Investigating the Origin of Mycobacterium chimaera Contamination in Heater-Cooler Units: Integrated Analysis with Fourier Transform Infrared Spectroscopy and Whole-Genome Sequencing.

Mycobacterium chimaera is ubiquitously spread in the environment, including factory and hospital water systems. Invasive cases of M. chimaera infection have been associated with aerosols produced by the use of heater-cooler units (HCU) during cardiac surgery. The aim of this study was to evaluate for the first time the performance of IR-Biotyper system on a large number of M. chimaera isolates collected from longitudinal environmental HCUs samples and water sources from hospitals located in three Italian provinces. In addition, IR-Biotyper results were compared with whole-genome sequencing (WGS) analysis, the reference method for molecular epidemiology, to investigate the origin of M. chimaera contamination of HCUs. From November 2018 to May 2021, 417 water samples from 52 HCUs (Stockert 3T, n = 41 and HCU40, n = 11) and 23 hospital taps (used to fill the HCU tanks) were concentrated, decontaminated, and cultured for M. chimaera. Positive cultures (n = 53) were purified by agar plate subcultures and analyzed by IR-Biotyper platform and Ion Torrent sequencing system. IR-Biotyper spectra results were analyzed using a statistical approach of dimensionality reduction by linear discriminant analysis (LDA), generating three separate clusters of M. chimaera, ascribable to each hospital. Furthermore, the only M. chimaera-positive sample from tap water clustered with the isolates from the HCUs of the same hospital, confirming that the plumbing system could represent the source of HCU contamination and, potentially, of patient infection. According to the genome-based phylogenies and following the classification proposed by van Ingen and collaborators in 2017, three distinct M. chimaera groups appear to have contaminated the HCU water systems: subgroups 1.1, 2.1, and branch 2. Most of the strains isolated from HCUs at the same hospital share a highly similar genetic profile. The nonrandom distribution obtained with WGS and IR-Biotyper leads to the hypothesis that M. chimaera subtypes circulating in the local plumbing colonize HCUs through the absolute filter, in addition with the current hypothesis that contamination occurs at the HCU production site. This opens the possibility that other medical equipment, such as endoscope reprocessing device or hemodialysis systems, could be contaminated by M. chimaera. IMPORTANCE Our manuscript focuses on interventions to reduce waterborne disease transmission, improve sanitation, and control infection. Sanitary water can be contaminated by nontuberculous Mycobacteria, including M. chimaera, a causative agent of invasive infections in immunocompromised patients. We found highly similar genetic and phenotypic profiles of M. chimaera isolated from heater-cooler units (HCU) used during surgery to thermo-regulate patients' body temperature, and from the same hospital tap water. These results lead to the hypothesis that M. chimaera subtypes circulating in the local plumbing colonize HCUs through the absolute filter, adding to the current hypothesis that contamination occurs at the HCU production site. In addition, this opens the possibility that other medical equipment using sanitized water, such as endoscope reprocessing devices or hemodialysis systems, could be contaminated by nontuberculous Mycobacteria, suggesting the need for environmental surveillance and associated control measures.

Development and validation of low-intensity pulsed ultrasound systems for highly controlled in vitro cell stimulation.

This work aims to describe the development and validation of two low-intensity pulsed ultrasound stimulation systems able to control the dose delivered to the biological target. Transducer characterization was performed in terms of pressure field shape and intensity, for a high-frequency range (500 kHz to 5 MHz) and for a low-frequency value (38 kHz). This allowed defining the distance, on the beam axis, at which biological samples should be placed during stimulation and to exactly know the intensity at the target. Carefully designed retaining systems were developed, for hosting biological samples. Sealing tests proved their impermeability to external contaminants. The assembly/de-assembly time of the systems resulted ~3 min. Time-domain acoustic simulations allowed to precisely estimate the ultrasound beam within the biological sample chamber, thus enabling the possibility to precisely control the pressure to be transmitted to the biological target, by modulating the transducer's input voltage. Biological in vitro tests were also carried out, demonstrating the sterility of the system and the absence of toxic and inflammatory effects on growing cells after multiple immersions in water, over seven days.

Inter-laboratory validation of a rapid assay for the detection and quantification of Legionella spp. in water samples.

