Pharmacokinetic-pharmacodynamic modeling of tuberculosis time to positivity and colony-forming unit to assess the response to dose-ranging linezolid.

According to the World Health Organization, the number of tuberculosis (TB) infections and the drug-resistant burden worldwide increased by 4.5% and 3.0%, respectively, between 2020 and 2021. Disease severity and complexity drive the interest for undertaking new clinical trials to provide efficient treatment to limit spread and drug resistance. TB Alliance conducted a phase 2 study in 106 patients to guide linezolid (LZD) dose selection using early bactericidal activity over 14 days of treatment. LZD is highly efficient for drug-resistant TB treatment, but treatment monitoring is required since serious adverse events can occur. The objective of this study was to develop a pharmacokinetic-pharmacodynamic (PKPD) model to analyze the dose-response relationship between linezolid exposure and efficacy biomarkers. Using time to positivity (TTP) and colony-forming unit (CFU) count data, we developed a PKPD model in six dosing regimens, differing on LZD dosing intensity. A one-compartment model with five transit absorption compartments and non-linear auto-inhibition elimination described best LZD pharmacokinetic characteristics. TTP and CFU logarithmic scaled [log(CFU)] showed a bactericidal activity of LZD against Mycobacterium tuberculosis. TTP was defined by a model with two significant covariates: the presence of uni- and bilateral cavities decreased baseline TTP value by 24%, and an increase on every 500 mg/L/h of cumulative area under the curve increased the rate at which TTP and CFU change from baseline by 20% and 11%, respectively. CLINICAL TRIALS: This study is registered with ClinicalTrials.gov as NCT02279875.

Alterations in cell arrangements of group B streptococcus due to virulence factor expression can bias estimates of bacterial populations based on colony count measures.

Group B streptococcus (GBS) is a chain-forming commensal bacterium and opportunistic pathogen that resides in the gastrointestinal and genitourinary tract of healthy adults. GBS can cause various infections and related complications in pregnant and nonpregnant women, adults, and newborns. Investigations of the mechanisms by which GBS causes disease pathogenesis often utilize colony count assays to estimate bacterial population size in experimental models. In other streptococci, such as group A streptococcus and pneumococcus, variation in the chain length of the bacteria that can occur naturally or due to mutation can affect facets of pathogenesis, such as adherence to or colonization of a host. No studies have reported a relationship between GBS chain length and pathogenicity. Here, we used GBS strain 874391 and several derivative strains displaying longer chain-forming phenotypes (874391pgapC, 874391ΔcovR, 874391Δstp1) to assess the impact of chain length on bacterial population estimates based on the colony-forming unit (c.f.u.) assay. Disruption of GBS chains via bead beating or sonication in conjunction with fluorescence microscopy was used to compare chaining phenotypes pre- and post-disruption to detect long- and short-chain forms, respectively. We used a murine model of GBS colonization of the female reproductive tract to assess whether chaining may affect bacterial colonization dynamics in the host during chronic infection in vivo. Overall, we found that GBS exhibiting long-chain form can significantly affect population size estimates based on the colony count assay. Additionally, we found that the length of chaining of GBS can affect virulence in the reproductive tract colonization model. Collectively, these findings have implications for studies of GBS that utilize colony count assays to measure GBS populations and establish that chain length can affect infection dynamics and disease pathogenesis for this important opportunistic pathogen.

Fluorescence Spectroscopy Based Identification of Pseudomonas Aeruginosa and Escherichia Coli Suspensions.

In this article, Fluorescence spectroscopy has been employed for the identification of Pseudomonas aeruginosa (PA) and Escherichia coli (E. coli) in water suspension. Emission spectra of PA and E. coli suspensions have been acquired by using excitation wavelengths from 270 to 420 nm with steps of 10 nm to explore their spectral features. It has been found that the emission spectra of tryptophan, tyrosine, NADH and FAD, being the intracellular biomolecules present in both bacteria, can be used as fingerprints for their identification, differentiation and quantification. Both bacterial strains can clearly be differentiated from water and from each other by using λex 270-290 nm through spectral analysis and from λex: 300-500 nm by applying statistical analysis. Furthermore, calibration curves for different bacterial loads of PA and E. coli suspensions have been produced between colonies forming units per ml (CFUs/ml) the integrated intensities of their emission spectra. CFUs/ml of both bacterial suspensions have been determined through plate count method which was used as cross-reference for the analysis of emission spectra of both bacterial suspensions. These curves may be used to estimate CFU/ml of both PA and E. coli in unknown water suspensions by determining the integrating intensity of their emission spectra.

