Technical challenges for complete implementation of automated growth-based methods for microbiological examination of advanced therapy medicinal products. What's wrong with Candida albicans?

BACKGROUND: Automated growth-based methods for sterility testing of cell-therapy products should be qualified to demonstrate that they are equivalent to, or better than, the conventional reference method. The aim of the present study was to assess the ability of the BACTEC FX40 system to detect low microbial contamination and to confirm the suitability of the method in the presence of seven different human mesenchymal cell-based products, according to Ph. Eur. 2.6.27. Additionally, a study to select the best vial to detect fungus contamination was performed. METHODS: Microorganisms representing Gram-negative, Gram-positive, aerobic, anaerobic, spore-forming, slow-growing bacteria, yeast and mold were prepared in either Dulbecco's PBS or seven biological matrices containing approximately 5, 10, and 15 colony-forming units (CFU) per sample. These preparations were inoculated to the specific media required for each test method: (i) BACTEC aerobic and anaerobic vials; (ii) aerobic and anaerobic media for direct inoculation; and (iii) Trypcase soy 3P or Brucella blood agar plates. Colonies from cultures were identified using MALDI-TOF mass spectrometry. RESULTS: The BACTEC FX40 system, in both Dulbecco's PBS and the biological matrices with a 5-CFU inoculum, detected most of the microorganisms significantly faster than the conventional method, despite the presence of a matrix containing gentamicin and several matrices containing 10% DMSO. Conversely, it showed an extremely delayed detection of Candida albicans compared with the conventional method. The addition of a Mycosis IC/F (MYC) vial decreased radically the time to detection (TTD) of C. albicans (28.2 ± 1.8 h) compared with the conventional method (36 h). CONCLUSIONS: When a MYC vial was added to the standard aerobic and anaerobic vials to test each sample, BACTEC FX40 was shown to be a superior alternative sterility method for cell-therapy products contaminated with low inocula, with a faster TTD for microbial growth compared with the conventional method (5 versus 14 days). The studies were carried out in different cell-based matrices with sensitivities and specificities of 100% for all the tested strains at 15-, 10- and 5-CFU inoculum, with the exception of Kocuria rhizophila at 5 CFU (90.48% sensitivity and 100% specificity).

Clinical and epidemiological features and prognosis of complicated pyelonephritis: a prospective observational single hospital-based study.

BACKGROUND: Complicated pyelonephritis (cPN), a common cause of hospital admission, is still a poorly-understood entity given the difficulty involved in its correct definition. The aim of this study was to analyze the main epidemiological, clinical, and microbiological characteristics of cPN and its prognosis in a large cohort of patients with cPN. METHODS: We conducted a prospective, observational study including 1325 consecutive patients older than 14 years diagnosed with cPN and admitted to a tertiary university hospital between 1997-2013. After analyzing the main demographic, clinical and microbiological data, covariates found to be associated with attributable mortality in univariate analysis were included in a multivariate logistic regression model. RESULTS: Of the 1325 patients, 689 (52%) were men and 636 (48%) women; median age 63 years, interquartile range [IQR] (46.5-73). Nine hundred and forty patients (70.9%) had functional or structural abnormalities in the urinary tract, 215 (16.2%) were immunocompromised, 152 (11.5%) had undergone a previous urinary tract instrumentation, and 196 (14.8%) had a long-term bladder catheter, nephrostomy tube or ureteral catheter. Urine culture was positive in 813 (67.7%) of the 1251 patients in whom it was done, and in the 1032 patients who had a blood culture, 366 (34%) had bacteraemia. Escherichia coli was the causative agent in 615 episodes (67%), Klebsiella spp in 73 (7.9%) and Proteus ssp in 61 (6.6%). Fourteen point one percent of GNB isolates were ESBL producers. In total, 343 patients (25.9%) developed severe sepsis and 165 (12.5%) septic shock. Crude mortality was 6.5% and attributable mortality was 4.1%. Multivariate analysis showed that an age >75 years (OR 2.77; 95% CI, 1.35-5.68), immunosuppression (OR 3.14; 95% CI, 1.47-6.70), and septic shock (OR 58.49; 95% CI, 26.6-128.5) were independently associated with attributable mortality. CONCLUSIONS: cPN generates a high morbidity and mortality and likely a great consumption of healthcare resources. This study highlights the factors directly associated with mortality, though further studies are needed in the near future aimed at identifying subgroups of low-risk patients susceptible to outpatient management.