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.     

Assessment of bioburden on human and animal tissues: Part 2—Results of testing of human tissue and qualification of a composite sample for routine bioburden determination

A quantitative method was developed and validated to assess bioburden on tissue from human donors and to compare bioburden determination results to swab culture results from the same donor. An initial study with allograft tissue from 101 donors showed a wide range of bioburden levels; values from no colony-forming units (CFU) detected to?>28,000?CFU were observed. Tissues from donors that had swab cultures negative for objectionable microorganisms generally had lower bioburden than tissues from donors where objectionable microorganisms were recovered by swab culturing. In a follow-up study with 1,445 donors, a wide range of bioburden levels was again observed on tissues from donors that were swab culture negative for objectionable microorganisms. Tissues from 885 (61%) of these donors had no recoverable bioburden (<2?CFU). Importantly, tissues from 560 (39%) of the donors had recoverable bioburden which ranged from 1 to?>24,000?CFU. Identification of bioburden isolates showed a diversity of genera and species. In compliance with the recent revision of the American Association of Tissue Banks K2.210 Standard, the quantitative bioburden determination method was validated with a composite tissue sample that contains bone and soft tissue sections tested together in one extraction vessel. A recovery efficiency of 68% was validated and the composite sample was shown to be representative of all of the tissues recovered from a donor. The use of the composite sample in conjunction with the quantitative bioburden determination method will facilitate an accurate assessment of the numbers and types of contaminating microorganisms on allografts prior to disinfection/sterilization. This information will ensure that disinfection/sterilization processes are properly validated and the capability of the overall allograft process is understood on a donor by donor basis.     

Validation of 15 kGy as a radiation sterilisation dose for bone allografts manufactured at the Queensland Bone Bank: application of the VD<Subscript>max</Subscript> 15 method

Background ISO 11137-2006 (ISO 11137-2a 2006) provides a VD_max 15 method for substantiation of 15 kGy as radiation sterilisation dose (RSD) for health care products with a relatively low sample requirement. Moreover, the method is also valid for products in which the bioburden level is less than or equal to 1.5. In the literature, the bioburden level of processed bone allografts is extremely low. Similarly, the Queensland Bone Bank (QBB) usually recovers no viable organisms from processed bone allografts. Because bone allografts are treated as a type of health care product, the aim of this research was to substantiate 15 kGy as a RSD for frozen bone allografts at the QBB using method VD_max 15—ISO 11137-2: 2006 (ISO 11137-2e, Procedure for method VD_max 15 for multiple production batches. Sterilisation of health care products – radiation – part 2: establishing the sterilisation dose, 2006; ISO 11137-2f, Procedure for method VD_max 15 for a single production batch. Sterilisation of health care products – radiation – part 2: establishing the sterilisation dose, 2006). Materials 30 femoral heads, 40 milled bone allografts and 40 structural bone allografts manufactured according to QBB standard operating procedures were used. Method Estimated bioburdens for each bone allograft group were used to calculate the verification doses. Next, 10 samples per group were irradiated at the verification dose, sterility was tested and the number of positive tests of sterility recorded. If the number of positive samples was no more than 1, from the 10 tests carried out in each group, the verification was accepted and 15 kGy was substantiated as RSD for those bone allografts. Results The bioburdens in all three groups were 0, and therefore the verification doses were 0 kGy. Sterility tests of femoral heads and milled bones were all negative (no contamination), and there was one positive test of sterility in the structural bone allograft. Accordingly, the verification was accepted. Conclusion Using the ISO validated protocol, VD_max 15, 15 kGy was substantiated as RSD for frozen bone allografts manufactured at the QBB.     

