International Journal of Pharmaceutics 485 (2015) 160–163
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Personalised medicine
Multiple withdrawals from single-use vials: A study on sterility Alba Ripoll Gallardo a, * , Grazia Meneghetti a , Luca Ragazzoni a , Vesselina Kroumova b , Daniela Ferrante c , Pier Luigi Ingrassia a , Paola Ruzza b , Angela Dell’Era b , Esther Boniolo a , Gjergji Koraqe a , Fabrizio Faggiano d, Francesco Della Corte a a
CRIMEDIM – Research Center in Emergency and Disaster Medicine, University of Piemonte Orientale A. Avogadro, Novara, Italy Department of Virology and Microbiology, Ospedale Maggiore della Carità, Novara, Italy CPO-Piemonte and Unit of Medical Statistics and Epidemiology, Department of Translational Medicine, University of Piemonte Orientale, Novara, Italy d Department of Translational Medicine, Università del Piemonte Orientale Amedeo Avogadro, Novara, Italy b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 January 2015 Accepted 5 March 2015 Available online 9 March 2015
Background: Reutilization of single-use vials containing medical drugs is still under discussion. This practice has been adopted as a standard to avoid drug wastage, particularly in developing countries and in the aftermath of disasters. Some studies have assessed sterility of medications stored in single-use vials after utilization as multiple doses; however, most of these were limited to one single drug, included a low number of samples and did not consider an intermediate transfer step from the vial to a disposable syringe. The purpose of this study was to assess microbial contamination of samples withdrawn over three days from disposable syringes prepared from single-use vials. Methods: A prospective sterility study was conducted. A total of 600 initial samples were prepared from six-hundred 10 mL single-use vials of physiological solution into six-hundred 20 mL disposable syringes. Samples were prepared in three different standard operating rooms, on six different days and by the same operator, using basic sterile technique. All syringes were capped, placed together in a non-sterile steel container, covered with a clean drape and stored in the refrigerator at 4 C under non-sterile conditions. Using basic sterile technique, four samples were withdrawn daily and cultured from each syringe over the next 3 days. Microbial growth was examined on Sabouraud agar and chocolate agar culture media. Results: A total of 7200 samples were collected and 14,400 cultures were performed. No evidence of microbial growth in any of the culture media plates was found. Conclusion: This study demonstrated that contents initially stored in single-use vials and subsequently transferred into disposable syringes in an operating room using sterile technique, maintain sterility after 4 withdrawals per day for a total of 3 days. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Single-use vial Drug packaging Drug storage Disposable equipment Drug contamination Syringes/microbiology
1. Introduction Ever since their first use in daily practice, reutilization of drugs contained in single-dose/use vials (SUVs)1 has been widely
* Corresponding author at: CRIMEDIM – Research Center in Emergency and Disaster Medicine, University of Piemonte Orientale A. Avogadro, Via Ferrucci 33/ Via Lanino 1, 28100 Novara, Italy. Tel.: +39 321660620; fax: +39 321660531. E-mail addresses: [email protected] (A. Ripoll Gallardo), [email protected] (G. Meneghetti), [email protected] (L. Ragazzoni), [email protected] (V. Kroumova), [email protected] (D. Ferrante), [email protected] (P.L. Ingrassia), [email protected] (P. Ruzza), [email protected] (A. Dell’Era), [email protected] (E. Boniolo), [email protected] (G. Koraqe), [email protected] (F. Faggiano), [email protected] (F. Della Corte). 1 SUV: single-dose/use vials. http://dx.doi.org/10.1016/j.ijpharm.2015.03.010 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
discussed (Stokowski, 2014). In its latest statement, the Center for Disease Control and prevention (CDC, 2011, 2012)2 reaffirmed that “Vials that are labeled as single-dose or single-use should be used for a single patient and single case/procedure/injection.” This assertion relies on the fact that SUVs contain neither antimicrobial agents nor preservatives and, therefore, contamination should be expected after initial vial entry. However, several studies have demonstrated that health professionals often do not comply with CDC recommendations (Stokowski 2014). For instance, the survey conducted by Woodbury et al. (2014) among anesthesia providers in Atlanta, showed that close to 60% admitted to having re-used SUVs for different patients. Similarly, Baniasadi et al. (2013) conducted a study in Iran in which 205 opened vials stored in
2
CDC: Center for Disease Control and Prevention.
