Bactec 9240

Sterility testing was achieved by sampling the Storage solution before the dehydration process, 6 days after the preservation in organ culture, and testing the Deswelling-Transport solution 24 h after the transfer of the dehydrated cornea prior to shipment to the surgical center. Microbial growth was screened in the media by means of two validated automated systems: Bactec 9240 (Becton-Dickinson, Franklin Lakes, NJ, USA) and HB&L (Alifax, Padua, Italy) (21 (link)).
Maintenance of sterile conditions during the dehydration process was assessed by a gelatin membrane filter (Gelatine Disposables, Sartorius, Gottingen, Germany) next to the Teflon base to collect airborne microbes that were then cultivated on blood agar plates for 7 days. In the event of positive results for microbial contamination of any of the tests, the cornea is discarded.

Conventional BC was performed in parallel by the Microbiology Department of the laboratory using BACTEC Plus Aerobic/F and BACTEC Plus Anaerobic/F bottles. All bottles were monitored by BACTEC 9240 blood culture system (Becton Dickinson and Company, Flanklin Lakes, New Jersey, USA). When a positive signal was obtained, BC bottles were removed from the instrument, Gram staining of the BC medium in the bottles was performed and the results rapidly reported to physicians. Samples were platted onto blood agar, chromogenic agar (chromID™ CPS™ bioMérieux) and anaerobic blood agar. Identification of bacterial or fungal species as well as antibiotic sensitivity tests were then carried out using the Vitek II system (bioMérieux, Marcy l'Etoile, France) and API 32 C (bioMérieux, Marcy l'Etoile, France) for yeast.

The concentration of serum CRP was measured with a Konelab 60i Clinical Chemistry Analyzer (Lab systems CLD, Konelab, Helsinki, Finland) or Cobas 6000 analyzer (Hitachi, Tokyo, Japan). The intra- and interassay CV% were 2.3–4.3%. The upper reference limit of serum or plasma CRP of a healthy reference population is 3 mg/L. Plasma PCT was measured from EDTA plasma using a Cobas 6000 analyzer (Hitachi, Tokyo, Japan) with a sensitivity of 0.06 μg/L. The CVs (intra- and interassay) were 1.4% and 3.0% for 0.46 μg/L and 1.1% and 2.6% for 9.4 μg/L PCT, respectively. Blood cultures (2-3 sets including two bottles/set) were drawn immediately at the beginning of neutropenic fever (day 0), and an additional sampling was done if fever persisted for 3–5 days. They were processed using the automated blood culture system Bactec 9240 (Becton Dickinson, Sparks, MD, USA). The incubation episode for aerobic and anaerobic bottles was 7 days and for MYCO F/Lytic bottles 42 days. The plasma samples for metabolomics assays were stored frozen at −80°C until analyzed.

GMP-compliant PL (Mesengen) was prepared and virally inactivated, as previously described [8 (link), 9 (link)]. Briefly, after obtaining informed consent, buffy coats from healthy volunteers were centrifuged and treated with InterSol solution (318 mg Na-citrate 2H₂O, 305 mg Na 2-phosphate anhydro, 105 mg Na dihydrogen phosphate 2H₂O, 442 mg Na-acetate 3H₂O, 452 mg NaCl, 100 mL H₂O, Fenwal Inc., Lake Zurich, Illinois) and subsequently with 20–30% human plasma. Potential pathogens were inactivated by using the Intercept Blood System for Platelets (Cerus Corporation, Concord, California, USA). PL was stored at −80°C for 24 hours before thawing at 37°C for 60 minutes. This procedure was repeated three times to enrich the pool of growth factors. Concentration and sterility of the preparation were determined by the haematology analyzer ABX Pentra DX 120 (Horiba ABX, Montpellier, France) and BACTEC 9240 (Becton and Dickinson), respectively. 5 U/mL heparin was added to cell culture media in order to avoid fibrin gel formation.

During the study period, the rate of antenatal steroid use in preterm deliveries was approximately 85%. In all centers, neonates with birth weight < 1000 g were given fluconazole prophylaxis. According to the center policy, a single (1 mL) or double blood culture was obtained before antibiotic treatment in each neonate with clinically suspected LOS. CSF culture was recommended in any case of suspected LOS but was deferred in unstable infants. Blood cultures were processed using automated systems (Bactec 9240; Becton Dickinson, Heidelberg, Germany; Bactalert; bioMérieux, Craponne, France).
Coagulase-negative Staphylococci (CoNS) were included in the list of pathogens causing LOS if infants had clinical signs of sepsis and received intravenous vancomycin or semisynthetic penicillin (e.g., oxacillin, nafcillin) for ≥ 5 days [8 (link),9 (link),23 ]. Two or more cultures identifying the same CoNS (with the same antimicrobial susceptibility) within 10 days were considered the same episode of LOS [12 (link)]. CoNS that did not meet these criteria (n = 244; 75.3%) were considered contaminants.
Micrococci, Aerococcus species, Corynebacterium species, Propionibacterium species, Bifidobacterium species, and Bacillus species grown in a single culture (n = 25) were also considered contaminants and excluded from analysis.

All blood culture bottles were incubated in the Bactec 9240 blood culture system (Becton Dickinson, Sparks, MD, USA) according to the manufacturer's instructions.