AIMS: To compare the standard culture method with a new, rapid test (ScanVIT-Legionella™) using fluorescently labelled gene probes for the detection and enumeration of Legionella spp. The new technique was validated through experiments conducted on both artificially and naturally contaminated water and through an inter-laboratory comparison. METHODS AND RESULTS: All samples were processed by the ScanVIT test according to the manufacturer's instructions and by a culture method (ISO 11731). ScanVIT detected significantly more positive samples, although concentrations were similar and a strong positive correlation between the two methods was observed (r = 0.888, P < 0.001). The new test was more accurate in identifying the co-presence of Legionella pneumophila and Leg. non-pneumophila. ScanVIT showed a slightly higher Legionella recovery from water samples artificially contaminated with Leg. pneumophila alone or together with Pseudomonas aeruginosa. Lastly, the inter-laboratory comparison revealed that the ScanVIT test exhibits a lower variability than the traditional culture test (mean coefficient of variation 8.7 vs 16.1%). CONCLUSIONS: The results confirmed that the ScanVIT largely overlaps the reference method and offers advantages in terms of sensitivity, quantitative reliability and reduced assay time. SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed method may represent a useful validated alternative to traditional culture for the rapid detection and quantification of Legionella spp. in water.

[Preventing and reducing comorbidity in candidates for kidney transplantation for the improvement of post-operative results]. / Prevenire e ridurre le comorbilita' nei candidati al trapianto di rene per migliorare i risultati post-trapianto.

The correct and constant management of transplant waiting lists is necessary for the optimal utilization of the limited number of organs available for transplantation. The guidelines regarding placement on transplant waiting lists (absolute and relative contraindications) are well documented, even though they are in constant development. The criteria for the monitoring of patients on waiting lists, however, are not so well defined; this aspect is subject to careful evaluation on account of the widening of the criteria for transplantation suitability, the increase in the average age of patients, a rise in the number of enrolments and, as a result, prolonged waiting time (in Italy, the average time spent on a waiting list is 37 months). During the waiting period, a greater risk of clinically significant comorbidities and mortality, above all from cardiovascular events, has been noted (the annual mortality is 5-7% in the US, 1.3% in Italy). An in-depth clinical and instrumental study of patients with chronic renal failure is necessary when screening eligible candidates for transplant programs, individualizing therapeutic strategies, and identifying patients for whom the risks outweigh the potential benefits. Clinical and instrumental monitoring, as well as adequate treatment of comorbidities during the waiting period, can help improve the post-transplant outcome. This work examines the study algorithms and monitoring procedures for patients on kidney transplant waiting lists.

[Monitoring and control of opportunistic bacteria in a spa water used for aerosol hydrotherapy]. / Monitoraggio e controllo della contaminazione da batteri opportunisti in un'acqua termale erogata per aerosol e docce nasali.

A bicarbonate-sulphate-calcic water of a therapeutic spa was monitored for the presence of Legionella, Pseudomonas and Mycobacteria. The water was analysed by taking samples from the well, the feed tank and from the final aerosol generating devices of two different water lines, the former at 21-23 degrees, the second at 36-38 degrees. The bacteria in question were always absent from the well. Legionellae were found in the water of aerosol equipment: Legionella micdadei was isolated from 75% of samples, L. bozemanii from 75% and 50% (respectively 36-38 degrees and 21-22 degrees water lines) and other species of environmental Legionellae from 25% of samples. The water of aerosol equipment presented high total bacterial counts (10(3)-10(4) cfu/ml) and exspecially high concentrations of Pseudomonadaceae (10(2)-10(3) cfu/100 ml). These bacteria, unlike the Legionellae, were also isolated from the feed tank at mean concentrations of about 10(2) cfu/100 ml. Mycobacteria were found in 75 and 50% of samples collected from final devices, respectively from the heated and not heated water lines. The isolates were M. gordonae (85% of isolates) and M. fortuitum (15%), but at concentrations very low. Both treatments with sodium hypochlorite (20 ppm of residual chlorine) and peracetic acid (20 ppm) resulted in the reduction of total bacterial counts and elimination of Pseudomonas from the water in the tank, but not in elimination of Pseudomonas and Legionellae from the nebulizers. The disinfectants were evidently not able to efficiently reach all the points where Pseudomonas and Legionella had settled and grown. In order to obtain total abatement it was necessary to carry out a radical restructuring of the plant, involving the replacement of the old nebulizer benches with new aerosol equipment that could be subjected to a new system of programmed thermal shock.

Analysis of mesenchymal stem cells grown on a three-dimensional HYAFF 11-based prototype ligament scaffold.