Fresh-frozen human bone allograft in vertical ridge augmentation: clinical and tomographic evaluation of bone formation and resorption

The aim of the current study is to evaluate fresh-frozen human bone allografts (FHBAs) used in vertical ridge augmentation clinically and by computed tomography, and to analyze the resulting bone formation and graft resorption. Sixteen FHBAs were grafted in the maxillae and mandibles of 9 patients. The FHBAs, which were provided by the Musculoskeletal Tissue Bank of Marilia Hospital (Unioss), were frozen at -80°C. After 7?months, dental implants were placed and bone parameters were evaluated. Vertical bone formation was measured by computerized tomography before (T0) and at 7?months (T1) after the surgical procedure. Bone graft resorption was measured clinically from a landmark screw head using a periodontal probe. The results were analyzed by Student's t-test. Significant differences existed in the bone formation values at T0 and T1, with an average change of 4.03?±?1.69?mm. Bone graft resorption values were 1.0?±?0.82?mm (20%). Implants were placed with varying insertion torque values (35-45?Ncm), and achieved primary stability. This study demonstrates that FHBAs promote satisfactory vertical bone formation with a low resorption rates, good density, and primary implant stability.     

Tissue banking in South Africa: a 19-year history

The establishment of a Tissue Bank and the science of Tissue Banking in South Africa started in the 1960s and is still developing. This article describes the development and growth of Tissue Banking in South Africa. The current emphasis is to supply viable bone products that have been produced under the best possible quality controlled circumstances; with the collaboration between different Organ Donation Organisations. Through collaboration, a dramatic increase in the number of donors was noted over the years. Concurrently, there was a dramatic increase in the usage of different allograft products and now necessitates the development of new graft materials for expanding patient options. As an ongoing concern, the Tissue Bank in South Africa experienced an ever increase in costs to enhance quality/safety controls: increase in historical patient information, documentation and serological testing in a population struggling to control HIV. To date, the South African Tissue Bank has not experienced any untoward patient incidence since the 1960s and currently is getting over 500 donors per year.     

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.     

Spatiotemporal Aspects of Hendra Virus Infection in Pteropid Bats (Flying-Foxes) in Eastern Australia

Hendra virus (HeV) causes highly lethal disease in horses and humans in the eastern Australian states of Queensland (QLD) and New South Wales (NSW), with multiple equine cases now reported on an annual basis. Infection and excretion dynamics in pteropid bats (flying-foxes), the recognised natural reservoir, are incompletely understood. We sought to identify key spatial and temporal factors associated with excretion in flying-foxes over a 2300 km latitudinal gradient from northern QLD to southern NSW which encompassed all known equine case locations. The aim was to strengthen knowledge of Hendra virus ecology in flying-foxes to improve spillover risk prediction and exposure risk mitigation strategies, and thus better protect horses and humans. Monthly pooled urine samples were collected from under roosting flying-foxes over a three-year period and screened for HeV RNA by quantitative RT-PCR. A generalised linear model was employed to investigate spatiotemporal associations with HeV detection in 13,968 samples from 27 roosts. There was a non-linear relationship between mean HeV excretion prevalence and five latitudinal regions, with excretion moderate in northern and central QLD, highest in southern QLD/northern NSW, moderate in central NSW, and negligible in southern NSW. Highest HeV positivity occurred where black or spectacled flying-foxes were present; nil or very low positivity rates occurred in exclusive grey-headed flying-fox roosts. Similarly, little red flying-foxes are evidently not a significant source of virus, as their periodic extreme increase in numbers at some roosts was not associated with any concurrent increase in HeV detection. There was a consistent, strong winter seasonality to excretion in the southern QLD/northern NSW and central NSW regions. This new information allows risk management strategies to be refined and targeted, mindful of the potential for spatial risk profiles to shift over time with changes in flying-fox species distribution.     

The impact of the International Atomic Energy Agency (IAEA) program on radiation and tissue banking in Malaysia

The IAEA was instrumental in developing the first Malaysian tissue bank at University Hospital of Universiti Sains Malaysia (HUSM), Kubang Kerian, Kelantan in early 1990s and it was officiated as National Tissue Bank in 1994. Up to date, 38 government and private hospitals have received a supply from the bank. Bone allografts in term of bone chips, morsalised bone and long bones are procured from Malaysian donors. Almost thirty students from Malaysia graduated in the training courses carried out in Singapore since 1998 at regional and interregional levels. Organ donation is more readily accepted by the public at the moments, perhaps due to the vast promotion and advertisement given by the local newspapers and other media, but gradually tissue donation is catching up as well.     