A. Ripoll Gallardo et al. / International Journal of Pharmaceutics 485 (2015) 160–163
different hospital wards were analyzed; of these, 80.49% were SUVs employed as multiple-use vials. Re-using SUVs has been adopted by health care providers as standard practice especially in developing countries, in the aftermath of disasters, or in complex humanitarian emergencies in an attempt to avoid drug wastage. For instance, although in the International Standards for a Safe Practice of Anesthesia (Merry et al., 2010) morphine and pethidine are both considered as mandatory medications, Hodges et al. (2007) found that among anesthesia practitioners in Uganda only 45% had uninterrupted access to this particular drugs. Often in these settings, the shortage of medications is not only a consequence of restrictive laws but also of high drug prices in comparison to Western health-care systems. The CDC recognized the motivations leading health providers to reutilize the contents of SUVs for more than one patient, and therefore an option was extended for the storage of contents of unopened SUVs into multiple-use syringes. However, this repackage procedure must be performed exclusively by qualified personnel and under specific conditions complying with the US Pharmacopeia (USP)3 797 standards (Stokowski, 2014), for instance, in pharmacies. Consequently, operating rooms and other clinical areas are considered inadequate environments for splitting and repacking vials regardless of the technique used (Stokowski, 2014). In recent years, some studies have been conducted to assess sterility of different medications stored into SUVs and used as multiple doses (Ornek et al., 2008; Reiter et al., 2003). However, most of these were restricted to a single drug, included a low number of samples and did not consider transferring contents into disposable syringes, a maneuver frequently performed when the dosage needed per patient is much lower than the total amount of drug contained in a single vial. Therefore, the purpose of this study was to determine microbial contamination of multiple samples withdrawn during several days from disposable syringes prepared from SUVs. The expected result was that contamination rate among all samples would not exceed that calculated for simple filling of syringes under the same environmental conditions (Stucki et al., 2009). 2. Materials and methods
161
performed using the Statistics Calculator software (nQuery Advisor, v5.05). 2.3. Sample preparation and storage A total of 600 samples were prepared into 20 mL disposable syringes from six-hundred 10 mL SUVs filled with PS (Fig. 2). The total volume was extended to 20 mL with additional PS to simulate drug dilution. All syringes were then capped with their needle, placed together in a non-sterile steel container, covered with a clean non-sterile drape and stored in the refrigerator at 4 C (Fig. 3). Using sterile technique, four samples were withdrawn daily from each syringe and cultured during the next 3 days (Fig. 4) yielding a total of 7200 samples to be examined for sterility. 2.4. Microbiological analysis 10 mL from each sample were cultured into Sabouraud agar and chocolate agar media according to the Difco & BBL Manual, BD guidelines for culture media (Difco and BBl Manual, 2014). Culture media plates were incubated at 30–37 C for 48 h and then checked for bacterial and yeast growth. Sabouraud media were kept at 25– 30 C for an additional 5 days to facilitate growth of filamentous fungi. Microbial contamination was defined in CFU/plate.6 Growth on the main inoculation site was considered significant. All cultures were performed by the same microbiologist. Both chocolate and Sabouraud culture media were received along with the manufacture’s Declaration of Conformity with the European In Vitro Diagnostic Directive. Media for microbial growth were validated with a promotion test according to the United Kingdom National External Quality Assessment Service for Microbiology7 (UK NEQAS) guidelines (UK NEQAS, 2014) by inoculating 0.1 CFU/mL of Escherichia coli,Staphylococcus epidermidis and Candida albicans. Positive growth was considered superior or equal to 10 CFU/plate. The same quality control procedure was performed at the opening of each new media lot. 3. Results A total of 7200 samples were collected. Overall, 14,400 cultures (7200 for bacteria and 7200 for fungi) were performed. No evidence of microbial growth in any of the culture media plates was found at the end of the observational period.
