Disinfection protocol for human musculoskeletal allografts in tissue banking using hydrogen peroxide 30.

Musculoskeletal allografts are used in reconstructive procedures, however, the risk of contamination with potential pathogens is possible, and safe transplantation requires multiple processing considerations. Hydrogen peroxide (H2O2) has commonly been used in bone washing because it can remove donor cells and eliminate antigens, pathogens, or cytotoxic agents from the matrix. The aim of this study was to evaluate the quantitative activity of H2O2 in a model of bone contamination with a high bacterial load to define the bioburden reduction. Twelve bone disc models were artificially contaminated with Staphylococcus aureus. The bones were treated with a washing process composed by antibiotics, 30% hydrogen peroxide, and 70% alcohol. Tryptic Soy Agar plates were directly inoculated with 100µL of each step of the washing process and colonies were counted in CFU/mL. Scanning electron microscopy was used for bone structural analysis before and after the washing process. After antibiotics, there was a drop of less than 1 log for cancellous bone and almost 1 log for cortical bone. However, after H2O2, there as a drop of 3 logs for cortical (p = 0.007), and 2 logs for cancellous bone (p = 0.063). The use of alcohol did not change the bioburden following H2O2 in cancellous and cortical bone. Despite the important drop of bacterial load, H2O2 was not enough to completely eradicate bacterial with this model of bioburden. H2O2 is useful in decontamination, but antibiotics have little activity, and alcohol is useless. The process is useful in decontamination up to 3 logs of bioburden.

Determination of antibiotics and detergent residues in decellularized tissue-engineered heart valves using LC-MS/MS.

Residual chemicals that are presented during tissue processing in human tissue banks can be a risk for the allograft recipient. Determine the residual concentrations of the antibiotics and detergent used in the process of human decellularized tissue-engineered heart valves stored in isotonic saline solution up to 18 months. A total of 24 human decellularized allografts were stored in sterile sodium chloride and analyzed immediately after the decellularization process (0 months) and after storage for 6, 12, and 18 months, which includes the use of sodium dodecyl sulfate (SDS) and antibiotics (cefoxitin, vancomycin hydrochloride, lincomycin hydrochloride, polymyxin B sulfate). These valves were used for suitability tests, the zone of inhibition evaluation, and direct contact cytotoxicity assay. The stock solution from 32 valves was used for LC-MS/MS analysis of antibiotics and SDS. Tissue samples from decellularized valves showed a zone of inhibition formation for S. aureus and B. subtilis, suggesting the presence of an inhibitory molecule in the tissue. Cytotoxicity tests were negative. Polymyxin B, vancomycin, and SDS were detected and quantified in human decellularized aortic and pulmonary allografts during all periods of the study. There were no traces of residual cefoxitin and lincomycin in the tissue stock solution. We found residual concentrations of the antibiotics and detergent used in the process of human decellularized tissue-engineered heart valves stored in isotonic saline solution up to 18 months.

Risk factors associated with contamination of allograft valves in a tissue bank.

The contamination of the transport solution used in cardiovascular allografts can occur from different sources. Risk factors associated with positive microbiological test of transport solution have not been reported previously. This study aimed to determine the risk factor for contamination of transport solution used in the heart valve allografts stored in a Brazilian tissue bank. This retrospective study was conducted on all donors of cardiovascular allografts stored in a tissue bank from December 2008 to December 2017. Microbiological cultures for aerobic and anaerobic bacteria, fungi/yeasts were carried out in TS. Clinical variables were included. From 1001 transport solution, 52% were contaminated. A total of 770 microorganisms were identified, and Staphylococcus spp. was identified in 248 isolates (32.2%). Skin bacteria from skin microbiota were the most commonly identified microorganisms (Staphylococcus spp., Cutibacterium spp., Corynebacterium spp., and Bacillus spp.), occurring in 49.6%. The presence of a diagnosis of healthcare-associated infection was not associated with skin contamination (odds ratio [OR] 0.62 [0.41-0.94]; p = 0.014). Conditions like fever, use of antibiotics, and leukocytosis were less likely associated with contamination of transport solution. A longer warm ischemic time was associated with higher frequency of contamination. In the multivariable analysis, warm ischemic time was independently associated with contamination, and antibiotic therapy was a factor that decreased the rate of contamination (p < 0.05). Contamination of transport solution is associated with modifiable risk factors, such as warm ischemic time. Measures to minimize contamination should be employed to avoid unnecessary tissue discharges.

