Molecular Diagnosis: A New Era in Wound Care
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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.
1. Sen CK, Gordillo GM, Roy S, et al. Human skin wounds: a major and snowballing threat to public health and the economy. Wound Repair Regen 2009;17(6):763-71.
2. Dowd SE, Wolcott RD, Sun Y, et al. Polymicrobial nature of chronic diabetic foot ulcer biofilm infections determined using bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP). PLoS One 2008;3(10):e3326.
3. Dowd SE, Sun Y, Secor PR, et al. Survey of bacterial diversity in chronic wounds using Pyrosequencing, DGGE, and full ribosome shotgun sequencing. BMC Microbiol 2008;8(1):43.
4. Leake JL, Dowd SE, Wolcott RD, Zischkau AM. Identification of yeast in chronic wounds using new pathogen-detection technologies. J Wound Care 2009;18(3):103-4, 106, 108.
5. Wolcott RD, Rhoads DD, Dowd SE. Biofilms and chronic wound inflammation. J Wound Care 2008;17(8):333-41.
6. Wolcott RD, Gontcharova V, Sun Y, Zischakau A, Dowd SE. Bacterial diversity in surgical site infections: not just aerobic cocci any more. J Wound Care 2009;18(8):317-23.
7. Wolcott RD, Gontcharova V, Sun Y, Dowd SE. Evaluation of the bacterial diversity among and within individual venous leg ulcers using bacterial tag-encoded FLX and titanium amplicon pyrosequencing and metagenomic approaches. BMC Microbiol 2009;9:226.
8. Wolcott RD, Gontcharova V, Sun Y, Zischakau A, Dowd SE. Bacterial diversity in surgical site infections: not just aerobic cocci any more. J Wound Care 2009;18(8):317-23.
9. Wolcott RD, Kennedy JP, Dowd SE. Regular debridement is the main tool for maintaining a healthy wound bed in most chronic wounds. J Wound Care 2009;18(2):54-6.
10. Wolcott RD, Dowd SE. A rapid molecular method for characterising bacterial bioburden in chronic wounds. J Wound Care 2008;17(12):513-6. 11. Fux CA, Costerton JW, Stewart PS, Stoodley P. Survival strategies of infectious biofilms. Trends Microbiol 2005;13(1):34-40.
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