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124 Lack of antimicrobial activity of ivacaftor against clinical CF respiratory isolates
S88
4. Microbiology
121 Extended HLA analysis in a cohort of patients hypersensitive to beta-lactam antibiotics P. Whitaker1 , A. Alfirevic2 , C. Etherington1 , I. Clifton1 , G. Fitch1 , M. Pirmohamed2 , D. Peckham1 . 1 Leeds Teaching Hospitals NHS Trust, Regional Adult CF Unit, Leeds, United Kingdom; 2 University of Liverpool, Institute of Translational Medicine, Liverpool, United Kingdom Objectives: Hypersensitivity reactions to beta-lactam antibiotics remain a major clinical problem in the management of patients with CF. Whilst reactions are more likely to occur in sicker patients receiving more frequent treatment this is not always the case and genetic predisposition may play a role. Methods: Patients were recruited from the Leeds Adult CF Centre. High resolution sequence based typing was performed on HLA A, B, C, DRB1, DQA1, and DQB1. The data was compared to our electronic patient records. Results: 263 patients took part in the study. 163 had a history of betalactam reactions and 100 were tolerant controls. 97 patients had a history of piperacillin hypersensitivity. No statistical differences in frequencies of single HLA alleles were seen between hypersensitive patients and controls. One haplotype (A*0201:B*4402:DRB1*0401) occurred at a higher frequency in patients with piperacillin reactions (OR 15.3; p = 0.0006). Conclusion: The high frequency of beta-lactam reactions in patients with CF makes it unlikely that a single HLA allele could account for increased predisposition alone. This study shows interesting data supporting an increased genetic risk in certain individuals and more work is needed to refine this further.
122 Successful Burkholderia spp. eradication with hypothiocyanite/ lactoferrin. In vitro study evidence over a worldwide collection of clinical strains Y. Sonmez1 , C. Bechetoille1 , S. Perrotto1 , A. Payet-Burin1 , V. Juarez-Perez1,2 . 1 Alaxia, Lyon, France; 2 Stragen France, Lyon, France Objectives: To test the efficacy of various combinations of OSCN− and bLF on clinical Burkholderia strains. Methods: We tested 165 Burkholderia spp. CF-clinical isolates from worldwide origin. MIC values were obtained according to CLSI guidelines with minor modifications and Time-kill curves with standard methods. All experiments were done in triplicate. Results: 100% of isolates tested were inhibited by OSCN− alone within a range of 30–157 mg/mL. In contrast, only 4.8% (8/165) of the tested strains are inhibited by bLF alone within a range of 0.25−96 mg/mL. The FIC analysis of the whole panel of strains reveals that inhibitory combinations are predominantly additive or synergistic for 73% of the collection. The combination of 118 mg/mL of OSCN− with 8 mg/mL of bLF inhibits 100% of tested isolates. Time-kill curves performed on 21 strains. Results show that the OSCN− /bLF combination (ALX-009) decreases the bacterial count by 2−4 logs at 6 h after inoculation when compared to the inoculum and by 4−6 logs if compared to the negative control. 24 h after inoculation, the bacterial counts were reduced by 4−6 logs if compared to the negative control. These results suggest that for time 0−6 h after inoculation OSCN− exerts an important antimicrobial activity that is then maintained by bLF over time. Conclusion: The data obtained by Alaxia on 165 clinical isolates of Burkholderia cepacia complex and other Burkholderia spp. demonstrates the high bactericidal potential of the OSCN− /bLF combination (ALX-009) and reinforces both the previous published data and the interest to developing this combination to respond to the unmet medical need of Burkholderia spp. infections in CF patients.
Posters
123 Twenty years of clinical studies on anti-Pseudomonas IgY to cystic fibrosis patients H. Kollberg1 . 1 Uppsala University, Pediatrics, Uppsala, Sweden Objectives: Clinical studies with CF-patients on Anti-psIgY (Anti-Pseudomonas IgY) to prevent infections with PA (Pseudomonas aeruginosa) in order to find out efficacy and adverse events have been running in Sweden for 20 years. AntipsIgY has got Orphan drug designation 2008. They are background for Phase III study started in 2011. Methods: Two studies for efficiency and advert events. Phase II 1995–2002: Two groups with intermittently PA-infected patients − one treated with Anti-psIgY, the other without IgY. Prolonged study 2002–now: Followed as first study but without controls. Two CF women (one pregnant twice) continued with Anti-psIgY during pregnancy. One boy transplanted 12 years ago after infections with PA and atyp. mycobact. Results: Group with Anti-psIgY: 2.35 pos PA cultures/100 months; Untreated group: 7 pos PA/100 months. Duration from first to second colonization was significantly prolonged for treated/control group (Kaplan–Meier p = 0.015). The time from first to chronic infection was prolonged in IgY group. Few infections minimized antibiotics. Lung function and BMI well preserved. Prolonged group: similar effects. Totally 33 patients included. Together they gargled >100,000 doses. No adverse events reported. All pregnancies went well and three healthy babies. Transplanted pat has not had any pseudomonas or atyp. mycobact. Discussion: Anti-PsIgY has good results in efficiency and absence of advert events. It reduces antibiotics and risk of resistance. Gargling is convenient. Treatment is cost effective. The cost for IgY less than for antibiotics. Costs for hospitalization is lower. Anti-psIgY might be registered, if phase III gives similar result.
