Broth versus solid agar culture of swab samples of cadaveric allograft musculoskeletal tissue

As part of the donor assessment protocol, bioburden assessment must be performed on allograft musculoskeletal tissue samples collected at the time of tissue retrieval. Swab samples of musculoskeletal tissue allografts from cadaveric donors are received at the microbiology department of the South Eastern Area Laboratory Services (Australia) to determine the presence of bacteria and fungi. This study will review the isolation rate of organisms from solid agar and broth culture of swab samples of cadaveric allograft musculoskeletal tissue over a 6-year period, 2006–2011. Swabs were inoculated onto horse blood agar (anaerobic, 35 °C) and chocolate agar (CO₂, 35 °C) and then placed into a cooked meat broth (aerobic, 35 °C). A total of 1,912 swabs from 389 donors were received during the study period. 557 (29.1 %) swabs were culture positive with the isolation of 713 organisms, 249 (34.9 %) from solid agar culture and an additional 464 (65.1 %) from broth culture only. This study has shown that the broth culture of cadaveric allograft musculoskeletal swab samples recovered a greater amount of organisms than solid agar culture. Isolates such as Clostridium species and Staphylococcus aureus would not have been isolated from solid agar culture alone. Broth culture is an essential part of the bioburden assessment protocol of swab samples of cadaveric allograft musculoskeletal tissue in this laboratory.     

Bacterial Contamination of Tissue Allografts – Experiences of the Donor Tissue Bank of Victoria

The aim of this study is to report the experience of the Donor Tissue Bank of Victoria with bacteria isolated from musculoskeletal, skin and cardiac allografts retrieved from cadaveric donors. The results of all quality control samples for bacterial culture, taken during retrieval and processing of allografts at the DTBV for a 12 month period, were extracted and analysed. It was found that 15.7% of skin, 15.1% of heart valves and 5.8% of musculoskeletal samples had positive culture results. The number and types of organisms isolated varied with tissue type. The most commonly isolated organisms were Staphylococcus species (including S. aureus). The identity of the isolate and the number of positive specimens from the same donor were considerations in the decision concerning the suitability of tissue for subsequent implantation.     

Effects of <Superscript>60</Superscript>Co gamma radiation dose on initial structural biomechanical properties of ovine bone—patellar tendon—bone allografts

Gamma radiation is established as a procedure for inactivating bacteria, fungal spores and viruses. Sterilization of soft tissue allografts with high dose ⁶⁰Co gamma radiation has been shown to have adverse effects on allograft biomechanical properties. In the current study, bone-patellar tendon-bone (BPTB) allografts from 32 mature sheep were divided into two treatment groups: low-dose radiation at 15 kGy (n = 16) and high-dose radiation at 25 kGy (n = 16) with the contralateral limb serving as a 0 kGy (n = 32) non-irradiated control. Half of the tendons from all treatment groups were biomechanically tested to determine bulk BPTB mechanical properties, cancellous bone compressive properties, and interference screw pull-out strength. The remaining tissues were prepared, implanted, and mechanically tested in an acute in vitro anterior crucial ligament (ACL) reconstruction. Low-dose radiation did not adversely affect mechanical properties of the tendon allograft, bone, or ACL reconstruction compared to internal non-irradiated control. However, high-dose radiation compromised bulk tendon load at failure and ultimate strength by 26.9 and 28.9%, respectively (P < 0.05), but demonstrated no negative effect on the cancellous bone compressive properties or interference screw pull-out strength. Our findings suggest that low dose radiation (15 kGy) does not compromise the mechanical integrity of the allograft tissue, yet high dose radiation (25 kGy) significantly alters the biomechanical integrity of the soft tissue constituent.     