Bioburden in transport solutions of human cardiovascular tissues: a comparative evaluation of direct inoculation and membrane filter technique.

All cardiac allograft tissues are under potential contamination, requiring a validated terminal sterilization process or a minimal bioburden. The bioburden calculation is important to determine the bacterial burden and further decontamination and disinfection strategies for the valve processing. The aim of this study was to determine the bioburden from transport solution (TS) of heart valves obtained from non-heart-beating and heart-beating donors in different culture methods. The bioburden from TS was determined in 20 hearts donated for valve allograft tissue using membrane filter (MF) and direct inoculation. Tryptic soy agar and Sabouraud plates were incubated and colonies were counted. Ninety-five percent of samples from this study were obtained from heart-beating donors. The warm ischemic time period for heart was 1.06 ± 0.74 h and the cold ischemic time period was 25.66 ± 11.16 h. The mean TS volume was 232.68 ± 96.67 mL (48.5-550 mL). From 20 samples directly inoculated on TSA agar plates, 2 (10%) were positive. However, when MF was used, from 20 samples in TSA, 13 (65%) were positive with a mean count of 1.36 ± 4.04 CFU/mL. In Sabouraud plates, the direct inoculation was positive in 5 samples (25%) with a mean count of 0.24 ± 0.56 CFU/mL. The use of MF increased the positivity to 50% (10 samples from a total of 20) with a mean of 0.28 ± 0.68 CFU/mL. The positivity was superior using MF in comparison with direct inoculation (p < 0.05). The bioburden of TS is low and MF is the technique of choice due to higher positivity.

Comparison of automated and conventional microbiological examination of donated human cardiac tissue in heart valve banking.

Most tissue banks use the conventional method; however, the automated method has advantages over the conventional method. The aim of this study was to compare the conventional and automated methods of culture in human cardiac tissue using an artificial contamination model. Myocardial samples were contaminated with sequential concentration (104 to 10-1 CFU/mL) with Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans. Cultures were obtained from solution were the fragment was immersed and minced tissue, before and after the routine decellularization solution, with automated and conventional culture methods. Automated and conventional methods were compared and a p value ≤ 0.05 was considered significant. Staphylococcus aureus presented a significantly higher growth in the automated method, as well as faster than the conventional (p < 0.05). The positivity for growth in the automated method was higher in concentrated inoculum (> 102 CFU/mL) (p < 0.05). The growth in the automated method was significantly faster than conventional when inoculum concentration was above 103 CFU/mL. The automated culture method is faster than conventional method with a higher positivity in a contaminated model of myocardial and transport solution used in tissue banks.

Validation of Lyophilized Human Fecal Microbiota for the Treatment of Clostridioides difficile Infection: A Pilot Study with Pharmacoeconomic Analysis of a Middle-Income Country-Promicrobioma Project.

BACKGROUND: Clostridioides difficile infection (CDI) represents a prevalent and potentially severe health concern linked to the usage of broad-spectrum antibiotics. The aim of this study was to evaluate a new lyophilized product based on human fecal microbiota for transplant, including cost-benefit analysis in the treatment of recurrent or refractory CDI. METHODS: The product for fecal microbiota transplant was obtained from two donors. Microbiological, viability, and genomic analysis were evaluated. After validation, a clinical pilot study including recurrent or refractory CDI with 24 patients was performed. Clinical response and 4-week recurrence were the outcome. Cost-benefit analysis compared the fecal microbiota transplant with conventional retreatment with vancomycin or metronidazole. RESULTS: The microbiota for transplant presented significant bacterial viability, with and adequate balance of Firmicutes and Bacteroidetes. The clinical response with the microbiota transplant was 92%. In financial terms, estimated expenditure for CDI solely related to recurrence, based on stochastic modeling, totals USD 222.8 million per year in Brazil. CONCLUSIONS: The lyophilized human fecal microbiota for transplant is safe and can be an important step for a new product with low cost, even with genomic sequencing. Fecal microbiota transplantation emerges as a more cost-effective alternative compared to antimicrobials in the retreatment of CDI.

