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Gross appearance of each model Control group
Colonisation group
signs and symptoms focus on either aspects of this balance, they are unsuitable for clinical use. Therefore, from this point, the authors focused on gene expression analysis because the biological responses to wound infection can be directly assessed at the messenger RNA (ribonucleic acid) (mRNA) level. Furthermore, the authors adopted the reverse transcription-polymerase chain reaction on the centrifugal precipitation of wound fluids (termed 'wound fluid RT-PCR'). Since wound fluid is easily collected and contains both
Infection group Day 0 Day 6
Figure 2. Unlike the control and colonisation group, the infection group shows obvious signs of infection, such as slough and necrotic tissue spreading to the periwound area, as well as differences in the level of the wound bed and edges. One representative result from the three groups is shown.
The authors claim that the 'gold standard' for diagnosing wound infection is counting the colony-forming units (CFUs). By using this method, wound infection is defined in international pressure ulcer guidelines as ’a bacterial bioburden of >105 CFU/g of tissue and⁄or the presence of beta-haemolytic streptococci'[1]
.
However, it is difficult to diagnose wound infection based solely on bacterial numbers because the virulence factor differs among bacterial species and interspecies. Bacterial synergy means that a mixed infection with two or more species of bacteria results in worse outcomes than single bacteria-induced infection. Furthermore, it is already well- known that more than 99% of bacteria cannot be cultured in vitro and are termed as 'viable but non-culturable'. This problem also hinders clinicians from performing bacterial counts to diagnose wound infection. As a result of these problems, it is recommended by the authors that clinicians assess the inflammatory signs and symptoms when diagnosing infection, even though this will not detect the critical colonisation.
Method for detecting wound infection and critical colonisation Delays in detecting wound infection or critical colonisation result in further complications. The authors, therefore, attempted to establish a new strategy for solving this clinical challenge. Since they already knew that wound infection is the result of an imbalance in bacterial virulence and host immunity, they postulated that analysing this balance would be a marker for detecting wound infection. However, because both the bacterial count and the clinical
14 Wounds International Vol 3 | Issue 2 | ©Wounds International 2012
200bp - 100bp -
200bp - 100bp -
200bp - 100bp -
bacterial and host cells, the authors thought this method may be promising when assessing the balance of bacteria and host relationship. Moreover, it is noteworthy that wound fluid is easily collected and RT-PCR is highly sensitive, thus clinicians can analyse the gene expression level from a small amount of samples, meaning this method would be noninvasive. To confirm its usefulness in diagnosing wound infection, the authors performed animal experiments. Different doses of bacteria were administered to the wound to make three wound states — control, colonisation, and infection. Using rat models featuring a wound that had not been inoculated (control), a wound with low dose bacterial load (colonisation), and a wound with high-dose bacterial load (infection), the authors extracted the mRNA from the centrifugal precipitation of the wound fluid leaking from the wound beds [Fig 2].
Wound fluid RT-PCR for detection of wound infection Bacteria
M NC 1
Control Colonisation Infection
2 3 4 5
toxA no-RT toxA (166 bp) rpoD (175 bp)
Host 40 cycles
M NC 1
200bp - 100bp -
200bp - 100bp -
M: size marker NC: negative control
Figure 3. While the expression of rpoD (a gene specific to Pseudomonas aeruginosa) indicates the presence of bacteria in colonised and infected wounds, only toxA was detected in infected wounds. Of the host maker genes, only Foxp3 was detected in colonised wounds. The combination of expression in bacterial and host genes can offer a useful clue to detecting the wound infection.
Control Colonisation Infection
2 3 4 5
Foxp3 (120 bp) Actb (168 bp)
40 cycles
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