Studies of chronic ulcers and larval therapy

Abstract: Our aims in this study were to learn more about chronic ulcer pathogenesis (Paper I) and larval therapy (Paper II-V).Materials and methods: Paper I: 26 bacterial isolates of P. aeruginosa from chronic ulcers were examined by PCR technique for detection of the genes of two virulence factors (elastase and alkaline proteinase). Zymography (Paper I & Paper III)) and azocasein assay (Paper I) were used for detection of excreted proteases. Paper II: The debridemental effect of the larvae of Lucilia sericata was investigated in 74 necrotic or sloughy ulcers. Cleaned ulcer area was estimated as thirds of the total area and documented photographically. Adverse side-effects were registered as yes or no - answers and pain was measured with visual analogue scale of pain. Paper III: Larval secretions were collected after the larvae had been emerged in different growth media, in phosphate buffered saline and in a wound. Paper IV: Bacterial cultures were taken before and after larval therapy from a leg ulcer colonised by methicillin-resistant Staphylococcus aureus. Paper V: Disposable equipment and mainly aseptically produced food were used for rearing fly larvae. The humidity (40%) and temperature (25oC) were kept at a constant level. The fly eggs were disinfected with chloramine solution 0.25%. Fifteen cultures were taken from disinfected larvae and single ones were taken from meat; fly drinking water and from non-disinfected larvae, under aerobic and anaerobic conditions. In the routine disinfection control disinfected eggs were streaked over a horse blood agar plate (Oxoid), which was incubated at 37oC in a thermostat for 24h under aerobic conditions. Results: Paper I: Both proteinase genes were detected in all the samples. The level of proteinase expression differed between the isolates, but was stable for each strain from time to time. Paper II: Some 86% of the wounds were well debrided. A single application of larvae for two or three days was sufficient for debridement in two-thirds of the ulcer cases, but in cases with a thick eschar two applications were required. The larvae seemed to thrive especially well in the wounds of diabetic patients. Most patients (41%) felt no difference in pain during larval therapy, while a quarter felt less and, although 34% felt an increase in pain, they generally wanted to continue the therapy. None of the patients experienced a tickling sensation or bleeding. Odour was mostly reduced or un-changed. Paper III: The larvae excreted predominantly serine proteases. Paper IV: MRSA was cleansed from the ulcer. Paper V: The larvae developed satisfactorily. Non-pathogenic spore-bearing bacteria were regularly detected on the larvae after disinfection. The disinfection control was sufficient for detecting the usual wound pathogens. Conclusions: Paper I: The bacterial phenotype should be taken into account in future leg ulcer studies. Paper II: Larval therapy is an effective and well tolerated debridemental therapy. Paper III: The larvae excrete proteases for their extra-corporal digestion. Paper IV: Larvae have an antibacterial effect. Paper V: The rearing technique is easy, reliable, safe and odour-free.