Multicenter study of the prevalence and the resistance mechanisms of carbapenem-resistant Enterobacteriaceae (CPE) in Belgium in

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1 Multicenter study of the prevalence and the resistance mechanisms of carbapenem-resistant Enterobacteriaceae (CPE) in Belgium in Study conducted by: 1. National Reference Center (NRC) for ESBL-producing and carbapenemaseproducing Enterobacteriaceae, CHU Mont-Godinne Université catholique de Louvain and Hôpital Erasme ULB, Belgium. Pr. Y. Glupczynski, Dr. O. Denis. 2. A National Multicenter Study Group (see appendix: participating centers) Study summary report (revised date: August 24, 2012; version 5) 1

2 1. Introduction Acquired and transferable carbapenemases have been reported extensively worldwide initially in non-fermenting Gram-negative bacilli (mainly in Pseudomonas spp.) and subsequently in Enterobacteriaceae [1, 2]. Asymptomatic carriage and subsequent infection caused by carbapenemase-producing Enterobacteriaceae (CPE) isolates currently represent a major public health threat for the individual therapeutic management and for the collective infection control issues [3]. The prevalence of CPE in Europe varies significantly from high (Greece and Italy) to low (Nordic countries). The types of carbapenemase vary among countries, partially depending on the cultural population exchange relationship between the European countries and the possible reservoirs of each carbapenemase [4]. In Belgium, a longitudinal survey conducted by the national reference center for multidrug-resistant Enterobacteriaceae over the past years has shown a significant increase since year 2010 of the number of carbapenemase-producing Enterobacteriaceae (CPE) [5]. The rapidly growing proportion of non-travel associated CPE isolates (which suggests the local establishment and spread of these isolates) is particularly worrisome and emphasizes the need for a strengthened national surveillance programme. In December 2011, guidelines for the detection and the control of transmission of CPE in and across hospitals were issued by the Scientific Council of Health (Advisory report n 8791 from the Superior Health Council). A call for action was also raised by the Ministry of Public Health on 7 June 2012 (Circulaire de la Direction Générale pour l Organisation des Etablissements de Soins) and the directions of all affiliated acute institutions were invited to participate in an active surveillance programme that had been launched by Public Institute of Health and the Belgian National Reference Center since January 2012 onwards (National Surveillance Report D/2012/2505/17 of CPE in Belgium, IPH, B. Jans & Y. Glupczynski). The present survey aimed to estimate a point prevalence of carbapenem-nonsusceptible Enterobacteriaceae (CNSE) and of CPE isolates among hospitalized patients in Belgium. 2. Objectives 1. To screen consecutive non-duplicate Enterobacteriaceae isolates originating from clinical specimens for decreased susceptibility (i.e.: intermediate or resistant) to several carbapenem agents (ertapenem, imipenem and meropenem) and to estimate a point prevalence (over a 1 to max 2-month period) of carbapenemnon-susceptible Enterobacteriaceae (CNSE) isolates in Belgian hospitals in To determine the resistance mechanisms of CNSE isolates by phenotypic and by molecular tests (detection of carbapenemase encoding genes by multiplex PCR) and to calculate a point prevalence of carbapenemase-producing Enterobacteriaceae (CPE) isolates in Belgian hospitals. 2