Micro-organisms isolated from cadaveric samples of allograft musculoskeletal tissue

Allograft musculoskeletal tissue is commonly used in orthopaedic surgical procedures. Cadaveric donors of musculoskeletal tissue supply multiple allografts such as tendons, ligaments and bone. The microbiology laboratory of the South Eastern Area Laboratory Services (SEALS, Australia) has cultured cadaveric allograft musculoskeletal tissue samples for bacterial and fungal isolates since 2006. This study will retrospectively review the micro-organisms isolated over a 6-year period, 2006–2011. Swab and tissue samples were received for bioburden testing and were inoculated onto agar and/or broth culture media. Growth was obtained from 25.1 % of cadaveric allograft musculoskeletal tissue samples received. The predominant organisms isolated were coagulase-negative staphylococci and coliforms, with the heaviest bioburden recovered from the hemipelvis. The rate of bacterial and fungal isolates from cadaveric allograft musculoskeletal tissue samples is higher than that from living donors. The type of organism isolated may influence the suitability of the allograft for transplant.     

Characterization of the mechanical properties of bovine cortical bone treated with a novel tissue sterilization process

Over the past decade chemical processing and engineering of musculoskeletal tissue (tendon and bone) has improved dramatically. The use of bone allograft and xenograft in reconstructive orthopedic and maxillofacial surgeries is increasing, yet severe complications can occur if the material is contaminated in any way. A novel tissue sterilization process, BioCleanse^®, has been developed to clean and sterilize musculoskeletal tissue for implantation. The present study was designed to determine the effect of this novel cleaning process on the biomechanical properties of bovine cortical bone prior to implantation. The mechanical properties of treated bovine bone material were compared to human samples with respect to failure under compression, shear and three-point bending. The data demonstrate that bovine bone treated with the novel sterilization procedure has favorable biomechanical properties compared to that of human bone treated in a similar fashion.     

Subcutaneous clostridial infection in Adelie penguins in Hope Bay,Antarctica

During the 2000–2001 breeding season in Hope Bay, Antarctica, two adult Adelie penguins were found dead with lesions compatible with subcutaneous clostridial infection. Clostridium cadaveris was isolated from the musculature and the subcutaneous tissue of one of these two penguins, whereas Clostridium sporogenes was isolated from the subcutaneous tissue of the other penguin. Escherichia coli and Staphylococcus spp. were isolated from both animals. This is the first report of subcutaneous clostridial infection in Antarctic Adelie penguins.     

Is ABO group incompatibility really the reason of accelerated failure of cryopreserved allografts in very young patients? – Echography assessment of the European Homograft Bank (EHB) cryopreserved allografts used for reconstruction of the right ventricular outflow tract<Superscript>*</Superscript>

Right ventricular outflow tract reconstruction (RVOTR) with cryopreserved allograft for Ross operation and other congenital or acquired cardiac malformation has become a routine and currently, the procedure of choice for children and young patients. A tendency of accelerated degeneration in the youngest recipients has been reported. Some authors advocate the ABO group incompatibility as the main reason for such failure. This retrospective monocentric study presents the long-term outcome of the European Homograft Bank (EHB) cryopreserved allografts, used for RVOTR in Ross operation (group one) and other congenital heart malformation-s (group two). The evaluation of the allograft performance was done by means of echography, considering the allografts with the transvalvular gradient of ≥40 mm Hg and/or regurgitation of ≥3+ as failed. Fifty-one patients of group one and 123 of group two were analysed after completed follow-up information. About 25.5% of patients of group one and 30.8% of group two had a compatible, whereas 74.5% of group one and 68.92 of group two an incompatible ABO group with the donor. The mean follow up was 45.77 and 68.88 months, respectively. In second group 22.76% received the aortic, while 77.24% pulmonary allograft. Only three cases of group one (5.88%) failed: one with a compatible (7.69%) and two with an incompatible ABO group (5.26%) (p = 0.1), whereas 39 patients (29.4%) of group two failed between 20.1 and 120.2 months (29.73% with and 29.07% without ABO compatibility, p = 0.03). Contrary, the age showed more importance in the allograft failure: out of 41 failed allografts, 24 (58.54%) were implanted in patients of 0–5 years (9 or 37.5% with compatible and 15 or 62.5% with incompatible ABO group). Generally, analysing both groups together, there was no influence of ABO mismatching on the allograft failure (p = 0.79). Contrary, there was a significant difference in survival between Ross and non-Ross group (p = 0.00082). This revised version was published online in July 2006 with corrections to the Cover Date.     