2.1. Study setting Samples were prepared from SUVs filled with physiological solution (PS)4 in three different standard operating rooms (International Organization for Standardization-ISO Class 7),5 (Cleanroom Cleaning Product Guide, 2014) on six different days, upon conclusion of all elective surgeries, and by the same operator (Fig. 1). The operator was a senior anesthetist, well-trained in the sterile preparation of drugs. Samples were prepared using exclusively basic principles of asepsis: hand-washing, mask, cap, sterile gloves and a sterile sheet. Air particulate contamination was not assessed in any of the three different operating rooms. 2.2. Power analysis and statistics Considering a 95% confidence interval for the proportion of contaminated samples, with a margin error of 4%, we initially prepared 600 syringes to test. Sample size calculations were
3 4 5
USP: US Pharmacopeia. PS: physiological solution. ISO: International Organization for Standardization.
4. Discussion To the best of our knowledge, this is the first attempt to systematically assess microbial contamination of SUVs after content withdrawal, dilution, storage and subsequent multiple withdrawals performed during several days. Our findings suggest that if initial withdrawal from SUVs into disposable syringes is performed in a standard operating room with sterile technique, and if syringes are stored under refrigeration, sterility is preserved for at least 3 days when four withdrawals per day are performed. It is remarkable that, even though samples were prepared in three different operating rooms at the end of all planned surgeries, and therefore with increased possibility of environmental contamination, no microbial growth was detected. Supporting our findings, Stucki et al. (2009) assessed contamination of syringes filled in different environments (horizontal laminar-airflow, cleanroom, operating room and ward) after different risk
6
CFU: colony forming unit. UK NEQAS: United Kingdom National External Quality Assessment Service for Microbiology. 7
162
A. Ripoll Gallardo et al. / International Journal of Pharmaceutics 485 (2015) 160–163
Fig. 1. Summary of sample preparation, storage, withdrawal and culture.
manipulations. None of the syringes were contaminated after simple-filling even when air was intentionally drawn into the syringe, indicating that direct aerobiological contamination rarely occurs even in uncontrolled air environments. Arguably, basic asepsis principles could guarantee sterility of drugs after withdrawal and dilution in an ISO Class 7 environment, even after storage in non-sterile conditions. Reiter et al. (2003) assessed sterility of Calfactant (exogenous surfactant) stored in refrigerated SUVs after 12 and 24 h from first vial entry. Similarly, Ornek et al. (2008) tested sterility of Bevacizumad (recombinant monoclonal antibody) after different storage procedures. In both studies, all samples collected resulted negative for microbial growth. The aforementioned studies focused on a single drug (Gonzalez et al., 2007). Since a number of articles have already demonstrated the antibacterial properties of some medications (Apan et al., 2007; Erden et al., 2013), we decided to use PS instead of a real drug to avoid misinterpretation in case of negative microbial growth. It is well-known that SUVs are commonly reutilized in daily clinical practice regardless of current CDC recommendations. These recommendations are based on the fact that, given the absence of preservative in the vials, contamination should be assumed after first entry. Our results do not support this assertion.
Fig. 2. Withdrawing PS stored in SUVs into disposable syringes.
CDC statements are not based on a systematic literature analysis but rely on a number of case reports (Manchikanti et al., 2012). Manchikanti et al. (2012) performed an accurate literature review to evaluate the role of re-utilized SUVs in infection development. When strict attention to infection control was ensured, no causality between re-utilization of SUVs and reported infection outbreaks could be established. Therefore, universally accepted guidelines for control and prevention of diseases have been demonstrated to be very weak in providing scientific evidence directly correlating infectious disease outbreaks with reutilization of SUVs. (Manchikanti et al., 2012). However, as a matter of fact, application of CDC guidelines for infection control has huge cost implications and may result in limited access to basic medications (Manchikanti et al., 2012) particularly in underserved areas. Cost reduction is widely acknowledged as the main factor leading to reutilization of SUVs even in high-income countries (Gonzalez et al., 2007; Ornek et al., 2008; Reiter et al., 2003; Woodbury et al., 2014). Unpublished data, collected by members of our research center among health providers operating in developing countries, demonstrate that cost constrains and drug saving are the most frequently stated reasons for re-using SUVs, in order to extend minimum standards of care (such as pain relief) to the highest
Fig. 3. Storage of disposable syringes in a non-sterile steel container at
4 C.
A. Ripoll Gallardo et al. / International Journal of Pharmaceutics 485 (2015) 160–163
163
Conflict of interest We declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Authors contribution
Fig. 4. Withdrawal from syringes stored in the refrigerator.