Improved automated spot counting and modeling with bias correction.

A complete workflow was presented for estimating the concentration of microorganisms in biological samples by automatically counting spots that represent viral plaque forming units (PFU) bacterial colony forming units (CFU), or spot forming units (SFU) in images, and modeling the counts. The workflow was designed for processing images from dilution series but can also be applied to stand-alone images. The accuracy of the methods was greatly improved by adding a newly developed bias correction method. When the spots in images are densely populated, the probability of spot overlapping increases, leading to systematic undercounting. In this paper, this undercount issue was addressed in an empirical way. The proposed empirical bias correction method utilized synthetic images with known spot sizes and counts as a training set, enabling the development of an effective bias correction function using a thin-plate spline model. Its application focused on the bias correction for the automated spot counting algorithm LoST proposed by Lin et al. Simulation results demonstrated that the empirical bias correction significantly improved spot counts, reducing bias for both fixed and random spot sizes and counts.

Fluorescence Spectroscopy Based Characterization of Pseudomonas Aeruginosa Suspension.

In this article, optical characterization of Pseudomonas aeruginosa (PA) suspension has been performed by using Fluorescence spectroscopy. Optical density (OD) and plate count methods have been employed as a reference for the analysis of emission spectra of Pseudomonas aeruginosa in water suspension. Emission spectra of PA suspension has been acquired by using excitation wavelengths from 270 to 420 nm with step of 10 nm to explore its spectral behavior. It has been found that emission spectra of tryptophan, tyrosine, NADH and FAD, the intracellular biomolecules of bacteria, can be used as finger prints for the detection of Pseudomonas aeruginosa. Furthermore, the effect of water matrix on the spectral emission of Pseudomonas aeruginosa has been investigated that might be one of the limitation of Fluorescence spectroscopy for complex water matrices. Moreover, a calibration curve has been produced between ODs600 of Pseudomonas aeruginosa suspensions of different bacterial load and integrated intensities of the emission spectra of same samples. These ODs600 and integrating intensities have been further vetted through plate count method by determining their corresponding colony forming units per ml (CFU/ml). This calibration curve may be used to determine CFU/ml of Pseudomonas aeruginosa in water sample by determining integrating intensity of its emission spectrum.

Microbial adhesion and viability on novel CAD/CAM framework materials for implant-supported hybrid prostheses.

The aim of the study was to investigate the adhesion and viability of Streptococcus oralis and Candida albicans under in vitro conditions on CAD/CAM framework materials for implant-supported hybrid prostheses. Twenty-nine specimens were prepared from each of three different materials: ZR (zirconia), PEEK (polyether ether ketone) and CoCr4 (CoCr4 alloy). The experimental part included surface roughness (SR) and contact angle of water (CAW) measurements, followed by colony forming unit (CFU), cell viability assay and scanning electron microscopy (SEM) analyses of Strep. oralis and C. albicans biofilms on the materials' surfaces. Kruskal-Wallis and one-way analysis of variance (ANOVA) tests were used for differences between materials, and the correlation between measurements was estimated using Spearman's correlation coefficient. PEEK specimens revealed higher SR, CAW and CFU mean values, than ZR and CoCr4 specimens. Strong positive correlation was found between SR and CFU and between CAW and CFU for both microbial species. Cell viability assay revealed similar values for both species across materials. Higher numbers of Strep. oralis and C. albicans on PEEK specimens confirm the impact of the higher surface roughness and contact angle values on the microbial adhesion and describes PEEK as less desirable than ZR and CoCr4 from microbiological aspect.

Hybrid Approach to Colony-Forming Unit Counting Problem Using Multi-Loss U-Net Reformulation.