Sponge swabs increase sensitivity of sterility testing of processed bone and tendon allografts

Sterility testing is the final, and critical, step in quality control of tissue banking. It informs the decision whether to release the tissue allografts for clinical use, or not. The most common method for sterility testing of structural bone and tendon allografts is to swab using cotton tip streaks. This method provides low recovery efficiency; and therefore may pass allografts with low bioburden, providing false negatives. Our pilot data revealed organism recovery efficiencies of 60, 30 and 100% from cotton swab, membrane filtration and sponge swaps, respectively. Our aim was to develop a high sensitivity sterility test for structural bone and tendon allografts using a sponge sampling method. Eighty-one bone and tendon allograft samples were inoculated with organism suspensions (10² or less organisms per 0.1 mL) of Clostridium sporogenes, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Bacillus subtilis, Aspergillus niger, Staphylococcus epidermidis and Micrococcus spp. Nasco sponges (4 × 8 cm) were used to aseptically sample the whole surface of allograft samples. The sponges were cut in half and cultured in either tryptone soya or fluid thioglycollate broths for 14 days. Positive culture samples were further examined for microbial morphology. The results showed that the sensitivity of the method, and negative predictive value, is 100% for all inoculated organisms incubated with thioglycollate. We conclude that this sponge sampling method should be applied as the standard for sterility testing of structural bone and tendon allografts.     

Assessment of tissue allograft safety monitoring with administrative healthcare databases: a pilot project using Medicare data

Assess whether Medicare data are useful for monitoring tissue allograft safety and utilization. We used health care claims (billing) data from 2007 for 35 million fee-for-service Medicare beneficiaries, a predominantly elderly population. Using search terms for transplant-related procedures, we generated lists of ICD-9-CM and CPT^® codes and assessed the frequency of selected allograft procedures. Step 1 used inpatient data and ICD-9-CM procedure codes. Step 2 added non-institutional provider (e.g., physician) claims, outpatient institutional claims, and CPT codes. We assembled preliminary lists of diagnosis codes for infections after selected allograft procedures. Many ICD-9-CM codes were ambiguous as to whether the procedure involved an allograft. Among 1.3 million persons with a procedure ascertained using the list of ICD-9-CM codes, only 1,886 claims clearly involved an allograft. CPT codes enabled better ascertainment of some allograft procedures (over 17,000 persons had corneal transplants and over 2,700 had allograft skin transplants). For spinal fusion procedures, CPT codes improved specificity for allografts; of nearly 100,000 patients with ICD-9-CM codes for spinal fusions, more than 34,000 had CPT codes indicating allograft use. Monitoring infrequent events (infections) after infrequent exposures (tissue allografts) requires large study populations. A strength of the large Medicare databases is the substantial number of certain allograft procedures. Limitations include lack of clinical detail and donor information. Medicare data can potentially augment passive reporting systems and may be useful for monitoring tissue allograft safety and utilization where codes clearly identify allograft use and coding algorithms can effectively screen for infections.     

Post-operative infection with fresh frozen allograft: reported outcomes of a hospital-based bone bank over 14 years

Femoral head bone allografts have traditionally been used to provide mechanical stability to areas of bony deficiency, or for its osteoinductive and osteoconductive properties. Concerns have been raised over increased infection rates following the use of fresh-frozen graft tissue. This retrospective study aims to investigate the outcomes of fresh frozen femoral heads kept in a regulated, non-commercial bone bank at a university teaching hospital.The local bone bank database was used to identify released femoral heads during a 14 year study period (September 1999–December 2013) whereby a retrospective review of patient records was undertaken to determine clinical outcome. During the observed study period, 427 femoral heads were released from cold storage. Of these, 270 femoral heads had a mean follow-up of 347 days. 157 femoral heads were excluded due to insufficient follow-up data (n = 132) or discarded due to breaks in the cold chain prior to use (n = 25). Of the 270 included femoral heads, 231 (85.6 %) had no reported complications with good graft incorporation. In the remaining 39 with reported complications, only 5 (2.6 %) developed a postoperative infection. Our findings suggest that the use of fresh frozen allograft does not materially increase the risk of post-operative bacterial infection. Our reported post-operative infection rates are comparable with infection rates of other similar studies on fresh frozen allograft use.     

