Journal of Cystic Fibrosis 13 (2014) 550 – 556 www.elsevier.com/locate/jcf Original Article Contamination of hypertonic saline solutions in use by cystic fibrosis patients in Israel☆,☆☆ Orit Peled a,1 , Vardit Kalamaro b,d,f,1 , Eitan Kerem c , David Shoseyov c , Hannah Blau d,e , Ori Efrati f , Colin Block g,⁎ a g Department of Pharmacy, Schneider Children's Medical Center, 14 Kaplan St., PO Box 559, Petah Tikva 49202, Israel b Cystic Fibrosis Foundation of Israel, 79 Krinitzy St., Ramat-Gan 52423, Israel c Department of Pediatrics and Cystic Fibrosis Center, Hadassah Hebrew University Hospital, PO Box 24035, Mount Scopus, Jerusalem 91240, Israel d Graub Cystic Fibrosis Center, Pulmonary Unit, Schneider Children's Medical Center, 14 Kaplan St., PO Box 559, Petah Tikva 49202, Israel e Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv 69978, Israel f Edmond and Lili Safra Children's Hospital, Chaim Sheba Medical Center, Tel Hashomer, Ramat Gan 52621, Israel Department of Clinical Microbiology and Infectious Diseases, Hadassah-Hebrew University Medical Center, PO Box 12000, Ein Kerem, Jerusalem 91120, Israel Received 29 October 2013; received in revised form 9 January 2014; accepted 10 January 2014 Available online 28 January 2014 Abstract Background: Treatment of cystic fibrosis (CF) patients with inhaled hypertonic saline (HS) solutions is safe, beneficial and reduces exacerbation rates. We studied contamination of solutions used by Israeli CF patients for prolonged periods. Methods: The study addressed whether daily opening of previously unopened solutions caused contamination, survival of 6 CF-associated bacteria in artificially inoculated solutions, in-use contamination of solutions and patterns of their use by patients. Results: Repeated opening did not contaminate solutions and survival of indicator bacteria was variable. Mycobacterium abscessus survived in 3% HS solution for 6 weeks and Burkholderia cenocepacia and Pseudomonas aeruginosa were longer. In 30/76 (39.5%) of used solutions 49 contaminants were found, none being common CF-associated pathogens. Conclusions: Most CF-related bacteria survived to some degree in HS. Approximately 40% of solutions used by patients were contaminated by organisms of uncertain significance. Our findings highlight the potential risk posed by contamination of HS solutions and support recommendations to use sterile unit-dose formulations. © 2014 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Keywords: Cystic fibrosis; Hypertonic saline; Sterility; Contamination; Unit dose 1. Introduction ☆ Data from the manuscript were presented at the following meetings: 1. The 4th Annual National Israeli CF Conference, 5th November 2010, Kfar-Blum, Israel. 2. The Annual Meeting of The Pharmaceutical Society of Israel 13th December 2011, Tel-Aviv, Israel .3. The 25th Annual North American Cystic Fibrosis Conference, Anaheim, California, November 3–5 2011. Poster no. 282. ☆☆ OP's contribution to this study was performed in partial fulfillment of the requirements for the degree of M.Sc. in Clinical Pharmacy at the School of Pharmacy in the Faculty of Medicine of the Hebrew University of Jerusalem, Israel. ⁎ Corresponding author. Tel.: +972 2 6776543; fax: +972 2 6419545. E-mail address: colinb@ekmd.huji.ac.il (C. Block). 1 Orit Peled and Vardit Kalamaro contributed equally to this work. Cystic fibrosis (CF) is caused by mutations in the CFTR gene that result in the absence or dysfunction of the protein that regulates ion transport across the apical membrane at the surface of certain epithelia. In the lungs, CFTR dysfunction leads to airway surface liquid (ASL) depletion and thickened and viscous mucus that adhere to airway surfaces [1]. The result is decreased mucociliary clearance (MCC) and impaired host defenses. Dehydrated, thickened secretions lead to endobronchial infection with a limited spectrum of distinctive bacteria mainly Staphylococcus aureus and Pseudomonas aeruginosa, and an 1569-1993/$ -see front matter © 2014 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcf.2014.01.001 551 O. Peled et al. / Journal of Cystic Fibrosis 13 (2014) 550–556 (3%, 7%, 14.6%) under clean conditions and at defined intervals over a one month period. 2) Survival of selected CF-associated bacteria in artificially inoculated hypertonic saline solutions. 