124 Lack of antimicrobial activity of ivacaftor against clinical CF respiratory isolates J. Payne1 , L. McIlreavey1 , S. McGrath1 , M.M. Tunney1 , J.S. Elborn1 . 1 Queen’s University Belfast, CF and Airways Microbiology Research Group, Belfast, United Kingdom Objectives: Ivacaftor contains a quinolone ring within its structure, similar to that of Ciprofloxacin. A previous study demonstrated that ivacaftor had some antimicrobial activity against laboratory and non-CF clinical isolates of S. aureus and S. pneumoniae. The aim of this study was to investigate the antimicrobial activity of ivacaftor against clinical respiratory CF isolates. Methods: The MIC of ivacaftor against clinical isolates (P. aeruginosa, S. aureus, Streptococcus sp.; n = 5 from each genus) was determined using a radial diffusion assay. Bactericidal activity of ivacaftor against selected isolates growing planktonically and in biofilm was determined using a time-kill and a microtitre tray biofilm assay, respectively. Results were compared with ciprofloxacin. Results: The radial diffusion assay indicated that ivacaftor had no bacteriostatic activity with an MIC of >50 mg/mL for all isolates tested. In time-kill assays bactericidal activity was observed for Streptococcus sp. only (Table 1). Ivacaftor had no effect on biofilm formation. Table 1. Log change in CFU/mL (±SD) over 24 hours in time-kill assays P. aeruginosa S. aureus Streptococcus sp. Achromobacter sp. Stenotrophomonas sp.
Ivacaftor (32 mg/mL)
Ciprofloxacin (5 mg/mL)
Control
4.44±0.30 3.00±0.24 −3.17±2.26 2.83±0.27 2.69±0.16
−5.78±0.40 −5.63±0.02 −4.50±1.35 −1.63±3.84 −6.29
4.45±0.33 3.39±0.60 3.75±0.51 3.01±0.17 3.11±0.01
Conclusion: Ivacaftor did not have a direct antimicrobial action against the clinical CF isolates tested, and did not slow the growth of isolates or inhibit biofilm formation. Work supported by a DEL NI studentship and a US-Ireland Project Partnership Grant.
4. Microbiology
121 Extended HLA analysis in a cohort of patients hypersensitive to beta-lactam antibiotics P. Whitaker1 , A. Alfirevic2 , C. Etherington1 , I. Clifton1 , G. Fitch1 , M. Pirmohamed2 , D. Peckham1 . 1 Leeds Teaching Hospitals NHS Trust, Regional Adult CF Unit, Leeds, United Kingdom; 2 University of Liverpool, Institute of Translational Medicine, Liverpool, United Kingdom Objectives: Hypersensitivity reactions to beta-lactam antibiotics remain a major clinical problem in the management of patients with CF. Whilst reactions are more likely to occur in sicker patients receiving more frequent treatment this is not always the case and genetic predisposition may play a role. Methods: Patients were recruited from the Leeds Adult CF Centre. High resolution sequence based typing was performed on HLA A, B, C, DRB1, DQA1, and DQB1. The data was compared to our electronic patient records. Results: 263 patients took part in the study. 163 had a history of betalactam reactions and 100 were tolerant controls. 97 patients had a history of piperacillin hypersensitivity. No statistical differences in frequencies of single HLA alleles were seen between hypersensitive patients and controls. One haplotype (A*0201:B*4402:DRB1*0401) occurred at a higher frequency in patients with piperacillin reactions (OR 15.3; p = 0.0006). Conclusion: The high frequency of beta-lactam reactions in patients with CF makes it unlikely that a single HLA allele could account for increased predisposition alone. This study shows interesting data supporting an increased genetic risk in certain individuals and more work is needed to refine this further.