number of patients. This leads inevitably to an ethical dilemma: should reutilization of SUVs be condemned where a changeless lack of drugs exists even if no direct evidence on its influence in causing infections is currently available? With the data at hand it seems impossible to address this issue with scientific rigor. This study was conducted to provide additional information about SUV handling and utilization. However, as Manchikanti et al. (2012) stated, “( . . . ) the most entrenched conflict of interest in medicine is a disinclination to reverse a previous opinion”. The Authors hope that these results will prompt further in vivo research in different settings (such as low-resource environments), which should include specific medications. This will supply a solid scientific basis for the development of an evidence-based regulatory framework on the utilization (or re-utilization) of SUVs, leading to either revision or confirmation of current international recommendations and, perhaps, extending basic standards of care to underserved populations. 5. Limitations This study was conducted without using any medication which, undoubtedly, limits the impact of our results. However, this choice was made to avoid introducing an additional variable which may have led to misinterpretation of microbial growth. Also, our results are only applicable to samples prepared in operating rooms complying with ISO Class 7 requirements, which could prevent the extension of our study to underserved areas. As previously stated, our conclusions should be interpreted carefully due to the in vitro design of our study; in vivo, our results may have changed significantly. 6. Conclusions This study has demonstrated that contents initially stored in SUVs and subsequently transferred into disposable syringes in a standard operating room, using sterile technique, maintain sterility after 4 withdrawals per day for a total of 3 days. Arguably, this could be considered as an important additional step for a future reformulation of CDC recommendations. We encourage research institutions to reproduce our study in other settings and overcome our limitations. This would provide additional scientific evidence on the definition of optimal utilization (or re-utilization) of SUVs. It would also have relevant cost-reduction implications and may lead to the extension of basic levels of care to underserved populations.
Each author has participated in the preparation of this manuscript. Alba Ripoll Gallardo and Grazia Meneghetti contributed in study concept and design, analysis and interpretation of the data, drafting of the manuscript, and critical revision of the manuscript; Luca ragazzoni, Pier Luigi Ingrassia, Francesco Della Corte and Fabrizio Faggiano contributed in the critical revision of the manuscript. Vesselina Kroumova, Paola Ruzza and Angela Dell’Era cultured the samples and assessed microbial growth. Daniela Ferrante performed the statistical analysis. Esther Boniolo and Gjergji Koraqe acted as assistants during the conduction of the study. References Apan, T.Z., Apan, A., Sahin, S., Cakirca, M., 2007. Antibacterial activity of remifentanil and mixtures of remifentanil and propofol. J. Clin. Anesth. 19, 346–350. Baniasadi, S., Dorudinia, A., Mobarhan, M., Karimi Gamishan, M., Fahimi, F., 2013. Microbial contamination of single- and multiple-dose vials after opening in a pulmonary teaching hospital. Braz. J. Infect. Dis. 17, 69–73. CDC, 2011. CDC guidelines for the prevention of intravascular catheter-related infections. http://www.cdc.gov/hicpac/pdf/guidelines/bsi-guidelines-2011.pdf (accessed 30.05.14.). CDC, 2012. Centers for disease control and prevention national center for emerging and zoonotic infectious diseases. Division of healthcare quality promotion single-dose/single-use vial position and messages May 2, 2012. http://www. cdc.gov/injectionsafety/CDCposition-SingleUseVial.html (accessed 12.05.14.). Cleanroom Cleaning Product Guide, 2014. http://www.bluethundertechnologies. com/wp-content/uploads/2013/03/class-10000-iso-7-cleanroom-cleaning. pdf?652038 (accessed 15.05.14.). Difco and BBl Manual, 2014. BD guidelines. http://www.bd.com/ds/technicalCenter/ misc/difcobblmanual_2nded_lowres.pdf (accessed 30.05.14). Erden, I.A., Gulmez, D., Pamuk, A.G., Akincia, S.B., Hascelik, G., Aypar, U., 2013. The growth of bacteria in infusion drugs: propofol 2% supports growth when remifentanil and pantoprazole do not. Braz. J. Anesthesiol. 