Validating a Low Dose Gamma Irradiation Process for Sterilizing Allografts Using ISO 11137 Method 2B

This paper describes the validation of an allograft sterilization method specifically designed for the processing methods used at AlloSource in Centennial, CO. The methods used for this validation followed ISO Standard 11137, Method 2B. Three hundred allografts, collected from three defined production batches were dosed using a series of five incremental doses, beginning at 1 kGy and increasing by 1 kGy until 5 kGy was achieved. Following sterilization dosing, each allograft test article was analyzed using a sterility test to identify any viable microorganisms. The number of positive sterility samples was used to calculate the verification dose (1.27 kGy), which was then verified by an additional batch of 100 allografts. The results from this validation indicate that sterility (10⁻⁶ SAL) on human allograft tissue using gamma ⁶⁰Co radiation can be achieved when a dose of at least 9.2 kGy is employed.     

Swab or biopsy samples for bioburden testing of allograft musculoskeletal tissue?

Swab and biopsy samples of allograft musculoskeletal tissue are most commonly collected by tissue banks for bacterial and fungal bioburden testing. An in vitro study was performed using the National Committee for Clinical Laboratory Standards standard ‘Quality control of microbiological transport systems’ (2003) to validate and evaluate the recovery of six challenge organisms from swab and biopsy samples of allograft musculoskeletal tissue. On average, 8.4 to >100 and 7.2 to >100 % of the inoculum was recovered from swab and biopsy samples respectively. A retrospective review of donor episodes was also performed, consisting of paired swab and biopsy samples received in this laboratory during the period 2001–2012. Samples of allograft femoral heads were collected from living donors during hip operations. From the 3,859 donor episodes received, 21 paired swab and biopsy samples each recovered an isolate, 247 swab samples only and 79 biopsy samples only were culture positive. Low numbers of challenge organisms were recovered from inoculated swab and biopsy samples in the in vitro study and validated their use for bioburden testing of allograft musculoskeletal tissue. Skin commensals were the most common group of organisms isolated during a 12-year retrospective review of paired swab and biopsy samples from living donor allograft femoral heads. Paired swab and biopsy samples are a suitable representative sample of allograft musculoskeletal tissue for bioburden testing.     

Molecular testing of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> contaminating tissue allografts recovered from deceased donors

Microbiological screening of tissue allografts is crucial to prevent the transmission of bacterial and fungal infections to transplant recipients. Klebsiella was the most prevalent and resistant contaminating microorganism observed in our setting in the Iranian Tissue Bank. This study was conducted to determine the presence of extended-spectrum β-lactamase (ESBL) genes, antimicrobial resistance patterns of Klebsiella pneumoniae isolates, and their clonal relationships in allograft materials. K. pneumoniae contaminating bone and other tissue allografts recovered from deceased donors were identified and ESBL isolates were detected using a phenotypic confirmatory method. Antimicrobial susceptibility testing was carried out using the disk diffusion method. Distribution of ESBL genes and molecular typing were performed using polymerase chain reaction (PCR) and Repetitive-element (rep-PCR) methods. Of 3828 donated tissues, 51 (1.3%) were found contaminated by K. pneumoniae isolates. Compared to tissue allografts from brain-dead, heart-beating tissue donors, allografts from donors with circulatory cessation were associated with a higher risk of K. pneumoniae contamination [odds ratio (OR), 1.2 (CI 95% 0.9–2.3) (P value < 0.001)]. Half of the isolates produced ESBL, and the rate of susceptibility to cephalosporins was 51%. Among isolates, 22 (43.1%) harbored CTX-M, 31 (60.8%) SHV, and 9 (17.6%) harbored TEM types. The rep-dendrogram indicated that clones having identical or related strains with a similar antibiotype were isolated in the same period. This study provides evidence that a single clone of K. pneumoniae contaminated tissue allografts recovered from many different donors. A single clone found on tissues from several donors suggests contamination of tissues from a single source such as the tissue recovery process and environment. Genomic DNA testing and clonality of contaminating bacteria using molecular methods can focus the epidemiologic investigation on the tissue allograft recovery process including a search for contamination of the tissue recovery room environment, recovery staff, recovery equipment, reagents, solutions and supplies.     