122 Successful Burkholderia spp. eradication with hypothiocyanite/ lactoferrin. In vitro study evidence over a worldwide collection of clinical strains Y. Sonmez1 , C. Bechetoille1 , S. Perrotto1 , A. Payet-Burin1 , V. Juarez-Perez1,2 . 1 Alaxia, Lyon, France; 2 Stragen France, Lyon, France Objectives: To test the efficacy of various combinations of OSCN− and bLF on clinical Burkholderia strains. Methods: We tested 165 Burkholderia spp. CF-clinical isolates from worldwide origin. MIC values were obtained according to CLSI guidelines with minor modifications and Time-kill curves with standard methods. All experiments were done in triplicate. Results: 100% of isolates tested were inhibited by OSCN− alone within a range of 30–157 mg/mL. In contrast, only 4.8% (8/165) of the tested strains are inhibited by bLF alone within a range of 0.25−96 mg/mL. The FIC analysis of the whole panel of strains reveals that inhibitory combinations are predominantly additive or synergistic for 73% of the collection. The combination of 118 mg/mL of OSCN− with 8 mg/mL of bLF inhibits 100% of tested isolates. Time-kill curves performed on 21 strains. Results show that the OSCN− /bLF combination (ALX-009) decreases the bacterial count by 2−4 logs at 6 h after inoculation when compared to the inoculum and by 4−6 logs if compared to the negative control. 24 h after inoculation, the bacterial counts were reduced by 4−6 logs if compared to the negative control. These results suggest that for time 0−6 h after inoculation OSCN− exerts an important antimicrobial activity that is then maintained by bLF over time. Conclusion: The data obtained by Alaxia on 165 clinical isolates of Burkholderia cepacia complex and other Burkholderia spp. demonstrates the high bactericidal potential of the OSCN− /bLF combination (ALX-009) and reinforces both the previous published data and the interest to developing this combination to respond to the unmet medical need of Burkholderia spp. infections in CF patients.
Posters
123 Twenty years of clinical studies on anti-Pseudomonas IgY to cystic fibrosis patients H. Kollberg1 . 1 Uppsala University, Pediatrics, Uppsala, Sweden Objectives: Clinical studies with CF-patients on Anti-psIgY (Anti-Pseudomonas IgY) to prevent infections with PA (Pseudomonas aeruginosa) in order to find out efficacy and adverse events have been running in Sweden for 20 years. AntipsIgY has got Orphan drug designation 2008. They are background for Phase III study started in 2011. Methods: Two studies for efficiency and advert events. Phase II 1995–2002: Two groups with intermittently PA-infected patients − one treated with Anti-psIgY, the other without IgY. Prolonged study 2002–now: Followed as first study but without controls. Two CF women (one pregnant twice) continued with Anti-psIgY during pregnancy. One boy transplanted 12 years ago after infections with PA and atyp. mycobact. Results: Group with Anti-psIgY: 2.35 pos PA cultures/100 months; Untreated group: 7 pos PA/100 months. Duration from first to second colonization was significantly prolonged for treated/control group (Kaplan–Meier p = 0.015). The time from first to chronic infection was prolonged in IgY group. Few infections minimized antibiotics. Lung function and BMI well preserved. Prolonged group: similar effects. Totally 33 patients included. Together they gargled >100,000 doses. No adverse events reported. All pregnancies went well and three healthy babies. Transplanted pat has not had any pseudomonas or atyp. mycobact. Discussion: Anti-PsIgY has good results in efficiency and absence of advert events. It reduces antibiotics and risk of resistance. Gargling is convenient. Treatment is cost effective. The cost for IgY less than for antibiotics. Costs for hospitalization is lower. Anti-psIgY might be registered, if phase III gives similar result.
124 Lack of antimicrobial activity of ivacaftor against clinical CF respiratory isolates J. Payne1 , L. McIlreavey1 , S. McGrath1 , M.M. Tunney1 , J.S. Elborn1 . 1 Queen’s University Belfast, CF and Airways Microbiology Research Group, Belfast, United Kingdom Objectives: Ivacaftor contains a quinolone ring within its structure, similar to that of Ciprofloxacin. A previous study demonstrated that ivacaftor had some antimicrobial activity against laboratory and non-CF clinical isolates of S. aureus and S. pneumoniae. The aim of this study was to investigate the antimicrobial activity of ivacaftor against clinical respiratory CF isolates. Methods: The MIC of ivacaftor against clinical isolates (P. aeruginosa, S. aureus, Streptococcus sp.; n = 5 from each genus) was determined using a radial diffusion assay. Bactericidal activity of ivacaftor against selected isolates growing planktonically and in biofilm was determined using a time-kill and a microtitre tray biofilm assay, respectively. Results were compared with ciprofloxacin. Results: The radial diffusion assay indicated that ivacaftor had no bacteriostatic activity with an MIC of >50 mg/mL for all isolates tested. In time-kill assays bactericidal activity was observed for Streptococcus sp. only (Table 1). Ivacaftor had no effect on biofilm formation. Table 1. Log change in CFU/mL (±SD) over 24 hours in time-kill assays P. aeruginosa S. aureus Streptococcus sp. Achromobacter sp. Stenotrophomonas sp.
Ivacaftor (32 mg/mL)
Ciprofloxacin (5 mg/mL)
Control
4.44±0.30 3.00±0.24 −3.17±2.26 2.83±0.27 2.69±0.16
−5.78±0.40 −5.63±0.02 −4.50±1.35 −1.63±3.84 −6.29
4.45±0.33 3.39±0.60 3.75±0.51 3.01±0.17 3.11±0.01
Conclusion: Ivacaftor did not have a direct antimicrobial action against the clinical CF isolates tested, and did not slow the growth of isolates or inhibit biofilm formation. Work supported by a DEL NI studentship and a US-Ireland Project Partnership Grant.