63, 466–472. Gonzalez, S., Miller, D., Murphy, S.P., 2007. Maintenance of sterility in 1 mL polypropylene syringes. Am. J. Health Syst. Pharm. 64, 1962–1964. Hodges, S.C., Mijumbi, C., Okello, M., McCormick, B.A., Walker, I.A., Wilson, I.H., 2007. Anaesthesia services in developing countries: defining the problems. Anaesthesia 62, 4–11. Manchikanti, L., Falco, F.J., Benyamin, R.M., Caraway, D.L., Helm Ii, S., Wargo, B.W., Hansen, H., Parr, A.T., Singh, V., Hirsch, J.A., 2012. Assessment of infection control practices for interventional techniques: a best evidence synthesis of safe injection practices and use of single-dose medication vials. Pain Physician 15, E573–E614. Merry, A.F., Cooper, J.B., Soyannwo, O., Wilson, I.H., 2010. International standards for a safe practice of anesthesia. Can. J. Anaesth. 57 (November (11)), 1027–1034. Ornek, K., Karahan, Z.C., Ergin, A., Tekeli, A., Tekeli, O., 2008. Bevacizumab sterility in multiple doses from a single-use vial. Ann. Pharmacother. 42, 1425–1428. Reiter, P.D., Sims, C., Harmes, L., Paisley, J., Rosenberg, A.A., Valuck, R.J., 2003. Calfactant sterility in multiple doses from single-use vials. Ann. Pharmacother. 37, 1219–1223. Stucki, C., Sautter, A.M., Favet, J., Bonnabry, P., 2009. Microbial contamination of syringes during preparation: the direct influence of environmental cleanliness and risk manipulations on end-product quality. Am. J. Health Syst. Pharm. 66, 2032–2036. Stokowski, L.A., 2014. Single-Use vials: Safety, Cost, and Availability. http://www. medscape.com/viewarticle/768187 (accessed 12.05.14.). United Kingdom National External Quality Assessment Service for Microbiology (UK NEQAS) guidelines, 2014. http://www.ukneqas.org.uk/content/PageServer. asp?S=331185320&C=1252&AID=16&IID=8 (accessed 01.06.14.). Woodbury, A., Knight, K., Fry, L., Margolias, G., Lynde, G.C., 2014. A survey of anesthesiologist and anesthetist attitudes toward single-use vials in an academic medical center. J. Clin. Anesth. 26, 125–130.
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Personalised medicine
Multiple withdrawals from single-use vials: A study on sterility Alba Ripoll Gallardo a, * , Grazia Meneghetti a , Luca Ragazzoni a , Vesselina Kroumova b , Daniela Ferrante c , Pier Luigi Ingrassia a , Paola Ruzza b , Angela Dell’Era b , Esther Boniolo a , Gjergji Koraqe a , Fabrizio Faggiano d, Francesco Della Corte a a
CRIMEDIM – Research Center in Emergency and Disaster Medicine, University of Piemonte Orientale A. Avogadro, Novara, Italy Department of Virology and Microbiology, Ospedale Maggiore della Carità, Novara, Italy CPO-Piemonte and Unit of Medical Statistics and Epidemiology, Department of Translational Medicine, University of Piemonte Orientale, Novara, Italy d Department of Translational Medicine, Università del Piemonte Orientale Amedeo Avogadro, Novara, Italy b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 January 2015 Accepted 5 March 2015 Available online 9 March 2015
Background: Reutilization of single-use vials containing medical drugs is still under discussion. This practice has been adopted as a standard to avoid drug wastage, particularly in developing countries and in the aftermath of disasters. Some studies have assessed sterility of medications stored in single-use vials after utilization as multiple doses; however, most of these were limited to one single drug, included a low number of samples and did not consider an intermediate transfer step from the vial to a disposable syringe. The purpose of this study was to assess microbial contamination of samples withdrawn over three days from disposable syringes prepared from single-use vials. Methods: A prospective sterility study was conducted. A total of 600 initial samples were prepared from six-hundred 10 mL single-use vials of physiological solution into six-hundred 20 mL disposable syringes. Samples were prepared in three different standard operating rooms, on six different days and by the same operator, using basic sterile technique. All syringes were capped, placed together in a non-sterile steel container, covered with a clean drape and stored in the refrigerator at 4 C under non-sterile conditions. Using basic sterile technique, four samples were withdrawn daily and cultured from each syringe over the next 3 days. Microbial growth was examined on Sabouraud agar and chocolate agar culture media. Results: A total of 7200 samples were collected and 14,400 cultures were performed. No evidence of microbial growth in any of the culture media plates was found. Conclusion: This study demonstrated that contents initially stored in single-use vials and subsequently transferred into disposable syringes in an operating room using sterile technique, maintain sterility after 4 withdrawals per day for a total of 3 days. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Single-use vial Drug packaging Drug storage Disposable equipment Drug contamination Syringes/microbiology