Histological and Biomechanical Characteristics of Human Decellularized Allograft Heart Valves After Eighteen Months of Storage in Saline Solution.

Background: The best storage preservation method for maintaining the quality and safety of human decellularized allograft heart valves is yet to be established. Objective: The aim of the present study was to evaluate the stability in terms of extracellular matrix (ECM) integrity of human heart valve allografts decellularized using sodium dodecyl sulfate-ethylenediaminetetraacetic acid (SDS-EDTA) and stored for 6, 12, and 18 months. Methods: A total of 70 decellularized aortic and pulmonary valves were analyzed across different storage times (0, 6, 12, and 18 months) for solution pH measurements, histological findings, cytotoxicity assay results, biomechanical test results, and microbiological suitability test results. Continuous data were analyzed using one-way analysis of variance comparing the follow-up times. Results: The pH of the stock solution did not change during the different time points, and no microbial growth occurred up to 18 months. Histological analysis showed that the decellularized allografts did not present deleterious outcomes or signs of structural degeneration in the ECM up to 12 months. The biomechanical properties showed changes over time in different aspects. Allografts stored for 18 months presented lower tensile strength and elasticity than those stored for 12 months (p < 0.05). The microbiological suitability test suggested no residual antimicrobial effects. Conclusion: Changes in the structure and functionality of SDS-EDTA decellularized heart valve allografts occur after 12 months of storage.

Anaerobic bioburden in transport solution of human cardiovascular tissues.

Although reports of infections caused by anaerobes after tissue transplantation are uncommon, contamination of allografts may result in substantial complications. Anaerobic incubation and testing of organ transport solution (TS) are not routine. The aim of this study was to determine the bioburden of strict anaerobic bacteria and oxygen tension of heart-TS. Forty TS from different donors were evaluated cultured using membrane filtration (MF), direct inoculation on broth and automated blood culture bottle (ABCB). Bacterial identification was performed by MALDI-TOF. The transport conditions were simulated to verify the bacterial recovery. A sterile bag fulfilled with 250 ml-1 of sterile saline was spiked with 100 CFU ml-1 of Clostridium perfringens and the fluid recovered 0 h, 1 h, 2 h, 6 h, 12 h, 24 h and 48 h for culture and oxygen measurement. Strict anaerobic bacteria were not isolated in heart-TS. The recovery of C.perfringens spiked in heart-TS was 100% using automated blood culture bottles. MF method detected >100 CFU only after 6 h of spiking. The manual culture was not able to recover C.perfringens after the process. The percentage of O2 measures varied from 77.6 to 87.9%. MF or ABCB are better than direct inoculation for recovery of anaerobes from heart-TS. Although all samples from heart donors were negative for anaerobes (probably due to low incidence of contamination), C.perfringens were all recovered in the simulated transport condition.

Conventional culture method and qPCR using 16S rDNA for tissue bank: a comparison using a model of cardiac tissue contamination.

Real-time polymerase chain reaction (qPCR) using 16S rDNA is an alternative to conventional culture-based tests. The aim of this study was to compare the conventional culture method with qPCR using 16S rDNA in a model of cardiac tissue contamination. Samples of cardiac tissue for artificial contamination with Escherichia coli and control samples were submitted for DNA extraction, which was conducted by selective and alkaline lysis and purification steps. A standard curve for 16S rDNA was constructed to determine the efficiency and analytical sensitivity of the assay in concentrations from 106 to 102 c.f.u. ml-1 using TaqMan Master Mix. 16S rDNA was detected in all contaminated samples; however, it was not detected in the the final washing step solution of the sample with a bioburden of 102 c.f.u. ml-1. Using qPCR is a potential alternative to conventional culture for microbiological safety testing of allograft tissues for biobanking, reducing the time and labour input required.

Mobile health application to assist doctors in antibiotic prescription - an approach for antibiotic stewardship.