3 3. Materials and Methods 3.1 Centers involved: 24 hospital-based laboratories (including tertiary care referral and general hospitals) serving medium-to-large size hospitals throughout Belgium (cf. list of participants in Appendix A) were required to test Enterobacteriaceae isolates. Two out of 26 labs that had been contacted refused to participate, both because of the added workload generated by this study coupled with a lack of financial incentives for participation. 3.2 Inclusion criteria and testing at participating centers: Isolates to screen for carbapenem susceptibility: Each laboratory was requested to collect consecutively 200 Enterobacteriaceae isolates (over 1 month period to a maximum of 2 months): From hospitalized patients only (hospitalization duration of <48 h was acceptable and did not constitute an exclusion criterion) whatever the hospitalization wards/unit Without any restriction on the type of specimens and sampling sites (screening samples were also allowed but specimens from environmental sources were not accepted) Only one (the first) isolate of the same species per patient was included (inclusion of duplicates was not allowed). The sample collection and culture, strain isolation and bacterial identification were performed using local standard procedures. All collected isolates were tested locally for carbapenem susceptibility using disk diffusion method according to a well standardized and common testing protocol: A 0.5 MacFarland density-adjusted suspension of colonies from pure culture was inoculated onto a Mueller-Hinton plate (Oxoid). Paper discs of meropenem (10-µg), imipenem (10-µg) and ertapenem (10-µg) (Oxoid) were placed onto a 9 mm diameter MH plate incubated for 18 hours before reading. All provided paper discs and Mueller-Hinton plates for the testing were purchased from the same manufacturer (Oxoid) and had the same batch number of manufacture. For each of the isolates, inhibition zone diameters of the three carbapenems tested, as well as the sample and demographic patient data were recorded on a registration form sent to the reference laboratory CNSE isolates to select for characterization of resistance mechanisms by the reference lab: All tested Enterobacteriaceae isolates showing a decreased susceptibility according to CLSI interpretative criteria (document M100-S22; January 2012) to at least one of the three carbapenems tested (meropenem (zone diameter <23 mm) and/or imipenem (zone diameter <23 mm) and/or ertapenem (zone diameter <22 mm)) were 3

4 retained and referred to the reference lab. For Proteus, Morganella and Providencia spp. which intrinsically display a lower susceptibility to imipenem, only meropenem and/or ertapenem zone sizes were considered for screening of decreased susceptibility to carbapenems Quality control (QC) tests: The disc diffusion susceptibility to carbapenems of the E. coli ATCC isolate and of three provided carbapenemase-positive K. pneumoniae (one VIM-1, one KPC- 2 and one OXA-48-producing) isolates was measured at separate intervals of times at least twice during the survey. MIC values of 14 antimicrobial agents including carbapenems were determined centrally at the reference laboratory by broth microdilution method using Sensititre panels (TREK Diagnostic Systems, East-Grinstead, UK) and MIC of temocillin by E- test diffusion method for all four control strains. 3.3 Characterization of resistance mechanisms at the reference lab and data analysis: The identification of all CNSE isolates (as defined in 3.2.2) sent to the reference laboratory was verified by MALDI-TOF method with Microflex LT (Bruker Daltonik, GmbH, Bremen, Germany); the isolates were retested for in vitro susceptibility to a panel of 16 antimicrobial agents by disk diffusion method and multiplex PCR targeting bla VIM, bla IMP, bla NDM, bla KPC and bla OXA-48 was performed for the detection and confirmation of carbapenemase encoding genes. For confirmed CPE isolates, MIC values of 14 antimicrobial agents including carbapenems were determined by broth microdilution method using Sensititre panels and MIC of temocillin was tested separately by E-test method (biomérieux, Brussels, Belgium). Susceptibility categorization was interpreted according to CLSI interpretative criteria (CLSI document M100-S22; January 2012) for all antimicrobial agents except for tigecycline (2012 EUCAST clinical breakpoints v2.0) and for temocillin (breakpoints according to Fuchs et al. Eur J Clin Mic. 1985;4:30-3). Susceptibility rates for the three carbapenems tested, prevalence of CNSE and of CPE isolates were calculated overall and for each center. Global prevalence data were also calculated by species. 4

5 4. Results 4.1 Bacterial isolates Overall, 4564 Enterobacteriaceae isolates were screened for decreased carbapenem susceptibility in the 24 participating laboratories from February to April The numbers of total isolates tested and the proportions of CNSE isolates detected per center are shown in Table 1. A mean number of 190 isolates per laboratory was tested, 16 centers reaching the expected number of 200 isolates; only one center screened less than 150 isolates (78 isolates for center C18). The calculated proportion of CNSE isolates representing the point prevalence of CNSE isolates was 3.5% (158/4564) overall and ranged per center from 0.5% to 8.5% (the average and the median proportion of CNSE were 3.4% and 3.7% respectively). Table 1. Distribution per center of Enterobacteriaceae isolates screened and of CNSE isolates detected (n isolates) Center n Screened CNSE %CNSE C % C % C % C % C % C % C % C % C % C % C % C % C % C % C % C % C % C % C % C % C % C % C % C % Total % The distribution of total screened and of CNSE isolates according to the sample collection sites, to the medical wards of the patients at the time of sampling and to the presumptive mode of acquisition of the isolate (within 48 h versus more than 48 h of hospitalization) are shown in Table 2. 5

