failure. Until recently, adequate tools to diagnose bioburden have been unavailable. While standard microbiology cultures could identify the organisms on the surface of the wound, in more than 80% of cases, they were unable to identify the organisms present in the bioburden1. We have conducted large numbers of studies revealing two important points about chronic wounds:
1) chronic wounds have polymicrobial infections, sometimes with dozens of microorganisms (bacteria and yeast) all existing as a cooperative community, and
2) this community often creates an extensive biofilm2-10.
Molecular diagnostics has allowed us to personalize the care of our wound patients, and dramatically improve our healing success rate.

For many years identifying the bacteria has meant culturing them in the laboratory. However, there are some limitations to this standard technique:
• Limited cultivation: Fewer than 2% of all known bacteria can be grown "routinely" in the clinical microbiology laboratory and only a few of those can grow within
24 hours.
• Yeast: such as Candida albicans, Candida parapsilosis etc. are rarely identified.
• Poor quantification: Swab cultures do not allow quantification of the bacteria (ie, we do not know if there were 10 bacteria or 10 million present in the wound).
• Poor confidence on identification: Biochemical identification of bacteria and yeast is 80-90% accurate but only for the 2% of bacteria we can culture.
• Viable but not culturable: Even the 2% of bacteria, which are easily cultured in a planktonic growth state, fail to grow rapidly in the laboratory, and 98% of all the other bacteria don’t grow at all11.
• Selection bias: Bacterial species that do grow efficiently in laboratory conditions out-compete other species (perhaps the bacteria actually promoting the infection). Thus, such diagnostic methods artificially “select” one species over another.

Molecular Techniques
What is the alternative to old fashioned culture techniques? The Polymerase Chain Reaction (PCR) is a molecular technique utilized to target and “amplify” a single or few copies of a piece of DNA, generating thousands to millions of copies of a particular DNA sequence thereby allowing them to be detected. The method relies on thermal cycling, cycles of repeated heating and cooling for DNA melting and then enzymatic replication of the DNA. So, bacteria are identified by “amplifying” their DNA, requiring only a tiny sample, without actually having to grow bacterial cultures in the microbiology laboratory. This technology is sensitive, specific and can be done in hours.
Since we began using this technique to evaluate chronic wound patients, we have seen some surprising things. Wounds cultured with standard techniques showed only bacteria such as Staphylococcus aureus but PCR analysis revealed that chronic wounds were actually colonized by many other bacteria and fungi (yeast), including anaerobes and other very difficult to culture organisms. Without PCR analysis, these anaerobic organisms, fungi and other microorganisms were missed. When antibiotics targeting these other bacteria are used, the patients respond to the therapy.

But What About Culture And Sensitivity?
The following concerns are frequently raised regarding the use of molecular methods of testing:
1) Don’t you need bacterial cultures to perform antibiotic sensitivities?
No. We can detect many of the major genetic antibiotic resistance factors using molecular methods. And since we are more likely to diagnose ALL the bacteria with molecular techniques, we are more likely to give you complete information.
2) Molecular methods detect non-viable bacteria and naked DNA.
In data that was recently prepared for scientific review we show that DNA and dead bacteria do not persist in chronic wounds.
3) Molecular methods detect contaminant bacteria:
This was one of our early concerns. At first we were puzzled when we saw how many wounds had 80% Corynebacterium spp. since our medical training indicated that Corynebacterium in wounds is a non-pathogen. However, our training was based on data from culture techniques. We now know that Corynebacterium and many other strange bacteria propagate in wounds and contribute to chronicity. If a microorganism is propagating and colonizing a wound it should never be considered a “contaminant,” it is a problem.
4) The biofilm clinical diagnostic reports are overwhelming:
This is definitely true! It is not uncommon to see 14 different organisms in a chronic wound.
5) Molecular methods are too expensive:
Molecular methods have prices only slightly higher than that of clinical culture ($200 vs. $300). Most insurance including Medicare reimburse the full cost of the test. Even hospitals and clinics can develop simple reference laboratory contracts to utilize the service. They are more rapid since they do not require days of culture for results, provide more accurate information, and may result in cost savings by improving antibiotic choice and healing trajectory. Taking into account molecular methods are more rapid, provide more accurate, less biased, and infinitely more comprehensive results the cost savings is dramatic. The back-end savings are even more impressive and can fully negate the slightly higher price. Correct antibiotic choice, improved healing trajectory, improved patient care, reduction in incorrectly prescribed antibiotics and the knowledge that you as the clinician are now controlling the bioburden because you know what it really is made up of.
Since implementing molecular methods we are healing wounds faster, almost completely eliminating amputations due to chronic wounds. Also our referrals have gone up because we are healing wounds not just maintaining them, we are spending less money on clinical supplies, and literally the air is fresher in our clinic. Once our clinicians get use to the complexity of the reports and begin realizing these are "real answers" they all demand the test, even the patients demand to be tested with this new technology.