3) In-use contamination of hypertonic saline solutions in different settings (out-patient clinics, patients' homes). 4) An additional objective was to document the manner of use of these solutions in the home and clinic settings, in order to identify handling habits that might affect bacterial contamination. exaggerated inflammatory response with subsequent development of bronchiectasis and progressive obstructive airways disease. Pulmonary insufficiency is responsible for most CF-related deaths [2,3]. Inhaled hyperosmolar agents such as hypertonic saline induce osmotic flow of water into the mucus layer, thereby rehydrating secretions and improving mucus rheology and transportability of sputum [4,5], and increased hydration of the airway surface [6,7]. Inhalations of hypertonic saline (4 ml BID following pretreatment with bronchodilators) improved MCC and lung function and reduced exacerbation rate in patients with CF [8–11]. This improvement in mucociliary function may reduce bacterial load and chronic inflammation within the airways with a concomitant stabilization of lung function. Hypertonic saline is inexpensive, safe, and well tolerated in young children [10]. Since HS inhalation has become an acceptable treatment for CF patients, Israeli CF patients have been using it in increasing numbers as long term therapy within the home setting. However, until very recently in Israel, as in many other countries, there were no standardized sterile unit-dose HS formulations. Compounding pharmacies produce large-volume bottled solutions of HS, which patients use for prolonged periods. Furthermore, some patients prepare the HS solution at home by diluting sterile solutions containing higher concentrations of sodium chloride (14.6% and above) with water for injection or normal saline (0.9%) to achieve the desired concentration for inhalations (3–7%). These solutions are used by patients, often for weeks, under non-sterile conditions, with no consideration of the potential for contamination [12–14]. Therefore, concerns regarding the acquisition of potential pathogens by patients with CF through repeated use of pre-prepared HS solutions for inhalations were raised. Based upon prevention as a primary goal for infection control in CF, it is self-evident that any inhaled medication treating CF patients should be sterile. Therefore, the potential for contamination of different HS solutions in use at CF clinics and patients' homes was considered worthy of scrutiny. The purpose of this study was to investigate bacterial contamination of hypertonic saline solutions under the following circumstances: 2. Settings 2.1. CF outpatient clinics at three large hospitals Patients were asked to bring HS solutions they used at home, and were almost finished, for submission to the laboratory. Some patients brought their solutions at several visits (Table 1). The questionnaire was completed once for each patient at a clinic visit. Inhalations were routinely performed at the clinics by CF nurses using the clinics' nebulizers and HS solutions. Each patient was given a decontaminated nebulizer with new solutions for these treatments. There was no standard type of nebulizer in use by all clinics and patients. 2.2. Clinical microbiology laboratory Laboratory experiments and cultures of solutions in use were performed at the Hadassah-Hebrew University Medical Centre clinical microbiology laboratory. 3. Methods The institutional review boards of the Hadassah-Hebrew University Medical Center at Mt. Scopus and the Schneider Children's Medical Center in Petah Tikva approved the protocol with verbal informed consent of the patients or their parents. At the Chaim Sheba Medical Center at Tel Hashomer, written informed consent was required. 1) The effect of simple daily opening and closing of previously unopened hypertonic saline solutions at various concentrations Table 1 Distribution of 76 HS solutions submitted by 43 patients. Number of HS solutions per patient Number of patients 1 2 3 4 7 8 Total 28 b 7 5 1 1 1 43 a b Number of patients × number of solutions tested. 