Purpose: A prospective pilot study was undertaken to determine the rate (if any) of microbiological contamination in vials of bevacizumab (Avastin; Genentech, Inc., South San Francisco, CA) being accessed for multiple intravitreal injections. Methods: Twenty-one unfractionated, 100-mg (4 mL) vials of bevacizumab were used for 321 intravitreal injections. Each vial was accessed multiple times under sterile conditions. Upon completion of each vial, the residual contents were stored for a further 2 weeks before being sent for microbiological contamination studies. Results: No evidence of microbiological contamination was detected in 21 vials sent for assessment. Of 321 intravitreal injections performed, none was associated with any sign of endophthalmitis. Conclusions: Using the same vial multiple times for intravitreal injections (stored for up to 4 weeks after opening) is a reasonable alternative to other methods of administering bevacizumab. Larger studies are required to determine the safety and efficacy of this practice. RETINAL CASES & BRIEF REPORTS 2:133–135, 2008

From *City Eye Centre, Brisbane, and †University of Queensland, St. Lucia, Queensland, Australia.

highlighted as potential drawbacks in using a preparation not specifically designed for intravitreal use.3 Bevacizumab is supplied in either a 100-mg (4 mL) or a 400-mg (16 mL) preservative-free vial. The inactive ingredients include ␣,␣-trehalose dihydrate, sodium phosphate (monobasic, monohydrate), sodium phosphate (dibasic, anhydrous), polysorbate 20, and water.4 Little information exists about this compound’s potential for microbial growth. Many ophthalmologists are currently contracting independent pharmacies to fractionate the larger bevacizumab vials into individual, single-use syringes, presumably for sterility and ease of administration. Professional fractionation usually yields up to 40 doses of 0.1 mL.4 In our study, the vials were not fractionated. Instead, the same vials were accessed for multiple injections. The aim of this pilot study was determine the rate of microbiological contamination within the individual vials of bevacizumab after the completion of their contents and to demonstrate a reasonable safety


he intravitreal use of bevacizumab (Avastin; Genentech, Inc., South San Francisco, CA) for the treatment of age-related macular degeneration despite its off-label status has surged into common ophthalmic practice. This is likely the result of its molecular similarity to ranibizumab (Lucentis; Genentech, Inc.)1 and relatively low cost. Intravitreal bevacizumab treatment of neovascular age-related macular degeneration was first reported by Rosenfeld et al.2 However, much debate remains as to the safety and efficacy of this practice despite some early case series data.2 In addition, optimal dosing, stability of the drug, and potential for contamination have all been None of the authors have propriety interest in any of the products mentioned in this report. Reprint requests: Timothy C. Smith, City Eye Centre, Level 10, 135 Wickham Terrace, Brisbane, Queensland, Australia 4018; email: [email protected]




for this method in preventing episodes of endophthalmitis, with the desire that larger studies will be undertaken to validate or disprove our initial results.

Materials and Methods A total of 21 unfractionated, 100-mg (4 mL) vials of bevacizumab were obtained from a local pharmacy as needed throughout the study period. The vials were stored in a standard retail refrigerator. The average refrigerator temperature over the study period was 7.2°C. All bottles were stored in the original carton before, during, and after use. Before each intravitreal injection, a standard sterile field was used to prepare for the procedure. The top of the vial was cleaned with a sterile alcohol swab. A sterile syringe was then expelled of air and attached to a 30-gauge needle. The rubber stopper was pierced without the aid of air for withdrawal. Approximately 0.1 mL was withdrawn for each injection. The excess was then discarded, and a new 30-gauge needle was attached followed by a standard pars plana injection of 0.05 mL. This procedure was repeated for each injection until the contents of the vials were emptied. A small amount of the drug was left in each vial to aid microbiological assessment. The dates for the start and finish of each vial were noted as was the number of injections yielded by each vial. On average, the contents of each vial were completed within 2 weeks. Each vial was then kept in the refrigerator for a further 2 weeks before being sent for microbiological assessment. The rationale was to maximize the opportunity for microbial growth before assessment. Microbiological studies were performed via a local commercial pathology company. According to the company, samples were accessed under a biohazard 2 unit. The rubber stopper was cleaned with a sterile alcohol swab. The used vials of bevacizumab were then pierced with a sterile insulin syringe. Aliquots of 200 ␮L were spread onto horse blood agar and chocolate agar plates and incubated for 7 days in CO2. Both media were custom made in house by the company. The combination of horse blood agar and chocolate agar was used to maximize the chance for growth of most organisms while still being able to track specific strains. Blood culture broth would have been more sensitive overall but was unfortunately not used by the laboratory. In addition, specific fungal cultures were not routinely part of the laboratory’s microbiological workup. It is also worth noting that initial vials sent during the study period were not being tested for anaerobes; however, this was corrected for subsequent vials in the study.