Colony-Forming Unit (CFU) counting is a complex problem without a universal solution in biomedical and food safety domains. A multitude of sophisticated heuristics and segmentation-driven approaches have been proposed by researchers. However, U-Net remains the most frequently cited and used deep learning method in these domains. The latter approach provides a segmentation output map and requires an additional counting procedure to calculate unique segmented regions and detect microbial colonies. However, due to pixel-based targets, it tends to generate irrelevant artifacts or errant pixels, leading to inaccurate and mixed post-processing results. In response to these challenges, this paper proposes a novel hybrid counting approach, incorporating a multi-loss U-Net reformulation and a post-processing Petri dish localization algorithm. Firstly, a unique innovation lies in the multi-loss U-Net reformulation. An additional loss term is introduced in the bottleneck U-Net layer, focusing on the delivery of an auxiliary signal that indicates where to look for distinct CFUs. Secondly, the novel localization algorithm automatically incorporates an agar plate and its bezel into the CFU counting techniques. Finally, the proposition is further enhanced by the integration of a fully automated solution, which comprises a specially designed uniform Petri dish illumination system and a counting web application. The latter application directly receives images from the camera, processes them, and sends the segmentation results to the user. This feature provides an opportunity to correct the CFU counts, offering a feedback loop that contributes to the continued development of the deep learning model. Through extensive experimentation, the authors of this paper have found that all probed multi-loss U-Net architectures incorporated into the proposed hybrid approach consistently outperformed their single-loss counterparts, as well as other comparable models such as self-normalized density maps and YOLOv6, by at least 1% to 3% in mean absolute and symmetric mean absolute percentage errors. Further significant improvements were also reported through the means of the novel localization algorithm. This reaffirms the effectiveness of the proposed hybrid solution in addressing contemporary challenges of precise in vitro CFU counting.

Decoding of miR-7-5p in Colony Forming Unit-Hill Colonies as a Biomarker of Subclinical Cardiovascular Disease-A MERIT Study.

Colony forming unit-Hill (CFU-Hill) colonies were established to serve as a sensitive biomarker for vascular health. In animals, the overexpression of miR-7-5p was shown to be pro-atherogenic and associated with increased cardiovascular disease (CVD) risk. In a MERIT study, we aimed to explore the role of miR-7-5p expression in CFU-Hill colonies in type 1 diabetes mellitus (T1DM) and the effect of metformin in subclinical CVD. The expression of miR-7-5p in CFU-Hill colonies in 29 T1DM subjects without CVD and 20 healthy controls (HC) was measured. Metformin was administered to T1DM subjects for eight weeks. MiR-7-5p was upregulated in T1DM whereas metformin reduced it to HC levels. MiR-7-5p was positively correlated with c-reactive protein, and C-X-C motif chemokine ligand 10. The receiver operating characteristic curve revealed miR-7-5p as a biomarker of CVD, and upregulated miR-7-5p, defining subclinical CVD at a HbA1c level of 44.3 mmol/mol. Ingenuity pathway analysis predicted miR-7-5p to inhibit the mRNA expression of Krüppel-like factor 4, epidermal growth factor receptor, insulin-like growth factor 1 receptor, v-raf-1 murine leukemia viral oncogene homolog 1 and insulin receptor substrate ½, and insulin receptor, while metformin activated these miRNAs via transforming growth factor-ß1 and Smad2/3. We proved the pro-atherogenic effect of miR-7-5p that maybe used as a prognostic biomarker.

Percutaneous autologous bone marrow concentrate for knee osteoarthritis: patient-reported outcomes and progenitor cell content.

PURPOSE: Knee osteoarthritis (OA) is a common, progressively debilitating joint disease, and the intra-articular injection of autologous bone marrow concentrate (BMC) may offer a minimally invasive method of harnessing the body's own connective tissue progenitor cells to counteract accompanying degenerative effects of the disease. However, the extent to which the progenitor cell content of BMC influences treatment outcomes is unclear. We sought to determine whether patient-reported outcome measures associated with BMC treatment for knee OA are related to the concentration of progenitor cells provided. METHODS: In the present study, 65 patients (72 knees) underwent treatment for knee OA with autologous BMC and self-reported their outcomes for up to one year using follow-up questionnaires tracking function, pain, and percent improvement. A small fraction of each patient's BMC sample was reserved for quantification with a haematological analyzer and cryopreserved for subsequent analysis of potential connective tissue progenitor cells using a colony-forming unit fibroblast (CFU-F) assay. RESULTS: Patients reported significant increases in function and overall percent improvement in addition to decreases in pain relative to baseline levels following treatment with autologous BMC that persisted through 12 months. Patients reporting improved outcomes (46 of 72 knees) received BMC injections having higher CFU-F concentrations than non-responding patients (21.1×103 ± 12.4×103 vs 14.3×103 ± 7.0 x103 CFU-F per mL). A progenitor cell concentration of 18×103 CFU-F per mL of BMC was found to best differentiate responders from non-responders. CONCLUSION: This study provides supportive evidence for using autologous BMC in the minimally invasive treatment of knee OA and suggests that increased progenitor cell content leads to improved treatment outcomes. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03011398, 1/7/17.