6 About one-third each of the 158 CNSE isolates were isolated from urine (n=53) and from lower respiratory tract samples (n=49). However, the highest proportion of CNSE isolates was found in respiratory tract (6.9%). Medical care units and intensive care units (ICUs) were the two wards with the highest number of CNSE isolates (59 and 51 respectively) and the highest proportion of CNSE isolates was found in ICU (6.9%). As anticipated, the majority of CNSE isolates (73%) were considered as probably nosocomially-acquired (based on the definition of patient hospitalized for more than 48 hours). Table 2. Distribution of total screened and of CNSE isolates per sample origin, per medical ward and per presumptive mode of acquisition (n isolates) Sample data Screened CNSE %CNSE Sample origin Urine % Respiratory % Pus % Other % Blood % Screening % Unknown 6 0.0% Ward Medecine % ICU % Surgery % Other % Pediatrics % Unknown 8 0.0% Nosocomial No % Yes % Unknown % Total % The number of tested isolates per species and their susceptibility results to carbapenems interpreted according to the 2012 CLSI breakpoints criteria are detailed in Table 3. Of the three carbapenems tested, meropenem appeared to be the most active agent against the screened isolates (99.0% of susceptibility), while ertapenem and imipenem displayed less potent activity with 97.8% and 93.6% of the isolates susceptible to these two drugs respectively. 6

7 Table 3. Distribution per species/genus and the susceptibility results to carbapenems according to the 2012 CLSI interpretative criteria of the screened isolates (n isolates) Meropenem Imipenem Ertapenem Species or genus Total S I/R S I/R S I/R E. coli K. pneumoniae P. mirabilis E. cloacae K. oxytoca M. morganii S. marcescens E. aerogenes C. freundii C. koseri P. vulgaris/penneri H. alvei Providencia spp Klebsiella spp Salmonella spp C. braakii S. liquefaciens E. asburiae Enterobacter spp Citrobacter spp Serratia spp E. kobei R. ornitholytica Proteus spp A. hydrophila Total % of all isolates 99.0% 1.0% 93.6% 6.4% 97.8% 2.2% S, susceptible; I/R, intermediate/resistant 4.2 CNSE isolates referred and characterized at the reference lab Of the 158 CNSE isolates detected at the local laboratories, 123 were referred to the reference laboratory for the detection of carbapenemase production. This implies that the results reported here should be considered as minimal estimate of prevalence. The number of tested CNSE isolates per species and of CPE isolates detected by PCR are shown in Figure 1. E. cloacae and E. aerogenes together accounted for half of the total CNSE isolates (62/123) including 44 (71%) of these 62 Enterobacter spp. isolates being resistant to ertapenem and susceptible to meropenem. Remarkably, none of these Enterobacter spp. isolates could be confirmed as CPE. On the other hand, two-thirds of the K. pneumoniae isolates not susceptible to a carbapenem (11/17; 65%) were subsequently confirmed as CPE (OXA-48 [n=7], KPC-type [n=3] and NDM-type [n=1]). One OXA-48-positive E. coli and one KPCpositive K. oxytoca carbapenemase-producing isolates were also detected by PCR. The proportion of CPE isolates per species among the total number of isolates tested was therefore 2.44%, 0.47% and 0.04% and for K. pneumoniae (11/451), K. oxytoca (1/211) and E. coli (1/2580), respectively. 7

8 Figure 1. Species distribution and carbapenemase enzyme detected in CNSE isolates referred and characterized at the reference lab (n=123) n CNSE isolates E. cloacae E. aerogenes K. pneumoniae Species E. coli C. freundii E. asburiae S. marcescens E. kobei C. braakii K. oxytoca H. alvei R. ornitholytica Negative OXA-48 KPC NDM The distribution of susceptibility to the three carbapenems tested and the type of carbapenemase encoding gene detected in the 123 CNSE isolates referred and characterized at the reference laboratory is summarized in Table 4. All but one of the 13 confirmed CPE isolates were intermediately-resistant or resistant to all three carbapenems disks tested. One K. pneumoniae OXA-48-positive isolate displayed an inhibition zone to meropenem of 23 mm and was therefore classified as meropenem susceptible according to the 2012 CLSI breakpoints. Table 4. Distribution of the susceptibility results of CNSE to the different carbapenems agents according to the 2012 CLSI interpretative criteria and of carbapenemase coding genes (n isolates) Susceptibility to carbapenem Carbapenemase enzyme detected Ertapenem Imipenem Meropenem Negative OXA-48 KPC NDM Total No No No Yes Yes No 7 7 Yes Yes No No 4 4 Yes Yes No 2 2 Total In total, 8 out of the 24 participating centers collected at least one CPE isolate; the distribution of the proportion of CPE by centre is listed in Table 5. The minimal estimated proportion of CPE isolates representing the point prevalence of CPE 8