2 solutions were obtained from CF outpatient clinics. Total HS solutions a Number of contaminated HS solutions per patient 0 1 2 13 b 2 2 1 15 4 3 1 4 1 18 23 1 1 1 28 14 15 4 7 8 76 552 O. Peled et al. / Journal of Cystic Fibrosis 13 (2014) 550–556 3.1. Hypertonic saline solutions The following HS solutions available in Israel were studied: 3% sodium chloride 500 ml and 7% sodium chloride 160 ml, both from compounding pharmacy A; 7% sodium chloride 100 ml from compounding pharmacy B; 7% sodium chloride 100 ml from compounding pharmacy C; and a commercial preparation of 14.6% sodium chloride for injection, USP, 40 ml. All solutions were labeled as sterile. The study was divided into 4 parts: Part 1: The effect of daily opening and closing of containers on the contamination of previously unopened HS solutions was investigated. The purpose of this was to provide a comparator for “real world” use in clinics or by patients in their homes. Triplicates of each HS solution were opened and closed aseptically once a day for 1 month inside a class IIA biological safety cabinet using ungloved hands after disinfection with an alcohol based hand sanitizer. The rubber diaphragm and closure of each container were disinfected with 70% ethanol before opening. Volumes of 0.4–2 ml of each solution were drawn daily according to the capacity of each container, simulating real-life practice. Larger volumes of 4–50 ml, depending on container capacity, were collected at first opening (baseline), 24 h, 72 h, 7 d, 14 d and 30 d. Quantitative bacterial cultures were set up for each specimen. In anticipation of low bacterial densities in this part of the study, quantitative cultures were performed using a membrane filtration method (Millipore Microfil®, Millitech, Israel). Samples were drawn through sterile 0.45 μm pore diameter membranes. All procedures were carried out according to the manufacturer's instructions in a class IIA biological safety cabinet. Filters were aseptically transferred to tryptic soy agar (TSA, Novamed, Jerusalem, Israel) and incubated in air at 35 ºC for 48 h and subsequently at room temperature for 5 more days. Any organisms grown were identified by routine clinical laboratory methods. All laboratory tests were conducted at the Clinical Microbiology Unit at the Hadassah-Hebrew University Medical Center. Part 2: Survival of well recognized CF-associated bacteria in the HS solutions mentioned above was tested for the following species: 1) Staphylococcus aureus (ATCC 25923) 2) Pseudomonas aeruginosa (ATCC 27853) 3) Haemophilus influenzae (ATCC 49247) 4) Stenotrophomonas maltophilia (clinical isolate from a non-CF patient) 5) Burkholderia cenocepacia (clinical isolate from CF patient) 6) Mycobacterium abscessus (clinical isolate from CF patient) Saline suspensions of 0.5 McFarland turbidity standard of each bacterium were introduced, at initial opening, into 6 replicates of each type of HS solution to generate a concentration of approximately 7.5 × 105 CFU/ml in each bottle. Viable counts of the inocula (baseline) and the bacterial density in each bottle were performed by the drop-plate method [15]. H. influenzae and M. abscessus were enumerated on chocolate agar and Middlebrook agar respectively (Novamed, Jerusalem, Israel). The other species were cultured on TSA. Viable counts were performed at defined time intervals: 24 h, 72 h, 7 d, 14 d, 30 d and 42 d. Part 3: Seventy-six unselected samples of HS solutions from CF patients, including two from outpatient clinics, were collected anonymously, close to their final use. For each, the entire remaining solution was drawn aseptically, and cultures were performed by the membrane filtration method as described above. Equal parts of each sample were filtered aseptically on separate membranes for culture on both TSA and Middlebrook agar. TSA plates were incubated in air at 35 °C for two days and 6 days for Middlebrook agar plates. The identification of any bacterial or fungal growth, irrespective of quantities, was carried out by routine clinical laboratory methods. In addition to the characteristics of the formulation tested, the following data were recorded for each sample: the patient's clinic, ethnic group, use of refrigeration and time interval from last use to processing at the laboratory. Part 4: In order to identify factors that might influence the sterility of the solutions, a questionnaire on the home use of HS solutions was filled out anonymously by patients from the different clinics. Issues examined included storage of the containers, duration of usage of each, frequency and duration of exposure of a solution to open air, method used for obtaining HS solution from containers, the use of