The purpose of this study was to validate the sterility test of corneal culture and deswelling/transport media using a device for removal of antimicrobials before incubation in BACTEC™ automated system in two Italian Eye Banks. Corneal culture medium, TISSUE-C, and deswelling/transport medium, CARRY-C, were inoculated with 10–100 cfu of six European Pharmacopoeia (EP) reference strains and either treated with medical device RESEP for removal of antimicrobials (RESEP+ group) or left untreated (RESEP− group) before injection into the BACTEC Plus bottles. The same steps were repeated in the absence of inocula with tryptone soy broth samples as negative controls, and the inocula were also directly injected in the BACTEC™ bottles as growth controls. All the samples were incubated in BACTEC™ automated system for 7 days, and the time to detection of microbial growth was recorded automatically. At both the Eye Banks, in the RESEP+ groups, microbial growth was detected in 100% of samples. In the RESEP− group, the method sensitivity ranged from 66.7 ± 21.1 to 88.9 ± 6.4% for TISSUE-C samples while for CARRY-C samples the method sensitivity ranged from 94.5 ± 5.1 to 100%. The method specificity corresponded to 100% for all the groups at both Eye Banks. This two-centre validation study showed that the use of RESEP increased the sensitivity of sterility test using BACTEC™ automated system up to 100% and, consequently, allowed validation of the method for sterility testing of corneal storage and deswelling/transport media according to the EP requirements. The test could not be validated without the use of RESEP.
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Alexander K, Bryans T (2006) Evaluation of the sterility test for detection of microbial contaminants of allografts. Cell Tissue Bank 7(1):23–28
Armitage WJ (2011) Preservation of human cornea. Transfus Med Hemother 38:143–147
Buzzi M, Guarino A, Gatto C, Manara S, Dainese L, Polvani G (2014) Tóthová JD (2014) Residual antibiotics in decontaminated human cardiovascular tissues intended for transplantation and risk of falsely negative microbiological analyses. PLoS ONE 9(11):e112679
Camposampiero D, Grandesso S, Zanetti E, Mazzucato S, Solinas M, Parekh M, Frigo AC, Gion M, Ponzin D (2013) Evaluation of the HB&L system for the microbiological screening of storage medium for organ-cultured corneas. J Ophthalmol. https://doi.org/10.1155/2013/670947
Chen JY, Jones MN, Srinivasan S, Neal TJ, Armitage WJ, Kaye SB, NHSBT Ocular Tissue Advisory Group and Contributing Ophthalmologists (OTAG Audit Study 18) (2015) Endophthalmitis after penetrating keratoplasty. Ophthalmology 122:25–30. https://doi.org/10.1016/S0140-6736(12)60437-1
Council of Europe (2013a) Sterility test. In: European Directorate For The Quality of Medicines and Healthcare (ed) European Pharmacopoeia, Eight edn, Vol I, Strasbourg Cedex, France, pp 175–178
Council of Europe (2013b) Microbiological control of cellular products. In: European Directorate For The Quality of Medicines and Healthcare (ed) European pharmacopoeia, Eight edn, Vol I, Strasbourg Cedex, France, p 216
EUROCET European Network of Competent Authorities for Tissues and Cells. Report 2017 TISSUE DATA Year 2016. http://www.eurocet.org/
European Eye Bank Association - EEBA (2018) Minimum medical standards. https://www.eeba.eu/files/EEBA_Minimum_Medical_Standards_2018-01_003.pdf. Accessed 2 May 2018
Flayhart D, Borek AP, Wakefield T, Dick J, Carroll KC (2007) Comparison of BACTEC Plus blood culture media to BacT/Alert FA blood culture media for detection of bacterial pathogens in samples containing therapeutic levels of antibiotics. J Clin Microbiol 45:816–821
Fontana C, Favaro M, Minelli S, Bossa MC, Altieri A, Favalli C (2009) A novel culturing system for fluid samples. Med Sci Monit 15(2):BR55–BR60
Gain P, Gilles G, Chiquet C, Vautrin AC, Carricajo A, Acquart S, Maugery J, Aubert G (2001) Use of a pair of blood culture bottles for sterility testing of corneal organ culture media. Br J Ophthalmol 85:1158–1162. https://doi.org/10.1136/bjo.85.10.1158
Gain P, Jullienne R, He Z, Aldossary M, Acquart S, Cognasse F, Thuret G (2016) Global survey of corneal transplantation and eye banking. JAMA Ophthalmol 134(2):167–173