Decreased neutrophil-mediated bacterial killing in COVID-19 patients.

The coronavirus disease COVID-19 was first described in December 2019. The peripheral blood of COVID-19 patients have increased numbers of neutrophils which are important in controlling the bacterial infections observed in COVID-19. We sought to evaluate the cytotoxic capacity of neutrophils in COVID-19 patients. 34 confirmed COVID-19 patients (29 severe, five mild disease), and nine healthy controls were recruited from the Masih Daneshvari Hospital (Tehran, Iran) from March to May 2020. Polymorphonuclear (PMN) cells were isolated from whole blood and incubated with green fluorescent protein (GFP)-labelled methicillin-resistant Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA). Bacterial growth was determined by measuring the florescence of co-cultures of bacteria and neutrophils and reported as the lag time before exponential growth. The number of viable bacteria was determined after 70 hours as colony-forming units (CFU). The immunophenotype of tested cells was evaluated by flow cytometry. Isolated neutrophils have higher surface expression of CD16 and CD62L with negative markers for PMN-MDSC. Bacterial growth in the presence of SA (22 ± 0.9 versus 9.2 ± 0.5 h, P < .01) and PA (12.4 ± 0.6 versus 4.5 ± 0.22, P < .01) was significantly reduced in COVID-19 patients. After 70 h incubation of PMN with bacteria (SA and PA), CFUs were significant increased in COVID-19 patients SA (2.6 ± 0.09 × 108 CFU/mL-severe patients and 1.4 ± 0.06 × 108 CFU/mL-mild patients, P < .001) and PA (2.2 ± 0.09 × 109 CFU/mL-severe patients and 1.6 ± 0.03 × 109 CFU/mL-mild patients, P < .001). Gentamycin proliferation assays confirmed the presence of intracellular bacteria. Reduced bacterial killing by neutrophils from COVID-19 patients may be responsible for the high bacterial yield seen in these patients.

Cryopreserved bone marrow aspirate concentrate as a cell source for the colony-forming unit fibroblast assay.

PURPOSE: The prevalence of connective tissue progenitor cells within a cell-based therapy is often quantified using the colony-forming unit fibroblast (CFU-F) assay. The present study investigates the feasibility of using cryopreserved bone marrow aspirate concentrate (BMAC) as an alternative cell source to fresh BMAC for CFU-F quantification. METHODS: Freshly prepared and corresponding cryopreserved BMAC samples from patients receiving autologous cell therapy (n = 98) were analyzed using the CFU-F assay for comparison. Cultures were established by directly plating BMAC at low cell densities and maintained for a 2-week growth period. Colonies were enumerated to determine CFU-F frequency, and a subset of cultures was imaged and analyzed to quantify colony area and density. RESULTS: A nonlinear relationship was observed between plating density and CFU-F frequency over a wide range in plating densities (~30-fold). Cryopreserved BMAC yielded recoverable (77 ± 23%) and viable (73 ± 9%) nucleated cells upon thawing. After cryopreservation, CFU-F frequencies were found to be significantly lower (56.6 ± 34.8 vs. 50.3 ± 31.7 colonies per million nucleated cells). Yet the number of CFU-F in fresh and cryopreserved BMAC were strongly correlated (r = 0.87) and had similar area and densities. Further, moderate correlations were observed between the number of CFU-F and nucleated cells, and both the mean colony area and density were negatively correlated with patient age. Notably, no relationship was found between CFU-F frequency and age, regardless of whether fresh or cryopreserved BMAC was used. CONCLUSIONS: Freshly prepared and cryopreserved BMAC yielded correlated results when analyzed using the CFU-F assay. Our findings support the cryogenic storage of patient BMAC samples for retrospective CFU-F analyses, offering a potential alternative for characterizing BMAC and furthering our understanding of progenitor cells in relation to clinical outcome.

Lipoaspirate Storage Time and Temperature: Effects on Stromal Vascular Fraction Quality and Cell Composition.