a syringe and frequency of its exchange if this method was used. It should be noted that since the questionnaires were gathered anonymously, contamination results could not be associated with usage conditions by specific patients. Statistical methods: associations between contaminants and handling of the solutions by patients and clinics were evaluated using the chi-square test and Fisher's exact test. 4. Results Part 1: HS solutions did not become contaminated after one month of repeated opening in the laboratory environment. This supported the assumption that with good aseptic practice, opening and closing the containers for a month were not associated with detection of contaminants in the solutions. Part 2: Survival of the bacteria in the different solutions was variable (Fig. 1). H. influenzae did not survive in any HS solution. Most organisms survived better in the 3% and the 14.6% solutions than the 7% solutions. Some survived in the 3% HS solution for 6 weeks (M. abscessus) or longer (B. cenocepacia and P. aeruginosa). Part 3: Out of 76 solutions from 41 CF patients (Table 1) and two CF outpatient clinics, 30 (39.5%) were contaminated with 49 organisms, with a mean of 1.6 organisms per sample (Table 2). Various contaminants were found: 67.3% gram-positive bacteria, 22.4% gram-negative bacteria O. Peled et al. / Journal of Cystic Fibrosis 13 (2014) 550–556 553 Fig. 1. Survival of six cystic fibrosis associated organisms over six weeks in different hypertonic saline formulations shown as % concentrations. A, B and C represent different manufacturers. and 10.2% fungi. None of the CF-associated bacteria mentioned above were isolated from any contaminated fluid. The two solutions from outpatient clinics yielded no growth. No statistically significant associations were found between contaminants and handling of the solutions by patients, clinic, type of solution, refrigeration, time elapsed since last use, frequency of needle and syringe change or ethnicity. Part 4: The questionnaire regarding the use of the HS solutions was completed by 91 patients. Forty-six (51.1%) were male with a mean age of 17.7 ± 12.2 (mean ± SD). Fifty-nine (64.8%) were from the Hadassah-Hebrew University Medical Center at Mt. Scopus, 24 (26.4%) were from the Schneider Children's Medical Center in Petah Tikva and 8 (8.8%) were from the Chaim Sheba Medical Center at Tel Hashomer. Forty-five patients (49.5%) used solutions in a simple plastic bottle with a screw-cap closure, while 46 (50.5%) used containers with a rubber diaphragm. Normal use of containers with a screw-cap closure offers many opportunities for contamination: 3/45 respondents (6.7%) reported that the solutions remained open to air for 10 min or more during each use. Withdrawal of solution using a syringe and needle after opening the cap was reported by 19/45 respondents (42.2%); 11/45 respondents (24.4%) pierced the plastic with the needle to withdraw the solution; 11/45 respondents (24.4%) simply poured the estimated volume needed after opening the bottle normally and 4/45 respondents (8.9%) used other means. The major inappropriate practices in handling the HS solutions are summarized in Table 3. Only 24 (26.4%) of the 91 respondents refrigerated their solutions between uses. Thirty (33.0%) respondents used their solutions for more than 1 month, 24 (26.4%) for 1 month, 15 (16.5%) for 3 weeks and 22 (24.2%) for 2 weeks. Needles were changed monthly or less frequently by 43 respondents (47.8%). Ninety respondents answered the question regarding syringes and needles. Six (6.7%) changed their equipment after each use; 3 (3.3%) after a day or two and 27 (30.0%) once every week or two. Eleven (12.2%) didn't use syringe and needle at all and simply poured the estimated volume needed after opening the plastic bottle with a screw-cap closure as described above. 