The emergence and control of overwintering Helicoverpa armigera pupae in southern New South Wales

Abstract  The extent and timing of the spring emergence of Helicoverpa armigera moths from overwintering pupae was measured in southern New South Wales (NSW). Survival was comparable to that in northern NSW, suggesting that the colder conditions did not significantly reduce the survival of overwintering pupae. Most adults emerged from mid-October to mid-November, approximately 2 weeks later than reported in northern NSW. The pattern of emergence is similar to that predicted by the HElicoverpa Armigera and Punctigera Simulation (HEAPS) model, although it tended to be earlier than predicted. Simulation of the pattern of emergence from 1990 to 2001 predicted a range of approximately 4 weeks in the date of emergence. This suggests that moths will not emerge in this part of southern NSW before October. The impact of locally used cultivation techniques on the survival of overwintering pupae supported previous findings highlighting the need for either plant line or full cultivation to ensure adequate control of overwintering pupae. Neither direct drilling nor shallow cultivation (Lillistons) provided consistently reliable control. Practical management of overwintering pupae in the region requires adequate cultivation before October     

NOTE PROROCENTRUM LIMA (DINOPHYCEAE) IN WATERS OF THE GREAT SOUTH CHANNEL NEAR GEORGES BANK

A 0–10 m plankton tow collected at one station in the Great South Channel near Georges Bank in June 1994 was rich in dinoflagellates, with the usually epibenthic Prorocentrum lima (Ehrenberg) Dodge present as the dominant species. Temperature and salinity profiles suggest that the phytoplankton population at this station may have been advected from waters on the southern flank of Georges Bank. This is the first unequivocal report of P. lima in the plankton community of the Great South Channel and its vicinity.     

西双版纳、白茫雪山、波罗的海南岸地衣纯培养放线菌多样性

【背景】在地衣共生系统中除了共生真菌、藻类以外,还蕴藏着丰富的放线菌资源。【目的】采集来自云南西双版纳、白茫雪山、德国波罗的海南岸3个地区的地衣,对获得的地衣纯培养放线菌进行多样性分析。【方法】采用3种放线菌选择分离培养基,通过平板稀释涂布法分离放线菌。通过比较16S rRNA基因序列相似性以及构建系统发育树,确定纯培养放线菌的分类地位。【结果】共分离菌株1 123株,鉴定417株。其中从西双版纳17份地衣样品中分离纯化到107株放线菌,分布在7个目14个科33个属,潜在新种18株,其中链霉菌为优势菌属;从白茫雪山7份地衣样品中分离纯化到103株放线菌,分布在4个目5个科9个属,潜在新种16株。其中链霉菌为优势菌属,占比39%;从波罗的海南岸5份地衣样品中分离纯化到65株放线菌,分布在4个目8个科18个属,潜在新种5株,潜在新种菌和链霉菌为优势菌属。【结论】在本研究条件下,西双版纳可培养地衣放线菌多样性较白茫雪山和波罗的海南岸丰富。白茫雪山地衣链霉菌居多,潜在新种占比15.5%。3个地区地衣放线菌的区系组成各不相同,这与3个地区地衣所处地理环境、完全不同的气候密切相关。     

A century of tobamovirus evolution in an Australian population of Nicotiana glauca.