BACKGROUND: Technologies applied to mobile devices can be an important strategy in antibiotic stewardship programs. OBJECTIVE: The aim of this study was to determine the impact of a decision-making application on antibiotic prescription. METHODS: This was an observational, analytical and longitudinal study on the implementation of an antimicrobial guide for mobile application. This study analyzed the period of 12 months before and 12 months after the app implementation at a university hospital based on local epidemiology, avoiding high cost drugs and reducing the potential for drug resistance including carbapenem. Antimicrobials consumption was evaluated in Daily Defined Dose/1000 patients-day and direct expenses converted into USD. RESULTS: The monthly average consumption of aminoglycosides and cefepime had a statistically significant increase (p<0.05), while the consumption of piperacillin/tazobactam and meropenem was significantly decreased (p<0.05). The sensitivity to meropenem as well as to polymyxin increased after the app implementation. A decrease in sensitivity to cefepime was observed after introduction of this antibiotic as a substitute of piperacillin/tazobactam for treating intra-hospital infections. There was a net saving of USD 296,485.90 (p<0.05). CONCLUSION: An antibiotic protocol in the app can help antibiotic stewardship reducing cost, changing the microbiological profile and antimicrobial consumption.

Mobile health application to assist doctors in antibiotic prescription - an approach for antibiotic stewardship

ABSTRACT Background: Technologies applied to mobile devices can be an important strategy in antibiotic stewardship programs. Objective: The aim of this study was to determine the impact of a decision-making application on antibiotic prescription. Methods: This was an observational, analytical and longitudinal study on the implementation of an antimicrobial guide for mobile application. This study analyzed the period of 12 months before and 12 months after the app implementation at a university hospital based on local epidemiology, avoiding high cost drugs and reducing the potential for drug resistance including carbapenem. Antimicrobials consumption was evaluated in Daily Defined Dose/1000 patients-day and direct expenses converted into USD. Results: The monthly average consumption of aminoglycosides and cefepime had a statistically significant increase (p < 0.05), while the consumption of piperacillin/tazobactam and meropenem was significantly decreased (p < 0.05). The sensitivity to meropenem as well as to polymyxin increased after the app implementation. A decrease in sensitivity to cefepime was observed after introduction of this antibiotic as a substitute of piperacillin/tazobactam for treating intra-hospital infections.There was a net saving of USD 296,485.90 (p < 0.05). Conclusion: An antibiotic protocol in the app can help antibiotic stewardship reducing cost, changing the microbiological profile and antimicrobial consumption.

Effects of cryopreservation and/or decellularization on extracellular matrix of porcine valves.

The objective of this study was to evaluate the morphology of decellularized and/or cryopreserved porcine pulmonary valves, to determine a solution capable of completely remove the cells without damaging the extracellular matrix. Porcine pulmonary valves were incubated for 24 hs in sodium deoxicholate 1% or sodium dodecyl sulfate 0.1 and 0.3%, with or without associated cryopreservation. Evaluation was done with optical microscopy (Hematoxilin-Eosin, Acetic Orcein and Gomori) and with morphometric analysis. The effectiveness of the solutions was variable, but the best results were obtained with the sodium dodecyl sulfate solution 0.1%.

Efeito da criopreservação e/ou da descelularização na matriz extracelular de condutos valvados porcinos / Effects of cryopreservation and/or decellularization on extracellular matrix of porcine valves

O objetivo desse estudo foi avaliar a morfologia de valvas pulmonares porcinas criopreservadas e/ou descelularizadas para determinar uma solução que remova as células, sem promover danos à matriz extracelular. Valvas pulmonares porcinas foram incubadas por 24h em soluções de deoxicolato de sódio 1% e de dodecil sulfato de sódio 0,1% e 0,3%, com ou sem criopreservação adicional. A avaliação foi feita com microscopia óptica (hematoxilina eosina, orceína acética ou Gomori) e por morfometria. A efetividade das soluções foi variável, mas os melhores resultados foram obtidos com enxertos frescos descelularizados com dodecil sulfato de sódio 0,1%.

The objective of this study was to evaluate the morphology of decelluarized and/or cryopreserved porcine pulmonary valves, to determine a solution capable of completely remove the cells without damaging the extracellular matrix. Porcine pulmonary valves were incubated for 24 hs in sodium deoxicholate 1% or sodium dodecyl sulfate 0.1 and 0.3%, with or without associated cryopreservation. Evaluation was done with optical microscopy (Hematoxilin-Eosin, Acetic Orcein and Gomori) and with morphometric analysis. The effectiviness of the solutions was variable, but the best results were obtained with the sodium dodecyl sulfate solution 0.1%.