5. Discussion and conclusions The issue of the survival of CF-associated bacteria in HS solutions has not been addressed before in a systematic way. 554 O. Peled et al. / Journal of Cystic Fibrosis 13 (2014) 550–556 Table 2 Distribution of the organisms found in hypertonic saline solutions. Organism Total (%) Gram-positive bacteria Bacillus sp. Micrococci Diphtheroids Coagulase negative staphylococcus Rhodococcus sp. Total gram-positive Gram-negative bacteria Enterobacter cloacae Pseudomonas spp. (non-P. aeruginosa) Myroides odoratum Escherichia coli Acinetobacter lwoffii Klebsiella oxytoca Klebsiella pneumoniae Total gram-negative Fungi Aspergillus niger Candida parapsilosis Acremonium kiliense Curvularia sp. Total fungi Total organisms Total positive samples Mean organisms per sample Total cultured solutions % contaminated solutions 12 (24.5%) 9 (18.4%) 7 (14.3%) 4 (8.2%) 1 (2%) 33 (67.3%) 3 (6.1%) 3 (6.1%) 1 (2%) 1 (2%) 1 (2%) 1 (2%) 1 (2%) 11 (22.4%) 2 (4.1%) 1 (2%) 1 (2%) 1 (2%) 5 (10.2%) 49 (100%) 30 1.6 76 39.5 We showed that the HS solutions were not especially hostile to many classical CF-associated species. While H. influenzae unsurprisingly did not survive for any length of time, most of the others survived from days to weeks in some of the formulations. The survival for at least 6 weeks in 3% HS by M. abscessus, B. cenocepacia and P. aeruginosa was particularly worrisome. The possible reasons for the better survival of most organisms in the 3% and the 14.6% solutions than the 7% solutions were beyond the scope of this study. These findings emphasize the concern that aerosolized solutions, initially contaminated by contact with respiratory secretions or insufficient hand hygiene or poor handling during use, may be reservoirs for the transmission of infectious organisms to this vulnerable group of patients. In-use contamination was found in about 40% of containers examined. No statistically significant association was found between this contamination of HS solutions and their characteristics and handling issues. However, it should be noted that none of classical bacterial hazards for CF patients, as included in Part 2 of this study, were found as contaminants. Nevertheless, it should Table 3 Summary of the main problematic practices in patients' use of HS solutions in their homes, n = 91. Description No. of patients % HS solution not refrigerated Prolonged use (1 month or longer) Same syringe used for 1 month or longer 67 54 43 74 59 48 be noted that three non-fermentative gram-negative rods were not fully identified and may potentially include taxa associated in recent years with CF. Molecular studies have indicated that many previously unrecognized bacteria are likely common inhabitants of the airways in CF [16]. The presence of some filamentous fungi also deserves comment. The organisms found were very varied and the number of samples examined was very limited, so associations between some of the variables studied may not have been evident. Our study has several limitations that should be considered. One is that more than one solution was collected from some of the patients. While this raises some questions as to the interpretation of the contamination data, especially in view of the difficulty in relating individual patients and their contamination results to their particular usage habits, it is still noteworthy that almost 40% of the solutions were contaminated. Another potential limitation is the use of simple aerobic bacterial and fungal cultures of planktonic organisms in a field in which much weight is given to biofilms and their associated microbiota. In looking at contamination of inhaled nebulized solutions we were primarily interested in organisms in suspension that might be incorporated in the droplets during the nebulization process. Specific evaluation of possible biofilm formation and microbiology in HS containers was not feasible in the framework of this study, so this question remains unanswered at present. We did not specifically assess patient education by health care providers such as primary care physicians, nurses and pharmacists, as to the correct usage of HS solutions. Education for infection control is a standard practice at the participating CF clinics. The types of contaminations demonstrated in this study (Table 2) and the main inappropriate handling practices for HS solutions in the domestic environment (Table 3) are almost certainly preventable by simple interventions such as adequate education of the patients and their families, coupled with use of unit-dose containers for these HS solutions. Single unit-dose medication vials are always preferred [17]. The microbial contamination of home and hospital nebulizers in CF patients has been assessed [18,19], as well as patients' hands and clinic environment [20]. O'Malley (2007) concluded that there is a low risk of growth of P. aeruginosa, S. aureus