The adipose tissue-derived stromal vascular fraction (SVF) is a promising candidate for use in cell therapy and tissue engineering due to its regenerative and immunomodulatory properties. Some therapies are based on using the complete SVF product, whereas others depend on the expansion of adipose-derived stromal cells (ASCs) in culture. The latter application often involves a time delay between adipose tissue harvest and SVF isolation. This study investigated how storage time and temperature affected cell quality and composition. Aliquots of lipoaspirate were stored cold (4°C), at room temperature (18-20°C), or at 37°C. SVF was isolated on sequential time points over a period of 48 h, and the following were assessed: cell viability, vitality, composition, and the proliferative potential of the ASCs. When the lipoaspirate was stored cold, the viability of the SVF remained stable for up to 48 h; however, the vitality of the SVF decreased significantly after 24 h. When stored at higher temperatures (room temperature or 37°C), the vitality of the SVF decreased after 8 h. The ASC fraction in the SVF decreased rapidly after 8 h when stored at higher temperatures, whereas this change was delayed significantly when the lipoaspirate was stored cold. Tendencies towards increases in the lag phase, population doubling time (PDt), and time to reach confluency were observed when the lipoaspirate was stored at higher temperatures. The vitality of the SVF was correlated significantly with the time of the lag phase and the time required to reach confluence, whereas no correlation was observed with the PDt. Both prolonged storage time and increased temperature during lipoaspirate storage negatively affected the quality of the obtained SVF. Our results suggest that lipoaspirate should be stored for no longer than 24 h at 4°C to maintain the optimal quality for the isolation of SVF and the expansion of ASCs.

Preeclampsia in pregnancy affecting the stemness and differentiation potency of haematopoietic stem cell of the umbilical cord blood.

BACKGROUND: Umbilical cord blood (UCB) has been proposed as the potential source of haematopoietic stem cells (HSC) for allogeneic transplantation. However, few studies have shown that a common disease in pregnancy such as preeclampsia would affect the quality of UCB-HSC. Total nucleated cell count (TNC) is an important parameter that can be used to predict engraftment including UCB banking. Colony forming unit (CFU) assay is widely used as an indicator to predict the success of engraftment, since direct quantitative assay for HSC proliferation is unavailable. The aim of this study is to investigate the effects of preeclampsia in pregnancy on the stemness and differentiation potency of UCB-HSC. METHODS: Mononuclear cells (MNC) were isolated from UCB and further enriched for CD34+ cells using immune-magnetic method followed by CFU assay. A panel of HSC markers including differentiated haematopoietic markers were used to confirm the differentiation ability of UCB-HSC by flow cytometry analysis. RESULTS/ DISCUSSION: The HSC progenitor's colonies from the preeclampsia group were significantly lower compared to the control. This correlates with the low UCB volume, TNC and CD34+ cells count. In addition, the UCB-enriched CD34+ population were lymphoid progenitors and capable to differentiate into natural killer cells and T-lymphocytes. CONCLUSION: These findings should be taken into consideration when selecting UCB from preeclamptic mothers for banking and predicting successful treatment related to UCB transplant.

Antifungal Effectiveness of Various Intracanal Medicaments against Candida albicans: An In Vitro Study.

AIM AND OBJECTIVE: To evaluate the antifungal efficiency of various intracanal medicaments against Candida albicans. MATERIALS AND METHODS: One-hundred and forty extracted human mandibular premolar teeth were decoronated, and the biomechanical preparation was done in crown-down technique. 10 µL culture suspension of C. albicans was placed into the prepared root canal space of all the teeth. After 21 days of incubation, all the teeth were randomly divided into 7 groups with 20 teeth per each group. Group I: triple antibiotic powder (TAP) mixed with 3% chitosan solution; group II: TAP mixed with macrogol-propylene (MP) glycol; group III: chlorhexidine-guttapercha (CHX-GP); group IV: Vitapex; group V: 2% chlorhexidine gel; group VI: calcium hydroxide paste; group VII: normal saline with cotton (positive control) were used as intracanal medicaments, and the samples were incubated for 14 days. Intracanal medicaments were then completely removed using the canal brush. Dentinal chips were harvested from the walls of the root canal space in all samples using Gates-Glidden drills, were transferred into test tubes containing saline, and were serially diluted and placed in 140 Sabouraud dextrose agar plates, incubated at 37°C for 48 hours. Colony forming units (CFUs) of C. albicans were then counted using the digital colony counter. RESULTS: One-way ANOVA test showed statistically significant difference among the seven groups, as the p value was < 0.001. Tukey's post hoc test showed intergroup comparison between group I and group V; group II and group III were statistically nonsignificant as p value was >0.05. CONCLUSION: 2% chlorhexidine gel and TAP mixed with 3% chitosan solution showed superior antifungal efficiency against C. albicans. CLINICAL SIGNIFICANCE: Chitosan solution's inherent antifungal efficiency and slow and controlled drug release make it as an effective alternate carrier in mixing it with TAP instead of mixing TAP with MP.