1. Introduction Ever since their first use in daily practice, reutilization of drugs contained in single-dose/use vials (SUVs)1 has been widely
* Corresponding author at: CRIMEDIM – Research Center in Emergency and Disaster Medicine, University of Piemonte Orientale A. Avogadro, Via Ferrucci 33/ Via Lanino 1, 28100 Novara, Italy. Tel.: +39 321660620; fax: +39 321660531. E-mail addresses: [email protected] (A. Ripoll Gallardo), [email protected] (G. Meneghetti), [email protected] (L. Ragazzoni), [email protected] (V. Kroumova), [email protected] (D. Ferrante), [email protected] (P.L. Ingrassia), [email protected] (P. Ruzza), [email protected] (A. Dell’Era), [email protected] (E. Boniolo), [email protected] (G. Koraqe), [email protected] (F. Faggiano), [email protected] (F. Della Corte). 1 SUV: single-dose/use vials. http://dx.doi.org/10.1016/j.ijpharm.2015.03.010 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
discussed (Stokowski, 2014). In its latest statement, the Center for Disease Control and prevention (CDC, 2011, 2012)2 reaffirmed that “Vials that are labeled as single-dose or single-use should be used for a single patient and single case/procedure/injection.” This assertion relies on the fact that SUVs contain neither antimicrobial agents nor preservatives and, therefore, contamination should be expected after initial vial entry. However, several studies have demonstrated that health professionals often do not comply with CDC recommendations (Stokowski 2014). For instance, the survey conducted by Woodbury et al. (2014) among anesthesia providers in Atlanta, showed that close to 60% admitted to having re-used SUVs for different patients. Similarly, Baniasadi et al. (2013) conducted a study in Iran in which 205 opened vials stored in
2
CDC: Center for Disease Control and Prevention.
A. Ripoll Gallardo et al. / International Journal of Pharmaceutics 485 (2015) 160–163
different hospital wards were analyzed; of these, 80.49% were SUVs employed as multiple-use vials. Re-using SUVs has been adopted by health care providers as standard practice especially in developing countries, in the aftermath of disasters, or in complex humanitarian emergencies in an attempt to avoid drug wastage. For instance, although in the International Standards for a Safe Practice of Anesthesia (Merry et al., 2010) morphine and pethidine are both considered as mandatory medications, Hodges et al. (2007) found that among anesthesia practitioners in Uganda only 45% had uninterrupted access to this particular drugs. Often in these settings, the shortage of medications is not only a consequence of restrictive laws but also of high drug prices in comparison to Western health-care systems. The CDC recognized the motivations leading health providers to reutilize the contents of SUVs for more than one patient, and therefore an option was extended for the storage of contents of unopened SUVs into multiple-use syringes. However, this repackage procedure must be performed exclusively by qualified personnel and under specific conditions complying with the US Pharmacopeia (USP)3 797 standards (Stokowski, 2014), for instance, in pharmacies. Consequently, operating rooms and other clinical areas are considered inadequate environments for splitting and repacking vials regardless of the technique used (Stokowski, 2014). In recent years, some studies have been conducted to assess sterility of different medications stored into SUVs and used as multiple doses (Ornek et al., 2008; Reiter et al., 2003). However, most of these were restricted to a single drug, included a low number of samples and did not consider transferring contents into disposable syringes, a maneuver frequently performed when the dosage needed per patient is much lower than the total amount of drug contained in a single vial. Therefore, the purpose of this study was to determine microbial contamination of multiple samples withdrawn during several days from disposable syringes prepared from SUVs. The expected result was that contamination rate among all samples would not exceed that calculated for simple filling of syringes under the same environmental conditions (Stucki et al., 2009). 2. Materials and methods