Results Of 21 vials of bevacizumab studied, none showed any evidence of microbiological contamination. A total of 321 intravitreal injections were performed. No patient receiving injections from the 21 vials had any evidence of endophthalmitis or external ocular infection throughout the study period. Complications included small subconjunctival hemorrhages and minor pain or irritation at the site of local anesthetic injection. Discussion Although emerging case series reports show great promise for the clinical effects of bevacizumab, many issues remain unresolved. The most common dose in current clinical practice seems to be 0.05 mL, but optimum dose and frequency are still to be determined. Other areas of interest are the storage and handling of the drug. To date, however, not much information exists about accessing individual vials of bevacizumab for multiple intravitreal injections. Genentech, Inc., advises against using vials of bevacizumab for multiple injections, citing potential for contamination.5 Although it is conceded that professional pharmacies may be able to yield more doses per vial than most ophthalmologists, this method may not be available in all locations. Indeed, it has been our experience that some pharmacies may be unwilling to fractionate the drug. In addition, the cost per dose may increase significantly when using professional compounding services. Using the same vial multiple times may prove more cost effective for patients receiving the drug. A recent study points to a favorable (6-month) stability of anti–vascular endothelial growth factor activity once vials of bevacizumab are opened. This appears to apply not only to prealiquoted syringes but also to previously pierced original vials.4 Therefore, the average 2-week period from start to finish of each vial is unlikely to alter the efficacy of the drug. From our study, it appears that using the same vial multiple times for intravitreal injections (stored for up to 4 weeks after opening) is probably a safe and effective method of administering bevacizumab. However, this is only a pilot study, and larger numbers are required for confirmation. In addition to size, the limitations of our study included the unavailability of specific fungal cultures and more sensitive culture media such as blood culture broth. In addition, there is the possibility that some “transient” microbial growth may have been missed in the 2-week storage period after the completion of each vial. Future studies will need to address these issues. Caution is advised in applying these results to clinical practice, and strict adherence to sterile procedure


is recommended when using this method. Intravitreal injection by itself, regardless of the medication used, is associated with some risk of endophthalmitis (0.2% of injections).6 The continual monitoring for microbiological contamination in vials of bevacizumab can serve as an adjunct to good medical record keeping. This may help alleviate confusion if complications do arise. Key words: bevacizumab (Avastin), choroidal neovascularization, intravitreal injection, microbiological contamination, vial.





References 1.

Heier JS, Antoszyk AN, Pavan PR, et al. Ranibizumab for the treatment of neovascular age-related macular degeneration: a



phase I/II multicenter, controlled, multidose study. Ophthalmology 2006;113:633– 642. Rosenfeld PJ, Moshfeghi AA, Puliafito CA. Optical coherence tomography findings after an intravitreal injection of bevacizumab (Avastin) for neovascular age-related macular degeneration. Ophthalmic Surg Lasers Imaging 2005;36:331–335. Rich RM, Rosenfeld PJ, Puliafito CA, et al. Short-term safety and efficacy of intravitreal bevacizumab (Avastin) for neovascular age-related macular degeneration. Retina 2006;26:495–511. Bakri SJ, Snyder MR, Pulido JS, et al. Six-month stability of bevacizumab (Avastin) binding to vascular endothelial growth factor after withdrawal into a syringe and refrigeration or freezing. Retina 2006;26:519–522. Janis A. Debate Arises Over Genentech’s Avastin [news release]. Basel, Switzerland: Bio Valley; June 26, 2006. Jager RD, Aiello LP, Patel SC, Cunningham ET Jr. Risks of intravitreous injection: a comprehensive review. Retina 2004; 24:676– 698.

Rate of microbiological contamination in vials of bevacizumab used for multiple intravitreal injections.

A prospective pilot study was undertaken to determine the rate (if any) of microbiological contamination in vials of bevacizumab (Avastin; Genentech, ...
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