The evolution over the past century of two tobamoviruses infecting populations of the immigrant plant Nicotiana glauca in New South Wales (NSW), Australia, has been studied. This plant species probably entered Australia in the 1870s. Isolates of the viruses were obtained from N. glauca specimens deposited in the NSW Herbarium between 1899 and 1972, and others were obtained from living plants in 1985 and 1993. It was found that the NSW N. glauca population was infected with tobacco mosaic tobamovirus (TMV) and tobacco mild green mosaic tobamovirus (TMGMV) before 1950 but only with TMGMV after that date. Half the pre-1950 infections were mixtures of the two viruses, and one was a recombinant. Remarkably, sequence analyses showed no increase in the genetic diversity among the TMGMV isolates over the period. However, for TMV, the genetic diversity of synonymous (but not of nonsynonymous) differences between isolates varied and was correlated with their time of isolation. TMV accumulated to smaller concentrations than TMGMV in N. glauca plants, and in mixed experimental infections, the accumulation of TMV, but not of TMGMV, was around 1/10 that in single infections. However, no evidence was found of isolate-specific interaction between the viruses. We conclude that although TMV may have colonized N. glauca in NSW earlier or faster than TMGMV, the latter virus caused a decrease of the TMV population below a threshold at which deleterious mutations were eliminated. This phenomenon, called Muller's ratchet, or a "mutational meltdown," probably caused the disappearance of TMV from the niche.     

Size of the first spring generation of Helicoverpa punctigera (Wallengren) (Lepidoptera: Noctuidae) and winter rain in central Australia

Serious infestations of Helicoverpa punctigera are experienced yearly in the eastern cropping regions of Australia. Regression analysis was used to determine whether the size of the first generation in spring (G₁), which is comprised mostly of immigrants from inland Australia, was related to monthly rainfall in inland winter breeding areas. Data from two long series of light-trap catches at Narrabri in New South Wales (NSW) and Turretfield in South Australia (SA) were used in the analyses. The size of G₁ at Narrabri in each year was significantly regressed on the amount of rainfall in western Queensland and NSW in May and June. The size of G₁ at Turretfield each year was significantly regressed on the amount of rain in May, June and July in western Queensland and NSW and also in the desert of central Western Australia. Low r ² values of the regressions suggest that rainfall data for more sites, as well as biological and other physical factors, such as temperature, evaporation, and prevailing wind systems, may need to be included to improve forecasts of the potential magnitude of the infestations in coastal cropping regions.     

Role of lipids in bone marrow-induced pulmonary edema

We examined the mechanism of the bone marrow-induced pulmonary edema in the isolated Ringer-perfused rabbit lung. Bone marrow administration (0.2 ml/kg body wt) increased pulmonary arterial pressure, capillary pressure, arterial resistance, and venous resistance within 2-4 min. Bone marrow also produced marked increases in lung wet weight and the capillary filtration coefficient but at later time points (90-120 min) during the perfusion. Only the triglyceride-containing lipid component of the bone marrow produced increases in pulmonary hemodynamics, lung wet weight, and the capillary filtration coefficient comparable to those observed after bone marrow. Bone marrow and the lipid component of bone marrow both produced increases in venous effluent lipoprotein lipase activity (the enzyme responsible for hydrolysis of triglycerides to free fatty acids). Bone marrow also stimulated the production of thromboxane B2 but not 6-ketoprostaglandin F1 alpha in the perfused lung. Both meclofenamate (1 microM), a cyclooxygenase inhibitor, and U-60,257 (10 microM), a lipoxygenase inhibitor, attenuated the bone marrow-induced pulmonary hemodynamic response, whereas only U-60,257 attenuated the increases in lung wet weight and the capillary filtration coefficient. In conclusion, pulmonary embolization induced by bone marrow results in increases in lung weight and the capillary filtration coefficient in the isolated Ringer-perfused rabbit lung. Pulmonary vasoconstriction is partially dependent on arachidonic acid metabolites but appears to be independent of circulating blood-formed elements. The lipid component of bone marrow or products derived from this component (e.g., free fatty acids and lipoxygenase products) may mediate the bone marrow-induced pulmonary edema.     

