Evaluation of a microbiological screening and acceptance procedure for cryopreserved skin allografts based on 14 day cultures

Viable donor skin is still considered the gold standard for the temporary covering of burns. Since 1985, the Brussels military skin bank supplies cryopreserved viable cadaveric skin for therapeutic use. Unfortunately, viable skin can not be sterilised, which increases the risk of disease transmission. On the other hand, every effort should be made to ensure that the largest possible part of the donated skin is processed into high-performance grafts. Cryopreserved skin allografts that fail bacterial or fungal screening are reworked into ‘sterile’ non-viable glycerolised skin allografts. The transposition of the European Human Cell and Tissue Directives into Belgian Law has prompted us to install a pragmatic microbiological screening and acceptance procedure, which is based on 14 day enrichment broth cultures of finished product samples and treats the complex issues of ‘acceptable bioburden’ and ‘absence of objectionable organisms’. In this paper we evaluate this procedure applied on 148 skin donations. An incubation time of 14 days allowed for the detection of an additional 16.9% (25/148) of contaminated skin compared to our classic 3 day incubation protocol and consequently increased the share of non-viable glycerolised skin with 8.4%. Importantly, 24% of these slow-growing microorganisms were considered to be potentially pathogenic. In addition, we raise the issue of ‘representative sampling’ of heterogeneously contaminated skin. In summary, we feel that our present microbiological testing and acceptance procedure assures adequate patient safety and skin availability. The question remains, however, whether the supposed increased safety of our skin grafts outweighs the reduced overall clinical performance and the increase in work load and costs

Molecular Epidemiology and Clinical Impact of Acinetobacter calcoaceticus-baumannii Complex in a Belgian Burn Wound Center.

Multidrug resistant Acinetobacter baumannii and its closely related species A. pittii and A. nosocomialis, all members of the Acinetobacter calcoaceticus-baumannii (Acb) complex, are a major cause of hospital acquired infection. In the burn wound center of the Queen Astrid military hospital in Brussels, 48 patients were colonized or infected with Acb complex over a 52-month period. We report the molecular epidemiology of these organisms, their clinical impact and infection control measures taken. A representative set of 157 Acb complex isolates was analyzed using repetitive sequence-based PCR (rep-PCR) (DiversiLab) and a multiplex PCR targeting OXA-51-like and OXA-23-like genes. We identified 31 rep-PCR genotypes (strains). Representatives of each rep-type were identified to species by rpoB sequence analysis: 13 types to A. baumannii, 10 to A. pittii, and 3 to A. nosocomialis. It was assumed that isolates that belonged to the same rep-type also belonged to the same species. Thus, 83.4% of all isolates were identified to A. baumannii, 9.6% to A. pittii and 4.5% to A. nosocomialis. We observed 12 extensively drug resistant Acb strains (10 A. baumannii and 2 A. nosocomialis), all carbapenem-non-susceptible/colistin-susceptible and imported into the burn wound center through patients injured in North Africa. The two most prevalent rep-types 12 and 13 harbored an OXA-23-like gene. Multilocus sequence typing allocated them to clonal complex 1 corresponding to EU (international) clone I. Both strains caused consecutive outbreaks, interspersed with periods of apparent eradication. Patients infected with carbapenem resistant A. baumannii were successfully treated with colistin/rifampicin. Extensive infection control measures were required to eradicate the organisms. Acinetobacter infection and colonization was not associated with increased attributable mortality

Demographics and clinical characteristics of patients with Acb complex (as identified by the VITEK 2 system) colonization or infection.

<p>Demographics and clinical characteristics of patients with Acb complex (as identified by the VITEK 2 system) colonization or infection.</p

Dendrogram based on UPGMA and Pearson correlation coefficient, representing genetic similarity among reference and clinical Acb complex isolates.

<p>Representative Acb complex isolates (Type strains) were typed by rep-PCR (DiversiLab) and analyzed (autocorrection, Pearson correlation coefficient, optimization and curve smoothing) with the BioNumerics DiversiLab plugin. Thirty-one rep-PCR type strains with <95% similarity and 6 reference strains are shown. Different colors indicate distinct species within the <i>Acinetobacter</i> genus. The red arrow indicates the band responsible for the clustering of <i>A</i>. <i>baumannii</i> strain P11-01 in the <i>A</i>. <i>pittii</i> cluster (not uncommon for Pearson correlations).</p

Unrooted neighbor-joining tree representing phylogenetic relationships between the 31 rep-PCR type strains and 6 reference strains.

<p>The tree was derived from partial <i>rpoB</i> gene sequences. Different colors indicate distinct species within the <i>Acinetobacter</i> genus.</p

Characteristics of 157 Acb complex isolates (as identified by the VITEK 2 system) from 48 patients admitted to the burn unit over a 52-month period and of 6 reference strains.

<p>Characteristics of 157 Acb complex isolates (as identified by the VITEK 2 system) from 48 patients admitted to the burn unit over a 52-month period and of 6 reference strains.</p

Time course of MDR <i>A</i>. <i>baumannii</i> colonization/infection in the BWC.

<p>Green diamonds indicate the admission of patients from countries known to have a high prevalence of resistance mechanisms. A black diamond marks the death of a patient. Grey bars represent the period of time patients were hospitalized in the BWC, while colored bars indicate the period of time during which they were colonized or infected with a particular MDR <i>A</i>. <i>baumannii</i> strain (rep-PCR genotype). P, patient.</p