161
performed using the Statistics Calculator software (nQuery Advisor, v5.05). 2.3. Sample preparation and storage A total of 600 samples were prepared into 20 mL disposable syringes from six-hundred 10 mL SUVs filled with PS (Fig. 2). The total volume was extended to 20 mL with additional PS to simulate drug dilution. All syringes were then capped with their needle, placed together in a non-sterile steel container, covered with a clean non-sterile drape and stored in the refrigerator at 4 C (Fig. 3). Using sterile technique, four samples were withdrawn daily from each syringe and cultured during the next 3 days (Fig. 4) yielding a total of 7200 samples to be examined for sterility. 2.4. Microbiological analysis 10 mL from each sample were cultured into Sabouraud agar and chocolate agar media according to the Difco & BBL Manual, BD guidelines for culture media (Difco and BBl Manual, 2014). Culture media plates were incubated at 30–37 C for 48 h and then checked for bacterial and yeast growth. Sabouraud media were kept at 25– 30 C for an additional 5 days to facilitate growth of filamentous fungi. Microbial contamination was defined in CFU/plate.6 Growth on the main inoculation site was considered significant. All cultures were performed by the same microbiologist. Both chocolate and Sabouraud culture media were received along with the manufacture’s Declaration of Conformity with the European In Vitro Diagnostic Directive. Media for microbial growth were validated with a promotion test according to the United Kingdom National External Quality Assessment Service for Microbiology7 (UK NEQAS) guidelines (UK NEQAS, 2014) by inoculating 0.1 CFU/mL of Escherichia coli,Staphylococcus epidermidis and Candida albicans. Positive growth was considered superior or equal to 10 CFU/plate. The same quality control procedure was performed at the opening of each new media lot. 3. Results A total of 7200 samples were collected. Overall, 14,400 cultures (7200 for bacteria and 7200 for fungi) were performed. No evidence of microbial growth in any of the culture media plates was found at the end of the observational period.
2.1. Study setting Samples were prepared from SUVs filled with physiological solution (PS)4 in three different standard operating rooms (International Organization for Standardization-ISO Class 7),5 (Cleanroom Cleaning Product Guide, 2014) on six different days, upon conclusion of all elective surgeries, and by the same operator (Fig. 1). The operator was a senior anesthetist, well-trained in the sterile preparation of drugs. Samples were prepared using exclusively basic principles of asepsis: hand-washing, mask, cap, sterile gloves and a sterile sheet. Air particulate contamination was not assessed in any of the three different operating rooms. 2.2. Power analysis and statistics Considering a 95% confidence interval for the proportion of contaminated samples, with a margin error of 4%, we initially prepared 600 syringes to test. Sample size calculations were
3 4 5
USP: US Pharmacopeia. PS: physiological solution. ISO: International Organization for Standardization.
4. Discussion To the best of our knowledge, this is the first attempt to systematically assess microbial contamination of SUVs after content withdrawal, dilution, storage and subsequent multiple withdrawals performed during several days. Our findings suggest that if initial withdrawal from SUVs into disposable syringes is performed in a standard operating room with sterile technique, and if syringes are stored under refrigeration, sterility is preserved for at least 3 days when four withdrawals per day are performed. It is remarkable that, even though samples were prepared in three different operating rooms at the end of all planned surgeries, and therefore with increased possibility of environmental contamination, no microbial growth was detected. Supporting our findings, Stucki et al. (2009) assessed contamination of syringes filled in different environments (horizontal laminar-airflow, cleanroom, operating room and ward) after different risk
6
CFU: colony forming unit. UK NEQAS: United Kingdom National External Quality Assessment Service for Microbiology. 7
162
A. Ripoll Gallardo et al. / International Journal of Pharmaceutics 485 (2015) 160–163
Fig. 1. Summary of sample preparation, storage, withdrawal and culture.