Radioprotection provides functional mechanics but delays healing of irradiated tendon allografts after ACL reconstruction in sheep

Successful protection of tissue properties against ionizing radiation effects could allow its use for terminal sterilization of musculoskeletal allografts. In this study we functionally evaluate Achilles tendon allografts processed with a previously developed radioprotective treatment based on (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) crosslinking and free radical scavenging using ascorbate and riboflavin, for ovine anterior cruciate ligament reconstruction. Arthroscopic anterior cruciate ligament (ACL) reconstruction was performed using double looped allografts, while comparing radioprotected irradiated and fresh frozen allografts after 12 and 24 weeks post-implantation, and to control irradiated grafts after 12 weeks. Radioprotection was successful at preserving early subfailure mechanical properties comparable to fresh frozen allografts. Twelve week graft stiffness and anterior-tibial (A-T) translation for radioprotected and fresh frozen allografts were comparable at 30 % of native stiffness, and 4.6 and 5 times native A-T translation, respectively. Fresh frozen allograft possessed the greatest 24 week peak load at 840 N and stiffness at 177 N/mm. Histological evidence suggested a delay in tendon to bone healing for radioprotected allografts, which was reflected in mechanical properties. There was no evidence that radioprotective treatment inhibited intra-articular graft healing. This specific radioprotective method cannot be recommended for ACL reconstruction allografts, and data suggest that future efforts to improve allograft sterilization procedures should focus on modifying or eliminating the pre-crosslinking procedure.     

Role of the global regulator Rex in control of NAD+‐regeneration in Clostridioides (Clostridium) difficile

For the human pathogen Clostridioides (also known as Clostridium) difficile, the ability to adapt to nutrient availability is critical for its proliferation and production of toxins during infection. Synthesis of the toxins is regulated by the availability of certain carbon sources, fermentation products and amino acids (e.g. proline, cysteine, isoleucine, leucine and valine). The effect of proline is attributable at least in part to its role as an inducer and substrate of D‐proline reductase (PR), a Stickland reaction that regenerates NAD⁺ from NADH. Many Clostridium spp. use Stickland metabolism (co‐fermentation of pairs of amino acids) to generate ATP and NAD⁺. Synthesis of PR is activated by PrdR, a proline‐responsive regulatory protein. Here we report that PrdR, in the presence of proline, represses other NAD⁺‐generating pathways, such as the glycine reductase and succinate‐acetyl CoA utilization pathways leading to butyrate production, but does so indirectly by affecting the activity of Rex, a global redox‐sensing regulator that responds to the NAD⁺/NADH ratio. Our results indicate that PR activity is the favored mechanism for NAD⁺ regeneration and that both Rex and PrdR influence toxin production. Using the hamster model of C. difficile infection, we revealed the importance of PrdR‐regulated Stickland metabolism in the virulence of C. difficile.     

Tissue banking in Mexico

Introduction: Here, we describe our Tissue Banking experiences of 4 years of activity in Mexico. Methods: Data of allografts provided by our Bank and bone retrievals performed by our teams between February of 2001 and August of 2004 were included. Results: There were 100 bone donors, a total of 1107 tissues were obtained with an average of 11 tissues by retrieval, samples from all tissues were obtained during retrieval and cultured for bacterial contamination, 250 tissues were positives to bacterial growth with an average of 22.58% of bacterial contamination of tissue by retrieval. A total of 4493 allografts were provided and were utilized in 3643 patients. The allografts were used mainly by orthopedic surgeons (62%) and dentists (30%). The most used allografts were morcellized cancellous bone 31%, pulverized 25% and chips of cancellous bone 20%. Among orthopedic patients the most frequent procedures were related with spine degenerative diseases 39.09%, followed by non-pathological fractures and its complications 28.67% and bone tumors and cystic bone lesions 11.59%. Conclusions: Sustained increase of allograft utilization in Mexico reflects a great necessity for them in our country. The increase in public awareness about tissue donation has allowed an increase in tissue donations and retrievals.     