or H. influenzae in small volume nebulizer medication cups over a 24-hour period, which supports the practice of replacing the nebulizer every 24 h rather than cleaning or disinfecting it between uses [18]. Blau et al. (2007) demonstrated that a majority of nebulizers used at home by CF patient are contaminated, particularly when patients do not recall cleaning instructions, and that pseudomonas constituted the major contaminant [19]. Clinically, this might be particularly important during the initial stages of intermittent airway infection with P. aeruginosa. A continuous cycle of reinfection may occur in these patients, where contaminated secretions are transmitted by contact to nebulizer equipment, and later are inhaled back into the airways, generally increasing the risk of permanent infection with concomitant poor prognosis [21]. In a review on the organisms causing chronic airway infections in the CF patient population, Gilligan noted that enteric bacilli and gram-negative glucose-non-fermenting bacilli other than P. aeruginosa and Pseudomonas cepacia O. Peled et al. / Journal of Cystic Fibrosis 13 (2014) 550–556 (Burkholderia cenocepacia and related species) are occasionally recovered from the respiratory secretions of CF patients [22]. None of these organisms seemed to persist for extended periods. Any role that they may have in lung disease of these patients is likely to be secondary to that of the organisms mentioned previously. Organisms represented in that review included Enterobacter cloacae (6.1% of our contaminants, Table 1), Escherichia coli, Acinetobacter lwoffii, Klebsiella oxytoca and Klebsiella pneumoniae (each comprising 2% of our contaminants). A report by Klinger et al. on the occurrence and antimicrobial susceptibility of gram-negative non-fermentative bacilli in CF patients concluded that the frequency of organisms other than P. aeruginosa in respiratory tract cultures from CF patients has increased [23]. In recent years, much attention has been paid to a range of additional organisms, especially glucose non-fermenting gramnegative rods, which are increasingly being encountered in CF sputum [24]. This should be seen in the context of the growing evidence that suggests that pulmonary infections in CF should be considered from a microbial community perspective, since CF airways contain highly complex, polymicrobial bacterial communities. The notion that has held sway until now, that a limited number of microorganisms contribute to the disease state, may well be an oversimplification [25–27]. Undoubtedly, modern genetic and bioinformatics methods will dramatically expand our knowledge of the microbiome in CF airways and inform radical changes in the prevention and treatment of CF lung disease in the next few years. Regarding fungi, Nagano et al. reported that Acremonium spp. are found relatively infrequently in CF sputum, and that their clinical significance is unknown [28]. Infection and allergic bronchopulmonary aspergillosis (ABPA) due to Aspergillus fumigatus have been associated with deterioration in CF [29]. We encountered Aspergillus niger among the contaminants of the hypertonic saline solutions. The only reported case of presumed severe A. niger infection in CF was found in a study by Quattrucci et al. who evaluated complications and survival after sequential bilateral lung transplantation in patients with end-stage cystic fibrosis [30]. While we acknowledge that the possible role of the organisms found in the solutions in our study is unclear, the contamination of multi-dose HS solutions used for inhalation demonstrated in our study is unacceptable in respiratory therapy. Therefore, HS solutions should be restricted to unit-dose vials only. Our findings were presented at various forums and subsequently, in January 2012, commercially compounded, sterile unit dose vials for CF patients were made available for the first time in Israel. The product contains 6% NaCl and has been added to the list of medications subsidized by the government of Israel. The interesting behaviors revealed in the questionnaire, together with our contamination findings suggest that more attention should be paid to instruction of patients and their families regarding the safe use of materials introduced into the compromised airways of CF patients [31]. 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