manipulations. None of the syringes were contaminated after simple-filling even when air was intentionally drawn into the syringe, indicating that direct aerobiological contamination rarely occurs even in uncontrolled air environments. Arguably, basic asepsis principles could guarantee sterility of drugs after withdrawal and dilution in an ISO Class 7 environment, even after storage in non-sterile conditions. Reiter et al. (2003) assessed sterility of Calfactant (exogenous surfactant) stored in refrigerated SUVs after 12 and 24 h from first vial entry. Similarly, Ornek et al. (2008) tested sterility of Bevacizumad (recombinant monoclonal antibody) after different storage procedures. In both studies, all samples collected resulted negative for microbial growth. The aforementioned studies focused on a single drug (Gonzalez et al., 2007). Since a number of articles have already demonstrated the antibacterial properties of some medications (Apan et al., 2007; Erden et al., 2013), we decided to use PS instead of a real drug to avoid misinterpretation in case of negative microbial growth. It is well-known that SUVs are commonly reutilized in daily clinical practice regardless of current CDC recommendations. These recommendations are based on the fact that, given the absence of preservative in the vials, contamination should be assumed after first entry. Our results do not support this assertion.
Fig. 2. Withdrawing PS stored in SUVs into disposable syringes.
CDC statements are not based on a systematic literature analysis but rely on a number of case reports (Manchikanti et al., 2012). Manchikanti et al. (2012) performed an accurate literature review to evaluate the role of re-utilized SUVs in infection development. When strict attention to infection control was ensured, no causality between re-utilization of SUVs and reported infection outbreaks could be established. Therefore, universally accepted guidelines for control and prevention of diseases have been demonstrated to be very weak in providing scientific evidence directly correlating infectious disease outbreaks with reutilization of SUVs. (Manchikanti et al., 2012). However, as a matter of fact, application of CDC guidelines for infection control has huge cost implications and may result in limited access to basic medications (Manchikanti et al., 2012) particularly in underserved areas. Cost reduction is widely acknowledged as the main factor leading to reutilization of SUVs even in high-income countries (Gonzalez et al., 2007; Ornek et al., 2008; Reiter et al., 2003; Woodbury et al., 2014). Unpublished data, collected by members of our research center among health providers operating in developing countries, demonstrate that cost constrains and drug saving are the most frequently stated reasons for re-using SUVs, in order to extend minimum standards of care (such as pain relief) to the highest
Fig. 3. Storage of disposable syringes in a non-sterile steel container at
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Conflict of interest We declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Authors contribution
Fig. 4. Withdrawal from syringes stored in the refrigerator.
number of patients. This leads inevitably to an ethical dilemma: should reutilization of SUVs be condemned where a changeless lack of drugs exists even if no direct evidence on its influence in causing infections is currently available? With the data at hand it seems impossible to address this issue with scientific rigor. This study was conducted to provide additional information about SUV handling and utilization. However, as Manchikanti et al. (2012) stated, “( . . . ) the most entrenched conflict of interest in medicine is a disinclination to reverse a previous opinion”. The Authors hope that these results will prompt further in vivo research in different settings (such as low-resource environments), which should include specific medications. This will supply a solid scientific basis for the development of an evidence-based regulatory framework on the utilization (or re-utilization) of SUVs, leading to either revision or confirmation of current international recommendations and, perhaps, extending basic standards of care to underserved populations. 5. Limitations This study was conducted without using any medication which, undoubtedly, limits the impact of our results. However, this choice was made to avoid introducing an additional variable which may have led to misinterpretation of microbial growth. Also, our results are only applicable to samples prepared in operating rooms complying with ISO Class 7 requirements, which could prevent the extension of our study to underserved areas. As previously stated, our conclusions should be interpreted carefully due to the in vitro design of our study; in vivo, our results may have changed significantly. 6. Conclusions This study has demonstrated that contents initially stored in SUVs and subsequently transferred into disposable syringes in a standard operating room, using sterile technique, maintain sterility after 4 withdrawals per day for a total of 3 days. Arguably, this could be considered as an important additional step for a future reformulation of CDC recommendations. We encourage research institutions to reproduce our study in other settings and overcome our limitations. This would provide additional scientific evidence on the definition of optimal utilization (or re-utilization) of SUVs. It would also have relevant cost-reduction implications and may lead to the extension of basic levels of care to underserved populations.
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