Remodeling of heat-treated cortical bone allografts for posterior lumbar interbody fusion: serial 10-year follow-up

We have selected heat-treated bone allografts as the graft material since the Tokai Bone Bank, the first regional bone bank in Japan, was established in 1992. In this study, we examined changes in bone mineral density (BMD), and morphology observed by magnetic resonance imaging (MRI), and histological findings of bone grafts in cases followed up for 7-10?years after bone grafting to grasp the remodeling of heat-treated cortical bone allografts for posterior lumber interbody fusion (PLIF). BMD of bone grafts was reduced by half at 10?years after grafting. MRI revealed that bone grafts were indistinguishable initially in only 22.2% of cases, whereas after a lengthy period of 10?years distinguishable in many cases. Histologically, new bone formation at the graft-host interface was observed earlier, at 1?year after grafting, than that at the periphery of canals in the specimens. The laminated structure of the cortical bone eroded over time, and fragmented bone trabeculae were observed in the specimens at 8?years or longer after grafting, though necrotic bone still remained in some sites.     

A stochastic bioburden model for spacecraft sterilization

An important factor in the calculation of sterilization cycles for spacecraft is the bioburden on the surface of the spacecraft at the start of the cycle. This bioburden must be predicted by the use of models. This paper presents a stochastic model for the prediction of the bioburden on the surfaces of spacecraft. This model has many desirable properties as well as being consistent with observations.This work was conducted under Contract No. W-12,853, Planetary Programs, Office, of Space Science and Applications, NASA Headquarters, Washington, D.C.     

In vitro expression of osteoblastic markers in cells isolated from normal fetal and postnatal human bone and from bone of patients with osteogenesis imperfecta

We studied the expression of osteoblastic markers in cultured cells isolated from the bone of 15 patients with different clinical forms of osteogenesis imperfecta (OI) and of seven fetal and postnatal controls. Cultured bone cells of ten OI patients produced abnormal collagen type I. Similar to controls, OI bone cells produced predominantly collagen type I with traces of collagen types III and V. The 1,25(OH)₂ vitamin D₃-stimulated synthesis of osteocalcin, a specific osteoblastic marker protein, was similar in OI bone cells and age-matched controls. Bone cells from fetal controls and from patients with the perinatal lethal OI type II produced less osteocalcin than bone cells from postnatal controls and surviving OI patients. OI bone cells responded to parath.yroid hormone (PTH) by increased production of cAMP similar to controls. Bone cells from fetal controls and from OI type II donors showed a decreased response to PTH. Activity of the bone-liver-kidney isoenzyme alkaline phosphatase (AP) was detected in all control and OI bone cells. The expression of all osteoblastic markers was similar in bone cells producing abnormal collagen type I. These observations show that OI bone cells in vitro express a pattern of osteoblastic markers similar to age-matched control bone cells indicating that osteoblastic differentiation is not altered by the underlying defects of collagen type I metabolism in OI bone cells. © 1993 Wiley-Liss, Inc.     

Evaluation of the establishment of Aphidius ervi Haliday (Hymenoptera: Braconidae) in lucerne aphid populations in New South Wales

The aphid parasitoid Aphidius ervi was released in the major lucerne-growing areas of New South Wales (NSW), Australia, between 1978 and 1981. With the collaboration of district agronomists of the New South Wales Department of Agriculture, five State-wide surveys were conducted in 1982–1983 to determine the success of the release program. In each survey, the distribution of the parasitoid was checked in relation to populations of the aphids Acyrthosiphon kondoi Shinji and Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae). The surveys confirmed the successful dispersal and establishment of A. ervi in the major lucerne-growing areas of NSW. They demonstrated its ability to survive and recover rapidly after a severe and widespread drought.     

Isolation and characterization of microsatellite markers for the waratah,Telopea speciosissima (Proteaceae)

Thirteen microsatellite loci were developed from an (AG)‐enriched library using Telopea speciosissima, a widespread species of dry‐sclerophyll forest in New South Wales (NSW, Australia). Genotyping of 30 seeds from one population of T. speciosissima and 15 seeds from one population of the related Telopea aspera (restricted to the Gibraltar Range in northern NSW) was successfully performed. The loci were also tested across seven other related species. The microsatellites will be used to compare population dynamics across a range of taxa representing different distribution patterns.     

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.