9 isolates was 0.28% (13/4564) overall and ranged per center from 0% (for 16 centers) to 1.5%. Table 5. Distribution per center of carbapenemase-producing Enterobacteriaceae isolates detected (n isolates) Carbapenemase enzyme detected Total Total Center n Negative OXA-48 KPC NDM referred screened % CPE/screened C ,0% C ,0% C ,5% C ,0% C ,0% C ,0% C ,0% C ,0% C ,0% C ,0% C ,0% C ,5% C ,6% C ,0% C ,5% C ,0% C ,5% C ,0% C ,5% C ,0% C ,0% C ,0% C ,5% C ,0% Total ,3% MIC and susceptibility results to 15 tested antimicrobials for the 13 CPE isolates detected (together with the three quality control CPE strains) are detailed in Table 6. Using 2012 CLSI interpretative criteria, all CPE isolates were found intermediately or fully resistant to ertapenem and to piperacillin/tazobactam, while 5 OXA-48-positive K. pneumoniae isolates had imipenem and/or meropenem MICs within the susceptible category. All but one (KPC-positive) of the CPE isolates were resistant to temocillin by E-test method including all OXA-48 carbapenemase-producing isolates displaying high level resistance to temocillin (MIC 512 µg/ml). Only one NDM-positive K. pneumoniae isolate was resistant to colistin and two OXA-48- positive K. pneumoniae isolates showed intermediately resistance to tigecycline according to the 2012 EUCAST clinical breakpoints. 9

10 Table 6. MIC (a) (b) and susceptibility categorization results (c) to 15 antimicrobials of CPE isolates (including the three quality control strains) Center n Origin Ward Nosocomial Isolate Bacterial species Carbapenemase reference n enzyme TMO CTX CAZ FEP PTZ ERT IMI MER AZT GEN AMI TOB CIP TIG COL C03 Urine Medecine No S K. pneumoniae OXA-48 > <=0.5 2 > <=0.5 <=1 <=4 <=1 <= C10 Urine Surgery Yes S K. pneumoniae OXA > > <=1 <=4 8 > C10 Urine Surgery No S K. pneumoniae OXA > <= <=1 <=4 8 > C12 Respiratory ICU Yes S K. oxytoca KPC >64 32 > > >8 > C12 Pus Medecine No S K. pneumoniae NDM 48 >64 >64 >64 > >64 >8 <=4 8 >2 0.5 >8 C12 Respiratory Medecine Yes S K. pneumoniae KPC 48 >64 >64 32 > >64 >8 >32 >8 > C13 Respiratory ICU Yes S E. coli OXA > >8 <= <= C15 Urine Other No S K. pneumoniae OXA > > >8 <=4 >8 > C17 Screening ICU Yes S K. pneumoniae OXA-48 >1024 >64 >64 >64 >128 > >64 >8 <=4 >8 >2 2 1 C17 Respiratory ICU Yes S K. pneumoniae OXA-48 > <=0.5 <= <=0.5 <=1 <=4 <=1 <= C17 Screening ICU Yes S K. pneumoniae OXA-48 >1024 >64 32 >64 >128 > >8 <=4 >8 > C19 Urine Medecine Yes S K. pneumoniae KPC 64 >64 >64 >64 >128 >32 >32 >32 > >8 <= C23 Pus Medecine Yes S K. Pneumoniae KPC 1024 >64 >64 >64 >128 >32 32 >32 > >8 > #08119 K. Pneumoniae OXA-48 >1024 <=0.5 <=0.5 <= <=0.25 <=0.5 <=1 <=4 <=1 > Quality control strains #08386 K. Pneumoniae VIM-1 >1024 >64 >64 >64 > > >8 >2 0.5 >8 #11323 K. Pneumoniae KPC >64 64 >128 > > >8 > Susceptible Intermediate Resistant (a) MIC determined by E-test method for TMO (temocillin) (b) MIC determined by broth microdilution method (Sensititre panels) for CTX (cefotaxime), CAZ (ceftazidime), FEP (Cefepime), PTZ (piperacillin/tazobactam), ERT (ertapenem), IMI (imipenem), MER (meropenem), AZT (aztreonam), GEN (gentamicin), AMI (amikacin), TOB (tobramycin),cip (ciprofloxacin), TIG (Tigecycline), COL (colistin) (c) Susceptibility categorization was interpreted according to 2012 CLSI breakpoints for all antimicrobial agents except tigecycline (2012 EUCAST breakpoints v2.0) and temocillin (Fuchs et al. Eur J Clin Mic 1985;4:30-3) 10