Cryopreservation and thawing of hematopoietic stem cell CD34-induced apoptosis through caspase pathway activation: Key role of granulocytes.

INTRODUCTION: Cell damage inescapably occurs during both the freezing and the thawing graft processes for autologous hematopoietic stem cell (HSC) transplantation. To estimate HSC injury, a quality control is performed including: (i) CD34+ quantification; (ii) percentage of CD34+ viability and (iii) evaluation of HSC functional ability to form colony forming unit-granulocyte macrophage (CFU-GM). Apoptosis involves complex pathways such as caspase enzymes. Here, we assess the extent of apoptosis that is caspase-dependent before and after cryoconservation of CD34+, using a Fluorescent Labeled Inhibitor of CAspases (FLICA). METHODS: Caspase pathway activation status was evaluated in 46 patients (multiple myeloma [n = 24], lymphoma [n = 22]), by flow cytometry, using a 7-aminoactinomycin-D (7AAD)/FLICA staining test, in CD34+, CD3+, CD14+ and CD56+ cells. Viable 7AAD-/FLICA+ cells were then correlated with various parameters. RESULTS: We showed a significant caspase pathway activation, with 23% CD34+/7AAD-/FLICA+ cells after thawing, compared with the 2% described in fresh CD34+ cells (P < 0.0001). Moreover, caspase pathway was significantly activated in thawing CD3+, CD56+ and CD14+ cells. We also report a significant correlation between the rate of CD34+/7AAD-/FLICA+ cells and post-thawing granulocytes count (P = 0.042) and their potential to be differentiated into CFU-GM (P = 0.004). DISCUSSION: Our results show substantial cell death, induced by the increase of caspase pathway activation, secondary to the thawing process, and across all study cell types. This observation may affect the immune response quality during recipient aplasia, without detecting a clinical impact. Moreover, caspase pathway activation through CD3+ and CD56+ subpopulations could modify the therapeutic result of donor lymphocytes infusion (DLI).

Comparison and correlation among in vitro and in vivo assays to assess cord blood quality according to delivery temperature and time after collection.

Cord blood (CB) has been used as an alternative source for unrelated allogeneic hematopoietic stem cell transplantation. To determine which assay was useful for predicting the successful outcome of CB transplantation, CBs were grouped according to the temperature (4 °C, 24 °C, and 37 °C) and time (24, 48, and 72 h) after collection. The viability, early apoptosis, and colony forming units (CFUs) were ascertained for the total nucleated cells (TNCs) and CD34+ cells; in addition, the engraftment of infused CD34+ cells in NSG mice was determined. The viability of the TNCs and CD34+ cells and total CFUs were significantly decreased whereas the early apoptosis was significantly increased in the 72 h group at 37 °C compared to that of the 24 h group at 24 °C. The viability and early apoptosis of the TNCs correlated with those of CD34+ cells. In addition, the viability and early apoptosis correlated with the number of granulocyte/monocyte progenitor CFUs. In transplanted NSG mice, the frequency of human CD45+ cells decreased in the 72 h group at 24 °C compared to that of the 24 h group at 24 °C and was negatively correlated with early apoptosis of TNCs and CD34+ cells. This study demonstrated that the early apoptosis of TNCs and CD34+ cells constitutes a useful marker for predicting the engraftment of HSCs and may provide helpful data for standard assessment regarding CB quality by analyzing the correlation between in vitro and in vivo assays using NSG mice.

Sonication of Extracted Implants Improves Microbial Detection in Patients With Orthopedic Implant-Associated Infections.