The effect of sterilization on mechanical properties of soft tissue allografts

One major concern regarding soft tissue allograft use in surgical procedures is the risk of disease transmission. Current techniques of tissue sterilization, such as irradiation have been shown to adversely affect the mechanical properties of soft tissues. Grafts processed using Biocleanse processing (a proprietary technique developed by Regeneration Technologies to sterilize human tissues) will have better biomechanical characteristics than tissues that have been irradiated. Fifteen pairs of cadaveric Achilles tendon allografts were obtained and separated into three groups of 10 each. Three treatment groups were: Biocleanse, Irradiated, and Control (untreated). Each specimen was tested to determine the biomechanical properties of the tissue. Specimens were cyclically preloaded and then loaded to failure in tension. During testing, load, displacement, and optical strain data were captured. Following testing, the cross sectional area of the tendons was determined. Tendons in the control group were found to have a higher extrinsic stiffness (slope of the load–deformation curve, p = .005), have a higher ultimate stress (force/cross sectional area, p = .006) and higher ultimate failure load (p = .003) than irradiated grafts. Biocleanse grafts were also found to be stiffer than irradiated grafts (p = .014) yet were not found to be statistically different from either irradiated or non-irradiated grafts in terms of load to failure. Biocleanse processing seems to be a viable alternative to irradiation for Achilles tendon allografts sterilization in terms of their biomechanical properties.     

The visual assessment of broth cultures for tissue bank samples

The bioburden screening process of allograft musculoskeletal tissue samples received at the South Eastern Area Laboratory Services includes the routine use of solid agar and cooked meat (CM) broth media. CM has been routinely sub-cultured onto solid agar plates after aerobic incubation at 35 °C. This study will evaluate whether a visual assessment of CM can replace sub-culture by an in vitro inoculation and a prospective study. Eight challenge organisms were serially diluted and inoculated into CM. The average inoculum of 0.5–5.5 CFU produced visible turbidity of CM after 24-h incubation for 7 of the challenge organisms with one organism producing turbidity after 48-h incubation. The prospective study evaluated 222 CM of which 213 were visually clear and no-growth on sub-culture and 9 turbid CM which were culture positive. Broth cultures are an integral part of the bioburden screening process of allograft musculoskeletal tissue and swab samples and visual assessment of CM can replace sub-culture.     

Culture methods of allograft musculoskeletal tissue samples in Australian bacteriology laboratories

Samples of allograft musculoskeletal tissue are cultured by bacteriology laboratories to determine the presence of bacteria and fungi. In Australia, this testing is performed by 6 TGA-licensed clinical bacteriology laboratories with samples received from 10 tissue banks. Culture methods of swab and tissue samples employ a combination of solid agar and/or broth media to enhance micro-organism growth and maximise recovery. All six Australian laboratories receive Amies transport swabs and, except for one laboratory, a corresponding biopsy sample for testing. Three of the 6 laboratories culture at least one allograft sample directly onto solid agar. Only one laboratory did not use a broth culture for any sample received. An international literature review found that a similar combination of musculoskeletal tissue samples were cultured onto solid agar and/or broth media. Although variations of allograft musculoskeletal tissue samples, culture media and methods are used in Australian and international bacteriology laboratories, validation studies and method evaluations have challenged and supported their use in recovering fungi and aerobic and anaerobic bacteria.     

Chitosan‐heparin polyelectrolyte multilayers on cortical bone: Periosteum‐mimetic,cytophilic, antibacterial coatings

Cortical bone allografts suffer from high rates of failure due to poor integration with host tissue, leading to non‐union, fracture, and infection following secondary procedures. Here, we report a method for modifying the surfaces of cortical bone with coatings that have biological functions that may help overcome these challenges. These chitosan‐heparin coatings promote mesenchymal stem cell attachment and have significant antibacterial activity against both S. aureus and E. coli. Furthermore, their chemistry is similar to coatings we have reported on previously, which effectively stabilize and deliver heparin‐binding growth factors. These coatings have potential as synthetic periosteum for improving bone allograft outcomes. Biotechnol. Bioeng. 2013; 110: 609–618. © 2012 Wiley Periodicals, Inc.