Ligaments are complex structures that maintain the mechanical stability of the joint. Healing of injured ligaments involves the interactions of different cell types, local cellular environment, and the use of devices. To gain new information on the complex interactions between mesenchymal stem cells (MSCs) and a specific hyaluronan-based prototype scaffold (HYAFF, useful for ligament tissue engineering, short time-course experiments were performed to analyze the proliferation, vitality, and phenotype of MSCs grown on the scaffold. MSC proliferation was analyzed using the MTT test, during the early time points (2, 4, 6, days). Viability was assessed using calcein/acetyloxymethylester immunofluorescence dye and confocal microscopy analysis. Hyaluronic acid receptor (CD44), typical matrix ligament proteins (collagen type I, type III, laminin, fibronectin, actin), and chondrogenic/osteogenic markers (collagen type II and bone sialoprotein) were evaluated by immunohistochemistry. Our data demonstrated that MSC growth and viability were cell density-dependent. MSCs completely wrapped the fibers of the scaffold, expressed CD44, collagen type I, type III, laminin, fibronectin, and actin, and were negative to collagen type II and bone sialoprotein. These data demonstrate that MSCs survive well in the hyaluronan-based prototype ligament scaffold, as assessed after 2 days from seeding, and express CD44, a receptor important for scaffold interaction, and proteins responsible for the functional characteristics of the ligaments.

[Long-term use of Profiler in patients with dialysis intolerance: 8 months follow-up]. / Impiego a lungo termine del Profiler nei pazienti con intolleranza dialitica: follow-up a 8 mesi.

PURPOSE: A new method of profiled dialysis has been set up for many years in the Department of Nephrology, Dialysis and Renal Transplantation at the University of Bologna. This profiled dialysis is based on the use of a new kinetic mathematical model, in collaboration with the Faculty of Engineering at the University of Bologna, for the elaboration of individual sodium and ultrafiltration profiles. OBJECTIVE: The profiled dialysis aims are: 1) to stabilize the intradialytic blood volume, boosting the refilling of plasma water from the intracellular and the extravascular to the extracellular/intravascular compartments, to balance the ultrafiltration; 2) to counteract the disequilibrium syndrome reducing the shift of water from the extra to the intracellular compartment. The pre-dialysis elaboration of profiles is completely automatic and supported by a computerized programme, Profiler, which has been included in the software of the dialysis machine Bellco Formula 2000 Plus. METHODS: In this prospective and multicenter study, this profiled dialysis, performed according to the Profiler, was continuously applied, for an 8-month period, in a group of 13 hemodialysis (HD) patients with an intolerance to previous dialysis treatment. During the study, the following parameters were evaluated, comparatively, with the patient's basal treatment: a) sodium and water balance; b) percentage incidence of intradialytic complications such as hypotensive events, cramps, headache, and vomiting and; c) metabolic and nutritional status. RESULTS: Results evaluated in comparison with the patient's previous dialysis treatments, demonstrated: a) plasma sodium from 136.8 +/- 3 to 136.8 +/- 1.7 mEq/L (p=ns), dry body weight from 72.2 +/- 19.3 to 71.7 +/- 19.5 kg (p=ns), heart index from 3.7 +/- 0.7 to 3.1 +/- 0.5 L/min/m2 (p=ns), reactance from 5.3 +/- 15 to 4.9 +/- 11 ohm (p<0.05); b) incidence of intradialytic hypotensive events reduced from 64 to 4% (p<0.001), cramps reduced from 8 to 1% (p<0.01); c) plasma albumin from 3.5 +/- 0.2 to 3.7 +/- 0.3 g/dL (p=ns), Kt/Veq from 1.3 +/- 0.1 to 1.36 +/- 0.2 (p=ns). CONCLUSIONS: Patients treated with profiled dialysis had a higher stability of intradialytic blood pressure (BP) achieving a reduction in the incidence of disequilibrium syndrome symptoms, in comparison with previous treatment. These clinical intradialytic improvements were not correlated to clinical, instrumental or biochemical indexes of sodium-water overload nor to a worst dialysis adequacy and nutritional state.

Hyaluronan-based biomaterial (Hyaff-11) as scaffold to support mineralization of bone marrow stromal cells.