A New Era For Wound Care Practitioners
Pathogenius wound bioburden diagnostics provides two levels of molecular analysis.
Level I:
• Rapid screening assay for the top eight well known pathogens that are so often identified by clinical culture methods. Results are returned the same day as received.
• Semi quantitative information: (low, moderate, high, very high bacteria levels).
• Information on MRSA and VRE presence or absence.
Level II:
• A comprehensive test that is able to identify nearly all bacteria and fungi present in a chronic wound sample.
• Results returned usually within 3 business days.
• Both bacteria and fungi (yeast) are detected and relative abundance determined.
• An interpretive report based upon known antibiotic resistance and sensitivity to act as a research tool for the physician.
• An overview that indicates to the clinician what the major and minor populations are in the specific wound.
• Experts that can help you
with understanding how to use
the reports.
How does it work?
• The clinician orders sample shipping supplies, free of charge, from Pathogenius Laboratories.
• The clinician obtains a swab or even better for diagnostic purposes a sharp debridement sample and places it in a sterile tube provided by Pathogenius.
• The test requisition is filled out and the transport and shipping package provided by Pathogenius is packed using the instructions provided.
• FedEx is called to pick up and deliver the sample.
• The very next day the Level I Pathogenius report is returned giving the clinician the first vital pieces
of information. Within 72 hours the Level II final report is provided and the clinician now knows what to
do for that patients exact wound bioburden.
When should I do a molecular analysis? Anytime you would otherwise to a culture analysis, such as when a patient first comes to your clinic or when the wound drainage changes or the wound fails to respond to standard interventions.

Interpreting PCR Information:
A Few Examples
Example #1: Staphylococcus aureus is present and 90% of the bacteria are MRSA (high numbers of bacteria were also indicated). These results suggest an MRSA infection. This type of molecular result in the clinical context of a high risk wound would require treatment with first line MRSA drugs.
Example #2: The presence of MRSA constitute than 20% of the total bacterial population (high bacterial load detected). These results in the context of a non-limb threatening wound might lead the clinician to treat this wound less aggressively with second line drugs such as trimethoprim-sulfa (Bactrim DS).
Example #3: MRSA present in very low amount. It may be appropriate to conservatively mange patients for whom MRSA constitute less than 3% of the biofilm. Molecular methods can help in surveillance and the MRSA can be addressed if it becomes more prevalent.
These examples highlight the power of quantitation and its ability to guide clinical decision making. But it also points out the limitations of agar cultures. Agar cultures could easily demonstrate methicillin resistant Staphylococcus aureus in all three of these situations but could not have helped us in determining if first line drugs, second line drugs or no antibiotics at all were necessary for this pathogen. Conversely, if present only in the biofilm, the Staphylococcus aureus MRSA might have been viable but not culturable and therefore missed altogether.
An amazing number of microorganisms can be identified from each clinical sample. The diversity identified often makes the clinician uncomfortable in formulating a treatment regimen because is difficult to know which organisms need to be directly targeted and how to fashion a strategy which utilizes a reasonable number of antibiotics with the broadest coverage of the bacteria identified. We see many new bacteria such as a previously uncharacterized Bacteroides that occurred in a majority of surgical site infections and was reported as “culture negative"6.

Conclusion
Current clinical cultures have limitations which render them almost irrelevant for the diagnosis of polymicrobial clinical and biofilm infections. Molecular methods on the other hand have many advantages over agar cultures including accuracy, rapid results, high sensitivity, and high benefit to expense ratio, specificity and availability; moreover, they are quantitative. These attributes all combine to make molecular methods singularly suited to diagnose polymicrobial and biofilm associated clinical infections.
It is now possible to “personalize” antimicrobial therapy in a wound care patient based on specific diagnostic data. It is our opinion that molecular methods will quickly replace most clinical cultures and will prove themselves indispensable in the management of chronic wounds specifically and infectious disease in general.

Scot E. Dowd Ph.D. is CEO and a Laboratory director for Pathogenius Laboratory, Director of Research and Testing Laboratory, and Director of SpiroStat Technologies. Pathogenius and SpiroStat are specialized pathogen diagnostics laboratories with Pathogenius being focused on polymicrobial infections and specifically wound care pathogen and biofilm diagnostics services. Research and Testing Laboratory is a collaborative and commercial service laboratory specializing in development and implementation of next and future generation molecular methods. All of these companies are located in Lubbock, TX.

Randy Wolcott MD is medical director for Southwest Regional Wound Care Center (SWRWCC) as well as heading the board of directors for Pathogenius Laboratory and Research and Testing Laboratory. SWRWCC located in Lubbock, TX has championed and led molecular pathogen and biofilm diagnostics into clinical reality under Dr. Wolcott's vision and guidance.