Germain D, Strong DM, Dowling G, Mohr J, Duong A, Garibaldi A, Simunovic N, Ayeni OR, on behalf of the Bioburden Steering Committee and Cardiac Tissue Working group (2016) Disinfection of human cardiac valve allografts in tissue banking: systematic review report. Cell Tissue Bank 17(4):593–601
Hughes JG, Vetter EA, Patel R, Schleck CD, Harmsen SL, Turgeant LT, Cockerill FR (2001) Culture with BACTEC Peds Plus/F bottle compared with conventional methods for detection of bacteria in synovial fluid. J Clin Microbiol 39:4468–4471
Kawasaki R, Yasuda M, Song SJ, Chen SJ, Jonas JB, Wang JJ, Mitchell P, Wong TY (2010) The prevalence of age-related macular degeneration in Asians: a systematic review and meta-analysis. Ophthalmology 117:921–927
Linke SJ, Mau-Thek E, Jürgen B (2013) Thirty years of cornea cultivation: long-term experience in a single eye bank. Acta Ophthalmol 91:571–578. https://doi.org/10.1111/j.1755-3768.2012.02471.x
Mistò R, Giurgola L, Pateri F, Frigerio E, Limongelli A, D’Amato Tóthová J (2017) Method for sterility testing of corneal storage and transport media after removal of interfering antimicrobials: prospective validation study in compliance with the European Pharmacopoeia. BMJ Open Ophthalmol 2(1):e000093. https://doi.org/10.1136/bmjophth-2017-000093
Navarro Martínez-Cantullera A, Calatayud Pinuaga M (2016) Obtaining corneal tissue for keratoplasty. Arch Soc Esp Oftalmol 91:491–500
Schallhorn JM, Rose-Nussbaumer JR (2015) Current concepts in the management of unique post-keratoplasty infections. Curr Ophthalmol Rep 3:184–191
Schroeter J, Wilkemeyer SR, Pruss A (2012) Validation of the microbiological testing of tissue preparations using the BACTECTM blood culture system. Transfus Med Hemother 39(6):387–390. https://doi.org/10.1159/000345812
Seiler TG, Tschopp M, Zimmerli S, Tappeiner C, Wittwer VV, Frueh BE (2016) Time course of antibiotic and antifungal concentrations in corneal organ culture. Cornea 35(1):127–131
Skenderi Z, Giurgola L, Gatto C, D’Amato Tóthová J, Pruß A, Schroeter J (2018) Increased sensitivity of microbiological testing of cornea organ culture medium by additional resin treatment. BMJ Open Ophthalmol 3(1):e000173
Tan DTH, Dart JKG, Holland EJ, Kinoshita S (2012) Corneal transplantation. Lancet 379:1749–1761 penetrating keratoplasty. Ophthalmology 122:25–30. https://doi.org/10.1016/S0140-6736(12)60437-1
Thuret G, Carricajo A, Chiquet C, Vautrin AC, Celle N, Boureille M, Acquart S, Aubert G, Maugery J, Gain P (2002) Sensitivity and rapidity of blood culture bottles in the detection of cornea organ culture media contamination by bacteria and fungi. Br J Ophthalmol 86(12):1422–1427. https://doi.org/10.1136/bjo.86.12.1422
Thuret G, Carricajo A, Vautrin AC, Raberi H, Acquart S, Garraud O, Gain P, Aubert G (2005) Efficiency of blood culture bottles for the fungal sterility testing of corneal organ culture media. Br J Ophthalmol 89(5):586–590. https://doi.org/10.1136/bjo.2004.053439
Van Dooren BTH, Mulder PGH, Nieuwedaal CP, Beekhuis Melles GRJ (2004) Endothelial cell density after posterior lamellar keratoplasty (Melles techniques): 3 years follow-up. Am J Ophthalmol 138:211–217. https://doi.org/10.1016/j.ajo.2004.02.016
Varettas K (2013) Culture methods of allograft musculoskeletal tissue samples in Australian bacteriology laboratories. Cell Tissue Bank 14(4):609–614
Wilhelmus KR, Hassan SS (2007) The prognostic role of donor corneoscleral rim cultures in corneal transplantation. Ophthalmology 114(3):440–445
We would like to thank Cristina Gianotti for the writing assistance. No external funding was received for this study.
Conflict of interest
J. D’Amato Tóthová and L. Giurgola are employed by the company Alchilife S.r.l., which was involved in the development of one medical device discussed in this article. R. Vignola, R. Mistò, F. Pateri and A. Pocobelli have no financial or proprietary interest in any material or method mentioned.
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Vignola, R., Mistò, R., Giurgola, L. et al. A two-centre validation study of sterility test of corneal storage media with elimination of interfering antimicrobials in compliance with the European Pharmacopoeia. Cell Tissue Bank 20, 275–285 (2019). https://doi.org/10.1007/s10561-019-09766-7
- Corneal culture media
- Antimicrobials removal neutralization
- Eye banking
- Microbiological testing
- Corneal storage and transplantation
- European Pharmacopoeia sterility test