BACKGROUND: We assessed whether combining the conventional culture and implant sonicate fluid culture (SFC) methods increased the diagnostic accuracy of orthopedic implant-associated infection (OIAI). METHODS: Consecutive patients (n = 66) undergoing implant removal (OIAI, 17; non-OIAI, 49) were evaluated prospectively. We analyzed 493 samples (39 preoperative joint aspirates, 243 peri-implant tissue specimens, 124 implant sonication, 67 controls, and 20 water bath samples). OIAI was preoperatively evaluated based on clinical evidence of infection or aspirate culture (AC). Conventional methods required positive results in either preoperative ACs or intraoperative tissue cultures (TCs), whereas the combination method required at least 1 positive culture among 3 sources (AC, TC, or SFC). The application of SFC and the detection rate, sensitivity, and specificity of the diagnostic methods were assessed. RESULTS: No controls or water bath samples harbored bacteria. Three patients (18%) were detected by SFC only and Peptostreptococcus and Corynebacterium species were isolated by SFC only. The OIAI detection rate attributable to SFC was significantly higher than that of TC (61% vs 36%; P = .02). The sensitivities of AC, TC, and SFC at a cutoff point of 1 colony-forming unit/plate and 1 positive culture were 60%, 59%, and 71%, respectively. At a cutoff point of 2 positive cultures, the combination (vs conventional) method showed significantly greater sensitivity (71% vs 47%; P = .008). CONCLUSION: By incorporating SFCs into conventional culture methods, the diagnostic accuracy in the context of OIAI was significantly improved.

The environmental monitoring in hospital pharmacy cleanroom and microbiota catalogue preparation.

Knowing the normal cleanroom microbiota is the basis for ensuring microbiological quality; assess changes and the introduction of new sampling methods. During our study, we prepared a catalogue of cleanroom microorganisms located in four different cleanrooms in University Clinical Centre Ljubljana Pharmacy. Catalogue is prepared as a basis for assessing the suitability of the new rapid microbiological method and subsequent correlation of the results of both methods. The results of our study showed that 78% of isolated bacteria are Gram-positive. However, in more than 70% isolated bacteria were the part of the normal human microbiota, 10-15% of the microorganisms originated from the air, mainly spore-forming bacteria of the genus Bacillus and fungi, and 5-10% of the Gram-negative microorganisms that originated from the water and represent the potential endotoxins (pyrogens).

Novel functionalized ß-nitrostyrenes: Promising candidates for new antibacterial drugs.

The process of searching for new antibacterial agents is more and more challenging due to the increasing drug resistance which has become a major concern in the field of infection management. Our study presents a synthesis and characterization by IR, UV, 1H NMR and 13C NMR spectra of a homogenous series of 1-EWG functionalized 2-aryl-1-nitroethenes which could prove good candidates for the replacement of traditional antibacterial drugs In vitro screening against a panel of the reference strains of bacteria and fungi and their cytotoxicity towards cultured human HepG2 and HaCaT cells was performed. Antimicrobial results indicated that four of the synthesized compounds exhibited a significant antimicrobial activity against all tested reference bacteria and fungi belonging to yeasts with a specific and strong activity towards B. subtilis ATCC 6633. Two of these compounds had no detectable cytotoxicity towards the cultured human cell lines, making them promising candidates for new antibacterial drugs.

Robust fit of Bayesian mixed effects regression models with application to colony forming unit count in tuberculosis research.

Early bactericidal activity of tuberculosis drugs is conventionally assessed using statistical regression modeling of colony forming unit (CFU) counts over time. Typically, most CFU counts deviate little from the regression curve, but gross outliers due to erroneous sputum sampling are occasionally present and can markedly influence estimates of the rate of change in CFU count, which is the parameter of interest. A recently introduced Bayesian nonlinear mixed effects regression model was adapted to offer a robust approach that accommodates both outliers and potential skewness in the data. At its most general, the proposed regression model fits the skew Student t distribution to residuals and random coefficients. Deviance information criterion statistics and compound Laplace-Metropolis marginal likelihoods were used to discriminate between alternative Bayesian nonlinear mixed effects regression models. We present a relatively easy method to calculate the marginal likelihoods required to determine compound Laplace-Metropolis marginal likelihoods, by adapting methods available in currently available statistical software. The robust methodology proposed in this paper was applied to data from 6 clinical trials. The results provide strong evidence that the distribution of CFU count is often heavy tailed and negatively skewed (suggesting the presence of outliers). Therefore, we recommend that robust regression models, such as those proposed here, should be fitted to CFU count.