Evaluation of the HB&L System for the Microbiological Screening of Storage Medium for Organ-Cultured Corneas

Abstract

Aims. To compare HB&L and BACTEC systems for detecting the microorganisms contaminating the corneal storage liquid preserved at 31°C. Methods. Human donor corneas were stored at 4°C followed by preservation at 31°C. Samples of the storage medium were inoculated in BACTEC Peds Plus/F (aerobic microorganisms), BACTEC Plus Anaerobic/F (anaerobic microorganisms), and HB&L bottles. The tests were performed (a) after six days of storage, (b) end of storage, and (c) after 24 hours of preservation in deturgescent liquid sequentially. 10,655 storage and deturgescent media samples were subjected to microbiological control using BACTEC (6-day incubation) and HB&L (24-hour incubation) systems simultaneously. BACTEC positive/negative refers to both/either aerobic and anaerobic positives/negatives, whereas HB&L can only detect the aerobic microbes, and therefore the positives/negatives depend on the presence/absence of aerobic microorganisms. Results. 147 (1.38%) samples were identified positive with at least one of the two methods. 127 samples (134 identified microorganisms) were positive with both HB&L and BACTEC. 14 HB&L+/BACTEC− and 6 BACTEC+/HB&L− were identified. Sensitivity (95.5%), specificity (99.8%), and positive (90.1%) and negative predictive values (99.9%) were high with HB&L considering a 3.5% annual contamination rate. Conclusion. HB&L is a rapid system for detecting microorganisms in corneal storage medium in addition to the existing methods.

Method for sterility testing of corneal storage and transport media after removal of interfering antimicrobials: prospective validation study in compliance with the European Pharmacopoeia

“…Limitations of the automated system in detecting filamentous fungi have also been observed by other authors. 15 In our study, the system showed positive readings for A. brasiliensis when the incubation time was extended to 5 days. Therefore, according to our findings, the incubation time of the sample should be extended from 48 hours to 5 days at least, in order to include the detection of filamentous fungi.…”

“…First, unlike other automated systems such as the BACTEC blood culture system, 18 the HB&L system does not implement any antimicrobial removal or neutralisation process; second, as reported by other authors, the sample volume is restricted to 500 µL. 15 Our findings showed that the presence of streptomycin sulfate and penicillin G, which are known to be effective in reducing the bioburden of contaminated tissues, 19 in the tested media clearly interfered with the microbial growth during sterility testing and resulted in FN results. In fact, the automated system did not detect the presence of four ( B. subtilis , C. sporogens , P. aeruginosa and S. aureus ) out of six microbial strains after inoculation in TISSUE-C and CARRY-C media at 1–10 and 2–20 cfu/mL, respectively.…”

“…The automated HB&L system for microbiological analysis of biological samples may provide an important advantage to tissue banks that perform sterility testing of samples that are free from antimicrobials, because it allows rapid analysis with high method sensitivity and high sample throughput. 15–17 However, the system showed relevant limitations for the analysis of corneal storage media that contained antimicrobials. First, unlike other automated systems such as the BACTEC blood culture system, 18 the HB&L system does not implement any antimicrobial removal or neutralisation process; second, as reported by other authors, the sample volume is restricted to 500 µL.…”

“…The system was originally designed for bacterial screening of urine and other biological samples and was recently reported to be useful for the analysis of fluid samples, 14 including corneal storage media. 15 However, the system does not eliminate or neutralise the antimicrobial residues present in the corneal storage media. Therefore, in this validation study, we included an additional treatment of the sample using the RESEP device in order to remove the factors that could interfere with microbial growth in the samples, as recommended by EP, before running the test in the automated system.…”

Next-generation sequencing for the detection of microorganisms present in human donor corneal preservation medium

“…Antibacterial agents such as penicillin and streptomycin, and antifungal agents such as amphotericin B are usually used as an empiric cocktail in OC corneal preservation media. Conventional microbiological controls are currently performed using standard bacteriological media in aerobic and anaerobic environments, whereas Sabouraud broth is a routine medium for detection of fungi 24. Other options include the use of Bactec blood bottles (Becton Dickinson) incubated in the Bactec instrument (based on the detection of CO 2 produced by microorganisms), which offer many advantages over the standard microbiological techniques 25–27.…”