11 4.3 Quality control testing: The average values of the zone inhibition diameters (ZIDs) of the three carbapenems 10-µg paper disks tested against the four control strains are shown and detailed per center in Table 7. A total of 20 out of 24 centers tested at least twice the four control strains as requested in the study protocol. One center (C21) tested 5 times each the E. coli ATCC strain and the K. pneumoniae #08119 OXA-48-positive strain. Three centers did not provide any data of quality control testing. Overall 59 measurements of the three carbapenems tested were obtained with K. pneumoniae #08386 VIM-1-positive strain and K. pneumoniae #11323 KPC-2-positive strain, while 64 measurements were recorded for K. pneumoniae #08119 OXA-48-positive strain and for E. coli ATCC reference strain. Of the 64 measurements in total with the E. coli ATCC reference strain, 10, 15 and 20 results were superior to the acceptable upper QC limit for ertapenem, imipenem and meropenem disks respectively, while 4, 3 and 2 results were inferior to the acceptable lower QC limit respectively for these three carbapenems (QC limits from CLSI 2012). The overall calculated mean values and one standard deviation (SD) of the three carbapenems ZIDs are summarized in Figure 2. The highest variation of reading results was observed with ertapenem disk to K. pneumoniae #08119 OXA-48-positive strain, having a SD of 3.8 mm and could be explained by the inconsistently resistant colonies growing within the zone of inhibition frequently observed. All centers but two (C09 and C11) had returned the three carbapenemase-positive control strains to the reference laboratory and all isolates underwent multiplex PCR and were confirmed to harbour the correct carbapenemase-encoding gene. Figure 2. Mean of carbapenem disks inhibition diameters (mm) to the four quality control strains (59 measures) 40 Mean of disk inhibition diameter (mm) K. pneumoniae #08386 (VIM-1) K. pneumoniae #11323 (KPC-2) K. pneumoniae #08119 (OXA-48) Ertapenem Imipenem Meropenem Quality control strains reference number (carbapenemase enzyme) E. coli ATCC (none) 11

12 Table 7. Distribution per center of the mean of carbapenems zone inhibition diameter results (mm) for the four quality control strains K. pneumoniae #08386 (VIM-1) K. pneumoniae #11323 (KPC-2) K. pneumoniae #08119 (OXA-48) E. coli ATCC (none) Center n (n measures) Ertapenem Imipenem Meropenem Ertapenem Imipenem Meropenem Ertapenem Imipenem Meropenem Ertapenem Imipenem Meropenem C01 (2) C02 (2) C03 (2) C04 (2) C05 (3) C06 (0) C07 (3) C08 (3) C09 (5) C10 (2) C11 (2) C12 (3) C13 (0) C14 (10) C15 (3) C16 (3) C17 (2) C18 (0) C19 (3) C20 (3) C21 (5) C22 (2) C23 (2) C24 (2) Mean of all centers (a) SD of all centers (a) Acceptable QC limits (CLSI 2012) Inferior to the lower QC limit Superior to the upper QC limit (a) 59 measures of the three carbapenems ZIDs for K. pneumoniae #08386 and K. pneumoniae #11323 strains ; 64 for K. pneumoniae #08119 and E. coli ATCC strains 12