Various techniques are widely used to repair bone defects, association of hyaluronan-based biodegradable polymers (Hyaff-11) with bone marrow stromal cells (BMSC) promises to provide successful cell scaffolds for tissue-engineered repair of bone tissue. We evaluate in vitro and in vivo the potential of Hyaff-11 to facilitate mineralization of BMSC. Rat BMSC were seeded on Hyaff-11 and their differentiation were assessed at different time points. Osteogenic differentiation was investigated in vitro analysing the expression of alkaline phosphatase and osteocalcin. Mineralization of bone defects was evaluated also in vivo implanting Hyaff-11 scaffold combined with BMSC in large segmental radius defects. In vitro, we found a decrease expression of alkaline phosphatase and an increase of osteocalcin. In vivo, our data showed that mineralization was induced and basic fibroblast growth factor contributed to this process. These results provide a demonstration to therapeutic potential of Hyaff-11 as appropriate carrier vehicle for differentiation and mineralization of BMSC and for the repair of bone defects.

Multidetector CT-urography in the study of urological complications in renal transplant.

PURPOSE: To evaluate the value of CT-urography in the diagnosis and follow-up of the urological complications of renal transplantation. MATERIALS AND METHODS: We performed 19 CT-urography examinations (3 of which were follow-up) on 15 patients by using a spiral multislice CT scanner and multiplanar reconstructions. The examinations were carried out directly after administration of 100 ml of iodinated contrast medium by slow iv infusion, with acquisitions starting 5 minutes after the end of the infusion. Surgery was regarded as the gold standard for the diagnosis of urological complications in the operated patients, whereas in patients who had undergone medical therapy or stent placement the gold standard was 1 month ultrasound and clinical follow-up with evaluation of diuresis and renal function. RESULTS: Between January 1999 and December 2001 a total of 210 kidney transplantations were performed at our hospital. There were 34 urological complications in 28 patients with a 16.1% prevalence, consistent with the major international studies. The complications detected were 14 urine leaks and 19 ureteral obstructions secondary to stones, oedema, blood clots and stricture. We observed one case of reflux in the allograft ureter. Fourteen out of 16 CT-urography examinations yielded important clues for the diagnosis of urological complications in kidney allografts, completely replacing standard urography. In particular, CT-urography correctly detected 5 urinary fistulas by demonstrating iodinated contrast material leaks along the ureteral tract, and 8 cases of obstructive uropathy due to different causes (1 submucosal tunnel edema, 1 blood clot, 1 stone and 5 cases of ureteral stricture). One case of urinary fistula and one of obstructive uropathy were not detected. DISCUSSION AND CONCLUSIONS: CT-urography proved to be an important diagnostic tool in the evaluation of urological complications of kidney allografts, showing a diagnostic accuracy over 90%; it is useful for confirming the type and site of urological lesions, and therefore to provide guidance for a targeted surgical approach. Compared with excretory urography, which is no longer used in this diagnostic field, CT-urography is more complete and precise as it provides information not only about the urinary tract, but also about the kidney parenchyma and pararenal fluid collections. The only disadvantage, which limits its use to selected cases, is the risk associated with the use of iodinated contrast agents in patients with impaired renal function.

Evaluation of the resistive index (RI) for the diagnosis of acute renal rejection in renal allografts from the same donor.

PURPOSE: To investigate the importance of the resistive index (RI) in the diagnosis of acute renal rejection, compared with the RI of the twin kidney from the same donor, transplanted in two different patients. MATERIAL AND METHODS: From January to December 2000, we studied retrospectively 25 pairs (50 patients) of renal allografts from the same donor considering the RI obtained with by eco color-Doppler ultrasound, daily diuresis and renal function (serum creatinine level) in the first six days following surgery. Improvement of diuresis and renal function after corticosteroid therapy was considered the gold standard for the diagnosis of acute rejection. RESULTS: Medical complications (acute renal rejection) in the first six days were occurred in three cases, two in the first transplanted kidney as first and one in the second; all three cases showed disappearance of the diastolic waveform component. Considering a RI variation >0.15 with respect to the initial value, the sensitivity, specificity and diagnostic accuracy in the Doppler diagnosis of acute rejection were 100%, 97.1% and 97.3% respectively, with a prevalence of 7.8%. There were no statistically significant correlations between the RI variation of the renal transplant and the twin kidney from the same donor. DISCUSSION AND CONCLUSIONS: Doppler ultrasound is an important diagnostic tool in the detection of medical complications in the immediate postoperative period and during renal transplant follow-up. RI analysis, when studied serially and in the right clinical settings, allows an early diagnosis of renal rejection with high sensibility and specificity.