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bacteria that grow easily, even if they are at low levels in the sample initially. But the fatal flaw for cultures in diagnosing wound bioburden, and one that no methodological improvements will overcome, is the inability of the culture to fully identify the diversity present in an individual wound. Even though most molecular methods for identifying
bacteria are in their pioneering stages, they are more sensitive and specific than cultures. PCR methods can identify bacteria with high accuracy down to a few bacterial cells in just two to three hours. Yet, PCR methods only identify the bacteria for which primers have been developed. To identify the vast number of bacteria that have not had primers developed, sequencing methods must be used[13]
. Sequencing methods Figure 2: Extracting microbial DNA for analysis from wound samples.
cytosine and guanine) for a specific gene. In the 168 wound samples evaluated, molecular
technology identified 338 unique genera, whereas culture methods revealed only 17 different genera. Cultures failed to grow the vast majority of micro-organisms present in the wound. It should also be noted that 41 samples out of the 168 wounds cultured identified bacteria that molecular methods could not confirm. Given the high level of accuracy of sequencing and PCR it seems most likely that culture reported erroneous results. The important generalisation from the study is that culture methods are not adequate in identifying bacteria in a polymicrobial infection.[12] A second study (recently submitted for publication by
the author's group) looked at clinical culture versus DNA identification for 51 chronic wounds. Molecular methods identified 17.7 genera per sample whereas clinical cultures grew only 1.8 genera per sample with a maximum of five in a single sample. The major finding was that up to 59% of the aerobic bacterial load was not identified by culture. Another important finding was that clinical cultures reported micro- organisms that were less than 1% of the wound bioburden 17% of the time. Far from avoiding the reporting of minor populations,
cultures amplify minor populations a significant portion of the time. Also, a crossover analysis using molecular methods to re-identify the micro-organisms present on the subculture plate shows that a culture identification misidentified 18% of the micro-organisms that grew. Given the findings of these two studies, cultures seem
inadequate for evaluating the bacteria present in wounds. As reported above, cultures sometimes (about 25% of the time) report bacteria that are not shown as present using molecular methods and misidentify bacteria 18% of the time. Thus, molecular methods are felt to be more reliable. However, since there is currently no widely accepted 'gold
standard' for microbial identification in medicine, individual clinicians have to decide which results — PCR, sequencing or culture — they feel are more reliable. Cultures have a significant selection bias, amplifying
12 Wounds International Vol 3 | Issue 2 | ©Wounds International 2012
can be used to determine the exact order of the DNA bases (adenine, thymine, cytosine, guanine) of a very specific region (ie 16S rDNA gene), to determine a 'bacterial fingerprint' for each bacteria present in the infection. This method allows for comprehensive identification of all
micro-organisms along with quantitation, or how many of the micro-organisms are present. The downside of sequencing is that it takes three to four days to complete. However, by combining PCR and sequencing methods, the clinician can receive rapid information as to important pathogens — Methicillin-resistant Staphylococcus aureus (MRSA); Vancomycin-resistant Enterococcus (VRE) and multi-drug resistant Pseudomonas aeruginosa (MDRPA) — in real time, and then comprehensive data can be returned in three to four days. A second strategy utilising molecular methods is to
combine different technologies. This has been performed using the rapid power of PCR to amplify the 16S fingerprint region in a clinical sample. The amplified 16S rDNA gene can then be examined by mass spectroscopy instead of sequencing to identify a very broad range of micro- organisms. This method provides results that are similar to sequencing, but, as with PCR, in only two to three hours[14]
.
Currently, however, this technology lacks the breadth and DNA certainty of sequencing. This hybrid technology also lacks the ability to evaluate
highly diverse infections. This highlights the difficulty of developing a single molecular diagnostic test that meets all the needs of the clinician. However, new molecular technologies, such as the hybrid
technology Plex-ID (Abbott), which combines PCR and mass spectroscopy, are emerging which may provide all the important microbial as well as host information from a chronic infection, such as a non-healing wound. Clinically, these new molecular methods mean a more rapid, accurate and comprehensive diagnosis of the wound microbiota. Currently, there is scientific method that can reject any
micro-organism as being unimportant in a clinical infection. Therefore, it is necessary to identify and quantify all microbes present in a chronic wound. It has been shown in large cohort studies that employing molecular methods to direct the use of commercially available wound care products (topical iodine, silver, methylene blue etc), along with antibiotics, improved healing at six months[15]
. Similarly, the use of
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