13 5. Conclusion Overall, a point prevalence of 3.5% of CNSE isolates was found in this multicentric national survey. The calculated (minimal estimated) prevalence of CPE isolates was 0.28% since roughly 10% of the referred CNSE isolates were found to carry a carbapenemase-encoding gene. It is however worrisome to observe that one third of the 24 participating laboratories had isolated at least one CPE isolate in their institution during the study period. Half of the CNSE isolates belonged to E. cloacae and E. aerogenes (the majority of these being ertapenem resistant and meropenem susceptible), but none of them could be confirmed as CPE. K. pneumoniae was by far the most encountered CPE species and OXA-48 was the most frequent carbapenemase enzyme detected which clearly confirms the data obtained from the national surveillance programme (National Surveillance Report D/2012/2505/17 of CPE in Belgium, IPH, B. Jans & Y. Glupczynski). Our study results confirmed the lack of specificity of ertapenem disk for the detection of CPE isolates, mostly in E. cloacae and E. aerogenes and suggest that Enterobacter spp. isolates resistant to ertapenem only should not be sent to the reference lab for unnecessary confirmation of CPE. Although in the present study we did only screen for CPE the subset of carbapenem non-susceptible Enterobacteriaceae isolates (i.e: not excluding the possible occurrence of poorly expressed carbapenemases in carbpenem-susceptible isolates), the very low overall point prevalence value of CPE obtained together with susceptibility results of CPE isolates collected by the national reference centre in the setting of the national surveillance programme (data not shown) question the accuracy (sensitivity) of the current carbapenems susceptibility breakpoint (CLSI or EUCAST). Further studies are necessary to improve the laboratory detection strategies of CPE including adjusted screening cut-offs for carbapenems. Pending such data are obtained, we suggest based on the experience of the NRC that all K. pneumoniae isolates with meropenem zone <27 mm (10-µg paper discs) or meropenem MIC of >0.25 µg/ml should be considered for screening of CPE. 6. References 1 Queenan AM, Bush K. Carbapenemases: The versatile beta-lactamases. Clin Microbiol Rev. 2007; 20: , table of contents. 2 Nordmann P, Naas T, Poirel L. Global spread of carbapenemase-producing enterobacteriaceae. Emerg Infect Dis. 2011; 17: Nordmann P, Cornaglia G. Carbapenemase-producing enterobacteriaceae: A call for action! Clin Microbiol Infect. 2012; 18: Canton R, Akova M, Carmeli Y, et al. Rapid evolution and spread of carbapenemases among enterobacteriaceae in europe. Clin Microbiol Infect. 2012; 18: Huang TD, Bogaerts P, Berhin C, et al. Rapid emergence of carbapenemaseproducing enterobacteriaceae isolates in belgium. Euro Surveill. 2011;

14 7. Appendix A List of participants Centers AZ St-Jan Brugge CHR Namur CHU Brugmann CHU Mont-Godinne CHU Sart-Tilman Liège CHU St-Pierre Bruxelles Clinique Saint-Elisabeth Namur Cliniques Braine-L alleud Cliniques St Joseph Charleroi Cliniques St Pierre Ottignies Cliniques Sud Luxembourg Arlon Cliniques Univ. St-Luc Bruxelles CHR citadelle Liège Hôpital Erasme OLV Aalst Imeldaziekenhuis Bonheiden St-Lucas Gent CHU Tivoli La Louviere UZ Antwerpen UZ Brussel UZ Leuven UZ RUG Gent ZOL Genk Virga Jesse Hasselt Investigator names Dr.E. Nulens Dr. M.-G. Garrino Dr. G. Mascart/ Pharm. Y. Miendje Dr. T.-D. Huang/Pr. Y. Glupczynski Prof. P. Melin Dr. Anne Dediste/Prof. O. Vandenberg Pharm. J. Caddrobi Dr. Y. DeGheldre Dr. B. Lissoir Pharm. Biol. V. Verbelen Dr. P. Goffinet/ Pharm. J.S. Goffinet Prof. A. Simon/ Dr H. Rodriguez Dr. J.M. Senterre Dr. O. Denis / C. Nonhoff Dr. K. Van Vaerenbergh /A. Boel Dr. J. Frans Dr.A.-M. Vandenabeele Dr. C. Potvliege/Mme A. Pernet Prof. G. Ieven/ Prof. H. Goossens Prof. D. Pierard Prof. J. Verhaegen Prof. G. Claeys Dr. G. Coppens Dr. K. Magerman 14